A linker-drug conjugate, its preparation method and application

Abstract

This invention discloses a linker-drug conjugate, its preparation method, and its applications. The invention provides a linker-drug conjugate with the general structural formula L4-L2-L1-D, and also provides an antibody-drug conjugate based on this linker-drug conjugate with the general structural formula Ab-(L3-L2-L1-D). x The invention also includes a small molecule ligand-conjugate drug based on this linker-drug conjugate, with the general structural formula SM-L3-L2-L1-D. The antibody-drug conjugate and small molecule ligand-conjugate drug of this invention exhibit excellent biological activity and plasma stability, as well as good targeting and safety. Based on this novel linker-drug conjugate, a variety of cytotoxic drugs can be realized, particularly the widespread application of camptothecin in ADCs and SMDCs for the treatment of tumor patients resistant to microtubule-based ADCs.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medicine, and specifically relates to a linker-drug conjugate, its preparation method and application. Background Technology

[0002] Ligand-drug conjugates (ADCs) have become a hot research area in anti-tumor drugs in recent years, encompassing antibody-drug conjugates (ADCs) and small molecule ligand-drug conjugates (SMDCs), with ADCs attracting particular attention. Currently, nine ADC drugs have been approved for marketing abroad. On May 17, 2000, the FDA approved Pfizer's Gemtuzumab Ozogamicin (trade name Mylotarg) for the treatment of first-relapsed, CD33+ patients with acute myeloid leukemia (AML) who are over 60 years of age, unsuitable for cytotoxic chemotherapy. Although the drug was withdrawn from the market in 2010, it was re-marketed in 2017. In the same year, Pfizer's Inotuzumab ozogamicin (trade name Besponsa) was also approved by the FDA for the treatment of relapsed / refractory B-cell ALL in adults. On August 19, 2011, the FDA approved Brentuximab Vedotin (trade name Adcetris), developed by Seattle Genetics, for the treatment of CD30-positive Hodgkin's lymphoma (HL) and the rare disease systemic anaplastic large cell lymphoma (SALCL). On February 22, 2013, ado-trastuzumab emtansine (T-DM1, trade name Kadcyla), developed by Genentech, was approved by the FDA for marketing, primarily for the treatment of HER2-positive advanced (metastatic) breast cancer. Notably, in 2019, polatuzumab vedotin (trade name Polivy), enfortumab vedotin (trade name Padcev), and fam-trastuzumab deruxtecan (trade name Enhertu) were successively approved for marketing. In addition, more than 100 ADC drugs are in clinical and preclinical development stages both domestically and internationally.

[0003] Linker-drug is the core component of ADCs. The range of active molecules that can be used in ADCs is broad, including various highly active compounds, clinical-stage drugs, and marketed cytotoxic drugs. However, designing suitable linkers for specific types of drug molecules remains a significant challenge. Currently, most clinical-stage and marketed ADCs use linker-drug technology from Adcetris in Seattle. This technology is only applicable to amino drugs; linkers for hydroxyl drugs are less common. IMMU-132 uses a hydroxyl linker, but this linker is very unstable. DS-8201 uses a stable linker, but this linker is uncommon, and in practice, it has been found to have a slow release rate after enzymatic cleavage. An ideal linker should possess the following characteristics: (1) stability in the bloodstream; (2) rapid release of the complete drug molecule after enzymatic cleavage; (3) broad applicability, capable of splicing with common hydroxyl, thiol, and amino drugs; and (4) ability to be activated by common lysosomal enzymes (such as cathepsins) to release the drug. From the perspective of existing technology, linkers that can simultaneously meet the above four requirements are very rare. This invention provides a linker molecule that meets the above three requirements. In practice, it has been found that this linker can be linked with common hydroxyl and thiol groups to form stable linker-drug conjugates, which can be well applied in the design of ADCs and SMDCs. Summary of the Invention

[0004] This invention addresses the current lack of ideal linker-drug conjugates in the prior art by providing a linker-drug conjugate that is stable in both mouse and human plasma, releases almost no drug, and rapidly releases intact drug molecules under the action of cathepsin B, achieving 100% release efficiency.

[0005] This invention also provides ADC and SMDC molecules based on this type of linker-drug conjugate, enabling the widespread application of cytotoxic drugs in the fields of ADCs and SMDCs. The ADC and SMDC molecules provided by this invention exhibit excellent biological activity, stability, and homogeneity, can inhibit mammalian tumor growth, and possess good safety profiles, making them suitable for treating various cancers.

[0006] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0007] This invention provides a linker-drug conjugate with the general structural formula: L4-L2-L1-D, wherein,

[0008] D is a cytotoxic drug;

[0009] The structure of L1 is shown in Formula I, II, or III, with its a-terminus connected to the cytotoxic drug and its e-terminus connected to L2.

[0010]

[0011] R 1 It is a C1-C6 alkyl group or hydrogen;

[0012] R 2 It is hydrogen, C1-C6 alkyl, C3-C 10 cycloalkyl, C6-C 14 Aryl or 5-14 heteroaryl; the heteroatom in the 5-14 heteroaryl is selected from one or more of N, O and S, and the number of heteroatoms is 1, 2, 3 or 4;

[0013] R 3 -N(R) 3-1 R 3-2 ) substituted C1-C6 alkyl groups, R 3-3 -S(O)2-substituted C1-C6 alkyl, C1-C6 alkyl, C3-C 10 cycloalkyl, C6-C 14 Aryl or 5-14 heteroaryl; the heteroatom in the 5-14 heteroaryl is selected from one or more of N, O and S, and the number of heteroatoms is 1, 2, 3 or 4;

[0014] The R mentioned 3-1 R 3-2 and R 3-3 Independently, it is a C1 to C6 alkyl group;

[0015] R 4 It is hydrogen, C1-C6 alkyl, C3-C 10 Cycloalkyl or carboxylic acid-substituted C1-C6 alkyl groups;

[0016] When R 3 for And R 4 When it is hydrogen, R 2 Not hydrogen;

[0017] In general formula III, m is an integer from 1 to 3; in general formula II, n is 0 or an integer from 1 to 3.

[0018] L is independently selected from 20 natural amino acids; p is an integer from 1 to 3; (L) p It is a fragment of 1-3 amino acid residues, with both the amino and carbonyl ends connected by amide bonds. For example, in formulas I, II and III, the N-terminus is connected to the carbonyl group on the left and the C-terminus is connected to the amino group on the right.

[0019] L2 is C2-C 12Straight-chain alkyl, or C3-C6 cycloalkyl, or (poly)ethylene glycol chain containing 1-12 ethylene glycol units, or any combination of two thereof; one end of L2 is connected to the e end of L1, and the other end is connected to the d end of L4.

[0020] L4 is The d end is connected to one end of the L2.

[0021] In a preferred embodiment of the present invention, certain groups in the above-mentioned linker-drug conjugate have the following definitions, and the definitions of groups not mentioned are as described in any of the above embodiments (hereinafter referred to as "in a preferred embodiment of the linker-drug conjugate of the present invention"):

[0022] The cytotoxic drug can be a conventional cytotoxic drug in the ADC field. In particular, the present invention prefers a drug containing hydroxyl or thiol groups. The drug containing hydroxyl or thiol groups is preferably a drug with the following structure, and more preferably Dxd (topoisomerase I inhibitor camptothecin derivative (DX-8951 derivative DXd)).

[0023]

[0024] In DM-X, y is an integer from 0 to 6, and X is a hydroxyl or thiol group.

[0025] In a preferred embodiment of the linker-drug conjugate of the present invention, either the hydroxyl or thiol group in the cytotoxic drug structure may be optionally linked to the a-terminus of L1, and preferably linked to L1 in the form of an ether bond. Taking L1 as formula I and Dxd linkage as an example, the -L1-D is:

[0026]

[0027] In a preferred embodiment of the linker-drug conjugate of the present invention

[0028] The R mentioned 1 It can be a C1 to C6 alkyl group (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl) or a C1 to C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and methyl, for example.

[0029] In a preferred embodiment of the linker-drug conjugate of the present invention

[0030] The R mentioned 2 It is hydrogen or C1-C6 alkyl;

[0031] The C1-C6 alkyl group (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl) may be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and methyl, for example;

[0032] For example, the R 2 It can be hydrogen or methyl.

[0033] In a preferred embodiment of the linker-drug conjugate of the present invention

[0034] The R mentioned 3 For R 3-1 R 3-2 N-substituted C1-C6 alkyl or R 3-3 -S(O)2-substituted C1 to C6 alkyl groups.

[0035] In a preferred embodiment of the linker-drug conjugate of the present invention

[0036] The R mentioned 3 For R 3-1 R 3-2 N-substituted C1-C6 alkyl groups;

[0037] The C1-C6 alkyl group (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl) may be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and ethyl, for example;

[0038] The R mentioned 3-1 and R 3-2 Each can be an independent C1 to C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, and methyl, for example;

[0039] For example, the R 3 for

[0040] In a preferred embodiment of the linker-drug conjugate of the present invention

[0041] The R mentioned 3 For R 3-3 -S(O)2-substituted C1- to C6 alkyl groups;

[0042] The C1-C6 alkyl group (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl) may be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and ethyl, for example;

[0043] The R mentioned 3-3It can be a C1 to C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and methyl, for example;

[0044] For example, the R 3 for

[0045] In a preferred embodiment of the linker-drug conjugate of the present invention

[0046] The R mentioned 4 It is hydrogen or C1-C6 alkyl;

[0047] The C1-C6 alkyl group (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl) may be a C1-C4 alkyl group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and methyl, for example;

[0048] For example, the R 4 It can be hydrogen or methyl.

[0049] In a preferred embodiment of the linker-drug conjugate of the present invention

[0050] The L is independently a phenylalanine residue, alanine residue, glycine residue, glutamic acid residue, aspartic acid residue, cysteine ​​residue, glutamic acid residue, histidine residue, isoleucine residue, leucine residue, lysine residue, methionine residue, proline residue, serine residue, threonine residue, tryptophan residue, tyrosine residue, or valine residue.

[0051] For example, valine residues, glycine residues, or phenylalanine residues, and again, valine residues.

[0052] In a preferred embodiment of the linker-drug conjugate of the present invention, n is 1.

[0053] In a preferred embodiment of the linker-drug conjugate of the present invention, m is 1 or 2.

[0054] In a preferred embodiment of the linker-drug conjugate of the present invention, p is 1.

[0055] In a preferred embodiment of the linker-drug conjugate of the present invention

[0056] The (L) mentioned above p for The amino terminus is connected to the carbonyl terminus in formula I, II, or III, and the carbonyl terminus is connected to the amino terminus in formula I, II, or III.

[0057] In a preferred embodiment of the linker-drug conjugate of the present invention

[0058] When the L1 structure is as shown in Equation I, the L1 is...

[0059] In a preferred embodiment of the linker-drug conjugate of the present invention

[0060] When the L1 structure is as shown in Formula II, the L1 is...

[0061] In a preferred embodiment of the linker-drug conjugate of the present invention

[0062] When the L1 structure is as shown in Formula III, the L1 is...

[0063] In a preferred embodiment of the linker-drug conjugate of the present invention

[0064] The L2 mentioned is C2-C 12 A straight-chain alkyl group or a (poly)glycol chain containing 1-12 glycol units, or a combination of both.

[0065] In a preferred embodiment of the linker-drug conjugate of the present invention

[0066] The L2 mentioned is C2-C 12 Straight-chain alkyl;

[0067] The C2-C 12 The straight-chain alkyl group can be a C3-C7 straight-chain alkyl group, such as n-propyl, n-butyl, n-pentyl, n-hexyl or n-heptyl, and n-pentyl for example.

[0068] In a preferred embodiment of the linker-drug conjugate of the present invention

[0069] The L2 mentioned is C2-C 12 A combination of a straight-chain alkyl group and a (poly)glycol chain containing 1-12 glycol units;

[0070] For example -C2-C 12 Straight-chain alkyl group -O-(CH2CH2O)q-C2-C 12 Straight-chain alkyl-, the C2-C 12 The straight-chain alkyl group is preferably a C2-C4 straight-chain alkyl group, such as ethyl, n-propyl, n-butyl, or ethyl, and q in -(CH2CH2O)q- is 1-7, such as 1 or 7, or 7.

[0071] The C2-C 12Combinations of straight-chain alkyl groups with (poly)glycol chains containing 1-12 ethylene glycol units are, for example... For example

[0072] In a preferred embodiment of the linker-drug conjugate of the present invention

[0073] When the L1 structure is as shown in Formula I, the L2 is n-pentyl or

[0074] In a preferred embodiment of the linker-drug conjugate of the present invention

[0075] When the L1 structure is as shown in Formula II, the L2 is n-pentyl or

[0076] In a preferred embodiment of the linker-drug conjugate of the present invention

[0077] When the L1 structure is as shown in Formula III, the L2 is n-pentyl, For example

[0078] In a preferred embodiment of the linker-drug conjugate of the present invention

[0079] The L4 mentioned above is

[0080] In a preferred embodiment of the linker-drug conjugate of the present invention

[0081] The cytotoxic drug is Dxd; the structure of L1 is shown in Formula I, II, or III, and the (L) p for The L2 mentioned is n-pentyl or The L4 mentioned above is

[0082] When L1 is Equation I, R 1 and R 2 Each is independently a C1-C6 alkyl group; the C1-C6 alkyl group is preferably a C1-C4 alkyl group, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and most preferably methyl; the R 3 For R 3-1 R 3-2 N-substituted C1-C6 alkyl or R 3-3S(O)2-substituted C1-C6 alkyl groups, wherein the C1-C6 alkyl groups are preferably C1-C4 alkyl groups, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, and most preferably ethyl; wherein R 3-1 R 3-2 and R 3-3 Each of the components is preferably a C1-C4 alkyl group, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, and most preferably methyl; the R... 3 Optimal The R mentioned 4 The R is hydrogen or a C1-C6 alkyl group, preferably a C1-C4 alkyl group, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, and most preferably methyl; 4 Preferably hydrogen or methyl;

[0083] When L1 is Equation II, R 1 It is a C1-C6 alkyl group; the R 2 and R 4 Each is independently C1-C6 alkyl or hydrogen; the C1-C6 alkyl is preferably C1-C4 alkyl, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and most preferably methyl; n is 1;

[0084] When L1 is Equation III, R 1 The alkyl group is C1-C6, preferably C1-C4, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, and most preferably methyl; the R 2 It is hydrogen; m is 1 or 2.

[0085] In a preferred embodiment of the linker-drug conjugate of the present invention

[0086] The cytotoxic drug is DM-X, where y is 1 and X is a hydroxyl or thiol group; the structure of L1 is shown in formula I, II, or III, and the (L) p for The L4 mentioned above is

[0087] When L1 is Equation I, R 1 The alkyl group is C1-C6; the C1-C6 alkyl group is preferably C1-C4 alkyl, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and most preferably methyl; the R 2 It is hydrogen; the R mentioned above 3 For R3-3 S(O)2-substituted C1-C6 alkyl groups, wherein the C1-C6 alkyl groups are preferably C1-C4 alkyl groups, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, and most preferably ethyl; wherein R 3-3 Preferably, it is a C1-C4 alkyl group, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, and most preferably methyl; the R 3 Optimal The R mentioned 4 It is hydrogen; the L2 is n-pentyl;

[0088] When L1 is Equation II, R 1 and R 2 Each is independently a C1-C6 alkyl group; the C1-C6 alkyl group is preferably a C1-C4 alkyl group, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, and most preferably methyl; the R 4 It is hydrogen; n is 1; L2 is

[0089] When L1 is Equation III, R 1 The alkyl group is C1-C6, preferably C1-C4, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, and most preferably methyl; the R 2 It is hydrogen; m is 1; L2 is

[0090] In a preferred embodiment of the linker-drug conjugate of the present invention

[0091] The cytotoxic drug mentioned is Tubulysin A;

[0092] The structure of L1 is shown in Equation I, II, or III, wherein R 1 The alkyl group is C1-C6, preferably C1-C4, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, and most preferably methyl; the R 2 The R is hydrogen or a C1-C6 alkyl group, preferably a C1-C4 alkyl group, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, and most preferably methyl; 2 Preferably hydrogen or methyl; the R 3 For R 3-3S(O)2-substituted C1-C6 alkyl groups, wherein the C1-C6 alkyl groups are preferably C1-C4 alkyl groups, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, and most preferably ethyl; wherein R 3-3 Preferably, it is a C1-C4 alkyl group, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, and most preferably methyl; the R 3 Optimal The R mentioned 4 The R is hydrogen or a C1-C6 alkyl group, preferably a C1-C4 alkyl group, more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, and most preferably methyl; 4 Preferably hydrogen or methyl; m is 1 or 2; n is 1; (L) p for

[0093] The L2 mentioned is n-pentyl or The L4 mentioned above is

[0094] In a preferred embodiment of the linker-drug conjugate of the present invention, the linker-drug conjugate is preferably any of the following compounds:

[0095]

[0096]

[0097]

[0098] The present invention also provides an antibody-drug conjugate obtained by conjugating an antibody with a linker-drug conjugate as described in any of the above embodiments, the general structural formula of which is: Ab-(L3-L2-L1-D). x ,

[0099] in,

[0100] Ab represents antibodies;

[0101] x ranges from 2 to 8;

[0102] L3 is For example The b-terminus is connected to the antibody, and the d-terminus is connected to L2.

[0103] D, L1, and L2 are defined as described in any of the previous implementation schemes.

[0104] In a preferred embodiment of the present invention, certain groups in the above-mentioned antibody-drug conjugates have the following definitions, and the definitions of groups not mentioned are as described in any of the above embodiments (hereinafter referred to as "in a preferred embodiment of the antibody-drug conjugates of the present invention"):

[0105] The antibody mentioned can be a conventional IgG1 type antibody in the field of anti-tumor ADCs, such as Herceptin (trastuzumab for injection).

[0106] In a preferred embodiment of the antibody-drug conjugate of the present invention,

[0107] The b-terminus of L3 and the thiol group on the antibody are linked by a thioether bond.

[0108] For example The linkage form with the cysteine ​​residues in the antibody is as follows:

[0109] In a preferred embodiment of the antibody-drug conjugate of the present invention,

[0110] The value of m is preferably 4 to 8, and more preferably 7 to 8 (e.g., 7.8, 7.9, 8.0).

[0111] In a preferred embodiment of the antibody-drug conjugate of the present invention, m is preferably 4 to 8, and more preferably 7 or 8.

[0112] In a preferred embodiment of the antibody-drug conjugate of the present invention, D is preferably Dxd.

[0113] In a preferred embodiment of the antibody-drug conjugate of the present invention, L2 is preferably n-pentyl.

[0114] In a preferred embodiment of the antibody-drug conjugate of the present invention, the antibody-drug conjugate is preferably any of the following compounds:

[0115]

[0116] Wherein, Ab is an IgG1 type antibody, x is 2-8, preferably 4-8, more preferably 7-8, for example 7.0, 7.8, 7.9 or 8.0.

[0117] In a preferred embodiment of the antibody-drug conjugate of the present invention, the antibody-drug conjugate is preferably any of the following compounds:

[0118]

[0119] Wherein, Ab is Herceptin; x is preferably 7 to 8, for example 7.8, 7.9 or 8.0.

[0120] In a preferred embodiment of the antibody-drug conjugate of the present invention, the antibody-drug conjugate is preferably any of the following compounds:

[0121]

[0122] Where Ab stands for Herceptin.

[0123] The present invention also provides a method for preparing the above-mentioned antibody-drug conjugate, which includes the step of conjugating the above-mentioned linker-drug conjugate and the above-mentioned antibody.

[0124] In this invention, the conditions and operations for coupling can be the conventional conditions and operations for coupling in the art.

[0125] This invention also provides a small molecule ligand-coupled drug obtained by coupling the linker-drug conjugate described in any of the above embodiments with a small molecule ligand, the general structural formula of which is: SM-L3-L2-L1-D.

[0126] in,

[0127] SM is a small molecule ligand;

[0128] D, L1, and L2 are defined as described in any of the preceding implementation schemes;

[0129] L3 is For example Its d-terminus is connected to L2, and its b-terminus is connected to a small molecule ligand via an ether bond or a thioether bond.

[0130] In a preferred embodiment of the present invention, in the above-mentioned small molecule ligand-coupled drug, certain groups have the following definitions, and the definitions of groups not mentioned are as described in any of the above schemes (hereinafter referred to as "in a preferred embodiment of the small molecule ligand-coupled drug of the present invention"): the small molecule ligand is a folic acid derivative containing a thiol group, a small molecule polypeptide containing a thiol group, or a polysaccharide derivative containing a thiol group.

[0131] In a preferred embodiment of the small molecule ligand-conjugated drug of the present invention, D is Dxd.

[0132] In a preferred embodiment of the small molecule ligand-conjugated drug of the present invention, L1 is... R 1 R 2 R 3 R 4 and (L) p As described in any of the previous implementation schemes.

[0133] In a preferred embodiment of the small molecule ligand-coupled drug of the present invention, L2 is n-pentyl.

[0134] In a preferred embodiment of the small molecule ligand-conjugated drug of the present invention, the small molecule ligand is a folic acid derivative containing a thiol group, preferably a compound having the following structure:

[0135]

[0136] In a preferred embodiment of the small molecule ligand-conjugated drug of the present invention, the small molecule ligand-conjugated drug has the following structure:

[0137]

[0138] In a preferred embodiment of the small molecule ligand-conjugated drug of the present invention, the small molecule ligand-conjugated drug has the following structure:

[0139]

[0140] The present invention also provides a method for preparing the above-mentioned small molecule ligand-conjugated drug, which includes the step of conjugating the above-mentioned linker-drug conjugate and the above-mentioned small molecule ligand.

[0141] In this invention, the conditions and operations for coupling can be the conventional conditions and operations for coupling in the art.

[0142] The present invention also provides a pharmaceutical composition comprising the above-described antibody-drug conjugate or small molecule ligand-conjugate and pharmaceutical excipients, or, the above-described antibody-drug conjugate or small molecule ligand-conjugate and pharmaceutical excipients in a pharmaceutically acceptable salt thereof.

[0143] In the pharmaceutical composition, the amount of the antibody-drug conjugate or the small molecule ligand-conjugated drug described above can be a therapeutically effective amount.

[0144] This invention also provides the use of the above-described antibody-drug conjugate, or the above-described small molecule ligand-drug conjugate, or the above-described pharmaceutical composition in the preparation of a medicament for the prevention or treatment of cancer. The cancer is preferably gastric cancer, breast cancer, non-small cell lung cancer, urothelial carcinoma, colon cancer, or pancreatic cancer.

[0145] The present invention also provides a method for preventing and / or treating cancer, comprising administering to a subject a therapeutically effective amount of the aforementioned antibody-drug conjugate, or the aforementioned small molecule ligand-drug conjugate, or the aforementioned pharmaceutical composition. The cancer is preferably gastric cancer, breast cancer, non-small cell lung cancer, urothelial carcinoma, colon cancer, or pancreatic cancer.

[0146] Unless otherwise stated, the following terms appearing in this specification and claims have the following meanings:

[0147] As used herein, the terms "containing" or "including (comprise)" can be open-ended, semi-closed, or closed. In other words, the terms also include "consistently made of" or "composed of". The term "comprising" is an open-ended expression, meaning it includes the contents specified in this invention but does not exclude other aspects.

[0148] In this specification, groups and their substituents may be selected by those skilled in the art to provide stable structural moieties and compounds. When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes chemically equivalent substituents obtained when the structural formula is written from right to left.

[0149] The section headings used in this specification are for organizational purposes only and should not be construed as limiting the subject matter. All references or portions thereof cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and papers, are incorporated herein by reference in their entirety.

[0150] Unless otherwise specified, all technical and scientific terms used herein have the standard meaning in the field to which the claimed subject matter pertains. Where multiple definitions exist for a term, the definition herein shall prevail.

[0151] It should be understood that the singular forms used in this invention, such as "a," include plural references unless otherwise specified. Furthermore, the term "comprising" is an open-ended limitation, not a closed one; that is, it includes the contents specified in this invention but does not exclude other aspects.

[0152] Unless otherwise stated, the following definitions shall apply as used herein. For the purposes of this invention, chemical elements are consistent with the CAS edition of the periodic table and the Handbook of Chemistry and Physics, 75th edition, 1994. Furthermore, general principles of organic chemistry can be found in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry" by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, the entire contents of which are incorporated herein by reference.

[0153] In this specification, groups and their substituents may be selected by those skilled in the art to provide stable structural moieties and compounds. When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes chemically equivalent substituents obtained when the structural formula is written from right to left.

[0154] Certain chemical groups defined herein are preceded by simplified symbols to indicate the total number of carbon atoms present in the group. For example, C1-C6 alkyl refers to an alkyl group having a total of 1, 2, 3, 4, 5, or 6 carbon atoms as defined below. The total number of carbon atoms in the simplified symbols does not include carbons that may be present in substituents of the group.

[0155] In this paper, the numerical ranges defined in the substituents, such as 0 to 4, 1-4, 1 to 3, etc., indicate the integers within that range, such as 1-6 being 1, 2, 3, 4, 5, 6.

[0156] The singular form used in this invention, such as "a," includes plural references unless otherwise specified.

[0157] The terms “one or more” or “one or more kinds” refer to 1, 2, 3, 4, 5, 6, 7, 8, 9 or more; for example, 1, 2, 3, 4 or 5.

[0158] The terms “part,” “structural part,” “chemical part,” “group,” and “chemical group” used in this article refer to specific segments or functional groups within a molecule. A chemical part is generally considered to be a chemical entity embedded in or attached to a molecule.

[0159] The term "substituted" refers to the substitution of one or more hydrogen atoms on a particular atom by a substituent, including deuterium and hydrogen variants, provided that the valence state of the particular atom is normal and the substituted compound is stable.

[0160] Generally, the term "substituted" indicates that one or more hydrogen atoms in a given structure are substituted by a specific substituent. Further, when the group is substituted by more than one of the substituents, the substituents are independent of each other; that is, the more than one substituent can be different or the same. Unless otherwise indicated, a substituent can be substituted at each substituted position of the substituted group. When more than one position in a given structural formula can be substituted by one or more substituents selected from a specific group, the substituents can be substituted at the same or different positions.

[0161] When a listed substituent does not specify which atom it is attached to in a compound included but not specifically mentioned in the general chemical formula, such a substituent may be bonded to any of its atoms. Combinations of substituents and / or their variants are permitted only if such combinations produce stable compounds.

[0162] When a listed group does not explicitly indicate that it has a substituent, the group refers only to the unsubstituted group. For example, when "C1-C4 alkyl" is not preceded by the qualifier "substituted or unsubstituted", it refers only to "C1-C4 alkyl" itself or "unsubstituted C1-C4 alkyl".

[0163] In various parts of this specification, the substituents of the compounds disclosed herein are disclosed according to the type or scope of the groups. In particular, this invention includes every independent secondary combination of the respective members of these group types and scopes. The term "C" x -C y "Alkyl" refers to a straight-chain or branched saturated hydrocarbon containing x to y carbon atoms. For example, the terms "C1-C6 alkyl" or "C 1-6 "alkyl" specifically refers to independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl; "C" 1-4 "Alkyl" specifically refers to independently disclosed methyl, ethyl, C3 alkyl (i.e. propyl, including n-propyl and isopropyl), and C4 alkyl (i.e. butyl, including n-butyl, isobutyl, sec-butyl, and tert-butyl).

[0164] In this invention, the term "C1-C6 alkyl" alone or in combination refers to a saturated straight-chain or branched alkyl group containing 1 to 6, particularly 1 to 4, carbon atoms, such as methyl, ethyl, propyl, butyl, pentyl, hexyl; in one embodiment, "C1-C6 alkyl" is preferably "C1-C4 alkyl", such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl; or, for example, methyl or ethyl.

[0165] Linking substituents are described in various parts of this invention. When the structure clearly requires a linking group, the Markush variable listed for that group should be understood as the linking group. For example, if the structure requires a linking group and the Markush group definition for that variable lists "alkyl", then it should be understood that "alkyl" represents a linked alkylene group.

[0166] In some specific structures, when an alkyl group is clearly indicated as a linking group, then the alkyl group represents a linked alkylene group. For example, the C1-C6 alkyl in the group “halogenated-C1-C6 alkyl” should be understood as C1-C6 alkylene.

[0167] In this application, unless otherwise specified, the term "cycloalkyl" as a group or part of other groups refers to a saturated monocyclic, polycyclic, or bridged carbocyclic substituent consisting only of carbon and hydrogen atoms, and which may be connected to the rest of the molecule via a single bond through any suitable carbon atom; when polycyclic, it may be a fused or spirocyclic system (i.e., the two geminal hydrogens on the carbon atom are replaced by alkylene groups) of either a bridged or spirocyclic system. The cycloalkyl substituent may be connected to the central molecule via any suitable carbon atom. In some embodiments, a ring having 3-10 carbon atoms may be represented as C3-C 10 Cycloalkyl groups. In some embodiments, C3-C6 cycloalkyl groups include cyclopropyl (C3), cyclobutyl (C4), cyclopentyl (C5), and cyclohexyl (C6). In some embodiments, C3-C6 cycloalkyl groups... 10 Examples of cycloalkyl groups include the aforementioned C3-C6 cycloalkyl groups along with cycloheptyl (C7), cyclooctyl (C8), cyclononyl (C9), and cyclodecyl (C1). 10 ).

[0168] In this application, as part of a group or other group, the term "aryl" refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 shared p electrons in a cyclic array) having 6-14 ring atoms and providing zero heteroatoms in the aromatic ring system ("C6-C"). 14 Aryl group ("aryl"). Examples of the above aryl units include phenyl, naphthyl, phenanthryl, or anthracene.

[0169] In this application, as part of a group or other group, the term "heteroaryl" refers to a group ("4-16-membered heteroaryl") having a carbon atom and providing 1-3 heteroatoms (each heteroatom independently selected from nitrogen, oxygen, and sulfur) in the aromatic ring system of a 4n+2 aromatic ring system (e.g., having 6 or 10 shared p electrons in a cyclic array). In a heteroaryl group containing one or more nitrogen atoms, the linking point can be a carbon or nitrogen atom, provided the valence allows.

[0170] In some embodiments, the heteroaryl group is a 4-6 membered heteroaryl group with one or more heteroatoms selected from N, O and S, and the number of heteroatoms is 1 to 3, preferably a 5-6 membered heteroaryl group.

[0171] Exemplary 5-membered heteroaryl groups include, but are not limited to: pyrrole, furanyl, thiophene, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiazolyl, furazolyl, oxtriazolyl, or tetrazolyl. Exemplary 6-membered heteroaryl groups include, but are not limited to: pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazinyl, or tetraazinyl.

[0172] Those skilled in the art will understand that, according to the conventions used in the art, the structural formulas of the descriptive groups described in this application... This refers to the fact that the corresponding group is connected to other fragments or groups in the compound through this site.

[0173] In this invention, x represents the molar ratio of cytotoxic drug molecules to antibodies (also known as DAR, i.e., drug-antibody conjugation ratio). Preferably, it is understood as the average molar ratio of drug molecules to monoclonal antibody molecules in the antibody-drug conjugate obtained after conjugating a monoclonal antibody molecule with a cytotoxic drug. This ratio can generally be determined using methods such as hydrophobic-interaction chromatography (HIC), polyacrylamide-SDS gel electrophoresis (SDS-PAGE), or liquid chromatography-mass spectrometry (LC-MS). For example, L-D are groups reactive to the conjugation sites on the antibody, L is a linker, D is a cytotoxic agent further conjugated to the antibody linked to L, and x represents the final number of D conjugates on each antibody, or the number of D conjugates on a single antibody. x can be an integer or a decimal. In some embodiments, x is actually the average of 2 to 8, 4 to 8, or 6 to 8, or x is an integer among 2, 3, 4, 5, 6, 7, or 8. In some embodiments, x is the average of 2, 4, 6, or 8. In other embodiments, x is the average of 2, 3, 4, 5, 6, 7, or 8.

[0174] The antibodies of this invention are interpreted in the broadest sense, specifically binding to targets such as carbohydrates, polynucleotides, fats, and polypeptides through at least one antigen recognition region located in the variable region of the immunoglobulin molecule. Specifically, they include complete monoclonal antibodies, polyclonal antibodies, bispecific antibodies, and antibody fragments, provided they possess the desired biological activity. The antibodies of this invention can be prepared using techniques well-known in the art, such as hybridoma methods, recombinant DNA technology, phage display technology, synthetic techniques, or combinations thereof, or other techniques known in the art.

[0175] Variants of the antibodies of this invention refer to amino acid sequence mutants and covalent derivatives of natural polypeptides, provided that they retain biological activity equivalent to that of the natural polypeptide. The difference between the amino acid sequence mutant and the natural amino acid sequence generally lies in the substitution of one or more amino acids in the natural amino acid sequence, or the deletion and / or insertion of one or more amino acids in the polypeptide sequence. Deletion mutants include fragments of the natural polypeptide and N-terminal and / or C-terminal truncation mutants. Typically, amino acid sequence mutants have at least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) homology to the natural sequence.

[0176] Monoclonal antibodies, or monoclonal antibodies, refer to antibodies derived from a group of essentially homogeneous antibodies. This means that the antibodies constituting the cluster are completely identical, except for a small number of natural mutations or isoforms generated during antibody expression and preparation. Monoclonal antibodies exhibit high specificity against a single antigen. Polyclonal antibodies, on the other hand, comprise different antibodies targeting different determinants of the antigen, with each monoclonal antibody targeting only one determinant of the antigen. In this invention, monoclonal antibodies also specifically include chimeric antibodies and their fragments, meaning that a portion of the antibody's heavy chain and / or light chain originates from one, a class, or a subclass, while the remaining portion is derived from another, a class, or a subclass.

[0177] Linkers refer to the direct or indirect connection between antibodies and drugs. Linking a linker to a mAb can be accomplished in many ways, such as via surface lysine residues, reductive coupling to oxidized carbohydrates, and via the release of cysteine ​​residues through the reduction of interchain disulfide bonds. Various ADC linker systems are known in this field, including hydrazone, disulfide, and peptide-based links.

[0178] The pharmaceutical excipients described herein are those widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods to facilitate the dissolution of the active ingredient at a desired rate after administration to a subject, or to promote the effective absorption of the active ingredient after administration of the composition to a subject. The pharmaceutical excipients may be inert fillers, or provide a function such as stabilizing the overall pH of the composition or preventing the degradation of the active ingredient. The pharmaceutical excipients may include one or more of the following: buffers, chelating agents, preservatives, solubilizers, stabilizers, excipients and surfactants, colorants, flavoring agents, and sweeteners.

[0179] The term "pharmaceutically acceptable salt" refers to a salt prepared from the compounds of the present invention with a relatively non-toxic, pharmaceutically acceptable acid or base. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting a sufficient amount of a pharmaceutically acceptable base with the neutral form of such compounds in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, lithium salts, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, zinc salts, bismuth salts, ammonium salts, and diethanolamine salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting a sufficient amount of a pharmaceutically acceptable acid with the neutral form of such compounds in a pure solution or a suitable inert solvent. 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, and sulfuric acid. 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, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acidic citric acid, oleic acid, tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentian acid, fumaric acid, gluconic acid, succinic acid, formic acid, ethanesulfonic acid, dihydroxynaphthyl acid (i.e., 4,4'-methylene-bis(3-hydroxy-2-naphthylcarboxylic acid)), amino acids (e.g., glutamic acid, arginine), etc. When the compounds of the present invention contain relatively acidic and relatively basic functional groups, they can be converted into base addition salts or acid addition salts. For details, see 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 Camille G. Wermuth, ed., Wiley-VCH, 2002).

[0180] The term "treatment" or its equivalents, when used, for example, in the context of cancer, refers to a procedure or process used to reduce or eliminate the number of cancer cells in a patient's body or to alleviate the symptoms of cancer. "Treatment" for cancer or other proliferative disorders does not necessarily mean that cancer cells or other disorders will actually be eliminated, that the number of cells or disorders will actually be reduced, or that the symptoms of cancer or other disorders will actually be alleviated. Often, methods for treating cancer are undertaken even with only a low probability of success, but are still considered to induce an overall beneficial effect, taking into account the patient's medical history and estimated survival expectations.

[0181] The term "prevention" refers to the reduction of the risk of acquiring or developing a disease or disorder.

[0182] Furthermore, it should be noted that, unless otherwise explicitly stated, the descriptive phrase "...independently" used in this invention should be interpreted broadly, meaning that the described entities are independent of each other and can independently be the same or different specific functional groups. More specifically, the descriptive phrase "...independently" can mean either that the specific options expressed by the same symbol in different functional groups do not affect each other, or that the specific options expressed by the same symbol in the same functional group do not affect each other.

[0183] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0184] Unless otherwise specified, room temperature in this invention refers to 20-30°C. All reagents and raw materials used in this invention are commercially available.

[0185] The positive and progressive effects of this invention are as follows:

[0186] 1. The linker-drug conjugate of the present invention can be conjugated with different antibodies to prepare antibody-drug conjugates with good targeting, or conjugated with different small molecule ligands to prepare small molecule ligand conjugates with good affinity for the receptors of the small molecule ligands.

[0187] 2. The antibody-drug conjugate and small molecule ligand conjugate of the present invention have excellent in vitro cell activity and in vivo antitumor activity.

[0188] 3. The antibody-drug conjugate of the present invention has excellent plasma stability and safety. Detailed Implementation

[0189] Table 1. Explanation of Abbreviations

[0190]

[0191]

[0192] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0193] Example 1: General synthetic route for compounds represented by formula I

[0194]

[0195] Step 1

[0196] I-1 and I-2 are commercially available raw materials, and their feeding ratio is 1:1-1:2. Under the condition of DMF as solvent and EEDQ as condensing agent (2 equivalents), the reaction is carried out for 6-8 hours, and pure I-3 is obtained by column chromatography.

[0197] Step 2

[0198] I-3 and NPC (2 equivalents) were reacted in DMF in the presence of triethylamine (2 equivalents) for 5-6 hours, and pure I-4 was obtained by column chromatography.

[0199] Step 3

[0200] I-4 was reacted with different primary amine fragments (1-2 equivalents, commercially available) in DMF for 1-2 hours, and purified by column chromatography to obtain pure I-5.

[0201] Step 4

[0202] I-5 reacts with different aldehydes (3-5 equivalents, commercially available) and trimethylchlorosilane (3-5 equivalents) in anhydrous dichloromethane for 10-12 hours to give the active intermediate I-6. Further reaction with the thiol or hydroxyl groups of different drug molecules in the presence of a base (usually piracetam) for 1-3 hours yields intermediate I-7, which is purified by column chromatography to obtain the pure product.

[0203] Step 5

[0204] I-7 was reacted with trimethylphosphine reagent in a tetrahydrofuran / acetic acid buffer (pH 5.0) mixture for 1-3 hours, and then purified by column chromatography to obtain intermediate I-8 containing a primary amino group.

[0205] Step 6

[0206] I-8, along with different carboxyl-containing maleamide fragments (commercial or custom-made), yields different linker-drugs (I-LD series). The final products can be purified by preparative chromatography.

[0207] Following the general method described in steps 1-6 above, and using the starting materials listed in Table 2, compounds with the following structures were prepared:

[0208]

[0209]

[0210]

[0211] Table 2. MS data of I-LD01 to I-LD10 and raw materials used in their preparation

[0212]

[0213] The structures of each maleamide fragment in this invention are as follows:

[0214] Segment 1: Segment 2:

[0215] Segment 3:

[0216] Example 2: General synthetic route for compounds represented by formula II

[0217]

[0218] Step 1

[0219] II-1 is obtained from commercially available raw materials or custom-made materials. The ratio of II-1 to I-2 is 1:2-1:4. Under the condition of DMF as solvent and EEDQ as condensing agent (4 equivalents), the reaction is carried out for 6-8 hours, and pure II-2 is obtained by column chromatography.

[0220] Step 2

[0221] Referring to step 4 of Example 1, intermediate II-3 can be obtained, which is further spliced ​​with a drug molecule containing a thiol or hydroxyl group to obtain II-4.

[0222] Step 3

[0223] Intermediate II-5 can be obtained by referring to step 5 in Example 1.

[0224] Step 4

[0225] Different linker-drugs (II-LD series) can be obtained by referring to step 6 in Example 1. The final product can be purified by preparative chromatography.

[0226] Following the general method described in steps 1-4 above, and using the starting materials listed in Table 3, compounds with the following structures were prepared:

[0227]

[0228]

[0229]

[0230] Table 3. MS data and raw materials used in the preparation of II-LD01 to II-LD08

[0231]

[0232]

[0233] Example 3: General synthetic route for compounds represented by formula III

[0234]

[0235] Step 1

[0236] I-4 reacts with different amino alcohols (commercially available) to give intermediate III-2, which is purified by column chromatography.

[0237] Step 2

[0238] III-2 was oxidized by PCC to obtain III-3, which was then purified by column chromatography.

[0239] Step 3

[0240] Following step 4 in Example 1, active chlorine intermediate III-4 was obtained, which was further spliced ​​with a drug molecule containing hydroxyl or thiol groups to obtain intermediate III-5, which was then purified by column chromatography.

[0241] Step 4

[0242] Referring to step 5 in Example 1, III-5 was reduced by trimethylphosphine and then purified by column chromatography to obtain intermediate III-6.

[0243] Step 5

[0244] III-6 was condensed with different carboxylic acid fragments and purified by column chromatography to obtain the final product (III-LD series).

[0245] Following the general method described in steps 1-5 above, and using the starting materials listed in Table 4, compounds with the following structures were prepared:

[0246]

[0247]

[0248] Table 4. MS data and raw materials used in the preparation of III-LD01 to II-LD07

[0249]

[0250] Example 4: pH stability experiment

[0251] Buffer solutions with different pH values ​​(pH 1.0, pH 5.0, pH 7.4) were prepared. The linker-drug conjugates prepared according to the methods in Examples 1-3 were diluted 100-fold with DMSO dissolution buffer to a final substrate concentration of 100 μM. The solutions were incubated at 25°C for 24 hours, and HPLC analysis was performed. Using the corresponding drug molecule as an external standard, the release ratio of the drug molecule in the buffer solution (relative to an equal volume of external standard) was calculated. The results (as shown in Table 5) indicate that the linker-drug conjugates of this invention exhibit good stability at pH 5.0 and 7.4.

[0252] Table 5. pH stability test of linker-drug conjugates

[0253]

[0254]

[0255] Example 5 Mouse plasma stability experiment

[0256] The selected compound was dissolved in DMSO and diluted 100-fold with mouse plasma to a final substrate concentration of 100 μM. The mixture was then incubated at 37°C for 24 hours. 200 μL of each sample was then taken and treated with 400 μL of methanol. After centrifugation and precipitation, the drug release was detected by HPLC. The drug was calculated using the corresponding drug as an external standard. The results (as shown in Table 6) indicate that the linker-drug conjugate of the present invention has good stability in mouse plasma.

[0257] Table 6. Results of mouse plasma stability test

[0258]

[0259]

[0260] Example 6: Human plasma stability experiment

[0261] The selected compound was dissolved in DMSO and diluted 100 times with human plasma to a final substrate concentration of 100 μM. The mixture was then incubated at 37°C for 24 hours. 200 μL of each sample was then taken and treated with 400 μL of methanol. After centrifugation and precipitation, the drug release was detected by HPLC. The drug was calculated using the corresponding drug as an external standard. The results (see Table 7) show that the linker-drug conjugate of the present invention has good stability in human plasma.

[0262] Table 7. Results of human plasma stability test

[0263]

[0264]

[0265] Example 7 Enzyme digestion and release experiment

[0266] The selected compound was dissolved in DMSO and then diluted 100-fold with a prepared solution containing cathepsin B (enzyme concentration: 0.1 mg / mL, 25 mM acetate buffer, pH 5.0) to a final substrate concentration of 100 μM. The solution was incubated at 37°C for 5 hours. Then, 200 μL of each sample was taken and treated with 400 μL of ice-cold acetonitrile. After centrifugation and precipitation, the drug release was detected by HPLC. The drug was calculated using the corresponding drug as an external standard. The results (see Table 8) show that the linker-drug conjugate of the present invention can be effectively enzymatically cleaved by cathepsin B to release cytotoxic drug molecules.

[0267] Table 8. Results of enzyme cleavage and release experiments

[0268]

[0269]

[0270] Example 8 Preparation of Antibody-Drug Conjugates

[0271] The commercially available antibody Herceptin was prepared into a 20 mg / mL solution (pH 7.4 phosphate buffer), and 8 equivalents of TCEP were added. The mixture was reacted at 25°C for 1 hour to fully open the disulfide bonds between the antibody chains. The solution was then passed through a G25 gel column and the buffer was changed to a weakly acidic buffer (pH 5.0 citrate buffer). 12 molar equivalents of the above linker-drug conjugate were dissolved in an appropriate amount of DMSO and added to the antibody solution after buffer change. The reaction was carried out for 30 minutes to obtain the ADC stock solution. The unconjugated small molecules were removed by passing the solution through a G25 gel column to obtain the ADC sample. The DAR value was determined by LC-MS, and the monomer purity was determined by SEC. The results are shown in Table 9.

[0272] Table 9. DAR value and purity test results of antibody-drug conjugates

[0273] ADC number Linker-drug conjugates DAR SEC purity ADC01 I-LD01 7.9 99.1％ ADC02 II-LD01 7.8 99.5％ ADC03 III-LD01 8.0 99.4％

[0274] Example 9: Preparation of Small Molecule Ligand Conjugate Drugs

[0275] A commercially available folic acid derivative (structure shown below) was mixed with a selected linker-drug (I-LD01) in an equimolar amount in a citrate buffer at pH 6.0 and reacted for 1 hour. The mixture was purified by reversed-phase preparative chromatography, and the main components were collected and lyophilized to obtain a solid powder, designated SMDC01. The molecular weight was 1699.81 (M+H) by mass spectrometry.

[0276]

[0277] Example 10: Evaluation of cytotoxic activity of ADC01, ADC02, and ADC03

[0278] NCI-N87 cells, which highly express the HER2 receptor, were selected as the cell line for in vitro activity assays in this experiment to observe the dose-response effect of different antibody-drug conjugates on cell killing. The seeding density for each cell type was 2 × 10⁻⁶ cells / cell. 3 Cells / well were collected, and cytotoxic activity was measured after 16–24 hours. Next, the final concentration of the prepared antibody-drug conjugate was set at 5000 nM as the initial concentration, and ten series of concentrations (4–10-fold serial dilutions) ranging from 5000 to 0.006 nM were designed. Changes in cytotoxicity (or inhibition) were observed after 144 hours. Luminescent Cell Viability Assay: Chemiluminescent staining, reading fluorescence data, and calculating IC50. 50 The results (as shown in Table 10) indicate that the ADC provided by the present invention has excellent cell-killing activity in vitro.

[0279] Table 10. Evaluation of cytotoxic activity of ADCs

[0280] Sample number <![CDATA[IC 50 (nM)]]> ADC01 0.451 ADC02 0.501 ADC03 0.489 Dxd (reference) 4.6

[0281] Example 11 Evaluation of SMDC01 Cytotoxic Activity

[0282] KB cells, which highly express folate receptors, were selected as the cell line for in vitro activity assays in this experiment to observe the dose-response effect of different small molecule ligand-conjugated drugs on cell killing. The seeding density for each cell type was 3 × 10⁻⁶. 3 Cells / well were collected, and cytotoxic activity was measured after 16–24 hours. Next, the final concentration of the prepared antibody-drug conjugate was set at 5000 nM as the initial concentration, and ten series of concentrations (4–10-fold serial dilutions) ranging from 5000 to 0.006 nM were designed. Changes in cytotoxicity (or inhibition) were observed after 96 hours. Luminescent Cell Viability Assay: Chemiluminescent staining, reading fluorescence data, and calculating IC50. 50 The results obtained (as shown in Table 11) indicate that...

[0283] Table 11. Evaluation of cytotoxic activity of SMDA01

[0284] Sample number <![CDATA[IC 50 (nM)]]> SMDC01 4.5 Dxd 8.8

[0285] Example 12: In vivo efficacy evaluation of ADC01

[0286] Female Balb / c nude mice aged 6-8 weeks were subcutaneously injected in the nape of their necks with 5 × 10⁶ human pancreatic cancer cells (Capan-1) dissolved in 100 μL PBS solution. The tumors reached an average volume of approximately 160 mm². 3 Thirty-six nude mice were randomly divided into six groups of six each, based on tumor size. The mice were administered ADC-01 via tail vein injection: group 01 was the blank control group, and group 02 received ADC-01 (5 mg / kg) once daily. Animal weight and tumor volume were measured twice weekly, and animal survival was observed. The results (as shown in Table 12) indicated that ADC-01 exhibited good in vivo antitumor activity, and no mice showed mortality or weight loss, demonstrating the good safety profile of ADC-01.

[0287] Table 12. In vivo efficacy evaluation results of ADC01

[0288]

[0289] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A linker-drug conjugate having the general structural formula: L4-L2-L1-D, wherein, D is a cytotoxic drug; The structure of L1 is shown in Formula II, with its a-terminus connected to the cytotoxic drug and its e-terminus connected to L2. ； R 1 It is a C1~C6 alkyl or hydrogen; R 2 It is hydrogen or C1~C6 alkyl; R 4 It is hydrogen or C1~C6 alkyl; n is 1; L is independently selected from 20 natural amino acids; p is an integer from 1 to 3; (L) p It is a fragment of 1-3 amino acid residues, with both the amino and carbonyl ends connected by amide bonds, the N-terminus connected to the left carbonyl group, and the C-terminus connected to the right amino group; L2 is C2-C 12 A straight-chain alkyl group, or a (poly)ethylene glycol chain containing 1-12 ethylene glycol units, or any combination of two thereof; one end of L2 is connected to the e-end of L1, and the other end is connected to the d-end of L4; L4 is or , wherein the d end is connected to one end of the L2.

2. The linker-drug conjugate as described in claim 1, characterized in that, in, The cytotoxic drug is a drug containing a hydroxyl group or a thiol group; the hydroxyl group or thiol group in the structure of the cytotoxic drug is optionally connected to the a-terminus of L1. And / or, the R 1 It is a C1~C6 alkyl group; And / or, the R 4 It is hydrogen or C1~C6 alkyl; And / or, wherein L is independently a phenylalanine residue, alanine residue, glycine residue, glutamic acid residue, aspartic acid residue, cysteine ​​residue, glutamic acid residue, histidine residue, isoleucine residue, leucine residue, lysine residue, methionine residue, proline residue, serine residue, threonine residue, tryptophan residue, tyrosine residue, or valine residue. And / or, p is 1; And / or, the L4 is .

3. The linker-drug conjugate as described in claim 2, characterized in that, in, The cytotoxic drug is In DM-X, y is an integer from 0 to 6, and X is a hydroxyl or thiol group; And / or, the hydroxyl group in the cytotoxic drug structure is linked to L1 in the form of an ether bond; And / or, when the R 1 When the alkyl group is C1 to C6, the C1 to C6 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl; And / or, when the R 2 When the alkyl group is C1 to C6, the C1 to C6 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl; And / or, when the R 4 When the alkyl group is C1 to C6, the C1 to C6 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl; And / or, the L is independently a valine residue, a glycine residue, or a phenylalanine residue; And / or, when the L2 is C2-C 12 When the chain is linear alkyl, the C2-C 12 The straight-chain alkyl group is n-propyl, n-butyl, n-pentyl, n-hexyl, or n-heptyl; And / or, the L2 is C2-C 12 When a straight-chain alkyl group is combined with a (poly)ethylene glycol chain containing 1-12 ethylene glycol units, the C2-C 12 The straight-chain alkyl group is ethyl, n-propyl, or n-butyl; And / or, the L2 is C2-C 12 When a straight-chain alkyl group is combined with a (poly)ethylene glycol chain containing 1-12 ethylene glycol units, the C2-C 12 The combination of a straight-chain alkyl group and a (poly)ethylene glycol chain containing 1-12 ethylene glycol units forms a -C2-C group. 12 Straight-chain alkyl group -O-(CH2CH2O)q-C2-C 12 Straight-chain alkyl; q is 1-7; And / or, the aforementioned (L) p for The amino terminus is connected to the carbonyl terminus in Formula II, and the carbonyl terminus is connected to the amino terminus in Formula II.

4. The linker-drug conjugate as described in claim 3, characterized in that, in, The cytotoxic drug is Tubulysin A, Dxd, or DM-X; And / or, when the R 1 When the alkyl group is C1-C6, the C1-C6 alkyl group is methyl; And / or, when the R 2 When the alkyl group is C1-C6, the C1-C6 alkyl group is methyl; And / or, when the R 4 When the alkyl group is C1-C6, the C1-C6 alkyl group is methyl; And / or, the L is independently a valine residue; And / or, when the L2 is C2-C 12 When the chain is linear alkyl, the C2-C 12 The straight-chain alkyl group is n-pentyl; And / or, the L2 is C2-C 12 When a straight-chain alkyl group is combined with a (poly)ethylene glycol chain containing 1-12 ethylene glycol units, the C2-C 12 The straight-chain alkyl group is ethyl; And / or, the L2 is C2-C 12 When a straight-chain alkyl group is combined with a (poly)ethylene glycol chain containing 1-12 ethylene glycol units, the C2-C 12 The combination of a straight-chain alkyl group and a (poly)ethylene glycol chain containing 1-12 ethylene glycol units forms a -C2-C group. 12 Straight-chain alkyl group -O-(CH2CH2O)q-C2-C 12 Straight-chain alkyl; q is 1 or 7.

5. The linker-drug conjugate as described in claim 3, characterized in that, in, The cytotoxic drug is Tubulysin A or Dxd; And / or, the L1 is or ; And / or, the L2 is n-pentyl or .

6. The linker-drug conjugate as described in claim 1, characterized in that, The options are as follows: Option 1, Option 2, and Option 3. Option 1 The cytotoxic drug mentioned is Dxd; the (L) mentioned... p for The L2 mentioned is n-pentyl or The L4 mentioned above is ; The R mentioned 1 It is a C1-C6 alkyl group; the R 2 and R 4 Each is independently a C1-C6 alkyl or hydrogen; n is 1; Option 2 The cytotoxic drug is DM-X, where y is 1 and X is a hydroxyl or thiol group; (L) p for The L4 mentioned above is ; The R mentioned 1 and R 2 Each is independently a C1-C6 alkyl group; the R 4 It is hydrogen; n is 1; L2 is ; Option 3 The cytotoxic drug mentioned is Tubulysin A; The R mentioned 1 It is a C1-C6 alkyl group; the R 2 It is hydrogen or C1~C6 alkyl; the R 4 It is hydrogen or a C1-C6 alkyl group; n is 1; (L) p for ; The L2 mentioned is n-pentyl or The L4 mentioned above is .

7. The linker-drug conjugate as described in claim 1, characterized in that, in, The linker-drug conjugate is any of the following compounds: , , , , , , or .

8. An antibody-drug conjugate having the general structural formula: Ab-(L3-L2-L1-D) x Its characteristics are, in, Ab represents antibodies; x is between 2 and 8; L3 is or The b-terminus is connected to the antibody, and the d-terminus is connected to L2. D, L1, and L2 are defined as described in any one of claims 1-7.

9. The antibody-drug conjugate as described in claim 8, characterized in that, in, The antibody in question is an IgG1 type antibody; And / or, the b-terminus of the L3 and the thiol group on the antibody are linked by a thioether bond; And / or, the x is 4~8; And / or, the D is Dxd; And / or, the L2 is n-pentyl; And / or, L3 is .

10. The antibody-drug conjugate as described in claim 9, characterized in that, in, The antibody mentioned is Herceptin; And / or, the x is 7~8.

11. The antibody-drug conjugate as described in claim 10, characterized in that, The x values ​​are 7.8, 7.9, and 8.

0.

12. The antibody-drug conjugate as described in claim 9, characterized in that, The antibody-drug conjugate is any of the following compounds: Where Ab is Herceptin, L, p, R 1 R 2 R 4 The definitions of n and x are as described in claim 9 or 10.

13. The antibody-drug conjugate as described in claim 12, characterized in that, The antibody-drug conjugate is a compound as shown below: 。 14. A small molecule ligand-conjugated drug, having the general structural formula SM-L3-L2-L1-D, characterized in that, in, SM is a small molecule ligand; L3 is or The b-terminus is connected to the antibody, and the d-terminus is connected to L2. D, L1, and L2 are defined as described in any one of claims 1-7.

15. The small molecule ligand-conjugated drug as described in claim 14, characterized in that, in, The small molecule ligands mentioned are folic acid derivatives containing thiol groups, small molecule polypeptides containing thiol groups, and polysaccharide derivatives containing thiol groups. And / or, the b-terminus of the L3 and the thiol group on the antibody are linked by a thioether bond; And / or, the D is Dxd; And / or, the L2 is n-pentyl; And / or, L3 is .

16. The small molecule ligand-conjugated drug as described in claim 15, characterized in that, The small molecule ligand is a folic acid derivative containing a thiol group; the folic acid derivative containing a thiol group is a compound having the following structure: 。 17. A method for preparing an antibody-drug conjugate as described in any one of claims 8-13 or a small molecule ligand-drug conjugate as described in any one of claims 14-16, characterized in that, It includes the following steps, The linker-drug conjugate as described in any one of claims 1-7 and the antibody as described in any one of claims 8-13 or the small molecule ligand as described in any one of claims 14-16 can be conjugated.

18. A pharmaceutical composition comprising an antibody-drug conjugate as described in any one of claims 8-13 or a small molecule ligand-drug conjugate as described in any one of claims 14-16, and a pharmaceutically acceptable carrier.

19. Use of an antibody-drug conjugate as described in any one of claims 8-13 or a small molecule ligand-drug conjugate as described in any one of claims 14-16 in the preparation of a medicament for the prevention and / or treatment of cancer; wherein the cancer is gastric cancer, breast cancer, non-small cell lung cancer, urothelial carcinoma, or pancreatic cancer.

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