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CN115192732A - DNA toxic dimer compound and conjugate thereof - Google Patents

DNA toxic dimer compound and conjugate thereof Download PDF

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Publication number
CN115192732A
CN115192732A CN202210299307.0A CN202210299307A CN115192732A CN 115192732 A CN115192732 A CN 115192732A CN 202210299307 A CN202210299307 A CN 202210299307A CN 115192732 A CN115192732 A CN 115192732A
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alkyl
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cancer
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朱义
万维李
卓识
张宜英
朱贵莉
于天姿
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Chengdu Bailidote Biological Pharmaceutical Co ltd
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Chengdu Bailidote Biological Pharmaceutical Co ltd
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    • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
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Abstract

A DNA toxic dimer compound and its conjugate or its pharmaceutically acceptable salt, including its preparation method and its application in preventing or treating cancer. The conjugates are capable of specifically binding to highly expressed receptors in tumor cells. Has good water solubility, stability and uniformity, and can be used for preventing or treating tumor.

Description

DNA toxic dimer compound and conjugate thereof
Technical Field
The present invention relates to novel cytotoxic compounds and medicaments comprising these cytotoxic compounds and cell binding agents. More particularly, the present invention relates to novel benzodiazepine dimer compounds, derivatives thereof, intermediates thereof, pharmaceutically acceptable salts thereof and conjugates thereof, which are useful as drugs, especially as antitumor agents.
Background
Ligand-drug conjugates (ADCs), as novel targeted drugs, generally consist of three parts: an antibody or antibody-like ligand, a small molecule drug and a linker coupling the ligand and the drug. The antibody drug conjugate utilizes the specific recognition of the antibody to the antigen to transport drug molecules to the vicinity of target cells and effectively release the drug molecules, thereby achieving the purpose of treatment. New ADC drug Adecteis developed by Seattle Gene company and approved by Food and Drug Administration (FDA) for treating Hodgkin lymphoma and recurrent degenerative large cell lymphoma (ALCL) in 8 months of 2011 TM On the market, clinical application has proved the safety and effectiveness of the medicine. With the development of ADC drugs, there is a need for more effective, newer mechanisms of action small molecule drugs.
Benzodiazepine derivatives, having the ability to recognize and bind to specific DNA sequences, are highly effective interchain cross-linking agents, which react with guanine in the DNA minor groove to form DNA adducts, interfering with DNA processing, and thus are useful as antitumor agents. (Rahman et al (2009) Jour.Amer.chem.Soc.131 (38): 13756-13766.
DNA minor groove alkylating agents of the 1- (chloromethyl) -2, 3-dihydro-1H-benzo [ e ] indole (CBI) class are potent cytotoxins (Atwell et al, (1999) j.med.chem., 42. CBI and benzodiazepine derivatives have been linked together by alkyl chains (CN 105636612A).
Imidazo [1,2-a ] pyridine derivatives are a powerful DNA binding unit that has been used in the synthesis of antitumor antibiotic duocarmycin (duocarmycins) derivatives and exhibit very potent cytotoxicity (Ronald C. Elgersma et al (2015) mol. Pharmaceuticals.12: 1813-1835).
The benzodiazepine derivatives disclosed in the prior art are extremely toxic and are toxic at very low doses, and therefore an improved, less toxic and still therapeutically active, benzodiazepine derivative with a high therapeutic window appears to be highly desirable.
Disclosure of Invention
The present invention aims to provide a benzodiazepine dimer derivative and an antibody conjugate thereof having cytotoxicity with a good therapeutic window. The small molecule drug in the conjugate of the novel cytotoxic benzodiazepine dimer derivative has two functional groups of Chloromethyl (CBI) and imine (PBD). Chloromethyl is a prodrug structure, enter in vivo can form three-membered ring structure, further can take place DNA alkylation. The presence of these two functional groups can enhance the crosslinking of DNA. The inventor unexpectedly finds that the benzodiazepine ADC medicine has good safety and high-efficiency antitumor activity.
The invention discloses a ligand-drug conjugate shown as a formula I, or pharmaceutically acceptable salts, deuterons and solvates thereof:
Ab-L-D
(I)
wherein:
ab is a ligand unit selected from an antibody, an antibody fragment, a targeting protein or an Fc-fusion protein;
l is a linking unit of D and Ab;
d is a drug unit selected from the following structures:
Figure BDA0003564748370000021
wherein:
the wavy line indicates the site where the drug is attached to L, and only one of the three sites is attached to L;
R 1 is H, deuterium, OH OR from OR 3 Ether, sulfite SO 3 - Or OSO 3 - Wherein R is 3 Is selected from C 1 -C 10 Linear, branched or cyclic alkyl, alkenyl or alkynyl groups of (a);
double line between N and C
Figure BDA0003564748370000031
Represents a single or double bond, provided that when it is a double bond, N is not attached to L and R 1 Is H; when it is a single bond, N is linked to L, R 1 Selected from OH OR from OR 3 Ether, sulfite SO 3 - Or OSO 3 - Wherein R is 3 Is selected from C 1 -C 10 Linear, branched or cyclic alkyl, alkenyl or alkynyl groups of (a);
R 2 is H or an alkyl substituent;
t is selected from C 2 -C 12 Hydrocarbyl, Z, (C) 1 -C 6 Alkylene) -Z- (C) 1 -C 6 Alkylene group), (C) 1 -C 6 Alkylene) -Z- (C) 1 -C 6 Alkylene) -Z- (C) 1 -C 6 Alkylene group), (C) 1 -C 6 Alkenylene) -Z- (C 1 -C 6 Alkenylene) or (C) 1 -C 6 Alkynylene) -Z- (C 1 -C 6 Alkynylene);
wherein:
z is independently selected from O, S, NR 4 Aryl or heteroaryl; wherein R is 4 Selected from H, P (O) 3 H 2 Or C (O) NR 5 R 6 (ii) a Wherein R is 5 And R 6 Selected from H, C 1 -C 6 Alkyl, C substituted by one or more F 1 -C 6 Alkyl, or R 5 And R 6 To form a five-or six-membered heterocyclyl group;
alkylene, alkenylene, aryl and heteroaryl are independently and optionally substituted with F, OH, O (C1-C6 alkyl), NH 2 、NHCH 3 、N(CH 3 ) 2 Or C 1 -C 6 Alkyl substituted, wherein alkyl is optionally substituted with one or more F;
y is selected from one or more of H or C 1 -C 4 Alkyl groups of (a);
x is independently selected from-O-, -N-, -S-, -OC (O) -CR 7 R 8 -(CR 9 R 10 )m-O-、-OC(O)-CR 7 R 8 -(CR 9 R 10 )m-NH-、-OC(O)-CR 7 R 8 -(CR 9 R 10 )m-S-、-NHC(O)-CR 7 R 8 -(CR 9 R 10 )m-O-、-NHC(O)-CR 7 R 8 -(CR 9 R 10 ) m-NH-or-NHC (O) -CR 7 R 8 -(CR 9 R 10 ) m-S-, etc.;
wherein:
R 7 、R 8 each independently is a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a deuterated alkyl group, a haloalkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group, a substituted aryl group, or a heteroaryl group; or, R 7 、R 8 And the carbon atoms to which they are attached form C 3 -C 6 Cycloalkyl, cycloalkylalkyl, or heterocyclyl;
R 9 、R 10 the same or different, and each independently is a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, or a heterocyclic group; or, R 9 、R 10 And the carbon atom to which they are attached forms C 3 -C 6 Cycloalkyl, cycloalkylalkyl, or heterocyclyl;
m is an integer from 0 to 4;
X 1 selected from halogen or OSO 2 R 11 Wherein R is 11 Can be independently selected from H and C 1 -C 4 A hydrocarbyl, phenyl or substituted phenyl group.
Preferably, ab is an antibody, which can form a linkage with the linker unit through a heteroatom thereof, and is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody, and a multispecific antibody.
Further preferably, the antibody, or antigen binding fragment thereof, is selected from, without limitation: anti-EGFR VIII antibody, anti-DLL-3 antibody, anti-PSMA antibody, anti-CD 70 antibody, anti-MUC 16 antibody, anti-ENPP 3 antibody, anti-TDGF 1 antibody, anti-ETBR antibody, anti-MSLN antibody, anti-TIM-1 antibody, anti-LRRC 15 antibody, anti-LIV-1 antibody, anti-CanAg/AFP antibody, anti-cladin 18.2 antibody, anti-Mesothelin antibody, anti-HER 2 (ErbB 2) antibody, anti-EGFR antibody, anti-c-MET antibody, anti-SLITRK 6 antibody, anti-KIT/CD 117 antibody, anti-STEAP 1 antibody, anti-SLAMF 7/CS1 antibody, anti-NaPi 2B/SLC34A2 antibody, anti-GPNMB antibody, anti-HER 3 (ErbB 3) antibody, anti-MUC 1/CD227 antibody, anti-AXL antibody, anti-CD 166 antibody, anti-B7-H3 (CD 276) antibody, anti-PTK 7/CCK4 antibody, PRNK 4 antibody, LR 4 antibody, anti-5T 4 EFNA antibody, anti-LIF5 antibody anti-NOTCH 3 antibody, anti-Nectin 4 antibody, anti-TROP-2 antibody, anti-CD 142 antibody, anti-CA 6 antibody, anti-GPR 20 antibody, anti-CD 174 antibody, anti-CD 71 antibody, anti-EphA 2 antibody, anti-LYPD 3 antibody, anti-FGFR 2 antibody, anti-FGFR 3 antibody, anti-fra antibody, anti-ceacasms antibody, anti-GCC antibody, anti-integran Av antibody, anti-CAIX antibody, anti-P-Cadherin antibody, anti-GD 3 antibody, anti-Cadherin 6 antibody, anti-LAMP 1 antibody, anti-FLT 3 antibody, anti-BCMA antibody, anti-CD 79B antibody, anti-CD 19 antibody, anti-CD 33 antibody, anti-CD 56 antibody, anti-CD 74 antibody, anti-CD 22 antibody, anti-CD 30 antibody, anti-CD 37 antibody, anti-CD 47 antibody, anti-CD 138 antibody, anti-CD 352 antibody, anti-CD 25 antibody, or anti-CD 123 antibody.
A DNA toxic dimeric compound and its conjugates or pharmaceutically acceptable salts, deuterons and solvates thereof, preferably: l is cleavable or non-cleavable.
A DNA toxic dimer compound and its conjugate or its pharmaceutically acceptable salt, deuteron and solvate, the pharmaceutically acceptable salt comprises sodium salt, potassium salt, calcium salt or magnesium salt formed with acidic functional group in structural formula; or acetate, trifluoroacetate, citrate, oxalate, tartrate, malate, nitrate, chloride, bromide, iodide, sulfate, bisulfate, phosphate, lactate, oleate, ascorbate, salicylate, formate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate or p-toluenesulfonate formed with a basic functional group in the structure.
A DNA toxic dimer compound and a conjugate thereof or a pharmaceutically acceptable salt thereof, and application thereof in preparing medicaments for treating or preventing tumors.
Preferably, the tumor is a solid tumor or a hematological tumor such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma, leukemia, or the like.
The invention also discloses compounds of formula II or III, or pharmaceutically acceptable salts or solvates thereof, linked to Ab:
Figure BDA0003564748370000051
wherein:
R 1 is H, OH OR OR 3 Ether, sulfite SO 3 - Or OSO 3 - Wherein R is 3 Is selected from C 1 -C 10 Linear, branched or cyclic alkyl, alkenyl or alkynyl groups of (a);
double line between N and C
Figure BDA0003564748370000052
Represents a single bond or a double bond, with the proviso that when it is a double bond, R 12 Is absent and R 1 Is H; when it is a single bond, R 12 is-C (O) O-L 3 Wherein L is 3 Is a connecting unit; r is 1 Selected from OH, by OR 3 Ether, sulfite SO 3 - Or OSO 3 - Wherein R is 3 Is selected from C 1 -C 10 Linear, branched or cyclic alkyl, alkenyl or alkynyl groups of (a);
R 2 is H or an alkyl substituent;
t is selected fromC 2 -C 12 Hydrocarbyl, Z, (C) 1 -C 6 Alkylene) -Z- (C) 1 -C 6 Alkylene group), (C) 1 -C 6 Alkylene) -Z- (C) 1 -C 6 Alkylene) -Z- (C) 1 -C 6 Alkylene group), (C) 1 -C 6 Alkenylene) -Z- (C 1 -C 6 Alkenylene) or (C) 1 -C 6 Alkynylene) -Z- (C 1 -C 6 Alkynylene);
wherein:
z is independently selected from O, S, NR 4 Aryl and heteroaryl; wherein R is 4 Selected from H, P (O) 3 H 2 ，C(O)NR 5 R 6 Wherein R is 5 And R 6 Selected from H, C 1 -C 6 Alkyl, C substituted by one or more F 1 -C 6 Alkyl, or R 5 And R 6 To form a five-or six-membered heterocyclyl group;
alkylene, alkenylene, aryl and heteroaryl are independently and optionally substituted with F, OH, O (C) 1 -C 6 Alkyl), NH 2 ，NHCH 3 ，N(CH 3 ) 2 And C 1 -C 6 Alkyl substituted, wherein alkyl is optionally substituted with one or more F;
y is selected from one or more of H or C 1 -C 4 Alkyl groups of (a);
x is independently selected from-O-, -N-, -S-, -OC (O) -CR 7 R 8 -(CR 9 R 10 )m-O-、-OC(O)-CR 7 R 8 -(CR 9 R 10 )m-NH-、-OC(O)-CR 7 R 8 -(CR 9 R 10 )m-S-、-NHC(O)-CR 7 R 8 -(CR 9 R 10 )m-O-、-NHC(O)-CR 7 R 8 -(CR 9 R 10 ) m-NH-or-NHC (O) -CR 7 R 8 -(CR 9 R 10 )m-S-；
Wherein:
R 7 、R 8 each independently is a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a deuterated alkyl group, a haloalkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aromatic hydrocarbonA group, substituted aryl or heteroaryl; or, R 7 、R 8 And the carbon atoms to which they are attached form C 3 -C 6 Cycloalkyl, cycloalkylalkyl, or heterocyclyl;
R 9 、R 10 the same or different, and each independently is a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, or a heterocyclic group; or, R 9 、R 10 And the carbon atoms to which they are attached form C 3 -C 6 Cycloalkyl, cycloalkylalkyl, or heterocyclyl;
m is an integer from 0 to 4;
X 1 selected from halogen or OSO 2 R 11 Wherein R is 11 Can be independently selected from H and C 1 -C 4 A hydrocarbyl group of (a), phenyl or substituted phenyl;
L 1 ，L 2 is a linking unit or a substituent.
Preferably, T is selected from C 2 -C 12 An alkylene group of (a).
More preferably, T is
Figure BDA0003564748370000061
As a preferred mode of execution, X is-O-, -N-or-NHC (O) -CR 7 R 8 -(CR 9 R 10 )m-O-。
Preferably, L is 3 Comprises the following steps:
Figure BDA0003564748370000071
wherein: the wavy line is connected with-C (O) O-; l is 4 Is a linking unit, linked to a ligand unit.
Preferably, L is 4 Selected from the following, without limitation:
Figure BDA0003564748370000072
wherein: at the wavy line, the left side (carbon end) is connected to the ligand unit, and the right side (nitrogen end or ester carbonyl end) is connected to X 2 Are connected.
Preferably, Q is:
Figure BDA0003564748370000073
wherein Q x Is an amino acid residue or a peptide residue composed of amino acids.
Preferably, X is 2 Comprises the following steps:
Figure BDA0003564748370000074
wherein a is selected from integers from 0 to 5, b is selected from integers from 0 to 16, c is selected from integers from 0 to 1, and d is selected from integers from 0 to 5.
Further preferably, L 3 Selected from, without limitation:
Figure BDA0003564748370000075
wherein: at the wavy line, the left side (succinimide end) is connected to the ligand unit and the right side is connected to-C (O) O-.
Preferably, the compound of formula II or III or a pharmaceutically acceptable salt or solvate thereof, L 1 And L 2 Each independently selected from:
structure a: a hydrogen atom, C (O) NR 'R', wherein R 'and R' are selected from H, C 1 -C 6 Alkyl one or more F-substituted C1-C6 alkyl groups, or R 'and R' form a five-or six-membered heterocyclyl group;
structure B: l is 4 -L 5 -，L 4 -L 6 -or L 4 -L 7 -L 8 -L 9 -, wherein L 4 、L 5 、L 6 、L 7 、L 8 And L 9 Are all connected units, L 4 To the ligand unit, L 5 、L 6 、L 9 Is linked to X.
It is further preferable that the concentration of the organic compound,
when N and C are a single bond, i.e. R 12 When present, L 1 、L 2 Each independently selected from structure A;
when a double bond is between N and C, i.e. R 12 In the absence of, L 1 Is of structure A or B, then L 2 Is structure B or A.
Further preferably, L 5 Is- ((CH) 2 )sO)r(CH 2 )sX 3 L 10 -or- ((CH) 2 )sO)r(CH 2 )sX 4 L 10 -；
L 6 Is- ((CH) 2 )sO)r(CH 2 )s-；
L 10 Is- (CH) 2 ) s-or- ((CH) 2 )sNHC(＝O)X 5 X 6 C(＝O)(CH 2 )s-；
Wherein:
X 3 selected from the following, without limitation:
Figure BDA0003564748370000081
wherein R is 13 Independently selected from hydrogen atom, C 1 -C 6 A hydrocarbon group, a halogen atom or a hydroxyl group;
X 4 selected from, without limitation:
Figure BDA0003564748370000082
wherein R is 13 Independently selected from hydrogen atoms, C 1 -C 6 A hydrocarbon group, a halogen atom or a hydroxyl group;
X 5 selected from, without limitation:
Figure BDA0003564748370000091
X 6 selected from peptide residues consisting of amino acids, selected without limitation from:
Figure BDA0003564748370000092
s is an integer from 1 to 10 and r is an integer from 1 to 14.
Further preferably, L 7 is-NC (R) 14 R 15 )C(O)、-NR 16 (CH 2 ) o C(O)-、-NR 16 (CH 2 CH 2 O) o CH 2 C(O)-、-S(CH 2 ) p C (O) -or a bond, wherein O is selected from an integer from 0 to 20; p is an integer from 0 to 20; r 14 And R 15 The same or different, and each is independently selected from hydrogen atom, deuterium atom, alkyl group, substituted alkyl group, deuterated alkyl group, heteroalkyl group, carboxyl group, amino group, substituted amino group; r 16 Selected from the group consisting of hydrogen, deuterium, halogen, alkyl, substituted alkyl, deuterated alkyl, cycloalkylalkyl, alkoxyalkyl, aryl, substituted aryl, or heteroaryl;
L 8 selected from peptide residues consisting of amino acids, preferably peptide residues formed of one, two or more amino acids selected from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E) or aspartic acid (D);
L 9 is-NR 17 (CR 18 R 19 ) q -、-C(O)NR 17 -、-C(O)NR 17 (CH 2 ) q -or a chemical bond, wherein q is selected from an integer from 0 to 6; r 17 、R 18 And R 19 Are the same or different and are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a substituted alkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group, a substituted aryl group, or a heteroaryl group.
Further preferably, L 1 、L 2 Independently from structure B is selected from the following structures without limitation:
Figure BDA0003564748370000093
Figure BDA0003564748370000101
even more preferably, the compound of formula II or III, or a pharmaceutically acceptable salt or solvate thereof, is selected from the following structures without limitation:
Figure BDA0003564748370000111
Figure BDA0003564748370000121
Figure BDA0003564748370000131
Figure BDA0003564748370000141
preferably, the antibody-drug conjugate, or pharmaceutically acceptable salts, deuterons and solvates thereof, is selected from the following structures, without limitation:
Figure BDA0003564748370000142
Figure BDA0003564748370000151
Figure BDA0003564748370000161
Figure BDA0003564748370000171
Figure BDA0003564748370000181
Figure BDA0003564748370000191
Figure BDA0003564748370000201
Figure BDA0003564748370000211
wherein u is selected from an integer of 1 to 10.
Detailed Description
Abbreviations and Definitions
As used herein, the following terms and phrases are intended to have the following meanings unless otherwise indicated. When a brand name is used herein, the brand name includes the product formulation, the general purpose drug, and the active ingredient of the brand name product, unless the context indicates otherwise.
Unless stated to the contrary, the terms used in the claims and specification herein have the following meanings.
The term "ligand" is a macromolecular compound capable of recognizing and binding to an antigen or receptor associated with a target cell. The role of the ligand is to present the drug to a target cell population to which the ligand binds, including but not limited to, a protein hormone, lectin, growth factor, antibody, or other molecule capable of binding to cells. In an embodiment of the invention, the ligand is represented as Ab, which may form a linkage with the linker unit through a heteroatom in the ligand, preferably an antibody or an antigen-binding fragment thereof, which is selected from the group consisting of a chimeric, humanized, fully human or murine antibody; preferably a monoclonal antibody.
The ligand unit is a targeting agent that specifically binds to the target moiety. The ligand is capable of specifically binding to a cellular component or to other target molecules of interest. The target moiety or target is typically on the cell surface. In some aspects, the ligand unit functions to deliver the drug unit to the particular target cell population with which the ligand unit interacts. Ligands include, but are not limited to, proteins, polypeptides and peptides, as well as non-proteins such as sugars. Suitable ligand units include, for example, antibodies, such as full-length (intact) antibodies and antigen-binding fragments thereof. In embodiments where the ligand unit is a non-antibody targeting agent, it may be a peptide or polypeptide, or a non-proteinaceous molecule. Examples of such targeting agents include interferons, lymphokines, hormones, growth and colony stimulating factors, vitamins, nutrient transport molecules, or any other cell binding molecule or substance. In some embodiments, the linker is covalently attached to the sulfur atom of the ligand. In some aspects, the sulfur atom is a sulfur atom of a cysteine residue, which forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom of a cysteine residue that has been introduced into a ligand unit, which forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom of a cysteine residue that has been introduced into a ligand unit (e.g., by site-directed mutagenesis or chemical reaction). In other aspects, the linker bound sulfur atom is selected from cysteine residues that form interchain disulfide bonds of the antibody or additional cysteine residues into which ligand units have been introduced (e.g., by site-directed mutagenesis or chemical reaction). In some embodiments, the numbering system is according to the EU index as in Kabat { [ Kabat E.A et al, (1991) ], sequences of Immunological Interest (Sequences of proteins of Immunological Interest), fifth edition, NIH publication 91-3242 }.
As used herein, "antibody" or "antibody unit" is within the scope of it, including any part of an antibody structure. This unit may bind, reactively associate, or complex with a receptor, antigen or other receptor unit present in the targeted cell population. An antibody can be any protein or proteinaceous molecule that can bind, complex, or otherwise react with a portion of a cell population to be treated or biologically engineered. The antibody constituting the antibody-drug conjugate of the present invention retains its antigen-binding ability in its original wild state. Thus, the antibodies of the present invention are capable of specifically binding to an antigen. Antigens contemplated include, for example, tumor Associated Antigens (TAA), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules associated with tissue growth and differentiation (e.g., known or predicted to be functional), lymphokines, cytokines, molecules involved in the regulation of cell circulation, molecules involved in angiogenesis, and molecules associated with angiogenesis (e.g., known or predicted to be functional). The tumor associated factor may be a cluster differentiation factor (e.g., a CD protein).
Antibodies useful in antibody drug conjugates include, but are not limited to, antibodies directed against cell surface receptors and tumor associated antigens. Such tumor-associated antigens are well known in the art and can be prepared by antibody preparation methods and information well known in the art. In order to develop effective cellular level targets for cancer diagnosis and treatment, researchers have sought transmembrane or other tumor-associated polypeptides. These targets are capable of being specifically expressed on the surface of one or more cancer cells, while expressing little or no expression on the surface of one or more non-cancer cells. Typically, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells relative to the surface of non-cancer cells. The confirmation of such tumor-associated factors can greatly improve the specific targeting property of antibody-based cancer treatment. For convenience, the information related to antigens, which is well known in the art, is indicated below, including name, other names, and GenBank accession numbers. Nucleic acid and protein sequences corresponding to tumor associated antigens can be found in public databases, such as Genbank. The antibodies target the corresponding tumor associated antigen including all amino acid sequence variants and species, having at least 70%,80%,85%,90% or 95% homology with the sequences identified in the references, or having biological properties and characteristics that are completely identical to the tumor associated antigen sequences in the cited references.
The term "inhibit" or "inhibition of" refers to a reduction in a detectable amount, or a complete prevention.
The term "cancer" refers to a physiological condition or disease characterized by unregulated cell growth. "tumor" includes cancer cells.
The term "autoimmune disease" is a disease or disorder that results from targeting an individual's own tissue or protein.
The term "drug" refers to a cytotoxic drug, denoted d, that has a chemical molecule within the tumor cell that is strong enough to disrupt its normal growth. Cytotoxic drugs can kill tumor cells in principle at a high enough concentration, but due to lack of specificity, while killing tumor cells, they can also cause apoptosis of normal cells, resulting in serious side effects. The term includes toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (e.g., at) 211 、I 131 、I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 And Lu 176 Radioactive isotopes of (a), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, preferably toxic drugs.
The term "linker" or "linker fragment" or "linker unit" refers to a chemical moiety or bond that is linked at one end to a ligand and at the other end to a drug, and may be linked to a drug following attachment of another linker.
Linkers, including extenders, spacers and amino acid units, can be synthesized by methods known in the art, such as those described in US2005-0238649A 1. The linker may be a "cleavable linker" that facilitates release of the drug in the cell. For example, acid labile linkers (e.g., hydrazones), protease sensitive (e.g., peptidase sensitive) linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al Cancer Research 52; U.S. Pat. No.5,208,020.
According to the mechanism of drug release in cells, as used herein, a "linker" or a "linker of an antibody drug conjugate" can be divided into two categories: non-cleavable linkers and cleavable linkers. For antibody-drug conjugates containing a non-cleavable linker, the drug release mechanism is: after the conjugate is combined with antigen and endocytosed by cells, the antibody is subjected to enzymolysis in lysosomes, and active molecules consisting of small-molecule drugs, linkers and antibody amino acid residues are released. The resulting structural change in the drug molecule does not reduce its cytotoxicity, but because the active molecule is charged (amino acid residues), it cannot penetrate into neighboring cells. Thus, such active drugs are unable to kill adjacent tumor cells that do not express the targeted antigen (antigen negative cells) (Ducry et al, 2010, bioconjugate chem.21.
The term "antibody-drug conjugate" refers to an antibody linked to a biologically active drug via a stable linking unit. In the present invention, the "ligand-drug conjugate" is preferably an Antibody Drug Conjugate (ADC), which refers to a monoclonal antibody or antibody fragment linked to a biologically active toxic drug through a stable linking unit.
The three letter codes and the one letter codes for amino acids used in this disclosure are as described in j.boil.chem.1968,243,3558.
The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, most preferably an alkyl group containing 1 to 6 carbon atoms. <xnotran> , , , , , , , , ,1,1- ,1,2- ,2,2- ,1- ,2- ,3- , ,1- -2- ,1,1,2- ,1,1- ,1,2- ,2,2- ,1,3- ,2- ,2- ,3- ,4- ,2,3- , ,2- ,3- ,4- ,5- ,2,3- ,2,4- ,2,2- ,3,3- ,2- ,3- , ,2,3- ,2,4- ,2,5- ,2,2- ,3,3- ,4,4- ,2- ,3- ,4- ,2- -2- ,2- -3- , ,2- -2- ,2- -3- ,2,2- , ,3,3- ,2,2- , </xnotran> And various branched chain isomers thereof, and the like. More preferred are lower alkyl groups having 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl and the like. Alkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably one or more substituents independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halo, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo.
The term "substituted alkyl" means that the hydrogen in the alkyl group is replaced with a substituent group, and unless otherwise indicated herein, the substituent group of the alkyl group may be a variety of groups selected from the group consisting of: -halogen, -OR ', -NR ' R ", -SR ', -SiR ' R" R ' ", -OC (O) R ', -C (O) R ', -CO 2 R’、-CONR’R”、-OC(O)NR’R”、-NR”C(O)R’、-NR’-C(O)NR”R”’、-NR”C(O) 2 R’、-NH-C(NH 2 )＝NH、-NR’C(NH 2 )＝NH、-NH-C(NH 2 )＝NR’、-S(O)R’、-S(O) 2 R’、-S(O) 2 NR’R”、-NR’S(O) 2 R ", -CN and-NO 2 The number of substituents is 0 to (2 m' + 1), whereinm' is the total number of carbon atoms in the group. R ', R ' and R ' each independently represent hydrogen, unsubstituted C 1-8 Alkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted C 1-8 Alkyl radical, C 1-8 Alkoxy or C 1-8 Thioalkoxy, or unsubstituted aryl-C 1-4 An alkyl group. When R 'and R' are attached to the same nitrogen atom, they may form a 3-,4-,5-, 6-or 7-membered ring together with the nitrogen atom. For example, -NR' R "includes 1-pyrrolidinyl and 4-morpholinyl.
The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O, or S, wherein alkyl is as defined above.
The term "alkylene" refers to a saturated straight or branched chain aliphatic hydrocarbon group having 2 residues derived from the parent alkane by removal of two hydrogen atoms from the same carbon atom or two different carbon atoms, and is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkylene group containing 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH) 2 -, 1-ethylene (-CH (CH) 3 ) -), 1, 2-ethylene (-CH) 2 CH 2 ) -, 1-propylene (-CH (CH) 2 CH 3 ) -), 1, 2-propylene (-CH) 2 CH(CH 3 ) -), 1, 3-propylene (-CH) 2 CH 2 CH 2 -) 1, 4-butylene (-CH 2 CH 2 CH 2 CH 2 -) and 1, 5-butylene (-CH) 2 CH 2 CH 2 CH 2 CH 2 -) and the like. Alkylene groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably independently optionally substituted with one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halo, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.
The term "alkoxy" refers to-O- (alkyl) and-O- (cycloalkyl), wherein alkyl or cycloalkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. Alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.
The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, more preferably from 3 to 10 carbon atoms, and most preferably from 3 to 8 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups.
The term "heterocyclyl" refers to a saturated or partially unsaturated mono-or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms wherein one or more of the ring atoms is selected from nitrogen, oxygen, or S (O) m (wherein m <xnotran> 0 2) , -O-O-, -O-S- -S-S- , . </xnotran> Preferably 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably the cycloalkyl ring comprises 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclic groups include spiro, fused and bridged heterocyclic groups.
The term "cycloalkylalkyl" means an alkyl group substituted with one or more cycloalkyl groups, preferably with one cycloalkyl group, wherein alkyl is as defined above, and wherein cycloalkyl is as defined above.
The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein the alkyl group is as defined above.
The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.
The term "hydroxy" refers to an-OH group.
The term "halogen" refers to fluorine, chlorine, bromine or iodine.
The term "amino" refers to the group-NH 2 . The term "nitro" means-NO 2 。
The term "amido" refers to-C (O) N (alkyl) or (cycloalkyl), wherein alkyl, cycloalkyl are as defined above.
The term "carboxylate" refers to-C (O) O (alkyl) or (cycloalkyl), wherein alkyl, cycloalkyl are as defined above.
The invention also includes various deuterated forms of formula I. Each available hydrogen atom attached to a carbon atom may be independently replaced with a deuterium atom. The person skilled in the art is able to synthesize the deuterated form of formula I with reference to the relevant literature. Commercially available deuterated starting materials can be used in preparing the deuterated forms of formula I, or they can be synthesized using conventional techniques using deuterated reagents, non-limiting examples of which include deuterated boranes, trideuteroborane tetrahydrofuran solutions, deuterated lithium aluminum hydrides, deuterated iodoethanes, and deuterated iodomethanes, among others.
The term "antibody" refers to an immunoglobulin, a tetrapeptide chain structure made up of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, i.e., igM, igD, igG, igA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, iggs can be classified into IgG1, igG2, igG3 and IgG4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. Each of the five classes of Ig may have either a kappa chain or a lambda chain. The antibodies of the invention are preferably specific for cell surface antigens on target cells, non-limiting examples being the following antibodies: <xnotran> EGFRvIII , DLL-3 , PSMA , CD70 , MUC16 , ENPP3 , TDGF1 , ETBR , MSLN , TIM-1 , LRRC15 , LIV-1 , CanAg/AFP , cladin 18.2 , Mesothelin , HER2 (ErbB 2) , EGFR , c-MET , SLITRK6 , KIT/CD117 , STEAP1 , SLAMF7/CS1 , NaPi2B/SLC34A2 , GPNMB , HER3 (ErbB 3) , MUC1/CD227 , AXL , CD166 , B7-H3 (CD 276) , PTK7/CCK4 , PRLR , EFNA4 , 5T4 , NOTCH3 , Nectin4 , TROP-2 , CD142 , CA6 , GPR20 , CD174 , CD71 , EphA2 , LYPD3 , FGFR2 , FGFR3 , FR α , CEACAMs , GCC , Integrin Av , CAIX , P-cadherin , GD3 , Cadherin 6 , LAMP1 , FLT3 , BCMA , CD79b , CD19 , CD33 , CD56 , CD74 , CD22 , CD30 , CD37 , CD138 , CD352 , CD25 CD123 . </xnotran>
The term "solvate" or "solvate compound" means that the ligand-drug conjugate of the present invention forms a pharmaceutically acceptable solvate with one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropanol, DMSO, ethyl acetate.
The term "drug loading" refers to the average number of cytotoxic drugs loaded per antibody in formula I and can also be expressed as the ratio of drug amount to antibody amount, and the drug loading can range from 0 to 12, preferably 1 to 10 cytotoxic drugs (D) attached per antibody (Ab). In an embodiment of the invention, the drug loading is expressed as n, and an exemplary average value may be 1,2,3,4,5,6,7,8,9, 10. The average amount of drug per ADC molecule after the conjugation reaction can be identified by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assays and HPLC characterization.
In one embodiment of the invention, the cytotoxic drug is conjugated to the open cysteine thiol-SH and/or site-directed mutated cysteine residues in the antibody chain via a linker unit, typically the number of drug molecules that can be conjugated to the antibody in the conjugation reaction will be less than or equal to the theoretical maximum.
The loading of the ligand cytotoxic drug conjugate can be controlled by the following non-limiting methods, including:
(1) Controlling the molar ratio of the connecting reagent to the monoclonal antibody,
(2) The reaction time and the temperature are controlled,
(3) Different reagents were selected.
The preparation of the conventional pharmaceutical composition is shown in Chinese pharmacopoeia.
The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to salts of the ligand-drug conjugates of the present invention, or salts of the compounds described herein, which are safe and effective for use in the body of a mammal and which possess the requisite biological activity, and the ligand-drug conjugates of the present invention contain at least one carboxyl group and thus may form salts with bases, non-limiting examples of which include: sodium, potassium, calcium or magnesium salts, and the like.
The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to salts of the antibody-drug conjugates of the invention, or salts of the compounds described herein, which are safe and effective for use in a mammalian body and which possess the requisite biological activity, the ligand-drug conjugate compounds of the invention contain at least one amino group and thus can form salts with acids, non-limiting examples of which include: hydrochloride, hydrobromide, hydroiodide, sulphate, hydrogen sulphate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate.
"acidic amino acid" means that the isoelectric point of the amino acid is less than 7, and acidic amino acid molecules often have one or more acidic groups such as carboxyl groups, and can be effectively ionized into negative ions in the structure to increase the hydrophilicity. The acidic amino acid may be a natural amino acid or an unnatural amino acid.
"Natural amino acid" refers to an amino acid that is biosynthesized. Natural amino acids are generally L-form, with a few exceptions, such as glycine, including both natural and biosynthesized.
"unnatural amino acid" refers to an amino acid obtained by synthetic means.
The invention will be further described with reference to specific examples, which are intended to illustrate the invention and not to limit the scope of the invention. Test methods without specific conditions noted in the following examples are generally performed under conventional conditions or under conditions recommended by the manufacturer. All percentages, ratios, or parts are by weight unless otherwise specified.
Example 1:
synthesis of Compound A:
Figure BDA0003564748370000291
compound a was synthesized according to the procedure provided in patent "CN109180681A" example 14 synthesis of compound 012 ".
Example 2:
synthesis of Compound B and Compounds B1-9:
1. preparation of Compound B
Figure BDA0003564748370000292
Compound B was prepared according to "general procedure E" of patent "CN 109180681A": the synthesis method of the CBI-benzodiazepine derivative is a method provided by the specification.
2. Preparation of Compound B1
Figure BDA0003564748370000293
In a 25mL single-necked flask, compound B (50mg, 0.072mmol), glycolic acid (11mg, 0.14mmol), EDCI (20mg, 0.10mmol), HOBt (13.5mg, 0.10mmol) and 3mLDMF were added, reacted at room temperature for 2h, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was directly subjected to high performance liquid purification to obtain compound B1 (42 mg) in 78% yield, LC-MS: [ M + H ]] + ＝747.2。
3. Preparation of Compound B2
Figure BDA0003564748370000294
Referring to the synthesis method of Compound B1, compound B (50mg, 0.072mmol), L-lactic acid (12.6mg, 0.14mmol), EDCI (20mg, 0.10mmol), HOBt (13.5mg, 0.10mmol) and 3mLDMF were added to a 25mL single-neck flask, reacted at room temperature for 2h, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was directly subjected to high performance liquid purification to obtain compound B2 (37 mg) in 68% yield, LC-MS: [ M + H ]] + ＝761.2。
4. Preparation of Compound B3
Figure BDA0003564748370000301
Referring to the synthesis method of the compound B2, L-lactic acid is changed into D-lactic acid to obtain a compound B3, LC-MS: [ M + H ]] + ＝761.2。
5. Preparation of Compounds B4 and B5
Figure BDA0003564748370000302
Compound B (60mg, 0.087mmol), 3-trifluoro lactic acid (25mg, 0.17mmol), EDCI (25mg, 0.13mmol), HOBt (17.6mg, 0.13mmol) and 3mL DMMF were added to a 25mL single vial, reacted at room temperature for 1.5h, and the reaction monitored by HPLC. After the reaction is finished, the reaction solution is directly purified by high performance liquid phase, and the product preparation solution is freeze-dried to respectively obtain the compoundsB4 (26 mg), compound B5 (22 mg), LC-MS: [ M + H ]] + ＝815.2。
6. Preparation of Compounds B6 and B7
Figure BDA0003564748370000303
Referring to the synthesis method of compounds B4 and B5, high performance liquid purification was used to obtain compounds B6 and B7, LC-MS: [ M + H ]] + ＝787.3。
7. Preparation of Compounds B8 and B9
Figure BDA0003564748370000311
Referring to the synthesis method of compounds B4 and B5, high performance liquid purification was used to obtain compounds B8 and B9, LC-MS: [ M + H ]] + ＝801.3。
Example 3:
synthesis of Compounds C and C1-9:
1. preparation of Compound C
Figure BDA0003564748370000312
Compound C was prepared according to "general procedure E of patent" CN109180681A ": the synthesis method of the CBI-benzodiazepine derivative is provided in the second section.
2. Preparation of Compound C1
Figure BDA0003564748370000313
Compound C (50mg, 0.075mmol), glycolic acid (11.4mg, 0.15mmol), EDCI (21.6mg, 0.11mmol), HOBt (15.2mg, 0.11mmol) and 3mLDMF were added to a 25mL single-neck flask, reacted at room temperature for 2h, and the reaction was monitored by HPLC. After the reaction, the reaction solution was directly subjected to high performance liquid purification to obtain compound C1 (35 mg) in 65% yield, LC-MS: [ M + H ]] + ＝723.2。
3. Preparation of Compound C2
Figure BDA0003564748370000321
Referring to the synthesis method of compound C1, compound C (50mg, 0.075mmol), L-lactic acid (13mg, 0.15mmol), EDCI (21.6mg, 0.11mmol), HOBt (15.2mg, 0.11mmol) and 3mLDMF were added to a 25mL single-neck flask, reacted at room temperature for 2h, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was directly subjected to high performance liquid purification to obtain compound C2 (33.5 mg) in a yield of 63%, LC-MS: [ M + H ]] + ＝737.2。
4. Preparation of compound C3:
Figure BDA0003564748370000322
referring to the synthesis method of the compound C2, L-lactic acid is changed into D-lactic acid to obtain a compound C3, LC-MS: [ M + H ]] + ＝737.2。
5. Preparation of compounds C4 and C5:
Figure BDA0003564748370000323
compound C (60mg, 0.09mmol), 3-trifluorolactic acid (26mg, 0.18mmol), EDCI (25mg, 0.13mmol), HOBt (17.6mg, 0.13mmol) and 3mLDMF were added to a 25mL single-necked flask, reacted at room temperature for 1.5h, and the reaction was monitored by HPLC. After the reaction is finished, the reaction solution is directly purified by high performance liquid phase, and the product preparation solution is lyophilized to respectively obtain a compound C4 (21 mg), a compound C5 (25 mg), LC-MS: [ M + H ]] + ＝791.2。
6. Preparation of compounds C6 and C7:
Figure BDA0003564748370000331
referring to the synthesis method of compounds C4 and C5, purifying with high performance liquid phase to obtain compounds C6 and C7, LC-MS：[M+H] + ＝763.3。
7. Preparation of compounds C8 and C9:
Figure BDA0003564748370000332
referring to the synthesis method of the compounds C4 and C5, high performance liquid purification is utilized to obtain the compounds C8 and C9, and LC-MS: [ M + H ]] + ＝777.3。
Example 4:
synthesis of Compounds D1 and D1-9:
1. preparation of Compound D
Figure BDA0003564748370000333
Compound D refers to "general procedure a of patent" CN109180681A ": synthesis of benzodiazepine derivatives first and general procedure E: the synthesis method provided by the second synthesis method of the CBI-benzodiazepine derivative.
2. Preparation of Compound D1
Figure BDA0003564748370000341
Compound D (50mg, 0.078mmol), glycolic acid (12mg, 0.11mmol), EDCI (22.4mg, 0.12mmol), HOBt (15.8mg, 0.12mmol) and 3mLDMF were charged into a 25mL single-neck flask, reacted at room temperature for 2h, and the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was directly subjected to high performance liquid purification to obtain compound D1 (34 mg) in 62.5% yield, LC-MS: [ M + H ]] + ＝697.2。
3. Preparation of Compound D2
Figure BDA0003564748370000342
A25 mL single-neck flask was charged with compound D (50mg, 0.078mmol), L-lactic acid (14mg, 0.16mmol), EDCI (22.4mg, 0.12mmol),HOBt (15.8mg, 0.12mmol) and 3mLDMF, reaction at room temperature for 2h, and monitoring the reaction by HPLC. After the reaction was completed, the reaction solution was directly subjected to high performance liquid purification to obtain compound D2 (33.8 mg), yield 71%, LC-MS: [ M + H ]] + ＝711.2。
4. Preparation of Compound D3
Figure BDA0003564748370000343
Referring to the synthesis method of the compound D2, D-lactic acid replaces L-lactic acid to obtain a compound D3, LC-MS: [ M + H ]] + ＝711.2。
4. Preparation of Compounds D4 and D5
Figure BDA0003564748370000344
Compound D (60mg, 0.094mmol), 3-trifluorolactic acid (27mg, 0.187mmol), EDCI (27mg, 0.14mmol), HOBt (19mg, 0.14mmol) and 3mL DMF were added to a 25mL single-neck flask, reacted at room temperature for 1.5h, and the reaction was monitored by HPLC. After the reaction is finished, the reaction solution is directly subjected to high performance liquid purification, and a product preparation solution is lyophilized to obtain a compound D4 (27 mg), a compound D5 (29 mg), an LC-MS: [ M + H ]] + ＝765.2。
6. Preparation of Compounds D6 and D7
Figure BDA0003564748370000351
Referring to the synthesis method of the compounds D4 and D5, high performance liquid purification is utilized to obtain the compounds D6 and D7, and LC-MS: [ M + H ]] + ＝737.3。
7. Preparation of Compounds D8 and D9
Figure BDA0003564748370000352
Referring to the synthesis method of the compounds D4 and D5, the compounds D8 and D9, LC-MS：[M+H] + ＝751.3。
Example 5:
synthesis of compound M1:
Figure BDA0003564748370000353
in a 5L single-neck flask were added Fmoc-glycine (100g, 28mmol, 1.0eq), lead tetraacetate (175g, 395mmol, 1.4eq), 2L tetrahydrofuran and 670mL toluene, stirred well, heated to 85 ℃ and reacted for 2.5h. TLC, after the starting material had reacted, was cooled to room temperature, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography (PE: EA =5, 1-2: [ M + NH ] 4 ] + ＝386.0。
Example 6:
synthesis of compound M3:
Figure BDA0003564748370000361
a1000 mL single vial was charged with SM-2 (synthesized according to the method disclosed in patent CN 108452321A) (40g, 96mmol, 1.0eq), triethylamine (26.7mL, 2.0eq), and toluene (400 mL), and the mixture was heated to 120 ℃ and refluxed for 2 hours. TLC monitoring is carried out to ensure that the reaction is basically completed, and the solvent is removed by rotary removal under reduced pressure when the temperature is reduced to 50 ℃. The mixture was dissolved in ethyl acetate (150 mL) and water (40 mL), adjusted to pH 2-3 with 1M HCl while stirring in an ice bath, and the solution was separated. The aqueous layer was extracted once more with ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate. After filtration, concentration gave a crude product as a light yellow oil which was purified by column chromatography (DCM: meOH = 40; LC-MS: [ M + H ]] + ＝399.3。
In a 1L single-neck flask, compound M2 (26.5g, 60.5mmol, 1.0eq), pentafluorophenol (12.2g, 66.5mmol, 1.1eq), DCC (13.7g, 66.5mmol, 1.1eq), and THF (300 mL) were added, reacted at room temperature for 30min (monitored by TLC), and insoluble material was filtered off. Directly purifying the reaction solution by water pump, concentrating the solution at 35 deg.C under reduced pressure in water bath to remove acetonitrile, and lyophilizing to obtain 31.5g of compound M3 with high yield64％；LC-MS：[M+H] + ＝565.1。
Example 7:
synthesis of Compound 1:
Figure BDA0003564748370000371
the first step is as follows: synthesis of Compound 1a
In a 250mL single-neck flask, M1 (6 g,16.3 mmol), 100mL THF, p-toluenesulfonic acid monohydrate (0.31g, 1.63mmol) were added, stirred and cooled to 0 ℃, benzyl glycolate (5.4 g,32.6 mmol) was added dropwise, and after completion of dropwise addition, the reaction was naturally warmed to room temperature (about 2-4 h), monitored by TLC. After the reaction is finished, saturated NaHCO is added 3 The solution, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue purified by a silica gel column (PE: EA = 10; LC-MS: [ M + H ]] + ＝475.2。
The second step: synthesis of Compound 1b
Adding compound 1a (2g, 4.2mmol) and 10mL DMF in a 25mL single-port bottle, stirring at 0 ℃, adding DBU (766mg, 5.04mmol), reacting for 1h, and monitoring by TLC to obtain the product after Fmoc deprotection is completed;
another 25mL single vial was charged with compound M4 (prepared by the method disclosed in CN 111051330A) (1.73g, 4.2mmol), pyBOP (2.61g, 5.04mmol), HOBt (680mg, 5.04mmol) and 10mL DMF, DIPEA (830uL, 5.04mmol) was added in an ice-water bath, and stirring was continued for 30min, and the reaction mixture was charged into a reaction flask and allowed to warm to room temperature for reaction. After HPLC monitoring reaction is finished, purifying a reaction solution by a preparation liquid phase to obtain a product preparation solution, extracting the preparation solution by dichloromethane, washing the preparation solution by a saturated sodium chloride solution, drying by anhydrous sodium sulfate, filtering, and concentrating the filtrate under reduced pressure to obtain a compound 1b (1.7 g) with a yield of 63%; LCMS: [ M + H ]] + ＝648.3。
The third step: synthesis of Compound 1c
After compound 1b (900mg, 1.39mmol) and 15mL of DMF had been dissolved in a 25mL single-necked flask, 900mg of 5% Pd/C was added, and the reaction was hydrogenated for 2 hours, followed by filtration to obtain a filtrate, which was used in the next reaction without purification.
The fourth step: synthesis of Compound 1
The DMF solution of compound 1c obtained in the above step was placed in an ice water bath, DIPEA (235uL, 1.39mmol) was added, compound M3 (784 mg, 1.39mmol) was then added, and the mixture was allowed to warm to room temperature for reaction for 1 hour. After the HPLC monitoring reaction is finished, purifying the reaction solution by high performance liquid chromatography to obtain a preparation solution, and freeze-drying the preparation solution to obtain a compound 1 (604 mg) with a yield of 54%; LC-MS: [ M + H ]] + ＝804.4。
Example 8:
synthesis of Compound 2:
Figure BDA0003564748370000381
the first step is as follows: synthesis of Compound 1c
After compound 1b (900mg, 1.39mmol) and 15mL of DMF had been dissolved in a 25mL single-necked flask, 900mg of 5% Pd/C was added, and the reaction was hydrogenated for 2 hours, followed by filtration to obtain a filtrate, which was used in the next reaction without purification.
The second step: synthesis of Compound 2
The DMF solution of compound 1c obtained in the above step was placed in an ice water bath, DIPEA (235uL, 1.39mmol) was added, mcOSu (428.5mg, 1.39mmol) was further added, and the mixture was allowed to warm to room temperature for 1 hour. After the HPLC monitoring reaction is finished, purifying the reaction solution by high performance liquid chromatography to obtain a preparation solution, and freeze-drying the preparation solution to obtain a compound 2 (500 mg) with a yield of 58%; LC-MS: [ M + H ]] + ＝617.3。
Example 9:
synthesis of compounds 3A and 3B:
1. preparation of Compound 3A
Figure BDA0003564748370000391
The first step is as follows: synthesis of Compound 3a
In a 25mL single-neck flask were added M1 (1.5g, 4.0mmol,1.0 eq), p-toluenesulfonic acid monohydrate (77 mg,0.4mmol, 0.1eq) and 15mL THF, stirring well, cooling to 0 deg.C, slowly adding L-benzyl lactate (2.2g, 12.0mmol, 3eq), and heating to room temperature for reaction. TLC monitoring, after the reaction is finished, saturated NaHCO is added 3 The solution was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by reverse phase column to give compound 3a (1.03 g) in 52% yield, LC-MS: [ M + NH ] 4 ] + ＝506.2。
The second step: synthesis of Compound 3b
Compound 3a (1g, 2.04mmol) and 8mL of DMF were added to a 25mL single-necked flask, stirred well, then cooled to 0 deg.C, DBU (373mg, 2.45mmol) was added slowly, and after the addition, the temperature was raised to room temperature for reaction. Monitoring by TLC, and marking as reaction liquid (1) after the reaction is finished;
another 25mL single vial was charged with M4 (846 mg, 2.04mmol), pyBOP (1.27g, 2.45mmol) and 6mL DMF, stirred at room temperature for 5min, added with reaction mixture (1), reacted at room temperature, and monitored by HPLC. After the reaction, the reaction solution was purified by high performance liquid chromatography and lyophilized to obtain compound 3b (913 mg) in 67.6% yield, LC-MS: [ M + NH ] 4 ] + ＝679.2。
The third step: synthesis of Compound 3c
Compound 3b (800mg, 1.21mmol, 1.0eq) and DMF (15 mL) were dissolved in a 100mL single-necked flask, and then 5% Pd/C (800 mg) was added thereto, followed by hydrogenation at room temperature for 2 hours (monitoring the progress of the reaction by HPLC). Pd/C was filtered and the filtrate was used in the next step without concentration.
The fourth step: synthesis of Compound 3A
The DMF solution of Compound 3c obtained in the above step was placed in an ice-water bath, DIPEA (219uL, 1.21mmol) was added thereto, compound M3 (683mg, 1.21mmol) was added thereto, and the mixture was allowed to warm to room temperature for 1 hour. After the reaction was monitored by HPLC, the reaction solution was purified by high performance liquid chromatography to obtain a preparation, which was lyophilized to obtain compound 3A (524 mg) with a yield of 53%, LC-MS: [ M-H ]] - ＝816.3。
2. Preparation of Compound 3B
Figure BDA0003564748370000401
Referring to the synthesis method of the compound 3A, the benzyl L-lactate in the first step is changed into benzyl D-lactate to obtain a compound 3B, and the mass ratio of LC-MS: [ M-H ]] - ＝816.3。
Example 10:
synthesis of compounds 4A and 4B:
1. preparation of Compound 4A
Figure BDA0003564748370000402
The first step is as follows: synthesis of Compound 3c
In a 100mL single-neck flask, 3b (800mg, 1.21mmol, 1.0eq) and DMF (15 mL) were added and dissolved, and 5% by volume of Pd/C (800 mg) was added, and the reaction was hydrogenated at room temperature for 2 hours (the progress of the reaction was monitored by HPLC). The Pd/C was filtered and the filtrate was used in the next step without concentration.
The second step: synthesis of Compound 4A
The DMF solution of compound 3c obtained in the above step was placed in an ice-water bath, DIPEA (219uL, 1.21mmol) was added, and McOSu (373mg, 1.21mmol) was further added, and the reaction was allowed to warm to room temperature for 1h. After the HPLC monitoring reaction is finished, purifying the reaction solution by high performance liquid chromatography to obtain a preparation solution, and freeze-drying the preparation solution to obtain a compound 4A (450 mg) with a yield of 59%; LC-MS: [ M + H ]] + ＝631.3。
2. Synthesis of Compound 4B
Figure BDA0003564748370000411
Referring to the synthesis of compound 4A, compound 4b, lc-MS: [ M + H ]] + ＝631.3。
Example 11:
synthesis of Compound 5:
Figure BDA0003564748370000412
the first step is as follows: synthesis of Compound 5a
M1 (10g, 27.1mmol), benzyl 3,3,3-trifluorolactate (prepared by the method disclosed in WO2020063673A 1) (12.7g, 54.3mmol), zinc acetate (9.96g, 54.3mmol) and 100mL of toluene were charged in a 250mL single-neck flask, and the mixture was heated to 100 ℃ and reacted for 4 hours. After the reaction is finished, cooling to room temperature, filtering to remove insoluble substances, and concentrating the filtrate to obtain a crude product. The crude product was purified by silica gel column chromatography (PE: EA = 10; LC-MS: [ M + H ]] + ＝543.2。
The second step is that: synthesis of Compound 5b
Adding the compound 5a (5 g, 9.2mmol) and 15mL of DMF into a 50mL single-neck bottle, dissolving and clearing, adding DBU (1.68g, 11mmol) under an ice water bath, and reacting for 1h to obtain a reaction solution (1);
another 50mL single-neck flask was charged with M4 (3.8g, 9.2mmol), pyBOP (5.75g, 11mmol), HOBt (1.49g, 11mmol) and 10mL DMF, and after dissolution, DIPEA (1.82mL, 111mmol) was added to the flask in an ice-water bath to continue the reaction for 30min, followed by addition of reaction mixture (1) and warming to room temperature for reaction for 2h. And (3) monitoring the reaction process by HPLC, and after the reaction is finished, purifying the reaction solution by high performance liquid chromatography to obtain a preparation solution. Extracting the preparation solution by dichloromethane, washing by saturated sodium chloride solution, drying by anhydrous sodium sulfate, filtering and concentrating to obtain a compound 5b (4.1) g, wherein the yield is 62.3%; LC-MS: [ M + H ]] + ＝716.3。
The third step: synthesis of Compound 5
After compound 5b (900mg, 1.26mmol) and 15mL of DMF had been dissolved in a 25mL single-necked flask, 900mg of 5% Pd/C was added, hydrogenation was carried out for 2 hours, and after completion of the reaction, filtration was carried out, the filtrate was placed in an ice-water bath, DIPEA (228uL, 1.38mmol) was added, compound M3 (712mg, 1.26mmol) was added, and the mixture was warmed to room temperature and reacted for 1 hour. After the HPLC monitoring reaction is finished, purifying the reaction solution by high performance liquid chromatography to obtain a preparation solution, and freeze-drying the preparation solution to obtain a product compound 5 (525) mg with a yield of 47.9%; LC-MS: [ M-H ]] - ＝870.3。
Example 12:
synthesis of Compound 6:
Figure BDA0003564748370000421
after compound 5b (900mg, 1.26mmol) and 15mL of DMF had been dissolved in a 25mL single-necked flask, 900mg of 5% Pd/C was added, hydrogenation was carried out for 2 hours, and after the reaction was completed, filtration was carried out, the filtrate was placed in an ice-water bath, DIPEA (228uL, 1.38mmol) was added, mcOSu (388mg, 1.26mmol) was further added, and the mixture was allowed to warm to room temperature and reacted for 1 hour. After the HPLC monitoring reaction is finished, purifying the reaction solution by using a high performance liquid chromatography to obtain a preparation solution, and freeze-drying the preparation solution to obtain a product compound 6 (450) mg with the yield of 52%; LC-MS: [ M-H ]] - ＝683.3。
Example 13:
synthesis of compound 7:
Figure BDA0003564748370000431
the first step is as follows: synthesis of Compound 7a
M1 (10g, 27.1mmol), benzyl 2-cyclopropyl-2-hydroxyacetate (prepared by the method disclosed in WO2020244657A 1) (11.2g, 54.3mmol), zinc acetate (9.96g, 54.3mmol) and 100mL of toluene were charged in a 250mL single-neck flask, and the mixture was heated to 100 ℃ to react for 4 hours. After the reaction is finished, cooling to room temperature, filtering to remove insoluble substances, and concentrating the filtrate to obtain a crude product. The crude product was purified by silica gel column chromatography (PE: EA =10 1-5; LC-MS: [ M + H ]] + ＝515.2。
The second step: synthesis of Compound 7b
A50 mL single-neck flask was charged with compound 7a (4 g, 7.8mmol) and 10mL of DMF, and after dissolution, DBU (1.42g, 9.3mmol) was added under an ice-water bath to conduct reaction for 1 hour to obtain reaction solution (1);
another 50mL single-neck flask was charged with M4 (3.2g, 7.8 mmol), pyBOP (4.5g, 8.6 mmol), HOBt (1.16g, 8.6 mmol) and 10mL DMF, and after dissolution, DIPEA (1.65mL, 10mmol) was added in an ice-water bath to continue the reaction for 30min, and the reaction mixture (1) was added and warmed to room temperature for 2h. And monitoring the reaction process by HPLC, and after the reaction is finished, purifying the reaction solution by high performance liquid chromatography to obtain a preparation solution. Extracting the preparation solution by dichloromethane, washing by saturated sodium chloride solution, drying by anhydrous sodium sulfate, filtering and concentrating to obtain a compound 7b (4.2 g), wherein the yield is 78%; LC-MS: [ M + H ]] + ＝688.3。
The third step: synthesis of Compound 7
After compound 7b (1000mg, 1.45mmol) and 15mL of DMF were dissolved in a 25mL single-necked flask, 1000mg of 5% Pd/C was added, hydrogenation was carried out for 2 hours, the reaction was completed, filtration was carried out, the filtrate was placed in an ice-water bath, DIPEA (248uL, 1.5 mmol) was added, compound M3 (720mg, 1.45mmol) was added, and the mixture was warmed to room temperature and reacted for 1 hour. After the HPLC monitoring reaction is finished, purifying the reaction solution by high performance liquid chromatography to obtain a preparation solution, and freeze-drying the preparation solution to obtain a product compound 7 (503 mg) with a yield of 41%; LC-MS: [ M-H ]] - ＝842.3。
Example 14:
synthesis of compound 8:
Figure BDA0003564748370000441
the first step is as follows: synthesis of Compound 8a
M1 (10g, 27.1mmol), benzyl 2-hydroxy-3-cyclopropylpropionate (synthesized according to the method disclosed in WO 2020063676A) (12.0g, 54.3mmol), zinc acetate (9.96g, 54.3mmol) and 100mL of toluene were charged in a 250mL single-neck flask, and the mixture was heated to 100 ℃ and reacted for 4 hours. After the reaction is finished, cooling to room temperature, filtering to remove insoluble substances, and concentrating the filtrate to obtain a crude product. The crude product was purified by silica gel column chromatography (PE: EA =10 1-5; LC-MS: [ M + H ]] + ＝529.2。
The second step is that: synthesis of Compound 8b
A50 mL single-neck flask was charged with compound 8a (4 g,7.6 mmol) and 10mL of DMF, and after dissolution, DBU (1.39g, 9.1 mmol) was added in an ice-water bath to react for 1 hour, which was designated as reaction solution (1);
another 50mL single-neck flask was charged with M4 (3.12g, 7.6 mmol), pyBOP (4.5g, 8.6 mmol), HOBt (1.16g, 8.6 mmol) and 10mL DMF, and after clearing, DIPEA (1.65mL, 10mmol) was added in an ice-water bath to continue the reaction for 30min, and the reaction mixture (1) was added and allowed to warm to room temperature for 2h. And monitoring the reaction process by HPLC, and after the reaction is finished, purifying the reaction solution by high performance liquid chromatography to obtain a preparation solution. Extracting the preparation solution with dichloromethane, washing with saturated sodium chloride solution, drying with anhydrous sodium sulfate, filtering, and concentratingCompound 8b (4.5 g) was obtained in 84% yield; LC-MS: [ M + H ]] + ＝702.3。
The third step: synthesis of Compound 8
After dissolving 8b (1000mg, 1.42mmol) and 15mL of DMF in a 25mL single-neck flask, 1000mg of 5% Pd/C was added, hydrogenation was carried out for 2 hours, after completion of the reaction, filtration was carried out, the filtrate was placed in an ice-water bath, DIPEA (248uL, 1.5 mmol) was added, compound M3 (708mg, 1.42mmol) was added, and the mixture was warmed to room temperature and reacted for 1 hour. After the HPLC monitoring reaction is finished, purifying the reaction solution by high performance liquid phase to obtain a preparation solution, and freeze-drying the preparation solution to obtain a product compound 8 (443 mg) with a yield of 36%; LC-MS: [ M-H ]] - ＝856.4。
Example 15:
synthesis of compound 9:
Figure BDA0003564748370000451
the first step is as follows: synthesis of Compound 9a
In a 500mL single-neck flask were added amino-octapolyethylene glycol-carboxyl (10g, 22.7mmol), 20mL of a supernatant solution of DMMF, mcOSu (8.38g, 27.2mmol), DIEA (5.6 mL, 3.4mmol), reacted at room temperature for 2h, and monitored by TLC. After the reaction was completed, the reaction solution was poured into 100mL of water, extracted three times with dichloromethane, the organic phases were combined, washed twice with a saturated sodium chloride solution, and dried over anhydrous sodium sulfate. Filtering, and concentrating under reduced pressure at 45 deg.C to obtain crude product. The crude product was purified by column chromatography on silica gel (DCM: meOH =30: 1-10): [ M-H ]] + ＝633.3。
The second step: synthesis of Compound 9b
In a 500mL single-necked flask, compound 9a (12.0g, 18.9mmol), pentafluorophenol (3.83g, 20.8mmol), DCC (4.28g, 20.8mmol) and THF (50 mL) were added, the reaction was carried out at room temperature for 1h, and TLC was carried out. After completion of the reaction, insoluble matter was filtered off by filtration. The filtrate was concentrated by a water pump under reduced pressure at 45 ℃ to remove the solvent to obtain a residue. The residue was purified by column chromatography on silica gel (PE: EA =5: 1-2): [ M + H ]] + ＝801.3。
The third step: synthesis of Compound 9
To compound 1c (220mg, 0.5mmol) was added 10mL DMF, added compound 9b (500mg, 0.6 mmol) in an ice water bath, cooled to 0 deg.C, added compound DIPEA (124uL, 0.75mmol), reacted at room temperature for 1h, and the reaction monitored by HPLC. After the reaction, the reaction solution was purified by preparative high performance liquid chromatography to give preparative liquid, which was lyophilized after removal of acetonitrile under reduced pressure to give compound 9 (430 mg) with yield 83% LCMS: [ M-H ]] + ＝1038.5。
Example 16:
synthesis of compound 10:
Figure BDA0003564748370000461
compound 2 (50mg, 0.081mmol), compound B (55.8mg, 0.081mmol), pyBOP (50.4mg, 0.097mmol), HOBt (13.1mg, 0.097mmol) and 5mL of DMF were charged in a 25mL single-neck flask, DIPEA (15.5mg, 0.12mmol) was added under ice-water bath, and the mixture was allowed to warm to room temperature for 2 hours. After the HPLC monitoring reaction was completed, the reaction solution was purified by high performance liquid chromatography to obtain a preparation solution, which was lyophilized to obtain compound 10 (80 mg) with a yield of 76.7%, LC-MS: [ M + H ]] + ＝1287.5。
Example 17:
synthesis of compound 11:
Figure BDA0003564748370000462
compound 9 (84.2mg, 0.081mmol), compound B (55.8mg, 0.081mmol), pyBOP (50.4mg, 0.097mmol), HOBt (13.1mg, 0.097mmol) and 5mL of DMF were charged into a 25mL single-neck flask, DIPEA (15.5mg, 0.12mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature for 2 hours. After the HPLC monitoring reaction is finished, purifying the reaction solution by high performance liquid chromatography to obtain a preparation solution of a product, and freeze-drying the preparation solution to obtain a compound 11 (100 mg) with the yield of 70.9%, wherein the mass ratio of LC-MS: [ M/2+ H] + ＝855.86。
Example 18:
synthesis of compounds 12A and 12B:
1. preparation of Compound 12A
Figure BDA0003564748370000471
Compound 4A (51mg, 0.081mmol), compound B (55.8mg, 0.081mmol), pyBOP (50.4mg, 0.097mmol), HOBt (13.1mg, 0.097mmol) and 5mL of DMF were charged in a 25mL single-neck flask, DIPEA (15.5mg, 0.12mmol) was added under ice-water bath, and the mixture was allowed to warm to room temperature for 2 hours. After the HPLC monitoring reaction was completed, the reaction solution was purified by high performance liquid chromatography to obtain a preparation solution, which was lyophilized to obtain compound 12A (80 mg) with a yield of 76.2%, LC-MS: [ M + H ]] + ＝1301.5。
2. Preparation of Compound 12B
Figure BDA0003564748370000472
Referring to the synthesis method of the compound 12A, the compound 12B, LC-MS: [ M + H ]] + ＝1301.5。
Example 19:
synthesis of compounds 13A and 13B:
Figure BDA0003564748370000473
in a 50mL single-neck flask, compound 6 (99.3mg, 0.145mmol), compound B (100mg, 0.145mmol), pyBOP (90.5mg, 0.174mmol), HOBt (23.5mg, 0.174mmol) and 10mL DMF were added, DIPEA (28.2mg, 0.218mmol) was added under ice-water bath, and the mixture was warmed to room temperature for 2h. After the HPLC monitoring reaction is finished, purifying the reaction solution by high performance liquid chromatography to obtain preparation solutions of a compound 13A and a compound 13B, respectively lyophilizing the preparation solutions to obtain a compound 13A (90 mg) and a compound 13B (80 mg), and performing LC-MS: [ M + H ]] + ＝1355.5。
Example 20:
synthesis of compound 14:
Figure BDA0003564748370000481
the first step is as follows: synthesis of Compound 14a
In a 50mL single neck flask were added compound 1 (500mg, 0.62mmol), compound B (427.3mg, 0.62mmol), pyBOP (385mg, 0.74mmol), HOBt (100mg, 0.74mmol) and 15mL DMF, DIPEA (120mg, 0.93mmol) was added under ice water bath, and the mixture was allowed to warm to room temperature for 2h. After the HPLC monitoring reaction was completed, the reaction solution was purified by high performance liquid chromatography to obtain a preparation of compound 14a, and the preparation was lyophilized to obtain solid compound 14a (550 mg) with a yield of 60%, LC-MS: [ M + H ]] + ＝1474.6。
The second step: synthesis of Compound 14
Compound 14a (200mg, 0.136mmol), zinc bromide (612mg, 2.72mmol) and 6mL nitromethane were added to a 25mL single vial and reacted at room temperature for 1h. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain a preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 14 (100 mg) with the yield of 56%, LC-MS: [ M + H ]] + ＝1318.5。
Example 21:
synthesis of compounds 15A and 15B:
1. preparation of Compound 15A
Figure BDA0003564748370000491
The first step is as follows: synthesis of Compound 15a
In a 50mL single neck flask were added compound 3A (507mg, 0.62mmol), compound B (427.3mg, 0.62mmol), pyBOP (385mg, 0.74mmol), HOBt (100mg, 0.74mmol) and 15mL DMF, DIPEA (120mg, 0.93mmol) was added under ice water bath and allowed to warm to room temperature for 2h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high performance liquid chromatography to obtain a preparation of compound 15a, and the preparation was lyophilized to obtain solid compound 15a (650 mg) with a yield of 70%, LC-MS: [ M + H ]] + ＝1488.6。
The second step: synthesis of Compound 15A
Adding into a 25mL single-mouth bottleThe compound 15a (200mg, 0.134mmol), zinc bromide (603.5mg, 2.68mmol) and 6mL nitromethane were added and reacted at room temperature for 1 hour. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product by high performance liquid chromatography to obtain a preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 15A (110 mg) with a yield of 62%, wherein the preparation solution comprises the following components in percentage by weight: [ M + H ]] + ＝1332.5。
2. Preparation of Compound 15B
Figure BDA0003564748370000501
Referring to the synthesis of compound 15A, compound 15b, lc-MS: [ M + H ]] + ＝1332.5。
Example 22:
synthesis of compounds 16A and 16B:
Figure BDA0003564748370000502
the first step is as follows: synthesis of Compounds 16a and 16b
In a 50mL single-neck flask, compound 5 (540mg, 0.62mmol), compound B (427.3mg, 0.62mmol), pyBOP (385mg, 0.74mmol), HOBt (100mg, 0.74mmol) and 15mL DMF were added, DIPEA (120mg, 0.93mmol) was added in an ice-water bath, and the mixture was allowed to warm to room temperature for 2 hours. After the HPLC monitoring reaction is finished, purifying the reaction solution by high performance liquid chromatography to obtain preparation solutions of the compound 16a and the compound 16b, respectively lyophilizing the preparation solutions to obtain the compound 16a (350 mg) and the compound 16b (300 mg), and performing LC-MS: [ M/2+ H] + ＝771.8。
The second step: synthesis of Compound 16A
Figure BDA0003564748370000511
A25 mL single-neck flask was charged with compound 16a (200mg, 0.13mmol), zinc bromide (585.5mg, 2.6mmol) and 6mL nitromethane and reacted at room temperature for 1h. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phaseTo obtain a preparation of the product, which was lyophilized to obtain solid compound 16A (120 mg) in 66% yield, LC-MS: [ M + H ]] + ＝1386.4。
The third step: synthesis of Compound 16B
Figure BDA0003564748370000512
Compound 16b (200mg, 0.13mmol), zinc bromide (585.5mg, 2.6mmol) and 6mL nitromethane were added to a 25mL single-neck flask and allowed to react at room temperature for 1h. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 16B (115 mg) with the yield of 64%, wherein the preparation solution comprises the following components in percentage by weight: [ M + H ]] + ＝1386.4。
Example 23:
synthesis of compounds 17A and 17B:
Figure BDA0003564748370000521
the first step is as follows: synthesis of Compounds 17a and 17b
In a 50mL single neck flask were added compound 7 (523mg, 0.62mmol), compound B (427.3mg, 0.62mmol), pyBOP (385mg, 0.74mmol), HOBt (100mg, 0.74mmol) and 15mL DMF, DIPEA (120mg, 0.93mmol) was added under ice water bath, and the mixture was allowed to warm to room temperature for 2h. After the HPLC monitoring reaction is finished, purifying the reaction solution by high performance liquid chromatography to obtain preparation solutions of a compound 17a and a compound 17b, respectively lyophilizing the preparation solutions to obtain a compound 17a (320 mg) and a compound 17b (280 mg), and performing LC-MS: [ M/2+ H] + ＝757.8。
The second step is that: synthesis of Compound 17A
Figure BDA0003564748370000522
In a 25mL single-neck flask, compound 17a (200mg, 0.132mmol), zinc bromide (594.5mg, 2.64mmol) and 6mL nitromethane were added and reacted at room temperature for 1h. After the reaction was completed, the reaction was monitored by HPLC,concentrating under reduced pressure to remove the solvent to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain a preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 17A (120 mg) with the yield of 67%, LC-MS: [ M + H ]] + ＝1358.5。
The third step: synthesis of Compound 17B
Figure BDA0003564748370000531
In a 25mL single-neck flask, compound 17b (200mg, 0.132mmol), zinc bromide (594.5mg, 2.64mmol) and 6mL nitromethane were added and reacted at room temperature for 1h. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. And purifying the crude product by using a high performance liquid phase to obtain a preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 17B (116 mg) with the yield of 65 percent, wherein the preparation solution comprises the following components in percentage by weight: [ M + H ]] + ＝1358.5。
Example 24:
synthesis of compounds 18A and 18B:
Figure BDA0003564748370000532
the first step is as follows: synthesis of Compounds 18a and 18b
Compound 8 (532mg, 0.62mmol), compound B (427.3mg, 0.62mmol), pyBOP (385mg, 0.74mmol), HOBt (1000.74mmol) and 15mL DMF were added to a 50mL single neck flask, DIPEA (120mg, 0.93mmol) was added to the flask under an ice water bath, and the mixture was allowed to warm to room temperature for 2 hours. After the reaction is monitored by HPLC, the reaction solution is purified by high performance liquid chromatography to obtain preparation solutions of a compound 18a and a compound 18b, the preparation solutions are respectively lyophilized to obtain a compound 18a (300 mg) and a compound 18b (280 mg), and LC-MS: [ M/2+ H] + ＝764.8。
The second step is that: synthesis of Compound 18A
Figure BDA0003564748370000541
In a 25mL single-necked flask, compound 18a (200mg, 0.131mmol), zinc bromide (590.0 mg, 2.62mm)ol) and 6mL nitromethane at room temperature for 1h. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain a preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 18A (125 mg) with the yield of 70%, LC-MS: [ M + H ]] + ＝1372.5。
The third step: synthesis of Compound 18B
Figure BDA0003564748370000542
Compound 18b (200mg, 0.131mmol), zinc bromide (590.0 mg, 2.62mmol) and 6mL nitromethane were added to a 25mL single-necked flask and reacted at room temperature for 1h. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product by high performance liquid chromatography to obtain a preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 18B (120 mg), wherein the yield is 67%, and the content of LC-MS: [ M + H ]] + ＝1372.5。
Example 25:
synthesis of compound 19:
Figure BDA0003564748370000551
the first step is as follows: synthesis of Compound 19a
In a 50mL single neck flask, compound 1 (500mg, 0.62mmol), compound C (412.4mg, 0.62mmol), pyBOP (385mg, 0.74mmol), HOBt (100mg, 0.74mmol) and 15mL DMF were added, DIPEA (120mg, 0.93mmol) was added under ice water bath, and the mixture was allowed to warm to room temperature for 2h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high performance liquid chromatography to obtain a preparation of compound 19a, which was lyophilized to obtain solid compound 19a (600 mg) with a yield of 66%, LC-MS: [ M + H ]] + ＝1450.6。
The second step is that: synthesis of Compound 19
A25 mL single-necked flask was charged with compound 19a (200mg, 0.138mmol), zinc bromide (621mg, 2.76mmol) and 6mL of nitromethane, and reacted at room temperature for 1 hour. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain a product preparation solution,the preparation was lyophilized to give compound 19 (120 mg) as a solid in 67% yield, LC-MS: [ M + H ]] + ＝1294.5。
Example 26:
synthesis of compound 20A and compound 20B:
1. preparation of Compound 20A
Figure BDA0003564748370000561
The first step is as follows: synthesis of Compound 20a
In a 50mL single neck flask, compound 3A (507mg, 0.62mmol), compound C (412.4mg, 0.62mmol), pyBOP (385mg, 0.74mmol), HOBt (100mg, 0.74mmol) and 15mL DMF were added, DIPEA (120mg, 0.93mmol) was added under ice water bath, and the mixture was allowed to warm to room temperature for 2 hours. After the HPLC monitoring reaction was completed, the reaction solution was purified by high performance liquid chromatography to obtain a preparation of compound 20a, which was lyophilized to obtain solid compound 20a (560 mg) with a yield of 62%, LC-MS: [ M + H ]] + ＝1464.6。
The second step: synthesis of Compound 20A
A25 mL single-necked flask was charged with compound 20a (200mg, 0.136mmol), zinc bromide (614.8mg, 2.73mmol) and 6mL of nitromethane, and reacted at room temperature for 1 hour. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain a preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 20A (120 mg) with the yield of 67%, LC-MS: [ M + H ]] + ＝1308.5。
2. Preparation of Compound 20B
Figure BDA0003564748370000571
Referring to the synthesis method of the compound 20A, the compound 20B is obtained, LC-MS: [ M + H ]] + ＝1308.5。
Example 27:
synthesis of compounds 21A and 21B:
Figure BDA0003564748370000572
the first step is as follows: synthesis of Compounds 21a and 21b
Compound 5 (540mg, 0.62mmol), compound C (412.4mg, 0.62mmol), pyBOP (385mg, 0.74mmol), HOBt (100mg, 0.74mmol) and 15mL of DMF were charged into a 50mL single-neck flask, DIPEA (120mg, 0.93mmol) was added thereto in an ice-water bath, and the mixture was allowed to warm to room temperature for 2 hours. After the HPLC monitoring reaction is finished, the reaction solution is purified by high performance liquid phase to obtain preparation solutions of the compound 21a and the compound 21b, the preparation solutions are respectively lyophilized to obtain the compound 21a (330 mg) and the compound 21b (300 mg), and LC-MS: [ M/2+ H] + ＝759.8。
The second step is that: synthesis of Compound 21A
Figure BDA0003564748370000581
In a 25mL single-neck flask, compound 21a (200mg, 0.132mmol), zinc bromide (594.5mg, 2.64mmol) and 6mL nitromethane were added and reacted at room temperature for 1h. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain a preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 21A (125 mg) with the yield of 69%, LC-MS: [ M + H ]] + ＝1362.4。
The third step: synthesis of Compound 21B
Figure BDA0003564748370000582
In a 25mL single-neck flask, compound 21b (200mg, 0.132mmol), zinc bromide (594.5mg, 2.64mmol) and 6mL nitromethane were added and reacted at room temperature for 1h. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 21B (112 mg) with a yield of 62%, wherein the preparation solution comprises the following components in percentage by weight: [ M + H ]] + ＝1362.4。
Example 28:
synthesis of compounds 22A and 22B:
Figure BDA0003564748370000591
the first step is as follows: synthesis of Compounds 22a and 22b
In a 50mL single neck flask were added compound 7 (523mg, 0.62mmol), compound C (412.4mg, 0.62mmol), pyBOP (385mg, 0.74mmol), HOBt (100mg, 0.74mmol) and 15mL DMF, DIPEA (120mg, 0.93mmol) was added under ice water bath, and the mixture was allowed to warm to room temperature for 2h. After the reaction is monitored by HPLC, the reaction solution is purified by high performance liquid chromatography to obtain preparation solutions of a compound 22a and a compound 22b, the preparation solutions are respectively lyophilized to obtain a compound 22a (330 mg) and a compound 22b (300 mg), and LC-MS: [ M + H ]] + ＝1490.6。
The second step is that: synthesis of Compound 22A
Figure BDA0003564748370000592
Compound 22a (200mg, 0.134mmol), zinc bromide (603.5mg, 2.68mmol) and 6mL of nitromethane were added to a 25mL single-necked flask and reacted at room temperature for 1 hour. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain a preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 22A (120 mg) with the yield of 67%, LC-MS: [ M + H ]] + ＝1334.5。
The third step: synthesis of Compound 22B
Figure BDA0003564748370000601
Compound 22b (200mg, 0.134mmol), zinc bromide (603.5mg, 2.68mmol) and 6mL of nitromethane were added to a 25mL single-necked flask and reacted at room temperature for 1 hour. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain a preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 22B (115 mg) with a yield of 65%, wherein the preparation solution comprises the following components in percentage by mass: [ M + H ] + =1334.5.
Example 29:
synthesis of compounds 23A and 23B:
Figure BDA0003564748370000602
the first step is as follows: synthesis of Compounds 23a and 23b
A50 mL single-neck flask was charged with Compound 8 (532mg, 0.62mmol), compound C (412.4mg, 0.62mmol), pyBOP (385mg, 0.74mmol), HOBt (100mg, 0.74mmol) and 15mL DMF, and then DIPEA (120mg, 0.93mmol) was added under ice-water bath and allowed to warm to room temperature for 2h. After the reaction is monitored by HPLC, the reaction solution is purified by high performance liquid chromatography to obtain preparation solutions of a compound 23a and a compound 23b, the preparation solutions are respectively lyophilized to obtain a compound 23a (320 mg) and a compound 23b (300 mg), and LC-MS: [ M/2+ H] + ＝752.8。
The second step is that: synthesis of Compound 23A
Figure BDA0003564748370000611
Compound 23a (200mg, 0.133mmol), zinc bromide (598.5mg, 2.66mmol) and 6mL nitromethane were added to a 25mL single-necked flask and reacted at room temperature for 1h. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain a preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 23A (122 mg) with a yield of 68%, LC-MS: [ M + H ]] + ＝1348.5。
The third step: synthesis of Compound 23B
Figure BDA0003564748370000612
Compound 23b (200mg, 0.133mmol), zinc bromide (598.5mg, 2.66mmol) and 6mL nitromethane were added to a 25mL single-neck flask and reacted at room temperature for 1h. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain a preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 23B (115 mg) with a yield of 64%, wherein the preparation solution comprises the following components in percentage by mass: [ M + H ]] + ＝1348.5。
Example 30:
synthesis of compound 24:
Figure BDA0003564748370000621
the first step is as follows: synthesis of Compound 24a
Compound 1 (500mg, 0.62mmol), compound D (396.3mg, 0.62mmol), pyBOP (385mg, 0.74mmol), HOBt (100mg, 0.74mmol) and 15mL DMF were added to a 50mL single-neck flask, DIPEA (120mg, 0.93mmol) was added under an ice-water bath, and the mixture was allowed to warm to room temperature for 2 hours. After the HPLC monitoring reaction was completed, the reaction solution was purified by high performance liquid chromatography to obtain a preparation of compound 24a, and the preparation was lyophilized to obtain solid compound 24a (600 mg) with a yield of 68%, LC-MS: [ M + H ]] + ＝1424.6。
The second step is that: synthesis of Compound 24
Compound 24a (200mg, 0.14mmol), zinc bromide (631mg, 2.8mmol) and 6mL of nitromethane were charged in a 25mL single-neck flask and reacted at room temperature for 1 hour. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain a preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 24 (120 mg) with a yield of 68%, wherein the preparation solution comprises the following components in percentage by mass: [ M + H ]] + ＝1268.4。
Example 31:
synthesis of compound 25A and compound 25B:
1. preparation of Compound 25A
Figure BDA0003564748370000631
The first step is as follows: synthesis of Compound 25a
In a 50mL single vial was added compound 3A (507mg, 0.62mmol), compound D (396.3mg, 0.62mmol), pyBOP (385mg, 0.74mmol), HOBt (100mg, 0.74mmol) and 15mL DMF, DIPEA (120mg, 0.93mmol) was added under ice water bath, and the mixture was allowed to warm to room temperature for 2h. After the reaction was completed by HPLC monitoring, the reaction solution was purified by high performance liquid chromatography to obtain a preparation of compound 25a, which was lyophilized to obtain solid compound 25a (560 mg) in 63% yield, LC-MS: [ M ]+H] + ＝1438.6。
The second step is that: synthesis of Compound 25A
A25 mL single-neck flask was charged with compound 25a (200mg, 0.139mmol), zinc bromide (626 mg, 2.78mmol) and 6mL nitromethane, and reacted at room temperature for 1 hour. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain a preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 25A (115 mg) with a yield of 65%, wherein the preparation solution comprises the following components in percentage by weight: m + H + =1282.5.
2. Preparation of Compound 25B
Figure BDA0003564748370000641
Referring to the synthesis of compound 25A, compound 26b, lc-MS: [ M + H ] + =1282.5.
Example 32:
synthesis of compounds 26A and 26B:
Figure BDA0003564748370000642
the first step is as follows: synthesis of Compounds 26a and 26b
In a 50mL single-neck flask, compound 5 (540mg, 0.62mmol), compound D (396.3mg, 0.62mmol), pyBOP (385mg, 0.74mmol), HOBt (100mg, 0.74mmol) and 15mL DMF were added, DIPEA (120mg, 0.93mmol) was added under an ice-water bath, and the mixture was allowed to warm to room temperature for 2 hours. After the reaction is monitored by HPLC, the reaction solution is purified by high performance liquid chromatography to obtain preparation solutions of the compound 26a and the compound 26b, the preparation solutions are respectively lyophilized to obtain the compound 26a (330 mg) and the compound 26b (300 mg), and LC-MS: [ M + H ]] + ＝1492.5。
The second step: synthesis of Compound 26A
Figure BDA0003564748370000651
In a 25mL single-neck flask, compound 26a (200mg, 0.134mmol), zinc bromide (603.5mg, 2.68mmol) were added) And 6mL of nitromethane, and reacting at room temperature for 1h. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain a preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 26A (125 mg) with the yield of 70%, LC-MS: [ M + H ]] + ＝1336.4。
The third step: synthesis of Compound 26B
Figure BDA0003564748370000652
Compound 26b (200mg, 0.134mmol), zinc bromide (603.5mg, 2.68mmol) and 6mL of nitromethane were added to a 25mL single-necked flask and reacted at room temperature for 1 hour. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 26B (112 mg) with a yield of 63%, wherein the preparation solution comprises the following components in percentage by weight: [ M + H ]] + ＝1336.4。
Example 33:
synthesis of compounds 27A and 27B:
Figure BDA0003564748370000661
the first step is as follows: synthesis of Compounds 27a and 27b
Compound 7 (523mg, 0.62mmol), compound D (396.3mg, 0.62mmol), pyBOP (385mg, 0.74mmol), HOBt (100100mg, 0.74mmol) and 15mL DMF were added to a 50mL single-neck flask, DIPEA (120mg, 0.93mmol) was added under an ice-water bath, and the mixture was allowed to warm to room temperature for 2 hours. After the reaction is monitored by HPLC, the reaction solution is purified by high performance liquid chromatography to obtain preparation solutions of the compound 27a and the compound 27b, the preparation solutions are respectively lyophilized to obtain the compound 27a (330 mg) and the compound 27b (300 mg), and LC-MS: [ M + H ]] + ＝1464.6。
The second step is that: synthesis of Compound 27A
Figure BDA0003564748370000662
In a 25mL single-mouth bottleCompound 27a (200mg, 0.136mmol), zinc bromide (614.8mg, 2.73mmol) and 6mL of nitromethane were added thereto and reacted at room temperature for 1 hour. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 27A (125 mg) with the yield of 70%, LC-MS: [ M + H ]] + ＝1308.5。
The third step: synthesis of Compound 27B
Figure BDA0003564748370000671
A25 mL single vial was charged with compound 27b (200mg, 0.136mmol), zinc bromide (614.8mg, 2.73mmol) and 6mL nitromethane and reacted at room temperature for 1h. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain preparation solution, lyophilizing the preparation solution to obtain solid compound 27B (115 mg) with yield of 64%, LC-MS: [ M + H ]] + ＝1308.5。
Example 34:
synthesis of compounds 28A and 28B:
Figure BDA0003564748370000672
the first step is as follows: synthesis of Compounds 28a and 28b
Compound 8 (532mg, 0.62mmol), compound D (396.3mg, 0.62mmol), pyBOP (385mg, 0.74mmol), HOBt (1005mg, 0.74mmol) and 15mL DMF were added to a 50mL single-neck flask, DIPEA (120mg, 0.93mmol) was added under an ice-water bath, and the mixture was allowed to warm to room temperature for 2 hours. After the reaction is monitored by HPLC, the reaction solution is purified by high performance liquid chromatography to obtain preparation solutions of a compound 28a and a compound 28b, the preparation solutions are respectively lyophilized to obtain a compound 28a (320 mg) and a compound 28b (300 mg), and LC-MS: [ M + H ]] + ＝1478.6。
The second step is that: synthesis of Compound 28A
Figure BDA0003564748370000681
Compound 28a (200mg, 0.135mmol), zinc bromide (608mg, 2.7mmol) and 6mL nitromethane were charged in a 25mL single vial and reacted at room temperature for 1h. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain a preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 28A (121 mg) with a yield of 68%, LC-MS: [ M + H ]] + ＝1322.5。
The third step: synthesis of Compound 28B
Figure BDA0003564748370000682
Compound 28b (200mg, 0.135mmol), zinc bromide (608mg, 2.7mmol) and 6mL nitromethane were added to a 25mL single-neck flask and reacted at room temperature for 1h. After the reaction was monitored by HPLC, the solvent was removed by concentration under reduced pressure to obtain a crude product. Purifying the crude product with high performance liquid phase to obtain preparation solution of the product, and freeze-drying the preparation solution to obtain a solid compound 28B (115 mg) with the yield of 64%, wherein the preparation solution comprises the following components in percentage by weight: [ M + H ]] + ＝1322.5。
Example 34:
compound 29 synthesis:
Figure BDA0003564748370000691
the first step is as follows: synthesis of Compounds 29a to 29d
Reference is made to the synthesis of "compound 26" in patent CN102933236A to give compound 29d.
The second step: synthesis of Compound 29e
In a 250mL three-necked flask, compound 29d (3.0g, 5.8mmol) and 50mL of anhydrous THF were added, an ice-water bath was maintained at 0 ℃ under nitrogen protection, sodium borohydride solid (329mg, 8.7mmol) was added, the reaction was carried out at 0 ℃ for 30min, the temperature was raised to room temperature, the reaction was carried out for 2h, and the end point of the reaction was monitored by TLC. After the reaction, the reaction solution was poured into ice water, 50mL of water was slowly added to quench the reaction, 1N diluted HCl was added until no bubbling occurred, ethyl acetate was added for extraction (60mL. About.3), the organic phases were combined and saturated chlorination was performedWashing with sodium solution twice, drying with anhydrous sodium sulfate, filtering, and concentrating to obtain crude product. The crude was purified by column chromatography (DCM/MeOH =20, 1-5) to give compound 29e (2.1 g, yellow solid) in 73.8% yield, LCMS: [ M + H ]] + ＝491.2。
The third step: synthesis of Compound 29f
Compound 29e (2.1g, 4.3mmol), TBSCl (1.3g, 8.6mmol), imidazole (580mg, 8.6mmol), 15mL of anhydrous DMF was added to a 100mL single-neck flask and dissolved, and the reaction was carried out at room temperature for 3h, and the end point of the reaction was monitored by TLC. After the reaction was complete the reaction was poured into 30mL of water, extracted with DCM (25ml × 3), the organic phases were combined, washed with water and brine, dried over anhydrous sodium sulphate and evaporated under reduced pressure to give the crude product. The crude product was purified by column chromatography (PE/EA =5: 1-1): [ M + H ]] + ＝605.3。
The fourth step: synthesis of Compound 29g
Compound 29f (2.4 g,4.0 mmol) and 30mL of 5% formic acid/methanol solution were added to a 100mL single-neck flask, cooled to 5 ℃ in an ice bath, and then zinc powder (5.2 g,80.0 mmol) was added slowly with stirring, warmed to room temperature for reaction for 1h, and the end of the reaction was monitored by TLC. Filtering while hot after the reaction is finished, washing a filter cake with a small amount of methanol, adjusting the pH of the filtrate to 7 by using a saturated sodium bicarbonate solution, then concentrating under reduced pressure at 45 ℃, removing the solvent to obtain a brown yellow oily substance, adding 60mL of DCM, separating an organic layer, washing with saturated salt water once, drying with anhydrous sodium sulfate, filtering, and concentrating under reduced pressure at 45 ℃ to obtain a crude product. The crude product was purified by column chromatography (PE: EA = 1-1: [ M + H ]] + ＝575.3。
The fifth step: synthesis of Compound 29h
A50 mL single vial was charged with 29g (1.9g, 3.3mmol) of the compound, fmoc-VCPABO-PNP (2.5g, 3.3mmol), 15mL of DMF solution, DIPEA (827uL, 5.0 mmol) was added, the reaction was allowed to proceed at room temperature for 2h, and the end of the reaction was monitored by HPLC. After the reaction is finished, the reaction solution is subjected to high performance liquid chromatography purification to obtain a product preparation solution, and then freeze-drying is carried out to obtain a compound 29h (3.3 g, light yellow solid), wherein the yield is 83.1%, and the LCMS: [ M + H ]] + ＝1202.5。
And a sixth step: synthesis of Compound 29i
Compound 29h (3.3 g,2.7 mmol), 8mL of LTHF and 8mL of water were added to a 50mL single-neck flask, 20mL of glacial acetic acid was added, the reaction was carried out at room temperature, and the end of the reaction was monitored by HPLC. After the reaction was completed, the reaction solution was slowly dropped into 400mL of a saturated sodium bicarbonate solution, extracted with ethyl acetate (100ml × 3), and the organic phases were combined, washed with water and brine, dried over anhydrous sodium sulfate and evaporated under reduced pressure to give a crude product. The crude was purified by column chromatography (DCM/MeOH = 10) to give compound 29i (2.1 g, light yellow solid) in 71.1% yield, LCMS: [ M + H ]] + ＝1088.5。
The seventh step: synthesis of Compound 29j
A100 mL three-necked flask was charged with compound 29i (2.1g, 1.9mmol), 40mL anhydrous DCM was dissolved, dess-Martin periodinane (0.88g, 2.1mmol) was added under nitrogen, the reaction was carried out at room temperature for 4h, and the reaction endpoint was monitored by HPLC. After the reaction is finished, filtering, washing the filtrate by using a saturated sodium bicarbonate solution, water and a saturated sodium chloride solution, drying by using anhydrous sodium sulfate, and concentrating to obtain a crude product. The crude product was purified by high performance liquid chromatography, and the product preparation was lyophilized to give compound 29j (1.6 g, white solid) with 77.6% yield, LCMS: [ M + H ]] + ＝1086.5。
The eighth step: synthesis of Compound 29k
A50 mL three-necked flask was charged with 29j (1.6 g,1.5 mmol) of compound, 5% barium palladium sulfate (0.29 g), ammonium formate (1.9 g, 30mmol) and 20mL of methanol, dissolved, reacted at 45 ℃ for 2h under nitrogen protection, and the end point of the reaction was monitored by HPLC. After the reaction, filtration was performed, the filtrate was concentrated, then, 20mL of DCM and 20mL of water were added, liquid separation was performed after stirring, the aqueous layer was extracted once more with DCM, the organic layers were combined, washed once with a saturated aqueous sodium chloride solution, liquid separation was performed, drying was performed with anhydrous sodium sulfate, and concentration was performed to obtain a crude product. The crude product was purified by high performance liquid chromatography to give a preparation of the product, which was lyophilized to give compound 29k (1.3 g, off-white solid) in 93.3% yield, LCMS: [ M + H ]] + ＝996.4。
The ninth step: synthesis of Compound 29l
3- ((p-toluenesulfonyloxy) methyl) bicyclo [1.1.1 ] is added to a 100mL single-neck bottle]Pentane-1-carboxylic acid benzyl ester (0.61g, 1.2mmol)Compound 29k (1.3g, 1.3mmol) and 20mL DMF were dissolved with stirring, cesium carbonate (0.78g, 2.4mmol) was added, after completion of the addition, the temperature was raised to 60 ℃ under nitrogen protection for 4h, and the end of the reaction was monitored by TLC. Adding 40mL of water into the reaction solution, extracting with 30mL of EA for 2 times, combining organic layers, washing with water and saturated saline solution in sequence once respectively, separating the organic layer, drying with anhydrous sodium sulfate, filtering, concentrating under reduced pressure at 45 ℃ to obtain a crude product, purifying the crude product with a high performance liquid phase to obtain a preparation solution of a product, and freeze-drying to obtain 29l of a compound (1.2 g, a white-like solid) with the yield of 84.0%, LCMS: [ M + H ]] + ＝1210.5。
The tenth step: synthesis of Compound 29m
A50 mL three-necked flask was charged with 29l (1.2 g,1.0 mmol) of compound, 5% palladium barium sulfate (0.24 g), ammonium formate (1.3 g,20 mmol), 20mL of methanol, dissolved, reacted at 45 ℃ for 2h under nitrogen protection, and the end of the reaction was monitored by HPLC. After the reaction, filtration, filtrate concentration, adding 20mL DCM and 20mL water, stirring uniformly, separating liquid, extracting the water layer with DCM once more, combining organic layers, washing with saturated sodium chloride aqueous solution once, separating liquid, drying with anhydrous sodium sulfate, and concentrating to obtain a crude product. The crude product was purified by high performance liquid chromatography to give a preparation of the product, which was lyophilized to give compound 29m (0.92 g, off-white solid) in 82.5% yield, LCMS: [ M + H ]] + ＝1120.5。
The eleventh step: synthesis of Compound 29n
A100 mL single vial was charged with compound 29m (920mg, 0.82mmol) and 15mL of DMF, and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (496mg, 2.6 mmol) and DMAP (313mg, 2.6 mmol), N, were added sequentially with stirring at room temperature 2 Stirring at room temperature for 1h, and adding compound CBI (refer to patent CN 109180681A) (376mg, 1.0 mmol), N 2 The reaction was left overnight at room temperature and HPLC showed the starting material to be essentially complete. 40mL of water was added to the reaction mixture, and the mixture was extracted 2 times with 30mL of DCM, the organic layers were combined, washed with water and saturated brine in this order once each, the organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 40 ℃ to obtain a crude product. The crude product was purified by high performance liquid chromatography to give a preparation of the product, which was lyophilized to give compound 29n (874 mg, off-white solid) in 74.5% yield, LCMS: [ M + H ]] + ＝1439.5。
A twelfth step: synthesis of Compound 29o
A50 mL three-necked flask was charged with compound 29n (874mg, 0.60mmol), 5% barium palladium sulfate (174 mg), ammonium formate (390mg, 6 mmol), 20mL methanol, reacted at 45 ℃ for 2h under nitrogen, and the end point of the reaction was monitored by HPLC. After the reaction, filtration was performed, the filtrate was concentrated, then, 20mL of DCM and 20mL of water were added, liquid separation was performed after stirring, the aqueous layer was extracted once more with DCM, the organic layers were combined, washed once with a saturated aqueous sodium chloride solution, liquid separation was performed, drying was performed with anhydrous sodium sulfate, and concentration was performed to obtain a crude product. The crude product was purified by high performance liquid chromatography to give a preparation of the product, which was lyophilized to give compound 29o (667 mg, off-white solid) in 82.4% yield, LCMS: [ M + H ]] + ＝1349.5。
And a thirteenth step of: synthesis of Compound 29p
A50 mL single vial was charged with compound 29o (667mg, 0.49mmol), 20% piperidine in DMF (15 mL), reacted at room temperature for 2h, and the end point monitored by HPLC. After the reaction is finished, filtering, purifying the filtrate by high performance liquid phase to obtain a product preparation solution, and freeze-drying to obtain a compound 29p (428 mg, off-white solid) with the yield of 77.5 percent, LCMS: [ M + H ]] + ＝1127.5。
A fourteenth step of: synthesis of Compound 29
Compound 29p (428mg, 0.38mmol), DIPEA (156uL, 0.95mmol), mcOSu (166mg, 0.57mmol), 20mL DMF were dissolved in a 50mL single-necked flask, reacted at room temperature for 2h, and the end point of the reaction was monitored by HPLC. After the reaction is finished, the reaction solution is purified by high performance liquid chromatography to obtain a product preparation solution, and the product preparation solution is lyophilized to obtain a compound 29 (386 mg, white-like solid), wherein the yield is 77.0%, and the content of LCMS: [ M + H ]] + ＝1320.5。
Example 35:
synthesis of Compound 30:
Figure BDA0003564748370000731
the first step is as follows: synthesis of Compound 30a
A50 mL single-necked flask was charged with compound 29p (560mg, 0.50mmol), DIPEA (161mg, 1.25mmol), and compound M3(423mg, 0.75mmol), 10ml of DMF was dissolved, the reaction was carried out at room temperature for 2h, and the end point of the reaction was monitored by HPLC. After the reaction is finished, the reaction solution is purified by high performance liquid phase to obtain a product preparation solution, and the product preparation solution is lyophilized to obtain a compound 30a (622 mg, off-white solid), wherein the yield is 82.6 percent, and LCMS: [ M + H ]] + ＝1507.6。
The second step is that: synthesis of Compound 30
Adding compound 30a (622mg, 0.41mmol), zinc bromide (922mg, 4.1mmol) and 10mL of nitromethane into a 50mL single-mouth bottle, dissolving, reacting at 25 ℃ for 45min, monitoring the reaction end point by HPLC, removing the solvent at 40 ℃ by decompression after the reaction is finished to obtain a concentrate, purifying by high performance liquid phase to obtain a product preparation solution, and freeze-drying to obtain compound 30 (260 mg, white-like solid), wherein the yield is 48%, LCMS: [ M + H ]] + ＝1322.5。
Example 36:
compound 31 synthesis:
Figure BDA0003564748370000732
Figure BDA0003564748370000741
the first step is as follows: synthesis of Compound 31a
In a 100mL single-neck flask, 5-bromobenzyl valerate (488mg, 1.8mmol), compound 29k (1.8g, 1.8mmol) and 20mL of DMF were charged, and after stirring and dissolution, potassium carbonate (372mg, 2.7 mmol) was added, and after completion of the charging, the temperature was raised to 60 ℃ for reaction for 4 hours, and the end of the reaction was monitored by HPLC. Adding 40mL of water into the reaction solution, extracting for 2 times by 30mL of EA, combining organic layers, washing with water and saturated saline solution in sequence, separating the organic layers, drying with anhydrous sodium sulfate, filtering, concentrating under reduced pressure at 45 ℃ to obtain a crude product, purifying the crude product by high performance liquid phase to obtain a product preparation solution, and freeze-drying to obtain a compound 31a (1.8 g, white-like solid), wherein the yield is 84.0%, and LCMS: [ M + H ]] + ＝1186.5。
The second step: synthesis of Compound 31b
In a 50mL three-necked flask, compound 31a (1.8g, 1.0mmol), 5% palladium barium sulfate was charged(0.36 g), ammonium formate (0.95g, 15mmol), 20mL methanol dissolved, under nitrogen, 45 ℃ reaction for 2h, HPLC monitoring the end of the reaction. After the reaction, filtration was performed, the filtrate was concentrated, then, 20mL of DCM and 20mL of water were added, liquid separation was performed after stirring, the aqueous layer was extracted once more with DCM, the organic layers were combined, washed once with a saturated aqueous sodium chloride solution, liquid separation was performed, drying was performed with anhydrous sodium sulfate, and concentration was performed to obtain a crude product. The crude product was purified by high performance liquid chromatography to give a preparation of the product, which was lyophilized to give compound 31b (0.85 g, off-white solid) in 78% yield, LCMS: [ M + H ]] + ＝1096.5。
The fourth step: synthesis of Compound 31c
Compound 31b (850mg, 0.85mmol) and 15mL of DMF were charged in a 100mL single-neck flask, and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (514mg, 2.7mmol) and DMAP (324mg, 2.7mmol), N, were added successively under stirring at room temperature 2 Stirring at room temperature for 1h, and adding compound CBI (refer to patent CN 109180681A) (376mg, 1.0 mmol), N 2 The reaction was left overnight at room temperature and HPLC showed the starting material to be essentially complete. 40mL of water was added to the reaction mixture, and the mixture was extracted 2 times with 30mL of DCM, the organic layers were combined, washed with water and saturated brine in this order once each, the organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 40 ℃ to obtain a crude product. The crude product was purified by high performance liquid chromatography to give a preparation of product, which was lyophilized to give compound 31c (941 mg, off-white solid) in 78.2% yield, LCMS: [ M + H ]] + ＝1415.6。
The fifth step: synthesis of Compound 31d
A50 mL three-necked flask was charged with compound 31c (941mg, 0.66mmol), 5% palladium barium sulfate (188 mg), ammonium formate (415mg, 6.6 mmol), 20mL methanol, dissolved, reacted at 45 ℃ for 2h under nitrogen protection, and the end point of the reaction was monitored by HPLC. After the reaction, filtration was performed, the filtrate was concentrated, then, 20mL of DCM and 20mL of water were added, liquid separation was performed after stirring, the aqueous layer was extracted once more with DCM, the organic layers were combined, washed once with a saturated aqueous sodium chloride solution, liquid separation was performed, drying was performed with anhydrous sodium sulfate, and concentration was performed to obtain a crude product. The crude product was purified by high performance liquid chromatography to give a preparation of product, which was lyophilized to give compound 31d (735 mg, off-white solid) in 84.0% yield, LCMS: [ M + H ]] + ＝1325.5。
And a sixth step: synthesis of Compound 31e
A50 mL single vial was charged with compound 31d (735mg, 0.55mmol), 20% piperidine DMF solution (15 mL), reacted at room temperature for 2h, and the end of the reaction was monitored by HPLC. After the reaction is finished, filtering, purifying the filtrate by high performance liquid phase to obtain a product preparation solution, and freeze-drying to obtain a compound 31e (428 mg, off-white solid), wherein the yield is 86.5%, and LCMS: [ M + H ]] + ＝1103.5。
The seventh step: synthesis of Compound 31f
Compound 31e (524mg, 0.48mmol), DIPEA (168mg, 1.30mmol), compound M3 (441mg, 0.78mmol), and 10mL DMF were dissolved in a 50mL single vial, reacted at room temperature for 2h, and the end point of the reaction was monitored by HPLC. After the reaction is finished, purifying the reaction solution by high performance liquid chromatography to obtain a product preparation solution, and lyophilizing to obtain a compound 31f (549 mg, white-like solid), wherein the yield is 77.2 percent, and the LCMS: [ M + H ]] + ＝1483.6。
The eighth step: synthesis of Compound 31
Adding a compound 31f (549mg, 0.37mmol), zinc bromide (832mg, 3.7mmol) and 10mL of nitromethane into a 50mL single-mouth bottle, dissolving, reacting at 25 ℃ for 45min, monitoring the reaction end point by HPLC (high performance liquid chromatography), removing the solvent at 40 ℃ under reduced pressure after the reaction is finished to obtain a concentrate, purifying by using a high performance liquid phase to obtain a product preparation solution, and freeze-drying to obtain a compound 31 (213 mg, white-like solid), wherein the yield is 43.5%, LCMS: [ M + H ]] + ＝1326.5。
Example 37:
compound 32 synthesis:
Figure BDA0003564748370000751
the first step is as follows: synthesis of Compound 32a
In a 50mL single-neck flask, the compound N-propargylmaleimide (500mg, 3.7 mmol), N 3 -PEG 4 -OH (811mg, 3.7 mmol), 10mL DMF and 5mL water, and CuSO 4 ·5H 2 O (1.01g, 4.1mmol), sodium ascorbate (806mg, 4.1mmol), reaction at room temperature 1h, and monitoring the end point of the reaction by HPLC. After the reaction is finished, the reaction liquid passes through the high-efficiency liquidPhase prep purification gave product prep which was lyophilized to give compound 32a (1.06 g) in 81% yield, LCMS: [ M + H ]] + ＝355.1。
The second step is that: synthesis of Compound 32
A50 mL three-necked flask was charged with 32a (20mg, 0.056 mmol), A (19.8mg, 0.028mmol), triphenylphosphine (29.6mg, 0.11mmol) and 8mL DMF, and then the mixture was cooled in a nitrogen atmosphere, and diisopropyl azodicarboxylate (28uL, 0.11mmol) was added dropwise to the flask over an ice-water bath, and then the mixture was heated to room temperature for reaction and monitored by HPLC. After the reaction, the preparation was purified and lyophilized to give compound 32 (18 mg) in 63% yield, LC-MS: [ M + H ]] + ＝1040.4。
Example 38:
synthesis of Compound 33:
Figure BDA0003564748370000761
the first step is as follows: synthesis of Compound 33a
The compound is prepared according to the synthesis method of the compound 22 in the patent CN 111686259A.
The second step is that: synthesis of Compound 33c
Compound 33a (500mg, 2.0 mmol) was added to a 50mL single-neck flask, dissolved in 8mL of DMF, added at 300mg5% by weight in terms of Pd/C, hydrogenated for 2h, and the end of the reaction was monitored by TLC. After the reaction is finished, the DMF solution of the compound 32b is obtained by filtration and is used for standby.
To the above DMF solution were added the compound Mc-tripeptide (992mg, 2.0mmol), pyBop (1.3g, 2.5mmol), HOBt (339mg, 2.5mmol) and DIEA (415uL, 2.5mmol), and the mixture was reacted at room temperature for 2h, and the end of the reaction was monitored by HPLC. And (2) purifying the reaction solution by high performance liquid chromatography to obtain a product preparation solution, and freeze-drying to obtain a compound 33c (580 mg) with a yield of 52%, wherein the ratio of LC-MS: [ M + H ]] + ＝559.2。
The third step: synthesis of Compound 33
In a 50mL three-necked flask, compound 33c (20mg, 0.035mmol), compound A (12.6mg, 0.018mmol), triphenylphosphine (18.4mg, 0.07mmol) and 5mL DMF were added, and diisopropyl azodicarboxylate (14.2uL, 0.07mmol) was added dropwise in an ice-water bath under nitrogen protection07 mmol), after dropping, the reaction was warmed to room temperature and monitored by HPLC. After the reaction is finished, the preparation purification is carried out, and the compound 33 (15 mg) is obtained by freeze-drying, the yield is 68 percent, and the LC-MS: [ M + H ]] + ＝1244.5。
Example 39 (comparative):
synthesis of compounds 34 and 35:
Figure BDA0003564748370000771
compound 34 was synthesized according to the synthesis method of "compound 56" in patent CN 105636612A.
Figure BDA0003564748370000772
Compound 35 was prepared according to the literature "ACS Med Chem lett.2016;7, SG3249, tesiline in' 983-987 by a synthetic method.
Example 40:
the following is the sequence of Trastuzumab:
light chains
MDMRVPAQLLGLLLLWLRGARC
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*
Heavy chain
MDMRVPAQLLGLLLLWLRGARC
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG。
Preparation of ligand-drug conjugates:
1) General coupling method
And (3) replacing the antibody molecules with the monomer rate of more than 95% after primary purification into phosphate buffer solution with the concentration of 10mg/mL by using an ultrafiltration centrifugal tube. TCEP was added in an amount of 20 mol times the amount of the antibody molecules, and the reaction was carried out at room temperature for 4 hours to open the interchain disulfide bonds of the antibody. A linker-drug compound (payload) was added in an amount of 20 mol times the amount of the antibody molecules, and the mixture was reacted at room temperature for 2 hours. After the reaction is finished, the solution is changed into PBS by using an ultrafiltration centrifugal tube with the molecular weight cutoff of 30KDa, and the uncoupled payload is removed. The ADC samples after the exchange were filtered using a 0.22 micron sterile filter and ready for use.
2) Determination of ligand-drug conjugate DAR values
Monomer rate detection conditions:
centrifuging the sample at 14000rpm for 5 minutes, and taking the supernatant for sample injection analysis;
the instrument comprises: waters e2695 (2489 UV/Vis);
and (3) chromatographic column: TSKgel G3000SWXL (7.8X 300mm,5 μm);
mobile phase: a:50mM PB,300mM NaCl,200mM Arg,5% IPA, pH 6.5;
isocratic elution of mobile phase a for 30min, flow rate: 0.714mL/min, column temperature 25 ℃, detection wavelength: 280nm.
And DAR detection conditions:
centrifuging the sample at 14000rpm for 5 minutes, and taking the supernatant for sample injection analysis;
the instrument comprises: waters H-class (TUV);
and (3) chromatographic column: proteomix HIC Butyl-NP5 (4.6X 35mm,5 μm);
mobile phase: a:1.5M ammonium sulfate, 0.025M anhydrous sodium phosphate, pH 7.0, B:0.025M sodium phosphate anhydrous, 25% IPA, pH 7.0;
the mobile phase A balances the chromatographic column, and the mobile phases A and B are eluted in a gradient way, and the flow rate is 0.8mL/min; column temperature 25 ℃, detection wavelength: 214nm.
Example 41:
ADC-1 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000791
example 42:
ADC-2 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000801
example 43:
ADC-3 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000802
example 44:
prepared according to the general coupling procedure of example 40 to provide ADC-4:
Figure BDA0003564748370000803
example 45:
ADC-5 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000811
example 46:
ADC-6 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000812
example 47:
ADC-7 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000813
example 48:
ADC-8 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000814
example 49:
ADC-9 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000821
example 50:
ADC-10 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000822
example 51:
ADC-11 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000823
example 52:
ADC-12 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000824
example 53:
ADC-13 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000825
example 54:
ADC-14 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000831
example 55:
ADC-15 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000832
example 56:
prepared according to the general coupling procedure of example 40 to give ADC-16:
Figure BDA0003564748370000833
example 57:
ADC-17 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000834
example 58:
ADC-18 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000841
example 59:
ADC-19 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000842
example 60:
ADC-20 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000843
example 61:
prepared according to the general coupling procedure of example 40 to provide ADC-21:
Figure BDA0003564748370000844
example 62:
ADC-22 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000851
example 63:
ADC-23 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000852
example 64:
prepared according to the general coupling procedure of example 40 to provide ADC-24:
Figure BDA0003564748370000853
example 65:
ADC-25 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000854
example 66:
prepared according to the general coupling procedure of example 40 to provide ADC-26:
Figure BDA0003564748370000861
example 67:
ADC-27 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000862
example 68:
ADC-28 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000863
example 69:
ADC-29 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000864
example 70:
ADC-30 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000871
example 71:
ADC-31 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000872
example 72:
ADC-32 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000873
example 73:
ADC-33 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000874
example 74:
ADC-34 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000881
example 75:
ADC-35 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000882
example 76:
ADC-36 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000883
example 77:
prepared according to the general coupling procedure of example 40 to afford ADC-37:
Figure BDA0003564748370000884
example 78:
ADC-38 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000891
example 79 (control):
ADC-39 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000892
example 80 (control):
ADC-40 was prepared according to the general coupling procedure of example 40:
Figure BDA0003564748370000893
example 81: plasma stability test
1. Operation of
Taking a certain amount of ADC sample, adding the ADC sample into human plasma from which human IgG is removed, repeating three tubes for each ADC, placing the ADC sample in water bath at 37 ℃ for incubation, respectively incubating for 72h and 144h, taking out the ADC sample, adding 100uL of protein A resin (MabSelect SuReTM LX Lot: #10221479GE, washed by PBS) into each tube, shaking and adsorbing for 2h by using a vertical mixer, and washing and eluting to obtain the incubated ADC. The ADC samples incubated for a specified period of time were subjected to RP-HPLC detection.
2. As a result, the
Table 1. Ligand-drug conjugate (ADC) DAR values and monomer ratio data are disclosed.
Figure BDA0003564748370000894
Figure BDA0003564748370000901
Table 2. The present invention discloses ligand-drug conjugate (ADC) plasma stability data.
Figure BDA0003564748370000902
3) Conclusion
As shown in Table 1, the ADC disclosed by the invention has the excellent properties of DAR value (7.5) and high monomer yield (97%).
As shown in table 2, the DAR value can still maintain a higher level after 7 days of incubation in the ADC plasma disclosed in the present invention, demonstrating the excellent stability of the ADC in plasma.
Example 82: in vitro Activity assay
1) Experimental materials
Cell: derived from the cell bank of the Chinese academy of sciences;
tumor cell culture medium: gibco;
FBS：BIOWEST；
2) Preparation of culture Medium
Growth medium (with 10% FBS, penicillin/streptomycin (100U/mL);
detection medium (with 1% FBS, penicillin/streptomycin (100U/mL);
3) Operation of
The ultraviolet lamp of the biological safety cabinet is turned on 30min in advance, and then the air is ventilated for 3min. The growth culture medium, the detection culture medium, the D-PBS and the pancreatin are put into a constant-temperature water bath kettle at 37 ℃ for preheating, then the surface is disinfected by alcohol, and the mixture is put into a biological safety cabinet. Selecting cells with confluence at-80% (logarithmic phase), placing in a biological safety cabinet, sucking away the old culture medium, rinsing with D-PBS, sucking away, digesting with pancreatin for 2-3 min, adding the growth culture medium to stop pancreatin, and centrifuging for 5min at 500 Xg. The supernatant was aspirated off, mixed well with 4mL of assay medium, and 100uL counted (50 uL of cell sap was removed, 50uL of 0.4% Trypan Blue Stain was added and mixed well, and counted after mixing well). The cells were plated according to the number of cells previously set, 80 uL/well at 9In 6-well plate, 80uL of detection medium was added to E11, F11, and G11, and 200uL of DPBS was added to the edge wells for edge sealing. After the plated cells are fully adherent (usually at least 4 hours), the test sample preparation and dilution are performed: preparing 1.0mL of test sample with 2.5. Mu.M (5 XTop Dose) of detection medium, and subpackaging the test sample in the first row of V-type 96-well plate with 200. Mu.L per well; adding 180 mu L of detection culture medium into the subsequent 2 nd to 8 th rows respectively, taking 30 mu L from the first row and adding into the second row, mixing uniformly for 10 times up and down by using a row gun, abandoning the gun head, operating the rest detection concentration points in sequence, and performing 7-time gradient concentration dilution. Adding test samples of gradient concentration to the cells in an amount of 20uL per well while adding only 20uL of the detection medium to column 11, setting 3 multiple wells per concentration, and subsequently placing the 96-well plate in 5% CO 2 And culturing in a cell culture box at 37 ℃ for 5 days.
4) Detection
Taking out MTS reagent after the tested sample acts for 5 days, thawing at normal temperature and in the dark, fully and uniformly swirling, adding 20 mu L of Cell Titer One Solution reagent MTS reagent into the side wall of a hole according to the Cell culture volume of every 100 mu L in a biosafety cabinet, slightly flapping the plate surface to uniformly mix the MTS Solution, putting the mixed MTS Solution into a Cell culture box, and reacting with 5 percent CO 2 And standing and incubating for 2h at 37 ℃ in a dark place. And after the reaction is finished, taking out the 96-well plate, detecting the OD490 nm light absorption value in an enzyme labeling instrument, and recording, sorting and storing data.
5) As a result, the
Table 3: IC50 values for inhibition of in vitro proliferation of N87 tumor cells by antibody drug conjugates and toxins.
Figure BDA0003564748370000911
Figure BDA0003564748370000921
Table 4: IC50 values for in vitro proliferation inhibition of SK-BR-3 tumor cells by antibody drug conjugates and toxins.
Sample IC50(nM)
Compound A 0.52
Compound B1 4.23
Compound C1 8.67
Compound D1 10.33
ADC-3 0.45
ADC-5 0.60
ADC-7 4.55
ADC-12 5.82
ADC-21 6.11
ADC-30 9.05
ADC-39 (control) 26.12
6) Conclusion
As shown in Table 3, the ligand-drug conjugate for HER2 target of the invention has obvious in vitro proliferation inhibition activity on HER2 positive cell N87, and is obviously superior to naked antibody (Trastuzumab) and control group ADC-39.
As shown in Table 4, compared with naked antibody (Trastuzumab) and ADC in a control group, the ADC and the single drug disclosed by the invention also have obvious in-vitro proliferation inhibition activity on HER2 positive cells SK-BR-3.
Example 83: in vivo safety testing
1) Experimental Material
Cell: derived from the cell bank of Chinese academy of sciences;
tumor cell culture medium: gibco;
balb/c-nu nude mice: female, 5-7 weeks (week old mice when tumor cells were inoculated), weighing 18.0-24.0g,180 mice (120 plus 60 spared mice). Purchased from Beijing Wittiulihua laboratory animal technology, inc.;
test and control:
and (3) testing the sample: ADC-3 and ADC-40 were provided by Doudot antibody drug, inc.
Histidine buffer, available from Doudoutt antibody drugs, inc.
0.9% sodium chloride injection: koran pharmaceutical Limited liability company.
2) Cell culture
NCI-H1975 (human non-small cell lung carcinoma adenocarcinoma cells) was cultured in RPMI1640 medium. NCI-H1975 cells in the exponential growth phase were collected, and the RPMI1640 medium was resuspended to an appropriate concentration for subcutaneous tumor inoculation in mice.
3) Animal modeling and random grouping
120 female nude mice were inoculated subcutaneously on the right shoulder at 5X 10 7 An NCI-H1975 cell. Average tumor volume of 170mm 3 On the left and right, they were randomly grouped according to tumor size. Selecting 120 tumor-bearing mice with appropriate tumor volume, and randomly selectingGrouping and starting administration (tail vein injection, administration volume at 0.1mL/10 g). Grouping day is defined as day 0.
4) Preparation of test and reference substances
TABLE 5 formulation of test and control solutions for anti-tumor effect studies in the NCI-H1975 (human non-small cell lung carcinoma adenocarcinoma cell) transplant tumor model.
Figure BDA0003564748370000931
Figure BDA0003564748370000941
Note: mix well before use to ensure the preparation is uniform.
5) Experimental observations and data Collection
In the experimental process, animal experiment operation is carried out according to the requirements of standard operation procedures of screening test in anti-tumor drugs. After tumor inoculation, routine monitoring included tumor growth (tumors measured 2 times per week) and the effect of treatment on the normal behavior of the animals, as exemplified by activity, feeding and drinking profiles, weight gain or loss (body weight measured 2 times per week), eyes, hair follicles, and other abnormalities in the experimental animals. Clinical symptoms observed during the experiment were recorded in the raw data. Tumor volume calculation formula: tumor volume (mm) 3 )＝1/2×(a×b 2 ) (wherein a represents a long diameter and b represents a short diameter). Manual data recording including the measurement of the length and the length of the tumor and the weighing of the animal weight is adopted in the experiment.
6) Results
Table 6: the effect of administration of the antibody drug conjugate (11.25 mg/kg) on body weight in mice with NCI-H1975 transplantable tumors.
Figure BDA0003564748370000942
Figure BDA0003564748370000951
Note: * Mice death was observed in the marker group
7) Conclusion
As shown in Table 6, the invention discloses that the effect of ADC-3 on the body weight of NCI-H1975 tumor-bearing mice at low dose and high dose is obviously smaller than the effect of ADC-40, and the mice death shown in a control group does not occur even under the high dose group, thereby proving that the ADC medicament has a remarkable advantage in safety.

Claims (25)

1. A ligand-drug conjugate of formula I, or a pharmaceutically acceptable salt, deuteride, solvate thereof:
Ab-L-D
(I)
wherein:
ab is a ligand unit selected from an antibody, an antibody fragment, a targeting protein or an Fc-fusion protein;
l is a linking unit of D and Ab;
d is a drug unit selected from the following structures:
Figure FDA0003564748360000011
wherein:
the wavy line indicates the site where the drug is attached to L, and only one of the three sites is present to be attached to L;
R 1 is H, deuterium, OH OR is OR 3 Ether, sulfite SO 3 - Or OSO 3 - Wherein R is 3 Is selected from C 1 -C 10 Linear, branched or cyclic alkyl, alkenyl or alkynyl groups of (a);
double line between N and C
Figure FDA0003564748360000012
Represents a single or double bond, provided that when it is a double bond, N is not attached to L and R 1 Is H; when it is a single bond, N is linked to L, R 1 Selected from OH OR from OR 3 Ether, sulfite SO 3 - Or OSO 3 - Wherein R is 3 Is selected from C 1 -C 10 Linear, branched or cyclic alkyl, alkenyl or alkynyl groups of (a);
R 2 is H or an alkyl substituent;
t is selected from C 2 -C 12 Hydrocarbyl, Z, (C) 1 -C 6 Alkylene) -Z- (C) 1 -C 6 Alkylene group), (C) 1 -C 6 Alkylene) -Z- (C) 1 -C 6 Alkylene) -Z- (C) 1 -C 6 Alkylene group), (C) 1 -C 6 Alkenylene) -Z- (C 1 -C 6 Alkenylene) or (C) 1 -C 6 Alkynylene) -Z- (C 1 -C 6 Alkynylene);
wherein:
z is selected from O, S, NR 4 Aryl or heteroaryl; wherein R is 4 Selected from H, P (O) 3 H 2 Or C (O) NR 5 R 6 (ii) a Wherein R is 5 And R 6 Selected from H, C 1 -C 6 Alkyl, C substituted by one or more F 1 -C 6 Alkyl, or R 5 And R 6 To form a five-or six-membered heterocyclyl group;
alkylene, alkenylene, aryl and heteroaryl groups selected from F, OH, O (C1-C6 alkyl), NH 2 、NHCH 3 、N(CH 3 ) 2 Or C 1 -C 6 Alkyl substituted, wherein alkyl is substituted with one or more F;
y is selected from one or more of H or C 1 -C 4 Alkyl groups of (a);
x is selected from-O-, -N-, -S-, -OC (O) -CR 7 R 8 -(CR 9 R 10 )m-O-、-OC(O)-CR 7 R 8 -(CR 9 R 10 )m-NH-、-OC(O)-CR 7 R 8 -(CR 9 R 10 )m-S-、-NHC(O)-CR 7 R 8 -(CR 9 R 10 )m-O-、-NHC(O)-CR 7 R 8 -(CR 9 R 10 )m-NH-or-NHC (O) -CR 7 R 8 -(CR 9 R 10 )m-S-；
Wherein:
R 7 、R 8 each independently is a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a deuterated alkyl group, a haloalkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group, a substituted aryl group, or a heteroaryl group; or, R 7 、R 8 And the carbon atoms to which they are attached form C 3 -C 6 Cycloalkyl, cycloalkylalkyl, or heterocyclyl;
R 9 、R 10 the same or different, and each independently is a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, or a heterocyclic group; or, R 9 、R 10 And the carbon atom to which they are attached forms C 3 -C 6 Cycloalkyl, cycloalkylalkyl, or heterocyclyl; m is an integer from 0 to 4;
X 1 selected from halogen or OSO 2 R 11 Wherein R is 11 Is selected from H and C 1 -C 4 A phenyl group or a substituted phenyl group.
2. The ligand-drug conjugate of claim 1, or a pharmaceutically acceptable salt, deuteride, solvate thereof, wherein: ab is an antibody, which may form a linkage with the linking unit through a heteroatom thereof, selected from the group consisting of murine, chimeric, humanized, fully human, antibody fragment, bispecific or multispecific antibody.
3. The ligand-drug conjugate of claim 1 or 2, or a pharmaceutically acceptable salt, deuterode, solvate thereof, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of, but not limited to: <xnotran> EGFR VIII , DLL-3 , PSMA , CD70 , MUC16 , ENPP3 , TDGF1 , ETBR , MSLN , TIM-1 , LRRC15 , LIV-1 , CanAg/AFP , cladin 18.2 , Mesothelin , HER2 (ErbB 2) , EGFR , c-MET , SLITRK6 , KIT/CD117 , STEAP1 , SLAMF7/CS1 , NaPi2B/SLC34A2 , GPNMB , HER3 (ErbB 3) , MUC1/CD227 , AXL , CD166 , B7-H3 (CD 276) , PTK7/CCK4 , PRLR , EFNA4 , 5T4 , NOTCH3 , Nectin4 , TROP-2 , CD142 , CA6 , GPR20 , CD174 , CD71 , EphA2 , LYPD3 , FGFR2 , FGFR3 , FR α , CEACAMs , GCC , Integrin Av , CAIX , P-cadherin , GD3 , Cadherin 6 , LAMP1 , FLT3 , BCMA , CD79b , CD19 , CD33 , CD56 , CD74 , CD22 , CD30 , CD37 , CD47 , CD138 , CD352 , CD25 CD123 . </xnotran>
4. The ligand-drug conjugate of claim 1 or 2, or a pharmaceutically acceptable salt, deutero-compound, solvate thereof, wherein: l is cleavable or non-cleavable.
5. The ligand-drug conjugate of claim 1 or 2, or a pharmaceutically acceptable salt, deutero-compound, solvate thereof, wherein: the pharmaceutically acceptable salt comprises sodium salt, potassium salt, calcium salt or magnesium salt formed by the acid functional group in the structural formula; or acetate, trifluoroacetate, citrate, oxalate, tartrate, malate, nitrate, chloride, bromide, iodide, sulfate, bisulfate, phosphate, lactate, oleate, ascorbate, salicylate, formate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate or p-toluenesulfonate formed with a basic functional group in the structure.
6. A ligand drug conjugate as claimed in any one of claims 1 to 5, or a pharmaceutically acceptable salt, deuteron or solvate thereof, for use in the manufacture of a medicament for the treatment or prophylaxis of cancer.
7. Use according to claim 6, characterized in that: the tumor is a solid tumor or a blood tumor.
8. Use according to claim 7, characterized in that: the tumor is breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma multiforme, sarcoma, lymphoma or leukemia.
9. A compound of formula II or III, or a pharmaceutically acceptable salt or solvate thereof, for attachment to the Ab of claim 1:
Figure FDA0003564748360000041
wherein:
R 1 is H, OH OR from OR 3 Ether, sulfite SO 3 - Or OSO 3 - Wherein R is 3 Is selected from C 1 -C 10 Linear, branched or cyclic alkyl, alkenyl or alkynyl groups of (a);
double line between N and C
Figure FDA0003564748360000042
Represents a single bond or a double bond, provided that, when it is a double bond, R 12 Is absent and R 1 Is H; when it is a single bond, R 12 is-C (O) O-L 3 Wherein L is 3 Is a connecting unit; r is 1 Is selected fromOH, by OR 3 Ether, sulfite SO 3 - Or OSO 3 - Wherein R is 3 Is selected from C 1 -C 10 Linear, branched or cyclic alkyl, alkenyl or alkynyl groups of (a);
R 2 is H or an alkyl substituent;
t is selected from C 2 -C 12 Hydrocarbyl, Z, (C) 1 -C 6 Alkylene) -Z- (C) 1 -C 6 Alkylene group), (C) 1 -C 6 Alkylene) -Z- (C) 1 -C 6 Alkylene) -Z- (C) 1 -C 6 Alkylene group), (C) 1 -C 6 Alkenylene) -Z- (C 1 -C 6 Alkenylene) or (C) 1 -C 6 Alkynylene) -Z- (C 1 -C 6 Alkynylene);
wherein:
z is selected from O, S, NR 4 Aryl and heteroaryl; wherein R is 4 Selected from H, P (O) 3 H 2 ，C(O)NR 5 R 6 Wherein R is 5 And R 6 Selected from H, C 1 -C 6 Alkyl, C substituted by one or more F 1 -C 6 Alkyl, or R 5 And R 6 To form a five-or six-membered heterocyclyl group;
alkylene, alkenylene, aryl and heteroaryl groups are selected from F, OH, O (C) 1 -C 6 Alkyl), NH 2 ，NHCH 3 ，N(CH 3 ) 2 And C 1 -C 6 Alkyl substituted, wherein alkyl is substituted with one or more F;
y is selected from one or more of H or C 1 -C 4 Alkyl groups of (a);
x is selected from-O-, -N-, -S-, -OC (O) -CR 7 R 8 -(CR 9 R 10 )m-O-、-OC(O)-CR 7 R 8 -(CR 9 R 10 )m-NH-、-OC(O)-CR 7 R 8 -(CR 9 R 10 )m-S-、-NHC(O)-CR 7 R 8 -(CR 9 R 10 )m-O-、-NHC(O)-CR 7 R 8 -(CR 9 R 10 ) m-NH-or-NHC (O) -CR 7 R 8 -(CR 9 R 10 )m-S-；
Wherein:
R 7 、R 8 each independently is a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a deuterated alkyl group, a haloalkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group, a substituted aryl group, or a heteroaryl group; or, R 7 、R 8 And the carbon atom to which they are attached forms C 3 -C 6 Cycloalkyl, cycloalkylalkyl, or heterocyclyl;
R 9 、R 10 the same or different, and each independently is a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, or a heterocyclic group; or, R 9 、R 10 And the carbon atom to which they are attached forms C 3 -C 6 Cycloalkyl, cycloalkylalkyl, or heterocyclyl;
m is an integer from 0 to 4;
X 1 selected from halogen or OSO 2 R 11 Wherein R is 11 Is selected from H, C 1 -C 4 A hydrocarbyl group of (a), phenyl or substituted phenyl;
L 1 ，L 2 is a linking unit or a substituent.
10. Compound II, III or a pharmaceutically acceptable salt or solvate thereof, according to claim 9, characterized in that: t is selected from C 2 -C 12 An alkylene group of (a).
11. Compound II, III or a pharmaceutically acceptable salt or solvate thereof, according to claim 9 or 10, characterized in that: t is
Figure FDA0003564748360000051
12. Compound II, III or a pharmaceutically acceptable salt or solvate thereof, according to claim 9 or 10, characterized in that: x is-O-, -N-or-NHC (O) -CR 7 R 8 -(CR 9 R 10 )m-O-。
13. The compound II, III or a pharmaceutically acceptable salt or solvate thereof according to claim 9 or 10, wherein L is L 3 Comprises the following steps:
Figure FDA0003564748360000052
wherein: the wavy line is connected with-C (O) O-; l is 4 Is a linking unit, linked to a ligand unit.
14. The compound II, III, or a pharmaceutically acceptable salt or solvate thereof, as claimed in claim 9 or 10, wherein L is L 4 Selected from, without limitation:
Figure FDA0003564748360000061
wherein: at the wavy line, the left carbon end is connected with the ligand unit, and the right nitrogen end or ester carbonyl end is connected with X 2 Are connected.
15. The compound II, III, or a pharmaceutically acceptable salt or solvate thereof, according to claim 9 or 10, wherein Q is:
Figure FDA0003564748360000062
wherein Q x Is an amino acid residue or a peptide residue consisting of amino acids.
16. Compound II, III, or a pharmaceutically acceptable salt or solvate thereof, according to claim 9 or 10, characterized in that X is 2 Comprises the following steps:
Figure FDA0003564748360000063
wherein a is selected from integers from 0 to 5, b is selected from integers from 0 to 16, c is selected from integers from 0 to 1, and d is selected from integers from 0 to 5.
17. The compound II, III, or a pharmaceutically acceptable salt or solvate thereof, as claimed in claim 9 or 10, wherein L is 3 Selected from the following, without limitation:
Figure FDA0003564748360000064
wherein: at the wavy line, the succinimide end on the left side is connected with the ligand unit, and the succinimide end on the right side is connected with-C (O) O-.
18. Compound II, III, or a pharmaceutically acceptable salt or solvate thereof, according to claim 9 or 10, characterized in that L is 1 And L 2 Each independently selected from:
structure a: a hydrogen atom, C (O) NR 'R', wherein R 'and R' are independently selected from H, C 1 -C 6 Alkyl, C substituted by one or more F 1 -C 6 An alkyl group; or R 'and R' form a five-or six-membered heterocyclyl group;
structure B: l is 4 -L 5 -，L 4 -L 6 -or L 4 -L 7 -L 8 -L 9 -, wherein L 4 、L 5 、L 6 、L 7 、L 8 And L 9 Are all connected units, L 4 To ligand units, L 5 、L 6 、L 9 Is linked to X.
19. The compound II, III, or a pharmaceutically acceptable salt or solvate thereof, according to claim 9 or 10, wherein:
when N and C are a single bond, i.e. R 12 When present, L 1 、L 2 Are each independently selected fromStructure A;
when a double bond is between N and C, i.e. R 12 In the absence of, L 1 Is of structure A or B, then L 2 Is structure B or A.
20. The compound II, III, or a pharmaceutically acceptable salt or solvate thereof, according to claim 18, wherein:
L 5 is- ((CH) 2 )sO)r(CH 2 )sX 3 L 10 -or- ((CH 2 )sO)r(CH 2 )sX 4 L 10 -；
L 6 Is- ((CH) 2 )sO)r(CH 2 )s-；
L 10 Is- (CH) 2 ) s-or- ((CH) 2 )sNHC(＝O)X 5 X 6 C(＝O)(CH 2 )s-；
Wherein:
X 3 selected from, without limitation:
Figure FDA0003564748360000071
wherein R is 13 Independently selected from hydrogen atom, C 1 -C 6 A hydrocarbon group, a halogen atom or a hydroxyl group;
X 4 selected from the following, without limitation:
Figure FDA0003564748360000072
wherein R is 13 Independently selected from hydrogen atoms, C 1 -C 6 A hydrocarbon group, a halogen atom or a hydroxyl group;
X 5 selected from, without limitation:
Figure FDA0003564748360000081
X 6 selected from peptide residues consisting of amino acids, selected without limitation from:
Figure FDA0003564748360000082
s is an integer from 1 to 10 and r is an integer from 1 to 14.
21. The compound II, III, or a pharmaceutically acceptable salt or solvate thereof, according to claim 18, wherein:
L 7 is-NC (R) 14 R 15 )C(O)、-NR 16 (CH 2 ) o C(O)-、-NR 16 (CH 2 CH 2 O) o CH 2 C(O)-、-S(CH 2 ) p C (O) -or a bond, wherein O is selected from an integer of 0 to 20; p is selected from an integer of 0 to 20; r 14 And R 15 The same or different, and each is independently selected from hydrogen atom, deuterium atom, alkyl group, substituted alkyl group, deuterated alkyl group, heteroalkyl group, carboxyl group, amino group, substituted amino group; r 16 Selected from the group consisting of hydrogen, deuterium, halogen, alkyl, substituted alkyl, deuterated alkyl, cycloalkylalkyl, alkoxyalkyl, aryl, substituted aryl, or heteroaryl;
L 8 selected from peptide residues consisting of amino acids;
L 9 is-NR 17 (CR 18 R 19 ) q -、-C(O)NR 17 -、-C(O)NR 17 (CH 2 ) q -or a chemical bond, wherein q is selected from an integer from 0 to 6; r 17 、R 18 And R 19 Are the same or different and are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a substituted alkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group, a substituted aryl group, or a heteroaryl group.
22. The compound II, III, or a pharmaceutically acceptable salt or solvate thereof, according to claim 21, wherein: said L 8 Is a peptide residue formed from one or more amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, or aspartic acid.
23. Compound II, III, or a pharmaceutically acceptable salt or solvate thereof, according to claim 19, wherein L is 1 、L 2 Independently from structure B, without limitation, are selected from:
Figure FDA0003564748360000083
Figure FDA0003564748360000091
24. compound II, III or a pharmaceutically acceptable salt or solvate thereof, according to claim 9, wherein is selected from the structures:
Figure FDA0003564748360000101
Figure FDA0003564748360000111
Figure FDA0003564748360000121
Figure FDA0003564748360000131
25. the antibody-drug conjugate of claim 1, or a pharmaceutically acceptable salt, deutero, solvate thereof, wherein the antibody-drug conjugate is selected from the following structures:
Figure FDA0003564748360000132
Figure FDA0003564748360000141
Figure FDA0003564748360000151
Figure FDA0003564748360000161
Figure FDA0003564748360000171
Figure FDA0003564748360000181
Figure FDA0003564748360000191
Figure FDA0003564748360000201
wherein u is selected from an integer of 1 to 10.
CN202210299307.0A 2021-04-01 2022-03-25 DNA toxic dimer compound and conjugate thereof Pending CN115192732A (en)

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