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CN114569738A - Antibody conjugated drug, intermediate and application thereof - Google Patents

Antibody conjugated drug, intermediate and application thereof Download PDF

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Publication number
CN114569738A
CN114569738A CN202011402991.8A CN202011402991A CN114569738A CN 114569738 A CN114569738 A CN 114569738A CN 202011402991 A CN202011402991 A CN 202011402991A CN 114569738 A CN114569738 A CN 114569738A
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residue
independently
antibody
substituted
alkyl
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王宝霞
张文伯
沈毅珺
杨彤
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SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL CO LTD
Taizhou Fudan Zhangjiang Pharmaceutical Co Ltd
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SHANGHAI FUDAN-ZHANGJIANG BIO-PHARMACEUTICAL CO LTD
Taizhou Fudan Zhangjiang Pharmaceutical Co Ltd
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Priority to CN202011402991.8A priority Critical patent/CN114569738A/en
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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Abstract

The invention discloses an antibody conjugate drug, an intermediate and application thereof. The antibody coupling drug is shown in a formula III. The antibody conjugate drug has good in vitro cell activity, in vivo anti-tumor activity, plasma stability and safety, and has good patent drug prospect.

Description

Antibody conjugated drug, intermediate and application thereof
Technical Field
The invention belongs to the field of biotechnology and medicine, and particularly relates to an antibody coupling drug, an intermediate thereof and application thereof.
Background
Antibody-conjugated drugs (ADCs) are one of the hot spots of interest to the pharmaceutical industry in recent years. Because of the unsatisfactory clinical efficacy of many antibody drugs, many industries are increasingly turning their eyes to ADC drugs. At present, seven ADC medicines are sold on the market abroad. Gemtuzumab Ozogamicin (trade name Mylotarg) approved by FDA at 17 days 5.2000 for fevery, was marketed for the treatment of Acute Myelogenous Leukemia (AML) patients with first relapse, above 60 years old, CD33+, who were not suitable for cytotoxic chemotherapy, although this drug was withdrawn from the market in 2010 but re-marketed in 2017, and Inotuzumab Ozogamicin (trade name bestonsa) of homophony was also approved by FDA for the treatment of adult relapse refractory B-cell ALL. Brentuximab Vedotin (trade name Adcetris) developed by the FDA approved Seattle Genetics company is marketed at 19.8.2011 for the treatment of CD30 positive Hodgkin Lymphoma (HL) and the rare disease Systemic Anaplastic Large Cell Lymphoma (SALCL). On day 22/2/2013, ado-trastuzumab emtansine (T-DM1, trade name Kadcyla) developed by Genentech corporation was approved by FDA for marketing, and was mainly used for treating Her2 positive advanced (metastatic) breast cancer. Particularly in 2019, polatuzumab vedotin (trade name Polivy), unfortuna vedotin (trade name padsev) and fam-trastuzumab hederxtecan (trade name enthusiast) are successively sold on the market, which further arouses the enthusiasm of drug research and development enterprises, and as far as the end of the first quarter of 2020, 311 ADC drugs in the global active state exist, and 33 development pipelines exist in the second and third clinical stages.
The basic module of the antibody coupling drug comprises an antibody, a linker and an effector molecule, and the effector molecule is transmitted to a tumor part by using the antibody to be enriched, so that tumor cells are killed. Most of the traditional effector molecules are high-activity tubulin inhibitors, and generally have larger toxic and side effects, so that the application of ADC is limited. Recently, immunoledics company invented a novel ADC drug IMMU-132(ZL200980156218) taking a camptothecin compound as an effector molecule, which shows a better anti-tumor effect, and the first three co-invented another ADC drug DS-8201a (ZL201380053256) taking the camptothecin compound as the effector molecule, which also shows a better anti-tumor effect. In the existing ADC technology, few researches are carried out on a linker for connecting a camptothecin compound and an antibody. Generally, the ideal linker in an ADC needs to satisfy the following requirements: firstly, ensuring that the micromolecular drug is not separated from the antibody in the plasma, and breaking a linker under proper conditions after entering cells to quickly release the active micromolecular drug; secondly, the linker also has better physicochemical property so as to be connected with the antibody to form a conjugate; and thirdly, the linker is easy to prepare, thereby laying a foundation for ADC scale production. IMMU-132 uses a pH sensitive linker and has poor stability. DS-8201a adopts a tetrapeptide structure containing glycine-phenylalanine-glycine (GGFG), has slow enzyme digestion reaction compared with a general cathepsin B substrate sequence (such as valine-citrulline), and has the problems of poor physicochemical properties, difficult synthesis and the like.
Disclosure of Invention
The invention aims to overcome the defect of single type of the existing antibody conjugate drug, provides a linker drug conjugate, and provides an antibody conjugate drug, an intermediate and application thereof on the basis of the linker drug conjugate. The antibody conjugate drug can realize the wide application of cytotoxic drugs in the field of ADC and treat tumor patients with drug resistance to microtubule ADC.
The antibody coupling drug with the specific structure connecting group provided by the invention can inhibit the growth of mammal tumors and can be used for treating various cancers. The antibody conjugate drug has good biological activity, stability and uniformity, and good safety.
The invention solves the technical problems through the following technical scheme.
The invention provides a connection base drug conjugate, which has a structural general formula as follows: l is3-L2-L1-D；
D is a cytotoxic drug;
-L1the structure of-is shown in formula I or II, the a end of the-is connected with the cytotoxic drug, the e end of the-is connected with the-L2-to end c;
Figure BDA0002813060180000021
wherein X and Y are each independently a phenylalanine residue, an alanine residue, a glycine residue, a valine residue, a glutamic acid residue, an aspartic acid residue, a cysteine residue, a glutamic acid residue, a histidine residue, an isoleucine residue, a leucine residue, a lysine residue, a methionine residue, a proline residue, a serine residue, a threonine residue, a tryptophan residue, or a tyrosine residue;
p and q are each independently any integer of 2-4;
R1and R2Each independently is C1～C6Alkyl or by 1, 2 or 3R1-aSubstituted C1～C6An alkyl group;
R1-aeach independently is R3R4N-or R5S(O)2-；
R3、R4And R5Each independently is C1～C6An alkyl group;
-L2-is of
Figure BDA0002813060180000022
And
Figure BDA0002813060180000023
one or more of;
wherein,
n is any integer of 1-12;
-L2end c of-and said-L1-is connected to the e-terminal of-and-L2End f of-and said L3-to the d-terminal;
L3-is of
Figure BDA0002813060180000031
Wherein d is the same as the-L2The f ends of-are connected.
In a preferred embodiment of the present invention, in the above-mentioned linked-base drug conjugates, certain groups have the following definitions, and the definition of the group not mentioned is as described in any of the embodiments of the present invention (hereinafter referred to as "in a preferred embodiment of the present invention" in this paragraph),
the cytotoxic drug is a conventional cytotoxic drug in the ADC field, and the invention particularly preferably relates to a hydroxyl-containing topoisomerase inhibitor, further preferably relates to a hydroxyl-containing topoisomerase I inhibitor, further preferably relates to a camptothecin compound, and further preferably relates to a hydroxyl-containing topoisomerase I inhibitor
Figure BDA0002813060180000032
In a preferred embodiment of the invention, when said cytotoxic agent is a hydroxyl-containing topoisomerase I inhibitor, said-L1-is bonded to the hydroxyl group in D in the form of an ether bond, more preferably
Figure BDA0002813060180000033
More preferably
Figure BDA0002813060180000034
The cytotoxic drug is
Figure BDA0002813060180000035
For example, the said-L1-after ligation with the cytotoxic drug, the fragment of the cytotoxic drug remaining in the linker-drug conjugate is preferably
Figure BDA0002813060180000041
In a preferred embodiment of the invention, when said R is1And R2Each independently is C1～C6When alkyl, said C1～C6Alkyl is C1～C4The alkyl group is more preferably a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tert-butyl group, and still more preferably a methyl group.
In a preferred embodiment of the invention, when said R is1And R2Each independently of the other is substituted by 1, 2 or 3R1-aSubstituted C1～C6When alkyl, said C1～C6Alkyl is C1～C4The alkyl group is more preferably a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tert-butyl group, and still more preferably an ethyl group.
In a preferred embodiment of the invention, when said R is3And R4Each independently is C1～C6When alkyl, said C1～C6Alkyl is C1～C4The alkyl group is more preferably a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tert-butyl group, and still more preferably a methyl group or an ethyl group.
In a preferred embodiment of the invention, when said R is5Is C1～C6When alkyl, said C1～C6Alkyl is C1～C4The alkyl group is more preferably a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tert-butyl group, and still more preferably a methyl group.
In a preferred embodiment of the present invention, when said R is1And R2Each independently of the other is substituted by 1, 2 or 3R1-aSubstituted C1～C6When alkyl, said group is substituted by 1, 2 or 3R1-aSubstituted C1～C6Alkyl is
Figure BDA0002813060180000042
Or
Figure BDA0002813060180000043
In a preferred embodiment of the present invention, when said-L is1When the structure of-is as shown in formula II, the structure of-L is2-is of
Figure BDA0002813060180000044
Or
Figure BDA0002813060180000045
In a preferred embodiment of the present invention, said- (X)p-is of
Figure BDA0002813060180000046
The amino terminal of the amino-substituted carbonyl group is connected with the carbonyl terminal of the formula I.
In a preferred embodiment of the present invention, said- (Y) q-is
Figure BDA0002813060180000047
Figure BDA0002813060180000051
The amino terminus of which is linked to said carbonyl terminus of formula II, preferably
Figure BDA0002813060180000052
In a preferred embodiment of the invention, each X is independently a valine residue or an alanine residue.
In a preferred embodiment of the invention, p is 2.
In a preferred embodiment of the present invention, each of said Y is independently a glycine residue or a phenylalanine residue.
In a preferred embodiment of the invention, q is 4.
In a preferred embodiment of the present invention, R is1And R2Each independently is 1, 2 or 3R1-aSubstituted C1～C6An alkyl group.
In a preferred embodiment of the present invention, R is1-aIs R5S(O)2-。
In a preferred embodiment of the present invention, said-L2-is of
Figure BDA0002813060180000053
Figure BDA0002813060180000054
Preference is given to
Figure BDA0002813060180000055
In a preferred embodiment of the present invention, n is any integer of 6 to 9.
In a preferred embodiment of the invention, -L1-is of
Figure BDA0002813060180000056
Figure BDA0002813060180000057
Figure BDA0002813060180000061
In a preferred embodiment of the present invention, said-L2-L1-is of
Figure BDA0002813060180000062
Figure BDA0002813060180000071
In a preferred embodiment of the present invention, said-L1D is
Figure BDA0002813060180000072
Figure BDA0002813060180000081
In a preferred embodiment of the present invention, said-L2-L1D is
Figure BDA0002813060180000091
Figure BDA0002813060180000101
Figure BDA0002813060180000111
In a preferred embodiment of the invention, D is
Figure BDA0002813060180000112
X and Y are each independently a phenylalanine residue, an alanine residue, a glycine residue, or a valine residue;
-L2-is of
Figure BDA0002813060180000117
Figure BDA0002813060180000114
One or more of (a).
In a preferred embodiment of the invention, D is
Figure BDA0002813060180000115
X and Y are each independently a phenylalanine residue, an alanine residue, a glycine residue, or a valine residue;
R1and R2Each independently of the other is substituted by 1, 2 or 3R1-aSubstituted C1～C6An alkyl group;
-L2-is of
Figure BDA0002813060180000118
Figure BDA0002813060180000121
In a preferred embodiment of the invention, D is
Figure BDA0002813060180000122
X and Y are each independently a phenylalanine residue, an alanine residue, a glycine residue, or a valine residue;
R1and R2Each independently of the other is substituted by 1, 2 or 3R1-aSubstituted C1～C6An alkyl group;
R1-ais R5S(O)2-；
-L2-is of
Figure BDA0002813060180000128
Figure BDA0002813060180000124
q is 3 or 4.
In a preferred embodiment of the invention, D is
Figure BDA0002813060180000125
X and Y are each independently a phenylalanine residue, an alanine residue, a glycine residue, or a valine residue;
R1and R2Each independently of the other is substituted by 1, 2 or 3R1-aSubstituted C1～C6An alkyl group;
R1-ais R5S(O)2-；
-L2-is of
Figure BDA0002813060180000129
Figure BDA0002813060180000127
q is 2 or 4.
In a preferred embodiment of the invention, D is
Figure BDA0002813060180000131
X and Y are each independently a phenylalanine residue, an alanine residue, a glycine residue, or a valine residue;
R1and R2Each independently of the other is substituted by 1, 2 or 3R1-aSubstituted C1～C6An alkyl group;
R1-ais R5S(O)2-；
-L2-is of
Figure BDA0002813060180000132
q is 4.
In a preferred embodiment of the present invention, the linking group drug conjugate is any one of the following compounds:
Figure BDA0002813060180000133
Figure BDA0002813060180000141
Figure BDA0002813060180000151
the invention also provides an antibody coupling drug shown as the formula III or pharmaceutically acceptable salt thereof:
Figure BDA0002813060180000152
wherein Ab is an antibody;
m is 2-8;
D、-L1-and-L2-the definitions are as described in any of the above embodiments.
In a preferred embodiment of the invention, the antibody is an antibody conventional in the field of anti-tumor ADCs, and the invention is preferably the anti-HER 2 antibody Trastuzumab or a variant thereof. The amino acid sequence of the light chain in the anti-HER 2 antibody Trastuzumab is preferably shown as SEQ ID No.1 in a sequence table, and the amino acid sequence of the heavy chain is preferably shown as SEQ ID No.2 in the sequence table. The anti-HER 2 antibody Trastuzumab variant has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or more than 99% homology to the anti-HER 2 antibody Trastuzumab sequence.
In a preferred embodiment of the present invention, said
Figure BDA0002813060180000161
The end b is connected with the sulfhydryl on the antibody in a thioether bond mode. For example
Figure BDA0002813060180000162
The connection form with cysteine residue in the antibody is
Figure BDA0002813060180000163
In a preferred embodiment of the present invention, m is 4 to 8, preferably 7 to 8, more preferably 7, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, and most preferably 7 or 8.
In a preferred embodiment of the present invention, the antibody-conjugated drug represented by formula III is any one of the following compounds:
Figure BDA0002813060180000164
Figure BDA0002813060180000171
Figure BDA0002813060180000181
Figure BDA0002813060180000191
wherein m is 7-8; ab is an anti-Her 2 antibody Trastuzumab, the amino acid sequence of a light chain in the anti-Her 2 antibody Trastuzumab is shown as SEQ ID No.1 in a sequence table, and the amino acid sequence of a heavy chain is shown as SEQ ID No.2 in the sequence table.
In a preferred embodiment of the present invention, the antibody-conjugated drug represented by formula III is any one of the following compounds:
Figure BDA0002813060180000192
Figure BDA0002813060180000201
Figure BDA0002813060180000211
wherein Ab is an anti-Her 2 antibody Trastuzumab, the amino acid sequence of a light chain in the anti-Her 2 antibody Trastuzumab is shown as SEQ ID No.1 in a sequence table, and the amino acid sequence of a heavy chain is shown as SEQ ID No.2 in the sequence table.
The invention also provides a compound shown as the formula IV:
Figure BDA0002813060180000221
wherein R is1The definitions of (a) and (b) are as described in any of the above schemes.
In a preferred embodiment of the present invention, the compound represented by formula IV is any one of the following compounds:
Figure BDA0002813060180000222
the invention provides a preparation method of an antibody conjugate drug shown in formula III or a pharmaceutically acceptable salt thereof, which comprises the step of conjugating the connecting base drug conjugate with the antibody.
In the present invention, the conditions and procedures for the coupling may be those conventional in the art.
The invention also provides a pharmaceutical composition, which comprises the antibody conjugate drug shown as the formula III or pharmaceutically acceptable salt thereof, and pharmaceutic adjuvants.
The invention also provides a pharmaceutical preparation, which comprises the antibody conjugate drug shown in the formula III and pharmaceutically acceptable salts thereof.
In a preferred embodiment of the present invention, the pharmaceutical preparation is an injection.
The invention also provides the use of a substance M in the manufacture of a medicament for the prevention or treatment of cancer; the substance M is the antibody coupling drug shown in the formula III, the pharmaceutically acceptable salt thereof, the pharmaceutical composition or the pharmaceutical preparation thereof, and the application of the substance M in preparing the drugs for preventing or treating cancers.
In a preferred embodiment of the present invention, the cancer is gastric cancer, breast cancer, non-small cell lung cancer, urothelial cancer or pancreatic cancer.
The invention also provides a method for preventing and/or treating cancer, which comprises the step of administering a therapeutically effective amount of the antibody coupling medicament shown in the formula III or the pharmaceutically acceptable salt thereof or the pharmaceutical composition to a subject.
In a preferred embodiment of the present invention, the cancer is preferably one or more of gastric cancer, breast cancer, non-small cell lung cancer, urothelial cancer and pancreatic cancer, preferably gastric cancer and/or breast cancer.
In a preferred embodiment of the present invention, the antibody conjugate drug represented by formula III, a pharmaceutically acceptable salt thereof or the pharmaceutical composition may be administered in a dose of 2-5mg/kg (per dose) according to the body weight of the subject.
In a preferred embodiment of the present invention, the antibody conjugate drug represented by formula III, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition is administered 1 time per week.
In a preferred embodiment of the present invention, the administration mode of the antibody-conjugated drug represented by formula III, the pharmaceutically acceptable salt thereof or the pharmaceutical composition is intravenous injection.
Unless otherwise indicated, the following terms appearing in the specification and claims of the invention have the following meanings:
in the present invention, m represents the molar ratio of cytotoxic drug molecule to Ab (also known as DAR, i.e. drug antibody coupling ratio), preferably understood as being: the average value of the molar ratio of the drug molecules to the monoclonal antibody molecules in the antibody-conjugated drug obtained by conjugating the monoclonal antibody molecules to the cytotoxic drug can be generally determined by using Hydrophobic-Interaction Chromatography (HIC), polyacrylamide-SDS gel electrophoresis (SDS-PAGE, electrophoresis), liquid Chromatography-mass spectrometer (LC-MS), and the like. For example, L-D is a group reactive with the conjugation site on the antibody, L is a linker, D is a cytotoxic agent further conjugated on the antibody to which L is attached, m represents the number of final conjugated D per antibody, or the number of conjugated D per antibody. m can be an integer or decimal, and in some embodiments, m is actually an average value between 2 and 8, 4 and 8, or 6 and 8, or m is some integer of 2, 3, 4, 5, 6, 7, or 8; in some embodiments, m is an average of 2, 4, 6, or 8; in other embodiments, m is an average of 2, 3, 4, 5, 6, 7, or 8.
The term "alkyl" refers to a straight or branched chain saturated hydrocarbon radical, e.g. C1～C4Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.
The term "antibody" is to be interpreted herein in its broadest sense as specifically binding to a target, such as a carbohydrate, polynucleotide, fat, polypeptide, etc., through at least one recognition region located on the variable region of the immunoglobulin molecule. Specifically included are intact monoclonal antibodies, polyclonal antibodies, bispecific antibodies, and antibody fragments, so long as they possess the desired biological activity. Antibodies of the invention can be prepared using techniques well known in the art, such as hybridoma methods, recombinant DNA techniques, phage display techniques, synthetic techniques, or combinations thereof, or other techniques known in the art.
The term "variant of an antibody" refers to amino acid sequence mutants, as well as covalent derivatives of a native polypeptide, provided that the biological activity equivalent to that of the native polypeptide is retained. Amino acid sequence mutants typically differ from the native amino acid sequence in that one or more amino acids are substituted for one or more amino acids in the native amino acid sequence or one or more amino acids are deleted and/or inserted in the polypeptide sequence. Deletion mutants include fragments of the native polypeptide and N-terminal and/or C-terminal truncation mutants. Typically, the amino acid sequence mutant is at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) homologous to the native sequence.
By monoclonal antibody or mab is meant that the antibody is derived from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor natural mutations or isoforms produced during the production of the antibody expression. Monoclonal antibodies have a high degree of specificity for a single antigen. Whereas polyclonal antibodies comprise different antibodies directed against different determinants, each monoclonal antibody is directed against only one determinant of the antigen. In the present invention, monoclonal antibodies also specifically include chimeric antibodies and fragments thereof, i.e., a portion of the heavy and/or light chain of an antibody is from one class, subclass, or class, and the remainder is from another class, subclass, or class.
The term "linker" refers to a direct or indirect linkage between an antibody and a drug. Attachment of the linker to the mAb can be accomplished via a number of means, such as via surface lysines, reductive coupling to oxidized carbohydrates, and via reduction of cysteine residues released by interchain disulfide bonds. A variety of ADC ligation systems are known in the art, including hydrazone, disulfide and peptide-based ligation.
The term "pharmaceutical adjuvant" refers to an adjuvant that is widely used in the field of pharmaceutical production. The excipients are used primarily to provide a safe, stable and functional pharmaceutical composition and may also provide methods for dissolving the active ingredient at a desired rate or for promoting the effective absorption of the active ingredient after administration of the composition by a subject. The pharmaceutical excipients may be inert fillers or provide a function such as stabilizing the overall pH of the composition or preventing degradation of the active ingredients of the composition. The pharmaceutical excipients may include one or more of the following excipients: buffers, chelating agents, preservatives, co-solvents, stabilizers, excipients and surfactant colorants, flavors and sweeteners.
The term "pharmaceutically acceptable salt" refers to salts prepared from the compounds of the present invention with relatively nontoxic, pharmaceutically acceptable acids or bases. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to: lithium salt, sodium salt, potassium salt, calcium salt, aluminum salt, magnesium salt, zinc salt, bismuth salt, ammonium salt, and diethanolamine salt. When compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable acid in neat solution or in a suitable inert solvent. The pharmaceutically acceptable acids include inorganic acids including, but not limited to: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, sulfuric acid, and the like. The pharmaceutically acceptable acids include organic acids including, but not limited to: acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citric acid, oleic acid, tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, saccharic acid, formic acid, ethanesulfonic acid, pamoic acid (i.e. 4, 4' -methylene-bis (3-hydroxy-2-naphthoic acid)), amino acids (e.g. glutamic acid, arginine), and the like. When the compounds of the present invention contain relatively acidic and relatively basic functional groups, they may be converted to base addition salts or acid addition salts. See in particular Berge et al, "Pharmaceutical Salts", Journal of Pharmaceutical Science 66:1-19(1977), or, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P.Heinrich Stahl and Camile G.Wermuth, ed., Wiley-VCH, 2002).
The term "treatment" or its equivalent when used with reference to, for example, cancer, refers to a procedure or process for reducing or eliminating the number of cancer cells in a patient or alleviating the symptoms of cancer. "treating" cancer or another proliferative disorder does not necessarily mean that the cancer cells or other disorder will actually be eliminated, that the number of cells or disorders will actually be reduced or that the symptoms of the cancer or other disorder will actually be alleviated. Generally, methods for treating cancer are performed even with a low likelihood of success, but are still considered to induce an overall beneficial course of action, given the patient's medical history and estimated survival expectations.
The term "prevention" refers to a reduced risk of acquiring or developing a disease or disorder.
In general, the term "substituted" means that 1 or more hydrogen atoms in a given structure are replaced with a particular substituent. Further, when the group is substituted with 1 or more of the substituents, the substituents are independent of each other, that is, the 1 or more substituents may be different from each other or the same. Unless otherwise indicated, a substituent group may be substituted at each substitutable position of the substituted group. When more than one position in a given formula can be substituted with one or more substituents selected from a particular group, the substituents may be substituted at each position, identically or differently.
Substituents in the context of the present invention, unless defined as "substituted by" certain substituents, are intended to indicate that the substituent is unsubstituted, e.g. "R1And R2Each independently is C1～C6Alkyl, by 1, 2 or 3R1-aSubstituted C1～C6Alkyl radical, C3～C10Cycloalkyl radical, C6～C14Aryl or 5-to 14-membered heteroaryl group' of which3～C10Cycloalkyl radical, C6～C14Aryl and 5-to 14-membered heteroaryl "are both understood as being unsubstituted by any substituent.
It will be appreciated by those skilled in the art that, in accordance with common practice used in the art, the present invention describes the structural formulae used in the structural formulae of the radicals
Figure BDA0002813060180000251
Means that the corresponding group is linked to other fragments, groups in the compound through this site.
The above preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention without departing from the common general knowledge in the art.
Unless otherwise specified, the room temperature in the present invention means 20 to 30 ℃. The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
1. the linker drug coupling of the invention can be coupled with an antibody to prepare the antibody coupling drug with good targeting property.
2. The antibody coupling drug of the invention has good in vitro cell activity and in vivo anti-tumor activity.
3. The antibody conjugate drug has good plasma stability and safety.
Detailed Description
TABLE 1 description of abbreviations
Figure BDA0002813060180000252
Figure BDA0002813060180000261
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1 Synthesis of linker-based drug conjugates
Example 1-1: preparation of LD001
Figure BDA0002813060180000262
Synthesis of Compound 1
Starting from L-prolinamide, compound 1 was obtained according to the method reported in the literature (Angew. chem.2020, 132, 4205-one 4210), ESI-MS m/z: 249.3(M + H).
Synthesis of Compound 2
Tert-butyl 2-glycolate (2.5g, 18.9mmol) and di (p-nitrophenyl) carbonate (6.3g, 20.8mmol) were mixed and dissolved in 200mL anhydrous DMF, 25mL triethylamine was added and the reaction was carried out at room temperature for 2 hours. After completion of the reaction of the starting materials as monitored by LC-MS, the trifluoroacetate salt of Compound 1 (3.4g, 9.5mmol) was added and the reaction was continued for 1 hour. After the reaction, most of the solvent was distilled off under reduced pressure, 150mL of water was added, ethyl acetate was extracted three times (100 mL each), the organic phases were combined after liquid separation, washed with saturated brine, dried over anhydrous sodium sulfate and concentrated, and the resulting crude product was subjected to silica gel column chromatography [ dichloromethane: purification of ethyl acetate ═ 10:1(v/v) ] yielded tert-butyl ester of compound 2 (2.8g, yield 81%), ESI-MS m/z: 363.5(M + H).
Tert-butyl ester of Compound 2 (2.5g,6.9mmol) was dissolved in 50mL of dichloromethane, and 4.5mL of trifluoroacetic acid was added to react at room temperature for 0.5 h. After the reaction was completed, the solvent was removed by distillation under the reduced pressure, and the crude product was purified by silica gel column chromatography [ dichloromethane: purification of methanol 10:1(v/v) ] yielded compound 2(1.6g, 76% yield), ESI-MS m/z: 307.4(M + H).
Synthesis of Compound 3
Compound 2(1.5g, 4.9mmol) was mixed with commercially available Exatecan mesylate (2.8g, 4.9mmol) in 20mL of anhydrous DMF, HATU (2.3g, 6mmol) and triethylamine (1.2 mL) were added, and after 2 hours at room temperature, most of the solvent was removed by distillation under reduced pressure, 50mL of water was added, and the mixture was extracted with ethyl acetateEach 100mL of the solution was separated, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The resulting crude product was added to a mixed solution of water and tetrahydrofuran (90mL, volume ratio 1:2), and acetic acid (6mL) and 10% Pd/C (500mg) were further added. The resulting mixture was at room temperature in H2After stirring for 4 hours (30psi), Pd/C was removed by filtration, the obtained filtrate was evaporated under reduced pressure to remove most of the solvent, then saturated aqueous sodium bicarbonate solution (50mL) was added, ethyl acetate was extracted three times (100 mL each), the organic phases after separation were combined and washed with saturated brine, dried over anhydrous sodium sulfate and concentrated, and the obtained crude product was purified by silica gel column chromatography [ dichloromethane: methanol 10:1(v/v)]Purification afforded compound 3(2.0g, 64% yield), ESI-MS m/z: 634.7(M + H).
Synthesis of Compound 4
Compound 4 was prepared according to the method reported in the literature (int.j.mol.sci.2017,18,1860) starting from commercially available succinimidyl 6- (N-maleimidomethyl) pentyl-1-carboxylate.
Synthesis of LD001
To a solution of compound 3(95mg, 0.15mmol) and 4(148mg, 0.22mmol) in DMF (5mL) was added HOBt (20mg, 0.15mmol) and DIPEA (55uL, 0.30mmol), and the resulting mixture was stirred overnight at room temperature, after addition of water (15mL), extracted three times with ethyl acetate (20 mL each), the organic phases after separation were combined and washed with saturated brine, dried over anhydrous sodium sulfate and concentrated, and the crude product was subjected to silica gel column chromatography [ ethyl acetate: petroleum ether ═ 2:1(v/v) ] to give the target compound LD001(120mg, yield 70%), ESI-MS m/z: 1147.2(M + H).
Examples 1 to 2: preparation of LD002
Figure BDA0002813060180000281
Synthesis of Compound 5
Similarly, the substitution of iodomethane from the last 2 step with 1-bromo-2-methylsulfonylethane gave compound 5, ESI-MS m/z, according to the method reported in the literature (Angew. chem.2020, 132, 4205-one 4210): 341.5(M + H).
Synthesis of Compound 6
Compound 5 was used as starting material instead of compound 1 to prepare compound 6, ESI-MS m/z: 726.8(M + H).
Synthesis of LD002
Referring to the synthetic method of the connection base drug conjugate LD-001, the compound 6 and the compound 4 are subjected to coupling reaction to obtain a target compound LD002, ESI-MS m/z: 1239.4(M + H).
Examples 1 to 3: preparation of LD003
Figure BDA0002813060180000291
Synthesis of Compound 7
Similarly, the iodomethane of the 2 last step was replaced by 2-bromo-N, N-dimethylethylamine to give the compound 7, ESI-MS m/z: 306.4(M + H).
Synthesis of Compound 8
Compound 8 was prepared by substituting compound 7 for compound 1 as starting material, according to the synthetic procedure for compound 3, ESI-MS m/z: 691.8(M + H).
Synthesis of LD003
Referring to the synthetic method of the linker drug conjugate LD-001, compounds 8 and 4 were coupled to give the target compound LD003, ESI-MS m/z: 1204.5(M + H).
Examples 1 to 4: preparation of LD004-LD006
Figure BDA0002813060180000292
Figure BDA0002813060180000301
Synthesis of Compounds 9-11
Compounds 9-11 were obtained according to the synthesis method of compound 4 starting from the appropriate maleimide. Structures of compounds 9-11 and the corresponding starting materials are shown in table 2.
TABLE 2 Compounds 9-11 and corresponding Maleimide starting Material structures
Figure BDA0002813060180000302
Synthesis of LD004-LD006
Referring to the synthesis method of compound LD002, compound 6 was condensed with compounds 9, 10 and 11, respectively, to give the target compound LD004-LD 006. LD004, ESI-MS m/z: 1241.3(M + H); LD005, ESI-MS m/z: 1265.4(M + H); LD006, ESI-MS m/z: 1319.4(M + H).
Examples 1 to 5: preparation of LD007
Figure BDA0002813060180000311
Referring to the methods of International publication No. WO2020050406A1, pages 626 to 631, Compound 13 is synthesized starting from commercially available Compound 12(CAS:13171-93-2), and then Compound 13 and Compound 6 are condensed to give the desired Compound LD007, ESI-MS m/z: 1238.3(M + H).
Examples 1 to 6: preparation of LD008
Figure BDA0002813060180000312
Referring to the synthesis of compound LD007, compound 14 was prepared by replacing the succinimidyl 6- (N-maleimidomethyl) pentyl-1-carboxylate used in the preparation of compound 13 with the commercially available maleimide-octapolyethylene glycol-acrylic acid succinimidyl ester (CAS: 2055033-05-9), and then condensing compound 14 with compound 8 to give the target compound LD008, ESI-MS m/z: 1203.3(M + H).
Examples 1 to 7: preparation of LD009 and LD010
Figure BDA0002813060180000321
Referring to the synthesis of compound LD007, compound 16 and compound 18 were prepared by substituting commercially available 15 and 17 for the substituted amino acid compound 12 used in the preparation of compound 13, and then condensing compound 16 and compound 18 with compound 6 to give the desired compounds LD009 and LD010, LD009, ESI-MS m/z: 1181.3(M + H); LD010, ESI-MS m/z: 1124.2(M + H).
Examples 1 to 8: preparation of GGFG-Dxd
GGFG-Dxd, ESI-MS m/z prepared by known methods reported in WO2015146132A 1: 1034.5(M + H).
Figure BDA0002813060180000331
Example 2 general procedure for linking drug-based conjugates to antibodies
anti-HER 2 antibody Trastuzumab (concentration 15mg/mL) was replaced into 50mM PB/1.0mM EDTA buffer (pH 7.0) using a G25 desalting column, 12 equivalents of TECP were added, and stirred at 37 ℃ for 2 hours to completely open the inter-antibody-chain disulfide bonds, followed by adjusting the pH of the reduced antibody solution to 6.0 using phosphoric acid and lowering the temperature of the water bath to 25 ℃ to prepare for the coupling reaction. The linker drug conjugates prepared in example 1 were dissolved in DMSO, and 12 equivalents of linker-drug conjugate linker were pipetted from the solution dropwise into the reduced antibody solution, and further DMSO was added to the solution to a final concentration of 10% (v/v), and the reaction was stirred at 25 ℃ for 0.5 hour, after the reaction was completed, the sample was filtered using a 0.22um membrane. Using a tangential flow ultrafiltration system to purify and remove unconjugated small molecules, the buffer solution is 50mM PB/1.0mM EDTA solution (pH 6.0), and after purification, sucrose with the final concentration of 6% is added and the mixture is placed in a refrigerator at the temperature of-20 ℃ for storage. The absorbance values were measured at 280nm and 370nm, respectively, using the UV method to calculate the DAR value, and the results are shown in Table 3 below. The amino acid sequence of a light chain in the anti-HER 2 antibody Trastuzumab is shown as SEQ ID No.1 in a sequence table, and the amino acid sequence of a heavy chain is shown as SEQ ID No.2 in the sequence table. In the experimental process, the coupling of the linking group drug conjugate and the antibody does not generate precipitate, and the proportion of the polymer is in a normal range (< 0.5%), which shows that the linking group drug conjugate provided by the invention has good physicochemical properties.
TABLE 3 DAR values measured under different antibody-conjugated drug (ADC) UV method
ADC numbering Linking base drug conjugates DAR value
FADC-LD001 LD001 7.7
FADC-LD002 LD002 7.8
FADC-LD003 LD003 7.8
FADC-LD004 LD004 7.6
FADC-LD005 LD005 8.0
FADC-LD006 LD006 7.7
FADC-LD007 LD007 7.5
FADC-LD008 LD008 7.9
FADC-LD009 LD009 7.8
FADC-LD010 LD010 7.4
FADC-8201 GGFG-Dxd 7.6
Effect example 1: in vitro cell Activity assay
SK-BR-3 and NCI-N87 cells stably transfected with high expression and high expression HER2 are selected as cell strains for in vitro activity detection in the experiment. And observing the dose-effect condition of different antibody conjugated drugs on cell killing. Initial selection of plate density for each cell: 2X 103cells/hole, and cell cytotoxic activity is measured after 16-24 hours; secondly, the final concentration of the antibody coupling drug prepared in the test example 2 after loading is set to be 5000nM as the initial concentration, 10 concentrations (4-10 times dilution) of 5000-0.006 nM design series are set, the change of killing (or inhibition) is observed for 96 hours,
Figure BDA0002813060180000343
luminescence staining of luminescene Cell visual Assay, reading fluorescence data and calculating IC50. From the beginningAs shown in the results of sexual tests (see Table 4), all ADCs show certain antitumor activity, and part of the ADC activity exceeds FADC-8201.
TABLE 4 in vitro cytotoxic Activity of different ADCs
Figure BDA0002813060180000341
Effect example 2 in vitro plasma stability
This example evaluates the stability of antibody conjugated drugs in human plasma. Specifically, in this example, the antibody conjugate drug of example 2 was added to human plasma, and the release amount of the free drug was measured by placing the drug in a water bath at 37 ℃ for 1, 3, 7, 14, 21, and 28 days with an internal standard (irinotecan as an internal standard substance) and extracting the drug, and then by high performance liquid chromatography, the results (as shown in table 5) showed that the antibody conjugate drug of the present invention had excellent plasma stability, and that the plasma stability of the ADC of the present invention placed at day 1 was superior to that of ADC-8201; the plasma stability of the partial ADC of the present invention was superior to that of ADC-8201 on days 3, 7, 14, 21 or 28 of storage.
TABLE 5 evaluation of the stability of different ADCs in human plasma
Figure BDA0002813060180000342
Figure BDA0002813060180000351
Effect example 3: in vivo evaluation of drug efficacy and safety evaluation
6-8 weeks old female Balb/c nude mice were injected subcutaneously on the back of the neck with 5X 10 dissolved in 100uL PBS solution6Human pancreatic cancer cells (Capan-1). Average tumor volume of about 160mm3Then, the nude mice are randomly divided into 6 groups according to the tumor size, and the nude mice are divided into 6 animals in groups for tail vein injection administration: 01 is blank control group, 02 is FADC-LD002(5mg/kg), 03 is FADC-LD002(2mg/kg), 04 is FADC-LD007(5mg/kg)FADC-LD007(2mg/kg) was designated 05, and ADC-8201(5mg/kg) was designated 06, and was administered 1 time per week. The body weight and tumor volume of the experimental animals were measured twice a week and the survival status of the animals during the experiment was observed. Experimental results (shown in Table 6) show that FADC-LD002 and FADC-LD007 have good in-vivo anti-tumor activity, and meanwhile, all experimental mice have no death condition or weight loss condition, which indicates that FADC-LD002 and FADC-LD007 have good safety.
TABLE 6 evaluation of in vivo potency of ADC
Figure BDA0002813060180000352
Figure BDA0002813060180000361
Although specific embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these are merely illustrative and that various changes or modifications may be made without departing from the principles and spirit of the invention. The scope of the invention is therefore defined by the appended claims.
SEQUENCE LISTING
<110> Shanghai Compound Dangjiang biomedical corporation
<120> antibody coupling drug, intermediate and application thereof
<130> P20016314C
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 214
<212> PRT
<213> Artificial Sequence
<220>
<223> Trastuzumab light chain sequence
<400> 1
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 2
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> Trastuzumab heavy chain sequence
<400> 2
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450

Claims (14)

1. A linker drug conjugate having the general structural formula: l is a radical of an alcohol3-L2-L1-D；
D is a cytotoxic drug;
-L1the structure of-is as shown in formula I or II, wherein the a end is connected with the cytotoxic drug, the e end is connected with the L2The end c is connected;
Figure FDA0002813060170000011
wherein X and Y are each independently a phenylalanine residue, an alanine residue, a glycine residue, a valine residue, a glutamic acid residue, an aspartic acid residue, a cysteine residue, a glutamic acid residue, a histidine residue, an isoleucine residue, a leucine residue, a lysine residue, a methionine residue, a proline residue, a serine residue, a threonine residue, a tryptophan residue, or a tyrosine residue;
p and q are each independently any integer of 2-4;
R1and R2Each independently is C1～C6Alkyl or by 1, 2 or 3R1-aSubstituted C1～C6An alkyl group;
R1-aeach independently is R3R4N-or R5S(O)2-；
R3、R4And R5Each independently is C1～C6An alkyl group;
-L2-is of
Figure FDA0002813060170000012
Figure FDA0002813060170000013
One or more of;
wherein,
n is any integer of 1-12;
-L2end c of-and said-L1E terminal of-is connected to-L2End f of-and said L3-to the d-terminal;
L3-is of
Figure FDA0002813060170000014
Wherein d is the same as the-L2The f ends of-are connected.
2. The linker drug conjugate of claim 1 wherein the cytotoxic drug is a topoisomerase inhibitor, preferably a hydroxyl-containing topoisomerase I inhibitor, more preferably a camptothecin compound, and most preferably a topoisomerase I inhibitor
Figure FDA0002813060170000021
And/or, when said R is1And R2Each independently is C1～C6When alkyl, said C1～C6Alkyl is C1～C4Alkyl, preferably methyl;
and/or, when said R is1And R2Each independently of the other is substituted by 1, 2 or 3R1-aSubstituted C1～C6When alkyl, said C1～C6Alkyl is C1～C4Alkyl, preferably ethyl;
and/or, when said R is3And R4Each independently is C1～C6When alkyl, said C1～C6Alkyl is C1～C4Alkyl, preferably methyl or ethyl;
and/or, when said R is5Is C1～C6When alkyl, said C1～C6Alkyl is C1～C4Alkyl, preferably methyl;
and/or, when said-L1When the structure of-is as shown in formula II, the structure of-L is2-is of
Figure FDA0002813060170000022
Figure FDA0002813060170000023
3. The linking-group drug conjugate of claim 2, wherein-L is a hydroxyl-containing topoisomerase I inhibitor when the cytotoxic drug is present1-is linked to the hydroxyl group of D in the form of an ether bond, preferably
Figure FDA0002813060170000024
More preferably
Figure FDA0002813060170000025
And/or, when said R is1And R2Each independently of the other is substituted by 1, 2 or 3R1-aSubstituted C1～C6When alkyl, said group is substituted by 1, 2 or 3R1-aSubstituted C1～C6Alkyl is
Figure FDA0002813060170000026
And/or, said- (X)p-is of
Figure FDA0002813060170000031
The amino end of the amino-substituted carbonyl group is connected with the carbonyl end in the formula I;
and/or, said- (Y) q-is
Figure FDA0002813060170000032
Figure FDA0002813060170000033
The amino terminus of which is linked to said carbonyl terminus of formula II, preferably
Figure FDA0002813060170000034
4. The linker drug conjugate of claim 1 wherein each X is independently a valine residue or an alanine residue;
and/or, p is 2;
and/or, each Y is independently a glycine residue or a phenylalanine residue;
and/or, q is 4;
and/or, said R1And R2Each independently is 1, 2 or 3R1-aSubstituted C1～C6An alkyl group;
and/or, said R1-aIs R5S(O)2-；
And/or, said-L2-is of
Figure FDA0002813060170000035
Preference is given to
Figure FDA0002813060170000036
And/or n is any integer of 6-9.
5. The linker drug conjugate of claim 1 wherein-L is1-is of
Figure FDA0002813060170000037
Figure FDA0002813060170000041
Or, -L2-L1-is of
Figure FDA0002813060170000042
Figure FDA0002813060170000043
Figure FDA0002813060170000051
Or, -L1D is
Figure FDA0002813060170000052
Figure FDA0002813060170000061
Figure FDA0002813060170000071
6. The linker drug conjugate of claim 1 wherein the linker drug conjugate is according to any one of the following schemes:
scheme 1:
d is
Figure FDA0002813060170000072
X and Y are each independently a phenylalanine residue, an alanine residue, a glycine residue, or a valine residue;
R1and R2Each independently of the other is substituted by 1, 2 or 3R1-aSubstituted C1～C6An alkyl group;
-L2-is of
Figure FDA0002813060170000073
Figure FDA0002813060170000074
Scheme 2:
d is
Figure FDA0002813060170000075
X and Y are each independently a phenylalanine residue, an alanine residue, a glycine residue, or a valine residue;
R1and R2Each independently is C1～C6Alkyl or by 1, 2 or 3R1-aSubstituted C1～C6An alkyl group;
R1-ais R5S(O)2-；
-L2-is of
Figure FDA0002813060170000081
Figure FDA0002813060170000082
Scheme 3:
d is
Figure FDA0002813060170000083
X and Y are each independently a phenylalanine residue, an alanine residue, a glycine residue, or a valine residue;
R1and R2Each independently of the other is substituted by 1, 2 or 3R1-aSubstituted C1～C6An alkyl group;
R1-ais R5S(O)2-；
-L2-is of
Figure FDA0002813060170000084
Figure FDA0002813060170000085
q is 3 or 4;
scheme 4:
d is
Figure FDA0002813060170000086
X and Y are each independently a phenylalanine residue, an alanine residue, a glycine residue, or a valine residue;
R1and R2Each independently of the other is substituted by 1, 2 or 3R1-aSubstituted C1～C6An alkyl group;
R1-ais R5S(O)2-；
-L2-is of
Figure FDA0002813060170000091
q is 2 or 4;
scheme 5:
d is
Figure FDA0002813060170000092
X and Y are each independently a phenylalanine residue, an alanine residue, a glycine residue, or a valine residue;
R1and R2Each independently of the other is substituted by 1, 2 or 3R1-aSubstituted C1～C6An alkyl group;
R1-ais R5S(O)2-；
-L2-is of
Figure FDA0002813060170000093
q is 4.
7. The linker drug conjugate of claim 1, wherein the linker drug conjugate is any one of the following compounds:
Figure FDA0002813060170000094
Figure FDA0002813060170000101
Figure FDA0002813060170000111
8. an antibody-conjugated drug represented by formula III:
Figure FDA0002813060170000112
wherein Ab is an antibody;
m is 2-8;
D、-L1-and-L2-are defined as in any one of claims 1 to 7.
9. The antibody conjugate of formula III, or a pharmaceutically acceptable salt thereof, of claim 8, wherein the antibody is the anti-HER 2 antibody Trastuzumab or a variant thereof; the amino acid sequence of the light chain in the anti-HER 2 antibody Trastuzumab is preferably shown as SEQ ID No.1 in a sequence table, and the amino acid sequence of the heavy chain is preferably shown as SEQ ID No.2 in the sequence table; said anti-HER 2 antibody Trastuzumab variant has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or more than 99% homology to the anti-HER 2 antibody Trastuzumab sequence;
and/or, said
Figure FDA0002813060170000121
The end is connected with the sulfhydryl on the antibody in a thioether bond mode;
and/or m is 4-8, preferably 7, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8, and most preferably 7 or 8.
10. The antibody-conjugated drug according to formula III, or a pharmaceutically acceptable salt thereof, according to claim 8, wherein the antibody-conjugated drug according to formula III is any one of the following compounds:
Figure FDA0002813060170000122
Figure FDA0002813060170000131
Figure FDA0002813060170000141
wherein m is 7-8; ab is an anti-Her 2 antibody Trastuzumab, the amino acid sequence of a light chain in the anti-Her 2 antibody Trastuzumab is shown as SEQ ID No.1 in a sequence table, and the amino acid sequence of a heavy chain is shown as SEQ ID No.2 in the sequence table;
preferably, the antibody conjugate drug shown in the formula III is any one of the following compounds:
Figure FDA0002813060170000151
Figure FDA0002813060170000161
Figure FDA0002813060170000171
wherein Ab is an anti-Her 2 antibody Trastuzumab, the amino acid sequence of a light chain in the anti-Her 2 antibody Trastuzumab is shown as SEQ ID No.1 in a sequence table, and the amino acid sequence of a heavy chain is shown as SEQ ID No.2 in the sequence table.
11. A compound of formula IV:
Figure FDA0002813060170000172
wherein R is1As defined in any one of claims 1 to 7;
the compound shown in the formula IV is preferably any one of the following compounds:
Figure FDA0002813060170000181
12. a pharmaceutical composition comprising the antibody conjugate shown in formula III or a pharmaceutically acceptable salt thereof according to any one of claims 8-10, and a pharmaceutical adjuvant.
13. A pharmaceutical formulation comprising an antibody conjugate according to formula III as claimed in any one of claims 8 to 10, a pharmaceutically acceptable salt thereof.
14. Use of a substance M for the manufacture of a medicament for the prevention or treatment of cancer, said substance M being an antibody conjugate according to formula III as defined in any one of claims 8 to 10, a pharmaceutically acceptable salt thereof, a pharmaceutical composition according to claim 12 or a pharmaceutical formulation according to claim 13; the cancer is preferably gastric cancer, breast cancer, non-small cell lung cancer, urothelial cancer or pancreatic cancer.
CN202011402991.8A 2020-12-02 2020-12-02 Antibody conjugated drug, intermediate and application thereof Pending CN114569738A (en)

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