阅读说明：本技术 化合物及其缀合物 (Compounds and conjugates thereof ) 是由 P·W·霍华德 N·迪金森 T·卡约 L·马斯特森 W·冈德里 于 2020-03-23 设计创作，主要内容包括：一种缀合物,该缀合物包含以下拓扑异构酶抑制剂衍生物(A*)：具有用于连接配体单元的接头,其中该接头以可切割的方式附接至氨基残基。该配体单元优选是抗体。还提供了具有附接的连接单元的A*、和用于其合成的中间体以及释放的弹头。(A conjugate comprising the following topoisomerase depressantFormulation derivative (a): having a linker for connecting the ligand units, wherein the linker is attached to the amino residue in a cleavable manner. The ligand unit is preferably an antibody. A with attached connection units, and intermediates for their synthesis and released warheads are also provided.)

1. A compound having the formula I:

and salts and solvates thereof, wherein R^LIs a linker for attachment to a ligand unit, the linker being selected from the group consisting of:

(ia)：

wherein

Q is:

wherein Q^XSuch that Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue;

x is:

wherein a is 0 to 5, b1 is 0 to 16, b2 is 0 to 16, c1 is 0 or 1, c2 is 0 or 1, d is 0 to 5, wherein at least b1 or b2 is 0 and at least c1 or c2 is 0;

G^Lis a linker for attachment to a ligand unit;

(ib)：

wherein R is^L1And R^L2Independently selected from H and methyl, or together with the carbon atom to which they are bonded form a cyclopropene or cyclobutene group; and is

e is 0 or 1.

2. The compound of claim 1, wherein R^LHas the formula Ia.

3. The compound of claim 2, wherein Q is:

(a) an amino acid residue selected from: phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp; or

(b) A dipeptide residue selected from:

^NH-Phe-Lys-^C＝O、

^NH-Val-Ala-^C＝O、

^NH-Val-Lys-^C＝O、

^NHAla-Lys-^C＝O、

^NH-Val-Cit-^C＝O、

^NH-Phe-Cit-^C＝O、

^NH-Leu-Cit-^C＝O、

^NH-Ile-Cit-^C＝O、

^NH-Phe-Arg-^C＝O、

^NH-Trp-Cit-^C＝Oand, and

^NH-Gly-Val-^C＝O(ii) a Or

(c) A tripeptide residue selected from:

^NH-Glu-Val-Ala-^C＝O、

^NH-Glu-Val-Cit-^C＝O、

^NH-αGlu-Val-Ala-^C＝Oand, and

^NH-αGlu-Val-Cit-^C＝O(ii) a Or

(d) A tetrapeptide residue selected from:

^NH-Gly-Gly-Phe-Gly^C＝O(ii) a And

^NH-Gly-Phe-Gly-Gly^C＝O。

4. a compound according to claim 2 or claim 3, wherein a is:

(a)0 to 3; or

(b)0 or 1; or

(c)0。

5. The compound according to any one of claims 2 to 4, wherein b1 is:

(a)0 to 8; or

(b) 0; or

(c) 2; or

(d) 3; or

(e) 4; or

(f) 5; or

(g)8。

6. The compound according to any one of claims 2 to 4, wherein b2 is:

(a)0 to 8; or

(b) 0; or

(c) 2; or

(d) 3; or

(e) 4; or

(f) 5; or

(g)8。

7. The compound according to any one of claims 2 to 6, wherein

(i) c1 is:

(a) 0; or

(b) 1; and is

(ii) c2 is:

(a) 0; or

(b)1；

Wherein at least one of c1 and c2 is 0.

8. The compound according to any one of claims 2 to 7, wherein d is:

(a)0 to 3; or

(b)1 or 2; or

(c) 2; or

(d)5。

9. The compound according to any one of claims 2 to 8, wherein:

(a) a is 0, b1 is 0, c1 is 1, c2 is 0 and d is 2, and b2 is 0, 2, 3, 4, 5 or 8; or

(b) a is 1, b2 is 0, c1 is 0, c2 is 0 and d is 0, and b1 is 0, 2, 3, 4, 5 or 8; or

(c) a is 0, b1 is 0, c1 is 0, c2 is 0 and d is 1, and b2 is 0, 2, 3, 4, 5 or 8; or

(d) b1 is 0, b2 is 0, c1 is 0, c2 is 0, one of a and d is 0, and the other of a and d is 1 or 5; or

(e) a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 is 0, 2, 3, 4, 5, or 8.

10. The compound according to any one of claims 2 to 9, wherein G^LIs selected from

Wherein Ar represents C_5-6An arylene group, and X represents C_1-4An alkyl group.

11. The compound of claim 10, wherein G^LIs selected from G^L1-1And G^L1-2。

12. The compound of claim 1, wherein R^LHas formula Ib, and:

(a)R^L1and R^L2Both are H; or

(b)R^L1Is H and R^L2Is methyl; or

(c)R^L1And R^L2Both are methyl; or

(d) Wherein R is^L1And R^L2Form a cyclopropene group together with the carbon atom to which they are bonded; or

(e) Wherein R is^L1And R^L2Together with the carbon atom to which they are bonded form a cyclobutene group.

13. A conjugate having the formula IV:

L-(D^L)_p (IV)

or a pharmaceutically acceptable salt or solvate thereof, wherein L is a ligand unit, D^LIs a drug linker unit having formula III:

R^LLis a linker attached to the ligand unit, the linker being selected from

(ia’)：

Wherein Q and X are as defined in any one of claims 1 to 9 and G^LLIs a linker attached to the ligand unit; and

(ib’)：

wherein R is^L1And R^L2Is as defined in any one of claim 1 or claim 12; and is

p is an integer from 1 to 20.

14. The conjugate of claim 13, wherein G is^LLSelected from:

wherein Ar represents C_5-6An arylene group, and X represents C_1-4An alkyl group.

15. The conjugate of claim 14, wherein G is^LLIs selected from G^LL1-1And G^LL1-2。

16. The conjugate according to any one of claims 13 to 15, wherein the ligand unit is an antibody or an active fragment thereof.

17. The conjugate of claim 16, wherein the drug loading (p) of drug (D) and antibody (Ab) is an integer from 1 to about 10.

18. A mixture of conjugates according to claim 16 or claim 17, wherein the average drug loading per antibody in the mixture of antibody-drug conjugates is from about 1 to about 10.

19. A pharmaceutical composition comprising a conjugate or mixture according to any one of claims 13 to 18 and a pharmaceutically acceptable diluent, carrier, or excipient.

20. A conjugate or mixture according to any one of claims 13 to 18, or a pharmaceutical composition according to claim 19, for use in the treatment of a proliferative disease in a subject.

21. A conjugate, mixture or pharmaceutical composition according to claim 20, wherein the disease is cancer.

22. Use of a conjugate or mixture according to any one of claims 13 to 18, or a pharmaceutical composition according to claim 19, in a method of medical treatment.

23. A method of medical treatment comprising administering to a patient a pharmaceutical composition according to claim 19.

24. The method according to claim 23, wherein the method of medical treatment is for the treatment of cancer.

25. A compound A:

in the form of individual enantiomers or in enantiomerically enriched form.

26. A compound having the formula VI:

wherein Q is as defined in any one of claims 1 or 3.

Background

Topoisomerase inhibitors

Topoisomerase inhibitors are chemical compounds that block the action of topoisomerases (topoisomerases I and II), a type of enzyme that controls changes in DNA structure by catalyzing the cleavage and re-ligation of the phosphodiester backbone of DNA strands in the normal cell cycle.

The following compounds:

(in racemic form) is disclosed in EP 0296597 (example 63). Also disclosed (compound 34 in racemic form) in Sugimori, M., et al, J Med Chem [ J. Pharmacology ], 1998, 41, 2308-2318 (DOI: 10.1021/jm970765q) are biological activities thereof and of some related compounds.

Various topoisomerase inhibitors, such as irinotecan and irinotecan derivatives and doxorubicin, have been included in antibody drug conjugates. For example, the first Sankyo corporation (Daiichi Sankyo) used DS-8201 a:

wherein the antibody is Her2(Takegawa, N., et al, Int J Cancer [ J.International J.carc., 2017, 141, 1682-1689 (DOI: 10.1002/ijc.30870.) the ADC releases an irinotecan derivative:

burke, p.j., et al, Bioconjugate Chem. [ Bioconjugate chemistry ], 2009, 20, 1242-1250, disclose the following conjugates:

they are linked via an amino group to the following structure:

they include the PABC (p-aminobenzyloxycarbonyl) group.

The immunologists used gaulthikang-safirtuzumab (IMMU-132) (Cardillo, T.M., et al, Bioconjugate Chem [ Bioconjugate chemistry ], 2015, 26(5), 919-

Disclosure of Invention

In a general aspect, the present invention provides a conjugate comprising the following topoisomerase inhibitor derivative (a;, drug unit):

having a linker for connecting the ligand units, wherein the linker is attached to the amino residue in a cleavable manner. The ligand unit is preferably an antibody. The invention also provides a with attached connecting units, and intermediates for their synthesis and released warheads.

A first aspect of the invention comprises a compound having formula I:

and salts and solvates thereof, wherein R^LIs a linker for attachment to a ligand unit, the linker being selected from the group consisting of:

(ia)：

wherein

Q is:

wherein Q^XSuch that Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue;

x is:

where a is 0 to 5, b1 is 0 to 16, b2 is 0 to 16, c1 is 0 or 1, c2 is 0 or 1, d is 0 to 5, where at least b1 or b2 is 0 (i.e., only one of b1 and b2 may not be 0) and at least c1 or c2 is 0 (i.e., only one of c1 and c2 may not be 0);

G^Lis a linker for attachment to a ligand unit;

(ib)：

wherein R is^L1And R^L2Independently selected from H and methyl, or together with the carbon atom to which they are bonded form a cyclopropene or cyclobutene group; and is

e is 0 or 1.

A second aspect of the invention provides a process for the preparation of a compound of the first aspect of the invention, the process comprising at least one of the process steps listed below.

In a third aspect, the invention provides a conjugate having formula IV:

L-(D^L)_p (IV)

or a pharmaceutically acceptable salt or solvate thereof, wherein L is a ligand unit (i.e., targeting agent), D^LIs a drug linker unit having formula III:

R^LLis a linker attached to the ligand unit, the linker being selected from

(ia’)：

Wherein Q and X are as defined in the first aspect and G^LLIs a linker attached to the ligand unit; and

(ib’)：

wherein R is^L1And R^L2Is as defined in the first aspect; and is

p is an integer from 1 to 20.

Thus, the conjugate comprises a ligand unit covalently linked to at least one drug unit (a) by a linker unit (i.e. a ligand unit to which one or more drug linker units are attached). The ligand unit, described more fully below, is a targeting agent that binds to the target moiety. The ligand unit may, for example, specifically bind to a cellular component (cell-binding agent) or other target molecule of interest. Thus, the invention also provides methods for treating, for example, various cancers and autoimmune diseases. These methods encompass the use of conjugates in which the ligand unit is a targeting agent that specifically binds to the target molecule. The ligand unit may be, for example, a protein, polypeptide or peptide, such as an antibody, an antigen-binding fragment of an antibody, or other binding agent, such as an Fc fusion protein.

Drug loading is represented by p (number of drug units per ligand unit (e.g. antibody)). The drug loading per ligand unit (e.g., Ab or mAb) may range from 1 to 20 drug units (D). For the compositions, p represents the average drug loading of the conjugate in the composition, and p ranges from 1 to 20.

A fourth aspect of the invention provides the use of a conjugate of the third aspect of the invention in the manufacture of a medicament for the treatment of a proliferative disease. The fourth aspect also provides a conjugate of the third aspect of the invention for use in the treatment of a proliferative disease.

One of ordinary skill in the art can readily determine whether a candidate compound treats a proliferative disorder of any particular cell type. For example, assays that can be conveniently used to assess the activity provided by a particular compound are described in the examples below.

A series of ADCs are discussed in Nakada, et al, Bioorg Med Chem Lett [ Rapid Bioorganic and medicinal chemistry ], 26(2016), 1542-:

and it was concluded that the reduced cytotoxicity of ADC (1) and (2) may be due to steric hindrance of the drug moiety released at the site of action of the degradative enzyme in the tumor cells. This document teaches the importance of separating peptidyl groups (peptidic groups) from the bulk of the released drug moiety. In contrast, in the present invention, the peptidyl group is directly linked to a large number of drug-releasing moieties.

A fifth aspect of the invention is compound a:

in the form of individual enantiomers or in enantiomerically enriched form.

A sixth aspect of the invention is a compound having formula VI:

wherein Q is as defined in the first aspect.

Definition of

C_5-6Arylene group: as used herein, the term "C_5-6Arylene "refers to a divalent moiety obtained by removing two hydrogen atoms from an aromatic ring atom of an aromatic compound.

In this context, a prefix (e.g., C)_5-6) Denotes the number of ring atoms or the range of ring atom numbers, whether carbon or heteroatoms.

The ring atoms may all be carbon atoms, as in a "carboarylene group", in which case the group is phenylene (C)₆)。

Alternatively, the ring atoms may include one or more heteroatoms, as in a "heteroarylene group". Examples of heteroarylene groups include, but are not limited to, those derived from:

N₁: pyrrole (azole) (C)₅) Pyridine (azine) (C)₆)；

O₁: furan (oxacyclopentadiene) (oxole)) (C)₅)；

S₁: thiophene (thiacyclopentadiene) (C)₅)；

N₁O₁: oxazole (C)₅) Isoxazole (C)₅) Isooxazine (C)₆)；

N₂O₁: oxadiazole (furazan) (C)₅)；

N₃O₁: oxatriazole (C)₅)；

N₁S₁: thiazole (C)₅) Isothiazole (C)₅)；

N2: imidazole (1, 3-diazole) (C)₅) Pyrazole (1, 2-diazole) (C)₅) Pyridazine (1, 2-diazine) (C)₆) Pyrimidine (1, 3-diazine) (C)₆) (e.g., cytosine, thymine, uracil), pyrazine (1, 4-diazine) (C₆) (ii) a And

N₃: triazole (C)₅) Triazine (C)₆)。

C_1-4Alkyl groups: as used herein, the term "C_1-4Alkyl "refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon-based compound having from 1 to 4 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g., partially unsaturated, fully unsaturated). As used herein, the term "C₁N-alkyl "relates to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon-based compound having from 1 to n carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). Thus, the term "alkyl" includes the subclasses discussed below: alkenyl, alkynyl, cycloalkyl, and the like.

Examples of saturated alkyl groups include, but are not limited to, methyl (C)₁) Ethyl (C)₂) Propyl group (C)₃) And butyl (C)₄)。

Examples of saturated straight chain alkyl groups include, but are not limited to, methyl (C)₁) Ethyl (C)₂) N-propyl (C)₃) And n-butyl (C)₄)。

Examples of saturated branched alkyl groups include isopropyl (C)₃) Isobutyl (C)₄) Sec-butyl (C)₄) And tert-butyl (C)₄)。

C_2-4An alkenyl group; as used herein, the term "C_2-4Alkenyl "refers to an alkyl group having one or more carbon-carbon double bonds.

Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (ethenyl, vinyl) (-CH ═ CH₂) 1-propenyl (-CH ═ CH-CH)₃) 2-propenyl (allyl, -CH-CH ═ CH)₂) Isopropenyl (1-methylethenyl, -C (CH)₃)＝CH₂) And butenyl (C)₄)。

C_2-4Alkynyl: as used herein, the term "C_2-4The X group "relates to an alkyl group having one or more carbon-carbon triple bonds.

Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (-C ≡ CH) and 2-propynyl (propargyl, -CH₂-C≡CH)。

C_3-4Cycloalkyl groups: as used herein, the term "C_3-4Cycloalkyl "refers to an alkyl group that is also a cyclic group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbyl (carbocyclic) compound, the moiety having from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, those derived from:

saturated monocyclic hydrocarbon compound:

cyclopropane (C)₃) And cyclobutane (C)₄) (ii) a And

unsaturated monocyclic hydrocarbon compound:

cyclopropene (C)₃) And cyclobutene (C)₄)。

Connecting labels: in-situ typeIn (b), the superscripts C (═ O) and NH represent the groups to which the atoms are bonded. For example, the NH group is shown to be bound to a carbonyl group (which is not part of the indicated moiety), and the carbonyl group is shown to be bound to an NH group (which is not part of the indicated moiety).

Salt (salt)

Corresponding salts, e.g., pharmaceutically acceptable salts, of the active compounds may be conveniently or desirably prepared, purified, and/or processed. Examples of pharmaceutically acceptable salts are discussed in Berge, et al, j.pharm.sci. [ journal of pharmaceutical science ], 66, 1-19 (1977).

For example, if the compound is anionic, or has a functional group that can be anionic (e.g., -COOH can be-COO^-) Salts may be formed with appropriate cations. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na⁺And K⁺Alkaline earth metal cations such as Ca²⁺And Mg²⁺And other cations such as Al⁺³. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH)₄ ⁺) And substituted ammonium ions (e.g. NH)₃R⁺、NH₂R₂ ⁺、NHR₃ ⁺、NR₄ ⁺). Some examples of suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine and tromethamine, and amino acids (such as lysine and arginine). An example of a common quaternary ammonium ion is N (CH)₃)₄ ⁺。

If the compound is cationic, or has a functional group which may be cationic (e.g. -NH)₂May be-NH₃ ⁺) Salts may be formed with appropriate anions. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid,Sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.

Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, ethylenediaminetetraacetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid (gluceptonic acid), gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthoic acid, hydroxyethanesulfonic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, benzenesulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, trifluoroacetic acid, and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

Solvates

The corresponding solvates of the active compounds may be conveniently or desirably prepared, purified, and/or handled. The term "solvate" is used herein in the conventional sense to refer to a complex of a solute (e.g., active compound, salt of active compound) and a solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, e.g., a monohydrate, dihydrate, trihydrate, and the like.

Isomers

Certain compounds of the present invention can exist in one or more specific geometric, optical, enantiomeric, diastereomeric, epimeric, atropisomeric, stereoisomeric, tautomeric, conformational or anomeric forms (anomeric form), including but not limited to cis and trans forms; e-and Z-forms; c-, t-, and r-forms; endo-and exo-forms; r-, S-, and meso-forms; d-and L-forms; d-and l-forms; the (+) and (-) forms; keto-, enol-, and enolate-forms; cis-and trans-forms; syncline-and anticline-forms; alpha-and beta-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and half-chair-forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").

The term "chiral" refers to a molecule that has the non-superimposability of a mirror partner, while the term "achiral" refers to a molecule that can be superimposed on its mirror partner.

The term "stereoisomers" refers to compounds having the same chemical composition, but differing with respect to the arrangement of atoms or groups in space.

"diastereomer" refers to a stereoisomer that has two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers can be separated under high resolution analytical procedures such as electrophoresis and chromatography.

"enantiomer" refers to two stereoisomers of a compound that are mirror images of each other that are not superimposable.

The stereochemical definitions and conventions used herein generally follow s.p. parker, editions, McGraw-high Dictionary of chemistry 1 Terms [ mcgral-Hill Dictionary of chemical Terms ] (1984) mcgral Hill press, new york; and Eliel, e, and Wilen, s., "stereoschemistry of Organic compounds" [ Stereochemistry of Organic compounds ], John Wiley & Sons, Inc., new york, 1994. The compounds of the invention may contain asymmetric or chiral centers and thus exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the invention (including but not limited to diastereomers, enantiomers, and atropisomers, and mixtures thereof, e.g., racemic mixtures) form part of the invention. Many organic compounds exist in an optically active form, i.e., they have the ability to rotate the plane of plane polarized light. In describing optically active compounds, the prefixes D and L, or R and S, are used to denote the absolute configuration of a molecule about one or more of its chiral centers. The prefixes d and l or (+) and (-) are used to denote the sign of the compound rotating plane polarized light, where (-) or l denotes that the compound is left-handed. Compounds with (+) or d prefixes are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. A particular stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is commonly referred to as a mixture of enantiomers. A50: 50 mixture of enantiomers is referred to as a racemic mixture or racemate, and may occur where there is no stereoselectivity or stereospecificity in the chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, without optical activity.

"enantiomerically enriched form" refers to a sample of a chiral material having an enantiomeric ratio greater than 50: 50 but less than 100: 0.

Note that as used herein, the term "isomer" specifically excludes structural (or constitutional) isomers (i.e., linkages between atoms rather than merely isomers that differ in the position of the atoms in space), except as discussed below with respect to tautomeric forms. For example, a p-methoxy group (-OCH)₃) The reference to (A) should not be interpreted as referring to its structural isomer hydroxymethyl-CH₂Reference is made to OH. Similarly, reference to an o-chlorophenyl group should not be construed as a reference to its structural isomer, m-chlorophenyl. However, references to a class of structures are likely to include structural isomeric forms (e.g., C) belonging to that class_1-7Alkyl groups include n-propyl and isopropyl; butyl includes n-, iso-, sec-and tert-butyl; methoxyphenyl includes o-, m-, and p-methoxyphenyl).

The above exclusion does not refer to tautomeric forms, such as keto, enol and enol acid ester forms, such as for example the following tautomeric pairs: ketone/enol (shown below), imine/enamine, amide/iminoalcohol (imino alcohol), amidine/enediamine (enediamine), nitroso/oxime, thioketone/enethiol, N-nitroso/hydroxyazo (hypoxyazo), and nitro/nitrolic acid (aci-nitro).

The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also referred to as prototropic tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions by recombination of some valence electrons.

Note that specifically included in the term "isomer" are compounds having one or more isotopic substitutions. For example, H may be in any isotopic form, including¹H、²H (D), and³h (T); c may be in any isotopic form, including¹²C、¹³C. And¹⁴c; o may be in any isotopic form, including¹⁶O and¹⁸o; and the like.

Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine and iodine, such as, but not limited to²H (deuterium, D),³H (tritium),¹¹C、¹³C、¹⁴C、¹⁵N、¹⁸F、³¹P、³²P、³⁵S、³⁶Cl, and¹²⁵I. various isotopically-labeled compounds of the present invention, for example, those into which radioactive isotopes, such as 3H, 13C, and 14C, are incorporated. Such isotopically labeled compounds are useful in metabolic studies, reaction kinetic studies, detection or imaging techniques (e.g., Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT), including drug or stromal tissue distribution assays), or in the radiation treatment of patients. In connection with distribution, metabolism and excretion (ADME), deuterium labeled or substituted therapeutic compounds of the present invention may have improved DMPK (drug metabolism and pharmacokinetics) properties. Substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages (due to greater metabolic stability), for example increased in vivo half-life or reduced dosage requirements. The 18F labeled compounds are useful for PET or SPECT studies. Isotopically-labeled compounds of the present invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below, with easeThe resulting isotopically labeled reagent replaces the non-isotopically labeled reagent. In addition, substitution with heavier isotopes, particularly deuterium (i.e., 2H or D), may afford certain therapeutic advantages (due to greater metabolic stability), for example increased in vivo half-life or reduced dosage requirements or improvement in therapeutic index. It is to be understood that deuterium is considered a substituent herein. The concentration of such heavier isotopes, in particular deuterium, can be defined by the isotopic enrichment factor. In the compounds of the present invention, any atom not specifically designated as a specific isotope represents any stable isotope of the atom.

Unless otherwise indicated, reference to a particular compound includes all such isomeric forms, including (in whole or in part) racemates and other mixtures thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallization and chromatographic means) of such isomeric forms are known in the art or are readily obtained by employing the methods taught herein or known methods in a known manner.

Ligand unit

The ligand unit may be of any kind and includes proteins, polypeptides, peptides and non-peptide agents that specifically bind to the target molecule. In some embodiments, the ligand unit may be a protein, polypeptide, or peptide. In some embodiments, the ligand unit may be a cyclic polypeptide. These ligand units may include antibodies or antibody fragments that contain at least one target molecule binding site, lymphokines, hormones, growth factors, or any other cell-binding molecule or substance that can specifically bind to a target.

The terms "specific binding" and "specific binding" refer to the binding of an antibody or other protein, polypeptide, or peptide to a predetermined molecule (e.g., an antigen). Typically, the antibody or other molecule is administered at a dose of at least about 1x10⁷M^-1And binds to the predetermined molecule with an affinity that is at least two times greater than its affinity for binding to a non-specific molecule (e.g., BSA, casein) other than the predetermined molecule or closely related molecules.

Examples of ligand units include those reagents described for use in WO 2007/085930 (which is incorporated herein).

In some embodiments, the ligand unit is a cell-binding agent that binds to an extracellular target on a cell. Such cell-binding agents may be protein, polypeptide, peptide or non-peptide agents. In some embodiments, the cell-binding agent can be a protein, polypeptide, or peptide. In some embodiments, the cell-binding agent can be a cyclic polypeptide. The cell binding agent can also be an antibody or antigen binding fragment of an antibody. Thus, in one embodiment, the invention provides antibody-drug conjugates (ADCs).

Cell binding agents

The cell binding agent may be of any kind and includes peptides and non-peptides. These may include antibodies or antibody fragments that contain at least one binding site, lymphokine, hormone mimetic, vitamin, growth factor, nutrient transport molecule, or any other cell binding molecule or substance.

Peptides

In one embodiment, the cell binding agent is a linear or cyclic peptide comprising 4-30, preferably 6-20 consecutive amino acid residues.

In one embodiment, the cell binding agent comprises a binding integrin alpha_vβ₆The peptide of (1). The peptide pair alpha_vβ₆Can exceed XYS.

In one embodiment, the cell-binding agent comprises an a20FMDV-Cys polypeptide. A20FMDV-Cys has the following sequence: NAVPNLRGDLQVLAQKVARTC are provided. Alternatively, variants of the a20FMDV-Cys sequence may be used in which one, two, three, four, five, six, seven, eight, nine or ten amino acid residues are substituted by another amino acid residue. In addition, the polypeptide may have the sequence NAVXXXXXXXXXXXXXRC.

Antibodies

The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), multivalent antibodies and antibody fragments so long as they exhibit the desired biological activity (Miller et al (2003) journal of Immunology 170: 4854-4861). The antibody may be murine, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system that are capable of recognizing and binding to a particular antigen. (Janeway, C., Travers, P., Walport, M., Sholomchik (2001) Immuno Biology, 5 th edition, Calif. (Garland Publishing, N.Y.). The target antigen typically has a number of binding sites, also referred to as epitopes, that are recognized by CDRs on multiple antibodies. Each antibody that specifically binds a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. Antibodies include full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules (i.e., molecules that contain an antigen binding site that immunospecifically binds to a target antigen of interest or a portion thereof), such targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulin may be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. The immunoglobulin may be derived from any species, including human, murine or rabbit origin.

An "antibody fragment" includes a portion of a full-length antibody, typically the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')₂And scFv fragments; a diabody; a linear antibody; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDRs (complementarity determining regions) and epitope-binding fragments of any of the foregoing (which fragments immunospecifically bind to a cancer cell antigen, a viral antigen, or a microbial antigen), single chain antibody molecules; and multispecific antibodies formed from antibody fragments.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies, which includes otherwise identical populations except for possible naturally occurring mutations (which may be present in minor amounts). Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the invention may be produced by first screening a monoclonal antibody produced by Kohler et al (1975) Nature [ Nature ] 256: 495 or may be prepared by recombinant DNA methods (see US 4816567). Monoclonal antibodies can be isolated from phage antibody libraries (using the techniques described in Clackson et al (1991) Nature [ Nature ], 352: 624-.

The monoclonal antibody herein specifically includes chimeric antibodies, humanized antibodies and human antibodies.

Examples of cell binding agents include those agents described for use in WO 2007/085930 (which is incorporated herein).

Tumor-associated antigens and cognate antibodies for use in embodiments of the invention are listed below and described in more detail on pages 14 to 86 of WO 2017/186894, which is incorporated herein.

(1) BMPR1B (bone morphogenetic protein receptor-IB type)

(2)E16(LAT1、SLC7A5)

(3) STEAP1 (prostate six transmembrane epithelial antigen)

(4)0772P(CA125、MUC16)

(5) MPF (MPF, MSLN, SMR, megakaryocyte stimulating factor, mesothelin)

(6) Napi3B (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium dependent phosphate Transporter 3B)

(7) Sema5B (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin (Semaphorin)5B Hlog, Sema domain, seven thrombospondin repeats (type 1 and type 1), transmembrane domain (TM) and short cytoplasmic domain, (Semaphorin) 5B)

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene)

(9) ETBR (endothelin B type receptor)

(10) MSG783(RNF124, hypothetical protein FLJ20315)

(11) STEAP2 (HGNC-8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1, six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein)

(12) TrpM4(BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation 5 channel subfamily M member 4)

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratoma-derived growth factor)

(14) CD21(CR2 (complement receptor 2) or C3DR (C3 d/Epstein-Barr virus receptor) or Hs.73792)

(15) CD79B (CD79B, CD79 beta, IGb (immunoglobulin related beta), B29)

(16) FcRH2(IFGP4, IRTA4, SPAP1A (phosphatase anchoring protein 1a containing SH2 domain), SPAP1B, SPAP1C)

(17)HER2(ErbB2)

(18)NCA(CEACAM6)

(19)MDP(DPEP1)

(20)IL20R-α(IL20Ra、ZCYTOR7)

(21) Brevicin (Brevican) (BCAN, BEHAB)

(22)EphB2R(DRT、ERK、Hek5、EPHT3、Tyro5)

(23)ASLG659(B7h)

(24) PSCA (prostate stem cell antigen precursor)

(25)GEDA

(26) BAFF-R (B cell activating factor receptor, BLyS receptor 3, BR3)

(27) CD22(B cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814)

(27a) CD22(CD22 molecule)

(28) CD79a (CD79A, CD79 α), immunoglobulin-associated α, B cell-specific protein covalently interacting with Ig β (CD79B) and forming a complex with Ig M molecules on the surface, transducing signals involved in B cell differentiation), pI: 4.84, MW: 25028 TM: 2[ P ] Gene chromosome: 19q 13.2).

(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptor activated by CXCL13 chemokines, functional in lymphocyte migration and humoral defense, functional in HIV-2 infection and possibly in the development of AIDS, lymphoma, myeloma, and leukemia); 372aa, pI: 8.54 MW: 41959 TM: 7[ P ] Gene chromosome: 11q23.3 of the total weight of the rubber,

(30) HLA-DOB (the β subunit of MHC class II molecules (Ia antigen) that bind peptides and present them to CD4+ T lymphocytes); 273aa, pI: 6.56, MW: 30820. TM: 1[ P ] Gene chromosome: 6p21.3)

(31) P2X5 (purinoceptor P2X ligand-gated ion channel 5 (ion channel gated by extracellular ATP) may be involved in synaptic transmission and neurogenesis, and defects may lead to pathophysiology of idiopathic detrusor instability); 422aa), pI: 7.63, MW: 47206 TM: 1[ P ] Gene chromosome: 17p 13.3).

(32) CD72(B cell differentiation antigens CD72, Lyb-2); 359aa, pI: 8.66, MW: 40225, TM: 15[ P ] Gene chromosome: 9p 13.3).

(33) LY64 (lymphocyte antigen 64(RP105), type I membrane protein of the Leucine Rich Repeat (LRR) family, regulates B cell activation and apoptosis, loss of function is associated with increased disease activity in systemic lupus erythematosus patients); 661aa, pI: 6.20, MW: 74147 TM: 1[ P ] Gene chromosome: 5q 12).

(34) FcRH1(Fc receptor-like protein 1 (a putative receptor for immunoglobulin Fc domains, containing both C2-type Ig-like and ITAM domains) may play a role in B lymphocyte differentiation); 429aa, pI: 5.28, MW: 46925 TM: 1[ P ] Gene chromosome: 1q21-1q22)

(35) IRTA2 (immunoglobulin superfamily receptor translocation related 2, putative immunoreceptors that may play a role in B cell development and lymphomata; gene dysregulation by translocation occurs in some B cell malignancies); 977aa, pI: 6.88, MW: 106468, TM: 1[ P ] Gene chromosome: 1q21)

(36) TENB2(TMEFF2, brain tumor suppressor protein (tomoregulin), TPEF, HPP1, TR, putative transmembrane proteoglycans, associated with EGF/heregulin family growth factors and follistatin); 374aa)

(37) PSMA-FOLH1 (folate hydrolase (prostate specific membrane antigen) 1)

(38) SST (somatostatin receptors; note 5 subtypes)

(38.1) SSTR2 (somatostatin receptor 2)

(38.2) SSTR5 (somatostatin receptor 5)

(38.3)SSTR1

(38.4)SSTR3

(38.5)SSTR4

AvB 6-two subunits (39+40)

(39) ITGAV (integrin, alpha V)

(40) ITGB6 (integrin, beta 6)

(41) CEACAM5 (carcinoembryonic antigen associated cell adhesion molecule 5)

(42) MET (MET proto-oncogene; hepatocyte growth factor receptor)

(43) MUC1 (mucin 1, cell surface related)

(44) CA9 (Carbonic anhydrase IX)

(45) EGFRvIII (epidermal growth factor receptor (EGFR), transcript variant 3,

(46) CD33(CD33 molecule)

(47) CD19(CD19 molecule)

(48) IL2RA (interleukin 2 receptor, α); NCBI reference sequence: NM _ 000417.2);

(49) AXL (AXL receptor tyrosine kinase)

(50) CD30-TNFRSF8 (tumor necrosis factor receptor superfamily member 8)

(51) BCMA (B cell maturation antigen) -TNFRSF17 (tumor necrosis factor receptor superfamily member 17)

(52) CT Ags-CTA (cancer testis antigen)

(53) CD174(Lewis Y) -FUT3 (fucosyltransferase 3 (galactoside 3(4) -L-fucosyltransferase, Lewis blood type)

(54) CLEC14A (C-type lectin domain family 14 member A; Genbank accession No. NM175060)

(55) GRP78-HSPA5 (Heat shock 70kDa protein 5 (glucose regulatory protein, 78kDa)

(56) CD70(CD70 molecule) L08096

(57) A stem cell specific antigen. For example:

5T4 (see item (63) below)

CD25 (see item (48) above)

·CD32

·LGR5/GPR49

·Prominin/CD133

(58)ASG-5

(59) ENPP3 (ectonucleotide pyrophosphatase/phosphodiesterase 3)

(60) PRR4 (Rich in proline 4 (tear))

(61) GCC-GUCY2C (guanylate cyclase 2C (heat stable enterotoxin receptor)

(62) Liv-1-SLC39A6 (solute carrier family 39 (Zinc transporter) member 6)

(63)5T4, trophoblast glycoprotein, TPBG-TPBG (trophoblast glycoprotein)

(64) CD56-NCMA1 (neural cell adhesion molecule 1)

(65) CanAg (tumor associated antigen CA242)

(66) FOLR1 (Folic acid receptor 1)

(67) GPNMB (glycoprotein (transmembrane) nmb)

(68) TIM-1-HAVCR1 (hepatitis A Virus cell receptor 1)

(69) RG-1/prostate tumor target Mindin-Mindin/RG-1

(70) B7-H4-VTCN1 (V-set domain-containing T cell activation inhibitor 1)

(71) PTK7(PTK7 protein tyrosine kinase 7)

(72) CD37(CD37 molecule)

(73) CD138-SDC1 (syndecan 1)

(74) CD74(CD74 molecule, major histocompatibility complex, class II invariant chain)

(75) Tight junction protein-CL (tight junction protein)

(76) EGFR (epidermal growth factor receptor)

(77) Her3(ErbB3) -ERBB3(v-erb-b2 erythroblastic leukemia virus oncogene homolog 3 (birds))

(78) RON-MST1R (macrophage stimulating 1 receptor (c-met associated tyrosine kinase))

(79) EPHA2(EPH receptor A2)

(80) CD20-MS4A1 (transmembrane 4 domain subfamily A member 1)

(81) Tenascin C (tenascin C) -TNC (tenascin C)

(82) FAP (fibroblast activation protein alpha)

(83) DKK-1(Dickkopf 1 homolog (Xenopus laevis))

(84) CD52(CD52 molecule)

(85) CS1-SLAMF7(SLAM family member 7)

(86) Endoglin (Endoglin) -ENG (Endoglin)

(87) Annexin A1-ANXA1 (annexin A1)

(88) V-CAM (CD106) -VCAM1 (vascular cell adhesion molecule 1)

Additional tumor-associated antigens and cognate antibodies of interest are:

(89) ASCT2(ASC transporter 2, also known as SLC1a 5).

ASCT2 antibodies are described in WO 2018/089393 (which is incorporated herein by reference).

The cell binding agent may be labelled, for example to aid detection or purification of the agent prior to incorporation as a conjugate or as part of a conjugate. The label may be a biotin label. In another example, the cell binding agent may be labeled with a radioisotope.

Method of treatment

The conjugates of the invention may be used in methods of treatment. Also provided are methods of treatment comprising administering to a subject in need thereof a therapeutically effective amount of a conjugate having formula IV. The term "therapeutically effective amount" is an amount sufficient to show benefit to a patient. Such benefit may be at least a reduction in at least one symptom. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the disease being treated. Treatment prescriptions (e.g., determination of dosages) are the responsibility of general practitioners and other physicians.

The conjugates may be administered alone or in combination with other therapies, either simultaneously or sequentially, depending on the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (including administration of active agents such as drugs); performing surgery; and radiation therapy.

Thus, the pharmaceutical composition according to the invention and for use according to the invention may comprise, in addition to the active ingredient (i.e. the conjugate having formula IV), a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other material well known to the person skilled in the art. These materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders or liquids. Tablets may contain solid carriers or adjuvants. Liquid pharmaceutical compositions generally comprise a liquid carrier, such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. Capsules may contain solid carriers such as gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. The person skilled in the art is fully enabled to prepare suitable solutions using, for example, isotonic vehicles, such as sodium chloride injection, ringer's injection, lactated ringer's injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included as desired.

The conjugates are useful for treating proliferative and autoimmune diseases. The term "proliferative disease" relates to unwanted or uncontrolled cellular proliferation of excess or abnormal cells, whether in vitro or in vivo, which is undesirable, e.g., neoplastic or proliferative growth.

Examples of proliferative disorders include, but are not limited to, benign, precancerous, and malignant cell proliferation, including, but not limited to, neoplasms and tumors (e.g., histiocytoma, glioma, astrocytoma, osteoma), cancer (e.g., lung cancer, small cell lung cancer, gastrointestinal cancer, intestinal cancer, colon cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma, osteosarcoma, kaposi's sarcoma, melanoma), leukemia, psoriasis, skeletal diseases, fibroproliferative disorders (e.g., disorders of connective tissue), and atherosclerosis. Other cancers of interest include, but are not limited to, hematologic malignancies, such as leukemias and lymphomas, such as non-hodgkin's lymphoma and subtypes (e.g., DLBCL, marginal zone lymphoma, mantle zone lymphoma, and follicular lymphoma), hodgkin's lymphoma, AML, and other cancers of B or T cell origin. Any type of cell can be treated, including but not limited to lung, gastrointestinal tract (including, for example, intestine, colon), breast (breast or mammary), ovary, prostate, liver (liver or liver), kidney (kidney or renal), bladder, pancreas, brain and skin.

Examples of autoimmune diseases include the following: rheumatoid arthritis, autoimmune demyelinating diseases (e.g. multiple sclerosis, allergic encephalomyelitis), psoriatic arthritis, endocrine ophthalmopathy, uveal retinitis, systemic lupus erythematosus, myasthenia gravis, Graves ' disease, glomerulonephritis, autoimmune liver disorders, inflammatory bowel diseases (e.g. crohn's disease), allergies, anaphylaxis, sjogren's syndrome [ (bsyndrome), type I diabetes, primary biliary cirrhosis, Wegener's granulomatosis, fibromuscular mycosisPain, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupus, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, dresler's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia areata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, phnomenophenon), esophageal dyskinesia, telangiectasia, and capillary hemorrhage, Autoimmune infertility in males and females, ankylosing spondylitis, ulcerative colitis, mixed connective tissue disease, polyarteritis nodosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas ' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, antiphospholipid syndrome, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, autoimmune chronic active hepatitis, avicularia lung, toxic epidermal necrolysis, Alport syndrome, alveolitis (alveolitis), allergic alveolitis, fibrotic alveolitis, interstitial lung disease, erythema nodosum, pyoderma, transfusion reaction, Takayas ' arteritis, rheumatic polymyalgia, temporalis, schistosomiasis, Giant cell arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Capra's syndrome, Kawasaki's disease, dengue fever, encephalomyelitis, endocarditis, endocardial myocardial fibrosis, endophthalmitis, persistent elevated erythema (erythroblast et diutanum), psoriasis, fetal erythroblastosis, eosinophilic fasciitis, Schumann syndrome (Shuul)man's syndrome), Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterogeneous cyclitis, Fuch cyclitis, IgA nephropathy, Henoch purpura (Henoch-Schonlein purpura), graft versus host disease, transplant rejection, cardiomyopathy, myasthenia-syndrome (Eaton-Lambert syndrome), recurrent polychondritis, cryoglobulinemia, fahrenheit macroglobulinemia (Waldenstrom's macrobulbemia), venturi syndrome (Evan's syndrome), and autoimmune gonadal failure.

In some embodiments, the autoimmune disease is a disorder of the following cells: b lymphocytes (e.g., systemic lupus erythematosus, goodpasture's syndrome, rheumatoid arthritis, and type I diabetes), Th 1-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, sjogren's syndrome, hashimoto's thyroiditis, graves' disease, primary biliary cirrhosis, wegener's granulomatosis, tuberculosis, or graft-versus-host disease), or Th 2-lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis (rhinoconjunctivitis), allergic rhinitis, omenna's syndrome, or chronic graft-versus-host disease). Generally, disorders involving dendritic cells involve disorders of Th1 lymphocytes or Th2 lymphocytes. In some embodiments, the autoimmune disorder is a T cell-mediated immunological disorder.

A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer, regardless of its mechanism of action. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spindle poison plant alkaloids (spindle poison plant alkaloids), cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in "targeted therapy" and conventional chemotherapy.

Examples of chemotherapeutic agents include: erlotinib (b)Gene technology company (Genentech)/OSI pharmaceutical company (OSI Pharm.)),Docetaxel (docetaxel: (b))Xenof-Anthrate group (Sanofi-Aventis)), 5-FU (Fluorouracil, 5-Fluorouracil, CAS number 51-21-8), Gemcitabine (Gemcitabine)Lilly (Lilly)), PD-0325901(CAS No. 391210-10-9, Pfizer (Pfizer)), cisplatin (cis-diamine, dichloroplatinum (II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel (Taxol: (Lilly))Bethes-Shi Guibao tumor company (Bristol-Myers Squibb Oncology), Princeton, N.J.), trastuzumab (Trastuzumab)Gene technology Co., Ltd. (Genentech)), temozolomide (4-methyl-5-oxo-2, 3, 4, 6, 8-pentaazabicyclo [4.3.0 ]]Nonane-2, 7, 9-triene-9-carboxamide, CAS number 85622-93-1,schering Plough), tamoxifen ((Z) -2- [4- (1, 2-diphenylbut-1-enyl) phenoxy]-N, N-dimethylethylamine,) And doxorubicinAkti-1/2, HPPD, and rapamycin.

Further examples of chemotherapeutic agents include: oxaliplatin (oxaliplatin) ((oxaliplatin))Cenofuran group), phenylbenzomide (bortezomib) ((iii)Millennium pharmaceuticals (Millennium Pharm.), and suotan (sutent) ((Millennium Pharm.))SU11248, pyroxene), letrozole (letrozole), (ltrozole)Novartis), imatinib mesylate (imatinib mesylate), (Novartis), and (I) and (II) salts of the sameNowa), XL-518(Mek inhibitor, Icelis (Exelixis), WO2007/044515), ARRY-886(Mek inhibitor, AZD6244, Eliza biopharmaceutical company (Array BioPharma), AstraZeneca), SF-1126(PI3K inhibitor, Semafore Pharmaceuticals (Semafore Pharmaceuticals)), BEZ-235(PI3K inhibitor, Nowa), XL-147(PI3K inhibitor, Icelis), PTK787/ZK 222584 (Nowa), fulvestrant (fulvestrant) ((furlvetrant))Asricon), leucovorin (leucovorin or folinic acid), rapamycin (rapamycin) (sirolimus),whitman (Wyeth)), lapatinib (1apatinib), (1)GSK572016, Glan Smith Kline, Lonafanib (Sarasar @, Inc.), and Salarar^TMSCH 66336, Xian Probaba corporation), Sorafenib (sorafenib) ((Schafenib)BAY43-9006, Bayer Lab (Bayer Labs)), Gefitinib (gefitinib) (bAslicon), irinotecan (irinotecan) ((R)CPT-11, Perey), tipifarnib (ZARNESTRA)^TMJohnson corporation (Johnxson)&Johnson))、ABRAXANE^TM(Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, sconberg, il), vandetanib (rINN, ZD6474,astrazep), chlorambucil (chlorembucil), AG1478, AG1571(SU 5271; threo root (Sugen)), temsirolimus (temsirolimus), (a) and (e (having a combination of at least one or a combination of one or (for exampleHewlett-packard company), pazopanib (pazopanib) (Kurarian Stecke company), Kamamide (canfosfamide) ((Hewlett-packard Co.)Telik), thiotepa (thiotepa) and cyclophosphamide (cyclophosphamide)Alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines), such as benzotepa (benzodopa), carboquone (carboquone), metotepipa (meturedopa), and uretepa (uredopa); ethyleneimine (ethylenimine) and methyl melamine (melamelamines), including altretamine (altretamine), triethylenemelamine (triethyleneemelamine), triethylenephosphoramide (triethylenephosphoramide), triethylenethiophosphoramide (triethylenephosphoramide), and trimethylmelamine (trimethylomelamine); polyacetogenin (especially bullatacin and bullatacin); camptothecin (camptothecin) (including the synthetic analogue topotecan (topo)tecan)); bryostatin; callystatin; CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin (dolastatin); duocarmycins (including synthetic analogs, KW-2189 and CB1-TM 1); shogaol (eleutherobin); coprinus atrata base (pancratistatin); sarcandra glabra alcohol (sarcodictyin); spongistatin (spongistatin); nitrogen mustards (nitrosamines), such as chlorambucil (chlorambucil), chlorambucil (chlorenaphazine), chlorophosphamide (chlorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), dichloromethyldiethanamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), neonebivochin (novembichin), benzene mustard cholesterol (phenylesterine), prednimustine, triamcinolone (trofosfamide), uracil mustard (uramustard); nitrosoureas such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine), and ramustine (ranirnustine); antibiotics, such as enediyne antibiotics (e.g., calicheamicin (calicheamicin), calicheamicin gamma1I (calicheamicin gamma1I), calicheamicin omega I1(calicheamicin omega I1) (Angew chem. intl. ed. Engl. [ applied chemistry-English International edition ]](1994)33: 183-186); nikkomycin (dynemicin), nikkomycin a (dynemicin a); diphosphonates, such as clodronate; esperamicin (esperamicin); and the neooncostatin chromophore and related chromoproteins enediyne antibiotic chromophores), aclacinomycin (aclacinomysins), actinomycin (actinomycin), amphenomycin (aurramycin), azaserine (azaserine), bleomycin (bleomycin), actinomycin C (cacinomycin), clarithromycin (carabicin), carminomycin (carminomycin), carcinomycin (carminomycin), chromomycin (chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), ditobicin (Detorubicin), 6-diazo-5-oxo-rpL-norleucine, morpholino-adriamycin (moxinorubiholn), cyanomorpholino-adriamycin (cyano-doxorubicin), (dactinomycin, norubicin, and related chromoproteinscyamorphophallno-doxorubicin, 2-pyrrolo-doxorubicin and deoxydoxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), nemorubicin (nemorubicin), mariomycin (marcellomycin), mitomycins (e.g. mitomycin C), mycophenolic acid (mycophenolic acid), nogomycin (nogalamycin), olivomycin (olivomycin), pelomycin (pelomomycin), porphyrinomycin (porfiromycin), puromycin (puromycin), triiron doxorubicin (melamycin), rodobicin (rodorubicin), streptomycin (streptanigrin), streptozomycin (streptamycin), tubercidin (ubulin), mexican (mexican), zorubicin (zorubicin); antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thioguanine (thiamirine), thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as carpoterone, drotandrosterone propionate, epithioandrostanol, meiandrane, testolactone; anti-adrenalines, such as aminoglutethimide, mitotane, trostane; folic acid supplements, such as folic acid (frilic acid); acetic acid glucurolactone; an aldehydic phosphoramide glycoside; (ii) aminolevulinic acid; eniluracil; amsacrine; (xxix) brassica rapa (bestrabucil); bisantrene; edatrexate (edatraxate); desphosphamide (defosfamide); dimecorsine (demecolcine); diazaquinone (diaziqutone); isoflurine (elfornithine); ammonium etitanium acetate; epothilone (epothilone); etoglut (etoglucid); gallium nitrate; a hydroxyurea; lentinan; lonidanine (lonidanine); maytansinoids such as maytansine and ansamitocins; mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidanol (mopidanmol); rhizobia (nitrarine); pentostatin (pentostatin); phenamet (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); podophyllumAcid (podophyllic acid); 2-ethyl hydrazide; procarbazine (procarbazine);polysaccharide complex (JHS Natural Products, uk, JHS Natural Products); propyleneimine (razoxane); rhizomycin (rhizoxin); sizofuran (sizofiran); germanium spiroamines (spirogyranium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2, 2' -trichlorotriethylamine; trichothecenes (trichothecenes), in particular the T-2 toxin, the wart A (verracutinin A), the bacillosporin A (roridin A) and the serpentines (anguidine)); urethane (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannitol mustard (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); gatifloxacin; cytarabine (arabine) ("Ara-C"); cyclophosphamide (cyclophosphamide); thiotepa (thiotepa); 6-thioguanine (6-thioguanine); mercaptopurine; methotrexate; platinum analogs, such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide (ifosfamide); mitoxantrone (mitoxantrone); vincristine; vinorelbineMitoxantrone (novantrone); teniposide (teniposide); edatrexate (edatrexate); daunomycin (daunomycin); aminopterin; capecitabine (Roche (Roche)); ibandronate sodium (ibandronate); CPT-11; topoisomerase inhibition RFS 2000; difluoromethylornithine (DMFO); tretinoin, such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

Also included in the definition of "chemotherapeutic agent" are: (i) anti-hormonal agents that act to modulate or inhibit the action of hormones on tumors, such as anti-estrogens and Selective Estrogen Receptor Modulators (SERMs), including, for exampleSuch as tamoxifen (includingTamoxifen citrate), raloxifene (raloxifene), droloxifene (droloxifene), 4-hydroxytamoxifen (4-hydroxytamoxifen), trioxifene (trioxifene), raloxifene (keoxifene), LY117018, onapristone (onapristone), and(toremifene citrate); (ii) aromatase inhibitors which inhibit the enzyme aromatase, which regulates the production of estrogen by the adrenal glands, such as 4(5) -imidazoles, aminoglutethimide (aminoglutethimide),(megestrol acetate)) (a salt of megestrol acetate),(exemestane (Aemestane); pyroxene Co.), formestane (formestanie), fadrozole (fadrozole),(vorozole) and (C) a salt thereof,(letrozole; Nowa Co.), and(anastrozole; Astrozole, Aslicon (III) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, goserelin, and goserelin (goserelin), and troxacitabine (1, 3-dioxolane nucleoside cytosine analogues), (iv) protein kinase inhibitors, such as MEK inhibitors (WO2007/044515), (v) lipid kinase inhibitors, (vi) antisense oligonucleotides, particularly inhibitors of signaling pathways associated with abnormal cell proliferationThe gene expression oligonucleotides of (1), e.g., PKC-alpha, Raf and H-Ras, e.g., oblimersen (oblimersen) ((ii))Genta corporation (Genta Inc.); (vii) ribozymes, e.g., VEGF expression inhibitors (e.g.) And inhibitors of HER2 expression; (viii) vaccines, e.g. gene therapy vaccines, e.g.And rIL-2; topoisomerase 1 inhibitors, e.g. rmRH; (ix) anti-angiogenic agents, e.g. bevacizumab (bevacizumab) ((R))Gene technology corporation); and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

Also included in the definition of "chemotherapeutic agent" are therapeutic antibodies, such as alemtuzumab (alemtuzumab) (Campath), bevacizumab (bevacizumab) (r)Gene technology corporation); cetuximab (cetuximab) (ii)English clone (Imclone)); parnitolMonoclonal antibody (panitumumab) (panitumumab)Amersham Anin (Amgen)), rituximab (rituximab), (b)Gene technologies Inc./Baijian Aidi Inc. (Biogen Idec)), Pertuzumab (pertuzumab) (OMNITARG^TM2C4, GeneTechnical Co.), trastuzumab (trastuzumab) ((R)Genetech), tositumomab (Bexxar, Corixia), and antibody drug conjugates, gemtuzumab ozogamicin (gemtuzumab ozogamicin) ((r)Hewlett packard).

Humanized monoclonal antibodies having therapeutic potential as chemotherapeutic agents in combination with the conjugates of the invention include: alemtuzumab (alemtuzumab), aprelizumab (apiolizumab), alemtuzumab (aselizumab), natalizumab (atlizumab), bapinezumab (bapineuzumab), bevacizumab (bevacizumab), moxin-bivatuzumab (bivatuzumab mertansine), moxin-pertuzumab (cantuzumab), cetrizumab (cedellizumab), pegge-sedrituzumab (cezutamumab pegol), cidfutuzumab, cidtuzumab (daclizumab), eculizumab (efuzumab), epratuzumab (epfectuzumab), everuzumab (epuzumab), epuzumab (epuzumab), epreduzumab), epulilizumab (epulilizumab), apelizumab (daplizumab), epulilizumab (epulilizumab), epulizumab (epuzumab), epuzumab (epuzumab), epuzumab (epuzumab), epuzumab (epuzumab), epuzumab (epuzumab) or epuzumab (epuzumab), epuzumab (epuzumab), epuzumab) or (epuzumab) or (peripheral) or (peripheral) or (peripheral) or (peripheral) or a (peripheral) or (peripheral), or (peripheral) or a (peripheral) or a (peripheral) or (peripheral), or a peripheral), or a peripheral (peripheral), a (peripheral), or a (peripheral), or a (peripheral), peripheral (peripheral), peripheral unit (peripheral), a (peripheral), or a (peripheral (, Meperilizumab (mepolizumab), motavizumab (motavizumab), motavizumab, natalizumab (natalizumab), nimotuzumab (nimotuzumab), nolovizumab (nolovizumab), noumazumab (numazumab), orelizumab (ocrelizumab), omalizumab (omalizumab), palivizumab (palivizumab), paclobuzumab (paclobuzumab), pertuzumab (pertuzumab), pemulilizumab (pexelizumab), rallizumab (rapulizumab), ranibizumab (ranibizumab), trastuzumab (resvivumab), revazuzumab (reslizumab), vellizumab (pexelizumab), rallizumab (raplizumab), ranibizumab (ranibizumab), trastuzumab (reslizumab), ranibizumab (rellizumab), trastuzumab (reslizumab), trastuzumab (rellizumab), trastuzumab (trastuzumab), trastuzumab (rellizumab), trastuzumab (relevalizumab), trastuzumab (trastuzumab), trastuzumab (rellizumab), trastuzumab (trastuzumab), trastuzumab (rellizumab), trastuzumab (trastuzumab), trastuzumab (rellizumab), trastuzumab (trastuzumab), trastuzumab (trastuzumab), trastuzumab (trastuzumab), trastuzumab (trastuzumab), trastuzumab (trastuzumab), trastuzumab (trastuzumab), trastuzumab (rit (trastuzumab), trastuzumab (trastuzumab), trastuzumab (trastuzumab), trastuzumab (rit (trastuzumab), trastuzumab (trastuzumab), and (rit (trastuzumab), trastuzumab (rit (trastuzumab, Tositulizumab (tocilizumab), tolrelizumab (toralizumab), trastuzumab (trastuzumab), semukulkin-2-tuotuzumab (tucotuzumab celeukin), tucusituzumab, umavivzumab (tutoxuzumab), ertozumab (urtoxazumab), and willizumab (visilizumab).

Formulations

Although the conjugate may be used (e.g., applied) alone, it is generally preferred that it be present in the form of a composition or formulation.

In one embodiment, the composition is a pharmaceutical composition (e.g., formulation, preparation, medicament) comprising a conjugate described herein and a pharmaceutically acceptable carrier, diluent, or excipient.

In one embodiment, the composition is a pharmaceutical composition comprising at least one conjugate described herein and one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, surfactants (e.g., wetting agents), masking agents, coloring agents, flavoring agents, and sweetening agents.

In one embodiment, the composition further comprises other active agents, such as other therapeutic or prophylactic agents.

Suitable carriers, diluents, excipients and the like can be found in the standard pharmaceutical literature. See, for example, Handbook of Pharmaceutical Additives, 2 nd edition (edited by m.ash and i.ash), 2001 (synthetic Information Resources, Inc.), endcocott (Endicott), new york, usa), Remington's Pharmaceutical Sciences, remmington's Pharmaceutical Sciences, 20 th edition, published by the trademarks, Williams and Wilkins (Lippincott, Williams & Wilkins), 2000; and Handbook of Pharmaceutical Excipients [ Handbook of Pharmaceutical Excipients ], 2 nd edition, 1994.

Another aspect of the invention relates to a method of preparing a pharmaceutical composition, the method comprising admixing at least one of the terms [2 ], [ as defined herein¹¹C]The radiolabeled conjugate or conjugate-like compound is mixed with one or more other pharmaceutically acceptable ingredients (e.g. carriers, diluents, excipients, etc.) well known to those skilled in the art. If formulated in discrete units (e.g., tablets, etc.), each unit contains a predetermined quantity (dose) of the active compound.

As used herein, the term "pharmaceutically acceptable" refers to compounds, ingredients, materials, compositions, dosage forms, and the like, which are, within the scope of sound medical judgment, suitable for contact with the tissues of the subject in question (e.g., a human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

The formulations may be prepared by any method well known in the pharmaceutical arts. Such methods include the step of bringing into association the active compound with the carrier which constitutes one or more accessory ingredients. In general, formulations are prepared by uniformly and intimately bringing into association the active compound with carriers (e.g., liquid carriers, finely divided solid carriers, and the like), and then shaping the product as necessary.

The formulation may be prepared to provide rapid or slow release; immediate, delayed, timed or sustained release; or a combination thereof.

Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). Such liquids may additionally contain other pharmaceutically acceptable ingredients, such as antioxidants, buffers, preservatives, stabilizers, bacteriostats, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other relevant bodily fluids) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include sodium chloride injection, ringer's solution, or lactated ringer's injection. Typically, the concentration of active ingredient in the liquid is from about 1ng/ml to about 10 μ g/ml, for example from about 10ng/ml to about 1 μ g/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

Dosage form

One skilled in the art will recognize that the appropriate dosage of the conjugate, and compositions comprising the conjugate, may vary from patient to patient. Determining the optimal dosage will generally involve balancing the level of therapeutic benefit with any risk or deleterious side effects. The selected dosage level will depend upon a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds and/or materials used in combination, the severity of the condition, and the ethnicity, sex, age, weight, condition, general health, and prior medical history of the patient. The amount of the compound and the route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although the dosage will generally be selected to achieve local concentrations at the site of action which will achieve the desired effect without causing substantial deleterious or toxic side effects.

Administration may be carried out continuously or intermittently (e.g., in divided doses at appropriate intervals) in a dosage throughout the course of treatment. Methods of determining the most effective mode of administration and dosage are well known to those skilled in the art and will vary depending on the formulation used for treatment, the purpose of the treatment, the target cell or cells being treated and the subject being treated. Single or multiple administrations may be carried out with the dose level and pattern being selected by the treating physician, veterinarian or clinician.

Typically, a suitable dose of the active compound is in the range of about 100ng to about 25mg per kilogram body weight of the subject per day (more typically about 1 μ g to about 10 mg). When the active compound is a salt, ester, amide, prodrug, or the like, the amount administered is calculated based on the parent compound, and thus the actual weight to be used is increased proportionally.

The above doses may be applied to the conjugate, or to an effective amount of the compound that is releasable upon cleavage of the linker.

For the prevention or treatment of disease, the appropriate dosage of an ADC of the invention will depend on the type of disease to be treated, the severity and course of the disease, whether the molecule is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician, as defined above. The molecule may suitably be administered to a patient at once or by a series of treatments. Depending on the type and severity of the disease, molecules of about 1 μ g/kg to 100mg/kg or more are initial candidates for administration to the patient, e.g., by one or more separate administrations or continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is continued until the desired suppression of disease symptoms occurs. Other dosage regimens may be useful. The progress of the therapy is readily monitored by conventional techniques and assays.

Drug loading

Drug loading (p) is the average number of drugs per ligand unit, which may be a cell binding agent, such as an antibody.

The average drug amount per antibody in the ADC preparation from the conjugation reaction can be characterized by conventional means such as UV, reverse phase HPLC, HIC, mass spectrometry, ELISA assays and electrophoresis. The quantitative distribution of ADCs in terms of p can also be determined. The mean value of p in a particular ADC preparation can be determined by ELISA (Hamblett et al (2004) Clin. cancer Res. [ clinical cancer research ] 10: 7063-. However, the distribution of p (drug) values cannot be distinguished by antibody-antigen binding and detection limitations of ELISA. Furthermore, ELISA assays for detecting antibody-drug conjugates cannot determine where the drug moiety is attached to the antibody, e.g., a heavy or light chain fragment or a particular amino acid residue. In some cases, separation, purification, and characterization of homogeneous ADCs may be achieved by methods such as reverse phase HPLC or electrophoresis, where p is some value from ADCs of other drug loadings. Such techniques are also applicable to other types of conjugates.

For some antibody-drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, an antibody may have only one or a few cysteine thiol groups, or may have only one or a few sufficiently reactive thiol groups through which a linker may be attached. Higher drug loading may lead to aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates.

Typically, during the conjugation reaction, the drug moiety conjugated to the antibody is less than the theoretical maximum. The antibody may contain, for example, a number of lysine residues that are not reactive with the drug linker. Only the most reactive lysine groups can react with the amine-reactive linker reagent. Furthermore, only the most reactive cysteine thiol group may react with the thiol-reactive linker reagent. Typically, antibodies do not contain many, if any, free and reactive cysteine thiol groups to which drug moieties may be attached. Most cysteine thiol residues in antibodies to the compounds exist as disulfide bridges and must be reduced with a reducing agent such as Dithiothreitol (DTT) or TCEP under partially or fully reducing conditions. The loading (drug/antibody ratio) of the ADC can be controlled in several different ways, including: (i) limiting the molar excess of drug linker relative to antibody; (ii) limiting conjugation reaction time or temperature; and (iii) cysteine thiol-modified partial or limiting reduction conditions.

Some antibodies have reducible interchain disulfide bonds, i.e., cysteine bridges. The antibody can be made reactive for conjugation with a linker reagent by treatment with a reducing agent such as DTT (dithiothreitol). Thus, each cysteine bridge will theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into the antibody by reacting lysine with 2-iminothiolane (yurt's reagent), resulting in the conversion of the amine to a thiol. Reactive thiol groups can be introduced into an antibody (or fragment thereof) by engineering one, two, three, four, or more cysteine residues (e.g., making a mutant antibody comprising one or more non-native cysteine amino acid residues). US7521541 teaches the engineering of antibodies by introducing reactive cysteine amino acids.

Cysteine amino acids may be engineered at the reactive site of an antibody and do not form intra-or intermolecular disulfide bonds (Junutula, et al, 2008b Nature Biotech. [ Nature Biotechnology ], 26 (8): 925-. The engineered cysteine thiol may be reacted with a drug linker of the present invention (i.e., a compound having formula I) having a thiol-reactive electrophilic group, such as a maleimide or an alpha-haloamide, to form an ADC with the cysteine-engineered antibody. Thus, the location of the drug units can be designed, controlled and known. Since the engineered cysteine thiol groups typically react with the drug linker reagent in high yield, drug loading can be controlled. IgG antibodies were engineered to introduce cysteine amino acids by making substitutions at a single site on either the heavy or light chain, giving two new cysteines on the symmetric antibody. Drug loading approaching 2 can be achieved, with the conjugate product ADC approaching homogeneity.

If more than one nucleophilic or electrophilic group of the antibody is reacted with a drug linker, the resulting product may be a mixture of ADC compounds in which the distribution of drug units attached to the antibody is, for example, 1, 2, 3, etc. Liquid chromatography methods such as polymer reverse phase (PLRP) and Hydrophobic Interaction (HIC) can separate compounds in a mixture by drug loading value. Formulations of ADCs with individual drug loading values (p) can be isolated, however, these individual loading values ADCs can still be heterogeneous mixtures in that the drug units can be attached to different sites on the antibody via linkers.

Thus, the antibody-drug conjugate compositions of the invention can include a mixture of antibody-drug conjugates, wherein the antibody has one or more drug moieties, and wherein the drug moieties can be attached to the antibody at various amino acid residues.

In one embodiment, the average drug amount per cell-binding agent is in the range of 1 to 20. In some embodiments, the range is selected from 1 to 10, 2 to 8, 2 to 6, and 4 to 10.

In some embodiments, there is one drug per cell-binding agent.

General synthetic route

A compound having the formula I (wherein R^LHaving the formula Ia) can

By linking a compound having formula 3:

or an activated version thereof, from a compound having formula 2 (wherein R is^L*is-QH).

Such a reaction may be carried out under amide coupling conditions.

The compound having formula 2 may be represented by a compound having formula 4:

(where RL^*protis-Q-Prot^NWherein Prot^NIs an amine protecting group).

The compound having formula 4 may be represented by a compound having formula 5:

coupling with Compound A3 was synthesized using the Friedlander reaction.

The compound having formula 5 may be derived from a compound having formula 6:

synthesized by removing the trifluoroacetamide protecting group.

The compound having formula 6 can be synthesized by coupling: r^L*prot-OH to compound I7.

A compound having the formula I (wherein R^LHaving the formula Ia or Ib) can be prepared from compound I11 by means of compound R^L-OH, or an activated form thereof.

Amine protecting groups

Amine protecting groups are well known to those skilled in the art. Reference is made in particular to the disclosure of suitable protecting groups in the following documents: greene's Protecting Groups in Organic Synthesis, fourth edition, John Wiley & Sons, 2007(ISBN 978-0-471-.

Further preference

The following preferences may apply to all aspects of the invention as described above, or may relate to individual aspects. These preferences can be combined together in any combination.

Q^X

In one embodiment, Q is an amino acid residue. The amino acid may be a natural amino acid or an unnatural amino acid.

In one embodiment, Q is selected from: phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, wherein Cit is citrulline.

In one embodiment, Q comprises a dipeptide residue. The amino acids in the dipeptide can be any combination of natural and unnatural amino acids. In some embodiments, the dipeptide comprises a natural amino acid. When the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide is then the recognition site for the cathepsin.

In one embodiment, Q is selected from:

^NH-Phe-Lvs-^C＝O、

^NH-Val-Ala-^C＝O、

^NH-Val-Lvs-^C＝O、

^NH-Ala-Lys-^C＝O、

^NH-Val-Cit-^C＝O、

^NH-Phe-Cit-^C＝O、

^NH-Leu-Cit-^C＝O、

^NH-Ile-Cit-^C＝O、

^NH-Phe-Arg-^C＝O、

^NH-Trp-Cit-^C＝Oand, and

^NH-Gly-Val-^C＝O；

wherein Cit is citrulline.

Preferably, Q is selected from:

^NH-Phe-Lvs-^C＝O、

^NH-Val-Ala-^C＝O、

^NH-Val-Lys-^C＝O、

^NH-Ala-Lys-^C＝Oand, and

^NH-Val-Cit-^C＝O。

most preferably, Q is selected from^NH-Phe-Lys-^C＝O、^NH-Val-Cit-^C＝OOr^NH-Val-Ala-^C＝O。

Other dipeptide combinations of interest include:

^NH-Gly-Gly-^C＝O、

^NH-Glv-Val-^C＝O

^NH-Pro-Pro-^C＝Oand, and

^NH-Val-Glu-^C＝O。

other dipeptide combinations may be used, including those described by Dubowchik et al, Bioconjugate Chemistry 2002, 13, 855-869, which is incorporated herein by reference.

In some embodiments, Q is a tripeptide residue. The amino acids in the tripeptides may be any combination of natural and unnatural amino acids. In some embodiments, the tripeptides comprise natural amino acids. When the linker is a cathepsin labile linker, the tripeptides are the site of action for cathepsin-mediated cleavage. The tripeptide is then the recognition site for cathepsin. Particularly interesting tripeptide linkers are:

^NH-Glu-Val-Ala-^C＝O

^NH-Glu-Val-Cit-^C＝O

^NH-αGlu-Val-Ala-^C＝O

^NH-αGlu-Val-Cit-^C＝O

in some embodiments, Q is a tetrapeptide residue. The amino acids in the tetrapeptide can be any combination of natural and unnatural amino acids. In some embodiments, the tetrapeptide comprises natural amino acids. When the linker is a cathepsin labile linker, the tetrapeptide is the site of action for cathepsin-mediated cleavage. The tetrapeptide is then the recognition site for cathepsin. Particularly interesting tetrapeptide linkers are:

^NH-Gly-Gly-Phe-Gly^C＝O(ii) a And

^NH-Gly-Phe-Gly-Gly^C＝O。

in some embodiments, the tetrapeptide is:

^NH-Gly-Gly-Phe-Gly^C＝O。

in the above-mentioned representation of the peptide residues,^NHdenotes the N-terminus of the residue, and^C＝Othe C-terminus of the residue is indicated. The C-terminus is bound to NH at a.

Glu represents the residue of glutamic acid, i.e.:

α Glu represents the residue of glutamic acid when bound via the α chain, i.e.:

in one embodiment, the amino acid side chain is chemically protected, where appropriate. The side chain protecting group may be a group as discussed above. The protected amino acid sequence may be cleaved by an enzyme. For example, a dipeptide sequence containing a Lys residue protected by a Boc side chain can be cleaved by a cathepsin.

Protecting groups for amino acid side chains are well known in the art and are described in the Novabiochem catalogue and described above.

G^L

G^LCan be selected from

Wherein Ar represents C_5-6Arylene radicals, e.g. phenylene, and X represents C_1-4An alkyl group.

In some embodiments, G^LIs selected from G^L1-1And G^L1-2. In some of these embodiments, G^LIs G^L1-1。

G^LL

G^LLMay be selected from:

wherein Ar represents C_5-6Arylene radicals, e.g. phenylene, and X represents C_1-4An alkyl group.

In some embodiments, G^LLIs selected from G^LL1-1And G^LL1-2. In some of these embodiments, G^LLIs G^LL1-1。

X

X is:

where a is 0 to 5, b1 is 0 to 16, b2 is 0 to 16, c is 0 or 1, d is 0 to 5, where at least b1 or b2 is 0 and at least c1 or c2 is 0.

a may be 0, 1, 2, 3, 4 or 5. In some embodiments, a is 0 to 3. In some of these embodiments, a is 0 or 1. In further embodiments, a is 0.

b1 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, b1 is 0 to 12. In some of these embodiments, b1 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8.

b2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, b2 is 0 to 12. In some of these embodiments, b2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8.

Only one of b1 and b2 may be other than 0.

c1 may be 0 or 1.

c2 may be 0 or 1.

Only one of c1 and c2 may be other than 0.

d may be 0, 1, 2, 3, 4 or 5. In some embodiments, d is 0 to 3. In some of these embodiments, d is 1 or 2. In further embodiments, d is 2. In a further embodiment, d is 5.

In some embodiments of X, a is 0, b1 is 0, c1 is 1, c2 is 0 and d is 2, and b2 may be from 0 to 8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8.

In some embodiments of X, a is 1, b2 is 0, c1 is 0, c2 is 0 and d is 0, and b1 may be from 0 to 8. In some of these embodiments, b1 is 0, 2, 3, 4, 5, or 8.

In some embodiments of X, a is 0, b1 is 0, c1 is 0, c2 is 0 and d is 1, and b2 may be from 0 to 8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8.

In some embodiments of X, b1 is 0, b2 is 0, c1 is 0, c2 is 0 and one of a and d is 0. The other of a and d is from 1 to 5. In some of these embodiments, the other of a and d is 1. In other of these embodiments, the other of a and d is 5.

In some embodiments of X, a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 may be from 0 to 8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8.

In some embodiments, R^LHaving formula Ib.

In some embodiments, R^LLHas the formula Ib'.

R^L1And R^L2Independently selected from H and methyl, or together with the carbon atom to which they are bonded form a cyclopropene group or a cyclobutene group.

In some embodiments, R^L1And R^L2Both are H.

In some embodiments, R^L1Is H and R^L2Is methyl.

In some embodiments, R^L1And R^L2Both are methyl.

In some embodiments, R^L1And R^L2Together with the carbon atom to which they are bonded form a cyclopropene group.

In some embodiments, R^L1And R^L2Together with the carbon atom to which they are bonded form a cyclobutene group.

In group Ib, in some embodiments, e is 0. In other embodiments, e is 1 and the nitro group may be at any available position on the ring. In some of these embodiments, it is located in the ortho position. In other of these embodiments, it is in the para position.

In some embodiments of the fifth aspect of the invention, the enantiomerically enriched form has an enantiomeric ratio of greater than 60: 40, 70: 30; 80: 20 or 90: 10. In further embodiments, the ratio of enantiomers is greater than 95: 5, 97: 3, or 99: 1.

In some embodiments, R^LSelected from:

in some embodiments, RL^LIs derived fromFrom the above R^LThe radical of (a).

In one embodiment of the first aspect of the invention, the compound having formula I is:

further preference

In some embodiments, the compound having formula I is of formula I^PThe compound of (1):

and salts and solvates thereof, wherein R^LPIs a linker for attachment to a cell binding agent, the linker being selected from the group consisting of:

(ia)：

wherein

Q^PThe method comprises the following steps:

wherein Q^XPSo that Q^PIs an amino acid residue, a dipeptide residue or a tripeptide residue;

X^Pthe method comprises the following steps:

where aP is 0 to 5, bP is 0 to 16, cP is 0 or 1, dP is 0 to 5;

G^Lis a linker for attachment to a ligand unit;

(ib)：

wherein R is^L1And R^L2Independently selected from H and methyl, or together with the carbon atom to which they are bonded form a cyclopropene or cyclobutene group; and is

e is 0 or 1.

aP may be 0, 1, 2, 3, 4 or 5. In some embodiments, aP is 0 to 3. In some of these embodiments, aP is 0 or 1. In further embodiments, aP is 0.

bP can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b is 0 to 12. In some of these embodiments, bP is 0 to 8, and can be 0, 2, 4, or 8.

cP can be 0 or 1.

dP may be 0, 1, 2, 3, 4 or 5. In some embodiments, dP is 0 to 3. In some of these embodiments, dP is 1 or 2. In further embodiments, dP is 2.

At X^PIn some embodiments, aP is 0, cP is 1 and dP is 2, and bP can be from 0 to 8. In some of these embodiments, bP is 0, 4, or 8.

Where appropriate, Q as described above for compounds of formula I^XThe preference of (A) can be applied to Q^XP。

G as described above for the compounds of formula I^L、R^L1、R^L2Preferences for e and e may apply to a compound having formula I^PThe compound of (1).

In some embodiments, the conjugate having formula IV is of formula IV^PThe conjugate of (1):

L-(D^LP)_p (IV^P)

or a pharmaceutically acceptable salt or solvate thereof, wherein L is a ligand unit (i.e., targeting agent), D^LPIs of the formula III^PThe drug linker unit of (1):

R^LLPis a linker attached to the ligand unit, the linker being selected from

(ia’)：

Wherein Q^PAnd X^PIs as defined above and G^LLIs a linker attached to the ligand unit; and

(ib’)：

wherein R is^L1And R^L2Is as defined above; and is

p is an integer from 1 to 20.

In some embodiments, the compound having formula I is of formula I^P2The compound of (1):

and salts and solvates thereof, wherein R^LP2Is a linker for attachment to a cell binding agent, the linker being selected from the group consisting of:

(ia)：

wherein

Q is:

wherein Q^XSuch that Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue;

X^P2the method comprises the following steps:

where aP2 ═ 0 to 5, b1P2 ═ 0 to 16, b2P2 ═ 0 to 16, cP2 ═ 0 or 1, dP2 ═ 0 to 5, where at least b1P2 or b2P2 ═ 0 (i.e. only one of b1 and b2 may not be 0);

G^Lis a linker for attachment to a ligand unit;

(ib)：

wherein R is^L1And R^L2Independently selected from H and methyl, or together with the carbon atom to which they are bonded form a cyclopropene or cyclobutene group; and is

e is 0 or 1.

aP2 may be 0, 1, 2, 3, 4, or 5. In some embodiments, aP2 is 0 to 3. In some of these embodiments, aP2 is 0 or 1. In further embodiments, aP2 is 0.

b1P2 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, b1P2 is 0 to 12. In some of these embodiments, b1P2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8.

b2P2 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, b2P2 is 0 to 12. In some of these embodiments, b2P2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8.

Only one of b1P2 and b2P2 may be other than 0.

cP2 can be either 0 or 1.

dP2 may be 0, 1, 2, 3, 4, or 5. In some embodiments, dP2 is 0 to 3. In some of these embodiments, dP2 is 1 or 2. In a further embodiment, dP2 is 2. In a further embodiment, dP2 is 5.

At X^P2Some experiments ofIn an embodiment, aP2 is 0, b1P2 is 0, cP2 is 1 and dP2 is 2, and b2P2 may be from 0 to 8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5, or 8.

At X^P2In some embodiments, aP2 is 1, b2P2 is 0, cP2 is 0 and dP2 is 0, and b1P2 may be from 0 to 8. In some of these embodiments, b1P2 is 0, 2, 3, 4, 5, or 8.

At X^P2In some embodiments, aP2 is 0, b1P2 is 0, cP2 is 0 and dP2 is 1, and b2P2 may be from 0 to 8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5, or 8.

At X^P2In some embodiments, b1P2 is 0, b2P2 is 0, cP2 is 0 and one of aP2 and dP2 is 0. The other of aP2 and d is from 1 to 5. In some of these embodiments, the other of aP2 and d is 1. In other of these embodiments, the other of aP2 and dP2 is 5.

Where appropriate, Q as described above for compounds of formula I^XThe preferences of (Ia) may apply to^P2Q in (1)^X。

G as described above for the compounds of formula I^L、R^L1、R^L2Preferences for e and e may apply to a compound having formula I^P2The compound of (1).

In some embodiments, the conjugate having formula IV is of formula IV^P2The conjugate of (1):

L-(D^LP2)_p (IV^P2)

or a pharmaceutically acceptable salt or solvate thereof, wherein L is a ligand unit (i.e., targeting agent), D^LP2Is of the formula III^P2The drug linker unit of (1):

R^LLP2is a linker attached to the ligand unit, the linker being selected from

(ia’)：

Wherein Q and X^P2Is as defined above and G^LLIs a linker attached to the ligand unit; and

(ib’)：

wherein R is^L1And R^L2Is as defined above; and is

p is an integer from 1 to 20.

Examples of the invention

General information

Use ofIsolera^TMFlash chromatography was performed and the purity of the fractions was checked using Thin Layer Chromatography (TLC). TLC was performed using Kieselgel 60F 254 silica gel (with fluorescent indicator on aluminum plate) at Merck (Merck). Visualization of TLC was achieved with UV light.

Extraction and chromatography solvents were purchased from VWR, uk and used without further purification.

All fine chemicals were purchased from Sigma Aldrich (Sigma-Aldrich) unless otherwise stated.

Pegylated reagents were obtained from Quanta biodesign, USA via Stratech, UK.

LC/MS conditions

Method A

Positive mode electrospray mass spectrometry was performed using Waters Aquity class H SQD 2.

The mobile phases used were solvent a (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid). The initial component 5% B remained unchanged for 25 seconds and then increased from 5% B to 100% B over a period of 1 minute and 35 seconds. The composition was held at 100% B for 50 seconds and then returned to 5% B within 5 seconds and so held for 5 seconds. Gradient operationThe total duration of (c) was 3.0 minutes. The flow rate was 0.8 mL/min. Detection was performed at 254 nm. Column: waters AcquityBEH Shield RP181.7 μm 2.1X50mm charged with Waters Acquity at 50 ℃BEH Shield RP18 VanGuard front column, 130A, 1.7 μm, 2.1mm x5 mm.

Method B

HPLC (Waters Alliance 2695) was run using mobile phases of water (a) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%).

The initial component 5% B remained unchanged for 25 seconds and then increased from 5% B to 100% B over a period of 1 minute and 35 seconds. The composition was held at 100% B for 50 seconds and then returned to 5% B within 5 seconds and so held for 5 seconds. The total duration of the gradient run was 3.0 minutes. The flow rate was 0.8 mL/min. Wavelength detection range: 190 to 800 nm. Column: waters AcquityBEH Shield RP181.7 μm 2.1X50mm charged with Waters Acquity at 50 ℃BEH Shield RP18 VanGuard front column, 130A, 1.7 μm, 2.1mm x5 mm.

Method C

HPLC (Waters Alliance 2695) was run using mobile phases of water (a) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%).

The initial component 5% B remained unchanged for 1 minute and then increased from 5% B to 100% B over a period of 9 minutes. The composition was held at 100% B for 2 minutes and then returned to 5% B at 0.10 minutes and held for 3min as such. The total gradient run time was equal to 15 min. The flow rate was 0.6 mL/min. Wavelength detection range: 190 to 800 nm. Oven temperature: at 50 ℃. Column: ACE Excel 2C18-AR, 2 μ, 3.0x100 mm.

HPLC conditions

Reversed phase Ultra Fast Liquid Chromatography (UFLC) in Shimadzu corporation (Shimadzu) science^TMUsing Phenomenex on machine^TMGemini NX 5. mu.C 18 column (at 50 ℃) size: 150 X21.2mm. The eluent used was solvent A (H containing 0.1% formic acid)₂O) and solvent B (CH with 0.1% formic acid)₃CN). All UFLC experiments were performed under the following gradient conditions: the initial component 13% B was increased to 30% B over a period of 3 minutes, then to 45% B over 8 minutes and to 100% over another 6 minutes, then to 13% over 2 minutes and held for 1 minute. The total duration of the gradient run was 20.0 minutes. The flow rate was 20.0 mL/min and was measured at 254 and 223 nm.

NMR method

Proton NMR chemical shift values were measured on the delta scale of 400MHz using Bruker AV 400. The following abbreviations have been used: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintuple peak; m, multiplet; br, broad peak. The coupling constants are in Hz.

Synthesis of key intermediates

a) N- (5, 6, 7, 8-tetrahydronaphthalen-1-yl) acetamide (I2)

5, 6, 7, 8-tetrahydronaphthalen-1-amine I1(8.54g, 58.0mmol) was dissolved in dichloromethane (80 mL). Triethylamine (18mL, 129mmol) was added and the mixture was cooled to 0 ℃. Acetic anhydride (11.5mL, 122mmol) was added dropwise and after the addition was complete, the reaction mixture was warmed to rt and stirred for 45min, followed by LCMS to show completion of the reaction. Subjecting the mixture to CH₂Cl₂Diluting with H₂O, saturated NaHCO₃10% citric acid, washing the organic phase over MgSO₄Dried and concentrated in vacuo. The off-white solid was mixed with 1: 3Et₂O/isohexane were triturated together to give I2 as a white solid (10.8g, 57.1mmol, 98% yield) which was used without further purification. LC/MS (method A): retention time 1.44min (ES +) M/z 190[ M + H ]]⁺

b) N- (4-nitro-5, 6, 7, 8-tetrahydronaphthalen-1-yl) acetamide (I3)

N- (5, 6, 7, 8-tetrahydronaphthalen-1-yl) acetamide I2(1.00g, 5.2840mmol) was added portionwise to sulfuric acid (15mL, 281mmol) at-5 ℃. Sodium nitrate (450mg, 5.2945mmol) was added portionwise to the reaction mixture and stirred at-5 ℃ for 30min, followed by LCMS to show no further reaction progress. The reaction mixture was poured into ice with external cooling, and the aqueous mixture was diluted with CH₂Cl₂Extracting, and removing organic phase with MgSO₄Dried and purified by Isolera (10% -80% EtOAc in isohexane) to give a mixture of N- (4-nitro-5, 6, 7, 8-tetrahydronaphthalen-1-yl) acetamide I3 and N- (2-nitro-5, 6, 7, 8-tetrahydronaphthalen-1-yl) acetamide as a white/yellow solid (956mg, 4.0811mmol, 77% yield). LC/MS (method A): retention time 1.53min (ES +) M/z 235[ M + H ]]⁺。

c) N- (4-Nitro-8-oxo-5, 6, 7, 8-tetrahydronaphthalen-1-yl) acetamide (I4)

N- (4-Nitro-5, 6, 7, 8-tetrahydronaphthalen-1-yl) acetamide I3(1.01g, 4.31mmol) was dissolved in acetone (30 mL). Magnesium sulfate (3.9mL, 5.9mmol, 1.5mol/L) in water was added and the mixture was cooled to 0 ℃. Potassium permanganate (2.07g, 13.0mmol) was added to the reaction mixture in portions and the mixture was warmed to rt and stirred for 50min, followed by TLC to show completion of the reaction. The reaction mixture was filtered through celite, the solid was washed with CHCl3 and the resulting organic mixture was washed with H₂O, brine, over MgSO₄Dried and purified by isolera (20% -50% EtOAc in isohexane) to give a mixture of N- (4-nitro-8-oxo-5, 6, 7, 8-tetrahydronaphthalen-1-yl) acetamide I4 and N- (2-nitro-8-oxo-5, 6, 7, 8-tetrahydronaphthalen-1-yl) acetamide as a white/yellow solid (709mg, 2.86mmol, 66%). LC/MS (method A): retention time 1.44min (ES +) M/z 190[ M + H ]]⁺

d) 8-amino-5-nitro-3, 4-dihydronaphthalen-1 (2H) -one (I5)

A mixture of N- (4-nitro-8-oxo-5, 6, 7, 8-tetrahydronaphthalen-1-yl) acetamide I4 and N- (2-nitro-8-oxo-5, 6, 7, 8-tetrahydronaphthalen-1-yl) acetamide (709mg, 2.8561mmol) and 6N hydrochloric acid (7mL) at 80 deg.CStir for 2.5h then LCMS indicated reaction completion. The reaction mixture was cooled in an ice bath and 6N NaOH solution was added until the pH was basic. Mixing the aqueous mixture with CH2Cl₂Extracting, and removing organic phase with MgSO₄Dried and concentrated in vacuo. Isolera (0% -50% EtOAc in isohexane) gave 8-amino-5-nitro-3, 4-dihydronaphthalen-1 (2H) -one I5(320mg, 1.552mmol, 54% yield) as a yellow/orange solid. LC/MS (method A): retention time 1.54min (ES +) M/z 207[ M + H +]⁺

e)2, 2, 2-trifluoro-N- (4-nitro-8-oxo-5, 6, 7, 8-tetrahydronaphthalen-1-yl) acetamide (I6)

8-amino-5-nitro-3, 4-dihydronaphthalen-1 (2H) -one I5(430mg, 2.0854mmol) was dissolved in dichloromethane (20 mL). Pyridine (340 μ L, 4.20mmol) was added and the mixture was cooled to 0 ℃. Trifluoroacetic anhydride (590 μ L, 4.197mmol) was added and stirred for 30min, followed by LCMS to show completion of the reaction. Subjecting the mixture to CH₂Cl₂Diluting with H₂O washing, the organic phase is MgSO₄Dried and concentrated in vacuo to give 2, 2, 2-trifluoro-N- (4-nitro-8-oxo-5, 6, 7, 8-tetrahydronaphthalen-1-yl) acetamide I6(630mg, 2.0846mmol, > 99% yield) as a yellow solid, which was used without further purification. LC/MS (method A): retention time 1.86min (ES +) M/z 301X [ M-H]^-

f) N- (4-amino-8-oxo-5, 6, 7, 8-tetrahydronaphthalen-1-yl) -2, 2, 2-trifluoroacetamide (I7)

Zinc (2.73g, 41.7mmol) was suspended in methanol (80mL), formic acid (4mL) and water (4mL) and the mixture was cooled to 0 ℃.2, 2, 2-trifluoro-N- (4-nitro-8-oxo-5, 6, 7, 8-tetrahydronaphthalen-1-yl) acetamide I6(568mg, 2.0865mmol) was added portionwise and the mixture was stirred at 0 ℃ for 30min, followed by LCMS to indicate completion of the reaction. The reaction mixture was filtered, the filtrate was diluted with EtOAc and saturated NaHCO₃And (6) washing. The organic phase is passed over MgSO₄Dried and concentrated in vacuo to give N- (4-amino-8-oxo-5, 6, 7, 8-tetrahydronaphthalen-1-yl) -2, 2, 2-trifluoroacetamide I7(568mg, 2.0865mmol, > 99% yield) as a yellow solid, which was used without further purification. LC/MS (method A):retention time 1.65 min (ES +) M/z 273[ M + H ]]⁺

g) N- (4-acetamido-8-oxo-5, 6, 7, 8-tetrahydronaphthalen-1-yl) -2, 2, 2-trifluoroacetamide (I8)

N- (8-amino-4-oxo-tetralin-5-yl) -2, 2, 2-trifluoro-acetamide I7(568mg, 2.0865mmol) was dissolved in dichloromethane (20 mL). Triethylamine (580. mu.L, 4.16mmol) and then acetyl chloride (297. mu.L, 4.173mmol) were added and the mixture was stirred for 30min, followed by LCMS to show completion of the reaction. Reacting the mixture with CH₂Cl₂Diluting with H₂O washing, the organic phase is MgSO₄Dried and concentrated in vacuo to afford N- (8-acetamido-4-oxo-tetralin-5-yl) -2, 2, 2-trifluoro-acetamide I8(655mg, 2.084mmol, > 99% yield) as a yellow solid, which was used without further purification. LC/MS (method A): retention time 1.55 min (ES +) M/z 315[ M + H ]]⁺

h) N- (4-amino-5-oxo-5, 6, 7, 8-tetrahydronaphthalen-1-yl) acetamide (I9)

N- (8-acetamido-4-oxo-tetralin-5-yl) -2, 2, 2-trifluoro-acetamide I8(2.77g, 8.81mmol) was dissolved in methanol (240mL) and water (17 mL). Potassium carbonate (4.88g, 35.3mmol) was added and the mixture was stirred at 50 ℃ for 1.5h followed by LCMS to show completion of the reaction. The reaction mixture was cooled, concentrated in vacuo, and dissolved in CH2Cl₂10% MeOH in (1), and H₂And O washing. The organic phase is passed over MgSO₄Dried and chromatographed by isolera (2% -15% MeOH in CH)₂Cl₂Solution of (b) to give N- (8-amino-1-oxo-tetralin-5-yl) acetamide I9(1.20g, 5.50mmol, 62.3% yield) as a yellow solid. LC/MS (method A): retention time 0.98 min (ES +) M/z 219[ M + H]⁺

i) (S) -N- (9-ethyl-9-hydroxy-10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H-benzo [ de ] pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-4-yl) acetamide (I10)

N- (8-amino-1-oxo-tetralin-5-yl) acetamide I9(641mg, 2.94mmol, 1.0 equiv), (S) -4-ethyl-4-hydroxy-7, 8-dihydro-1H-pyrano [3, 4-f)]Indolizine-3, 6, 10(4H) -trione a3(840mg, 3.19mmol, 1.1 equiv.) and PPTS (740mg, 2.95mmol, 1.0 equiv.) were dissolved in toluene (60mL) and stirred at reflux for 3H, after which LCMS indicated that I9 had been consumed. The reaction mixture was cooled and concentrated in vacuo. The resulting solid was triturated with acetonitrile, then acetone to give I10 as a brown solid with slight TsOH contamination (1.26g, 96%). LC/MS (method A): retention time 1.32min (ES +) M/z 447[ M + H ]]⁺

j) (S) -4-amino-9-ethyl-9-hydroxy-l, 2, 3, 9, 12, 15-hexahydro-10H, 13H-benzo [ de ] pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinoline-10, 13-dione (I11)

Reacting (S) -N- (9-ethyl-9-hydroxy-10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H-benzo [ de ]]Pyrano [3 ', 4': 6,7]Indoxazino [1, 2-b ] s]Quinolin-4-yl) acetamide (I10) (1.26g, 2.83mmol, 1.0 equiv.) was dissolved in H₂O (12mL) in hydrochloric acid (6mol/L) and the mixture was stirred at 80 ℃ for 5h, followed by LCMS to show that I10 had been consumed. Subjecting the reaction mixture to hydrogenation with H₂O dilution and concentration in vacuo to give (S) -4-amino-9-ethyl-9-hydroxy-1, 2, 3, 9, 12, 15-hexahydro-10H, 13H-benzo [ de ] as a red crystalline solid]Pyrano [3 ', 4': 6,7]Indoxazino [1, 2-b ] s]Quinoline-10, 13-dione I11(1.51g, 2.85mmol, 90% by mass, 101% yield). LC/MS (method A): retention time 1.36min (ES +) M/z 405[ M + H ]]⁺。

Alternative Synthesis of I11

IPC, purity and determination method of the synthesis

a) 5-bromo-8-nitro-tetralin-1-one (I13)

A solution of potassium nitrate (1.15 equiv., 13.83g) dissolved in sulfuric acid (concentrated, 5.0 rel vol., 160mL) was added (addition time 4-12h, temperature maintained below 10 ℃) to a solution of 5-bromotetralin-1-one (I12) (1.0 equiv., 26.77g) in sulfuric acid (concentrated, 5.0 rel vol., 160mL) under nitrogen. When the reaction was complete, the reaction mixture was transferred to a flask containing water (36 relative volumes, 1.15L) and the transfer rate was adjusted to maintain the temperature below 10 ℃. The resulting solid was filtered, washed three times with water (4.0 relative volume, 128mL) and then dried at about 40 ℃ for 24 h. The dried filter cake was dissolved in a mixture (heated to about 75 ℃) of acetone (2.5 relative volume, 80mL) and water (0.38 relative volume, 12.2mL) and then cooled to about 20 ℃. The resulting solid was removed by filtration. The solvent was exchanged for ethanol by distillation and the solution volume was reduced to 2.0 relative volume (64 mL). The solution was cooled to about 25 ℃ and the resulting solid was collected by filtration. The solid was washed with ethanol (1.0 relative volume, 32mL) and then dried under vacuum at 40 ℃ to give 5-bromo-8-nitro-tetralin-1-one I13(15.36g, 40%) as a brown solid; RT 14.0min

Method 1 IPC, purity and assay method for bromo-8-nitro-tetralin-1-one.

b) N- (8-Nitro-1-oxo-tetrahydronaphthalen-5-yl) acetamide (I14)

A solution of bromo-8-nitro-tetralin-1-one (I13) (1.0 eq, 18.0g, 90.6% ww), acetamide (1.2 eq, 4.72g), tris (dibenzylideneacetone) dipalladium (0) (0.01 eq, 0.61g) potassium phosphate (1.4 eq, 19.8g) in dioxane (15 relative volumes, 270mL) was heated to about 70 ℃ under nitrogen. When the reaction was complete, the solution was cooled to about 20 ℃ and diluted with dioxane (5 relative volumes, 90.0mL) and filtered. The solvent was exchanged for ethanol and the volume was reduced to a total reaction volume of 3 relative volumes (54.0 mL). The solution was cooled to about 20 ℃ and the resulting solid was collected by filtration and washed with MTBE (methyl tert-butyl ether) (1.0 relative volume, 18.0 mL). The solid was dried under vacuum at 40 ℃ to give N- (8-nitro-1-oxo-tetralin-5-yl) acetamide I14(10.0g, 60.6%) as a dark yellow solid; RT 8.86 min.

c) N- (8-amino-1-oxo-tetrahydro-naphthalen-5-yl) acetamide (I15)

Palladium on carbon (20% w/w, 0.15 eq, 5.25g) was added to a solution of N- (8-nitro-1-oxo-tetrahydronaphthalen-5-yl) acetamide (I14) (1.0 eq, 32.6g) in methanol (40 relative volumes, 1250 mL). The reaction mixture was placed under an atmosphere of hydrogen (about 40psi) at about 40 ℃ for 8 h. The hydrogen was removed and replaced with nitrogen and the catalyst was removed by filtration through cellulose, washing the cellulose with methanol (4.0 relative volume, 130 mL). The solution volume was reduced to 4.0 relative volume by distillation and then diluted with MTBE (4 relative volume, 130 mL). The resulting solid was collected by filtration, washed with MTBE (2 relative volumes, 65mL) and dried under vacuum at 40 ℃ to give N- (8-amino-1-oxo-tetralin-5-yl) acetamide I15(21.1g, 77.8%; RT 5.44min as a grey-green solid.

d)5, 8-Diaminotetralin-1-one (I16)

A solution of N- (8-amino-1-oxo-tetralin-5-yl) acetamide (I15) (1.0 eq, 10.0g) in hydrochloric acid (5M, 6.0 rel vol, 60mL) was maintained at about 90 ℃ for 3 h. The temperature was reduced to 25 ℃ and sodium hydroxide (2M, 4.0 relative volume, 40mL) was added until a pH of 10.0 was reached, maintaining the temperature at 25 ℃. The resulting solid was collected by filtration and washed with water (2.0 relative volume, 20 mL). The wet cake was dissolved in tetrahydrofuran (60 relative volume, 600mL) and filtered. The solution was concentrated to 5.0 relative volume and heptane (20 relative volume, 200mL) was added. The solution was concentrated to 10.0 relative volume and heptane (20 relative volume, 200mL) was further added, and then the volume was again reduced to 10.0 relative volume. The resulting solid was collected by filtration and washed with heptane (5.0 relative volume, 50 mL). The solid was dried under vacuum at 40 ℃ for 17h to give 5, 8-diaminotetralin-1-one (I16) (6.90g, 82.7%) as a yellow solid; 1H NMR (400MHz DMSO-d6) δ ppm 1.82(m, 2H), 2.38(t, J ═ 2.0Hz, 2H), 2.47(t, J ═ 2.0Hz, 2H), 6.34(d, J ═ 2.0Hz, 1H), 6.68(d, J ═ 2.0Hz, 1H); RT 3.90

e) (S) -4-amino-9-ethyl-9-hydroxy-1, 2, 3, 9, 12, 15-hexahydro-10H, 13H-benzo [ de ] pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinoline-10, 13-dione (I11)

A solution of 5, 8-diaminotetralin-1-one (I16) (1.0 eq, 5.0g), (4S) -4-ethyl-4-hydroxy-7, 8-dihydro-1H-pyrano [3, 4-f ] indolizine-3, 6, 10-trione (A3) (1.06 eq, 7.9g), and pyridinium p-toluenesulfonate (1.0 eq, 7.2g) in toluene (50.0 rel vol, 250mL) was maintained at 120 ℃ for 15H. The volume of the solution was reduced to 2.0 relative volume and then diluted with acetonitrile (20 relative volume, 100mL) and water (20 relative volume, 100 mL). The resulting slurry was filtered and the solid was washed with aqueous acetonitrile (1: 1, 20 relative volumes, 100 mL). The solid was slurried with aqueous methanol (water: MeOH 3: 1, 40 relative volumes, 200mL), filtered, and washed with aqueous methanol (1: 1, 20 relative volumes, 100 mL). The solid was slurried with water (60 relative volume, 300mL) at 50 ℃, filtered and washed with water (10 relative volume, 50 mL). The solid was slurried with aqueous acetonitrile (water: acetonitrile, 1: 3, 40 relative volume, 200mL) at 30 ℃, filtered and washed with aqueous acetonitrile (water: acetonitrile, 1: 3, 5 relative volume, 50mL) and then dried under vacuum at 40 ℃ to give (S) -4-amino-9-ethyl-9-hydroxy-1, 2, 3, 9, 12, 15-hexahydro-10H, 13H-benzo [ de ] pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinoline-10, 13-dione (I11) (5.0g, 43.7%); RT 5.13.

Synthesis of I18

a) Tert-butyl (S) - (2- ((2- ((1- ((2- ((4-amino-5-oxo-5, 6, 7, 8-tetrahydronaphthalen-1-yl) amino) -2-oxoethyl) amino) -1-oxo-3-phenylpropan-2-yl) amino) -2-oxoethyl) carbamate (I17)

Boc-GGFG-OH (227mg, 0.52mmol) and EEDQ (157mg, 0.634mmol) were dissolved in CH₂Cl₂(25mL) and the mixture was stirred for 15min until the peptide dissolved in the solution. Compound I16(100mg, 0.56747mmol) was then added and the mixture was stirred until completion. After 1h, LVMC showed that the reaction was 90% complete. As the product is pressed out (crasing out), the mixture becomes thicker. The mixture was allowed to stand for an additional hour and then dried by applying vacuum. The crude product was taken up in Et₂O (50 mL). The solid was filtered and then taken up in CH₂Cl₂(50mL) for further purification. The solid was filtered and dried to give product I17(273mg, 0.459mmol, 80.9% yield) as a grey solid. Analyzing data: LCMS 3 min: ES (ES)⁺＝1.46min，m/z 595.7[M+H]^.+。

b) (S) -2- (2- (2-Aminoacetamido) acetamido) -N- (2- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H-benzo [ de ] pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-4-yl) amino) -2-oxoethyl) -3-phenylpropionamide (I18)

Aniline I17(450mg, 1.045mMol), lactone A5(280mg, 1.064mMol), and pyridinium p-toluenesulfonate (273mg, 1.086mMol) were dissolved in toluene (20mL) and the mixture was heated to 150 deg.C (high reflux). MeOH (4mL) was added to help dissolve the mixture. For 7h, the crude reaction was vacuumed (vacuumed) to dry. The crude product was chromatographed on silica gel (CHCl)₃MeOH, 100% to 65: 35) to give product I18(259mg, 0.359mMol, 78.1 yield). Analyzing data: LCMS 3 min: ES (ES)⁺＝1.17min，m/z 722.8[M+H]^.+。

Alternative Synthesis of I16

a) 5-fluoro-8-nitro-tetralin-1-one (I20)

At the 3 neckIn a round-bottomed flask, 5-fluorotetralin-1-one I19(4.7g, 29mmol) was dissolved in 1/2 amount of sulfuric acid (120 mL). The mixture was stirred until all solids dissolved and then cooled to 0-5 ℃. In the dropping funnel, potassium nitrate (3g, 29.6730mmol) was dissolved in the remaining half of sulfuric acid (120mL) at 0 deg.C-5 deg.C. Slowly added to the SM mixture, ensuring to keep the solution cold (45 min). Stirring at 0-5 deg.C until completion. The reaction mixture was then quenched with water (250mL) and stirred at 0 deg.C-5 deg.C. The solid was filtered and washed with water (50 mL). The solid was dried in a vacuum oven at 50 ℃ for 2 h. The crude solid is taken up in Et₂Slurried in O overnight, then cooled to 0 ℃ and filtered. The wet cake was taken up in more cold Et₂O (50mL) was washed and dried in a vacuum oven at 50 ℃ to give pure product I20(5.5g, 26mmol, 92% yield) as a light pink fine powder. LCMS (method B): ES (ES)⁺＝1.55min，m/z 210.1[M+H]^.+。

b) 5-amino-8-nitro-tetralin-1-one (I21)

Compound I20(2.7g, 13mmol) was dissolved in CH₃CN (2.5mL), and NH in H2O (8mL, 40mmol)₄OH (21 mass%) was added to the sealed pressure tube and heated to 185 ℃. Once complete, the mixture was transferred to a round bottom flask and evacuated. Subjecting the crude product to silica gel column chromatography (CHC)_l3/MeOH; 100 to 99: 1) to give the pure product I21(1.1g, 5.3mmol, 41% yield) as a black solid. LCMS (method B): ES (ES)⁺＝1.34min，m/z 207.1[M+H]^.+。

c)5, 8-Diaminotetralin-l-one (I16)

Compound I21(1.35g, 6.55mmol) was dissolved in methanol (20mL), H at 0 deg.C₂O (1mL) and formic acid (1 mL). Zinc (8.5g, 130mmol) was added slowly, ensuring that the temperature was kept below 40 ℃. Addition of more formic acid/H₂O (0.5mL) to drive the reaction to completion. The reaction mixture was filtered, and the filtrate was washed with EtOAc and CH2C1₂Diluting and then vacuumizing. The crude product was dried and loaded onto silica gel column chromatography (CHCl)₃/EtOAc; 100 to 7: 3 and then CHCl₃(ii) MeOH; 99: 1 to 98: 2)To give pure product I16(1.015g, 5.760mmol, 88.0% yield). LCMS (method B): ES (ES)⁺0.2min, no m/z was observed.

Example 1

a) Allyl ((S) -3-methyl-1-oxo-1- (((S) -1-oxo-1- ((5-oxo-4- (2, 2, 2-trifluoroacetamido) -5, 6, 7, 8-tetrahydronaphthalen-1-yl) amino) propan-2-yl) amino) butan-2-yl) carbamate (A1)

DCC (6.54g, 31.7mMol) and HOPO (3.36g, 30.2mMol) were added to allyloxycarbonyl-Val-Ala-OH (9.09g, 31.7mMol) and I7(7.85g, 28.8mMol) in CH at 25 deg.C₂Cl₂(300 mL). The resulting mixture was stirred overnight. The white solid formed during the reaction was filtered off and washed with cold CH₂Cl₂And (6) washing. The filtrate was washed with water (150mL) and brine (150 mL). The organic layer was purified over MgSO₄Dried, filtered and evaporated. The crude product was purified by chromatography on silica gel (Hex/EtOAc, 60: 40). The isolated product A1 was contaminated with co-eluted DCU (21.1g, 140% yield). LC/MS (method B): ES (ES)⁺＝1.81min，m/z 527.6[M+H]^.+。

b) Allyl ((S) -1- (((S) -l- ((4-amino-5-oxo-5, 6, 7, 8-tetrahydronaphthalen-1-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) carbamate (A2)

The protected aniline A1(18g, 34.19mMol) was dissolved in MeOH and H₂O10: 1(165mL) mixture and addition of K₂CO₃(10g, 72.36 mMol). The mixture was stirred at 50 ℃ until completion. The mixture was vacuumed almost dry and the residue was taken up in CH₂Cl₂Absorb and use H₂O and brine, then MgSO₄Dried, filtered and evaporated. The crude product was chromatographed on silica gel (CHCl)₃MeOH, 100% to 7: 3). The isolated product a2 was contaminated with co-eluting impurities (10.71g, 73% yield). LC/MS (method B): ES (ES)⁺＝1.46min，m/z 431.7[M+H]^.+。

c) Allyl ((S) -1- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H-benzo [ de ] pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-4-yl) amino) -1-oxoprop-2-yl) amino) -3-methylbut-2-yl) carbamate (A4)

Aniline a2(450mg, 1.045mMol), lactone A3(280mg, 1.064mMol) and pyridinium p-toluenesulfonate (273mg, 1.086mMol) were dissolved in toluene (20mL) and the mixture was heated to 130 ℃ (high reflux). Several drops of MeOH were added from time to help dissolve the mixture. For 7h, the crude reaction was vacuumed (vacuumed) to dry. The crude product was chromatographed on silica gel (CHCl)₃MeOH, 100% to 95: 5) to give product A4(360mg, 52.3% yield). LC/MS (method B): ES (ES)⁺＝1.51min，m/z 658.8[M+H]^.+。

d) Allyl (S) -2-amino-N- ((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H-benzo [ de ] pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-4-yl) amino) -l-oxoprop-2-yl) -3-methylbutanamide (A5)

An excess of piperidine (642 μ L) was added to A4(543mg, 0.82mMol) and PdP (Ph)₃)₄(89mg, 0.08mMol) in CH₂Cl₂(15 mL). The mixture was allowed to stir at room temperature for 20min, at which point the reaction had completed (as monitored by LC/MS). Reacting the mixture with CH₂Cl₂(25mL) diluted and the organic phase was taken up in H₂O (25mL) and brine (25 mL). The organic phase is passed over MgSO₄Drying, filtration and removal of excess solvent by rotary evaporation under reduced pressure gave the crude product a5, which was used as such in the next step. LC/MS (method B): ES (ES)⁺＝1.15min，m/z 574.6[M+H]^.+。

e)1- (3- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) propylamino) -N- ((S) -1- (((S) -1- ((((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H-benzo [ de ] pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-4-yl) amino) -1-oxoprop-2-yl) amino) -3-methyl-1-oxobutan-2-yl) -3, 6, 9, 12, 15, 18, 21, 24-octaoxaheptacosane-27-amide (1)

Pyridine (83. mu.L, 1.03mMol) and Mal-dPEG were added under an argon atmosphere₈OTFP (767mg, 1.03mMol) was added to crude A5 (assumed to be 1.03mMol) in dry CH₂Cl₂(50 mL). The reaction was stirred overnight and due to incomplete reaction 0.5 equivalents of Mal-dPEG were added₈OTFP in an attempt to drive the reaction. Reacting with CH₂Cl₂(25mL) diluted and the organic phase was taken up in H₂O (2X50mL) and brine, then MgSO₄Dry, filter and remove excess solvent by rotary evaporation under reduced pressure. The crude product is passed through reverse phase HPLC (H)₂O/CH₃CN + 0.05% FA gradient) were purified and freeze-dried to give 1(1.189g, 31% yield over 2 steps). LC/MS (method B): ES (ES)⁺＝1.43min，m/z 1149.3[M+H]^.+. LC/MS (method C): ES (ES)⁺＝5.37min，m/z 1149.4[M+H]^.+。

Example 2

6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N- ((S) -1- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H-benzo [ de ] pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) hexanamide (2)

Mal-hexanoic acid (56mg, 0.26mMol) and EDCI.HCl (51mg, 0.26mMol) were added to crude A5 (assumed to be 0.26mMol) in dry CH under an argon atmosphere₂Cl₂(20 mL). The reaction was stirred overnight and, due to incomplete reaction, an additional 0.5 equivalents of Mal-hexanoic acid and edci.hcl were added. Reacting with CH₂Cl₂(25mL) diluted and the organic phase was taken up in H₂O (2X50mL) and brine, then MgSO₄Drying, filtering and evaporating the excess solvent by rotary evaporation under reduced pressureHair is removed. The crude product was chromatographed on silica gel (CHCl)₃MeOH 95: 5) to give 2(31.6mg, 20% yield over 2 steps). LC/MS (method B): ES (ES)⁺＝1.56min，m/z 767.8[M+H]^.+. LC/MS (method C)15 min: ES (ES)⁺＝6.05min，m/z 767.8[M+H]^.+。

Example 3

(S) -2- (2- (2- (2- (2-azidoethoxy) ethoxy) acetamido) -N- ((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H-benzo [ de ] pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) -3-methylbutanamide (3)

In an argon atmosphere, nitrine-dPEG₃Acid (77.5mg, 0.31mMol) and EDCI.HCl (60mg, 0.31mMol) were added to crude A5 (assumed to be 0.31mMol) in dry CH₂Cl₂(20 mL). The reaction was stirred overnight and, due to incomplete reaction, an additional 0.5 equivalents of azide-dPEG was added₃-OH and edci.hc1. Reacting with CH₂Cl₂(25mL) diluted and the organic phase was taken up in H₂O (2X50mL) and brine, then MgSO₄Dry, filter and remove excess solvent by rotary evaporation under reduced pressure. The crude product was purified by preparative HPLC and fractions were freeze dried to give pure 3(92.2mg, 24.7% yield over 2 steps). LC/MS (method B): ES (ES)⁺＝1.69min，m/z 789.9[M+H]^.+. LC/MS (method C): ES (ES)⁺＝6.68min，m/z 790.0[M+H]^.+。

Example 4

N- ((S) -1- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H-benzo [ de ] pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-4-yl) amino) -1-oxoprop-2-yl) amino) -3-methyl-1-oxobutan-2-yl) -4, 7, 10, 13, 16-pentaoxanonadecane-18-ynamide (4)

Under argon atmosphere, propargyl-dPEG₅Acid (56mg, 0.19mMol) and EDCI.HCl (37mg, 0.19mMol) were added to crude A5 (assumed to be 0.19mMol) in anhydrous CH₂Cl₂(10 mL). The reaction was stirred overnight and, due to incomplete reaction, an additional 0.5 equivalents of propargyl-dPEG was added₅-OH and edci.hcl. Reacting with CH₂Cl₂(25mL) diluted and the organic phase was taken up in H₂O (2X50mL) and brine, then MgSO₄Dry, filter and remove excess solvent by rotary evaporation under reduced pressure. The crude product was purified by preparative HPLC and fractions were freeze dried to give pure 4(22mg, 16.7% yield over 2 steps). LC/MS (method B): ES (ES)⁺＝1.54min，m/z 860.9[M+H]^.+. LCMS (method C): ES (ES)⁺＝5.57min，m/z 860.9[M+H]^.+。

Example 5

(S) -2- (2- (4- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) phenyl) acetamido) -N- ((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H-benzo [ de ] pyrano [3 ', 4': 6, ] indolizino [1, 2-b ] quinolin-4-yl) amino) -1-oxopropan-2-yl) -3-methylbutanamide (5)

PM-acetic acid-OSu (64mg, 0.19mMol) was added to crude A5 (assumed to be 0.19mMol) in dry CH under an argon atmosphere₂Cl₂(10 mL). Since the reaction did not proceed, DIPEA (51. mu.L, 0.28mMol) was added. The reaction was stirred until completion. Subjecting the mixture to CH₂Cl₂(25mL) diluted and the organic phase was taken up in H₂O (2X50mL) and brine, then MgSO₄Drying, filtering and passing the excess solvent under reduced pressureAnd then removed by rotary evaporation. The crude product was purified by preparative HPLC and fractions were freeze dried to give pure 5(2.5mg, 1.6% yield over 2 steps). LC/MS (method B): ES (ES)⁺＝1.54min，m/z 787.7[M+H]^.+. LC/MS (method C): ES (ES)⁺＝5.61min，m/z 787.8[M+H]^.+。

Example 6

(R) -2- ((3-Nitropyridin-2-yl) disulfanyl) propyl ((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H-benzo [ de ] pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-4-yl) carbamate (6)

(i) Reacting (2R) -2- [ (3-nitro-2-pyridyl) dithioalkyl]Propan-1-ol A6(25mg, 0.1015mmol, 1.0 equiv.) was dissolved in dichloromethane (1 mL). Pyridine (8.5 μ L, 0.11mmol, 1.0 equiv.) is added followed by triphosgene (11mg, 0.0370685mmol, 0.33 equiv.) and the mixture stirred under Ar for 45min, followed by LCMS (Et)₂NH quenching) indicated the formation of the corresponding carbamate.

(ii) Reacting (S) -4-amino-9-ethyl-9-hydroxy-1, 2, 3, 9, 12, 15-hexahydro-10H, 13H-benzo [ de ]]Pyrano [3 ', 4': 6,7]Indoxazino [1, 2-b ] s]Quinoline-10, 13-dione (I11) (43mg, 0.09026mmol, 1.0 equiv.) was dissolved in dichloromethane (2mL), N-diisopropylethylamine (42. mu.L, 0.241mmol, 2.7 equiv.), and pyridine (25. mu.L, 0.309mmol, 3.4 equiv.). The reaction mixture from step (i) was added and the mixture was stirred for 30min, followed by LCMS to show completion of the reaction. The reaction mixture was concentrated in vacuo and chromatographed by isolera (0% -4% MeOH in CH)₂Cl₂Medium) to give 6(22mg, 0.03256mmol, 36% yield, QC ═ 96.8%) as a yellow solid. LC/MS (method B): RT 1.86min, 676.6[ M + H [ ]]⁺。

Example 7

6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -N- (2- ((2- (((S) -1- ((2- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H-benzo [ de ] pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-4-yl) amino) -2-oxoethyl) amino) -1-oxo-3-phenylprop-2-yl) amino) -2-oxoethyl) hexanamide (7).

Compound I18(259mg, 0.3588mmol) was dissolved in CH₂Cl₂(25 mL). The starting material was completely insoluble, so DMA (1mL) was added. Since no improvement was observed, DIPEA (68 μ L, 0.390mmol) was added and all solids went into solution. Maleimidocaproic acid (69mg, 0.358mmol) was added and the mixture was stirred at r.t. overnight at which time LCMS analysis indicated that the reaction was complete. The reaction mixture was quenched with MeOH (2mL) and evacuated to dryness. The crude product was purified by preparative HPLC and then lyophilized to give compound 7 as an ochre solid (38.2mg, 11% yield). Analyzing data: LCMS 3 min: ES (ES)⁺＝1.47min，m/z 916.2[M+H]^.+LCMS 15min：ES⁺＝5.46min，m/z 916.1[M+H]^.+。

Example 8

1- (3- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) propylamino) -N- (2- ((2- (((S) -1- ((2- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H-benzo [ de ] pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-4-yl) amino) -2-oxoethyl) amino) -1-oxo-3-phenylprop-2-yl) amino) -2-oxoethyl) -3, 6, 9, 12, 15, 18, 21, 24-octaoxaheptacosane-27-carboxamide (8)

Compound I18(70mg, 0.096mmol) was dissolved in CH₂Cl₂(5 mL). The starting material was completely insoluble, so DMA (0.5mL) was added. Due to unperceivedImprovement was observed, so DIPEA (19 μ L, 0.106mmol) was added and all solids went into solution. Adding Mal-dPEG₈-OH (63mg, 0.106mMol) and edci.hcl (19mg, 0.099mMol) and the mixture was stirred at r.t. overnight when LCMS analysis indicated that the reaction was complete. The reaction mixture was quenched with MeOH (2mL) and evacuated to dryness. The crude product was purified by preparative HPLC and then lyophilized to give 8 as an ochre solid (30mg, 24% yield). LCMS 3 min: ES (ES)⁺＝1.44min，m/z 1297.6[M+H]^.+。

Alternative Synthesis of example 9-1

Reacting (S) -4-amino-9-ethyl-9-hydroxy-1, 2, 3, 9, 12, 15-hexahydro-10H, 13H-benzo [ de ]]Pyrano [3 ', 4': 6,7]Indoxazino [1, 2-b ] s]Quinoline-10, 13-dione I11(371mg, 0.779mmol, 1.0 equiv.) was dissolved in dichloromethane (30 mL). N, N-diisopropylethylamine (69. mu.L, 0.396mmol, 0.51 equiv.) and (2S) -2- [ [ (2S) -2- [3- [2- [2- [2- [2- [2- [3- (2, 5-dioxopyrrol-1-yl) propionylamino ] added to N, N-dimethylacetamide (10mL)]Ethoxy radical]Ethoxy radical]Ethoxy radical]Ethoxy radical]Ethoxy radical]Ethoxy radical]Ethoxy radical]Ethoxy radical]Propionyl amino group]-3-methyl-butyryl]Amino group]Propionic acid (664mg, 0.871mmol, 1.1 equiv.), followed by EDCI. HCl (226mg, 1.18mmol, 1.5 equiv.) and the mixture stirred for 2h, then LCMS indicated good conversion, but the reaction had stalled. The reaction mixture was warmed to 30 ℃ and stirred for 30min, LCMS showed no change, so CH was removed in vacuo₂Cl₂And Et is added₂O was added to the resulting DMA solution. The precipitated oil was collected and Et removed in vacuo₂O and repeating the precipitation process. The combined precipitates were purified by HPLC (10% -60% B in A for 13min) to give 1 as a yellow residue after lyophilization (200mg, 0.174mmol, 98% purity, 22% yield). LC/MS (method A): retention time 1.44min (ES +) M/z 1149[ M + H ]]⁺

¹H NMR(600MHz, chloroform-d) δ 8.81(s, 1H), 7.83(s, 2H), 7.48(s, 1H), 7.18(dd, J ═ 18.7, 7.5Hz, 2H), 6.69(s, 2H), 6.43(s, 1H), 5.68(d, J ═ 16.1Hz, 1H), 5.27(d, J ═ 16.1Hz, 1H), 5.03(d, J ═ 18.4Hz, 1H), 4.90(d, J ═ 18.4Hz, 1H), 4.75(p, J ═ 7.2Hz, 1H), 4.32(dd, J ═ 7.4, 5.8Hz, 1H), 4.05(s, 1H), 3.83(t, J ═ 7.2, 3H), 3.68 (3.68, 3.78, 3.4, 5.8Hz, 1H), 3.06 (d, 3.06H), 3.83(t, 3.2, 3.3.3, 3.8H), 3.3.8, 3.8H, 3, 3.8 (d, 3.3, 3, 3.06, 3.3.3.3, 3, 3.3, 3, 3.3.3, 3.8, 3, 3.3, 3, etc.),4, etc., 2.57-2.44(m, 4H), 2.30(dq, J ═ 13.4, 6.7Hz, 1H), 2.10(p, J ═ 6.4Hz, 3H), 1.91(ddt, J ═ 16.8, 14.3, 7.2Hz, 3H), 1.54(d, J ═ 7.1Hz, 3H), 1.02(dd, J ═ 15.5, 6.9Hz, 10H).

Alternative Synthesis of example 10-A2

Allyl ((S) -1- (((S) -l- ((4-amino-5-oxo-5, 6, 7, 8-tetrahydronaphthalen-1-yl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) carbamate (A2)

EDCI.HCl (7.71g, 31.2mMol) was added to allyloxycarbonyl-Val-Ala-OH (8.49g, 31.2mMol) in CH₂Cl₂(200mL) and stirred for 15min or until dissolved. I16(5g, 28.3mMol) was then added and the resulting mixture was stirred until the reaction was complete. The volatiles were removed under reduced pressure. The crude product was taken up in Et₂O (50mL) and the mixture was sonicated for 3 min. The solid was filtered and reabsorbed into CH2C12(50mL), sonicated for 3min and filtered again to give pure product a2 as a grey solid (12.21g, 79% yield). LC/MS (method B): ES (ES)⁺＝1.47min，m/z 431.5[M+H]^.+。

Example 11

a) (9H-fluoren-9-yl) methyl N2- (1- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-oxo-7, 10, 13, 16, 19, 22, 25, 28-octaoxo-4-aminoundecane-31-yl) -N5- ((S) -1- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H-benzo [ de ] pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-4-yl) amino) -1-oxoprop-2-yl) amino) - 3-methyl-1-oxobut-2-yl) -L-glutamine (A7)

EDCI.HCl (0.10mmol, 1.2 equiv.) was added to A5(0.087mmol, 1.0 equiv.) and Mal-PEG₈A solution of-Glu-OH (0.10mmol, 1.2 equiv.) in DCM (5mL) and the resulting mixture was stirred at room temperature overnight. The reaction mixture was evaporated to dryness and purified by column (8% -12% MeOH in DCM) to give the product as a white solid. Yield 80mg (63%). LC/MS (method B) rt 1.66min M/z (1456.2) M + H.

b) N2- (1- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) -3-oxo-7, 10, 13, 16, 19, 22, 25, 28-octaoxo-4-aminoundecane-31-acyl) -N5- ((S) -1- (((S) -1- (((S) -9-ethyl-9-hydroxy-10, 13-dioxo-2, 3, 9, 10, 13, 15-hexahydro-1H, 12H-benzo [ de ] pyrano [3 ', 4': 6, 7] indolizino [1, 2-b ] quinolin-4-yl) amino) -1-oxoprop-2-yl) amino) -3-methyl-1-oxobutandin-e -2-yl) -L-glutamine (9)

1-Methylpyrrolidine (200. mu.L) was added to a solution of A7(0.06mmol) in DMF (0.8mL) and stirred at room temperature for 10 min. The solvent was removed in vacuo and the residue was purified by preparative HPLC (30% MeCN/water + 0.05% formic acid over 8.5 min). The product containing fractions were freeze dried to give the product as a white solid. Yield 23mg (30%). LC/MS (method B) rt 1.43min M/z (1278.4) M + H.

Example 12-conjugation

Herceptin (Herceptin) -C239i antibody

The herceptin antibody was engineered to have a cysteine inserted between positions 239 and 240 generated according to the method described in Dimasi, N.et al, Molecular pharmaceuticals [ Molecular pharmacy ], 2017, 14, 1501-1516 (DOI: 510.1021/acs. molpharmaceut.6b00995).

ConjA

A solution of 50mM DL-Dithiothreitol (DTT) in phosphate buffered saline (pH 7.4) (PBS) (150 molar equivalents/antibody, 40 micromolar, 800 μ L) was added to a solution of 10mL of herceptin-C239 i antibody (40mg, 267 nanomolar) in reduction buffer (containing PBS and 1mM ethylenediaminetetraacetic acid (EDTA)), and the final antibody concentration was 4.0 mg/mL. The reduction mixture was allowed to react for 4 hours 45 minutes in an orbital shaker at room temperature with gentle (60rpm) shaking (or until complete reduction was observed by UHPLC). The reduced antibody buffer was exchanged to a re-oxidation buffer containing PBS and 1mM EDTA via spin-filter centrifugation to remove any excess reducing agent. A solution of 50mM dehydroascorbic acid (DHAA, 20 molar equivalents per antibody, 5.33 micromolar, 106.7 μ L) in DMSO was added and the reoxidation mixture was allowed to react for 16 hours at room temperature with gentle (60rpm) shaking at an antibody concentration of 4mg/mL (or more DHAA was added for a longer time until complete reoxidation of the cysteine thiol to reform interchain cysteine disulfide was observed by UHPLC). The reoxidation mixture was then sterile filtered and diluted in conjugation buffer containing PBS and 1mM EDTA to a final antibody concentration of 3.6 mg/mL. Compound 1 was added as a DMSO solution (10 molar equivalents per antibody, 1.33 micromolar in 0.55mL DMSO) to 5.0mL of the reoxidized antibody solution (20mg, 133 nanomolar) at a final DMSO concentration of 10% (v/v). The solution was mixed at room temperature for 2 hours, then the conjugate was quenched by addition of N-acetyl cysteine (6.67 micromolar, 67 μ Ι at 100 mM), then purified by rotary filtration into PBS using a 15mL Amicon Ultracell 30kDa MWCO rotary filter, sterile filtered and analyzed.

UHPLC analysis was performed on the shimeji science system using a seemer femtology Scientific MAbPac 50mm x2.1mm column, gradient elution with water and acetonitrile, showing unconjugated light chains at 214nm and 330nm, and the attachment of a mixture of unconjugated heavy and heavy chains to individual molecules of compound 1 on a reduced sample of ConjA (compound 1 specific), consistent with a drug/antibody ratio (DAR) of compound 1 of 1.89 molecules per antibody.

UHPLC analysis was performed on the Shimadzu Promins System using a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6X150mM column (with a4 μm 3.0X20mM guard column), eluting with 0.3 mL/min sterile filtered SEC buffer containing 200mM potassium phosphate pH 6.95, 250mM potassium chloride and 10% isopropanol (v/v)), and showed 98% monomer purity at 280nm on a ConjA sample. UHPLC SEC analysis gave a final ConjA concentration of 2.14mg/mL in 6.5mL and a ConjA mass of 13.9mg (70% yield) was obtained.

ConjA*

A solution of 10mM tris (2-carboxyethyl) phosphine (TCEP) (10 molar equivalents per antibody, 400 nanomoles, 40 μ L) in phosphate buffered saline pH 7.4(PBS) was added to a solution of 2.4mL trastuzumab antibody (6mg, 40 nanomoles) in reduction buffer (PBS and 1mM ethylenediaminetetraacetic acid (EDTA)) and the final antibody concentration was 2.5 mg/mL. The reduction mixture was allowed to react for 16 hours (or until complete reduction was observed by UHPLC) in an orbital shaker at room temperature with gentle (60rpm) shaking. The reduced antibody solution was buffer exchanged (to remove any excess reducing agent) via spin filter centrifugation to a conjugation buffer containing PBS and 1mM EDTA at a final antibody concentration of 2.0 mg/mL. Compound 1 was added as a DMSO solution (20 molar equivalents per antibody, 400 nanomoles, in 0.15mL DMSO) to 1.35mL of the reduced antibody solution (3mg, 20 nanomoles) at a final DMSO concentration of 10% (v/v). The solution was mixed at room temperature for 2 hours, then the conjugate was quenched by addition of N-acetyl cysteine (2 micromolar, 20 μ Ι at 100 mM), then purified via spin filter centrifugation using a 15mL Amicon Ultracell 30KDa MWCO spin filter, sterile filtered and analyzed.

UHPLC analysis was performed on the shimadzu technologies MAbPac 50mm × 2.1mm column on shimadzu corporation, gradient elution with water and acetonitrile, showing single molecule attachment of the unconjugated light chain, mixture of light chains to compound 1, and up to three molecule attachments of unconjugated heavy and heavy chains to compound 1, on a reduced sample of ConjA (compound 1 specific) at 214nm and 330nm, consistent with a drug/antibody ratio (DAR) of 7.89 molecules per antibody compound 1.

UHPLC analysis was performed on the Shimadzu Promin System using a Tosoh bioscience TSKgel SuperSW mAb HTP 4 μm 4.6x150mM column (with a4 μm 3.0x20mM guard column), eluting with 0.3 mL/min sterile filtered SEC buffer (containing 200mM potassium phosphate pH 6.95, 250mM potassium chloride and 10% isopropanol (v/v)), showing a monomer purity of 98.5% at 280nm on ConjA samples. The UHPLC SEC analysis gave a final ConjA concentration of 2.02mg/mL in 1.25mL and a mass of 2.5mg of ConjA was obtained (84% yield).

ConjB

A solution of 10mM tris (2-carboxyethyl) phosphine (TCEP) in phosphate buffered saline pH 7.4(PBS) (10 molar equivalents per antibody, 3.56 micromoles, 356 μ L) was added to a solution of 11.1mL trastuzumab antibody (53.4mg, 356 nanomoles) in reduction buffer containing PBS, pH 7.4 and 1mM ethylenediaminetetraacetic acid (EDTA), and the final antibody concentration was 4.84 mg/mL. The reduction mixture was allowed to react for 1 hour 30 minutes in an orbital shaker at 37 ℃ with gentle (60rpm) shaking (or until complete reduction was observed by UHPLC). Compound 2 was added as a DMSO solution (15 molar equivalents per antibody, 5.1 micromolar in 1.2mL DMSO) to 10.5mL of the reduced antibody solution (50.8mg, 339 nanomolar) at a final DMSO concentration of 10% (v/v). The solution was mixed for 1 hour 30 minutes at room temperature, then the conjugate was quenched by addition of N-acetyl cysteine (25.4 micromolar, 254. mu.L at 100 mM) and then the general electro-medical group (GE Healthcare) HiLoadT was used^M26/600 column (packed with Superdex 200PG) in AKTA^TMPurification was performed on Start FPLC eluting with 2.6mL/min PBS. Fractions corresponding to the ConjB monomer peak were combined, concentrated, and buffer exchanged into 25mM histidine 205mM sucrose pH6.0 buffer using a 15mL Amicon Ultracell 50kDa MWCO spin filter, sterile filtered and analyzed.

UHPLC analysis was performed on the shimadzu scientific MAbPac 50mm x2.1mm column on shimadzu promience system, gradient elution with water and acetonitrile, showing a mixture of unconjugated light and light chains attached to a single molecule of compound 2, and unconjugated heavy and heavy chains attached to up to three molecules of compound 2, at 214nm and 330nm, on a reduced sample of ConjB (compound 2 specificity), consistent with a drug/antibody ratio (DAR) of 7.93 molecules of compound 2 per antibody.

UHPLC analysis was performed on the Shimadzu Promin System using a Tosoh bioscience TSKgel SuperSW mAb HTP 4 μm 4.6x150mM column (with a4 μm 3.0x20mM guard column), eluting with 0.3 mL/min sterile filtered SEC buffer (containing 200mM potassium phosphate pH 6.95, 250mM potassium chloride and 10% isopropanol (v/v)), showing a monomer purity of 98.9% at 280nm on a ConjB sample. UHPLC SEC analysis gave a final ConjB concentration of 2.4mg/mL in 16mL and a ConjB mass of 38.4mg (84% yield) was obtained.

ConjC

A solution of 10mM tris (2-carboxyethyl) phosphine (TCEP) in phosphate buffered saline pH 7.4(PBS) (40 molar equivalents per antibody, 11.2 micromoles, 1.12mL) was added to a solution of 20mL of herceptin-C239 i antibody (42mg, 280 nanomoles) in reduction buffer containing PBS and 1mM ethylenediaminetetraacetic acid (EDTA), and the final antibody concentration was 2.1 mg/mL. The reduction mixture was allowed to react for 16 hours (or until complete reduction was observed by UHPLC) in an orbital shaker at room temperature with gentle (60rpm) shaking. The reduced antibody buffer was exchanged to a re-oxidation buffer containing PBS and 1mM EDTA via spin-filter centrifugation to remove any excess reducing agent. A solution of 50mM dehydroascorbic acid (DHAA, 30 molar equivalents per antibody, 7.0 micromolar, 141 μ L) in DMSO was added to 22mL of this reducing buffer exchanged antibody (35.2mg, 235 nanomolar) and the re-oxidation mixture was allowed to react for 2 hours 30 minutes at room temperature with gentle (60rpm) shaking at an antibody concentration of 1.6mg/mL (or more DHAA was added, the reaction time was longer until complete re-oxidation of the cysteine thiol to re-form interchain cysteine disulfide was observed by UHPLC). The reoxidation mixture is then sterile filtered. Compound 6 was added as a DMSO solution (20 molar equivalents per antibody, 2.3 micromoles in 1.36mL DMSO) to 11.0mL of this reoxidized antibody solution (17.6mg, 117 nanomoles), adjusted in pH with 1.22mL of 1M sodium bicarbonate, with final DMSO concentrations of 10% (v/v) and 10% (v/v)1M sodium bicarbonate. The solution was allowed to react for 2 hours at room temperature with gentle shaking. The conjugate was then quenched by addition of N-acetyl cysteine (12 micromolar, 117 μ Ι at 100 mM), then purified and buffer exchanged using a 15mL Amicon Ultracell 50kDa MWCO spin filter into 25mM histidine 205mM sucrose pH6.0 buffer, sterile filtered and analyzed.

UHPLC analysis was performed on an shimadzu progression system using a Sepax Proteomix HIC butyl np54.6x35mm 5 μ M column, eluting with a gradient of 25mM sodium phosphate, 1.5M ammonium sulfate pH 7.4 buffer and 20% acetonitrile (v/v) in 25mM sodium phosphate pH 7.4 buffer, intact samples of ConjC showing attachment of unconjugated and conjugated antibodies to one or two molecules of compound 6 at 214nm and 330nm (compound 6 specific), consistent with a drug/antibody ratio (DAR) of 1.42 molecules of compound 6 per antibody.

UHPLC analysis was performed on the Shimadzu Promin System using a Tosoh bioscience TSKgel SuperSW mAb HTP 4 μm 4.6x150mM column (with a4 μm 3.0x20mM guard column), eluting with 0.3 mL/min sterile filtered SEC buffer (containing 200mM potassium phosphate pH 6.95, 250mM potassium chloride and 10% isopropanol (v/v)), showing a monomer purity of 98% at 280nm on a ConjC sample. UHPLC SEC analysis gave a final ConjC concentration of 1.06mg/mL in 10.1mL and a ConjC mass of 10.7mg (61% yield) was obtained.

Example 13 in vitro assay

Solid test materials were dissolved in DMSO as 2mM stock solutions from which eight serial dilutions were made in DMSO at a ratio of 1: 10 and stored at-20 ℃ until use.

Adherent NCI-N87 cells were washed with D-PBS and detached with trypsin-EDTA, followed by use of an automatic cell counter (LUNA-II)^TM) Cell density and viability were determined in duplicate by trypan blue exclusion assay. The cell suspension was grown in growth medium (containing Glutamax + 10% (v/v) HyClone)^TMRPMI 1640 of fetal bovine serum) to 1x10⁵cells/mL and vortexed, then 2mL per well was dispensed into sterile 3mL polypropylene plates. The bullet dilutions were then dispensed into appropriate wells at 10 μ l/well and mixed by repeat pipetting. For control wells, 10 μ l DMSO was dispensed into 2mL cell suspension and mixed well. However, the device is not suitable for use in a kitchenThen 100. mu.l of the sample was aliquoted into 2 replicate wells of a sterile 96-well microplate and CO at 37 ℃₂Air (5%) filled incubator. At the end of the incubation period (7 days), the cells were passed through CellTiter 96^TMCell viability was measured by the aquous One (MTS) assay, which was dispensed at 20. mu.l/well and 5% CO at 37 ℃₂Incubate for 4 hours. Then in EnVision^TMPlates were read on a multi-label microplate reader (Perkin Elmer) using absorbance at 490 nm.

Cell viability was calculated by comparing the average absorbance from 2 replicate wells of each sample to the average absorbance (100%) of two control wells treated with DMSO only. IC was determined by fitting each data set to a sigmoidal dose-response curve with variable slope using a non-linear curve fitting algorithm on GraphPad Prism software (san Diego, Calif.)₅₀。

All experiments in this report were performed and tested in three separate experiments. Data are reported as the average of three independent replicates.

	IC50(nM)
		I11	0.3854

EXAMPLE 14 ADC in vitro assay

Concentration and viability of cells from sub-confluent (80% -90% confluent) T75 flasks were measured by trypan blue staining and LUNA-II was used^TMAn automated cell counter counts. Cells were diluted to 2x10⁵One/ml, dispensed (50. mu.l per well) in a 96-well flat-bottom plate.

Stock solutions (1ml) of Antibody Drug Conjugates (ADCs) (20 μ g/ml) were prepared by diluting filter sterilized ADCs into cell culture media. A set of 8x 10-fold diluted stock ADCs were prepared in 24-well plates by serially transferring 100 μ Ι into 900 μ Ι cell culture medium. Dilutions of ADC (50 μ Ι per well) were dispensed into 4 replicate wells of a 96-well plate containing 50 μ Ι of cell suspension previously seeded. Control wells received 50 μ l of cell culture medium. 96-well plates containing cells and ADC were incubated at 37 ℃ in CO₂Incubate exposure time in an aerated incubator.

At the end of the incubation period, cell viability was measured by MTS assay. MTS (Promega) was dispensed (20. mu.l per well) to each well and in CO₂The aerated incubator was incubated at 37 ℃ for 4 hours. The absorbance of the wells was measured at 490 nm. Percent cell survival was calculated from the average absorbance of 4 ADC-treated wells versus 4 control untreated wells (100%). Determination of IC from dose response data Using GraphPad Prism Using a non-Linear Curve fitting Algorithm (sigmoidal dose response Curve with variable slope)₅₀。

The ADC incubation time for MDA-MB-468 was 4 days and for NCI-N87 was 7 days. In a sample with Glutamax + 10% (v/v) HyClone^TMMDA-MB-468 and NCI-N87 were cultured in RPMI 1640 of fetal bovine serum. NCI-N87 is an expressed Her2 cell line, while MDA-MB-468 is a Her2 negative cell line.

EC50(μg/ml)	NCI-N87	MDA-MB-468
			ConjA	0.1176	＞10
ConjA*	0.01634	＞10
			ConjB	0.01857	＞10
ConjC	0.1452	＞10

Example 15 ADC in vivo assay

Method and material

Mouse

Female severe combined immunodeficient mice (Fox Chase SCID)^TMCB 17/Icr-Prkdcscid/icoicrcl, charles river) was eight weeks old and Body Weight (BW) ranged from 14.5 to 20.0 grams on day 1 of the study. Animals were fed ad libitum water (reverse osmosis, 1ppm Cl), and modified NIH 31 and Irradiated Lab Diet^TM(consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crude fiber). Mice were housed in irradiated Enrich-o' cobs (TM) laboratory animal beds in static mini-isolators and subjected to 12-hour light cycles at 20 ℃ -22 ℃ and 40% -60% humidity. CR Discovery Services are specifically performed following guidelines for laboratory animal care and use with recommendations for containment, feeding, surgery, feed and fluid administration, and veterinary care. Animal care and use programs for CR Discovery Services have received approval from the international association for laboratory animal care assessment and certification (AAALAC), which ensures compliance with accepted laboratory animal care and use standards.

Tumor cell culture

In a medium supplemented with 10% fetal bovine serum, 2mM glutamine, 100 units/mL penicillin, 100. mu.g/mL streptomycin sulfate, and 25. mu.g/mLHuman NCI-N87 gastric carcinoma lymphoma cells were cultured in RPMI-1640 medium of gentamicin. Cells were incubated at 37 ℃ in 5% CO₂And 95% air atmosphere.

In vivo implantation and tumor growth

NCI-N87 cells were harvested for implantation during the logarithmic growth phase and resuspended in a medium containing 50% Matrigel^TM(BD Biosciences) phosphate buffered saline (PBS.) on the day of tumor implantation, the right flank of each test mouse was injected subcutaneously with 1x10⁷Individual cells (0.1mL cell suspension), and when the average size is close to 100 to 150mm³The growth of the tumor is monitored at the target range of (a). After 12 days (designated day 1 of the study), mice were divided into 14 groups according to the calculated tumor size, 7 groups were designated for efficacy assessment (n-10) and 7 groups were designated for sample collection (n-3), each group ranging from 108 to 172mm of individual tumor volume³And the group mean tumor volume is 120-124mm³. Tumors were measured in two dimensions using calipers and volume was calculated using the following formula:

tumor volume (mm)³)＝(w²xl)/2

Where w is the width of the tumor and l is the length of the tumor in mm. It can be assumed that 1mg equals 1mm³To estimate tumor weight.

Therapeutic agents

ConjA was stored at 4 ℃ in the dark. Sterile PBS was used for administration to the vehicle control group.

Treatment of

Female SCID mice bearing established NCI-N87 xenografts were grouped on study day 1. Aliquots of the stock were diluted to appropriate concentrations with PBS. The agent was administered via tail vein injection once on day 1. The dose volume was 0.2mL per 20 grams body weight (10 mL/kg) and was scaled to the body weight of each animal.

Group 1 mice received PBS vehicle and served as a control group. Group 2 received 4mg/kg ConjA.

Twice a week makeThe tumors were measured with calipers, when the tumors reached 800mm per animal³Either at the end of the study (day 68) or (subject to first arrival), each animal was euthanized. Animals that exited the study with tumor volume endpoints were scored as euthanized by Tumor Progression (TP) and the date of euthanization was scored.

Criteria for resolution reaction

Treatment efficacy can be determined by the incidence and magnitude of the regression response observed during the study. Treatment may result in Partial Regression (PR) or Complete Regression (CR) of the tumor in the animal. In the PR response, three consecutive measurements of tumor volume of 50% or less of the day 1 tumor volume were taken over the course of the study, and in one or more of these three measurements, the tumor volume was equal to or greater than 13.5mm³. In the CR reaction, tumor volumes of less than 13.5mm were measured in triplicate during the study³. Animals were scored for PR or CR events only one during the study, and CR was scored if both PR and CR criteria were met. Animals with CR response at study termination were additionally classified as tumor-free survivors (TFS). Animals were monitored for regression responses.

Toxicity

Animals were weighed daily on days 1-5 and then twice weekly until the study was complete. Mice are often observed for significant signs of any adverse, treatment-related (TR) side effects, and recorded when clinical signs are observed. The individual body weight was monitored according to the protocol and animals with weight loss of more than 30% measured at any one time or more than 25% measured three consecutive times were euthanized as TR deaths. Group mean weight loss was also monitored according to the CR Discovery Services protocol. Acceptable toxicity was defined as a group mean Body Weight (BW) loss of less than 20% and TR death of no more than 10% during the study.

Results

Figure 1 shows a mean tumor growth plot, wherein:

media	●
		ConjA*	◆

Group 1 mice received i.v.qd x1 PBS vehicle and served as a control group. The median TTE for group 1 was 24.8 days. All control tumors reached 800mm³And (4) finishing.

Group 2 received ConjA at 4mg/kg i.v.qd x 1. CR was observed in all 10 mice at the end of the study, which were otherwise classified as TFS.

The lowest point of body weight in the treatment group was-9.5% on study day 50. No TR death was observed.

Statement of the invention

1. A compound having the formula I:

and salts and solvates thereof, wherein R^LIs a linker for attachment to a ligand unit, the linker being selected from the group consisting of:

(ia)：

wherein

Q is:

wherein Q^XSuch that Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue;

x is:

wherein a is 0 to 5, b1 is 0 to 16, b2 is 0 to 16, c1 is 0 or 1, c2 is 0 or 1, d is 0 to 5, wherein at least b1 or b2 is 0 and at least c1 or c2 is 0;

G^Lis a linker for attachment to a ligand unit;

(ib)：

wherein R is^L1And R^L2Independently selected from H and methyl, or together with the carbon atom to which they are bonded form a cyclopropene or cyclobutene group; and is

e is 0 or 1.

2. The compound according to statement 1, wherein R^LHas the formula Ia.

3. The compound according to statement 2, wherein Q is an amino acid residue.

4. A compound according to statement 3, wherein Q is selected from: phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp.

5. The compound of statement 2, wherein Q is a dipeptide residue.

6. The compound of statement 5, wherein Q is selected from:

^NH-Phe-Lys-^C＝O、

^NH-Val-Ala-^C＝O、

^NH-Val-Lys-^C＝O、

^NH-Ala-Lys-^C＝O、

^NH-Val-Cit-^C＝O、

^NH-Phe-Cit-^C＝O、

^NH-Leu-Cit-^C＝O、

^NH-Ile-Cit-^C＝O、

^NH-Phe-Arg-^C＝O、

^NH-Trp-Cit-^C＝^O、and

^NH-Gly-Val-^C＝O。

7. the compound of statement 6, wherein Q is selected from^NH-Phe-Lys-^C＝O、^NH-Val-Cit-^C＝OAnd^NH-Val-Ala-^C＝O。

8. the compound of statement 2, wherein Q is a tripeptide residue.

9. The compound of statement 8, wherein Q is selected from:

^NH-Glu-Val-Ala-^C＝O、

^NH-Glu-Val-Cit-^C＝O、

^NH-αGlu-Val-Ala-^C＝Oand, and

^NH-αGlu-Val-Cit-^C＝O。

10. the compound of statement 2, wherein Q is a tetrapeptide residue.

11. The compound of statement 10, wherein Q is selected from:

^NH-Gly-Gly-Phe-Gly^C＝O(ii) a And

^NH-Gly-Phe-Gly-Gly^C＝O。

12. the compound according to statement 11, wherein Q is:

^NH-Gly-Gly-Phe-Gly^C＝O。

13. the compound according to any one of statements 2 to 12, wherein a is 0 to 3.

14. The compound according to statement 13, wherein a is 0 or 1.

15. The compound according to statement 13, wherein a is 0.

16. The compound according to any one of statements 2 to 15, wherein b1 is 0 to 8.

17. The compound of statement 16, wherein b1 is 0.

18. The compound of statement 16, wherein b1 is 2.

19. The compound of statement 16, wherein b1 is 3.

20. The compound of statement 16, wherein b1 is 4.

21. The compound of statement 16, wherein b1 is 5.

22. The compound of statement 16, wherein b1 is 8.

23. The compound according to any one of statements 2 to 15 and 17, wherein b2 is 0 to 8.

24. The compound of statement 23, wherein b2 is 0.

25. The compound of statement 23, wherein b2 is 2.

26. The compound of statement 23, wherein b2 is 3.

27. The compound of statement 23, wherein b2 is 4.

28. The compound of statement 23, wherein b2 is 5.

29. The compound of statement 23, wherein b2 is 8.

30. The compound according to any one of statements 2 to 29, wherein c1 is 0.

31. The compound according to any one of statements 2 to 29, wherein c1 is 1.

32. The compound according to any one of statements 2 to 31, wherein c2 is 0.

33. The compound according to any one of statements 2 to 30, wherein c2 is 1.

34. The compound according to any one of statements 2 to 33, wherein d is 0 to 3.

35. The compound of statement 34, wherein d is 1 or 2.

36. The compound of statement 34, wherein d is 2.

37. The compound according to any one of statements 2 to 33, wherein d is 5.

38. The compound according to any one of statements 2 to 12, wherein a is 0, b1 is 0, c1 is 1, c2 is 0 and d is 2, and b2 is from 0 to 8.

39. The compound of statement 38, wherein b2 is 0, 2, 3, 4, 5, or 8.

40. The compound according to any one of statements 2 to 12, wherein a is 1, b2 is 0, c1 is 0, c2 is 0 and d is 0, and b1 is from 0 to 8.

41. The compound of statement 40, wherein b1 is 0, 2, 3, 4, 5, or 8.

42. The compound according to any one of statements 2 to 12, wherein a is 0, b1 is 0, c1 is 0, c2 is 0 and d is 1, and b2 is from 0 to 8.

43. The compound of statement 42, wherein b2 is 0, 2, 3, 4, 5, or 8.

44. The compound according to any one of statements 2 to 12, wherein b1 is 0, b2 is 0, c1 is 0, c2 is 0, one of a and d is 0, and the other of a and d is from 1 to 5.

45. The compound of statement 44, wherein the other of a and d is 1 or 5.

46. The compound according to any one of statements 2 to 12, wherein a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 is from 0 to 8.

47. The compound of statement 46, wherein b1 is 0, 2, 3, 4, 5, or 8.

48. The compound according to any one of statements 2 to 47, wherein G^LIs selected from

Wherein Ar represents C_5-6An arylene group, and X represents C_1-4An alkyl group.

49. The compound of statement 48, wherein G^LIs selected from G^L1-1And G^L1-2。

50. The compound of statement 48, wherein G^LIs G^L1-1。

51. The compound according to statement 1, wherein R^LHaving formula Ib.

52. The compound of statement 51, wherein R^L1And RL²Both are H.

53. The compound of statement 51, wherein R^L1Is H and R^L2Is methyl.

54. The compound of statement 51, wherein R^L1And RL²Both are methyl.

55. The compound of statement 51, wherein R^L1And R^L2Together with the carbon atom to which they are bonded form a cyclopropene group.

56. The compound of statement 51, wherein R^L1And R^L2Together with the carbon atom to which they are bonded form a cyclobutene group.

57. The compound according to any one of statements 51 to 56, wherein e is 0.

58. The compound according to any one of statements 51 to 56, wherein e is 1.

59. A conjugate having the formula IV:

L-(D^L)_p (IV)

or a pharmaceutically acceptable salt or solvate thereof, wherein L is a ligand unit (i.e., targeting agent), D^LIs a drug linker unit having formula III:

R^LLis a linker attached to the ligand unit, the linker being selected from

(ia’)：

Wherein Q and X are as in any of statements 1 to 47A is as defined and G^LLIs a linker attached to the ligand unit; and

(ib’)：

wherein R is^L1And R^L2Is as defined in any one of statements 1 and 52 to 56; and is

p is an integer from 1 to 20.

60. The conjugate of statement 59, wherein G^LLSelected from:

wherein Ar represents C_5-6An arylene group, and X represents C_1-4An alkyl group.

61. The conjugate of statement 60, wherein G^LLIs selected from G^LL1-1And G^LL1-2。

62. The conjugate of statement 61, wherein G^LLIs G^LL1-1。

63. A conjugate according to any of statements 59 to 62, wherein the ligand unit is a cell binding agent.

64. A conjugate according to any of statements 59 to 62, wherein the ligand unit is an antibody or an active fragment thereof.

65. The conjugate of statement 64, wherein the antibody or antibody fragment is an antibody or antibody fragment to a tumor-associated antigen.

66. The conjugate of statement 65, wherein the antibody or antibody fragment is an antibody that binds to one or more tumor associated antigens or cell surface receptors selected from the group consisting of (1) - (89) below:

(1)BMPR1B；

(2)E16；

(3)STEAP1；

(4)0772P；

(5)MPF；

(6)Napi3b；

(7)Sema 5b；

(8)PSCA hlg；

(9)ETBR；

(10)MSG783；

(11)STEAP2；

(12)TrpM4；

(13)CRIPTO；

(14)CD21；

(15)CD79b；

(16)FcRH2；

(17)HER2；

(18)NCA；

(19)MDP；

(20)IL20R-α；

(21) a short peptide;

(22)EphB2R；

(23)ASLG659；

(24)PSCA；

(25)GEDA；

(26)BAFF-R；

(27)CD22；

(28)CD79a；

(29)CXCR5；

(30)HLA-DOB；

(31)P2X5；

(32)CD72；

(33)LY64；

(34)FcRH1；

(35)IRTA2；

(36)TENB2；

(37)PSMA-FOLH1；

(38)SST；

(38.1)SSTR2；

(38.2)SSTR5；

(38.3)SSTR1；

(38.4)SSTR3；

(38.5)SSTR4；

(39)ITGAV；

(40)ITGB6；

(41)CEACAM5；

(42)MET；

(43)MUC1；

(44)CA9；

(45)EGFRvIII；

(46)CD33；

(47)CD19；

(48)IL2RA；

(49)AXL；

(50)CD30-TNFRSF8；

(51)BCMA-TNFRSF17；

(52)CT Ags-CTA；

(53)CD174(Lewis Y)-FUT3；

(54)CLEC14A；

(55)GRP78-HSPA5；

(56)CD70；

(57) a stem cell specific antigen;

(58)ASG-5；

(59)ENPP3；

(60)PRR4；

(61)GCC-GUCY2C；

(62)Liv-1-SLC39A6；

(63)5T4；

(64)CD56-NCMA1；

(65)CanAg；

(66)FOLR1；

(67)GPNMB；

(68)TIM-1-HAVCR1；

(69) RG-1/prostate tumor target Mindin-Mindin/RG-1;

(70)B7-H4-VTCN1；

(71)PTK7；

(72)CD37；

(73)CDl38-SDC1；

(74)CD74；

(75) claudin-CL;

(76)EGFR；

(77)Her3；

(78)RON-MST1R；

(79)EPHA2；

(80)CD20-MS4A1；

(81) tenascin-C-TNC;

(82)FAP；

(83)DKK-1；

(84)CD52；

(85)CS1-SLAMF7；

(86) endoglin-ENG;

(87) annexin A1-ANXA 1;

(88)V-CAM(CD106)-VCAM1；

(89)ASCT2(SLC1A5)。

67. the conjugate according to any one of statements 64 to 66, wherein the antibody or antibody fragment is a cysteine engineered antibody.

684. The conjugate of any one of statements 64 to 67, wherein the drug load (p) of drug (D) and antibody (Ab) is an integer from 1 to about 10.

69. The conjugate of statement 68, wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

70. A mixture of conjugates according to any one of statements 64 to 69, wherein the average drug loading per antibody in the mixture of antibody-drug conjugates is from about 1 to about 10.

71. A conjugate or mixture according to any one of statements 59 to 70 for use in therapy.

72. A pharmaceutical composition comprising a conjugate or mixture according to any one of statements 59 to 70 and a pharmaceutically acceptable diluent, carrier, or excipient.

73. The conjugate or mixture of any one of statements 59 to 70, or the pharmaceutical composition of statement 72, for use in the treatment of a proliferative disease in a subject.

74. The conjugate, mixture, or pharmaceutical composition of statement 73, wherein the disease is cancer.

75. Use of the conjugate or mixture of any one of statements 59 to 70, or the pharmaceutical composition of statement 72, in a method of medical treatment.

76. A method of medical treatment comprising administering to a patient a pharmaceutical composition according to statement 72.

77. The method according to statement 76, wherein the method of medical treatment is for treating cancer.

78. The method according to statement 77, wherein the chemotherapeutic agent is administered to the patient in combination with the conjugate.

79. Use of a conjugate or mixture of any of statements 59 to 70 in a method of manufacture of a medicament for treating a proliferative disease.

80. A method of treating a mammal having a proliferative disease, comprising administering an effective amount of a conjugate or mixture according to any of statements 59 to 70, or a pharmaceutical composition according to statement 72.

81. A compound A:

in the form of individual enantiomers or in enantiomerically enriched form.

82. A compound having the formula VI:

wherein Q is as defined in any one of statements 1 and 3 and 12.

Statement of invention from priority 1 application (P1)

P1-1. a compound having the formula I:

and salts and solvates thereof, wherein R^LIs a linker for attachment to a cell binding agent, the linker being selected from the group consisting of:

(ia)：

wherein

Q is:

wherein Q^XSuch that Q is an amino acid residue, a dipeptide residue, or a tripeptide residue;

x is:

wherein a is 0 to 5, b is 0 to 16, c is 0 or 1, d is 0 to 5;

G^Lis a linker for attachment to a ligand unit;

(ib)：

wherein R is^L1And R^L2Independently selected from H and methyl, or together with the carbon atom to which they are bonded form a cyclopropene or cyclobutene group; and is

e is 0 or 1.

P1-2. the compound according to statement P1-1, wherein R^LHas the formula Ia.

P1-3 the compound according to statement P1-2, wherein Q is an amino acid residue.

P1-4. the compound according to statement P1-3, wherein Q is selected from: phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp.

P1-5. the compound according to statement P1-2, wherein Q is a dipeptide residue.

P1-6. the compound according to statement P1-5, wherein Q is selected from:

^NH-Phe-Lys-^C＝O、

^NH-Val-Ala-^C＝O、

^NH-Val-Lys-^C＝O、

^NHAla-Lys-^C＝O、

^NH-Val-Cit-^C＝O、

^NH-Phe-Cit-^C＝O、

^NH-Leu-Cit-^C＝O、

^NH-Ile-Cit-^C＝O、

^NH-Phe-Arg-^C＝O、

^NH-Trp-Cit-^C＝Oand, and

^NH-Gly-Val-^C＝O。

p1-7 the conjugate according to statement P1-6, wherein Q is selected from^NH-Phe-Lys-^C＝O、^NH-Val-Cit-^C＝OAnd^NH-Val-Ala-^C＝O。

p1-8 the compound according to statement P1-2, wherein Q is a tripeptide residue.

P1-9 the compound according to any one of statements P1-2 to P1-8, wherein a is 0 to 3.

P1-10. the compound according to statement P1-9, wherein a is 0 or 1.

P1-11. the compound according to statement P1-9, wherein a is 0.

P1-12 the compound according to any one of statements P1-2 to P1-11, wherein b is 0 to 8.

P1-13. the compound according to statement P1-12, wherein b is 0.

P1-14. the compound according to statement P1-12, wherein b is 4.

P1-15. the compound according to statement P1-12, wherein b is 8.

P1-16 the compound according to any one of statements P1-2 to P1-15, wherein c is 0.

Pl-17. the compound according to any one of statements P1-2 to P1-15, wherein c is 1.

P1-18 the compound according to any one of statements P1-2 to P1-17, wherein d is 0 to 3.

P1-19. the compound according to statement P1-18, wherein d is 1 or 2.

P1-20. the compound according to statement P1-19, wherein d is 2.

Pl-21. the compound according to any one of statements P1-2 to P1-8, wherein a is 0, c is 1 and d is 2, and b is 0, 4 or 8.

P1-22 the compound according to any one of statements P1-2 to P1-21, wherein G^LIs selected from

Wherein Ar represents C_5-6An arylene group, and X represents C_1-4An alkyl group.

P1-23. the compound according to statement P1-22, wherein G^LIs selected from G^L1-1And G^L1-2。

P1-24. the compound according to statement P1-22, wherein G^LIs G^L1-1。

P1-25. the compound according to statement P1-1, wherein R^LHaving formula Ib.

P1-26. the compound according to statement P1-25, wherein R^L1And R^L2Both are H.

P1-27. the compound according to statement P1-25, wherein R^L1Is H and R^L2Is methyl.

P1-28. the compound according to statement P1-25, wherein R^L1And R^L2Both are methyl.

P1-29, the compound according to statement P1-25,wherein R is^L1And R^L2Together with the carbon atom to which they are bonded form a cyclopropene group.

P1-30. the compound according to statement P1-25, wherein R^L1And R^L2Together with the carbon atom to which they are bonded form a cyclobutene group.

P1-31 the compound according to any one of statements P1-25 to P1-30, wherein e is 0.

P1-32 the compound according to any one of statements P1-25 to P1-30, wherein e is 1.

P1-33 a conjugate having formula IV:

L-(D^L)_p (IV)

or a pharmaceutically acceptable salt or solvate thereof, wherein L is a ligand unit (i.e., targeting agent), D^LIs a drug linker unit having formula III:

R^LLis a linker attached to the ligand unit, the linker being selected from

(ia’)：

Wherein Q and X are as defined in any one of statements P1-1 to P1-21 and G^LLIs a linker attached to the ligand unit; and

(ib’)：

wherein R is^L1And R^L2Is as defined in any one of statements P1-1 and P1-25 to P1-30; and is

p is an integer from 1 to 20.

P1-34 the conjugate of statement P1-33, wherein G^LLSelected from:

wherein Ar represents C_5-6An arylene group, and X represents C_1-4An alkyl group.

P1-35 the conjugate of statement P1-34, wherein G^LLIs selected from G^LL1-1And G^LL1-2。

P1-36 the conjugate of statement P1-35, wherein G^LLIs G^LL1-1。

P1-37 the conjugate according to any one of statements P1-33 to P1-36, wherein the cell binding agent is an antibody or an active fragment thereof.

P1-38 the conjugate according to statement P1-37, wherein the antibody or antibody fragment is an antibody or antibody fragment to a tumor associated antigen.

P1-39 the conjugate according to statement P1-38, wherein the antibody or antibody fragment is an antibody that binds to one or more tumor associated antigens or cell surface receptors selected from (1) - (88) below:

(1)BMPR1B；

(2)E16；

(3)STEAP1；

(4)0772P；

(5)MPF；

(6)Napi3b；

(7)Sema 5b；

(8)PSCA hlg；

(9)ETBR；

(10)MSG783；

(11)STEAP2；

(12)TrpM4；

(13)CRIPTO；

(14)CD21；

(15)CD79b；

(16)FcRH2；

(17)HER2；

(18)NCA；

(19)MDP；

(20)IL20R-α；

(21) a short peptide;

(22)EphB2R；

(23)ASLG659；

(24)PSCA；

(25)GEDA；

(26)BAFF-R；

(27)CD22；

(28)CD79a；

(29)CXCR5；

(30)HLA-DOB；

(31)P2X5；

(32)CD72；

(33)LY64；

(34)FcRH1；

(35)IRTA2；

(36)TENB2；

(37)PSMA-FOLH1；

(38)SST；

(38.1)SSTR2；

(38.2)SSTR5；

(38.3)SSTR1；

(38.4)SSTR3；

(38.5)SSTR4；

(39)ITGAV；

(40)ITGB6；

(41)CEACAM5；

(42)MET；

(43)MUC1；

(44)CA9；

(45)EGFRvIII；

(46)CD33；

(47)CD19；

(48)IL2RA；

(49)AXL；

(50)CD30-TNFRSF8；

(51)BCMA-TNFRSF17；

(52)CT Ags-CTA；

(53)CD174(Lewis Y)-FUT3；

(54)CLEC14A；

(55)GRP78-HSPA5；

(56)CD70；

(57) a stem cell specific antigen;

(58)ASG-5；

(59)ENPP3；

(60)PRR4；

(61)GCC-GUCY2C；

(62)Liv-1-SLC39A6；

(63)5T4；

(64)CD56-NCMA1；

(65)CanAg；

(66)FOLR1；

(67)GPNMB；

(68)TIM-1-HAVCR1；

(69) RG-1/prostate tumor target Mindin-Mindin/RG-1;

(70)B7-H4-VTCN1；

(71)PTK7；

(72)CD37；

(73)CD138-SDC1；

(74)CD74；

(75) claudin-CL;

(76)EGFR；

(77)Her3；

(78)RON-MST1R；

(79)EPHA2；

(80)CD20-MS4A1；

(81) tenascin-C-TNC;

(82)FAP；

(83)DKK-1；

(84)CD52；

(85)CS1-SLAMF7；

(86) endoglin-ENG;

(87) annexin A1-ANXA 1;

(88)V-CAM(CD106)-VCAM1；

(89)ASCT2(SLC1A5)。

p1-40 the conjugate according to any one of statements P1-37 to P1-39, wherein the antibody or antibody fragment is a cysteine engineered antibody.

P1-41 the conjugate of any one of statements P1-37 to P1-40, wherein the drug load (P) of drug (D) and antibody (Ab) is an integer from 1 to about 10.

P1-42 the conjugate of statement P1-41, wherein P is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

P1-43, a mixture of conjugates according to any one of statements P1-33 to P1-42, wherein the average drug load per antibody in the mixture of antibody-drug conjugate compounds is from about 1 to about 10.

P1-44 the conjugate or mixture of any one of statements P1-33 to P1-43, for use in therapy.

P1-45 a pharmaceutical composition comprising a conjugate or mixture according to any one of statements P1-33 to P1-43 and a pharmaceutically acceptable diluent, carrier, or excipient.

P1-46 the conjugate or mixture of any one of statements P1-33 to P1-43, or the pharmaceutical composition of statements P1-45, for use in the treatment of a proliferative disease in a subject.

P1-47 the conjugate or mixture according to statement P1-46, wherein the disease is cancer.

P1-48 use of the conjugate or mixture according to any one of statements P1-33 to P1-43, or the pharmaceutical composition according to statements P1-45, in a method of medical treatment.

P1-49 a method of medical treatment, the method comprising administering to a patient a pharmaceutical composition as described in statement P1-45.

P1-50 the method according to statement P1-49, wherein the method of medical treatment is for the treatment of cancer.

P1-51. the method according to statement P1-50, wherein the patient is administered a chemotherapeutic agent in combination with the conjugate.

P1-52 use of a conjugate or mixture according to any one of statements P1-33 to P1-43 in a method of manufacturing a medicament for the treatment of a proliferative disease.

P1-53 a method of treating a mammal suffering from a proliferative disease, the method comprising administering an effective amount of a conjugate or mixture according to any of statements P1-33 to P1-43, or a pharmaceutical composition according to statements P1-45.

P1-54 compound a:

in the form of individual enantiomers or in enantiomerically enriched form.

Statement of invention from the 2 nd priority application (P2)

P2-1. a compound having the formula I:

and salts and solvates thereof, wherein R^LIs a linker for attachment to a cell binding agent, the linker being selected from the group consisting of:

(ia)：

wherein

Q is:

wherein Q^XSuch that Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue;

x is:

wherein a is 0 to 5, b1 is 0 to 16, b2 is 0 to 16, c is 0 or 1, d is 0 to 5, wherein at least b1 or b2 is 0;

G^Lis a linker for attachment to a ligand unit;

(ib)：

wherein R is^L1And R^L2Independently selected from H and methyl, or together with the carbon atom to which they are bonded form a cyclopropene or cyclobutene group; and is

e is 0 or 1.

P2-2. the compound according to statement P2-1, wherein R^LHas the formula Ia.

P2-3 the compound according to statement P2-2, wherein Q is an amino acid residue.

P2-4. the compound according to statement P2-3, wherein Q is selected from: phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp.

P2-5. the compound according to statement P2-2, wherein Q is a dipeptide residue.

P2-6. the compound according to statement P2-5, wherein Q is selected from:

^NH-Phe-Lys-^C＝O、

^NH-Val-Ala-^C＝O、

^NH-Val-Lys-^C＝O、

^NH-Ala-Lys-^C＝O、

^NH-Val-Cit-^C＝O、

^NH-Phe-Cit-^C＝O、

^NH-Leu-Cit-^C＝O、

^NH-Ile-Cit-^C＝O、

^NH-Phe-Arg-^C＝O、

^NH-Trp-Cit-^C＝Oand, and

^NH-Gly-Val-^C＝O。

p2-7. the compound according to statement P2-6, wherein Q is selected from^NH-Phe-Lys-^C＝O、^NH-Val-Cit-^C＝OAnd^NH-Val-Ala-^C＝O。

p2-8 the compound according to statement P2-2, wherein Q is a tripeptide residue.

P2-9. the compound according to statement P2-8, wherein Q is selected from:

^NH-Glu-Val-Ala-^C＝O、

^NH-Glu-Val-Cit-^C＝O、

^NH-αGlu-Val-Ala-^C＝Oand, and

^NH-αGlu-Val-Cit-^C＝O。

p2-10. the compound according to statement P2-2, wherein Q is a tetrapeptide residue.

P2-11. the compound according to statement P2-10, wherein Q is selected from:

^NH-Gly-Gly-Phe-Gly^C＝O(ii) a And

^NH-Gly-Phe-Gly-Gly^C＝O。

p2-12. the compound according to statement P2-11, wherein Q is:

^NH-Gly-Gly-Phe-Gly^C＝O。

p2-13 the compound according to any one of statements P2-2 to P2-12, wherein a is 0 to 3.

P2-14. the compound according to statement P2-13, wherein a is 0 or 1.

P2-15. the compound according to statement P2-13, wherein a is 0.

P2-16 the compound of any one of statements P2-2 to P2-15, wherein b1 is 0 to 8.

P2-17. the compound according to statement P2-16, wherein b1 is 0.

P2-18. the compound according to statement P2-16, wherein b1 is 2.

P2-19. the compound according to statement P2-16, wherein b1 is 3.

P2-20. the compound according to statement P2-16, wherein b1 is 4.

P2-21. the compound according to statement P2-16, wherein b1 is 5.

P2-22. the compound according to statement P2-16, wherein b1 is 8.

P2-23 the compound of any one of statements P2-2 to P2-15 and P2-17, wherein b2 is 0 to 8.

P2-24. the compound according to statement P2-23, wherein b2 is 0.

P2-25. the compound according to statement P2-23, wherein b2 is 2.

P2-26. the compound according to statement P2-23, wherein b2 is 3.

P2-27. the compound according to statement P2-23, wherein b2 is 4.

P2-28. the compound according to statement P2-23, wherein b2 is 5.

P2-29. the compound according to statement P2-23, wherein b2 is 8.

P2-30 the compound according to any one of statements P2-2 to P2-29, wherein c is 0.

P2-31 the compound according to any one of statements P2-2 to P2-29, wherein c is 1.

P2-32 the compound according to any one of statements P2-2 to P2-31, wherein d is 0 to 3.

P2-33. the compound according to statement P2-32, wherein d is 1 or 2.

P2-34. the compound according to statement P2-32, wherein d is 2.

P2-35 the compound of any one of statements P2-2 to P2-12, wherein a is 0, b1 is 0, c is 1 and d is 2, and b2 is from 0 to 8.

P2-36. the compound according to statement P2-35, wherein b2 is 0, 2, 3, 4, 5 or 8.

P2-37 the compound of any one of statements P2-2 to P2-12, wherein a is 1, b2 is 0, c is 0 and d is 0, and b1 is from 0 to 8.

P2-38. the compound according to statement P2-37, wherein b1 is 0, 2, 3, 4, 5 or 8.

P2-39 the compound of any one of statements P2-2 to P2-12, wherein a is 0, b1 is 0, c is 0 and d is 1, and b2 is from 0 to 8.

P2-40. the compound according to statement P2-39, wherein b2 is 0, 2, 3, 4, 5 or 8.

P2-41 the compound of any one of statements P2-2 to P2-12, wherein b1 is 0, b2 is 0, c is 0, one of a and d is 0, and the other of a and d is from 1 to 5.

P2-42. the compound according to statement P2-41, wherein the other of a and d is 1 or 5.

P2-43 the compound according to any one of statements P2-2 to P2-42, wherein G^LIs selected from

Wherein Ar represents C_5-6An arylene group, and X represents C_1-4An alkyl group.

P2-44 the compound according to statement P2-43, wherein G^LIs selected from G^L1-1And G^L1-2。

P2-45. the compound according to statement P2-43, wherein G^LIs G^L1-1。

P2-46. the compound according to statement P2-1, wherein R^LHaving formula Ib.

P2-47 the compound according to statement P2-46, wherein R^L1And R^L2Both are H.

P2-48 the compound according to statement P2-46, wherein R^L1Is H and R^L2Is methyl.

P2-49 the compound according to statement P2-46, wherein R^L1And R^L2Both are methyl.

P2-50 the compound according to statement P2-46, wherein R^L1And R^L2Together with the carbon atom to which they are bonded form a cyclopropene group.

P2-51. the compound according to statement P2-46, wherein R^L1And R^L2Together with the carbon atom to which they are bonded form a cyclobutene group.

P2-52 the compound according to any one of statements P2-46 to P2-51, wherein e is 0.

P2-53 the compound according to any one of statements P2-46 to P2-51, wherein e is 1.

P2-54 a conjugate having formula IV:

L-(D^L)_p (IV)

or a pharmaceutically acceptable salt or solvate thereof, wherein L is a ligand unit (i.e., targeting agent), D^LIs a drug linker unit having formula III:

R^LLis a linker attached to the ligand unit, the linker being selected from

(ia’)：

Wherein Q and X are as defined in any one of statements P2-1 to P2-42 and G^LLIs a linker attached to the ligand unit; and

(ib’)：

wherein R is^L1And R^L2Is as defined in any one of statements P2-1 and P2-47 to P2-51; and is

p is an integer from 1 to 20.

P2-55. The conjugate of statement P2-54, wherein G^LLSelected from:

wherein Ar represents C_5-6An arylene group, and X represents C_1-4An alkyl group.

P2-56 the conjugate according to statement P2-55, wherein G^LLIs selected from G^LL1-1And G^LL1-2。

P2-57 the conjugate according to statement P2-56, wherein G^LLIs G^LL1-1。

P2-58 the conjugate according to any one of statements P2-54 to P2-57, wherein the ligand unit is an antibody or an active fragment thereof.

P2-59 the conjugate according to statement P2-58, wherein the antibody or antibody fragment is an antibody or antibody fragment to a tumor associated antigen.

P2-60 the conjugate according to statement P2-59, wherein the antibody or antibody fragment is an antibody that binds to one or more tumor associated antigens or cell surface receptors selected from (1) - (89) below:

(1)BMPR1B；

(2)E16；

(3)STEAP1；

(4)0772P；

(5)MPF；

(6)Napi3b；

(7)Sema 5b；

(8)PSCA hlg；

(9)ETBR；

(10)MSG783；

(11)STEAP2；

(12)TrpM4；

(13)CRIPTO；

(14)CD21；

(15)CD79b；

(16)FcRH2；

(17)HER2；

(18)NCA；

(19)MDP；

(20)IL20R-α；

(21) a short peptide;

(22)EphB2R；

(23)ASLG659；

(24)PSCA；

(25)GEDA；

(26)BAFF-R；

(27)CD22；

(28)CD79a；

(29)CXCR5；

(30)HLA-DOB；

(31)P2X5；

(32)CD72；

(33)LY64；

(34)FcRH1；

(35)IRTA2；

(36)TENB2；

(37)PSMA-FOLH1；

(38)SST；

(38.1)SSTR2；

(38.2)SSTR5；

(38.3)SSTR1；

(38.4)SSTR3；

(38.5)SSTR4；

(39)ITGAV；

(40)ITGB6；

(41)CEACAM5；

(42)MET；

(43)MUC1；

(44)CA9；

(45)EGFRvIII；

(46)CD33；

(47)CD19；

(48)IL2RA；

(49)AXL；

(50)CD30-TNFRSF8；

(51)BCMA-TNFRSF17；

(52)CT Ags-CTA；

(53)CD174(Lewis Y)-FUT3；

(54)CLEC14A；

(55)GRP78-HSPA5；

(56)CD70；

(57) a stem cell specific antigen;

(58)ASG-5；

(59)ENPP3；

(60)PRR4；

(61)GCC-GUCY2C；

(62)Liv-1-SLC39A6；

(63)5T4；

(64)CD56-NCMA1；

(65)CanAg；

(66)FOLR1；

(67)GPNMB；

(68)TIM-1-HAVCR1；

(69) RG-1/prostate tumor target Mindin-Mindin/RG-1;

(70)B7-H4-VTCN1；

(71)PTK7；

(72)CD37；

(73)CDl38-SDC1；

(74)CD74；

(75) claudin-CL;

(76)EGFR；

(77)Her3；

(78)RON-MST1R；

(79)EPHA2；

(80)CD20-MS4A1；

(81) tenascin-C-TNC;

(82)FAP；

(83)DKK-1；

(84)CD52；

(85)CS1-SLAMF7；

(86) endoglin-ENG;

(87) annexin A1-ANXA 1;

(88)V-CAM(CD106)-VCAM1；

(89)ASCT2(SLC1A5)。

p2-61 the conjugate according to any one of statements P2-58 to P2-60, wherein the antibody or antibody fragment is a cysteine engineered antibody.

P2-62 the conjugate according to any one of statements P2-58 to P2-61, wherein the drug load (P) of drug (D) and antibody (Ab) is an integer from 1 to about 10.

P2-63 the conjugate of statement P2-62, wherein P is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

P2-64 a mixture of conjugates according to any one of statements P2-58 to P2-63, wherein the average drug load per antibody in the mixture of antibody-drug conjugates is from about 1 to about 10.

P2-65 the conjugate or mixture of any one of statements P2-54 to P2-64 for use in therapy.

P2-66 a pharmaceutical composition comprising a conjugate or mixture according to any one of statements P2-54 to P2-64 and a pharmaceutically acceptable diluent, carrier, or excipient.

P2-67 the conjugate or mixture of any one of statements P2-54 to P2-64, or the pharmaceutical composition of statements P2-66, for use in the treatment of a proliferative disease in a subject.

P2-68 the conjugate, mixture or pharmaceutical composition according to statement P2-67, wherein the disease is cancer.

P2-69 use of the conjugate or mixture according to any one of statements P2-54 to P2-64, or the pharmaceutical composition according to statements P2-66, in a method of medical treatment.

P2-70 a method of medical treatment, the method comprising administering to a patient a pharmaceutical composition as described in statement P2-66.

P2-71 the method according to statement P2-70, wherein the method of medical treatment is for the treatment of cancer.

P2-72 the method according to statement P2-71, wherein the patient is administered a chemotherapeutic agent in combination with the conjugate.

P2-73 use of a conjugate or mixture according to any one of statements P2-54 to P2-64 in a method of manufacturing a medicament for the treatment of a proliferative disease.

P2-74 a method of treating a mammal suffering from a proliferative disease, the method comprising administering an effective amount of a conjugate or mixture according to any one of statements P2-54 to P2-64, or a pharmaceutical composition according to statements P2-66.

P2-75 compound a:

in the form of individual enantiomers or in enantiomerically enriched form.