阅读说明：本技术 Ezh2抑制剂及其用途 (Ezh2 inhibitor and application thereof ) 是由 J.E.布拉德纳 J.齐 K.K.王 于 2015-11-06 设计创作，主要内容包括：本申请提供式(I)和(II)中任一个的化合物。本文描述的化合物是组蛋白甲基转移酶(例如,zeste增强子同源物1(EZH1)和zeste增强子同源物2(EZH2))的抑制剂并用于治疗和/或预防宽范围的疾病(例如,增殖性疾病)。本申请还提供药物组合物、试剂盒、方法和用途,包括或使用本文描述的化合物。本申请还提供鉴别EZH1和/或EZH2抑制剂的方法。(The present application provides compounds of any one of formulas (I) and (II). The compounds described herein are inhibitors of histone methyltransferases (e.g., enhancer of zeste homolog 1(EZH1) and enhancer of zeste homolog 2(EZH2)) and are useful in the treatment and/or prevention of a wide range of diseases (e.g., proliferative diseases). The present application also provides pharmaceutical compositions, kits, methods and uses, including or using the compounds described herein. The present application also provides methods of identifying EZH1 and/or EZH2 inhibitors.)

1. A compound of formula (II), or a pharmaceutically acceptable salt thereof:

wherein:

R^B1is a substituted or unsubstituted heterocyclic radical, -CN, -C (═ O) N (R)^b)₂Or

Each R^bIndependently hydrogen or a substituted or unsubstituted acyl group;

R^Ais hydrogen, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R^Bis hydrogen, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

or R^AAnd R^BAre linked to form a substituted or unsubstituted carbocyclic ring or a substituted or unsubstituted heterocyclic ring;

R^Cis hydrogen;

R^B2is a substituted or unsubstituted heterocyclic group or a warhead;

wherein the warhead is:

wherein:

L³is a bond or substituted or unsubstituted C_1-4A hydrocarbon chain;

R^E1、R^E2and R^E3Each is hydrogen; and

y is O;

R^B3is hydrogen;

R^B4is hydrogen; and

R^B5is of the formula:

wherein:

R^B6is substituted or unsubstituted C_1-6An alkyl group;

R^B7is substituted or unsubstituted C_2-6An alkyl group;

R^B8is substituted or unsubstituted C_1-6An alkyl group;

R^B9is substituted or unsubstituted C_1-6An alkyl group;

R^B10is-N (R)^b)₂；

Each R^B11Independently is halogen;

u is 0, 1, 2, 3 or 4;

R^B12is hydrogen;

each R^B13Independently is substituted or unsubstituted C_1-6An alkyl group; and

v is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9.

2. The compound of claim 1, wherein R ^B5Is of the formula:

3. the compound of claim 1 or 2, wherein R^B5Is of the formula:

4. the compound of claim 1 or 2, wherein R^B6Is Me.

5. The compound of claim 1 or 2, wherein R^B7Is n-Pr.

6. The compound of claim 1 or 2, wherein R^B2Is warhead and L³Is substituted or unsubstituted C_1-4A hydrocarbon chain.

7. The compound of claim 6, wherein R^B1Is a substituted or unsubstituted 3-to 7-membered monocyclic heterocyclyl containing 0, 1 or 2 double bonds in the heterocyclyl system, wherein 1, 2 or 3 atoms in the heterocyclyl system are independently nitrogen, oxygen or sulfur.

8. The compound of claim 7, wherein R^B1Is a substituted or unsubstituted piperazinyl group.

9. The compound of claim 8, wherein R^B1Is of the formula:

10. the compound of claim 9, wherein R^B6Is Me and/or R^B7Is n-Pr.

11. The compound of claim 1 or 2, wherein R^B1Is a substituted or unsubstituted piperazinyl, -CN or-C (═ O) N (R)^b)₂。

12. The compound of claim 1 or 2, wherein R^B1is-C (═ O) NH₂Or of the formula:

13. the compound of claim 1 or 2, wherein R^B2Is a substituted or unsubstituted tetrahydropyranyl group.

14. The compound of claim 1 or 2, wherein R^B2Is composed of

15. The compound of claim 1, wherein R^B5Is of the formula:

16. The compound of claim 15, wherein R^B5Is of the formula:

17. the compound of claim 1, wherein R^B5Is of the formula:

18. the compound of claim 1, wherein R^B5Is of the formula:

19. the compound of claim 18, wherein R^B5Is of the formula:

20. the compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is of the formula:

21. the compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound is of the formula:

22. the compound of claim 1 or 2, wherein R^B1Is a substituted or unsubstituted 3-to 7-membered monocyclic heterocyclyl containing 0, 1 or 2 double bonds in the heterocyclyl system, wherein 1, 2 or 3 atoms in the heterocyclyl system are independently nitrogen, oxygen or sulfur.

23. The compound of claim 1 or 2, wherein R^B1Is a substituted or unsubstituted piperazinyl group.

24. The compound of claim 1 or 2, wherein R^B1Is of the formula:

25. a compound, or a pharmaceutically acceptable salt thereof, which compound is:

26. a pharmaceutical composition comprising a compound of any one of claims 1-2, 7-10, 15-21, or 25, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

27. Use of a compound according to any one of claims 1-2, 7-10, 15-21 or 25 in the manufacture of a medicament for the treatment of a disease associated with aberrant or increased activity of histone methyltransferase.

28. The use of claim 27, wherein the disease is a genetic disease, a proliferative disease, a benign tumor, pathological angiogenesis, an inflammatory disease, an autoimmune disease, a hematologic disease, a neurological disease, a painful condition, a psychiatric disease, a metabolic disorder, a hyperplasia, brain cancer, breast cancer, lymphoma, leukemia, weber's syndrome, lung cancer and/or non-small cell lung cancer.

29. Use of a compound according to any one of claims 1-2, 7-10, 15-21 or 25 in the manufacture of a medicament for inhibiting the activity of histone methyltransferase.

30. An in vitro method of inhibiting histone methyltransferase activity in a cell, the method comprising contacting the cell with an effective amount of a compound of any one of claims 1-2, 7-10, 15-21, or 25.

31. A method of making chromatin, comprising: mouse lung Tissue was comminuted using the Covaris Tissue Smasher model CP02 according to CryoPrep pulserizin manual; lung tissue was pulverized 1-2 times in tissue TUBE (Covaris #520071) at setting 4; approximately 50mg of comminuted lung tissue was cross-linked with pre-warmed 1% formaldehyde (ThermoScientific #28906, diluted in PBS) at 37 ℃ for 20 minutes; the tissue was centrifuged at 1000rpm for 2 minutes and quenched with 0.125M glycine in PBS + 0.5% BSA for 20 minutes at room temperature, centrifuged at 1,000rpm for 2 minutes and washed with PBS +2x Protease Inhibitor Cocktail (PIC) (Roche #11873580001) +5mM sodium butyrate (Millipore #19-137) and then centrifuged at 1,000rpm for 2 minutes; the crosslinked tissue was then lysed with 390 μ L CHIP lysis buffer (1% SDS, 10mM EDTA pH8.0, 50mM Tris-HCl pH8.0, 2 XPIC and 5mM sodium butyrate) on ice for 1 hour; the lysate was aliquoted into 3 microtubes (Covaris #520045) and sheared on the Covaris E210 line at 5% duty cycle, 5 intensity, 200 cycles per burst (burst) for a total of 27 minutes; the sheared chromatin was centrifuged at 14,000rpm for 15 min at 4 ℃; the added aliquots were stored while the remaining chromatin was rapidly frozen and stored at-80 ℃; the addition was increased to 100. mu.l with TE, 10. mu.g of RNAseA (Roche) was added and incubated for 30 min at 37 ℃ followed by 100. mu.g of proteinase K (Roche) and incubated overnight at 65 ℃; the addition was purified and quantified using Qiagen PCR purification kit (# 28104).

32. A method for an intracellular EZH2 binding assay, comprising: MDA-MB-231 cells (ATCC) were cultured in Ham's F12K medium supplemented with 10% (v/v) FBS and 100UmL-1 penicillin-streptomycin; cells were diluted at 1e5 cells/mL in culture medium and seeded at 40. mu.L/well in black, clear-bottomed 384-well plates (Aurora IQ-EB 384) at 37 ℃/5% CO₂Incubating for 24 hours; to assess overall involvement of endogenous EZH2, cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.3% Triton X-100 in PBS for 20 minutes; cells were then incubated with 40 μ L of 1nM Hoechst 33342(Life Technologies), 5 μ M TAMRA-EZ05 and various concentrations of competitor compound; after incubation at room temperature for 1 hour, wash with 40 μ L PBSCells 4X, then imaged; to assess cell permeability and involvement of endogenous EZH2, TAMRA-EZ05 and competitor compounds were needle transferred into culture media and allowed to stand at 37 ℃/5% CO₂Incubation with cells for 2 hours followed by fixation and counterstaining (fig. 38, fig. 39, fig. 40); all plates were imaged on an ImageXpress Micro automatic microscope (Molecular Devices) using a 10X objective with laser based focusing; image analysis was performed using the Cell sorting module in metaxpress (molecular devices) to determine mean nuclear fluorescence and nuclear counts per well; calculate mean and standard deviation (STDEV) for all DMSO wells in each assay plate; data were analyzed and plotted using GraphPad PRISM v6, IC determined using a 'log (inhibitor) versus normalized response-variable slope' analysis module ₅₀The value is obtained.

Background

Chromatin structure contains important regulatory information for all DNA-based processes such as transcription, repair and replication. In eukaryotes, polycomb group (PcG) and drosophila Trx group (TrxG) protein complexes regulate chromatin structure through evolutionarily conserved mechanisms for gene silencing or activation, respectively (Schuettengruber et al, 2007). Polycombin assembles into at least two distinct complexes, polycombin inhibitory complexes 1 and 2 (PRC 1 and PRC2, respectively). Several lines of evidence demonstrate that PCR2 is involved in recruiting the PRC1 complex into the promoter of their common target genes. The precise balance of PcG protein levels ensures proper cell proliferation and normal tissue homeostasis, while abnormal expression patterns or genomic changes in PcG proteins can lead to transcriptional dysregulation and cause a variety of diseases including cancer (Laugesen and hellin, 2014).

The histone tail extends from the nucleosome core and undergoes a variety of modifications including phosphorylation, acetylation, methylation, ubiquitination and sumoylation. Histone modifications have a major impact on transcriptional regulation by modulating higher chromatin structure. Chromatin has two functional states: euchromatin and heterochromatin, which are transcriptionally activated and transcriptionally inactivated, respectively. Some histone modifications, such as tri-methylation at histone 4 lysine 20(H4K20me3), histone 3 lysine 9(H3K9me3), or lysine 79(H3K79me3), occur primarily in constitutive heterochromatin domains; while other modifications such as tri-methylation at histone 3 lysine 4(H3K4me3) and acetylation at histone 3 lysine 27(H3K27ac) are considered markers of actively transcribed regions in euchromatin. Trimethylation at histone 3 lysine 27(H3K27 me3) is often associated with transcriptional expression in higher eukaryotes (Cao et al, 2002; Czermin et al, 2002; Muller et al, 2002). The bivalent domain (contradictory coexistence called the suppressor H3K27me3 and the activator H3K4me3) keeps the developmental genes in a silent but balanced state for activation upon differentiation (Chen and Dent, 2014).

Enhancer of Zeste homolog 2(EZH2) is the core component of PRC2, which catalyzes the di-and tri-methylation of lysine 27 of histone H3 (H3K27me 2/3). EZH2 plays a key role in normal development, and EZH2 deficient mice die from early embryonic stages due to implantation and failure of gastrulation (O' Carroll et al, 2001). Somatic mutations in the SET domain of EZH2 (e.g., Y641N) that lead to hyperactivation of enzymes have been identified in most follicular and diffuse large B-cell lymphomas, suggesting a driver function for EZH2 in cancer development (Beguelin et al, 2013; Morin et al, 2010). Recently a GEM model with conditional expression of EZH2(Y641N) with mutations was developed which, in cooperation with BCL2, induces Germinal Center (GC) proliferation and accelerates lymphomata development (Beguelin et al, 2013).

Disclosure of Invention

In one aspect, described herein are compounds of any one of formulas (I) and (II) and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives and prodrugs thereof. The compounds described herein are inhibitors of histone methyltransferases (HMTs, e.g., enhancer of zeste homolog 1(EZH1), enhancer of zeste homolog 2(EZH 2)). The compounds are useful for treating and/or preventing a disease associated with aberrant activity or increased activity of HMT, such as a proliferative disease, an inflammatory disease, an autoimmune disease, a genetic disease, a hematological disease, a neurological disease, a painful disorder, a psychiatric disease, or a metabolic disorder in a subject in need thereof. The compounds may also be used to induce apoptosis in a cell. The present application also provides pharmaceutical compositions, kits, methods and uses comprising the compounds described herein.

In one aspect, the present application provides compounds of formula (I), and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives and prodrugs thereof:

wherein R is^A1、R^A2、R^A3、R^A4And R^A5As defined herein.

Exemplary compounds of formula (I) include, but are not limited to:

and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, and prodrugs thereof.

In some embodiments, the compound of formula (I) is a compound of the formula:

in some embodiments, the compound of formula (I) is a compound of the formula:

in another aspect, the present application provides compounds of formula (II), and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives and prodrugs thereof:

Wherein R is^B1、R^B2、R^B3、R^B4And R^B5As defined herein.

Exemplary compounds of formula (II) include, but are not limited to:

and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, and prodrugs thereof.

In some embodiments, the compound of formula (II) is a compound of the formula:

in some embodiments, the compound of formula (II) is a compound of the formula:

in some embodiments, the compound of formula (II) is a compound of the formula:

in another aspect, described herein are pharmaceutical compositions comprising a compound described herein and optionally a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition described herein comprises a therapeutically or prophylactically effective amount of a compound described herein. In some embodiments, the pharmaceutical compositions described herein further comprise an additional pharmaceutical agent. The pharmaceutical compositions can be used to modulate (e.g., inhibit) the activity of HMT in a subject, a biological sample, a tissue, or a cell, to treat a disease (e.g., a proliferative disease) in a subject in need thereof, or to prevent a disease in a subject in need thereof.

In some embodiments, the disease is a disease associated with aberrant activity of HMT. In some embodiments, the aberrant activity of HMT is increased HMT activity. In some embodiments, the disease is a disease associated with increased HMT activity compared to normal cells. In some embodiments, the disease is a proliferative disease (e.g., cancer, benign tumor, pathological angiogenesis), an inflammatory disease, an autoimmune disease, a genetic disease, a hematological disease, a neurological disease, a painful condition, a psychiatric disease, or a metabolic disorder.

In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, the cell is in vitro. In some embodiments, the cell is in vivo.

In another aspect, described herein is a kit comprising a container containing a compound or pharmaceutical composition described herein. The kits described herein may include a single dose or multiple doses of the compound or pharmaceutical composition. The kits can be used to inhibit the activity of HMT in a subject, a biological sample, a tissue, or a cell, for treating a disease associated with aberrant activity of HMT in a subject in need thereof, for preventing a disease associated with aberrant activity of HMT in a subject in need thereof, for treating a disease (e.g., a proliferative disease, an inflammatory disease, an autoimmune disease, a genetic disease, a hematological disease, a neurological disease, a painful disorder, a psychiatric disease, or a metabolic disorder) in a subject in need thereof, and/or for preventing a disease (e.g., a proliferative disease, an inflammatory disease, an autoimmune disease, a genetic disease, a hematological disease, a neurological disease, a painful disorder, a psychiatric disease, or a metabolic disorder) in a subject in need thereof. In some embodiments, the kits described herein further comprise instructions for using the compounds or pharmaceutical compositions contained in the kit.

In another aspect, the present application provides a method of modulating (e.g., inhibiting) an aberrant activity of HMT in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition described herein.

In another aspect, the present application provides a method of modulating (e.g., inhibiting) the activity of HMT in a biological sample, tissue or cell comprising contacting the biological sample, tissue or cell with an effective amount of a compound or pharmaceutical composition described herein.

In some embodiments, the compounds are administered or used to selectively inhibit the activity of a particular HMT (e.g., EZH1, EZH2, DOT 1).

In another aspect, the present application provides a method of inducing apoptosis in a cell, the method comprising contacting the cell with an effective amount of a compound or pharmaceutical composition described herein.

Another aspect of the present application relates to a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition described herein.

In another aspect, the present application provides a method of preventing a disease in a subject in need thereof, the method comprising administering to the subject a prophylactically effective amount of a compound or pharmaceutical composition described herein.

Another aspect of the present application relates to a method of reducing methylation of a histone in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound or pharmaceutical composition described herein.

Another aspect of the present application relates to a method of reducing histone methylation in a biological sample, tissue or cell, comprising contacting the biological sample, tissue or cell with an effective amount of a compound or pharmaceutical composition described herein.

Another aspect of the present application relates to a method of modulating (e.g., down-regulating or up-regulating) gene expression in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound or pharmaceutical composition described herein.

Another aspect of the present application relates to a method of modulating (e.g., down-regulating or up-regulating) gene expression in a biological sample, tissue or cell, the method comprising contacting the biological sample, tissue or cell with an effective amount of a compound or pharmaceutical composition described herein.

Another aspect of the disclosure relates to methods of screening libraries of compounds to identify compounds useful in the methods described herein.

Another aspect of the present application relates to methods of identifying EZH1 and/or EZH2 inhibitors.

In another aspect, the present application provides compounds and pharmaceutical compositions described herein for use in the methods of the present application (e.g., methods of inhibiting aberrant activity of HMT, methods of inducing apoptosis, methods of treating a disease (e.g., a proliferative disease), or methods of preventing a disease (e.g., a proliferative disease)).

Definition of

The definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are defined in the periodic Table of the elements, CAS edition, Handbook of Chemistry and Physics, 75 th edition, inner cover, and the specific functional groups are generally defined as described herein. Furthermore, the general principles of Organic Chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausaltito, 1999; smith and March, March's Advanced Organic Chemistry, 5 th edition, John Wiley & Sons, Inc., New York, 2001; larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and carrousers, Some model Methods of Organic Synthesis, 3 rd edition, Cambridge University Press, Cambridge, 1987. This application is not intended to be limited in any way by the exemplary substituents listed herein.

The compounds described herein may contain one or more asymmetric centers and may therefore exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein may be in the form of a single enantiomer, diastereomer, or geometric isomer, or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral High Pressure Liquid Chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis. See, e.g., Jacques et al, eneriomers, Racemates and solutions (Wiley Interscience, New York, 1981); wilen et al, Tetrahedron 33: 2725 (1977); eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, tablets of solving Agents and Optical solutions, page 268 (edited by E.L. Eliel, Univ.of Notre Dame Press, Notre Dame, IN 1972). The invention also includes the compounds described herein as a single isomer substantially free of other isomers, and alternatively, as mixtures of different isomers.

When a range of values is recited, each value and subrange within the range is intended to be encompassed. E.g. "C_1-6"is intended to cover C₁、C₂、C₃、C₄、C₅、C₆、C_1–6、C_1–5、C_1–4、C_1–3、C_1–2、C_2–6、C_2–5、 C_2–4、C_2–3、C_3–6、C_3–5、C_3–4、C_4–6、C_4–5And C_5–6。

The term "aliphatic" includes saturated and unsaturated, straight-chain (i.e., unbranched), branched, acyclic, cyclic, or polycyclic aliphatic hydrocarbons, which may be optionally substituted with one or more functional groups. As will be understood by those skilled in the art, "aliphatic" is intended herein to include, but is not limited to: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. Thus, the term "alkyl" includes straight chain, branched chain and cyclic alkyl groups. Similar rules apply to other general terms such as "alkenyl", "alkynyl", and the like. Furthermore, the terms "alkyl", "alkenyl", "alkynyl", and the like encompass both substituted and unsubstituted groups. In some embodiments, "lower alkyl" is used to denote those alkyl groups having 1 to 6 carbon atoms (cyclic, acyclic, substituted, unsubstituted, branched, or unbranched).

In some embodiments, alkyl, alkenyl, and alkynyl groups used in the present invention contain 1-20 aliphatic carbon atoms. In some other embodiments, alkyl, alkenyl, and alkynyl groups used in the present invention contain 1-10 aliphatic carbon atoms. In still other embodiments, alkyl, alkenyl, and alkynyl groups useful herein contain 1-8 aliphatic carbon atoms. In still other embodiments, alkyl, alkenyl, and alkynyl groups useful herein contain 1-6 aliphatic carbon atoms. In still other embodiments, alkyl, alkenyl, and alkynyl groups useful herein contain 1 to 4 carbon atoms. Thus, exemplary aliphatic groups include, but are not limited to: for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, -CH ₂-cyclopropyl, vinyl, allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl, -CH₂-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl, cyclopentyl, -CH₂-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl、-CH₂Cyclohexyl and the like, which likewise may bear one or more substituents. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

The term "alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 10 carbon atoms ("C_1-10Alkyl "). In some embodiments, the alkyl group has 1 to 9 carbon atoms ("C)_1-9Alkyl "). In some embodiments, the alkyl group has 1 to 8 carbon atoms ("C)_1-8Alkyl "). In some embodiments, the alkyl group has 1 to 7 carbon atoms ("C)_1-7Alkyl "). In some embodiments, the alkyl group has 1 to 6 carbon atoms ("C)_1-6Alkyl "). In some embodiments, the alkyl group has 1 to 5 carbon atoms ("C)_1-5Alkyl "). In some embodiments, the alkyl group has 1 to 4 carbon atoms ("C) _1-4Alkyl "). In some embodiments, the alkyl group has 1 to 3 carbon atoms ("C)_1-3Alkyl "). In some embodiments, the alkyl group has 1 to 2 carbon atoms ("C)_1-2Alkyl "). In some embodiments, the alkyl group has 1 carbon atom ("C")₁Alkyl "). In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C)_2-6Alkyl "). C_1-6Examples of alkyl groups include methyl (C)₁) Ethyl (C)₂) Propyl group (C)₃) (e.g., n-propyl, isopropyl), butyl (C)₄) (e.g., n-butyl, t-butyl, sec-butyl, isobutyl), pentyl (C)₅) (e.g., n-pentyl, 3-pentyl, neopentyl, 3-methyl-2-butyl, tert-pentyl) and hexyl (C)₆) (e.g., n-hexyl). Other examples of alkyl groups include n-heptyl (C)₇) N-octyl (C)₈) And the like. Unless otherwise indicated, an alkyl group is independently unsubstituted in each instance ("unsubstituted alkyl") or substituted with one or more substituents (e.g., halogen, e.g., F) ("substituted alkyl"). In some embodiments of the present invention, the substrate is,the alkyl radical being unsubstituted C_1-10Alkyl (e.g. unsubstituted C)_1-6Alkyl radicals, e.g., -CH₃(Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g., unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (n-Bu), unsubstituted t-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl (sec-Bu), unsubstituted isobutyl (i-Bu)). In some embodiments, the alkyl group is substituted C _1-10Alkyl (e.g. substituted C)_1-6Alkyl radicals, e.g., -CF₃、Bn)。

"alkenyl" means a group of straight or branched hydrocarbon radicals having 2 to 20 carbon atoms, one or more carbon-carbon double bonds and no triple bonds ("C)_2-20Alkenyl "). In some embodiments, alkenyl groups have 2-10 carbon atoms ("C)_2-10Alkenyl "). In some embodiments, alkenyl groups have 2-9 carbon atoms ("C)_2-9Alkenyl "). In some embodiments, alkenyl groups have 2-8 carbon atoms ("C)_2-8Alkenyl "). In some embodiments, alkenyl groups have 2-7 carbon atoms ("C)_2-7Alkenyl "). In some embodiments, alkenyl groups have 2-6 carbon atoms ("C)_2-6Alkenyl "). In some embodiments, alkenyl groups have 2-5 carbon atoms ("C)_2-5Alkenyl "). In some embodiments, alkenyl groups have 2-4 carbon atoms ("C)_2-4Alkenyl "). In some embodiments, alkenyl groups have 2-3 carbon atoms ("C)_2-3Alkenyl "). In some embodiments, alkenyl has 2 carbon atoms ("C)₂Alkenyl "). The one or more carbon-carbon double bonds may be internal (e.g., in a 2-butenyl group) or terminal (e.g., in a 1-butenyl group). C_2-4Examples of the alkenyl group include vinyl (C)₂) 1-propenyl (C)₃) 2-propenyl (C)₃) 1-butenyl (C) ₄) 2-butenyl (C)₄) Butadienyl radical (C)₄) And the like. C_2-6Examples of the alkenyl group include the above-mentioned C_2-4Alkenyl, and pentenyl (C)₅) Pentadienyl (C)₅) Hexenyl (C)₆) And the like. Additional examples of alkenyl groups include heptenyl (C)₇) Octenyl (C)₈) Octrienyl (C)₈) And the like. Unless otherwise specified, an alkenyl group is optionally independently substituted in each instance, i.e., unsubstituted (an "unsubstituted alkenyl") or substituted (a "substituted alkenyl") with one or more substituents. In some embodiments, the alkenyl group is unsubstituted C_2-10An alkenyl group. In some embodiments, the alkenyl is substituted C_2-10An alkenyl group. In the alkenyl group, a stereochemically unspecified C ═ C double bond (e.g., -CH ═ CHCH₃Or) May be an (E) -or (Z) -double bond.

"alkynyl" refers to a group of straight or branched hydrocarbon radicals having 2 to 20 carbon atoms, one or more carbon-carbon triple bonds and optionally one or more double bonds ("C)_2-20Alkynyl "). In some embodiments, alkynyl groups have 2-10 carbon atoms ("C)_2-10Alkynyl "). In some embodiments, alkynyl groups have 2-9 carbon atoms ("C)_2-9Alkynyl "). In some embodiments, alkynyl groups have 2-8 carbon atoms ("C)_2-8Alkynyl "). In some embodiments, alkynyl groups have 2-7 carbon atoms ("C) _2-7Alkynyl "). In some embodiments, alkynyl groups have 2-6 carbon atoms ("C)_2-6Alkynyl "). In some embodiments, alkynyl groups have 2-5 carbon atoms ("C)_2-5Alkynyl "). In some embodiments, alkynyl groups have 2-4 carbon atoms ("C)_2-4Alkynyl "). In some embodiments, alkynyl groups have 2-3 carbon atoms ("C)_2-3Alkynyl "). In some embodiments, alkynyl has 2 carbon atoms ("C)₂Alkynyl "). The one or more carbon-carbon triple bonds may be internal (e.g., in 2-butynyl) or terminal (e.g., in 1-butynyl). C_2-4Examples of alkynyl groups include, but are not limited to, ethynyl (C)₂) 1-propynyl (C)₃) 2-propynyl (C)₃) 1-butynyl (C)₄) 2-butynyl (C)₄) And the like. C_2-6Examples of alkynyl groups include the above-mentioned C_2-4Alkynyl and pentynyl (C)₅) To have business turnAlkynyl (C)₆) And the like. Other examples of alkynyl groups include heptynyl (C)₇) (C) octynyl group₈) And the like. Unless otherwise specified, an alkynyl group is optionally independently substituted in each instance, i.e., unsubstituted (an "unsubstituted alkynyl") or substituted (a "substituted alkynyl") with one or more substituents. In some embodiments, the alkynyl group is unsubstituted C_2-10Alkynyl. In some embodiments, the alkynyl is substituted C _2-10Alkynyl.

"carbocyclyl" or "carbocyclic" refers to a group of nonaromatic cyclic hydrocarbon radicals having from 3 to 10 ring carbon atoms ("C)_3-10Carbocyclyl ") and no heteroatoms in the non-aromatic ring system. In some embodiments, carbocyclyl has 3 to 8 ring carbon atoms ("C)_3-8Carbocyclyl "). In some embodiments, carbocyclyl has 3-6 ring carbon atoms ("C)_3-6Carbocyclyl "). In some embodiments, carbocyclyl has 3-6 ring carbon atoms ("C)_3-6Carbocyclyl "). In some embodiments, carbocyclyl has 5 to 10 ring carbon atoms ("C)_5-10Carbocyclyl "). Exemplary C_3-6Carbocyclyl groups include, but are not limited to: cyclopropyl (C)₃) Cyclopropenyl group (C)₃) Cyclobutyl (C)₄) Cyclobutenyl radical (C)₄) Cyclopentyl (C)₅) Cyclopentenyl group (C)₅) Cyclohexyl (C)₆) Cyclohexenyl (C)₆) Cyclohexadienyl (C)₆) And the like. Exemplary C_3-8Carbocyclyl includes, but is not limited to, C as described above_3-6Carbocyclyl and cycloheptyl (C)₇) Cycloheptenyl (C)₇) Cycloheptadienyl (C)₇) Cycloheptatrienyl (C)₇) Cyclooctyl (C)₈) Cyclooctenyl (C)₈) Bicyclo [2.2.1]Heptyl (C)₇) Bicyclo [2.2.2]Octyl radical (C)₈) And the like. Exemplary C_3-10Carbocyclyl includes, but is not limited to, C as described above_3-8Carbocyclyl and cyclononyl (C)₉) Cyclononenyl (C) ₉) Cyclodecyl (C)₁₀) Cyclodecenyl (C)₁₀) octahydro-1H-indenyl (C)₉) Decahydronaphthyl (C)₁₀) Spiro [4.5 ]]Decyl (C)₁₀) And the like. As illustrated by the examples above, in some instancesIn embodiments, the carbocyclyl group is monocyclic ("monocyclic carbocyclyl") or contains a fused, bridged or spiro ring system, for example a bicyclic system ("bicyclic carbocyclyl") and may be saturated or partially unsaturated. "carbocyclyl" may also include ring systems wherein the carbocycle as defined above is fused to one or more aryl or heteroaryl groups wherein the point of attachment is on the carbocycle and in such cases carbon number still refers to the number of carbons in the carbocyclic ring system. Unless otherwise specified, a carbocyclyl group is optionally independently substituted in each instance, i.e., unsubstituted (an "unsubstituted carbocyclyl") or substituted (a "substituted carbocyclyl") with one or more substituents. In some embodiments, the carbocyclyl is unsubstituted C_3-10A carbocyclic group. In some embodiments, the carbocyclyl is substituted C_3-10A carbocyclic group.

In some embodiments, "carbocyclyl" is a monocyclic, saturated carbocyclyl ("C") having 3 to 10 ring carbon atoms_3-10Cycloalkyl "). In some embodiments, cycloalkyl groups have from 3 to 8 ring carbon atoms ("C) _3-8Cycloalkyl "). In some embodiments, cycloalkyl groups have 3 to 6 ring carbon atoms ("C)_3-6Cycloalkyl "). In some embodiments, cycloalkyl groups have 5 to 6 ring carbon atoms ("C)_5-6Cycloalkyl "). In some embodiments, cycloalkyl groups have from 5 to 10 ring carbon atoms ("C)_5-10Cycloalkyl "). C_5-6Examples of cycloalkyl include cyclopentyl (C)₅) And cyclohexyl (C)₆)。C_3-6Examples of the cycloalkyl group include the above-mentioned C_5-6Cycloalkyl and cyclopropyl (C)₃) And cyclobutyl (C)₄)。C_3-8Examples of the cycloalkyl group include the above-mentioned C_3-6Cycloalkyl and cycloheptyl (C)₇) And cyclooctyl (C)₈). Unless otherwise specified, a cycloalkyl group is independently in each instance unsubstituted (an "unsubstituted cycloalkyl") or substituted (a "substituted cycloalkyl") with one or more substituents. In some embodiments, the cycloalkyl is unsubstituted C_3-10A cycloalkyl group. In some embodiments, the cycloalkyl is substituted C_3-10A cycloalkyl group.

"Heterocyclyl" or "heterocyclic" refers to a group of a 3-to 10-membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("3-10 membered heterocyclyl"). In heterocyclic groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can be monocyclic ("monocyclic heterocyclyl") or a fused, bridged or spiro ring system, for example a bicyclic system ("bicyclic heterocyclyl"), and can be saturated or can be partially unsaturated. The heterocyclyl bicyclic ring system may contain one or more heteroatoms in one or both rings. "heterocyclyl" also includes ring systems in which a heterocyclic ring as defined above is fused to one or more carbocyclic groups in which the point of attachment is on the carbocyclic or heterocyclic ring, or ring systems in which a heterocyclic ring as defined above is fused to one or more aryl or heteroaryl groups in which the point of attachment is on the heterocyclic ring, and in which case the number of ring atoms still refers to the number of ring atoms in the heterocyclic ring system. Unless otherwise specified, a heterocyclyl group is optionally independently substituted in each instance, i.e., unsubstituted (an "unsubstituted heterocyclyl") or substituted (a "substituted heterocyclyl") with one or more substituents. In some embodiments, the heterocyclyl is an unsubstituted 3-10 membered heterocyclyl. In some embodiments, the heterocyclyl is a substituted 3-10 membered heterocyclyl.

In some embodiments, heterocyclyl is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("5-10 membered heterocyclyl"). In some embodiments, heterocyclyl is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-8 membered heterocyclyl"). In some embodiments, heterocyclyl is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-6 membered heterocyclyl"). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to, aziridinyl, oxacyclopropaneyl, thietanepropyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl and thietanyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to: tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2, 5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to: dioxolanyl, oxathiolanyl, dithiolanyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thiacyclohexyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithiino-cyclohexyl, and dioxanyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to, triazinylalkyl groups. Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to, azepanyl, oxepinyl, and thiepanyl. Exemplary 8-membered heterocyclic groups containing one heteroatom include, but are not limited to, azacyclooctyl, oxocyclooctyl, and thiepinyl. And C ₆Exemplary 5-membered heterocyclic groups fused to the aromatic ring (also referred to herein as 5, 6-bicyclic heterocycles) include, but are not limited to: indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolonyl, and the like. Exemplary 6-membered heterocyclic groups fused to the aromatic ring (also referred to herein as 6, 6-bicyclic heterocycles) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

"aryl" means a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n +2 aromatic ring system(e.g., having 6, 10, or 14 pi electrons shared in a ring array) having 6-14 ring carbon atoms in the aromatic ring system and no heteroatoms ("C)_6-14Aryl "). In some embodiments, an aryl group has 6 ring carbon atoms ("C)₆Aryl "; for example, phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C)₁₀Aryl "; e.g., naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms ("C)₁₄Aryl "; for example, an anthracene group). "aryl" also includes ring systems in which an aryl ring, as defined above, is fused to one or more carbocyclic or heterocyclic groups in which the groups or points of attachment are on the aryl ring, and in which case the number of carbon atoms still refers to the number of carbon atoms in the aryl ring system. Unless otherwise specified, an aryl group is optionally independently substituted in each instance, i.e., unsubstituted (an "unsubstituted aryl") or substituted (a "substituted aryl") with one or more substituents. In some embodiments, the aryl group is unsubstituted C _6-14And (4) an aryl group. In some embodiments, the aryl is substituted C_6-14And (4) an aryl group.

"aralkyl" is a subset of alkyl and aryl, and refers to optionally substituted alkyl substituted with optionally substituted aryl. In some embodiments, the aralkyl group is an optionally substituted benzyl group. In some embodiments, the aralkyl group is benzyl. In some embodiments, the aralkyl group is an optionally substituted phenethyl group. In some embodiments, the aralkyl group is phenethyl.

"heteroaryl" refers to a group of a 5-10 membered monocyclic or bicyclic 4n +2 aromatic ring system (e.g., having 6 or 10 pi electrons shared in the ring array) having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems may contain one or more heteroatoms in one or both rings. "heteroaryl" includes ring systems in which a heteroaryl ring as defined above is fused to one or more carbocyclyl or heterocyclyl groups, wherein the point of attachment is on the heteroaryl ring, and in which case the number of ring atoms still refers to the number of ring atoms in the heteroaryl ring system. "heteroaryl" also includes ring systems in which a heteroaryl ring as defined above is fused to one or more aryl groups, wherein the point of attachment is on the aryl or heteroaryl ring, and in which case the number of ring atoms refers to the number of ring atoms in the fused (aryl/heteroaryl) ring system. In bicyclic heteroaryl groups (e.g., indolyl, quinolinyl, carbazolyl, and the like) in which one ring does not contain a heteroatom, the point of attachment can be on either ring, i.e., on the heteroatom-containing ring (e.g., 2-indolyl) or on the heteroatom-free ring (e.g., 5-indolyl).

In some embodiments, heteroaryl is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"). In some embodiments, heteroaryl is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-8 membered heteroaryl"). In some embodiments, heteroaryl is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-6 membered heteroaryl"). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified, heteroaryl groups are in each case optionally independently substituted, i.e., unsubstituted (an "unsubstituted heteroaryl") or substituted (a "substituted heteroaryl") with one or more substituents. In some embodiments, the heteroaryl is an unsubstituted 5-14 membered heteroaryl. In some embodiments, the heteroaryl is a substituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thienyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to: imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5, 6-bicyclic heteroaryls include, but are not limited to: indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothienyl, isobenzothienyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzooxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indolizinyl, and purinyl. Exemplary 6, 6-bicyclic heteroaryls include, but are not limited to: naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl and quinazolinyl.

"heteroarylalkyl" is a subset of alkyl and heteroaryl and refers to optionally substituted alkyl substituted with optionally substituted heteroaryl.

"unsaturated" or "partially unsaturated" refers to a group that contains at least one double or triple bond. "partially unsaturated" ring systems are further intended to encompass rings having multiple sites of unsaturation, but are not intended to include aromatic groups (e.g., aryl or heteroaryl). Likewise, "saturated" refers to groups that do not contain double or triple bonds, i.e., contain only single bonds.

Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl, as divalent bridging groups when the prefix arylene is used, further indicate, for example, alkylene, alkenylene, alkynylene, carbocyclylene, heterocyclylene, arylene and heteroarylene.

Unless explicitly stated otherwise, an atom, moiety or group described herein may be unsubstituted or substituted, as valency permits. The term "optionally substituted" refers to substituted or unsubstituted.

Unless otherwise specifically stated, groups are optionally substituted. The term "optionally substituted" refers to substituted or unsubstituted. In some embodiments, alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl are optionally substituted (e.g., a "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" carbocyclyl, "substituted" or "unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl, or "substituted" or "unsubstituted" heteroaryl). In general, the term "substituted", whether preceded by the term "optionally" or not, means that at least one hydrogen atom present on a group (e.g., a carbon or nitrogen atom) is replaced with an allowable substituent (e.g., a substituent that is substituted to form a stable compound, e.g., the compound does not spontaneously undergo transformation, such as rearrangement, cyclization, elimination or other reaction). Unless otherwise specified, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position is substituted in any given structure, the substituents are the same or different at each position. The term "substituted" is intended to include substitution with all permissible substituents of organic compounds (any of the substituents described herein that result in the formation of stable compounds). The present invention includes any and all such combinations to provide stable compounds. For purposes of the present invention, a heteroatom (e.g., nitrogen) can have a hydrogen substituent and/or any suitable substituent described herein that satisfies the valence of the heteroatom and forms a stable group. In some embodiments, the substituent is a carbon atom substituent. In some embodiments, the substituent is a nitrogen atom substituent. In some embodiments, the substituent is an oxygen atom substituent. In some embodiments, the substituent is a sulfur atom substituent.

Exemplary carbon atom substituents include, but are not limited to: halogen, -CN, -NO₂、–N₃、–SO₂H、–SO₃H、–OH、–OR^aa、–ON(R^bb)₂、–N(R^bb)₂、–N(R^bb)₃ ⁺X^–、–N(OR^cc)R^bb、–SH、–SR^aa、–SSR^cc、–C(＝O)R^aa、–CO₂H、–CHO、–C(OR^cc)₂、–CO₂R^aa、–OC(＝O)R^aa、–OCO₂R^aa、–C(＝O)N(R^bb)₂、–OC(＝O)N(R^bb)₂、–NR^bbC(＝O)R^aa、–NR^bbCO₂R^aa、–NR^bbC(＝O)N(R^bb)₂、–C(＝NR^bb)R^aa、–C(＝NR^bb)OR^aa、–OC(＝NR^bb)R^aa、–OC(＝NR^bb)OR^aa、–C(＝NR^bb)N(R^bb)₂、–OC(＝NR^bb)N(R^bb)₂、–NR^bbC(＝NR^bb)N(R^bb)₂、–C(＝O)NR^bbSO₂R^aa、–NR^bbSO₂R^aa、–SO₂N(R^bb)₂、–SO₂R^aa、–SO₂OR^aa、–OSO₂R^aa、–S(＝O)R^aa、–OS(＝O)R^aa、–Si(R^aa)₃、–OSi(R^aa)₃–C(＝S)N(R^bb)₂、–C(＝O)SR^aa、–C(＝S)SR^aa、–SC(＝S)SR^aa、–SC(＝O)SR^aa、–OC(＝O)SR^aa、–SC(＝O)OR^aa、–SC(＝O)R^aa、–P(＝O)(R^aa)₂、-P(＝O)(OR^cc)₂、–OP(＝O)(R^aa)₂、–OP(＝O)(OR^cc)₂、–P(＝O)(N(R^bb)₂)₂、–OP(＝O)(N(R^bb)₂)₂、 -NR^bbP(＝O)(R^aa)₂、–NR^bbP(＝O)(OR^cc)₂、–NR^bbP(＝O)(N(R^bb)₂)₂、–P(R^cc)₂、–P(R^cc)₃ ⁺X^-、-P(OR^cc)₃ ⁺X^-、-P(R^cc)₄、-P(OR^cc)₄、-P(OR^cc)₂、–OP(R^cc)₂、–OP(R^cc)₃、–B(R^aa)₂、–B(OR^cc)₂、–BR^aa(OR^cc)、C_1–10Alkyl radical, C_1–10Perhaloalkyl, C_2–10Alkenyl radical, C_2–10Alkynyl, C_3–10Carbocyclyl, 3-14 membered heterocyclyl, C_6–14Aryl and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5R^ddSubstituted by a group; wherein X^-Is a counter ion; or two geminal hydrogens on a carbon atom are replaced by a group ═ O, ═ S, ═ NN (R)^bb)₂、＝NNR^bbC(＝O)R^aa、＝NNR^bbC(＝O)OR^aa、＝NNR^bbS(＝O)₂R^aa、＝NR^bbOr as NOR^ccReplacement;

each R^aaIndependently selected from C_1–10Alkyl radical, C_1–10Perhaloalkyl, C_2–10Alkenyl radical, C_2–10Alkynyl, C_3–10Carbocyclyl, 3-14 membered heterocyclyl, C_6–14Aryl and 5-14 membered heteroaryl, or two R^aaThe groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5R^ddSubstituted by a group;

each R^bbIndependently selected from hydrogen, -OH, -OR^aa、–N(R^cc)₂、–CN、–C(＝O)R^aa、–C(＝O)N(R^cc)₂、–CO₂R^aa、–SO₂R^aa、–C(＝NR^cc)OR^aa、–C(＝NR^cc)N(R^cc)₂、–SO₂N(R^cc)₂、–SO₂R^cc、–SO₂OR^cc、–SOR^aa、–C(＝S)N(R^cc)₂、–C(＝O)SR^cc、–C(＝S)SR^cc、–P(＝O)(R^aa)₂、-P(＝O)(OR^cc)₂、–P(＝O)(N(R^cc)₂)₂、C_1–10Alkyl radical, C_1–10Perhaloalkyl, C_2–10Alkenyl radical, C_2–10Alkynyl, C_3–10Carbocyclyl, 3-14 membered heterocyclyl, C_6–14Aryl and 5-14 membered heteroaryl, or two R^bbThe groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5R ^ddSubstituted by a group; wherein X^-Is a counter ion;

each R^ccIndependently selected from hydrogen, C_1–10Alkyl radical, C_1–10Perhaloalkyl, C_2–10Alkenyl radical, C_2–10Alkynyl, C_3–10Carbocyclyl, 3-14 membered heterocyclyl, C_6–14Aryl and 5-14 membered heteroaryl, or two R^ccThe groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5R^ddSubstituted by a group;

each R^ddIndependently selected from halogen, -CN, -NO₂、–N₃、–SO₂H、–SO₃H、–OH、–OR^ee、–ON(R^ff)₂、–N(R^ff)₂、–N(R^ff)₃ ⁺X^–、–N(OR^ee)R^ff、–SH、–SR^ee、–SSR^ee、–C(＝O)R^ee、–CO₂H、–CO₂R^ee、–OC(＝O)R^ee、–OCO₂R^ee、–C(＝O)N(R^ff)₂、–OC(＝O)N(R^ff)₂、–NR^ffC(＝O)R^ee、–NR^ffCO₂R^ee、–NR^ffC(＝O)N(R^ff)₂、–C(＝NR^ff)OR^ee、–OC(＝NR^ff)R^ee、–OC(＝NR^ff)OR^ee、–C(＝NR^ff)N(R^ff)₂、–OC(＝NR^ff)N(R^ff)₂、–NR^ffC(＝NR^ff)N(R^ff)₂、–NR^ffSO₂R^ee、–SO₂N(R^ff)₂、–SO₂R^ee、–SO₂OR^ee、–OSO₂R^ee、–S(＝O)R^ee、–Si(R^ee)₃、–OSi(R^ee)₃、–C(＝S)N(R^ff)₂、–C(＝O)SR^ee、–C(＝S)SR^ee、–SC(＝S)SR^ee、–P(＝O)(OR^ee)₂、–P(＝O)(R^ee)₂、–OP(＝O)(R^ee)₂、–OP(＝O)(OR^ee)₂、C_1–6Alkyl radical, C_1–6Perhaloalkyl, C_2–6Alkenyl radical, C_2–6Alkynyl, C_3–10Carbocyclyl, 3-10 membered heterocyclyl, C_6–10Aryl, 5-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5R^ggSubstituted by radicals, or two geminal R^ddSubstituents may be linked to form ═ O or ═ S; wherein X^-Is a counter ion;

each R^eeIndependently selected from C_1–6Alkyl radical, C_1–6Perhaloalkyl, C_2–6Alkenyl radical, C_2–6Alkynyl, C_3–10Carbocyclyl, C_6–10Aryl, 3-10 membered heterocyclyl and 3-10 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5R ^ggSubstituted by a group;

each R^ffIndependently selected from hydrogen, C_1–6Alkyl radical, C_1–6Perhaloalkyl, C_2–6Alkenyl radical, C_2–6Alkynyl, C_3–10Carbocyclyl, 3-10 membered heterocyclyl, C_6–10Aryl and 5-10 membered heteroaryl, or two R^ffThe groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5R^ggSubstituted by a group; and

each R^ggIndependently halogen, -CN, -NO₂、–N₃、–SO₂H、–SO₃H、–OH、–OC_1–6Alkyl, -ON (C)_1–6Alkyl radical)₂、–N(C_1–6Alkyl radical)₂、–N(C_1–6Alkyl radical)₃ ⁺X^–、–NH(C_1–6Alkyl radical)₂ ⁺X^–、–NH₂(C_1–6Alkyl radical)⁺X^–、–NH₃ ⁺X^–、–N(OC_1–6Alkyl) (C_1–6Alkyl), -N (OH) (C)_1–6Alkyl), -NH (OH), -SH, -SC_1–6Alkyl, -SS (C)_1–6Alkyl), -C (═ O) (C)_1–6Alkyl), -CO₂H、–CO₂(C_1–6Alkyl), -OC (═ O) (C)_1–6Alkyl), -OCO₂(C_1–6Alkyl), -C (═ O) NH₂、–C(＝O)N(C_1–6Alkyl radical)₂、–OC(＝O)NH(C_1–6Alkyl), -NHC (═ O) (C)_1–6Alkyl), -N (C)_1–6Alkyl) C (═ O) (C)_1–6Alkyl), -NHCO₂(C_1–6Alkyl), -NHC (═ O) N (C)_1–6Alkyl radical)₂、–NHC(＝O)NH(C_1–6Alkyl), -NHC (═ O) NH₂、–C(＝NH)O(C_1–6Alkyl), -OC (═ NH) (C)_1–6Alkyl), -OC (═ NH) OC_1–6Alkyl, -C (═ NH) N (C)_1–6Alkyl radical)₂、–C(＝NH)NH(C_1–6Alkyl), -C (═ NH) NH₂、–OC(＝NH)N(C_1–6Alkyl radical)₂、–OC(NH)NH(C_1–6Alkyl), -OC (NH) NH₂、–NHC(NH)N(C_1–6Alkyl radical)₂、–NHC(＝NH)NH₂、–NHSO₂(C_1–6Alkyl), -SO₂N(C_1–6Alkyl radical)₂、–SO₂NH(C_1–6Alkyl), -SO₂NH₂、–SO₂C_1–6Alkyl, -SO₂OC_1–6Alkyl, -OSO ₂C_1–6Alkyl, -SOC_1–6Alkyl, -Si (C)_1–6Alkyl radical)₃、–OSi(C_1–6Alkyl radical)₃、–C(＝S)N(C_1–6Alkyl radical)₂、C(＝S)NH(C_1–6Alkyl), C (═ S) NH₂、–C(＝O)S(C_1–6Alkyl), -C (═ S) SC_1–6Alkyl, -SC (═ S) SC_1–6Alkyl, -P (═ O)₂(C_1–6Alkyl), -P (═ O) (C)_1–6Alkyl radical)₂、–OP(＝O)(C_1–6Alkyl radical)₂、–OP(＝O)(OC_1–6Alkyl radical)₂、C_1–6Alkyl radical, C_1–6Perhaloalkyl, C_2–6Alkenyl radical, C_2–6Alkynyl, C_3–10Carbocyclyl, C_6–10Aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two geminal R^ggSubstituents may be linked to form ═ O or ═ S; wherein X^–Are counter ions.

"counterions" or "anionic counterions" are negatively charged groups associated with positively charged groups to maintain electrical neutrality. The anionic counterion can be monovalent (i.e., include one formal negative charge). Anionic counterions can also be multivalent (i.e., include more than one formal negative charge), such as divalent or trivalent. Exemplary antibodiesCounter ions include halide ions (e.g., F)^–、Cl^–、Br^–、I^–)、NO₃ ^–、 ClO₄ ^–、OH^–、H₂PO₄ ^–、HCO₃ ^-、HSO₄ ^–Sulfonate ions (e.g., methanesulfonate, trifluoromethanesulfonate, p-toluenesulfonate, benzenesulfonate, 10-camphorsulfonate, naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate, ethane-1-sulfonic acid-2-sulfonate, etc.), carboxylate ions (e.g., acetate, propionate, benzoate, glycerate, lactate, tartrate, glycolate, gluconate, etc.), BF ₄ ^-、PF₄ ^–、PF₆ ^–、AsF₆ ^–、SbF₆ ^–、B[3,5-(CF₃)₂C₆H₃]₄]^–、B(C₆F₅)₄ ^-、BPh₄ ^–、 Al(OC(CF₃)₃)₄ ^–And carborane anions (e.g., CB)₁₁H₁₂ ^–Or (HCB)₁₁Me₅Br₆)^–). Exemplary counterions that can be multivalent include CO₃ ^2-、HPO₄ ^2-、PO₄ ^3-、B₄O₇ ^2-、SO₄ ^2-、S₂O₃ ^2-Carboxylate anions (e.g., tartrate, citrate, fumarate, maleate, malate, malonate, gluconate, succinate, glutarate, adipate, pimelate, suberate, azelate, sebacate, salicylate, phthalate, aspartate, glutamate, and the like) and carboranes.

"halo" or "halogen" refers to fluoro (fluoro, -F), chloro (chloro, -Cl), bromo (bromo, -Br), or iodo (iodo, -I).

"acyl" means a radical selected from-C (═ O) R^aa、-CHO、-CO₂R^aa、-C(＝O)N(R^bb)₂、 -C(＝NR^bb)R^aa、-C(＝NR^bb)OR^aa、-C(＝NR^bb)N(R^bb)₂、-C(＝O)NR^bbSO₂R^aa、 -C(＝S)N(R^bb)₂、-C(＝O)SR^aaor-C (═ S) SR^aaWherein R is^aaAnd R^bbAs defined herein.

The nitrogen atoms may be substituted or unsubstituted, as valency permits, and include primary, secondary, tertiary, and quaternary nitrogen atoms. Exemplary nitrogen atom substituents include, but are not limited to: hydrogen, -OH, -OR^aa、–N(R^cc)₂、–CN、–C(＝O)R^aa、–C(＝O)N(R^cc)₂、–CO₂R^aa、–SO₂R^aa、–C(＝NR^bb)R^aa、–C(＝NR^cc)OR^aa、–C(＝NR^cc)N(R^cc)₂、–SO₂N(R^cc)₂、–SO₂R^cc、–SO₂OR^cc、–SOR^aa、–C(＝S)N(R^cc)₂、–C(＝O)SR^cc、–C(＝S)SR^cc、–P(＝O)(OR^cc)₂、–P(＝O)(R^aa)₂、–P(＝O)(N(R^cc)₂)₂、C_1–10Alkyl radical, C_1–10Perhaloalkyl, C_2–10Alkenyl radical, C_2–10Alkynyl, C_3–10Carbocyclyl, 3-14 membered heterocyclyl, C_6–14Aryl and 5-14 membered heteroaryl, or two R attached to a nitrogen atom^ccThe groups combine to form a 3-14 membered heterocyclyl or 5-14 membered heteroaromatic ring wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5R ^ddIs substituted by radicals, and wherein R^aa、R^bb、R^ccAnd R^ddAs defined above.

In some embodiments, the substituent present on the nitrogen atom is a nitrogen protecting group (also referred to as an amino protecting group). Nitrogen protecting groups include, but are not limited to: -OH, -OR^aa、–N(R^cc)₂、–C(＝O)R^aa、–C(＝O)N(R^cc)₂、–CO₂R^aa、–SO₂R^aa、–C(＝NR^cc)R^aa、–C(＝NR^cc)OR^aa、–C(＝NR^cc)N(R^cc)₂、–SO₂N(R^cc)₂、–SO₂R^cc、–SO₂OR^cc、–SOR^aa、–C(＝S)N(R^cc)₂、–C(＝O)SR^cc、–C(＝S)SR^cc、C_1–10Alkyl (e.g., aralkyl, heteroaralkyl), C_2–10Alkenyl radical, C_2–10Alkynyl, C_3–10Carbocyclyl, 3-14 membered heterocyclyl, C_6–14Aryl and 5-14 membered heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl and heteroaryl is independently substituted with 0, 1, 2, 3, 4 or 5R^ddIs substituted by radicals, and wherein R^aa、R^bb、R^ccAnd R^ddAs defined herein. Nitrogen Protecting Groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T.W.Greene and P.G.M.Wuts, third edition, John Wiley&Sons, 1999, which is incorporated herein by reference.

For example, a nitrogen protecting group such as an amide group (e.g., -C (═ O) R^aa) Including but not limited to: formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropionamide, pyridine-2-carboxamide, pyridine-3-carboxamide, N-benzoylphenylalanyl derivative, benzamide, p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenyloxyacetamide, acetoacetamide, (N' -dithiobenzyloxyacylamino) acetamide, 3- (p-hydroxyphenyl) propionamide, 3- (o-nitrophenyl) propionamide, 2-methyl-2- (o-nitrophenyloxy) propionamide, 2-methyl-2- (o-phenylazophenoxy) propionamide, 4-chlorobutyramide, 3-methyl-3-nitrobutyramide, o-nitrocinnamamide, or a salt thereof, N-acetylmethionine derivatives, o-nitrobenzamide and o- (benzoyloxymethyl) benzamide.

Nitrogen protecting groups such as carbamate (e.g., -C (═ O) OR^aa) Including but not limited to: methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9- (2-sulfo) fluorenylmethyl carbamate, 9- (2, 7-dibromo) fluorenylmethyl carbamate, 2, 7-di-tert-butyl- [9- (10, 10-dioxo-10, 10,10, 10-tetrahydrothioxanthyl) carbamate]Methyl ester (DBD-Tmoc), 4-methoxybenzoyl methyl carbamate (Phenoc), 2,2, 2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1- (1-adamantyl) -1-methylethyl carbamate (Adpoc), 1-dimethyl-2-haloethyl carbamate, 1-dimethyl-2, 2-dibromoethyl carbamate (DB-t-BOC), 1-dimethyl-2, 2, 2-trichloroethyl carbamate (TCBOC), 1-methyl-1- (4-biphenylyl) ethyl carbamate (Bpoc), 1- (3, 5-di-tert-butylphenyl) -1-methylethyl carbamate (t-Bumeoc), 2- (2 '-and 4' -pyridyl) ethyl carbamate (Pyoc), 2- (N, N-dicyclohexyl-carboxamide) ethyl carbamate, tert-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiol carbamate, benzyl carbamate (Cbz), P-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2, 4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2- (p-toluenesulfonyl) ethyl carbamate, [2- (1, 3-dithianyl) carbamate) ]Methyl ester (Dmoc), 4-methylthiophenyl carbamate (Mtpc), 2, 4-dimethylthienyl carbamate (Bmpc), 2-phosphonium ethyl carbamate (Peoc), 2-triphenylphosphonium isopropyl carbamate (m-PGM)Ppoc), 1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p- (dihydroxyboryl) benzyl carbamate, 5-benzisoxazolyl methyl carbamate, 2- (trifluoromethyl) -6-chromonylmethyl carbamate (Tcroc), m-nitrophenyl carbamate, 3, 5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3, 4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl) methyl carbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, m-acyloxybenzyl carbamate, p-nitrobenzyl carbamate, p-cyclohexyl carbamate, p-cyclopentyl carbamate, p-nitrobenzyl ester, cyclohexyl carbamate, p-nitrobenzyl ester, p-n-nitrobenzyl ester, p-n-nitrobenzyl ester, p-n-, Cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2-dimethoxyacylvinyl carbamate, o- (N, N-dimethylcarboxamido) benzyl carbamate, 1-dimethyl-3- (N, N-dimethylcarboxamido) propyl carbamate, 1-dimethylpropynyl carbamate, bis (2-pyridyl) methyl carbamate, 2-furylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinoyl carbamate, p- (p ' -methoxyphenylazo) benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, p- (p ' -methoxyphenylazo) benzyl carbamate, o- (N, N-dimethylcarboxamido) benzyl carbamate, 1-furylmethyl carbamate, 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinoyl ester, p- (p ' -methoxyphenylazo) benzyl carbamate, cyclohexyl ester, N-methyl carbamate, N-methyl-carbamate, N-methyl-ethyl-methyl-carbamate, N-methyl-ethyl-methyl-ethyl-methyl-ethyl-methyl-ethyl-methyl-ethyl-methyl-ethyl-methyl-ethyl-methyl-ethyl-methyl-ethyl-methyl-ethyl-methyl-ethyl-methyl-, 1-methyl-1-cyclopropylmethyl carbamate, 1-methyl-1- (3, 5-dimethoxyphenyl) ethyl carbamate, 1-methyl-1- (p-phenylazophenyl) ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1-methyl-1- (4-pyridyl) ethyl carbamate, phenyl carbamate, p- (phenylazo) benzyl carbamate, 2,4, 6-tri-tert-butylphenyl carbamate, 4- (trimethylammonium) benzyl carbamate and 2,4, 6-trimethylbenzyl carbamate.

A nitrogen protecting group such as a sulfonamide group (e.g., -S (═ O)₂R^aa) Including but not limited to: p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6, -trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4, 6-trimethoxybenzenesulfonamide (Mtb), 2, 6-dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5, 6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs),2,4, 6-trimethylbenzenesulfonamide (Mts), 2, 6-dimethoxy-4-methylbenzenesulfonamide (iMds), 2,5,7, 8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β -trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4- (4 ', 8' -dimethoxynaphthylmethyl) benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide and benzoylmethanesulfonamide.

Other nitrogen protecting groups include, but are not limited to: phenothiazinyl- (10) -acyl derivatives, N '-p-toluenesulfonylaminoacyl derivatives, N' -phenylaminothioacyl derivatives, N-benzoylphenylalanyl derivatives, N-acetylmethionine derivatives, 4, 5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiosuccinimide (Dts), N-2, 3-diphenylmaleimide, N-2, 5-dimethylpyrrole, N-1,1,4, 4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1, 3-dimethyl-1, 3, 5-triazacyclohexan-2-one, 5-substituted 1, 3-dibenzyl-1, 3, 5-triazacyclohex-2-one, 1-substituted 3, 5-dinitro-4-pyridone, N-methylamine, N-allylamine, N- [2- (trimethylsilyl) ethoxy ]Methylamine (SEM), N-3-acetoxypropylamine, N- (1-isopropyl-4-nitro-2-oxo-3-pyrrolidin-3-yl) amine, quaternary ammonium salt, N-benzylamine, N-bis (4-methoxyphenyl) methylamine, N-5-dibenzocycloheptylamine, N-triphenylmethylamine (Tr), N- [ (4-methoxyphenyl) diphenylmethyl]Amines (MMTr), N-9-phenylfluorenylamine (PhF), N-2, 7-dichloro-9-fluorenylmethylidene amine, N-ferrocenylmethylamino (Fcm), N-2-pyridylmethylamino N' -oxide, N-1, 1-dimethylthiomethylidene amine, N-benzylidene amine, N-p-methoxybenzylideneamine, N-diphenylmethylidene amine, N- [ (2-pyridyl)Base of]Methylamine, N- (N ', N ' -dimethylaminomethylene) amine, N ' -isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylidene amine, N-5-chlorosalicylideneamine, N- (5-chloro-2-hydroxyphenyl) phenylmethylidene amine, N-cyclohexylidene amine, N- (5, 5-dimethyl-3-oxo-1-cyclohexenyl) amine, N-borane derivatives, N-diphenylboronic acid derivatives, N- [ phenyl ] amine(Pentaacylchromium-or tungsten) acyl]Amines, N-copper chelates, N-zinc chelates, N-nitramines, N-nitrosamines, amine N-oxides, diphenylphosphinamides (Dpp), dimethylthiophosphonamides (Mpt), diphenylphosphinamides (Ppt), dialkylphosphoramidates, dibenzylphosphoramidates, diphenylphosphinates, benzenesulfenamides, o-nitrobenzenesulfinamides (Nps), 2, 4-dinitrobenzenesulfenamides, pentachlorobenzenesulfinamides, 2-nitro-4-methoxybenzenesulfinamides, triphenylmethylsulfinamides, and 3-nitropyridine sulfenamides (Npys).

Exemplary oxygen atom substituents include, but are not limited to: -R^aa、–C(＝O)SR^aa、–C(＝O)R^aa、–CO₂R^aa、–C(＝O)N(R^bb)₂、–C(＝NR^bb)R^aa、–C(＝NR^bb)OR^aa、–C(＝NR^bb)N(R^bb)₂、–S(＝O)R^aa、–SO₂R^aa、–Si(R^aa)₃、–P(R^cc)₂、–P(R^cc)₃ ⁺X^-、-P(OR^cc)₂、-P(OR^cc)₃ ⁺X^-、–P(＝O)(R^aa)₂、–P(＝O)(OR^cc)₂and-P (═ O) (N (R)^bb)₂)₂Wherein X is^-、R^aa、R^bbAnd R^ccAs defined herein. In some embodiments, the oxygen atom substituent present on the oxygen atom is an oxygen protecting group (also referred to as a hydroxyl protecting group). Oxygen Protecting Groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, T.W.Greene and P.G.M.Wuts, third edition, John Wiley&Sons, 1999, which is incorporated herein by reference. Exemplary oxygen protecting groups include, but are not limited to: methyl, tert-butyloxycarbonyl (BOC or Boc), methoxymethyl (MOM), methylthiomethyl (MTM), tert-butylthiomethyl, (phenyldimethylsilyl) methoxymethyl (SMOM), Benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy) methyl (p-AOM), Guaiacolmethyl (GUM), tert-butyloxycarbonyl (BOC or Boc), tert-butyloxymethyl (MOM), methylthiomethyl (MTM), tert-butylthiomethyl (SMOM), Benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy) methyl (p-AOM), Guaiacolmethyl (GUM), tert-butylthiomethyl (GUM), and N-butylthiomethyl (GUM)Oxymethyl, 4-Pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2, 2-trichloroethoxymethyl, bis (2-chloroethoxy) methyl, 2- (trimethylsilyl) ethoxymethyl (SEMOR), Tetrahydropyranyl (THP), 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-Methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl S, S-dioxide, 1- [ (2-chloro-4-methyl) phenyl ]-4-methoxypiperidin-4-yl (CTMP), 1, 4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothienyl, 2,3,3a,4,5,6,7,7 a-octahydro-7, 8, 8-trimethyl-4, 7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1- (2-chloroethoxy) ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2,2, 2-trichloroethyl, 2-trimethylsilylethyl, 2- (phenylseleno) ethyl, tert-butyl, allyl, methyl, 2-methoxy-2-fluoroethyl, 2,2, 2-trichloroethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 2- (phenylseleno) ethyl, tert-butyl, 2-methyl, 4-methyl, 5-methyl-4, 7-methano-2-yl, 2-methyl-2-fluoroethyl, 1-methyl-1-methoxy-1-methoxy-2-ethyl, 2-methyl-1-methoxy-seleno-ethyl, 2-methyl-1-ethyl, 2-methyl-4-methyl-ethyl, 2-methyl-4-methoxy-ethyl, 2-methyl-4-methyl-ethyl, 2-methyl-4-methyl-4-ethyl, 2-methyl-ethyl, 2-methyl-ethyl, 2-methyl, 2-ethyl, 2-methyl, 2-ethyl, 2-methyl, 2-ethyl, 2, 2-ethyl, 2-methyl, 2-ethyl, 2,2, or a, 2,2,2, P-chlorophenyl, p-methoxyphenyl, 2, 4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3, 4-dimethoxybenzyl, o-nitrobenzyl, p-halobenzyl, 2, 6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxide, diphenylmethyl, p ' -dinitrobenzhydryl, 5-dibenzosuberenyl, triphenylmethyl, alpha-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di (p-methoxyphenyl) phenylmethyl, tri (p-methoxyphenyl) methyl, 4- (4 ' -bromobenzoylmethoxyphenyl) diphenylmethyl, 4 ', 4 '-tris (4, 5-dichlorophthalimidophenyl) methyl group, 4' -tris (levulinoyloxyphenyl) methyl group, 4 '-tris (benzoyloxyphenyl) methyl group, 3- (imidazol-1-yl) bis (4', 4 '-dimethoxyphenyl) methyl group, 1-bis (4-methoxyphenyl) -1' -pyrenylmethyl group, 9-anthryl group, 9- (9-phenyl) group Xanthyl, 9- (9-phenyl-10-oxo) anthracenyl, 1, 3-benzodithiolan (benzodithiolan) -2-yl, benzisothiazolyl S, S-dioxide, Trimethylsilyl (TMS), trisubstitutedEthylsilyl (TES), Triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), Diethylisopropylsilyl (DEIPS), dimethylhexylsilyl, tert-butyldimethylsilyl (TBDMS), tert-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, Diphenylmethylsilyl (DPMS), tert-butylmethoxyphenylsilyl (TBMPS), formic acid ester, benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate (levulinate), 4- (ethylenedithio) pentanoate (levulinyl dithioacetal), Pivalate, adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4, 6-trimethylbenzoate (mesitooate), alkylmethylcarbonate, 9-fluorenylmethylcarbonate (Fmoc), alkylethylcarbonate, alkyl 2,2, 2-trichloroethylcarbonate (Troc), 2- (trimethylsilyl) ethylcarbonate (TMSEC), 2- (phenylsulfonyl) ethylcarbonate (Psec), 2- (triphenylphosphonio) ethylcarbonate (Peoc), alkylisobutylcarbonate, alkylvinylcarbonate, alkylallylcarbonic ester, alkyl p-nitrophenylcarbonate, alkylbenzylcarbonate, alkyl p-methoxybenzylcarbonate, alkyl 3, 4-dimethoxybenzylcarbonate, methyl-p-hydroxybenzoate, methyl-p-ethylcarbonate, methyl-n-butyl-ethyl-carbonate, methyl-ethyl-2- (trimethylsilyl) ethylcarbonate, methyl-2, 2-ethylmethyl-2-ethyl-2- (trimethylsilyl) ethylcarbonate, 2-ethylmethyl-carbonate, 2- (phenylsulfonyl-ethyl-carbonate, 2-methyl-carbonate, 2-methyl-ethyl-carbonate, 2-methyl-carbonate, 2-methyl-ethyl-carbonate, 2-methyl-carbonate, 2-methyl-ethyl-methyl-carbonate, 2-methyl-carbonate, 2-ethyl-methyl-carbonate, and/ethyl-carbonate, Alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzylthiocarbonate, 4-ethoxy-1-naphthyl carbonate, methyldithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o- (dibromomethyl) benzoate, 2-formylbenzenesulfonate, 2- (methylthiomethoxy) ethyl, 4- (methylthiomethoxy) butyrate, 2- (methylthiomethoxymethyl) benzoate, 2, 6-dichloro-4-methylphenoxyacetate, 2, 6-dichloro-4- (1,1,3, 3-tetramethylbutyl) phenoxyacetate, 2, 4-bis (1, 1-dimethylpropyl) phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinate, (E) -2-methyl-2-butenoate, o- (methoxyacyl) phenoxyacetate ) Benzoate esters, α -naphthoates, nitrates, alkyl N, N' -tetramethylphosphorodiamidites, alkyl N-phenylcarbamates, borate esters, dimethylphosphinothioates, alkyl 2, 4-dinitrophenylsulfenamates, sulfates, methanesulfonates (methanesulfonates), benzylsulfonates and tosylates (Ts).

Exemplary sulfur atom substituents include, but are not limited to: -R^aa、–C(＝O)SR^aa、–C(＝O)R^aa、–CO₂R^aa、–C(＝O)N(R^bb)₂、–C(＝NR^bb)R^aa、–C(＝NR^bb)OR^aa、–C(＝NR^bb)N(R^bb)₂、–S(＝O)R^aa、–SO₂R^aa、–Si(R^aa)₃、-P(R^cc)₂、-P(R^cc)₃ ⁺X^-、-P(OR^cc)₂、-P(OR^cc)₃ ⁺X^-、 -P(＝O)(R^aa)₂、-P(＝O)(OR^cc)₂and-P (═ O) (N (R)^bb)₂)₂Wherein R is^aa、R^bbAnd R^ccAs defined herein. In some embodiments, the sulfur atom substituent present on the sulfur atom is a sulfur protecting group (also referred to as a mercapto-protecting group). Sulfur Protecting Groups are well known in the art and include Protecting Groups in Organic Synthesis, T.W.Greene and P.G.M.Wuts, 3 rd edition, John Wiley&Sons, 1999, which is incorporated herein by reference.

The term "leaving group" is given its ordinary meaning in the field of synthetic organic chemistry and refers to an atom or group capable of being displaced by a nucleophile. See, for example, Smith, March Advanced Organic Chemistry 6th ed. (501-502). Exemplary leaving groups include, but are not limited to: halogen (e.g. chloro, bromo, iodo) and activated substituted hydroxy (e.g., -OC (═ O) SR ^aa、–OC(＝O)R^aa、–OCO₂R^aa、–OC(＝O)N(R^bb)₂、–OC(＝NR^bb)R^aa、–OC(＝NR^bb)OR^aa、–OC(＝NR^bb)N(R^bb)₂、–OS(＝O)R^aa、–OSO₂R^aa、–OP(R^cc)₂、–OP(R^cc)₃、–OP(＝O)₂R^aa、–OP(＝O)(R^aa)₂、–OP(＝O)(OR^cc)₂、–OP(＝O)₂N(R^bb)₂and-OP (═ O) (NR)^bb)₂Wherein R is^aa、R^bbAnd R^ccAs defined herein). In some cases, the leaving group is a sulfonate ester, such as tosylate (-OTs), mesylate (-OMs), p-bromophenylsulfonyloxy (-OBs), -OS (═ O)₂(CF₂)₃CF₃(perfluorobutylsulfonic acid group, -ONf), or trifluoromethanesulfonic acid ester (-OTf). In some cases, the leaving group is a p-bromobenzenesulfonate, such as p-bromobenzenesulfonyloxy. In some cases, the leaving group is m-nitrobenzenesulfonate, such as 2-nitrobenzenesulfonyloxy. In some embodiments, the leaving group is a sulfonate-containing group. In some embodiments, the leaving group is a tosylate group. The leaving group may also be a phosphine oxide (e.g., formed in a Mitsunobu reaction) or an internal leaving group such as an epoxide or cyclic sulfate. Other non-limiting examples of leaving groups are water, ammonia, alcohols, ether moieties, thioether moieties, zinc halides, magnesium moieties, diazonium salts, and copper moieties.

"Hydrocarbon chain" refers to a substituted or unsubstituted divalent alkyl, alkenyl, or alkynyl group. The hydrocarbon chain comprises (1) one or more chains of carbon atoms directly between two groups of the hydrocarbon chain; (2) optionally one or more hydrogen atoms in a chain of carbon atoms; and (3) optionally one or more substituents on the chain of carbon atoms ("non-chain substituents", which are not hydrogen). The chain of carbon atoms consists of carbon atoms ("chain atoms") that are continuously connected and does not include hydrogen atoms or heteroatoms. However, the non-chain substituents of the hydrocarbon chain may contain any atom, including hydrogen atoms, carbon atoms, and heteroatoms. For example, the hydrocarbon chain-C ^AH(C^BH₂C^CH₃) Containing a chain atomC^AOne is at C^AHydrogen atom and non-chain substituents (C)^BH₂C^CH₃). The term "C_xHydrocarbon chain ", wherein x is a positive integer, refers to a hydrocarbon chain comprising x chain atoms between two groups of the hydrocarbon chain. If x has more than one possible value, the minimum possible value of x is used for the definition of the hydrocarbon chain. For example, -CH (C)₂H₅) -is C₁A hydrocarbon chain, andis C₃A hydrocarbon chain. When a range of values is used, the range is intended to have the meaning described herein. E.g. C_3-10A hydrocarbon chain refers to a hydrocarbon chain having 3, 4, 5, 6, 7, 8, 9, or 10 chain atoms in the shortest chain of carbon atoms directly connected between two groups of the hydrocarbon chain. The hydrocarbon chain can be saturated (e.g., - (CH)₂)₄-). The hydrocarbon chain may also be unsaturated and contain one or more C ═ C and/or C ≡ C bonds at any position of the hydrocarbon chain. For example, -CH ═ CH- (CH)₂)₂–、–CH₂–C≡C–CH₂-and-C ≡ C-CH ═ CH-are both examples of unsubstituted and unsaturated hydrocarbon chains. In certain embodiments, the hydrocarbon chain is unsubstituted (e.g., -C.ident.C-or- (CH)₂)₄-). In certain embodiments, the hydrocarbon chain is substituted (e.g., -CH (C)₂H₅) -and-CF₂-). Any two substituents on the hydrocarbon chain can be joined to form an optionally substituted carbocyclyl, an optionally substituted heterocyclyl, an optionally substituted aryl, or an optionally substituted heteroaryl ring. For example,

Are examples of hydrocarbon chains. In contrast, in some embodiments, the first and second electrodes,outside the range of hydrocarbon chains described herein. When C is present_xWhen a chain atom of the hydrocarbon chain is replaced by a heteroatom, the resulting group is said to beIs C in which the chain atoms are replaced by hetero atoms_xHydrocarbon chain, this and C_x-1Hydrocarbon chains are used for comparison. For example,is C₃A hydrocarbon chain in which one chain atom is replaced by an oxygen atom.

The term "pharmaceutically acceptable salts" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, the pharmaceutically acceptable salts described in detail in J.pharmaceutical Sciences, 1977, 66, 1-19 by Berge et al, which are incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or using other methods known in the art (e.g. ion exchange). Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, glucoheptonates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxy-ethanesulfonates, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, salts of benzoic acid, bisulfates, salts of hydrogen, salts of glycerol with a glucuronate, hemisulfates, heptanoates, caproates, 2-hydroxy-ethanesulfonates, lactobionates, lactates, laurylsulfonates, methosulfonates, citrates, salts of hydrogen propionates, and mixtures thereof, Pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-formazan Benzenesulfonate, undecanoate, valerate, and the like. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N⁺(C_1-4Alkyl radical)₄ ^-And (3) salt. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like. Other pharmaceutically acceptable salts include, where appropriate, salts of non-toxic ammonium, quaternary ammonium and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.

The term "solvate" refers to a form of a compound or salt thereof associated with a solvent, typically formed by a solvolysis reaction. Such physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, ether, and the like. The compounds described herein can be prepared, for example, in crystalline form, and can be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric and non-stoichiometric solvates. In some cases, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "solvate" includes solvates in a solution state and isolatable solvates. Representative solvates include hydrates, ethanolates, and methanolates.

The term "hydrate" refers to a compound associated with water. Generally, the ratio of the number of water molecules contained in a hydrate of a compound to the number of molecules of the compound in the hydrate is determined. Thus, hydrates of the compounds can be used, for example, of the formula R. x H₂O represents, wherein R is the compound and x is a number greater than 0. A given compound may form more than one hydrate type, including, for example, monohydrate (x is 1), lower hydrates (x is a number greater than 0 and less than 1), e.g., hemihydrate (R0.5H)₂O)) and polyhydrates (x is a number greater than 1, e.g. dihydrate (R.2H)₂O) and hexahydrate (R.6H)₂O)。

The term "tautomer" or "tautomeric" refers to two or more interconvertible compounds resulting from formal migration of at least one hydrogen atom and at least one change in valence (e.g., single bond to double bond, triple bond to single bond, or vice versa). The exact ratio of tautomers depends on a variety of factors including temperature, solvent and pH. Tautomerization (i.e., the reaction that provides a tautomeric pair) can be catalyzed by either an acid or a base. Exemplary tautomerism includes tautomerism of keto-enols, amide-imides, lactam-lactams, enamine-imines, and enamines- (different enamines).

It is also understood that compounds having the same molecular formula but differing in nature or in the order of bonding of their atoms or in the arrangement of their atoms in space are referred to as "isomers". Isomers in which the spatial arrangement of their atoms is different are referred to as "stereoisomers".

Stereoisomers that are not in mirror image relationship with each other are referred to as "diastereomers", while those that are not in overlapping mirror image relationship with each other are referred to as "enantiomers". When a compound has an asymmetric center, for example, when it is bonded to four different groups, a pair of enantiomers is possible. Enantiomers can be characterized by the absolute configuration of their asymmetric centers and can be described by the R-and S-ordering rules of Cahn and Prelog, or by the fact that the molecule rotates in the plane of polarized light and is designated dextrorotatory or levorotatory (i.e., the (+) or (-) -isomers, respectively). The chiral compound may exist as a single enantiomer or as a mixture thereof. Mixtures containing equal proportions of enantiomers are referred to as "racemic mixtures".

The term "polymorph" refers to a crystalline form of a compound (or a salt, hydrate, or solvate thereof). All polymorphs have the same elemental composition. Different crystalline forms typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shape, optoelectronic properties, stability and solubility. Recrystallization solvent, crystallization rate, storage temperature, and other factors may cause a crystalline form to dominate. Various polymorphs of a compound may be prepared by crystallization under different conditions.

The term "prodrugs"refers to a compound that has a cleavable group and that is pharmaceutically active in vivo by solvolysis or under physiological conditions to a compound as described herein. Examples of such include, but are not limited to: choline ester derivatives and the like, N-alkyl morpholine esters and the like. Other derivatives of the compounds of the invention are active in their acid or acid derivative forms, but generally offer advantages in solubility, histocompatibility or delayed release in mammalian organisms in acid-sensitive forms (see Bundgard, h., Design of produgs, pages 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs include acid derivatives well known to practitioners in the art, for example, esters prepared by reaction of the parent acid with a suitable alcohol, or amides prepared by reaction of the parent acid compound with a substituted or unsubstituted amine, or anhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters, amides and anhydrides derived from acidic groups on the compounds of the present invention are specific prodrugs. In some cases, it is desirable to prepare diester-type prodrugs, such as (acyloxy) alkyl esters or ((alkoxycarbonyl) oxy) alkyl esters. C of the Compounds described herein ₁-C₈Alkyl ester, C₂-C₈Alkenyl esters, C₂-C₈Alkynyl ester, aryl ester, C₇-C₁₂Substituted aryl esters and C₇-C₁₂Arylalkyl esters may be preferred.

The term "small molecule" refers to a molecule having a relatively low molecular weight, whether naturally occurring or artificially generated (e.g., by chemical synthesis). Typically, the small molecule is an organic compound (i.e., it contains carbon). The small molecules can contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyls, carbonyls, and heterocycles, etc.). In some embodiments, the small molecule has a molecular weight of no more than about 1,000g/mol, no more than about 900g/mol, no more than about 800g/mol, no more than about 700g/mol, no more than about 600g/mol, no more than about 500g/mol, no more than about 400g/mol, no more than about 300g/mol, no more than about 200g/mol, or no more than about 100 g/mol. In some embodiments, the small molecule has a molecular weight of at least about 100g/mol, at least about 200g/mol, at least about 300g/mol, at least about 400g/mol, at least about 500g/mol, at least about 600g/mol, at least about 700g/mol, at least about 800g/mol, or at least about 900g/mol, or at least about 1,000 g/mol. Combinations of the above ranges (e.g., at least about 200g/mol and no more than about 500g/mol) are also possible. In some embodiments, the small molecule is a therapeutically active agent, such as a drug (e.g., a molecule approved by the U.S. food and drug administration and provided in the U.S. federal regulations compilation (c.f.r)). The small molecule may also be complexed with one or more metal atoms and/or metal ions. In the present application, the small molecules are also referred to as "small organometallic molecules". Preferred small molecules are biologically active, which produce a biological effect in an animal, preferably a mammal, more preferably a human. Small molecules include, but are not limited to, radionuclides and imaging agents. In some embodiments, the small molecule is a drug. Preferably, but not necessarily, the medicament is one that has been deemed safe and effective for use in humans or animals by a suitable governmental or regulatory agency. For example, drugs approved for use in humans are listed by the FDA in 21c.f.r. § 330.5, 331 to 361 and 440 to 460, which are incorporated herein by reference; drugs for veterinary use are listed by the FDA in 21c.f.r. § 500 to 589, which are incorporated herein by reference. All listed drugs are considered to be acceptable for use in the present invention.

The term "small molecule drug" refers to a small molecule or group of small molecules that has been approved by a governmental agency (e.g., FDA) for administration to a subject (e.g., a human or non-human animal).

The term "small molecule tag" refers to a small molecule or group of such small molecules that can be detected. Exemplary small molecule tags include, but are not limited to, biotin, radioisotopes, enzymes, luminescent agents, precipitating agents, fluorophores, and dyes.

The term "small molecule fluorophore" refers to a small molecule that fluoresces, e.g., is capable of re-emitting light upon photoexcitation. Exemplary small molecule fluorophores include, but are not limited to, fluorescein, rhodamine, coumarin, cyanine, and derivatives thereof.

"protein", "peptide" or "polypeptide" includes polymers of amino acid residues joined together by peptide bonds. The term refers to proteins, polypeptides and peptides of any size, structure or function. Typically, the protein will be at least 3 amino acids in length. A protein may refer to an individual protein or a collection of proteins. The proteins of the invention preferably contain only natural amino acids, but may alternatively employ unnatural amino acids (i.e., compounds that do not occur in nature but which can be incorporated into polypeptide chains) and/or amino acid analogs known in the art. In addition, one or more amino acids in a protein can be modified, for example, by the addition of a chemical entity, such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation or functionalization, or other modification. The protein may also be a single molecule or may be a multi-molecule complex. The protein may be a fragment of a naturally occurring protein or peptide. The protein may be naturally occurring, recombinant, synthetic or any combination of these.

The terms "polynucleotide", "nucleotide sequence", "nucleic acid molecule", "nucleic acid sequence" and "oligonucleotide" refer to a series of nucleotide bases (also referred to as "nucleotides") in DNA and RNA, and to any strand of two or more nucleotides. The polynucleotide may be a chimeric mixture of single or double strands or a derivative or modified form thereof. Oligonucleotides may be modified on the base moiety, sugar moiety or phosphate backbone, for example, to improve the stability of the molecule, its hybridization parameters, and the like. The antisense oligonucleotide may comprise a modified base moiety selected from the group including, but not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β -D-galactosylQ nucleoside (β -D-galactosylqueosine), inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosyl Q nucleoside, 5' -methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, wybutoxosine, pseudouracil, Q nucleoside, 2-thiocytosine, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, thioguanine and 2, 6-diaminopurine. Nucleotide sequences typically carry genetic information, including information used to produce proteins and enzymes by cellular mechanisms. These terms include double-or single-stranded genomes and cdnas, RNAs, any synthetic and genetically manipulated polynucleotides, and sense and antisense polynucleotides. This includes single-and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, and "protein nucleic acids" (PNAs) formed by binding bases to amino acid backbones. This also includes nucleic acids containing carbohydrates or lipids. Exemplary DNAs include single-stranded DNA (ssdna), double-stranded DNA (dsdna), plasmid DNA (pdna), genomic DNA (gdna), complementary DNA (cdna), antisense DNA, chloroplast DNA (ctDNA or cpDNA), microsatellite DNA, mitochondrial DNA (mtDNA or mDNA), kinetoplast DNA (kdna), provirus, lysogen, repetitive DNA, satellite DNA, and viral DNA. Exemplary RNAs include single-stranded RNA (ssrna), double-stranded RNA (dsrna), small interfering RNA (sirna), messenger RNA (mRNA), pre-messenger RNA (pre-mRNA), small or short hairpin RNA (shrna), micro RNA (mirna), guide RNA (grna), transfer RNA (trna), antisense RNA (asrna), heterologous nuclear RNA (hnrna), coding RNA, non-coding RNA (ncRNA), long non-coding RNA (long ncRNA or lncRNA), satellite RNA, viral satellite RNA, signal-recognition-particle RNA, small cytoplasmic RNA, small nuclear RNA (snrna), ribosomal RNA (rrna), Piwi-interacting RNA (pirna), polyinosinic acid, ribozymes, flexzyme, small nucleolar RNA (snorna), lead-spliced RNA, viral RNA, and viral satellite RNA.

Polynucleotides described herein can be synthesized by standard methods known in the art, for example, by using an automated DNA synthesizer (e.g., those commercially available from Biosearch, Applied Biosystems, etc.). By way of example, phosphorothioate oligonucleotides can be prepared by the method of Stein et al, Nucl. acids Res.,16,3209, (1988), and methylphosphonate oligonucleotides can be prepared by using controlled pore glass polymer supports (Sarin et al, Proc. Natl. Acad. Sci. U.S. A.85, 7448-7451, (1988)). A number of methods have been developed for delivering antisense DNA or RNA to cells, for example, antisense molecules can be injected directly into a tissue site or modified antisense molecules (antisense linked to a peptide or antibody that specifically binds to a receptor or antigen expressed on the surface of a target cell) designed to target the desired cells can be administered systemically. Alternatively, RNA molecules can be produced by in vitro and in vivo transcription of DNA sequences encoding antisense RNA molecules. Such DNA sequences may be incorporated into a variety of vectors, which incorporate a suitable RNA polymerase promoter such as the T7 or SP6 polymerase promoter. Alternatively, an antisense cDNA construct that synthesizes antisense RNA constitutively or inducibly depending on the promoter used can be stably introduced into the cell line. However, it is often difficult to achieve intracellular concentrations of the antisense sufficient to inhibit translation of endogenous mRNA. Thus, a preferred method utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong promoter. Transfection of target cells in a patient with such a construct will result in transcription of a sufficient amount of single-stranded RNA that will form complementary base pairs with the endogenous target gene transcript, thereby preventing translation of the target gene mRNA. For example, the vector can be introduced in vivo, allowed to be taken up by cells and direct transcription of antisense RNA. Such vectors may remain episomal or become chromosomally integrated so long as they can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA techniques standard in the art. The vector may be a plasmid, virus or other vector known in the art for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be effected in mammalian, preferably human, cells by any promoter known in the art. Such promoters may be inducible or constitutive. Any type of plasmid, cosmid, yeast artificial chromosome, or viral vector can be used to prepare a recombinant DNA construct that can be introduced directly into a tissue site.

The polynucleotide may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, Internal Ribosome Entry Sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5 '-and 3' -noncoding regions, and the like. Nucleic acids can also be modified by a number of methods known in the art. Non-limiting examples of such modifications include methylation, "caps," substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications, such as those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). The polynucleotide may contain one or more additional covalently linked moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidized metals, etc.), and alkylators. Polynucleotides may be derivatized by forming methyl or ethyl phosphotriester or phosphoramidate alkyl ester linkages. In addition, the polynucleotides herein may also be modified with a tag capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, isotopes (e.g., radioisotopes), biotin, and the like.

The term "inhibit" or "inhibitor" refers to the ability of a compound to reduce, slow, stop, or prevent the activity of a particular biological process (e.g., the activity of a bromodomain and/or bromodomain-containing protein) in a cell relative to a vehicle.

When a compound, pharmaceutical composition, method, use, or kit is said to "selectively", "specifically", or "competitively" inhibit HMT, the compound, pharmaceutical composition, method, use, or kit inhibits the HMT to a greater extent (e.g., not less than 2-fold, not less than 5-fold, not less than 10-fold, not less than 30-fold, not less than 100-fold, not less than 1,000-fold, or not less than 10,000-fold; and/or not more than 2-fold, not more than 5-fold, not more than 10-fold, not more than 30-fold, not more than 100-fold, not more than 1,000-fold, or not more than 10,000-fold) than inhibits a different HMT.

The term "aberrant activity" refers to an activity that deviates from the normal activity. In some embodiments, the aberrant activity is increased activity. In some embodiments, the aberrant activity is decreased activity. The term "increased activity" refers to an activity that is higher than normal. The term "reduced activity" refers to an activity that is lower than normal.

The terms "composition" and "formulation" are used interchangeably.

A "subject" for which administration is contemplated refers to a human (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., an infant, child, or adolescent) or an adult subject (e.g., a young adult, a middle aged adult, or an elderly adult)) or a non-human animal. In some embodiments, the non-human animal is a mammal (e.g., a primate (e.g., a cynomolgus monkey, a rhesus monkey), a commercially relevant mammal (e.g., a cow, pig, horse, sheep, goat, cat, or dog), or a bird (e.g., a commercially relevant avian species such as a chicken, duck, goose, or turkey). The plant is a seed plant. In some embodiments, the plant is a cultivated plant. In some embodiments, the plant is a dicot. In some embodiments, the plant is a monocot. In some embodiments, the plant is a flowering plant. In some embodiments, the plant is a cereal, for example, maize (maize), maize (corn), wheat, rice, oats, barley, rye, or millet. In some embodiments, the plant is a leguminous plant, e.g., a legume plant, e.g., a soybean plant. In some embodiments, the plant is a tree or shrub.

The term "biological sample" refers to any sample, including tissue samples (e.g., tissue sections and needle biopsies of tissue); a cell sample (e.g., a cytological smear (such as a Pap or blood smear) or a cell sample obtained by microdissection); whole biological samples (such as yeast or bacterial samples); or a cell fraction, fragment, or organelle (e.g., obtained by lysing the cells and separating components thereof by centrifugation or other means). Other examples of biological samples include blood, serum, urine, semen, stool, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsy tissue (e.g., obtained by surgical biopsy or needle biopsy), nipple aspirate, milk, vaginal fluid, saliva, a swab (e.g., a buccal swab), or any material containing a biomolecule derived from a biological sample.

The terms "administering," "administering," or "administering" refer to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing into a subject a compound described herein, or a composition thereof.

The terms "treat," "treating," and "treatment" refer to reversing, alleviating, delaying the onset of, or inhibiting the progression of the diseases described herein. In some embodiments, treatment may be administered after the disease has appeared or one or more signs or symptoms have been observed. In other embodiments, the treatment may be administered without signs or symptoms of the disease. For example, treatment can be administered to a susceptible subject prior to the onset of symptoms (e.g., based on history of symptoms and/or based on exposure to pathogens). Treatment may also be continued after the symptoms have disappeared, e.g., to delay and/or prevent relapse.

The term "prevention" refers to prophylactic treatment of a subject who has not had a disease but is at risk of developing the disease or who has had a disease that is not the disease but is at risk of relapse of the disease. In some embodiments, the patient is at a higher risk of developing the disease or of relapse of the disease than a healthy member of the population.

The terms "condition," "disease," and "disorder" are used interchangeably.

An "effective amount" of a compound as described herein is an amount sufficient to elicit the desired biological response. The effective amount of the compounds described herein may vary depending on the following factors: the biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject are contemplated. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount is prophylactic treatment. In some embodiments, an effective amount is the amount of a compound of the present invention in a single dose. In some embodiments, an effective amount is a combined amount of the compounds of the present invention in multiple doses.

A "therapeutically effective amount" of a compound described herein is an amount sufficient to provide a therapeutic benefit in treating a disorder or to delay or minimize one or more symptoms associated with the disorder. A therapeutically effective amount of a compound refers to the amount of a therapeutic agent that alone, or in combination with other therapies, provides a therapeutic benefit for treating the condition. The term "therapeutically effective amount" can include an amount that improves the overall treatment, reduces or eliminates the symptoms, signs, or causes of the disorder, and/or enhances the efficacy of another therapeutic agent.

A "prophylactically effective amount" of a compound as described herein is an amount effective to prevent the disorder or one or more symptoms associated with the disorder, or prevent the recurrence thereof. A prophylactically effective amount of a compound refers to the amount of a therapeutic agent that alone, or in combination with other drugs, provides a prophylactic benefit in preventing the condition. The term "prophylactically effective amount" can include an amount that increases the overall prophylactic or prophylactic effect of another prophylactic agent.

The term "histone" refers to an overbased protein found in the nucleus of eukaryotic cells, which packages and arranges DNA into structural units known as nucleosomes. They are the major protein component of chromatin, act as spools of DNA windings, and play a role in gene regulation. In some embodiments, the histone is histone H1 (e.g., histone H1F, histone H1H 1). In some embodiments, the histone is histone H2A (e.g., histone H2AF, histone H2a1, histone H2a 2). In some embodiments, the histone is histone H2B (e.g., histone H2BF, histone H2B1, histone H2B 2). In some embodiments, the histone is histone H3 (e.g., histone H3a1, histone H3a2, histone H3 A3). In some embodiments, the histone is histone H4 (e.g., histone H41, histone H44).

A "histone methyltransferase" or "HMT" is a histone modifying enzyme that catalyzes the transfer of one, two or three methyl groups to lysine and/or arginine residues of histone proteins. HMT modifies histones at specific sites by methylation. Methylation of histones is of biological interest, as such methylation is the major epigenetic modification of chromatin that determines gene expression, genomic stability, stem cell maturation, cell lineage development, genetic imprinting, DNA methylation, and/or cellular mitosis. In some embodiments, the HMT described herein is histone-lysine N-methyltransferase. In some embodiments, the HMT described herein is a histone-arginine N-methyltransferase. In some embodiments, the HMT described herein is EZH 1. In some embodiments, the HMT described herein is EZH 2. In some embodiments, the HMT described herein is DOT 1. In some embodiments, the HMT described herein is G9a, GLP, MLL1, MLL2, MLL3, MLL4, NSD2, PRMT1, PRMT3, PRMT4, PRMT5, PRMT6, SET1b, SET7/9, SET8, setmr, SMYD2, SUV39H1, or SUV39H 2.

The terms "enhancer of zeste homolog 1", "enhancer of zeste 2 polycombin inhibitory complex 1 subunit", "EZH 1", "EZH 1 enzyme", "histone-lysine N-methyltransferase EZH 1" refer to the enzyme encoded by the EZH1 gene. ENSEMBL of the human EZH1 gene: ENSG 00000108799.

The terms "enhancer of zeste homolog 2", "enhancer of zeste 2 polycombin inhibitory complex 2 subunit", "EZH 2", "EZH 2 enzyme", "histone-lysine N-methyltransferase EZH 2" refer to an enzyme encoded by the EZH2 gene. EZH2 is the core catalytic component of the polycomb group (PcG) protein complex family. EZH2 is a histone methyltransferase that catalyzes di-and tri-methylation (H3K27me2/3) in histone H3 lysine 27, silencing gene expression. The catalytic site of EZH2 is present within the SET domain, a highly conserved sequence motif found in several chromatin-associated proteins. EZH2 plays a key role in normal development, and EZH2 deficient mice die early in the embryo due to implantation and failure of gastrulation. EZH2 is known to be associated with embryonic ectodermal developmental proteins, VAV1 oncoprotein and X-linked nucleoprotein (XNP). EZH2 may also play a role in the hematopoietic and central nervous systems. ENSEMBL of the human EZH2 gene: ENSG 00000106462.

The term "genetic disease" refers to a disease caused by one or more abnormalities in the genome of a subject, such as that which exists at birth from the subject. Genetic diseases may be inherited and may be transmitted from parental genes. Genetic diseases may also be caused by mutations or changes in the DNA and/or RNA of a subject. In this case, if the genetic disease occurs in the germ line, the genetic disease will be heritable. Exemplary genetic diseases include, but are not limited to: alzheimer's syndrome, Abset syndrome, achondroplasia, acrodysplasia, addiction, adrenoleukodystrophy, albinism, blepharoedema-macrokolema, Alarvillea syndrome, homogentisate urine, alpha-1 antitrypsin deficiency, Alport syndrome, Alzheimer's disease, asthma, autoimmune polyglandular syndrome, androgen-insensitive syndrome, Angerman syndrome, ataxia telangiectasia, atherosclerosis, Attention Deficit Hyperactivity Disorder (ADHD), autism, baldness, Batten disease (Batten disease), Behcet-Wilson syndrome, Behcet's disease, bipolar disorder, brachial finger malformation, breast cancer, Burkitt lymphoma, chronic myeloid leukemia, Charcot-Marie disease, Crohn's disease, Graves disease, Crohn's disease, and Crohn's disease, Cheilosis, kekan syndrome, kolo syndrome, colon cancer, congenital adrenal hyperplasia, delaunar's syndrome, Costello syndrome, cowden syndrome, craniofacial nasal dysplasia, creutzfeldt-jakob syndrome, creutzfeldt-jakob disease, cystic fibrosis, deafness, depression (dexsuppression), diabetes, dysplasia bone malformations, deguerg syndrome, down syndrome, reading disorders, duchenne muscular dystrophy, dobuzzetts syndrome, ectoderm dysplasia, eh-vandyr syndrome, eh-dongo syndrome, epidermolysis bullosa, epilepsy, essential tremor, familial hypercholesterolemia, familial mediterranean fever, fragile X syndrome, friedreich's ataxia, gaucher disease, glaucoma, glucose galactose malabsorption, glutaric urine, gyrocopheresis, gaucher's syndrome (soft palate-heart-face syndrome), Golan syndrome, blackish-black disease, hemizygous hypertrophy, hemochromatosis, hemophilia, Hereditary Motor and Sensory Neuropathy (HMSN), hereditary nonpolyposive colorectal cancer (HNPCC), Huntington's chorea, immunodeficiency with high-IgM, juvenile diabetes mellitus, Klinefelter's syndrome, Goshiisha syndrome, Leisha disease, Long QT syndrome, malignant melanoma, manic depression, Marfan syndrome, Mengkins syndrome, abortion, mucopolysaccharidoses, multiple endocrine adenomas, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis, myotonic dystrophy, neurofibromatosis, Nino-Pierci disease, Noonan syndrome, obesity, ovarian cancer, pancreatic cancer, Parkinson's disease, paroxysmal nocturnal hemoglobinuria, Pendulley syndrome, amyotrophic lateral sclerosis, Phenylketonuria (PKU), Polycystic kidney disease, Par-widie syndrome, primary biliary cirrhosis, prostate cancer, REAR syndrome, Rafstrom's disease, retinitis pigmentosa, retinoblastoma, Rett syndrome, Sanfilippo syndrome, schizophrenia, severe combined immunodeficiency, sickle cell anemia, spina bifida, spinal muscular atrophy, spinocerebellar atrophy, sudden adult death syndrome, Danger's disease, Thai-saxophone disease, thrombocytopenia, radius deficiency syndrome, Tang-Bu syndrome, tuberous sclerosis, Turner's syndrome, Uscher syndrome, (Von) Hill-Lindi syndrome, Waldens syndrome, Wefter's syndrome, Voroner's syndrome, Williams' syndrome, Wilson's disease, xeroderma pigmentosum and Zerewing's syndrome.

"proliferative disease" refers to a disease caused by abnormal growth or expansion due to cell proliferation (Walker, Cambridge Dictionary of Biology; Cambridge University Press: Cambridge, UK, 1990). Proliferative diseases may be associated with: 1) pathological proliferation of normal resting stage cells; 2) pathological migration of cells from their normal location (e.g., metastasis of tumor cells); 3) Pathological expression of proteolytic enzymes such as matrix metalloproteinases (e.g., collagenase, gelatinase, and elastase); or 4) pathological angiogenesis in proliferative retinopathies and tumor metastases. Exemplary proliferative diseases include cancer (i.e., "malignant tumor"), benign tumor, pathological angiogenesis, inflammatory diseases, and autoimmune diseases.

The term "angiogenesis" refers to the physiological process of forming new blood vessels from pre-existing blood vessels. Angiogenesis is distinct from angiogenesis (vasculogenesis), which is the de novo formation of endothelial cells from mesodermal cell precursors. After the first blood vessels of the developing embryo are formed by angiogenesis, angiogenesis is primarily responsible for the vast majority of blood vessel growth during normal or abnormal development. Angiogenesis is an important process in growth and development, as well as wound healing and the formation of granulation tissue. However, angiogenesis is also a fundamental step in the transition of tumors from a benign state to a malignant state, resulting in the use of angiogenesis inhibitors in the treatment of cancer. Angiogenesis can be chemically stimulated by angiogenic proteins, such as growth factors (e.g., VEGF). "pathological angiogenesis" refers to abnormal (e.g., excessive or insufficient) angiogenesis that progresses to and/or is associated with a disease.

The terms "neoplasms" and "tumors" are used interchangeably and refer to abnormal masses of tissue in which the growth of the mass exceeds and is not coordinated with the growth of normal tissue. A neoplasm or tumor may be "benign" or "malignant," depending on the following characteristics: degree of cell differentiation (including morphology and function), growth rate, local invasion and metastasis. "benign tumors" are generally well differentiated, have significantly slower growth than malignant tumors, and remain localized at the site of origin. In addition, benign tumors do not have the ability to infiltrate, invade, or metastasize to distant locations. Exemplary benign tumors include, but are not limited to: lipoma, chondroma, adenoma, acrochordon, senile hemangioma, seborrheic keratosis, lentigo and sebaceous hyperplasia. In some cases, some "benign" tumors may later cause malignant tumors, which may be due to additional genetic changes in a subpopulation of tumor cells of the tumor, and these tumors are referred to as "pre-malignant neoplasms". An exemplary pre-cancerous tumor is a teratoma. In contrast, "malignant tumors" are generally poorly differentiated (anaplasia) and have significantly rapid growth with progressive infiltration, invasion and destruction of surrounding tissue. In addition, malignant tumors often have the ability to metastasize to distant locations. The terms "metastasis," "metastatic," or "migration" refer to the spread or metastasis of cancer cells from a primary or original tumor to another organ or tissue and is typically determined by: in the organ or tissue where the secondary (metastatic) tumor is located, there is a "secondary tumor" or "secondary cell mass" of the tissue type of the primary or original tumor and not of the organ or tissue where it is located. For example, prostate cancer that has metastasized to bone is referred to as metastatic prostate cancer and includes cancerous prostate cancer cells that grow in bone tissue.

The term "cancer" refers to a class of diseases characterized by the development of abnormal cells that proliferate uncontrollably and have the ability to infiltrate and destroy normal body tissues. See, e.g., Stedman's Medical Dictionary,25th ed.; hensyl ed.; williams & Wilkins: philiadelphia, 1990. Exemplary cancers include, but are not limited to, hematological malignancies. Other exemplary cancers include, but are not limited to: lung cancer (e.g., bronchial cancer, Small Cell Lung Cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); kidney cancer (e.g., nephroblastoma, also known as wilms' tumor, renal cell carcinoma); acoustic neuroma; adenocarcinoma; adrenal cancer; anal cancer; angiosarcoma (angiosarcoma) (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, angiosarcoma); adnexal cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; adenocarcinoma (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, breast cancer (mammary cancer), medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastoma, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchial cancer; carcinoid tumors; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial cancer; ependymoma; endothelial sarcoma (e.g., kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, barrett's adenocarcinoma); ewing's sarcoma; ocular cancers (e.g., intraocular melanoma, retinoblastoma); familial hypereosinophyllia (familiar hypereosinophyllia); gallbladder cancer; stomach cancer (e.g., gastric adenocarcinoma); gastrointestinal stromal tumors (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral disorders (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); heavy chain diseases (e.g., alpha chain disease, gamma chain disease, mu chain disease; hemangioblastoma; laryngopharyngeal cancer; inflammatory myofibroblast tumor; immune cell amyloidosis; liver cancer (e.g., hepatocellular carcinoma (HCC), malignant hepatoma; Leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorders (MPD) (e.g., Polycythemia Vera (PV), Essential Thrombocytosis (ET), causative myeloid metaplasia (AMM), also known as Myelofibrosis (MF), chronic idiopathic myelofibrosis, Chronic Myelogenous Leukemia (CML), Chronic Neutrophilic Leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibromas (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis (schwannomatosis)); neuroendocrine tumors (e.g., gastroenteropancreatic neuroendocrine tumor (GEP-NET), carcinoid tumors); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic adenocarcinoma (pancreatic andenocicepma), Intraductal Papillary Mucinous Neoplasm (IPMN), pancreatic islet cell carcinoma); penile cancer (e.g., paget's disease of the penis and scrotum); pineal tumor; primitive Neuroectodermal Tumors (PNT); a plasmacytoma; paraneoplastic syndromes (paraneoplastic syndromes); intraepithelial tumors; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., Squamous Cell Carcinoma (SCC), Keratoacanthoma (KA), melanoma, Basal Cell Carcinoma (BCC)); small bowel cancer (e.g., appendiceal cancer); soft tissue sarcomas (e.g., Malignant Fibrous Histiocytoma (MFH), liposarcoma, Malignant Peripheral Nerve Sheath Tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland cancer; small bowel cancer; sweat gland cancer; a synovial tumor; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, Papillary Thyroid Carcinoma (PTC), medullary thyroid carcinoma); cancer of the urethra; vaginal cancer; and vulvar cancer (e.g., paget's disease of the vulva).

"hematological disorders" include disorders affecting hematopoietic cells or tissues. Hematological disorders include disorders associated with abnormal blood content and/or function. Exemplary hematological diseases include those resulting from bone marrow irradiation or chemotherapy treatment of cancer, such as the following: pernicious anemia, hemorrhagic anemia, hemolytic anemia, aplastic anemia, sickle cell anemia, iron-granulocyte anemia, anemia associated with chronic infections such as malaria, trypanosomiasis, HTV, hepatitis virus or other viruses, myeloid anemia arising from bone marrow defects, renal failure resulting from anemia, polycythemia, infectious mononucleosis (EVI), acute non-lymphocytic leukemia (ANLL), Acute Myelogenous Leukemia (AML), Acute Promyelocytic Leukemia (APL), acute myelomonocytic leukemia (AMMoL), polycythemia vera, lymphoma, Acute Lymphocytic Leukemia (ALL), chronic lymphocytic leukemia, Wilms 'tumor, Ewing's sarcoma, retinoblastoma, hemophilia, diseases associated with increased risk of thrombosis, herpes, thalassemia, antibody-mediated diseases such as transfusion reactions and erythroblastosis, mechanical trauma of erythrocytes such as microvascular hemolytic anemia, thrombotic thrombocytopenic purpura and disseminated intravascular coagulation, infections caused by parasites such as plasmodium, chemical damage caused by, for example, lead poisoning, and splenic hyperactivity. In some embodiments, the hematologic disease is a hematologic malignancy. The term "hematologic malignancy" refers to a tumor that affects the blood, bone marrow, and/or lymph nodes. Exemplary hematological malignancies include, but are not limited to, leukemias, such as Acute Lymphocytic Leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), Acute Myelogenous Leukemia (AML) (e.g., B-cell AML, T-cell AML), Chronic Myelogenous Leukemia (CML) (e.g., B-cell CML, T-cell CML), and Chronic Lymphocytic Leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphomas such as Hodgkin's Lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin's lymphoma (NHL) (e.g., B-cell NHL such as Diffuse Large Cell Lymphoma (DLCL) (e.g., diffuse large B cell lymphoma (DLBCL such as Activated B Cell (ABC) DLBCL (ABC-DLBCL))), follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), Mantle Cell Lymphoma (MCL), marginal zone B cell lymphoma (e.g., mucosa-associated lymphoid tissue (MALT) lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt's lymphoma, Waldenstrom's macroglobulinemia (WM, lymphoplasmacytic lymphoma), hairy Cell Leukemia (HCL), immune cell large cell lymphoma, precursor B-lymphocytic lymphoma, Central Nervous System (CNS) lymphoma (e.g., primary CNS lymphoma and secondary CNS lymphoma); and T-cell NHLs such as precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous tonsillitis-like T-cell lymphoma, and anaplastic large cell lymphoma); immunospecific site lymphoma (e.g., brain lymphoma, ocular lymphoma, placental lymphoma, fetal lymphoma, testicular lymphoma); a mixture of one or more leukemias/lymphomas as described above; myelodysplasia; and Multiple Myeloma (MM).

The term "inflammatory disease" refers to a disease caused by, or resulting in inflammation. The term "inflammatory disease" may also refer to a dysregulated inflammatory response that results in exaggerated responses by macrophages, granulocytes, and/or T-lymphocytes, resulting in abnormal tissue damage and/or cell death. Inflammatory diseases may be acute or chronic inflammatory disorders and may result from infectious or non-infectious causes. Inflammatory diseases include, but are not limited to: atherosclerosis, arteriosclerosis, autoimmune disorders, multiple sclerosis, systemic lupus erythematosus, polymyalgia rheumatica (PMR), gouty arthritis, degenerative arthritis, tendonitis, bursitis, psoriasis, cystic fibrosis, osteoarthritis, rheumatoid arthritis, inflammatory arthritis, sjogren's syndrome, giant cell arteritis, progressive systemic sclerosis (scleroderma), ankylosing spondylitis, polymyositis, dermatomyositis, pemphigus, pemphigoid, diabetes (e.g., type I), myasthenia gravis, hashimoto's thyroiditis, Graves 'disease, Goodpasture's disease, mixed connective tissue disease, sclerosing cholangitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, pernicious anemia, inflammatory skin disease, common interstitial pneumonia (UIP), asbestosis, silicosis, bronchiectasis, berylliosis, talc disease, inflammatory bowel disease, scleroderma, inflammatory bowel disease, Crohn's disease, ulcerative colitis, pernicious anemia, inflammatory skin disease, common interstitial pneumonia (UIP), asbestosis, pneumonitis, bronchodilation, berylliosis, talcosmopathy, talc disease, and inflammatory bowel disease, Pneumoconiosis, sarcoidosis, desquamative interstitial pneumonia, lymphoid interstitial pneumonia, giant cell interstitial pneumonia, intercellular pneumonia, extrinsic allergic alveolitis, Wegener's granulomatosis and related forms of vasculitis (temporal arteritis and polyarteritis nodosa), inflammatory dermatoses, hepatitis, delayed-type hypersensitivity reactions (e.g., poison ivy dermatitis), pneumonia, airway inflammation, Adult Respiratory Distress Syndrome (ARDS), encephalitis, immediate hypersensitivity reactions, asthma, pollinosis, allergy, acute hypersensitivity reactions, rheumatic fever, glomerulonephritis, pyelonephritis, cellulitis, cystitis, chronic cholecystitis, ischemia (ischemic injury), reperfusion injury, appendicitis, arteritis, blepharitis, bronchiolitis, bronchitis, cervicitis, cholangitis, chorioamnionitis, conjunctivitis, dacryadenitis, dermatomyositis, Endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis, gingivitis, ileitis, iritis, laryngitis, myelitis, myocarditis, nephritis, omphalitis, oophoritis, orchitis (orchitis), osteitis, otitis, pancreatitis, parotitis, pericarditis, pharyngitis, pleuritis, phlebitis, pneumonia, proctitis, prostatitis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, orchitis (testitis), tonsillitis, urethritis, cystitis, uveitis, vaginitis, vasculitis, vulvitis, vulvovaginitis, vasculitis, chronic bronchitis, osteomyelitis, optic neuritis, temporal arteritis, transverse myelitis, necrotizing fasciitis and necrotizing enterocolitis. Inflammatory diseases of the eye include, but are not limited to, inflammation following surgery.

"autoimmune disease" refers to a disease that results from an inappropriate immune response of a subject's body to substances and tissues that are normally present in the body. In other words, the immune system mistreats a portion of the body as a pathogen and attacks its own cells. This can be restricted to specific organs (e.g., in autoimmune thyroiditis) or to specific tissues involved in different locations (e.g., goodpasture's disease, which may affect the basement membrane of the lung and kidney). Treatment of autoimmune diseases is often with immunosuppression, e.g., drugs that reduce the immune response. Exemplary autoimmune diseases include, but are not limited to: glomerulonephritis, goodpasture's syndrome, necrotizing vasculitis, lymphadenitis, periarteritis nodosa, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus, psoriasis, ulcerative colitis, systemic sclerosis, dermatomyositis/polymyositis, antiphospholipid antibody syndrome, scleroderma, pemphigus vulgaris, ANCA-associated vasculitis (e.g., wegener's granulomatosis, microscopic polyangiitis), uveitis, sjogren's syndrome, crohn's disease, reiter's syndrome, ankylosing spondylitis, lyme disease, guillain-barre syndrome, hashimoto's thyroiditis, and cardiomyopathy.

The term "neurological disease" refers to any disease of the nervous system, including diseases involving the central nervous system (brain, brainstem and cerebellum), the peripheral nervous system (including cranial nerves) and the autonomic nervous system (parts of which are located in the central and peripheral nervous systems). Neurodegenerative diseases refer to a neurological disease characterized by loss of nerve cells, including but not limited to: alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, tauopathy (including frontotemporal dementia) and Huntington's chorea. Examples of neurological diseases include, but are not limited to: headache, numbness and coma, dementia, epilepsy, sleep disorders, trauma, infection, tumor, neuroophthalmology, movement disorders, demyelinating diseases, spinal cord diseases and diseases of the peripheral nerve, muscle and neuromuscular junction. Addiction and psychiatric disorders include, but are not limited to, bipolar disorder and schizophrenia, and are also included in the definition of neurological disorders. Other examples of neurological disorders include acquired epilepsy-like aphasia; acute disseminated encephalomyelitis; adrenoleukodystrophy; agenesis of the corpus callosum; agnosia; an Aikadi syndrome; alexander disease; alper's disease; cross acroparalysis; alzheimer's disease; amyotrophic lateral sclerosis; no brain malformation; anglerman syndrome; vascular tumor diseases; hypoxia; aphasia; disuse disease; arachnoid cyst; arachnoiditis; a malformation of Ashi; arteriovenous malformation; asperger syndrome; dyskinetic telangiectasia; attention deficit with hyperactivity disorder; autism disorder; autonomic dysfunction; back pain; barnyard disease; behcet's disease; bell paralysis; benign idiopathic blepharospasm; benign lesions; muscle atrophy; benign intracranial hypertension; guest schwanger disease; blepharospasm; bur-threo syndrome; damage to brachial plexus; brain abscess; brain damage; brain tumors (including glioblastoma multiforme); spinal cord tumors; brown-seekar syndrome; kanaiwan disease; carpal Tunnel Syndrome (CTS); causalgia; central pain; central pontine myelination; head disorders; a cerebral aneurysm; cerebral arteriosclerosis; atrophy of the brain; cerebral gigantism; cerebral palsy; charcot-horse-charcot-tooth disease; chemotherapy-induced neuropathy and neuropathic pain; a Chiari deformity; chorea; chronic Inflammatory Demyelinating Polyneuropathy (CIDP); chronic pain; chronic regional pain syndrome; koehler syndrome; coma, including persistent vegetative human status; congenital facial paralysis; corticobasal degeneration (corticobasal degeneration); cerebral arteritis; craniosynostosis; Creutzfeldt-Jakob disease; cumulative traumatic disorders; cushing's syndrome; eosinophilic giant Cell Inclusion Body Disease (CIBD); cytomegalovirus infection; dancing eyes-feet syndrome (dancing eyes-dancing heel syndrome); dan-wade syndrome; a disease of the dawn; DemosieeEx syndrome; dethermrilina-cloned pocky paralysis; dementia; dermatomyositis; diabetic neuropathy; diffuse hardening; autonomic dysfunction; difficulty in writing; dyslexia; tension imbalance; early childhood epileptic encephalopathy; empty pommel syndrome; encephalitis; a brain hernia; cerebral trigeminal neurovascular tumor disease; epilepsy; eupatorium paralysis (Erb's palsy); essential tremor; fabry disease; fahr's syndrome; syncope; familial spastic paralysis; febrile convulsions; -fichil syndrome; friedreich ataxia; frontotemporal dementia and other "tauopathies"; gaucher disease; guerbem syndrome; giant cell arteritis; giant cell inclusion body disease; globular cellular leukodystrophy; guillain-barre syndrome; HTLV-1 related myelopathy; hashimoto's disease (Hallervorden-Spatz disease); head injury; headache; hemifacial spasm; hereditary spastic paraplegia; hereditary polyneuritis-like ataxia (heredopathia atactica polyneuritifidomi); herpes zoster of the ear; herpes zoster; mountain syndrome (Hirayama syndrome); HIV-associated dementia and neuropathy (see also the neurological manifestations of AIDS); no fissure in forebrain; huntington's disease and other polyglutamine diseases; hydrocele anocephaly; hydrocephalus; hypercortisolism; hypoxia; immune-mediated encephalomyelitis; inclusion body myositis; pigment disorders; infantile phytic acid storage disease; refsum disease in infants; infantile spasms; inflammatory myopathy; an intracranial cyst; intracranial hypertension; a syndrome of umber; cahns-seoul syndrome; kennedy disease; chorea eye syndrome; creutzfeldt-jakob disease; krabbe's disease; (iii) kuv-wess disease; kuru disease; lafula disease; lang-erzian myasthenia syndrome; Landau-Kleffner syndrome; lateral medullary (Wallenberg) syndrome; learning disability; lei's disease; lun-plus syndrome; Lei-Naphthalenedis syndrome; leukodystrophy; dementia with lewy bodies; no gyria; a latch-up syndrome; lou Gehrig's disease (also known as motor neuron disease or amyotrophic lateral sclerosis); intervertebral disc lesions; lyme disease-neurological sequelae; macchardol-joseph disease; brain hypertrophy; the megacephalum; mei-luodi syndrome; meniere's disease; meningitis; menkes disease; metachromatic leukodystrophy; small head deformity; migraine headache; miller-phillips syndrome; minor stroke; mitochondrial myopathy (mitochondrial myopathie); mobius syndrome; atrophy of single limb muscles; motor neuron diseases; smoke disease; mucopolysaccharidosis; multi-infarct dementia; multifocal motor neuropathy; multiple sclerosis and other demyelinating diseases; multiple system atrophy with postural hypotension; muscular dystrophy; myasthenia gravis; demyelinating diffuse sclerosis (myelocystic fibrosis diffuse sclerosis); infantile myoclonic encephalopathy; myoclonus; myopathy; myotonia congenita; narcolepsy; neurofibromatosis; malignant syndrome of nerve blocking agents; neurological manifestations of aids; neurological sequelae of lupus; nervous muscle rigidity; neuronal ceroid lipofuscinosis; neuronal migration disorder; niemann-pick disease; o' Sullivan-McLeod syndrome; occipital neuralgia; recessive spinal neural tube insufficiency sequence signature; a landiological syndrome; olivopontocerebellar atrophy; strabismus ocular clonus-myoclonus; optic neuritis (optic neuriti); orthostatic hypotension; overuse syndrome; paresthesia; parkinson's disease; paramyxia congenita; paraneoplastic disease; paroxysmal attacks; Par-Roche syndrome; peyrexia disease; periodic paralysis; peripheral neuropathy; painful neuropathy and neuropathic pain; a persistent vegetative human state; comprehensive mental development disorder; photosensitive sneeze reflex; phytanic acid storage disease; pick's disease; pinching nerve; pituitary tumors; polymyositis; a brain punch through malformation; late poliomyelitis syndrome; postherpetic neuralgia (PHN); post-infection encephalomyelitis; orthostatic hypotension; par-widmans syndrome; primary lateral sclerosis; prion diseases; progressive unilateral atrophy; progressive multifocal leukoencephalopathy; progressive sclerosing gray matter atrophy; progressive supranuclear palsy; pseudocerebroma; lamqir-hunter syndrome (types I and II); rasmussen encephalitis; reflex sympathetic dystrophy syndrome; refsum disease; repetitive dyskinesia; repetitive stress injury; restless leg syndrome; retroviral-related myelopathy; rett syndrome; -leishmaniasis; chorea; sandhoff disease; sherde's disease; splitting the brain; hyaline septal-optic nerve dysplasia; startle infant syndrome; herpes zoster; xiayi-dererg syndrome; sicca syndrome; sleep apnea; soxhlet syndrome; spasticity; spina bifida; spinal cord injury; spinal cord tumors; spinal muscular atrophy; stiff person syndrome; stroke; stecke-weber syndrome; subacute sclerosing panencephalitis; subarachnoid hemorrhage; subcortical arteriosclerotic encephalopathy; sydenham chorea; syncope (syncope); syringomyelia; tardive dyskinesia; tay-saxophone disease; temporal arteritis; spinal cord tethering syndrome; thomson disease; thoracic outlet syndrome; trigeminal neuralgia; paralysis of tuoder; tourette's syndrome; transient ischemic attacks; transmissible spongiform encephalopathy; transverse myelitis (transverse myeliti); traumatic brain injury; shaking; trigeminal neuralgia; spinal cord tethering syndrome; nodular sclerosis; vascular dementia (multi-infarct dementia); vasculitis, including temporal arteritis; Hill-Lindersonis disease (VHL); valencerg syndrome; Werdnig-Hoffman disease; west syndrome; acute neck sprain; williams syndrome; wilson's disease; and zellweger syndrome.

"painful conditions" include, but are not limited to, neuropathic pain (e.g., peripheral neuropathic pain), central pain, afferent blocking pain, chronic pain (e.g., chronic nociceptive pain and other forms of chronic pain such as post-operative pain (e.g., pain that occurs after hip, knee, or other replacement surgery), pre-operative pain, stimulation of nociceptors (nociceptive pain), acute pain (e.g., phantom pain and transient acute pain), non-inflammatory pain, pain associated with cancer, wound pain, burn pain, post-operative pain, pain associated with medical procedures, pain caused by pruritus, painful bladder syndrome, pain associated with premenstrual anxiety disorder and/or premenstrual syndrome, pain associated with chronic fatigue syndrome, pain associated with pre-term addiction, pain associated with withdrawal symptoms of medications, pain associated with withdrawal symptoms of medications, pain associated with post-operative pain, post-operative pain, post-operative pain, post-operative post-, Joint pain, arthritis pain (e.g., pain associated with crystalline arthritis, osteoarthritis, psoriatic arthritis, gouty arthritis, reactive arthritis, rheumatoid arthritis, or reiter's arthritis), lumbosacral pain, musculoskeletal pain, headache, migraine, muscle pain, lumbago, neck pain, dental/maxillofacial pain, visceral pain, and the like. The one or more painful conditions contemplated herein may include a mixture of the various types of pain provided above and herein (e.g., nociceptive pain, inflammatory pain, neuropathic pain, etc.). In some embodiments, one specific pain predominates. In other embodiments, the painful condition comprises two or more types of pain, and not predominantly one. A skilled clinician can determine the dosage to achieve a therapeutically effective amount for a particular subject based on the painful condition.

The term "psychiatric disorders" refers to psychiatric disorders including those listed in the american psychiatric society (Washington d.c.) (1994) "diagnostic and statistics manual for psychotic disorders fourth edition (DSM-IV)". Psychiatric disorders include, but are not limited to, anxiety disorders (e.g., acute stress disorder, agoraphobia, generalized anxiety disorder, obsessive-compulsive disorder, panic disorder, post-traumatic stress disorder, separation anxiety disorder, social phobia, and specific phobia), childhood disorders (e.g., attention-deficit/hyperactivity disorder, behavioral disorders, and oppositional defiant disorder), eating disorders (e.g., anorexia nervosa and bulimia nervosa), mood disorders (e.g., depression disorder, bipolar disorder, cyclothymic disorder, dysthymic disorder, and major depression), personality disorders (e.g., antisocial personality disorder, avoidant personality disorder, borderline personality disorder, dependent personality disorder, performance personality disorder, self-loved personality disorder, obsessive-compulsive personality disorder, paranoid personality disorder, schizoid personality disorder, and schizotypal personality disorder), Psychotic disorders (e.g., brief psychotic disorder, delusional disorder, schizoaffective disorder, schizophreniform disorder, schizophrenia and shared psychosis), substance-related disorders (e.g., alcohol dependence, amphetamine dependence, cannabis dependence, cocaine dependence, hallucinogen dependence, inhalant dependence, nicotine dependence, opioid dependence, phencyclidine dependence and sedative dependence), adaptive disorders, autism, delirium, dementia, multi-infarct dementia, learning and memory disorders (e.g., amnesia and age-related memory loss), and tourette's psychotic disorder.

The term "metabolic disorder" refers to any condition involving alteration of the normal metabolism of carbohydrates, lipids, proteins, nucleic acids, or combinations thereof. Metabolic disorders are associated with a deficiency or excess in metabolic pathways, resulting in an imbalance in the metabolism of nucleic acids, proteins, lipids and/or carbohydrates. Factors that affect metabolism include, but are not limited to, endocrine (hormone) control systems (e.g., insulin pathway, enteroendocrine hormones including GLP-1, PYY, etc.), nervous control systems (e.g., GLP-1 in the brain), and the like. Examples of metabolic disorders include, but are not limited to: diabetes (e.g., type I diabetes, type II diabetes, gestational diabetes), hyperglycemia, hyperinsulinemia, insulin resistance, and obesity.

Drawings

FIG. 1 shows exemplary Western blot results of H3K27me3 treated with compound 5 at 1 μ M (upper panel) and 0.1 μ M (lower panel) for 72 hours. And uM: and mu.M.

Figure 2 shows exemplary dimethylation and trimethylation of H3K27 treated with compound 5 at 1 μ M for 72 hours.

Figure 3 shows exemplary cell viability results for selected cell lines treated with compound 5 for 72 hours. The upper diagram: ly18(EZH2 wt (wild type)) cell line. The following figures: k422(EZH2 mutant) cell line. Log10[ M ]: log (molar concentration of compound 5).

Figure 4 shows exemplary dimethylation and trimethylation of H3K27 treated with compound 5, measured using ALPHALISA assay. The upper diagram: H3K27me 2. The following figures: H3K27me 3. Log10 [ M ]: log (molar concentration of compound 5).

FIG. 5 shows exemplary activities of compound EZ-05 on WT (wild type) and mutant EZH2 PRC2 complexes. log [ EZ-05 ]: log (molar concentration of compound EZ-05).

Fig. 6 shows exemplary Surface Plasmon Resonance (SPR) results of compound JQEZ6 obtained by moving compound JQEZ6 on a surface to capture a PRC2 five-component complex.

Figure 7 shows exemplary inhibitory activity of one set of compounds described herein (top panel) and another set of compounds described herein (bottom panel) on PRC 25 component complexes. log (um): log (micromolar concentration of compound). log [ Cpd ], nM: log (nanomolar concentration of compound). % activity: % PRC2 activity.

Figure 8 shows the chemical structure of selected compounds.

FIGS. 9A-9E: overexpression of EZH2 induced murine lung cancer. (FIG. 9A) a LSL-EZH2 genetically engineered mouse model using 3 different strategies to express Cre recombinase to induce overexpression of EZH2 is depicted in graphical form. (FIG. 9B) Kaplan-Meier Lung cancer free survival Profile of LSL-EZH2 transgenic mice (EZH2) versus wild type mice (WT). (FIG. 9C) histology of sections of wild type lung (upper) and EZH 2-induced lung adenocarcinoma (bottom) stained with hematoxylin and eosin. The right panel shows immunostaining with Ki-67, which is a marker for proliferation. Scale bar indicates 50 um. (FIG. 9D) hematoxylin and eosin staining or immunostaining of EZH2, p-AKT and p-ERK1/2 in EZH 2-induced mouse lung tumors (top) and KRAS-induced mouse lung tumors (bottom). Scale bar indicates 50 um. (FIG. 9E) Western blot of EZH2, AKT, p-AKT, ERK1,2 and p-ERK1,2 expression in normal lung tissue from mice, EZH 2-induced tumor lung tissue, and KRAS-induced tumor lung tissue (top). Relative protein expression levels quantified with ImageJ (bottom). See also fig. 15.

FIGS. 10A-10C: EZH 2-driven lung cancer, which is a molecularly diverse entity. (FIG. 10A) heat map of log2 fold change (LFC) gene expression in EZH2 over-expressed (OE) normal lung, KRAS mutant lung tumors, EGFR mutant lung tumors, and EZH2-OE lung tumors relative to corresponding normal lung tissue. The top 500 most variable genes were selected for aggregation in all samples. Dark grey areas indicate down-regulated genes in mutant or over-expressed tissues relative to controls; the light grey areas indicate up-regulated genes in mutant or over-expressed tissues relative to controls. (fig. 10B) distribution of EZH2 expression from 471 human lung adenocarcinomas (dark grey) versus 58 normal lungs (light grey) from a cancer genomic map (TCGA). The mean EZH2 expression level for human lung adenocarcinoma was 394.8 and 56.7 in normal lung. (FIG. 10C) is a block diagram of ssGSEA comparing the enrichment of MEK (left) and mTOR (right) gene sets in TCGA lung adenocarcinomas with specific driver mutations (KRAS, EGFR, unknown) or high EZH2 levels.

FIGS. 11A-11I: overexpression of EZH2 modulates super-enhancer-related transcriptional profiles in mouse lungs. (FIG. 11A) heatmap showing hierarchical cluster analysis of H3K27ac Super Enhancer (SE) regions in Wild Type (WT) and tumor lung tissue. The relative H3K27ac level is represented by color depth. (FIG. 11B) heatmap showing the aggregation of super-enhancer similarity between WT and tumor tissue. The depth of color indicates an increase in similarity. (FIG. 11C) is a waterfall plot showing the rank change of H3K27ac signal in the super-enhancer containing region between tumor and wild-type (WT) lung. The X-axis depicts LFC in the H3K27ac signal. Enhancers rank with LFCs in the signal, with the region that gains the most H3K27ac in the tumor at the top. The super enhancer's change in H3K27ac levels was colored by a change in intensity from light gray to dark gray. (FIG. 11D) block diagram of RNA-seq expression in RPKM units of genes associated with super-enhancers that are gain, invariant or missing in tumors compared to WT lung tissue. The significance of the distribution differences was calculated using the two-tailed t-test. P <2e-4，***p<2E-6 (FIG. 11E) scatter plot of normalized enrichment fraction (NES) versus False Discovery Rate (FDR) q-value, MSigDB-engineered genomic enrichment in tumors was compared to WT super enhancer-related genes. The X-axis shows NES for the assessed genome. The Y-axis shows the false FDR q values for each genome. The up-regulated genome in tumors has a high positive NES, while the down-regulated genome has a negative NES. The dashed line represents a significance cutoff q value of 0.05. Dark gray dots indicate PRC 2-related features that were down-regulated in the tumor. For the PRC2 signature, n is 8. (FIG. 11F) super enhancer-associated gene set enrichment analysis showed down-regulation of EED target in tumors relative to WT tissue. (FIG. 11G) is a solution containingRegion of super enhancer, heatmap of LFC in H3K27ac/H3K27me3 signal. Blue or dark grey areas indicate super enhancers with strong gain of H3K27me3 in the tumor relative to WT, while red or light grey areas indicate those with strong loss. (FIG. 11H) Gene access to the super enhancer region at log₁₀Dashed line plot of RNA-seq expression in RPKM units, H3K27me3 with strong gain in tumor relative to WT. The significance of the distribution differences was calculated by the two-tailed t-test. P <1 e-5. (FIG. 11I) block diagram of ssGSEA, comparing the enrichment of our mouse H3K27me3 genome in TCGA lung adenocarcinoma with high EZH2 levels and in normal lung tissue. The H3K27me3 genome consists of 32 mouse genes adjacent to the SE region, which had a strong H3K27me3 gain in murine EZH2 overexpressing tumors. See also fig. 16.

FIGS. 12A-12G: EZH2 overexpresses transformed human lung epithelial cells. (FIG. 12A) SDS-PAGE and Western blot analysis of human tracheobronchial epithelial (hTBE) cells expressing either control (ctl) or EZH2(oeEZH2) constructs. (FIG. 12B) colony formation assay hTBE cells overexpressing either the control (left) or EZH2 (right) constructs. (FIG. 12C) hTBE cells expressing either control (ct1) or EZH2(oeEZH2) were cultured in vitro for 10 or 20 passages and then seeded on soft agar for colony formation assays. Error bars represent SEM, # p <0.05, # p < 0.001. (FIG. 12D) the expression of Ezh2 in H661 (left) and H292 (right) cells expressing non-targeting control shRNA (NT) or two different shRNAs targeting EZH2(shEZH2-A and shEZH2-B) was analyzed by Western blotting. (FIG. 12E) relative cell growth was measured by MTS assay in H661 cells expressing control (NT) or EZH 2-targeted shRNA. Error bars represent SEM, n ═ 3. P < 0.001. (F-G) H661 cells infected with lentivirus containing control (NT) or ShEZH2 were injected subcutaneously into the flanks of nude mice. When the size of the largest tumor reached approximately 5mm in diameter, the mice were euthanized and the tumors were measured. (fig. 12F) relative shEZH2 tumor sizes were quantified compared to shNT tumor sizes (mean ± SEM, n 3/treatment). (FIG. 12G) H661 and H292 cells infected with lentivirus containing control (NT) or ShEZH2 were injected subcutaneously into the flanks of nude mice. When the largest tumor size reached approximately 5mm in diameter, mice were euthanized and tumors were recorded (mean ± SEM, n ═ 3 per treatment). See also fig. 17.

FIGS. 13A-13C: development of small molecule EZH2 inhibitors. (FIG. 13A) chemical structure of negative control compound JQEZ 23. (FIG. 13B) the small molecule inhibitory activity of JQEZ5, JQEZ23, GSK-126 and UNC1999 was measured in a five-component PRC2 complex radioactive Scintillation Proximity Assay (SPA) using radiolabeled S-adenosylmethionine (SAM). (fig. 13C) IC50 of JQEZ5 was measured as SAM concentration increased to confirm its SAM competitive binding activity. (FIG. 13D) computational docking model of JQEZ5 in combination with EZH 2. (FIG. 13E) ligand interaction plot (LID) describes how JQEZ5 interacts with EZH2 residues. (FIG. 13F) Selectivity profiles of the methyltransferase panel show that JQEZ5 selectively binds EZH2 in 22 methyltransferase assay panels. See also fig. 18.

FIGS. 14A-14G: JQEZ5 inhibited the in vitro and in vivo growth of lung cancer. (FIG. 14A) H661 lung cancer cells were incubated with increasing concentrations of JQEZ 5. Cell lysates were prepared and subjected to SDS-PAGE and Western blot analysis with the indicated antibodies. (FIG. 14B) Western blot of methylation levels in lung cancer cell line H661, treated with increasing concentrations of JQEZ23 for 72H. H3 is a loading control. (FIG. 14C) Western blot of methylation levels in H661 lung cancer cell line after 48H or 72H treatment with increasing concentrations of JQEZ 5. H3 is a loading control. (FIG. 14D) H661 and (E) H292 lung cancer cells were incubated with increasing concentrations of JQEZ5 and relative cell growth was assessed by MTS assay. Error bar indicates SD, n is 3. (panel F) relative mouse tumor volumes were quantified based on MRI. Relative tumor sizes were compared before JQEZ5 treatment and after three weeks of treatment (mean ± SEM, n ═ 2). (FIG. 14G) MRI showed that in vivo treatment of mice with JQEZ5 caused lung tumor regression as shown by the circle. H, heart. See also fig. 19.

Fig. 15A to 15E, refer to fig. 9. Production of LSL-Ezh2 conditional transgenic mice. (FIG. 15A) schematic representation of the generation of EZH2 transgenic mice by Frt-mediated homologous recombination. Targeting vectors carrying EZH2 cDNA and pgkATGfrt were used to target the modification site located about 500bp downstream of the 3' untranslated region of the ColA1 locus with the FLPe transient expression vector by co-electroporation. The LOX-STOP-LOX (LSL) cassette placed between the CAG promoter and EZH2 cDNA ensures that the EZH2 transgene is only expressed when Cre-mediated LSL cassette excision is present. E ═ EcoRI; p ═ PstI; S-SpeI (fig. 15B) mouse tail DNA was genotyped using two different primer sets-P1 and P2 or P3 and P4. The target of the first primer set spans the integration site, ensuring correct recombination. (FIG. 15C) schematic representation of the opening of the EZH2 transgene by Cre-mediated excision of the LSL cassette. (FIG. 15D) the protein from wild type and actin-Cre: lungs from LSL-Ezh2 mice were sectioned and stained for Ezh2 expression by immunohistochemistry. Scale bar indicates 50 um. (fig. 15E) expression of actin-Ire: LSL-Ezh2 and UBC: lung from LSL-Ezh2 mice were prepared as lysates. And (3) mixing UBC: LSL-Ezh2 mice were treated with tamoxifen at 6 weeks of age and the tissues harvested two weeks later. Lysates were analyzed by Western blot for Ezh2 protein expression levels.

FIGS. 16A-16F, related to FIG. 11. H3K27ac Super Enhancer (SE) analysis in EZH2 mouse lung tumors. (FIG. 16A) 32 mouse genes adjacent to the SE region with strong H3K27me3 gain in tumors with over-expression of EZH 2. (FIG. 16B) genome-wide traces of ChIP-seq signals in WT and tumor lung tissue, in units of rpm/bp, H3K27ac and H3K27me 3. (FIG. 16C) Gene traces of ChIP-Seq signals in rpm/bp in WT and tumor lung tissue for H3K27ac and H3K27me3 in the Super Enhancer (SE) region. (FIG. 16D) Gene traces of ChIP-seq signals in rpm/bp in WT or tumor lung tissue for H3K27ac and H3K27me3 at the Foxf1a locus. (FIG. 16E) Gene traces of ChIP-seq signals in rpm/bp in WT or tumor lung tissue for H3K27ac and H3K27me3 at the DUSP4 locus. (FIG. 16F) Western blot analysis of lysates prepared from normal lung (N-1, N-2 and N-3) and lung tumor (T-1, T-2 and T-3) samples.

FIGS. 17A-17B, related to FIG. 12. Cells with low levels of EZH2 were not sensitive to disruption by EZH 2. (FIG. 17A) Western blotting to compare the expression level of EZH2 between NSCLC cell lines H661 and H292. Actin is a loading control. (FIG. 17B) relative cell growth of H292 cells expressing either a non-targeting control shRNA (NT) or two different shRNAs targeting EZH2 (shEZH2-A and shEZH2-B) was determined by MTS assay. Error bars represent s.e.m, n ═ 3.

FIGS. 18A-18D, related to FIG. 13. Characterization of JQEZ 5. (FIG. 18A) chemical structures reported for EZH2 inhibitor, GSK-126 and UNC 1999. (figure 18B) IC50 values of JQEZ5, negative control compound, JQEZ23, and literature reported EZH2 inhibitor, determined in a radioactive Scintillation Proximity Assay (SPA) for measurement of PRC2 activity. (FIG. 18C) the inhibitory activity of JQEZ5 against PRC2 was measured by SPA assay with increasing concentrations of S-adenosylmethionine (SAM). (FIG. 18D) JQEZ5 was assayed for activity against a panel of 22 methyltransferases. The IC50 values of JQEZ5 and a control compound, i.e., S-adenosyl-homocysteine (SAH), are listed.

FIGS. 19A-19C, related to FIG. 14. In vivo properties of JQEZ 5. (fig. 19A) half-life of JQEZ5 in mice revealed by pharmacokinetic analysis of mean whole blood concentration over time after intraperitoneal injection of 75mg/kg in male CD1 mice (n ═ 3). (fig. 19B) MRI scans from animal #1 showed that in vivo treatment with JQEZ5 resulted in remission of lung tumors from week 0 to week 3. (fig. 19C) MRI scans from animal #2 showed that in vivo treatment with JQEZ5 resulted in remission of lung tumors from week 0 to week 3.

Figure 20 shows the chemical structure of selected comparative compounds. "EZH 2-F" is the same as "EZ-F".

Figure 21 shows the chemical structures of exemplary aldehydes and ketones that can be used to prepare the hydrazides described herein.

Fig. 22 shows exemplary results of the biochemical selectivity of selected compounds described herein for EZH2 versus EZH 1.

Fig. 23A and 23B illustrate exemplary results of Western blots of two-color H3K27me 3. 293T cells were treated with different concentrations of any of the test compounds for 3 days. FIG. 23A: H3K27me3(Ms) is shown as green or light gray; histone H3 (Rb; C-terminal Ab) is shown in red or dark grey. "uM" means. mu.M. Fig. 23B shows the quantization results of an exemplary H3K27me 3. "[ CPD ]" indicates the molar concentration of the test compound.

Fig. 24 shows exemplary results of biochemical selectivity of EZH2(WT) versus EZH2(Y641) for selected compounds described herein.

Fig. 25A to 25C show exemplary results of Rhabdoid viability assays. The assay was performed in 384-well format. There were 625G401 cells/well and 313BT16 cells/well. Cells were treated with either test compound for 4 days. Fig. 25A shows Z factor values for G401 cells and BT16 cells treated with 10 μ M EZ 05. Fig. 25B shows Z factor values for G401 cells and BT16 cells treated with 3.3 μ M EZ 05. Fig. 25C shows% viability of G401 cells. DMSO was used as a control.

Fig. 26A and 26B show exemplary results of DLBCL cell viability. OCI-LY-18(WT EZH2) cells (FIG. 26A) and Karpas 422 (mutant EZH2) cells (FIG. 26B) were treated with different concentrations of either test compound for 6 days.

Fig. 27A shows an exemplary average whole blood concentration-time curve for EZ26 following an IV dose of 5mg/kg or a PO dose of 10mg/kg (N ═ 3) in male CD1 mice. Fig. 27B shows an exemplary individual whole blood concentration-time curve of EZ26 following a PO dose of 10mg/kg (N ═ 3) in male CD1 mice. Fig. 27C shows an exemplary average whole blood concentration-time curve for EZ27 following an IV dose of 25mg/kg or an IP dose of 50mg/kg (N ═ 3) in male CD1 mice. Fig. 27D shows an exemplary mean whole blood concentration-time curve for EZ27 after a PO dose of 50mg/kg (N-3) in male CD1 mice. Fig. 27E shows the mean whole blood concentration-time curve of JQE5(EZ-005) after an IP dose of 75mg/kg (N ═ 3) in male CD1 mice.

Figure 28A shows the percent activity of different concentrations of EZ05 on wild-type (WT) EZH2 or EZH2 with a Y641C mutation, a Y641N mutation, or a Y641S mutation. FIG. 28B shows the fluorescence intensity of different concentrations of EZ05 in EZH2+/+ (WT) or Y641F/+ mutants.

FIG. 29 shows exemplary IC of selected compounds against Wild Type (WT) EZH2 or EZH2 with Y641C, Y641N, or Y641S mutations₅₀Values (in nM).

Fig. 30A shows a compound described herein that includes a warhead, such as the depicted EZ05 analog with an acyl amide (covalently attached to an acyl amide moiety) and Cys641 of EZH forming a covalent bond. FIG. 30B shows energy minimization of EZH complexes and described EZ05 analogs with acyl amidesExemplary docking results of the structure. FIG. 30C shows exemplary IC of selected compounds against Wild Type (WT) EZH2 or EZH2 with Y641C, Y641N, or Y641S mutations₅₀Values (in nM).

Fig. 31 depicts the binding of EZ06 to PRC2 using AlphaAssay.

Fig. 32 depicts competitive AlphaScreen for PRC2 SAM competitive inhibitors.

FIG. 33 depicts PRC2-EZH2 competitive fluorescence polarization assay using EZ 05-FITC.

FIG. 34 depicts PRC2-EZH2 fluorescence polarization assay using EZ 05-FITC.

Figure 35 depicts binding isotherms for all compounds screened in an EZH2 ligand-displacement fluorescence polarization assay.

Figure 36 depicts binding isotherms for the most and least potent compounds screened in an EZH2 ligand displacement fluorescence polarization assay. A compound "X #", wherein X is a letter, # is an integer between 1 and 12, inclusive, and represents a compound prepared by the process of:

WhereinAn aldehyde or ketone as shown in panel 1, fig. 21; "X" indicates the number of rows, and "#" indicates the number of columns.

FIG. 37 reports the calculated IC of selected compounds screened in an EZH2 ligand displacement fluorescence polarization assay₅₀And (4) data. A compound "X #", wherein X is a letter, # is an integer between 1 and 12, inclusive, that represents the compound as shown in figure 36.

FIG. 38 depicts the structure of EZ-TAMRA and EZ-TOM.

Figure 39 depicts an intracellular EZH2 binding assay.

Figure 40 depicts an intracellular competitive EZH2 binding assay.

Detailed Description

Described herein are compounds of formulae (I) and (II) and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives and prodrugs thereof. The compounds described herein bind to protein methyltransferases (HMTs, e.g., EZH1 and EZH2) and are useful for modulating (e.g., inhibiting) the activity (e.g., aberrant activity) of HMTs in a subject, biological sample, tissue, or cell. The compounds are useful for treating or preventing a disease (e.g., a proliferative disease, an inflammatory disease, an autoimmune disease, a genetic disease, a hematological disease, a neurological disease, a painful disorder, a psychiatric disease, or a metabolic disorder) in a subject in need thereof and/or for treating or preventing a disease associated with aberrant activity or increased activity of HMT in a subject. Also provided are pharmaceutical compositions, kits and uses comprising the compounds described herein. Further provided in the present disclosure are methods of identifying EZH1 and/or EZH2 inhibitors.

Compound (I)

One aspect of the present application relates to compounds described herein. The compounds described herein are inhibitors of HMT (e.g., EZH1, EZH2, DOT 1). In some embodiments, the compounds described herein are compounds of formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof. In some embodiments, the compounds described herein are compounds of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the compounds described herein are compounds of formula (II) or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof. In some embodiments, the compounds described herein are compounds of formula (II) or a pharmaceutically acceptable salt thereof.

In one aspect, the present application provides compounds of formula (I), and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives and prodrugs thereof:

Wherein:

R^A1is halogen, substituted OR unsubstituted alkyl, substituted OR unsubstituted alkenyl, substituted OR unsubstituted alkynyl, substituted OR unsubstituted carbocyclyl, substituted OR unsubstituted heterocyclyl, substituted OR unsubstituted aryl, substituted OR unsubstituted heteroaryl, -OR^a、–N(R^a)₂、–SR^a、–CN、–SCN、–C(＝NR^a)R^a、–C(＝NR^a)OR^a、–C(＝NR^a)N(R^a)₂、–C(＝O)R^a、–C(＝O)OR^a、–C(＝O)N(R^a)₂、–NO₂、–NR^aC(＝O)R^a、–NR^aC(＝O)OR^a、–NR^aC(＝O)N(R^a)₂、–OC(＝O)R^a、–OC(＝O)OR^a、–OC(＝O)N(R^a)₂A marker or

Each R^aIndependently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two R^aLinked to form a substituted or unsubstituted heterocyclic ring or a substituted or unsubstituted heteroaromatic ring;

R^Ais hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstitutedUnsubstituted aryl or substituted or unsubstituted heteroaryl;

R^Bis hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

Or R^AAnd R^BAre linked to form a substituted or unsubstituted carbocyclic ring or a substituted or unsubstituted heterocyclic ring;

R^Cis hydrogen, substituted or unsubstituted C_1-6An alkyl or nitrogen protecting group;

R^A2is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, a nitrogen protecting group, a label, or a warhead;

R^A3is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^a、–N(R^a)₂Or a warhead;

R^A4is hydrogen, substituted or unsubstituted C_1-6An alkyl or nitrogen protecting group; and

R^A5is of the formula: wherein:

R^A6is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^aor-N (R)^a)₂；

R^A7Is hydrogen, halogen, substituted or unsubstituted C_2-6Alkyl, substituted OR unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 OR 2 double bonds in the carbocyclic ring system, -OR^aor-N (R)^a)₂；

R^A8Is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^aOr-N(R^a)₂；

R^A9Is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, substituted OR unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 OR 2 double bonds in the carbocyclic ring system, -OR^aor-N (R)^a)₂；

R^A10is-OR^a–N(R^a)₂Or a warhead;

each R^A11Independently is halogen, substituted or unsubstituted C_1-6Alkyl, substituted OR unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 OR 2 double bonds in the carbocyclic ring system, -OR ^aor-N (R)^a)₂；

n is 0, 1, 2, 3 or 4;

R^A12is hydrogen, substituted or unsubstituted C_1-6Alkyl, nitrogen protecting group or warhead;

each R^A13Independently is halogen, substituted or unsubstituted C_1-6Alkyl, substituted OR unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 OR 2 double bonds in the carbocyclic ring system, -OR^aor-N (R)^a)₂；

m is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9;

R^A14is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^aor-N (R)^a)₂；

R^A15Is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^aor-N (R)^a)₂；

R^A16Is hydrogen, halogen, substituted or unsubstituted C_2-6Alkyl, substituted OR unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 OR 2 double bonds in the carbocyclic ring system, -OR^aor-N (R)^a)₂(ii) a And

R^A17is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted C_1-6Alkyl, nitrogen protecting group or warhead.

In some embodiments, the EZH2 inhibitor is a compound of formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof:

wherein:

R^A1is halogen, substituted OR unsubstituted alkyl, substituted OR unsubstituted alkenyl, substituted OR unsubstituted alkynyl, substituted OR unsubstituted carbocyclyl, substituted OR unsubstituted heterocyclyl, substituted OR unsubstituted aryl, substituted OR unsubstituted heteroaryl, -OR ^a、–N(R^a)₂、–SR^a、–CN、–SCN、–C(＝NR^a)R^a、–C(＝NR^a)OR^a、–C(＝NR^a)N(R^a)₂、–C(＝O)R^a、–C(＝O)OR^a、–C(＝O)N(R^a)₂、–NO₂、–NR^aC(＝O)R^a、–NR^aC(＝O)OR^a、–NR^aC(＝O)N(R^a)₂、–OC(＝O)R^a、–OC(＝O)OR^aor-OC (═ O) N (R)^a)₂；

Each R^aIndependently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two R^aLinked to form a substituted or unsubstituted heterocyclic ring or a substituted or unsubstituted heteroaromatic ring;

R^A2is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl or a nitrogen protecting group;

R^A3is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^aor-N (R)^a)₂；

R^A4Is hydrogen, substituted or unsubstituted C_1-6An alkyl or nitrogen protecting group; and

R^A5is of the formula: wherein:

R^A6is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^aor-N (R)^a)₂；

R^A7Is hydrogen, halogen, substituted or unsubstituted C_2-6Alkyl, substituted OR unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 OR 2 double bonds in the carbocyclic ring system, -OR^aor-N (R)^a)₂；

R^A8Is hydrogen, halogen, substituted or unsubstituted C _1-6Alkyl, -OR^aor-N (R)^a)₂；

R^A9Is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, substituted OR unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 OR 2 double bonds in the carbocyclic ring system, -OR^aor-N (R)^a)₂；

R^A10is-OR^aor-N (R)^a)₂；

Each R^A11Independently is halogen, substituted or unsubstituted C_1-6Alkyl, substituted OR unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 OR 2 double bonds in the carbocyclic ring system, -OR^aor-N (R)^a)₂；

n is 0, 1, 2, 3 or 4;

R^A12is hydrogen, substituted or unsubstituted C_1-6An alkyl or nitrogen protecting group;

each R^A13Independently is halogen, substituted or unsubstituted C_1-6Alkyl, substituted OR unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 OR 2 double bonds in the carbocyclic ring system, -OR^aor-N (R)^a)₂；

m is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9;

R^A14is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^aor-N (R)^a)₂；

R^A15Is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^aor-N (R)^a)₂；

R^A16Is hydrogen, halogen, substituted or unsubstituted C_2-6Alkyl, substituted OR unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 OR 2 double bonds in the carbocyclic ring system, -OR^aor-N (R)^a)₂(ii) a And

R^A17is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted C _1-6Alkyl or nitrogen protecting groups.

Formula (I) includes a substituent R on the pyridine ring^A. In some embodiments, R^A1Is halogen (e.g., F, Cl, Br, or I). In some embodiments, R^A1Is a substituted or unsubstituted alkyl group (e.g., substituted or unsubstituted C)_1-6Alkyl groups). In some embodiments, R^A1Is Me. In some embodiments, R^A1is-CF₃Bn, Et, perfluoroethyl, Pr, perfluoropropyl, Bu or perfluorobutyl. In some embodiments, R^A1Is a substituted or unsubstituted alkenyl group (e.g., substituted or unsubstituted C_2-6Alkenyl). In some embodiments, R^A1Is a substituted or unsubstituted alkynyl group (e.g., substituted or unsubstituted C_1-6Alkynyl). In some embodiments, R^A1Is a substituted or unsubstituted carbocyclyl (e.g., a substituted or unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1, or 2 double bonds in the carbocyclic ring system). In some embodiments, R^A1Is a substituted or unsubstituted heterocyclic radical(e.g., a substituted or unsubstituted 3-to 7-membered monocyclic heterocyclic group containing 0, 1, or 2 double bonds in the heterocyclic ring system, wherein 1, 2, or 3 atoms in the heterocyclic ring system are independently nitrogen, oxygen, or sulfur). In some embodiments, R ^A1Is a substituted or unsubstituted piperazinyl group. In some embodiments, R^A1Is of the formula:wherein R is^A14Is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or a nitrogen protecting group. In some embodiments, R^A1Is of the formula:in some embodiments, R^A1Is of the formula:wherein R is^A14Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^A1Is of the formula:in some embodiments, R^A1Is of the formula:wherein L is^AIs a bond, or substituted or unsubstituted C_1-100A hydrocarbon chain, optionally wherein one or more chain atoms in the hydrocarbon chain are independently-O-, -S-, or-NR^a-replacing; and X^AIs a small molecule, peptide, protein or polynucleotide. In some embodiments, R^A1Is of the formula:wherein r is 0 or 1 and k is 0 to 11Integers, inclusive, p is an integer from 0 to 10, inclusive, and q is an integer from 0 to 10, inclusive. In some embodiments, k is an integer from 3 to 11, inclusive, p is 0 and q is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, X ^AIs a small molecule. In some embodiments, X^AIs a small molecule drug (e.g., wherein Z^Ais-O-or-NH-or other drug described herein as a small molecule). In some embodiments, X^AIs a small molecule tag (e.g., a biotin moiety (e.g.,) Or a small molecule fluorophore). In some embodiments, R^A1Is a substituted or unsubstituted oxetanyl group, a substituted or unsubstituted tetrahydrofuryl group, a substituted or unsubstituted pyrrolidinyl group, a substituted or unsubstituted tetrahydropyranyl group, a substituted or unsubstituted piperidinyl group, a substituted or unsubstituted morpholinyl group, a substituted or unsubstituted azepanyl group, or a substituted or unsubstituted diazepanyl group. In some embodiments, R^A1Is of the formula:

wherein each R^aIndependently is unsubstituted C_1-6Alkyl (e.g., Me)). In some embodiments, R^A1Is a substituted or unsubstituted aryl group (e.g., a substituted or unsubstituted 6-to 10-memberedAryl). In some embodiments, R^A1Is a substituted or unsubstituted phenyl group. In some embodiments, R^A1Is a substituted or unsubstituted heteroaryl (e.g., a substituted or unsubstituted 5-to 6-membered monocyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In some embodiments, R ^A1is-OR^a(e.g., -OH, -O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe, -OEt, -OPr, -OBu, or-OBn) or-O (substituted or unsubstituted phenyl) (e.g., -OPh)). In some embodiments, R^A1is-SR^a(e.g., -SH, -S (substituted or unsubstituted C)_1-6Alkyl) (e.g., -SMe, -SEt, -SPr, -SBu, or-SBn) or-S (substituted or unsubstituted phenyl) (e.g., -SPh)). In some embodiments, R^A1is-N (R)^a)₂(e.g., -NH)₂-NH (substituted or unsubstituted C_1-6Alkyl) (e.g., -NHMe) or-N (substituted or unsubstituted C)_1-6Alkyl) - (substituted or unsubstituted C_1-6Alkyl) (e.g., -NMe)₂)). In some embodiments, R^A1is-CN. In some embodiments, R^A1is-SCN or-NO₂. In some embodiments, R^A1is-C (═ NR)^a)R^a、–C(＝NR^a)OR^aor-C (═ NR)^a)N(R^a)₂. In some embodiments, R^A1is-C (═ O) R^a(e.g., -C (═ O) (substituted or unsubstituted alkyl) or-C (═ O) (substituted or unsubstituted phenyl)). In some embodiments, R^A1is-C (═ O) OR^a(e.g., -C (═ O) OH, -C (═ O) O (substituted or unsubstituted alkyl) (e.g., -C (═ O) OMe) or-C (═ O) O (substituted or unsubstituted phenyl)). In some embodiments, R ^A1is-C (═ O) N (R)^a)₂(e.g., -C (═ O) NH₂C (═ O) NH (substituted or unsubstituted alkyl), -C (═ O) NH (substituted or unsubstituted phenyl), -C (═ O) N (substituted or unsubstituted alkyl) - (substituted or unsubstituted alkyl), or-C (═ O) N (substituted or unsubstituted alkyl)Or unsubstituted phenyl) - (substituted or unsubstituted alkyl)). In some embodiments, R^A1is-NR^aC(＝O)R^a(e.g., -NHC (═ O) Me). In some embodiments, R^A1is-NR^aC(＝O)OR^aor-NR^aC(＝O)N(R^a)₂. In some embodiments, R^A1is-OC (═ O) R^a、–OC(＝O)OR^aor-OC (═ O) N (R)^a)₂. In some embodiments, R^A1Is substituted OR unsubstituted alkyl, -OR^a、–N(R^a)₂、–C(＝O)OR^aor-NR^aC(＝O)R^a. In some embodiments, R^A1Is unsubstituted C_1-6Alkyl, -OMe, -NH₂、–N(Me)₂-C (═ O) OH, -C (═ O) OMe or-NHC (═ O) Me. In some embodiments, R^A1Is composed ofDescribed herein as including one or more partsThe compound of (1) is a hydrazide.

A moiety of any of formulae (I) and (II)Comprising a substituent R^A、R^BAnd R^C. In some embodiments, R^AIs H. In some embodiments, R^AIs a substituted acyl group. In some embodiments, R^AIs an unsubstituted acyl group. In some embodiments, R^AIs acetyl. In some embodiments, R ^Ais-C (═ O) R^c(e.g., -C (═ O) (substituted or unsubstituted alkyl)). In some embodiments, R^Ais-C (═ O) OR^c(e.g., -C (═ O) O (substituted or unsubstituted alkyl) or-C (═ O) OH). In some embodiments, R^Ais-C (═ O) N (R)^c)₂(for example,–C(＝O)NH₂-C (═ O) NH (substituted or unsubstituted alkyl) or-C (═ O) N (substituted or unsubstituted alkyl)₂). In some embodiments, R^AIn some embodiments, R is an unsubstituted alkyl group^AIs a substituted alkyl group. In some embodiments, R^AIs unsubstituted C_1-6An alkyl group. In some embodiments, R^AIs substituted C_1-6An alkyl group. In some embodiments, R^AIs C substituted by at least one halogen_1-6An alkyl group. In some embodiments, R^Ais-CH₃. In some embodiments, R^AIs a substituted methyl group. In some embodiments, R^Ais-CH₂F、–CHF₂or-CF₃. In some embodiments, R^AEt, substituted ethyl, Pr, substituted propyl, Bu or substituted butyl. In some embodiments, R^AIs unsubstituted alkenyl. In some embodiments, R^AIs a substituted alkenyl group. In some embodiments, R^AIs unsubstituted C_1-6An alkenyl group. In some embodiments, R ^AIs substituted C_1-6An alkenyl group. In some embodiments, R^AIs unsubstituted alkynyl. In some embodiments, R^AIs a substituted alkynyl group. In some embodiments, R^AIs unsubstituted C_1-6Alkynyl. In some embodiments, R^AIs substituted C_1-6Alkynyl. In some embodiments, R^AIs a substituted carbocyclyl. In some embodiments, R^AIs unsubstituted carbocyclyl. In some embodiments, R^AIs a saturated carbocyclyl group. In some embodiments, R^AIs an unsaturated carbocyclic group. In some embodiments, R^AIs a 3-to 8-membered monocyclic carbocyclic group optionally including 1, 2 or 3 double bonds in the carbocyclic ring system. In some embodiments, R^AIs a 5-to 14-membered bicyclic carbocyclic group optionally including 1, 2, 3 or 4 double bonds in the carbocyclic ring system. In some embodiments, R^AIs a 5-to 20-membered tricyclic carbocyclic group optionally including 1, 2, 3, 4 or 5 double bonds in the carbocyclic ring system. At one endIn some embodiments, R^AIs a 5-to 26-membered tetracyclic carbocyclic group optionally including 1, 2, 3, 4, 5 or 6 double bonds in the carbocyclic ring system. In some embodiments, R^AIs a substituted heterocyclic group. In some embodiments, R^AIs an unsubstituted heterocyclic group. In some embodiments, R ^AIs a saturated heterocyclic group. In some embodiments, R^AIs an unsaturated heterocyclic group. In some embodiments, R^AIs a 3-to 8-membered monocyclic heterocyclyl optionally including 1 or 2 double bonds in the heterocyclic ring system, wherein 1, 2 or 3 atoms in the heterocyclic ring system are independently nitrogen, oxygen or sulfur. In some embodiments, R^AIs a 5-to 14-membered bicyclic heterocyclic group optionally comprising 1, 2 or 3 double bonds in the heterocyclic ring system, wherein 1, 2, 3 or 4 atoms in the heterocyclic ring system are independently nitrogen, oxygen or sulfur. In some embodiments, R^AIs a 5-to 20-membered tricyclic heterocyclyl group optionally including 1, 2, 3 or 4 double bonds in the heterocyclyl system, wherein 1, 2, 3, 4 or 5 atoms in the heterocyclyl system are independently nitrogen, oxygen or sulfur. In some embodiments, R^AIs a substituted aryl group. In some embodiments, R^AIs an unsubstituted aryl group. In some embodiments, R^AIs 6-14 membered aryl. In some embodiments, R^AIs a 6-to 10-membered aryl group. In some embodiments, R^AIs a substituted phenyl group. In some embodiments, R^AIs unsubstituted phenyl. In some embodiments, R^AIs a substituted naphthyl group. In some embodiments, R ^AIs unsubstituted naphthyl. In some embodiments, R^AIs a substituted heteroaryl group. In some embodiments, R^AIs unsubstituted heteroaryl. In some embodiments, R^AIs a 5-to 6-membered monocyclic heteroaryl group, wherein 1, 2, 3 or 4 atoms in the heteroaryl ring system are independently nitrogen, oxygen or sulfur. In some embodiments, R^AIs an 8-to 10-membered bicyclic heteroaryl group, wherein 1, 2, 3 or 4 atoms in the heteroaryl ring system are independently nitrogen, oxygen or sulfur.

In some embodiments, R^AIs a watch1A. In some embodiments, R^AAre the groups shown in Table 1B.

TABLE 1A. exemplary R^ARadical (I)

TABLE 1B exemplary R^AAnd R^BRadical (I)

R^AThe radicals may independently comprise one or more substituents R^c. In some embodiments, all R are^cAre all the same. In some embodiments, two R are^cAre different from each other. In some embodiments, at least one R is^cIs H. In some embodiments, each R is^cIs H. In some embodiments, at least one R is^cIs a substituted or unsubstituted acyl group (e.g., acetyl). In some embodiments, at least one R is^cIs a substituted or unsubstituted alkyl group (e.g., substituted or unsubstituted C) _1-6Alkyl groups). In some embodiments, at least one R is^cis-CH₃. In some embodiments, at least one R is^cis-CF₃Unsubstituted ethyl, perfluoroethyl, unsubstituted propyl, perfluoropropyl, unsubstituted butyl or perfluorobutyl. In some embodiments, at least one R is^cIs a substituted or unsubstituted alkenyl group (e.g., substituted or unsubstituted C_1-6Alkenyl). In some embodiments, at least one R is^cIs a substituted or unsubstituted alkynyl group (e.g., substituted or unsubstituted C_1-6Alkynyl). In some embodiments, at least one R is^cIs a substituted or unsubstituted carbocyclyl (e.g., a substituted or unsubstituted 3-to 8-membered monocyclic carbocyclyl optionally including 1, 2, or 3 double bonds in the carbocyclic system, or a substituted or unsubstituted 5-to 14-membered bicyclic carbocyclyl optionally including 1, 2, 3, or 4 double bonds in the carbocyclic system). In some embodiments, at least one R is^cIs a substituted or unsubstituted heterocyclyl group (e.g., a substituted or unsubstituted 3-to 8-membered monocyclic heterocyclyl group optionally including 1 or 2 double bonds in the heterocyclic ring system, wherein 1, 2, or 3 atoms in the heterocyclic ring system are independently nitrogen, oxygen, or sulfur, or a substituted or unsubstituted 5-to 14-membered bicyclic heterocyclyl group optionally including 1, 2, or 3 double bonds in the heterocyclic ring system, wherein 1, 2, 3, or 4 atoms in the heterocyclic ring system are independently nitrogen, oxygen, or sulfur). In that In some embodiments, at least one R is^cIs a substituted or unsubstituted aryl group (e.g., a substituted or unsubstituted 6-to 10-membered aryl group). In some embodiments, at least one R is^cIs a substituted or unsubstituted phenyl group. In some embodiments, at least one R is^cIs a substituted or unsubstituted heteroaryl (e.g., a substituted or unsubstituted 5-to 6-membered monocyclic heteroaryl, or a substituted or unsubstituted 8-to 10-membered bicyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In some embodiments, at least one R when attached to a nitrogen atom^cIs a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R when attached to an oxygen atom^cIs an oxygen protecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl). In some embodiments, R when attached to the sulfur atom^cIs a sulfur protecting group (e.g., acetamidomethyl, t-Bu, 3-nitro-2-pyridylthio, 2-pyridylthio or triphenylmethyl). In some embodiments, two R are ^cLinked to form a substituted or unsubstituted heterocyclic ring (e.g., a substituted or unsubstituted 3-to 8-membered monocyclic heterocyclic ring optionally including 1 or 2 double bonds in the heterocyclic ring system, wherein 1, 2, or 3 atoms in the heterocyclic ring system are independently nitrogen, oxygen, or sulfur). In some embodiments, two R are^cLinked to form a substituted or unsubstituted heteroaryl ring (e.g., a substituted or unsubstituted 5-to 6-membered monocyclic heteroaryl ring, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur).

In some embodiments, R^BIs H. In some embodiments, R^BIs a substituted acyl group. In some embodiments, R^BIs an unsubstituted acyl group. In some embodiments, R^BIs acetyl. In some embodiments, R^Bis-C (═ O) R^d(e.g., -C (═ O) (substituted or unsubstituted alkyl)). In some embodiments, R^Bis-C (═ O) OR^d(e.g., -C (═ C)O) O (substituted or unsubstituted alkyl) or — C (═ O) OH). In some embodiments, R^Bis-C (═ O) N (R)^d)₂(e.g., -C (═ O) NH₂-C (═ O) NH (substituted or unsubstituted alkyl) or-C (═ O) N (substituted or unsubstituted alkyl)₂). In some embodiments, R ^BIs an unsubstituted alkyl group. In some embodiments, R^BIs a substituted alkyl group. In some embodiments, R^BIs unsubstituted C_1-6An alkyl group. In some embodiments, R^BIs substituted C_1-6An alkyl group. In some embodiments, R^BIs C substituted by at least one halogen_1-6An alkyl group. In some embodiments, R^Bis-CH₃. In some embodiments, R^BIs a substituted methyl group. In some embodiments, R^Bis-CH₂F、–CHF₂or-CF₃. In some embodiments, R^BIs ethyl. In some embodiments, R^BIs propyl. In some embodiments, R^BIs butyl. In some embodiments, R^BIs pentyl. In some embodiments, R^BIs hexyl. In some embodiments, R^BIn some embodiments, R is an unsubstituted alkenyl^BIs a substituted alkenyl group. In some embodiments, R^BIs unsubstituted C_1-6An alkenyl group. In some embodiments, R^BIs substituted C_1-6An alkenyl group. In some embodiments, R^BIs unsubstituted alkynyl. In some embodiments, R^BIs a substituted alkynyl group. In some embodiments, R^BIs unsubstituted C_1-6Alkynyl. In some embodiments, R^BIs substituted C_1-6Alkynyl. In some embodiments, R ^BIs a substituted carbocyclyl. In some embodiments, R^BIs unsubstituted carbocyclyl. In some embodiments, R^BIs a saturated carbocyclyl group. In some embodiments, R^BIs an unsaturated carbocyclic group. In some embodiments, R^BIs 3-to 3-optionally comprising 1, 2 or 3 double bonds in the carbocyclic ring systemAn 8-membered monocyclic carbocyclic group. In some embodiments, R^BIs a 5-to 14-membered bicyclic carbocyclic group optionally including 1, 2, 3 or 4 double bonds in the carbocyclic ring system. In some embodiments, R^BIs a 5-to 20-membered tricyclic carbocyclic group optionally including 1, 2, 3, 4 or 5 double bonds in the carbocyclic ring system. In some embodiments, R^BIs a 5-to 26-membered tetracyclic carbocyclic group optionally including 1, 2, 3, 4, 5 or 6 double bonds in the carbocyclic ring system. In some embodiments, R^BIs a substituted heterocyclic group. In some embodiments, R^BIs an unsubstituted heterocyclic group. In some embodiments, R^BIs a saturated heterocyclic group. In some embodiments, R^BIs an unsaturated heterocyclic group. In some embodiments, R^BIs a 3-to 8-membered monocyclic heterocyclyl optionally including 1 or 2 double bonds in the heterocyclic ring system, wherein 1, 2 or 3 atoms in the heterocyclic ring system are independently nitrogen, oxygen or sulfur. In some embodiments, R ^BIs a 5-to 14-membered bicyclic heterocyclic group optionally comprising 1, 2 or 3 double bonds in the heterocyclic ring system, wherein 1, 2, 3 or 4 atoms in the heterocyclic ring system are independently nitrogen, oxygen or sulfur. In some embodiments, R^BIs a 5-to 20-membered tricyclic heterocyclyl group optionally including 1, 2, 3 or 4 double bonds in the heterocyclyl system, wherein 1, 2, 3, 4 or 5 atoms in the heterocyclyl system are independently nitrogen, oxygen or sulfur. In some embodiments, R^BIs a substituted aryl group. In some embodiments, R^BIs an unsubstituted aryl group. In some embodiments, R^BIs 6-14 membered aryl. In some embodiments, R^BIs a 6-to 10-membered aryl group. In some embodiments, R^BIs a substituted phenyl group. In some embodiments, R^BIs unsubstituted phenyl. In some embodiments, R^BIs a substituted naphthyl group. In some embodiments, R^BIs unsubstituted naphthyl. In some embodiments, R^BIs a substituted heteroaryl group. In some embodiments, R^BIs unsubstituted heteroaryl. In some embodiments, R^BIs a 5-to 6-membered monocyclic heteroaryl group, wherein 1, 2, in the heteroaryl ring system,3 or 4 atoms are independently nitrogen, oxygen or sulfur. In some embodiments, R ^BIs an 8-to 10-membered bicyclic heteroaryl group, wherein 1, 2, 3 or 4 atoms in the heteroaryl ring system are independently nitrogen, oxygen or sulfur.

In some embodiments, R^BAre groups as shown in Table 1A. In some embodiments, R^BAre groups as shown in Table 1B.

R^BThe radicals may independently comprise one or more substituents R^d. In some embodiments, all R are^dAre all the same. In some embodiments, two R are^dAre different from each other. In some embodiments, at least one R is^dIs H. In some embodiments, each R is^dIs H. In some embodiments, at least one R is^dIs a substituted or unsubstituted acyl group (e.g., acetyl). In some embodiments, at least one R is^dIs a substituted or unsubstituted alkyl group (e.g., substituted or unsubstituted C)_1-6Alkyl groups). In some embodiments, at least one R is^dis-CH₃. In some embodiments, at least one R is^dis-CF₃Unsubstituted ethyl, perfluoroethyl, unsubstituted propyl, perfluoropropyl, unsubstituted butyl or perfluorobutyl. In some embodiments, at least one R is^dIs a substituted or unsubstituted alkenyl group (e.g., substituted or unsubstituted C_1-6Alkenyl). In some embodiments, at least one R is ^dIs a substituted or unsubstituted alkynyl group (e.g., substituted or unsubstituted C_1-6Alkynyl). In some embodiments, at least one R is^dIs a substituted or unsubstituted carbocyclyl (e.g., a substituted or unsubstituted 3-to 8-membered monocyclic carbocyclyl optionally including 1, 2, or 3 double bonds in the carbocyclic system, or a substituted or unsubstituted 5-to 14-membered bicyclic carbocyclyl optionally including 1, 2, 3, or 4 double bonds in the carbocyclic system). In some embodiments, at least one R is^dIs a substituted or unsubstituted heterocyclic group (e.g., a substituted or unsubstituted 3-to 8-membered monocyclic heterocyclic group optionally including 1 or 2 double bonds in the heterocyclic ring system, wherein 1, b, c, d, a, d, a, d, a, d,2 or 3 atoms are independently nitrogen, oxygen or sulfur; or a substituted or unsubstituted 5-to 14-membered bicyclic heterocyclic group optionally comprising 1, 2 or 3 double bonds in the heterocyclic ring system, wherein 1, 2, 3 or 4 atoms in the heterocyclic ring system are independently nitrogen, oxygen or sulfur). In some embodiments, at least one R is^dIs a substituted or unsubstituted aryl group (e.g., a substituted or unsubstituted 6-to 10-membered aryl group). In some embodiments, at least one R is^dIs a substituted or unsubstituted phenyl group. In some embodiments, at least one R is ^dIs a substituted or unsubstituted heteroaryl (e.g., a substituted or unsubstituted 5-to 6-membered monocyclic heteroaryl, or a substituted or unsubstituted 8-to 10-membered bicyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In some embodiments, at least one R is^dIs a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts) when attached to a nitrogen atom. In some embodiments, R^dAn oxygen protecting group when attached to an oxygen atom (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl). In some embodiments, R^dIs a sulfur protecting group when attached to a sulfur atom (e.g., acetamidomethyl, t-Bu, 3-nitro-2-pyridylthio, 2-pyridylthio or triphenylmethyl). In some embodiments, two R are^dLinked to form a substituted or unsubstituted heterocyclic ring (e.g., a substituted or unsubstituted 3-to 8-membered monocyclic heterocyclic ring optionally including 1 or 2 double bonds in the heterocyclic ring system, wherein 1, 2, or 3 atoms in the heterocyclic ring system are independently nitrogen, oxygen, or sulfur). In some embodiments, two R are ^dLinked to form a substituted or unsubstituted heteroaryl ring (e.g., a substituted or unsubstituted 5-to 6-membered monocyclic heteroaryl ring, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur).

In some embodiments, R^AAnd R^BAre all the same. In some embodiments, R^AAnd R^BAre different from each other. In some embodiments, R^AIs a watch1B a group shown in item Table, and R^BAre other groups shown in the item tables.

In some embodiments, R^AAnd R^BLinked to form a substituted or unsubstituted, saturated or unsaturated carbocyclic ring. In some embodiments, R^AAnd R^BJoined to form a 3-to 8-membered monocyclic carbocyclic ring, optionally including 1, 2 or 3 double bonds in the carbocyclic ring system. In some embodiments, R^AAnd R^BLinked to form a 5-to 14-membered bicyclic carbocyclic ring, optionally including 1, 2, 3 or 4 double bonds in the carbocyclic ring system. In some embodiments, R^AAnd R^BJoined to form a 5-to 20-membered tricyclic carbocycle, optionally including 1, 2, 3, 4 or 5 double bonds in the carbocyclic ring system.

In some embodiments, R^AAnd R^BLinked to form a substituted or unsubstituted, saturated or unsaturated heterocyclic ring. In some embodiments, R ^AAnd R^BLinked to form a 3-to 8-membered monocyclic heterocycle, optionally including 1 or 2 double bonds in the heterocyclic ring system, wherein 1, 2 or 3 atoms in the heterocyclic ring system are independently nitrogen, oxygen or sulfur. In some embodiments, R^AAnd R^BLinked to form a 5-to 14-membered bicyclic heterocycle, optionally including 1, 2 or 3 double bonds in the heterocyclic ring system, wherein 1, 2, 3 or 4 atoms in the heterocyclic ring system are independently nitrogen, oxygen or sulfur. In some embodiments, R^AAnd R^BAre linked to form a 5-to 20-membered tricyclic heterocycle, optionally including 1, 2, 3, 4, or 5 double bonds in the heterocyclic ring system, wherein 1, 2, 3, 4, or 5 atoms in the heterocyclic ring system are independently nitrogen, oxygen, or sulfur.

In some embodiments, R^AAnd R^BThe linkage formed the groups shown in table 1C.

Table 1c. exemplaryRadical (I)

In some embodiments, R^CIs H. In some embodiments, R^CIs substituted or unsubstituted C_1-6Alkyl (e.g., Me, -CF)₃Unsubstituted ethyl, perfluoroethyl, unsubstituted propyl, perfluoropropyl, unsubstituted butyl or perfluorobutyl). In some embodiments, R^CIs Me. In some embodiments, R^CIs a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts).

In some embodiments, R^A1Is a label (e.g., a biotin derivative, a radiolabel, or a fluorophore). Either of formulas (I) and (II) can include a label. The marker is a label. The term "tag" includes any moiety that enables the compound to which it is attached to be captured, detected or made visible. The label may be directly detectable (i.e., it is detectable without any further reaction or manipulation, e.g., a fluorophore or chromophore is directly detectable) or it may be indirectly detectable (i.e., it is made detectable by reaction or binding with another detectable entity, e.g., a hapten is detectable by immunostaining after reaction with a suitable antibody comprising a receptor, e.g., a fluorophore). Labels suitable for use in the present invention can be detected by a variety of means including, but not limited to, spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Suitable labels include, but are not limited to, affinity tags, radioactive labels (e.g., radionuclides (e.g.,³²P、³⁵S、³H、¹⁴C、¹²⁵I、¹³¹i, etc.), fluorescent dyes, phosphorescent dyes, chemiluminescent agents (e.g., acridinium esters, stabilized dioxetanes, etc.), spectrally-resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, and platinum), or Nanoclusters, enzymes (e.g., those used in ELISA, i.e., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), colorimetric tags (e.g., dyes, colloidal gold, etc.), magnetic tags (e.g., Dynabeads)^TM) And a hapten.

In some embodiments, the tag comprises a fluorescent moiety. A wide variety of known fluorescent labeling moieties of chemical structure and physical properties are suitable for use in the practice of the present invention. Suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., Fluorescein Isothiocyanate (FITC), naphthalocyanine, 4 ', 5' -dichloro-2 ', 7' -dimethoxy-fluorescein, 6-carboxyfluorescein, or FAM), carbocyanines, merocyanines, styrene dyes, cyanine dyes, phycoerythrins, erythrocyanins, eosins, rhodamine dyes (e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-Rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine, or TMR), methoxycoumarins, dialkylaminocoumarins, hydroxycoumarins, and aminomethylcoumarins or AMCAs), oregon green dyes (e.g., oregon green 488, oregon Green 500, Oregon Green 514), Texas Red-X, Spectrum Red ^TM、Spectrum Green^TMCyanine dyes (e.g., Cy-3)^TM,Cy-5^TM,Cy-3.5^TM,Cy-5.5^TM) Alexa Fluor dyes (e.g., Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), IRDys (e.g., IRD40, IRD 700, IRD 800), and the like. Further examples of suitable Fluorescent dyes and methods for coupling Fluorescent dyes to other chemical entities are found, for example, in The Handbook of Fluorescent Probes and Research Products,9th ed., Molecular Probes, inc.

The term "luminescence" or "luminescent" refers to any method of light emission including fluorescence, phosphorescence, scintillation, chemiluminescence, and bioluminescence.

The terms "chemiluminescent", "chemiluminescent" or "chemiluminescent substrate" refer to a chemical substance that generates light as a result of a chemical reaction. Commonly used chemiluminescent substrates include, but are not limited to, luminol (5-amino-2, 3-dihydro-1, 4-phthalazinedione), lopinine (Lophine) (2,4, 5-triphenylimidazole), lucigenin (bis-N-methylacridinium), other acridinium esters, luciferin-luciferase and dimethylthiophene derivatives. For example, ECL from Amersham, which is well known in the art ^TMIn the detection system, the acridinium substrate is oxidized by horseradish peroxidase to produce an acridinium ester, which reacts with excess peroxide at alkaline pH to produce chemiluminescence visible at 430 nm.

In some embodiments, the tag comprises an affinity tag. The term "affinity tag" includes any moiety (e.g., antigens and antibodies, enzymes and substrates, receptors and ligands) that participates in an interaction that facilitates capture and/or purification of a molecule. Examples of such affinity moieties include small chemical compounds (e.g., biotin and its derivatives), short amino acid sequences (e.g., 2 to 20 amino acids in length, 4 to 12 amino acids in length), e.g., (His)₆Marker, (Leu)₃A marker or a FLAG marker. The affinity moiety may also be a fluorine label, which is a fluorinated alkyl (e.g., perfluoroalkyl) group that allows recovery of the molecule through its interaction with a fluorine phase (e.g., fluorine liquid phase, fluorine solid). Other affinity moieties are known in the art. In some embodiments, the affinity moiety is selected from (His)₆Marker (His)₄Marker (His)₃Marker (His)₂Marker, (Leu)₄Marker, (Leu)₃Marker, (Leu)₂Markers, HA tags, FLAG markers, VSV-G markers, HSV markers, V5 markers, biotin and derivatives thereof, carbohydrates and glycans. In some embodiments, the affinity moiety is C ₄-C₂₀Perfluoroalkyl (e.g., C)₆-C₁₂Perfluoroalkyl group, C₆-C₈Perfluoroalkyl group, C₄Perfluoroalkyl group, C₅Perfluoroalkyl group, C₆Perfluoroalkyl group, C₇Perfluoroalkyl group, C₈Perfluoroalkyl group, C₉Perfluoroalkyl group, C₁₀Perfluoroalkyl group, C₁₁Perfluoroalkyl group, C₁₂Perfluoroalkyl group, C₁₃Perfluoroalkyl group, C₁₄Perfluoroalkyl group, C₁₅Perfluoroalkyl group, C₁₆Perfluoroalkyl group, C₁₇Perfluoroalkyl group, C₁₈Perfluoroalkyl group, C₁₉Perfluoroalkyl or C₂₀Perfluoroalkyl). In some embodiments, the affinity moiety is biotin. In some embodiments, the affinity moiety is C₈A perfluoroalkyl group.

The tag may include a divalent linking group. In some embodiments, the divalent linking group is optionally substituted C_1-60A hydrocarbon chain, optionally wherein one or more carbon units in the hydrocarbon chain are independently replaced by: -C ═ O-, -S-, -NR^L3a-、-NR^L3aC(＝O)-、-C(＝O)NR^L3a-、-SC(＝O)-、 -C(＝O)S-、-OC(＝O)-、-C(＝O)O-、-NR^L3aC(＝S)-、-C(＝S)NR^L3a-, trans-CR^L3b＝CR^L3b-, cis-CR^L3b＝CR^L3b-、-C≡C-、-S(＝O)-、-S(＝O)O-、-OS(＝O)-、 -S(＝O)NR^L3a-、-NR^L3aS(＝O)-、-S(＝O)₂-、-S(＝O)₂O-、-OS(＝O)₂-、-S(＝O)₂NR^L3a-or-NR^L3aS(＝O)₂-. In some embodiments, the divalent linking group is a PEG moiety (e.g., - (PEG)_1-20–、–(PEG)_1-12–、–(PEG)_1-6–、–(PEG)_6-12-). In some embodiments, the divalent linking group is- (CH)₂)_1-40- (e.g., - (CH)₂)_1-20–、–(CH₂)_1-10-). In some embodiments, the divalent linking group is one or more PEG moieties (e.g., independently is- (PEG)_1-20–、–(PEG)_1-12–、–(PEG)_1-6–、–(PEG)_6-12-) and one or more- (CH) ₂)_1-40A moiety (e.g. independently-(CH₂)_1-20–、–(CH₂)_1-10-, optionally wherein said PEG moiety and/or said- (CH)₂)_1-40-one or more methylene units of the moiety are independently replaced by-C (═ O) NH-or-NHC (═ O) -.

The formula (I) may comprise one or more substituents R^a. When formula (I) includes two or more R^aWhen there are two arbitrary R^aMay be the same as or different from each other. In some embodiments, at least one R is^aIs H. In some embodiments, each R is^aIs H. In some embodiments, at least one R is^aIs substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts), an oxygen protecting group when attached to an oxygen atom (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl or benzoyl) or a sulfur protecting group when attached to a sulfur atom (e.g., acetamidomethyl, t-Bu, 3-nitro-2-pyridylthio, 2-pyridylthio or triphenylmethyl), or both R. ^aJoined to form a substituted or unsubstituted heterocyclic ring or a substituted or unsubstituted heteroaromatic ring.

Formula (I) includes a substituent R at the 1-position of the 2, 7-diazaindolyl ring^A2. In some embodiments, R^A2Is H. In some embodiments, R^A2Is a substituted or unsubstituted acyl group. In some embodiments, R^A2is-C (═ O) R^aOptionally wherein R is^aIs substituted or unsubstituted C_1-6Alkyl (e.g. Me) or substituted or unsubstituted C_2-6An alkenyl group. In some embodiments, R^A2is-C (═ O) R^aWherein R is^aIs a substituted or unsubstituted vinyl group. In some embodiments, R^A2is-C (═ O) CH ═ CH₂. In some implementationsIn the scheme, R^A2is-C (═ O) OR^aOptionally wherein R is^aIs H, substituted or unsubstituted C_1-6Alkyl (e.g., Me) or an oxygen protecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl). In some embodiments, R^A2is-C (═ O) N (R)^a)₂Optionally wherein each R is^aIndependently is H, substituted or unsubstituted C_1-6Alkyl (e.g., Me) or nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts). In some embodiments, R ^A2Is a substituted or unsubstituted alkyl group (e.g., substituted or unsubstituted C)_1-6Alkyl groups). In some embodiments, R^A2Is Me. In some embodiments, R^A2Is Et. In some embodiments, R^A2Is n-Pr. In some embodiments, R^A2Is i-Pr. In some embodiments, R^A2Is Bu (e.g., n-Bu, i-Bu, sec-Bu or t-Bu). In some embodiments, R^A2Is an unsubstituted pentyl group (e.g., unsubstituted n-pentyl group, unsubstituted t-pentyl group, unsubstituted neopentyl group, unsubstituted isopentyl group, unsubstituted sec-pentyl group, or unsubstituted 3-pentyl group). In some embodiments, R^A2sec-Bu, t-Bu or unsubstituted 3-pentyl. In some embodiments, R^A2is-CF₃Bn, perfluoroethyl, perfluoropropyl, perfluorobutyl or perfluoropentyl. In some embodiments, R^A2is-CH₂C(＝O)–NH–N＝C(R^a)₂. In some embodiments, R^A2Is a substituted or unsubstituted carbocyclyl (e.g., a substituted or unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1, or 2 double bonds in the carbocyclic ring system). In some embodiments, R^A2Is a substituted or unsubstituted cyclopropyl. In some embodiments, R^A2Is unsubstituted cyclopropyl. In some embodiments, R ^A2Is a substituted or unsubstituted cyclobutyl group. In some embodiments, R^A2Is a substituted or unsubstituted cyclopentyl group. In some embodimentsIn, R^A2Is unsubstituted cyclopropyl, unsubstituted cyclobutyl or unsubstituted cyclopentyl. In some embodiments, R^A2Is a substituted or unsubstituted heterocyclyl group (e.g., a substituted or unsubstituted 3-to 7-membered monocyclic heterocyclyl group containing 0, 1, or 2 double bonds in the heterocyclic ring system, wherein 1, 2, or 3 atoms in the heterocyclic ring system are independently nitrogen, oxygen, or sulfur). In some embodiments, R^A2Is a substituted or unsubstituted tetrahydropyranyl group. In some embodiments, R^A2Is of the formula:in some embodiments, R^A2Is of the formula:in some embodiments, R^A2Is a substituted or unsubstituted oxetanyl group, a substituted or unsubstituted tetrahydrofuranyl group, a substituted or unsubstituted pyrrolidinyl group, a substituted or unsubstituted piperidinyl group, a substituted or unsubstituted morpholinyl group, or a substituted or unsubstituted piperazinyl group. In some embodiments, R^A2Is of the formula: wherein each R^aIndependently is unsubstituted C_1-6Alkyl (e.g., Me)). In some embodiments, R^A2Is a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R ^A2Is Boc. In some embodiments, R^A2Is a warhead.

Any one of formulas (I) and (II) may include one or more warheads independently selected from:

wherein:

L³is a bond or optionally substituted C_1-4A hydrocarbon chain, optionally wherein one or more carbon units in the hydrocarbon chain are independently replaced by: -C ═ O-, -S-, -NR^L3a-、-NR^L3aC(＝O)-、 -C(＝O)NR^L3a-、-SC(＝O)-、-C(＝O)S-、-OC(＝O)-、-C(＝O)O-、-NR^L3aC(＝S)-、 -C(＝S)NR^L3a-, trans-CR^L3b＝CR^L3b-, cis-CR^L3b＝CR^L3b-、-C≡C-、-S(＝O)-、 -S(＝O)O-、-OS(＝O)-、-S(＝O)NR^L3a-、-NR^L3aS(＝O)-、-S(＝O)₂-、-S(＝O)₂O-、 -OS(＝O)₂-、-S(＝O)₂NR^L3a-or-NR^L3aS(＝O)₂-, wherein R^L3aIs hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups, and wherein each occurrence of R^L3bIndependently hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl, or two R^L3bThe groups are linked to form an optionally substituted carbocyclic ring or an optionally substituted heterocyclic ring;

L⁴is a bond or an optionally substituted branched or unbranched C_1-6A hydrocarbon chain;

R^E1、R^E2and R^E3Each independently is hydrogen, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, -CN, -CH₂OR^EE、-CH₂N(R^EE)₂、-CH₂SR^EE、-OR^EE、-N(R^EE)₂、-Si(R^EE)₃or-SR^EEWherein each occurrence of R^EEIndependently is hydrogen, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl, or two R ^EEThe groups are linked to form an optionally substituted heterocyclic ring; or R^E1And R^E3Or R is^E2And R^E3Or R is^E1And R^E2Linked to form an optionally substituted carbocyclic ring or an optionally substituted heterocyclic ring;

R^E4is a leaving group;

R^E5is halogen;

R^E6is hydrogen, substituted or unsubstituted C_1-6An alkyl or nitrogen protecting group;

each Y is independently O, S or NR^E7Wherein R is^E7Is hydrogen, substituted or unsubstituted C_1-6An alkyl or nitrogen protecting group;

a is 1 or 2; and

where the chemical bond permits, each z is independently 0, 1, 2, 3, 4, 5, or 6.

In some embodiments, either of formulas (I) and (II) does not include a warhead. In some embodiments, either of formulas (I) and (II) comprises a warhead. In some embodiments, either of formulas (I) and (II) includes two or more warheads. When formula (I) or (II) includes two or more warheads, two of the warheads may be the same as or different from each other. In some embodiments, at least one warhead is of formula (i-1 a):in some embodiments, at least one warhead is of formula (i-1 b):in some embodiments, at least one warhead is of formula (i-1 c): in some embodiments, at least one warhead is of formula (i-1 d):in some embodiments, at least one warhead is of formula (i-1 e): In some embodiments, at least one warhead is of formula (i-1 f):in some embodiments, at least one warhead is of formula (i-1 g):in some embodiments, at least one warhead isIn some embodiments, at least one warhead is(for example,). In some embodiments, at least one warhead isIn some embodiments, at least one warhead isIn some embodiments, at least one warhead is of formula (i-1 h):(for example,in some embodiments, at least one warhead is(for example,). In some embodiments, at least one warhead is(for example,). In some embodiments, at least one warhead is(for example,)。

in some embodiments, R^A2Is a label (e.g., a biotin derivative, a radiolabel, or a fluorophore).

Formula (I) includes a substituent R at the 3-position of the 2, 7-diazaindolyl ring^A3. In some embodiments, R^A3Is H. In some embodiments, R^A3Is halogen (e.g., F, Cl, Br, or I). In some embodiments, R^A3Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^A3Is Me. In some embodiments, R^A3is-CF₃Bn, Et, perfluoroethyl, Pr, perfluoropropyl, Bu or perfluorobutyl. In some embodiments, R ^A3is-OR^aOptionally wherein R is^aIs H, substituted or unsubstituted C_1-6The alkyl group (for example,me), substituted or unsubstituted acyl or oxygen protecting groups (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl). In some embodiments, R^A3is-OC (═ O) R^aOptionally wherein R is^aIs H, substituted or unsubstituted C_1-6Alkyl (e.g. Me) or substituted or unsubstituted C_2-6Alkenyl (e.g., substituted or unsubstituted vinyl). In some embodiments, R^A3is-OC (═ O) CH ═ CH₂. In some embodiments, R^A3is-N (R)^a)₂Optionally wherein each R is^aIndependently is H, substituted or unsubstituted C_1-6Alkyl (e.g., Me), substituted or unsubstituted acyl, or nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts). In some embodiments, R^A3is-N (R)^a)C(＝O)R^aOptionally wherein each R is^aIndependently is H, substituted or unsubstituted C_1-6Alkyl (e.g. Me) or substituted or unsubstituted C_2-6Alkenyl (e.g., substituted or unsubstituted vinyl). In some embodiments, R^A3is-NHC (═ O) CH ═ CH₂. In some embodiments, R ^A3Is a warhead.

Formula (I) includes a substituent R on the nitrogen atom^A4. In some embodiments, R^A4Is H. In some embodiments, R^A4Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^A4Is Me. In some embodiments, R^A4is-CF₃Bn, Et, perfluoroethyl, Pr, perfluoropropyl, Bu or perfluorobutyl. In some embodiments, R^A4Is a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts).

Formula (I) includes a substituent R^A5. In some embodiments, R^A5Is of the formula:(for example,wherein R is^A7Et, Pr or Bu). In some embodiments, R^A5Is of the formula:in some embodiments, R^A5Is of the formula:in some embodiments, R^A6Is H. In some embodiments, R^A6Is halogen (e.g., F, Cl, Br, or I). In some embodiments, R^A6Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^A6Is Me. In some embodiments, R^A6Being substituted methyl (e.g., -CF)₃Or Bn). In some embodiments, R^A6Is Et, substituted ethyl (e.g., perfluoroethyl), Pr, substituted propyl (e.g., perfluoropropyl), Bu, or substituted butyl (e.g., perfluorobutyl). In some embodiments, R ^A6is-OR^a(e.g., -OH or-O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe)). In some embodiments, R^A6is-N (R)^a)₂Optionally wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R^A6is-NH₂-NHMe or-N (Me)₂. In some embodiments, R^A7Is H. In some embodiments, R^A7Is halogen (e.g., F, Cl, Br, or I). In some embodiments, R^A7Is substituted or unsubstituted C_2-6An alkyl group. In some embodiments, R^A7Is Et. In some embodiments, R^A7Is a substituted ethyl group (e.g., perfluoroethyl). In some embodiments, R^A7Is n-Pr. In some casesIn embodiments, R^A7Is i-Pr. In some embodiments, R^A7Is a substituted propyl group (e.g., perfluoropropyl group). In some embodiments, R^A7Bu or unsubstituted pentyl. In some embodiments, R^A7Is a substituted butyl group (e.g., perfluorobutyl group) or a substituted pentyl group (e.g., perfluoropentyl group). In some embodiments, R^A7Is a substituted or unsubstituted 3-to 7-membered monocyclic carbocyclic group containing 0, 1 or 2 double bonds in the carbocyclic ring system. In some embodiments, R ^A7Is a substituted or unsubstituted cyclopropyl, substituted or unsubstituted cyclobutyl, or substituted or unsubstituted cyclopentyl. In some embodiments, R^A7is-OR^a(e.g., -OH or-O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe)). In some embodiments, R^A7is-N (R)^a)₂Optionally wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R^A7is-NH₂、–NHMe、–NHEt、–N(Me)₂or-N (Et)₂. In some embodiments, R^A7Is a substituted or unsubstituted cyclopropyl or-N (R)^a)₂Wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts).

In some embodiments, R^A5Is of the formula:(for example,wherein R is^A9Me, Et, Pr or Bu). A moiety of the formula:also included are tautomers thereofStructural formIn some embodiments, R^A5Is of the formula:in some embodiments, R^A5Is of the formula:in some embodiments, R^A5Is of the formula:in some embodiments, R^A8Is H. In some embodiments, R ^A8Is halogen (e.g., F, Cl, Br, or I). In some embodiments, R^A8Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^A8Is Me. In some embodiments, R^A8Being substituted methyl (e.g., -CF)₃Or Bn). In some embodiments, R^A8Is Et, substituted ethyl (e.g., perfluoroethyl), Pr, substituted propyl (e.g., perfluoropropyl), Bu, or substituted butyl (e.g., perfluorobutyl). In some embodiments, R^A8is-OR^a(e.g., -OH or-O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe)). In some embodiments, R^A8is-N (R)^a)₂Optionally wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R^A8is-NH₂-NHMe or-N (Me)₂. In some embodiments, R^A9Is H. In some embodiments, R^A9Is halogen (e.g., F, Cl, Br, or I). In some embodiments, R^A9Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^A9Is Me. At one endIn some embodiments, R^A9Being substituted methyl (e.g., -CF) ₃Or Bn). In some embodiments, R^A9Is Et. In some embodiments, R^A9Is a substituted ethyl group (e.g., perfluoroethyl). In some embodiments, R^A9Is n-Pr. In some embodiments, R^A9Is i-Pr. In some embodiments, R^A9Is a substituted propyl group (e.g., perfluoropropyl group). In some embodiments, R^A9Bu or unsubstituted pentyl. In some embodiments, R^A9Is a substituted butyl group (e.g., perfluorobutyl group) or a substituted pentyl group (e.g., perfluoropentyl group). In some embodiments, R^A9Is a substituted or unsubstituted 3-to 7-membered monocyclic carbocyclic group containing 0, 1 or 2 double bonds in the carbocyclic ring system. In some embodiments, R^A9Is a substituted or unsubstituted cyclopropyl, substituted or unsubstituted cyclobutyl, or substituted or unsubstituted cyclopentyl. In some embodiments, R^A9is-OR^a(e.g., -OH or-O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe)). In some embodiments, R^A9is-N (R)^a)₂Optionally wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R ^A9is-NH₂、–NHMe、–NHEt、–N(Me)₂or-N (Et)₂. In some embodiments, R^A9Is a substituted OR unsubstituted cyclopropyl, -OR^aor-N (R)^a)₂Wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl, an oxygen protecting group when attached to an oxygen atom, or a nitrogen protecting group when attached to a nitrogen atom.

In some embodiments, R^A5Is of the formula:in some embodiments, R^A5Is of the formula:(for example,). In some embodiments, R^A5Is of the formula:(for example,). In some embodiments, R^A5Is of the formula:(for example, ). In some embodiments, R^A5Is of the formula: in some embodiments, R^A5Is of the formula:in some embodiments, R^A10is-OR^a(e.g., -OH). In some embodiments, R^A10is-N (R)^a)₂. In some embodiments, R^A10is-NH₂. In some embodiments, R^A10is-NHR^aWherein R is^aIs substituted or notSubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R^A10Is a warhead. In some embodiments, R^A10is-NHC (═ O) R^aOptionally wherein R is^aIs a substituted or unsubstituted vinyl group. In some embodiments, R ^A10is-NHC (═ O) CH ═ CH₂. When formula (I) includes two or more R^A11When there are two arbitrary R^A11May be the same as or different from each other. In some embodiments, at least one R is^A11Is halogen. In some embodiments, at least one R is^A11Is Br. In some embodiments, at least one R is^A11F, Cl or I. In some embodiments, at least one R is^A11Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, at least one R is^A11Is Me. In some embodiments, at least one R is^A11Being substituted methyl (e.g., -CF)₃Or Bn). In some embodiments, at least one R is^A11Is Et. In some embodiments, at least one R is^A11Is a substituted ethyl group (e.g., perfluoroethyl). In some embodiments, at least one R is^A11Is n-Pr. In some embodiments, at least one R is^A11Is i-Pr. In some embodiments, at least one R is^A11Is a substituted propyl group (e.g., perfluoropropyl group). In some embodiments, at least one R is^A11Me, Et or n-Pr. In some embodiments, at least one R is^A11Bu or unsubstituted pentyl. In some embodiments, at least one R is^A11Is a substituted butyl group (e.g., perfluorobutyl group) or a substituted pentyl group (e.g., perfluoropentyl group). In some embodiments, at least one R is ^A11Is a substituted or unsubstituted 3-to 7-membered monocyclic carbocyclic group containing 0, 1 or 2 double bonds in the carbocyclic ring system. In some embodiments, at least one R is^A11Is a substituted or unsubstituted cyclopropyl, substituted or unsubstituted cyclobutyl, or substituted or unsubstituted cyclopentyl. In some embodiments, at least one R is^A11is-OR^a(e.g. in-OH or-O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe)). In some embodiments, at least one R is^A11is-N (R)^a)₂Optionally wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, at least one R is^A11is-NH₂、–NHMe、–NHEt、–N(Me)₂or-N (Et)₂. In some embodiments, at least one R is^A11Is substituted or unsubstituted C_1-6Alkyl, substituted OR unsubstituted cyclopropyl, -OR^aor-N (R)^a)₂Wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl, an oxygen protecting group when attached to an oxygen atom, or a nitrogen protecting group when attached to a nitrogen atom. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4.

In some embodiments, R^A5Is of the formula:in some embodiments, R^A5Is of the formula:(for example,for example). In some embodiments, R^A12Is H. In some embodiments, R^A12Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^A12Is Me. In some embodiments, R^A12is-CF₃Bn, Et, perfluoroethyl, Pr, perfluoropropyl, Bu orA perfluorobutyl group. In some embodiments, R^A12Is a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R^A12Is a warhead. When formula (I) includes two or more R^A13When there are two arbitrary R^A13May be the same as or different from each other. In some embodiments, at least one R is^A13Is halogen (e.g., F, Cl, Br, or I). In some embodiments, at least one R is^A13Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, at least one R is^A13Is Me. In some embodiments, at least one R is^A13Being substituted methyl (e.g., -CF)₃Or Bn). In some embodiments, at least one R is^A13Is Et. In some embodiments, at least one R is^A13Is a substituted ethyl group (e.g., perfluoroethyl). In some embodiments, at least one R is ^A13Is n-Pr. In some embodiments, at least one R is^A13Is i-Pr. In some embodiments, at least one R is^A13Is a substituted propyl group (e.g., perfluoropropyl group). In some embodiments, at least one R is^A13Me, Et or n-Pr. In some embodiments, at least one R is^A13Bu or unsubstituted pentyl. In some embodiments, at least one R is^A13Is a substituted butyl group (e.g., perfluorobutyl group) or a substituted pentyl group (e.g., perfluoropentyl group). In some embodiments, at least one R is^A13Is a substituted or unsubstituted 3-to 7-membered monocyclic carbocyclic group containing 0, 1 or 2 double bonds in the carbocyclic ring system. In some embodiments, at least one R is^A13Is a substituted or unsubstituted cyclopropyl, substituted or unsubstituted cyclobutyl, or substituted or unsubstituted cyclopentyl. In some embodiments, at least one R is^A13is-OR^a(e.g., -OH or-O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe)). In some embodiments, at least one R is^A13is-N (R)^a)₂Optionally wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g. B)n, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts). In some embodiments, at least one R is ^A13is-NH₂、–NHMe、–NHEt、–N(Me)₂or-N (Et)₂. In some embodiments, at least one R is^A13Is halogen, substituted OR unsubstituted cyclopropyl, -OR^aor-N (R)^a)₂Wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl, an oxygen protecting group when attached to an oxygen atom, or a nitrogen protecting group when attached to a nitrogen atom. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3, 4, 5, 6, 7, or 8. In some embodiments, m is 9.

Formula (I) includes a substituent R^A5. In some embodiments, R^A5Is of the formula:(for example,optionally wherein R is^A16Et, Pr or Bu). In some embodiments, R^A5Is of the formula:in some embodiments, R^A5Is of the formula:in some embodiments, R^A14Is H. In some embodiments, R^A14Is halogen (e.g., F, Cl, Br, or I). In some embodiments, R^A14Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^A14Is Me. In some embodiments, R^A14Being substituted methyl (e.g., -CF)₃Or Bn). In some embodiments, R^A14Is Et, substituted ethyl (e.g., perfluoroethyl), Pr, substituted propyl (e.g., perfluoroethyl) Such as perfluoropropyl), Bu, or a substituted butyl (e.g., perfluorobutyl). In some embodiments, R^A14is-OR^a(e.g., -OH or-O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe)). In some embodiments, R^A14is-N (R)^a)₂Optionally wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R^A14is-NH₂-NHMe or-N (Me)₂. In some embodiments, R^A15Is H. In some embodiments, R^A15Is halogen (e.g., F, Cl, Br, or I). In some embodiments, R^A15Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^A15Is Me. In some embodiments, R^A15Being substituted methyl (e.g., -CF)₃Or Bn). In some embodiments, R^A15Is Et, substituted ethyl (e.g., perfluoroethyl), Pr, substituted propyl (e.g., perfluoropropyl), Bu, or substituted butyl (e.g., perfluorobutyl). In some embodiments, R^A15is-OR^a(e.g., -OH or-O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe)). In some embodiments, R ^A15is-N (R)^a)₂Optionally wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R^A15is-NH₂-NHMe or-N (Me)₂. In some embodiments, R^A16Is H. In some embodiments, R^A16Is halogen (e.g., F, Cl, Br, or I). In some embodiments, R^A16Is substituted or unsubstituted C_2-6An alkyl group. In some embodiments, R^A16Is Et. In some embodiments, R^A16Is a substituted ethyl group (e.g., perfluoroethyl). In some embodiments of the present invention, the substrate is,R^A16is n-Pr. In some embodiments, R^A16Is i-Pr. In some embodiments, R^A16Is a substituted propyl group (e.g., perfluoropropyl group). In some embodiments, R^A16Bu or unsubstituted pentyl. In some embodiments, R^A16Is a substituted butyl group (e.g., perfluorobutyl group) or a substituted pentyl group (e.g., perfluoropentyl group). In some embodiments, R^A16Is a substituted or unsubstituted 3-to 7-membered monocyclic carbocyclic group containing 0, 1 or 2 double bonds in the carbocyclic ring system. In some embodiments, R^A16Is a substituted or unsubstituted cyclopropyl, substituted or unsubstituted cyclobutyl, or substituted or unsubstituted cyclopentyl. In some embodiments, R ^A16is-OR^a(e.g., -OH or-O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe)). In some embodiments, R^A16is-N (R)^a)₂Optionally wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R^A16is-NH₂、–NHMe、–NHEt、–N(Me)₂or-N (Et)₂. In some embodiments, R^A16Is a substituted or unsubstituted cyclopropyl or-N (R)^a)₂Wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R^A17Is H. In some embodiments, R^A17Is a substituted or unsubstituted acyl group. In some embodiments, R^A17is-C (═ O) R^aOptionally wherein R is^aIs substituted or unsubstituted C_1-6Alkyl (e.g. Me) or substituted or unsubstituted C_2-6An alkenyl group. In some embodiments, R^A17Is a warhead. In some embodiments, R^A17is-C (═ O) R^aWherein R is^aIs a substituted or unsubstituted vinyl group. In some embodiments, R^A17is-C (═ O) CH ═ CH₂. In some embodiments, R ^A17is-C (═ O) OR^aOptionally wherein R is^aIs H, substituted or unsubstituted C_1-6Alkyl (e.g., Me) or an oxygen protecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl). In some embodiments, R^A17is-C (═ O) N (R)^a)₂Optionally wherein each R is^aIndependently is H, substituted or unsubstituted C_1-6Alkyl (e.g., Me) or nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts). In some embodiments, R^A17Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^A17Is Me. In some embodiments, R^A17is-CF₃Bn, Et, perfluoroethyl, Pr, perfluoropropyl, Bu or perfluorobutyl. In some embodiments, R^A17Is a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts).

In some embodiments, the compound of formula (I) is a compound of the formula:

In some embodiments, the compound of formula (I) is a compound of the formula:

in some embodiments, the compound of formula (I) is a compound of the formula:

in some embodiments, the compound of formula (I) is a compound of the formula:

in some embodiments, the compound of formula (I) is a compound of the formula:

in some embodiments, the compound of formula (I) is a compound of the formula:

Wherein R is^A14Is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstitutedAryl, substituted or unsubstituted heteroaryl, or a nitrogen protecting group.

In some embodiments, the compound of formula (I) is a compound of the formula:

in some embodiments, the compound of formula (I) is a compound of the formula:

in some embodiments, the compound of formula (I) is a compound of the formula:

in some embodiments, the compound of formula (I) is a compound of the formula:

In some embodiments, the compound of formula (I) is a compound of the formula:

wherein R is^A2Is a nitrogen protecting group (e.g., Boc).

In some embodiments, the compound of formula (I) is a compound of the formula:

in some embodiments, the compound of formula (I) is a compound of the formula:

optionally wherein each R is^A2Is i-Pr.

In some embodiments, the compound of formula (I) is a compound of the formula:

wherein each k is independently an integer from 3 to 11, inclusive.

In some embodiments, the compound of formula (I) is a compound of the formula:

in some embodiments, the compound of formula (I) is a compound of the formula:

in some embodiments, the compound of formula (I) is a compound of the formula:

in some embodiments, the compound of formula (I) is a compound of the formula:

in some embodiments, the compound of formula (I) is a compound of the formula:

In some embodiments, the compound of formula (I) is a compound of the formula:

in some embodiments, the compound of formula (I) is a compound of the formula:

in some embodiments, the compound of formula (I) is a compound of the formula:

exemplary compounds of formula (I) include, but are not limited to:

and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, and prodrugs thereof.

Exemplary compounds of formula (I) further include, but are not limited to:

and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, and prodrugs thereof.

In some embodiments, the compound of formula (I) is a compound of the formula:

in some embodiments, the compound of formula (I) is EZ05-TOM, EZ05-FITC, EZ-TAMRA, or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

In some embodiments, the compound of formula (I) is EZ05 — biotinylated, or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

In another aspect, the present application provides compounds of formula (II), and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives and prodrugs thereof:

wherein:

R^B1is halogen, substituted OR unsubstituted alkyl, substituted OR unsubstituted alkenyl, substituted OR unsubstituted alkynyl, substituted OR unsubstituted carbocyclyl, substituted OR unsubstituted heterocyclyl, substituted OR unsubstituted aryl, substituted OR unsubstituted heteroaryl, -OR ^b、–N(R^b)₂、–SR^b、–CN、–SCN、–C(＝NR^b)R^b、–C(＝NR^b)OR^b、–C(＝NR^b)N(R^b)₂、–C(＝O)R^b、–C(＝O)OR^b、–C(＝O)N(R^b)₂、–NO₂、–NR^bC(＝O)R^b、–NR^bC(＝O)OR^b、–NR^bC(＝O)N(R^b)₂、–OC(＝O)R^b、–OC(＝O)OR^b、–OC(＝O)N(R^b)₂A label or

Each R^bIndependently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two R^bLinked to form a substituted or unsubstituted heterocyclic ring or a substituted or unsubstituted heteroaromatic ring;

R^Ais hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstitutedA substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl group of (a);

R^Bis hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

or R^AAnd R^BAre linked to form a substituted or unsubstituted carbocyclic ring, or a substituted or unsubstituted heterocyclic ring;

R^CIs hydrogen, substituted or unsubstituted C_1-6An alkyl or nitrogen protecting group;

R^B2is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, a nitrogen protecting group, a label, or a warhead; and

R^B3is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^b、–N(R^b)₂Or a warhead;

R^B4is hydrogen, substituted or unsubstituted C_1-6An alkyl or nitrogen protecting group; and

R^B5is of the formula:

wherein:

R^B6is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl or-N (R)^b)₂；

R^B7Is hydrogen, halogen, substituted or unsubstituted C_2-6Alkyl, or substituted or unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 or 2 double bonds in the carbocyclic ring system;

R^B8is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl or-N (R)^b)₂；

R^B9Is hydrogen, halogen, substituted orUnsubstituted C_1-6Alkyl, or substituted or unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 or 2 double bonds in the carbocyclic ring system;

R^B10is-OR^b–N(R^b)₂Or a warhead;

each R^B11Independently is halogen, substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 or 2 double bonds in the carbocyclic ring system, or-N (R)^b)₂；

u is 0, 1, 2, 3 or 4;

R^B12is hydrogen, substituted or unsubstituted C _1-6Alkyl, nitrogen protecting group or warhead;

each R^B13Independently is halogen, substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 or 2 double bonds in the carbocyclic ring system, or-N (R)^b)₂；

v is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9;

R^B14is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^bor-N (R)^b)₂；

R^B15Is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^bor-N (R)^b)₂；

R^B16Is hydrogen, halogen, substituted or unsubstituted C_2-6Alkyl, substituted OR unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 OR 2 double bonds in the carbocyclic ring system, -OR^bor-N (R)^b)₂(ii) a And

R^B17is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted C_1-6Alkyl, nitrogen protecting group or warhead.

In some embodiments, the EZH2 inhibitor is a compound of formula (II), or a pharmaceutically acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof:

wherein:

R^B1is halogen, substituted OR unsubstituted alkyl, substituted OR unsubstituted alkenyl, substituted OR unsubstituted alkynyl, substituted OR unsubstituted carbocyclyl, substituted OR unsubstituted heterocyclyl, substituted OR unsubstituted aryl, substituted OR unsubstituted heteroaryl, -OR ^b、–N(R^b)₂、–SR^b、–CN、–SCN、–C(＝NR^b)R^b、–C(＝NR^b)OR^b、–C(＝NR^b)N(R^b)₂、–C(＝O)R^b、–C(＝O)OR^b、–C(＝O)N(R^b)₂、–NO₂、–NR^bC(＝O)R^b、–NR^bC(＝O)OR^b、–NR^bC(＝O)N(R^b)₂、–OC(＝O)R^b、–OC(＝O)OR^bor-OC (═ O) N (R)^b)₂；

Each R^bIndependently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or two R^bLinked to form a substituted or unsubstituted heterocyclic ring, or a substituted or unsubstituted heteroaromatic ring;

R^B2is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl or a nitrogen protecting group;

R^B3is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^bor-N (R)^b)₂；

R^B4Is hydrogen,Substituted or unsubstituted C_1-6An alkyl or nitrogen protecting group; and

R^B5is of the formula:

wherein:

R^B6is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl or-N (R)^b)₂；

R^B7Is hydrogen, halogen, substituted or unsubstituted C_2-6Alkyl or substituted or unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 or 2 double bonds in the carbocyclic ring system;

R^B8is hydrogen, halogen, substituted or unsubstituted C _1-6Alkyl or-N (R)^b)₂；

R^B9Is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl or substituted or unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 or 2 double bonds in the carbocyclic ring system;

R^B10is-OR^bor-N (R)^b)₂；

Each R^B11Independently is halogen, substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 or 2 double bonds in the carbocyclic ring system, or-N (R)^b)₂；

u is 0, 1, 2, 3 or 4;

R^B12is hydrogen, substituted or unsubstituted C_1-6An alkyl or nitrogen protecting group;

each R^B13Independently is halogen, substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 or 2 double bonds in the carbocyclic ring system, or-N (R)^b)₂；

v is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9;

R^B14is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^bor-N (R)^b)₂；

R^B15Is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^bor-N (R)^b)₂；

R^B16Is hydrogen, halogen, substituted or unsubstituted C_2-6Alkyl, substituted OR unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 OR 2 double bonds in the carbocyclic ring system, -OR^bor-N (R)^b)₂(ii) a And

R^B17is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups.

Formula (II) includes a substituent R on the pyridine ring^B1. In some embodiments, R^B1Is halogen (e.g., F, Cl, Br, or I). In some embodiments, R^B1Is a substituted or unsubstituted alkyl group (e.g., substituted or unsubstituted C)_1-6Alkyl groups). In some embodiments, R^B1Is Me. In some embodiments, R^B1is-CF₃Bn, Et, perfluoroethyl, Pr, perfluoropropyl, Bu or perfluorobutyl. In some embodiments, R^B1Is a substituted or unsubstituted alkenyl group (e.g., substituted or unsubstituted C_2-6Alkenyl). In some embodiments, R^B1Is a substituted or unsubstituted alkynyl group (e.g., substituted or unsubstituted C_1-6Alkynyl). In some embodiments, R^B1Is a substituted or unsubstituted carbocyclyl (e.g., a substituted or unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1, or 2 double bonds in the carbocyclic ring system). In some embodiments, R^B1Is a substituted or unsubstituted heterocyclyl group (e.g., a substituted or unsubstituted 3-to 7-membered monocyclic heterocyclyl group containing 0, 1, or 2 double bonds in the heterocyclic ring system, wherein 1, 2, or 3 atoms in the heterocyclic ring system are independently nitrogen, oxygen, or sulfur). In some embodiments, R^B1Is a substituted or unsubstituted piperazinyl group. In some embodiments, R ^B1Is of the formula:wherein R is^B14Is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or a nitrogen protecting group. In some embodiments, R^B1Is of the formula:in some embodiments, R^B1Is of the formula:wherein R is^B14Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^B1Is of the formula:in some embodiments, R^B1Is of the formula:wherein L is^BIs a bond or substituted or unsubstituted C_1-100A hydrocarbon chain, optionally wherein one or more chain atoms in the hydrocarbon chain are independently-O-, -S-, or-NR^b-replacing; and X^BIs a small molecule, peptide, protein or polynucleotide. In some embodiments, R^B1Is of the formula:wherein z is 0 or 1, w is an integer from 0 to 11, inclusive, x is an integer from 0 to 10, inclusive, and y is an integer from 0 to 10, inclusive. In some embodiments, w is an integer from 3 to 11, inclusive, x is 0 and y is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, X ^BIs a small molecule. In some embodiments, X^BIs a small molecule drug (e.g., wherein Z^Bis-O-or-NH-or other drug described herein as a small molecule). In some embodiments, X^BIs a small molecule tag (e.g., a biotin moiety (e.g.,) Or a small molecule fluorophore). In some embodiments, R^B1Is a substituted or unsubstituted oxetanyl group, a substituted or unsubstituted tetrahydrofuryl group, a substituted or unsubstituted pyrrolidinyl group, a substituted or unsubstituted tetrahydropyranyl group, a substituted or unsubstituted piperidinyl group, a substituted or unsubstituted morpholinyl group, a substituted or unsubstituted azepanyl group, or a substituted or unsubstituted diazepanyl group. In some embodiments, R^B1Is of the formula: wherein each R^bIndependently is unsubstituted C_1-6Alkyl (e.g., Me)). In some embodiments, R^B1Is a substituted or unsubstituted aryl group (e.g., a substituted or unsubstituted 6-to 10-membered aryl group). In some embodiments, R^B1Is a substituted or unsubstituted phenyl group. In some embodiments, R^B1Is a substituted or unsubstituted heteroaryl (e.g., a substituted or unsubstituted 5-to 6-membered monocyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in the heteroaryl ring system are independently nitrogen, oxygen, or sulfur). In some embodiments, R ^B1is-OR^b(e.g., -OH, -O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe, -OEt, -OPr, -OBu, or-OBn) or-O(substituted or unsubstituted phenyl) (e.g., -OPh)). In some embodiments, R^B1is-SR^b(e.g., -SH, -S (substituted or unsubstituted C)_1-6Alkyl) (e.g., -SMe, -SEt, -SPr, -SBu, or-SBn) or-S (substituted or unsubstituted phenyl) (e.g., -SPh)). In some embodiments, R^B1is-N (R)^b)₂(e.g., -NH)₂-NH (substituted or unsubstituted C_1-6Alkyl) (e.g., -NHMe) or-N (substituted or unsubstituted C)_1-6Alkyl) - (substituted or unsubstituted C_1-6Alkyl) (e.g., -NMe)₂)). In some embodiments, R^B1is-CN. In some embodiments, R^B1is-SCN or-NO₂. In some embodiments, R^B1is-C (═ NR)^b)R^b、–C(＝NR^b)OR^bor-C (═ NR)^b)N(R^b)₂. In some embodiments, R^B1is-C (═ O) R^b(e.g., -C (═ O) (substituted or unsubstituted alkyl) or-C (═ O) (substituted or unsubstituted phenyl)). In some embodiments, R^B1is-C (═ O) OR^b(e.g., -C (═ O) OH, -C (═ O) O (substituted or unsubstituted alkyl) (e.g., -C (═ O) OMe) or-C (═ O) O (substituted or unsubstituted phenyl)). In some embodiments, R ^B1is-C (═ O) N (R)^b)₂(e.g., -C (═ O) NH₂C (═ O) NH (substituted or unsubstituted alkyl), -C (═ O) NH (substituted or unsubstituted phenyl), -C (═ O) N (substituted or unsubstituted alkyl) - (substituted or unsubstituted alkyl), or-C (═ O) N (substituted or unsubstituted phenyl) - (substituted or unsubstituted alkyl)). In some embodiments, R^B1is-NR^bC(＝O)R^b(e.g., -NHC (═ O) Me). In some embodiments, R^B1is-NR^bC(＝O)OR^bor-NR^bC(＝O)N(R^b)₂. In some embodiments, R^B1is-OC (═ O) R^b、–OC(＝O)OR^bor-OC (═ O) N (R)^b)₂. In some embodiments, R^B1Is substituted OR unsubstituted alkyl, -OR^b、–N(R^b)₂、–C(＝O)OR^bor-NR^bC(＝O)R^b. In some embodiments, R^B1Is unsubstituted C_1-6Alkyl, -OMe, -NH₂、–N(Me)₂-C (═ O) OH, -C (═ O) OMe or-NHC (═ O) Me. In some embodiments, R^B1Is composed ofIn some embodiments, R^B1Is a label (e.g., a biotin derivative, a radiolabel, or a fluorophore).

The formula (II) may comprise one or more substituents R^b. When formula (II) includes one or more R^bWhen there are two R^bMay be the same as or different from each other. In some embodiments, at least one R is^bIs H. In some embodiments, each R is^bIs H. In some embodiments, at least one R is ^bIs substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts), an oxygen protecting group when attached to an oxygen atom (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl or benzoyl) or a sulfur protecting group when attached to a sulfur atom (e.g., acetamidomethyl, t-Bu, 3-nitro-2-pyridylthio, 2-pyridylthio or triphenylmethyl), or both R.^bJoined to form a substituted or unsubstituted heterocyclic ring, or a substituted or unsubstituted heteroaromatic ring.

Formula (II) includes a substituent R at the 1-position of the indole ring^B2. In some embodiments, R^B2Is H. In some embodiments, R^B2Is a substituted or unsubstituted acyl group. In some embodiments, R^B2is-C (═ O) R^bOptionally wherein R is^bIs substituted or unsubstituted C_1-6Alkyl (e.g. Me) or substituted or unsubstituted C _2-6An alkenyl group. In some embodiments, R^B2is-C (═ O) R^bWherein R is^bIs a substituted or unsubstituted vinyl group. In some embodiments, R^B2is-C (═ O) CH ═ CH₂. In some embodiments, R^B2is-C (═ O) OR^bOptionally wherein R is^bIs H, substituted or unsubstituted C_1-6Alkyl (e.g., Me) or an oxygen protecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl). In some embodiments, R^B2is-C (═ O) N (R)^b)₂Optionally wherein each R is^bIndependently is H, substituted or unsubstituted C_1-6Alkyl (e.g., Me) or nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts). In some embodiments, R^B2Is a substituted or unsubstituted alkyl group (e.g., substituted or unsubstituted C)_1-6Alkyl groups). In some embodiments, R^B2Is Me. In some embodiments, R^B2Is Et. In some embodiments, R^B2Is n-Pr. In some embodiments, R^B2Is i-Pr. In some embodiments, R^B2Is Bu (e.g., n-Bu, i-Bu, sec-Bu or t-Bu). In some embodiments, R^B2Is an unsubstituted pentyl group (e.g., unsubstituted n-pentyl group, unsubstituted t-pentyl group, unsubstituted neopentyl group, unsubstituted isopentyl group, unsubstituted sec-pentyl group, or unsubstituted 3-pentyl group). In some embodiments, R ^B2sec-Bu, t-Bu or unsubstituted 3-pentyl. In some embodiments, R^B2is-CF₃Bn, perfluoroethyl, perfluoropropyl, perfluorobutyl or perfluoropentyl. In some embodiments, R^B2is-CH₂C(＝O)–NH–N＝C(R^b)₂. In some embodiments, R^B2Is a substituted or unsubstituted carbocyclyl (e.g., substituted or unsubstituted with 0, 1 or 2 double bonds in the carbocyclic ring system)3-to 7-membered monocyclic carbocyclic groups of (a). In some embodiments, R^B2Is a substituted or unsubstituted cyclopropyl. In some embodiments, R^B2Is unsubstituted cyclopropyl. In some embodiments, R^B2Is a substituted or unsubstituted cyclobutyl group. In some embodiments, R^B2Is a substituted or unsubstituted cyclopentyl group. In some embodiments, R^B2Is unsubstituted cyclopropyl, unsubstituted cyclobutyl or unsubstituted cyclopentyl. In some embodiments, R^B2Is a substituted or unsubstituted heterocyclyl group (e.g., a substituted or unsubstituted 3-to 7-membered monocyclic heterocyclyl group containing 0, 1, or 2 double bonds in the heterocyclic ring system, wherein 1, 2, or 3 atoms in the heterocyclic ring system are independently nitrogen, oxygen, or sulfur). In some embodiments, R^B2Is a substituted or unsubstituted tetrahydropyranyl group. In some embodiments, R ^B2Is of the formula:in some embodiments, R^B2Is of the formula:in some embodiments, R^B2Is a substituted or unsubstituted oxetanyl group, a substituted or unsubstituted tetrahydrofuranyl group, a substituted or unsubstituted pyrrolidinyl group, a substituted or unsubstituted piperidinyl group, a substituted or unsubstituted morpholinyl group, or a substituted or unsubstituted piperazinyl group. In some embodiments, R^B2Is of the formula: wherein each R^bIndependently is unsubstituted C_1-6Alkyl (e.g., Me)). In some embodiments, R^B2Is a nitrogen protecting group (e.g. Bn, Boc)Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R^B2Is Boc. In some embodiments, R^B2Is a warhead. In some embodiments, R^B2Is a label (e.g., a biotin derivative, a radiolabel, or a fluorophore).

Formula (II) includes a substituent R at the 3-position of the indole ring^B3. In some embodiments, R^B3Is H. In some embodiments, R^B3Is halogen (e.g., F, Cl, Br, or I). In some embodiments, R^B3Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^B3Is Me. In some embodiments, R^B3is-CF₃Bn, Et, perfluoroethyl, Pr, perfluoropropyl, Bu or perfluorobutyl. In some embodiments, R ^B3is-OR^bOptionally wherein R is^bIs H, substituted or unsubstituted C_1-6Alkyl (e.g., Me), substituted or unsubstituted acyl, or oxygen protecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl). In some embodiments, R^B3is-OC (═ O) R^bOptionally wherein R is^bIs H, substituted or unsubstituted C_1-6Alkyl (e.g. Me) or substituted or unsubstituted C_2-6Alkenyl (e.g., substituted or unsubstituted vinyl). In some embodiments, R^B3is-OC (═ O) CH ═ CH₂. In some embodiments, R^B3is-N (R)^b)₂Optionally wherein each R is^bIndependently is H, substituted or unsubstituted C_1-6Alkyl (e.g., Me), substituted or unsubstituted acyl, or nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts). In some embodiments, R^B3is-N (R)^b)C(＝O)R^bOptionally wherein each R is^bIndependently is H, substituted or unsubstituted C_1-6Alkyl (e.g. Me) or substituted or unsubstituted C_2-6Alkenyl (e.g., substituted or unsubstituted vinyl). In some casesIn embodiments, R^B3is-NHC (═ O) CH ═ CH₂. In some embodiments, R ^B3Is a warhead.

Formula (II) includes a substituent R on the nitrogen atom^B4. In some embodiments, R^B4Is H. In some embodiments, R^B4Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^B4Is Me. In some embodiments, R^B4is-CF₃Bn, Et, perfluoroethyl, Pr, perfluoropropyl, Bu or perfluorobutyl. In some embodiments, R^B4Is a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts).

Formula (II) includes substituent R^B5. In some embodiments, R^B5Is of formula (ii-1): (for example,wherein R is^B7Et, Pr or Bu). In some embodiments, R^B5Is of the formula:in some embodiments, R^B5Is of the formula:in some embodiments, R^B6Is H. In some embodiments, R^B6Is halogen (e.g., F, Cl, Br, or I). In some embodiments, R^B6Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^B6Is Me. In some embodiments, R^B6Being substituted methyl (e.g., -CF)₃Or Bn). In some embodiments, R^B6Is Et,Substituted ethyl (e.g., perfluoroethyl), Pr, substituted propyl (e.g., perfluoropropyl), Bu, or substituted butyl (e.g., perfluorobutyl). In some embodiments, R ^B6is-OR^b(e.g., -OH or-O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe)). In some embodiments, R^B6is-N (R)^b)₂Optionally wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R^B6is-NH₂-NHMe or-N (Me)₂. In some embodiments, R^B7Is H. In some embodiments, R^B7Is halogen (e.g., F, Cl, Br, or I). In some embodiments, R^B7Is substituted or unsubstituted C_2-6An alkyl group. In some embodiments, R^B7Is Et. In some embodiments, R^B7Is a substituted ethyl group (e.g., perfluoroethyl). In some embodiments, R^B7Is n-Pr. In some embodiments, R^B7Is i-Pr. In some embodiments, R^B7Is a substituted propyl group (e.g., perfluoropropyl group). In some embodiments, R^B7Bu or unsubstituted pentyl. In some embodiments, R^B7Is a substituted butyl group (e.g., perfluorobutyl group) or a substituted pentyl group (e.g., perfluoropentyl group). In some embodiments, R^B7Is a substituted or unsubstituted 3-to 7-membered monocyclic carbocyclic group containing 0, 1 or 2 double bonds in the carbocyclic ring system. In some embodiments, R ^B7Is a substituted or unsubstituted cyclopropyl, substituted or unsubstituted cyclobutyl, or substituted or unsubstituted cyclopentyl. In some embodiments, R^B7is-OR^b(e.g., -OH or-O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe)). In some embodiments, R^B7is-N (R)^b)₂Optionally wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g. Bn, Boc, Cbz, Fmoc, trifluoroacetylMesityl, triphenylmethyl, acetyl or Ts). In some embodiments, R^B7is-NH₂、–NHMe、–NHEt、–N(Me)₂or-N (Et)₂. In some embodiments, R^B7Is a substituted or unsubstituted cyclopropyl or-N (R)^b)₂Wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts).

In some embodiments, R^B5Is of the formula:(for example,wherein R is^B9Me, Et, Pr or Bu). A moiety of the formula:also included are tautomeric forms thereofIn some embodiments, R^B5Is of the formula:in some embodiments, R^B5Is of the formula:in some embodiments, R^B5Is of the formula:in some embodiments, R^B8Is H. In some embodiments, R ^B8Is halogen (e.g., F, Cl, Br, or I). In some embodiments, R^B8Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^B8Is Me. In some casesIn embodiments, R^B8Being substituted methyl (e.g., -CF)₃Or Bn). In some embodiments, R^B8Is Et, substituted ethyl (e.g., perfluoroethyl), Pr, substituted propyl (e.g., perfluoropropyl), Bu, or substituted butyl (e.g., perfluorobutyl). In some embodiments, R^B8is-OR^b(e.g., -OH or-O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe)). In some embodiments, R^B8is-N (R)^b)₂Optionally wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R^B8is-NH₂-NHMe or-N (Me)₂. In some embodiments, R^B9Is H. In some embodiments, R^B9Is halogen (e.g., F, Cl, Br, or I). In some embodiments, R^B9Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^B9Is Me. In some embodiments, R^B9Being substituted methyl (e.g., -CF) ₃Or Bn). In some embodiments, R^B9Is Et. In some embodiments, R^B9Is a substituted ethyl group (e.g., perfluoroethyl). In some embodiments, R^B9Is n-Pr. In some embodiments, R^B9Is i-Pr. In some embodiments, R^B9Is a substituted propyl group (e.g., perfluoropropyl group). In some embodiments, R^B9Bu or unsubstituted pentyl. In some embodiments, R^B9Is a substituted butyl group (e.g., perfluorobutyl group) or a substituted pentyl group (e.g., perfluoropentyl group). In some embodiments, R^B9Is a substituted or unsubstituted 3-to 7-membered monocyclic carbocyclic group containing 0, 1 or 2 double bonds in the carbocyclic ring system. In some embodiments, R^B9Is a substituted or unsubstituted cyclopropyl, substituted or unsubstituted cyclobutyl, or substituted or unsubstituted cyclopentyl. In some embodiments, R^B9is-OR^b(e.g., -OH or-O (substituted or unsubstituted)Substituted C_1-6Alkyl) (e.g., -OMe)). In some embodiments, R^B9is-N (R)^b)₂Optionally wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R ^B9is-NH₂、–NHMe、–NHEt、–N(Me)₂or-N (Et)₂. In some embodiments, R^B9Is a substituted OR unsubstituted cyclopropyl, -OR^bor-N (R)^b)₂Wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl, an oxygen protecting group when attached to an oxygen atom, or a nitrogen protecting group when attached to a nitrogen atom.

In some embodiments, R^B5Is of the formula:in some embodiments, R^B5Is of the formula:(for example,). In some embodiments, R^B5Is of the formula:(for example,). In some embodiments, R^B5Is of the formula:(for example,). In some embodiments, R^B5Is of the formula: in some embodiments, R^B5Is of the formula:in some embodiments, R^B10is-OR^b(e.g., -OH). In some embodiments, R^B10is-N (R)^b)₂. In some embodiments, R^B10is-NH₂. In some embodiments, R^B10is-NHR^bWherein R is^bIs substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R^B10Is a warhead. In some embodiments, R^B10is-NHC (═ O) R^bOptionally wherein R is^bIs a substituted or unsubstituted vinyl group. In some embodiments, R ^B10is-NHC (═ O) CH ═ CH₂. When formula (II) includes two or more R^B11When any two R are present^B11May be the same as or different from each other. In some embodiments, at least one R is^B11Is halogen. In some embodiments, at least one R is^B11Is Br. In some embodiments, at least one R is^B11F, Cl or I. In some embodiments, at least one R is^B11Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, at least one R is^B11Is Me. In some embodiments, at least one R is^B11Being substituted methyl (e.g., -CF)₃Or Bn). In some embodiments, at least one R is^B11Is Et. In some embodiments, at least one R is^B11Is a substituted ethyl group (e.g. allFluoroethyl). In some embodiments, at least one R is^B11Is n-Pr. In some embodiments, at least one R is^B11Is i-Pr. In some embodiments, at least one R is^B11Is a substituted propyl group (e.g., perfluoropropyl group). In some embodiments, at least one R is^B11Me, Et or n-Pr. In some embodiments, at least one R is^B11Bu or unsubstituted pentyl. In some embodiments, at least one R is^B11Is a substituted butyl group (e.g., perfluorobutyl group) or a substituted pentyl group (e.g., perfluoropentyl group). In some embodiments, at least one R is ^B11Is a substituted or unsubstituted 3-to 7-membered monocyclic carbocyclic group containing 0, 1 or 2 double bonds in the carbocyclic ring system. In some embodiments, at least one R is^B11Is a substituted or unsubstituted cyclopropyl, a substituted or unsubstituted cyclobutyl, or a substituted or unsubstituted cyclopentyl. In some embodiments, at least one R is^B11is-OR^b(e.g., -OH or-O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe)). In some embodiments, at least one R is^B1is-N (R)^b)₂Optionally wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, at least one R is^B11is-NH₂、–NHMe、–NHEt、–N(Me)₂or-N (Et)₂. In some embodiments, at least one R is^B11Is substituted or unsubstituted C_1-6Alkyl, substituted OR unsubstituted cyclopropyl, -OR^bor-N (R)^b)₂Wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl, an oxygen protecting group when attached to an oxygen atom, or a nitrogen protecting group when attached to a nitrogen atom. In some embodiments, u is 0. In some embodiments, u is 1. In some embodiments, u is 2. In some embodiments, u is 3. In some embodiments, u is 4.

In some embodiments, R^B5Is as followsFormula (II):in some embodiments, R^B5Is of the formula:(for example,such as). In some embodiments, R^B12Is H. In some embodiments, R^B12Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^B12Is Me. In some embodiments, R^B12is-CF₃Bn, Et, perfluoroethyl, Pr, perfluoropropyl, Bu or perfluorobutyl. In some embodiments, R^B12Is a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R^B12Is a warhead. When formula (II) includes two or more R^B13When there are two arbitrary R^B13May be the same as or different from each other. In some embodiments, at least one R is^B13Is halogen (e.g., F, Cl, Br, or I). In some embodiments, at least one R is^B13Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, at least one R is^B13Is Me. In some embodiments, at least one R is^B13Being substituted methyl (e.g., -CF)₃Or Bn). In some embodiments, at least one R is^B13Is Et. In some embodiments, at least one R is^B13Is a substituted ethyl group (e.g., perfluoroethyl). In some embodiments, at least one R is ^B13Is n-Pr. In some embodiments, at least one R is^B13Is i-Pr. In some embodiments, at least one R is^B13Is a substituted propyl group (e.g., perfluoropropyl group). In some embodiments, at least one R is^B13Me, Et or n-Pr. In some embodiments, at least one R is^B13Bu or unsubstituted pentyl. In some embodiments, at least one R is^B13Is a substituted butyl group (e.g., perfluorobutyl group) or a substituted pentyl group (e.g., perfluoropentyl group). In some embodiments, at least one R is^B13Is a substituted or unsubstituted 3-to 7-membered monocyclic carbocyclic group containing 0, 1 or 2 double bonds in the carbocyclic ring system. In some embodiments, at least one R is^B13Is a substituted or unsubstituted cyclopropyl, substituted or unsubstituted cyclobutyl, or substituted or unsubstituted cyclopentyl. In some embodiments, at least one R is^B13is-OR^b(e.g., -OH or-O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe)). In some embodiments, at least one R is^B13is-N (R)^b)₂Optionally wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, at least one R is ^B13is-NH₂、–NHMe、–NHEt、–N(Me)₂or-N (Et)₂. In some embodiments, at least one R is^B13Is halogen, substituted OR unsubstituted cyclopropyl, -OR^bor-N (R)^b)₂Wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl, an oxygen protecting group when attached to an oxygen atom, or a nitrogen protecting group when attached to a nitrogen atom. In some embodiments, v is 0. In some embodiments, v is 1. In some embodiments, v is 2. In some embodiments, v is 3, 4, 5, 6, 7, or 8. In some embodiments, v is 9.

Formula (II) includes substituent R^B5. In some embodiments, R^B5Is of the formula:(for example,optionally wherein R is^B16Et, Pr or Bu). In some embodiments, R^B5Is of the formula:in some embodiments, R^B5Is of the formula:in some embodiments, R^B14Is H. In some embodiments, R^B14Is halogen (e.g., F, Cl, Br, or I). In some embodiments, R^B14Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^B14Is Me. In some embodiments, R^B14Being substituted methyl (e.g., -CF)₃Or Bn). In some embodiments, R^B14Is Et, substituted ethyl (e.g., perfluoroethyl), Pr, substituted propyl (e.g., perfluoropropyl), Bu, or substituted butyl (e.g., perfluorobutyl). In some embodiments, R ^B14is-OR^b(e.g., -OH or-O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe)). In some embodiments, R^B14is-N (R)^b)₂Optionally wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R^B14is-NH₂-NHMe or-N (Me)₂. In some embodiments, R^B15Is H. In some embodiments, R^B15Is halogen (e.g., F, Cl, Br, or I). In some embodiments, R^B15Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^B15Is Me. In some embodiments, R^B15Being substituted methyl (e.g., -CF)₃Or Bn). In some embodiments, R^B15Is Et, substituted ethyl (e.g., perfluoroethyl), Pr, substituted propyl (e.g., perfluoropropyl), Bu, or substituted butyl (e.g., perfluoropropyl)Perfluorobutyl). In some embodiments, R^B15is-OR^b(e.g., -OH or-O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe)). In some embodiments, R^B15is-N (R)^b)₂Optionally wherein each R is^bIndependently hydrogen, substituted or unsubstituted C _1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R^B15is-NH₂-NHMe or-N (Me)₂. In some embodiments, R^B16Is H. In some embodiments, R^B16Is halogen (e.g., F, Cl, Br, or I). In some embodiments, R^B16Is substituted or unsubstituted C_2-6An alkyl group. In some embodiments, R^B16Is Et. In some embodiments, R^B16Is a substituted ethyl group (e.g., perfluoroethyl). In some embodiments, R^B16Is n-Pr. In some embodiments, R^B16Is i-Pr. In some embodiments, R^B16Is a substituted propyl group (e.g., perfluoropropyl group). In some embodiments, R^B16Bu or unsubstituted pentyl. In some embodiments, R^B16Is a substituted butyl group (e.g., perfluorobutyl group) or a substituted pentyl group (e.g., perfluoropentyl group). In some embodiments, R^B16Is a substituted or unsubstituted 3-to 7-membered monocyclic carbocyclic group containing 0, 1 or 2 double bonds in the carbocyclic ring system. In some embodiments, R^B16Is a substituted or unsubstituted cyclopropyl, substituted or unsubstituted cyclobutyl, or substituted or unsubstituted cyclopentyl. In some embodiments, R ^B16is-OR^b(e.g., -OH or-O (substituted or unsubstituted C)_1-6Alkyl) (e.g., -OMe)). In some embodiments, R^B16is-N (R)^b)₂Optionally wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R^B16is-NH₂、–NHMe、–NHEt、–N(Me)₂or-N (Et)₂. In some embodiments, R^B16Is a substituted or unsubstituted cyclopropyl or-N (R)^b)₂Wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts). In some embodiments, R^B17Is H. In some embodiments, R^B17Is a substituted or unsubstituted acyl group. In some embodiments, R^B17is-C (═ O) R^bOptionally wherein R is^bIs substituted or unsubstituted C_1-6Alkyl (e.g. Me) or substituted or unsubstituted C_2-6An alkenyl group. In some embodiments, R^B17Is a warhead. In some embodiments, R^B17is-C (═ O) R^bWherein R is^bIs a substituted or unsubstituted vinyl group. In some embodiments, R^B17is-C (═ O) CH ═ CH₂. In some embodiments, R ^B17is-C (═ O) OR^bOptionally wherein R is^bIs H, substituted or unsubstituted C_1-6Alkyl (e.g., Me) or an oxygen protecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl). In some embodiments, R^B17is-C (═ O) N (R)^b)₂Optionally wherein each R is^bIndependently is H, substituted or unsubstituted C_1-6Alkyl (e.g., Me) or nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts). In some embodiments, R^B17Is substituted or unsubstituted C_1-6An alkyl group. In some embodiments, R^B17Is Me. In some embodiments, R^B17is-CF₃Bn, Et, perfluoroethyl, Pr, perfluoropropyl, Bu or perfluorobutyl. In some embodiments, R^B17Is a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl or Ts).

In some embodiments, the compound of formula (II) is a compound of the formula:

In some embodiments, the compound of formula (II) is a compound of the formula:

in some embodiments, the compound of formula (II) is a compound of the formula:

in some embodiments, the compound of formula (II) is a compound of the formula:

in some embodiments, the compound of formula (II) is a compound of the formula:

in some embodiments, the compound of formula (II) is a compound of the formula:

Wherein R is^B14Is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or a nitrogen protecting group.

In some embodiments, the compound of formula (II) is a compound of the formula:

in some embodiments, the compound of formula (II) is a compound of the formula:

in some embodiments, the compound of formula (II) is a compound of the formula:

in some embodiments, the compound of formula (II) is a compound of the formula:

In some embodiments, the compound of formula (II) is a compound of the formula:

wherein R is^B2Is a nitrogen protecting group (e.g., Boc).

In some embodiments, the compound of formula (II) is a compound of the formula:

in some embodiments, the compound of formula (II) is a compound of the formula:

optionally wherein each R is^B2Is i-Pr.

In some embodiments, the compound of formula (II) is a compound of the formula:

wherein each w is independently an integer from 3 to 11, inclusive.

In some embodiments, the compound of formula (II) is a compound of the formula:

in some embodiments, the compound of formula (II) is a compound of the formula:

or an acceptable salt, solvate, hydrate, polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled derivative, or prodrug thereof.

In some embodiments, the compound of formula (II) is a compound of the formula:

in some embodiments, the compound of formula (II) is a compound of the formula:

In some embodiments, the compound of formula (II) is a compound of the formula:

in some embodiments, the compound of formula (II) is a compound of the formula:

in some embodiments, when R^B2Is i-Pr, R^B3Is hydrogen and R^B5Is of formula (ii-1), then R^B1Is not Me,-OMe or-NH (═ O) Me. At one endIn some embodiments, when R^B2Is unsubstituted C_3-5Alkyl radical, R^B3Is Me or halogen and R^B5Is of formula (ii-1), then R^B1Is not Me,–OMe、–NH₂、–N(Me)₂-C (═ O) OH, -C (═ O) OMe or-NH (═ O) Me. In some embodiments, when R^B2Is i-Pr, R^B3Is hydrogen and R^B5Is of formula (ii-1), then R^B1Not being unsubstituted C_1-6Alkyl, -OR^b、–NH(＝O)R^bOr unsubstituted or substituted by one unsubstituted C_1-6An alkyl-substituted saturated 6-membered monocyclic heterocyclyl, wherein two atoms in the heterocyclic ring system are independently oxygen or nitrogen. In some embodiments, when R ^B2Is unsubstituted C_3-5Alkyl radical, R^B3Is Me or halogen, and R^B5Is of formula (ii-1), then R^B1Not being unsubstituted C_1-6Alkyl, -OR^b、–N(R^b)₂、–C(＝O)OR^b、–NH(＝O)R^bOr unsubstituted or substituted by one unsubstituted C_1-6An alkyl-substituted saturated 6-membered monocyclic heterocyclyl, wherein two atoms in the heterocyclic ring system are independently oxygen or nitrogen; wherein each R^bIndependently is H or unsubstituted C_1-6An alkyl group. In some embodiments, when R^B5Is of formula (ii-1), then R^B2Not being unsubstituted C_3-5An alkyl group. In some embodiments, when R^B3Is hydrogen and R^B5Is of formula (ii-1), then R^B2Is not i-Pr. In some embodiments, the compound of formula (II) is not a compound of the formula:

exemplary compounds of formula (II) include, but are not limited to:

and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, and prodrugs thereof.

Exemplary compounds of formula (II) further include, but are not limited to:

and pharmaceutically acceptable salts, solvates, hydrates, polymorphs, co-crystals, tautomers, stereoisomers, isotopically labeled derivatives, and prodrugs thereof.

In some embodiments, the compound of formula (II) is a compound of the formula:

in some embodiments, the compound of formula (II) is a compound of the formula:

in some embodiments, the compounds of formula (I) or (II) are reversible EZH2 inhibitors. In some embodiments, the compound of formula (I) or (II) excluding the warhead is a reversible EZH2 inhibitor. In some embodiments, the compound of formula (I) or (II), excluding the warhead, does not form a covalent bond with EZH 2.

In some embodiments, the compounds of formula (I) or (II) are irreversible EZH2 inhibitors. In some embodiments, the compound of formula (I) or (II) comprising one or more warheads is an irreversible EZH2 inhibitor. In some embodiments, a compound of formula (I) or (II) comprising one or more warheads forms one or more covalent bonds with EZH2 (e.g., the cysteine residue of EZH 2). In some embodiments, a compound of formula (I) or (II) comprising one or more warheads forms one or more covalent bonds with EZH2 (e.g., the cysteine residue of EZH 2) via a reaction (e.g., michael addition) of EZH2 with at least one of the warheads.

In some embodiments, the irreversible EZH2 inhibitor is a compound of the formula:

in some embodiments, the compound of formula (II) is a compound of the formula:

pharmaceutical compositions, kits and administrations

The present application also provides pharmaceutical compositions comprising a compound described herein and optionally a pharmaceutically acceptable excipient.

In some embodiments, the compounds described herein are provided in the pharmaceutical composition in an effective amount. In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, the effective amount is a prophylactically effective amount. In some embodiments, a therapeutically effective amount is an amount effective to inhibit aberrant activity of HMT (e.g., EZH1, EZH2, DOT 1). In some embodiments, a therapeutically effective amount is an amount effective to treat a disease, e.g., a disease associated with aberrant activity of HMT (e.g., a proliferative disease). In some embodiments, a therapeutically effective amount is an amount effective to inhibit abnormal activity of HMT (e.g., EZH1, EZH2, DOT1) and treat a disease (e.g., a disease associated with abnormal activity of HMT (e.g., a proliferative disease)). In some embodiments, a therapeutically effective amount is an amount effective to induce apoptosis in a cell. In some embodiments, a prophylactically effective amount is an amount effective to inhibit aberrant activity of HMT (e.g., EZH1, EZH2, DOT 1). In some embodiments, a prophylactically effective amount is an amount effective to prevent or reduce a disease (e.g., a disease associated with aberrant activity of HMT (e.g., a proliferative disease)) in a subject in need thereof. In some embodiments, a prophylactically effective amount is an amount effective to inhibit abnormal activity of HMT (e.g., EZH1, EZH2, DOT1) and an amount effective to prevent or reduce a disease in a subject in need thereof (e.g., a disease associated with abnormal activity of HMT (e.g., a proliferative disease)). In some embodiments, a prophylactically effective amount is an amount effective to induce apoptosis in a cell.

In some embodiments, an effective amount is an amount effective to inhibit the activity of HMT (e.g., EZH1, EZH2, DOT1) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98%. In some embodiments, the effective amount is an amount effective to inhibit the activity of HMT (e.g., EZH1, EZH2, DOT1) by no more than 10%, no more than 20%, no more than 30%, no more than 40%, no more than 50%, no more than 60%, no more than 70%, no more than 80%, no more than 90%, no more than 95%, or no more than 98%.

In some embodiments, the subject is an animal. The animal may be of any sex and may be at any stage of development. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a domestic animal, such as a dog, cat, cow, pig, horse, sheep, or goat. In some embodiments, the subject is a companion animal, such as a dog or cat. In some embodiments, the subject is a livestock animal, such as a cow, pig, horse, sheep, or goat. In some embodiments, the subject is a zoo animal. In other embodiments, the subject is a research animal, such as a rodent (e.g., mouse, rat), dog, pig, or non-human primate. In some embodiments, the animal is a genetically engineered animal. In some embodiments, the animal is a transgenic animal (e.g., a transgenic mouse and a transgenic pig). In some embodiments, the subject is a fish or a reptile.

In some embodiments, the cell is present in vitro. In some embodiments, the cell is present in vivo.

The pharmaceutical compositions described herein may be prepared by any method known in the art of pharmacology. Generally, such a preparation method comprises the steps of: the compounds described herein (i.e., the "active ingredient") are combined with a carrier or excipient, and/or one or more other adjuvants, and the product is then shaped and/or packaged, if necessary and/or desired, into single-or multi-dose units as desired.

The pharmaceutical compositions may be prepared, packaged and/or sold in bulk in a single unit dose and/or in multiple single unit doses. A "unit dose" is a discrete amount (discrete amount) of a pharmaceutical composition comprising a predetermined amount of active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient administered to the subject and/or a simple fraction of this dose, e.g., one half or one third of this dose

The relative amounts of the active ingredient, pharmaceutically acceptable excipient and/or any additional ingredients in the pharmaceutical compositions of the invention will vary depending on the identity, size and/or condition of the subject being treated and further depending on the route of administration of the composition. The composition may comprise from 0.1% to 100% (w/w) of the active ingredient.

Pharmaceutically acceptable excipients used in preparing the provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surfactants and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents and/or oils. Excipients such as cocoa butter and suppository waxes, colorants, coatings, sweeteners, flavoring agents and flavoring agents may also be present in the composition.

Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, dicalcium phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, sugar powder, and mixtures thereof.

Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly (vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (cross-linked carboxymethyl cellulose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

Exemplary surfactants and/or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondlux, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes, and lecithin), bentonites (e.g., bentonite (aluminum silicate) and Veegum (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, glyceryl monostearate (triacetin monostearate), ethylene glycol distearate, glyceryl monostearate and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene (polyacrylic acid), acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, carboxymethylcellulose, sodium alginate, pectin, and sodium alginate), long chain amino acid derivatives, high molecular weight alcohols (e.g., sodium alginate, sodium, and sodium alginate, Hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate)Polyoxyethylene sorbitanPolyoxyethylene sorbitan monooleateSorbitan monopalmitate Sorbitan monostearateSorbitan tristearateGlyceryl monooleate, sorbitan monooleatePolyoxyethylene esters (e.g., polyoxyethylene monostearate)Polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxyl stearate and) Sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g.,) Polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether)) Poly (vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, and sodium lauryl sulfate,F-68, poloxamer P-188, cetrimide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium and/or mixtures thereof.

Exemplary binders include starches (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, and the like), natural and synthetic gums (e.g., acacia, sodium alginate, Irish moss extract, pangami, and the like)Wall gum (panwar Rum), gum ghatti (ghatti gum), viscose of the isabgol shell (mucous of isabgol husks), carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, poly (vinyl-pyrrolidone), magnesium aluminium silicate And larch arabinogalactan (larch arabinogalactan), alginate, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylate, wax, water, ethanol, and/or mixtures thereof.

Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, antiprotozoal preservatives, ethanol preservatives, acidic preservatives, and other preservatives. In some embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, thioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium ethylenediaminetetraacetate, disodium ethylenediaminetetraacetate, trisodium ethylenediaminetetraacetate, disodium calcium ethylenediaminetetraacetate, dipotassium ethylenediaminetetraacetate, etc.), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethanol.

Exemplary acidic preservatives include vitamin a, vitamin C, vitamin E, beta carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopherol acetate, dexemethylamine mesylate, cetrimide, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), ethylenediamine, Sodium Lauryl Sulfate (SLS), Sodium Lauryl Ether Sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Sodium Lauryl Ether Sulfate (SLES), sodium metabisulfite, sodium lauryl ether Sulfate (SLE), sodium metabisulfite, sodium lauryl ether sulfate (SLS), sodium lauryl ether Sulfate (SLE), sodium lauryl ether sulfate (SLS), sodium metabisulfite, sodium salt,Plus、methyl p-hydroxybenzoate, 115、II、And

exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium glucoheptonate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propionic acid, calcium pentanoate, valeric acid, calcium hydrogen phosphate, phosphoric acid, calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, ringer's solution, ethanol, and mixtures thereof.

Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

Exemplary natural oils include almond oil (almond), apricot kernel oil (apricot kernel), avocado oil, babassu oil (babassu), bergamot oil, blackcurrant seed oil, borage oil, juniper tar (cade), chamomile oil (chamomile), canola oil, caraway oil, palm olein, castor oil, cinnamon oil, cocoa butter, coconut oil, cod liver oil, coffee oil, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, linseed oil, geranium oil, cucurbit oil, grape seed oil, hazelnut oil, hyssop oil (hyssop), isopropyl myristate oil, jojoba oil, kukukui nut oil (kukukui nut), lavandin oil (landavalin oil), lavender oil (lavender), lemon oil, litsea cubeba oil, macadamia oil (macadamia oil), mallow oil, meadowfoam oil, mink oil (marmordant oil), mink oil (macadamia oil), mink oil (laevigat oil), and the like, Olive oil, orange oil, deep sea fish oil (orange roughy), palm oil, palm kernel oil, peach kernel oil, peanut oil, poppy seed oil, pumpkin seed oil, rapeseed oil, rice bran oil, rosemary oil, safflower oil, sandalwood oil, camellia oil (sasquana), savory oil (savoury oil), sea buckthorn oil, sesame oil, shea butter, silicone oil, soybean oil, sunflower oil, tea tree oil, thistle oil, cedrela oil (tsubaki), vetiver oil, walnut oil, and wheat germ oil. Exemplary synthetic oils include, but are not limited to: butyl stearate oils, caprylic triglyceride oils, capric triglyceride oils, cyclomethicone oils, diethyl sebacate, dimethicone 360, isopropyl myristate oils, mineral oils, octyl dodecanol oils, oleyl alcohol, silicone oils and mixtures thereof.

Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils such as cottonseed, groundnut, corn, germ, olive, castor, and sesame oils, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the oral compositions can contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In some embodiments of parenteral administration, the conjugates of the invention are mixed with a solubilizing agent, e.g., a solubilizing agentAlcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the methods known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol. Acceptable vehicles and solvents that can be used are water, u.s.p. ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids (e.g., oleic acid) can be used in the preparation of injectables.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by the addition of sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the therapeutic effect of a drug, it is often desirable to slow the absorption of the drug by subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous material which is poorly water soluble. The rate of absorption of the drug depends on its rate of dissolution, which itself may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form may be achieved by dissolving or suspending the drug in an oily vehicle.

Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the conjugates of the invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which is solid at ordinary temperatures but liquid at body temperature and therefore melts in the rectum or vaginal cavity and releases the active ingredient.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In the solid dosage form, the active ingredient is admixed with at least one inert, pharmaceutically acceptable excipient or carrier (e.g., sodium citrate or dicalcium phosphate) and/or (a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, such as glycerol, (d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents, such as paraffin, (f) absorption promoters, such as quaternary ammonium compounds, (g) wetting agents, such as cetyl alcohol and glycerol monostearate, (h) absorbents, such as kaolin and bentonite, and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may contain buffering agents.

Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmacological arts. They may optionally comprise opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferably, in a specific part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulation compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

The active ingredient may be in microencapsulated form with one or more of the excipients mentioned above. The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings, controlled release coatings and other coatings well known in the art of pharmaceutical formulation. In such solid dosage forms, the active ingredient may be mixed with at least one inert diluent (e.g., sucrose, lactose or starch). Such dosage forms may contain, as is common practice, other substances in addition to inert diluents, e.g., tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may contain buffering agents. They may optionally comprise opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferably, in a specific part of the intestinal tract, optionally, in a delayed manner. Examples of encapsulating agents that may be used include polymeric substances and waxes.

Dosage forms for topical and/or transdermal administration of a compound of the invention may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. Typically, the active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier or excipient and/or any required preservatives and/or required buffers. In addition, the present invention may employ transdermal patches, which generally have the added advantage of controllably delivering the active ingredient to the body. Such dosage forms may be prepared, for example, by dissolving and/or dispersing the active ingredient in the appropriate medium. Alternatively or additionally, the rate may be controlled by providing a rate controlling membrane and/or dispersing the active ingredient in a polymer matrix and/or gel.

Suitable devices for delivering the intradermal pharmaceutical compositions described herein include short needle devices. Intradermal compositions may be administered using a device that limits the effective penetration length of the needle into the skin. Alternatively or additionally, conventional syringes may be used for the classical mantoux method of intradermal administration. A rapid injection device that delivers the liquid formulation to the dermis by means of a liquid jet syringe and/or by means of a needle that pierces the stratum corneum and produces a jet that reaches the dermis is suitable. A propellant powder (balistic powder)/particle delivery device that uses compressed gas to accelerate the passage of a compound in powder form through the outer layers of the skin to the dermis is suitable.

Formulations suitable for topical administration include, but are not limited to, liquid and/or semi-liquid formulations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions such as creams, ointments and/or pastes, and/or solutions and/or suspensions. Formulations for topical administration may, for example, contain from about 1% to about 10% (w/w) of the active ingredient, but the concentration of the active ingredient may be up to the solubility limit of the active ingredient in the solvent. Formulations for topical administration may also include one or more additional ingredients as described herein.

The pharmaceutical compositions of the present invention may be prepared, packaged and/or sold in formulations suitable for pulmonary administration through the oral cavity. Such formulations may include dry particles that contain the active ingredient and which have a diameter in the range of about 0.5 to about 7 nanometers, or about 1 to about 6 nanometers. Such compositions are conveniently administered in the form of a dry powder using a device comprising a dry powder reservoir into which a propellant stream can be directed to disperse the powder, and/or using a self-propelled solvent/powder dispensing container, e.g. a device comprising an active ingredient dissolved and/or suspended in a low boiling point propellant in a closed container. Such powders comprise particles wherein at least 98% by weight of the particles have a diameter greater than 0.5 nm and at least 95% by number of the particles have a diameter less than 7 nm. Alternatively, at least 95% by weight of the particles have a diameter greater than 1 nanometer and at least 90% by number of the particles have a diameter less than 6 nanometers. The dry powder composition may include a solid finely divided diluent (e.g. sugar) and is conveniently provided in unit dosage form.

Low boiling propellants typically include liquid propellants having a boiling point of less than 65 ° F at atmospheric pressure. Typically, the propellant may constitute 50-99.9% (w/w) of the composition and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as liquid non-ionic and/or solid anionic surfactants and/or solid diluents (which may be of the same order of particle size as the particles comprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonary delivery can provide the active ingredient in the form of droplets of solution and/or suspension. Such formulations may be prepared, packaged and/or sold as aqueous and/or diluted alcohol solutions and/or suspensions, optionally sterile, containing the active ingredient and conveniently administered using any spraying and/or atomising device. Such formulations may also contain one or more additional ingredients, including but not limited to: flavoring agents, such as sodium saccharin, volatile oils, buffers, surfactants, and/or preservatives, such as methyl hydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range of about 0.1 to about 200 nanometers.

The formulations for pulmonary delivery described herein are used for intranasal delivery of the pharmaceutical compositions described herein. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle size of about 0.2 to 500 microns. Such formulations are administered by rapid inhalation into the nasal passage from a powder container held near the nostril.

Formulations for nasal administration may, for example, comprise from about at least 0.1% (w/w) to at most 100% (w/w) of the active ingredient, and may comprise one or more of the additional ingredients described herein. The pharmaceutical compositions described herein may be prepared, packaged and/or sold in an orally administrable formulation. Such formulations may, for example, be in the form of tablets and/or lozenges prepared using conventional methods, and may comprise, for example, 0.1 to 20% (w/w) of the active ingredient, the balance comprising the orally-dissolvable and/or degradable composition, and optionally one or more additional ingredients as described herein. Alternatively, formulations for oral administration may comprise powders and/or aerosolized solutions and/or suspensions containing the active ingredient. Such powdered, aerosolized and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size ranging from about 0.1 to about 200 nanometers, and may further comprise one or more additional ingredients described herein.

The pharmaceutical compositions described herein may be prepared, packaged and/or sold in an ophthalmic delivery formulation. Such formulations may, for example, be in the form of eye drops comprising, for example, a solution and/or suspension of 0.1-1.0% (w/w) of the active ingredient in an aqueous or oily liquid carrier or vehicle. Such drops may also comprise buffers, salts and/or one or more other additional ingredients as described herein. Other useful formulations for ocular administration include those containing the active ingredient in microcrystalline and/or liposomal formulation. Ear drops and/or eye drops are also intended to be encompassed within the scope of the present disclosure.

Although the description of the pharmaceutical compositions provided herein primarily refers to pharmaceutical compositions suitable for administration to humans, those skilled in the art will appreciate that such compositions are generally suitable for administration to a variety of animals. It is well known to modify pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to various animals, and veterinarians of ordinary skill in the art can design and/or practice such modifications with ordinary experimentation.

The compounds provided herein are generally formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily dosage of the compositions of the present invention will be determined by a physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disease being treated and the severity of the disease; the activity of the particular active ingredient employed; the specific composition employed; the age, weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the particular active ingredient employed; the duration of the treatment; drugs used in combination or concomitantly with the specific active ingredient employed; and similar factors well known in the medical arts.

The compounds and compositions provided herein can be administered by any route, including enterally (e.g., oral), parenterally, intravenously, intramuscularly, intraarterially, intramedullary, intrathecally, subcutaneously, intraventricularly, transdermally, intradermally, rectally, intravaginally, intraperitoneally, topically (e.g., by powders, ointments, creams, and/or drops), mucosally, nasally, buccally; by intratracheal instillation, bronchial instillation and/or inhalation; and/or as an oral spray, nasal spray and/or aerosol. Routes of particular concern are oral administration, intravenous administration (e.g., systemic intravenous injection), regional administration via the blood and/or lymphatic supply, and/or direct administration to the affected site. Generally, the most suitable route of administration will depend on various factors, including the nature of the agent (e.g., stability in the gastrointestinal environment), and/or the condition of the subject (e.g., whether the subject can tolerate oral administration). In some embodiments, the compound or pharmaceutical composition described herein is suitable for topical administration to the eye of a subject.

The exact amount of a compound required to achieve an effective amount varies from subject to subject, depending, for example, on the species, age, and general condition of the subject, the severity of the side effect or disorder, the identity of the particular compound, the mode of administration, and the like. An effective amount may be included in a single dose (e.g., a single oral dose) or in multiple doses (e.g., multiple oral doses). In some embodiments, when multiple doses are administered to a subject or applied to a biological sample, tissue, or cell, any two of the multiple doses include different amounts or substantially the same amount of a compound described herein. In some embodiments, when multiple doses are administered to the subject or multiple doses are applied to the biological sample, tissue, or cells, the frequency of administering multiple doses to the subject or multiple doses to the biological sample, tissue, or cells is three doses a day, two doses a day, one dose every other day, one dose every three days, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In some embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to a biological sample, tissue, or cell is one dose per day. In some embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to a biological sample, tissue, or cell is two doses a day. In some embodiments, the frequency of administering multiple doses to a subject or applying multiple doses to a biological sample, tissue, or cell is three doses a day. In some embodiments, when administering multiple doses to a subject or applying multiple doses to a biological sample, tissue, or cell, the time span between the first and last doses of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject, tissue, or cell. In some embodiments, the time span between the first and last doses of the plurality of doses is three months, six months, or one year. In some embodiments, the time span between the first and last doses of the plurality is the lifetime of the subject, tissue, or cell. In some embodiments, a dose described herein (e.g., a single dose or multiple doses of any dose) independently includes between 0.1 μ g and 1 μ g, between 0.001mg and 0.01mg, between 0.01mg and 0.1mg, between 0.1mg and 1mg, between 1mg and 3mg, between 3mg and 10mg, between 10mg and 30mg, between 30mg and 100mg, between 100mg and 300mg, between 300mg and 1,000mg, or between 1g and 10g (inclusive) of a compound described herein. In some embodiments, the doses described herein independently include between 1mg and 3mg (inclusive) of a compound described herein. In some embodiments, the doses described herein independently include between 3mg and 10mg (inclusive) of a compound described herein. In some embodiments, the doses described herein independently include between 10mg and 30mg (inclusive) of a compound described herein. In some embodiments, the doses described herein independently include between 30mg and 100mg (inclusive) of the compound described herein.

The dosage ranges described herein provide guidance for administering the provided pharmaceutical compositions to adults. The amount administered to, for example, a child or adolescent can be determined by a physician or one skilled in the art and can be slightly less than or the same as the amount administered to an adult.

The compounds or compositions of the present invention may be administered in combination with one or more other drugs (e.g., therapeutically and/or prophylactically active drugs). The compounds or compositions can be administered in combination with other drugs to increase their activity (e.g., potency and/or effectiveness) in a subject, biological sample, tissue, or cell, to treat a disease in a subject in need thereof, to prevent a disease in a subject in need thereof, to inhibit the activity of HMT in a subject, biological sample, tissue, or cell), to increase bioavailability, to increase safety, to reduce drug resistance, to decrease and/or improve metabolism, to inhibit excretion, and/or to improve distribution. It will also be appreciated that the applied treatment may achieve the desired effect on the same condition, and/or may achieve different effects. In some embodiments, a pharmaceutical composition of the invention comprising a compound of the invention and an additional agent exhibits a synergistic effect as compared to a pharmaceutical composition comprising a compound of the invention or an additional agent, but not both.

The compound or composition may be administered simultaneously with, before or after one or more additional agents, which may be used, for example, as a combination therapy. The medicament includes a therapeutically active agent. The medicament also includes a prophylactically active agent. Agents include small organic molecules, such as pharmaceutical compounds (e.g., human or veterinary compounds approved by the U.S. food and drug administration as provided in the federal regulations Compilation (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucins, lipoproteins, synthetic polypeptides or proteins, small molecules linked proteins, glycoproteins, steroids, nucleic acids, DNA, RNA, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins and cells. In some embodiments, the additional agent is an agent for treating and/or preventing a disease (e.g., a proliferative disease, an inflammatory disease, an autoimmune disease, a genetic disease, a hematological disease, a neurological disease, a painful condition, a psychiatric disease, or a metabolic disorder). Each additional agent may be administered at a dose and/or on a schedule determined by the agent. The additional agents may also be administered together with each other and/or with the compounds or compositions described herein, in a single dose or separately in different doses. The particular combination employed in this regimen will take into account the compatibility of the compounds described herein with additional agents and/or the desired therapeutic and/or prophylactic effect that will be achieved. Generally, it is contemplated that the additional agents when used in combination are at levels not exceeding those when used individually. In some embodiments, the level used in the combination will be lower than they are used alone.

Such additional agents include, but are not limited to: antiproliferative agents, anticancer agents, antiangiogenic agents, anti-inflammatory agents, immunosuppressive agents, antibacterial agents, antiviral agents, cardiovascular agents, lipid lowering agents, antidiabetic agents, antiallergic agents, contraceptive agents, pain relieving agents, and combinations thereof. In some embodiments, the additional agent is an antiproliferative agent (e.g., an anticancer agent). In some embodiments, the additional agent is an anti-leukemic agent. In some embodiments, the additional agent is ABITREXATE (methotrexate), ADE, doxorubicin RDF (doxorubicin hcl), ambochloririn (chlorambucil), arron (nelarabine), ARZERRA (alfuzumab), BOSULIF (bosutinib), BUSULFEX (busulfan), CAMPATH (alemtuzumab), CERUBIDINE (daunorubicin hcl), CLAFEN (cyclophosphamide), CLOFAREX (clofarabine), CLOLAR (clofarabine), CVP, CYTOSAR-U (cytarabine), CYTOXAN (cyclophosphamide), ERWINE (Chrysanthemum morifolium asparaginase), FLUDARA (fludarabine phosphate), FOLEX (methotrexate), FOLEX PFS (methotrexate), GAZYVA (orbitumumab), GLVEVEC (imatinib mesylate), Hypoper-SIG (CVLUBRICAD), vincamine hydrochloride (FOURINA), vincamine hydrochloride (FOURALIA), vincalexin (FOURINA), vincamine sulfate, vincalexin (FOURETINA), vincalexin (FOLIBORATE), VICINAVICINAN (VICINAN), VICINAN, VICINAVICINAN (VICINATE (VICITAB), VICINAVITAB), VITAB, VICITAB, VITAB, VITAMINA (VITAB, VITAN, VITAB, VITAX PFS, VITAX (VITAB, VITAX (C (VITAB, VITAX (VITAB, VITAX (VITAB, VITAX (C (VITAB, VITAX (VITAB, VITAX (C, VITAB, VITAX (C, VITAB), VITAB, VITAX (VITAB, VITAX (C, VITAX (C, VITAX (VITA, METHOTREXATE LPF (METHOTREXATE), MEXATE-AQ (METHOTREXATE), mitoxantrone hydrochloride, mustagen (nitrogen mustard hydrochloride), myreran (busulfan), NEOSAR (cyclophosphamide), ONCASPAR (peimepirnase), PURINETHOL (mercaptopurine), PURIXAN (mercaptopurine), Rubidomycin (daunorubicin hydrochloride), SPRYCEL (dasatinib), homoharobo (homoharringtonine), TARABINE PFS (cytarabine), TASIGNA (nilotinib), trenda (bendamustine hydrochloride), TRISENOX (arsenic trioxide), VINCASAR PFS (vincristine sulfate), ZYDELIG (ilarris), or combinations thereof. In some embodiments, the additional agent is an anti-lymphoma drug. In some embodiments, the additional agent is ABITREXATE (methotrexate), ABVD, ABVE-PC, ADCETRIS (brentuximab vedotin), ADRIAMYCIN PFS (doxorubicin hydrochloride), ADRIAMYCIN RDF (doxorubicin hydrochloride), AMBOCHLORIN (chlorambucil), AMBOCLORIN (chlorambucil), ARRANON (nelarabine), BEACOPP, BECENUM (carmustine), BELEODAQ (belinostat), BEXXAR (tositumomab and I131 tositumomab), BICNU (carmustine), BLENOXANE (bleomycin), CARMUBRIS (carmustine), CLAFEN (cyclophosphamide), COPP-ABV, CVP, CYXAN (cyclophosphamide), DECYPOT (cytarabine), DTIC-DOME (EPOCAROME), FOPHOSPHAX (FOURACH), FOURACEPIN (FOBROAD), FOURACETINA 2, FORCONITAX (FORCONITAX), FORCONITON (FORCONITON), FORCONITAX 2, FORCONITON (FORCONITX), FORCONITX-2, FORCONITAX (FORCA), FORCONITON, FORCONITA, FORCONITX (FORCONITON, FORCONITX), FORCAT), BENCA, FORCAT, FORCONITON (FORCAT), BENCT, FORCONITAX, FORCAT, FOX, FORCAT, FORC, Isotaxx (romidepsin), LEUKERAN (chlorambucil), LINFOLIZIN (chlorambucil), lomustine, MATULANE (procarbazine hydrochloride), METHOTREXATE LPF (METHOTREXATE), MEXATE-AQ (METHOTREXATE), MOPP, MOZOBIL (plerixafof), MUSTARGEN (mechlorethamine hydrochloride), NEOSAR (cyclophosphamide), OEPA, ONTAK (dinil 2), OPPA, R-CHOP, revalid (lenalidomide), rituximab (rituximab), STANFORD V, TREANDA (bendamustine hydrochloride), VAMP, VELBAN (vinblastine sulfate), VELCADE (bortezomib), velsa (vinblastine sulfate), VINCASAR PFS (vincristine sulfate), zetimumab), zovorozolinarin (vinela), linylilin (elvalvanil), or combinations thereof. In some embodiments, the additional agent is revalimid (lenalidomide), DACOGEN (decitabine), vidaka (azacitidine), cytar-U (cytarabine), IDAMYCIN (idarubicin), CERUBIDINE (daunorubicin), LEUKERAN (chlorambucil), NEOSAR (cyclophosphamide), FLUDARA (fludarabine), LEUSTATIN (cladribine), or a combination thereof. In some embodiments, the additional agent is ABITREXATE (methotrexate), ABRAXANE (paclitaxel albumin-stabilized nanoparticulate formulation), AC-T, ADE, ADRIAMYCIN PFS (doxorubicin hydrochloride), ADRUCIL (fluorouracil), AFINITOR (everolimus), AFINITOR DISPERZ (everolimus), ALDARA (imiquimod), ALIMTA (disodium pemetrexed), AREDIA (disodium pamidronate), ARANIDEX (anastrozole), AROMASIN (exemestane), AVASTIN (bevacizumab), BECENUM (carmustine), BEECAP, BICNU (carmustine), BLENOXANOX (bleomycin), CAF, CAMPAR (irinotecan hydrochloride), CAPRELA (vandetanib), BOPLATIN-TAXOL, CARBRMUMUIS (carmustine), CASOX (DEMOX), BILUX (CLARITUIX (HPV), HOMORPE-HCI (CINOX), HOMORPE (CINOX), HARCEMA (CINOX), HALOX (CIT-E), HALOX (CITRACEQ), HALOX (CIT-E), HALOX (CIT), HALOX), HAS (CIT-E), HALOX (CIE), HALOX (CALCOME), HAS (CALCE), HAS (CALCO-C (CALCE), HAS (CALCE), CALCE (CALCC (CALCO-C (CALCE), CALCE (CALCC (CALCE), CALCC (CALCE) and CALCE) wherein, COSMEGEN (Actinomycin D), CYFOS (ifosfamide), CYRAMZA (ramucirumab), CYTOSAR-U (cytarabine), CYTOXAN (cyclophosphamide), DACOGEN (decitabine), DEGARELIX, DOXIL (doxorubicin hydrochloride liposome), doxorubicin hydrochloride, DOX-SL (doxorubicin hydrochloride liposome), DTIC-DOME (dacarbazine), EFUDEX (fluorouracil), ELLENCE (epirubicin hydrochloride), ELOXATIN (oxaliplatin), ERBITIX (CETUXIMAB), ERIVEGEL (Virgimod), ETOPHOS (etoposide phosphate), EVACET (doxorubicin hydrochloride liposome), FARESTON (toremifene), FASLODEX (fulvestrant), FEC, CITRAZOLAB (lyxate), FLUOROPELEX (fluorouracil), FOLEX (methotrexate), FOLFLEX (FOLFLEI), FOVARDFOVALVI, FOXIDOXIDOXIFU-FO-FOLIX (FOIRIFO), FOIRIFO-B, FOIRIFO-S (FOIRIFOLV), FOIROX, FOIRIFOLI, FOIRE (FOIRE-S), FOIRIFO-S (FOIRFOIRFOIRFOIRE), FOIRFOIRE), FOIRE-S (FOLIX, FOIRE, FOLIX (FOIRE), FOIRE, FOLIX, FOIRE, FOLIX, GEMCITABINE-CISPLATIN, GEMCITABINE-OXALLATIN, GEMZAR (GEMCITABINE hydrochloride), GILOTRIF (Afatinib dimaleate), GLEEVEC (imatinib mesylate), GLIADEL (carmustine implant), GLIADEL WAFER (carmustine implant), HERCEPTIN (trastuzumab), HYCAMTIN (topotecan hydrochloride), IFEX (ifosfamide), IFOSFAMIDUM (ifosfamide), INLYTA (acitinib), INTRON A (recombinant interferon alpha-2 b), SSA (gefitinib), IXEMPRA (ixabepilone), KAFI (ruxotinib phosphate), JEVTANA (cabazitaxel), KADCYLA (ado-trastuzumab emsine), KEYTRULEKYL, PROLIS (carfilzomib), ODVTLULOX (Doxil hydrochloride), DEMONLULULULULULIN (DEMONLULIN), DELULIN (DELULIN-4), LIP (DEMONLULIN-4-L-, LUPRON DEPOT-PED (leuprolide acetate), MEGACE (megestrol acetate), MEKINIST (triametinib), METHAZOLASTONE (temozolomide), METHOTREE LPF (METHOTREXATE), MEXATE-AQ (METHOTREXATE), mitoxantrone hydrochloride, MITOTZYTREX (mitomycin c), MOZOBIL (plexafof), MUSTARGEN (mechlorethamine hydrochloride), MUTAMYCIN (mitomycin c), MYLOSAR (azacitidine), NAVSBILINE (vinorelbine tartrate), NEOSAR (cyclophosphamide), NEXAVAR (mefenanib tosylate), NOLVADEX (tamoxifen citrate), NOVALDEX (tamoxifen citrate), PAD, PARLAT (carboplatin), PARAPAPMAUX (carboplatin), PEG-IN (pegylated interferon alpha-2 b), pemetrexed (polyethylene glycol), disodium (prednisone), AQP-sodium (prednisolone (prednisone), cisplatin), and cisplatin, PROLEUKIN (Addison), PROLIA (dinolizumab), PROVENGE (sipuleucel-t), REVLIMID (lenalid), RUBIDOMYCIN (daunorubicin hydrochloride), SPRYCEL (dasatinib), STIVARGA (regorafenib), SUTENT (sunitinib malate), SYLATRON (pegylated interferon alpha-2 b), SYLVANT (cetuximab), SYNOVIR (thalidomide), TAC, TAFINLAR (dalanib), TARABIPFS (cytarabine), TARCEVA (erlotinib hydrochloride), TAGNSIA (nilotinib), TAXOL (paclitaxel), TAXOTERE (docetaxel), TEMODAR (temozolomide), THALOMID (thalidomide), TOPOSOSOSOSP (etoposide), TOSEN (cilobovansen), TPF (TRIKEarsenic Trioxide (TRIKEASE), vinorelbine sulfate (L), vincamine sulfate (L), L-L (L), TAXLOT-L (L-L), TAXLOT-L (L-, VEPESID (etoposide), VIADUR (leuprorelin acetate), VIDAZA (azacitidine), VINCASAR PFS (vincristine sulfate), VOTRIENT (pazopanib hydrochloride), WELLCOVERIN (calcium folinate), XALKORI (crizotinib), XELODA (capecitabine), XELOX, XGAEVA (dinomab), XOFIGO (radium dichloride 223), XTANDI (enzalutamide), YERVOY (ipilimumab), ZALTRAP (zip-Abelicept), ZELBORAF (Verofenib), ZOLADEX (goserelin acetate), ZOMETA (zoledronic acid), ZKAYKADIA (ceritinib), ZYTIGA (abiraterone acetate), or combinations thereof. In some embodiments, the additional agent is a binder or inhibitor of HMT (e.g., EZH1, EZH2, DOT 1). In some embodiments, the additional agent is a protein kinase inhibitor (e.g., a tyrosine protein kinase inhibitor). In some embodiments, the additional agent is selected from epigenetic or transcriptional modulators (e.g., DNA methyltransferase inhibitors, histone deacetylase inhibitors (HDAC inhibitors), lysine methyltransferase inhibitors), antimitotic drugs (e.g., taxanes and vinca alkaloids), hormone receptor modulators (e.g., estrogen receptor modulators and androgen receptor modulators), cell signaling pathway inhibitors (e.g., tyrosine protein kinase inhibitors), modulators of protein stability (e.g., proteasome inhibitors), Hsp90 inhibitors, glucocorticoids, all-trans retinoic acid, and other differentiation promoting drugs. In particular embodiments, the compounds or pharmaceutical compositions of the present invention may be administered in combination with anti-cancer therapies, including but not limited to: surgery, radiation therapy, transplantation (e.g., stem cell transplantation, bone marrow transplantation), immunotherapy, and chemotherapy.

The invention also includes kits (e.g., pharmaceutical packages). Kits are provided that can include a pharmaceutical composition or compound of the invention, and a container (e.g., a vial, ampoule, bottle, syringe, and/or dispensing package, or other suitable container). In some embodiments, the provided kits may also optionally include a second container comprising a pharmaceutical excipient for diluting or suspending a pharmaceutical composition or compound of the present invention. In some embodiments, a pharmaceutical composition or compound of the invention is provided in a first container and the second container is combined to form a unit dosage form.

Thus, in one aspect, the present application provides a kit comprising a first container comprising a compound or pharmaceutical composition described herein. In some embodiments, the kit is for treating a disease (e.g., a proliferative disease, an inflammatory disease, an autoimmune disease, a genetic disease, a hematological disease, a neurological disease, a painful disorder, a psychiatric disease, or a metabolic disorder) in a subject in need thereof. In some embodiments, the kit is for preventing a disease (e.g., a proliferative disease, an inflammatory disease, an autoimmune disease, a genetic disease, a hematological disease, a neurological disease, a painful disorder, a psychiatric disease, or a metabolic disorder) in a subject in need thereof. In some embodiments, the kit is for inhibiting an activity (e.g., an aberrant activity such as increased activity) of HMT in a subject, biological sample, tissue, or cell. In some embodiments, the kit is for inducing apoptosis in a cell.

In some embodiments, the kits described herein further comprise instructions for using the compounds or pharmaceutical compositions included in the kits. The kits described herein also include information required by regulatory agencies such as the u.s.food and Drug Administration (FDA). In some embodiments, the information included in the kit is prescription information. In some embodiments, the kits and instructions provide for treating a disease (e.g., a proliferative disease, an inflammatory disease, an autoimmune disease, a genetic disease, a hematological disease, a neurological disease, a painful condition, a psychiatric disease, or a metabolic disorder) in a subject in need thereof. In some embodiments, the kits and instructions provide for preventing a disease (e.g., a proliferative disease, an inflammatory disease, an autoimmune disease, a genetic disease, a hematological disease, a neurological disease, a painful condition, a psychiatric disease, or a metabolic disorder) in a subject in need thereof. In some embodiments, the kits and instructions provide for modulating (e.g., inhibiting) the activity (e.g., aberrant activity such as increased activity) of HMT in a subject, biological sample, tissue, or cell. In some embodiments, the kits and instructions provide for inducing apoptosis. The kits described herein may include one or more additional agents described herein as separate compositions.

Methods of treatment and uses

The compounds described herein are capable of binding (e.g., reversibly binding or irreversibly binding) HMT and modulating (e.g., reversibly modulating or irreversibly modulating) the activity of HMT. Accordingly, the present disclosure also provides methods of modulating (e.g., inhibiting or increasing) the activity (e.g., aberrant activity such as increased or decreased activity) of HMT in a subject, biological sample, tissue or cell. The present disclosure further provides methods for treating a wide range of diseases, such as diseases associated with aberrant or increased HMT activity, proliferative diseases, inflammatory diseases, autoimmune diseases, genetic diseases, hematological diseases, neurological diseases, painful disorders, psychiatric diseases, and metabolic disorders, in a subject in need thereof.

In another aspect, the present application provides a method of modulating (e.g., inhibiting) the activity of HMT in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound or pharmaceutical composition described herein.

In another aspect, the present application provides a method of modulating (e.g., inhibiting) the activity of HMT in a biological sample, tissue or cell, the method comprising contacting the biological sample, tissue or cell with an effective amount of a compound or pharmaceutical composition described herein.

In some embodiments, the activity of HMT in a subject, biological sample, tissue, or cell is inhibited by at least 1%, at least 3%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of a compound, pharmaceutical composition, kit, use, or method described herein. In some embodiments, the activity of HMT in a subject, biological sample, tissue, or cell is inhibited by a compound, pharmaceutical composition, kit, use, or method described herein by no more than 1%, no more than 3%, no more than 10%, no more than 20%, no more than 30%, no more than 40%, no more than 50%, no more than 60%, no more than 70%, no more than 80%, or no more than 90%. In some embodiments, the activity of HMT in a subject, biological sample, tissue, or cell is selectively inhibited by the compound, pharmaceutical composition, kit, use, or method. In some embodiments, the activity of EZH2 in a subject, biological sample, tissue, or cell is selectively inhibited by the compound, pharmaceutical composition, kit, use, or method as compared to a different HMT (e.g., EZH 1). In some embodiments, the activity of EZH1 in a subject, biological sample, tissue, or cell is selectively inhibited by the compound, pharmaceutical composition, kit, use, or method as compared to a different HMT (e.g., EZH 2). In some embodiments, the activity of HMT in a subject, biological sample, tissue, or cell described herein is reversibly inhibited by the compound, pharmaceutical composition, kit, use, or method. In some embodiments, the activity of HMT in a subject, biological sample, tissue, or cell described herein is irreversibly inhibited by the compound, pharmaceutical composition, kit, use, or method. In some embodiments, the compound, pharmaceutical composition, kit, use or method inhibits the activity of a mutant form of HMT (e.g., a mutant form of EZH1 or a mutant form of EZH 2). In some embodiments, the compound, pharmaceutical composition, kit, use or method reduces methylation of a histone.

Another aspect of the present disclosure relates to a method of reducing methylation of a histone in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound or pharmaceutical composition described herein.

Another aspect of the present disclosure relates to a method of reducing methylation of histones in a biological sample, tissue or cell, the method comprising contacting the biological sample, tissue or cell with an effective amount of a compound or pharmaceutical composition described herein.

Another aspect of the present disclosure relates to a method of modulating (e.g., down-regulating or up-regulating) gene expression in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound or pharmaceutical composition described herein.

Another aspect of the present disclosure relates to a method of modulating (e.g., down-regulating or up-regulating) gene expression in a biological sample, tissue or cell, the method comprising contacting the biological sample, tissue or cell with an effective amount of a compound or pharmaceutical composition described herein.

In some embodiments, the gene described herein is a gene encoding an HMT described herein (e.g., a gene encoding EZH1, EZH2, or DOT 1).

Another aspect of the present disclosure relates to a method of treating a disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition described herein.

HMT is implicated in a variety of diseases. For example, changes in the EZH2 gene have been associated with various types of cancer. Mutations in this gene have been identified in hematological malignancies (e.g., lymphomas, leukemias). These mutations may be described as "gain-of-function" because they appear to enhance the activity of the EZH2 enzyme and/or to gain the enzyme a new atypical function. In addition, increased activity (overexpression) of the EZH2 gene has been identified in cancerous tumors of the prostate, breast and other organs. Changes involving the EZH2 gene may compromise the normal control of cell division (proliferation), cause cells to grow and divide too quickly, or grow and divide in an uncontrolled manner and lead to the development of cancer. In addition, at least 20 EZH2 gene mutations have been identified in patients with wevern syndrome. Signs and symptoms of this condition include bone overgrowth, a distinctive facial appearance, and joint problems. Patients with weaver syndrome are at increased risk of developing cancer. Mutations in the EZH2 gene may disrupt methylation and impair the regulation of some genes in the organs and tissues of many subjects, leading to the abnormal features of wevern's syndrome. Weverer syndrome is also associated with abnormal activity of EZH 1. DOT1 has also been reported to be involved in leukemias (e.g., AML, ALL). In patients with leukemia, DOT1 may be mislocalized on chromatin, affecting local H3K79 methylation status. The overall level of H3K79 methylation was unaffected, but the local level of H3K79 methylation in specific regions was abnormally altered, resulting in dysregulated transcription that could be an important participant in leukemia.

Mutations in tyrosine 641 (e.g., Y641F, Y641N, Y641S, Y641H) in the SET domain of EZH2 have been found to be associated with some types of cancer (e.g., non-hodgkin's lymphoma). These mutations have been shown to affect the substrate specificity of EZH2 for some methylation states of lysine 27 on histone H3. A subject may be selected for treatment with an EZH2 inhibitor based on the presence or absence of an EZH2 mutation in the subject. For example, in some embodiments, if the subject has a mutation at tyrosine 641 in the SET domain of EZH2, the subject is selected for treatment with an EZH2 inhibitor.

In some embodiments, the diseases described herein are HMT-associated diseases. In some embodiments, the diseases described herein are diseases associated with aberrant activity (e.g., increased activity) of HMT. In some embodiments, the disease described herein is a proliferative disease. In some embodiments, the disease described herein is cancer. In some embodiments, the disease described herein is hyperplasia (e.g., Germinal Center (GC) hyperplasia). In some embodiments, the disease described herein is brain, breast or prostate cancer. In some embodiments, the disease described herein is a benign tumor. In some embodiments, the disease described herein is pathological angiogenesis or a correlation thereof. In some embodiments, the disease described herein is an inflammatory disease. In some embodiments, the disease described herein is an autoimmune disease. In some embodiments, the disease described herein is a genetic disease. In some embodiments, the disease described herein is weber's syndrome. In some embodiments, the disease described herein is a hematological disease. In some embodiments, the disease described herein is lymphoma (e.g., follicular large B-cell lymphoma, diffuse large B-cell lymphoma). In some embodiments, the disease described herein is leukemia (e.g., CML). In some embodiments, the disease described herein is a neurological disease. In some embodiments, the disease described herein is a painful condition. In some embodiments, the disease described herein is a psychiatric disease. In some embodiments, the disease described herein is a metabolic disorder.

In another aspect, the present application provides a method of preventing a disease described herein in a subject in need thereof, the method comprising administering to the subject a prophylactically effective amount of a compound or pharmaceutical composition described herein.

In another aspect, the application provides a compound described herein for use in a method described herein (e.g., a method of inhibiting HMT activity, a method of treating a disease (e.g., a proliferative disease), a method of preventing a disease (e.g., a proliferative disease), a method of inducing apoptosis, or a method of screening a library of compounds).

In another aspect, the present application provides a pharmaceutical composition described herein for use in a method described herein (e.g., a method of inhibiting the activity of HMT, a method of treating a disease (e.g., a proliferative disease), a method of preventing a disease (e.g., a proliferative disease), a method of inducing apoptosis, or a method of screening a library of compounds).

Method for screening compound libraries

Other aspects of the invention relate to methods of screening libraries of compounds and pharmaceutically acceptable salts thereof to identify compounds or pharmaceutically acceptable salts thereof that are useful in the methods of the invention. In some embodiments, the method of screening a library comprises obtaining at least two different compounds described herein; and performing at least one test using the different compounds of the present application. In some embodiments, at least one test can be used to identify compounds for use in the methods of the invention.

Typically, the method of screening a library of compounds comprises at least one test. In some embodiments, the test is performed to detect one or more of the following characteristics: features associated with the treatment and/or prevention of the diseases described herein, or features associated with modulating (e.g., inhibiting) the activity of HMT. The characteristic may be a desired characteristic (e.g., the disease has been treated, the disease has been prevented, the activity of HMT has been modulated, and/or apoptosis has been induced). The characteristic may be an undesirable characteristic (e.g., the disease is not treated, the disease is not prevented, the activity of HMT is not modulated, and/or apoptosis is not induced). The test may be an immunoassay, such as a sandwich test, a competitive binding test, a one-step direct test, a two-step test, or a western blot test. The step of performing at least one test may be performed automatically or manually. In some embodiments, the testing comprises (a) contacting a library of compounds with HMT; and (b) detecting binding of the library of compounds to HMT. In some embodiments, the assay comprises detecting specific binding of the library of compounds to HMT. In some embodiments, the detected binding of the library of compounds to HMT is used to identify compounds useful in the methods of the invention. In some embodiments, the step of detecting binding comprises using Differential Scanning Fluorimetry (DSF), Isothermal Titration Calorimetry (ITC), and/or light activated chemiluminescence immunoassay (ALPHA). The step of performing at least one test may be performed in vitro or in vivo in a cell.

Measurement of

Enhancer of zeste homolog 2(EZH2) is the core component of PRC2, which catalyzes the di-and tri-methylation of histone H3 lysine 27 (H3K27me 2/3). Somatic mutations in the SET domain of EZH2 (e.g., Y641N) result in high activity of enzymes that have been identified in most follicular and diffuse large B-cell lymphomas, implying driver functions of EZH2 in cancer development (Beguelin et al, 2013; Morin et al, 2010).

The compounds described herein are inhibitors of histone methyltransferase (HMT, e.g., enhancer of zeste homolog 1(EZH1), enhancer of zeste homolog 2(EZH 2)). The compounds are useful for treating and/or preventing a disease associated with aberrant activity or increased activity of HMT, e.g., a proliferative disease, an inflammatory disease, an autoimmune disease, a genetic disease, a hematological disease, a neurological disease, a painful disorder, a psychiatric disease, or a metabolic disorder, in a subject in need thereof.

In some embodiments, the compounds of the present invention or those identified by the methods and systems of the present invention include those compounds of the following:

exhibit the ability to inhibit EZH1 binding and/or activity,

exhibit the ability to inhibit EZH2 binding and/or activity,

Exhibit the ability to compete with compounds known to bind EZH1 for binding EZH1,

exhibit the ability to compete with compounds known to bind EZH2 for binding EZH2,

exhibits the ability to bind PRC2 and disrupt the binding of the EZH1 binding site,

exhibits the ability to bind PRC2 and disrupt the binding of the EZH2 binding site,

exhibit the ability to bind PRC2 and displace compounds known to bind to EZH1 in cells, e.g., displace compounds from nuclear locations, or

Exhibit the ability to bind PRC2 and displace compounds known to bind EZH2 in cells, e.g., displace compounds from nuclear localization.

According to one aspect of the invention, there is provided a method for identifying EZH1 and/or EZH2 binding compounds. In some embodiments, the method comprises an ALPHA assay. The method is based on the detection of EZH1 and/or EZH 2-compound complex formation by labeling EZH1 and/or EZH2 and the compounds with luminescent probes (including fluorophores) and chemiluminescent substrates. The physical proximity between EZH1 and/or EZH2 and the labeled compound in the protein-compound complex provides for a change in the fluorescence signal, or the formation of chemiluminescent products associated with the formation of the protein-labeled compound complex, particularly the proximity of EZH1 and/or EZH2 to the labeled compound. In the presence of a competitor compound that binds to EZH1 and/or EZH2, protein-labeled compound complex formation is disrupted, resulting in a corresponding decrease in the expected luminescence detection signal.

The methods generally comprise providing EZH1 and/or EZH2 binding compounds labeled with a fluorescence donor and EZH1 and/or EZH2 labeled with a fluorescence acceptor, wherein binding of the labeled compounds to EZH1 and/or EZH2 is detected by a proximity-based luminescence detection method; binding the labeled compound to EZH1 and/or EZH2 in the presence of a test compound; and identifying the test compound as an EZH1 and/or EZH2 inhibitor when the proximity-based luminescence detection signal is decreased in the presence of the test compound relative to when the signal is in the absence of the test compound. The amount of decrease in the measured detection signal required for a test compound to be identified as an EZH1 and/or EZH2 inhibitor depends on the type of proximity-based luminescence detection assay used. Typically, a decrease of 5% or greater relative to an assay performed in the absence of the test compound indicates that the test compound is an EZH1 and/or EZH2 inhibitor. In some embodiments, the test compound stimulates a decrease in the test signal by at least 10%, 25%, 50%, 75% or 100%.

Any proximity-based luminescence detection method may be used in the present invention. Embodiments of proximity-based luminescence detection methods include, but are not limited to: fluorescence resonance energy transfer ("FRET") (Stryer, L.Ann.Rev. biochem.47,819-846,1978), Fluorescence resonance energy transfer ("LRET") (Mathis, G.Clin. chem.41,1391-1397,1995), Fluorescence correlation Spectroscopy ("FCCS") (Maiti et al Proc. Nat' l Acad Sci USA 94,11753 11757,1997), scintillation proximity analysis ("SPA") (Hart and Greenwald, Molecular Immunology 16: 265. 267, 1979; U.S. Pat. No. 4,658,649), direct quenching (Tyagi et al, Nature Biotechnology 16,49-53,1998), chemiluminescence energy transfer ("CRET" (Campbell, A.K., and Patel, A.Biom.J.216, 185-194, 1983), bioluminescence energy transfer ("BRIC. Ack. Schaft, Inc., Kloen. Schaft, Inc., 55, 92, Klaus. forming molecule of Fluorescence resonance energy transfer (" John, P.J.1999, P.J.216, J.P.P.. It should be understood that one skilled in the art will recognize alternative proximity-based luminescence detection methods that may be applied to and used with the present invention.

The term "luminescence" or "luminescent" refers to any method of light emission including fluorescence, phosphorescence, scintillation, chemiluminescence, and bioluminescence.

The term fluorescent donor or fluorescence donor refers to a light-emitting molecule whose emitted light is absorbed by a fluorescence acceptor. The term fluorescent acceptor or fluorescence acceptor refers to a second luminescent molecule or a quencher molecule that absorbs light emitted by the fluorescence donor. The second fluorophore absorbs light emitted from the fluorescence donor and emits light of a different wavelength than the light emitted by the fluorescence donor. The quenching molecule absorbs the light emitted by the fluorescence donor. It is contemplated that any light emitting molecule may be used in the practice of the present invention.

Examples of fluorophores and quenchers include, but are not limited to: alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, 7-diethylaminocoumarin-3-carboxylic acid, fluorescein, Oregon Green (Oregon Green)488, Oregon Green (Oregon Green)514, tetramethylrhodamine, rhodamine X, Texas Red QS dye, QSY 7, QSY33, Dabcyl, BODIPY FL, BODIPY 630/650, BODIPY 650/665, BODIPY TMR-X, BODIPY TR-X, dialkylaminocoumarin, Cy5.5, 5, Cy3.5, CyDIPY 3, DTPA (AMCy 3) and AMTTEu (AMTTHA-3).

The terms "chemiluminescent", "chemiluminescent" or "chemiluminescent substrate" refer to a chemical substance that generates light as a result of a chemical reaction. Commonly used chemiluminescent substrates include, but are not limited to, luminol (5-amino-2, 3-dihydro-1, 4-phthalazinedione), loxol (2,4, 5-triphenylimidazole), lucigenin (bis-N-methylacridinium) other acridinium esters, luciferin-luciferase and dimethylthiophene derivatives. For example, ECL from Amersham, which is well known in the art^TMIn the detection system, the acridinium substrate is oxidized by horseradish peroxidase to produce an acridinium ester, which reacts with excess peroxide at alkaline pH to produce chemiluminescence visible at 430 nm.

In some embodiments, the AlphaLISA TruHits kit of PerkinElmer, well known in the art, is used. The kit includes AlphaLISA BSA-biotin acceptor beads and streptavidin alpha donor beads that interact to generate an AlphaLISA signal. Excitation of the donor beads causes the release of a unimodal oxygen molecule, which triggers an energy transfer cascade in the acceptor beads, resulting in a spike in luminescence at 615 nm.

It is to be understood that one skilled in the art will recognize that any compatible fluorescence donor-acceptor pair will function in the present invention, and that the aforementioned fluorophores and quenchers are exemplary and not limiting.

In one embodiment, the labeled compound and/or EZH1 and/or EZH2 are in solution and diffuse freely in all directions. In another embodiment, the labeled compound and/or EZH1 and/or EZH2 are immobilized on a solid phase substrate, such as a microtiter plate, microarray slide, membrane, or microsphere. In some embodiments, the labeled compound and/or EZH1 and/or EZH2 are attached to the solid substrate by covalent or non-covalent interactions, such as biotin/avidin interactions.

In some embodiments, the method comprises a Fluorescence Polarization (FP) assay. Fluorescence Polarization (FP) assays can be used to study molecular interactions (e.g., Lea, w.a., Simeonov, a.expert opin. Drug discov.,6,17-32,2011). Generally, in an FP assay, a fluorescently labeled molecule will emit fluorescence with a polarization angle that is inversely related to its rotation rate when excited by plane polarized light. In solution, many phenomena (e.g., drag, diffusion, brownian motion) cause smaller particles to have a greater rotational velocity than larger particles. Thus, when a complex comprising a protein (e.g., EZH1 and/or EZH2) and a fluorescently labeled compound (e.g., a compound described herein) is excited by plane polarized light, the emitted light remains highly polarized because the fluorophore is restricted from rotating for the time that the light is absorbed and emitted. When the unbound fluorescently labeled compound is excited by plane polarized light, its spin rate is faster than that of the corresponding complex. As a result, the light emitted from unbound fluorescently labeled compounds is depolarized to a greater extent than the same molecule bound by complexation. In the context of small molecule compound screening, fluorescent tags may be attached to compounds known to form binding interactions with proteins (e.g., EZH1 and/or EZH 2). Binding of the labeled compound to the protein can be monitored by FP. In the presence of unlabeled compound bound to the protein, the complex comprising the labeled compound and the protein is displaced. As a result, the concentration of unbound labeled compound increases and the FP signal reflects the subsequent increase in depolarizing light.

In some embodiments, the methods comprise an intracellular competitive binding assay, such as an intracellular competitive EZH2 binding assay. For example, cells in culture may be incubated with labeled EZH 2-binding compounds, which may be localized to the nucleus to bind endogenous EZH 2. The cells may be incubated in the presence of an unlabeled test compound (e.g., a candidate EZH2 inhibitor compound). If the test compound binds to exogenous EZH2, it can compete for binding with the labeled EZH2 binding compound, thereby localizing the labeled EZH2 binding compound to a location outside of the nucleus. Such binding and localization can be detected by detecting the label. Thus, in the presence of unlabeled compound bound to EZH2, the complex comprising the labeled compound and endogenous EZH2 is displaced. As a result, the labeled compound localizes to the nucleus at a lower level than the localization of the labeled compound in the absence of unlabeled compound.

Candidate test compounds for use in the present invention include many chemical classes, but generally they are small organic compounds. The term "small molecule" is used to refer to a naturally occurring or artificially produced (e.g., by chemical synthesis) molecule having a relatively low molecular weight. Typically, the small molecule is an organic compound (i.e., it contains carbon). Small molecules can contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyl groups, carbonyl groups, heterocycles, etc.). In some embodiments, the small molecule is monomeric and has a molecular weight of less than about 1500 g/mol. In some embodiments, the small molecule has a molecular weight of less than about 1000g/mol or less than about 500 g/mol. In some embodiments, the small molecules are biologically active in that they produce a biological effect in an animal, preferably a mammal, more preferably a human. Small molecules include, but are not limited to, radionuclides and imaging agents. In some embodiments, the small molecule is a drug. Preferably, but not necessarily, the medicament has been deemed safe and effective for use in humans or animals by appropriate governmental agencies or regulatory bodies. For example, human pharmaceuticals are listed by the FDA in accordance with 21c.f.r. § 330.5,331 to 361, and 440 to 460, which are incorporated by reference; veterinary drugs are listed by FDA in accordance with 21c.f.r. § 500 to 589, which is incorporated by reference. All listed drugs are considered acceptable according to the present invention.

Candidate test compounds contain functional chemical groups necessary for structural interaction with proteins and/or nucleic acids, and typically include at least an amine, carbonyl, hydroxyl, or carboxyl group, preferably at least two functional chemical groups, and more preferably at least three functional chemical groups. Candidate test compounds may comprise cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate test compounds may also be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs thereof, or combinations thereof, and the like.

Candidate test compounds are obtained from a variety of sources, including synthetic or natural compound libraries (e.g., without limitation, commercial libraries, historical libraries/collections). For example, a number of methods are available for the random and directed synthesis of a variety of organic compounds and biomolecules, including expression of random oligonucleotides, synthesis of organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be obtained or readily produced. In addition, natural and synthetically produced libraries and compounds can be readily modified by conventional chemical, physical and biochemical means. In addition, known pharmacological agents may undergo directed or random chemical modifications, such as acylation, alkylation, esterification, amidation, and the like, to produce structural analogs of the agents.

Various other reagents may also be included in the reaction mixture. These include reagents such as salts, buffers, proteins (e.g., albumin), detergents and polymers, which can be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such reagents may also reduce non-specific or background interactions of reaction components. Other reagents that increase the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like, can also be used.

The order of addition of the components, the incubation temperature, the incubation time and other parameters of the assay can be determined by one skilled in the art. Such experiments typically involve optimization of assay parameters, rather than the basic composition of the assay. The incubation temperature is typically between 4 ℃ and 40 ℃. The incubation time is preferably minimized to facilitate rapid, high throughput screening, and may be between 0.1 and 10 hours.

The invention also relates to kits and compositions comprising a marker compound and/or EZH1 and/or EZH 2. The kit may contain other compounds, such as enzymes and/or buffers, for performing the methods of the invention. The kit may further comprise instructions for performing the methods of the invention to identify an EZH1 and/or EZH2 inhibitor as described herein. The kit may also include a package containing one or more containers containing one or more reagents for performing the methods of the invention.

In some embodiments, a method of identifying an EZH1 and/or EZH2 inhibitor comprises performing a high throughput proximity-based luminescence detection assay to identify compounds having potential EZH1 and/or EZH2 inhibitory activity; re-testing the identified potential EZH1 and/or EZH2 inhibitor compounds with different concentrations of a potential EZH1 and/or EZH2 inhibitor by a proximity-based luminescence detection assay to identify at least one panel of compounds having potential EZH1 and/or EZH2 inhibitory activity; and performing two and three assays to confirm the ability of the identified compounds to inhibit EZH1 and/or EZH2, and optionally to determine the mode of action of the identified compounds. In some embodiments, the secondary assay is a cell-based and/or biochemical assay.

Examples

In order that the disclosure may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are intended to illustrate the compounds, pharmaceutical compositions and methods provided herein and are not to be construed in any way as limiting the scope thereof.

Preparation of Compounds described herein

The compounds provided herein can be prepared from readily available starting materials using the following general methods and procedures. For example, the compound of formula (I) may be prepared according to any one of schemes 1 to 3, and the compound of formula (II) may be prepared according to a method similar to that shown in any one of schemes 1 to 3. Alternatively, the compounds described herein may be prepared using methods similar to those described in U.S. patent application publication No. US 2013/0040906 and international PCT application publications WO 2013/067302, WO 2013/039988, WO 2012/118812, WO 2012/005805, WO 2014/100665, WO 2013/138361, WO 2013/067300, WO 2013/067296, WO 2013/049770, and WO 2011/140324. Given typical or preferred process conditions (i.e., reaction temperature, time, molar ratios of reactants, solvents, pressures, etc.), other process conditions may also be used unless otherwise specified. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art by routine optimization methods.

Scheme 1 exemplary preparation of Compound EZ-005

Scheme 2 exemplary preparation of Compounds JQEZ6 and AVC-1-018

Scheme 3 exemplary preparation of other Compounds described herein

In some embodiments, the hydrazides described herein are prepared according to the methods described in international PCT application PCT/US2015/044303, which is incorporated herein by reference. In some embodiments, the hydrazides described herein are prepared according to the methods shown in scheme 4 or 5.

Scheme 4 exemplary preparation of Compounds described herein that are hydrazides

Scheme 5 exemplary preparation of Compounds described herein that are hydrazides

In some embodiments of the present invention, the substrate is,is an aldehyde or ketone as shown in figure 21.

GSK126 and UNC1999 were purchased directly from Sigma-Aldrich, Inc. The structure and purity of the two compounds was further confirmed by NMR and LCMS. The detailed synthesis of compounds JQEZ5 and JQEZ23 is described in detail below. The reaction was performed as described in each run, using standard dual manifold and syringe techniques; the glassware was dried by baking in an oven at 130 ℃ for 12 hours and then used. The solvent used for the reaction was purchased from Sigma-Aldrich anhydrous and used as received; the only exception is EtOH, which is stored in On a molecular sieve. HPLC grade solvents were used for aqueous workup and chromatography. The reagents were used as received. By using EMD silica gel 60F254(250 μm) glass plates (quenched by UV fluorescence and quenched with KMnO₄Staining to visualize) and by LCMS using a Waters Aquity BEH C182X 50mm 1.7 μm particle column (50 ℃ C.) using H₂O/acetonitrile [ addition of 0.2% v/v formic acid or concentrated NH₄OH (aqueous solution) solution, 95: 5(0min) → 1: 99(3.60min) → 1: 99(4.00min)]Eluting at 1 mL/min; using alternating positive/negativeElectrospray ionization (125-. Flash column chromatography using Merck grade 9385 silica gelThe aperture (230-. Melting points were obtained using a capillary melting point apparatus, uncorrected. 1H NMR spectra were recorded at 400MHz on a Bruker spectrometer and reported in ppm using the residual solvent signal (dimethyl sulfoxide-d 6 ═ 2.50 ppm; chloroform-d ═ 7.27 ppm; methanol-d 4 ═ 3.31 ppm; dichloromethane-d 2 ═ 5.32ppm) as internal standard. The data are reported as: { (δ displacement), [ (s ═ singlet, d ═ doublet, dd, doublet, ddd ═ doublet, t ═ triplet, quin ═ quintet, sept ═ septenat, br ═ broad, ap ═ sharp), (J ═ coupling constant, in Hz), and (integral) ]}. Proton-decoupled 13C NMR spectra were recorded at 100MHz on a Bruker spectrometer and the residual solvent signal (chloroform-d 77.0 ppm; dimethyl sulfoxide-d 6 39.51 ppm; methanol-d 4 49.15ppm) was used as internal standard. The infrared spectrum was recorded using an ATR-FTIR instrument. By flow injection with resolutionTo obtain high resolution mass spectra on an ES + ionized qTOF Premiere mass spectrometer.

Scheme 6. synthesis of intermediate 4.

A mixture of potassium tert-butoxide (BuOK,4g,35.7mmol), 2-cyanoacetamide (1) (3.3g,39.2mmol) and 3-hepten-2-one (2) (4g,35.7mmol) in DMSO (60mL) was stirred at room temperature for 30 min. More t-BuOK (12g,107mmol) was then added and the reaction mixture was stirred under oxygen for 1 h. The reaction mixture was purged with nitrogen and slowly diluted with water (250mL) and aqueous HCl (4N, 300 mL). The reaction mixture was filtered to collect a yellow precipitate, which was washed with water and dried to give 1.5g of 3 (24% yield) as a yellow solid. MS: m/z 177(M + H)⁺。

To the above mixture of 3(1.5g) in THF (20mL) was added Pd/C (10%, 1.5g) and concentrated HCl (1 mL). The mixture was stirred at room temperature overnight under a hydrogen atmosphere. The mixture was filtered through Celite and the filtrate was concentrated in vacuo and the residue was recrystallized from PE/EA (v/v ═ 10) to give 1.4g of 4 (91% yield) as a tan solid. MS: m/z 181.1(M + H) ⁺；164.1(M-NH3+H)⁺；¹H NMR (500MHz,DMSO_d6)δ11.87(br s,1H),8.12(s,3H),6.49(br s,1H),6.00(s,1H), 3.78(m,2H),2.18(s,3H),1.50(m,2H),0.92(t,J＝7.0Hz,3H)ppm.

Scheme 7 Synthesis of intermediate 12

A mixture of ethyl (ethoxymethylene) cyanoacetate (5) (5g,29.6mmol), isopropylhydrazine hydrochloride (6) (3.9g,35.5mmol) and potassium carbonate (8.2g,59.2mmol) in ethanol (100mL) was refluxed for 16 h. The volatiles were removed in vacuo to give crude 7 as a yellow solid containing inorganic salts, which was used in the next step without further purification. MS: m/z 198.1(M + H)⁺。

A suspension of crude 7 above in aqueous sodium hydroxide (4N,50mL) was refluxed for 16 h. The mixture was cooled and acidified to pH-3.5 with concentrated HCl. To the reaction mixture was added HCl/dioxane (4N, 2mL to pH<1) And refluxed for 16 h. The organic layer was separated off and the aqueous solution was brought to pH with aqueous sodium hydroxide (4N, pH)>10) And (4) neutralizing. The mixture was then extracted with dichloromethane. The combined organic layer solutions were washed with brine, dried over sodium sulfate and concentrated to give about 2g of 8 as an orange oil (54% yield from 3 steps), which was used in the next step without further purification. MS: m/z 126.1(M + H)⁺。

A mixture of 8(1.00g,7.99mmol) and diethyl oxaloacetate (9) (2.26g,12.0mmol) in toluene (20mL) was refluxed for 16 h. The volatiles were removed in vacuo and the residue was dissolved in acetic acid (10 mL) and refluxed for 4 hours. The mixture was then diluted with water (20mL) and extracted with ethyl acetate. The combined organic solution was washed with water and brine, washed with sulfur The sodium salt was dried and concentrated. The crude product was recrystallized from dichloromethane to give 0.9g of 10 (45% yield) as a white solid. MS: m/z 250 (M + H)⁺。

To a solution of 10(0.90g,3.61mmol) in THF (10mL) was added aqueous sodium hydroxide (4N,5mL) and stirred at room temperature overnight. The resulting mixture was then acidified to pH-2 with concentrated HCl and extracted with dichloromethane. The organic solution was washed with brine, dried over sodium sulfate and concentrated in vacuo. The crude product was recrystallized from dichloromethane to give 0.7g of 11 (88% yield) as a yellow solid. MS: m/z 222(M + H)⁺。

A mixture of 11(600mg,2.71mmol) in phosphorus oxychloride (15mL) was stirred in a sealed tube at 120 ℃ overnight. Most of the phosphorus oxychloride was removed in vacuo and the residue was quenched with water at 0 ℃. The mixture was extracted with dichloromethane. The combined organic solutions were washed with water, dried over sodium sulfate and concentrated in vacuo. The residue was purified by silica gel column chromatography (PE/EA, v/v ═ 1) to give 110mg of 12 (17% yield) as a white solid. MS: m/z 240(M + H) +. MS: m/z 240.0 (M + H) +; 1H NMR (500MHz, CDCl3) δ 8.51(s,1H),7.83(s,1H),5.34(m,1H), 1.64(d, J ═ 6.5Hz,6H) ppm.

Scheme 8 Synthesis of Compound JQEZ5

A mixture of 12(70mg,0.29mmol), HATU (277mg,0.73mmol) and Diethylpropylethylamine (DIPEA) (1mL) in dichloromethane (10mL) was stirred for 10min, then 4 (127mg,0.58mmol) was added. The reaction mixture was stirred at room temperature for 3 hours. The mixture was diluted with dichloromethane (50mL) and washed with water and brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography (50% EA/PE) to give 60mg of 13 (51% yield) as a white solid. MS: m/z 402.1(M + H)⁺；¹H NMR(500MHz,CDCl3)δ11.91(br s, 1H),8.38(s,1H),8.24(t,J＝5.0Hz,1H),7.43(s,1H),6.00(s,1H),5.26(m, 1H),4.65(d,J＝5.5Hz,2H),2.69(t,J＝7.5Hz,2H),2.28(s,3H),1.64(m,2H), 1.55(d,J＝6.5Hz,6H),1.02(t,J＝7.0Hz,3H)ppm.

A mixture of 14(2.37g,10mmol) and N-methylpiperazine (4g,40mmol) in N-BuOH (25mL) was refluxed for 96 h. The volatiles were removed in vacuo and the crude product was purified by silica gel column chromatography (PE/EA, v/v 3 to EA) to give 1.8g of 15 (70% yield) as a yellow semisolid. MS: m/z 256.0(M + H)⁺,258.0(M+H,Br)⁺。

15(1.5g,5.9mmol), bis (pinacolato) diboron (1.6g,6.5mmol), potassium acetate (1.8 g,18mmol) and Pd (dppf)₂Cl₂[CH₂Cl₂]A mixture (0.73g,0.89mmol) in DMSO (20mL) was blanketed with nitrogen and stirred at 80 deg.C overnight. The mixture was diluted with water and extracted with EA. The organic solution was concentrated in vacuo and the residue was purified by silica gel column chromatography (PE/EA, v/v 3 to EA) to give 1.0g of 16 (56% yield) as a brown solid. MS: m/z 304(M + H) ⁺.

Mixing 13(133mg,0.33mmol), 16(200mg,0.66mmol), Pd (dppf)₂Cl₂[CH₂Cl]₂(41mg,0.050mmol) and Cs₂CO₃A mixture of (215mg,0.66mmol) in DMF (5mL) was blanketed with argon and microwave irradiated at 140 ℃ for 30 minutes. The mixture was diluted with ethyl acetate and filtered through Celite. The filtrate was concentrated and the residue was purified by flash chromatography and preparative-HPLC (HCOOH system) to give 55mg of JQEZ5 (31% yield) as a black solid. MS: m/z 543.4(M + H) +; 1H NMR (500MHz, CDCl3) δ 9.00(d, J ═ 2.5Hz,1H),8.33 (s,1H),8.31(dd, J ═ 9.0 and 2.5Hz,1H),8.14(t, J ═ 6.0Hz,1H),7.87(s,1H), 6.75(d, J ═ 9.0Hz,1H),6.02(s,1H),5.38(m,1H),4.69(d, J ═ 6.0Hz,2H),3.88 (m,4H),2.92(m,4H),2.75(m,2H),2.62(s,3H),2.27(s,3H),1.67(m,2H), 1.62(d, J ═ 6.5, 6H),1.05(t, 0 ppm), 7.05 (t, 3H).

Bioassays for compounds described herein

Example 1 inhibitory Activity of Compound 5 against selected HMTs

Compound 5 was characterized with a panel of 22 HMTs (methyltransferases listed in table 1) and showed that compound 5 was not only active against EZH2, but also 10-fold selective for EZH2 over EZH1 or DOT1 (table 1). Compound 5 inhibited EZH2 at 1 μ M after 72 hours treatment, which resulted in a decrease in trimethylation of H3K27 at 1 μ M in the EZH2 mutant line.

Table 1. inhibitory activity of compound 5 on selected HMTs.

Example 2 surface plasmon resonance experiment of Compound JQEZ6 to identify binding of Compound to PRC2

Compound EZ06 was immobilized on SPR chips by biotin-streptavidin interaction. The PRC2 five-component complex solution was then slowly flowed across the chip, followed by SPR to detect binding between EZ06 and 5-component complexes. Exemplary results are shown in fig. 6. The data show that compound JQEZ6 binds to PRC2 complex. The compound JQEZ6 was also used to develop an ALPHA assay for assessing the activity of small molecule inhibitors of EZH 2. The ALPHA assay is advantageous over known assays for HMT, since the ALPHA assay is a non-radiometric assay, whereas the known HMT assay is a radiometric assay, which requires special radioactive reagents and results in low throughput. The ALPHA assay described herein allows for high throughput screening. For the ALPHA assay, JQEZ6 was immobilized on the surface of the donor beads, and the PRC 25 component complexed with Flag tag was immobilized on the acceptor beads via Flag anti-Flag tag interaction. The mixture of these two beads produces a signal upon excitation. Effective inhibitors would disrupt this interaction and cause a decrease in signal, which is used to evaluate all compounds.

Example 3 oncogenic dysregulation of EZH2 as an opportunity for targeted therapy of Lung cancer

SUMMARY

As a major regulator of chromatin structure and function, EZH2 lysine methyltransferase initiates transcriptional silencing of a developmental gene network. Overexpression of EZH2 is commonly observed in human epithelial cancers such as non-small cell lung cancer (NSCLC), but there is no clear evidence for malignant transformation due to deregulated EZH 2. The causal relationship of EZH2 overexpression in NSCLC is demonstrated herein with a novel genetically engineered mouse model of lung adenocarcinoma. Deregulated EZH2 silences the normal developmental pathway leading to epigenetic transformation, independent of classical growth factor pathway activation. Thus, tumors were characterized as having transcriptional programs distinct from KRAS and EGFR mutant mouse lung cancers, but shared with human lung adenocarcinoma exhibiting high EZH2 expression. To target EZH 2-dependent cancers, a new and potent EZH2 inhibitor was developed, which is derived from a simple synthesis and has improved pharmacological properties. JQEZ5 promotes in vivo EZH2 driven tumor regression, confirms oncogene addiction to EZH2 in established tumors, and provides a theoretical basis for epigenetic therapy for a defined subset of lung cancers.

Targeted therapy for the treatment of lung cancer has significantly improved overall survival in defined patient subpopulations; however, many of the carcinogenic drivers of lung cancer remain unknown. Dysregulation of chromatin-associated enzymes is pathogenic in many cancers and, because they are reversible, represents a potential therapeutic target. Here, it has been shown that overexpression of EZH2 can induce lung cancer in mice with a phenotype similar to human lung cancer with high EZH2 expression. Murine and human lung cancers with EZH2 overexpression show low levels of phosphorylated AKT and ERK, suggesting that there may be other biomarkers for tumors that are sensitive to EZH2 inhibitors. Finally, a new small molecule inhibitor, JQEZ5, was developed that selectively inhibits EZH2 and promotes regression of these tumors, revealing the potential role of anti-EZH 2 treatment in lung cancer.

Introduction to

Lung cancer is one of the most common and deadliest cancers in the world (Jemal et al, 2011). Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, including the heterogeneous disease group (Chen et al, 2014). The identification of recurrent mutations and amplifications in many potentially targetable oncogenes has significantly improved the overall survival of a subpopulation of NSCLC patients. Activating mutations in BRAF, KRAS and Epidermal Growth Factor Receptor (EGFR) and fusions involving Anaplastic Lymphoma Kinase (ALK) are associated with responses to kinase inhibition (Lynch et al, 2004; Paez et al, 2004; Pao et al, 2004; Soda et al, 2007). Furthermore, with the advent of improved genomic analysis and next generation sequencing, recurrent mutations and amplifications have been identified in HER2, MET, fibroblast growth factor receptor 1(FGFR1) and FGFR2, ROS1 receptor tyrosine kinase, neuregulin 1(NRG1), neurotrophic tyrosine kinase receptor type 1 (NTRK1), and RET for a review see (Chen et al, 2014). Although these changes are considered for the majority of the pool of lung adenocarcinoma cases, a significant number of NSCLC patients lack identifiable genetic lesions in a therapeutically tractable target.

In addition to growth factor signaling pathways, chromatin-associated complexes have recently been identified as recurrent alterations or transcriptional deregulation in NSCLC, including TET methylcytosine dioxygenase (TET2), DNA methyltransferase 3A (DNMT3A), and homolog 2 of the enhancer of zeste (EZH2) (Kandoth et al, 2013). Notably, each of these factors affects heterochromatin structure and each is associated with the coordinated regulation of normal developmental transcriptional pathways (Chen and Chan, 2014; Hamdi et al 2015; Simon and Kingston, 2009; Wu and Zhang, 2011). These data suggest that disruption of chromatin structure is a common event in lung cancer pathogenesis, compatible or distinct from oncogenic signaling pathways, functioning to deregulate transcriptional programs associated with cell differentiation.

The dynamic structure of chromatin is influenced by post-translational modification of DNA (PTM) and unstructured amino-terminal tails of histones within the nucleoprotein granule. Recruitment of chromatin-associated enzyme complexes conferring covalent PTM of chromatin is influenced by the control of gene expression pathways by DNA-bound transcriptional activators and suppressors. In general, side chain acetylation of lysine residues in histone tails is associated with active euchromatin, in particular histone 3 lysine 27, which is associated with an active cis-regulatory enhancer element (H3K27ac) (Zhou et al, 2011). The modification of H3K27 exhibited switch-like behavior, as mono-, di-, tri-methylation of H3K27 (H3K27me1, -me2, -me3) was associated with inhibitory, facultative heterochromatin (Margueron and Reinberg, 2011). H3K27 methylation is primarily mediated by polycomb inhibitory complex 2(PRC2), and PRC2 is a multiprotein assembly with the function of activating and directing the core catalytic enzyme (EZH2), which mediates S-adenosylmethionine-dependent lysine methylation.

Recurrent changes in EZH2 were observed in solid tumors and hematologic malignancies, underscoring the unexpected centrality of chromatin structure in cancer pathogenesis. Activation (recurrent mutations) and inactivation (deletion, inactivating mutations) of EZH2 have been characterized, which supports the tissue-specific role of EZH2 as an oncogene or tumor suppressor. Activating mutations have been characterized in B-cell lymphomas to promote efficient H3K27 trimethylation (Morin et al, 2010; sneerringer et al, 2010). Alterations in inactivation have been identified in T-cell acute lymphocytic leukemia and malignant myeloid disease (Ernst et al, 2010; Nikoloski et al, 2010; nzhiachristos et al, 2012). More broadly than these genetic events of interest, overexpression of EZH2 has been found in a variety of cancers (Bracken et al, 2003; Simon and Lange, 2008; Varampallly et al, 2002). Like gain function mutations, overexpression is associated with increased total H3K27me3, contributes to silencing of tumor suppressors and developmental regulators, and often confers poor prognosis (Alford et al, 2012; Bachmann et al, 2006; Gong et al, 2011; Kleer et al, 2003; Varambally et al, 2002). Associated with lung adenocarcinoma, several recent studies have repeatedly demonstrated a correlation between increased EZH2 expression and poor outcome (Behrens et al, 2013; Kikuchi et al, 2010; Lv et al, 2012).

EZH2 has therefore become an urgent target for cancer therapy development. Strategies have been undertaken to develop disruptors of complex assemblies (Kim et al, 2013), as well as SAM competitive inhibitors of the typical SET lysine methyltransferase domain (Knutson et al, 2012; McCabe et al, 2012; Qi et al, 2012). Selective EZH2 inhibition using these chemical probes has established EZH2 as context-specific tumor-dependency, while providing pharmacological target validation in B-cell lymphomas (Knutson et al, 2012; McCabe et al, 2012; Qi et al, 2012; Zhao et al, 2013) and in established soft tissue sarcomas (cianica et al, 2014; Knutson et al, 2013; Li et al, 2013). Therefore, human clinical studies have begun using drug-like EZH2 inhibitors administered by oral and intravenous administration (clinical trial. gov identifier: NCT01897571, NCT02082977, NCT 02395601).

The apparent overexpression of EZH2 in lung adenocarcinoma and the feasibility of clinical studies prompted the present inventors to attempt to characterize the effects of transcriptional dysregulation of EZH2 on lung cancer pathogenesis. Using genetic and chemogenetic methods, both the carcinogenic role of wild-type EZH2 over-expression in lung cancer and the opportunity for epigenetic treatment in the disease were demonstrated. Specifically, a Genetically Engineered Mouse Model (GEMM) was generated that overexpresses wild-type EZH2 in the whole body and particularly in the lungs. Systemic and lung-specific EZH2 overexpression promotes the formation of lung tumors, which exhibit biochemical and transcriptional characteristics similar to the human tumor subtype that expresses high levels of EZH 2. Analysis of chromatin state in EZH 2-overexpressed lung tumors revealed abnormal spreading of H3K27me3, particularly at developmental regulator gene sites, many of which are known tumor suppressors in lung cancer. To overcome the limitations of existing EZH2 inhibitors in potency, availability and in vivo potency, a new and open source EZH2 chemical probe JQEZ5 has been developed and characterized. In GEMM and human NSCLC models, JQEZ5 showed excellent exposure and pharmacodynamic target modulation. Long-term treatment of EZH2 addicted tumor bearing mice with JQEZ5 consistently resulted in a reduction in tumor burden. Together, these studies reveal the role of EZH2 as a driver gene for NSCLC and the opportunity for targeted epigenetic therapy.

Overexpression of EZH2 results in murine lung cancer

To investigate the causal relationship of EZH2 overexpression in cancer, two different strategies were commonly used to boost the expression of human EZH2 in mice to control temporal specificity, since EZH2 is critical for embryogenesis and early development. All mice were engineered to carry one transgene expressing full-length murine EZH2 containing a STOP cassette flanked by loxP sites between the CAG promoter and the EZH2 gene (LSL-Ezh2) (fig. 15A-15C). Two different strategies were used to induce EZH2 overexpression using Cre recombinase (fig. 9A). First, EZH2 was constitutively overexpressed in all tissues of the mouse using actin-Cre (actin-Cre: LSL-EZH 2). Second, Ezh2 was ubiquitously overexpressed using ubiquitin-Cre-ERT 2 (UBC: LSL-EZH2) after treatment with tamoxifen at 6 weeks of age. This approach was designed to circumvent any lethality due to overexpression of EZH2 at key stages of embryogenesis and early development.

actin-Cre: LSL-Ezh2 mice overexpressed EZH2, as demonstrated by Immunohistochemistry (IHC) and Western blotting (fig. 15D and 15E). The animal is viable, fertile, normally developed, and indistinguishable from a littermate that does not express Cre recombinase in adulthood. These results indicate that overexpression of EZH2 is tolerated during embryonic and developmental growth. With actin-Cre: LSL-EZH2 mice entered adulthood and multiple tumor types were observed, including lymphomas and histiocytomas of the liver. However, most mice (6/11 mice; 55%) developed lung adenomas/adenocarcinomas with no significant metastasis at week ages averaging 64.8 ± 3.3 (table 2 and fig. 9B). In addition, UBC of tamoxifen administered at 6 weeks of age: LSL-Ezh2 mice also developed lung adenocarcinoma with an exonic rate of 40% (4/10 mice) at an average of 84.8 ± 10.1 weeks. In contrast, wild-type mice had no apparent phenotype and all harvested lungs were normal at 80 weeks, suggesting a causal relationship for EZH2 overexpression in lung tumorigenesis (table 3 and fig. 9B).

Table 2: summary of the LSL-EZH2 mouse model

^aOne mouse had both lung adenocarcinoma and histiocytic sarcoma in the liver.

^bTwo mice had both lung adenocarcinoma and histiocytic sarcoma in the liver.

Table 3: summary of lung adenocarcinoma in the LSL-EZH2 mouse model

To extend these findings, EZH2 overexpression was restricted to the lungs in the third GEMM using inhaled Adeno-Cre virus to direct Cre expression to the lung epithelium of LSL-EZH2 mice (fig. 9A) (DuPage et al, 2009). The virus Adeno-Cre was administered to 6 week old animals by inhalation, and 42% (5/12) of these animals developed lung adenocarcinoma at 88.2 ± 14.6 weeks, indicating that overexpression of EZH2 in lung epithelial cells is sufficient to induce cancer. Taken together, the data showed that 45% of mice overexpressing EZH2 developed lung adenocarcinoma with a mean survival time of 73.6 weeks of age (fig. 9B).

Histology of all generated mouse lung tumors revealed characteristics of human grade 1-2 lung adenomas/adenocarcinomas. Compared to staining of normal mouse lungs (fig. 9C, top panel), EZH 2-overexpressed lung adenocarcinomas showed high cell density and less differentiation, all consistent with low and medium grade adenocarcinomas. Immunohistochemical (IHC) analysis of Ezh 2-overexpressing tumors showed an increase in proliferation marker Ki67 compared to normal lung tissue (fig. 9C). Ezh2 expression was significantly higher in EZH 2-driven lung tumors compared to murine lung cancer driven by Kras expression (fig. 9D). Analysis of the expression of pathway markers commonly identified in Kras-driven lung cancer, such as phosphorylated AKT (p-AKT) and phosphorylated ERK (p-ERK), showed low p-AKT and p-ERK expression in mouse lung tumors induced by EZH2 (fig. 9D). Western blot analysis further confirmed that EZH2 mouse lung tumors had significantly reduced p-AKT and p-ERK compared to KRAS-induced mouse lung tumors and normal mouse lung (FIG. 9E). Taken together, these data suggest that lung tumors driven by EZH 2-mediated epigenetic disorders are histologically similar, but molecularly distinct from lung tumors driven by KRAS-dependent oncogenic signaling.

EZH 2-driven lung cancer as a molecularly distinct entity

To determine whether EZH 2-induced mouse lung cancer reflects the molecular characteristics of human disease, unsupervised comparison of gene expression profiles of mouse lung, GEMM tumor and human cancer was performed. First, RNA sequencing (RNA-seq) was performed to compare gene expression profiles of pre-cancerous normal lung tissue over-expressed by EZH2 and of lung adenocarcinoma tumors over-expressed by EZH2 from mice. Gene expression profiles from these samples were compared to the expression profiles of EGFR-mutated and KRAS-mutated lung adenocarcinoma mouse tumors using unsupervised hierarchical cluster analysis (fig. 10A). Tumors overexpressing EZH2 were isolated that are transcriptionally different from lung tumors overexpressing EZH2 for normal lung, EGFR and KRAS mutations. These data suggest that chromatin EZH2 regulation leads to activation/inhibition of transcriptional pathways, which is different from typical lung adenocarcinomas driven by EGFR and KRAS hyperactivating mutations, providing further transcriptional evidence for the above-mentioned measurement of different upstream signaling pathway activation states.

EZH 2-dependent and tumor-specific transcriptional states were defined in murine lung adenocarcinoma, followed by an assessment of the presence or absence of an equivalent subgroup of human NSCLC. Using published data from cancer genomic profiling (TCGA), a group of lung adenocarcinoma patients with elevated tumor EZH2 expression was determined (fig. 10B). Expression of EZH2 was found to be widely distributed in > 50-fold range in human lung cancer, and high EZH2 levels were not mutually exclusive with KRAS or EGFR mutations. Tumors with high EZH2 overexpression of wild-type KRAS and EGFR (top 20%) were further selected to mimic the genetics of the murine model. Pathway enrichment was assessed by Gene Set Enrichment Analysis (GSEA) (Subramanian et al, 2005). Compared to tumors with low EZH2 expression, the transcriptional signature associated with MEK and mTOR activation was inhibited in EZH2 overexpressed tumors, whether in the presence or absence of oncogenic KRAS or EGFR mutations (fig. 10C), again demonstrating that EZH2 driven tumors are molecularly distinct from tumors driven by classical signaling pathways.

Effect of EZH2 overexpression on chromatin Structure of Lung cancer

The polycomb inhibitory complex exerts a broad influence on the cis-regulatory chromatin domain, thereby affecting the epigenomic cell state (Ku et al, 2008). Acetylation and trimethylation of H3K27 are mutually exclusive biochemical events, consistent with the pivot points for transcriptional activation (H3K27ac) and inhibition (H3K27me3) observed in developmental transitions (Riising et al, 2014). Whole genome assessment of enhancer promoter activity by H3K27ac chromatin immunoprecipitation using massively parallel DNA sequencing (ChIP-seq) has been used to comparatively study malignant and inflammatory cell states (Brown et al, 2014; Chapuy et al, 2013). Focused epigenetic analysis of large-scale H3K27 ac-enriched regions, the so-called super-enhancer (SE), has provided inferences for oncogenic signaling (Hnisz et al, 2015) and subcategories of human tumors (Chapuy et al, 2013). It is believed that over time, EZH2 overexpression in the lung epithelium results in stable trimethylation and silencing of developmental transcription factors.

To understand the dynamic effect of EZH2 overexpression on chromatin structure in the context of malignant transformation, comparative epigenetic analyses were performed on normal and malignant lung tissues overexpressing EZH 2. First, the position of the active enhancer in two groups of tumor-normal pairs was determined by H3K27ac ChIP-seq, and the region of asymmetric hyperacetylation (SE) was determined, as previously reported (Chapuy et al, 2013; Loven et al, 2013). Unsupervised hierarchical cluster analysis isolated EZH 2-overexpressed tumors and lung epithelial cells (WT) exhibited markedly different euchromatin epigenomic structures (fig. 11A, 11B). Differential analysis of the highly occupied H3K27ac region in EZH 2-overexpressed tumors and normal lungs revealed global redistribution of H3K27ac, with 1244 individual sites showing greater than log in H3K27ac ₂1.5 times (432 losses, 812 gains, fig. 11C). Changes in H3K27ac at SE resulted in reciprocal changes in gene expression of adjacently expressed genes (measured by RNA-seq), indicating that modulation of chromatin affected gene expression in these tumors (fig. 11D). Unbiased frontier analysis of genes adjacent to the region lacking H3K27ac in tumors identified polycombin inhibitory target gene signatures, suggesting PRC 2-mediated inhibition of these regulatory elements (fig. 11E, 11F).

To correlate EZH2 function with missing SEs, all samples were next subjected to H3K27me3 ChIP-seq. In the region of deletion of H3K27ac SE, different subgroups of 33 cis-regulatory regions were identified, wherein the deletion of H3K27ac was accompanied by a strong increase of the polycombin H3K27me3 marker (fig. 11G). Functionally, the genes associated with these 33 regulatory regions showed a reduction in gene expression in tumors caused by RNA-seq (fig. 11H) and consisted of a variety of developmental transcriptional regulators including Foxf1a, Irf8, Hoxa9 and Meis1, as well as other chromatin factors such as Aff3 (fig. S2A). Inhibition of Hoxa9, Meis1, Irf8 and Foxf1 has been observed in NSCLC and their activity has been functionally correlated with reduced tumorigenesis, suggesting tumor suppression by these gene-regulated substances (Hwang et al, 2014; Li et al, 2014; Suzuki et al, 2014). Visual examination of the major developmental TF inhibited in tumor samples confirmed the epigenomic shift from the large hyperacetylated enhancer element to the broad H3K27 trimethylated region (fig. 16B-16D).

Among SE-associated regions obtained in tumor samples, genes encoding well-characterized negative regulators of the MAPK-ERK pathway were noted: dusp4, Spry1, Spry2 and Errfi1 (FIG. 11C). Visual inspection confirmed that all four genes were characterized by a strong gain in cis regulatory elements of H3K27ac (fig. 16E, 16F), and that RNA-seq confirmed elevated expression in tumors. These data confirm that enhancer remodeling due to overexpressed EZH2 during progression to lung adenocarcinoma specifically silences normal differentiation genes and activates negative regulators of MAPK-ERK signaling, consistent with the signaling immunophenotype of GEMM driven by EZH 2.

To explore the relevance of these findings to human lung cancer pathophysiology, we next asked whether down-regulation of the SE-associated gene identified in murine tumors as hypermethylated PRC2 was observed in human lung adenocarcinoma overexpressed by EZH 2. A strong down-regulation of functional 32 genomes was observed in human lung cancers with the highest (top 20%) expression of EZH2 (fig. 11I). In summary, comprehensive epigenetic analysis suggests that there is a distinct subset of human lung cancers characterized by 1) high levels of EZH2, 2) low activation of Ras effectors, and 3) a distinct group of inhibition of EZH2 target genes.

Overexpression of EZH2 facilitates cell transformation of human cells

To examine the oncogenic potential of EZH2 in human cells, EZH2 was overexpressed in an immortalized normal human lung epithelial cell line (human airway bronchial epithelium (hTBE) cells) and their oncogenic potential was monitored (fig. 12A). To monitor transformation, we performed a soft agar colony formation assay. hTBE cells overexpressing EZH2 (hTBE-EZH2) formed colonies on soft agar after four weeks of inoculation compared to cells expressing the control vector (hTBE-ct1) (FIG. 12B). Since many transformed cell lines can form colonies on the less delayed soft agar plates, and considering the epigenetic mechanism of EZH2 driven transformation, it is believed that hTBE-EZH2 cells may acquire higher transformation capacity over time. Therefore, hTBE-EZH2 cells were serially passaged 20 times (p20) before plating onto soft agar. hTBE-oeEZH2 cells at p20 formed colonies within two weeks and had greater transformation potential than cells at p10 (FIG. 12C). In addition, hTBE-ctl cells from passage 10 or 20 failed to form colonies on soft agar (FIG. 12C). Thus, the data indicate that constitutive overexpression of EZH2 promotes cellular transformation of human cells over time, consistent with the long latency for lung cancer development in EZH2 transgenic mice (fig. 9C).

Human NSCLC with high EZH2 expression is sensitive to deletion of EZH2

Overexpression of EZH2 was observed to produce lung cancer in mice and to transform normal human lung epithelial cells, suggesting that EZH2 may play an important role in a subset of human NSCLC. Using two previously validated EZH2 shrnas (shEZH2-a and shEZH2-B), EZH2 expression was knocked out in human NSCLC cells expressing high levels of EZH2, without other known oncogenic mutations (H661) (Fillmore et al, 2015; Tzatsos et al, 2013; Xu et al, 2012). Both shrnas showed almost complete inhibition of EZH2 protein expression compared to cells with non-targeting shRNA (shnt) controls (fig. 12D). Cell proliferation assays indicated that H661 cells showed more than 50% growth inhibition in response to the shEZH2 knock-out compared to control cells (fig. 12E). Finally, it was tested whether EZH2 expression is required for tumor formation in a mouse xenograft model of human NSCLC. H661 cells expressing control or shEZH2 vector were injected subcutaneously into mice and tumor formation was monitored every two weeks. The EZH2 knockout significantly inhibited the growth of H661 tumors in vivo (fig. 12F, 12G). Human H292 lung cancer cells expressed lower levels of EZH2 compared to H661 cells expressing high levels of EZH2 protein (fig. 17A). In agreement, H292 cell growth was not affected by EZH2 knockdown (fig. 12D and 17B). Likewise, EZH2 knockdown also did not affect the growth of these cells in the mouse xenograft model (fig. 12G).

Dependence of EZH2 overexpression of lung cancer on catalytically active EZH2

To assess the dependence of EZH2 overexpressing lung tumors on sustained EZH2 enzyme activity, chemogenetic methods were used. Recently, several pyridone-based small molecule EZH2 inhibitors have been reported as chemical probes (e.g., GSK-126 and UNC 1999; FIG. 18A) (Konze et al, 2013; McCabe et al, 2012). These structurally similar analogs are both potent and selective inhibitors of EZH2, however the broad application of these probes in vivo biological studies may be limited by low potency (high dose administration), limited bioavailability (twice daily administration) and uncertain availability (cost of synthesis, transfer of drug material). A novel EZH2 inhibitor was developed as an open source chemical probe for the scientific community. The structure-activity relationship was derived empirically from iterative simulated synthesis and biochemical testing, absent guidance from crystallographic data.

Discovered from subsequent chemistry is JQEZ5, characterized by a pyrazolopyridine core and a retained pyridone warhead that exhibit 6-substituted solubilization characteristics. The synthesis of JQEZ5 is nine linear steps, high yield and scalable to support broad distribution. As a matched control, JQEZ23 replaced the active pyridone with the predicted inactive pyridinium ring (highlighted in grey; FIG. 13A). Both compounds were evaluated in an enzymatic assay using a five-component PRC2 complex with radiolabeled S-adenosylmethionine (SAM). JQEZ5 inhibited the enzymatic function of PRC2 with a biochemical IC 50 of 80nM, similar to GSK-126 and UNC1999, while JQEZ23 had little inhibitory activity on purified PRC2 (fig. 13B, 18B). JQEZ5 exhibited SAM competitive inhibition of PRC2 as determined by assessing biochemical inhibition in the presence of increasing concentrations of unlabeled SAM cofactor (fig. 13C, 18C). To understand the putative way in which inhibitors recognize the EZH2 molecule, the binding of JQEZ5 to EZH2 was modeled using a recently reported computational model (fig. 13D) (Kalinic et al, 2014). Our established binding model showed that the pyridone ring of JQEZ5 binds to Asn78 on EZH2, and the pyrazolopyridine ring is also deeply buried in the SAM-binding pocket of EZH 2. Ligand interaction maps (LID) of JQEZ5 and EZH2 also predict that the piperazine ring on JQEZ5 extends out of the SAM binding pocket of EZH2 and can therefore be further modified as required (fig. 13E). The specificity of JQEZ5 for EZH2 was evaluated and confirmed by parallel studies of a panel of recombinant purified lysine methyltransferases (fig. 18D) (Horiuchi et al, 2013).

After biochemical validation of the specificity and potency of JQEZ5, human NSCLC cells were treated in dose-varying biochemical and cellular studies (H661). Cells treated with increasing concentrations of JQEZ5 demonstrated a dramatic drop in H3K27me3 levels without affecting mono-or di-methylation of H3K27, as assessed by Western blotting (fig. 14A). Treatment with the negative control compound JQEZ23 had no effect on H3K27 methylation status (fig. 14B). The decrease in H3K27me3 correlated with compound concentration and length of treatment time (fig. 14C). Similar to the EZH2 shRNA study, JQEZ5 inhibited the proliferation of H661 cells over-expressed by EZH2 after 4 days of treatment without affecting the proliferation of H292 cells characterized by lower EZH2 expression (fig. 14D-14E).

To investigate the translational relevance of this study, a treatment trial of JQEZ5 was performed in GEMM with tumors. JQEZ5 was formulated for Intraperitoneal (IP) administration, and repeated dosing studies established 75mg/kg IP daily as a tolerated dose and dosing regimen. Pharmacokinetic studies confirmed excellent exposure of JQEZ5 without twice weekly dosing (fig. 19A). To prepare for the treatment study, actin-Cre was monitored weekly: LSL-EZH2 mice and UBC: LSL-Ezh2 mice (treated with tamoxifen at 6 weeks of age) were monitored for the onset of symptoms of lung adenocarcinoma (dyspnea). At this time (t ═ 0), lung cancer was visualized and diagnosed by MRI (fig. 19B-19C). Mice bearing tumors were then treated with JQEZ5 for three weeks (75 mg/kg IP daily) and tumors were made relatively visible by MRI. Animals treated with JQEZ5 showed rapid and significant tumor regression during the three weeks of treatment, as evidenced by two-dimensional MRI and volume measurements (fig. 14F-14G, 19B-19C).

Discussion of the related Art

As a major cause of death for cancer, lung cancer has a far-unmet medical need. Over the past decade, a small percentage of lung cancer patients benefit from targeted therapy, but most do not benefit from these approaches, and all metastatic disease remains incurable. There are a large number of patients who cannot be effectively treated due to the lack of a pharmaceutically acceptable oncogenic driver (e.g., KRAS). Therefore, finding new actionable drivers and tumor dependencies in these remaining tumors is an urgent and important task.

Although not influenced by changes in the receptor cells, dysregulated overexpression of EZH2 was observed in a subset of human lung cancers (Behrens et al, 2013; Kikuchi et al, 2010; Lv et al, 2012). To date, no causal relationship in the development of lung tumors has been identified. The oncogenic potential of EZH2 dysregulation was explored above by generating a series of Genetically Engineered Mouse Models (GEMMs) with conditional EZH2 overexpression. Three GEMMs established strong oncogenic activity of EZH2 in NSCLC formation. Indeed, 45% of mice were engineered to overexpress lung adenocarcinoma developed by EZH 2. These data are in concert with previous studies that illustrate the transforming activity of mutant EZH2 in B-cell lymphomas (Morin et al, 2010; sneerringer et al, 2010). Previous studies have shown that overexpression of EZH2 in the medullary compartment elucidates myeloproliferative diseases, but to date there is also a lack of solid evidence that EZH2 overexpression leads to neoplastic transformation (Herrera-Merchan et al, 2012). This work is a demonstration that overexpression of wild-type EZH2 in vivo caused cancer. Over-expression of EZH2 is a common feature of many solid tumors and the reagents generated in this study will help to further define more broadly the role of EZH2 in cancer pathogenesis.

The resulting cell biology studies and integrated epigenome analysis of EZH 2-driven murine tumors determined a subset of cytoplasmic and transcriptional signaling of human disease. Lung adenocarcinomas induced by EZH2 overexpression show low levels of p-AKT and p-ERK, which are accompanied by an increased expression of known negative regulatory substances of the MAPK-ERK pathway, such as bispecific protein phosphatase 4(DUSP4) and sprouting homologs (SPRY homologies) 1 and 2(SPRY1& SPRY 2). Consistent with these findings, the transcriptional signature associated with MEK and mTOR activation is inhibited in human tumors that express high levels of EZH 2. Given that many known oncogenes in lung cancer activate these pathways, EZH2 appears to promote tumorigenesis through mechanisms not involved in these typical pathways, which have important mechanisms and therapeutic implications. However, some EGFR and KRAS mutated human cancers also express high levels of EZH2, suggesting that in some cases these pathways may be coordinated. Consistent with this concept, DUSP4 is considered to be a growth inhibitory factor in EGFR mutant lung adenocarcinoma (Chitale et al, 2009) and a positive activator of ERK in EGFR mutant lung carcinoma cell lines (Britson et al, 2009). A unified model for EZH 2-mediated malignant transformation based on these findings may be the reorganization of chromatin structure into a dedifferentiated cellular state that promotes proliferative transformation by additional genetic drivers.

The dependence of EZH 2-overexpressed human and murine lung cancer models on EZH2 was demonstrated using functional genetic (shRNA) and chemogenetic methods. Aiming at pharmacological target validation, a novel SAM competitive inhibitor is created, which establishes a clear therapeutic index for targeting EZH2 overexpressing tumors in vivo. Successful clinical translation of therapies targeting lung cancer is facilitated by genomic or immunohistochemical biomarkers. Here, EZH2 was shown to be important for the growth of human lung cancer cell lines expressing high levels of EZH2, while not being necessary for cell lines with lower levels of EZH 2. Thus, EZH2 expression may be a useful biomarker for patient selection or planning stratification in downstream clinical trials. These findings support previous work with prostate cancer, where EZH2 was found to be essential for cell growth in LNCaP-abl cells with higher EZH2 levels, but not in LNCaP cells with lower EZH2 levels (Xu et al, 2012).

EZH2 inhibition has been previously proposed as a therapeutic strategy for NSCLC in the context of BRG1 or EGFR mutations (Fillmore et al, 2015). The present study found that EZH2 inhibitors can sensitize NSCLC cells with EGFR or BRG1 mutations to chemotherapy and suggests a combination of EZH2 inhibition and Topo II inhibition. Here, EZH2 inhibition was determined to be an effective monotherapy in a defined subgroup of NSCLC that overexpresses EZH2 without other known concurrent oncogenic mutations. In summary, this work established the oncogenic role of EZH2 dysregulation in lung adenocarcinoma, created a reliable model of a subset of human diseases, described and characterized a novel chemical probe for studying EZH2 function in vivo, and provided a theoretical basis for human clinical investigations.

Experimental procedures

Mice were housed in a pathogen-free animal facility, and all experiments were approved by the Harvard medical institute and Dana-Farber cancer institute animal Care and use Committee.

Human EZH2 cDNA was cloned into a transgene targeting vector and co-electroporated into V6.5C 57BL/6(F) x 129/sv (m) embryonic stem cells (Open Biosystems, # MES 1402) with a plasmid expressing the FLP recombinase (Beard et al, 2006). Embryonic stem cells were selected for integration of the transgene by PCR. Correctly targeted embryonic stem cells were injected into black 6 blastocysts and the resulting chimeras propagated with BALB/c strain wild type mice for germline transmission of transgenes. EZH2 transgenic mice were crossed with ubiquitin-Cre-ERT2(Ruzankina et al, 2007) or actin-Cre mice (Jackson Laboratories). Mice were kept in mixed background strains and 6-week-old double transgenic mice were given 2mg of 4-hydroxytamoxifen (4-OHT) (Sigma, # 1054029-01) for 4 consecutive days.

Immunohistochemistry

Tissues were fixed in 10% buffered formalin overnight and paraffin embedded (FFPE) overnight for two days. Staining was performed as previously described (Xu et al, 2014).

Gene expression profiling

Total RNA was extracted using trizol (invitrogen) and RNA clearance was performed (Qiagen, # 74204). Due to the platform differences (gene expression levels in EZH2-OE tumors and control lungs were from RNA-seq, whereas gene expression levels in KRAS mutant tumors and EGFR mutant tumors were derived from microarrays (Carretero et al, 2010)), log2 fold change in gene expression was calculated using the expression values of tumors divided by the average of the corresponding normal values. The top 500 most variable genes were selected for clustering in all samples.

Single sample gene set enrichment analysis

Expression profiles of TCGA lung adenocarcinoma tumor and normal lung were used to perform ssGSEA (cancerrgeneme. nih. gov /). For each tumor, driver mutations were determined based on TCGA mutation analysis. All ALK and NF1 mutant tumors were removed from the analysis. High EZH2 tumors were defined as the first 20% of tumors with the highest EZH2 expression and lacking any mutations in KRAS, EGFR, ALK or NF 1. The KRAS and EGFR mutant classes were defined as mutants in KRAS or EGFR and had low EZH2 expression (tumors with 20% of the lowest EZH2 expression). Analysis was performed with MEK (MEK _ up.v1_ UP) and mTOR (mTOR _ up.n4.v1_ DN) gene lists and our own gene list of H3K27Me3 binding genes.

Chromatin immunoprecipitation (ChIP)

According to the ThruPLEX-FD Prep kit (Rubicon # R40048), chromatin pools were prepared with 10-20ng of Input or ChIP'd DNA and sequenced with SE75 Next-Seq.

Preparation of chromatin: mouse lung Tissue was comminuted using the Covaris Tissue Smasher model CP02 following CryoPrep pulserizin manual. Lung tissue was pulverized 1-2 times in tissue TUBE (Covaris # 520071) at setting 4. Approximately 50mg of comminuted lung tissue was cross-linked with pre-warmed 1% formaldehyde (Thermoscientific #28906, diluted in PBS) at 37 ℃ for 20 minutes. Tissues were centrifuged at 1000rpm for 2 minutes and quenched with 0.125M glycine in PBS + 0.5% BSA for 20 minutes at room temperature, centrifuged at 1,000rpm for 2 minutes, washed with PBS +2 Xprotease inhibitor cocktail (PIC) (Roche #11873580001) +5mM sodium butyrate (Millipore #19-137), and then centrifuged at 1,000rpm for 2 minutes. The crosslinked tissue was then lysed with 390. mu.L of ChIP lysis buffer (1% SDS, 10mM EDTA pH8.0, 50mM Tris-HCl pH8.0, 2 XPIC and 5mM sodium butyrate) on ice for 1 hour. The lysate was aliquoted into 3 microtubes (Covaris #520045) and sheared on the Covaris E210 line at 5% duty cycle, 5 intensity, 200 cycles per burst (burst) for a total of 27 minutes. Sheared chromatin was centrifuged at 14,000rpm for 15 min at 4 ℃. The added aliquot was stored, while the remaining chromatin was rapidly frozen and stored at-80 ℃. The addition was increased to 100. mu.l with TE, 10. mu.g of RNAseA (Roche) was added and incubated at 37 ℃ for 30 minutes, followed by 100. mu.g of proteinase K (Roche) and incubated overnight at 65 ℃. The addition was purified and quantified using Qiagen PCR purification kit (# 28104).

Preparation of the library and CHIP: prepared chromatin was thawed on ice while 10 μ G of anti-H3K 27ac (Abcam # Ab4729) or H3K27me3(Cell Signaling # CS9733S) antibody was conjugated to a mixture of magnetic protein a and protein G coupled beads (Invitrogen #100.02D and #100.04D, respectively) in the presence of 0.5% BSA in PBS while rotating at 4 ℃ for 2 hours. The beads were washed 3 times with 0.5% BSA in PBS and 5. mu.g of chromatin was added to H3K27ac ChIP or 10. mu.g of chromatin was added to H3K27me3 ChIP and spun overnight at 4 ℃. The beads were washed 2 times with Tris-base RIPA buffer (0.1% SDS, 1% Triton X-100, 10mM Tris-HCl pH7.4, 1mM EDTA pH8.0, 0.1% sodium deoxycholate), 2 times with 0.3M NaCl RIPA (0.1% SDS, 1% Triton X-100, 10mM Tris-HCl pH7.4, 1mM EDTA pH8.0, 0.1% sodium deoxycholate, 0.3M NaCl), 2 times with LiCl buffer (250mM LiCl, 0.5% NP-40, 0.5% deoxycholate, 1mM EDTA pH8.0,10mM Tris-HCl pH8.0) and 2 times with TE buffer pH7.6(Fisher Scientific catalog number BP 2474-1). The beads were resuspended in 100 μ L TE and RNAseA and PK digestion/reverse cross-linking and purification performed as described in chromatin preparation.

Docking and ligand interaction assays

All structural modeling was performed by SBgrid using Schrodinger calculation software. Ligands of interest were imported into Maestro and exhaustive conformational searches were performed by Monte-Carlo simulations using the MM5 force field. The resulting constellations are clustered by 3D similarity, and a sample structure is selected from each structure for subsequent docking. Docking was performed on a previously reported published binding model in which ligand binding sites were used to define the receptor lattice (Kalinic et al, 2014). The protein was pre-treated with Maestro and gridded and docked with Glide using standard input parameters. All docking was performed with XP level precision and results were ranked using Glide Score. The ligand interaction map was generated by Maestro for the top docking pose.

Measurement results of general synthetic procedures and biochemical assays

All reactions were carried out in an oven dried or flame dried round bottom flask. The flask was fitted with a rubber septum and the reaction was run under positive pressure nitrogen. Flash column chromatography using silica gel as described by Still et al (Pore diameter of 40-63 μm, 4-6% H₂O content, Zeochem). Analytical thin layer chromatography was performed using glass plates pre-coated with 0.25mm 230-400 mesh silica gel (254 nm) impregnated with a fluorescent indicator. The thin layer chromatography plate was observed by exposure to iodine vapor. All intermediates and final products were NMR-ed with protons and carbon-13: ( ¹H NMR and¹³c NMR) spectrum and Mass Spectrum (MS).

PRC2 methyltransferase assay: recombinant five-component PRC2

(EZH2/EED/SUZ12/RBBP4/AEBP2) was co-expressed and purified in Sf9 as described (Cao and Zhang, 2004). PRC2 activity was measured using a radioactive Scintillation Proximity Assay (SPA) performed in 384-well optiplates (perkin elmer). For determination of IC50, 2.3nM PRC2 was incubated with 1. mu.M histone H3(21-44) -lys (biotin) (Anaspec), 1.5. mu.MSAM (NEB)) and 500nM 3H-SAM in 20. mu.L of reaction buffer containing compound or DMSO (50mM Tris pH 8.5, 5mM DTT and 0.01% Tween-20) for 90 minutes at room temperature. The reaction was quenched with TCA, followed by addition of PVT streptavidin-coated SPA beads (Perkin Elmer; 40. mu.L 140ng diluted in PBS) and incubation at room temperature for 1 hour. CPM values were measured using a TopCount NXT reader. Percent activity values were calculated by setting the average background (no enzyme wells) to 0% and the average DMSO wells to 100% activity. The standard deviation was determined from four replicate measurements of each compound concentration. Data were analyzed and plotted using GraphPad PRISM v6, IC calculated using a 'log (inhibitor) vs. normalized response-variable slope' analysis module ₅₀。

To determine the mechanism of action and Ki values of JQEZ5, reactions were carried out in the presence of various concentrations of SAM/3H-SAM (1: 20 ratio) with a fixed concentration of 1. mu.M histone H3 peptide as described above. The data were analyzed and plotted using the "enzyme kinetics-inhibition" and "enzyme kinetics-substrate versus velocity" analysis modules in GraphPad PRISM v 6.

Exemplary results regarding the biochemical selectivity of EZH2 versus EZH1 for selected compounds herein are shown in fig. 22. The compounds EZH2-16, EZ-27 and EZ-005 were about 10-fold selective for EZH2 over EZH 1. The compounds EZ-26 and EZ-20 are about 20 to about 40 times more selective for EZH2 than for EZH 1.

The compounds EZ-41 and EZH2 were used for docking experiments. See, McCabe et al, Nature,492, 108-. The docking results show that EZ-41 and EZH2 can form a covalent bond.

EZH1/2 emissive methyltransferase assay

The recombinant five-component PRC2-EZH2 (EZH2/EED/SUZ12/RBBP4/AEBP2) and PRC2-EZH1 complex (EZH1/EED/SUZ12/RBBP4/AEBP2) were co-expressed in Sf9 and purified as described in (Cao and Zhang, 2004). Methyltransferase activity was measured using a radioactive Scintillation Proximity Assay (SPA) performed in 384-well optiplates (perkin elmer). To determine IC50, 2.3nM PRC2-EZH1/2 was incubated with 1. mu.M histone H3 (21-44) -lys (biotin) (Anaspec), 1.5. mu.M SAM (NEB) and 500nM 3H-SAM in 20. mu.L of reaction buffer containing compound or DMSO (50mM Tris pH 8.5,5mM DTT and 0.01% Tween-20) for 90 minutes at room temperature. The reaction was quenched with TCA, and PVT streptavidin-coated SPA beads (Perkin Elmer; 40. mu.L 140ng diluted in PBS) were added and incubated at room temperature for 1 hour. CPM values were measured using a TopCount NXT reader. Percent activity values were calculated by setting the average background (no enzyme wells) to 0% and the average DMSO wells to 100% activity. The standard deviation was determined from four replicate measurements of each compound concentration. Data were analyzed and plotted using GraphPad PRISM v6, using a 'log (inhibitor) versus normalized response-variable slope' analysis module to calculate IC ₅₀(FIG. 32 and FIG. 33).

To determine the MOA and Ki values, the reaction was carried out as described above in the presence of various concentrations of SAM/3H-SAM (1: 20 ratio) with a fixed concentration of 1. mu.M histone H3 peptide. The data were analyzed and plotted using the "enzyme kinetics-inhibition" analysis module in GraphPad PRISM v 6.

EZH2 ligand-displacement AlphaScreen assay

We used a novel ligand competition AlphaScreen assay to assess the binding affinity of SAMs to EZH2 and determined compound IC50 in nonradioactive form (fig. 31 and 32). In 384-well AlphaPlates (Perkin Elmer), 61.5nM PRC2-EZH2 and 62.5nM EZ-06 were diluted in 20. mu.L reaction buffer (50mM Tris pH 8.5, 5mM DTT and 0.01% Tween-20) containing competitor compound or DMSO. After 30 min incubation, donor beads containing streptavidin diluted to 20 ng/. mu.L in 1 Xepigenetics buffer (Perkin Elmer) and20 μ l of detection solution of anti-FLAG receptor beads. After 1 hour incubation at room temperature, luminescence was measured on an Envision 2104 plate reader. Percent activity values were calculated by setting the average background (no enzyme wells) to 0% and the average DMSO wells to 100% activity. The standard deviation was determined from four replicate measurements of compound concentration (figure 32). Data were analyzed and plotted using GraphPad PRISM v6, IC calculated using a 'log (inhibitor) versus normalized response-variable slope' analysis module ₅₀(FIG. 32).

EZH2 ligand-displacement FP assay

In a 384-well blackboard (Nunc), 43nM PRC2-EZH2 and 32nM EZ05-FITC were diluted in 20. mu.L of reaction buffer (50mM Tris pH8.5, 5mM DTT and 0.01% Tween-20) containing competitor compound or DMSO. After 30 minutes of incubation, fluorescence polarization (mP) was measured using a Wallac Envision 2104 Multilabel Reader (FP FITC Dual-light Module; excitation: 480nm, emission of S and P channels: 535nm) (FIG. 34). Normalized mP values were calculated by setting the average background (no enzyme wells) to 0% and the average DMSO wells to 100% activity. Standard deviation was determined from four replicate measurements of compound concentration (fig. 35, fig. 36, fig. 33). Data were analyzed and plotted using GraphPad PRISM v6, IC calculated using a 'log (inhibitor) versus normalized response-variable slope' analysis module₅₀(FIG. 37 and FIG. 33).

Intracellular EZH2 binding assay

MDA-MB-231 cells (ATCC) were cultured in Ham's F12K medium supplemented with 10% (v/v) FBS and 100UmL-1 penicillin-streptomycin. Cells were diluted at 1e5 cells/mL in culture medium and seeded at 40. mu.L/well in black, clear-bottomed 384-well plates (Aurora IQ-EB 384) at 37 ℃/5% CO₂Incubate for 24 hours. To assess overall involvement of endogenous EZH2, cells were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.3% Triton X-100 in PBS for 20 minutes. Cells were then incubated with 40. mu.L of 1nM Hoechst 33342(Life Technologies), 5. mu.M TAMRA-EZ05 and various concentrations of competitor compound. After 1 hour incubation at room temperature, cells were washed 4X with 40 μ L PBS, followed by imaging. To assess cell permeability and involvement of endogenous EZH2, TAMRA-EZ05 and competitor compounds were needle transferred into culture media and allowed to stand at 37 ℃/5% CO ₂The cells were incubated for 2 hours next, then fixed and counterstained (fig. 38, fig. 39, fig. 40). All plates were imaged on an ImageXpress Micro automatic microscope (Molecular Devices) using a 10X objective with laser based focusing. Image analysis was performed using the Cell sorting module in metaxpress (molecular devices) to determine mean nuclear fluorescence and nuclear counts per well. The mean and standard deviation (STDEV) were calculated for all DMSO wells in each assay plate. Data were analyzed and plotted using GraphPad PRISM v6, IC determined using a 'log (inhibitor) versus normalized response-variable slope' analysis module₅₀The value is obtained.

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Equivalents and scope

In the claims, for example, "a," "an," and "the" may mean one or more than one unless the context clearly dictates otherwise. For claims or descriptions that include an "or" between one or more members of a group, if one, more than one, or all of the members of the group are present, employed, or otherwise relevant to a given product or process, then such claim or description is deemed to be satisfied unless the context clearly dictates otherwise. The invention includes embodiments in which only one member of the group is present, employed, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one or all members of the group are present, employed or otherwise relevant in connection with a given product or process.

Furthermore, the present invention includes all variations, combinations, and permutations in which one or more limitations, elements, phrases, and descriptive terms from one or more of the listed claims are introduced into another claim. For example, any claim that is dependent on another claim may be modified to include one or more of the definitions of any other claim that is dependent on the same base claim. When elements are presented as a list, for example, in the form of a markush group, each subset of the elements is also disclosed and any element can be removed from the group. It will be understood that in general, when the invention or aspects of the invention are specified to include a particular element and/or feature, some embodiments of the invention or aspects of the invention consist of, or consist essentially of, such element and/or feature. For the sake of brevity, those embodiments are not specifically mentioned herein with words. It should also be noted that the terms "comprising" and "comprises" are intended to be open-ended and allow for the inclusion of other elements or steps. When ranges are given, endpoints are included. Furthermore, unless otherwise indicated or clearly indicated by context and understanding of one of ordinary skill in the art, values expressed as ranges can take any specific value or subrange within the ranges set forth in the various embodiments of the invention, up to one tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

This application refers to various published patents, published patent applications, periodicals, and other publications, which are incorporated herein by reference in their entirety. In the event of conflict between any of the references cited and the present application, the present specification shall control. Furthermore, any particular embodiment of the invention falling within the prior art may be explicitly excluded from any one or more claims. Since such embodiments are considered to be known to those skilled in the art, they may be excluded even if the exclusion is not explicitly mentioned herein. Any particular embodiment of the invention may be excluded from any claim for any reason, whether or not related to the presence of prior art.

Those skilled in the art will understand, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the specific embodiments described herein is not intended to be limited by the foregoing description, but is instead set forth in the following claims. Those skilled in the art will appreciate that various changes and modifications may be made to the disclosure without departing from the spirit or scope of the disclosure as defined by the claims.

The invention comprises the following contents:

1. a compound of formula (I), or a pharmaceutically acceptable salt thereof:

wherein:

R^A1is halogen, substituted OR unsubstituted alkyl, substituted OR unsubstituted alkenyl, substituted OR unsubstituted alkynyl, substituted OR unsubstituted carbocyclyl, substituted OR unsubstituted heterocyclyl, substituted OR unsubstituted aryl, substituted OR unsubstituted heteroaryl, -OR^a、–N(R^a)₂、–SR^a、–CN、–SCN、–C(＝NR^a)R^a、–C(＝NR^a)OR^a、–C(＝NR^a)N(R^a)₂、–C(＝O)R^a、–C(＝O)OR^a、–C(＝O)N(R^a)₂、–NO₂、–NR^aC(＝O)R^a、–NR^aC(＝O)OR^a、–NR^aC(＝O)N(R^a)₂、–OC(＝O)R^a、–OC(＝O)OR^a、–OC(＝O)N(R^a)₂A marker or

Each R^aIndependently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or both R^aLinked to form a substituted or unsubstituted heterocyclic ring or a substituted or unsubstituted heteroaromatic ring;

R^Ais hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R^BIs hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

or R^AAnd R^BAre linked to form a substituted or unsubstituted carbocyclic ring or a substituted or unsubstituted heterocyclic ring;

R^Cis hydrogen, substituted or unsubstituted C_1-6An alkyl or nitrogen protecting group;

R^A2is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, a nitrogen protecting group, a label, or a warhead;

R^A3is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^a、–N(R^a)₂Or a warhead;

R^A4is hydrogen, substituted or unsubstituted C_1-6An alkyl or nitrogen protecting group; and

R^A5is of the formula: wherein:

R^A6is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^aor-N (R)^a)₂；

R^A7Is hydrogen, halogen, substituted or unsubstituted C_2-6Alkyl, substituted OR unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 OR 2 double bonds in the carbocyclic ring system, -OR^aor-N (R)^a)₂；

R^A8Is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^aor-N (R)^a)₂；

R^A9Is hydrogen, halogen, substituted or unsubstituted C _1-6Alkyl, substituted OR unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 OR 2 double bonds in the carbocyclic ring system, -OR^aor-N (R)^a)₂；

R^A10is-OR^a、–N(R^a)₂Or a warhead;

each R^A11Independently is halogen, substituted or unsubstituted C_1-6Alkyl, substituted OR unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 OR 2 double bonds in the carbocyclic ring system, -OR^aor-N (R)^a)₂；

n is 0, 1, 2, 3 or 4;

R^A12is hydrogen, substituted or unsubstituted C_1-6Alkyl, nitrogen protecting group or warhead;

each R^A13Independently is halogen, substituted or unsubstituted C_1-6Alkyl, substituted OR unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 OR 2 double bonds in the carbocyclic ring system, -OR^aor-N (R)^a)₂；

m is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9;

R^A14is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^aor-N (R)^a)₂；

R^A15Is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^aor-N (R)^a)₂；

R^A16Is hydrogen, halogen, substituted or unsubstituted C_2-6Alkyl, substituted OR unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 OR 2 double bonds in the carbocyclic ring system, -OR^aor-N (R)^a)₂(ii) a And

R^A17is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted C_1-6Alkyl, nitrogen protecting group or warhead.

2. The compound of embodiment 1, wherein the compound is of the formula:

3. The compound of embodiment 1, wherein the compound is of the formula:

4. the compound of embodiment 1, wherein the compound is of the formula:

5. the compound of embodiment 1, wherein the compound is of the formula:

6. the compound of embodiment 1, wherein the compound is of the formula:

7. the compound of embodiment 1, wherein the compound is of the formula:

wherein R is^A14Is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or a nitrogen protecting group.

8. The compound of embodiment 7, wherein the compound is of the formula:

9. the compound of embodiment 7, wherein the compound is of the formula:

10. the compound of embodiment 7, wherein the compound is of the formula:

11. The compound of embodiment 7, wherein the compound is of the formula:

12. the compound of embodiment 1, wherein the compound is of the formula:

13. the compound of embodiment 1, wherein the compound is of the formula:

14. the compound of embodiment 1, wherein the compound is of the formula:

15. the compound of embodiment 1, wherein the compound is of the formula:

16. the compound of embodiment 1, wherein the compound is of the formula:

17. the compound of embodiment 1, wherein the compound is of the formula:

18. the compound of embodiment 1, wherein the compound is of the formula:

19. the compound of embodiment 1, wherein the compound is of the formula:

20. a compound of any of embodiments 1-6 and 12-19 wherein R^A1Is a substituted or unsubstituted 3-to 7-membered monocyclic heterocyclyl containing 0, 1 or 2 double bonds in the heterocyclyl system, wherein 1, 2 or 3 atoms in the heterocyclyl system are independently nitrogen, oxygen or sulfur.

21. A compound of embodiment 20 wherein R^A1Is a substituted or unsubstituted piperazinyl group.

22. A compound of embodiment 20 wherein R^A1Is of the formula:

23. a compound of embodiment 20 wherein R^A1Is of the formula:wherein:

L^Ais a bond or substituted or unsubstituted C_1–100A hydrocarbon chain, optionally wherein one or more chain atoms in the hydrocarbon chain are independently-O-, -S-, or-NR^a-replacing; and

X^Ais a small molecule, peptide, protein or polynucleotide.

24. A compound of embodiment 23 wherein R^A1Is of the formula:

wherein:

r is 0 or 1;

k is an integer from 0 to 11, inclusive;

p is an integer from 0 to 10, inclusive; and

q is an integer from 0 to 10, inclusive.

25. A compound of embodiment 23 or 24 wherein X^AA small molecule of the formula:

wherein Z^Ais-O-or-NH-.

26. A compound of embodiment 20 wherein R^A1Is a substituted or unsubstituted oxetanyl group, a substituted or unsubstituted tetrahydrofuryl group, a substituted or unsubstituted pyrrolidinyl group, a substituted or unsubstituted tetrahydropyranyl group, a substituted or unsubstituted piperidinyl group, a substituted or unsubstituted morpholinyl group, a substituted or unsubstituted azepanyl group, or a substituted or unsubstituted diazepanyl group.

27. A compound of any of embodiments 1-6 and 12-19 wherein R^A1is-CN.

28. A compound of any of embodiments 1-6 and 12-19 wherein R^A1is-C (═ O) N (R)^a)₂。

29. A compound of embodiment 28 wherein R^A1is-C (═ O) NH₂。

30. A compound of any of embodiments 1-6 and 12-19 wherein R^A1Is substituted OR unsubstituted alkyl, -OR^a、–N(R^a)₂、–C(＝O)OR^aor-NR^aC(＝O)R^a。

31. A compound of embodiment 30 wherein R^A1Is unsubstituted C_1-6Alkyl, -OMe, -NH₂、–N(Me)₂-C (═ O) OH, -C (═ O) OMe or-NHC (═ O) Me.

32. A compound of any of embodiments 1-5, 7-10 and 12-31 wherein R^A2Is a substituted or unsubstituted acyl group.

33. A compound of embodiment 32 wherein R^A2is-C (═ O) R^aWherein R is^aIs a substituted or unsubstituted vinyl group.

34. A compound of any of embodiments 1-5, 7-10 and 12-31 wherein R^A2Is a substituted or unsubstituted alkyl group.

35. A compound of embodiment 34 wherein R^A2Is substituted or unsubstituted C_1-6An alkyl group.

36. A compound of embodiment 34 wherein R^A2Is i-Pr.

37. A compound of embodiment 34 wherein R^A2sec-Bu, t-Bu or unsubstituted 3-pentyl.

38. A compound of embodiment 34 wherein R^A2is-CH₂C(＝O)–NH–N＝C(R^a)₂。

39. A compound of any of embodiments 1-5, 7-10 and 12-31 wherein R ^A2Is a substituted or unsubstituted carbocyclyl.

40. A compound of embodiment 39 wherein R^A2Is a substituted or unsubstituted 3-to 7-membered monocyclic carbocyclic group containing 0, 1 or 2 double bonds in the carbocyclic ring system.

41. A compound of embodiment 39 wherein R^A2Is unsubstituted cyclopropyl, unsubstituted cyclobutyl orSubstituted cyclopentyl groups.

42. A compound of any of embodiments 1-5, 7-10 and 12-31 wherein R^A2Is a substituted or unsubstituted heterocyclic group.

43. A compound of embodiment 42 wherein R^A2Is a substituted or unsubstituted 3-to 7-membered monocyclic heterocyclyl containing 0, 1 or 2 double bonds in the heterocyclyl system, wherein 1, 2 or 3 atoms in the heterocyclyl system are independently nitrogen, oxygen or sulfur.

44. A compound of embodiment 42 wherein R^A2Is a substituted or unsubstituted tetrahydropyranyl group.

45. A compound of embodiment 42 wherein R^A2Is of the formula:

46. a compound of embodiment 42 wherein R^A2Is a substituted or unsubstituted oxetanyl group, a substituted or unsubstituted tetrahydrofuranyl group, a substituted or unsubstituted pyrrolidinyl group, a substituted or unsubstituted piperidinyl group, a substituted or unsubstituted morpholinyl group, or a substituted or unsubstituted piperazinyl group.

47. A compound of any of embodiments 1-46 wherein R ^A3Is hydrogen.

48. A compound according to any one of embodiments 1-3, 7-8, 12, 14, 16, 18 and 20-47 wherein R^A3Is substituted or unsubstituted C_1-6An alkyl group.

49. A compound of embodiment 48 wherein R^A3Is Me.

50. A compound according to any one of embodiments 1-3, 7-8, 12, 14, 16, 18 and 20-47 wherein R^A3Is halogen.

51. A compound according to any one of embodiments 1-3, 7-8, 12, 14, 16, 18 and 20-47 wherein R^A3is-N (R)^a)₂。

52. A compound of embodiment 51 wherein R^A3is-N (R)^a)C(＝O)R^aWherein R is^aIs a substituted or unsubstituted vinyl group.

53. A compound of any of embodiments 1-52 wherein R^A4Is hydrogen.

54. A compound of any of embodiments 1 and 20-53 wherein R^A5Is of the formula:

55. a compound of embodiment 54 wherein R^A5Is of the formula:

56. a compound of any of embodiments 1-4, 7-9 and 20-54 wherein R^A6Is substituted or unsubstituted C_1-6An alkyl group.

57. A compound of embodiment 56 wherein R^A6Is Me.

58. A compound of any of embodiments 1-4, 7-9 and 20-54 wherein R^A6is-N (R)^a)₂Wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups.

59. A compound of any one of embodiments 1-4, 7-9, 20-54 and 56-58 wherein R ^A7Is substituted or unsubstituted C_2-6An alkyl group.

60. A compound of embodiment 59 wherein R^A7Is n-Pr.

61. A compound of any one of embodiments 1-4, 7-9, 20-54 and 56-58 wherein R^A7Is a substituted or unsubstituted cyclopropyl or-N (R)^a)₂Wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups.

62. A compound of any of embodiments 1 and 20-53 wherein R^A5Is of the formula:

63. a compound of embodiment 62 wherein R^A5Is of the formula:

64. a compound of any of embodiments 1, 12-13 and 20-53 wherein R^A8Is substituted or unsubstituted C_1-6An alkyl group.

65. A compound of embodiment 64 wherein R^A8Is Me.

66. A compound of any of embodiments 1, 12-13 and 20-53 wherein R^A8is-N (R)^a)₂Wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups.

67. A compound of any one of embodiments 1, 12-13, 20-53, 62, and 64-66 wherein R^A9Is substituted or unsubstituted C_1-6An alkyl group.

68. A compound of embodiment 67 wherein R^A9Me, Et or n-Pr.

69. A compound of any one of embodiments 1, 12-13, 20-53, 62, and 64-66 wherein R^A9Is a substituted OR unsubstituted cyclopropyl, -OR ^aor-N (R)^a)₂Wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6An alkyl group, an oxygen protecting group when attached to an oxygen atom, or a nitrogen protecting group when attached to a nitrogen atom.

70. A compound of any of embodiments 1 and 20-53 wherein R^A5Is of the formula:

71. a compound of embodiment 70 wherein R^A5Is of the formula:

72. a compound of embodiment 70 wherein R^A5Is of the formula:

73. a compound of embodiment 70 wherein R^A5Is of the formula:

74. a compound of any one of embodiments 1, 14-15, 20-53, 70-71 and 73 wherein R^A10is-OR^a。

75. A compound of embodiment 74 wherein R^A10is-OH.

76. A compound of any one of embodiments 1, 14-15, 20-53, 70-71 and 73 wherein R^A10is-N (R)^a)₂。

77. A compound of embodiment 76 wherein R^A10is-NHC (═ O) R^aWherein R is^aIs a substituted or unsubstituted vinyl group.

78. A compound of any one of embodiments 1, 14-15, 20-53, 70-71 and 73-77 wherein at least one R^A11Is halogen.

79. A compound of any one of embodiments 1, 14-15, 20-53, 70-71 and 73-77 wherein at least one R^A11Is substituted or unsubstituted C_1-6An alkyl group.

80. A compound of any one of embodiments 1, 14-15, 20-53, 70-71 and 73-77 wherein at least one R ^A11Is substituted or unsubstituted C_1-6Alkyl, substituted OR unsubstituted cyclopropyl, -OR^aor-N (R)^a)₂Wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6An alkyl group, an oxygen protecting group when attached to an oxygen atom, or a nitrogen protecting group when attached to a nitrogen atom.

81. A compound of any of embodiments 1 and 20-53 wherein R^A5Is of the formula：

82. A compound of embodiment 81 wherein R^A5Is of the formula:

83. a compound of embodiment 81 wherein R^A5Is of the formula:

84. a compound of any of embodiments 1, 16-17, 20-53 and 81-82 wherein R^A12Is hydrogen.

85. A compound according to any one of embodiments 1, 16-17, 20-53, 81-82 and 84 wherein at least one R^A13Is substituted or unsubstituted C_1-6An alkyl group.

86. A compound according to any one of embodiments 1, 16-17, 20-53, 81-82 and 84 wherein at least one R^A13Is halogen, substituted OR unsubstituted cyclopropyl, -OR^aor-N (R)^a)₂Wherein each R is^aIndependently hydrogen, substituted or unsubstituted C_1-6An alkyl group, an oxygen protecting group when attached to an oxygen atom, or a nitrogen protecting group when attached to a nitrogen atom.

87. A compound of any of embodiments 1 and 20-53 wherein R^A5Is of the formula:

88. a compound of embodiment 87 wherein R^A5Is of the formula:

89. A compound of any of embodiments 1, 18-53 and 87 wherein R^A14Is H.

90. A compound of any of embodiments 1, 18-53, 87 and 89 wherein R^A15Is substituted or unsubstituted C_1-6An alkyl group.

91. A compound of embodiment 90 wherein R^A15Is Me.

92. A compound of any of embodiments 1, 18-53, 87, and 89-91 wherein R^A16Is substituted or unsubstituted C_2-6An alkyl group.

93. A compound of embodiment 92 wherein R^A16Is n-Pr.

94. A compound of any of embodiments 1, 18-53, 87, and 89-93 wherein R^A17Is H.

95. A compound of any of embodiments 1, 18-53, 87, and 89-93 wherein R^A17is-C (═ O) R^aWherein R is^aIs a substituted or unsubstituted vinyl group.

96. The compound of embodiment 1, wherein the compound is of the formula:

97. the compound of embodiment 1, wherein the compound is of the formula:

98. a compound of formula (II), or a pharmaceutically acceptable salt thereof:

wherein:

R^B1is halogen, substituted OR unsubstituted alkyl, substituted OR unsubstituted alkenyl, substituted OR unsubstituted alkynyl, substituted OR unsubstituted carbocyclyl, substituted OR unsubstituted heterocyclyl, substituted OR unsubstituted aryl, substituted OR unsubstituted heteroaryl, -OR ^b、–N(R^b)₂、–SR^b、–CN、–SCN、–C(＝NR^b)R^b、–C(＝NR^b)OR^b、–C(＝NR^b)N(R^b)₂、–C(＝O)R^b、–C(＝O)OR^b、–C(＝O)N(R^b)₂、–NO₂、–NR^bC(＝O)R^b、–NR^bC(＝O)OR^b、–NR^bC(＝O)N(R^b)₂、–OC(＝O)R^b、–OC(＝O)OR^b、–OC(＝O)N(R^b)₂A marker or

Each R^bIndependently hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, a nitrogen protecting group when attached to a nitrogen atom, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom, or both R^bAre linked to form a substituted or unsubstituted heterocyclic ringOr a substituted or unsubstituted heteroaryl ring;

R^Ais hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R^Bis hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

or R^AAnd R ^BAre linked to form a substituted or unsubstituted carbocyclic ring or a substituted or unsubstituted heterocyclic ring;

R^Cis hydrogen, substituted or unsubstituted C_1-6An alkyl or nitrogen protecting group;

R^B2is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, a nitrogen protecting group, a label, or a warhead; and

R^B3is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^bor-N (R)^b)₂；

R^B4Is hydrogen, substituted or unsubstituted C_1-6An alkyl or nitrogen protecting group; and

R^B5is of the formula:

wherein:

R^B6is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl or-N (R)^b)₂；

R^B7Is hydrogen, halogen, substituted or unsubstituted C_2-6Alkyl or substituted or unsubstituted containing 0, 1 or 2 double bonds in the carbocyclic ring systemA substituted 3-to 7-membered monocyclic carbocyclic group;

R^B8is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl or-N (R)^b)₂；

R^B9Is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, or substituted or unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 or 2 double bonds in the carbocyclic ring system;

R^B10is-OR^b、–N(R^b)₂Or a warhead;

each R^B11Independently is halogen, substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 or 2 double bonds in the carbocyclic ring system, or-N (R) ^b)₂；

u is 0, 1, 2, 3 or 4;

R^B12is hydrogen, substituted or unsubstituted C_1-6Alkyl, nitrogen protecting group or warhead;

each R^B13Independently is halogen, substituted or unsubstituted C_1-6Alkyl, substituted or unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 or 2 double bonds in the carbocyclic ring system, or-N (R)^b)₂；

v is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9;

R^B14is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^bor-N (R)^b)₂；

R^B15Is hydrogen, halogen, substituted or unsubstituted C_1-6Alkyl, -OR^bor-N (R)^b)₂；

R^B16Is hydrogen, halogen, substituted or unsubstituted C_2-6Alkyl, substituted OR unsubstituted 3-to 7-membered monocyclic carbocyclyl containing 0, 1 OR 2 double bonds in the carbocyclic ring system, -OR^bor-N (R)^b)₂(ii) a And

R^B17is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted C_1-6Alkyl, nitrogen protecting groups or warheads；

With the proviso that when R^B2Is i-Pr, R^B3Is hydrogen and R^B5Is of formula (ii-1), then R^B1Is not Me, -OMe or-NH (═ O) Me; and

with the proviso that when R^B2Is unsubstituted C_3-5Alkyl radical, R^B3Is Me or halogen, R^B5Is of formula (ii-1), then R^B1Is not Me,–OMe、–NH₂、–N(Me)₂-C (═ O) OH, -C (═ O) OMe or-NH (═ O) Me.

99. The compound of embodiment 98, wherein the compound is of the formula:

100. The compound of embodiment 98, wherein the compound is of the formula:

101. the compound of embodiment 98, wherein the compound is of the formula:

102. the compound of embodiment 98, wherein the compound is of the formula:

103. the compound of embodiment 98, wherein the compound is of the formula:

104. the compound of embodiment 98, wherein the compound is of the formula:

wherein R is^B14Is hydrogen, substituted or unsubstituted acyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or a nitrogen protecting group.

105. The compound of embodiment 104, wherein the compound is of the formula:

106. the compound of embodiment 104, wherein the compound is of the formula:

107. The compound of embodiment 104, wherein the compound is of the formula:

108. the compound of embodiment 104, wherein the compound is of the formula:

109. the compound of embodiment 98, wherein the compound is of the formula:

110. the compound of embodiment 98, wherein the compound is of the formula:

111. the compound of embodiment 98, wherein the compound is of the formula:

112. the compound of embodiment 98, wherein the compound is of the formula:

113. the compound of embodiment 98, wherein the compound is of the formula:

114. the compound of embodiment 98, wherein the compound is of the formula:

115. the compound of embodiment 98, wherein the compound is of the formula:

116. the compound of embodiment 98, wherein the compound is of the formula:

117. The compound of any one of embodiments 98-103 and 109-116 wherein R^B1Is a substituted or unsubstituted 3-to 7-membered monocyclic heterocyclyl containing 0, 1 or 2 double bonds in the heterocyclyl system, wherein 1, 2 or 3 atoms in the heterocyclyl system are independently nitrogen, oxygen or sulfur.

118. A compound of embodiment 117 wherein R^B1Is a substituted or unsubstituted piperazinyl group.

119. A compound of embodiment 117 wherein R^B1Is of the formula:

120. a compound of embodiment 117 wherein R^B1Is of the formula:wherein:

L^Bis a bond or substituted or unsubstituted C_1–100A hydrocarbon chain, optionally wherein one or more chain atoms in the hydrocarbon chain are independently-O-, -S-, or-NR^b-replacing; and

X^Bis a small molecule, peptide, protein or polynucleotide.

121. A compound of embodiment 120 wherein R^B1Is of the formula:

wherein:

z is 0 or 1;

w is an integer from 0 to 11, inclusive;

x is an integer from 0 to 10, inclusive; and

y is an integer from 0 to 10, inclusive.

122. A compound of embodiment 120 or 121 wherein X^BA small molecule of the formula:

wherein Z^Bis-O-or-NH-.

123. A compound of embodiment 117 wherein R^B1Is a substituted or unsubstituted oxetanyl group, a substituted or unsubstituted tetrahydrofuryl group, a substituted or unsubstituted pyrrolidinyl group, a substituted or unsubstituted tetrahydropyranyl group, a substituted or unsubstituted piperidinyl group, a substituted or unsubstituted morpholinyl group, a substituted or unsubstituted azepanyl group, or a substituted or unsubstituted diazepanyl group.

124. The compound of any one of embodiments 98-103 and 109-116 wherein R^B1is-CN.

125. Embodiment 98A compound of any one of-103 and 109-116, wherein R^B1is-C (═ O) N (R)^b)₂。

126. A compound of embodiment 125 wherein R^B1is-C (═ O) NH₂。

127. The compound of any one of embodiments 98-103 and 109-116 wherein R^B1Is substituted OR unsubstituted alkyl, -OR^b、–N(R^b)₂、–C(＝O)OR^bor-NR^bC(＝O)R^b。

128. A compound of embodiment 127 wherein R^B1Is unsubstituted C_1-6Alkyl, -OMe, -NH₂、–N(Me)₂-C (═ O) OH, -C (═ O) OMe or-NHC (═ O) Me.

129. The compound according to any one of embodiments 98-102, 104-107 and 109-128, wherein R^B2Is a substituted or unsubstituted acyl group.

130. A compound of embodiment 129 wherein R^B2is-C (═ O) R^bWherein R is^bIs a substituted or unsubstituted vinyl group.

131. The compound according to any one of embodiments 98-102, 104-107 and 109-128, wherein R^B2Is a substituted or unsubstituted alkyl group.

132. A compound of embodiment 131 wherein R^B2Is substituted or unsubstituted C_1-6An alkyl group.

133. A compound of embodiment 131 wherein R^B2Is i-Pr.

134. A compound of embodiment 131 wherein R^B2Unsubstituted sec-Bu, t-Bu or unsubstituted 3-pentyl.

135. A compound of embodiment 131 wherein R^B2is-CH₂C(＝O)–NH–N＝C(R^b)₂。

136. The compound according to any one of embodiments 98-102, 104-107 and 109-128, wherein R^B2Is a substituted or unsubstituted carbocyclyl.

137. A compound of embodiment 136 wherein R^B2To contain 0, 1 or 2 bis in a carbocyclic ring systemA bonded substituted or unsubstituted 3-to 7-membered monocyclic carbocyclic group.

138. A compound of embodiment 136 wherein R^B2Is unsubstituted cyclopropyl, unsubstituted cyclobutyl or unsubstituted cyclopentyl.

139. The compound according to any one of embodiments 98-102, 104-107 and 109-128, wherein R^B2Is a substituted or unsubstituted heterocyclic group.

140. A compound of embodiment 139 wherein R^B2Is a substituted or unsubstituted 3-to 7-membered monocyclic heterocyclyl containing 0, 1 or 2 double bonds in the heterocyclyl system, wherein 1, 2 or 3 atoms in the heterocyclyl system are independently nitrogen, oxygen or sulfur.

141. A compound of embodiment 139 wherein R^B2Is a substituted or unsubstituted tetrahydropyranyl group.

142. A compound of embodiment 139 wherein R^B2Is of the formula:

143. a compound of embodiment 139 wherein R^B2Is a substituted or unsubstituted oxetanyl group, a substituted or unsubstituted tetrahydrofuranyl group, a substituted or unsubstituted pyrrolidinyl group, a substituted or unsubstituted piperidinyl group, a substituted or unsubstituted morpholinyl group, or a substituted or unsubstituted piperazinyl group.

144. A compound of any of embodiments 98-143 wherein R^B3Is hydrogen.

145. The compound of any one of embodiments 98-100, 104-, 105, 109, 111, 113, 115 and 117-, 144 wherein R^B3Is substituted or unsubstituted C_1-6An alkyl group.

146. A compound of embodiment 145 wherein R^B3Is Me.

147. The compound of any one of embodiments 98-100, 104-, 105, 109, 111, 113, 115 and 117-, 144 wherein R^B3Is halogen.

148. The compound of any one of embodiments 98-100, 104-, 105, 109, 111, 113, 115 and 117-, 144 wherein R^B3is-N (R)^b)₂。

149. A compound of embodiment 148 wherein R^B3is-N (R)^b)C(＝O)R^bWherein R is^bIs a substituted or unsubstituted vinyl group.

150. A compound of any of embodiments 98-149 wherein R^B4Is hydrogen.

151. The compound of any one of embodiments 98 and 117-150 wherein R^B5Is of the formula:

152. a compound of embodiment 151 wherein R^B5Is of the formula:

153. the compound according to any one of embodiments 98-101, 104-106 and 117-151, wherein R is^B6Is substituted or unsubstituted C_1-6An alkyl group.

154. A compound of embodiment 153 wherein R^B6Is Me.

155. The compound according to any one of embodiments 98-101, 104-106 and 117-151, wherein R is^B6is-N (R) ^b)₂Wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups.

156. The compound according to any one of embodiments 98-101, 104-106 and 117-155, wherein R is^B7Is substituted or unsubstituted C_2-6An alkyl group.

157. A compound of embodiment 156 wherein R^B7Is n-Pr.

158. The compound according to any one of embodiments 98-101, 104-106, 117-151 and 153-155, wherein R is^B7Is a substituted or unsubstituted cyclopropyl or-N (R)^b)₂Wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups.

159. The compound according to any one of embodiments 98 and 117 and 150,wherein R is^B5Is of the formula:

160. a compound of embodiment 159 wherein R^B5Is of the formula:

161. the compound of any one of embodiments 98, 109-110 and 117-150, wherein R^B8Is substituted or unsubstituted C_1-6An alkyl group.

162. A compound of embodiment 161 wherein R^B8Is Me.

163. The compound of any one of embodiments 98, 109-110 and 117-150, wherein R^B8is-N (R)^b)₂Wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6Alkyl or nitrogen protecting groups.

164. The compounds according to any of embodiments 98, 109-110, 117-150, 159 and 161-163, wherein R is^B9Is substituted or unsubstituted C _1-6An alkyl group.

165. A compound of embodiment 164 wherein R^B9Me, Et or n-Pr.

166. The compounds according to any of embodiments 98, 109-110, 117-150, 159 and 161-163, wherein R is^B9Is a substituted OR unsubstituted cyclopropyl, -OR^bor-N (R)^b)₂Wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6An alkyl group, an oxygen protecting group when attached to an oxygen atom, or a nitrogen protecting group when attached to a nitrogen atom.

167. The compound of any one of embodiments 98 and 117-150 wherein R^B5Is of the formula:

168. a compound of embodiment 167 wherein R^B5Is of the formula:

169. a compound of embodiment 167 wherein R^B5Is of the formula:

170. a compound of any of embodiments 167 wherein R^B5Is of the formula:

171. compounds according to any of embodiments 98, 111-112, 117-150, 167-168 and 170, wherein R is^B10is-OR^b。

172. A compound of embodiment 171 wherein R^B10is-OH.

173. Compounds according to any of embodiments 98, 111-112, 117-150, 167-168 and 170, wherein R is^B10is-N (R)^b)₂。

174. A compound of embodiment 173 wherein R^B10is-NHC (═ O) R^bWherein R is^bIs a substituted or unsubstituted vinyl group.

175. Compounds as claimed in any of embodiments 98, 111-112, 117-150, 167-168 and 170-174, where at least one R is ^B11Is halogen.

176. Compounds as claimed in any of embodiments 98, 111-112, 117-150, 167-168 and 170-174, where at least one R is^B11Is substituted or unsubstituted C_1-6An alkyl group.

177. The compounds in any of the embodiments 98, 111, 112, 117, 150, 167, 168 and 170, 174A compound of formula (I), wherein at least one R^B11Is substituted or unsubstituted C_1-6Alkyl, substituted OR unsubstituted cyclopropyl, -OR^bor-N (R)^b)₂Wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6An alkyl group, an oxygen protecting group when attached to an oxygen atom, or a nitrogen protecting group when attached to a nitrogen atom.

178. The compound of any one of embodiments 98 and 117-150 wherein R^B5Is of the formula:

179. a compound of embodiment 178 wherein R^B5Is of the formula:

180. a compound of embodiment 178 wherein R^B5Is of the formula:

181. compounds according to any one of embodiments 98, 113, 117, 150 and 178, 179, wherein R^B12Is hydrogen.

182. Compounds according to any of embodiments 98, 113, 117, 150, 178, 179 and 181, wherein at least one R is^B13Is substituted or unsubstituted C_1-6An alkyl group.

183. Compounds according to any of embodiments 98, 113, 117, 150, 178, 179 and 181, wherein at least one R is^B13Is halogen, substituted OR unsubstituted cyclopropyl, -OR ^bor-N (R)^b)₂Wherein each R is^bIndependently hydrogen, substituted or unsubstituted C_1-6An alkyl group, an oxygen protecting group when attached to an oxygen atom, or a nitrogen protecting group when attached to a nitrogen atom.

184. The compound of any one of embodiments 98 and 117-150 wherein R^B5Is of the formula:

185. a compound of embodiment 184 wherein R^B5Is of the formula:

186. a compound according to any one of embodiments 98, 115-150 and 184, wherein R^B14Is H.

187. A compound according to any one of embodiments 98, 115, 150, 184 and 186, wherein R^B15Is substituted or unsubstituted C_1-6An alkyl group.

188. A compound of embodiment 187 wherein R^B15Is Me.

189. The compound of any one of embodiments 98, 115, 150, 184 and 186, 188, wherein R^B16Is substituted or unsubstituted C_2-6An alkyl group.

190. A compound of embodiment 189 wherein R^B16Is n-Pr.

191. The compound of any one of embodiments 98, 115, 150, 184 and 186, 190, wherein R^B17Is H.

192. The compound of any one of embodiments 98, 115, 150, 184 and 186, 190, wherein R^B17is-C (═ O) R^bWherein R is^bIs a substituted or unsubstituted vinyl group.

193. The compound of embodiment 98, wherein the compound is of the formula:

194. The compound of embodiment 98, wherein the compound is of the formula:

195. a pharmaceutical composition comprising a compound of any one of embodiments 1-194, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient.

196. The pharmaceutical composition of embodiment 195, further comprising an additional agent.

197. A method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of embodiments 1-194 or the pharmaceutical composition of embodiment 195 or 196.

198. The method of embodiment 197, wherein the disease is a disease associated with aberrant activity of histone methyltransferases.

199. The method of embodiment 197, wherein the disease is a genetic disease.

200. The method of embodiment 197, wherein the disease is a proliferative disease.

201. The method of embodiment 197, wherein the disease is cancer.

202. The method of embodiment 201, wherein said cancer is lung cancer.

203. The method of embodiment 202, wherein said lung cancer is non-small cell lung cancer.

204. The method of embodiment 197, wherein the disease is a benign tumor.

205. The method of embodiment 197, wherein the disease is pathological angiogenesis.

206. The method of embodiment 197, wherein the disease is an inflammatory disease.

207. The method of embodiment 197, wherein the disease is an autoimmune disease.

208. The method of embodiment 197, wherein the disease is a hematological disease.

209. The method of embodiment 197, wherein the disease is a neurological disease.

210. The method of embodiment 197, wherein the disease is a painful condition.

211. The method of embodiment 197, wherein the disease is a psychiatric disease.

212. The method of embodiment 197, wherein the disease is a metabolic disorder.

213. The method of embodiment 197, wherein the disease is hyperplasia, brain cancer, breast cancer, prostate cancer, lymphoma, leukemia, or weverer's syndrome.

214. A method of inhibiting the activity of a histone methyltransferase in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of any one of embodiments 1-194 or the pharmaceutical composition of embodiment 195 or 196.

215. The method of any one of embodiments 197-214, wherein the subject is a human.

216. A method of inhibiting the activity of a histone methyltransferase in a cell, the method comprising contacting the cell with an effective amount of a compound of any one of embodiments 1-194 or a pharmaceutical composition of embodiment 195 or 196.

217. The method of any one of embodiments 198 and 214-216, wherein said histone methyltransferase is enhancer of zeste homolog 1(EZH 1).

218. The method of any one of embodiments 198 and 214-216, wherein said histone methyltransferase is enhancer of zeste homolog 2(EZH 2).

219. A method of inducing apoptosis in a cell, the method comprising contacting the cell with an effective amount of a compound of any one of embodiments 1-194 or a pharmaceutical composition of embodiment 195 or 196.

220. The method of any one of embodiments 216-219, wherein the cell is in vitro.

221. The method of any one of embodiments 216-219, wherein the cell is in vivo.

222. A kit, comprising:

a compound of any one of embodiments 1-194 or a pharmaceutical composition of embodiment 195 or 196; and

instructions for using the compound or pharmaceutical composition.

223. A method of identifying an EZH1 and/or EZH2 inhibitor, the method comprising:

(a) providing cells in culture with EZH1 and/or EZH2 binding compounds labeled with a detectable tag, wherein the cells express endogenous EZH1 and/or EZH2,

(b) providing unlabeled test compound to the cells in culture, and

(c) Identifying a test compound as an EZH1 and/or EZH2 inhibitor when the signal from the detectable tag in the nucleus of the cell is reduced in the presence of the test compound relative to the signal in the absence of the test compound.

224. The method of embodiment 223, further comprising detecting the level of the tag in the cell.

225. The method of embodiment 223 or 224, further comprising detecting the level of the tag in the nucleus of said cell.

226. The method of any one of embodiments 223-225, wherein the cell is immobilized prior to detecting the tag in the cell.

227. The method of any one of embodiments 223-226, wherein the detectable label is a fluorescent label.

228. The method of any one of embodiments 223-227, wherein the test compound is a small molecule.

229. The method of any one of embodiments 223-228, wherein the method is a high throughput assay.

Sequence listing

<110> Dana-Faber CANCER INSTITUTE, Inc. (DANA-FARBER CANCER INSTITUTE, INC.)

<120> Ezh2 inhibitor and use thereof

<130> D0504.70080WO00

<140> PCT/US2015/059622

<141> 2015-11-06

<150> US 62/076,410

<151> 2014-11-06

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic Polypeptide

<400> 1

His His His His His His

1 5

<210> 2

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic Polypeptide

<400> 2

His His His His

1

<210> 3

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<400> 3

Leu Leu Leu Leu

1

<210> 4

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<221> misc_feature

<222> (5)..(5)

<223> Xaa is epsilon protonated neutral histidine

<400> 4

Arg Phe Ala Asn Xaa Ser Val

1 5