# US 10,501,412 B2 · Conjugates of cell binding molecules with cytotoxic agents

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,501,412 B2
- Document ID: US10501412B2
- Status: Granted
- Kind: B2
- Application number: 14/430,230
- Filed: 2012-07-12
- Published: 2017-06-08
- Granted: 2019-12-10
- Earliest claimed: 2012-07-12
- Assignees: Hangzhou Dac Biotech Co., Ltd.
- Applicants: R. Yongxin Zhao; Yue Zhang; Yourang Ma
- Inventors: R. Yongxin Zhao; Yue Zhang; Yourang Ma
- Web page: https://patentgrep.com/patent/US10501412B2

## CPC

- C07D 207/40: 2,5-Pyrrolidine-diones
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61K 47/64: Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent (peptidic linkers A61K47/65)
- A61K 47/6803: {Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates}
- A61K 47/6811: {the drug being a protein or peptide, e.g. transferrin or bleomycin}
- A61K 47/6849: {the antibody targeting a receptor, a cell surface antigen or a cell surface determinant}
- A61K 47/6851: {the antibody targeting a determinant of a tumour cell}
- A61K 47/6855: {the tumour determinant being from breast cancer cell}
- A61K 47/6863: {the tumour determinant being from stomach or intestines cancer cell}
- A61K 47/6867: {the tumour determinant being from a cell of a blood cancer}
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 31/04: Antibacterial agents
- A61P 35/00: Antineoplastic agents
- A61P 37/00: Drugs for immunological or allergic disorders
- A61P 37/02: Immunomodulators
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- 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
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

A conjugate of a potent cytotoxic agent with a cell-surface receptor binding molecule having a formula (I), wherein T, L, m, n, Y, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, R12, and R13 are defined herein, can be used for targeted treatment of cancer, autoimmune disease, and infectious disease.

## Claims

### Claim 1

A conjugate having a structure of Formula (I):
or a pharmaceutically acceptable salt or solvate thereof,
wherein T is a cell-binding ligand; L is a releasable linker; is a linkage bond that L connects to an atom in a moiety inside the bracket independently; n is 1˜20 and m is 1˜10;
wherein the cell-binding ligand T is selected from the group consisting of an antibody, a single chain antibody, an antibody fragment that binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody fragment that binds to the target cell, a chimeric antibody, a chimeric antibody fragment that binds to the target cell, a domain antibody, a domain antibody fragment that binds to the target cell, an adnectin that mimics antibody, DARPins, a lymphokine, a hormone, a vitamin, a growth factor, a colony stimulating factor, a nutrient-transport molecule, and a binding peptide, protein, small molecule attached on albumin, a polymer, a dendrimer, a liposome, a nanoparticle, a vesicle, or a (viral) capsid;
wherein the releasable linker L has a formula of: -Ww-(Aa)r-Vv-, wherein: -W- is a stretcher unit, which links the moiety inside the bracket to an amino acid unit (Aa) when present, or V when an Aa is not present, and W is independently a self-immolative spacer, a peptidyl unit, a hydrazone, a disulfide, a thioether, an ester, or an amide bond; w is 0 or 1; wherein Aa is independently a natural or unnatural amino acid unit; r is independently an integer ranging from 0 to 12; (Aa)r represents a natural or unnatural amino acid, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit; wherein V is a spacer unit and is independently H, O, NH, S, C1-C8 alkyl, C2-C8 heteroalkyl, alkenyl, or alkynyl, C3-C8 aryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroaralkyl, heteroalkylcycloalkyl, or alkylcarbonyl, or one to four amino acid units, or (CH2CH2O)r, r is an integer ranging from 0 to 12; and v is 0, 1 or 2;
wherein R1, R3, and R4 are independently H, C1˜C8 alkyl; C2˜C8 heteroalkyl, or heterocyclic; C3˜C8 aryl, Ar-alkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, carbocyclic, or alkylcarbonyl; R2 is independently absent, C1˜C8 alkyl; C2˜C8 heteroalkyl, or heterocyclic; C3˜C8 aryl, Ar-alkyl, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, carbocyclic, or alkylcarbonyl; or R1 and R2, R1 and R3, R2 and R3, or R3 and R4 form a 3˜7 membered carbocyclic, cycloalkyl, heterocyclic, or heterocycloalkyl ring system;
wherein Y is N or CH;
wherein R5, R6, R8 and R10 are independently H, or C1˜C4 alkyl or heteroalkyl, or R5 and R6 form a 3˜7 membered carbocyclic, cycloalkyl, heterocyclic, or heterocycloalkyl ring system;
wherein R7 is independently absent, H, R14, —R14C(═O)X1R15, —R14X1R15, —R14C(═O)X1R15—, or —R14X1R15—; X1 is O, S, S—S, NH, or NR14;
wherein R9 is independently absent, H, —O—, —OH, —OR14, —OR14—, —OC(═O)R14—, —OC(═O)NHR14—, —OC(═O) R14SSR15—, OP(═O)(OR14)—, OP(═O)(OR14)O—, OP(═O)(OR14)2, or OR14OP(═O)(OR15)2;
wherein R11 is independently H, R14, —R14C(═O)R16, —R14X2R16, —R14C(═O)NHR14, wherein X2 is —O—, —S—, —NH—, —N(R14)—, —O—R14—, —S—R14—, or —S(═O)—R14—;
wherein R12 is independently R14, —OH, —SH, —NH2, ═NH, ═NNH2, —NH(R14), —OR14, —COR16, —C(O)NH2, —C(O)NHR14, —SR14, —S(═O)R14, —P(═O)(OR16)2, —OP(═O)(OR16)2, —CH2OP(═O)(OR16)2, or —SO2R16;
wherein R13 is C1˜C10 alkyl, heteroalkyl, alkyl acid, alkyl amide, alkyl amine, or Ar; Ar is an aromatic or hetero aromatic group, composed of one or several rings, and comprising four to ten carbon atoms; the hetero aromatic group is an aromatic group that has one or several carbon atoms replaced by hetero atoms; Ar is an aromatic group, wherein one or several H atoms are optionally replaced independently by R17, F, Cl, Br, I, OR16, SR16, NR16R17, N═NR16, N═R16, NR16R17, NO2, SOR16R17, SO2 R16, SO3R16, OSO3R16, PR16R17, POR16R17, PO2R16R17, OP(O)OR17)2, OCH2OP(O)(OR17)2, OC(O)OP(O)(OR17)2, PO(OR16)(OR17), OP(O)(OR17)OP(O)(OR17)2, OC(O)R17 or OC(O)NHR17;
wherein R14 and R15 are independently H, C1˜C8 alkyl, or heteroalkyl; C2˜C8 alkenyl, alkynyl, or heterocyclic; C3˜C8 aryl, heterocycloalkyl, heteroalkylcycloalkyl, carbocyclic, cycloalkyl, heteroaralkyl, or alkylcarbonyl, or one to four amino acid units;
wherein R16 and R17 are independently H, OH, C1˜C8 alkyl, or heteroalkyl; C2˜C8 alkenyl, alkynyl, or heterocyclic; C3˜C8 aryl, cycloalkyl, alkylcycloalkyl, carbocyclic, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, alkylcarbonyl or C4˜C12 glycoside, or a pharmaceutical salt;
or R12 and R13 form a 3˜7 membered carbocyclic, cycloalkyl, heterocyclic, or heterocycloalkyl ring system;
provided that: (i) R12 is H when R10 is not H,
or (ii) R12 is H when R13 is:
wherein Z1 is H, CH2OP(O)(OR18)2, C(O)OP(O)(OR18)2, PO(OR18)2, P(O)(OR18)OP(O)(OR18)2, C(O)R18, C(O)NHR18, SO2(OR18), C4-C12 glycoside, C1-C8 alkyl, or C3-C8 carboxyalkyl, or heterocyclic;
wherein R18 is H, C1˜C8 alkyl, or heteroalkyl; C2˜C8 carboxyalkyl, alkenyl, alkynyl, or heterocyclic; C3˜C8 aryl, or alkylcarbonyl; wherein R19 is H, OH, NH2, OSO2(OR18), XCH2OP(O)OR18)2, XPO(OR18), XC(O)OP(O)(OR18)2, XC(O)R18, XC(O)NHR18, C1˜C8 alkyl, carboxylic acid derivative; C2˜C8 alkenyl, alkynyl, heterocyclic, or carboxyalkyl; C3˜C8 aryl, or alkylcarbonyl; or a pharmaceutical salt; X is O, S, or NH; Y1 and Y2 are N or CH independently;
or (iii) R12 is H when R11 is:
wherein X2 is O, S, or N—R8; R8 is H, C1˜C6 alkyl or heteroalkyl; R8, is R8,

### Claim 2 (depends on 1)

The conjugate according to claim 1, having a structure of Formula (II):
or a pharmaceutical acceptable salt or solvate thereof,
wherein T, L, n, m, Y, R1, R2, R3, R4, R5, R6, R8, R10, R11, R12 and R13 are defined the same as in claim 1;
wherein R7 is independently H, R14, —R14C(═O)X1R15, or —R14X1R15; X1 is O, S, S—S, NH, or NR14;
wherein R9 is independently H, —OH, —OR14, OP(═O)(OR14)2, or OR14OP(═O)(OR15)2;
wherein R14, and R15 are defined the same as in claim 1.

### Claim 3 (depends on 1)

The conjugate according to claim 1, having a structure of Formula (IIII):
or a pharmaceutically acceptable salt or solvate thereof,
wherein T, L, n, m, Y, R1, R2, R3, R4, R5, R6, R8, R10, R11, R12 and R13 are defined the same as in claim 1;
wherein R7 is independently absent, R14, —R14C(═O)X1R15—, or —R14X1R15—; wherein X1 is O, S, S—S, NH, or NR14; and R14 and R15 are independently H, C1-C8 alkyl, or heteroalkyl; C2-C8 alkenyl, alkynyl, or heterocyclic; C3˜C8 aryl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, carbocyclic, cycloalkyl, or alkylcarbonyl;
wherein R9 is independently H, —OH, —OR14, OP(═O)(OR14)2, or OR14OP(═O)(OR15)2;
wherein R14, and R15 are defined the same as in claim 1.

### Claim 4 (depends on 1)

The conjugate according to claim 1, having a structure of Formula (IV):
or a pharmaceutically acceptable salt or solvate thereof,
wherein T, L, n, m, Y, R1, R2, R3, R4, R5, R6, R8, R10, R11, R12 and R13 are defined the same as in claim 1;
wherein R7 is independently H, R14, —R14C(═O)X1R15, or —R14X1R15; X1 is O, S, S—S, NH, or NR14;
wherein R9 is independently absent, —O—, —OR14—, —OC(═O)R14—, —OC(═O)NHR14—, —OC(═O)R14SSR15—, or OP(═O)(OR14)O—, wherein R14, R15 are independently H, C1˜C8 alkyl, or heteroalkyl; C3˜C8 aryl, heteroaryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, heteroalkylcycloalkyl, heteroaralkyl, or alkylcarbonyl or a pharmaceutical salt.

### Claim 5

A conjugate having a structure of formula IIa, IIb, IIc, IId, IIe, IIf, IIg, IIh, IIi, IIj, IIk, IIl, IIm, IIn, IIo, IIp, IIq, IIr, IIIa, IIIb, IIIc, IIId, IIIe, IIIf, IIIg, IIIh, IIIi, IIIj, IVa, IVb, IVc, IVd, IVe, IVf, mAb-TZ02, mAB-TZ03, mAb-TZ06, mAb-TZ08, mAb-TZ09, mAb-TZ15, mAb-TZ16, or mAb-TZ18, which are illustrated below:
wherein T is a cell-binding ligand; Aa is a natural or unnatural amino acid; Ar is aryl; n is 1˜20; q=1˜5; X′, Y′ and Z′ are independently CH, O, S, NH, or NR22; R22, R23 and R24 are independently C1˜C8 alkyl; C2˜C8 alkenyl, alkynyl, or heteroalkyl; C3˜C8 aryl, heterocyclic, carbocyclic, cycloalkyl, alkylcycloalkyl, heterocycloalkyl, Ar-alkyl, heteroalkylcycloalkyl, or heteroaralkyl, or —(OCH2CH2)n—; R′ and R″ are independently H or CH3;
wherein mAb is an antibody.

### Claim 6

A method for preparing a conjugate, comprising reacting a cell binding agent with a molecule having a structure of formula 308a, 308b, 309a, 309b, 290, 299a, or 299b, which are illustrated below:

### Claim 7 (depends on 1)

The conjugate of claim 1, wherein the cell-binding ligand T is capable of binding to target cells selected from the group consisting of tumor cells, virus infected cells, microorganism infected cells, parasite infected cells, autoimmune cells, activated cells, myeloid cells, activated T-cells, B cells, or melanocytes, cells expressing one or more of Apo2, ASLG659, BAFF-R, BMPR1B (bone morphogenetic protein receptor), IGF-IR, CA125, CanAg, E16, EGFR, EphA2 receptor, ErbB2, interleukin, MUC1, MUC16, Napi3b, VEGF, TF, MY9, anti-B4, EpCAM, FcRH2, C242, CD2, CD3, CD4, CD5, CD6, CD11, CD11a, CD18, CD19, CD20, CD21, CD22, CD26, CD30, CD33, CD37, CD38, CD40, CD44, CD56, CD70, CD72, CD79, CD79a, CD79b, CD90, CD105, CD138, CRIPTO, endothelin B receptor (ETBR), EphA receptors, EphB receptors, EGFRvIII, FCRH1, HER2, HER2/neu, HER3, HLA-DOB (MHC class II molecule Ia antigen), integrins, IRTA2, LIV-1, MPF, GEDA, Mesothelin, NaPi2b, PDL1, Sema 5b, PSCA, PSMA, STEAP1 (six transmembrane epithelial antigen of prostate 1), and STEAP2; and cells expressing insulin growth factor receptor, and folate receptor.

### Claim 8 (depends on 1)

The conjugate according to claim 1, wherein when n in Formula (I) is 2, the linker L has a 3-bromo-maleimido, or 3, 4-dibromo-maleimido terminus as illustrated in following formula:
wherein R is C1˜C8 alkyl or C2H4(OC2H4)n, n is 1˜20; X3 is H or Br.

### Claim 9 (depends on 1)

The conjugate of claim 1, wherein when n in Formula (I) is 2, the conjugate has following formula:
wherein R is C1˜C8 alkyl or C2H4(OC2H4)n, n is 1˜20; D is a cytotoxic agent, which is a moiety inside the bracket of Formula (I), T is a cell-binding ligand.

### Claim 10 (depends on 1)

The conjugate of claim 1, wherein when n in Formula (I) is 2, the conjugate has following formula:
wherein R is C1˜C8 alkyl or C2H4(OC2H4)n, n is 1˜20; D is a cytotoxic agent, which is a moiety inside the bracket of Formula (I), T is a cell-binding ligand.

### Claim 11 (depends on 9)

The conjugate of claim 9, wherein at least one of Ds is selected from the group consisting of calicheamicins, auristatins, maytansinoids, dolastatins, CC-1065 analogs, doxorubicins, taxanes, pyrrolobenzodiazepine dimmer, siRNA and a combination thereof, or a pharmaceutically acceptable salt, acid or derivatives thereof.

### Claim 12 (depends on 10)

The conjugate of claim 10, wherein at least one of Ds is selected from the group consisting of calicheamicins, auristatins, maytansinoids, dolastatins, CC-1065 analogs, doxorubicins, taxanes, pyrrolobenzodiazepine dimmer, siRNA and a combination thereof, or a pharmaceutically acceptable salt, acid or derivatives thereof.

### Claim 13 (depends on 1)

A pharmaceutical composition comprising a therapeutically effective amount of the conjugate of claim 1, or a pharmaceutically acceptable salt thereof, and a carrier, diluent, or excipient therefor.

### Claim 14 (depends on 1)

The conjugate of claim 1, wherein the cell-binding ligand T is selected from the group consisting of an antibody, a single chain antibody, an antibody fragment that binds to a target cell, a monoclonal antibody, a single chain monoclonal antibody, a monoclonal antibody fragment that binds to the target cell, a chimeric antibody, a chimeric antibody fragment that binds to the target cell, a domain antibody, and a domain antibody fragment that binds to the target cell.