Antibody drug conjugate containing protein degradation agent bioactive compound and preparation method and application thereof

Abstract

The invention relates to an antibody drug conjugate of a protein degradation agent bioactive compound as shown in a formula I, a preparation method of the antibody drug conjugate, and application of the antibody drug conjugate in prevention and / or treatment of diseases related to abnormal cell activity, including but not limited to prevention and / or treatment of tumor diseases. # imgabs0 #

Description

Antibody-drug conjugates containing protein degrader-type bioactive compounds and preparation methods and uses thereof Technical Field

[0001] The present disclosure belongs to the field of medical technology and relates to antibody-drug conjugates of protein degrader-type bioactive compounds, protein degrader-type bioactive compounds, drug-linker conjugates and preparation methods thereof, as well as their use in preventing and / or treating diseases associated with abnormal cell activity, including but not limited to preventing and / or treating tumor diseases. Background Art

[0002] Many diseases are associated with abnormal protein function within cells, and the primary treatment for these diseases is small molecule compounds. However, over 80% of proteins lack druggable sites, making these targets considered undruggable with traditional small molecule drugs.

[0003] Targeted protein degradation agents (TPDs), such as protein degradation targeting chimeras (PROTACs) and molecular glue degraders (MGDs), mediate the degradation of target proteins through the ubiquitin-proteasome pathway. They can work without tightly binding to sites that affect protein activity, making the aforementioned "undruggable" proteins new drug targets. At the same time, targeted protein degradation therapy can continuously induce the rapid and efficient degradation of pathogenic proteins, reducing the development of drug resistance in target proteins.

[0004] Targeted protein degraders are currently in clinical development. C4 Therapeutics presented preclinical data for its IKZF1 / 3-targeting molecular glue, CFT7455, at the 2021 AACR. The company also presented clinical results for CFT7455 at the April 2022 AACR meeting. While CFT7455 demonstrated clinical benefits from deep target degradation, neutropenia was dose-limiting.

[0005] Currently, in the field of antibody-drug conjugates (ADCs), maleimide is widely used for coupling to antibody thiols through Michael addition reactions to form ADC molecules due to its high selectivity, high reactivity, and good stability. Of the 15 ADC drugs already on the market, 9 are coupled via maleimide. Numerous literature reports indicate that sulfosuccinimide bonds are unstable in the presence of thiol-containing substances (such as in plasma), and can break through reverse Michael reactions or exchange with endogenous thiols such as albumin (HSA) and glutathione (GSH), resulting in unsatisfactory pharmacodynamics, pharmacokinetics, and safety.

[0006] Summary of the Invention

[0007] The present invention relates to an antibody-drug conjugate (ADC) as shown in Formula I and its use. ADC drugs combine the tumor-targeting effect of antibodies with the high activity of bioactive compounds, becoming a biological missile with highly anticipated efficacy and safety advantages. Antibodies guide ADCs to bind to target cells, achieving tumor tissue enrichment, reducing exposure to non-target tissues, and reducing the toxicity that may be caused by systemic administration of bioactive compounds. ADCs bound to tumor cells can be cleaved in the tumor microenvironment to release bioactive molecules, killing tumor cells. ADCs can also be internalized by tumor cells and undergo enzymatic hydrolysis under the action of specific enzymes within the cells to release small molecule drugs for disease treatment. Therefore, it can be expected that ADCs composed of a protein degrader and a tumor-targeting antibody can achieve tumor enrichment, eliminate or reduce the toxic side effects caused by the protein degrader acting on non-disease tissues, and improve the therapeutic effect. Utilizing ADC technology to achieve tumor targeting of protein degraders and reduce their toxic side effects will have high clinical value.

[0008] To this end, in the first aspect of the present disclosure, the present disclosure provides an antibody drug conjugate as shown in Formula I,

[0009] or a stereoisomer of the antibody drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof,

[0010] in,

[0011] Tb is an antibody or an antigen-binding fragment thereof;

[0012] q is the number of Tb couplings L1-L2-L3-L4-D;

[0013] D is a bioactive compound fragment of a protein degrader;

[0014] L1 is the connector unit;

[0015] L2 is the connection unit;

[0016] L3 is selected from AA 1 The amino acid residues shown may contain AA 1 A short peptide consisting of 2-10 amino acid residues of the amino acid residues shown; the amino acid residues are selected from natural amino acid residues, non-natural amino acid residues, AA 1 the indicated amino acid residue or its stereoisomers;

[0017] AA 1 The structures of the indicated amino acid residues are shown below,

[0018] in:

[0019] R a 、Rb Each independently selected from hydrogen or And R a 、R b Not simultaneously hydrogen;

[0020] Or, R a With R b Together with the carbon atoms to which they are attached, they form a 4-10 membered heterocyclic ring, which is optionally substituted by one or more R 0 replaced by;

[0021] r、r 1 Each is independently selected from any integer between 0 and 20;

[0022] R m1 、R n1 Each is independently selected from hydrogen, C1-6 alkyl or C3-6 cycloalkyl;

[0023] Or, R m1 With R n1 Together with the nitrogen atom to which they are attached, they form a 4-10 membered heterocyclic ring, which is optionally substituted by one or more R 0’ replaced by;

[0024] R 0 、R 0’ Each independently selected from C1-6 alkyl, C3-6 cycloalkyl, -NR m2 R n2 or a 4-10 membered heterocyclic group optionally substituted by a C1-6 alkyl group;

[0025] R m2 、R n2 Each is independently selected from hydrogen or C1-6 alkyl;

[0026] L4 is absent or present. When L4 is present, L4 is selected from Bit 1 is connected to L3 and bit 2 is connected to D.

[0027] In some embodiments, the protein degrader bioactive compound is selected from: a protein degradation targeting chimera or a molecular glue.

[0028] In some embodiments, the protein degrader bioactive compound is selected from one or more of the following:

[0029] (1) Protein degradation targeting chimeras that can cause degradation of GSPT1, IKZF1 and / or 3, CK1α and other family proteins.

[0030] (2) Molecular glue that can cause the binding and degradation of GSPT1, IKZF1 and / or 3, CK1α and other family proteins.

[0031] In some embodiments, the protein degrader bioactive compound is a molecule that can cause degradation of GSPT1 family proteins.

[0032] In some embodiments, q is selected from any value between 0.1-12.0.

[0033] In some embodiments, q is selected from any value between 1.0-10.0.

[0034] In some embodiments, q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0035] In some embodiments, q is selected from any value between 2-8.

[0036] In some embodiments, q is selected from 2, 4, 6, or 8.

[0037] In some embodiments, L1 is selected from Position 1 is connected to Tb via an S atom, and position 2 is connected to L2.

[0038] In some embodiments, L1 is selected from Position 1 is connected to Tb via an S atom, and position 2 is connected to L2.

[0039] In some embodiments, L2 is selected from Bit 1 is connected to L1 and bit 2 is connected to L3.

[0040] In some embodiments, L2 is selected from Bit 1 is connected to L1, and bit 2 is connected to L3.

[0041] In some embodiments, Y is selected from -CH2- or -OCH2CH2-.

[0042] In some embodiments, Y is -CH2-.

[0043] In some embodiments, n is selected from any integer between 0-10.

[0044] In some embodiments, n is selected from 0, 1, 2, or 3.

[0045] In some embodiments, n is selected from 1, 2, or 3.

[0046] In some embodiments, X is selected from -CR m R n -or-NR m -.

[0047] In some embodiments, X is selected from -CH2-, -C(CH3)2-, -N(CH3)-, or -NH-.

[0048] In some embodiments, R m 、R n Each is independently selected from hydrogen, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-6 cycloalkyl or 3-6 membered heterocyclyl;

[0049] Or, R m With R n Together with the carbon atoms to which they are attached, they form a 3-6 membered carbocyclic ring or a 3-6 membered heterocyclic ring.

[0050] In some embodiments, R m 、R n are each independently selected from hydrogen or methyl.

[0051] In some embodiments, L1-L2 are selected from Position 1 is connected to Tb via an S atom, and position 2 is connected to L3.

[0052] In some embodiments, L1-L2 are selected from

[0053] Position 1 is connected to Tb via an S atom, and position 2 is connected to L3.

[0054] In some embodiments, L1-L2 are selected from

[0055] Position 1 is connected to Tb via an S atom, and position 2 is connected to L3.

[0056] In some embodiments, L3 is selected from AA 1 The amino acid residues shown may contain AA 1 A dipeptide, tripeptide or tetrapeptide of the indicated amino acid residues.

[0057] In some embodiments, L3 is selected from the group consisting of an amino acid residue, a dipeptide, or a tripeptide as shown below: AA 1 , Val-Ala, Val-AA 1 、Ala-AA 1 , Gly-AA 1 AA 1 -Gly, AA 1 -Ala, Val-AA 1 -Gly, Ala-AA 1 -Gly, Gly-AA 1 -Gly or Val-AA1 -Ala.

[0058] In some embodiments, L3 is selected from the group consisting of an amino acid residue, a dipeptide, or a tripeptide as shown below: AA 1 、Val-AA 1 、Ala-AA 1 , Gly-AA 1 AA 1 -Gly, AA 1 -Ala, Val-AA 1 -Gly, Ala-AA 1 -Gly, Gly-AA 1 -Gly or Val-AA 1 -Ala.

[0059] In some embodiments, L3 is selected from the dipeptide or tripeptide shown below: Val-AA 1 、Val-AA 1 -Ala or Val-AA 1 -Gly.

[0060] In some embodiments, L3 is Val-AA 1 -Gly tripeptide.

[0061] In some embodiments, L3 is selected from Bit 1 is connected to L2, and bit 2 is connected to L4 or D.

[0062] In some embodiments, L3 is selected from Bit 1 is connected to L2, and bit 2 is connected to L4 or D.

[0063] In some embodiments, L3 is selected from Bit 1 is connected to L2, and bit 2 is connected to L4 or D.

[0064] In some embodiments, R a 、R b In which one is hydrogen and the other is

[0065] In some embodiments, R a With R b Together with the carbon atoms they are connected to, they form the 0 Substituted 5-6 membered heterocycle.

[0066] In some embodiments, R a With R bTogether with the carbon atoms they are connected to, they form the 0 a substituted piperidine ring or a piperazine ring.

[0067] In some embodiments, R a With R b Together with the carbon atoms they are connected to, they form the 0 Substituted piperidine ring.

[0068] In some embodiments, R a With R b Together with the carbon atoms they are connected to, they form Carbon atom No. 1 is R a and R b carbon atoms that are linked together.

[0069] In some embodiments, r, r 1 Each is independently selected from 0, 1, 2, 3, 4 or 5.

[0070] In some embodiments, r, r 1 are each independently selected from 0 or 4.

[0071] In some embodiments, r is 0, r 1 is 4.

[0072] In some embodiments, R m1 、R n1 Each is independently selected from hydrogen or C1-6 alkyl.

[0073] In some embodiments, R m1 、R n1 Each is independently selected from C1-6 alkyl.

[0074] In some embodiments, R m1 、R n1 Each is independently selected from hydrogen, methyl, ethyl, n-propyl or n-butyl.

[0075] In some embodiments, R m1 、R n1 Each is independently selected from methyl, ethyl, n-propyl or n-butyl.

[0076] In some embodiments, R m1 With R n1 Together with the nitrogen atom to which they are attached, they form an optionally R 0’ Substituted 5-6 membered heterocycle.

[0077] In some embodiments, R m1 With R n1 Together with the nitrogen atom to which they are attached, they form an optionally R0’ a substituted piperidine ring or a piperazine ring.

[0078] In some embodiments, R m1 With R n1 Together with the nitrogen atoms they are connected to, they form Nitrogen atom No. 1 is R m1 and R n1 Commonly linked nitrogen atoms.

[0079] In some embodiments, R 0 、R 0’ Each independently selected from C1-6 alkyl, -NR m2 R n2 or a 5-6 membered heterocyclic group optionally substituted by a C1-6 alkyl group.

[0080] In some embodiments, R 0 is selected from C1-6 alkyl or a 5-6 membered heterocyclic group substituted by C1-6 alkyl, wherein the 5-6 membered heterocyclic group is selected from piperidinyl and piperazinyl.

[0081] In some embodiments, R 0 is selected from methyl, ethyl or a 5-6 membered heterocyclic group substituted by methyl, wherein the 5-6 membered heterocyclic group is piperidinyl.

[0082] In some embodiments, R 0 Selected from methyl, ethyl or

[0083] In some embodiments, R 0’ Selected from C1-6 alkyl or -NR m2 R n2 .

[0084] In some embodiments, R 0’ Selected from methyl or -NR m2 R n2 .

[0085] In some embodiments, R m2 、R n2 It is a methyl group.

[0086] In some embodiments, AA 1 The structures of the indicated amino acid residues are shown below,

[0087] in:

[0088] R a 、R b In, either one is H and the other is r 1is 4;

[0089] Or, R a With R b Together with the carbon atoms they are connected to, they form the 0 substituted 5-6 membered heterocycle;

[0090] R m1 、R n1 Each is independently selected from hydrogen, C1-6 alkyl or C3-6 cycloalkyl;

[0091] R 0 Selected from C1-6 alkyl, C3-6 cycloalkyl, -NR m2 R n2 or a 5-6 membered heterocyclic group optionally substituted by a C1-6 alkyl group;

[0092] R m2 、R n2 Each is independently selected from hydrogen or C1-6 alkyl.

[0093] In some embodiments, AA 1 The amino acid residues shown are selected from

[0094] In some embodiments, AA 1 The amino acid residues shown are selected from

[0095] In some embodiments, AA 1 The amino acid residues shown are selected from

[0096] In some embodiments, L4 is absent.

[0097] In some embodiments, L4 is selected from Bit 1 is connected to L3 and bit 2 is connected to D.

[0098] In some embodiments, L4 is selected from Bit 1 is connected to L3 and bit 2 is connected to D.

[0099] In some embodiments, Selected from

[0100] 1 is connected to L2, 2 is connected to D, AA 1 The definition of is as described in any embodiment of the present disclosure.

[0101] In some embodiments, Selected from 1 is connected to L2, 2 is connected to D, AA 1 The definition of is as described in any embodiment of the present disclosure.

[0102] In some embodiments, Selected from the following structures:

[0103] Bit 1 is connected to L2 and bit 2 is connected to D.

[0104] In some embodiments, Selected from the following:

[0105] Bit 1 is connected to L2 and bit 2 is connected to D.

[0106] In some embodiments, Selected from:

[0107] Position 1 is connected to Tb via S atom, position 2 is connected to D, and Y, X, AA 1 and n are as defined in any embodiment of the present disclosure.

[0108] In some embodiments, Selected from:

[0109] Position 1 is connected to Tb via S atom, position 2 is connected to D, and Y, X, AA 1 and n are as defined in any embodiment of the present disclosure.

[0110] In some embodiments, Selected from:

[0111] Position 1 is connected to Tb via an S atom, and position 2 is connected to D.

[0112] In some embodiments, Selected from:

[0113] Position 1 is connected to Tb via an S atom, and position 2 is connected to D.

[0114] In some embodiments, D is a structural unit represented by Formula II, and D is connected to L4 or L3 through A;

[0115] in,

[0116] R 1is selected from hydrogen, deuterium, halogen, cyano, amino, nitro, C1-4 alkoxy or C1-4 alkyl;

[0117] Z is selected from -NH-, -CR 2a R 3a -or-C(O)-;

[0118] R 2a 、R 3a are each independently selected from F or OH;

[0119] U 1 、U 2 are each independently selected from -CH2- or -C(O)-, and U 1 、U 2 Not at the same time -CH2-;

[0120] A is selected from O, S, NR 9 、 The 1 position is connected to L4 or L3, and the 2 position is connected to the benzene ring; or the 1 position is connected to the benzene ring, and the 2 position is connected to L4 or L3;

[0121] R2, R3, R4, R5, R6, R7, and R8 are each independently selected from hydrogen, deuterium, C1-4 alkyl, C3-6 cycloalkyl, or 3-6 membered heterocyclyl;

[0122] Alternatively, R2, R3 and the carbon atom to which they are attached form a 3-7 membered carbocyclic ring or a 3-7 membered heterocyclic ring;

[0123] R4, R5 and the carbon atom and nitrogen atom to which they are connected form a 4-7 membered heterocyclic ring;

[0124] R5, R6 and the carbon atom to which they are attached form a 3-7 membered carbocyclic ring or a 3-7 membered heterocyclic ring;

[0125] R7, R8 and the carbon atom to which they are attached form a 3-7 membered carbocyclic ring or a 3-7 membered heterocyclic ring;

[0126] m, p, and y are each independently selected from any integer between 0 and 8;

[0127] W and V are each independently selected from a direct bond, O, S or NR 9 ;

[0128] R 9 Selected from hydrogen, deuterium, C1-4 alkyl or C3-6 cycloalkyl.

[0129] In some embodiments, R 1 Selected from hydrogen, halogen or C1-4 alkyl.

[0130] In some embodiments, R 1 It is a halogen.

[0131] In some embodiments, R 1 is selected from hydrogen, methyl or chlorine.

[0132] In some embodiments, R 1 For chlorine.

[0133] In some embodiments, Z is -NH-.

[0134] In some embodiments, U 1 、U 2 are each independently selected from -CH2- or -C(O)-, and U 1 、U 2 Not the same.

[0135] In some embodiments, U 1 -CH2-, U 2 It is -C(O)-.

[0136] In some embodiments, A is Position 1 is connected to L4 or L3, and position 2 is connected to a benzene ring; or position 1 is connected to a benzene ring, and position 2 is connected to L4 or L3; the definitions of W, V, R2, R3, R4, R5, R6, R7, R8, m, p and y are as described in any scheme in the present disclosure.

[0137] In some embodiments, A is The 1 position is connected to L4 or L3, and the 2 position is connected to the benzene ring; the definitions of W, V, R2, R3, R4, R5, R6, R7, R8, m, p and y are as described in any scheme in the present disclosure.

[0138] In some embodiments, A is selected from O, S, -NH-, -N(CH3)-, -O(CH2) m -、-S(CH2) m -、-NH(CH2) m -、-N(CH3)(CH2) m -、-O(CH2) m O-, -S(CH2) m O-, -S(CH2) m S-, -NH(CH2) m O-, -N(CH3)(CH2) m O-, -NH(CH2) m S-, -N(CH3)(CH2) m S-, -NH(CH2) m NH-, -N(CH3)(CH2) m NH-, -O(CH2) m C(O)NH(CH2) P-、-O(CH2) m C(O)N(CH3)(CH2) P -、-O(CH2) m C(O)NH(CH2) P O-, -O(CH2) m C(O)N(CH3)(CH2) P O-, -S(CH2) m C(O)NH(CH2) P -、-S(CH2) m C(O)N(CH3)(CH2) P -、-NH(CH2) m C(O)NH(CH2) P -、-NH(CH2) m C(O)N(CH3)(CH2) P -、-O(CH2) m C(O)NH(CH2) P O(CH2) y -or-O(CH2) m C(O)N(CH3)(CH2) P O(CH2) y -.

[0139] In some embodiments, A is selected from -O(CH2) m C(O)NH(CH2) P -、-O(CH2) m C(O)N(CH3)(CH2) P -、-O(CH2) m C(O)NH(CH2) P O-, -O(CH2) m C(O)N(CH3)(CH2) P O-, -S(CH2) m C(O)NH(CH2) P -、-S(CH2) m C(O)N(CH3)(CH2) P -、-NH(CH2) m C(O)NH(CH2) P -、-NH(CH2) m C(O)N(CH3)(CH2) P -、-O(CH2) m C(O)NH(CH2) P O(CH2) y -or-O(CH2) mC(O)N(CH3)(CH2) P O(CH2) y -.

[0140] In some embodiments, A is selected from -N(CH3)(CH2) m -、-O(CH2) m C(O)N(CH3)(CH2) P -or-O(CH2) m C(O)N(CH3)(CH2) P O(CH2) y -.

[0141] In some embodiments, A is selected from -O(CH2) m C(O)N(CH3)(CH2) P -or-O(CH2) m C(O)N(CH3)(CH2) P O(CH2) y -.

[0142] In some embodiments, A is selected from -N(CH3)(CH2)3-, -OCH2C(O)N(CH3)(CH2)3-, or -OCH2C(O)N(CH3)(CH2)2O(CH2)2-.

[0143] In some embodiments, A is selected from -OCH2C(O)N(CH3)(CH2)3- or -OCH2C(O)N(CH3)(CH2)2O(CH2)2-.

[0144] In some embodiments, R2, R3, R4, R5, R6, R7, and R8 are each independently selected from hydrogen, deuterium, or C1-4 alkyl.

[0145] In some embodiments, W is selected from O, S, or NR 9 .

[0146] In some embodiments, W is O.

[0147] In some embodiments, V is selected from a direct bond, O, S, or NR 9 .

[0148] In some embodiments, V is selected from a direct bond or O.

[0149] In some embodiments, m, p, y are each independently selected from 0, 1, 2, 3, or 4. In some embodiments, m, p, y are each independently selected from 1, 2, 3, or 4.

[0150] In some embodiments, R9 is selected from hydrogen, deuterium, or methyl.

[0151] In some embodiments, D is preferably a structural unit shown in II-1, II-2 or II-3:

[0152] in,

[0153] R 1 , A, and Z are defined as described in any embodiment of the present disclosure, and position 1 is connected to L4 or L3.

[0154] In some embodiments, D is selected from the following structures, and position 1 is connected to L4 or L3:

[0155] In some embodiments, D is selected from the following structures, and position 1 is connected to L4 or L3:

[0156] In some embodiments, the antibody drug conjugate is a structure shown in Formula III,

[0157] Among them, Tb, q, L1, L2, L3, L4, R 1 , A, Z, U 1 and U 2 The definition of is as described in any embodiment of the present disclosure.

[0158] In some embodiments, the antibody drug conjugate is a structure shown in Formula III-1:

[0159] Among them, Tb, L1, L2, L3, L4, W, V, R2, R3, R4, R5, R6, R7, R8, Z, U 1 、U 2 , m, p, y and q are defined as described in any embodiment of the present disclosure.

[0160] In some embodiments, the antibody drug conjugate is selected from the following:

[0161] q is selected from any value between 1.0 and 10.0.

[0162] In some preferred embodiments, the antibody drug conjugate is selected from the following:

[0163] q is selected from any value between 1.0 and 10.0.

[0164] In some preferred embodiments, q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0165] In some more preferred embodiments, q is selected from 2, 4, 6 or 8.

[0166] In some embodiments, the Tb is a targeting moiety, such as an antibody and its antigen-binding fragment, a ligand, a protein, a polypeptide, a non-protein agent (such as sugar, RNA or DNA), an antibody analog, etc.

[0167] In some embodiments, Tb is an antibody or an antigen-binding fragment thereof.

[0168] In some embodiments, Tb is an antibody or an antigen-binding fragment thereof having endocytic activity.

[0169] In some embodiments, Tb is an antibody or an antigen-binding fragment thereof that has the activity of binding to tumor cell surface antigens.

[0170] In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that has tumor cell surface antigen binding activity and tumor cell endocytosis activity.

[0171] In some embodiments, the antibody or antigen-binding fragment thereof has antigen-binding activity and no or weak tumor cell endocytosis activity.

[0172] In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that binds to a non-endocytosed antigen on the surface of a tumor cell.

[0173] In some embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that does not have tumor cell endocytosis activity.

[0174] In some preferred embodiments, the antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that has the activity of binding to tumor cell surface antigens and has the activity of tumor cell endocytosis.

[0175] In some embodiments, the antibody or antigen-binding fragment thereof comprises Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single-chain antibody (e.g., scFv), non-human antibody, humanized antibody, chimeric antibody, fully human antibody, probody, bispecific antibody or multispecific antibody.

[0176] In some embodiments, the antibody or antigen-binding fragment thereof is a non-human antibody, a humanized antibody, a chimeric antibody, or a fully human antibody.

[0177] In some embodiments, the antibody or antigen-binding fragment thereof is a probody, a bispecific antibody, or a multispecific antibody.

[0178] In some embodiments, the antibody or antigen-binding fragment thereof includes Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single chain antibody (eg, scFv).

[0179] In some embodiments, Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof, an anti-Trop-2 antibody or an antigen-binding fragment thereof, an anti-Her 2 antibody or an antigen-binding fragment thereof, an anti-Her 3 antibody or an antigen-binding fragment thereof, or an anti-EGFR antibody or an antigen-binding fragment thereof.

[0180] In some embodiments of the present disclosure, Tb is a monoclonal antibody against B7H3, or an antigen-binding fragment thereof. In some embodiments, the anti-B7H3 antibody includes all anti-B7H3 antibodies in the prior art, for example, see CN112521512, WO2021027674, WO2021021543, WO2021006619, CN111662384, CN111454357, WO2020151384, WO2020140094, WO2020103100, WO2020102779, WO2020063673, WO2020047257, WO2020041626, CN110684790, CN110 642948, WO2019225787, WO2019226017, US20190338030, CN110305213, WO2018209346, WO2018177393, US9150656, WO2016106004, WO20 16044383, WO2016033225, WO2015181267, US20120294796, WO2011109400, CN101104639, WO2004093894, WO2002010187, WO2001018021.

[0181] In some embodiments, Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof, such as 1D1-01, 2E3-02 antibody, enoblituzumab, mirzotamab, omburtamab or an antigen-binding fragment thereof. In some preferred embodiments, Tb is 2E3-02 antibody.

[0182] In some embodiments, the VH sequence of 1D1-01 is shown in SEQ ID NO: 1, and the VL sequence is shown in SEQ ID NO: 2. In some embodiments, the heavy chain sequence of 1D1-01 is shown in SEQ ID NO: 3, and the light chain sequence is shown in SEQ ID NO: 4.

[0183] In some embodiments, the VH sequence of 2E3-02 is shown in SEQ ID NO: 5, and the VL sequence is shown in SEQ ID NO: 6. In some embodiments, the heavy chain sequence of 2E3-02 is shown in SEQ ID NO: 7, and the light chain sequence is shown in SEQ ID NO: 8.

[0184] In some embodiments, the Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof having a VH sequence as shown in SEQ ID NO: 1 and a VL sequence as shown in SEQ ID NO: 2. In some preferred embodiments, the Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof having a heavy chain sequence as shown in SEQ ID NO: 3 and a light chain sequence as shown in SEQ ID NO: 4.

[0185] In some embodiments, the Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof with a VH sequence as shown in SEQ ID NO: 5 and a VL sequence as shown in SEQ ID NO: 6. In some preferred embodiments, the Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof with a heavy chain sequence as shown in SEQ ID NO: 7 and a light chain sequence as shown in SEQ ID NO: 8.

[0186] In some embodiments, Tb is an anti-Trop-2 antibody or an antigen-binding fragment thereof, such as datopotamab, sacituzumab, or an antigen-binding fragment thereof.

[0187] In some embodiments, Tb is an anti-Her 2 antibody or an antigen-binding fragment thereof, such as anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, pertuzumab, timigutuzumab, zanidatamab, trastuzumab, pertuzumab or an antigen-binding fragment thereof. In some preferred embodiments, Tb is trastuzumab or pertuzumab.

[0188] In some embodiments, Tb is an anti-Her 2 antibody or an antigen-binding fragment thereof.

[0189] In some embodiments, Tb is an anti-Her 2 monoclonal antibody or an antigen-binding fragment thereof.

[0190] In some embodiments, Tb is a bispecific antibody or an antigen-binding fragment thereof having Her 2 binding activity.

[0191] In some embodiments, Tb is a multispecific antibody or an antigen-binding fragment thereof having Her 2 binding activity.

[0192] In some preferred embodiments, Tb is Trastuzumab or an antigen-binding fragment thereof.

[0193] In some preferred embodiments, Tb is Pertuzumab or an antigen-binding fragment thereof.

[0194] In some embodiments, Tb is an anti-Her3 antibody or an antigen-binding fragment thereof.

[0195] In some embodiments, Tb is an anti-EGFR antibody or an antigen-binding fragment thereof, such as demupitamab, depatuxizumab, futuximab, imgatuzumab, laprituximab, losatuxizumab, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, pimurutamab, serclutamab, tomozutuximab, zalutumumab, Cetuximab, or an antigen-binding fragment thereof.

[0196] In some embodiments, the antibody has an antibody that binds to the B7H3 antigen but does not have endocytosis activity, such as INV721 and I7-01 of WO2021168379A1. More specifically, the anti-B7H3 antibody has a VH of SEQ ID NO: 2 and a VL of SEQ ID NO: 1 as described in WO2021168379A1.

[0197] In some embodiments, the antibody has the ability to bind to the CD20 antigen but lacks endocytosis activity. Although so-called "type II" CD20-specific antibodies have been shown to be poorly internalized by CD20-positive target cells, other so-called "type I" CD20-specific antibodies have been found to be internalized and degraded to some extent, depending on the expression levels of activating and inhibitory FcγRs on the target cells with which they interact. In some embodiments, the antibody has the ability to bind to the CD20 antigen but lacks endocytosis activity is a "type II" CD20-specific antibody, such as obinutuzumab.

[0198] In some embodiments, the antibody has the activity of binding to non-endocytic antigens (e.g., ALCAM / CD166). In some embodiments, the antibody is an antibody comprising the VH of SEQ ID No: 73 and the VL of SEQ ID No: 74, the VH of SEQ ID No: 75 and the VL of SEQ ID No: 76, the VH of SEQ ID No: 77 and the VL of SEQ ID No: 78, or the VH of SEQ ID No: 79 and the VL of SEQ ID No: 88 in EP3911682A1.

[0199] In some embodiments, the antibody drug conjugate is:

[0200] Wherein, Tb1 is an anti-B7H3 antibody or an antigen-binding fragment thereof; L1, L2, L3, L4, W, V, R2, R3, R4, R5, R6, R7, R8, Z, U 1 、U 2 , m, p, y and q are defined as described in any embodiment of the present disclosure.

[0201] In some preferred embodiments, Tb1 is the 2E3-02 antibody.

[0202] In some embodiments, the antibody drug conjugate is:

[0203] The DAR value is 8.0.

[0204] In some embodiments, the antibody drug conjugate is:

[0205] Wherein, Tb2 is an anti-Her 2 antibody or an antigen-binding fragment thereof; L1, L2, L3, L4, W, V, R2, R3, R4, R5, R6, R7, R8, Z, U 1 、U 2 , m, p, y and q are defined as described in any embodiment of the present disclosure.

[0206] In some preferred embodiments, Tb2 is Trastuzumab.

[0207] In some embodiments, the antibody drug conjugate is:

[0208] The DAR value is 8.0.

[0209] In some embodiments, the antibody drug conjugate is:

[0210] The DAR value is 8.0.

[0211] In some embodiments, the antibody drug conjugate is:

[0212] The DAR value is 8.0.

[0213] In some embodiments, the antibody drug conjugate is:

[0214] The DAR value is 7.0.

[0215] In some embodiments, the antibody drug conjugate is:

[0216] The DAR value is 8.0.

[0217] In the second aspect of the present disclosure, the present disclosure provides a drug-linker conjugate represented by Formula IV, Lg-L1-L2-L3-L4- D Formula IV

[0218] or a stereoisomer of the drug-linker conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof,

[0219] in,

[0220] When L1 is When , bit 1 is connected to Lg, bit 2 is connected to L2;

[0221] Lg is a leaving group when reacting with an antibody; L2, L3, L4 and D are as defined in any embodiment of the present disclosure;

[0222] When L1 is When Lg-L1 is The definitions of L2, L3, L4 and D are as described in any embodiment of the present disclosure.

[0223] In some embodiments, Lg is selected from halogen, sulfone, tertiary amine salt (Me3N + 、Et3N + ), diazonium salt, -OMs, MeSO2- or CF3SO3-.

[0224] In some embodiments, Lg is selected from F, Cl or MeSO2-; more preferably; Lg is selected from F or MeSO2-.

[0225] In some embodiments, the drug-linker conjugate is a structure represented by Formula V,

[0226] Among them, Lg, L1, L2, L3, L4, R 1, A, Z, U 1 and U 2 The definition of is as described in any embodiment of the present disclosure.

[0227] In some embodiments, the drug-linker conjugate has a structure represented by Formula V-1:

[0228] Among them, Lg, L1, L2, L3, L4, W, V, R2, R3, R4, R5, R6, R7, R8, Z, U 1 、U 2 The definitions of m, p and y are as described in any embodiment of the present disclosure.

[0229] In some embodiments, the drug-linker conjugate is selected from:

[0230] In a third aspect of the present disclosure, the present disclosure provides a protein degrader-type bioactive compound represented by Formula IIA,

[0231] or a stereoisomer of the biologically active compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof,

[0232] Among them, R 1 , A, Z, U 1 and U 2 The definition of is as described in any embodiment of the present disclosure.

[0233] In some embodiments, the protein degrader bioactive compound has a structure represented by Formula IIA-1:

[0234] Among them, W, V, R2, R3, R4, R5, R6, R7, R8, Z, U 1 、U 2 The definitions of m, p and y are as described in any embodiment of the present disclosure.

[0235] In some embodiments, the biologically active compound is selected from the group consisting of:

[0236] In a fourth aspect of the present disclosure, the present disclosure provides a linker in an antibody drug conjugate, the structure of which is shown below:

[0237] Wherein, position 1 is connected to the antibody, position 2 is connected to the protein degrader-type bioactive compound fragment; L1, L2, L3, and L4 are defined as described in any embodiment of the present disclosure;

[0238] Preferably, the definitions of the antibody and the protein degrader-type bioactive compound fragment are as described in Tb and D in any embodiment of the present disclosure, respectively.

[0239] In a fifth aspect of the present disclosure, the present disclosure provides a method for preparing an antibody drug conjugate of Formula I, comprising:

[0240] Conducting a coupling reaction between Tb and the drug-linker conjugate Lg-L1-L2-L3-L4-D represented by formula IV;

[0241] in,

[0242] Tb, L1, L2, L3, L4, Lg and D are as defined in any embodiment of the present disclosure.

[0243] Specifically, the method includes the step of performing a coupling reaction between Tb and a drug-linker conjugate Lg-L1-L2-L3-L4-D represented by formula IV in a suitable solvent and conditions to form a CS bond.

[0244] In some embodiments, the ratio of the amount of Tb to the amount of the drug-linker conjugate is 1:(1-20), such as 1:2, 1:4, 1:6, 1:8, 1:10, 1:12, 1:14, 1:16, 1:18, 1:(10-20), 1:(12-20), 1:(14-20), 1:(16-20) or 1:(18-20).

[0245] In some embodiments, the coupling reaction is carried out in water and / or an organic solvent.

[0246] In some embodiments, the organic solvent is selected from N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, nitriles (such as acetonitrile), alcohols (such as methanol, ethanol) or any combination thereof.

[0247] In some embodiments, the method further comprises the step of purifying the coupling product.

[0248] In some embodiments, the coupled product is purified by chromatography.

[0249] In some embodiments, the chromatography method comprises one or more of ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, or affinity chromatography.

[0250] In a sixth aspect of the present disclosure, the present disclosure provides a method for preparing a drug-linker conjugate represented by Formula IV:

[0251] The compounds represented by the general formula IV-E-1 and the general formula IV-E-2 are reacted as follows;

[0252] Among them, L4' is W, R2, R3, and m are defined as described in any embodiment of the present disclosure, position 1 is connected to L4, and position 2 is connected to Lg2;

[0253] Lg2 is selected from hydroxyl, halogen, activated hydroxyl, such as hydroxyl, chlorine, bromine,

[0254] D2 is a structure shown in Formula VI, R1, R4, R5, R6, R7, R8, V, Z, U 1 、U 2 , p, and y are defined as described in any embodiment of the present disclosure.

[0255] Lg, L1, L2, L3 and L4 are defined as described in any embodiment of the present disclosure.

[0256] In some embodiments, the present disclosure provides a method for preparing a drug-linker conjugate of Formula V-1, comprising: reacting the compounds of Formula VA-1 and Formula VI as follows:

[0257] Among them, Lg, Lg2, L1, L2, L3, L4, W, V, R1, R2, R3, R4, R5, R6, R7, R8, V, Z, U 1 、U 2 The definitions of m, p, and y are as described in any embodiment of the present disclosure.

[0258] In some embodiments, the present disclosure provides a method for preparing a drug-linker conjugate represented by Formula DL-1.

[0259] Specifically:

[0260] The compound INT1 shown in the general formula undergoes an amide condensation reaction with the compound DL-1-1 to obtain the target compound (DL-1). Drug-linker conjugates of various structures can be obtained by coupling analogs of INT1 with analogs of DL-1-1.

[0261] In a seventh aspect of the present disclosure, the present disclosure provides a linker represented by formula VA-1,

[0262] Wherein, Lg, L1, L2, L3, L4, W, R2, R3 and m are as defined in any embodiment of the present disclosure, and Lg2 is selected from hydroxyl, halogen, activated hydroxyl, such as hydroxyl, chlorine, bromine,

[0263] In some embodiments, the linker is selected from:

[0264] The present disclosure provides the use of the aforementioned protein degrader-type bioactive compound, or a stereoisomer of the compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof in the preparation of an antibody-drug conjugate (ADC).

[0265] The present disclosure provides a pharmaceutical composition comprising the aforementioned antibody-drug conjugate, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; or the aforementioned protein degrader bioactive compound, or a stereoisomer of the bioactive compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; or the aforementioned drug-linker conjugate, or a stereoisomer of the drug-linker conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, and optionally one or more pharmaceutical excipients.

[0266] In some embodiments, the drug to antibody ratio (DAR) is any value between 1.0-12.0.

[0267] In some embodiments, the drug to antibody ratio (DAR) is any value between 1.0-10.0.

[0268] In some embodiments, the drug to antibody ratio (DAR) is any value between 1.0-9.0.

[0269] In some embodiments, the drug to antibody ratio (DAR) is any value between 1.0-8.5.

[0270] In some embodiments, the drug to antibody ratio (DAR) is any value between 1.5-8.5.

[0271] In some embodiments, the drug to antibody ratio (DAR) is any value between 2.0-8.0.

[0272] In some embodiments, the drug to antibody ratio (DAR) is any value within the range of 2±0.4, 4±0.4, 6±0.4, or 8±0.4.

[0273] In some embodiments, the drug to antibody ratio (DAR) is about 2.0, 4.0, 6.0, or 8.0.

[0274] The term "drug to antibody ratio" or "DAR" refers to the amount of drug, e.g., a small molecule toxin, attached to the antibody of an ADC. The DAR of an ADC can range from 1 to 16, but higher loadings (e.g., 20) are possible depending on the number of attachment sites on the antibody. The term DAR can be used when referring to the amount of drug loaded onto a single antibody, or alternatively, when referring to the average or mean DAR of a group of ADCs. In some embodiments, the ADC comprises ADCs having a distribution of DARs from 1 to 8, e.g., 1.5, 2, 4, 6, and 8 (i.e., drug loading species of 1.5, 2, 4, 6, and 8). Notably, degradation products can be produced such that the ligand drug conjugate can also contain DARs of 1, 3, 5, and 7. In addition, the ADC can also have a DAR greater than 8. The ADC is produced by interchain disulfide reduction followed by conjugation. In some embodiments, the ADC comprises both: the ADC having a DAR of 4 or less (ie, 4 or less drug loading species) and the ADC having a DAR of 6 or more (ie, 6 or more drug loading species).

[0275] The present disclosure provides the aforementioned antibody-drug conjugate, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; or the aforementioned protein degrader-type bioactive compound, or a stereoisomer of the bioactive compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; or the aforementioned drug-linker conjugate, or a stereoisomer of the drug-linker conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; or the aforementioned pharmaceutical composition for use in the preparation of a medicament for treating and / or preventing diseases associated with abnormal cell activity (e.g., cancer diseases).

[0276] The present disclosure provides the aforementioned antibody-drug conjugate, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; or the aforementioned protein degrader bioactive compound, or a stereoisomer of the bioactive compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; or the aforementioned drug-linker conjugate, or a stereoisomer of the drug-linker conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; or the aforementioned pharmaceutical composition, for use in treating and / or preventing diseases associated with abnormal cell activity (e.g., cancer diseases).

[0277] The present disclosure provides a method for preventing and / or treating diseases associated with abnormal cell activity (e.g., cancer), comprising: administering to an individual in need thereof a preventive and / or therapeutically effective amount of the aforementioned antibody-drug conjugate, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; or the aforementioned protein degrader-type bioactive compound, or a stereoisomer of the bioactive compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; or the aforementioned drug-linker conjugate, or a stereoisomer of the drug-linker conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; or the aforementioned pharmaceutical composition.

[0278] In some embodiments, the cancer disease described in the present disclosure is selected from esophageal cancer (e.g., esophageal adenocarcinoma or esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, urothelial carcinoma, solid tumors, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

[0279] In some embodiments, the cancer disease is a cancer disease associated with HER2, TROP2, B7H3, HER3, or EGFR.

[0280] In some embodiments, the cancer disease is a solid tumor or a hematological tumor.

[0281] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures for cell culture, molecular genetics, nucleic acid chemistry, and immunology used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0282] As used in the present disclosure, the term "protein degrader" refers to a class of compounds that can induce ubiquitination of a protein of interest by E3 ligase and then degradation by the proteasome.

[0283] As used in this disclosure, the term "protein degradation targeting chimera (PROTAC)" generally includes three parts: an E3 ubiquitin ligase ligand and a target protein ligand. The two active ligands are connected together by a specially designed linker structure, which recruits the E3 ubiquitin ligase to the target protein, ubiquitinates the target protein, and then degrades the target protein.

[0284] As used in this disclosure, the term "molecular glue degrader (MGD)" refers to a class of small molecules that can induce a novel interaction between E3 ubiquitin ligase and target protein, thereby leading to degradation of the target protein.

[0285] As used herein, the term "pharmaceutically acceptable salt" is exemplified by organic acid addition salts formed from organic acids that form pharmaceutically acceptable anions, including but not limited to formates, acetates, propionates, benzoates, maleates, fumarates, succinates, tartrates, citrates, ascorbates, α-ketoglutarate, α-glycerophosphates, alkylsulfonates, or arylsulfonates; preferably, the alkylsulfonate is methylsulfonate or ethylsulfonate; and the arylsulfonate is benzenesulfonate or p-toluenesulfonate. Suitable inorganic salts may also be formed, including but not limited to hydrochlorides, hydrobromides, hydroiodides, nitrates, bicarbonates, carbonates, sulfates, or phosphates.

[0286] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with a suitable acid affording a pharmaceutically acceptable anion.

[0287] As used in this disclosure, the term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds with one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers can be produced. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of the present invention can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-ketone tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is understood that the scope of this disclosure encompasses all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0288] Solid lines (-), solid wedges, or dashed wedges may be used in this disclosure to depict carbon-carbon bonds of the compounds of the present invention. The use of solid lines to depict bonds to asymmetric carbon atoms is intended to indicate that all possible stereoisomers at that carbon atom are included (e.g., specific enantiomers, racemic mixtures, etc.). The use of solid or dashed wedges to depict bonds to asymmetric carbon atoms is intended to indicate that the indicated stereoisomers exist. When present in a racemic mixture, solid and dashed wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise indicated, the compounds of the present invention are intended to exist in the form of stereoisomers (which include cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, atropisomers, and mixtures thereof). The compounds of the present invention may exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).

[0289] The compounds of the present disclosure may exist in the form of solvates (preferably hydrates), wherein the compounds of the present disclosure contain a polar solvent as a structural element of the crystal lattice of the compound, in particular water, methanol or ethanol. The amount of the polar solvent, in particular water, may be present in a stoichiometric or non-stoichiometric ratio.

[0290] The present disclosure further includes within its scope prodrugs of the compounds of the present invention. Typically, such prodrugs will be functional group derivatives of the compounds that are readily converted into the desired therapeutically active compound in vivo. Therefore, in these cases, the term "administering" as used in the methods of treatment of the present disclosure should include treating various diseases or conditions with one or more prodrug forms of the claimed compounds, but the prodrug forms are converted into the aforementioned compounds in vivo after administration to the individual. For example, conventional methods for selecting and preparing suitable prodrug derivatives are described in "Design of Prodrug", ed. H. Bundgaard, Elsevier, 1985.

[0291] For the purposes of this disclosure, pharmaceutical excipients refer to excipients and additives used in the production of pharmaceuticals and the preparation of prescriptions. These substances, in addition to the active ingredients, have been reasonably evaluated for safety and are included in pharmaceutical preparations. In addition to providing shape, acting as carriers, and improving stability, pharmaceutical excipients also have important functions such as solubilization, dissolution enhancement, and sustained-release control. They are important components that may affect the quality, safety, and efficacy of drugs. Based on their source, they can be categorized as natural, semi-synthetic, and fully synthetic. According to their functions and uses, they can be divided into: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc. According to their route of administration, they can be divided into oral, parenteral, mucosal, transdermal or topical administration, nasal or oral inhalation administration, and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations for different routes of administration and have different functions and uses.

[0292] The pharmaceutical composition can be prepared into various suitable dosage forms according to the route of administration, such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder sprays, sprays, etc. The pharmaceutical composition or suitable dosage form can contain 0.01 mg to 1000 mg of the compound of the present disclosure or a pharmaceutically acceptable salt or conjugate thereof, preferably 0.1 mg to 800 mg, preferably 0.5-500 mg, preferably 0.5 to 350 mg, and particularly preferably 1-250 mg.

[0293] The pharmaceutical composition can be administered in the form of an injection, including an injection solution, sterile powder for injection, and concentrated solution for injection. Usable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, non-volatile oils, such as monoglycerides or diglycerides, can also be used as solvents or suspending media. The pharmaceutical composition can be administered in the form of an infusion.

[0294] As used herein, the term "treat" generally refers to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms; and / or therapeutic, in terms of partially or completely stabilizing or curing a disease and / or causing side effects due to the disease. As used herein, "treat" encompasses any treatment of a disease in a patient, including: (a) preventing the disease or symptoms from occurring in a patient who is susceptible to the disease or symptoms but has not yet been diagnosed with the disease; (b) suppressing the symptoms of a disease, i.e., preventing its development; or (c) alleviating the symptoms of a disease, i.e., causing the disease or symptoms to regress.

[0295] In the present disclosure, the term "individual" includes humans and non-human animals. Exemplary human individuals include human individuals suffering from a disease (e.g., a disease described in the present disclosure) (referred to as a patient) or a normal individual. The term "non-human animal" in the present disclosure includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and / or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

[0296] As used herein, the term "effective dose" refers to an amount of an antibody drug conjugate, drug-linker conjugate, compound, or composition that, when administered, will alleviate to some extent one or more symptoms of the condition being treated.

[0297] In the present disclosure, the terms "antibody drug conjugate" and "ADC" refer to a substance obtained by connecting a bioactive compound fragment (drug molecule) to an antibody or its antigen-binding fragment. In some embodiments of the present disclosure, the bioactive compound fragment is connected to the targeting portion via a linker. The linker can be broken in a specific environment (such as a low pH environment in the cell) or under a specific action (such as the action of a lysosomal protease), thereby separating the bioactive compound fragment from the targeting portion or the antibody or its antigen-binding fragment. In some embodiments of the present disclosure, the linker comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond. In some embodiments of the present disclosure, the bioactive compound fragment is directly connected to the targeting portion or the antibody or its antigen-binding fragment through a covalent bond, and the covalent bond can be broken under a specific environment or action, thereby separating the bioactive compound fragment from the antibody or its antigen-binding fragment.

[0298] In the present disclosure, the term "drug" refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0299] In the present disclosure, when it is stated that “the 1-position of L1 is connected to Tb via an S atom”, it will be understood by those skilled in the art that the 1-position of L1 is connected to the sulfhydryl group contained in Tb (e.g., an antibody) after the disulfide bond is opened (for example, the disulfide bond can be opened by reducing the disulfide bond with the reducing agent TCEP to generate a sulfhydryl group -SH). In other words, the -S- between L1 and Tb is not an additional external sulfur atom. For example, the -S- is not an additional external sulfur atom, but rather the sulfhydryl group contained in Tb after the disulfide bond is opened and connected to L1, for example -S- is formed by connecting the 1 bits of

[0300] In the present disclosure, the term "linker" refers to a fragment that connects a biologically active compound fragment (drug molecule) to an antibody portion.

[0301] In this disclosure, the "bioactive compound fragment" refers to a portion (fragment or group) of an antibody-drug conjugate (or antibody-drug conjugate, ADC) known in the art, which can form a bioactive drug (e.g., a small molecule cytotoxic drug, including a group after losing an atom or atomic group) or its derivative (e.g., a precursor) after the linker cleavage / degradation / enzymatic cleavage between tumor tissues or within tumor cells. For the avoidance of doubt, "drug" does not only refer to "drugs" approved by medical regulatory authorities, but also includes any compound with potential therapeutic biological activity in clinical practice, or in research and development and academic research.

[0302] In this disclosure, the term "antibody" is used in its broadest sense, including complete monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed by at least two complete antibodies, as long as they have the desired biological activity. In this disclosure, "antibody" and "immunoglobulin" can be used interchangeably.

[0303] In this disclosure, the term "monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations. Monoclonal antibodies have high specificity for a single determinant (epitope) of an antigen, whereas polyclonal antibodies, in contrast, comprise different antibodies directed against different determinants (epitopes). In addition to their specificity, monoclonal antibodies have the advantage of being synthesized without contamination by other antibodies. The modifier "monoclonal" herein indicates that the antibody is characterized by being derived from a substantially homogeneous population of antibodies and should not be construed as requiring production by a specific method.

[0304] In some embodiments of the present disclosure, monoclonal antibodies also specifically include chimeric antibodies, that is, a portion of the heavy chain and / or light chain is identical or homologous to a certain type, class, or subclass of antibody, and the remaining portion is identical or homologous to another type, class, or subclass of antibody, as long as they have the desired biological activity (see, for example, US 4,816,567; and Morrison et al., 1984, PNAS, 81: 6851-6855). Chimeric antibodies that can be used in the present disclosure include primatized antibodies, which contain variable region antigen-binding sequences from non-human primates (e.g., monkeys, orangutans, etc.) and human constant region sequences.

[0305] The term "antibody fragment" refers to a portion of an antibody, preferably the antigen binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region fragments, diabodies, linear antibodies and single-chain antibody molecules.

[0306] The term "bispecific antibody", also known as "bifunctional antibody conjugate", refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) through a coupling arm. The conjugate retains the activity of each antibody and thus has bifunctionality and bispecificity.

[0307] The term "multispecific antibody" includes, for example, trispecific antibodies, which are antibodies with three different antigen-binding specificities, and tetraspecific antibodies, which are antibodies with four different antigen-binding specificities.

[0308] The term "intact antibody" refers to an antibody comprising an antigen-binding variable region and a light chain constant region (CL), a heavy chain constant region (CH1, CH2, and CH3). The constant region may be a native sequence (e.g., a human native constant region sequence) or an amino acid sequence variant thereof. An intact antibody is preferably an intact antibody having one or more effector functions.

[0309] The term "probody" refers to a modified antibody, including an antibody or an antibody fragment, that can specifically bind to its target and can be coupled to a masking group, wherein the masking group refers to a cleavage constant for the binding ability of the antibody or antibody fragment to its target that is at least 100 times, 1000 times, or 10,000 times greater than the cleavage constant for the binding ability of the antibody or antibody fragment to its target without the coupled masking group.

[0310] In the present disclosure, the "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that contains a minimal amount of non-human immunoglobulin sequence. Most humanized antibodies are those in which the hypervariable region residues of a human recipient immunoglobulin are replaced with non-human (e.g., mouse, rat, rabbit, or non-human primate) hypervariable region residues (donor antibody) having the desired specificity, affinity, and function. In some embodiments, the framework region (FR) residues of the human immunoglobulin are also replaced with non-human residues. Moreover, the humanized antibody may also include residues that are not present in the recipient antibody or the donor antibody. These modifications are to further optimize the performance of the antibody. Humanized antibodies generally include at least one, typically two, variable regions in which all or nearly all of the hypervariable loops correspond to those of a non-human immunoglobulin, while the FRs are entirely or almost entirely human immunoglobulin sequences. The humanized antibody may also include at least a portion of an immunoglobulin constant region (Fc, typically a human immunoglobulin Fc). For details, see, for example, Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; and Presta, 1992, Curr Op Struct Bwl 2:593-596.

[0311] Intact antibodies can be divided into different "classes" based on the amino acid sequence of the constant region of their heavy chains. The five main classes are IgA, IgD, IgE, IgG, and IgM, and several of these classes are further divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of the different antibody classes are called α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0312] Although amino acid substitutions in antibodies are generally made with L-amino acids in the present disclosure, this is not limiting. In some embodiments, the antibody peptide chain may include one or more D-amino acids. Peptides containing D-amino acids are more stable and less susceptible to degradation in the oral cavity, intestinal tract, or plasma than peptides containing only L-amino acids.

[0313] Monoclonal antibodies used in the present disclosure can be produced by many methods. For example, monoclonal antibodies for the present disclosure can be obtained by hybridoma methods using cells from many species (including mice, hamsters, rats and people) (see, for example, Kohler et al., 1975, Nature, 256:495), or obtained by recombinant DNA technology (see, for example, US 4,816,567), or isolated from phage antibody libraries (see, for example, Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, Journal of Molecular Biology, 222:581-597).

[0314] In this disclosure, unless otherwise explicitly stated, the descriptions “each independently selected from” and “each independently selected from” used throughout this disclosure are interchangeable and should be understood in a broad sense. They can mean that in different groups, the specific options expressed by the same or different symbols do not affect each other, or that in the same group, the specific options expressed by the same or different symbols do not affect each other.

[0315] AA 1 The structures of the indicated amino acid residues If r is 0, it will be understood by those skilled in the art that AA 1 The structure of the indicated amino acid residues will become

[0316] AA 1 The structures of the indicated amino acid residues In, if R a With R b Together with the carbon atoms to which they are attached, they form a 4-10 membered heterocyclic ring, which is optionally substituted by one or more R 0 substituted, wherein the term "the 4-10 membered heterocyclic ring is optionally substituted by one or more R 0 "substituted" means that the 4-10 membered heterocyclic ring may be unsubstituted or may be replaced by one or more R 0 and the plurality of R 0 In each R 0 The definitions of can be the same or different. Other similar definitions can be understood with reference to the above content.

[0317] Throughout this specification, substituents of the disclosed compounds are disclosed by group class or range. It is specifically noted that the disclosure includes every independent subcombination of the individual members of these group classes and ranges. For example, the term "C1-6 alkyl" specifically refers to the independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0318] In the present disclosure, the term "direct bond" means that the indicated substituent does not exist, and both end portions of the substituent are directly connected to form a bond.

[0319] In the present disclosure, the terms "comprises," "comprising," "having," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0320] In the present disclosure, the term "C1-6 alkyl" refers to a straight-chain or branched alkyl group containing 1 to 6 carbon atoms, including, for example, "C1-3 alkyl" or "C1-4 alkyl", methyl, ethyl, etc. Specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.

[0321] In the present disclosure, the term "C2-6 alkenyl" refers to a linear, branched, or cyclic alkenyl group containing at least one double bond and having 2 to 6 carbon atoms, including, for example, "C2-4 alkenyl," etc. Examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hexadienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, 1,4-cyclohexadienyl, etc.

[0322] In the present disclosure, the term "C2-6 alkynyl" refers to a straight or branched alkynyl group containing at least one triple bond and having 2 to 6 carbon atoms, including, for example, "C2-4 alkynyl". Examples include, but are not limited to, ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl, and the like.

[0323] In the present disclosure, the term "halogen" includes fluorine, chlorine, bromine, and iodine.

[0324] In this disclosure, the term "C3-6 cycloalkyl" refers to a saturated cyclic alkyl group containing 3 to 6 carbon atoms. Optionally, the carbon atoms in the cyclic structure may be oxo-substituted. Specific examples include, but are not limited to, cyclopropane (i.e., cyclopropyl), cyclobutane (i.e., cyclobutyl), cyclopentane (i.e., cyclopentyl), and cyclohexyl.

[0325] In the present disclosure, the term "C1-6 alkoxy" refers to an alkyl group as defined above attached to the parent molecular moiety through an oxygen atom, including, for example, "C1-3 alkoxy" or "C1-4 alkoxy." Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

[0326] In the present disclosure, the term "C1-6 haloalkyl" refers to an alkyl group as defined above that is substituted by one or more halogen groups as defined above, including, for example, "C1-3 haloalkyl" or "C1-4 haloalkyl." Specific examples include, but are not limited to, chloromethyl, fluoroethyl, bromopropyl, and the like.

[0327] In the present disclosure, the term "4-10 membered heterocycle" refers to a ring containing 4-10 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom). The term "3-6 membered heterocycle" refers to a ring containing 3-6 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom). The term "3-7 membered heterocycle" refers to a ring containing 3-7 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom). The term "5-6 membered heterocycle" refers to a ring containing 5-6 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom). Optionally, the ring atoms (such as carbon atoms, nitrogen atoms or sulfur atoms) in the cyclic structure can be oxoed, and specific examples include but are not limited to rings such as pyrrolidine, tetrahydrofuran, piperidine, piperazine, tetrahydropyran, pyrrolidone.

[0328] In this disclosure, the term "3-10 membered carbocycle" refers to a ring having 3-10 carbon atoms. Optionally, the carbon atoms in the ring structure may be oxo-substituted. Examples include 3-6 membered carbocycles, 3-7 membered carbocycles, 5-8 membered carbocycles, and the like. Specific examples include, but are not limited to, cyclopentane, cyclohexane, and the like.

[0329] In the present disclosure, the term "4-10 membered heterocyclic radical" refers to a cyclic group containing 4-10 ring atoms (wherein at least one ring atom is a heteroatom, such as an oxygen atom, a nitrogen atom or a sulfur atom). The term "4-6 membered heterocyclic radical" refers to a cyclic group containing 4-6 ring atoms (wherein at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom). The term "3-6 membered heterocyclic radical" refers to a cyclic group containing 3-6 ring atoms (wherein at least one ring atom is a heteroatom, such as an oxygen atom, a nitrogen atom or a sulfur atom). Optionally, the ring atoms (such as carbon atoms, nitrogen atoms or sulfur atoms) in the cyclic structure can be oxoed. “4-8 membered heterocyclic group” includes, for example, “4-8 membered nitrogen-containing heterocyclic group”, “4-8 membered oxygen-containing heterocyclic group”, “4-7 membered heterocyclic group”, “4-7 membered oxygen-containing heterocyclic group”, “4-7 membered heterocyclic group”, “4-6 membered heterocyclic group”, “5-7 membered heterocyclic group”, “5-6 membered heterocyclic group”, “5-6 membered nitrogen-containing heterocyclic group”, and specific examples include but are not limited to oxocyclobutane, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, homopiperazinyl and the like.

[0330] In this disclosure, the term "aryl" refers to a monocyclic or polycyclic hydrocarbon group having aromatic properties, such as a 6-10 membered aryl group, a 5-8 membered aryl group, and the like. Specific examples include, but are not limited to, phenyl, naphthyl, anthracenyl, and phenanthrenyl. The "6-10 membered aryl" refers to an aryl group containing 6-10 ring atoms. The "C6-10 aryl" refers to an aryl group containing 6-10 carbon atoms.

[0331] In the present disclosure, term " heteroaryl " refers to a cyclic group with aromaticity, wherein at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom. Optionally, the ring atoms (such as carbon atoms, nitrogen atoms or sulfur atoms) in the ring structure can be oxoed. Specific examples include but are not limited to 5-10 yuan heteroaryl, 5-6 yuan heteroaryl, 5-10 yuan nitrogen-containing heteroaryl, 6-10 yuan oxygen-containing heteroaryl, 6-8 yuan nitrogen-containing heteroaryl, 5-8 yuan oxygen-containing heteroaryl, such as furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3, 4-oxadiazolyl, pyridinyl, 2-pyridonyl, 4-pyridonyl, pyrimidinyl, 1,4-dioxadienyl, 2H-1,2-oxazinyl, 4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 4H-1,4-oxazinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetrazinyl, azacycloheptatrienyl, 1,3-diazacycloheptatrienyl, azacyclooctatetraenyl, and the like.

[0332] The bonds in the structural formulae of the present disclosure represented by a wavy line “~~” are intended to indicate that the structure represents a cis or trans isomer, or a mixture of cis and trans isomers in any ratio.

[0333] In this disclosure, the term "Linker Unit" refers to a component of an antibody-drug conjugate or drug-linker conjugate or linker that connects the target-binding antibody or antigen-binding fragment thereof to the remainder of the antibody-drug conjugate. Linker Units can connect the Tb unit to L2. Specific examples include, but are not limited to, (wherein position 1 is connected to the antibody or antigen-binding fragment thereof, and position 2 is connected to L2 or L3):

[0334] In this disclosure, the term "Linker" refers to a component of an antibody-drug conjugate or drug-linker conjugate or linker that is used to bind the Linker to an amino acid residue or a short peptide consisting of 2-10 amino acid residues. When present, the Linker can connect L1 to L3. Specific examples include, but are not limited to, (wherein position 1 is connected to the Linker and position 2 is connected to L3):

[0335] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0336] The reagents and raw materials used in the present disclosure are commercially available.

[0337] Advantageous Effects of the Invention

[0338] Through extensive research, the present disclosure has obtained a novel GSPT1 protein degrader (bioactive compound or payload) and its antibody-drug conjugate (ADC), both of which have extremely strong bioactivity and can achieve the following multiple technical effects:

[0339] (1) GSPT1 protein degrader, showing extremely strong anti-proliferation activity on different cells, significantly better than the control compound (3-40 times), and has the potential to have a strong bystander effect after forming an ADC. The molecule has no basic groups, which can reduce or eliminate the lysosomal capture phenomenon (i.e., molecules containing basic groups cannot function due to being bound to the highly acidic lysosomes), and on the other hand, it will not be rapidly distributed to other non-tumor tissues and cause "off-target" toxicity.

[0340] (2) Overcoming the stability problem of traditional maleimide-coupled linkers, ADC has high stability in the circulatory system and can reduce the shedding of bioactive compounds in non-target tissues.

[0341] (3) ADC can release payload simultaneously inside and outside tumor cells, increasing the effective release of bioactive compounds at the tumor site. Bioactive compounds have a higher intratumoral and blood concentration ratio, and therefore have a higher therapeutic index.

[0342] (4) ADC showed extremely strong proliferation inhibitory activity on cells; in terms of in vivo efficacy, it showed a significantly better tumor inhibition effect than the control compound; during the administration period, the animals in each group had no obvious weight loss and obvious drug toxicity, and had good safety.

[0343] Therefore, the GSPT1 protein degrader and ADC of the present invention have high clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0344] FIG1A shows the results of ELISA binding assay of anti-B7H3 antibody 1D1-01 and human B7H3-4 IgG-His protein;

[0345] FIG1B shows the results of ELISA binding assay of anti-B7H3 antibody 2E3-02 and human B7H3-4 IgG-His protein;

[0346] FIG2A shows the results of ELISA binding assay of anti-B7H3 antibody 1D1-01 and monkey B7H3-4 IgG-His protein;

[0347] FIG2B shows the results of ELISA binding assay of anti-B7H3 antibody 2E3-02 and monkey B7H3-4 IgG-His protein;

[0348] FIG3A shows the flow cytometry results of the binding of anti-B7H3 antibody 1D1-01 to MCF-7 tumor cells;

[0349] FIG3B shows the flow cytometry results of the anti-B7H3 antibody 2E3-02 binding to MCF-7 tumor cells;

[0350] FIG4A shows the flow cytometry results of the binding of anti-B7H3 antibody 1D1-01 to A549 tumor cells;

[0351] FIG4B shows the flow cytometry binding assay results of anti-B7H3 antibody 2E3-02 and A549 tumor cells;

[0352] FIG5 shows the flow cytometry results of the anti-B7H3 antibody 2E3-02 binding to PC-3 tumor cells;

[0353] FIG6A shows the results of the endocytosis detection of the candidate antibody 1D1-01 by tumor cells A549;

[0354] FIG6B shows the results of the endocytosis detection of the candidate antibody 2E3-02 by tumor cells A549;

[0355] FIG7A shows one of the test results of anti-Her2-ADC inhibiting tumor growth in the NCI-N87 xenograft tumor model;

[0356] Figure 7B shows the changes in body weight of the test animals in each group at a dose of 1 mg / kg;

[0357] FIG8A shows the second test result of anti-Her2-ADC inhibiting tumor growth in the NCI-N87 xenograft tumor model;

[0358] FIG8B shows the changes in body weight of the test animals in the 2 mg / kg and 6 mg / kg dosage groups. DETAILED DESCRIPTION

[0359] The present disclosure will be further described below by way of specific embodiments, but this is not intended to limit the present disclosure. Those skilled in the art may make various modifications or improvements based on the teachings of the present disclosure without departing from the basic concept and scope of the present disclosure. Reagents or instruments used without indicating the manufacturer are conventional products that can be purchased commercially.

[0360] The abbreviations in this disclosure have the following meanings:

[0361] Sequence and its specific information

[0362] Preparation plan

[0363] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0364] Nuclear magnetic resonance (NMR) 1 H NMR was measured using a Bruker 400 MHz nuclear magnetic resonance instrument; the measurement solvent was deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0365] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

[0366] s: singlet, d: doublet, t: triplet, q: quartet, dd: double doublet, qd: quartet doublet, ddd: double double doublet, ddt: double double triplet, dddd: double double double doublet, m: multiplet, br: broad, J: coupling constant, Hz: hertz, DMSO-d6: deuterated dimethyl sulfoxide. δ values ​​are expressed in ppm.

[0367] The mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0368] The ultra-performance liquid chromatography (UPLC) instrument used was AB SCIEX, model ExionLC.

[0369] The high-resolution mass spectrometer used was AB SCIEX, model X500B.

[0370] Example 1. Intermediate Synthesis

[0371] Example 1.1: Synthesis of (10S,13S)-10-(4-(dipropylamino)butyl)-13-isopropyl-20-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazaeicosane-19-ynoic acid (INT1)

[0372] Step 1:

[0373] Compound INT1-1 (117 g, 206.2 mmol) was added to dioxane (1.5 L) and stirred for 10 minutes. HCl (0.75 L, 4N in dioxane) was then added and allowed to react overnight at room temperature. Methyl tert-butyl ether (2 L) was added to the reaction solution, stirred for 30 minutes, and filtered. The solid was then dried in a forced air drying oven to obtain the hydrochloride salt of the target compound INT1-2 (94 g, yield: 91%, purity: 94%).

[0374] LCMS (ESI) [M+H] + =468.3.

[0375] 1H NMR(400MHz,DMSO-d6)δ8.24(d,J=7.3Hz,1H),8.07(brs,3H),7.90(d,J=7.5 Hz,2H),7.77(dd,J=7.1,3.4Hz,2H),7.47–7.37(m,3H),7.34(td,J=7.4,1.7Hz,2H),4.34–4.17(m,4H),3.96(dd,J=8.6 ,7.4Hz,1H),2.83–2.65(m,2H),2.04(dd,J=13.5,6.7Hz,1H),1.82–1.52(m,4H),1.51–1.30(m,2H),0.96–0.84(m,6H).

[0376] Step 2:

[0377] The hydrochloride of compound INT1-2 (55.0 g, 109.1 mmol) and acetic acid (10 ml) were dissolved in methanol (500 ml). Under an ice-water bath, n-propanal (25.35 g, 436.4 mmol) was added to the reaction solution and stirred at room temperature for 30 minutes. Sodium cyanoborohydride (27.43 g, 436.4 mmol) was then added to the reaction solution under an ice-water bath and the reaction was stirred at room temperature for 1 hour. Then, n-propanal (12.67 g, 218.2 mmol) and sodium cyanoborohydride (13.72 g, 218.2 mmol) were added to the reaction solution and the reaction was continued for 0.5 hours. The reaction solution was filtered, the filtrate was concentrated, and the concentrate was added with purified water (300 ml) and extracted with DCM (300 ml*2). The organic phase was washed with saturated brine, dried over anhydrous Na2SO4, and concentrated to obtain a crude product. The crude product was purified by column chromatography (eluent: MeOH and DCM, wherein MeOH:DCM (V / V) = 1:4-2:3) to obtain compound INT1-3 (39 g, yield: 65%, purity 98%).

[0378] LCMS (ESI) [M+H] + =552.3.

[0379] 1H NMR(400MHz, DMSO-d6)δ7.95–7.85(m,3H),7.76(t,J=7.3Hz,2H),7.51–7.40(m,3H),7.3 5(t,J=7.4Hz,2H),4.34–4.22(m,3H),4.13(dd,J=12.6,7.2Hz,1H),3.91(dd,J=8.8,7.1H z,1H),2.52–2.41(m,6H),2.03(dt,J=13.5,6.7Hz,1H),1.79–1.67(m,1H),1.66–1.56(m ,1H),1.42-1.36(m,6H),1.36–1.21(m,2H),0.90(t,J=6.9Hz,6H),0.82(t,J=6.9Hz,6H).

[0380] Step 3:

[0381] Compound INT1-3 (6.6 g, 12 mmol) was dissolved in DMF (50 mL), and compound INT1-4 (3 g, 12 mmol), HATU (4 g, 11 mmol), and N,N-diisopropylethylamine (3.5 g, 30 mmol) were added in sequence. The mixture was stirred at room temperature for 1 h, and then water (100 mL) was added to the reaction solution. The mixture was extracted with ethyl acetate (200 mL*3). The combined organic phases were washed with saturated brine (200 mL*2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (methanol: dichloromethane = 5%-10%) to obtain the target compound INT1-5 (9 g).

[0382] LCMS (ESI) [M+H] + =787.0;

[0383] 1H NMR(400MHz, DMSO-d6)δ8.87(s,1H),8.70(t,J=6.5Hz,1H),8.27–8.18(m,1H),8.06(d,J=7.4Hz,1H),7.90 (d,J=7.5Hz,2H),7.73(d,J=7.2Hz,2H),7.47–7.40(m,3H),7.38–7.30(m,6H),5.15(s,2H),4.71–4.55(m, 2H),4.37–4.27(m,2H),4.26–4.19(m,2H),4.15(s,2H),3.92–3.83(m,1H),3.73(d,J=5.5Hz,2H),3.04–2. 89(m,6H),2.01–1.94(m,1H),1.81–1.63(m,2H),1.61–1.52(m,6H),1.36–1.27(m,2H),0.91–0.82(m,12H).

[0384] Step 4:

[0385] Compound INT1-5 (3 g, 3.8 mmol) was dissolved in a mixed solvent of ethanol (20 mL) and ethyl acetate (10 mL). Pd / C (1.5 g) was added under nitrogen protection, and hydrogen was replaced three times. The reaction solution was stirred at room temperature under a hydrogen atmosphere overnight. The reaction solution was filtered and the filtrate was concentrated under reduced pressure to obtain the target compound INT1-6 (2.8 g), which was directly used in the next reaction.

[0386] LCMS (ESI) [M+H] + =696.4.

[0387] 1 H NMR (400MHz, DMSO-d6) δ8.67(t,J=6.5Hz,1H),8.25(t,J=5.7Hz,1H),8.06(d,J=7.5Hz,1H),7.90(d,J=7.5Hz,2H),7.73(d,J=7. 3Hz,2H),7.49–7.39(m,3H),7.33(t,J=7.4Hz,2H),4.65–4.55(m,2H),4.35–4.26(m,2H),4.24–4.14(m,2H),3.97(s,2H),3.92– 3.83(m,1H),3.73(d,J=5.6Hz,2H),2.94–2.84(m,6H),1.73–1.64(m,1H),1.63–1.47(m,8H),1.34–1.26(m,3H),0.89–0.82(m,12H).

[0388] Step 5:

[0389] Compound INT1-6 (1 g, 1.43 mmol) was dissolved in THF (30 mL), and diethylamine (1.0 g, 14.3 mmol) was added. The mixture was stirred at room temperature for 1 h. The reaction solution was concentrated under reduced pressure to obtain the target compound INT1-7 (680 mg), which was directly used in the next reaction.

[0390] LCMS (ESI) [M+H] + =474.1.

[0391] Step 6:

[0392] Compound INT1-8 (340 mg, 1.27 mmol) was dissolved in DMF (50 mL), and HATU (4 g, 11 mmol) and N,N-diisopropylethylamine (3.5 g, 30 mmol) were added in sequence. After stirring at room temperature for 30 min, compound INT1-7 (0.6 g, 1.27 mmol) was added. After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction solution was purified by preparative HPLC (acetonitrile: water = 5%-50%) to obtain the target compound INT1 (200 mg).

[0393] LCMS (ESI) [M+H] + =724.8;

[0394] 1 H NMR(400MHz, DMSO-d6)δ9.13(s,2H),8.72–8.67(m,1H),8.20(t,J=5.1Hz,1H),8.11(d,J=7.3Hz ,1H),8.01(d,J=8.7Hz,1H),4.62(d,J=6.2Hz,2H),4.26–4.21(m,1H),4.18–4.14(m,1H),3.90( s,2H),3.70(d,J=5.6Hz,2H),3.42(s,3H),3.17(s,2H),2.42–2.32(m,4H),2.05–1.94(m,3H),1 .87–1.78(m,2H),1.75–1.67(m,2H),1.63–1.53(m,2H),1.48–1.40(m,9H),0.87–0.84(m,12H).

[0395] Example 1.2: Synthesis of 1-(3-chloro-4-(3-(methylamino)propyl)phenyl)-3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)urea (INT2)

[0396] Step 1:

[0397] Compound INT2-1 (4.0 g, 23.65 mmol) and triethylamine (0.33 mL, 2.36 mmol) were dissolved in compound INT2-2 (3.18 g, 24.84 mmol), and zirconocene hydrochloride (691 mg, 2.36 mmol) was added. The reaction mixture was stirred at 60°C under nitrogen for 8 h. The reaction was monitored by LCMS. The reaction mixture was directly concentrated under reduced pressure to obtain the crude product, which was then purified by column chromatography (petroleum ether:ethyl acetate = 40:1) to obtain the target compound INT2-3 (2.92 g).

[0398] 1 H NMR (400MHz, DMSO-d6) δ6.38(dt,J=18.0,4.3Hz,1H),5.32(d,J=18.1Hz,1H),3.83(d,J=3.7Hz,2H),2.74(s,3H),1.39(s,9H),1.20(s,12H).

[0399] Step 2:

[0400] Compound INT2-4 (1.86 g, 7.88 mmol), compound INT2-3 (2.34 g, 7.88 mmol), potassium phosphate (5.02 g, 23.64 mmol), and 2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl (368 mg, 0.788 mmol) were dissolved in a mixture of 1,4-dioxane and water (V:V = 5:1, 50 mL). Palladium acetate (88 mg, 0.394 mmol) was added, and the reaction mixture was stirred at 80°C under nitrogen for 5 h. The reaction was monitored by LCMS. The reaction mixture was concentrated under reduced pressure to obtain a crude product, which was then purified by column chromatography (petroleum ether:ethyl acetate = 40:1) to obtain the target compound INT2-5 (1.0 g).

[0401] 1 H NMR(400MHz,DMSO-d6)δ8.29(d,J=2.1Hz,1H),8.16–8.14(m,1H),8.02(d,J=8.7Hz,1H),6 .79(d,J=16.0Hz,1H),6.19–6.05(m,1H),3.78(d,J=5.7Hz,2H),2.84(s,3H),1.39(s,9H).

[0402] Step 3:

[0403] Compound INT2-5 (500 mg, 1.53 mmol) was dissolved in a mixture of tetrahydrofuran, anhydrous methanol, and water (V:V:V = 2:2:1, 6 mL). Ammonium chloride (821 mg, 15.30 mmol) and zinc powder (997 mg, 15.30 mmol) were added sequentially. The mixture was stirred at 50°C for 2 h and monitored by LCMS. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to obtain the crude product, which was purified by column chromatography (petroleum ether:ethyl acetate = 10:1) to obtain the target compound INT2-6 (320 mg).

[0404] LCMS (ESI) [M+H-56] + =241.0.

[0405] 1 H NMR(400MHz,DMSO-d6)δ7.36(d,J=8.5Hz,1H),6.62-6.58(m,2H),6.51(d,J=8.5Hz,1 H),5.95-5.93(m,1H),5.50(s,2H),3.89(d,J=5.4Hz,2H),2.77(s,3H),1.40(s,9H).

[0406] Step 4:

[0407] Compound INT2-6 (300 mg, 1.01 mmol) was dissolved in THF (10 mL). Platinum dioxide (50 mg) was added to the reaction mixture, and the mixture was stirred under a hydrogen atmosphere for 1 hour. The reaction was monitored by LCMS. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound INT2-7 (200 mg), which was used directly in the next step.

[0408] LCMS (ESI) [M+H-100] + =199.0.

[0409] 1 H NMR (400MHz, DMSO-d6) δ6.95(d,J=8.2Hz,1H),6.59(d,J=2.2Hz,1H),6.45(dd,J=8.2,2.2Hz,1H ),5.16(s,2H),3.17(t,J=7.1Hz,2H),2.77(s,3H),2.48–2.43(m,2H),1.66(s,2H),1.37(s,9H).

[0410] Step 5:

[0411] Compound INT2-7 (200 mg, 0.67 mmol) was dissolved in tetrahydrofuran (5 mL), and trichloromethyl chloroformate (265 mg, 0.14 mmol) was added. The reaction mixture was stirred at room temperature for 1.5 h. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in DMF (5 mL), and a DMF solution of the hydrochloride salt of compound INT2-8 (221 mg, 0.88 mmol) and triethylamine (670 mg, 6.70 mmol) was added. The reaction was monitored by LCMS. The reaction mixture was added with water (50 mL) and extracted with ethyl acetate (25 mL*3). The organic phases were combined, washed with saturated brine (30 mL*3), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (DCM:MeOH = 10:1) to obtain the target compound INT2-9 (150 mg).

[0412] LCMS (ESI) [M+H-100] + =498.2.

[0413] 1 H NMR (400MHz, DMSO-d6) δ10.98(s,1H),8.81(s,1H),7.69(d,J=7.9Hz,1H),7.66(s,1H),7.5 1(s,1H),7.44(d,J=8.2Hz,1H),7.20–7.14(m,2H),6.84(t,J=5.7Hz,1H),5.17–5.03(m,1H ),4.43–4.39(m,2H),4.34–4.27(m,1H),3.21–3.17(m,2H),2.96–2.86(m,1H),2.77(s,3H) ,2.64–2.52(m,4H),2.43–2.33(m,1H),2.03–1.98(m,1H),1.76–1.67(m,2H),1.37(s,9H).

[0414] Step 6:

[0415] Compound INT2-9 (120 mg, 0.20 mmol) was dissolved in DCM (6 mL), followed by the addition of trifluoroacetic acid (2 mL). The reaction mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction mixture was concentrated under reduced pressure to afford the trifluoroacetic acid salt of the target compound INT2 (100 mg).

[0416] LCMS (ESI) [M+H] + =498.1.

[0417] Example 1.3: Synthesis of 2-(3-chloro-4-(3-(methylamino)propyl)phenyl)-N-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide (INT3)

[0418] Step 1:

[0419] Under nitrogen, compound INT3-1 (10.0 g, 31.50 mmol) was dissolved in dimethyl sulfoxide (70 mL). Ethyl difluorobromoacetate (12.79 g, 63.00 mmol) and copper powder (4.0 g, 63.00 mmol) were added sequentially to the reaction solution. The reaction solution was stirred at room temperature for 48 h and the reaction was monitored by TLC. The reaction solution was slowly poured into saturated aqueous ammonium chloride (200 mL) and extracted with petroleum ether (200 mL*2). The combined organic phases were washed with saturated brine (200 mL*2), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 10:1) to obtain the target compound INT3-2 (7.4 g).

[0420] 1 H NMR (400MHz, DMSO-d6) δ7.98(d,J=8.3Hz,1H),7.83(s,1H),7.51(d,J=8.3Hz,1H),4.35–4.30(m,2H),1.24(t,J=7.1Hz,3H).

[0421] Step 2:

[0422] Under nitrogen, compound INT3-2 (3.0 g, 9.57 mmol) was dissolved in DMF (50 mL). Methyl(propyl-2-yn-1-yl)carbamic acid tert-butyl ester (3.2 g, 19.14 mmol), triethylamine (4.8 g, 47.84 mmol), cuprous iodide (182 mg, 0.96 mmol), and bis(triphenylphosphine)palladium dichloride (672 mg, 0.96 mmol) were added sequentially. The reaction mixture was stirred at 60°C for 4 h. The reaction was monitored by LCMS. The reaction mixture was cooled to room temperature, and water (150 mL) was added. The mixture was extracted with ethyl acetate (150 mL x 3). The combined organic phases were washed with saturated brine (200 mL x 3), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 10:1) to obtain the target compound INT3-3 (2.0 g).

[0423] LCMS (ESI) [M+H-56] + =346.0.

[0424] 1 H NMR (400MHz, DMSO-d6) δ7.75(d,J=7.4Hz,2H),7.58(d,J=8.2Hz,1H),4.38–4.29(m,4H),2.91(s,3H),1.43(s,9H),1.24(t,J=7.1Hz,3H).

[0425] Step 3:

[0426] Compound INT3-3 (1.2 g, 2.99 mmol) was dissolved in methanol (10 mL). Raney nickel (300 mg) was added under nitrogen. The atmosphere was replaced with hydrogen three times. The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 6 h. The reaction was monitored by LCMS. The reaction mixture was filtered through celite and the filtrate was concentrated under reduced pressure to obtain the target compound INT3-4 (930 mg), which was used directly in the next step.

[0427] LCMS (ESI) [M+H-56] + =350.0.

[0428] 1 H NMR (400MHz, DMSO-d6) δ7.64–7.54(m,2H),7.52(s,1H),4.32–4.30(m,2H),3.29–3.16(m ,2H),2.78(s,3H),2.74–2.67(m,2H),1.87–1.70(m,2H),1.41(s,9H),1.27–1.22(m,3H).

[0429] Step 4:

[0430] Compound INT3-4 (900 mg, 2.22 mmol) was dissolved in a mixture of methanol (15 mL) and water (3 mL). Lithium hydroxide monohydrate (212 mg, 8.87 mmol) was added and stirred at room temperature for 2 h. The reaction was monitored by LCMS. Water (20 mL) was added to the reaction solution, which was then concentrated under reduced pressure to remove the methanol. The pH was adjusted to 4 with 1N aqueous hydrochloric acid. The solution was extracted with ethyl acetate (20 mL x 2). The combined organic phases were washed with saturated brine (20 mL x 3), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to obtain the target compound INT3-5 (700 mg), which was used directly in the next step.

[0431] LCMS (ESI) [M+H-56] + =322.2.

[0432] 1H NMR (400MHz, DMSO-d6) δ7.49(s,1H),7.40(s,2H),3.23-3.20(m,2H),2.78(s,3H),2.68-2.64(m,2H),1.77-1.73(m,2H),1.36(s,9H).

[0433] Step 5:

[0434] Compound INT3-5 (670 mg, 1.77 mmol) and 3-(6-aminomethylene-3-oxo-1H-isoindol-2-yl)piperidine-2,6-dione hydrochloride (500 mg, 1.61 mmol) were dissolved in dichloromethane (20 mL). Triethylamine (765 mg, 7.57 mmol) and propylphosphonic tricyclic anhydride in ethyl acetate (2.87 g, 4.51 mmol, 50% by mass) were added. The reaction mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with saturated brine (20 mL x 2), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain the target compound INT3-6 (270 mg).

[0435] LCMS (ESI) [M+Na] + =655.6.

[0436] 1 H NMR (400MHz, DMSO-d6) δ10.99(s,1H),9.68(t,J=5.9Hz,1H),7.68(d,J=7.8Hz,1H) ,7.60(s,1H),7.52(d,J=11.0Hz,2H),7.43(s,1H),7.37(d,J=7.8Hz,1H),5.13–5.0 9(m,1H),4.45–4.27(m,4H),3.22–3.20(m,3H),2.94–2.91(m,1H),2.78(s,3H),2.6 9(d,J=7.8Hz,2H),2.67–2.62(m,1H),2.42–2.35(m,1H),1.77(s,2H),1.33(s,9H).

[0437] Step 6:

[0438] Dissolve compound INT3-6 (205 mg, 0.32 mmol) in HCl / 1,4-dioxane solution (5 mL). Stir the reaction mixture at room temperature for 2 hours. Monitor the reaction by LCMS. Concentrate the reaction mixture under reduced pressure to obtain the hydrochloride salt of the target compound INT3 (165 mg).

[0439] LCMS (ESI) [M+H] + =532.9.

[0440] Example 1.4: Synthesis of 1-(3-chloro-4-(3-(methylamino)propyl)phenyl)-3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)methyl)urea (INT4)

[0441] Step 1:

[0442] Compound INT2-7 (200 mg, 0.67 mmol) was dissolved in THF (5 mL), and trichloromethyl chloroformate (265 mg, 1.34 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to obtain the crude intermediate. The crude intermediate was dissolved in DMF (5 mL), followed by the addition of a solution of compound INT4-1 (193 mg, 0.67 mmol) and triethylamine (670 mg, 6.70 mmol) in DMF (5 mL). The reaction was monitored by LCMS. The reaction mixture was added with water (25 mL) and extracted with EA (30 mL x 3). The organic phases were combined, washed with saturated brine (30 mL x 2), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (dichloromethane:methanol = 10:1) to obtain the target compound INT4-2 (200 mg).

[0443] LCMS (ESI) [M+H-100] + =512.3.

[0444] 1H NMR (400MHz, DMSO-d6) δ11.12(s,1H),8.86(s,1H),7.89(d,J=7.7Hz,1H),7.85- 7.75(m,2H),7.64(s,1H),7.18(s,2H),6.91(t,J=6.1Hz,1H),5.14(dd,J=12.9, 5.3Hz,1H),4.46(d,J=6.1Hz,2H),3.18(t,J=7.1Hz,2H),2.94-2.85(m,1H),2.7 7(s,3H),2.64-2.52(m,4H),2.09-2.03(m,1H),1.77-1.64(m,2H),1.36(s,9H).

[0445] Step 2:

[0446] Compound INT4-2 (120 mg, 0.20 mmol) was dissolved in DCM (6 mL), and trifluoroacetic acid (2 mL) was added. The reaction solution was stirred at room temperature for 1 h; the reaction was monitored by LCMS; the reaction solution was concentrated under reduced pressure to obtain the trifluoroacetate salt of the target compound INT4 (100 mg).

[0447] LCMS (ESI) [M+H] + =512.3.

[0448] 1 H NMR (400MHz, DMSO-d6) δ11.12(s,1H),8.98(s,1H),8.34(s,2H),7.90(d,J=7.7Hz,1H ),7.85-7.78(m,2H),7.67(d,J=1.7Hz,1H),7.23-7.17(m,2H),7.04(t,J=6.0Hz,1H), 5.15(dd,J=12.8,5.3Hz,1H),4.47(d,J=5.8Hz,2H),2.95-2.85(m,3H),2. 70-2.57(m,4H),2.57-2.54(m,3H),2.10-2.01(m,1H),1.87-1.79(m,2H).

[0449] Example 1.5: Synthesis of (5S,8S)-8-(4-(dimethylamino)butyl)-1-(9H-fluoren-9-yl)-5-isopropyl-3,6,9,12-tetraoxo-2,15-dioxa-4,7,10,13-tetraazaheptadecan-17-oic acid (INT5)

[0450] Step 1:

[0451] Compound INT1-2 (50.0 g, 107.07 mmol) was dissolved in MeOH (1000 mL), and sodium acetate (17.6 g, 214.1 mmol) was added. Under ice bath conditions, paraformaldehyde (57.8 g, 642.4 mmol) was added and stirred for 30 min. Then, sodium cyanoborohydride (13.5 g, 214.1 mmol) was added to the reaction system. After the addition was completed, the temperature was returned to room temperature, and the reaction solution was stirred at room temperature overnight; the reaction was monitored by LCMS; the reaction solution was concentrated under reduced pressure, and the crude product was directly purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 5%-70%) to obtain the target compound INT5-1 (25 g).

[0452] LCMS (ESI) [M+H] + =496.6.

[0453] 1 H NMR(400MHz, DMSO-d6)δ9.96(s,1H),8.19(d,J=7.5Hz,1H),7.89(d,J=7.5Hz,2H),7.75( d,J=7.4Hz,2H),7.42-7.40(m,3H),7.37-7.27(m,2H),4.33-4.25(m,1H),4.24-4.18(m,2 H),3.94-3.85(m,1H),3.01-2.89(m,2H),2.68(d,J=4.7Hz,6H),2.06-1.91(m,2H),1.78- 1.68(m,1H),1.65-1.58(m,2H),1.38-1.28(m,2H),1.27-1.22(m,1H),0.93-0.83(m,6H).

[0454] Step 2:

[0455] Compound INT5-1 (4.7 g, 9.5 mmol) and compound INT1-4 (2.4 g, 9.5 mmol) were dissolved in DMF (50 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (3.2 g, 8.6 mmol) and N,N-diisopropylethylamine (3.7 g, 28.5 mmol) were added. The reaction solution was stirred at room temperature for 1 h; the reaction was monitored by LCMS; water (200 mL) was added to the reaction solution, and EA (200 mL*3) was used for extraction. The organic phases were combined, washed with saturated brine (150 mL*3), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure, and the crude product was purified by column chromatography (DCM:MeOH=10:1) to obtain the target compound INT5-2 (1.5 g).

[0456] LCMS (ESI) [M+H] + =730.4.

[0457] 1 H NMR (400MHz, DMSO-d6) δ8.70(t,J=6.6Hz,1H),8.25(t,J=6.2Hz,1H),8.08(d,J=7.3Hz,1 H),7.90(d,J=7.5Hz,2H),7.74(d,J=7.3Hz,2H),7.43-7.32(m,10H),5.15(s,2H),4.66- 4.60(m,2H),4.33-4.24(m,2H),4.24-4.19(m,2H),4.16(s,2H),3.92-3.86(m,1H),3.73 (d,J=5.9Hz,2H),2.91-2.82(m,2H),2.66(s,6H),2.02-1.94(m,1H),1.74-1.64(m,1H), 1.60-1.51(m,3H),1.31-1.26(m,2H),0.85(t,J=6.0Hz,6H).

[0458] Step 3:

[0459] The compound INT5-2 (1.3 g, 1.7 mmol) was dissolved in a mixed solvent of EtOH (40 mL) and EA (20 mL), Pd / C (400 mg) was added, and the hydrogen gas was replaced three times. The reaction solution was stirred under a hydrogen atmosphere for 16 h; the reaction was monitored by LCMS; the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound INT5 (780 mg).

[0460] LCMS (ESI) [M+H] + =640.4.

[0461] Example 1.6: Synthesis of 1-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-5-yl)methyl)-3-(4-(3-(methylamino)propyl)phenyl)urea (INT6)

[0462] Step 1:

[0463] Compound INT6-1 (3.0 g, 17.60 mmol) was dissolved in DMF (100 mL), and INT6-2 (5.0 g, 17.60 mmol), triethylamine (8.9 g, 88.20 mmol), cuprous iodide (335 mg, 1.76 mmol) and triphenylphosphine palladium dichloride (1.2 g, 1.76 mmol) were added to the reaction solution in sequence. The reaction solution was stirred at room temperature for 4 h under nitrogen protection; the reaction was monitored by TLC; water (100 mL) was added to the reaction solution to quench the reaction, and the solution was extracted with ethyl acetate (100 mL*3). The organic phases were combined and washed with saturated brine (100 mL*2) and water (100 mL*2), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain the target compound INT6-3 (3.5 g).

[0464] 1 H NMR (400MHz, DMSO-d6) δ8.39(d,J=2.3Hz,1H),8.18(dd,J=8.8Hz,2.4Hz,1H),7.84(d,J=8.6Hz,1H),4.38(s,2H),2.91(s,3H),1.42(s,9H).

[0465] Step 2:

[0466] Compound INT6-3 (1.0 g, 3.09 mmol) was dissolved in anhydrous MeOH (20 mL), and THF (20 mL), distilled water (10 mL), zinc powder (2.0 g, 30.86 mmol) and ammonium chloride (1.6 g, 30.86 mmol) were added to the reaction solution in sequence, and the reaction solution was stirred at 50°C for 4 h; the reaction was monitored by LCMS; the reaction solution was filtered, the filtrate was concentrated under reduced pressure, and EA was added for extraction (50 mL×3), the combined organic phases were washed with saturated brine (50 mL×2) and water (50 mL×2), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by chromatography (petroleum ether: ethyl acetate = 5:1) to obtain the target compound INT6-4 (900 mg).

[0467] LCMS (ESI) [M+H-56] + =239.0.

[0468] 1H NMR (400MHz, DMSO-d6) δ7.16(d,J=8.4Hz,1H),6.64(d,J=2.1Hz,1H),6.45(dd,J=8.4Hz,2.0Hz,1H),5.78(s,2H),4.22(s,2H),2.86(s,3H),1.41(s,9H).

[0469] Step 3:

[0470] Compound INT6-4 (5.0 g, 17.00 mmol) was dissolved in MeOH (50 mL), and ammonium formate (5.36 g, 85.00 mmol) and palladium carbon (360 mg, 3.40 mmol) were added in sequence. The mixture was replaced with hydrogen three times, and the reaction solution was reacted at 60°C under a hydrogen atmosphere overnight; the reaction was monitored by LCMS; the reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 5%-50%) to obtain the target compound INT6-5 (2.6 g).

[0471] LCMS (ESI) [M+H-100] + =165.1.

[0472] 1 H NMR (400MHz, CDCl3) δ6.97 (d, J = 8.2 Hz, 2H), 6.62 (d, J = 8.3 Hz, 2H), 3.18-3. 23(m,2H),2.83(s,3H),2.52-2.44(m,2H),1.83-1.72(m,2H),1.45(s,9H).

[0473] Step 4:

[0474] Compound INT6-5 (1.0 g, 3.78 mmol) was dissolved in THF (20 mL), and triphosgene (1.50 g, 7.56 mmol) was added. After the addition, the reaction mixture was stirred at room temperature for 30 min. LCMS detected the formation of the reaction intermediate. The reaction solution was concentrated under reduced pressure and DMF (5 mL) was added for dissolution. The hydrochloride of compound INT2-8 (1.04 g, 3.78 mmol) and triethylamine (3.03 g, 30.04 mmol) were dissolved in DMF (5 mL) and slowly added dropwise to the above-mentioned reaction solution. After the addition, the reaction mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. Water (10 mL) was added to the reaction solution to quench the reaction, and the mixture was extracted with EA (10 mL*3). The organic phases were combined, washed with saturated brine (10 mL*3), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by chromatography (methanol: dichloromethane = 0%~7%) to obtain the target compound INT6-6 (800 mg).

[0475] LCMS (ESI) [M+H-100] + =464.3.

[0476] 1 H NMR(400MHz, CDCl3)δ8.97(s,1H),7.57-7.45(m,2H),7.29(s,1H),7.26-7.21(m,2H) ,7.18(d,J=7.8Hz,1H),7.04(d,J=7.7Hz,2H),5.95(s,1H),5.04(dd,J=13.1,4.8Hz,1 H),4.43-4.30(m,2H),4.24-4.11(m,2H),3.21(t,J=7.0Hz,2H),2.81(s,3H),2.80-2 .63(m,2H),2.50(t,J=7.1Hz,2H),2.27-2.05(m,2H),1.79-1.74(m,2H),1.43(s,9H).

[0477] Step 5:

[0478] Compound INT6-6 (200 mg, 0.35 mmol) was dissolved in DCM (5 mL), and trifluoroacetic acid (1 mL) was slowly added dropwise to the reaction solution. After the addition, the reaction was stirred at room temperature for 30 min; the reaction was monitored by LCMS; the reaction solution was concentrated under reduced pressure to obtain the trifluoroacetate salt of the target compound INT6 (150 mg).

[0479] LCMS (ESI) [M+H] + =464.2.

[0480] 1 H NMR(400MHz,DMSO-d6)δ10.90(s,1H),8.54(s,1H),8.25(s,2H),7.63(d,J=7.8Hz,1H),7 .44(s,1H),7.37(d,J=7.9Hz,1H),7.27(d,J=8.4Hz,2H),7.00(d,J=8.4Hz,2H),6.69(t, J=6.0Hz,1H),5.03(dd,J=13.3,4.9Hz,1H),4.42-4.24(m,4H),2.90-2.83(m,2H),2.82- 2.75(m,2H),2.62-2.47(m,5H),2.37-2.24(m,1H),1.96-1.90(m,1H),1.81-1.70(m,2H).

[0481] Example 1.7: Synthesis of 2,2-dimethyl-4-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)butanoic acid (INT7)

[0482] Step 1:

[0483] Compound INT7-1 (500 mg, 4.39 mmol) was dissolved in ultra-dry DCM (5 mL). Boron tribromide (1.15 g, 4.6 mmol) was added dropwise at 0°C. The mixture was warmed to room temperature and stirred for 24 hours. The temperature was then lowered to 0°C and methanol (5 mL) was added dropwise to quench the reaction. The reaction mixture was stirred at room temperature for another 24 hours. Saturated aqueous sodium bicarbonate (30 mL) was added to the reaction mixture, followed by extraction with DCM (30 mL x 3). The combined organic phases were dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound INT7-2 (830 mg).

[0484] 1 H NMR (400MHz, CDCl3) δ3.69(s,3H),3.40-3.27(m,2H),2.20-2.09(m,2H),1.21(s,6H).

[0485] Step 2:

[0486] Dissolve compound INT7-2 (650 mg, 3.13 mmol) in DMF (10 mL), add sodium azide (1.02 g, 15.63 mmol), raise the temperature to 80°C, stir overnight, then cool to room temperature, add water (30 mL) to the reaction solution, and then extract with MTBE (30 mL*3), combine the organic phases, dry over anhydrous Na2SO4, filter, and concentrate the filtrate under pressure to obtain the target compound INT7-3 (470 mg).

[0487] 1 H NMR (400MHz, CDCl3) δ3.69(s,3H),3.31-3.23(m,2H),1.89-1.81(m,2H),1.22(s,6H).

[0488] Step 3:

[0489] Compounds INT7-3 (365 mg, 2.13 mmol) and INT7-4 (320 mg, 2.13 mmol) were dissolved in tert-butanol (5 mL) and water (5 mL). Sodium ascorbyl sulfate (84 mg, 0.43 mmol) and anhydrous copper sulfate (34 mg, 0.21 mmol) were added, and the reaction mixture was stirred at room temperature for 1 h. Water (25 mL) was added to the reaction mixture, followed by extraction with ethyl acetate (30 mL*3). The organic phases were combined, dried over anhydrous Na2SO4, filtered, and the filtrate was evaporated to dryness. The crude product was purified by column chromatography (PE:EA=3:1) to obtain the target compound INT7-5 (600 mg).

[0490] LCMS (ESI) [M+H] + =322.1.

[0491] 1 H NMR (400MHz, CDCl3) δ8.95(s,2H),7.85(s,1H),4.53-4.39(m,2H),3.69(s,3H),2.61(s,3H),2.29-2.17(m,2H),1.31(s,6H).

[0492] Step 4:

[0493] Compound INT7-5 (500 mg, 1.60 mmol) was dissolved in THF (5 mL) and water (5 mL), and lithium hydroxide (112 mg, 4.70 mmol) was added. The reaction mixture was stirred at room temperature for 5 h. 1N hydrochloric acid was added to adjust the pH to 3-4. A large amount of white solid precipitated, which was filtered and the filter cake dried to obtain the target compound INT7-6 (450 mg).

[0494] LCMS (ESI) [M+H] + =308.0.

[0495] 1 H NMR (400MHz, DMSO-d6) δ12.43(s,1H),9.06(s,2H),8.76(s,1H),4.55-4.31(m,2H),2.56(s,3H),2.15-2.04(m,2H),1.20(s,6H).

[0496] Step 5:

[0497] Dissolve compound INT7-6 (1.5 g, 4.90 mmol) in THF (10 mL) and water (10 mL), add Oxane (15.0 g, 24.5 mmol), and stir the reaction mixture at room temperature overnight. Pour the reaction mixture into water (200 mL), filter, and dry the filter cake to obtain the target compound INT7 (1.6 g).

[0498] LCMS (ESI) [M+H] + =340.1.

[0499] 1 H NMR (400MHz, DMSO-d6) δ9.48(s,2H),8.98(s,1H),4.54-4.48(m,2H),3.44(s,3H),2.16-2.09(m,2H),1.21(s,6H).

[0500] Example 2. Synthesis of biologically active compounds (Payload)

[0501] Example 2.1: Synthesis of N-(3-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)ureido)phenyl)propyl)-2-hydroxy-N-methylacetamide (PL-1)

[0502] Step 1:

[0503] The trifluoroacetic acid salt of compound INT2 (20 mg, 0.04 mmol) and glycolic acid (3 mg, 0.04 mmol) were dissolved in DMF (3 mL). Triethylamine (12 mg, 0.12 mmol) and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (17 mg, 0.06 mmol) were added. The reaction mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction mixture was directly purified by preparative HPLC (acetonitrile:H2O containing 0.05% FA = 5%-50%) to obtain the target compound PL-1 (10 mg).

[0504] LCMS (ESI) [M+H] + =556.2;

[0505] 1H NMR (400MHz, DMSO-d6) δ10.97(s,1H),8.95–8.86(m,1H),7.69(d,J=7.8Hz,1H),7.66(s,1H),7.5 1(s,1H),7.44(d,J=7.7Hz,1H),7.22–7.15(m,2H),6.98–6.90(m,1H),5.18–5.03(m,1H),4.46–4. 36(m,4H),4.33–4.28(m,1H),4.09–4.01(m,2H),3.24–3.17(m,1H),2.96–2.86(m,1H),2.84(s,3H ),2.64–2.58(m,1H),2.58–2.54(m,2H),2.44–2.32(m,1H),2.02–1.97(m,1H),1.80–1.66(m,2H).

[0506] Example 2.2: Synthesis of 2-(3-chloro-4-(3-(2-hydroxy-N-methylacetamido)propyl)phenyl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)-2,2-difluoroacetamide (PL-2)

[0507] Step 1:

[0508] The hydrochloride salt of compound INT3 (40 mg, 0.08 mmol) and glycolic acid (6 mg, 0.08 mmol) were dissolved in DMF (2 mL). Triethylamine (24 mg, 0.24 mmol) and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (33 mg, 0.12 mmol) were added. The reaction mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction mixture was directly purified by preparative HPLC (CH3CN:H2O containing 0.05% FA = 5%-50%) to obtain the target compound PL-2 (15 mg).

[0509] LCMS (ESI) [M+H] + =591.2;

[0510] 1H NMR (400MHz, DMSO-d6) δ10.98(s,1H),9.69(t,J=5.8Hz,1H),7.68(d,J=7.8Hz,1H),7.59(s,1H),7.56(d,J =8.1Hz,1H),7.49(d,J=8.0Hz,1H),7.42(s,1H),7.37(d,J=7.9Hz,1H),5.16–5.05(m,1H),4.45(d,J=6.1Hz ,2H),4.41–4.38(m,1H),4.40–4.30(m,1H),4.07(t,J=4.8Hz,2H),3.30–3.23(m,2H),2.97–2.88(m,1H),2 .86(s,3H),2.74–2.67(m,2H),2.64–2.57(m,1H),2.44–2.32(m,1H),2.03–1.98(m,1H),1.84–1.72(m,2H).

[0511] Example 2.3: Synthesis of N-(3-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl)methyl)ureido)phenyl)propyl)-2-hydroxy-N-methylacetamide (PL-3)

[0512] Step 1:

[0513] Compound INT4 (50.0 mg, 0.10 mmol) was dissolved in DMF (1 mL), and glycolic acid (7.0 mg, 0.10 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (38 mg, 0.10 mmol), and N,N-diisopropylethylamine (32 mg, 0.25 mmol) were added sequentially. The reaction solution was stirred at room temperature for 1 h; the reaction was monitored by LCMS; the reaction solution was directly purified by preparative HPLC (acetonitrile: H2O containing 0.05% FA = 5%-50%) to obtain the target compound PL-3 (15 mg).

[0514] LCMS (ESI) [M+H] + =570.3;

[0515] 1H NMR (400MHz, DMSO-d6) δ11.11(s,1H),8.86(d,J=5.0Hz,1H),7.89(d,J=7.7Hz,1H),7.82(s,1H),7.79(d,J= 7.7Hz,1H),7.63(d,J=1.6Hz,1H),7.24-7.15(m,2H),6.92(t,J=6.4Hz,1H),5.14(dd,J=13.0,5.4Hz,1H),4. 51-4.44(m,2H),4.44-4.32(m,1H),4.08-3.99(m,2H),3.37-3.35(m,1H),3.25-3.16(m,1H),2.95-2.86(m, 1H),2.84(d,J=5.1Hz,3H),2.65(d,J=19.8Hz,1H),2.59-2.53(m,3H),2.11-2.01(m,1H),1.81-1.65(m,2H).

[0516] Example 2.4: Synthesis of N-(2-chloro-4-(3-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-methyl)ureido)phenethoxy)ethyl-2-hydroxy-N-methylacetamide (PL-4)

[0517] Step 1:

[0518] Compound PL-4-1 (50.0 mg, 0.09 mmol, prepared with reference to patent document WO2021198965) was dissolved in DMF (1 mL), and glycolic acid (6.9 mg, 0.09 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (37 mg, 0.09 mmol), and N,N-diisopropylethylamine (31 mg, 0.22 mmol) were added sequentially. The reaction solution was stirred at room temperature for 1 h; the reaction was monitored by LCMS; the reaction solution was directly purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 5%-50%) to obtain the target compound PL-4 (23 mg).

[0519] LCMS (ESI) [M+H] + =586.4;

[0520] 1H NMR (400MHz, DMSO-d6) δ10.97(s,1H),8.77(s,1H),7.68(dd,J=9.5,4.8Hz,2H),7.51(s,1H),7.44(d, J=7.9Hz,1H),7.20-7.11(m,2H),6.80(t,J=5.8Hz,1H),5.10(dd,J=13.3,5.1Hz,1H),4.48-4.26(m,4 H),4.05(d,J=17.8Hz,2H),3.57-3.53(m,2H),3.52-3.48(m,2H),3.46-3.41(m,2H),3.36-3.30(m,1H ),2.96-2.84(m,2H),2.83(d,J=4.6Hz,3H),2.60-2.51(m,1H),2.45-2.30(m,1H),2.02-1.95(m,1H).

[0521] Example 2.5: Synthesis of N-(3-(4-(3-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-methyl)ureido)phenyl)propyl)-2-hydroxy-N-methylacetamide (PL-5)

[0522] Step 1:

[0523] The trifluoroacetate of compound INT6 (50.0 mg, 0.10 mmol) was dissolved in DMF (1 mL), and glycolic acid (8.0 mg, 0.10 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (41 mg, 0.10 mmol), and N,N-diisopropylethylamine (35 mg, 0.25 mmol) were added sequentially. The reaction solution was stirred at room temperature for 1 h; the reaction was monitored by LCMS; the reaction solution was directly purified by preparative HPLC (acetonitrile: H2O containing 0.05% FA = 5%-50%) to obtain the target compound PL-5 (18 mg).

[0524] LCMS (ESI) [M+H] + =522.4.

[0525] 1H NMR (400MHz, DMSO-d6) δ10.97(s,1H),8.52(d,J=5.7Hz,1H),7.69(d,J=7.8Hz,1H),7.51(s,1H),7.44(d,J=7 .8Hz,1H),7.35-7.27(m,2H),7.07(t,J=6.8Hz,2H),6.68(t,J=5.6Hz,1H),5.10(dd,J=13.3,5.0Hz,1H),4.4 8-4.27(m,4H),4.03-3.99(m,2H),3.85(s,1H),3.31(t,J=7.3Hz,1H),3.20-3.10(m,1H),2.97-2.86(m,1H), 2.83(s,3H),2.61-2.57(m,1H),2.49-2.42(m,2H),2.41-2.30(m,1H),2.04-1.93(m,1H),1.82-1.67(m,2H).

[0526] Example 3. Synthesis of Toxin-Linker

[0527] Example 3.1: Synthesis of N-((16S,19S)-1-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)ureido)phenyl)-16-(4-(dipropylamino)butyl)-6,20-dimethyl-7,12,15,18-tetraoxo-3,9-dioxa-6,11,14,17-tetraazaheneicosan-19-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (DL-1)

[0528] Method 1:

[0529] Compound INT1 (22 mg) was dissolved in DMF (1 mL), and HATU (15 mg) and N,N-diisopropylethylamine (100 μl) were added in sequence. After stirring at room temperature for 5 min, compound PL-4-1 (16 mg) was added. After the addition was completed, the mixture was stirred at room temperature for 1 h. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: water = 5%-50%) to obtain the formate salt of the target compound DL-1 (18 mg).

[0530] LCMS (ESI) [M+H] + =1233.6.

[0531] 1H NMR(400MHz,DMSO-d6)δ10.97(s,1H),9.11(s,2H),8.82(s,1H),8.63-8.57(m, 1H),8.16(s,2H),8.03(d,J=7.2Hz,1H),7.93(d,J=8.4Hz,1H),7.70-7.66(m,2H ),7.51(s,1H),7.44(d,J=8.1Hz,1H),7.19-7.14(m,2H),6.85(s,1H),5.10(dd ,J=13.3,5.0Hz,1H),4.60-4.55(m,2H),4.48-4.38(m,4H),4.34-4.28(m,1H),4 .24-4.16(m,2H),4.11(d,J=15.5Hz,2H),3.74-3.68(m,2H),3.56-3.45(m,6H) ,3.41(s,3H),2.96-2.89(m,1H),2.87-2.78(m,6H),2.62(s,1H),2.59-2.51(m, 4H),2.42-2.29(m,4H),2.02-1.96(m,2H),1.85-1.78(m,2H),1.70-1.65(m,1H ),1.59-1.53(m,1H),1.45-1.38(m,7H),1.33-1.27(m,2H),0.85-0.81(m,12H).

[0532] Method 2:

[0533] Step 1:

[0534] Compound PL-4-1 (200 mg, 0.38 mmol) was dissolved in DMF (2 mL), and compound IN1-6 (263 mg, 0.38 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (144 mg, 0.38 mmol) and N,N-diisopropylethylamine (122 mg, 0.95 mmol) were added in sequence. After the addition, the reaction solution was stirred at room temperature for 1 h; the reaction was monitored by LCMS; methyl tert-butyl ether (80 mL) was slowly added to the reaction solution for dilution, and after standing for 10 min, the supernatant was poured out and the residue was directly concentrated under reduced pressure on an oil pump to remove excess solvent. The crude product was purified by preparative TLC (dichloromethane: methanol = 8:1) to obtain the target compound DL-1-1 (280 mg).

[0535] LCMS (ESI) [M+H] + =1205.5.

[0536] 1 H NMR (400MHz, DMSO-d6) δ10.99(s,1H),8.81(s,1H),8.65(t,J=8.3Hz,1H),8.30-8.18(m ,1H),8.04(d,J=7.7Hz,1H),7.89(d,J=7.5Hz,2H),7.74-7.70(m,2H),7.67(d,J=5.7Hz ,2H),7.51(s,1H),7.47-7.39(m,4H),7.32(t,J=7.4Hz,2H),7.20-7.13(m,2H),6.83(t ,J=5.4Hz,1H),5.10(dd,J=13.3,5.1Hz,1H),4.61-4.54(m,2H),4.43-4.39(m,2H),4.3 5-4.29(m,2H),4.21(d,J=6.8Hz,2H),4.11(d,J=15.8Hz,2H),3.92-3.85(m,1H),3.72( t,J=6.0Hz,2H),3.57-3.45(m,6H),2.96-2.87(m,2H),2.86(s,2H),2.83(d,J=6.2Hz,2 H),2.78(s,2H),2.61(d,J=4.5Hz,1H),2.59-2.56(m,1H),2.42-2.32(m,2H),2.04-1.9 4(m,3H),1.74-1.51(m,4H),1.50-1.37(m,6H),1.32-1.25(m,3H),0.87-0.81(m,12H).

[0537] Step 2:

[0538] Compound DL-1-1 (100 mg, 0.08 mmol) was dissolved in DMF (2 mL), and diethylamine (25.6 mg, 0.32 mmol) was added to the reaction solution. The reaction solution was stirred at room temperature for 1 h; the reaction was monitored by LCMS; the reaction solution was directly placed under an oil pump and concentrated under reduced pressure to obtain the target compound DL-1-2 (60 mg), which was directly used in the next reaction.

[0539] LCMS (ESI) [M+H] + =983.5.

[0540] Step 3:

[0541] Compound DL-1-2 (60 mg, 0.06 mmol), compound INT1-8 (18 mg, 0.06 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (25 mg, 0.06 mmol), and N,N-diisopropylethylamine (21 mg, 0.15 mmol) were dissolved in DMF (3 mL), and the reaction solution was stirred at room temperature for 1 h; the reaction was monitored by LCMS; the reaction solution was directly purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 5%-50%) to obtain the formate salt of the target compound DL-1 (23 mg).

[0542] LCMS (ESI) [M+H] + =1233.9.

[0543] 1 H NMR(400MHz,DMSO-d6)δ10.97(s,1H),9.11(s,2H),8.82(s,1H),8.63-8.57(m, 1H),8.16(s,2H),8.03(d,J=7.2Hz,1H),7.93(d,J=8.4Hz,1H),7.70-7.66(m,2H ),7.51(s,1H),7.44(d,J=8.1Hz,1H),7.19-7.14(m,2H),6.85(s,1H),5.10(dd ,J=13.3,5.0Hz,1H),4.60-4.55(m,2H),4.48-4.38(m,4H),4.34-4.28(m,1H),4 .24-4.16(m,2H),4.11(d,J=15.5Hz,2H),3.74-3.68(m,2H),3.56-3.45(m,6H) ,3.41(s,3H),2.96-2.89(m,1H),2.87-2.78(m,6H),2.62(s,1H),2.59-2.51(m, 4H),2.42-2.29(m,4H),2.02-1.96(m,2H),1.85-1.78(m,2H),1.70-1.65(m,1H ),1.59-1.53(m,1H),1.45-1.38(m,7H),1.33-1.27(m,2H),0.85-0.81(m,12H).

[0544] Example 3.2: Synthesis of N-(14S,17S)-1-(2-chloro-4-(3-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)ureido)phenyl)-14-(4-(dipropylamino)butyl)-4,18-dimethyl-5,10,13,16-tetraoxo-7-oxa-4,9,12,15-tetraazanonadecan-17-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexane-5-hexanamide (DL-2)

[0545] Step 1:

[0546] The trifluoroacetic acid salt of compound INT2 (100 mg, 0.20 mmol) and compound INT1-6 (139 mg, 0.20 mmol) were dissolved in DMF (5 mL), and N,N-diisopropylethylamine (77 mg, 0.60 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (76 mg, 0.20 mmol) were added. The reaction solution was stirred at room temperature for 1 h. The reaction was monitored by LCMS. Water (45 mL) was added to the reaction solution and extracted with ethyl acetate (25 mL*3). The combined organic phases were washed with saturated brine (30 mL*3), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative thin-layer chromatography (DCM:MeOH=10:1) to obtain the target compound DL-2-1 (100 mg).

[0547] LCMS (ESI) [M+H] + =1175.3.

[0548] 1H NMR(400MHz,DMSO-d6)δ10.98(s,1H),8.85–8.73(m,1H),8.68–8.56(m,1H) ),8.31–8.18(m,1H),8.07–7.97(m,1H),7.88(d,J=7.5Hz,2H),7.74–7.64 (m,4H),7.51(s,1H),7.46–7.39(m,4H),7.31(t,J=6.2Hz,2H),7.20–7.15 (m,2H),6.86–6.77(m,1H),5.17–5.04(m,1H),4.61–4.54(m,2H),4.47–4.3 8(m,4H),4.34–4.27(m,2H),4.23(d,J=6.8Hz,2H),4.10(s,2H),3.92–3.8 4(m,1H),3.76–3.67(m,2H),2.97–2.86(m,2H),2.81(d,J=21.5Hz,4H),2.6 3–2.53(m,6H),2.40–2.33(m,2H),2.05–1.94(m,4H),1.77–1.64(m,4H),1 .60–1.48(m,2H),1.47–1.34(m,6H),0.87–0.83(m,6H),0.83–0.77(m,6H).

[0549] Step 2:

[0550] Compound DL-2-1 (50 mg, 0.04 mmol) was dissolved in DMF (2 mL), and diethylamine (15 mg, 0.20 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction mixture was concentrated under reduced pressure to obtain the target compound DL-2-2 (50 mg), which was used directly in the next step.

[0551] LCMS (ESI) [M+H] + =953.5.

[0552] Step 3:

[0553] Compound DL-2-2 (50 mg, 0.04 mmol) and compound INT1-8 (14 mg, 0.04 mmol) were dissolved in DMF (3 mL). N,N-diisopropylethylamine (15 mg, 0.12 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (15 mg, 0.04 mmol) were added. The reaction mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction mixture was directly purified by preparative HPLC (CH3CN:H2O containing 0.05% FA = 5%-50%) to obtain the formate salt of the target compound DL-2 (10 mg).

[0554] LCMS (ESI) [M+H] + =1203.6.

[0555] 1 H NMR (400MHz, DMSO-d6) δ10.97 (s, 1H), 9.11 (s, 2H), 8.80 (d, J = 5.4Hz, 1H), 8. 61(t,J=6.5Hz,1H),8.20–8.15(m,2H),8.05–8.03(m,1H),7.93(d,J=8.3Hz,1 H),7.69(d,J=7.8Hz,1H),7.65(s,1H),7.51(s,1H),7.44(d,J=7.9Hz,1H),7. 21–7.16(m,2H),6.86–6.80(m,1H),5.10(dd,J=13.4,5.0Hz,1H),4.61–4.56( m,2H),4.47–4.29(m,4H),4.22–4.14(m,2H),4.10(s,2H),3.72–3.69(m,2H), 3.41(s,3H),2.95–2.88(m,2H),2.86(s,2H),2.79(s,1H),2.63–2.54(m,12H) ,2.41–2.31(m,4H),2.02–1.97(m,2H),1.84–1.78(m,2H),1.70–1.66(m,2H), 1.59–1.54(m,1H),1.47–1.42(m,6H),1.31–1.26(m,2H),0.86–0.82(m,12H).

[0556] Example 3.3: Synthesis of N-((14S,17S)-1-(2-chloro-4-(2-(((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)amino)-1,1-difluoro-2-oxoethyl)phenyl)-14-(4-(dipropylamino)butyl)-4,18-dimethyl-5,10,13,16-tetraoxo-7-oxa-4,9,12,15-tetraazanonadecan-17-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (DL-3)

[0557] Step 1:

[0558] The hydrochloride salt of compound INT3 (165 mg, 0.31 mmol) was dissolved in DMF (8 mL), and compound INT1-6 (216 mg, 0.31 mmol), N,N-diisopropylethylamine (80 mg, 0.62 mmol), and 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (129 mg, 0.47 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. Water (45 mL) was added to the reaction mixture, and the mixture was extracted with ethyl acetate (25 mL*3). The combined organic phases were washed with saturated brine (30 mL*3), dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative thin-layer chromatography (DCM:MeOH = 10:1) to obtain the target compound DL-3-1 (150 mg).

[0559] LCMS (ESI) [M+H] + =1210.2.

[0560] 1H NMR(400MHz, DMSO-d6)δ10.98(s,1H),9.68(t,J=6.0Hz,1H),8.70–8.58(m,1H),8.29–8.19( m,1H),8.02(t,J=9.1Hz,1H),7.88(d,J=7.6Hz,2H),7.75–7.70(m,2H),7.67(d,J=7.9Hz,1H) ,7.58(s,1H),7.56–7.52(m,1H),7.51–7.44(m,2H),7.44–7.37(m,4H),7.31(t,J=7.0Hz,2H) ,5.33(t,J=4.6Hz,1H),5.13–5.06(m,1H),4.61–4.55(m,2H),4.45(d,J=5.9Hz,2H),4.30(s, 1H),4.23–4.20(m,2H),4.11–4.09(m,1H),3.87(t,J=8.2Hz,1H),3.77–3.65(m,2H),2.94–2. 89(m,1H),2.87–2.79(m,3H),2.69–2.66(m,2H),2.64–2.60(m,1H),2.59–2.56(m,1H),2.43– 2.35(m,2H),2.34–2.32(m,1H),2.28–2.22(m,2H),2.05–1.93(m,6H),1.78–1.71(m,2H),1.6 8–1.61(m,2H),1.56–1.52(m,1H),1.47–1.44(m,1H),1.37–1.26(m,8H),0.86–0.78(m,12H).

[0561] Step 2:

[0562] Dissolve compound DL-3-1 (100 mg, 0.08 mmol) in DMF (2 mL) and add diethylamine (2 mg, 0.24 mmol). Stir the reaction mixture at room temperature for 2 h. Monitor the reaction by LCMS. The reaction mixture was concentrated under reduced pressure to obtain the target compound DL-3-2 (70 mg), which was used directly in the next step.

[0563] LCMS (ESI) [M+H] + =988.3.

[0564] Step 3:

[0565] Compound DL-3-2 (70 mg, 0.06 mmol) and compound INT1-8 (15 mg, 0.06 mmol) were dissolved in DMF (2 mL). N,N-diisopropylethylamine (23 mg, 0.18 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (23 mg, 0.06 mmol) were added. The reaction mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction mixture was purified by preparative HPLC (CH3CN:H2O containing 0.05% FA = 5%-50%) to obtain the formate salt of the target compound DL-3 (15 mg).

[0566] LCMS (ESI) [M+H] + =1238.4.

[0567] 1 H NMR (400MHz, DMSO-d6) δ10.98(s,1H),9.67(t,J=6.4Hz,1H),9.11(s,2H),8.59(t,J=6. 0Hz,1H),8.23(s,1H),8.17(t,J=5.8Hz,1H),8.02(t,J=7.2Hz,1H),7.94(d,J=8.5Hz,1 H),7.68(d,J=7.8Hz,1H),7.60–7.57(m,1H),7.57–7.53(m,1H),7.52–7.47(m,1H),7.4 2(d,J=5.9Hz,1H),7.37(d,J=7.9Hz,1H),5.16–5.04(m,1H),4.62–4.55(m,2H),4.47–4 .39(m,3H),4.29–4.25(m,1H),4.20–4.14(m,2H),4.12(d,J=5.5Hz,2H),3.75–3.66(m, 2H),3.41(s,3H),2.96–2.89(m,1H),2.87(s,3H),2.72–2.69(m,1H),2.68–2.66(m,1H) ,2.64–2.58(m,1H),2.57–2.52(m,4H),2.42–2.36(m,2H),2.35–2.31(m,2H),2.31–2.2 5(m,6H),2.04–1.96(m,3H),1.84–1.72(m,4H),1.40–1.28(m,8H),0.85–0.78(m,12H).

[0568] Example 3.4: Synthesis of N-((13S,16S)-1-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)ureido)phenoxy)-13-(4-(dipropylamino)butyl)-3,17-dimethyl-4,9,12,15-tetraoxo-6-oxa-3,8,11,14-tetraazanonadecan-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (DL-4)

[0569] Step 1:

[0570] Compound INT6 (150 mg, 0.32 mmol) was dissolved in DMF (2 mL), and compound INT1-6 (192 mg, 0.32 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (123 mg, 0.32 mmol) and N,N-diisopropylethylamine (104 mg, 0.80 mmol) were added in sequence. After the addition, the reaction solution was stirred at room temperature for 1 h; the reaction was monitored by LCMS; methyl tert-butyl ether (80 mL) was slowly added to the reaction solution for dilution, and the supernatant was poured out after standing for 10 min. The residue was directly concentrated under reduced pressure to remove excess solvent. The crude product was purified by preparative TLC (dichloromethane: methanol = 8:1) to obtain the target compound DL-4-1 (250 mg).

[0571] LCMS (ESI) [M+H] + =1141.7.

[0572] 1H NMR (400MHz, DMSO-d6) δ10.98(s,1H),8.65(t,J=6.2Hz,1H),8.54(d,J=5.3Hz,1 H),8.27-8.19(m,1H),8.08-8.01(m,1H),7.89(d,J=7.6Hz,2H),7.75-7.66(m,3 H),7.51(s,1H),7.46-7.39(m,4H),7.35-7.29(m,4H),7.07(t,J=9.1Hz,2H),6. 74-6.67(m,1H),5.10(dd,J=13.3,5.0Hz,1H),4.63-4.53(m,2H),4.41(d,J=6.2H z,2H),4.35-4.20(m,5H),4.11-4.05(m,2H),3.88(t,J=8.2Hz,1H),3.73(d,J=2 .5Hz,2H),3.31-3.24(m,4H),3.21-3.15(m,2H),2.88-2.85(m,1H),2.80-2.77( m,3H),2.64-2.56(m,2H),2.48-2.42(m,2H),2.40-2.33(m,1H),2.04-1.93(m,3 H),1.82-1.60(m,4H),1.56-1.42(m,7H),1.33-1.27(m,2H),0.88-0.83(m,12H).

[0573] Step 2:

[0574] Compound DL-4-1 (100 mg, 0.08 mmol) was dissolved in DMF (2 mL), and diethylamine (25.6 mg, 0.32 mmol) was added to the reaction solution. The reaction solution was stirred at room temperature for 1 h; the reaction was monitored by LCMS; the reaction solution was concentrated under reduced pressure to obtain the target compound DL-4-2 (55 mg), which was directly used in the next reaction.

[0575] LCMS (ESI) [M+H] + =919.6.

[0576] Step 3:

[0577] Compound DL-4-2 (55 mg, 0.06 mmol), compound INT1-8 (18 mg, 0.06 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (25 mg, 0.06 mmol), and N,N-diisopropylethylamine (21 mg, 0.15 mmol) were added to DMF (2 mL), and the reaction solution was stirred at room temperature for 1 h; the reaction was monitored by LCMS; the reaction solution was directly purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.05% FA = 5%-50%) to obtain the formate salt of the target compound DL-4 (15 mg).

[0578] LCMS (ESI) [M+H] + =1169.7.

[0579] 1 H NMR(400MHz,DMSO-d6)δ10.97(s,1H),9.11(s,2H),8.67-8.57(m,2H),8.23-8.17(m, 2H),8.09-8.03(m,1H),7.95(d,J=8.5Hz,1H),7.69(d,J=7.8Hz,1H),7.51(s,1H),7. 44(d,J=7.9Hz,1H),7.35-7.29(m,2H),7.07(t,J=8.6Hz,2H),6.85-6.78(m,1H),5.1 0(dd,J=13.2,5.0Hz,1H),4.62-4.54(m,2H),4.44-4.28(m,4H),4.23-4.14(m,2H),4. 10-4.07(m,2H),3.74-3.69(m,4H),3.41(s,3H),3.30-3.23(m,2H),3.20-3.14(m,1H ),2.96-2.89(m,1H),2.81-2.73(m,3H),2.60-2.57(m,1H),2.56-2.52(m,6H),2.48- 2.43(m,2H),2.38-2.32(m,2H),2.02-1.95(m,2H),1.86-1.75(m,3H),1.72-1.66(m, 2H),1.60-1.52(m,1H),1.48-1.40(m,6H),1.33-1.25(m,2H),0.84(t,J=6.8Hz,12H).

[0580] Example 3.5: Synthesis of (2S)-N-(2-(((2-((3-(2-chloro-4-(3-((2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)methyl)ureido)phenyl)propyl)(methyl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)-2-((S)-2-(2,2-dimethyl-4-(4)-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)butanamido)-3-methylbutanamido)-6-(dimethylamino)hexanamide (DL-5)

[0581] Step 1:

[0582] Compound INT2 (100 mg, 0.20 mmol) was dissolved in DMF (2 mL), and then compound INT5 (128 mg, 0.20 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (76 mg, 0.20 mmol) and N,N-diisopropylethylamine (26 mg, 0.50 mmol) were added in sequence. After the addition, the reaction solution was stirred at room temperature for 1 h; the reaction was monitored by LCMS; methyl tert-butyl ether (80 mL) was added to the reaction solution, and the supernatant was poured out after standing for 10 min. The residue was directly concentrated under reduced pressure on an oil pump to remove excess solvent. The crude product was directly purified by preparative TLC (DCM:MeOH=8:1) to obtain the target compound DL-5-1 (150 mg).

[0583] LCMS (ESI) [M+H] + =1119.5.

[0584] 1H NMR (400MHz, DMSO-d6) δ10.97 (s, 1H), 8.85 (t, J = 8.3Hz, 1H), 8.67-8.61 (m, 1H), 8.38-8.21 ( m,2H),8.19-8.10(m,1H),8.07-7.98(m,1H),7.89(d,J=7.5Hz,2H),7.84(d,J=7.5Hz,1H),7 .75-7.70(m,1H),7.70-7.64(m,2H),7.51(s,1H),7.44(d,J=7.9Hz,1H),7.42-7.40(m,1H), 7.36(d,J=7.5Hz,1H),7.33-7.29(m,1H),7.20-7.16(m,2H),6.92-6.83(m,1H),5.10(dd,J= 13.6,5.2Hz,1H),4.61-4.56(m,2H),4.41(d,J=5.8Hz,2H),4.35-4.28(m,2H),4.24-4.20(m ,1H),4.10(s,2H),3.92-3.84(m,1H),3.75-3.68(m,3H),3.63-3.59(m,2H),2.94-2.90(m,1 H),2.86(s,2H),2.79(s,1H),2.59-2.54(m,2H),2.41-2.37(m,2H),2.29-2.26(m,6H),2.02 -1.97(m,2H),1.71-1.66(m,2H),1.45-1.41(m,2H),1.31-1.28(m,2H),0.91-0.78(m,12H).

[0585] Step 2:

[0586] Compound DL-5-1 (100 mg, 0.09 mmol) was dissolved in DMF (2 mL), and diethylamine (26 mg, 0.36 mmol) was added to the reaction solution. The reaction solution was stirred at room temperature for 1 h; the reaction was monitored by LCMS; the reaction solution was directly placed under an oil pump and concentrated under reduced pressure to obtain the target compound DL-5-2 (80 mg), which was directly used in the next reaction.

[0587] LCMS (ESI) [M+H] + =897.5;

[0588] Step 3:

[0589] Compound DL-5-2 (80 mg, 0.09 mmol) and compound INT7 (23 mg, 0.09 mmol) were dissolved in DMF (3 mL), and N,N-diisopropylethylamine (21 mg, 0.22 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (25 mg, 0.09 mmol) were added. The reaction mixture was stirred at room temperature for 1 h; the reaction was monitored by LCMS. The reaction mixture was directly purified by preparative HPLC (acetonitrile:H2O containing 0.05% FA = 5%-50%) to obtain the formate salt of the target compound DL-5 (13 mg).

[0590] LCMS (ESI) [M+H] + =1218.8.

[0591] 1 H NMR(400MHz,DMSO-d6)δ10.98(s,1H),9.47(s,2H),8.93(s,1H),8.85(d,J=7.0Hz,1H),8 .64(t,J=6.4Hz,1H),8.27(t,J=5.3Hz,1H),8.20(s,1H),8.06(d,J=7.0Hz,1H),7.69(d,J =7.8Hz,1H),7.65(s,1H),7.51(s,1H),7.43(t,J=6.7Hz,2H),7.23-7.13(m,2H),6.93-6 .84(m,1H),5.10(dd,J=13.2,5.0Hz,1H),4.62-4.52(m,2H),4.44-4.39(m,4H),4.31(d,J =17.5Hz,1H),4.28-4.22(m,1H),4.14(t,J=7.9Hz,1H),4.09(s,2H),3.74-3.69(m,2H), 3.44(s,3H),2.96-2.89(m,1H),2.82(d,J=29.0Hz,3H),2.64-2.53(m,4H),2.43-2.36(m, 2H),2.35(s,6H),2.21-2.10(m,2H),2.05-1.96(m,2H),1.80-1.72(m,1H),1.71-1.63(m ,2H),1.62-1.38(m,4H),1.30-1.23(m,4H),1.22(d,J=3.2Hz,6H),0.87(t,J=7.1Hz,6H).

[0592] Example 4. Preparation and identification of antibodies

[0593] 4.1.1 Preparation of B7H3 antigen and anti-B7H3 antibody

[0594] The nucleic acid sequence of amino acids 29-245 of human 2Ig B7H3 (NCBI protein number NP_001316557.1) was selected, and a 6×His tag was added to the C-terminus for detection, and the result was named human B7H3-2IgG-his; the nucleic acid sequence of amino acids 27-461 of human 4Ig B7H3 (Uniprot protein number Q5ZPR3-1) was selected, and a 6×His tag was added to the C-terminus for purification, and the result was named human B7H3-4IgG-his; the nucleic acid sequence of amino acids 27-465 of monkey B7H3 4Ig (Uniprot protein number F7G5V3) was selected, and a 6×His tag was added to the C-terminus for purification, and the result was named monkey B7H3-4IgG-his; B7H3-C1 was selected based on CN The antibody numbered M30-H1-L4 in 103687945B; B7H3-C2 refers to the antibody numbered mAb-C-DUBA in CN109069633A.

[0595] Genes for the three B7H3 antigens, as well as codon-optimized genes for the B7H3-C1 and B7H3-C2 antibodies (Shanghai Sangon Biotechnology Co., Ltd.), were synthesized and incorporated into the PTT5 expression vector. The plasmids were then extracted in large quantities. The extracted expression vectors were transiently expressed in HEK293F cells for 7 days. The expression supernatants were then protein-A-conjugated to produce the antigenic proteins and the B7H3-C1 and B7H3-C2 antibodies.

[0596] The VH amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 5 were codon-optimized and gene-synthesized (Shanghai Sangon Biotechnology Co., Ltd.) with the constant region amino acid sequence of IgG1 (SEQ ID NO: 9), and then constructed into the PTT5 vector, named PTT5-01-CH and PPT5-02-CH, respectively. The VL amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 6 were codon-optimized and gene-synthesized (Shanghai Sangon Biotechnology Co., Ltd.), and then constructed into the PTT5 vector, named PTT5-01-CL and PTT5-02-CL, respectively. The antibody B7H3 expression plasmids PTT5-01-CH / PTT5-01-CL and PTT5-02-CH / PTT5-02-CL were co-transfected into HEK293F cells for expression using max reagent. The expression was carried out at 37°C with 5% CO2 shaking for 7 days. The supernatant was collected and purified using ProA magnetic beads to obtain anti-B7H3 antibodies, which were named 1D1-01 (whose heavy chain sequence is SEQ ID NO: 3, and the light chain sequence is SEQ ID NO: 4) and 2E3-02 (whose heavy chain sequence is SEQ ID NO: 7, and the light chain sequence is SEQ ID NO: 8).

[0597] 4.1.2 Anti-B7H3 Antibody Protein Affinity Detection

[0598] The affinity of the fully human B7H3 antibody to human B7H3-4IgG-his and monkey B7H3-4IgG-his proteins was detected by ELISA. Specific experimental procedures: Human B7H3-4IgG-his or monkey B7H3-4IgG-his protein was diluted with carbonate buffer (pH 9.6) to a final concentration of 1 μg / mL and added to 96-well enzyme-labeled wells at 100 μL / well and coated at 4°C overnight; the coating solution was discarded, the wells were washed once with PBST, 100 μL PBS (containing 2% BSA) was added to each well, and blocked at 37°C for 1 hour; then 100 μL PBS (containing 2% BSA) diluted B7H3 fully human antibody (10 μg starting, 3-fold dilution, 2 replicates) was added to each well, incubated at 37°C for 2 hours; washed three times with PBST, and horseradish peroxidase-labeled anti-human Fc antibody (diluted 1:10,000 with PBS) was added at 100 μL / well and incubated at 37°C for 1 hour; the plate was washed five times with PBST. TMB colorimetric solution was added for color development (100 μL / well) at 37°C for 7 min. Stop reaction was terminated by adding stop solution (50 μL / well) and measuring absorbance at 450 nm. EC50 values ​​were calculated based on the raw data.

[0599] The results are shown in Figures 1A, 1B, 2A and 2B, and the detailed results are shown in Tables 1-1 and 1-2. 1D1-01 and 2E3-02 both have high affinities for human B7H3-4 IgG, which are 43.56 pM and 17.54 pM, respectively. Among them, 2E3-02 has a stronger affinity than B7H3-C1, which is 51.85 pM. 1D1-01 and 2E3-02 have high binding abilities for monkey B7H3-4 IgG, which are 35.82 pM and 27.72 pM, respectively.

[0600] Table 1-1.1D1-01 antibody protein affinity test results

[0601] Table 1-2. 2E3-02 Antibody Protein Affinity Test Results

[0602] 4.1.3 Anti-B7H3 Antibody Cell Affinity Detection

[0603] B7H3 protein is highly expressed on a variety of solid tumor cells. The affinity of B7H3 fully human antibodies to tumor cells was detected by FACS. Specific experimental procedures: MCF-7 breast cancer cells (from Nanjing Kebai, catalog number CBP60380), A549 human non-small cell lung cancer cells (from ATCC, catalog number CRM-CCL-185), and PC3 human prostate cancer cells (from ATCC, catalog number CRL-1435) grown to the logarithmic phase were digested with trypsin and resuspended in PBS buffer containing 2% BSA to 5*10^ 5 For every milliliter of cells, 100 μL of cell suspension was added to a 96-well plate; B7H3-C1, B7H3-C2, 1D1-01, and 2E3-02 were diluted 3-fold in PBS buffer containing 2% BSA to a starting concentration of 40 μg / mL, and then 100 μL of antibody suspension was added to the 96-well plate and mixed thoroughly; the plate was incubated at 4°C for 1 hour, washed twice with pre-cooled PBS, 300 μL / well each time, and centrifuged at 500 g for 5 minutes; 100 μL / well of 1:50 fluorescent secondary antibody APC anti-human IgG or PE anti-human IgG (purchased from Biolegend) was added and mixed thoroughly; the plate was incubated at 4°C for 0.5 hour, washed twice with pre-cooled PBS, 300 μL / well each time, and centrifuged at 500 g for 5 minutes. The plate was re-selected with 500 μL PBS, and the average fluorescence signal value was detected using a Beckman flow cytometer. The EC50 value was calculated based on the average fluorescence signal value. The results are shown in Figures 3A, 3B, 4A, 4B and Figure 5. The specific data are shown in Tables 2-1 and 2-2.

[0604] Table 2-1. 1D1-01 Antibody Cell Affinity Test Results

[0605] Table 2-2. 2E3-02 Antibody Cell Affinity Test Results

[0606] As shown in Tables 2-1 and 2-2, on MCF-7 tumor cells with high B7H3 expression, the affinities of 1D1-01 and 2E3-02 were 0.524 nM and 0.398 nM, respectively, both significantly higher than B7H3-C1. The maximum fluorescence signal values ​​were 12% and 23% higher than those of B7H3-C1, respectively. The maximum fluorescence signal value of 2E3-02 was 2.78-fold higher than that of control antibody 2. On A549 tumor cells with moderate B7H3 expression, the affinities of 1D1-01 and 2E3-02 were 0.322 nM and 0.150 nM, respectively, both significantly higher than those of B7H3-C1. The maximum fluorescence signal values ​​were 36% and 77% higher than those of B7H3-C1, respectively. On PC-3 cells with low B7H3 expression, 2E3-02's affinity of 0.06 nM was significantly higher than that of B7H3-C1 (0.618 nM), which also exceeded the affinity of B7H3-C1 (0.167 nM). The maximum fluorescence signal values ​​were 1.51 and 4.48 times those of B7H3-C1 and B7H3-C2, respectively. Overall, the anti-B7H3 antibodies 1D1-01 and 2E3-02 exhibited superior tumor cell affinity compared to both B7H3-C1 and B7H3-C1.

[0607] 4.1.4 Anti-B7H3 Antibody Endocytosis Activity Detection

[0608] The ability of A549 human non-small cell lung cancer cells to internalize anti-B7H3 antibodies was detected by FACS. A549 cells grown to the logarithmic growth phase were trypsinized, collected by centrifugation, and washed three times with pre-cold PBS. The cells were resuspended in PBS (containing 1% BSA), and the anti-B7H3 antibodies to be tested were added at a concentration of 10 μg / mL, and incubated at 4°C for 1 hour. The cells were collected by centrifugation, washed three times with PBS, and resuspended in DMEM + 10% FBS. The resuspended cells were divided into four parts and incubated at 37°C for 0, 1, 2, and 4 hours, respectively. After the incubation phase, the cells were collected by centrifugation, washed three times with pre-cold PBS, and resuspended in 50 μL 1% BSA (in PBS). 1 μL of fluorescent secondary antibody APC anti-human IgG (Biolegend) was added to each well, mixed, and incubated at 4°C for 0.5 hour. The cells were collected by centrifugation, washed three times with pre-cold PBS, and resuspended in 200 μL PBS for detection. According to the formula: endocytosis rate (%) = [1-(average fluorescence value of the test sample at that time point-average fluorescence value of the negative control sample at that time point) / (average fluorescence value of the test sample at 0 hour-average fluorescence value of the negative control sample at 0 hour)] * 100. The results of 1D1-01 and 2E3-02 are shown in Figures 6A and 6B, respectively. The anti-B7H3 antibodies 1D1-01 and 2E3-02 have strong endocytosis activity on A549 tumor cells, reaching about 40% in 4 hours, which is comparable to the antibody B7H3-C1.

[0609] Example 5. Synthesis of Antibody-Drug Conjugates (ADCs)

[0610] Example 5.1 Preparation of B7H3-ADC

[0611] Example 5.1.1 Preparation of B7H3-ADC-001

[0612] 25 ml of 2E3-02 antibody (anti-B7H3, concentration 28.5 mg / ml, 20 mM acetate buffer) was diluted with 25 ml of 20 mM acetate buffer, followed by the addition of 1 ml of an aqueous solution containing 0.25 M EDTA and mixing. The sample pH was adjusted to 7.6 with 0.5 M sodium hydrogen phosphate solution, followed by the addition of a 20 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride) solution (4.5 equivalents of the antibody) and mixing. The reaction was allowed to proceed at room temperature for 90 min. Finally, 10 equivalents of compound DL-1 dissolved in DMSO were added, mixed, and the reaction was continued at room temperature for 2 h. After the reaction, the sample was purged into a 10 mM histidine buffer at pH 5.5 using a 30 kDa ultrafiltration tube to remove low-molecular-weight substances. The sample was then concentrated to obtain a solution containing the anti-B7H3 antibody ADC, B7H3-ADC-001. The DAR value was determined by mass spectrometry to be 8.0.

[0613] Example 5.2 Preparation of HER2-ADC

[0614] Example 5.2.1: Preparation of HER2-ADC-001

[0615] 0.8 mL of trastuzumab (20.4 mg / mL) was diluted with 0.008 mL of 20 mM PB + 100 mM edetate disodium solution (pH 7.6). The pH was adjusted to 7.81 with 0.5 M Na2HPO4 solution. 20 mM TCEP (tris(2-carboxyethyl)phosphine, 0.0303 mL, 0.606 μmol) solution was added, mixed, and allowed to stand at room temperature for 90 minutes. A solution of DL-2 (1.654 mg) in dimethyl sulfoxide (0.1323 mL) was added to the above solution, mixed, and allowed to stand at room temperature for 2 hours. After completion, the solution was exchanged using a centrifugal ultrafiltration tube (Merck, Amicon Ultra-15). This yielded the DL-2-trastuzumab conjugate, HER2-ADC-001. The DAR value, determined by mass spectrometry, was 8.0.

[0616] In the table, LC represents the antibody light chain; HC represents the antibody heavy chain; DAR1 represents a conjugate comprising a light chain or heavy chain coupled to one drug-linker; DAR2 represents a conjugate comprising a light chain or heavy chain coupled to two drug-linkers; and DAR3 represents a conjugate comprising a light chain or heavy chain coupled to three drug-linkers. LC, HC, DAR1, DAR2, and DAR3 are as described above.

[0617] The trastuzumab light chain was conjugated to one toxin molecule (LC+DAR1 ratio was 100%) and the heavy chain was conjugated to three toxin molecules (HC+DAR3 ratio was 100%). Based on this, the antibody-drug conjugation ratio (DAR value) of HER2-ADC-001 (DAR8) was calculated to be 8.0.

[0618] Antibody-drug coupling ratio (DAR value) determination method:

[0619] Take 50 μg of ADC sample, dilute it to 0.5 mg / ml with ultrapure water, then add 1 μl of 1M DTT, mix well, centrifuge and take the supernatant for injection.

[0620] Liquid phase parameters:

[0621] Mass spectrometry parameters:

[0622] Example 5.2.2: Preparation of HER2-ADC-002

[0623] The same method as in Example 5.2.1 was used, replacing DL-2 with DL-3, to obtain the trastuzumab-conjugated product HER2-ADC-002. The DAR value determined by mass spectrometry was 8.0.

[0624] Example 5.2.3: Preparation of HER2-ADC-003

[0625] The same method as in Example 5.2.1 was used, replacing DL-2 with DL-1, to obtain the trastuzumab conjugate HER2-ADC-003. The DAR value determined by mass spectrometry was 8.0.

[0626] Example 5.2.4: Preparation of HER2-ADC-004

[0627] The same method as in Example 5.2.1 was used, replacing DL-2 with DL-5, to obtain the trastuzumab conjugate HER2-ADC-004. The DAR value determined by mass spectrometry was 8.0.

[0628] Example 5.2.5: Preparation of HER2-ADC-005

[0629] The same method as in Example 5.2.1 was used, replacing DL-2 with DL-4, to obtain the trastuzumab-conjugated product HER2-ADC-005. The DAR value determined by mass spectrometry was 7.0.

[0630] Example 5.2.6: Preparation of HER2-ADC-006

[0631] DL-6 (preparation method, see patent document WO2021198965)

[0632] The same method as in Example 5.2.1 was used, replacing DL-2 with DL-6, to obtain the trastuzumab-conjugated product HER2-ADC-006. The DAR value determined by mass spectrometry was 9.0.

[0633] Example 6. In vitro cell activity test of biologically active compounds (Payload)

[0634] The structure of reference compound 1 is as follows:

[0635] Reference compound 1 (preparation method refer to patent document WO2021198965)

[0636] Example 6.1 Test of the inhibitory activity of bioactive compounds (Payload) on N87, MCF7, and NUGC4 cell proliferation

[0637] 1. Test method:

[0638] Resuscitated N87 (1640 + 10% FBS), MCF7 (DMEM + 10% FBS + 10 μg / ml of insulin), and NUGC4 (1640 + 10% FBS) cells were plated in 96-well flat-bottom plates at a density of 5000 / well and 100 μl / well, and cultured in a 37 ° C incubator overnight. Dissolve the payload to be tested with DMSO, aliquot and freeze at -80 ° C. The next day, dilute the payload to be tested with each complete culture medium to a maximum concentration of 10000 nM, and dilute 4 times for 10 points. Add 100 μl of diluted drug to the cell culture wells (2 parallels per well). After culturing at 37 ° C for 4 days, add 50 μl of Cell titer glo substrate to each well and detect the fluorescence value after incubation for 5 minutes. Among them, the sources of key materials are shown in Table 1.

[0639] Table 1 Sources of key materials

[0640] 2. Experimental Conclusions

[0641] The test results show that the disclosed payloads have significant proliferation inhibitory activity on N87 cells, MCF7 cells, and NUGC4 cells, among which PL-1 and PL-4 have significantly better proliferation inhibitory activity than the control compound 1. The test results are shown in Table 2.

[0642] Table 2 Test results of the proliferation inhibition activity of Payload on N87, MCF7 and NUGC4 cells

[0643] Example 7. In vitro cell activity test of antibody-drug conjugates (ADCs)

[0644] Example 7.1 ADC Inhibitory Activity Test on Cell (NCI-H358 Tumor Cells) in Vitro

[0645] NCI-H358 tumor cells were digested using trypsin by conventional methods, the cell count was collected, the cells were resuspended with the corresponding detection medium (containing 2% FBS), and 5000 cells / well were added to a 96-well plate. 100 μL of ADC diluted with 2% FBS medium was added to a 96-well plate, starting at a concentration of 150 μg / ml and diluted 3 times (12 concentration gradients). Cultured at 37°C 5% CO2 for 7 days, 20 μL of CCK8 reagent was then added to each well, reacted for 4 hours, and read on a microplate reader (detection wavelength was 450 nm). The test results showed that the ADC molecules disclosed herein have tumor cell killing effects.

[0646] Example 7.2: In vitro cell (N87 tumor cell) proliferation inhibitory activity test of ADC

[0647] 1. Test method:

[0648] N87 cells were revived and cultured with 1640+10% FBS, plated in a 96-well flat-bottom plate at a density of 5000 / well and 100μl / well, and cultured in a 37°C incubator overnight. The next day, 1640+10% FBS was used to dilute the ADC molecules to be tested. The highest concentration of the ADC molecule to be tested -002 was 200nM, and the highest concentration of the other four samples was 2000nM, with 10 points diluted 4 times. Add 100μl of diluted drug to the cell culture wells. After culturing at 37°C for 4 days, 50μl of Cell titer glo substrate was added to each well, and the fluorescence value was detected after incubation for 5 minutes. The sources of key materials are shown in Table 3

[0649] Table 3 Sources of key materials

[0650] 2. Experimental Conclusions

[0651] The test results show that the ADC molecules disclosed herein have significant proliferation inhibitory activity on the N87 tumor cell line. The test results are shown in Table 4.

[0652] Table 4 ADC inhibition activity test results on N87 tumor cells

[0653] Example 8. In vivo activity test of antibody-drug conjugates (ADCs)

[0654] Example 8.1 Antibody-drug conjugate (ADC) efficacy test on NCI-N87 xenograft tumors (dose 1 mg / kg)

[0655] 1. Experimental Materials

[0656] Test compounds: HER2-ADC-001, HER2-ADC-003, HER2-ADC-006, and normal saline as a negative control.

[0657] Experimental cells: NCI-N87 cells were purchased from Nanjing Kebai Biological.

[0658] Experimental animals: Balb / c nude mice, female, 5-6 weeks old, were purchased from Weitonglihua Laboratory Animal Co., Ltd.

[0659] 2. Experimental Plan

[0660] Cell treatment

[0661] NCI-N87 cells were cultured in 15 cm diameter culture dishes using 1640 medium containing 10% FBS. When the confluence reached about 80-90%, they were digested with trypsin-EDTA, washed twice with PBS, and then centrifuged and resuspended in pre-cooled PBS. The cells were counted using a cell counter and diluted with PBS to a cell concentration of 5 × 10 7 / ml.

[0662] 2.2. Tumor cell transplantation

[0663] Balb / c nu mice were acclimated to the laboratory environment for 2-5 days and then inoculated subcutaneously with 5×10 NCI-N87 cells in the right rib cage. 6 / mouse, the inoculation volume was 0.2 ml (containing 50% Matrigel), and the tumor was grown to 200 mm 3 When about , conduct the experiment.

[0664] 2.3. Animal Dosing and Testing

[0665] The tumor-bearing nude mice were dosed according to the following regimen:

[0666] Table 5

[0667] 2.4. Tumor volume and body weight determination:

[0668] Tumor volume and body weight were measured twice a week, and T / C% and TGI (%) were calculated using the following formulas: relative tumor proliferation rate (T / C%) = TRTV / CRTV × 100% (TRTV: mean RTV of the treatment group; CRTV: mean RTV of the control group; RTV = Vt / V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment); relative tumor inhibition rate (TGI%) = (1-T / C) × 100% (T and C are the relative tumor volumes (RTV) of the treatment and control groups, respectively, at a specific time point).

[0669] The formula for calculating tumor volume (V) is: V = 1 / 2 × L length × L short 2 , where Llong and Lshort represent the long diameter and short diameter of the tumor, respectively.

[0670] 3. Experimental Results

[0671] Test results showed that the ADCs disclosed herein all exhibited significant tumor-suppressing effects, with HER2-ADC-001 and HER2-ADC-003 demonstrating significantly better tumor suppression than the control compound, HER2-ADC-006. During dosing, no significant weight loss or drug toxicity was observed in any of the animal groups. Detailed results are shown in Table 6 and Figures 7A and 7B.

[0672] Table 6 NCI-N87 transplant tumor model data

[0673] Example 8.2 Antibody-drug conjugate (ADC) efficacy test on NCI-N87 xenograft tumors (dosages 2 mg / kg and 6 mg / kg)

[0674] 1. Experimental Materials

[0675] Test compounds: HER2-ADC-001, HER2-ADC-003, and normal saline as a negative control.

[0676] Experimental cells: NCI-N87 cells were purchased from Nanjing Kebai Biological.

[0677] Experimental animals: Balb / c nude mice, female, 5-6 weeks old, were purchased from Weitonglihua Laboratory Animal Co., Ltd.

[0678] 2. Experimental Plan

[0679] Cell treatment

[0680] NCI-N87 cells were cultured in 15 cm diameter culture dishes using 1640 medium containing 10% FBS. When the confluence reached about 80-90%, they were digested with trypsin-EDTA, washed twice with PBS, and then centrifuged and resuspended in pre-cooled PBS. The cells were counted using a cell counter and diluted with PBS to a cell concentration of 5 × 10 7 / ml.

[0681] 2.2. Tumor cell transplantation

[0682] Balb / c nu mice were acclimated to the laboratory environment for 2-5 days and then inoculated subcutaneously with 5×10 NCI-N87 cells in the right rib cage. 6 / mouse, the inoculation volume was 0.2 ml (containing 50% Matrigel), and the tumor was grown to 165 mm 3 When about , conduct the experiment.

[0683] 2.3. Animal Dosing and Testing

[0684] The tumor-bearing nude mice were dosed according to the following regimen:

[0685] Table 7

[0686] 2.4. Tumor volume and body weight determination

[0687] Tumor volume and body weight were measured twice a week, and T / C% and TGI (%) were calculated using the following formulas: relative tumor proliferation rate (T / C%) = TRTV / CRTV × 100% (TRTV: mean RTV of the treatment group; CRTV: mean RTV of the control group; RTV = Vt / V0, where V0 is the tumor volume of the animal at the time of grouping and Vt is the tumor volume of the animal after treatment); relative tumor inhibition rate (TGI%) = (1-T / C) × 100% (T and C are the relative tumor volumes (RTV) of the treatment and control groups, respectively, at a specific time point).

[0688] The formula for calculating tumor volume (V) is: V = 1 / 2 × L length × L short 2 , where Llong and Lshort represent the long diameter and short diameter of the tumor, respectively.

[0689] 3. Experimental Results

[0690] Test results showed that all of the ADCs disclosed herein exhibited significant tumor inhibition effects. HER2-ADC-001 exhibited tumor inhibition rates of 62.47% at 2 mg / kg and 98.30% at 6 mg / kg, while HER2-ADC-003 exhibited tumor inhibition rates of 44.08% at 2 mg / kg and 94.15% at 6 mg / kg. During dosing, no significant weight loss or drug toxicity was observed in any of the animal groups. Detailed results are shown in Table 8 and Figures 8A and 8B.

[0691] Table 8 NCI-N87 transplant tumor model data

[0692] Example 9 ADC plasma stability test

[0693] Solution preparation: Each ADC prepared in Example 5 was diluted with PBS to a concentration of 2 mg / mL.

[0694] Sample incubation: Add the antibacterial agent ProClin to human plasma at a final concentration of 0.1% under aseptic conditions. Each ADC prepared in Example 5 was added to the sterile plasma at a final concentration of 200 μg / ml and incubated in a 37°C cell culture incubator at 80 shakes / minute. After 0 and 14 days of incubation, remove the ADC samples, add 100 μl of Protein A to each tube, allow adsorption for 2 hours, and elute to obtain the incubated ADCs. Perform DAR value testing on the incubated ADCs (testing and calculation methods are the same as in Example 5.2.1) to determine the plasma stability of the test samples.

[0695] Test results showed that the DAR values ​​of the ADCs prepared in Example 5 remained largely unchanged during plasma incubation, while the DAR value of the ADC with a maleimide linker (e.g., the control compound HER2-ADC-006) decreased significantly after 14 days of incubation. This demonstrates that the ADCs provided by the present invention exhibit improved plasma stability, thereby reducing the shedding of bioactive compounds in non-target tissues.

[0696] Example 10 Stability Test of Antibody Drug Conjugates in Tumor Tissue Homogenates and Muscle Tissue Homogenates

[0697] Referring to Example 6.11 of WO 2022 / 170971A1, the stability of each ADC prepared in Example 5 was investigated in vitro in 22RV1 Balb / c nude mouse tumor tissue homogenate, Balb / c mouse muscle tissue homogenate, and homogenate matrix saline, simulating the lysis of each ADC prepared in Example 5 in tumor tissue / tumor microenvironment. The payload was measured in an incubation system. The results for tumor tissue homogenate were compared with the stability results for plasma to assess the efficiency of payload release in tumor tissue / tumor microenvironment.

[0698] Test results showed that each ADC prepared in Example 5 released a significant amount of payload in tumor tissue homogenates, but no significant release was observed in normal tissue homogenates. This demonstrates that the ADCs provided by the present invention can enhance the effective release of bioactive compounds at the tumor site, thereby exhibiting a higher therapeutic index.

[0699] Unless otherwise defined, the terms used in the present invention have the same meanings as those commonly understood by those skilled in the art.

[0700] The embodiments described in the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art may make various other substitutions, changes and improvements within the scope of the present invention. Therefore, the present invention is not limited to the above-mentioned embodiments, but is only limited by the claims.

Claims

1. The antibody drug conjugate of formula I, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; in, Tb is an antibody or an antigen-binding fragment thereof; q is the number of Tb couplings L1-L2-L3-L4-D; D is a bioactive compound fragment of a protein degrader; L1 is the connector unit; L2 is the connection unit; L3 is selected from AA 1 The amino acid residues shown may contain AA 1 A short peptide consisting of 2-10 amino acid residues of the amino acid residues shown; the amino acid residues are selected from natural amino acid residues, non-natural amino acid residues, AA 1 the indicated amino acid residue or its stereoisomers; AA 1 The structures of the indicated amino acid residues are shown below, in: R a 、R b Each independently selected from hydrogen or And R a 、R b Not simultaneously hydrogen; Or, R a With R b Together with the carbon atoms to which they are attached, they form a 4-10 membered heterocyclic ring, which is optionally substituted by one or more R 0 replaced by; r、r 1 Each is independently selected from any integer between 0 and 20; R m1 、R n1 Each is independently selected from hydrogen, C1-6 alkyl or C3-6 cycloalkyl; Or, R m1 With R n1 Together with the nitrogen atom to which they are attached, they form a 4-10 membered heterocyclic ring, which is optionally substituted by one or more R 0’ replaced by; R 0 、R 0’ Each independently selected from C1-6 alkyl, C3-6 cycloalkyl, -NR m2 R n2 or a 4-10 membered heterocyclic group optionally substituted by a C1-6 alkyl group; R m2 、R n2 Each is independently selected from hydrogen or C1-6 alkyl; L4 is absent or present. When L4 is present, L4 is selected from Bit 1 is connected to L3 and bit 2 is connected to D.

2. The antibody-drug conjugate, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to claim 1, wherein: Meet one or more of the following conditions: (1) q is selected from any value between 0.1 and 12.0, preferably, q is selected from any value between 1.0 and 10.0; more preferably, q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; (2) L1 is selected from Position 1 is connected to Tb via an S atom, and position 2 is connected to L2; (3) L2 is selected from Bit 1 is connected to L1, bit 2 is connected to L3; Preferably, said L2 is selected from Bit 1 is connected to L1, bit 2 is connected to L3; (4) n is selected from any integer between 0 and 10; preferably, n is selected from 0, 1, 2 or 3; (5) Y is selected from -CH2- or -OCH2CH2-; (6) X is selected from -CR m R n -or-NR m -; (7) R m 、R n Each is independently selected from hydrogen, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, C3-6 cycloalkyl or 3-6 membered heterocyclyl; Alternatively, the R m With R n Together with the carbon atoms to which they are attached, they form a 3-6 membered carbocyclic ring or a 3-6 membered heterocyclic ring; Preferably, the R m 、R n are each independently selected from hydrogen or methyl; (8) L3 is selected from AA 1 The amino acid residues shown may contain AA 1 Preferably, L3 is selected from the following amino acid residues, dipeptides or tripeptides: AA 1 、Val-AA 1 、Ala-AA 1 、Gly-AA 1 AA 1 -Gly, AA 1 -Ala, Val-AA 1 -Gly, Ala-AA 1 -Gly, Gly-AA 1 -Gly or Val-AA 1 -Ala; More preferably, said L3 is selected from the dipeptide or tripeptide shown below: Val-AA 1 、Val-AA 1 -Ala or Val-AA 1 -Gly; particularly preferably, L3 is Val-AA 1 -A tripeptide represented by Gly; (9) R a 、R b In, either one is H and the other is Alternatively, the R a With R b Together with the carbon atoms they are connected to, they form the 0 substituted 5-6 membered heterocycle; (10) r, r 1 Each independently selected from 0, 1, 2, 3, 4 or 5; Preferably, said r, r 1 Each independently selected from 0 or 4; More preferably, the r is 0, the r 1 is 4; (11) R m1 、R n1 are each independently selected from hydrogen or C1-6 alkyl; or, said R m1 With R n1 Together with the nitrogen atom to which they are attached, they form an optionally R 0’ substituted 5-6 membered heterocycle; Preferably, the R m1 、R n1 are each independently selected from hydrogen, methyl, ethyl, n-propyl or n-butyl; or, the R m1 With R n1 Together with the nitrogen atom to which they are attached, they form an optionally R 0’ substituted piperidine ring or piperazine ring; more preferably, the R m1 、R n1 each independently selected from methyl, ethyl, n-propyl or n-butyl; (12) R 0 、R 0’ Each independently selected from C1-6 alkyl, -NR m2 R n2 or a 5-6 membered heterocyclic group optionally substituted by a C1-6 alkyl group; (13) R m2 、R n2 is methyl; (14) The protein degrader-type bioactive compound is a molecule that can cause degradation of GSPT1 family proteins.

3. The antibody-drug conjugate according to claim 1 or 2, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein: Meet one or more of the following conditions: (1) L1-L2 are selected from Position 1 is connected to Tb via an S atom, and position 2 is connected to L3; Preferably, said L1-L2 are selected from Position 1 is connected to Tb via an S atom, and position 2 is connected to L3; (2) L3 is selected from Bit 1 is connected to L2, bit 2 is connected to L4 or D; Preferably, said L3 is selected from 1 bit is connected to L2, 2 bits are connected to L4 or D connected; (3) The AA 1 The amino acid residues shown are selected from Preferably, the AA 1 The amino acid residues shown are selected from 4. The antibody-drug conjugate according to any one of claims 1 to 3, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, characterized in that: Selected from: 1 is connected to L2, 2 is connected to D, AA 1 The definition is as defined in any one of claims 1 to 3; Preferably, Selected from: Bit 1 is connected to L2, bit 2 is connected to D; More preferably, Selected from: Position 1 is connected to Tb via S atom, position 2 is connected to D, and Y, X, AA 1 and n is as defined in any one of claims 1 to 3; More preferably, Selected from: Position 1 is connected to Tb via an S atom, and position 2 is connected to D.

5. The antibody-drug conjugate according to any one of claims 1 to 4, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, characterized in that: The D is a structural unit shown in formula II, wherein position 1 is connected to L4 or L3; in, R 1 is selected from hydrogen, deuterium, halogen, cyano, amino, nitro, C1-4 alkoxy or C1-4 alkyl; Z is selected from -NH-, -CR 2a R 3a -or-C(O)-; R 2a 、R 3a are each independently selected from F or OH; U 1 、U 2 are each independently selected from -CH2- or -C(O)-, and U 1 、U 2 Not at the same time -CH2-; A is selected from O, S, NR 9 、 Position 1 is connected to L4 or L3, and position 2 is connected to a benzene ring; or position 1 is connected to a benzene ring, and position 2 is connected to L4 or L3; R2, R3, R4, R5, R6, R7, and R8 are each independently selected from hydrogen, deuterium, C1-4 alkyl, C3-6 cycloalkyl, or a 3-6 membered heterocyclic group; Alternatively, R2, R3 and the carbon atom to which they are attached form a 3-7 membered carbocyclic ring or a 3-7 membered heterocyclic ring; R4, R5 and the carbon atom and nitrogen atom to which they are connected form a 4-7 membered heterocyclic ring; R5, R6 and the carbon atom to which they are attached form a 3-7 membered carbocyclic ring or a 3-7 membered heterocyclic ring; R7, R8 and the carbon atom to which they are attached form a 3-7 membered carbocyclic ring or a 3-7 membered heterocyclic ring; m, p, and y are each independently selected from any integer between 0 and 8; W and V are each independently selected from a direct bond, O, S or NR 9 ; R 9 Selected from hydrogen, deuterium, C1-4 alkyl or C3-6 cycloalkyl.

6. The antibody-drug conjugate, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to claim 5, wherein: Meet one or more of the following conditions: (1) R 1 is selected from hydrogen, halogen or C1-4 alkyl; preferably, the R 1 is a halogen; (2) Z is -NH-; (3) The U 1 、U 2 are each independently selected from -CH2- or -C(O)-, and U 1 、U 2 Not the same; preferably, the U 1 is -CH2-, the U 2 is -C(O)-; (4) A is The 1 position is connected to L4 or L3, and the 2 position is connected to the benzene ring; the definitions of W, V, R2, R3, R4, R5, R6, R7, R8, m, p and y are as defined in claim 5; preferably, A is selected from -O(CH2) m C(O)NH(CH2) P -、-O(CH2) m C(O)N(CH3)(CH2) P -、-O(CH2) m C(O)NH(CH2) P O-, -O(CH2) m C(O)N(CH3)(CH2) P O-, -S(CH2) m C(O)NH(CH2) P -、-S(CH2) m C(O)N(CH3)(CH2) P -、-NH(CH2) m C(O)NH(CH2) P -、-NH(CH2) m C(O)N(CH3)(CH2) P -、-O(CH2) m C(O)NH(CH2) P O(CH2) y -or-O(CH2) m C(O)N(CH3)(CH2) P O(CH2) y -; Preferably, said A is selected from -O(CH2) m C(O)N(CH3)(CH2) P -or-O(CH2) m C(O)N(CH3)(CH2) P O(CH2) y -; Preferably, A is selected from -OCH2C(O)N(CH3)(CH2)3- or -OCH2C(O)N(CH3)(CH2)2O(CH2)2-; (5) W is selected from O, S or NR 9 , preferably, said W is O; (6) V is selected from direct bond, O, S or NR 9 , preferably, said V is selected from a direct bond or O; (7) R2, R3, R4, R5, R6, R7, and R8 are each independently selected from hydrogen, deuterium, or C1-4 alkyl; (8) m, p, and y are each independently selected from 0, 1, 2, 3, or 4; (9) R9 is selected from hydrogen, deuterium or methyl.

7. The antibody-drug conjugate according to claim 5 or 6, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein: The D is a structural unit shown in II-1, II-2 or II-3: Among them, R 1 , A, Z are as defined in claim 5 or 6, and bit 1 is connected to L4 or L3.

8. The antibody-drug conjugate according to any one of claims 1 to 7, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, characterized in that: D is selected from the following structures, and position 1 is connected to L4 or L3:

9. The antibody-drug conjugate according to any one of claims 1 to 8, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, characterized in that: The antibody-drug conjugate has a structure shown in Formula III, Among them, Tb, q, L1, L2, L3, L4, R 1 , A, Z, U 1 and U 2 As defined in any one of claims 1 to 8.

10. The antibody-drug conjugate according to any one of claims 1 to 9, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, characterized in that: The antibody drug conjugate is selected from: Wherein, q is selected from any value between 1.0-10.0; preferably, q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; more preferably, q is selected from 2, 4, 6 or 8.

11. The antibody drug conjugate according to any one of claims 1 to 10, or a stereoisomer of the antibody drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein the Tb is selected from one or more of the following: (1) The Tb is an antibody or an antigen-binding fragment thereof having endocytosis activity; (2) The Tb is an antibody or an antigen-binding fragment thereof that has the activity of binding to tumor cell surface antigens; (3) the Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof, an anti-Trop-2 antibody or an antigen-binding fragment thereof, an anti-Her2 antibody or an antigen-binding fragment thereof, an anti-Her3 antibody or an antigen-binding fragment thereof, or an anti-EGFR antibody or an antigen-binding fragment thereof; (4) The Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof, such as 1D1-01, 2E3-02 antibodies, enoblituzumab, mirzotamab, omburtamab or an antigen-binding fragment thereof; (5) The Tb is an anti-Her 2 antibody or an antigen-binding fragment thereof, such as anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, pertuzumab, timigutuzumab, zanidatamab, trastuzumab, pertuzumab or an antigen-binding fragment thereof; preferably, the Tb is trastuzumab or pertuzumab; (6) The Tb is an anti-EGFR antibody or an antigen-binding fragment thereof, such as demupitamab, depatuxizumab, futuximab, imgatuzumab, laprituximab, losatuxizumab, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, pimurutamab, serclutamab, tomozutuximab, zalutumumab, cetuximab or an antigen-binding fragment thereof; (7) The Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof whose VH sequence is shown in SEQ ID NO: 1 and whose VL sequence is shown in SEQ ID NO: 2; preferably, the Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof whose heavy chain sequence is shown in SEQ ID NO: 3 and whose light chain sequence is shown in SEQ ID NO: 4; (8) The Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof having a VH sequence as shown in SEQ ID NO: 5 and a VL sequence as shown in SEQ ID NO: 6; preferably, the Tb is an anti-B7H3 antibody or an antigen-binding fragment thereof having a heavy chain sequence as shown in SEQ ID NO: 7 and a light chain sequence as shown in SEQ ID NO:

8.

12. The drug-linker conjugate of Formula IV, Lg-L1-L2-L3-L4-D Formula IV or a stereoisomer of the drug-linker conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable Accepted solvates, in, When L1 is When , bit 1 is connected to Lg, bit 2 is connected to L2; Lg is a leaving group when reacting with an antibody; L2, L3, L4 and D are as defined in any one of claims 1 to 10; When L1 is When Lg-L1 is L2, L3, L4 and D are as defined in any one of claims 1-10.

13. The drug-linker conjugate according to claim 12, or a stereoisomer of the drug-linker conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein: The Lg is selected from halogen, sulfone, tertiary amine salt (Me3N + 、Et3N + ), diazonium salt, -OMs, MeSO2- or CF3SO3-; preferably, said Lg is selected from F, Cl or MeSO2-; more preferably, said Lg is selected from F or MeSO2-.

14. The drug-linker conjugate according to claim 12 or 13, or a stereoisomer of the drug-linker conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein: The drug-linker conjugate has a structure shown in Formula V, Among them, Lg, L1, L2, L3, L4, R 1 , A, Z, U 1 and U 2 As defined in any one of claims 1 to 13.

15. The drug-linker conjugate according to any one of claims 12 to 14, or a stereoisomer of the drug-linker conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, characterized in that: The drug-linker conjugate is selected from:

16. The biologically active compound of formula IIA, or a stereoisomer of the biologically active compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, in, R 1 is selected from hydrogen, halogen or C1-4 alkyl; preferably, the R 1 is a halogen; Z is -NH-; U 1 、U 2 are each independently selected from -CH2- or -C(O)-, and U 1 、U 2 Not the same; preferably, the U 1 is -CH2-, the U 2 is -C(O)-; A is The 1 position is connected to L4 or L3, and the 2 position is connected to the benzene ring; preferably, the A is selected from -O(CH2) m C(O)NH(CH2) P -、-O(CH2) m C(O)N(CH3)(CH2) P -、-O(CH2) m C(O)NH(CH2) P O-, -O(CH2) m C(O)N(CH3)(CH2) P O-, -S(CH2) m C(O)NH(CH2) P -、-S(CH2) m C(O)N(CH3)(CH2) P -、-NH(CH2) m C(O)NH(CH2) P -、-NH(CH2) m C(O)N(CH3)(CH2) P -、-O(CH2) m C(O)NH(CH2) P O(CH2) y -or-O(CH2) m C(O)N(CH3)(CH2) P O(CH2) y -; Preferably, said A is selected from -O(CH2) m C(O)N(CH3)(CH2) P -or-O(CH2) m C(O)N(CH3)(CH2) P O(CH2) y -; Preferably, A is selected from -OCH2C(O)N(CH3)(CH2)3- or -OCH2C(O)N(CH3)(CH2)2O(CH2)2-; W is selected from O, S or NR 9 , preferably, said W is O; V is selected from a direct bond, O, S or NR 9 , preferably, said V is selected from a direct bond or O; R2, R3, R4, R5, R6, R7, and R8 are each independently selected from hydrogen, deuterium, or C1-4 alkyl; m, p, y are each independently selected from 0, 1, 2, 3 or 4; R9 is selected from hydrogen, deuterium or methyl.

17. The bioactive compound according to claim 16, or a stereoisomer of the bioactive compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein: The biologically active compound is selected from:

18. A method for preparing an antibody-drug conjugate of formula I, comprising: Tb is coupled with the drug-linker conjugate Lg-L1-L2-L3-L4-D represented by formula IV; wherein Tb, L1, L2, L3, L4, Lg and D are as defined in any one of claims 1-15.

19. The method according to claim 18, wherein The method comprises the steps of coupling Tb with the drug-linker conjugate Lg-L1-L2-L3-L4-D represented by formula IV to form a CS bond; The molar ratio of Tb to the drug-linker conjugate is 1:(1-20), such as 1:(2-20), 1:(4-20), 1:(6-20), 1:(8-20), 1:(10-20), 1:(12-20), 1:(14-20), 1:(16-20) or 1:(18-20); The coupling reaction is preferably carried out in water and / or an organic solvent; the organic solvent is preferably one or more of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, nitrile solvents and alcohol solvents.

20. A linker represented by formula VA-1, in, Lg, L1, L2, L3, L4, W, R2, R3 and m are as defined in any one of claims 12 to 15, Lg2 is selected from hydroxyl, halogen, activated hydroxyl, preferably selected from hydroxyl, chlorine, bromine, 21. The connector according to claim 20, wherein: The linker is selected from:

22. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1 to 11, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; Or the drug-linker conjugate according to any one of claims 12 to 15, or a stereoisomer of the drug-linker conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; Or the biologically active compound according to any one of claims 16 to 17, or the stereoisomer of the biologically active compound, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, and optionally one or more pharmaceutical excipients.

23. The pharmaceutical composition of claim 22, wherein the antibody-drug conjugate has a drug-to-antibody ratio (DAR) of 1.0-12.0; preferably, the drug-to-antibody ratio (DAR) is 2±0.4, 4±0.4, 6±0.4, or 8±0.

4.

24. The antibody-drug conjugate according to any one of claims 1 to 11, or a stereoisomer of the antibody-drug conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; Or the drug-linker conjugate according to any one of claims 12 to 15, or a stereoisomer of the drug-linker conjugate, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; or the biologically active compound according to any one of claims 16 to 17, or a stereoisomer of the biologically active compound, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; Or use of the pharmaceutical composition according to claim 22 or 23 in the preparation of a medicament for treating and / or preventing a disease associated with abnormal cell activity (e.g., cancer); Preferably, the cancer disease is selected from esophageal cancer (e.g., esophageal adenocarcinoma or esophageal squamous cell carcinoma), brain tumor, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, urothelial carcinoma, solid tumors, non-Hodgkin's lymphoma, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma or sarcoma), prostate cancer or thyroid cancer.

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