# US 10,307,391 B2 · Disulfur bridge linkers for conjugation of a cell-binding molecule

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- Number: US 10,307,391 B2
- Document ID: US10307391B2
- Status: Granted
- Kind: B2
- Application number: 15/479,903
- Filed: 2017-04-05
- Published: 2017-07-20
- Granted: 2019-06-04
- Earliest claimed: 2015-07-15
- Assignees: Hangzhou Dac Biotech Co., Ltd.
- Applicants: Suzhou M-Conj Biotech Co., Ltd.
- Inventors: Robert Yongxin Zhao
- Web page: https://patentgrep.com/patent/US10307391B2

## CPC

- A61K 31/337: having four-membered rings, e.g. taxol
- A61K 31/404: Indoles, e.g. pindolol
- A61K 31/5365: ortho- or peri-condensed with heterocyclic ring systems
- A61K 31/551: having two nitrogen atoms, e.g. dilazep
- A61K 31/5513: 1,4-Benzodiazepines, e.g. diazepam {or clozapine}
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61K 47/6803: {Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates}
- A61K 47/68031: {the drug being an auristatin}
- A61K 47/68033: {the drug being a maytansine}
- A61K 47/6889: {Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment}
- A61K 49/0002: {General or multifunctional contrast agents, e.g. chelated agents}
- C07C 243/28: to hydrogen atoms or to carbon atoms of a saturated carbon skeleton
- C07C 275/50: Y being a hydrogen or an acyclic carbon atom
- C07C 311/55: having sulfur atoms of the sulfonylurea groups bound to acyclic carbon atoms
- C07D 207/452: with hydrocarbon radicals, substituted by hetero atoms, directly attached to the ring nitrogen atom
- C07D 213/71: to which a second hetero atom is attached
- C07D 403/14: containing three or more hetero rings
- C07D 405/14: containing three or more hetero rings
- C07D 413/14: containing three or more hetero rings
- C07D 417/14: containing three or more hetero rings
- C07D 493/08: Bridged systems
- C07D 498/14: Ortho-condensed systems
- C07D 519/00: Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
- C07K 5/06078: {and aromatic or cycloaliphatic}
- C07K 5/06139: {with the first amino acid being heterocyclic}
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention relates to novel disulfur bridge linkers containing hydrazine used for the specific conjugation of compounds/cytotoxic agents to a cell-binding molecule, through bridge linking a pair of thiols on the cell-binding molecule. The invention also relates to methods of making such linkers, and of using such linkers in making homogeneous conjugates, as well as of application of the conjugates in treatment of cancers, infections and autoimmune disorders.

## Claims

### Claim 1

A compound of Formula (III):
wherein:
Cb represents an antibody;
n is 1-20;
Z1 and Z2 are the same or different and represent a thiol; disulfide; amino; carboxy; aldehyde; ketone; maleimido; haloacetyl; hydrazine; alkoxyamino; hydroxy group; carboxy halide; N-hydroxysuccinimide ester; p-nitrophenyl ester; dinitrophenyl ester; pentafluorophenyl ester; ester formed with tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, triflate, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, tosylate, mesylate, or 2-ethyl-5-phenylisoxazolium-3′-sulfonate; or anhydride;
R1, R2, R3 and R4 are the same or different, and are absent, a linear alkyl having from 1 to 8 carbon atoms, branched or cyclic alkyl having from 3 to 8 carbon atoms, linear, branched or cyclic alkenyl or alkynyl having from 2 to 8 carbon atoms, an ester, ether or amide having 2 to 8 carbon atoms, a polyethyleneoxy unit of formula (OCH2CH2)p, wherein p is an integer from 1 to about 1000, 6-maleimidocaproyl, carbonyl, sulfonyl, thioether, azide, amine, imine, polyamine, hydrazine, hydrazone, urea, semicarbazide, carbazide, alkoxylamine, urethane, amino acid, peptide having 1 to 20 natural or unnatural amino acids, acyloxylamine, hydroxamic acid, or combination thereof, or a combination thereof with a disulfide, hydrozone, triazole, or alkoxime unit.

### Claim 2 (depends on 1)

The compound of claim 1, wherein R1, R2, R3 or R4 is one or more selected from the group consisting of 6-maleimidocaproyl, maleimido propanoyl, valine-citrulline, alanine-phenylalanine, lysine-phenylalanine, p-aminobenzyloxycarbonyl, 4-thiopentanoate, 4-(N-maleimidomethyl)cyclo-hexane-1-carboxylate, 4-thio-butyrate, maleimidoethyl, 4-thio-2-hydroxysulfonyl-butyrate, pyridinyl-dithiol, alkoxy amino, ethyleneoxy, 4-methyl-4-dithio-pentanoic, azido, alkynyl, dithio, and (4-acetyl)aminobenzoate.

### Claim 3 (depends on 1)

The compound of claim 1, wherein R1, R2, R3 or R4 is a peptide of 1 to 20 units of natural or unnatural amino acids, or a p-aminobenzyl unit, or a 6-maleimidocaproyl unit, or a disulfide unit, or a thioether unit, or a hydrozone unit, or a triazole unit, or an alkoxime unit.

### Claim 4 (depends on 1)

The compound of claim 1, wherein Z1 and Z2 are respectively a thiol, disulfide, amino, carboxy, aldehyde, ketone, maleimido, haloacetyl, hydrazine, alkoxyamino, or hydroxy group.

### Claim 5 (depends on 1)

The compound of claim 1, wherein Z1 and Z2 are respectively a carboxy halide; N-hydroxysuccinimide ester; p-nitrophenyl ester; dinitrophenyl ester, pentafluorophenyl ester; ester formed with tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, triflate, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, tosylate, mesylate, 2-ethyl-5-phenylisoxazolium-3′-sulfonate; or anhydride.

### Claim 6 (depends on 5)

The compound of claim 5, wherein the carboxy halide is acyl halide, or the anhydride is acetyl anhydride or formyl anhydride.

### Claim 7 (depends on 1)

A method for preparing the compound of claim 1, comprising reacting a compound of Formula (I) with a cell-binding molecule having a pair of free thiols:
wherein Y1 and Y2 are the same or different and represent a functional group that is capable of reacting with the free thiol of the cell-binding molecule.

### Claim 8 (depends on 1)

A method for preparing a conjugate of a cell-binding molecule with a cytotoxic drug, comprising reacting the compound of claim 1 with the cytotoxic drug.

### Claim 9 (depends on 1)

The compound of claim 1, wherein the antibody is a monoclonal antibody.

### Claim 10 (depends on 1)

The compound of claim 1, wherein Z1 and Z2 are the same and represent a carboxy, aldehyde, ketone, ester, haloacetyl, or hydrazine.

### Claim 11 (depends on 1)

The compound of claim 1, wherein Z1 and Z2 are respectively a maleimdo, a haloacetyl, an ethylsulfonyl, a pyridyldithio, a carboxyl, a carboxyl ester of imidazole, nitrophenol, N-hydroxysuccinimide, phenol, dinitrophenol, pentafluorophenol, tetrafluorophenol, difluorophenol, monofluorophenol, pentachlorophenol, triflate, imidazole, dichlorophenol, tetrachlorophenol, 1-hydroxybenzotriazole, tosylate; mesylate, or 2-ethyl-5-phenylisoxazolium-3′-sulfonate.

### Claim 12 (depends on 1)

The compound of claim 1, wherein Z1 and Z2 are the same and represent —COO—C(CH3)3, —COCl, —COOH, —CONHNH2,

### Claim 13 (depends on 1)

The compound of claim 1, wherein R1, R2, R3 or R4 is absent, or contains a linear alkyl having from 1 to 8 carbon atoms, branched or cyclic alkyl having from 3 to 8 carbon atoms, amide having 2 to 8 carbon atoms, (OCH2CH2)1-100, 6-maleimidocaproyl, CO, SO2, or S.

### Claim 14 (depends on 1)

The compound of claim 1, wherein R1 and R2 are the same and represent —CO-alkyl-, —SO2-alkyl-, —CO-alkyl-S—,
wherein alkyl is —(CH2)1-4—.

### Claim 15 (depends on 1)

The compound of claim 1, wherein R3 and R4 are the same and represent -alkyl-, -alkyl-(CH2OCH2)m-alkyl-, —CO-alkyl-, -alkyl-CONH—CH(-alkyl-Ph)-CONH—CH(-alkyl-NH2)—, -alkyl-CONH-alkyl-(CH2OCH2)m-alkyl-, -alkyl-CON[-alkyl-(CH2OCH2)m-alkyl]2, —CH(-alkyl-NH2)—, or -alkyl-CO—, wherein m is 1 to 100, and alkyl is —(CH2)1-4—.

### Claim 16 (depends on 1)

The compound of claim 1, wherein the compound is
wherein U is OC(CH3)3 or OH, V is OH or
W is OH or Cl, m is 0 to 100, n is 1 to 20, and mAb is antibody.