Thiocycloyne derivatives and uses thereof
阅读说明：本技术 硫代环庚炔衍生物及其用途 (Thiocycloyne derivatives and uses thereof ) 是由 约瑟夫斯·约翰尼斯·韦特里格斯 克里斯蒂安尼·约翰娜·费迪南·里耶肯 于 2019-07-12 设计创作，主要内容包括：本发明涉及通式(I)的新型硫代环庚炔衍生物,并且具体地涉及硫杂环炔烃磺酰亚胺衍生物及其合成。本发明还涉及新型硫代环庚炔衍生物在与连接体和药物的偶联反应中的用途。本发明还涉及新型硫代环庚炔在生物正交(无铜)点击反应中的用途。本发明还涉及新型硫代环庚炔衍生物在产生高级多功能药物递送系统(载药)纳米颗粒中的用途。(The present invention relates to novel thiacycloheptyne derivatives of the general formula (I), and in particular to thiacycloalkyne sulfonimide derivatives and their synthesis. The invention also relates to the use of novel thiocycloheptyne derivatives in coupling reactions with linkers and drugs. The invention also relates to the use of novel thiocycloheptynes in bio-orthogonal (copper-free) click reactions. The invention also relates to the use of novel thio-cycloheptyne derivatives for the production of advanced multifunctional drug delivery system (drug loaded) nanoparticles.)
1. A compound of formula (I):
wherein:
n and m are independently 0, 1 or 2, and n + m is 2;
x is O or NR9，
Y is NR10，
R1、R2、R3、R4Independently selected from hydrogen, halogen (F, Cl, Br, I), O, N, P and S, C1-C24Alkyl radical, C6-C24(hetero) aryl, C7-C24Alkyl (hetero) aryl and C7-C24(hetero) arylalkyl, wherein O, N, P and S are further independently coupled to hydrogen, halogen (F, Cl, Br, I), C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy, said alkyl group being optionally interrupted by one of more heteroatoms selected from O, N, P and S, wherein said alkyl, (hetero) aryl, alkyl (hetero) aryl and (hetero) arylalkyl groups are independently optionally substituted with one or more substituents independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy, halogen (F, Cl, Br, I), amino, oxo and silyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkoxy are optionally substituted, the alkyl, the alkoxy, the cycloalkyl and the cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S, wherein the silyl is represented by formula (R)11)3Si-represents, wherein R11Independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy and C3-C12Cycloalkoxy, wherein said alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy, and cycloalkoxy are optionally substituted, said alkyl, said alkoxy, said cycloalkyl, and said cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S;
R5、R6、R7、R8independently selected from hydrogen, halogen (F, Cl, Br, I), O, N, P and S, C1-C24Alkyl radical, C6-C24(hetero) aryl, C7-C24Alkyl (hetero) aryl and C7-C24(hetero) arylalkyl, wherein O, N, P and S are further independently coupled to hydrogen, halogen (F, Cl, Br, I), C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy, said alkyl group being optionally interrupted by one of more heteroatoms selected from O, N, P and S, wherein said alkyl, (hetero) aryl, alkyl (hetero) aryl and (hetero) arylalkyl groups are independently optionally substituted with one or more substituents independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy, halogen (F, Cl, Br, I), amino, oxo and silyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkoxy are optionally substituted, the alkyl, the alkoxy, the cycloalkyl and the cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S, wherein the silyl is represented by formula (R)11)3Si-represents, wherein R11Independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy and C3-C12Cycloalkoxy, wherein said alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy, and cycloalkoxy are optionally substituted, said alkyl, said alkoxy, said cycloalkyl, and said cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S;
wherein, optionally, R1 and R7, R1 and R8, R2 and R7, R2 and R8, R3 and R5, R3 and R6, R4 and R5 and/or R4 and R6 independently form a fused ring system, such as a cycloalkyl-system, a cyclo (hetero) aryl-system, a cycloalkyl (hetero) aryl-system, a cyclo (hetero) arylalkyl-system,
wherein the alkyl groups of the fused ring system are optionally interrupted by one of more heteroatoms selected from O, N, P and S, wherein the alkyl, (hetero) aryl, alkyl (hetero) aryl, and (hetero) arylalkyl groups of the fused ring system are independently optionally substituted with one or more substituents independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy, halogen, amino, oxo, and silyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy, and cycloalkoxy are optionally substituted, the alkyl, the alkoxy, the cycloalkyl, and the cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S, wherein the silyl is represented by formula (R)11)3Si-represents, wherein R11Independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy and C3-C12Cycloalkoxy, wherein said alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy, and cycloalkoxy are optionally substituted, said alkyl, said alkoxy, said cycloalkyl, and said cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S;
R9、R10independently selected from hydrogen, halogen (F, Cl, Br, I), R12、-CH＝C(R12)2、-C≡CR12Wherein q is 1 to 200- [ C (R)12)2C(R12)2O]q-R12、-CN、-N3、-NCX、-XCN、-XR12、-N(R12)2、-+N(R12)3、-C(X)N(R12)2、-C(R12)2XR12、-C(X)R12、-C(X)XR12、-S(O)R12、-S(O)2R12、-S(O)OR12、-S(O)2OR12、-S(O)N(R12)2、-S(O)2N(R12)2、-OS(O)R12、-OS(O)2R12、-OS(O)OR12、-OS(O)2OR12、-P(O)(R12)(OR12)、-P(O)(OR12)2、-OP(O)(OR12)2、-Si(R12)3、-XC(X)R12、-XC(X)XR12、-XC(X)N(R12)2、-N(R12)C(X)R12、-N(R12)C(X)XR12and-N (R)12)C(X)N(R12)2Wherein X is oxygen or sulfur, and wherein R12Independently selected from hydrogen, halogen (F, Cl, Br, I), C1-C24Alkyl radical, C6-C24(hetero) aryl, C7-C24Alkyl (hetero) aryl and C7-C24(hetero) arylalkyl.
2. The compound of claim 1, wherein n is 1 and m is 1, and the compound is of formula (II)
And R is1-R8X, Y is as defined in claim 1.
3. A compound according to claim 1 or 2, wherein X is O, and/or R10Is H.
4. The compound of any one of claims 1 to 3, wherein R1、R2、R3、R4、R5、R6、R7、R8Independently H, halogen (F, Cl, Br, I) or C1-C24Alkyl, preferably wherein R1、R2、R3、R4、R5、R6、R7、R8Independently is H or C1-C4An alkyl group.
5. The compound of claim 4, wherein:
-R1、R2、R3、R4、R5、R6、R7、R8is methyl, or
-R1、R2、R3、R4Is methyl and R5、R6、R7、R8Is H.
6. The compound of any one of claims 1 to 5, wherein one atom adjacent to the S atom in the X and/or Y and/or thiocycloacetylene ring of the compound of formula (I) is independently coupled to an optional linking group (L) and a functional group (Q) to yield a compound of formula (III):
(formula I) -L-Q
(III)
Wherein the linking group (L) is absent or selected from linear or branched C1-C24Alkylene radical, C2-C24Alkenylene radical, C2-C24Alkynylene, C3-C24Cycloalkylene radical, C5-C24Cycloalkenylene group, C5-C24Cycloalkynylene, C7-C24Alkyl (hetero) arylene, C7-C24(hetero) arylalkylene radical, C5-C24(hetero) arylalkenylene, C9-C24(hetero) arylalkynylene, said alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkyl (hetero) arylene, (hetero) arylalkylene, (hetero) arylalkenylene, and (hetero) arylalkynylene optionally substituted with one or more substituents independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C5-C12Cycloalkenyl radical, C5-C12Cycloalkynyl group, C8-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy, halo (F, Cl, Br, I), amino, oxo, and silyl, wherein the silyl can be represented by the formula (R)11)3Si-represents, wherein R11As defined above;
wherein the functional group Q is selected from hydrogen, halogen (F, Cl, Br, I), R12、-CH＝C(R12)2、-C≡CR12Wherein q is 1 to 200- [ C (R)12)2C(R12)2O]q-R12、-CN、-N3、-NCX、-XCN、-XR12、-N(R12)2、-+N(R12)3、-C(X)N(R12)2、-C(R12)2XR12、-C(X)R12、-C(X)XR12、-S(O)R12、-S(O)2R12、-S(O)OR12、-S(O)2OR12、-S(O)N(R12)2、-S(O)2N(R12)2、-OS(O)R12、-OS(O)2R12、-OS(O)OR12、-OS(O)2OR12、-P(O)(R12)(OR12)、-P(O)(OR12)2、-OP(O)(OR12)2、-Si(R12)3、-XC(X)R12、-XC(X)XR12、-XC(X)N(R12)2、-N(R12)C(X)R12、-N(R12)C(X)XR12and-N (R)12)C(X)N(R12)2Wherein X is oxygen or sulfur, and wherein R12Independently selected from hydrogen, halogen (F, Cl, Br, I), C1-C24Alkyl radical, C6-C24(hetero) aryl, C7-C24Alkyl (hetero) aryl and C7-C24(hetero) arylalkyl.
7. The compound of claim 6, wherein L is absent or- [ C (R) wherein q is 1 to 20012)2C(R12)2O]q-R12、-CN、-NCX、-XCN、-XR12、-N(R12)2、-+N(R12)3、-C(X)N(R12)2、-C(R12)2XR12、-C(X)R12、-C(X)XR12、-S(O)R12、-S(O)2R12、-S(O)OR12、-S(O)2OR12、-S(O)N(R12)2、-S(O)2N(R12)2、-OS(O)R12、-OS(O)2R12、-OS(O)OR12、-OS(O)2OR12、-P(O)(R12)(OR12)、-P(O)(OR12)2、-OP(O)(OR12)2、-Si(R12)3、-XC(X)R12、-XC(X)XR12、-XC(X)N(R12)2、-N(R12)C(X)R12、-N(R12)C(X)XR12and-N (R)12)C(X)N(R12)2Wherein X is oxygen or sulfur, and wherein R12As defined in claim 6, and/or
Wherein Q is selected from-OR12、-N(R12)2、-+N(R12)3、-C(O)N(R12)2、-C(O)OR12、-OC(O)R12、-OC(O)OR12、-OC(O)N(R12)2、-N(R12)C(O)R12、-N(R12)C(O)OR12and-N (R)12)C(O)N(R12)2Wherein R is12As defined in claim 6.
8. The compound of claim 6 or 7, wherein the functional group Q is coupled to one or more of a drug, an antibody, a protein, a peptide, a ligand, an imaging label, a targeting ligand, a delivery agent, a nanoparticle, a carrier.
9. The compound of any one of claims 6 to 8, consisting of a compound of formula (I) coupled to an optional linking group (L) and a functional group (Q) to yield a compound of formula (III):
(formula I) -L-Q
(III)，
Wherein
Formula (I) is coupled at atom Y to an optional linking group (L) and a functional group (Q),
m and n are both 1 and n are,
x is a group selected from the group consisting of O,
R1、R2、R3、R4are the same and are selected from H and C1-C4An alkyl group, a carboxyl group,
R5、R6、R7、R8are the same and are selected from H and C1-C4An alkyl group, a carboxyl group,
y is NR10Wherein R is10Is replaced by L-Q, and then,
l is absent or is a chain of 1-25 atoms, preferably 1-15 atoms in length, and comprises one or more, preferably 1-6, independently selected from-S (O)2A moiety of-, -S-, -S-S-, -C (O) NH-, -NHC (O) -, -C (O) O-, and phenylene, wherein the chain length is determined by the number of atoms in the longest linear chain of atoms, and
q includes alcohols, amines, thiols, carboxylates, carboxylic acids or activated esters, ketones, aldehydes, nitriles, maleimides, alkenes, alkynes, heteroaromates, leaving groups and phosphoramidites.
10. A compound comprising a compound according to any one of claims 6 to 9, wherein functional group Q is reactive with a target molecule, preferably selected from the group consisting of a drug, an antibody, a protein, a peptide, a ligand, an imaging label, a targeting ligand, a delivery agent, a nanoparticle and a carrier.
11. Compound comprising a compound according to any one of claims 1 to 10 coupled to a 1, 3-dipole or 1,3- (hetero) diene containing compound, wherein the alkynyl group of the thiocycloheptyne of formula (I) is coupled to the 1, 3-dipole or 1,3- (hetero) diene containing compound, wherein preferably the 1, 3-dipole or 1,3- (hetero) diene containing compound is an azide containing compound, a nitrone containing compound or a nitrile oxide containing compound, more preferably an azide containing compound, and azide-alkyne coupling preferably results in the formation of a triazole compound, preferably wherein the 1, 3-dipole or 1,3- (hetero) diene containing compound comprises a drug, an antibody, a protein, a peptide, One or more of a peptide, a ligand, an imaging label, a targeting ligand, a delivery agent, a nanoparticle, and a carrier.
12. A method for coupling a compound according to claims 1 to 10 with a 1, 3-dipole or 1,3- (hetero) diene containing compound, preferably wherein the 1, 3-dipole or 1,3- (hetero) diene containing compound is an azide containing compound, a nitrone containing compound or a nitrile oxide containing compound, preferably an azide containing compound, preferably wherein the coupling is a copper free click reaction, and preferably wherein the 1, 3-dipole or 1,3- (hetero) diene containing compound comprises one or more of a drug, an antibody, a protein, a peptide, a ligand, an imaging label, a targeting ligand, a delivery agent, a nanoparticle, a carrier.
13. A method for preparing a construct comprising a nanoparticle and an active compound, preferably wherein the nanoparticle is a self-assembling polymeric micelle, preferably from a thermosensitive block copolymer; the active compound is selected from the group consisting of a drug, an antibody, a protein, a peptide, a ligand, an imaging label, a targeting ligand, a delivery agent, a nanoparticle and a carrier, wherein coupling of the nanoparticle and the active compound comprises coupling a compound of claims 1 to 8 with an azide-containing compound, preferably to form a triazole compound, wherein the nanoparticle is the azide-containing compound and the active compound comprises the compound of claims 1 to 8, or wherein the nanoparticle comprises the compound of claims 1 to 8 and the active compound comprises the azide-containing compound.
14. A process for the preparation of a compound of formula (I) wherein X is O and Y is NH, m and n are both 1, R1-R4Is methyl and R5-R8Is hydrogen, the process comprising the steps of:
a. conversion of the bishydrazone (2) to the Iminodidehydrosulfonylimino (3)
b. The resulting iminodidehydrosulfonylimino (3) is isolated.
15. The method of claim 13, wherein the conversion comprises an oxidation reaction in which the dihydrazone (2) reacts with an oxidizing agent to oxidize a sulfur functional group to a sulfonylimino functional group, preferably wherein the conversion further comprises an oxidation reaction in which the dihydrazone (2) reacts with an oxidizing agent to oxidize a hydrazone functional group to an alkyne functional group.
16. The process as set forth in claim 14 wherein the oxidation is a two-step reaction, preferably wherein the oxidation is a one-pot reaction with an oxidizing agent, preferably iodobenzene diacetate.
17. Use of a compound of claims 1 to 9 in a method for coupling two target molecules, wherein optionally the molecules are independently selected from the group consisting of drugs, antibodies, proteins, peptides, ligands, imaging labels, targeting ligands, delivery agents, nanoparticles and carriers.
18. Use of a compound according to claims 1 to 9 in a bio-orthogonal, optionally copper-free, click reaction.
19. Use of a compound of claims 1 to 9 in a method for coupling a nanoparticle to one or more of a drug, an antibody, a protein, a peptide, a ligand, an imaging label, a targeting ligand, a delivery agent, a nanoparticle, and a carrier using a copper-free click reaction.
Technical Field
The invention relates to novel thiacycloheptyne derivatives, in particular to thiacycloalkyne sulfimide derivatives and synthesis thereof. The present invention also relates to the use of the novel thiocycloheptyne derivatives in various coupling reactions, e.g., coupling reactions involving linkers, drug and drug delivery systems, proteins, imaging agents, dyes, chromophores, ligands, and the like. The invention also relates to the use of the novel thiocycloheptynes in copper-free click reactions. The present invention also relates to the use of novel thiocycloheptyne derivatives in the production of various systems, e.g., targeted and/or labeled delivery systems, such as nanoparticles, proteins, hydrogels, liposomes, antibody-drug conjugates, drug polymer conjugates, and the like.
Background
Bio-orthogonal chemistry is used in the study of biomolecules and physiological processes. Bio-orthogonal chemistry may be used when other more conventional research tools or medical treatments are unavailable or inadequate. Generally, the protocol begins by labeling a target biomolecule in a cell or living organism with bio-orthogonal functional groups. Probe molecules with complementary functions are provided to the system, and bio-orthogonal chemical reactions deliver the probes specifically to the target of interest. Kinetic optimization and yield optimization (e.g. by making purification easier) are considered as key factors for the further development of this useful process. In the past, it has been found that the strain-promoted cycloaddition reaction of azide and cyclooctyne is a well-tolerated bio-orthogonal reaction. The tension-promoted cycloaddition reaction of azides and cyclooctynes, also known as Cu-free (or copper-free) click chemistry, was inspired by the classical work of Krebs and Kimling in the 70's of the 20 th century. They observed that cyclooctyne reacts readily with the same substrate at room temperature compared to the unactivated linear alkyne which undergoes 1, 3-dipolar cycloaddition with azide only at elevated temperature. The increased reactivity of cyclooctyne is due to the ring tension resulting from bond angle deformation of the alkyne.
Almeida et al have described a group of cyclooctynes which have been developed for years and used in bioorthogonal coupling reactions. Almeida investigated the effect of the endocyclic sulfur on cyclooctyne activity and synthesized a number of thiocyclines. The 3,3,6, 6-tetramethylthiepin (TMTH) was found to be more reactive in cycloaddition reactions than previous cyclooctyne compounds. King et al (Chem Commun 2102, 48, 9308) describe the reasonable stability of TMTH only when derivatized at the sulfur atom with benzyl bromide. It was also found that other derivatives of TMTH are difficult to synthesize and sulfur is difficult to further derivatize. Dommerthot et al (NATURE COMMUNICATIONS 2014, 5, 5378, Top Curr Chem (Z)2016, 374:16) show that TMTH is poorly stable and cannot be isolated in pure form. Li et al (Molecules, 2016, 21, 1393) show that TMTH is the fastest SPAAC reported to date, but cannot be equipped with markers that hinder the application to bioorthogonal reactions.
Taking advantage of the advantages of TMTH in copper-free click reactions, there is a need for more compounds for copper-free click reactions that are novel, readily available and reactive, and have at least the same reactivity in 1,3 dipolar cycloadditions to azides, nitrones and other 1,3 dipoles.
Disclosure of Invention
The present inventors have discovered a novel class of sulfur-containing cycloalkynes, particularly thiocycloheptynes. The cycloalkynes of the present invention form a novel class of compounds that can be used for a variety of purposes, one of which is their reactivity in copper-free click chemistry.
In a first aspect, the invention relates to compounds of formula (I)
Wherein:
n and m are independently 0, 1 or 2, and n + m is 2;
x is O or NR9，
Y is NR10，
R1、R2、R3、R4Independently selected from hydrogen, halogen (F, Cl, Br, I), O, N, P and S, C1-C24Alkyl radical, C6-C24(hetero) aryl, C7-C24Alkyl (hetero) aryl and C7-C24(hetero) arylalkyl, wherein O, N, P and S are further independently from hydrogen, halogen (F, Cl, Br, I), C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy coupling, said alkyl being optionally interrupted by one of more heteroatoms selected from O, N, P and S, wherein said alkyl, (hetero) aryl, alkyl (hetero) aryl and (hetero) arylalkyl are independently optionally substituted with one or more substituents independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy, halogen(F, Cl, Br, I), amino, oxo, and silyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy, and cycloalkoxy are optionally substituted, the alkyl, the alkoxy, the cycloalkyl, and the cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S, wherein the silyl is represented by formula (R, Cl, Br, I), wherein the alkyl, the alkoxy, the cycloalkyl, and the cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S11)3Si-represents, wherein R11Independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy and C3-C12Cycloalkoxy, wherein said alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy, and cycloalkoxy are optionally substituted, said alkyl, said alkoxy, said cycloalkyl, and said cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S;
R5、R6、R7、R8independently selected from hydrogen, halogen (F, Cl, Br, I), O, N, P and S, C1-C24Alkyl radical, C6-C24(hetero) aryl, C7-C24Alkyl (hetero) aryl and C7-C24(hetero) arylalkyl, wherein O, N, P and S are further independently from hydrogen, halogen (F, Cl, Br, I), C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy coupling, said alkyl being optionally interrupted by one of more heteroatoms selected from O, N, P and S, wherein said alkyl, (hetero) aryl, alkyl (hetero) aryl and (hetero) arylalkyl are independently optionally substituted with one or more substituents independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy, halogen (F, Cl, Br, I), amino, oxo and silyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkoxy are optionally substituted, the alkyl, the alkoxy, the cycloalkyl and the cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S, wherein the silyl is represented by formula (R)11)3Si-represents, wherein R11Independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy and C3-C12Cycloalkoxy, wherein said alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy, and cycloalkoxy are optionally substituted, said alkyl, said alkoxy, said cycloalkyl, and said cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S;
wherein, optionally, R1And R7、R1And R8、R2And R7、R2And R8、R3And R5、R3And R6、R4And R5And/or R4And R6Independently form a fused ring system, such as cycloalkyl-, cyclo (hetero) aryl-, cycloalkyl (hetero) aryl, -cyclo (hetero) arylalkyl systems,
wherein the alkyl groups of the fused ring system are optionally interrupted by one of more heteroatoms selected from O, N, P and S, wherein the alkyl, (hetero) aryl, alkyl (hetero) aryl and (hetero) arylalkyl groups of the fused ring system are independently optionally substituted with one or more substituents independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy, halogen, amino, oxo, and silyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy, and cycloalkoxy are optionally substituted, the alkyl, the alkoxy, the cycloalkyl, and the cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S, wherein the silyl is represented by formula (R)11)3Si-represents, wherein R11Independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy and C3-C12Cycloalkoxy, wherein said alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy, and cycloalkoxy are optionally substituted, said alkyl, said alkoxy, said cycloalkyl, and said cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S;
R9、R10independently selected from hydrogen, halogen (F, Cl, Br, I), R12、-CH＝C(R12)2、-C≡CR12、-[C(R12)2C(R12)2O]q-R12(wherein q is 1 to 200), -CN, -N3、-NCX、-XCN、-XR12、-N(R12)2、-+N(R12)3、-C(X)N(R12)2、-C(R12)2XR12、-C(X)R12、-C(X)XR12、-S(O)R12、-S(O)2R12、-S(O)OR12、-S(O)2OR12、-S(O)N(R12)2、-S(O)2N(R12)2、-OS(O)R12、-OS(O)2R12、-OS(O)OR12、-OS(O)2OR12、-P(O)(R12)(OR12)、-P(O)(OR12)2、-OP(O)(OR12)2、-Si(R12)3、-XC(X)R12、-XC(X)XR12、-XC(X)N(R12)2、-N(R12)C(X)R12、-N(R12)C(X)XR12and-N (R)12)C(X)N(R12)2Wherein X is oxygen or sulfur, and wherein R12Independently selected from hydrogen, halogen (F, Cl, Br, I), C1-C24Alkyl radical, C6-C24(hetero) aryl, C7-C24Alkyl (hetero) aryl and C7-C24(hetero) arylalkyl.
In a second aspect, the present invention relates to a process for the preparation of a compound of formula (I), said process comprising the steps of:
a. converting the dihydrazone (2) to an iminodidehydrosulfonylimino (3);
b. the resulting iminodidehydrosulfonylimino (3) is isolated.
In a third aspect, the present invention relates to compounds wherein one atom in the X and/or Y and/or the thiocycloheptyne ring adjacent to the S atom of the compound of formula (I) is independently coupled with an optional linking group (L) and a functional group (Q) to yield compounds of formula (III):
(formula I) -L-Q
(III)
Wherein the linking group (L) is absent or selected from linear or branched C1-C24Alkylene radical, C2-C24Alkenylene radical, C2-C24Alkynylene, C3-C24Cycloalkylene radical, C5-C24Cycloalkenylene group, C5-C24Cycloalkynylene, C7-C24Alkyl (hetero) arylene, C7-C24(hetero) arylalkylene radical, C5-C24(hetero) arylalkenylene, C9-C24(hetero) arylalkynylene, alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkyl (hetero) arylene, (hetero) arylalkyleneA group, (hetero) arylalkenylene and (hetero) arylalkynylene, which is optionally independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C5-C12Cycloalkenyl radical, C5-C12Cycloalkynyl group, C8-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy, halogen (F, Cl, Br, I), amino, oxo and silyl, wherein the silyl may be substituted by one or more substituents of formula (R)11)3Si-represents, wherein R11As defined above;
wherein the functional group Q is selected from hydrogen, halogen (F, Cl, Br, I), R12、-CH＝C(R12)2、-C≡CR12、-[C(R12)2C(R12)2O]q-R12(wherein q is 1 to 200), -CN, -N3、-NCX、-XCN、-XR12、-N(R12)2、-+N(R12)3、-C(X)N(R12)2、-C(R12)2XR12、-C(X)R12、-C(X)XR12、-S(O)R12、-S(O)2R12、-S(O)OR12、-S(O)2OR12、-S(O)N(R12)2、-S(O)2N(R12)2、-OS(O)R12、-OS(O)2R12、-OS(O)OR12、-OS(O)2OR12、-P(O)(R12)(OR12)、-P(O)(OR12)2、-OP(O)(OR12)2、-Si(R12)3、-XC(X)R12、-XC(X)XR12、-XC(X)N(R12)2、-N(R12)C(X)R12、-N(R12)C(X)XR12and-N (R)12)C(X)N(R12)2Wherein X is oxygen or sulfur, and wherein R12Independently selected from hydrogen, halogen (F, Cl, Br, I), C1-C24Alkyl radical, C6-C24(hetero) aryl, C7-C24Alkyl (hetero) aryl and C7-C24(hetero) arylalkyl.
In another aspect, the present invention provides compounds comprising a compound of formula (I) according to the present invention coupled with an optional linking group (L) and a functional group (Q) at atom X, Y and/or one atom in the thiocycloheptyne ring adjacent to the S atom, preferably Y, to yield compounds of formula (III):
(formula I) -L-Q
(III)，
Wherein L and Q are as defined herein above.
In another aspect, the invention relates to a method for coupling a compound of formula (I) or formula (III) with a compound comprising a 1, 3-dipole or a 1,3- (hetero) diene, preferably wherein the compound comprising a 1, 3-dipole or a 1,3- (hetero) diene is an azide-containing compound, a nitrone-containing compound or a nitrile oxide-containing compound, preferably an azide-containing compound. Coupling of the compounds of the invention with azides typically results in triazoles. Compounds comprising a 1, 3-dipole or 1,3- (hetero) diene preferably include drugs, antibodies, proteins, peptides, ligands, imaging labels, targeting ligands, delivery agents, nanoparticles, and/or carriers.
In another aspect, the present invention relates to a compound comprising a compound of formula (III) as defined herein, wherein the functional group Q is reactive with a target molecule, preferably said target molecule is selected from the group consisting of a drug, an antibody, a protein, a peptide, a ligand, an imaging label, a targeting ligand, a delivery agent, a nanoparticle and a carrier.
In another aspect, the present invention relates to a method for the preparation of various systems, e.g. targeted and/or labeled delivery systems, such as nanoparticles, proteins, hydrogels, liposomes, antibody-drug conjugates, drug polymer conjugates, etc., wherein a (functionalized) compound of formula (I) or formula (III) is coupled to an azide.
In another aspect, the invention relates to the use of a compound of formula (I) or formula (III) in a method for coupling two target molecules, more specifically, coupling a targeted and/or labeled delivery system (e.g., nanoparticle, protein, hydrogel, liposome, antibody-drug conjugate) to one or more of a drug, an antibody, a protein, a peptide, a ligand, an imaging label, a targeting ligand, a delivery agent, a nanoparticle, and a carrier using a copper-free click reaction.
Other aspects include methods for making a construct comprising a nanoparticle and an active compound selected from a drug, an antibody, a protein, a peptide, a ligand, an imaging label, a targeting ligand, a delivery agent, a nanoparticle, and a carrier, wherein coupling of the nanoparticle and the active compound comprises coupling a compound of formula I or formula III with an azide-containing compound to form a triazole compound.
In another aspect, the invention relates to the use of a compound according to the invention in a method for coupling two target molecules, wherein optionally said molecules are independently selected from the group consisting of a drug, an antibody, a protein, a peptide, a ligand, an imaging label, a targeting ligand, a delivery agent, a nanoparticle and a carrier. Preferably, one of the target molecules comprises a cycloalkyne according to the invention and the other target molecule is a compound comprising a 1, 3-dipole or a 1,3- (hetero) diene, preferably an azide-containing compound, a nitrone-containing compound or a nitrile oxide-containing compound, more preferably an azide-containing compound. Compounds comprising a 1, 3-dipole or 1,3- (hetero) diene preferably include drugs, antibodies, proteins, peptides, ligands, imaging labels, targeting ligands, delivery agents, nanoparticles, and/or carriers.
In another aspect, the invention relates to a compound of formula (I) as defined herein, wherein the alkynyl group is coupled to a 1, 3-dipole or 1,3- (hetero) diene-containing compound, wherein preferably said 1, 3-dipole or 1,3- (hetero) diene-containing compound is an azide-containing compound, a nitrone-containing compound or a nitrile oxide-containing compound, more preferably an azide-containing compound, and the azide-alkyne coupling preferably results in the formation of a triazole compound. The 1, 3-dipole or 1,3- (hetero) diene containing compound preferably comprises a drug, an antibody, a protein, a peptide, a ligand, an imaging label, a targeting ligand, a delivery agent, a nanoparticle and/or a carrier.
In another aspect, the present invention relates to a compound comprising a compound of formula (I) as defined herein, coupled to a compound comprising a 1, 3-dipole or a 1,3- (hetero) diene, wherein the alkynyl group of the thiocycloheptyne of formula (I) is coupled to a compound comprising a 1, 3-dipole or a 1,3- (hetero) diene, wherein preferably the compound comprising a 1, 3-dipole or a 1,3- (hetero) diene is an azide-containing compound, a nitrone-containing compound or a nitrile oxide-containing compound, more preferably an azide-containing compound, and the azide-alkyne coupling preferably results in the formation of a triazole compound. The 1, 3-dipole or 1,3- (hetero) diene containing compound preferably comprises a drug, an antibody, a protein, a peptide, a ligand, an imaging label, a targeting ligand, a delivery agent, a nanoparticle and/or a carrier.
In another aspect, the invention relates to the use of a compound according to the invention in a bio-orthogonal, optionally copper click-free reaction. Preferably, the compounds according to the invention are coupled with a compound comprising a 1, 3-dipole or a 1,3- (hetero) diene, preferably an azide-containing compound, a nitrone-containing compound or a nitrile oxide-containing compound, more preferably an azide-containing compound. The 1, 3-dipole or 1,3- (hetero) diene containing compound preferably comprises a drug, an antibody, a protein, a peptide, a ligand, an imaging label, a targeting ligand, a delivery agent, a nanoparticle and/or a carrier.
In another aspect, the invention relates to the use of a compound according to the invention in a method of coupling a nanoparticle to one or more of a drug, an antibody, a protein, a peptide, a ligand, an imaging label, a targeting ligand, a delivery agent, a nanoparticle and a carrier using a copper-free click reaction. Preferably, the nanoparticle comprises a cycloalkyne compound according to the invention and the drug, antibody, protein, peptide, ligand, imaging label, targeting ligand, delivery agent, nanoparticle or carrier comprises a 1, 3-dipole or a 1,3- (hetero) diene, preferably an azide, nitrone or nitrile oxide, more preferably an azide. Alternatively, the drug, antibody, protein, peptide, ligand, imaging label, targeting ligand, delivery agent, nanoparticle or carrier comprises a cycloalkyne compound according to the invention and the nanoparticle comprises a 1, 3-dipole or a 1,3- (hetero) diene, preferably an azide, a nitrone or a nitrile oxide, more preferably an azide.
Description of the drawings
FIG. 1: dihydrazone 2 analytical data
GCMS Agilent 6890N/column: rxi-5MS 20m, ID 180 μm, df 0.18 μm. Average velocity 50 cm/s/carrier gas: and (e) He. Initial temperature 100 ℃/initial time: 1.5 min/solvent delay: 1.3 min. Rate 75 ℃/min, final temperature: 250 ℃, final time: 4.5 min. The split ratio is 20: 1/sample introduction temperature: 250 ℃, injection volume: 1 μ l. And (3) detection: MSD (EI-positive)/detection temperature: 280 ℃/mass range: 50-550. And (3) detection: FID/detector temperature: at 300 ℃.
FIG. 2: dihydrazone 2 analytical data
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: 298.7411K. The scanning times are as follows: 16. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 3[ example 2 Compound 3] TMTHSI analytical data
Column: waters XSelect CSH C18(30X2.1mm 3.5. mu.). Flow rate: 1 ml/min; column temperature: at 25 ℃. Eluent A: 95% acetonitrile + 5% 10mM aqueous ammonium bicarbonate. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210nm and 220 nm). And (3) detection: PDA (210-320 nm). And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 4[ example 2 Compound 3] TMTHSI analytical data
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: 297.5603K. The scanning times are as follows: 16. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 5[ example 2 Compound 3] TMTHSI analytical data
Bruker Biospin GmbH; method of producing a composite materialPulse sequence: zgpg 30. Relaxation delay: and 8 s. Solvent: CDCl3. The temperature is 300.5658K. The scanning times are as follows: 1024. frequency: 100.622829328806 MHz. And (3) nucleus: 13C.
FIG. 6[ example 2 Compound 3] TMTHSI analytical data
Bruker Biospin GmbH; method pulse sequence: jmod. Relaxation delay: and 8 s. Solvent: CDCl3. Temperature: 301.3172K. The scanning times are as follows: 1024. the frequency is 100.622829802853 MHz. And (3) nucleus: 13C.
FIG. 7[ example 3 Compound 4] TMTHSI-Suc-NHS analytical data
Column: waters XSelect CSH C18(30X2.1mm, 3.5. mu.). Flow rate: 1 ml/min; column temperature: 35 ℃ is carried out. Eluent A: 0.1% formic acid in acetonitrile. Eluent B: 0.1% aqueous formic acid. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210nm and 220 nm). PDA (210-400nm) was detected. And (3) detection: MSD ESI pos/neg (mass range: 100-. And (3) detection: ELSD (Alltech 3300) gas flow rate 1.5ml/min, gas temperature 40 ℃.
FIG. 8[ example 3 Compound 4] TMTHSI-Suc-NHS analytical data.
Bruker BioSpin GmbH method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: 298.2043K. The scanning times are as follows: 16. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 9[ example 3 Compound 4] TMTHSI-Suc-NHS analytical data
Column: waters XSelect CSH C18(30X2.1mm 3.5. mu.). Flow rate: 1 ml/min; column temperature: at 25 ℃. Eluent A: and (3) acetonitrile. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient t ═ 0min 5% a, t ═ 1.6min 98% a, t ═ 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210-220 nm). And (3) detection: PDA (210-320 nm). And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 10[ example 3 Compound 4] TMTHSI-Suc-NHS analytical data
Bruker BioSpin GmbH method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature:298.5264K. The scanning times are as follows: 64. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 11[ example 5 Compound 6] PoC TMTHSI analytical data
Column: waters XSelect CSH C18(30X2.1mm 3.5. mu.). Flow rate: 1 ml/min; column temperature: at 25 ℃. Eluent A: 95% acetonitrile + 5% 10mM ammonium bicarbonate in water. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210nm and 220nm) detects PDA (210-320 nm). And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 12[ example 5 Compound 6] PoC TMTHSI analytical data
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: 297.6677K. The scanning times are as follows: 16. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 13[ example 6 Compound 9] PoC TMTHSI-Suc-NHBn analytical data
Column: waters XSelect CSH C18(30X2.1mm, 3.5. mu.). Flow rate: 1 ml/min; column temperature: 35 ℃ is carried out. Eluent A: 0.1% formic acid in acetonitrile. Eluent B: 0.1% aqueous formic acid. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. DAD (220-320nm, 210nm and 220nm) was detected. And (3) detection: PDA (210-400 nm). And (3) detection: MSD ESI pos/neg (mass range: 100-. And (3) detection: ELSD (Alltech 3300) gas flow rate: 1.5ml/min, gas temperature: at 40 ℃.
FIG. 14[ example 6 Compound 9] PoC TMTHSI-Suc-NHBn analytical data
Column: waters XSelect CSH C18(30X2.1mm 3.5. mu.). Flow rate: 1 ml/min; column temperature: at 25 ℃. Eluent A: and (3) acetonitrile. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210nm and 220 nm). And (3) detection: PDA (210-320 nm). And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 15[ example 6 Compound 9] PoC TMTHSI-Suc-NHBn analytical data
Bruker BioSpin GmbH method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: 298.097K. The scanning times are as follows: 16. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 16[ example 8 Compound 8] TMTHSI-HER2 analytical data
Column: waters XSelect CSH C18(50X2.1mm 3.5. mu.). Flow rate: 0.8 ml/min; column temperature: at 25 ℃. Eluent A: and (3) acetonitrile. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient: t is 0min 5% a, t is 3.5min 98% a, t is 3min 98% a. Post-run time: and 2 min. DAD (220-320nm, 210nm and 220nm) was detected. PDA (210-320nm) was detected. And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 17: FIG. 17A comparison of the reaction kinetics of TMTHSI with BCN-OH. TMTHSI (upper curve) is significantly faster than BCN-OH (lower curve). FIG. 17B magnification of measurement of TMTHSI.
FIG. 18: [ example 11 Compound 11]N1- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -N4- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiepin-1-ylidene) succinamide, LCMS and mass spectral data.
Column: waters XSelect CSH C18(50X2.1mm 3.5. mu.). Flow rate: 1 ml/min; column temperature: at 25 ℃. Eluent A: and (3) acetonitrile. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient: t is 0min 5% a, t is 3.5min 98% a, t is 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210nm and 220 nm). PDA (210-320nm) was detected. And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 19: [ example 11 Compound 11]N1- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -N4- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiepin-1-ylidene) succinamide.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: 298.9557K. The scanning times are as follows: 16. the frequency is 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 20: [ example 12 Compound 12](3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiepin-1-ylidene) LCMS and mass spectral data for 2, 5-dioxopyrrolidin-1-yl carbamate.
Column: waters XSelect CSH C18(50X2.1mm 3.5. mu.). Flow rate: 1 ml/min; column temperature: at 25 ℃. Eluent A: and (3) acetonitrile. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210nm and 220 nm). And (3) detection: PDA (210-320 nm). And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 21: [ example 12 Compound 12](3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiepin-1-ylidene) carbamic acid 2, 5-dioxopyrrolidin-1-yl ester.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: 298.8484K. The scanning times are as follows: 16. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 22: [ example 14 Compound 14]1- (2- (2-hydroxyethoxy) ethyl) -3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6LCMS and mass spectral data for thiepin-1-ylidene) urea.
Column: waters XSelect CSH C18(50X2.1mm 3.5. mu.). Flow rate: 1 ml/min; column temperature: at 25 ℃. Eluent A: and (3) acetonitrile. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210nm and 220 nm). And (3) detection: PDA (210-320 nm). And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 23: [ example 14 Compound 14]1- (2- (2-hydroxyethoxy) ethyl) -3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6NMR data for thiepin-1-ylidene) urea.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: 299.7071K. The scanning times are as follows:16. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 24: [ example 15 Compound 15]3- (N- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiepin-1-ylidene) sulfamoyl) benzoic acid methyl ester LCMS and mass spectral data.
Column: waters XSelect CSH C18(50X2.1mm 3.5. mu.). Flow rate: 1 ml/min; column temperature: at 25 ℃. Eluent A: and (3) acetonitrile. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210nm and 220 nm). And (3) detection: PDA (210-320 nm). And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 25: [ example 15 Compound 15]3- (N- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiepin-1-ylidene) sulfamoyl) benzoic acid methyl ester.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: 299.8145K. The scanning times are as follows: 16. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 26: [ example 16 Compound 16]3- (N- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiepin-1-ylidene) sulfamoyl) benzoic acid, LCMS and mass spectral data.
Column: waters XSelect CSH C18(50X2.1mm 3.5. mu.). Flow rate: 1 ml/min; column temperature: at 25 ℃. Eluent A: and (3) acetonitrile. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210nm and 220 nm). And (3) detection: PDA (210-320 nm). And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 27 is a schematic view showing: [ example 16 Compound 16]3- (N- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiepin-1-ylidene) sulfamoyl) benzoic acid.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature ofDegree: 300.1365K. The scanning times are as follows: 16. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 28: [ example 17 Compound 17]3,3,6, 6-tetramethyl-1- (methylimino) -4, 5-didehydro-2, 3,6, 7-tetrahydro-1H-1 lambda6LCMS and Mass Spectroscopy data for thiepin 1-oxide.
Column: waters XSelect CSH C18(50X2.1mm 3.5. mu.). Flow rate: 1 ml/min; column temperature: at 25 ℃. Eluent A: and (3) acetonitrile. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210nm and 220 nm). And (3) detection: PDA (210-320 nm). And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 29: [ example 17 Compound 17]3,3,6, 6-tetramethyl-1- (methylimino) -4, 5-didehydro-2, 3,6, 7-tetrahydro-1H-1 lambda6NMR data of thiepin 1-oxide.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: 300.1365K. The scanning times are as follows: 16. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 30: [ example 18 Compound 18]4- ((3,3,6, 6-tetramethyl-1- (methylimino) -1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1H-1. lambda6-thiepin-2-yl) methyl) benzoate and mass spectral data.
Column: waters XSelect CSH C18(50X2.1mm 3.5. mu.). Flow rate: 1 ml/min; column temperature: at 25 ℃. Eluent A: and (3) acetonitrile. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210nm and 220 nm). And (3) detection: PDA (210-320 nm). And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 31: [ example 18 Compound 18]4- ((3,3,6, 6-tetramethyl-1- (methylimino) -1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1H-1. lambda6-thiepin-2-yl) methyl) benzoate.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent:CDCl3. Temperature: 299.9218K. The scanning times are as follows: 16. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 32: [ example 19 Compound 19]4- (((3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiepin-1-ylidene) amino) methyl) benzoate and mass spectral data.
Column: waters XSelect CSH C18(50X2.1mm 3.5. mu.). Flow rate: 1 ml/min; column temperature: at 25 ℃. Eluent A: and (3) acetonitrile. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210nm and 220 nm). And (3) detection: PDA (210-320 nm). And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 33: [ example 19 Compound 19]4- (((3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiepin-1-ylidene) amino) methyl) benzoate.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: 299.9218K. The scanning times are as follows: 16. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 34: [ example 13 Compound 13]2,2, 2-trifluoro-N- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1 lambda6LCMS and mass spectral data of-thiepin-1-ylidene) acetamide.
Column: waters XSelect CSH C18(50X2.1mm 3.5. mu.). Flow rate: 1 ml/min; column temperature: at 25 ℃. Eluent A: and (3) acetonitrile. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210nm and 220 nm). And (3) detection: PDA (210-320 nm). And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 35: [ example 13 Compound 13]2,2, 2-trifluoro-N- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1 lambda6NMR data for thiepin-1-ylidene) acetamide.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation ofDelaying the relaxation: 1 s. Solvent: CDCl3. Temperature: 299.1845K. The scanning times are as follows: 16. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 36: LCMS and Mass Spectroscopy data for tert-butyl [ example 20 Compound 21] (2- ((2-aminoethyl) disulfanyl) ethyl) carbamate.
Column: waters XSelect CSH C18(50X2.1mm 3.5. mu.). Flow rate: 1 ml/min; column temperature: at 25 ℃. Eluent A: and (3) acetonitrile. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210nm and 220 nm). And (3) detection: PDA (210-320 nm). And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 37: [ NMR data for tert-butyl 2- ((2-aminoethyl) disulfanyl) ethyl) carbamate, Compound 21 of example 20.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: 295.7355K. The scanning times are as follows: 16. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 38: [ example 21 Compound 22] LCMS and Mass Spectrometry data for 2, 2-dimethyl-4, 13-dioxo-3-oxa-8, 9-dithia-5, 12-diazahexadecane-16-oic acid.
Column: waters XSelect CSH C18(30X2.1mm, 3.5. mu.). Flow rate: 1 ml/min; column temperature: 35 ℃ is carried out. Eluent A: 0.1% formic acid in acetonitrile. Eluent B: 0.1% aqueous formic acid. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. DAD (220-320nm, 210nm and 220nm) was detected. And (3) detection: PDA (210-400 nm). And (3) detection: MSD ESI pos/neg (mass range: 100-. And (3) detection: ELSD (Alltech 3300) gas flow rate: 1.5ml/min, gas temperature: at 40 ℃.
FIG. 39: [ example 21 Compound 22] NMR data for 2, 2-dimethyl-4, 13-dioxo-3-oxa-8, 9-dithia-5, 12-diazahexadecane-16-oic acid.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: 295.6282K. The scanning times are as follows: 16. Frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 40: [ example 22 Compound 23] NMR data for 4- ((2- ((2-aminoethyl) disulfanyl) ethyl) amino) -4-oxobutanoic acid trifluoroacetate.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: and (4) MeOD. Temperature: 298.097K. The scanning times are as follows: 16. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 41: [ example 23 Compound 24]4-oxo-4- ((2- ((2- (3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda.))6LCMS and mass spectral data for-thiepin-1-ylidene) ureido) ethyl) disulfanyl) ethyl) amino) butyric acid.
Column: waters XSelect CSH C18(50X2.1mm 3.5. mu.). Flow rate: 1 ml/min; column temperature: at 25 ℃. Eluent A: and (3) acetonitrile. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210nm and 220 nm). And (3) detection: PDA (210-320 nm). And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 42: [ example 23 Compound 24]4-oxo-4- ((2- ((2- (3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda.))6-thiepin-1-ylidene) ureido) ethyl) disulfanyl) ethyl) amino) butanoic acid.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: 298.097K. The scanning times are as follows: 16. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 43: [ example 24 Compound 25]4-oxo-4- ((2- ((2- (3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda.))6LCMS and mass spectral data for thiepin-1-ylidene) ureido) ethyl) disulfanyl) ethyl) amino) butanoic acid 2, 5-dioxopyrrolidin-1-yl ester.
Column: waters XSelect CSH C18(30X2.1mm, 3.5. mu.). Flow rate: 1 ml/min; column temperature: 35 ℃ is carried out. Eluent A: 0.1% formic acid in acetonitrile. Eluent B: 0.1% aqueous formic acid. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. DAD (220-320nm, 210nm and 220nm) was detected. And (3) detection: PDA (210-400 nm). And (3) detection: MSD ESI pos/neg (mass range: 100-. And (3) detection: ELSD (Alltech 3300) gas flow rate: 1.5ml/min, gas temperature: at 40 ℃.
FIG. 44: [ example 24 Compound 25]4-oxo-4- ((2- ((2- (3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda.))6NMR data for thiepin-1-ylidene) ureido) ethyl) disulfanyl) ethyl) amino) butanoic acid 2, 5-dioxopyrrolidin-1-yl ester.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: CDCl3. Temperature: 298.097K. The scanning times are as follows: 16. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 45: [ example 29 Compound 30] (S) -15- (4- (((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) amino) benzoylamino) -2, 2-dimethyl-4, 12-dioxo-3, 8-dioxa-5, 11-diazahexadecane-16-oic acid LCMS and mass spectrometry data.
Column: waters XSelect CSH C18(50X2.1mm 3.5. mu.). Flow rate: 1 ml/min; column temperature: at 25 ℃. Eluent A: and (3) acetonitrile. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210nm and 220 nm). And (3) detection: PDA (210-320 nm). And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 46: [ example 29 Compound 30] (S) -15- (4- (((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) amino) benzoylamino) -2, 2-dimethyl-4, 12-dioxo-3, 8-dioxa-5, 11-diazahexadecane-16-oic acid NMR data.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: DMSO-d6. Temperature: 295.7355K. The scanning times are as follows: 64. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 47: [ example 30 Compound 31]N2- (4- (((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) amino) benzoyl) -N5- (2- (2-Ammonia)Ylethoxy) ethyl) -L-glutamine.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: DMSO-d6. Temperature: 298.097K. The scanning times are as follows: 64. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 48: [ Compound 32 of example 31]N2- (4- (((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) amino) benzoyl) -N5- (2- (2- (3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda.))6LCMS and mass spectral data for-thiepin-1-ylidene) ureido) ethoxy) ethyl) -L-glutamine.
Column: waters XSelect CSH C18(50X2.1mm 3.5. mu.). Flow rate: 1 ml/min; column temperature: at 25 ℃. Eluent A: and (3) acetonitrile. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210nm and 220 nm). And (3) detection: PDA (210-320 nm). And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 49: [ Compound 32 of example 31]N2- (4- (((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) amino) benzoyl) -N5- (2- (2- (3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda.))6NMR data for thiepin-1-ylidene) ureido) ethoxy) ethyl) -L-glutamine.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: DMSO-d6. Temperature: 298.097K. The scanning times are as follows: 128. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 50: [ example 33 Compound 34]1- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6LCMS and mass spectral data for thiepin-1-ylidene) urea.
Column: waters XSelect CSH C18(50X2.1mm 3.5. mu.). Flow rate: 1 ml/min; column temperature: at 25 ℃. Eluent A: and (3) acetonitrile. Eluent B: 10mM ammonium bicarbonate in water. Lin, gradient: t is 0min 5% a, t is 1.6min 98% a, t is 3min 98% a. Post-run time: 1.3 min. And (3) detection: DAD (220-320nm, 210nm and 220 nm). And (3) detection: PDA (210-320 nm). And (3) detection: MSD ESI pos/neg (mass range 100-.
FIG. 51: [ example 33 Compound 34]1- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6NMR data for thiepin-1-ylidene) urea.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: DMSO-d6. Temperature: 297.86K. The scanning times are as follows: 128. frequency: 400.232471584084 MHz. And (3) nucleus: 1H.
FIG. 52: [ example 34 Compound 35]1- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6LCMS and mass spectral data of the formate salt of the-thiepin-1-ylidene) urea-Cy 7 adduct.
Column: waters XSelect CSH C18(50X2.1mm, 3.5. mu.). Flow rate: 0.8 ml/min; column temperature: 35 ℃ is carried out. Eluent A: 0.1% formic acid in acetonitrile. Eluent B: 0.1% aqueous formic acid. Lin, gradient: t is 0min 5% a, t is 3.5min 98% a, t is 6min 98% a. Post-run time: and 2 min. DAD (220-320nm, 210nm and 220nm) was detected. And (3) detection: PDA (210 and 800 nm). And (3) detection: MSD ESI pos/neg (mass range: 100-. And (3) detection: ELSD (Alltech 3300) gas flow rate: 1.5ml/min, gas temperature: at 40 ℃.
FIG. 53: [ example 34 Compound 35]1- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6NMR data for formate salt of the-thiepin-1-ylidene) urea-Cy 7 adduct.
Bruker Biospin GmbH; method pulse sequence: zg 30. Relaxation delay: 1 s. Solvent: and (4) MeOD. Temperature: 296.1649K. The scanning times are as follows: 64. frequency: 400.132470966543 MHz. And (3) nucleus: 1H.
FIG. 54: 1-imino-3, 3,6, 6-tetramethyl-4, 5-didehydro-2, 3,6, 7-tetrahydro-1H-1 lambda produced according to the invention6General description of thiepin 1-oxide (TMTHSI) derivatives.
FIG. 55: reaction kinetics of TMTHSI with benzyl azide to determine the reaction rate constant value kt.
FIG. 56: the following UPLC chromatograms:
A) siRNA PLK1 oligonucleotide reference
B) Amino-modified siRNA PLK1 was completely converted to the tmphsi functionalized siRNA PLK1 oligonucleotide, example 25, compound 26
C) Complete conversion of TMTHSI-functionalized siRNA oligonucleotides in reaction with azide-functionalized CPP1 peptide, resulting in CPP1-siRNA PLK1 conjugate, example 26, Compound 27
D) Complete conversion of the acid-labile linker NHS ester in reaction with amine-CPP 1-siRNA conjugate yielded the L7-CPP1-siRNA PLK1 conjugate, example 27, compound 28.
Detailed Description
Definition of
The verb "to comprise" and its conjugations as used in this specification and claims is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
In addition, the modification of an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that one and only one of the elements be present. Thus, the indefinite article "a" or "an" usually means "at least one".
The compounds disclosed in the present specification and claims may be described as thiocycloheptyne compounds, i.e. cycloheptyne compounds in which a combination of sulfur and triple bonds are present in the ring structure. The triple bond of the cycloheptyne moiety may be located in any of three possible positions relative to sulfur (numbering according to the IUPAC organic chemistry nomenclature "rule a 31.2). The description of any cycloheptyne compound in this specification and claims is meant to include all three separate regioisomers of the cycloheptyne moiety.
The compounds disclosed in the specification and claims may contain one or more asymmetric centers and may exist in different diastereomers and/or enantiomers of the compound. The description of any compound in this specification and claims is meant to include all diastereomers and mixtures thereof, unless otherwise indicated. Furthermore, the description of any compound in this specification and claims is meant to include individual enantiomers, as well as any mixture of enantiomers (racemic or other mixtures), unless otherwise specified. When the structure of a compound is described as a specific enantiomer, it is understood that the invention of the present application is not limited to that specific enantiomer.
The compounds may exist in different tautomeric forms. The compounds according to the invention are meant to include all tautomeric forms, unless otherwise indicated.
The compounds disclosed in the present specification and claims may also exist as exo and endo regioisomers. Unless otherwise indicated, the description of any compound in the specification and claims is intended to include the individual exo and endo regioisomers of the compound, as well as mixtures thereof.
Furthermore, the compounds disclosed in the present specification and claims may exist as cis-isomer and trans-isomer. Unless otherwise indicated, the description of any compound in the specification and claims is intended to include the individual cis-isomer and the individual trans-isomer of the compound as well as mixtures thereof. As an example, when the structure of a compound is depicted as a cis isomer, it is understood that the corresponding trans isomer or a mixture of cis and trans isomers is not excluded from the invention of the present application.
Unsubstituted alkyl has the formula CnH2n+1And may be straight chain or branched. Unsubstituted alkyl groups may also contain cyclic moieties and thus have the accompanying formula CnH2n-1. Optionally, the alkyl is substituted with one or more substituents further specified herein. Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, propyl, 2-propyl, t-butyl, 1-hexyl, 1-dodecyl and the like.
Unsubstituted alkenyl radicalsHas the general formula CnH2n-1And may be straight chain or branched. Examples of suitable alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, pentenyl, decenyl, octadecenyl, eicosenyl, and the like. Unsubstituted alkenyl groups may also contain cyclic moieties and thus have the accompanying formula CnH2n-3。
The unsubstituted olefin has the formula CnH2nAnd unsubstituted alkynes have the general formula CnH2n-2。
Aryl groups contain at least six carbon atoms and may include monocyclic, bicyclic, and polycyclic structures. Optionally, the aryl group may be substituted with one or more substituents further specified herein. Examples of aryl groups include groups such as phenyl, naphthyl, anthracenyl, and the like.
Arylalkyl and alkylaryl groups contain at least seven carbon atoms and can include monocyclic and bicyclic ring structures. Optionally, the aryl group may be substituted with one or more substituents further specified herein. Arylalkyl is, for example, benzyl and the like. The alkylaryl group is, for example, 4-tert-butylphenyl and the like.
In the case where aryl is represented as (hetero) aryl, this notation is intended to include both aryl and heteroaryl. Similarly, alkyl (hetero) aryl is meant to include alkylaryl and alkylheteroaryl, and (hetero) arylalkyl is meant to include arylalkyl and heteroarylalkyl.
Heteroaryl groups contain one to four heteroatoms selected from oxygen, phosphorus, nitrogen and sulfur.
Compound (I)
Starting with the thiomeptyne compound of Almeida, the present inventors propose to find improved compounds that can be used in copper-free click reactions. TMTH itself was found to be unsuitable for practical use due to inherent instability (Dommerthot et al, Nature Communications5, particle number:5378 (2014)). Derivatization of the sulfur in the TMTH ring structure via direct alkylation has been described in King et al (chem.comm., 2012, 9308-9309). Other derivatizations were described as indirect, since King has observed that the ring tension should not be stretched beyond its limit to prevent reactivity of the alkyne bond. Furthermore, any derivatization should provide compounds suitable for reacting under aqueous conditions (high reactivity) and which are resistant to biological media in subsequent applications to produce suitable bio-orthogonal reagents.
The inventors have now surprisingly found that other derivatisations at sulphur are possible. The resulting compounds combine high reactivity in cycloaddition with good relative stability. Sulfonimides of the invention (>S (═ O) (═ NH) and/or sulfondiimine (S) (S) (═ O) (S) (-))) and/or sulfondiimine (S) ((>S(＝NR)2) Are a novel class of compounds that can be used as bio-orthogonal labels or conjugation agents. The sulfonimides and/or sulfondiimides of the invention can be readily derivatized with other functional groups and linkers using known chemistry, and can be further functionalized, for example, for bioorthogonal labeling, imaging, or modification (e.g., surface modification of target molecules). The compounds of the present invention may be conjugated to a variety of biologically active compounds and/or drug delivery systems.
The derivatives of TMTH and TMTH disclosed in King et al (chem. Comm., 2012, 9308-one 9309) have shown poor stability and cannot be used as such for further syntheses (Li, Molecules, 2016, 21, 1393, Krebs, Tet. Lett., 1970, 761-764). However, the TMTHSI compounds and derivatives according to the invention can be synthesized and isolated and are stable to both basic and acidic purification and can therefore be used for further synthesis. The TMTHSI and TMTHSI derivatives thereof according to the present invention have demonstrated extended shelf life over a year as shown in the examples. The TMTHSI and TMTHSI derivatives can be reacted with azides in acidic/neutral/basic aqueous environments and their combinations with organic solvents.
In a first aspect, the present invention therefore relates to compounds of formula (I)
Wherein:
n and m are independently 0, 1 or 2, and n + m is 2;
x is O or NR9，
Y is NR10，
R1、R2、R3、R4Independently selected from hydrogen, halogen (F, Cl, Br, I), O, N, P and S, C1-C24Alkyl radical, C6-C24(hetero) aryl, C7-C24Alkyl (hetero) aryl and C7-C24(hetero) arylalkyl, wherein O, N, P and S are further independently from hydrogen, halogen (F, Cl, Br, I), C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy coupling, said alkyl being optionally interrupted by one of more heteroatoms selected from O, N, P and S, wherein said alkyl, (hetero) aryl, alkyl (hetero) aryl and (hetero) arylalkyl are independently optionally substituted with one or more substituents independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy, halogen (F, Cl, Br, I), amino, oxo and silyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkoxy are optionally substituted, the alkyl, the alkoxy, the cycloalkyl and the cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S, wherein the silyl is represented by formula (R)11)3Si-represents, wherein R11Independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy and C3-C12Cycloalkoxy, wherein said alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy, and cycloalkoxy are optionally substituted, said alkyl, said alkoxy, said cycloalkyl, and said cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S;
R5、R6、R7、R8independently selected from hydrogen, halogen (F, Cl, Br, I), O, N, P and S, C1-C24Alkyl radical, C6-C24(hetero) aryl, C7-C24Alkyl (hetero) aryl and C7-C24(hetero) arylalkyl, wherein O, N, P and S are further independently from hydrogen, halogen (F, Cl, Br, I), C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy coupling, said alkyl being optionally interrupted by one of more heteroatoms selected from O, N, P and S, wherein said alkyl, (hetero) aryl, alkyl (hetero) aryl and (hetero) arylalkyl are independently optionally substituted with one or more substituents independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy, halogen (F, Cl, Br, I), amino, oxo and silyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy and cycloalkoxy are optionally substituted, the alkyl, the alkoxy, the cycloalkyl and the cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S, wherein the silyl is represented by formula (R)11)3Si-represents, wherein R11Independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy and C3-C12Cycloalkoxy, wherein said alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy, and cycloalkoxy are optionally substituted, said alkyl, said alkoxy, said cycloalkyl, and said cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S;
wherein, optionally, R1And R7、R1And R8、R2And R7、R2And R8、R3And R5、R3And R6、R4And R5And/or R4And R6Independently form a fused ring system, such as a cycloalkyl system, a cyclo (hetero) aryl system, a cycloalkyl (hetero) aryl system, a cyclo (hetero) arylalkyl system,
wherein the alkyl groups of the fused ring system are optionally interrupted by one of more heteroatoms selected from O, N, P and S, wherein the alkyl, (hetero) aryl, alkyl (hetero) aryl and (hetero) arylalkyl groups of the fused ring system are independently optionally substituted with one or more substituents independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy, halogen, amino, oxo, and silyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy, and cycloalkoxy are optionally substituted, the alkyl, the alkoxy, the cycloalkyl, and the cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S, wherein the silyl is represented by formula (R)11)3Si-represents, wherein R11Independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C1-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy and C3-C12Cycloalkoxy, wherein said alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkenyloxy, alkynyloxy, and cycloalkoxy are optionally substituted, said alkyl, said alkoxy, said cycloalkyl, and said cycloalkoxy are optionally interrupted by one of more heteroatoms selected from O, N, P and S;
R9、R10independently selected from hydrogen, halogen (F, Cl, Br, I), R12、-CH＝C(R12)2、-C≡CR12、-[C(R12)2C(R12)2O]q-R12(wherein q is 1 to 200), -CN, -N3、-NCX、-XCN、-XR12、-N(R12)2、-+N(R12)3、-C(X)N(R12)2、-C(R12)2XR12、-C(X)R12、-C(X)XR12、-S(O)R12、-S(O)2R12、-S(O)OR12、-S(O)2OR12、-S(O)N(R12)2、-S(O)2N(R12)2、-OS(O)R12、-OS(O)2R12、-OS(O)OR12、-OS(O)2OR12、-P(O)(R12)(OR12)、-P(O)(OR12)2、-OP(O)(OR12)2、-Si(R12)3、-XC(X)R12、-XC(X)XR12、-XC(X)N(R12)2、-N(R12)C(X)R12、-N(R12)C(X)XR12and-N (R)12)C(X)N(R12)2Wherein X is oxygen or sulfur, and wherein R12Independently selected from hydrogen, halogen (F, Cl, Br, I), C1-C24Alkyl radical, C6-C24(hetero) aryl, C7-C24Alkyl (hetero) aryl and C7-C24(hetero) arylalkyl.
In a preferred embodiment, the integers n and m are both 1, and the compound has formula (II):
R1-R8x, Y are as defined elsewhere herein. Preferably, the alkyne is located at the 4-5 position relative to sulfur (1-position). The resulting thiocycloheptyne is a symmetric compound having an axis of symmetry through the sulfur and alkyne bonds. Compared to known BCN compounds containing cis-trans isomerism and being chiral (E/Z), the current TMTHSI-compounds exhibit only E/Z isomerism, but remain the same. The use of symmetric thiocycloheptynes as click-reaction reagents in this application avoids mixtures of isomeric compounds with possible physicochemical or bio-variable behavior.
In a preferred embodiment, X is O. The resulting sulfonimides were successfully prepared with good stability and were successfully derivatized as shown in the examples.
In a preferred embodiment, R10Is H or comprises a radical selected from the group consisting of alcohols, amines, esters, C1-4Alkyl, carboxylic acid, trifluoroacetyl and n-hydroxysuccinimide (NHS) ester.
In a preferred embodiment, R10Is H. The resulting S (═ O) NH functionality is stable and has good reactivity at the NH position for further derivatization.
In a preferred embodiment, R1、R2、R3、R4Independently selected from H, halogen (F, Cl, Br, I) and C1-C4Alkyl, preferably methyl or ethyl, or R1And R2And/or R3And R4Form C3-C6Cycloalkyl, preferably cyclopropyl. In other preferred embodiments, R1、R2、R3、R4Independently selected from C1-C4Alkyl, preferably methyl or ethyl.
In other preferred embodiments, R1、R2、R3、R4Are the same and are selected from H, halogen and C1-C4Alkyl, or R1And R2And R3And R4Form C3-C6Cycloalkyl, preferably cyclopropyl. In other preferred embodiments, R1、R2、R3、R4Are the same and are selected from H, C1-C4Alkyl, preferably methyl or ethyl (preferably methyl), or R1And R2And R3And R4Forming a cyclopropyl group. In other preferred embodiments, R1、R2、R3、R4Are the same and are selected from C1-C4Alkyl, preferably methyl or ethyl, most preferably methyl.
In one embodiment, R1、R2、R3、R4、R5、R6、R7、R8Independently H, halogen (F, Cl, Br, I) or C1-C24Alkyl, and preferably independently is H or lower alkyl, i.e. C1-C4Alkyl, i.e. methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl or tert-butyl. In a particularly preferred embodiment, R1-R4Are the same lower alkyl groups. In a more preferred embodiment, R1-R4Are both methyl groups.
In a preferred embodiment, R5、R6、R7、R8Independently selected from H, halogen (F, Cl, Br, I) and C1-C4An alkyl group. More preferably, R5、R6、R7、R8Identical and selected from H, halogen (F, Cl, Br, I) and C1-C4Alkyl, preferably methyl or ethyl. More preferably, R5、R6、R7、R8Are the same and are selected from H and C1-C4Alkyl, preferably methyl or ethyl. In one embodiment, R5-R8Are all H.
In a particularly preferred embodiment, the compound of formula (I) is 1-imino-3, 3,6, 6-propan-3 of formula (III)Tetramethyl-4, 5-didehydro-2, 3,6, 7-tetrahydro-1H-1 lambda6-thiepin 1-oxide:
synthesis of
In another aspect, the present invention relates to the synthesis of the compounds of the present invention, and in particular to a process for the preparation of a compound of formula (I), said process comprising the steps of:
a. converting the dihydrazone (2) to an iminodidehydrosulfonylimino (3);
b. the resulting iminodidehydrosulfonylimino (3) is isolated.
The synthesis is based in part on the synthesis of TMTH by Almeida et al, using thiepinedione (thiophanate) 1 as an intermediate in the synthesis of TMTH, followed by the one-step conversion of dihydrazone 2 to sulfonimide 3 in the present invention.
The synthesis of diketo 1 and dihydrazone 2 has been described in detail in the literature [ Almeida et al ]. The synthesis starts with the conversion of diketone 1 to dihydrazone 2. The reaction is carried out in the presence of one or more hydrazine, preferably hydrazine sulfate and/or hydrazine monohydrate, and preferably under pressure.
The obtained bis-hydrazine 2 is converted to the target compound 3 in a two-step oxidation reaction. One step of the conversion involves an oxidation reaction (addition) in which the dihydrazone (2) is reacted with an oxidizing agent to react sulfur: (a)>S) oxidation of the functional group to a sulfonylimino group (S)>An S (═ O) (═ NR) functional group. Another step in the conversion is an oxidation reaction (elimination), in which the dihydrazone (2) is reacted with an oxidizing agent to react the (bis) hydrazine (bis)>C＝N-NH2)2The functional group is oxidized to an alkyne (-C ≡ C-) functional group. In a preferred embodiment, this two-step reaction is carried out in a one-step synthesis, preferably with the same oxidizing agent, preferably iodobenzene diacetate:
coupling of linker and functional group
In another aspect of the invention, a linker and/or functional group is coupled to a functionalized thiocycloheptyne of the invention. Thus, in another aspect, the present invention relates to compounds wherein one atom of the X and/or Y and/or the thiocycloheptyne ring of the compound of formula (I) adjacent to the S atom is independently coupled with an optional linking group (L) and a functional group (Q) to yield compounds of formula (III):
(formula I) -L-Q;
(III)
wherein the linking group (L) is absent or selected from linear or branched C1-C24Alkylene radical, C2-C24Alkenylene radical, C2-C24Alkynylene, C3-C24Cycloalkylene radical, C5-C24Cycloalkenylene group, C5-C24Cycloalkynylene, C7-C24Alkyl (hetero) arylene, C7-C24(hetero) arylalkylene radical, C5-C24(hetero) arylalkenylene, C9-C24(hetero) arylalkynylene, said alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkyl (hetero) arylene, (hetero) arylalkylene, (hetero) arylalkenylene, and (hetero) arylalkynylene optionally substituted with one or more substituents independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C5-C12Cycloalkenyl radical, C5-C12Cycloalkynyl group, C8-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy, halo (F, Cl, Br, I), amino, oxo, and silyl, wherein the silyl can be represented by the formula (R)11)3Si-represents, wherein R11As defined above;
officer thereinThe functional group Q is selected from hydrogen, halogen (F, Cl, Br, I), R12、-CH＝C(R12)2、-C≡CR12、-[C(R12)2C(R12)2O]q-R12(wherein q is 1 to 200), -CN, -N3、-NCX、-XCN、-XR12、-N(R12)2、-+N(R12)3、-C(X)N(R12)2、-C(R12)2XR12、-C(X)R12、-C(X)XR12、-S(O)R12、-S(O)2R12、-S(O)OR12、-S(O)2OR12、-S(O)N(R12)2、-S(O)2N(R12)2、-OS(O)R12、-OS(O)2R12、-OS(O)OR12、-OS(O)2OR12、-P(O)(R12)(OR12)、-P(O)(OR12)2、-OP(O)(OR12)2、-Si(R12)3、-XC(X)R12、-XC(X)XR12、-XC(X)N(R12)2、-N(R12)C(X)R12、-N(R12)C(X)XR12and-N (R)12)C(X)N(R12)2Wherein X is oxygen or sulfur, and wherein R12Independently selected from hydrogen, halogen (F, Cl, Br, I), C1-C24Alkyl radical, C6-C24(hetero) aryl, C7-C24Alkyl (hetero) aryl and C7-C24(hetero) arylalkyl. Preferably, R12Is hydrogen or C1-C6An alkyl group.
If the optional linking group (L) and the functional group (Q) are attached to an atom of the thiocycloheptyne ring adjacent to the S atom5、R6、R7Or R8One of which is replaced by an optional linking group (L) attached to the functional group (Q). R if the optional linking group (L) and the functional group (Q) are attached to X or Y, respectively9Or R10Each is replaced by an optional linking group (L) attached to the functional group (Q).
Preferably, the optional linking group (L) and functional group (Q) are attached to one of X or Y, preferably to Y, and/or to an atom of the thiocycloheptyne ring adjacent to the S atom. More preferably, the optional linking group (L) and functional group (Q) are attached to one of X or Y, preferably to Y.
In one embodiment, compounds are provided that constitute the following: the compounds of formula (I) according to the invention are coupled at atom X or Y, preferably Y and/or at one atom in the thiocycloheptyne ring adjacent to the S atom, with an optional linking group (L) and a functional group (Q) to give compounds of formula (III):
(formula I) -L-Q
(III)，
Wherein the linking group (L) is absent or selected from linear or branched C1-C24Alkylene radical, C2-C24Alkenylene radical, C2-C24Alkynylene, C3-C24Cycloalkylene radical, C5-C24Cycloalkenylene group, C5-C24Cycloalkynylene, C7-C24Alkyl (hetero) arylene, C7-C24(hetero) arylalkylene radical, C5-C24(hetero) arylalkenylene, C9-C24(hetero) arylalkynylene, said alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkyl (hetero) arylene, (hetero) arylalkylene, (hetero) arylalkenylene, and (hetero) arylalkynylene optionally substituted with one or more substituents independently selected from C1-C12Alkyl radical, C2-C12Alkenyl radical, C2-C12Alkynyl, C3-C12Cycloalkyl radical, C5-C12Cycloalkenyl radical, C5-C12Cycloalkynyl group, C8-C12Alkoxy radical, C2-C12Alkenyloxy radical, C2-C12Alkynyloxy, C3-C12Cycloalkoxy, halo (F, Cl, Br, I), amino, oxo, and silyl, wherein the silyl can be represented by the formula (R)11)3Si-represents, wherein R11As defined above;
wherein is functionallyThe group Q is selected from hydrogen, halogen (F, Cl, Br, I), R12、-CH＝C(R12)2、-C≡CR12、-[C(R12)2C(R12)2O]q-R12(wherein q is 1 to 200), -CN, -N3、-NCX、-XCN、-XR12、-N(R12)2、-+N(R12)3、-C(X)N(R12)2、-C(R12)2XR12、-C(X)R12、-C(X)XR12、-S(O)R12、-S(O)2R12、-S(O)OR12、-S(O)2OR12、-S(O)N(R12)2、-S(O)2N(R12)2、-OS(O)R12、-OS(O)2R12、-OS(O)OR12、-OS(O)2OR12、-P(O)(R12)(OR12)、-P(O)(OR12)2、-OP(O)(OR12)2、-Si(R12)3、-XC(X)R12、-XC(X)XR12、-XC(X)N(R12)2、-N(R12)C(X)R12、-N(R12)C(X)XR12and-N (R)12)C(X)N(R12)2Wherein X is oxygen or sulfur, and wherein R12Independently selected from hydrogen, halogen (F, Cl, Br, I), C1-C24Alkyl radical, C6-C24(hetero) aryl, C7-C24Alkyl (hetero) aryl and C7-C24(hetero) arylalkyl.
A variety of linkers can be used, and the choice of linker can be based on other criteria, such as criteria substantially related to the end use disclosed elsewhere herein.
In a preferred embodiment, L is absent or selected from- [ C (R)12)2C(R12)2O]q-R12(wherein q is 1 to 200), -CN, -NCX, -XCN, -XR12、-N(R12)2、-+N(R12)3、-C(X)N(R12)2、-C(R12)2XR12、-C(X)R12、-C(X)XR12、-S(O)R12、-S(O)2R12、-S(O)OR12、-S(O)2OR12、-S(O)N(R12)2、-S(O)2N(R12)2、-OS(O)R12、-OS(O)2R12、-OS(O)OR12、-OS(O)2OR12、-P(O)(R12)(OR12)、-P(O)(OR12)2、-OP(O)(OR12)2、-Si(R12)3、-XC(X)R12、-XC(X)XR12、-XC(X)N(R12)2、-N(R12)C(X)R12、-N(R12)C(X)XR12and-N (R)12)C(X)N(R12)2Wherein X is oxygen or sulfur, and wherein R12As defined elsewhere.
In other preferred embodiments, L is absent or is a chain of 1 to 25 atoms, preferably 1 to 15 atoms in length, and comprises one or more, preferably 1 to 6, independently selected from-S (O)2-, -S-, -S-, -C (O) NH-, -NHC (O) -, -C (O) O-and phenylene, whereby the chain length is determined by the number of atoms in the longest linear chain of atoms. The longest straight chain may contain one or more other heteroatoms, such as O, N, S and P. Preferably, it may contain one or more further O atoms. In preferred embodiments, L comprises 1,2, 3,4, or 5 substituents independently selected from-S (O)2-, -S-, -C (O) NH-, -NHC (O) -, -C (O) O-and phenylene. More preferably, said chain having a length of 1 to 25 atoms, preferably 1 to 15 atoms, is not branched, i.e. said chain is linear, optionally except for one or more parts not comprised in the linear chain (-S (O))2A heteroatom of-C (O) NH-, -NHC (O) -, -C (O) O-.
In one embodiment, Q is selected from-OR12、-N(R12)2、-+N(R12)3、-C(O)N(R12)2、-C(O)OR12、-OC(O)R12、-OC(O)OR12、-OC(O)N(R12)2、-N(R12)C(O)R12、-N(R12)C(O)OR12and-N (R)12)C(O)N(R12)2Wherein R is12As defined elsewhere herein.
In a preferred embodiment, Q comprises an alcohol, amine, thiol, carboxylate, carboxylic acid or activated ester, ketone, aldehyde, nitrile, maleimide, alkene, alkyne, heteroaromatic ester, leaving group, and phosphoramidite. In a particularly preferred embodiment, Q is an alcohol, amine, carboxylic acid ester, carboxylic acid or N-hydroxysuccinimide (NHS) ester.
In one embodiment, the coupling of the linker L and/or the functional group Q is at the functionalized sulfur of the thiocycloheptyne, i.e., at X and/or Y of the S (═ X) (═ Y) portion of the molecule. Coupling is performed with a functional group, optionally via a linker. The functional group Q may be attached to any target molecule.
In certain embodiments, Q may be linked to one or more of a drug, an antibody, a protein, a peptide, a ligand, an imaging label, a targeting ligand, a delivery agent, a nanoparticle, a carrier, or a combination thereof. The chemical reactions for such coupling are well developed and one skilled in the art can select an appropriate coupling chemistry to couple Q to the target molecule.
Click reaction
The alkynyl groups of the compounds of the invention are reactive and can be functionalized using, for example, a cycloaddition reaction. In embodiments, the alkynyl group may be reacted with a compound comprising a 1, 3-dipole or a 1,3- (hetero) diene. In certain embodiments, the compound comprising a 1, 3-dipole or 1,3- (hetero) diene is an azide-containing compound, a nitrone-containing compound, or a nitrile oxide-containing compound. Most preferred are azide-containing compounds. In embodiments, the azide-containing compound may be coupled using a copper-free click reaction. Coupling gives rise to triazole type structures.
The compounds of the present invention are bifunctional compounds having reactive sites at the functionalized sulfur sites and at the alkyne functional group. The two functional groups may be functionalized independently, one via the optional linker (L) and functional group (Q), and the other via click chemistry. The functionalization can be independently with drugs (e.g., small molecules, genetic material, etc.), antibodies, proteins, peptides, ligands, imaging labels, targeting ligands, delivery agents, nanoparticles, vectors.
Thus, in one embodiment, the functional group Q is optionally coupled to one or more of a drug, an antibody, a protein, a peptide, a ligand, an imaging label, a targeting ligand, a delivery agent, a nanoparticle, a carrier via a linker L.
In another embodiment, the alkyne can be coupled to one or more of a drug, an antibody, a protein, a peptide, a ligand, an imaging label, a targeting ligand, a delivery agent, a nanoparticle, a carrier. The coupling of the alkyne is preferably carried out via reaction of the alkyne with a compound comprising a 1, 3-dipole or a 1,3- (hetero) diene. In certain embodiments, the compound comprising a 1, 3-dipole or 1,3- (hetero) diene is an azide-containing compound, a nitrone-containing compound, or a nitrile oxide-containing compound, more preferably an azide-containing compound.
Thus, the compounds of the present invention are suitable for generating a combination of two target molecules/functional groups via a 1, 3-dipole/1, 3 (hetero) diene/thiepin derivative/(linker)/functional group. Schematically, the general principles of this application of the present thiepine are illustrated in the following table:
the linker used may be biodegradable, i.e. initially stable, but cleavable over time or under a particular environment (e.g. physiological environment).
In certain embodiments, combinations are preferred wherein the nanoparticle is coupled via an optional linker/1, 3-dipole or 1, 3-heterodiene/thiocycloheptyne/optional linker to a functional group/target molecule selected from a drug, an antibody, a protein, a peptide, a ligand, an imaging label, a targeting ligand, a delivery agent, a nanoparticle, a carrier. The nanoparticles themselves may include drugs, antibodies, proteins, peptides. The preferred 1, 3-dipole or 1, 3-heterodiene for coupling nanoparticles to the thiocycloheptyne derivatives of the present invention is azide (N3).
Nanoparticles
Drug delivery systems are increasingly used in pharmaceutical science. Through carefully designed derivatization, drugs can be delivered, targeted, monitored, and provided to patients with increased effectiveness and efficacy. The use of these drug delivery systems (e.g., nanoparticles, proteins, hydrogels, liposomes, antibody-drug conjugates, drug polymer conjugates) is rapidly evolving in pharmaceutical science.
Pharmaceutical science uses these systems to reduce toxicity and side effects of drugs, improve delivery and add imaging ligands to aid in monitoring of effectiveness.
Nanoparticles, typically with diameters <100nm, are made from a variety of materials, and the intended applications in medicine include drug delivery (including in vitro and in vivo diagnostics), nutraceuticals, and the production of improved biocompatible materials. Nanoparticles can be tailored for specific purposes and a wide variety of materials are available. Most drug target nanoparticles are based on (bio) polymer materials, which can be in various forms. The source material may be of biological origin (e.g. phospholipids, lipids, lactic acid, dextran, chitosan), or have more "chemical" properties (e.g. various (co) polymers). These nanoparticles are usually functionalized by linking, coupling, (covalently) binding of active ingredients (drugs, ligands, imaging ligands, etc.). The functionalization of many of these nanoparticles can be achieved by various chemical methods, such as copper-free click chemistry using the inventive thiocycloheptyne derivatives.
In certain embodiments, the nanoparticles are self-assembled polymeric micelles, preferably from thermosensitive block copolymers. In particular, copolymers based on PEG-b-poly (N-hydroxyalkyl methacrylamide-oligolactate) with partially methacrylated oligolactate units are preferred, but other (meth) acrylamide esters can also be used to construct thermosensitive blocks of HPMAm (hydroxypropyl methacrylamide) and HEMAm (hydroxyethyl methacrylamide) and N- (meth) acryloyl amino acid esters, such as esters and optionally (oligo) lactate. Also preferred are thermosensitive block copolymers derived from monomers containing functional groups that can be modified with derivatized and underivatized methacrylate groups, such as HPMAm-lactate polymers; that is, such modifications include the incorporation of linker moieties.
Other types of functional thermo-sensitive (co) polymers that can be used are hydrophobically modified poly (N-hydroxyalkyl) (meth) acrylamides, copolymer compositions of N-isopropylacrylamide (NIPAAm) with monomers containing reactive functional groups such as acidic acrylamides and other moieties such as N-acryloxysuccinimide, or similar copolymers of poly (alkyl) 2-oxazines, and the like. Also preferred thermosensitive groups may be based on NIPAAm and/or alkyl-2-oxazoline, which may be reacted with monomers containing reactive functional groups, such as (meth) acrylamide or (meth) acrylate containing hydroxyl, carboxyl, amine or succinimide groups.
Suitable heat-sensitive polymers are described in US-B-7,425,581 and EP-A-1776400. Furthermore, in WO2010/033022 and WO 2013/002636.
WO2012/039602 describes drug-polymer matrix particles using such polymers. Furthermore, in WO2012/039602, biodegradable linker molecules are described which can be used in these known polymer matrix particles.
Typically, nanoparticles based on thermosensitive block copolymers as outlined above can be attached to the compounds of the present invention using azide-alkyne copper-free coupling. Examples thereof are described in WO 2017086794.
Thus, in certain embodiments of the invention, nanoparticles are prepared in which the inventive thiocycloheptyne derivatives are coupled to azide-containing nanoparticles. In certain embodiments, the nanoparticles are self-assembled polymeric micelles, preferably from thermosensitive block copolymers.
The invention also relates to the use of the inventive thiocycloheptyne derivatives in a method of coupling two target molecules, wherein optionally the molecules are independently selected from the group consisting of drugs, antibodies, proteins, peptides, ligands, imaging labels, targeting ligands, delivery agents, nanoparticles, and carriers. The invention also relates to the use of the inventive thiocycloheptyne derivatives in bio-orthogonal, optionally copper-free click reactions, and in methods of coupling nanoparticles to one or more of drugs, antibodies, proteins, peptides, ligands, imaging labels, targeting ligands, delivery agents, nanoparticles, and carriers using copper-free click reactions. In particular, the coupling of azide-containing thermosensitive polymers with the compounds of the present invention is advantageous because the click chemistry between the alkyne-containing compounds of the present invention and azides is faster than conventional alkynes used in click chemistry, especially in the preparation of biodegradable nanoparticles and conjugated/embedded drugs.
For purposes of clarity and conciseness of description, features may be described herein as part of the same or separate aspects or embodiments of the invention. Those skilled in the art will appreciate that the scope of the present invention may include embodiments having a combination of all or some of the features described herein as being part of the same or separate embodiments.
The invention will be explained in more detail in the following non-limiting examples.
Examples
FIG. 54 provides 1-imino-3, 3,6, 6-tetramethyl-4, 5-didehydro-2, 3,6, 7-tetrahydro-1H-1 λ produced according to the invention6An overview of the thiepin 1-oxide (TMTHSI) derivatives, the synthesis of which is described in the examples below.
The core structure of the tmshsi (example 2) can be functionalized in various ways.
Example 2, X ═ O, Y ═ N, nuclear tmshsi structure, R1-R4 ═ methyl, R5-R8 ═ hydrogen
Example 3, X ═ O, Y ═ N, is example 2 where L ═ succinic acid linker, Q ═ activated NHS ester, R1-R4 ═ methyl, R5-R8 ═ hydrogen
Example 5 is example 3, where the NHS ester is replaced in the reaction with benzylamine, R1-R4 ═ methyl, R5-R8 ═ hydrogen
Example 8, X ═ O, Y ═ N, is example 3 where the NHS ester was replaced in the reaction with HER2 peptide, R1-R4 ═ methyl, R5-R8 ═ hydrogen
Example 11, X ═ O, Y ═ N, is example 3 where the NHS ester was replaced in the reaction with diamino-PEG spacer (═ L), Q is an amine, R1-R4 ═ methyl, R5-R8 ═ hydrogen
Example 12, X ═ O, Y ═ N,
R1-R4 ═ methyl, R5-R8 ═ hydrogen
Example 13, X ═ O, Y ═ N-trifluoroacetamide, L ═ no linker, and Q ═ no functional group. R1-R4 ═ methyl, R5-R8 ═ hydrogen. The compounds are intended for alpha-sulfur substitution
Example 14, X ═ O, Y ═ N, is example 12 where the NHS ester was replaced in the reaction with amino-PEG-hydroxy spacer (═ L), Q is hydroxy, R1-R4 ═ methyl, R5-R8 ═ hydrogen
Example 15, X ═ O, Y ═ N, is example 2 where L ═ S (O)2) Phenyl-, Q ═ 3-methoxycarbonyl, R1 to R4 ═ methyl, R5 to R8 ═ hydrogen
Example 16, X ═ O, Y ═ N, is example 2 where L ═ S (O)2) Phenyl, Q ═ 3-hydroxycarbonyl, R1 to R4 ═ methyl, R5 to R8 ═ hydrogen
Example 17, X ═ O, Y ═ N-methyl, L ═ no linker, and Q ═ no functional group. R1-R4 ═ methyl, R5-R8 ═ hydrogen. The compounds are intended for alpha-sulfur substitution
Example 18, X ═ O, Y ═ N-methyl, L ═ no linker, Q ═ no functional group. R1-R4 ═ methyl, R5-R8 ═ one hydrogen replaced by L ═ methylenephenyl-, Q ═ 4-methoxycarbonyl. The compounds are alpha-sulfur substituted variants of TMTHSI
Example 19 where X ═ O, Y ═ N, is example 2 where L ═ methylenephenyl-, Q ═ 4-methoxycarbonyl, R1-R4 ═ methyl, R5-R8 ═ hydrogen
Example 23, X ═ O, Y ═ N, is example 12, where the NHS moiety is replaced with a cystamine-based amine linker, L ═ -C (O) NHC2H4SSC2H4NHC (O) C3H6-, Q ═ COOH,
R1-R4 ═ methyl, R5-R8 ═ hydrogen
Example 24, X ═ O, Y ═ N, example 23 activated as an NHS ester, L ═ -C (O) NHC2H4SSC2H4NHC (O) C3H6-, Q ═ ((2, 5-dioxopyrrolidin-1-yl) oxy) carbonyl, C (O) OSu.
R1-R4 ═ methyl, R5-R8 ═ hydrogen
Example 25, X ═ O, Y ═ N, is example 24 where the NHS ester was replaced in the reaction with amino-C6 functionalized siRNA oligonucleotides, R1-R4 ═ methyl, R5-R8 ═ hydrogen
Example 31, X ═ O, Y ═ N, is example 12 where the NHS ester was replaced in the reaction with amino PEG-functionalized folic acid, R1-R4 ═ methyl, R5-R8 ═ hydrogen
Example 33, X ═ O, Y ═ N, is example 12 where the NHS ester was replaced in the reaction with diamino-PEG spacer (═ L), Q is an amine, R1-R4 ═ methyl, R5-R8 ═ hydrogen
Example 34, X ═ O, Y ═ N, is example 33 reacted with Cy7 NHS label. L-diamino-PEG spacer, R1-R4-methyl, R5-R8-hydrogen
One skilled in the art would be able to proficiently provide other derivatives having other variables of R1-R8, L, and/or Q, and coupled to other target molecules, such as drugs, antibodies, proteins, peptides, ligands, imaging labels, targeting ligands, delivery agents, nanoparticles, or carriers, using reactions known in the art, in addition to the structures prepared in the examples below and/or the structures shown in fig. 54.
Example 1: synthesis of (3,3,6, 6-tetramethylthiaheptane-4, 5-diylidene) bis (hydrazine).
A glass autoclave (300ml) was charged with 3,3,6, 6-tetramethylthiaheptane-4, 5-dione (6.17g, 30.8mmol), ethanol (2.4ml) and ethylene glycol (60 ml). To the stirred mixture was added hydrazine sulfate (16.03g, 123mmol, 4 equiv.) and hydrazine monohydrate (3ml, 61.6mmol, 2 equiv.). The autoclave was closed and heated in an oil bath at 140 ℃ for 20 hours.
After cooling to room temperature, the mixture was partitioned between water (500ml) and diethyl ether (200ml) and transferred to a separatory funnel while being filtered through a piece of cotton to remove some white solid residue (probably hydrazine sulfate, as it slowly dissolved in water). The layers were separated and the aqueous layer was extracted with diethyl ether (2X 100 ml). The combined ether layers were washed with water (2X 75ml) and brine (100ml) and dried over Na2SO4Dried and concentrated under reduced pressure to give 5.75g of crude product as a viscous yellow oil.
The residue was purified by flash column chromatography (silica 40 g; 30% EtOAc in heptane); the product fractions were combined and concentrated under reduced pressure. The residue was coevaporated with diethyl ether to give 0.9g (12%) of the product as an off-white solid.
GC/MS (method _ A) tR 4.47min，M+＝228。
*1H NMR (400MHz, chloroform-d) δ 5.24(s, 4H), 2.54(d, J ═ 14.4Hz, 2H), 2.48(d, J ═ 14.4Hz, 2H), 1.34(s, 6H), 1.21(s, 6H).
Example 2: 1-imino-3, 3,6, 6-tetramethyl-4, 5-didehydro-2, 3,6, 7-tetrahydro-1H-1 lambda6-synthesis of thiepin 1-oxide (TMTHSI).
An 8ml screw cap vial was loaded with (3,3,6, 6-tetramethylthiaheptane-4, 5-diylidene) bis (hydrazine) (100mg, 0.44mmol) and ammonium acetate (270mg, 3.5mmol, 8 equiv.) and suspended in methanol (0.4 ml). The mixture was cooled in ice/water. Iodobenzene diacetate (494mg, 1.53mmol, 3.5 equiv.) in a mixture of MeOH (0.4ml) and dichloromethane (0.6ml) was added dropwise to control the exotherm and gas formation (the reaction showed gas evolution upon addition of each drop). After the addition was complete, the mixture was stirred at room temperature for 1 hour.
The mixture was diluted with dichloromethane (2ml) and quenched with brine (1 ml). The organic layer was removed by pipette and passed through a phase separator. The aqueous residue was extracted with dichloromethane (2 ×), the organic phase being passed through a phase separator each time and combined with the previous extract. The organic extract was concentrated under reduced pressure and the evaporation was carefully monitored as the product may be volatile to give 427mg of the crude product as a yellow oil. The residue was passed through preparative RP-MPLC (Reveleria, XSelect; 10-50% MeCN in water, 10mM NH)4HCO3pH 9.5). The product fractions were combined and extracted with dichloromethane (3 ×), and each organic extract was passed through a phase separator and concentrated under reduced pressure. The residue was coevaporated with heptane and diethyl ether to give 33mg (37%) of a crystalline residue.
LC/MS(SC_BASE)tR1.628min, purity 98.1%, measured value of mass [ M + H ]]+200。
1H NMR (400MHz, chloroform-d) δ 3.26(d, J ═ 14.1Hz, 2H), 3.18(d, J ═ 14.1Hz, 2H), 2.76(s, 1H), 1.46(s, 6H), 1.30(s, 6H).
TMTHSI proved to be stable during storage. Compound 3 was stored as a powder in the light and dark locations at atmospheric pressure.1H-NMR and LC/MS (SC _ BASE) confirmed that the compound was still intact after 10 months of storage.
Example 3: is coupled to a linker. 4-oxo-4- ((3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiepin-1-ylidene) amino) butyric acid 2, 5-dioxopyrrolidin-1-yl ester.
Diisopropylethylamine (266 μ l, 1.52mmol, 2 equiv.) was added to 1-imino-3, 3,6, 6-tetramethyl-4, 5-didehydro-2, 3,6, 7-tetrahydro-1H-1. lambda6-thiepin 1-oxide (152mg, 0.76mmol) and succinic anhydride (114mg, 1.14mmol, 1.5 equiv.) in dichloromethane (5ml) and the resulting mixture was stirred at room temperature for 20 hours.
The reaction mixture was concentrated under reduced pressure and redissolved in dichloromethane and again concentrated under reduced pressure. The residue was redissolved in dichloromethane (5ml) and N-hydroxysuccinimide (220mg, 1.91mmol, 2.5 equivalents) was added. The mixture was stirred for 5 minutes, then a suspension of N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (366mg, 1.91mmol, 2.5 equivalents) in dichloromethane (0.5ml) was added via a pipette. The resulting solution was stirred at room temperature for 4 hours.
Mixing the mixture with 2M KHSO4The aqueous solution was quenched and the organic phase was passed through a phase separator. The filtrate was concentrated under reduced pressure to give 420mg of a crude mixture in the form of a foam.
The residue was purified by flash column chromatography (silica 12g, 50-75% EtOAc in heptane), the product fractions were pooled and concentrated under reduced pressure to give 141mg (46%) of the product as a white solid.
LC/MS(SC_ACID)tR1.84min, purity 85%, measured value of mass [ M + H ]]+397。
1H NMR (400MHz, chloroform-d) δ 3.79(d, J ═ 14.1Hz, 2H), 3.63(d, J ═ 14.2Hz, 2H), 2.97-2.90 (m, 2H), 2.88-2.81 (m, 4H), 2.79-2.71 (m, 2H), 1.55(s, 6H), 1.28(s, 6H).
Example 4: is coupled to a linker. N is a radical of1-benzyl-N4- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiepin-1-ylidene) succinamide synthesis.
To 4-oxo-4- ((3)3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6To a solution of thiepin-1-ylidene) amino) butanoic acid 2, 5-dioxopyrrolidin-1-yl ester (15.8mg, 0.040mmol) in dichloromethane (1ml) was added benzylamine (8.71. mu.l, 0.080mmol, 2 equiv.). The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was then purified by flash column chromatography (silica, 12 g; 70-100% EtOAc in heptane), the product fractions were combined and concentrated under reduced pressure to give 13.1mg of product with a purity of 83% by LC/MS.
By preparative RP-MPLC (Reveleries, XSelect; 20-60% MeCN in water, 10mM NH)4HCO3pH 9.5), the product fractions were pooled and concentrated under reduced pressure to yield 6.2mg (40%) of the title compound as a white solid.
LC/MS(SC_BASE)tR2.01min, purity 96.8%, measured value of mass [ M + H ]]+389。
1H NMR (400MHz, chloroform-d) δ 7.36-7.22 (m, 5H), 6.25(s, 1H), 4.44(d, J ═ 5.7Hz, 2H), 3.60(d, J ═ 14.1Hz, 2H), 3.53(d, J ═ 14.1Hz, 2H), 2.73(dd, J ═ 6.8, 6.2Hz, 2H), 2.52(dd, J ═ 6.6, 6.4Hz, 2H), 1.49(s, 6H), 1.24(s, 6H).
Example 5: and (4) click reaction. 1-benzyl-6-imino-4, 4,8, 8-tetramethyl-1, 4,5,6,7, 8-hexahydro-6. lambda4-thiaheptino [4,5-d][1,2,3]Synthesis of triazole 6-oxide.
LC/MS:
Loading of 1-imino-3, 3,6, 6-tetramethyl-4, 5-didehydro-2, 3,6, 7-tetrahydro-1H-1. lambda. into LC/MS vials6Thiepin 1-oxide (5mg, 0.025mmol) and dissolved in MeCN (1ml), benzyl azide was added and the reaction mixture was analyzed by LC/MS. LC/MS analysis showed complete consumption of starting material and formation of product as a mixture with unreacted benzyl azide.
LC/MS(SC_BASE)tR1.728min, purity 64%, measured value of mass [ M + H ]]+333。
1H-NMR:
1-imino-3, 3,6, 6-tetramethyl-4, 5-didehydro-2, 3,6, 7-tetrahydro-1H-1 lambda6Thiepinone 1-oxide (5mg, 0.025mmol) in CDCl3(0.6ml) and benzyl azide was added. Recording after about 15 minutes reaction time1H-NMR：
1H NMR (400MHz, chloroform-d) δ 7.40-7.29 (m, 3H), 7.06-7.01 (m, 2H), 5.73(s, 2H), 3.45(d, J ═ 8.9Hz, 4H), 2.69(s, 1H), 1.70(d, J ═ 2.1Hz, 6H), 1.44(d, J ═ 2.7Hz, 6H).
The starting material was completely consumed and there was-35 mol% excess benzyl azide.
Example 6: and (4) click reaction. N is a radical of1-benzyl-N4- (1-benzyl-4, 4,8, 8-tetramethyl-6-oxo-4, 5,7, 8-tetrahydro-1H-6. lambda4-thiaheptino [4,5-d][1,2,3]Triazol-6-ylidene) succinamide synthesis.
To the solution containing CDCl3N in (1)1-benzyl-N4- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6NMR tube of (4.6mg, 12. mu. mol) Thiepin-1-ylidene) succinamide with CDCl of benzylazide3Solution (10 vol%, 15.5. mu.L, 1.05 eq.). The mixture was shaken and allowed to react after about 5 minutes1H-NMR analysis.
1H NMR (400MHz, chloroform-d) δ 7.43-7.27 (m, 8H), 7.06-7.00 (m, 2H), 6.18(s, 1H), 5.72(s, 2H), 4.42(d, J ═ 5.8Hz, 2H), 3.96(d, J ═ 15.5Hz, 1H), 3.82(d, J ═ 15.0Hz, 1H), 3.70(d, J ═ 15.3Hz, 1H), 3.57(d, J ═ 15.1Hz, 1H), 2.74-2.67 (m, 2H), 2.49(t, J ═ 6.7Hz, 2H), 1.70(s, 3H), 1.67(s, 3H), 1.44(s, 3H), 1.36(s, 3H).
The mixture was concentrated to dryness and the residue was analyzed by LC/MS:
LC/MS (SC _ BASE) showed a peak of the product (t) with the target massR1.947min, purity 75%, [ M + H ]]+522)。
LC/MS (SC _ ACID) showed two product peaks [ t respectivelyR1.90 (57%) and 1.94min (23%)]Target mass ([ M + H)]+522)。
Example 7: coupling of a TMTHSI-containing linker to the HER 2-peptide. Preparation of TMTHSI-succinyl-Fcycle [ CGDGFYAC ] YMDV.
Fcycle [ CGDGFYAC ] was loaded into 8ml screw cap vials]YMDV (20mg, 13. mu. mol) and dissolved in DMSO (1 ml). Adding 4-oxo-4- ((3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1 lambda)6Thiepin-1-ylidene) amino) butanoic acid 2, 5-dioxopyrrolidin-1-yl ester (6.5mg, 14. mu. mol, 1.04 eq.) followed by addition of diisopropylethylamine (13. mu.l, 75. mu. mol, 5.6 eq.). The mixture was stirred at room temperature for 16 hours. The reaction mixture was directly purified by preparative RP-MPLC (revieris, LUNA-C18, 20-60% MeCN in water, 0.1% formic acid), and the product fractions were pooled and lyophilized to give 16.6mg (69%) of product.
LC/MS(AN_BASE_M1800)tR2.57min, purity 96%, measured value of quality [ M-H ]]-1768，[M-2H]2-883。
Analytical method
GC/MS method:
method A, apparatus: GC: Agilent 6890N, FID: detection temperature: 300 ℃ and MS:5973MSD, EI-positive, detection temperature: 280 ℃ mass range: 50-550 parts of; column: rxi-5MS 20m, ID 180 μm, df 0.18 μm; average speed: 50 cm/s; sample introduction volume: 1 mul; sample introduction temperature: 250 ℃; the split ratio is as follows: 20/1, respectively; carrier gas: he; initial temperature: 100 ℃; initial time: 1.5 min; solvent retardation: 1.3 min; the speed is 75 ℃/min; the final temperature is 250 ℃; final time 2.5 min.
LC/MS method:
SC _ ACID, device: agilent 1260 Bin. A pump: G1312B, degasser; an autosampler, ColCom, DAD Agilent G1315D, 220-; column: waters XSelectTM C18, 30X2.1mm, 3.5. mu. temperature: 35 ℃, flow rate: 1mL/min, gradient: t is t0＝5％A，t1.6min＝98％A，t3min98% a, post run time: 1.3min, eluent A: 0.1% formic acid in acetonitrile, eluent B: 0.1% aqueous formic acid).
SC _ BASE, device: agilent 1260 Bin. A pump: G1312B, degasser; an autosampler, ColCom, DAD Agilent G1315C, 220- "320 nm, MSD Agilent LC/MSD G6130B ESI, pos/neg 100-" 1000; column: waters XSelectTM CSH C18, 30X2.1mm, 3.5. mu. temperature: 25 ℃, flow rate: 1mL/min, gradient: t is t0＝5％A，t1.6min＝98％A，t3min98% a, post run time: 1.3min, eluent A: acetonitrile, eluent B: 10mM ammonium bicarbonate in water (pH 9.5).
AN _ BASE _ M1800, device: agilent 1260 Bin. A pump: G1312B, degasser; an autosampler, ColCom, DAD Agilent G1315C, 220- & 320nm, MSD Agilent LC/MSD G6130B ESI, pos/neg 800- & 1800; column: waters XSelectTM CSH C18, 50X2.1mm, 3.5. mu. temperature: 25 ℃, flow rate: 0.8mL/min, gradient: t is t0＝5％A，t3.5min＝98％A，t6min98% a, post run time: 2min, eluent A: acetonitrile, eluent B: 10mM ammonium bicarbonate in water (pH 9.5).
1H-NMR:
Using CDCl3Or DMSO-d6 as solvent, all recorded on a Bruker Avance-400 ultra-shielded NMR spectrometer1H-NMR spectrum and reported in ppm using TMS (0.00ppm) as internal standard.
Example 8: coupling of linker HER 2-peptide containing TMTHSI with Azide-containing nanoparticles (CriPec)
HER2 peptide-labeled core cross-linked polymeric micelles were generated by conjugating HER2 peptide to the surface of the polymeric micelle shell and used for targeting studies. To allow HER2 peptide conjugation via click chemistry, the protocol reported was essentially followed [ Hu et al, biomaterials.2015; 53:370-8]To make an azide-functionalized polymer micelle, but adding a fraction of the azide-functionalized block copolymer (7.5kDa, with 10 mol% crosslinker L2[ 1)]Derivatized, 5 w% of total block copolymer), but not (95 w%) non-functionalized block copolymer (7.5kDa, derivatized with 10 mol% crosslinker L2[ biomaterials.2015; 53:370-8]95 w% of the total block copolymer). The two block copolymers were synthesized following the same procedure, but for the former, use was made of azide-PEG5000-OH-synthesized (azide-PEG)5000)2-ABCPA initiator substitution. The resulting azide-functionalized polymer micelles were then purified in 20mM ammonium acetate pH5 buffer containing 130mM NaCl, the buffer was exchanged for 20mM sodium phosphate pH7.4 buffer containing 130mM NaCl and concentrated to about 30mg/mL polymer equivalents using Tangential Flow Filtration (TFF) equipped with a modified polyethersulfone (mPES)100kDa module (Spectrumlabs). Then, HER2 peptide was conjugated to concentrated azide-functionalized core-crosslinked polymer micelles via copper-free click chemistry using the following method:
ACN (86 μ L) was added to 200 μ L of azide-functionalized modified core-crosslinked polymer micelle (0.4 μmol azide equivalent) while stirring in an amber UPLC vial (300rpm) at room temperature. HER2 peptide (1.0 eq, 0.4 μmol, 86 μ L, Mercachem) was added dropwise to the reaction mixture as the heat dissipated. The progress of the conjugation reaction was monitored by UPLC-UV for 24 hours, after which the reaction was stopped. Conversion of the HER2 peptide was determined to be 43% based on UPLC, which converted to 2.2% HER2 peptide-labeled core-crosslinked polymer micelles. After the conjugation reaction, the HER2 peptide-labeled polymer micelles were purified by TFF against 10 v% ethanol to remove unreacted HER2 peptide.
Example 9: linker HER 2-peptide containing tmphsi was coupled to azide containing nanoparticles (CriPec) at acidic pH.
To allow HER2 peptide conjugation via click chemistry, the protocol reported was essentially followed [ Hu et al, biomaterials.2015; 53:370-8]To make an azide-functionalized polymer micelle, but adding a fraction of the azide-functionalized block copolymer (7.5kDa, with 10 mol% crosslinker L2[ 1)]Derivatized, 5 w% of total block copolymer), but not (95 w%) non-functionalized block copolymer (7.5kDa, derivatized with 10 mol% crosslinker L2[ biomaterials.2015; 53:370-8]95 w% of the total block copolymer). The two block copolymers were synthesized following the same procedure, but for the former, use was made of azide-PEG5000-OH-synthesized (azide-PEG)5000)2-ABCPA initiator substitution. The resulting azide-functionalized polymer micelles were then purified in 20mM ammonium acetate pH5 buffer containing 130mM NaCl and concentrated to about 60mg/mL polymer equivalent using Tangential Flow Filtration (TFF) equipped with a modified polyethersulfone (mPES)100kDa module (Spectrumlabs). Then, HER2 peptide was conjugated to concentrated azide-functionalized core-crosslinked polymer micelles via copper-free click chemistry using the following method:
150mM ammonium acetate pH5 (130. mu.L) was added to 1500. mu.L of azide-functionalized modified core-crosslinked polymer micelles (0.57. mu. mol azide equivalents) while stirring (300rpm) in an amber UPLC vial at room temperature. HER2 peptide (5.0 equiv., 2.85 μmol, 842 μ L of 2mg/ml HER2-TMTH stock solution) was added dropwise to the reaction mixture as the heat dissipated. The progress of the conjugation reaction was monitored by UPLC-UV for 24 hours, after which the reaction was stopped. Conversion of HER2 peptide was determined to be 23% based on UPLC, which converted to 5% HER2 peptide-labeled core cross-linked polymer micelles. After the conjugation reaction, the HER2 peptide-labeled polymer micelles were purified by TFF against 10 v% ethanol to remove unreacted HER2 peptide.
Example 10: comparison of reaction kinetics of model click reactions between TMTHSI and BCNOH and benzyl azide.
Reaction of an alkyne (TMTSHI or BCN-OH) with 1.3 equivalents of benzylAzide reaction, and NMR (CDCl) was used3) Reaction kinetics were measured. The conversion of TMTHSI with benzyl azide reached 79% after 225 seconds and the reaction with BCN-OH took about 40 times longer (9312 seconds). The reaction conversion (%) was calculated based on the triazole signal and the level of triazole signal at the end of the reaction was taken as the 100% level when all the tmssi or BCN-OH had reacted completely with the benzyl azide. The measurement results are included in fig. 17A. TMTHSI (upper curve) is significantly faster than BCN-OH (lower curve). Fig. 17B shows an amplification of the measurement of tmshsi.
Example 11: n is a radical of1- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -N4- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiaheptane-1-ylidene) succinamide synthesis.
4-oxo-4- ((3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1 lambda)6A solution of thiepin-1-ylidene) amino) butanoic acid 2, 5-dioxopyrrolidin-1-yl ester (89mg, 0.22mmol) in dichloromethane (2ml) was added dropwise over a period of 5 minutes to a solution of 1, 2-bis (2-aminoethoxy) ethane (131. mu.l, 0.90mmol, 4 equiv.) in dichloromethane (5 ml). After the addition was complete, the mixture was allowed to stir at room temperature for 15 minutes. The mixture was then saturated with NH4Aqueous Cl (5ml) was quenched and the organic phase was pipetted onto a phase separator. The aqueous residue was extracted with dichloromethane (2 ×) each time and the organic phase was pipetted onto the phase separator. The combined filtrates were concentrated under reduced pressure to give 47.7mg of crude product. The residue was passed through preparative RP-MPLC (Reveleries, XSelect 10-50% MeCN in water, 10mM NH)4HCO3pH 9.5), the product fractions were combined and concentrated under reduced pressure to-15 ml and transferred to a 50ml round bottom flask and lyophilized to give 26mg (27%) of the product as a white solid.
LC/MS(SC_BASE)tR1.77min, purity 99%, measured value of mass [ M + H ]]+430. FIG. 18
1H NMR (400MHz, chloroform-d) δ 6.73(t, J ═ 5.4Hz, 1H), 3.68(d, J ═ 14.1Hz, 2H), 3.63(s, 4H), 3.59(d, J ═ 10.3Hz, 2H), 3.57-3.52 (m, 4H), 3.45(q, J ═ 5.3Hz, 2H), 2.90(t, J ═ 5.1Hz, 2H), 2.69(t, J ═ 6.9Hz, 2H), 2.48(t, J ═ 6.9Hz, 2H), 2.16(bs, 2H), 1.51(s, 6H), 1.27(s, 6H).
Example 12: (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiaheptan-1-ylidene) carbamic acid 2, 5-dioxopyrrolidin-1-yl ester.
An 8ml screw cap vial was loaded with N, N' -disuccinimidyl carbonate (52mg, 0.20mmol, 2 equiv.) and dissolved in acetonitrile (2 ml). For this purpose, 1-imino-3, 3,6, 6-tetramethyl-4, 5-didehydro-2, 3,6, 7-tetrahydro-1H-1. lambda. was added dropwise using a syringe6-thiepin 1-oxide (20mg, 0.1mmol) in acetonitrile (1 ml). The mixture was stirred at room temperature for one hour. The mixture was diluted with a mixture of diethyl ether (15ml) and ethyl acetate (15ml) and quenched with water. The organic layer was washed again with water, then brine, over Na2SO4Dried and concentrated under reduced pressure to give 117mg of crude product as a white solid. The residue was purified by flash column chromatography (silica 4 g; 20-50% EtOAc in heptane), the product fractions were combined and concentrated under reduced pressure to give 49mg (43%) of the product as a white fluffy solid.
LC/MS(SC_BASE)tR1.97min, purity 95%, measured value of quality [ M + Na%]+363，[2M+Na]+703。
1H NMR (400MHz, chloroform-d) δ 3.92(d, J ═ 14.2Hz, 2H), 3.45(d, J ═ 14.2Hz, 2H), 2.80(s, 4H), 1.52(s, 6H), 1.29(s, 6H).
TMTHSI proved to be stable during storage. Compound 12 was stored as a powder in the light and dark locations at atmospheric pressure.1H-NMR and LC/MS (SC _ BASE) confirmed that the compound was still intact after 4 months of storage.
Example 13: 2,2, 2-trifluoro-N- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1 lambda6Synthesis of thiepin-1-ylidene) acetamide
An 8ml screw cap vial was loaded with TMTHSI (50mg, 0.25mmol) and the material was dissolved in acetonitrile (2 ml). Addition of K2CO3(69mg, 0.50mmol, 2 equiv.) followed by the addition of trifluoroacetic anhydride (38. mu.l, 0.27mmol, 1.1 equiv.). The mixture was stirred at room temperature for one hour. The mixture was diluted with diethyl ether and washed with water (2 ×). The organic layer was washed with brine, over Na2SO4Dried and concentrated under reduced pressure to give 56mg of crude product as a white solid.
The residue was purified by preparative RP-MPLC (Reveleria Prep, LUNA-C18, 20-60% MeCN in water, 0.1% (v/v) formic acid). The product fractions were combined and lyophilized to give 35mg (47%) of the product as a white solid.
LC/MS(SC_BASE)tR2.18min, purity 99.0%, measured value of mass [ M + H ]]+296。
1H NMR (400MHz, chloroform-d) δ 3.86(d, J ═ 14.1Hz, 2H), 3.66(d, J ═ 14.1Hz, 2H), 1.57(s, 6H), 1.33(s, 6H).
Example 14: 1- (2- (2-hydroxyethoxy) ethyl) -3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-synthesis of thiepin-1-ylidene) urea.
An 8ml screw cap vial was loaded with 2- (2-amino-ethoxy) ethanol (29 μ l, 0.29mmol, 2 equiv.) and diisopropylethylamine (63 μ l, 0.36mmol, 2.5 equiv.) and dissolvedIn dichloromethane (2 ml). (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda. was added slowly via pipette6-Thiepin-1-ylidene) carbamic acid 2, 5-dioxopyrrolidin-1-yl ester (49mg, 0.14mmol) in dichloromethane (1ml) and the mixture stirred at room temperature for 16 hours. The mixture was diluted with water and extracted with dichloromethane (3 ×), and the organic layers were filtered through a phase separator. The combined organic extracts were concentrated under reduced pressure to give 43mg of crude product. The residue was passed through preparative RP-MPLC (Reveleries, XSelect 10-50% MeCN in water, 10mM NH)4HCO3pH 9.5), the product fractions were combined and lyophilized to give 12mg (25%) of the product as a fluffy solid.
LC/MS(SC_BASE)tR1.73min, purity 94%, found value of mass [ M + H%]+331。
1H NMR (400MHz, chloroform-d) δ 5.41-5.30 (m, 1H), 3.84-3.75 (m, 2H), 3.75-3.70 (m, 2H), 3.60-3.53 (m, 4H), 3.48(d, J ═ 14.1Hz, 2H), 3.43-3.34 (m, 2H), 1.49(s, 6H), 1.27(s, 6H).
Example 15: 3- (N- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiepin-1-ylidene) sulfamoyl) benzoic acid methyl ester.
A8 ml screw cap vial was charged with 1-imino-3, 3,6, 6-tetramethyl-4, 5-didehydro-2, 3,6, 7-tetrahydro-1H-1. lambda6Thiepin 1-oxide (57mg, 0.28mmol) and dissolved in dichloromethane (1.5ml), pyridine (50 μ l, 0.62mmol, 2.16 equiv.) was added and the mixture was stirred for 15 min. Methyl 3- (chlorosulfonyl) benzoate (81mg, 0.34mmol, 1.5 eq) was added, and the mixture was stirred at room temperature for 3 hours. The mixture was quenched in 0.1N aqueous HCl (15ml) and extracted with dichloromethane (3 ×), and each organic extract was filtered through a phase separator. The combined extracts were concentrated under reduced pressure to give 100mg of an oily residueAnd (3) obtaining a crude product. The residue was purified by preparative RP-MPLC (Reveleria, LUNA-C18, 30-70% MeCN in water, 0.1% (v/v) formic acid). The product fractions were combined and lyophilized to give 39mg (34%) of the product as a white fluffy solid.
LC/MS(SC_BASE)tR2.16min, purity 100%, measured value of mass [ M + H ]]+398。
1H NMR (400MHz, chloroform-d) δ 8.65-8.61 (m, 1H), 8.21(d, J ═ 7.8Hz, 1H), 8.17(d, J ═ 7.9Hz, 1H), 7.58(t, J ═ 7.8Hz, 1H), 4.08(d, J ═ 14.3Hz, 2H), 3.95(s, 3H), 3.41(d, J ═ 14.2Hz, 2H), 1.49(s, 6H), 1.28(s, 6H).
Example 16: 3- (N- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiepin-1-ylidene) sulfamoyl) benzoic acid.
A8 ml screw cap vial was charged with 3- (N- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiepin-1-ylidene) sulfamoyl) benzoic acid methyl ester (29mg, 73. mu. mol), and the material was dissolved in THF (0.5 ml). LiOH. H2O (7mg, 167. mu. mol, 2.3 equivalents) in water (0.5 ml). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was purified by preparative RP-MPLC (Reveleria, LUNA-C18, 30-70% MeCN in water, 0.1% (v/v) formic acid). The product fractions were combined and lyophilized to give 26mg (93%) of the product as a white fluffy solid.
LC/MS(SC_BASE)tR1.70min, purity 98%, measured value of mass [ M + H ]]+384。
1H NMR (400MHz, chloroform-d) δ 8.70(t, J ═ 1.8Hz, 1H), 8.26(d, J ═ 7.7Hz, 1H), 8.22(d, J ═ 7.8Hz, 1H), 7.62(t, J ═ 7.8Hz, 1H), 4.10(d, J ═ 14.3Hz, 2H), 3.42(d, J ═ 14.2Hz, 2H), 1.49(s, 6H), 1.29(s, 6H).
Example 17: 3,3,6, 6-tetramethyl-1- (methylimino) -4, 5-didehydro-2, 3,6, 7-tetrahydro-1H-1 lambda6Synthesis of 1-oxide of thiepin.
A8 ml screw cap vial was charged with 1-imino-3, 3,6, 6-tetramethyl-4, 5-didehydro-2, 3,6, 7-tetrahydro-1H-1. lambda6Thiepin 1-oxide (50mg, 0.25mmol) and dissolved in THF (1ml), the solution was treated with KOtBu (1.7M in THF; 200. mu.l, 1.35 eq). The resulting suspension was stirred for 5min, then iodomethane (30 μ l, 0.48mmol, 1.9 eq) was added and the mixture was stirred at room temperature for 2 h. The mixture was diluted with diethyl ether and quenched with water. The layers were separated and the aqueous layer was extracted with diethyl ether (2 ×). The combined organic layers were washed with brine, over Na2SO4Dried and concentrated under reduced pressure to give 44mg of the crude product as a clear oil. The residue was passed through preparative RP-MPLC (Reveleries, XSelect 10-50% MeCN in water, 10mM NH)4HCO3pH 9.5), the product fractions are combined and concentrated under reduced pressure. The residue was diluted with acetonitrile and lyophilized to give 31.3mg (58%) of the product as a white solid.
LC/MS(SC_BASE)tR1.79min, purity 99%, measured value of mass [ M + H ]]+214。
1H NMR (400MHz, chloroform-d) δ 3.32(d, J ═ 13.9Hz, 2H), 3.07(d, J ═ 14.0Hz, 2H), 2.84(s, 3H), 1.43(s, 6H), 1.28(s, 6H).
Example 18: 4- ((3,3,6, 6-tetramethyl-1- (methylimino) -1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1H-1. lambda6-thiepin-2-yl) methyl) benzoate.
A8 ml screw cap vial was charged with 3,3,6, 6-tetramethyl-1- (methylimino) -4, 5-didehydro-2, 3,6, 7-tetrahydro-1H-1. lambda6Thiepin 1-oxide (31mg, 0.14mmol) and dissolved in anhydrous THF (1 ml). LiHMDS (1M in THF; 174. mu.l, 0.17mmol, 1.2 equiv.) is added and the mixture is stirred at room temperature for 15 minutes, then methyl 4- (bromomethyl) benzoate (50mg, 0.22mmol, 1.5 equiv.) is added and the mixture is stirred at room temperature for 20 hours. The mixture was quenched in water and extracted with diethyl ether (3 × 10 ml). The combined organic layers were washed with brine and concentrated under reduced pressure to give a semi-solid residue. The residue was purified by preparative RP-MPLC (Reveleria, LUNA-C18, 30-70% MeCN in water, 0.1% (v/v) formic acid). The product fractions were combined and lyophilized to give 9mg (17%) of the product as a white fluffy solid.
LC/MS(SC_BASE)tR2.20min, purity 97%, measured value of quality [ M + H%]+362。
1H NMR (400MHz, chloroform-d) δ 7.97(d, J ═ 8.2Hz, 2H), 7.43(d, J ═ 8.2Hz, 2H), 6.72(d, J ═ 15.7Hz, 1H), 6.25(d, J ═ 15.8Hz, 1H), 3.91(s, 3H), 3.81-3.69 (m, 1H), 3.02(d, J ═ 12.8Hz, 1H), 2.83-2.76 (m, 4H), 1.44(s, 3H), 1.39(s, 3H), 1.37(s, 6H).
Example 19: 4- (((3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiepin-1-ylidene) amino) methyl) benzoate.
A8 ml screw cap vial was charged with 1-imino-3, 3,6, 6-tetramethyl-4, 5-didehydro-2, 3,6, 7-tetrahydro-1H-1. lambda6Thiepin 1-oxide (75mg, 0.37mmol) and dissolved in anhydrous THF (1.5 ml). KOtBu (1.7M in THF; 280. mu.l, 0.47mmol, 1.26 equiv.) was added and the resulting mixture was stirred at room temperature for 15 min. Methyl 4- (bromomethyl) benzoate (129mg, 0.56mmol,1.5 equivalents) was added to the resulting suspension, and the mixture was stirred at room temperature for 6 hours. The mixture was quenched in water and extracted with diethyl ether (3X 15ml), the combined organic extracts were extractedThe material was washed with brine, over Na2SO4Dried and concentrated under reduced pressure to give 128mg of crude product as a semi-solid residue. The residue was purified by preparative RP-MPLC (Reveleria, LUNA C18, 30-70% MeCN in water, 0.1% (v/v) formic acid). The product fractions were combined and lyophilized to give 28mg (21%) of the product as an oily residue.
LC/MS(SC_BASE)tR2.14min, purity 86%, measured value of mass [ M + H ]]+348。
1H NMR (400MHz, chloroform-d) δ 7.99(d, J ═ 8.3Hz, 2H), 7.46(d, J ═ 8.0Hz, 2H), 4.39(s, 2H), 3.91(s, 3H), 3.35(d, J ═ 14.0Hz, 2H), 3.18(d, J ═ 14.1Hz, 2H), 1.45(s, 6H), 1.28(s, 6H).
Example 20: synthesis of tert-butyl (2- ((2-aminoethyl) disulfanyl) ethyl) carbamate.
A250 ml round bottom flask was charged with bis- (2-aminoethyl) disulfide dihydrochloride (1.49g, 6.62mmol) and MeOH (75 ml). Triethylamine (2.89ml, 20.8mmol, 3.15 equivalents) was added, and when all solids were dissolved, di-tert-butyl dicarbonate (1.45g, 6.64mmol, 1 equivalent) was added. The mixture was stirred at room temperature for about 1 hour.
By adding 1M NaH2PO4The mixture was quenched with aqueous solution (50ml) and the organic solvent was removed by evaporation. The aqueous residue was washed with diethyl ether (3X 30ml) and then made basic using 1N aqueous NaOH (. about.70 ml). The aqueous mixture was extracted with diethyl ether (3X 60 ml). The combined organic extracts are passed over Na2SO4Dried and concentrated under reduced pressure to give 0.71g of a colorless oil. The aqueous layer was further extracted with dichloromethane (3X 50ml) and the combined organic extracts were extracted over Na2SO4Dried and concentrated in the amount previously isolated to give 0.78g (46%) of the product as a clear viscous oil.
LC/MS(SC_BASE)tR 1.83min，Purity 98%, measured value of mass [ M + H ]]+253。
1H NMR (400MHz, chloroform-d) δ 4.93(s, 1H), 3.53-3.39 (m, 2H), 3.03(t, J ═ 6.2Hz, 2H), 2.84-2.74 (m, 4H), 1.62(s, 2H), 1.45(s, 9H).
Example 21: synthesis of 2, 2-dimethyl-4, 13-dioxo-3-oxa-8, 9-dithia-5, 12-diazahexadecane-16-oic acid.
A250 ml round bottom flask was loaded with tert-butyl (2- ((2-aminoethyl) disulfanyl) ethyl) carbamate (0.78g, 3.09mmol) and the material was dissolved in dichloromethane (25 ml). DMAP (0.11g, 0.93mmol, 0.3 equiv) was added followed by succinic anhydride (0.3g, 3.09mmol, 1 equiv). The resulting mixture was stirred at room temperature for 1 hour. With 1N KHSO4The mixture was quenched with aqueous solution (30ml), the layers were separated and washed with 1N KHSO4The organic layer was washed with aqueous solution (. about.30 ml). The combined aqueous layers were extracted with dichloromethane (30ml) and the combined organic layers were washed with brine, over Na2SO4Dried and concentrated under reduced pressure to give 1.02g (94%) of the crude product as a solid.
LC/MS(SC_ACID)tR1.80min, purity 100%, measured value of quality [ M + Na%]+375，[M-H]-351。
1H NMR (400MHz, chloroform-d) δ 11.57(bs, 1H), 7.02(t, J ═ 5.9Hz, 1H), 5.07(t, J ═ 6.3Hz, 1H), 3.59(q, J ═ 5.9Hz, 2H), 3.53-3.34 (m, 2H), 2.86(t, J ═ 6.0Hz, 2H), 2.77(t, J ═ 7.2Hz, 2H), 2.73-2.66 (m, 2H), 2.64-2.47 (m, 2H), 1.45(s, 9H).
Example 22: synthesis of 4- ((2- ((2-aminoethyl) disulfanyl) ethyl) amino) -4-oxobutanoic acid trifluoroacetate.
A250 ml round bottom flask was charged with 2, 2-dimethyl-4, 13-dioxo-3-oxa-8, 9-dithia-5, 12-diazahexadecane-16-oic acid (1.02g, 2.89 mmol). The material was dissolved in dichloromethane (5ml) and treated with trifluoroacetic acid (3ml, 39.2mmol, 13.5 equiv). The reaction was stirred at room temperature for about 1 hour. The reaction was concentrated under reduced pressure and co-evaporated with toluene, dichloromethane and diethyl ether to give 1.4g (132%). The residue was dissolved in water and washed with dichloromethane (3 ×). The aqueous layer was lyophilized to give 1.04g (98%) of the product as a viscous clear oil.
1H NMR (400MHz, methanol-d)4) δ 3.51(dt, J ═ 8.4, 6.4Hz, 2H), 3.36-3.23 (m, 2H, with methanol-d)4Residual peaks were identical), 2.99(dq, J ═ 8.4, 6.1, 5.6Hz, 2H), 2.86(dt, J ═ 8.8, 6.6Hz, 2H), 2.61(dt, J ═ 8.5, 6.4Hz, 2H), 2.49(dt, J ═ 8.7, 6.5Hz, 2H).
Example 23: 4-oxo-4- ((2- ((2- (3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda.))6-thiepin-1-ylidene) ureido) ethyl) disulfanyl) ethyl) amino) butanoic acid.
A8 ml screw cap vial was charged with (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiaheptan-1-ylidene) carbamic acid 2, 5-dioxopyrrolidin-1-yl ester (98mg, 0.29mmol), and the material was suspended/dissolved in acetonitrile (1 ml). DIPEA (115 μ L, 0.66mmol, 2.3 equivalents) was added followed by a solution of 4- ((2- ((2-aminoethyl) disulfanyl) ethyl) amino) -4-oxobutanoic acid TFA salt (113mg, 0.31mmol, 1.07 equivalents) in water (0.5ml) to form a suspension. An additional amount of acetonitrile (0.5ml) was added, and the mixture was stirred at room temperature for 20 hours.
The reaction mixture was purified by preparative RP-MPLC (Reveleria Prep, LUNA-C18, 10-50% MeCN in water, 0.1% (v/v) formic acid). The product fractions were combined and lyophilized to give 38mg (28%) of product.
LC/MS(SC_ACID)tR1.77min, purity 96%, measured value of mass [ M + H ]]+478。
1H NMR (400MHz, chloroform-d) Δ 7.73(s, 1H), 6.52(s, 0.2H), 6.00(s, 0.8H), 3.91-3.76 (m, 2H), 3.58-3.38 (m, 6H), 2.92-2.73 (m, 4H), 2.59-2.36 (m, 4H), 1.49(s, 6H), 1.31(s, 6H).
Example 24: 4-oxo-4- ((2- ((2- (3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda.))6Synthesis of thiepin-1-ylidene) ureido) ethyl) disulfanyl) ethyl) amino) butanoic acid 2, 5-dioxopyrrolidin-1-yl ester.
An 8ml screw cap vial was charged with 4-oxo-4- ((2- ((2- (3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda.)6-thiepin-1-ylidene) ureido) ethyl) disulfanyl) ethyl) amino) butanoic acid (38.8mg, 0.081mmol) and dichloromethane (1 ml). N-hydroxysuccinimide (13mg, 0.11mmol, 1.4 equivalents) was added followed by EDC (22mg, 0.11, 1.4 equivalents). The mixture was stirred at room temperature for about 2 hours. The mixture was concentrated under reduced pressure and the resulting white foam was passed through preparative RP-MPLC (Reveleria Prep, LUNA-C18, 20-60% aqueous MeCN, 0.1% (v/v) formic acid). The product fractions were combined and lyophilized. The freeze-dried material was dissolved in dichloromethane and transferred to an 8ml brown glass vial in N2The solvent was evaporated under flow and co-evaporated with diethyl ether to give 35mg (76%) of product.
LC/MS(SC_ACID)tR1.86min, purity 93%, measured value of mass [ M + H%]+575。
1H NMR (400MHz, chloroform-d) δ 6.73(s, 1H), 5.32(t, J ═ 6.1Hz, 1H), 3.78(d, J ═ 14.1Hz, 2H), 3.58(q, J ═ 6.1Hz, 2H), 3.53-3.41 (m, 4H), 3.00(t, J ═ 7.1Hz, 2H), 2.89-2.76 (m, 8H), 2.67(t, J ═ 7.1Hz, 2H), 1.47(s, 6H))，1.28(s，6H)。
Example 25: conjugation of TMTHSI-disulfide NHS ester to siRNA oligonucleotides. Preparation of TMTHSI-NH-siRNA PLK 1.
A2 ml UPLC vial was loaded with siRNA PLK1 oligonucleotide (5mg, 0.36. mu. mol) and borate buffer pH8.4 (250. mu.l) was added. Preparation of 20mM 4-oxo-4- ((2- ((2- (3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda.))6-thiepin-1-ylidene) ureido) ethyl) disulfanyl) ethyl) amino) butyric acid 2, 5-dioxopyrrolidin-1-yl ester in DMSO and 5 equivalents (90 μ l, 1.04mg, 1.8 μmol) of the stock solution were added to the solution containing siRNA while stirring. The mixture was stirred at room temperature and monitored by UPLC. After addition of an additional 5 equivalents of 4-oxo-4- ((2- ((2- (3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda.)6After-thiepin-1-ylidene) ureido) ethyl) disulfanyl) ethyl) amino) butanoic acid 2, 5-dioxopyrrolidin-1-yl ester (90. mu.l, 1.04mg, 1.8. mu. mol) and additional stirring, the reaction showed>90% conversion to TMTHSI-NH-siRNA PLK 1. Purification by PD-10 (buffer exchange to phosphate buffer, pH7.4) and Vivaspin (5000Da MWCO, 4000g, 3 hypophosphite buffer, pH7.4, final concentration step) to remove excess TMTHSI NHS 4-oxo-4- ((2- ((2- (3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiepin-1-ylidene) ureido) ethyl) disulfanyl) ethyl) amino) butanoic acid 2, 5-dioxopyrrolidin-1-yl ester. The final solution of TMTHSI-NH-siRNA PLK1 (1000. mu.L) was transferred to a 2ml UPLC vial and stored at + 4C.
Example 26: coupling of the azide-CPP 1 peptide to TMTHSI-NH-siRNA oligonucleotides. Preparation of CPP1-TMTHSI-NH-siRNA PLK1 conjugate.
First, a stock solution of azide-CPP 1 peptide (0.49mg, 0.35. mu. mol) in acetonitrile/water (1:1, 100. mu.l volume) was prepared. Initially, 50. mu.l (0.5 equiv., 0.25mg, 0.18. mu. mol) of the stock solution was added to a 2ml UPLC vial containing TMTHSI-NH-siRNA PLK1 (0.35. mu. mol in 1000. mu.l of phosphate buffer pH 7.4). The mixture was stirred at room temperature and monitored by UPLC. After 30 minutes, all azide-CPP 1 peptide was converted, then the second half of the azide-CPP 1 peptide stock solution (0.5 eq, 0.25mg, 0.18 μmol) was added. After stirring for an additional 30 minutes, the reaction to CPP1-TMTHSI-NH-siRNA PLK1 reached complete conversion. The final crude product was washed (5000Da MWCO, 4000g, 3 hypophosphite buffer ph7.4, final concentration step) and concentrated in Vivaspin centrifuge tubes. The final solution of CPP1-TMTHSI-NH-siRNA PLK1 (750. mu.l) was transferred to a 2ml UPLC vial.
Example 27: coupling of acid-labile linker 7 to CPP1-TMTHSI-siRNA oligonucleotide. Preparation of linker 7-CPP1-TMTHSI-NH-siRNA PLK1 conjugate.
A solution of CPP1-TMTHSI-NH-siRNA PLK1 (750. mu.l) was transferred to a Vivaspin centrifuge tube and the buffer exchanged to borate buffer pH8.4(5000Da MWCO, 4000g, 3 times borate buffer pH8.4, final concentration step). Final volume after Vivaspin: 1200. mu.l. Stock solutions of 20mM hydrazone MA linker NHS ester (linker 7) were prepared in DMSO. While stirring, a stock solution of 5 equivalents hydrazone MA linker NHS ester (66 μ Ι, 0.73mg, 1.6 μmol) in DMSO was added to CPP 1-thtisi-siRNA (0.32 μmol) in borate buffer (1000 μ Ι) at room temperature and monitored by UPLC. After 45min, the reaction showed complete conversion to linker 7-CPP 1-ththi-siRNA PLK1 conjugate. The final crude conjugate was purified by PD-10(5000Da MWCO, 4000g, buffer exchange to phosphate buffer ph7.4) and Vivaspin (5000Da MWCO cut-off, 4000g, 3 hypophosphite buffer ph7.4, final concentration step) to remove excess hydrazone MA linker NHS ester.
Example 29: synthesis of (S) -15- (4- (((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) amino) benzoylamino) -2, 2-dimethyl-4, 12-dioxo-3, 8-dioxa-5, 11-diazahexadecane-16-oic acid. (separation of the product into a mixture of regioisomers)
Folic acid (1.84g, 4.17mmol) was suspended in anhydrous dimethyl sulfoxide (15ml) and the flask was placed in a sand bath at 100 ℃ until a solution formed. Then, the solution was allowed to cool to room temperature and N-hydroxysuccinimide (503mg, 4.37mmol, 1.05 eq) was added followed by N-Boc-2- (2-amino-ethoxy) -ethylamine (852mg, 4.17mmol) in anhydrous dimethylsulfoxide (3 ml). The mixture was stirred for 10 min, then EDC (840mg, 4.38mmol, 1.05 eq) was added and the mixture was stirred at room temperature for 17 h.
By preparative RP-MPLC (Reveleries, XSelect 5-30% MeCN in water, 10mM NH)4HCO3pH 9.5) to purify (in 7 batches) the reaction mixture. The product fractions were combined and partially concentrated under reduced pressure. The resulting residue was lyophilized to give 0.91g (34%) of product.
LC/MS(SC_BASE)tR1.37min, purity 100%, measured value of mass [ M-H%]-626。
1H NMR(400MHz，DMSO-d6+D2O) δ 8.66(s, 1H), 7.99(q, J ═ 4.9Hz, 1H), 7.65(dd, J ═ 11.6, 8.5Hz, 2H), 6.75(d, J ═ 6.1Hz, 1H), 6.67(dd, J ═ 9.0, 2.7Hz, 2H), 4.52(s, 2H), 4.33(dd, J ═ 8.9, 4.8Hz, 0.4H), 4.19(dd, J ═ 8.1, 4.7Hz, 0.6H), 3.45-3.31 (m, 4H), 3.26-3.13 (m, 2H), 3.11-3.01 (m, 2H), 2.26(t, J ═ 7.1, 6.4Hz, 1H), 2.15(t, 8.9, 2H), 1.82 (t, 2.08H), 1.35 (m, 2H), 2.2.2.2.26 (t, J ═ 7.1, 6.4Hz, 1H), 1H, 2.15(t, 2.2H), and 2x s (H).
Example 30: n is a radical of2- (4- (((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) amino) benzoyl) -N5Synthesis of- (2- (2-aminoethoxy) ethyl) -L-glutamine. (isolation of the product as a mixture of α/β regioisomers)
(S) -15- (4- (((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) amino) benzoylamino) -2, 2-dimethyl-4, 12-dioxo-3, 8-dioxa-5, 11-diazahexadecane-16-oic acid (403mg, 0.64mmol) was dissolved in trifluoroacetic acid (2ml, 0.64mmol), and the resulting mixture was stirred at room temperature for 25 minutes. The reaction mixture was concentrated under reduced pressure and the residue was co-evaporated with dichloromethane (2 ×). The residue was taken up in N, N-dimethylformamide (1ml) and warmed until complete dissolution, then allowed to cool to room temperature. The solution was treated with triethylamine (3X 200. mu.l) to give an orange precipitate. The mixture was diluted with acetone and the solid was collected by filtration. The residue was washed with acetone (3 ×) and air dried to give 346mg (97%) of the product as an orange solid.
1H NMR(400MHz，DMSO-d6)δ8.66–8.57(m，1H)，8.24(t，J＝5.5Hz，0.6H)，8.07(t，J＝5.6Hz，0.4H)，7.72–7.62(m，1H)，7.62–7.51(m，1H)，7.30(bs，2H)，6.91(q，J＝6.1Hz，1H)，6.63(t，J＝7.6Hz，2H)，4.52–4.38(m，2H)，4.30(q，J＝6.3Hz，0.4H)，4.10(q，J＝6.4Hz，0.6H)，3.55(q，J＝6.2，5.6Hz，3H)，3.43(bs，3H)，3.31–3.13(m，4H)，2.94(q，J＝6.2，5.8Hz，2H)，2.21–2.11(m，2H)，2.06–1.79(m，3H)，0.97(t，J＝7.2Hz，1H。
NMR showed very broad HOD signals at signals between 4.5ppm and 2.5 ppm.
Example 31: n is a radical of2- (4- (((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) amino) benzoyl) -N5-(2-(2-(3-(3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1 lambda6-thiepin-1-ylidene) ureido) ethoxy) ethyl) -L-glutamine. (isolation of the product as a mixture of α/β regioisomers)
Will N2- (4- (((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) amino) benzoyl) -N5- (2- (2-aminoethoxy) ethyl) -L-glutamine (90.3mg, 0.13mmol) was dissolved in hot dimethyl sulfoxide (1.5ml) and allowed to cool to room temperature. Triethylamine (107. mu.l, 0.77mmol, 6 equivalents) was added, and the resulting mixture was added to (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6Thiepin-1-ylidene) carbamic acid 2, 5-dioxopyrrolidin-1-yl ester (52mg, 0.15mmol, 1.2 equiv.). The reaction mixture was stirred at room temperature for 4 days.
By preparative RP-MPLC (Reveleries, XSelectrode 5-40% MeCN in water, 10mM NH)4HCO3pH 9.5) to purify the reaction mixture. The product fractions were combined and lyophilized to give 51mg (53%) of product.
LC/MS(SC_BASE)tR1.45min, purity 99%, measured value of mass [ M + H ]]+753。
1H NMR(400MHz，DMSO-d6+D2O)δ8.66(s，1H)，8.02–7.90(m，1H)，7.70–7.59(m，2H)，6.72–6.61(m，3H)，4.50(s，2H)，4.37-4.32(m，0.4H)，4.22–4.13(m，0.6H)，3.90–3.79(m，2H)，3.59(dd，J＝14.0，4.5Hz，2H)，3.44–3.30(m，5H)，3.25–3.14(m，2H)，3.09(t，J＝5.8Hz，2H)，2.26(t，J＝7.7Hz，1H)，2.15(t，J＝7.6Hz，1H)，2.05–1.93(m，1H)，1.90(d，J＝13.5Hz，1H)，1.33(s，6H)，1.19(s，6H)。
Example 32: coupling of linker folate containing TMTHSI to azide containing nanoparticles (CriPec).
By conjugating folic acid to the surface of the polymeric micelle shellFolate-labeled core-crosslinked polymer micelles were generated and used for targeting studies. To allow folate conjugation via click chemistry, the protocol reported is essentially followed [ Hu et al, biomaterials.2015; 53; 370-8]To make an azide-functionalized polymer micelle, but adding a fraction of the azide-functionalized block copolymer (22kDa, with 10 mol% crosslinker L2[ 1)]Derivatized, 5 w% of total block copolymer), but not (95 w%) non-functionalized block copolymer (22kDa, derivatized with 10 mol% crosslinker L2[ biomaterials.2015; 53:370-8]95 w% of the total block copolymer). The two block copolymers were synthesized following the same procedure, but for the former, use was made of azide-PEG5000-OH-synthesized (azide-PEG)5000)2-ABCPA initiator substitution. The resulting azide-functionalized polymer micelles were then purified in 20mM ammonium acetate pH5 buffer containing 130mM NaCl, the buffer was exchanged into 20mM sodium phosphate pH7.4 buffer containing 130mM NaCl and concentrated to about 96mg/mL polymer equivalents using Tangential Flow Filtration (TFF) equipped with a modified polyethersulfone (mPES)100kDa module (Spectrumlabs). Then, folic acid was conjugated to the concentrated azide-functionalized core-crosslinked polymer micelle via copper-free click chemistry using the following method:
DMSO (93 μ L) was added to 200 μ L of the azide-functionalized modified core-crosslinked polymer micelle (0.13 μmol azide equivalent) while stirring (300rpm) in an amber UPLC vial at room temperature. Folic acid (1.0 eq, 0.13 μmol, 5 μ L, Mercachem) was added dropwise to the reaction mixture as the heat dissipated. The progress of the conjugation reaction was monitored by UPLC-UV for 4 hours, after which the reaction was stopped. The conversion of folic acid was determined to be 45% based on UPLC, which converted to 2.3% folate-labeled core-crosslinked polymer micelles.
Example 33: 1- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-synthesis of thiepin-1-ylidene) urea.
In N22, 5-Dioxopyrrolidin-1-yl (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1, 6-thiaheptan-1-ylidene) carbamate (197mg, 0.58mmol) was dissolved in dichloromethane (12.5ml) under an atmosphere and 2,2' - (ethane-1, 2-diylbis (oxo)) bis (ethane-1-amine) (0.42ml, 2.89mmol, 5 equiv.) was added. The reaction was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure. The residue was taken up in a 1:1 mixture of acetonitrile/water (4ml) and purified by preparative RP-MPLC (Reveleries, XSelectric 10-50% MeCN in water, 10mM NH)4HCO3pH 9.5) (in two batches). The product fractions were combined and lyophilized to give 156mg (72%) of the product as a white fluffy solid.
LC/MS(SC_BASE)tR1.67min, purity 99%, measured value of mass [ M + H ]]+374。
1H NMR(400MHz，DMSO-d6+D2O)δ3.86(d，J＝14.0Hz，2H)，3.61(d，J＝13.9Hz，2H)，3.54–3.46(m，4H)，3.40(q，J＝6.0Hz，4H)，3.15–3.04(m，3H)，2.67(t，J＝5.7Hz，1H)，1.34(s，6H)，1.21(s，6H)。
Example 34: 1- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6Synthesis of formate salt of the-thiepin-1-ylidene) urea-Cy 7 adduct.
An 8ml screw cap vial was charged with Cy7-NHS ester (95mg, 0.13mol) and MeCN (1.5ml), 1- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -3- (3,3,6, 6-tetramethyl-1-oxo-4, 5-didehydro-2, 3,6, 7-tetrahydro-1. lambda6-thiepin-1-ylidene) urea (66.5mg, 0.18mmol, 1.37 equiv.) in MeCN (1.5ml) and the mixture was stirred at room temperature for 2 hours. The mixture was then stored at-20 ℃ overnight. The mixture was allowed to warm to room temperature and then passed through preparative MPLC (revieris Prep, LU)NA-C18, 20-60% MeCN in water, 0.1% (v/v) formic acid), the product fractions were concentrated under reduced pressure, and the residue was diluted with a small amount of MeCN and lyophilized overnight to give 95mg (77%) of the product as a dark green solid.
LC/MS(AN_ACID_DAY_UV800)tR3.46min, purity 88%, measured value of mass [ M]+905，[M+H]2+453。
1H NMR (400MHz, methanol-d)4)δ8.37(s，1H)，8.02-7.95(m，0.5H)，7.75(dd，J＝14.0，8.5Hz，1.5H)，7.47(d，J＝7.5Hz，2H)，7.43–7.34(m，1H)，7.31–7.20(m，3H)，7.15–6.92(m，1H)，6.81–6.34(m，2H)，6.24–6.09(m，1H)，4.11(t，J＝7.3Hz，1H)，3.86(d，J＝14.1Hz，2H)，3.66–3.55(m，8H)，3.55–3.45(m，5H)，3.39–3.32(m，3H)，3.29–3.21(m，2H)，2.57(q，J＝5.6Hz，3H)，2.50–2.28(m，1H)，2.27–2.15(m，2H)，2.00–1.89(m，2H)，1.89–1.74(m，3H)，1.75–1.61(m，10H)，1.59–1.52(m，2H)，1.51–1.42(m，2H)，1.40(s，7H)，1.33–1.20(m，8H)，1.03(s，1H)。
AN _ ACID _ DAY _ UV800, equipment: agilent 1260Bin, pump: G1312B, degasser; an autosampler, ColCom, DAD Agilent G1315D, 210-; column: waters XSelectTMC18, 50 × 2.1mm, 3.5 μ, temperature: 35 ℃, flow rate: 0.8mL/min, gradient: t is t0＝5％A，t3.5min＝98％A，t6min98% a, post run time: 2 min; eluent A: 0.1% formic acid in acetonitrile, eluent B: 0.1% aqueous formic acid).
MS parameters
ESI, capillary voltage 3000V, dry gas flow rate: 12L/min, nebulizer pressure 60psig, dry gas temperature: 350 deg.C, Fragmentor 70, MS scanning, MS range 100-.
Example 35: reaction kinetics of TMTHSI with benzyl azide to determine the reaction rate constant value kt.
The reaction was monitored by mass spectrometry and kt values for the conversion of benzyl azide to BCN-OH and TMTHSI were investigated. In this study, QTOF mass spectrometry with ESI sources coupled thereto was used as the measurement method. In detail, alkynes (TMTSHI or BCN-OH) were reacted with 1.0 equivalent of benzyl azide in ACN/water/acetic acid (3:1: 0.01%) and reaction kinetics were measured using MS. The measurement results are included in fig. 55. The kt value of TMTHSI measured in the reaction with benzylazide was 0.83M-1S-1The ratio of BCN-OH (0.12M)-1S-1) About 7 times faster.
Cited documents
Krebs and H.Kimling, Tetrahedron Lett., 1970, 761-764.
-de Almeida，G.，Sletten，E.M.，Nakamura，H.，Palaniappan，K.K.，Bertozzi，C.R.，Angew.Chem.Int.Ed.，2012，51，2443-2447。
Li et al, Molecules, 2016, 21, 1393.
-Tota，A.，Zenzola，M.，Chawner，S.J.，St John-Campbell，S.，Carlucci，C.Romanazzi，G.，Degennaro，L.，Bull，J.A.，Luisi，R.，Chem.Commun.，2017，53，348-351。
Hu et al, biomaterials.2015; 53:370-8.
King et al, Chem Comm, 2012, 48, 9308-.
Dommerthot et al, Nature Communications5, particle number:5378 (2014. RTM.).
Dommeerhot et al, Top Curr Chem (Z)2016, 374: 16.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：CDK抑制剂及其用途