Drug-linker conjugate containing protein degrader, preparation method therefor, and use thereof

Abstract

The present application provides a drug-linker conjugate containing a protein degrader and an antibody-drug conjugate thereof. The present application also provides a preparation method for the drug-linker conjugate and the antibody-drug conjugate, as well as a use of the antibody-drug conjugate in the prevention and / or treatment of diseases associated with abnormal cellular activity, including but not limited to a use in the prevention and / or treatment of tumor diseases.

Description

A drug linker conjugate containing a protein degrading agent, its preparation method and uses

[0001] This application claims priority to Chinese patent application 2024115178566, filed on October 29, 2024. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This application belongs to the field of compounds and relates to a class of drug linker conjugates and their antibody-drug conjugates consisting of an indegradable linker and a protein degrading agent, methods for preparing the drug linker conjugates and their antibody-drug conjugates, pharmaceutical compositions of the antibody-drug conjugates, and the use of the antibody-drug conjugates in the prevention and / or treatment of diseases related to abnormal cell activity, including but not limited to their use in the prevention and / or treatment of tumor diseases. Background Technology

[0003] Many diseases are related to abnormal intracellular protein function, and the main approach to treating these diseases is with small molecule compounds. However, more than 80% of proteins lack sites that can produce drug-like effects, and these targets are considered unsuitable for traditional small molecule drug development.

[0004] Targeted protein degradation (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 exert their effects without tightly binding to sites affecting protein activity, allowing previously "undruggable" proteins to become novel drug targets. Simultaneously, targeted protein degradation therapy can continuously induce rapid and efficient degradation of pathogenic proteins, reducing the development of drug resistance in target proteins.

[0005] Several targeted protein degradation agents have entered the clinical development stage. For example, C4 Therapeutics' molecular gel CFT7455, which targets IKZF1 / 3, was presented at the AACR meeting in April 2022. Although CFT7455 showed clinical benefits of deep target degradation, neutropenia was dose-limited, with 3 out of 5 patients experiencing grade 4 neutropenia and 1 experiencing grade 3 neutropenia.

[0006] Antibody-drug conjugates (ADCs) combine the tumor-targeting activity of antibodies with the high activity of bioactive compounds, becoming a kind of biological missile with promising therapeutic and safety advantages. Antibodies guide ADCs to bind to target cells, achieving tumor tissue enrichment, reducing exposure to non-target tissues, and mitigating the toxicity that may result from systemic administration of bioactive compounds. ADCs bound to tumor cells are internalized, releasing small molecule drugs intracellularly to treat the disease. Therefore, it is expected that ADCs composed of protein degraders and tumor-targeting antibodies can achieve tumor enrichment, eliminate or reduce the toxic side effects caused by protein degraders acting on non-disease tissues, and improve therapeutic efficacy. Utilizing ADC technology to achieve tumor targeting of protein degraders and reduce their toxic side effects will have high clinical value.

[0007] ADC linkers are classified as either cleavable or non-cleavable. Cleavable linkers release toxin molecules directly through chemical bond cleavage; non-cleavable linkers do not involve specific bond cleavage but release small molecules related to the toxin molecules through complete or partial hydrolysis of the antibody. Compared to cleavable linkers, using non-cleavable linkers allows for targeted killing effects at tumor or tissue sites. This is because non-cleavable linkers effectively reduce the entry of toxins into the circulatory system due to linker cleavage, thus preventing systemic toxicity. In general, non-cleavable linkers provide a larger therapeutic window than cleavable linkers. Summary of the Invention

[0008] In a first aspect, this application provides a drug linker conjugate of Formula A or a pharmaceutically acceptable salt thereof;

[0009] in,

[0010] Lg is the leaving group that reacts with antibodies or their antigen-binding fragments;

[0011] Y is selected from direct bonds and -O-;

[0012] X is selected from direct bond, -CR m R n -、-NR m -、-C(O)-、-CR m R n C(O)- and 4-6 membered heterocyclic groups;

[0013] R m R n Each is independently selected from hydrogen and C. 1-4 Alkyl, the C 1-4 The alkyl group may optionally be replaced by one or more hydroxyl, phosphate, sulfonic acid, and sugar groups;

[0014] Z1 is selected from -NH-, -CF2-, or -C(O)-;

[0015] Z2 is selected from -CH2-, -O-, or -NH-;

[0016] U 1 U 2 Each is independently selected from -CH2- and -C(O)-, and U 1 U 2 Not both -CH2-;

[0017] A is selected from -NR 1 (CH2) m -、-NR 1 (CH2) m O-、-NR 1 (CH2) m S-、-NR 1 (CH2) m NR 2 -、-NR 1 (CH2) m O(CH2) P -、-NR 1 (CH2) m S(CH2) P -、-NR 1 (CH2) m NR 2 (CH2) P -、-(O)C(CH2) m -、-S(O)2(CH2) m -、 and -4-6-membered heterocyclic group (CH2) m - and 4-6 membered heterocyclic groups, with X attached at position 1 and benzene ring attached at position 2;

[0018] R 1 R 2 Each is independently selected from hydrogen and C. 1-4 Alkyl, the C 1-4 The alkyl group may optionally be substituted with one or more hydroxyl, sulfonic acid, sulfonyl, phosphate, and sugar groups;

[0019] m and p are each independently selected from any integer between 1 and 5.

[0020] In some implementations, Lg is selected from halogens, sulfones, and tertiary amine salts (Me3N). + Et3N + ), diazonium salts, -OMs, MeSO2-, MeS-, CF3SO3-, p-toluenesulfonyl, The substituted phenoxy group is selected from halogens and nitro groups.

[0021] In some implementations, Lg is selected from halogens, sulfones, and tertiary amine salts (Me3N). + Et3N + Diazonium salts, -OMs, MeSO2-, CF3SO3-, p-toluenesulfonyl groups, The substituted phenoxy group is selected from halogens and nitro groups.

[0022] In some embodiments, Lg is selected from fluorine, chlorine, bromine, iodine, -OMs, MeSO2-, MeS-, CF3SO3-, and p-toluenesulfonyl.

[0023] In some embodiments, Lg is selected from fluorine, chlorine, bromine, iodine, -OMs, MeSO2-, CF3SO3-, and p-toluenesulfonyl.

[0024] In some implementations, Lg is selected from MeS-.

[0025] In some implementations, Lg is MeSO2-.

[0026] In some implementations, Y is a direct key.

[0027] In some implementations, X is selected from direct bonds, -CH2-, -C(CH3)2-, -N(CH3)-, -NH-, -C(O)-, -CH2C(O)-, Bit 1 is connected to Y, and bit 2 is connected to A.

[0028] In some implementation schemes, R m R n Each is independently selected from hydrogen, C1-4 alkyl, C1-4 alkyl hydroxyl, n is selected from 1, 2, and 3, with position 1 connected to the corresponding carbon or nitrogen atom.

[0029] In some implementation schemes, R m R n Each is independently selected from hydrogen, methyl, -CH2CH2OH and One position is connected to the corresponding carbon or nitrogen atom.

[0030] In some implementations, X is selected from -N(CH3)- and -C(O)-, bit 1 is connected to Y, and bit 2 is connected to A.

[0031] In some implementation schemes, Selected from direct keys, In this configuration, position 1 is connected to a carbon atom, and position 2 is connected to an atom.

[0032] In some implementation schemes, Selected from direct keys, In this configuration, position 1 is connected to a carbon atom, and position 2 is connected to an atom.

[0033] In some implementation schemes, Selected from 1 is connected to A.

[0034] In some implementations, Z1 is -NH-; Z2 is -NH-.

[0035] In some implementations, Z1 is -NH-; Z2 is -O-.

[0036] In some implementations, Z1 is -CF2-; Z2 is -NH-.

[0037] In some implementation schemes, U 1 For -CH2-, U 2 For -C(O)- or U 1 For -C(O)-, U 2 It is -CH2-.

[0038] In some implementations, Z1 is -NH-, Z2 is -NH-, U 1 For -CH2-, U 2 It is -C(O)-.

[0039] In some implementations, Z1 is -NH-, Z2 is -O-, and U 1 For -C(O)-, U 2 It is -CH2-.

[0040] In some implementations, A is selected from -NR 1 (CH2) m -、-NR 1 (CH2) m O-、-NR 1 (CH2) m S-、-NR 1 (CH2) m NR 2 -、-NR 1 (CH2) m O(CH2) P -、-NR 1 (CH2) m S(CH2) P -、-NR 1 (CH2)m NR 2 (CH2) P -、-(O)C(CH2) m -、-S(O)2(CH2) m -、 It has a 4-6 membered heterocyclic group, with X attached at position 1 and benzene attached at position 2.

[0041] In some implementations, A is selected from -NR 1 (CH2)3-、-NR 1 (CH2)2O(CH2)2-, -(O)C(CH2)2-, -(O)C(CH2)3-, -S(O)2(CH2)3-, Position 1 is connected to X, and position 2 is connected to the benzene ring.

[0042] In some implementations, A is selected from -NR 1 (CH2)3-、-NR 1 (CH2)2O(CH2)2-, -(O)C(CH2)2-, -(O)C(CH2)3-, -S(O)2(CH2)3-, Position 1 is connected to X, and position 2 is connected to the benzene ring.

[0043] In some implementation schemes, R 1 It is hydrogen, methyl, methanesulfonyl or The 1st position is connected to the corresponding nitrogen atom.

[0044] In some implementation schemes, R 2 It can be hydrogen or methyl.

[0045] In some implementations, m is selected from 1, 2, and 3.

[0046] In some implementations, p is selected from 1, 2, and 3.

[0047] In some implementation schemes, Select from the following structure, with one bit connected to X;

[0048] In some embodiments, the drug linker conjugate has the structure shown in Formula A-1.

[0049] Wherein, Lg, X, Y and A are defined as described in any of the embodiments of this disclosure.

[0050] In some embodiments, this application provides a drug linker conjugate of Formula I or a pharmaceutically acceptable salt thereof;

[0051] in,

[0052] Lg is the leaving group that reacts with antibodies or their antigen-binding fragments;

[0053] X is selected from direct bond, -NR m -or -C(O)-;

[0054] R m Selected from hydrogen or C 1-4 Alkyl, the C 1-4 The alkyl group is optionally substituted by one or more substituents selected from hydroxyl, phosphoric acid, sulfonic acid and sugar groups;

[0055] A is selected from -NR 1 (CH2) m -、-NR 1 (CH2) m Q(CH2) p -、-(O)C(CH2) m -、-(O)C(CH2) m Q(CH2) p -、-S(O)2(CH2) m -、-S(O)2(CH2) m Q(CH2) p -、-S(O)(CH2) m -or -S(O)(CH2) m Q(CH2) p -;

[0056] Q is selected independently from -O-, -S-, or -NR. 2 -;

[0057] R 1 Independently selected from hydrogen, sulfonyl, or C 1-4 Alkyl, the C 1-4 The alkyl group is optionally substituted by one or more substituents selected from hydroxyl, sulfonic acid, phosphate and sugar groups;

[0058] R 2 Selected from hydrogen or C 1-4 alkyl;

[0059] m and p are each independently selected from any integer between 1 and 5.

[0060] In some implementations, Lg is selected from halogens, sulfones, and tertiary amine salts (Me3N). + Et3N + ), diazo

[0061] Base, -OMs, MeSO2-, MeS-, CF3SO3-, p-toluenesulfonyl, Or a substituted phenoxy group, wherein the substituent is selected from halogens and / or nitro groups.

[0062] In some implementations, Lg is selected from halogens, sulfones, and tertiary amine salts (Me3N). + Et3N + Diazonium salts, -OMs, MeSO2-, CF3SO3-, p-toluenesulfonyl groups, Or a substituted phenoxy group, wherein the substituent is selected from halogens and / or nitro groups.

[0063] In some embodiments, Lg is selected from fluorine, chlorine, bromine, iodine, -OMs, MeSO2-, MeS-, CF3SO3- or p-toluenesulfonyl.

[0064] In some embodiments, Lg is selected from fluorine, chlorine, bromine, iodine, -OMs, MeSO2-, CF3SO3- or p-toluenesulfonyl.

[0065] In some implementations, Lg is MeS-.

[0066] In some implementations, Lg is MeSO2-.

[0067] In some implementations, X is selected from direct bonds, -N(CH3)-, Or -C(O)-, with position 1 connected to the corresponding carbon atom and position 2 connected to A.

[0068] In some implementation schemes, R m Selected from hydrogen, C 1-4 Alkyl, hydroxyl substituted C 1-4 alkyl and phosphate substituted C 1-4 C substituted with alkyl or sulfonic acid groups 1-4 alkyl or sugar-substituted C 1-4 alkyl.

[0069] In some implementation schemes, R m Selected from C 1-4 Alkyl, hydroxyl substituted C 1-4 alkyl, n is selected from 1, 2, 3, and 4, with position 1 connected to the corresponding nitrogen atom.

[0070] In some implementation schemes, R m Selected from methyl, -CH2CH2OH or The 1st position is connected to the corresponding nitrogen atom.

[0071] In some implementation schemes, Selected from 1 is connected to A.

[0072] In some implementations, Q is -O-.

[0073] In some implementations, A is selected from -NR 1 (CH2) m -、-NR 1 (CH2) m O(CH2) p -、-NR 1 (CH2) m S(CH2) p -、-NR 1 (CH2) m NR 2 (CH2) p -、-(O)C(CH2) m -、-(O)C(CH2) m O(CH2) p -、-(O)C(CH2) m S(CH2) p -、-(O)C(CH2) m NR 2 (CH2) p -、-S(O)2(CH2) m -、-S(O)2(CH2) m O(CH2) p -、-S(O)2(CH2) m S(CH2) p -、-S(O)2(CH2) m NR 2 (CH2) p -、-S(O)(CH2) m -、-S(O)(CH2) m O(CH2) p -、-S(O)(CH2) m S(CH2) p -or -S(O)(CH2) m NR 2 (CH2) p -

[0074] In some implementations, A is selected from -NR 1 (CH2) m -、-NR 1 (CH2) m O(CH2) p -、-(O)C(CH2) m-、-S(O)2(CH2) m -or -S(O)(CH2) m -

[0075] In some implementations, A is selected from -NR 1 (CH2)3-、-NR 1 (CH2)2O(CH2)2-, -(O)C(CH2)2-, -(O)C(CH2)3-, -S(O)2(CH2)3- or -S(O)(CH2)3-.

[0076] In some implementation schemes, R 1 Selected from hydrogen, sulfonyl, C 1-4 Alkyl, hydroxyl substituted C 1-4 alkyl and phosphate substituted C 1-4 C substituted with alkyl or sulfonic acid groups 1-4 alkyl or sugar-substituted C 1-4 alkyl.

[0077] In some implementation schemes, R 1 Independently selected from sulfonyl, C 1-4 Alkyl, hydroxyl substituted C 1-4 alkyl, n is selected from 1, 2, 3, and 4, with position 1 connected to the corresponding nitrogen atom.

[0078] In some implementation schemes, R 1 Independently selected from methyl, methanesulfonyl, -CH2CH2OH, The 1st position is connected to the corresponding nitrogen atom.

[0079] In some implementation schemes, R 2 It can be hydrogen or methyl.

[0080] In some implementations, m is selected from 1, 2, and 3.

[0081] In some implementations, p is selected from 1, 2, and 3.

[0082] In some implementation schemes, Select from the following structure, with one bit connected to X;

[0083] In some implementations, X is a direct bond; A is selected from -S(O)2(CH2). m -、-S(O)2(CH2) m Q(CH2) p -、-S(O)(CH2) m -、S(O)(CH2) m Q(CH2)p -、-NR 1 (CH2) m -or-NR 1 (CH2) m Q(CH2) p -

[0084] In some implementations, X is -NR m -; A is selected from -(O)C(CH2) m -or -(O)C(CH2) m Q(CH2) p -

[0085] In some implementations, X is -C(O)-; A is selected from -NR 1 (CH2) m -or-NR 1 (CH2) m Q(CH2) p -

[0086] In some embodiments, the drug linker conjugate or a pharmaceutically acceptable salt thereof has the structure shown in Formula I-1.

[0087] in,

[0088] A is selected from -S(O)2(CH2) m -、-S(O)2(CH2) m Q(CH2) p -、-S(O)(CH2) m -、-S(O)(CH2) m Q(CH2) p -、-NR 1 (CH2) m -and-NR 1 (CH2) m Q(CH2) p -;

[0089] Q, R 1 The definitions of Lg, m, and p are as described in any of the embodiments of this disclosure.

[0090] In some embodiments, the drug linker conjugate or a pharmaceutically acceptable salt thereof has the structure shown in Formula I-2.

[0091] Where A is selected from -NR 1 (CH2) m -or-NR 1 (CH2) m Q(CH2) p-;

[0092] R 1 The definitions of Q, Lg, m, and p are as described in any of the embodiments of this disclosure.

[0093] In some embodiments, the drug linker conjugate or a pharmaceutically acceptable salt thereof has the structure shown in Formula I-3.

[0094] Wherein, A is selected from -(O)C(CH2). m -or -(O)C(CH2) m Q(CH2) p -;

[0095] R m The definitions of Q, Lg, m, and p are as described in any of the embodiments of this disclosure.

[0096] In some embodiments, the drug linker conjugate is selected from:

[0097] In some embodiments, the above-described drug linker conjugate may optionally be replaced by one or more suitable substituents.

[0098] Secondly, this application provides the antibody-drug conjugate of Formula B or a pharmaceutically acceptable salt thereof.

[0099] in,

[0100] Tb is an antibody or its antigen-binding fragment;

[0101] q is any integer between 1 and 12;

[0102] X, Y, A, Z1, Z2, U 1 and U 2 The definition is as described in any of the embodiments in this disclosure.

[0103] In some embodiments, the antibody-drug conjugate has the structure shown in Formula B-1.

[0104] X, Y, A, and Tb are defined as described in any of the embodiments of this disclosure.

[0105] In some embodiments, this application provides the antibody-drug conjugate of Formula II or a pharmaceutically acceptable salt thereof.

[0106] in,

[0107] Tb is an antibody or its antigen-binding fragment;

[0108] q is any integer between 1 and 12;

[0109] X and A are defined as described in any of the embodiments of this disclosure.

[0110] In some implementations, Tb is an antibody or its antigen-binding fragment that has endocytic activity.

[0111] In some implementations, Tb is an antibody or its antigen-binding fragment that has the activity of binding to tumor cell surface antigens.

[0112] In some implementations, Tb is an antibody or its antigen-binding fragment that has the activity of binding to surface antigens of solid or hematologic tumor cells.

[0113] In some embodiments, the antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment that has tumor cell surface antigen-binding activity and tumor cell endocytosis activity.

[0114] In some embodiments, the antibody or its antigen-binding fragment includes 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.

[0115] In some embodiments, the antibody or its antigen-binding fragment is a non-human antibody, a humanized antibody, a chimeric antibody, or a fully human antibody.

[0116] In some embodiments, the antibody or its antigen-binding fragment probody, bispecific antibody, or multispecific antibody is used.

[0117] In some embodiments, the antibody or its antigen-binding fragment includes Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, and single-chain antibody (e.g., scFv).

[0118] In some implementations, the targets of Tb are selected from CD19, CD20, CD21, CD22, CD23, CD30, CD123, CD33, FLT3, Her2, and Trop-2.

[0119] In some implementations, Tb is an anti-Trop-2 antibody or its antigen-binding fragment, or an anti-Her2 antibody or its antigen-binding fragment.

[0120] In some embodiments, Tb is an anti-Her2 antibody or its antigen-binding fragment, such as anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, pertuzumab, timigutuzumab, zanidatamab, trastuzumab, pertuzumab or its antigen-binding fragment; preferably, Tb is trastuzumab or pertuzumab; for example, Tb is trastuzumab.

[0121] In some implementations, Tb is an anti-Trop-2 antibody or its antigen-binding fragment, such as datopotamab, sacituzumab or its antigen-binding fragment.

[0122] In some implementations, the targets for Tb are selected from CD123 and CD33.

[0123] In some implementations, Tb is an anti-CD123 or CD33 antibody or its antigen-binding fragment.

[0124] In some implementations, Tb is an antibody against CD123 or its antigen-binding fragment, such as Pivekimab, Talacotuzumab, h12F1, G4723A, mAb-01 or its antigen-binding fragment.

[0125] In some implementations, Tb is an antibody against CD33 or its antigen-binding fragment, such as Gemtuzumab, Vadastuximab, Lintuzumab, YLAb-36 or its antigen-binding fragment.

[0126] In some embodiments, Tb is an antibody or antigen-binding fragment thereof that binds to the IgC domain of CD33. In some preferred embodiments, the antibody or antigen-binding fragment thereof that binds to the IgC domain of CD33 has a light chain as shown in SEQ ID NO. 223 of US_20190382481_A1 and a heavy chain as shown in SEQ ID NO. 191 of US_20190382481_A1.

[0127] In some embodiments, Tb is an antibody or antigen-binding fragment thereof that binds to the IgV domain of CD33. In some preferred embodiments, the antibody or antigen-binding fragment thereof that binds to the IgV domain of CD33 is selected from Gemtuzumab, Vadastuximab, and Lintuzumab.

[0128] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:3 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:4, wherein the CDRs are defined according to the Chothia, AbM, Kabat, IMGT, Contact scheme or any combination thereof.

[0129] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:3 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:4, wherein the CDRs are defined according to the Chothia scheme.

[0130] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:3 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:4, wherein the CDRs are defined according to the Abm scheme.

[0131] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:3 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:4, wherein the CDRs are defined according to the Kabat scheme.

[0132] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:3 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:4, wherein the CDRs are defined according to the IMGT scheme.

[0133] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises CDR-H1 as shown in SEQ ID NO:5, CDR-H2 as shown in SEQ ID NO:10, CDR-H3 as shown in SEQ ID NO:15, CDR-L1 as shown in SEQ ID NO:18, CDR-L2 as shown in SEQ ID NO:21, and CDR-L3 as shown in SEQ ID NO:24, according to the Chothia definition scheme.

[0134] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises CDR-H1 as shown in SEQ ID NO:6, CDR-H2 as shown in SEQ ID NO:11, CDR-H3 as shown in SEQ ID NO:15, CDR-L1 as shown in SEQ ID NO:18, CDR-L2 as shown in SEQ ID NO:21, and CDR-L3 as shown in SEQ ID NO:24, according to the Abm definition scheme.

[0135] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises CDR-H1 as shown in SEQ ID NO:7, CDR-H2 as shown in SEQ ID NO:12, CDR-H3 as shown in SEQ ID NO:15, CDR-L1 as shown in SEQ ID NO:18, CDR-L2 as shown in SEQ ID NO:21, and CDR-L3 as shown in SEQ ID NO:24, according to the Kabat definition scheme.

[0136] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises CDR-H1 as shown in SEQ ID NO:9, CDR-H2 as shown in SEQ ID NO:14, CDR-H3 as shown in SEQ ID NO:17, CDR-L1 as shown in SEQ ID NO:20, CDR-L2 as shown in SEQ ID NO:23, and CDR-L3 as shown in SEQ ID NO:24, as defined in the IMGT scheme.

[0137] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises CDR-H1 shown in SEQ ID NO:8, CDR-H2 shown in SEQ ID NO:13, CDR-H3 shown in SEQ ID NO:16, CDR-L1 shown in SEQ ID NO:19, CDR-L2 shown in SEQ ID NO:22, and CDR-L3 shown in SEQ ID NO:25, as defined in the Contact scheme.

[0138] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises: the heavy chain variable region (VH) of SEQ ID NO:3 and the light chain variable region (VL) of SEQ ID NO:4.

[0139] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:3, and a light chain variable region (VL) having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:4.

[0140] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) having at least 95%, at least 98%, at least 99%, or 100% sequence identity with respect to SEQ ID NO:3, and a light chain variable region (VL) having at least 95%, at least 98%, at least 99%, or 100% sequence identity with respect to SEQ ID NO:4.

[0141] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises: the heavy chain of SEQ ID NO:1 and the light chain of SEQ ID NO:2.

[0142] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises: a heavy chain having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with respect to SEQ ID NO:1, and a light chain having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with respect to SEQ ID NO:2.

[0143] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises: a heavy chain having at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:1, and a light chain having at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:2.

[0144] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:28 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:29, wherein the CDRs are defined according to the Chothia, ABM, Kabat, IMGT, Contact scheme or any combination thereof.

[0145] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises: the heavy chain variable region (VH) of SEQ ID NO:28 and the light chain variable region (VL) of SEQ ID NO:29;

[0146] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises: a heavy chain as shown in SEQ ID NO:26 and a light chain as shown in SEQ ID NO:27.

[0147] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:32 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:33, wherein the CDRs are defined according to the Chothia, Abm, Kabat, IMGT, Contact scheme or any combination thereof.

[0148] In some embodiments, the CD123 antibody or its antigen-binding fragment described in Tb comprises: the heavy chain variable region (VH) of SEQ ID NO:32 and the light chain variable region (VL) of SEQ ID NO:33;

[0149] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment comprises: a heavy chain as shown in SEQ ID NO:30, and a light chain as shown in SEQ ID NO:31.

[0150] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:36 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:37, wherein the CDRs are defined according to the Chothia, Abm, Kabat, IMGT, Contact scheme or any combination thereof.

[0151] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:36 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:37, wherein the CDRs are defined according to the Chothia scheme.

[0152] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:36 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:37, wherein the CDRs are defined according to the Abm scheme.

[0153] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:36 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:37, wherein the CDRs are defined according to the Kabat scheme.

[0154] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:36 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:37, wherein the CDRs are defined according to the IMGT scheme.

[0155] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises CDR-H1 as shown in SEQ ID NO:38, CDR-H2 as shown in SEQ ID NO:43, CDR-H3 as shown in SEQ ID NO:48, CDR-L1 as shown in SEQ ID NO:51, CDR-L2 as shown in SEQ ID NO:54, and CDR-L3 as shown in SEQ ID NO:57, according to the Chothia definition scheme.

[0156] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises CDR-H1 as shown in SEQ ID NO:39, CDR-H2 as shown in SEQ ID NO:44, CDR-H3 as shown in SEQ ID NO:48, CDR-L1 as shown in SEQ ID NO:51, CDR-L2 as shown in SEQ ID NO:54, and CDR-L3 as shown in SEQ ID NO:57, according to the Abm definition scheme.

[0157] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises CDR-H1 as shown in SEQ ID NO:40, CDR-H2 as shown in SEQ ID NO:45, CDR-H3 as shown in SEQ ID NO:48, CDR-L1 as shown in SEQ ID NO:51, CDR-L2 as shown in SEQ ID NO:54, and CDR-L3 as shown in SEQ ID NO:57, according to the Kabat definition scheme.

[0158] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises CDR-H1 as shown in SEQ ID NO:42, CDR-H2 as shown in SEQ ID NO:47, CDR-H3 as shown in SEQ ID NO:50, CDR-L1 as shown in SEQ ID NO:53, CDR-L2 as shown in SEQ ID NO:56, and CDR-L3 as shown in SEQ ID NO:57, as defined in the IMGT scheme.

[0159] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises CDR-H1 as shown in SEQ ID NO:41, CDR-H2 as shown in SEQ ID NO:46, CDR-H3 as shown in SEQ ID NO:49, CDR-L1 as shown in SEQ ID NO:52, CDR-L2 as shown in SEQ ID NO:55, and CDR-L3 as shown in SEQ ID NO:67, as defined in the Contact scheme.

[0160] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: the heavy chain variable region (VH) of SEQ ID NO:36 and the light chain variable region (VL) of SEQ ID NO:37.

[0161] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:36, and a light chain variable region (VL) having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:37.

[0162] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) having at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:36, and a light chain variable region (VL) having at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:37.

[0163] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: the heavy chain of SEQ ID NO:34 or 66 and the light chain of SEQ ID NO:35.

[0164] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: the heavy chain of SEQ ID NO:34 and the light chain of SEQ ID NO:35.

[0165] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: the heavy chain of SEQ ID NO:66 and the light chain of SEQ ID NO:35.

[0166] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: a heavy chain having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:34, and a light chain having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:35.

[0167] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: a heavy chain having at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:66, and a light chain having at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:35.

[0168] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:60 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:61, wherein the CDRs are defined according to the Chothia, ABM, Kabat, IMGT, Contact scheme or any combination thereof.

[0169] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:60 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:61, wherein the CDRs are defined according to the Chothia scheme.

[0170] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:60 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:61, wherein the CDRs are defined according to the AbM scheme.

[0171] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:60 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:61, wherein the CDRs are defined according to the Kabat scheme.

[0172] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:60 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:61, wherein the CDRs are defined according to the IMGT scheme.

[0173] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:60, and a light chain variable region (VL) having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:61.

[0174] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) having at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:60, and a light chain variable region (VL) having at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:61.

[0175] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: the heavy chain of SEQ ID NO:58 and the light chain of SEQ ID NO:59.

[0176] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: a heavy chain having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:58, and a light chain having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:59.

[0177] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: a heavy chain having at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:58, and a light chain having at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:59.

[0178] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:64 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:65, wherein the CDRs are defined according to the Chothia, ABM, Kabat, IMGT, Contact scheme or any combination thereof.

[0179] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:64 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:65, wherein the CDRs are defined according to the Chothia scheme.

[0180] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:64 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:65, wherein the CDRs are defined according to the AbM scheme.

[0181] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:64 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:65, wherein the CDRs are defined according to the Kabat scheme.

[0182] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:64 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:65, wherein the CDRs are defined according to the IMGT scheme.

[0183] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:64, and a light chain variable region (VL) having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:65.

[0184] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: a heavy chain variable region (VH) having at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:64, and a light chain variable region (VL) having at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:65.

[0185] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: the heavy chain of SEQ ID NO:62 and the light chain of SEQ ID NO:63.

[0186] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: a heavy chain having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:62, and a light chain having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:63.

[0187] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment comprises: a heavy chain having at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:62, and a light chain having at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity compared to SEQ ID NO:63.

[0188] In some implementations, q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0189] In some implementations, q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0190] In some implementations, q is selected from 2, 4, 6, and 8.

[0191] In some implementations, q is 2.

[0192] In some implementations, q is 4.

[0193] In some implementations, q is 6.

[0194] In some implementations, q is 8.

[0195] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof has the structure shown in Formula II-1.

[0196] in,

[0197] A is selected from -S(O)2(CH2) m -、-S(O)2(CH2) m Q(CH2) p -、-S(O)(CH2) m -、-S(O)(CH2) m Q(CH2) p -、-NR 1 (CH2) m -or-NR 1 (CH2) m Q(CH2) p -;

[0198] Q, R 1 The definitions of Tb, m, p, and q are as described in any of the embodiments of this disclosure.

[0199] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof has the structure shown in Formula II-2.

[0200] Where A is selected from -NR 1 (CH2) m -or-NR 1 (CH2) m Q(CH2) p -;

[0201] R 1 The definitions of Q, Tb, m, p, and q are as described in any of the embodiments of this disclosure.

[0202] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof has the structure shown in Formula II-3.

[0203] Wherein, A is selected from -(O)C(CH2). m -or -(O)C(CH2) m Q(CH2)p -;

[0204] R m The definitions of Q, Tb, m, p, and q are as described in any of the embodiments of this disclosure.

[0205] In some implementations, the antibody-drug conjugate is selected from:

[0206] Wherein, Tb and q are defined as described in any of the schemes in this application.

[0207] In some embodiments, the antibody-drug conjugate has any of the following structures:

[0208] Where Tb1 is the antibody against CD123 or its antigen-binding fragment, and q is any integer between 1 and 12.

[0209] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment is the anti-CD123 antibody or its antigen-binding fragment described in any of the embodiments of this disclosure.

[0210] In some embodiments, the anti-CD123 antibody or its antigen-binding fragment is selected from mAb-01, G4723A, and h12F1.

[0211] In some embodiments, the antibody-drug conjugate has any of the following structures:

[0212] Where Tb2 is the antibody against CD33 or its antigen-binding fragment, and q is any integer between 1 and 12.

[0213] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment is the anti-CD33 antibody or its antigen-binding fragment as described in any of the embodiments of this disclosure.

[0214] In some embodiments, the anti-CD33 antibody or its antigen-binding fragment is selected from Gemtuzumab, mAb-2, Lintuzumab, and YLAb-36.

[0215] In some embodiments, the antibody-drug conjugate has any of the following structures:

[0216] Where Tb4 is the anti-HER2 antibody or its antigen-binding fragment, and q is any integer between 1 and 12.

[0217] In some embodiments, the anti-HER2 antibody or its antigen-binding fragment is the anti-HER2 antibody or its antigen-binding fragment described in any of the embodiments of this disclosure.

[0218] In some embodiments, the anti-HER2 antibody or its antigen-binding fragment is Trastuzumab.

[0219] Thirdly, this application provides a bioactive compound of formula C or a pharmaceutically acceptable salt thereof.

[0220] Among them, X, Y, A, Z1, Z2, U 1 and U 2 The definition is as described in any of the embodiments in this disclosure.

[0221] In some embodiments, the bioactive compound has the structure shown in formula C-1.

[0222] Wherein, X, Y and A are defined as described in any of the embodiments of this disclosure.

[0223] Thirdly, this application provides a bioactive compound of formula III or a pharmaceutically acceptable salt thereof.

[0224] Wherein, X and A are defined as described in any of the embodiments of this disclosure.

[0225] In some embodiments, the bioactive compound is selected from:

[0226] Fourthly, this application provides compounds represented by Formula IV or pharmaceutically acceptable salts thereof.

[0227] Where A is selected from -NR 1 (CH2) m -、-NR 1 (CH2) m Q(CH2) p -、-(O)C(CH2) m -、-(O)C(CH2) m Q(CH2) p -、-S(O)2(CH2) m -、-S(O)2(CH2) m Q(CH2) p -、-S(O)(CH2) m -or -S(O)(CH2)m Q(CH2) p -;

[0228] Q is selected independently from -O-, -S-, or -NR. 2 -;

[0229] R 1 Independently selected from hydrogen, sulfonyl, and C 1-4 Alkyl, the C 1-4 The alkyl group is optionally substituted by one or more substituents selected from hydroxyl, sulfonic acid, phosphate and sugar groups;

[0230] R 2 Selected from hydrogen or C 1-4 alkyl;

[0231] m and p are each independently selected from any integer between 1 and 5;

[0232] B is hydrogen, hydroxyl group, or C. 1-4 alkyl;

[0233] And R 1 It is not methyl.

[0234] In some implementations, A is selected from -S(O)2(CH2). m -、-S(O)2(CH2) m Q(CH2) p -、-S(O)(CH2) m -or -S(O)(CH2) m Q(CH2) p -, B is C 1-4 alkyl.

[0235] In some implementations, A is selected from -NR 1 (CH2) m -or-NR 1 (CH2) m Q(CH2) p -, B is hydrogen.

[0236] In some implementations, A is selected from -(O)C(CH2). m -or -(O)C(CH2) m Q(CH2) p -, B is a hydroxyl group.

[0237] In some implementations, Q is -O-.

[0238] In some implementations, A is selected from -NR 1 (CH2) m -、-NR 1 (CH2) mO(CH2) p -、-NR 1 (CH2) m S(CH2) p -、-NR 1 (CH2) m NR 2 (CH2) p -、-(O)C(CH2) m -、-(O)C(CH2) m O(CH2) p -、-(O)C(CH2) m S(CH2) p -、-(O)C(CH2) m NR 2 (CH2) p -、-S(O)2(CH2) m -、-S(O)2(CH2) m O(CH2) p -、-S(O)2(CH2) m S(CH2) p -、-S(O)2(CH2) m NR 2 (CH2) p -、-S(O)(CH2) m -、-S(O)(CH2) m O(CH2) p -、-S(O)(CH2) m S(CH2) p -or -S(O)(CH2) m NR 2 (CH2) p -

[0239] In some implementations, A is selected from -NR 1 (CH2) m -、-NR 1 (CH2) m O(CH2) p -、-(O)C(CH2) m -、-S(O)2(CH2) m -or -S(O)(CH2) m -

[0240] In some implementations, A is selected from -NR 1 (CH2)3-、-NR 1(CH2)2O(CH2)2-, -(O)C(CH2)2-, -(O)C(CH2)3-, -S(O)2(CH2)3- or -S(O)(CH2)3-.

[0241] In some implementation schemes, R 1 Independently selected from hydrogen, sulfonyl, C 1-4 Alkyl, hydroxyl substituted C 1-4 alkyl and phosphate substituted C 1-4 C substituted with alkyl or sulfonic acid groups 1-4 alkyl or sugar-substituted C 1-4 alkyl.

[0242] In some implementation schemes, R 1 Independently selected from sulfonyl, C 1-4 Alkyl, hydroxyl substituted C 1-4 alkyl, n is selected from 1, 2, 3, and 4, with position 1 connected to the corresponding nitrogen atom.

[0243] In some implementation schemes, R 1 Independently selected from methanesulfonyl, -CH2CH2OH, The 1st position is connected to the corresponding nitrogen atom.

[0244] In some implementation schemes, R 2 It can be hydrogen or methyl.

[0245] In some implementations, m is selected from 1, 2, and 3.

[0246] In some implementations, p is selected from 1, 2, and 3.

[0247] In some embodiments, the compound is selected from:

[0248] This application provides the use of the above-mentioned compounds or pharmaceutically acceptable salts thereof in the preparation of drug linker conjugates.

[0249] This application provides the use of the above-mentioned compounds or pharmaceutically acceptable salts thereof in the preparation of antibody-drug conjugates (ADCs).

[0250] This application provides the use of the above-mentioned compound or a pharmaceutically acceptable salt thereof in the preparation of antibody-drug conjugates (ADCs) containing GSPT1 protein degraders.

[0251] Fifthly, this application provides a non-splitterable connector as shown in Formula V.

[0252] Wherein, X is defined as described in any of the embodiments of this disclosure, with position 1 linked to an antibody or its antigen-binding fragment, and position 2 linked to a bioactive molecule fragment.

[0253] In some embodiments, X is defined as described in any of the embodiments of this disclosure, with position 1 linked to Tb via an S atom and position 2 linked to a bioactive molecular fragment.

[0254] In some implementations, the non-splittable linkers are selected from:

[0255] Position 1 is linked to Tb via an S atom, and position 2 is linked to a bioactive molecular fragment.

[0256] This application provides the use of the above-mentioned non-cleavable linker in the preparation of drug linker conjugates.

[0257] This application provides the use of the above-mentioned non-cleavable linker in the preparation of antibody-drug conjugates (ADCs).

[0258] This application provides the use of the above-mentioned non-cleavable linker in the preparation of antibody-drug conjugates (ADCs) containing GSPT1 protein degraders.

[0259] Sixthly, this application provides a method for preparing a drug linker conjugate.

[0260] In some embodiments, the compound shown in Formula I-1 is obtained by reacting Formula I-1-1 and Formula I-1-2.

[0261] in,

[0262] A is selected from -S(O)2(CH2) m -、-S(O)2(CH2) m Q(CH2) p -、-S(O)(CH2) m -、-S(O)(CH2) m Q(CH2) p -、-NR 1 (CH2) m -or-NR 1 (CH2) m Q(CH2) p -;

[0263] Lg1 is selected from halogens, preferably bromine or iodine;

[0264] Lg, Q, R 1 The definitions of m and p are as described in any of the embodiments of this disclosure.

[0265] In some embodiments, the compound shown in formula I-2 is obtained by reacting formulas I-2-1 and I-2-2.

[0266] in,

[0267] Lg2 is selected from hydroxyl groups, halogens,

[0268] A is selected from -NR 1 (CH2) m -or-NR 1 (CH2) m Q(CH2) p -;

[0269] Lg, Q, R 1 The definitions of m and p are as described in any of the embodiments of this disclosure.

[0270] In some embodiments, the compound shown in formula I-3 is obtained by reacting formulas I-3-1 and I-3-2.

[0271] in,

[0272] G is selected from hydroxyl, halogen,

[0273] A is selected from -(O)C(CH2) m -or -(O)C(CH2) m Q(CH2) p -;

[0274] The definitions of Lg, Q, m, and p are as described in any of the embodiments of this disclosure.

[0275] In a seventh aspect, this application provides a method for preparing an antibody-drug conjugate, the method comprising: coupling Tb with the above-mentioned drug linker conjugate; wherein Tb and q are defined as described in any of the embodiments of this application.

[0276] Specifically, the method includes the step of coupling Tb with the above-mentioned drug linker conjugate in a solvent to form CS bonds.

[0277] In some embodiments, the molar ratio of Tb to the drug linker conjugate is 1:(1-20), such as 1:(2-16), 1:(2-14), 1:(2-12), or 1:(2-10).

[0278] In some embodiments, the coupling reaction is carried out in water and / or an organic solvent.

[0279] In some embodiments, the organic solvent is selected from one or more of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone, and nitrile compounds (e.g., acetonitrile).

[0280] In some embodiments, the method further includes a step of purifying the coupling product.

[0281] In some implementations, the coupling product is purified by chromatography.

[0282] In some embodiments, the chromatography method includes one or more of ion exchange chromatography, hydrophobic chromatography, reversed-phase chromatography, or affinity chromatography.

[0283] In some embodiments, the method is carried out at -20 to 100°C, for example 0 to 50°C, preferably at room temperature.

[0284] Eighthly, this application provides a group of antibody-drug conjugates comprising the aforementioned antibody-drug conjugates or pharmaceutically acceptable salts thereof or combinations thereof, wherein the antibody-drug conjugates have one, two or more q values.

[0285] In some implementations, when a q-value of an antibody-drug conjugate accounts for the majority (e.g., 80%, 85%, 90%, 95%, 95%, 97%, 98%, 99%) of the antibody-drug conjugate group, the q-value and the average DAR are close.

[0286] In some implementations, when there is only one q-value antibody-drug conjugate in the antibody-drug conjugate group, the q-value and the average DAR are equal.

[0287] In some embodiments, when the antibody-drug conjugates of the antibody-drug conjugate group have two or more q values, the proportion of the antibody-drug conjugate with one particular q value among all antibody-drug conjugates in the composition is greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0288] In some embodiments, the average drug-to-antibody ratio (average DAR) in the antibody-drug conjugate group is selected from an integer or decimal of 1-12, preferably an integer or decimal of 1-10.

[0289] In some embodiments, the average drug-to-antibody ratio (average DAR) of the antibody-drug conjugate group is selected from 1.5-2.5, 3.5-4.5, 5.5-6.5, or 7.5-8.5.

[0290] In some embodiments, the average drug-to-antibody ratio (average DAR) of the antibody-drug conjugate group is selected from about 2.0, 4.0, 6.0, or 8.0.

[0291] In some embodiments, the average drug-to-antibody ratio (average DAR) in the antibody-drug conjugate group is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9, 9, or 9.7.

[0292] In some embodiments, the antibody-drug conjugate group contains ADCs with a DAR distribution of 1 to 8, for example, 1.5, 2, 4, 6, and 8 (i.e., drug loadings of 1.5, 2, 4, 6, and 8). It is noteworthy that degradation products can be generated, such that the mixture may also contain DARs of 1, 3, 5, and 7. Furthermore, the antibody-drug conjugate group may also have an average DAR greater than 8. The antibody-drug conjugates are produced by reduction and subsequent coupling of interchain disulfides. In some embodiments, the antibody-drug conjugates comprise both: antibody-drug conjugates with a DAR of 4 or lower (i.e., drug loadings of 4 or lower) and antibody-drug conjugates with a DAR of 6 or higher (i.e., drug loadings of 6 or higher).

[0293] Ninthly, this application provides a pharmaceutical composition comprising the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof; or the aforementioned group of antibody-drug conjugates and optionally one or more pharmaceutical excipients.

[0294] In a tenth aspect, this application provides the use of the aforementioned drug-conjugates or pharmaceutically acceptable salts thereof; or the aforementioned antibody-drug conjugates or pharmaceutically acceptable salts thereof; or the aforementioned bioactive compounds or pharmaceutically acceptable salts thereof; or the aforementioned compounds or pharmaceutically acceptable salts thereof; or the aforementioned antibody-drug conjugate groups; or the aforementioned pharmaceutical compositions in the preparation of medicaments for treating and / or preventing diseases (e.g., cancer) related to abnormal cellular activity.

[0295] This application provides the aforementioned drug-conjugate or pharmaceutically acceptable salt thereof; or the aforementioned antibody-drug conjugate or pharmaceutically acceptable salt thereof; or the aforementioned bioactive compound or pharmaceutically acceptable salt thereof; or the aforementioned compound or pharmaceutically acceptable salt thereof; or the aforementioned group of antibody-drug conjugates; or the aforementioned pharmaceutical composition thereof, which are used as pharmaceuticals.

[0296] This application provides the aforementioned drug-conjugates or pharmaceutically acceptable salts thereof; or the aforementioned antibody-drug conjugates or pharmaceutically acceptable salts thereof; or the aforementioned bioactive compounds or pharmaceutically acceptable salts thereof; or the aforementioned compounds or pharmaceutically acceptable salts thereof; or the aforementioned groups of antibody-drug conjugates; or the aforementioned pharmaceutical compositions for the treatment and / or prevention of diseases (e.g., cancer) associated with abnormal cellular activity.

[0297] This application provides a method for preventing and / or treating diseases (e.g., cancer) associated with abnormal cellular activity, comprising: administering to an individual in need a preventive and / or therapeutically effective amount of the aforementioned drug-conjugate or a pharmaceutically acceptable salt thereof; or the aforementioned antibody-drug conjugate or a pharmaceutically acceptable salt thereof; or the aforementioned bioactive compound or a pharmaceutically acceptable salt thereof; or the aforementioned compound or a pharmaceutically acceptable salt thereof; or the aforementioned group of antibody-drug conjugates; or the aforementioned pharmaceutical composition.

[0298] In some implementations, the cancer is a solid tumor and / or a hematologic malignancy.

[0299] In some embodiments, the cancers described in this application are selected from esophageal cancer (e.g., esophageal adenocarcinoma or esophageal squamous cell carcinoma), lung cancer (e.g., small cell lung cancer or non-small cell lung 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, central nervous system tumors (e.g., glioma, glioblastoma multiforme, glioma, or sarcoma), prostate cancer or thyroid cancer, and hematologic malignancies (e.g., acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, plasmacytoid dendritic cell tumor).

[0300] In some implementations, the cancer is a hematologic malignancy.

[0301] In some implementations, the cancer is a cancer associated with targets of HER2, CD123, and / or CD33.

[0302] In some implementations, the cancerous diseases described in this application are selected from acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, multiple myeloma, plasmacytoid dendritic cell tumor, non-Hodgkin lymphoma, and Hodgkin lymphoma.

[0303] Without violating common sense in the field, the above preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this application.

[0304] All reagents and raw materials used in this application are commercially available.

[0305] definition

[0306] In this application, unless otherwise stated, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used in this application are all standard procedures widely used in their respective fields. To better understand this application, definitions and explanations of relevant terms are provided below.

[0307] As used herein, the term "drug-to-antibody ratio" or "DAR" refers to the amount of drug, such as a small molecule toxin attached to an antibody in 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 binding sites on the antibody. The term DAR may 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 contains an ADC with a DAR distribution of 1 to 8, for example, 1.5, 2, 4, 6, and 8 (i.e., drug loadings of 1.5, 2, 4, 6, and 8). Notably, degradation products can be generated, such that the ligand-drug conjugate may also contain DARs of 1, 3, 5, and 7. Furthermore, the ADC may also have a DAR greater than 8. The ADC is produced by reduction and subsequent coupling of an interchain disulfide. In some implementations, the ADC comprises both of the following: the ADC with a DAR of 4 or lower (i.e., a drug class of 4 or lower) and the ADC with a DAR of 6 or higher (i.e., a drug class of 6 or higher).

[0308] As used in this application, examples of the term "pharmaceutically acceptable salt" are organic acid adduct salts formed from organic acids that form pharmaceutically acceptable anions, including but not limited to formate, acetate, propionate, benzoate, maleate, fumarate, succinate, tartrate, citrate, ascorbate, α-ketoglutarate, α-glycerophosphate, alkyl sulfonates, or aryl sulfonates; preferably, the alkyl sulfonate is a methanesulfonate or ethyl sulfonate; and the aryl sulfonate is a benzenesulfonate or p-toluenesulfonate. Suitable inorganic salts may also be formed, including but not limited to hydrochlorides, hydrobromides, hydroiodates, nitrates, bicarbonates and carbonates, sulfates, or phosphates.

[0309] Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example, by reacting an adequate amount of a basic compound with a suitable acid that provides a pharmaceutically acceptable anion.

[0310] In this application, solid lines (—), solid wedges, or dashed wedges may be used to depict the carbon-carbon bonds of the compounds of this application. The use of solid lines to depict bonds to asymmetric carbon atoms indicates that all possible stereoisomers (e.g., specific enantiomers, racemic mixtures, etc.) are included at that carbon atom. The use of solid or dashed wedges to depict bonds to asymmetric carbon atoms indicates the presence of the indicated stereoisomers. When present in racemic mixtures, solid and dashed wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise specified, the compounds of this application are intended to exist as stereoisomers (including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, trans-isomers, and mixtures thereof). The compounds of this application may exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereomer pairs).

[0311] In this application, pharmaceutical excipients refer to the excipients and additives used in the production of pharmaceuticals and the formulation of prescriptions. They are substances, other than the active ingredient, that have undergone reasonable safety assessments and are included in the pharmaceutical preparation. Besides acting as a formifier, carrier, and improving stability, pharmaceutical excipients also have important functions such as solubilization, co-solubilization, and sustained-release. They are important components that may affect the quality, safety, and efficacy of pharmaceuticals. Based on their origin, they can be classified as natural substances, semi-synthetic substances, and fully synthetic substances. Based on their function and use, pharmaceutical excipients can be classified as follows: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc. Based on their route of administration, they can be classified as oral, injection, mucosal, transdermal or local, nasal or oral inhalation, and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations with different routes of administration and has different functions and uses.

[0312] The pharmaceutical composition can be formulated into various suitable dosage forms depending on the route of administration. Examples include tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder inhalers, and sprays. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of the compound of this application or its pharmaceutically acceptable salts or conjugates, preferably 0.1 mg to 800 mg, preferably 0.5-500 mg, more preferably 0.5-350 mg, and particularly preferably 1-250 mg.

[0313] The pharmaceutical composition can be administered in injectable form, including injection solutions, sterile powders for injection, and concentrated solutions for injection. Suitable carriers and solvents include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile non-volatile oils, such as monoglycerides or diglycerides, can also be used as solvents or suspension media. The pharmaceutical composition can also be administered via intravenous infusion.

[0314] As used in this application, the term "treatment" generally refers to achieving a desired pharmacological and / or physiological effect. This effect may be preventative based on the complete or partial prevention of a disease or its symptoms; and / or therapeutic based on the partial or complete stabilization or cure of a disease and / or side effects resulting from the disease. As used in this application, "treatment" encompasses any treatment of a patient's disease, including: (a) preventing a disease or symptoms occurring in a patient who is susceptible to the disease or its symptoms but has not yet been diagnosed with the disease; (b) suppressing the symptoms of the disease, i.e., preventing its progression; or (c) alleviating the symptoms of the disease, i.e., causing the disease or its symptoms to regress.

[0315] In this application, the term "individual" includes humans or non-human animals. Exemplary human individuals include individuals suffering from a disease (such as the disease described in this application) (referred to as patients) or healthy individuals. The term "non-human animal" in this application 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.).

[0316] In this application, the term "effective dose" refers to the amount of an antibody-drug conjugate, drug-linked conjugate, compound, or composition that, when administered, will alleviate one or more symptoms of the treated condition to a certain extent.

[0317] In this application, the terms "antibody-drug conjugate" and "ADC" refer to substances obtained by linking a bioactive compound fragment (drug molecule) or its GSPT1 protein degrader to an antibody or its antigen-binding fragment. In some embodiments of this application, the bioactive compound fragment or its GSPT1 protein degrader is linked to the target portion via an inclevable linker, which is completely stable under physiological conditions, regardless of the organ or biological compartment where the ADC drug resides. Therefore, ADCs with inclevable linkers depend on the complete (lysosomal) degradation of the antibody after the ADC is internalized into the cell. Due to this degradation, the payload is released, but still carries the linker, as well as peptide fragments and / or amino acids from which the linker was initially attached to the antibody, for example, the following ADC. After endocytosis into the lysosome, the antibody Tb is completely degraded, yielding a payload carrying the linker, as well as cysteine ​​fragments from the linker initially attached to the antibody.

[0318] In this application, the term "position 1 linked to Tb via an S atom" means, as those skilled in the art will understand, that position 1 is linked to the thiol group already present in Tb (such as an antibody) after the disulfide bond has been opened (e.g., by reducing the disulfide bond with the reducing agent TCEP to generate a thiol group -SH). In other words, the -S- linker between the linker and Tb is not an additional external sulfur atom. For example... In this context, -S- is not an additional external sulfur atom, but rather the thiol group and linker inherent in Tb itself after the disulfide bond is broken. -S- is formed by concatenating the 1st bit.

[0319] In this application, the term "antibody" is used in its broadest sense, including intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, provided they possess the desired biological activity. In this application, "antibody" and "immunoglobulin" are used interchangeably.

[0320] In this application, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that the antibodies constituting the cluster are identical except for a small number of possible natural mutations. Monoclonal antibodies possess high specificity against a single determinant (epitope) of an antigen, while polyclonal antibodies, in contrast, contain different antibodies targeting different determinants (epitopes). Besides specificity, the advantage of monoclonal antibodies is that their synthesis is unaffected by contamination from other antibodies. The modifier "monoclonal" here indicates that the antibody is characterized by originating from a substantially homogeneous group of antibodies, and should not be construed as requiring special methods for preparation.

[0321] In some embodiments of this application, monoclonal antibodies further specifically include chimeric antibodies, i.e., a portion of the heavy chain and / or light chain is identical or homologous to a certain type, class, or subclass of antibody, while the remainder is identical or homologous to another type, class, or subclass of antibody, provided they possess 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 this application include primatized antibodies, which comprise a variable region antigen-binding sequence from a non-human primate (e.g., ancient monkey, chimpanzee, etc.) and a human constant region sequence.

[0322] In this application, the term "antibody fragment" refers to a portion of an antibody, preferably an antigen-binding region or a variable region. Examples of antibody fragments include Fab, Fab′, F(ab′)2, Fd, Fv, dab, and complementarity-determining region fragments, diantibodies, linear antibodies, and single-chain antibody molecules.

[0323] In this application, the term "bispecific antibody" is also referred to as "bifunctional antibody conjugate," which refers to a conjugate formed by a first antibody (fragment) and a second antibody (fragment) through a conjugate arm. This conjugate retains the activity of each antibody and thus has both bifunctionality and bispecificity.

[0324] In this application, the term "multispecific antibody" includes, for example, trispecific antibodies and tetraspecific antibodies, the former being antibodies with three different antigen-binding specificities and the latter being antibodies with four different antigen-binding specificities.

[0325] In this application, the term "intact antibody" refers to an antibody comprising an antigen-binding variable region and a light chain constant region (CL), and heavy chain constant regions (CH1, CH2, and CH3). The constant regions can be natural sequences (e.g., human natural constant region sequences) or amino acid sequence variants thereof. Intact antibodies are preferably intact antibodies having one or more effector functions.

[0326] In this application, the term "probody" is a modified antibody, including an antibody or an antibody fragment that is specifically designed to bind to its target and is coupled to a masking group, wherein the masking group refers to a cleavage constant that is at least 100 times, 1000 times, or 10000 times greater than the cleavage constant that is not coupled to the target of an antibody or antibody fragment.

[0327] In this application, the “humanized” form of a non-human (e.g., mouse) antibody refers to a chimeric antibody containing a minimal amount of non-human immunoglobulin sequence. Most humanized antibodies are donor antibodies (e.g., mouse, rat, rabbit, or non-human primate) hypervariable region residues of human recipient immunoglobulins that have been replaced with non-human (e.g., mouse, rat, rabbit, or non-human primate) hypervariable region residues having the desired specificity, affinity, and function. In some embodiments, framework region (FR) residues of human immunoglobulins are also replaced with non-human residues. Furthermore, humanized antibodies may also contain residues not present in the recipient or donor antibody. These modifications are intended to further optimize antibody performance. Humanized antibodies generally contain at least one, typically two, variable regions, where all or almost all hypervariable loops correspond to those of the non-human immunoglobulin, while the FR is entirely or almost entirely a sequence of the human immunoglobulin. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc, typically 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.

[0328] Intact antibodies can be classified into different "classes" based on the amino acid sequence of their heavy chain constant regions. The five main classes are IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into different "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions of different antibody classes are referred to as α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different immunoglobulin classes are well known in the art.

[0329] In this application, although in most cases the amino acid substitutions in the antibody are L-amino acids, this is not the only possibility. 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 prone to degradation in the oral cavity, intestines, or plasma than peptides containing only L-amino acids.

[0330] The monoclonal antibodies used in this application can be produced by many methods. For example, the monoclonal antibodies used in this application can be obtained by hybridoma methods using cells from many species, including mice, hamsters, rats, and humans (see, for example, Kohler et al., 1975, Nature, 256:495), or 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).

[0331] In this application, unless otherwise expressly indicated, the descriptive phrases “each…independently selected” and “…independently selected” used throughout this application are interchangeable and should be interpreted broadly. They can mean that the specific options expressed by the same or different symbols in different groups do not affect each other, or that the specific options expressed by the same or different symbols in the same group do not affect each other.

[0332] In this application, the terms “comprising,” “including,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0333] In this application, in the structures shown by formulas A, A-1, I, I-1, I-2, I-3, B, B-1, II, II-1, II-2, II-3, C, C-1, III, IV, and V, the bonds at both ends of each variable group are connected to the chemical structures at their respective ends. For example, in the structure shown by formula A, when A is selected from -NR1(CH2)... m When -, the left-hand bond "-" in A is connected to the left-hand X in A, and the right-hand bond "-" in A is connected to the right-hand benzene ring in A.

[0334] In this application, the term "optional substitution" refers to the presence or absence of substituents.

[0335] In this application, the term "direct bond" refers to a covalent bond.

[0336] In this application, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0337] In this application, the term "glycosyl" refers to the group obtained by removing the hemiacetal hydroxyl group of a cyclic monosaccharide or oligosaccharide, including furanyl and pyranyl groups, such as glucosyl, galactosyl, mannosyl, etc.

[0338] In this application, the term "C1-4 alkyl" refers to a linear or branched aliphatic hydrocarbon group with 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl.

[0339] In this application, the term "sulfonyl" refers to -S(O)2R, where R represents a C1-4 alkyl group.

[0340] In this application, the term "phosphate group" refers to

[0341] In this application, the term "sulfonic acid group" refers to

[0342] In this application, the term "heterocyclic group" refers to a monocyclic or bicyclic system with 3 to 9 ring atoms that is monovalently saturated or partially unsaturated, containing 1, 2, or 3 heteroatoms selected from N, O, and S, with the remaining ring atoms being carbon. The term "4-6 membered heterocyclic group" refers to a monocyclic system with 4 to 6 ring atoms that is monovalently saturated, containing 1, 2, or 3 ring heteroatoms selected from N, O, and S, with the remaining ring atoms being carbon. Examples of monocyclic saturated heterocyclic groups are aziridinyl, ethylene oxide, aziridine, oxadiazinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydro-thiophenyl, pyrazolyl, imidazoyl, oxazolyl, isoxazolyl, thiazoyl, piperidinyl, tetrahydropyranyl, tetrahydrothiaranyl, piperazinyl, morpholinyl, thiomorpholinyl, and 1,1-dioxo-thiomorpholin-4-yl. Examples of bicyclic saturated heterocyclic groups are 8-aza-bicyclo[3.2.1]octyl, quininecyclo, 8-oxa-3-aza-bicyclo[3.2.1]octyl, 9-aza-bicyclo[3.3.1]nonyl, 3-oxa-9-aza-bicyclo[3.3.1]nonyl, or 3-thia-9-aza-bicyclo[3.3.1]nonyl. Examples of partially unsaturated heterocyclic groups are dihydrofuranyl, imidazolinyl, dihydrooxazolyl, tetrahydropyridyl, or dihydropyranyl. The term "sub-heterocyclic group" refers to a divalent heterocyclic group.

[0343] In this application, the term "antibody-drug conjugate group" refers to a group or cluster of antibody-drug conjugates disclosed herein, their stereoisomers, their prodrugs, their pharmaceutically acceptable salts, their tautomers, or their pharmaceutically acceptable solvates, wherein the q of the antibody-drug conjugates may be the same or different. Alternatively, it may be referred to as a "mixture of antibody-drug conjugates".

[0344] Beneficial effects of the invention

[0345] This application, through extensive research, has developed a novel drug linker conjugate and its antibody-drug conjugate composed of a non-cleavable linker and a protein degrading agent. Compared with antibody-drug conjugates obtained by drug linker conjugates composed of cleavable linkers and protein degrading agents in the prior art, it has more unexpected advantages:

[0346] 1. The antibody-drug conjugate has good stability under physiological conditions. Compared with commonly used cleavable linkers, the use of non-cleavable linkers can target the killing effect to the tumor or tissue site, reduce the entry of toxins into the circulatory system due to linker cleavage, and thus prevent systemic toxicity.

[0347] 2. The active molecules released by the antibody-drug conjugate in tumor cells have poor cell permeability, which can reduce side effects on normal tissues.

[0348] Therefore, the novel drug linker conjugates and their antibody-drug conjugates provided in this application have high clinical application value. Detailed Implementation

[0349] The following description of specific embodiments further illustrates this application, but it is not intended to limit the scope of the application. Those skilled in the art can make various modifications or improvements based on the teachings of this application without departing from its basic ideas and scope. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0350] The abbreviations used in this application have the following meanings:

[0351] Preparation scheme

[0352] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance (NMR). 1 It can be determined by 1H NMR or mass spectrometry (MS).

[0353] Nuclear magnetic resonance (NMR) 1 The H NMR (H2N) assay was performed using a Bruker 400MHz NMR spectrometer; the assay solvents were deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0354] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the embodiments are shown below.

[0355] 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: dimethyl sulfoxide deuterated. δ values ​​are expressed in ppm.

[0356] The mass spectrometry (MS) measurements were performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0357] The ultra-high performance liquid chromatography (UPLC) instrument used was AB SCIEX, model ExionLC.

[0358] The high-resolution mass spectrometer used for the measurements was an AB SCIEX X500B.

[0359] Methods for determining the antibody-drug conjugate ratio (DAR value):

[0360] Take 50 μg of ADC sample, dilute it with ultrapure water to 0.5 mg / ml, then add 1 μl of 1M DTT, mix well, centrifuge and take the supernatant for injection.

[0361] Liquid phase parameters:

[0362] Mass spectrometry parameters:

[0363] Example 1. Intermediate Synthesis

[0364] Example 1.1 Compound Synthesis

[0365] Example 1.1.1: Synthesis of compound INT1

[0366] Step 1:

[0367] Sodium hydride (4.6 g, 114.6 mmol, 60% purity) was dissolved in N,N-dimethylformamide (50 mL), and dimethyl malonate (13.8 g, 104.2 mmol) was added dropwise under ice bath. Then, INT1-1 (10.0 g, 52.1 mmol) dissolved in N,N-dimethylformamide (50 mL) was added to the reaction system. The reaction mixture was stirred at 70 °C for 4 h under nitrogen protection. The reaction was monitored by LCMS. The reaction was quenched by slowly adding saturated ammonium chloride solution (100 mL), extracted with ethyl acetate (200 mL * 3), and the combined organic phases were washed with saturated sodium chloride solution (200 mL * 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 5:1) to obtain the target compound INT1-2 (11.7 g).

[0368] LCMS(ESI)[M+H] + =287.9.

[0369] 1 H NMR (400MHz, DMSO-d6) δ8.37(d,J=2.3Hz,1H),8.24(dd,J=8.6,2.3Hz,1H),7.74(d,J=8.6Hz,1H),5.45(s,1H),3.74(s,6H).

[0370] Step Two:

[0371] Compound INT1-2 (5.0 g, 17.4 mmol) was dissolved in anhydrous methanol (100 mL), methyl acrylate (4.5 g, 52.3 mmol) was added, and sodium methoxide (94 mg, 1.74 mmol) was slowly added. The reaction mixture was stirred overnight at 50 °C under nitrogen protection. The reaction was monitored by LCMS. The reaction mixture was quenched with water (100 mL), extracted with dichloromethane (80 mL * 3), and the combined organic phases were washed with saturated sodium chloride solution (120 mL * 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 10:1) to give the target compound INT1-3 (2.6 g).

[0372] LCMS(ESI)[M+H] + =374.1.

[0373] 1H NMR(400MHz,DMSO-d6)δ8.32(d,J=2.4Hz,1H),8.20(dd,J=8.8,2.4Hz,1H),7.66( d,J=8.8Hz,1H),3.77(s,6H),3.55(s,3H),2.72–2.66(m,2H),2.30–2.24(m,2H).

[0374] Step 3:

[0375] Compound INT1-3 (2.6 g, 7.0 mmol) was dissolved in dimethyl sulfoxide (25 mL) and water (5 mL), and sodium chloride (1.2 g, 21.0 mmol) was added. The reaction mixture was stirred overnight at 150 °C. The reaction was monitored by TLC (petroleum ether: ethyl acetate = 15:1). The reaction mixture was quenched with water (30 mL), extracted with ethyl acetate (40 mL * 3), and the combined organic phases were washed with saturated sodium chloride solution (50 mL * 3). The organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 20:1) to give the target compound INT1-4 (720 mg).

[0376] 1 H NMR (400MHz, DMSO-d6) δ8.26 (d, J=2.3Hz, 1H), 8.15 (dd, J=8.4, 2.3Hz, 1H), 7.65 (d, J= 8.5Hz,1H),3.59(s,3H),2.86–2.81(m,2H),2.39(t,J=7.3Hz,2H),1.90–1.82(m,2H).

[0377] Step Four:

[0378] Compound INT1-4 (2 g, 7.76 mmol) was dissolved in methanol (10 mL), and platinum dioxide (352 mg, 1.55 mmol) was added under nitrogen protection. After the addition was complete, the mixture was purged three times with hydrogen, and the reaction was stirred at room temperature for 2 h under a hydrogen atmosphere. The reaction was monitored by LC-MS. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by normal-phase column chromatography (PE:EA = 10:1) to obtain the target compound INT1-5 (1.3 g).

[0379] LCMS(ESI)[M+H] + =228.12.

[0380] 1H NMR (400MHz, DMSO-d6) δ6.95(d,J=8.2Hz,1H),6.62(d,J=2.3Hz,1H),6.49(dd,J=8.2,2.3Hz ,1H),5.21(s,2H),3.61(s,3H),2.58–2.54(m,2H),2.32(t,J=7.4Hz,2H),1.81–1.71(m,2H).

[0381] Step 5:

[0382] Compound INT1-5 (1.2 g, 5.27 mmol) was dissolved in tetrahydrofuran (5 mL), and diphosgene (1.56 g, 7.90 mmol) was added. The mixture was stirred at room temperature for 30 min. The reaction solution was concentrated under reduced pressure, dissolved in DMF (5 mL), and then a DMF (5 mL) suspension of 3-[5-(aminomethyl)-1-oxoisoindololin-2-yl]piperidine-2,6-dione (1.73 g, 6.32 mmol) and triethylamine (5.33 g, 52.70 mmol) was added. After the addition was complete, the reaction was continued at room temperature for 1 h, and the reaction was monitored by LCMS. The reaction solution was diluted with water (50 mL), extracted with ethyl acetate (50 mL * 3), and the combined organic phases were concentrated under reduced pressure. The crude product was purified by normal phase column chromatography (DCM:MeOH = 10:1) to obtain the target compound INT1-6 (2 g).

[0383] LCMS(ESI)[M+H] + =527.23.

[0384] 1 H NMR(400MHz,DMSO-d6)δ11.00(s,1H),8.79(s,1H),7.99(s,1H),7.79–7.68(m,2H), 7.55(s,1H),7.47(dd,J=7.9,1.4Hz,1H),7.19(s,2H),6.83(t,J=6.0Hz,1H),5.13(d d,J=13.3,5.1Hz,1H),4.54–4.29(m,4H),3.61(s,3H),2.99–2.93(m,1H),2.68–2.64 (m,2H),2.45–2.39(m,1H),2.38–2.32(m,2H),2.07–1.99(m,1H),1.87–1.77(m,2H).

[0385] Step Six:

[0386] Compound INT1-6 (2 g, 3.8 mmol) was dissolved in concentrated hydrochloric acid (20 mL, 12 M), and the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. Water (100 mL) was added to the reaction solution, and the mixture was stirred for 30 min. The mixture was then filtered, and the filtrate was washed with water (10 mL) and dried to obtain the target compound INT1 (1.7 g).

[0387] LCMS(ESI)[M+H] + =513.20.

[0388] 1 H NMR(400MHz,DMSO-d6)δ11.00(s,1H),8.83(s,1H),7.75–7.65(m,2H),7.54(s, 1H),7.47(d,J=7.9Hz,1H),7.19(d,J=2.3Hz,2H),6.85(t,J=6.1Hz,1H),5.13( dd,J=13.3,5.1Hz,1H),4.53–4.28(m,4H),3.00–2.87(m,1H),2.68–2.60(m,3H ),2.47–2.33(m,1H),2.28–2.21(m,2H),2.08–1.97(m,1H),1.85–1.72(m,2H).

[0389] Example 1.1.2: Synthesis of compound INT2

[0390] Step 1:

[0391] Compound INT2-1 (10.0 g, 62.0 mmol), DMAP (1.52 g, 12.4 mmol), and imidazole (12.7 g, 186.1 mmol) were dissolved in DCM (250 mL). TBSCl (11.2 g, 74.4 mmol) was added at 0 °C. The reaction solution was stirred at room temperature for 2 h. The reaction solution was washed twice with water (250 mL * 2). The organic phase was concentrated under reduced pressure. The crude product was purified by normal phase column chromatography (ethyl acetate: petroleum ether = 0-30%) to obtain the target compound INT2-2 (14 g).

[0392] LCMS(ESI)[M+H-100] + =176.32.

[0393] 1H NMR (400MHz, DMSO-d6) δ6.70(t,J=5.4Hz,1H),3.53(t,J=6.3Hz,2H),3.05-2.93(m,2H),1.37(s,9H),0.85(s,9H),0.02(s,6H).

[0394] Step Two:

[0395] Compound INT2-2 (10 g, 36.3 mmol) was dissolved in DMF (100 mL). Sodium hydride (2.90 g, 72.6 mmol, 60% purity) was slowly added in portions at 0 °C. After the addition was complete, the reaction mixture was brought back to room temperature and stirred for 1 h. Then, the reaction system was cooled to 0 °C and bromopropyne (8.64 g, 72.6 mmol) was added. After the addition was complete, the reaction mixture was brought back to room temperature and stirred for 1 h. The reaction mixture was quenched with saturated ammonium chloride (50 mL), diluted with dichloromethane (250 mL), and washed twice with water (250 mL * 2). The organic phase was concentrated under reduced pressure. The crude product was purified by normal phase column chromatography (ethyl acetate: petroleum ether = 0-30%) to obtain the target compound INT2-3 (9 g, 50%).

[0396] LCMS(ESI)[M+H-100] + =214.20.

[0397] 1 H NMR (400MHz, DMSO-d6) δ4.04(s,2H),3.74–3.64(m,2H),3.30–3.28(m,2H),3.17(t,J=2.4Hz,1H),1.40(s,9H),0.86(s,9H),0.04(s,6H).

[0398] Step 3:

[0399] Compound INT2-3 (9.0 g, 28.7 mmol) was dissolved in tetrahydrofuran (90 mL), and bis(triphenylphosphine)palladium dichloride (2.01 g, 2.87 mmol), cuprous iodide (1.09 g, 5.74 mmol), and triethylamine (8.71 g, 86.1 mmol) were added sequentially. After the addition was complete, the reaction solution was stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure, and the crude product was purified by reversed-phase column chromatography (acetonitrile:H2O containing 0.1% FA = 10-90%) to obtain the target compound INT2-4 (4.1 g).

[0400] LCMS(ESI)[M+H-100] + =369.20.

[0401] 1H NMR (400MHz, DMSO-d6) δ8.39(d,J=2.3Hz,1H),8.18(dd,J=8.6,2.4Hz,1H),7.81(d,J=8.5Hz,1H),4 .49–4.35(m,2H),3.80–3.69(m,2H),3.41(t,J=6.2Hz,2H),1.42(s,9H),0.85(s,9H),0.03(s,6H).

[0402] Step Four:

[0403] Compound INT2-4 (200 mg, 0.43 mmol) was dissolved in methanol (5 mL), and platinum dioxide (20 mg, 10% w / w) was added under nitrogen protection. The mixture was purged with hydrogen three times and stirred at room temperature for 1 h. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.1% FA = 10-90%) to obtain the target compound INT2-5 (120 mg).

[0404] LCMS(ESI)[M+H-100] + =343.24.

[0405] Step 5:

[0406] Compound INT2-5 (120 mg, 0.27 mmol) was dissolved in tetrahydrofuran (3 mL), cooled to 0 °C, and diphosgene (80 mg, 0.41 mmol) was added. The reaction solution was stirred at room temperature for 1 h. After concentration under reduced pressure, the solution was dissolved in DMF (1 mL), and then a DMF (2 mL) solution of 3-(5-aminomethyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione hydrochloride (88.8 mg, 0.32 mmol) and triethylamine (274 mg, 2.71 mmol) was added. The reaction solution was stirred at room temperature for another 2 h. The reaction solution was then purified directly by reversed-phase column chromatography (acetonitrile: H2O containing 0.1% FA = 10-90%) to obtain the target compound INT2-6 (130 mg).

[0407] LCMS(ESI)[M+H-100] + =642.38.

[0408] Step Six:

[0409] Compound INT2-6 (60 mg, 0.08 mmol) was dissolved in a mixed solution of dichloromethane and trifluoroacetic acid (2 mL, 10 / 1, v / v), and the reaction solution was stirred at room temperature for 1 h. The reaction solution was concentrated under reduced pressure and distilled three times with dichloromethane (5 mL * 3) to obtain the trifluoroacetic acid salt of the target compound INT2 (40 mg).

[0410] LCMS(ESI)[M+H] + =528.27.

[0411] Example 1.1.3: Synthesis of compound INT3

[0412] Step 1:

[0413] Compound INT2-4 (4.3 g, 9.17 mmol) was dissolved in a mixed solution of acetic acid:tetrahydrofuran:water (40 mL, 3 / 1 / 1, v / v / v). The reaction solution was heated to 50 °C and stirred for 2 h. The reaction solution was concentrated under reduced pressure to remove the organic solvent. The resulting aqueous solution was purified by reversed-phase column chromatography (acetonitrile:H2O containing 0.1% FA = 10-90%) to obtain the target compound INT3-1 (3.3 g).

[0414] LCMS(ESI)[M+H-100] + =255.11.

[0415] 1 H NMR (400MHz, DMSO-d6) δ8.39 (d, J=2.3Hz, 1H), 8.18 (dd, J=8.6, 2.3Hz, 1H), 7.82 (d, J= 8.6Hz,1H),4.74(s,1H),4.42(s,2H),3.57(s,2H),3.36(t,J=6.3Hz,2H),1.42(s,9H).

[0416] Step Two:

[0417] Compounds INT3-1 (1.0 g, 2.82 mmol) and INT3-2 (2.24 g, 5.64 mmol) were dissolved in dichloromethane (30 mL). The reaction solution was cooled to 0 °C, and silver carbonate (946 mg, 5.64 mmol) and silver trifluoromethanesulfonate (1.45 g, 5.64 mmol) were added. After the addition was complete, the reaction solution was stirred at 0 °C for 2 h. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.1% FA = 10-90%) to obtain the target compound INT3-3 (570 mg).

[0418] LCMS(ESI)[M+H-100] + =571.16.

[0419] Step 3:

[0420] Compound INT3-3 (420 mg, 0.43 mmol) was dissolved in methanol (12 mL), and platinum dioxide (20 mg, 10% w / w) was added under nitrogen protection. The mixture was purged with hydrogen three times, and then stirred at room temperature for 2 h. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by Prep-TLC (methanol:tetrahydrofuran:dichloromethane = 1:5:50) to obtain the target compound INT3-4 (177 mg).

[0421] LCMS(ESI)[M+H-100] + =545.23.

[0422] 1 H NMR(400MHz,DMSO-d6)δ6.93(d,J=8.3Hz,1H),6.59(d,J=2.3Hz,1H),6.45(dd,J=8.2,2.3Hz,1H), 5.33(t,J=9.6Hz,1H),5.16(s,1H),4.96(t,J=9.8Hz,1H),4.88(d,J=7.9Hz,1H),4.80(dd,J=9.6, 7.9Hz,1H),4.45(d,J=9.9Hz,1H),4.07(s,1H),3.77(s,1H),3.64(s,3H),3.58(s,1H),3.17(s,3H ),3.13–3.06(m,1H),2.43(t,J=7.8Hz,2H),1.97(d,J=8.8Hz,9H),1.64(s,2H),1.39–1.34(m,9H).

[0423] Step Four:

[0424] Compound INT3-4 (60 mg, 0.27 mmol) was dissolved in tetrahydrofuran (2 mL), and diphosgene (27.9 mg, 0.14 mmol) was added at 0 °C. The reaction solution was stirred at room temperature for 1 h. After concentration under reduced pressure, the solution was dissolved in DMF (0.5 mL), and then a DMF (1 mL) solution of 3-(5-aminomethyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione hydrochloride (30.5 mg, 0.11 mmol) and triethylamine (94.1 mg, 0.93 mmol) was added. The reaction solution was stirred at room temperature for another 2 h. The reaction solution was then purified directly by reversed-phase column chromatography (acetonitrile: H2O containing 0.1% FA = 10-90%) to obtain the target compound INT3-5 (55 mg).

[0425] LCMS(ESI)[M+H-100] + =844.43.

[0426] 1 H NMR (400MHz, DMSO-d6) δ10.97(s,1H),8.79(s,1H),7.72(d,J=7.9Hz,1H),7.68(t,J=1.3Hz,1H),7.54(s,1H),7.47(dd,J=7.9,1.5Hz,1H),7.20(s ,2H),6.84(t,J=6.3Hz,1H),5.36(t,J=9.5Hz,1H),5.13(dd,J=13.3,5.1Hz,1H),4.99(t,J=9.7Hz,1H),4.91(d,J=7.9Hz,1H),4.83(dd,J=9.6,7. 9Hz, 1H), 4.50 (d, J = 2.7Hz, 1H), 4.45 (dd, J = 8.7, 5.4Hz, 3H), 4.34 (d, J = 1 7.5Hz,1H),3.80(s,1H),3.67(s,3H),3.63(s,1H),3.32(s,3H),3.14(dt ,J=13.7,7.1Hz,1H),3.01–2.87(m,1H),2.68–2.54(m,3H),2.43(td,J=1 3.2, 4.3Hz, 1H), 2.00 (d, J = 8.8Hz, 10H), 1.72 (s, 2H), 1.44–1.32 (m, 9H).

[0427] Step 5:

[0428] Compound INT3-5 (45 mg, 0.048 mmol) was dissolved in tetrahydrofuran (1 mL), and then hydrochloric acid aqueous solution (1 mL, 6 M) was added. After the addition was complete, the mixture was heated to 45 °C and stirred for 2 h. The reaction solution was concentrated under reduced pressure to remove tetrahydrofuran. The resulting aqueous solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.1% FA = 10-90%) to obtain the target compound INT3 (28 mg).

[0429] LCMS(ESI)[M+H] + =704.27.

[0430] 1H NMR (400MHz, DMSO-d6) δ10.96(s,1H),8.90(s,1H),7.69(dd,J=4.9,3.0Hz,2H),7.51(s,1H),7.44(dd,J =7.8,1.4Hz,1H),7.18(d,J=2.2Hz,2H),6.92(t,J=6.1Hz,1H),5.10(dd,J=13.3,5.1Hz,1H),4.98(s,1H ),4.47–4.28(m,4H),4.23(d,J=7.7Hz,1H),4.08(dd,J=10.6,3.9Hz,1H),3.76–3.70(m,1H),3.20–3.06 (m,6H),3.04–2.80(m,6H),2.67–2.61(m,3H),2.61–2.55(m,1H),2.46–2.31(m,1H),2.05–1.85(m,3H).

[0431] Example 1.1.4: Synthesis of compound INT4

[0432] Step 1:

[0433] Compound INT4-1 (10.0 g, 35.3 mmol) was dissolved in N,N-dimethylformamide (100 mL), followed by the addition of tert-butyl acrylate (5.4 g, 42.4 mmol), triethylamine (7.1 g, 70.7 mmol), and palladium acetate (1.2 g, 7.07 mmol). The reaction mixture was heated to 120 °C and stirred for 3 h under nitrogen protection. The reaction was monitored by TLC (petroleum ether: ethyl acetate = 5:1). The reaction mixture was quenched with water (100 mL), extracted with ethyl acetate (80 mL * 3), and the combined organic phases were washed with saturated sodium chloride aqueous solution (120 mL * 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 20:1) to obtain the target compound INT4-2 (7.6 g).

[0434] 1 H NMR(400MHz, DMSO-d6)) δ8.36(d,J=2.1Hz,1H),8.24–8.15(m,2H),7.82(d,J=15.9Hz,1H),6.80(d,J=15.9Hz,1H),1.51(s,9H).

[0435] Step Two:

[0436] Compound INT4-2 (500 mg, 1.76 mmol) was dissolved in MeOH, and platinum dioxide (250 mg) was added under nitrogen protection. The reaction was stirred at room temperature for 3 h under hydrogen atmosphere, and the reaction was monitored by LCMS. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound INT4-3 (380 mg).

[0437] LCMS(ESI)[M+H-56] + =200.21.

[0438] 1 H NMR (400MHz, DMSO-d6) δ6.93(d,J=8.2Hz,1H),6.58(d,J=2.3Hz,1H),6.43(dd,J=8.2 ,2.3Hz,1H),5.18(s,2H),2.72(t,J=7.6Hz,2H),2.40(t,J=7.6Hz,2H),1.37(s,9H).

[0439] Step 3:

[0440] Compound INT4-3 (100 mg, 0.39 mmol) was dissolved in THF (1 mL). A THF (1 mL) solution containing 77.15 mg, 0.39 mmol of diphosgene was added with stirring at room temperature. After the addition was complete, the reaction mixture was stirred at room temperature for 30 min. The reaction solution was concentrated under reduced pressure, dissolved in DMF (1 mL), and then a DMF (1 mL) solution containing 106.58 mg, 0.39 mmol of 3-(5-(aminomethyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione hydrochloride (106.58 mg, 0.39 mmol) was added. The reaction mixture was stirred at room temperature for 1 h, and the reaction was monitored by LC-MS. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.1% FA = 20%-90%) to obtain the target compound INT4-4 (56 mg).

[0441] LCMS(ESI)[M+H-56] + =499.30.

[0442] Step Four:

[0443] Compound INT4-4 (120 mg, 0.22 mmol) was dissolved in DCM (1 mL), and TFA (1 mL) was added with stirring at room temperature. The reaction was continued with stirring at room temperature for 1 h, and the reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and DCM (3 mL * 3) was added and concentrated under reduced pressure to remove TFA, yielding the target compound INT4 (108 mg), which was directly used in the next reaction.

[0444] LCMS(ESI)[M+H] + =499.19.

[0445] Example 1.1.5: Synthesis of intermediate INT5

[0446] Step 1:

[0447] Compound INT4-1 (5 g, 17.64 mmol), cuprous iodide (340 mg, 1.76 mmol), palladium dichloride (620 mg, 0.88 mmol), triethylamine (5.35 g, 52.92 mmol), and propargyl alcohol (1.19 g, 21.17 mmol) were dissolved in acetonitrile (50 mL). The reaction mixture was stirred at room temperature for 2 h under nitrogen protection, and the reaction was monitored by LC-MS. The reaction solution was directly purified by normal-phase column chromatography (DCM:MeOH = 1:1) to obtain the target compound INT5-1 (3.3 g).

[0448] 1 H NMR (400MHz, DMSO-d6) δ8.42(d,J=2.3Hz,1H),8.20(dd,J=8.6,2.4Hz,1H),7.85(d,J=8.6Hz,1H),5.56(t,J=6.0Hz,1H),4.44(d,J=6.0Hz,2H).

[0449] Step Two:

[0450] Compound INT5-1 (500 mg, 2.36 mmol) was dissolved in THF (8 mL), and acetic acid (2 mL) and platinum dioxide (250 mg) were added under nitrogen protection. The mixture was purged with hydrogen three times, and the reaction was stirred at room temperature for 3 h under a hydrogen atmosphere. The reaction was monitored by LCMS. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound INT5-2 (380 mg).

[0451] LCMS(ESI)[M+H] + =186.13.

[0452] 1 H NMR (400MHz, DMSO-d6) δ6.95(d,J=8.2Hz,1H),6.61(d,J=2.3Hz,1H),6.47(dd,J=8.2,2.3Hz,1H ),5.16(s,2H),4.46(t,J=5.1Hz,1H),3.47–3.38(m,2H),2.58–2.54(m,2H),1.70–1.59(m,2H).

[0453] Step 3:

[0454] Compound INT5-2 (360 mg, 1.94 mmol) was dissolved in methanol (5 mL), and di-tert-butyl dicarbonate (847 mg, 3.88 mmol) was added with stirring at room temperature. After the addition was complete, the reaction was continued with stirring at room temperature for 2 h, and the reaction was monitored by LCMS. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.1% FA = 20%-90%) to obtain the target compound INT5-3 (460 mg).

[0455] LCMS(ESI)[M+H-56] + =230.14.

[0456] Step Four:

[0457] Compound INT5-3 (100 mg, 0.35 mmol) was dissolved in DCM (4 mL). p-Toluenesulfonyl chloride (133.45 mg, 0.70 mmol), TEA (106.25 mg, 1.05 mmol), and DMAP (8.55 mg, 0.070 mmol) were added sequentially with stirring at room temperature. After the additions were complete, the reaction mixture was stirred at room temperature for 2 h, and the reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was purified by normal-phase column chromatography (EA:PE = 1:4) to obtain the target compound INT5-4 (110 mg).

[0458] LCMS(ESI)[M+Na] + =462.16.

[0459] 1 H NMR (400MHz, DMSO-d6) δ9.49 (s, 1H), 7.80 (d, J = 8.3Hz, 2H), 7.58 (d, J = 2.2Hz, 1H), 7.50 (d, J = 8.1Hz, 2H), 7.25 (dd, J = 8. 4,2.2Hz,1H),7.04(d,J=8.4Hz,1H),4.09–4.03(m,2H),2.64–2.56(m,2H),2.46(s,3H),1.90–1.79(m,2H),1.50(s,9H).

[0460] Step 5:

[0461] Compound INT5-4 (630 mg, 1.43 mmol), sodium metabisulfite (543.71 mg, 2.86 mmol), potassium formate (300.73 mg, 3.57 mmol), cesium carbonate (931.85 mg, 2.86 mmol), and tetrabutylammonium bromide (691.48 mg, 2.15 mmol) were dissolved in DMSO (5 mL). 5-Iodo-1-pentyne (693.59 mg, 3.57 mmol) was added with stirring at room temperature. After the addition was complete, the mixture was heated to 130 °C under nitrogen protection and stirred for 2 h. The reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H₂O containing 0.1% FA = 20%-90%) to obtain the target compound INT5-5 (80 mg).

[0462] LCMS(ESI)[M+Na] + =422.14.

[0463] 1 H NMR (400MHz, DMSO-d6) δ9.52(s,1H),7.63(d,J=2.1Hz,1H),7.34(dd,J=8.4,2.2Hz,1H),7.25(d,J=8.4Hz,1H),3. 22–3.12(m,4H),2.90(t,J=2.6Hz,1H),2.77(t,J=7.7Hz,2H),2.38–2.30(m,2H),2.03–1.82(m,4H),1.50(s,9H).

[0464] Step Six:

[0465] Compound INT5-5 (65 mg, 0.16 mmol) was dissolved in DCM (2 mL), and TFA (0.2 mL) was added with stirring at room temperature. After the addition was complete, the reaction was stirred at room temperature for 1 h, and the reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and DCM (3 mL * 3) was added to concentrate under reduced pressure to remove excess TFA, yielding the target compound INT5-6 (49 mg), which was directly used in the next reaction.

[0466] LCMS(ESI)[M+H] + =300.13.

[0467] Step Seven:

[0468] Compound INT5-6 (70 mg, 0.23 mmol) was dissolved in THF (1 mL). A THF (1 mL) solution containing 45 mg, 0.23 mmol of diphosgene was added with stirring at room temperature. After the addition was complete, the reaction mixture was stirred at room temperature for 30 min. The reaction solution was concentrated under reduced pressure, dissolved in DMF (1 mL), and then a DMF (1 mL) suspension of 3-(5-(aminomethyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione hydrochloride (71 mg, 0.23 mmol) and TEA (233 mg, 2.3 mmol) was added. After the addition was complete, the reaction mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was purified by reversed-phase column chromatography (acetonitrile: H2O containing 0.1% FA = 20%-90%) to obtain the target compound INT5 (80 mg).

[0469] LCMS(ESI)[M+H] + =599.20.

[0470] 1 H NMR(400MHz,DMSO-d6)δ11.00(s,1H),8.82(s,1H),7.77–7.65(m,2H),7.54(s, 1H),7.47(dd,J=7.9,1.4Hz,1H),7.27–7.19(m,2H),6.83(t,J=6.0Hz,1H),5.13 (dd,J=13.3,5.1Hz,1H),4.55–4.29(m,4H),3.22–3.11(m,4H),3.01–2.87(m,2 H),2.80–2.73(m,2H),2.67–2.54(m,2H),2.45–2.31(m,3H),2.04–1.85(m,4H).

[0471] Example 1.1.6: Synthesis of intermediate INT6

[0472] Step 1:

[0473] Compound INT6-1 (500 mg, 1.524 mmol) was dissolved in dichloromethane (10 mL), and phenyl chloroformate (497 mg, 3.048 mmol) was added. The mixture was heated to 100 °C for 5 h under nitrogen protection. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was purified by column chromatography (PE:EA = 5:1) to obtain the target compound INT6-2 (400 mg).

[0474] LCMS(ESI)[M+H-100] + =349.0.

[0475] 1H NMR (400MHz, DMSO-d6) δ10.36(s,1H),7.62(d,J=1.6Hz,1H),7.46-7.41(m,2H),7.39-7.35(m,1H),7.31(d,J=8.4Hz,1H),7.29-7.26(m,1H), 7.25-7.21(m,2H),3.58(t,J=6.8Hz,2H),3.48(t,J=5.6Hz,2H),3.28(t,J=5.7Hz,2H),2.87(t,J=6.6Hz,2H),2.77-2.74(m,3H),1.38(s,9H).

[0476] Step Two:

[0477] Compounds INT6-2 (320 mg, 0.714 mmol) and INT6-3 (234 mg, 0.857 mmol) were dissolved in N,N-dimethylformamide (3 mL), and sodium hydride (32 mg, 0.334 mmol, 60% purity suspended in paraffin oil) was added at 0 °C and reacted for 30 min. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and the crude product was purified by column chromatography (DCM:EA = 3:1) to obtain the target product INT6-4 (450 mg).

[0478] LCMS(ESI)[M+H-100] + =529.1.

[0479] 1 H NMR(400MHz,DMSO-d6)δ10.98(s,1H),9.94(s,1H),7.79(s,1H),7.69-7.58(m,3H),7.34-7.29 (m,1H),7.26(d,J=8.4Hz,1H),5.27(s,2H),5.13(dd,J=13.2,5.0Hz,1H),4.50-4.32(m,2H),3. 57(t,J=6.7Hz,2H),3.47(t,J=5.4Hz,2H),3.27(t,J=5.6Hz,2H),2.97-2.88(m,1H),2.85(t,J= 6.5Hz,2H),2.75(s,3H),2.64-2.57(m,1H),2.45-2.37(m,1H),2.05-2.00(m,1H),1.37(s,9H).

[0480] Step 3:

[0481] Compound INT6-4 (450 mg, 0.720 mmol) was dissolved in dichloromethane / trifluoroacetic acid (5 mL / 1 mL) and stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was directly concentrated under reduced pressure to obtain the trifluoroacetate salt of the target compound INT6 (450 mg), which was directly used in the next step of the reaction.

[0482] LCMS(ESI)[M+H] + =529.1.

[0483] 1 H NMR(400MHz,DMSO-d6)δ11.00(s,1H),9.98(s,1H),8.44(brs,1H),7.79(s,1H),7.70-7.6 2(m,2H),7.61(d,J=1.6Hz,1H),7.37-7.33(m,1H),7.31(t,J=8.4Hz,1H),5.28(s,2H),5. 12(dd,J=13.3,5.1Hz,1H),4.49-4.30(m,2H),3.67-3.60(m,4H),3.12-3.05(m,2H),2.96 -2.86(m,3H),2.66-2.58(m,1H),2.57-2.54(m,3H),2.46-2.36(m,1H),2.04-1.99(m,1H).

[0484] Example 1.1.7: Synthesis of intermediate INT7

[0485] Step 1:

[0486] Compound INT7-1 (500 mg, 1.67 mmol) was dissolved in dichloromethane (20 mL), and then phenyl chloroformate (523 mg, 3.34 mmol) and triethylamine (339 mg, 3.34 mmol) were added sequentially. After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction was monitored by TLC. The reaction solution was quenched with water (20 mL), extracted with dichloromethane (20 mL * 3), and the combined organic phases were washed with saturated brine (20 mL) and water (20 mL). The mixture was dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (ethyl acetate: petroleum ether = 0%-20%) to obtain the target compound INT7-2 (640 mg).

[0487] LCMS(ESI)[M+H-56] + =363.4.

[0488] Step Two:

[0489] Compounds INT7-2 (640 mg, 1.2 mmol) and INT6-3 (350 mg, 1.2 mmol) were dissolved in N,N-dimethylformamide (5 mL). Sodium hydroxide (77 mg, 1.9 mmol, 60% purity suspended in paraffin oil) was slowly added under ice bath conditions. After the addition was complete, the reaction was stirred under ice bath conditions for 30 min. The reaction was monitored by LCMS. The reaction solution was quenched with water (20 mL), extracted with ethyl acetate (20 mL * 3), and the combined organic phases were washed with saturated brine (20 mL) and water (20 mL). The mixture was dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (ethyl acetate: petroleum ether = 0%-95%) to obtain the target compound INT7-3 (700 mg).

[0490] LCMS(ESI)[M+H-100] + =499.5.

[0491] 1 H NMR (400MHz, CDCl3) δ8.33 (s, 1H), 7.89 (s, 1H), 7.57 (d, J = 7.3Hz, 1H), 7.52 ( s,1H),7.45(d,J=7.8Hz,1H),7.21(s,2H),7.12(d,J=8.3Hz,1H),5.25(s,2H ),5.21(dd,J=13.3,5.1Hz,1H),4.50-4.30(m,2H),3.26(s,2H),2.90-2.76( m,5H),2.68-2.60(m,2H),2.40-2.15(m,2H),1.83-1.75(m,2H),1.45(s,9H).

[0492] Step 3:

[0493] Compound INT7-3 (300 mg, 0.50 mmol) was dissolved in dichloromethane (5 mL), and then trifluoroacetic acid (1 mL) was added. After the addition was complete, the mixture was stirred at room temperature for 30 min. The reaction was monitored by LCMS. The reaction solution was directly concentrated under reduced pressure to obtain trifluoroacetate of the target compound INT7 (240 mg), which was directly used in the next step of the reaction.

[0494] LCMS(ESI)[M+H] + =499.5.

[0495] Example 1.1.8: Synthesis of intermediate INT8

[0496] Step 1:

[0497] Propylene alcohol (1.70 g, 30.0 mmol) was dissolved in ultra-dry tetrahydrofuran (20 mL), and sodium hydrogen (728 mg, 18.0 mmol, 60% purity suspended in paraffin oil) was slowly added. After the addition was complete, the mixture was stirred at room temperature for 30 min. Then, compound INT8-1 (2.6 g, 9.0 mmol) was added, and the mixture was stirred at room temperature for 30 min. The reaction was monitored by TLC. Water (20 mL) was added to the reaction solution to quench the reaction. The mixture was extracted with ethyl acetate (20 mL * 3). The combined organic phases were washed with saturated brine (20 mL) and water (20 mL). The mixture was dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (ethyl acetate: petroleum ether = 0%–10%) to obtain the target compound INT8-2 (1.8 g).

[0498] 1 H NMR (400MHz, DMSO-d6) δ9.34(s,1H),7.50(s,1H),7.33(d,J=8.3Hz,1H),7.22(t,J=7.8Hz,1H) ,6.91(d,J=7.5Hz,1H),4.46(s,2H),4.16(d,J=2.4Hz,2H),3.47(t,J=2.4Hz,1H),1.47(s,9H).

[0499] Step Two:

[0500] Compound INT8-2 (1.3 g, 5.0 mmol) was dissolved in acetonitrile (30 mL), and INT8-3 (1.5 g, 5.5 mmol), cuprous iodide (95 mg, 0.5 mmol), bis(triphenylphosphine)palladium dichloride (350 mg, 0.5 mmol), and triethylamine (1.7 g, 17.5 mmol) were added sequentially. After the addition was complete, the mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The reaction solution was filtered through diatomaceous earth and then concentrated under reduced pressure. The crude product was purified by column chromatography (ethyl acetate: petroleum ether = 0%–20%) to obtain the target compound INT8-4 (1.5 g).

[0501] LCMS(ESI)[M+Na] + =439.1.

[0502] 1H NMR(400MHz, DMSO-d6)δ9.36(s,1H),8.40(d,J=2.0Hz,1H),8.24-8.14(m,1H),7.87(d,J=8.6Hz,1H),7.57(s,1 H),7.35(d,J=8.4Hz,1H),7.24(t,J=7.8Hz,1H),6.96(d,J=7.5Hz,1H),4.60(s,2H),4.55(s,2H),1.47(s,9H).

[0503] Step 3:

[0504] Compound INT8-4 (1.5 g, 3.6 mmol) was dissolved in tetrahydrofuran (30 mL), and Raney nickel (3.0 g, 52 mmol) was added under nitrogen protection. The mixture was purged with hydrogen three times, and the reaction solution was stirred at room temperature for 48 h under a hydrogen atmosphere. The reaction was monitored by LCMS. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain the target compound INT8-5 (1.2 g).

[0505] LCMS(ESI)[M+H] + =391.1.

[0506] 1 H NMR (400MHz, DMSO-d6) δ9.33 (s, 1H), 7.50 (s, 1H), 7.34 (d, J = 8.0Hz, 1H), 7.21 (t, J = 7.8Hz, 1H), 6.96-6.88 (m, 2H), 6.59 (d, J = 2.2Hz, 1H),6.44(dd,J=8.2,2.2Hz,1H),5.16(s,2H),4.39(s,2H),3.41(t,J=6.3Hz,2H),2.63-2.53(m,2H),1.80-1.69(m,2H),1.48(s,9H).

[0507] Step Four:

[0508] Compound INT8-5 (500 mg, 1.28 mmol) was dissolved in dichloromethane (20 mL), followed by the addition of phenyl chloroformate (392 mg, 1.92 mmol) and triethylamine (259 mg, 2.56 mmol) to the reaction solution. The reaction solution was stirred at room temperature for 1 h; the reaction was monitored by TLC. The reaction solution was directly concentrated under reduced pressure to obtain a crude product (700 mg). The crude product was dissolved in N,N-dimethylformamide (10 mL), followed by the addition of 3-(5-aminomethyl)-1-oxoisoindololin-2-yl)piperidine-2,6-dione. Hydrochloride (305 mg, 1.1 mmol) and triethylamine (259 mg, 2.56 mmol) were added. After the addition, the reaction solution was heated to 60 °C and stirred for 5 h. The reaction was monitored by LCMS. Water (40 mL) was added to the reaction solution to quench the reaction. The solution was extracted with ethyl acetate (20 mL * 3). The combined organic phases were washed with saturated brine (20 mL) and water (20 mL). The solution was dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (methanol: dichloromethane = 0%–10%) to obtain the target compound INT8-6 (500 mg).

[0509] LCMS(ESI)[M+H-100] + =590.3.

[0510] 1 H NMR (400MHz, DMSO-d6) δ10.97(s,1H),9.32(d,J=4.2Hz,1H),7.77-7.62(m,2H),7.54-7.47(m,2H),7.44(d,J=7.9Hz,1H ),7.41-7.30(m,2H),7.20(dd,J=16.1,8.1Hz,2H),7.15(s,1H),7.10(d,J=8.2Hz,1H),6.89(dd,J=12.3,7.1Hz,1H),5. 10(dd,J=13.2,5.0Hz,1H),4.50-4.26(m,6H),4.14(d,J=6.0Hz,1H),3.45-3.36(m,2H),3.31-3.25(m,1H),2.97-2.85( m,1H),2.71-2.64(m,1H),2.60(d,J=16.8Hz,1H),2.45-2.31(m,1H),2.04-1.95(m,1H),1.84-1.73(m,1H),1.47(s,9H).

[0511] Step 5:

[0512] Compound INT8-6 (500 mg, 0.72 mmol) was dissolved in dichloromethane (10 mL), and then trifluoroacetic acid (2.5 mL) was added. After the addition was complete, the mixture was stirred at room temperature for 30 min. The reaction was monitored by LCMS. The reaction solution was directly concentrated under reduced pressure to remove excess trifluoroacetic acid, yielding the target compound INT8 (400 mg).

[0513] LCMS(ESI)[M+H] + =590.3.

[0514] 1 H NMR (400MHz, DMSO-d6) δ10.98 (s, 1H), 8.77 (s, 1H), 7.68 (d, J = 12.2Hz, 2H), 7.48 (d,J=27.0Hz,2H),7.15(s,2H),7.02-6.79(m,2H),6.62-6.48(m,3H),5.83-5.23 (m,2H),5.19-5.02(m,1H),4.51-4.25(m,6H),3.36-3.30(m,2H),3.02-2.85(m, 1H),2.76-2.61(m,3H),2.44-2.31(m,1H),2.09-1.90(m,1H),1.87-1.68(m,2H).

[0515] Example 1.2 Linker Synthesis

[0516] Example 1.2.1: Synthesis of connector L1

[0517] Step 1:

[0518] Sodium hydride (327 mg, 8.19 mmol, 60%) was dissolved in DMF (10 mL). A DMF solution of L1-1 (1.0 g, 5.46 mmol) in 6 mL was added at 0 °C. After the addition was complete, the reaction mixture was brought to room temperature and stirred for 1 h. Then, iodomethane (1.01 g, 7.09 mmol) was added, and the reaction mixture was stirred for another 2 h at room temperature. The reaction was quenched by adding saturated ammonium chloride aqueous solution (1 mL), diluted with ethyl acetate (50 mL), and washed three times with water (50 mL x 3). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound L1-2 (980 mg), which was directly used in the next reaction.

[0519] LCMS(ESI)[M+H-56] + =142.24.

[0520] 1H NMR (400MHz, DMSO-d6) δ3.26–3.16(m,2H),2.83–2.72(m,4H),2.16–2.09(m,2H),1.62(s,2H),1.39(s,9H).

[0521] Step Two:

[0522] Compound L1-2 (250 mg, 1.27 mmol), 5-bromo-2-(methylsulfonyl)pyrimidine (250 mg, 1.06 mmol), Pd(PPh3)2Cl2 (74 mg, 0.11 mmol), and cuprous iodide (20 mg, 0.11 mmol) were dissolved in tetrahydrofuran (5 mL), followed by the addition of triethylamine (320 mg, 3.17 mmol). After the addition was complete, the reaction mixture was microwave-heated to 65 °C for 30 min. The reaction mixture was diluted with ethyl acetate (30 mL), washed with saturated brine (30 mL x 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was dissolved in DCM (10 mL), loaded onto a normal-phase column chromatography (ethyl acetate: petroleum ether = 0%–50%), and purified to obtain the target compound L1-3 (250 mg).

[0523] LCMS(ESI)[M+H-56] + =298.14.

[0524] 1 H NMR (400MHz, DMSO-d6) δ9.11(s,2H),3.41(s,3H),3.32–3.28(m,2H),2.80(s,3H),2.58–2.52(m,2H),1.83–1.73(m,2H),1.38(s,9H).

[0525] Step 3:

[0526] Compound L1-3 (35 mg, 0.099 mmol) was dissolved in DCM (2 mL), and TFA (0.2 mL) was added under stirring at room temperature. The reaction was continued for 30 min with stirring. The reaction was monitored by LCMS. The reaction solution was concentrated under reduced pressure, and residual TFA was removed by distillation with DCM (5 x 3 mL) to obtain trifluoroacetate of the target compound L1 (36.4 mg, 0.099 mmol), which was directly used in the next step of the reaction.

[0527] LCMS(ESI)[M+H] + =254.16.

[0528] Example 1.2.2: Synthesis of connector L2

[0529] Step 1:

[0530] Compound DL001-2 (5.0 g, 18.6 mmol), N-hydroxysuccinimide (2.57 g, 22.4 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.29 g, 22.4 mmol) were dissolved in dichloromethane (100 mL), and the reaction solution was stirred at room temperature for 2 h. The reaction solution was then purified directly by normal-phase column chromatography (methanol:dichloromethane = 0-10%) to give the target compound L2 (5.8 g).

[0531] LCMS(ESI)[M+H] + =366.13.

[0532] Example 1.2.3: Synthesis of connector L3

[0533] Step 1:

[0534] Compound L3-1 (5.0 g, 50.9 mmol) was dissolved in toluene (170 mL), tetrabutylammonium bromide (6.3 g, 19.6 mmol) was added, the mixture was cooled to 0 °C, sodium hydroxide (61.2 g, 535.5 mmol) was added, followed by tert-butyl bromoacetate (34.78 g, 178.32 mmol), and the reaction was allowed to proceed overnight at room temperature. Water (80 mL) was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate (80 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the target compound L3-2 (10.2 g).

[0535] 1 H NMR (400MHz, DMSO-d6) δ8.67(s,2H),3.98(s,2H),3.57(t,J=6.2Hz,2H),2.57–2.54(m,2H),2.52(s,3H),1.83–1.76(m,2H),1.42(s,9H).

[0536] Step Two:

[0537] Compound L3-2 (10 g, 50.5 mmol) was dissolved in tetrahydrofuran (100 mL), and L3-3 (10.35 g, 50.5 mmol), bis(triphenylphosphine)palladium dichloride (3.5 g, 5.05 mmol), cuprous iodide (1.92 g, 10.1 mmol), and triethylamine (15.3 g, 151.5 mmol) were added. The mixture was stirred overnight at 70 °C under a nitrogen atmosphere. The reaction solution was cooled and filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (PE:EA = 5:1) to obtain the target compound L3-4 (5.2 g).

[0538] LCMS(ESI)[M+H] + =323.0;

[0539] 1 H NMR (400MHz, DMSO-d6) δ8.67(s,2H),3.98(s,2H),3.57(t,J=6.2Hz,2H),2.57–2.54(m,2H),2.52(s,3H),1.83–1.76(m,2H),1.42(s,9H).

[0540] Step 3:

[0541] Compound L3-4 (1.0 g, 3.1 mmol) was dissolved in DCM (50 mL), and TFA (3.54 g, 31 mmol) was added. The mixture was stirred overnight at room temperature. The reaction solution was then concentrated under reduced pressure to give compound L3-5 (1.5 g).

[0542] LCMS(ESI)[M+H] + =267.0;

[0543] 1 H NMR (400MHz, DMSO-d6) δ8.67(s,2H),4.03(s,2H),3.59(t,J=6.2Hz,2H),2.58–2.53(m,2H),2.53(s,3H),1.84–1.77(m,2H).

[0544] Step Four:

[0545] Compound L3-5 (1.5 g, 5.6 mmol) was dissolved in THF (20 mL) and H2O (20 mL), and Oxone (10.3 g, 16.8 mmol) was added. The mixture was stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure to remove tetrahydrofuran. The aqueous phase was extracted three times with EA (50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by reversed-phase chromatography (acetonitrile: water containing 0.05% HCl = 5%-50%) to obtain the target compound L3 (0.4 g).

[0546] LCMS(ESI)[M+H] + =299.1;

[0547] 1 H NMR (400MHz, DMSO-d6) δ12.60(s,1H),9.11(s,2H),4.03(s,2H),3.60(t,J=6.2Hz,2H),3.41(s,3H),2.63(t,J=7.1Hz,2H),1.87–1.80(m,2H).

[0548] Example 2. Synthesis of drug linker conjugates

[0549] Example 2.1: Synthesis of DL001

[0550] Step 1:

[0551] Compound DL001-2 (100 mg, 0.201 mmol) was dissolved in DMF (2 mL), and then compound DL001-1 (52 mg, 0.201 mmol, synthesis method according to patent WO2023221975), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (76 mg, 0.201 mmol), and DIPEA (0.06 mL, 0.381 mmol) were added sequentially. After the addition was complete, the mixture was stirred at room temperature for 2 h, and the reaction was monitored by LC-MS. The reaction solution was directly purified by preparative high performance liquid chromatography (acetonitrile: water containing 0.05% FA = 5%-90%) to obtain the target compound DL001 (25.73 mg).

[0552] LCMS(ESI)[M+H] + =748.1.

[0553] 1 H NMR (400MHz, DMSO-d6) δ10.98(s,1H),9.10(d,J=11.9Hz,2H),8.76(s,1H),7.69(d,J=7.9Hz,1 H),7.66–7.61(m,1H),7.51(s,1H),7.44(d,J=7.4Hz,1H),7.21–7.13(m,2H),6.80(t,J=6.1Hz, 1H),5.10(dd,J=13.2,5.0Hz,1H),4.48–4.30(m,4H),3.40(d,J=3.5Hz,3H),2.99–2.80(m,4H), 2.68–2.53(m,5H),2.47–2.29(m,4H),2.05–1.93(m,2H),1.87–1.73(m,3H),1.72–1.62(m,1H).

[0554] Example 2.2: Synthesis of DL002

[0555] Step 1:

[0556] Compounds DL001-2 (61.17 mg, 0.23 mmol) and DL002-1 (100 mg, 0.19 mmol, synthetic method according to patent WO2021198965) were dissolved in DMF (2.5 mL), and HATU (86.69 mg, 0.23 mmol) and DIPEA (73.67 mg, 0.57 mmol) were added sequentially. The reaction solution was stirred at room temperature for 2 h. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: water containing 0.1% FA = 10%-90%) to obtain the target compound DL002 (28.16 mg).

[0557] LCMS(ESI)[M+H] + =778.24.

[0558] 1 H NMR (400MHz, DMSO-d6) δ10.97(s,1H),9.09(d,J=9.4Hz,2H),8.76(s,1H),7.69(d,J=7.8Hz,1H),7 .65(t,J=2.1Hz,1H),7.51(s,1H),7.43(d,J=7.8Hz,1H),7.23–7.09(m,2H),6.82–6.75(m,1H),5.1 0(dd,J=13.3,5.2Hz,1H),4.49–4.27(m,4H),3.59–3.50(m,3H),3.50–3.44(m,2H),3.43–3.38(m,4 H),2.97–2.78(m,6H),2.64–2.52(m,3H),2.48–2.31(m,3H),2.04–1.95(m,1H),1.84–1.75(m,2H).

[0559] Example 2.3: Synthesis of DL003

[0560] Step 1:

[0561] Compound INT1 (50.78 mg, 0.099 mmol) was dissolved in DMF (3 mL). Compounds L1 (25.08 mg, 0.099 mmol), HATU (47.05 mg, 0.12 mmol), and DIPEA (63.97 mg, 0.49 mmol) were added sequentially with stirring at room temperature. After the additions were complete, the reaction mixture was stirred at room temperature for 1 h, and the reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile:water containing 0.1% FA) to obtain the target compound DL003 (28.26 mg).

[0562] LCMS(ESI)[M+H] +=748.47.

[0563] 1 H NMR (400MHz, DMSO-d6) δ11.00(s,1H),9.13(d,J=4.5Hz,2H),8.77(d,J=6.7Hz,1H),7 .75–7.63(m,2H),7.54(s,1H),7.47(d,J=7.9Hz,1H),7.24–7.12(m,2H),6.86–6.78( m,1H),5.13(dd,J=13.3,5.1Hz,1H),4.55–4.31(m,4H),3.49–3.42(m,5H),3.01–2.8 3(m,4H),2.67–2.55(m,5H),2.44–2.30(m,3H),2.08–1.98(m,1H),1.89–1.72(m,4H).

[0564] Example 2.4: Synthesis of DL004

[0565] Step 1:

[0566] Compounds INT2 (40 mg, 0.08 mmol), DL001-2 (15.3 mg, 0.09 mmol), and HATU (34.6 mg, 0.09 mmol) were dissolved in DMF (1 mL), and then DIPEA (29.4 mg, 0.23 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.1% FA = 10-90%) to obtain the target compound DL004 (8.4 mg).

[0567] LCMS(ESI)[M+H] + =778.36.

[0568] 1H NMR (400MHz, DMSO-d6) δ10.96(s,1H),9.09(d,J=8.2Hz,2H),8.80(s,1H),7.69(d,J=7.8Hz,1H),7.65–7.61(m ,1H),7.51(s,1H),7.46–7.41(m,1H),7.20–7.13(m,2H),6.85(t,J=5.9Hz,1H),5.10(dd,J=13.3,5.1Hz,1H),4 .86–4.59(m,1H),4.48–4.27(m,4H),3.56-3.51(m,1H),3.50-3.42(m,1H),3.40(d,J=3.7Hz,3H),3.37(dd,J=6 .8,0.7Hz,3H),2.96–2.85(m,1H),2.64–2.51(m,7H),2.45–2.35(m,2H),2.04–1.95(m,1H),1.86–1.65(m,4H).

[0569] Example 2.5: Synthesis of DL005

[0570] Step Six:

[0571] Compounds INT3 (28.0 mg, 0.04 mmol) and L2 (21.8 mg, 0.06 mmol) were dissolved in DMF (2 mL), and then DIPEA (51.4 mg, 0.40 mmol) was added. The reaction solution was stirred at room temperature for 2 h. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.1% FA = 10-90%) to obtain the target compound DL005 (11 mg).

[0572] LCMS(ESI)[M+H] + =954.35.

[0573] 1H NMR (400MHz, DMSO-d6) δ10.96(s,1H),9.09(d,J=10.9Hz,2H),8.93(d,J=4.8Hz,1H),7.68(d,J=7.8Hz,1H),7.64(dd,J=7.1,2. 1Hz,1H),7.51(s,1H),7.44(d,J=8.1Hz,1H),7.22–7.12(m,2H),7.01–6.92(m,1H),5.09(dd,J=13.2,5.1Hz,1H),4.99–4.89(m, 2H),4.49–4.27(m,4H),4.19(dd,J=16.9,7.6Hz,1H),3.81–3.72(m,1H),3.60(dd,J=48.9,5.7Hz,2H),3.39(d,J=3.1Hz,7H),3 .18(d,J=9.3Hz,3H),2.99–2.72(m,3H),2.58(dd,J=16.2,9.8Hz,5H),2.44–2.35(m,2H),2.04–1.95(m,1H),1.85–1.65(m,4H).

[0574] Example 2.6: Synthesis of DL006

[0575] Step 1:

[0576] Compound INT4 (29.94 mg, 0.06 mmol) was dissolved in DMF (3 mL). L1 (18.24 mg, 0.072 mmol), HATU (28.52 mg, 0.075 mmol), and DIPEA (38.77 mg, 0.30 mmol) were added sequentially with stirring at room temperature. After the additions were complete, the reaction mixture was stirred at room temperature for 1 h, and the reaction was monitored by LCMS. The reaction solution was purified by preparative high-performance liquid chromatography (acetonitrile: H2O containing 0.1% FA = 20%-90%) to obtain the target compound DL006 (20 mg).

[0577] LCMS(ESI)[M+H] + =734.14.

[0578] 1H NMR(400MHz,DMSO-d6)δ10.98(s,1H),9.15–9.04(m,2H),8.77(d,J=17.1Hz,1H),7.8 0–7.62(m,2H),7.56–7.53(m,1H),7.50–7.44(m,1H),7.24–7.10(m,2H),6.86–6.77( m,1H),5.12(dd,J=13.3,5.1Hz,1H),4.53–4.29(m,4H),3.49–3.40(m,6H),3.01–2.8 3(m,5H),2.72–2.54(m,5H),2.47–2.34(m,1H),2.08–2.00(m,1H),1.93–1.76(m,2H).

[0579] Example 2.7: Synthesis of DL007

[0580] Step 8:

[0581] Compound INT5 (50 mg, 0.083 mmol), 5-bromo-2-methanesulfonylpyrimidine (19.68 mg, 0.083 mmol), bis(triphenylphosphine) palladium dichloride (5.83 mg, 0.0083 mmol), and cuprous iodide (1.58 mg, 0.0083 mmol) were dissolved in THF (3 mL). Triethylamine (25.20 mg, 0.25 mmol) was added with stirring at room temperature. The mixture was filtered, washed with DMF (2 mL), and the filtrate was purified by preparative high performance liquid chromatography (acetonitrile: H2O containing 0.1% FA = 20%-90%) to obtain the target compound DL007 (9.5 mg).

[0582] LCMS(ESI)[M+H] + =755.29.

[0583] 1H NMR(400MHz,DMSO-d6)δ10.99(s,1H),9.16(s,2H),8.90(s,1H),7.74–7.68(m,2H),7.54 (s,1H),7.47(d,J=7.9Hz,1H),7.25–7.19(m,2H),6.91(t,J=5.9Hz,1H),5.13(dd,J=13.3 ,5.1Hz,1H),4.53–4.29(m,4H),3.43(s,3H),3.30–3.27(m,3H),3.22–3.16(m,2H),2.98– 2.89(m,1H),2.82–2.72(m,4H),2.66–2.59(m,1H),2.47–2.39(m,1H),2.05–1.97(m,4H).

[0584] Example 2.8: Synthesis of DL008

[0585] Step 1:

[0586] Compound INT7 (100 mg, 0.20 mmol) was dissolved in DMF (1 mL), followed by the sequential addition of compound DL001-2 (54 mg, 0.20 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (76 mg, 0.20 mmol), and DIPEA (65 mg, 0.50 mmol). After addition, the mixture 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:water containing 0.05% FA = 5%-50%) to obtain the target compound DL008 (30 mg).

[0587] LCMS(ESI)[M+H] + =748.8.

[0588] 1H NMR (400MHz, DMSO-d6) δ10.99(s,1H),9.94(s,1H),9.10(d,J=13.0Hz,2H),7.79(s,1H),7.66(dd,J=18.7,7.9 Hz,2H),7.60–7.55(m,1H),7.32(dd,J=8.3,1.8Hz,1H),7.28–7.22(m,1H),5.27(s,2H),5.13(dd,J=13.3,5.1H z,1H),4.50–4.25(m,2H),3.40(d,J=2.7Hz,3H),3.33(d,J=7.3Hz,2H),2.95(d,J=6.3Hz,2H),2.95–2.85(m,1H ),2.81(s,1H),2.64–2.55(m,5H),2.48–2.37(m,3H),2.06–1.96(m,1H),1.85–1.76(m,3H),1.73–1.65(m,1H).

[0589] Example 2.9: Synthesis of DL009

[0590] Step 1:

[0591] Compound INT6 (120 mg, 0.23 mmol) was dissolved in DMF (2 mL), followed by the sequential addition of compound DL001-2 (61 mg, 0.23 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (86 mg, 0.23 mmol), and DIPEA (73 mg, 0.58 mmol). After addition, the mixture was stirred at room temperature for 1 h. The reaction was monitored by LC-MS. The reaction solution was directly purified by preparative high-performance liquid chromatography (acetonitrile:water containing 0.05% FA = 5%-50%) to obtain the target compound DL009 (30 mg).

[0592] LCMS(ESI)[M+H] + =779.2.

[0593] 1H NMR (400MHz, DMSO-d6) δ10.99(s,1H),9.95(s,1H),9.09(d,J=9.4Hz,2H),7.79(s,1H),7.70–7.61(m,2H),7.58(s ,1H),7.35–7.29(m,1H),7.28–7.20(m,1H),5.27(s,2H),5.12(dd,J=13.3,5.1Hz,1H),4.50–4.28(m,2H),3.60–3 .51(m,3H),3.49–3.44(m,2H),3.42(d,J=5.1Hz,1H),3.40(d,J=5.5Hz,3H),2.94(s,1H),2.94–2.89(m,1H),2.89 –2.82(m,2H),2.80(d,J=7.2Hz,2H),2.64–2.52(m,4H),2.46–2.37(m,2H),2.05–1.96(m,1H),1.85–1.74(m,2H).

[0594] Example 2.10: Synthesis of DL010

[0595] Step 1:

[0596] The trifluoroacetate salt of compound DL001-1 (38.56 mg, 0.063 mmol) and DL010-1 (33.54 mg, 0.13 mmol) were dissolved in DMF (2 mL). DIPEA (24.43 mg, 0.19 mmol) was added with stirring at room temperature, and the reaction was continued with stirring 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: water containing 0.1% FA = 20%-90%) to obtain the target compound DL010 (12.06 mg).

[0597] LCMS(ESI)[M+H] + =649.18.

[0598] 1H NMR (400MHz, DMSO-d6) δ10.99(s,1H),8.88–8.82(m,1H),7.72(d,J=7.8Hz,1H),7.69–7.65(m,1H) ,7.54(s,1H),7.47(d,J=7.9Hz,1H),7.25–7.16(m,2H),7.02(d,J=7.7Hz,2H),6.92–6.85(m,1H), 5.13(dd,J=13.3,5.1Hz,1H),4.58–4.31(m,4H),3.68–3.60(m,2H),3.33–3.28(m,2H),3.00–2.87 (m,3H),2.80(s,1H),2.68–2.55(m,5H),2.47–2.35(m,1H),2.07–1.98(m,1H),1.83–1.64(m,2H).

[0599] Example 2.11: Synthesis of DL011

[0600] Step 1:

[0601] Compounds INT8 (11 mg, 0.019 mmol) and L3 (6.23 mg, 0.021 mmol) were dissolved in DMF (0.3 mL), and HATU (8.7 mg, 0.023 mmol) and DIPEA (7.4 mg, 0.057 mmol) were added sequentially. The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was purified by preparative high performance liquid chromatography (acetonitrile:water containing 0.1% FA = 10%-90%) to obtain the target compound DL011 (7.84 mg).

[0602] LCMS(ESI)[M+H] + =870.27.

[0603] 1H NMR (400MHz, DMSO-d6) δ10.97(s,1H),9.69(s,1H),9.06(s,2H),8.75(s,1H),7.69(d,J=8.0Hz,1H),7.66–7.61(m,2H),7.56 (d,J=8.1Hz,1H),7.51(s,1H),7.47–7.40(m,1H),7.26(t,J=7.8Hz,1H),7.14(d,J=1.4Hz,2H),7.00(d,J=7.4Hz,1H),6.79(t ,J=6.0Hz,1H),5.10(dd,J=13.1,5.2Hz,1H),4.48–4.28(m,6H),4.07(s,2H),3.66(t,J=6.1Hz,2H),3.43(t,J=6.4Hz,2H),3. 39(s,3H),2.96–2.86(m,1H),2.72–2.64(m,4H),2.63–2.55(m,1H),2.44–2.34(m,1H),2.04–1.85(m,3H),1.83–1.74(m,2H).

[0604] Example 2.12: Synthesis of DL012

[0605] Step 1:

[0606] Compounds DL001-1 (40.0 mg, 0.08 mmol, synthetic method according to patent WO2023221975) and DL012-1 (41.6 mg, 0.096 mmol) were dissolved in DMF (1.5 mL), and HATU (36.5 mg, 0.096 mmol) and DIPEA (31.0 mg, 0.24 mmol) were added. The reaction solution was stirred at room temperature for 2 h. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile:water containing 0.1% FA = 10%-90%) to obtain the target compound DL012 (25.3 mg).

[0607] LCMS(ESI)[M+H] + =913.22.

[0608] 1H NMR (400MHz, DMSO-d6) δ10.96(s,1H),8.79(d,J=5.5Hz,1H),7.69(d,J=7.9Hz,1H),7.65(dd,J=4.7,2.1Hz,1H),7.51(s,1H), 7.44(d,J=9.1Hz,1H),7.23–7.12(m,2H),7.01(s,2H),6.86–6.78(m,1H),5.10(dd,J=13.3,5.2Hz,1H),4.49–4.25(m,4H),3.6 4–3.58(m,2H),3.58–3.53(m,2H),3.53–3.50(m,2H),3.49–3.42(m,21H),3.32–3.28(m,2H),2.94(s,2H),2.92–2.85(m,1H),2 .79(s,1H),2.64–2.56(m,2H),2.56–2.52(m,2H),2.44–2.32(m,1H),2.05–1.94(m,1H),1.81–1.72(m,1H),1.72–1.62(m,1H).

[0609] Example 2.13: Synthesis of DL013

[0610] Step 1:

[0611] Compounds INT7 (20.0 mg, 0.040 mmol) and DL013-1 (8.09 mg, 0.040 mmol) were dissolved in DMF (1 mL), and then HATU (16.73 mg, 0.044 mmol) and DIPEA (15.51 mg, 0.12 mmol) were added. The mixture was stirred at 30 °C until the reaction was complete. The reaction solution was concentrated under reduced pressure. The crude product was purified by preparative high performance liquid chromatography (acetonitrile: water containing 0.05% FA = 5%-90%) to obtain the target compound DL013 (7.8 mg).

[0612] LCMS(ESI)[M+H] + =683.15.

[0613] 1H NMR(400MHz,DMSO-d6)δ10.98(s,1H),10.00–9.86(m,1H),9.20–9.11(m,2H),8.60(s,1 H),7.79(s,1H),7.71–7.66(m,1H),7.66–7.53(m,2H),7.35–7.26(m,1H),5.27(d,J=3. 0Hz,2H),5.17–5.08(m,1H),4.51–4.31(m,2H),3.58–3.50(m,1H),3.43(s,3H),3.03–2 .84(m,4H),2.73–2.56(m,3H),2.44–2.33(m,2H),2.06–1.98(m,1H),1.93–1.74(m,2H).

[0614] Example 2.14: Synthesis of DL014

[0615] Step 1:

[0616] Compounds DL001-1 (20.0 mg, 0.040 mmol) and DL013-1 (8.09 mg, 0.040 mmol) were dissolved in DMF (1 mL), and then HATU (16.73 mg, 0.044 mmol) and DIPEA (15.51 mg, 0.12 mmol) were added. The mixture was stirred at 30 °C until the reaction was complete. The reaction solution was concentrated under reduced pressure, and the crude product was purified by preparative high performance liquid chromatography (acetonitrile: water containing 0.05% FA = 5%-90%) to obtain the target compound DL014 (2.8 mg).

[0617] LCMS(ESI)[M+H] + =682.16.

[0618] 1H NMR(400MHz,DMSO-d6)δ10.96(s,1H),9.25–9.08(m,2H),8.84–8.69(m,1H),7.72–7.61(m,2 H),7.51(s,1H),7.49–7.42(m,1H),7.28–7.17(m,1H),7.15–7.07(m,1H),6.82(s,1H),5.15– 5.05(m,1H),4.49–4.27(m,4H),3.57–3.50(m,1H),3.46–3.41(m,3H),3.06–2.95(m,3H),2. 93–2.85(m,1H),2.73–2.56(m,3H),2.46–2.35(m,2H),2.05–1.95(m,1H),1.93–1.71(m,2H).

[0619] Example 3. Synthesis of Bioactive Compounds

[0620] Example 3.1: Synthesis of D1

[0621] Step 1:

[0622] Compounds DL002 (50.0 mg, 0.063 mmol) and D1-1 (15.3 mg, 0.13 mmol) were dissolved in a mixed solution of acetonitrile (2 mL) and water (2 mL), and then TEA (19.0 mg, 0.19 mmol) was added. The reaction solution was stirred at room temperature for 2 h. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile:water containing 0.1% FA = 10%-90%) to obtain the target compound D1 (13.9 mg).

[0623] LCMS(ESI)[M+H] + =819.22.

[0624] 1H NMR (400MHz, DMSO-d6) δ10.96(s,1H),9.52(d,J=16.6Hz,1H),8.61(d,J=13.8Hz,2H),7.70–7. 40(m,6H),7.20–7.01(m,3H),5.09(dd,J=13.3,5.1Hz,1H),4.49–4.26(m,5H),3.83–3.72(m,1 H),3.69–3.63(m,1H),3.55–3.52(m,3H),3.48–3.41(m,5H),2.96(s,2H),2.93–2.84(m,1H),2 .84–2.74(m,4H),2.64–2.56(m,1H),2.48–2.34(m,3H),2.05–1.96(m,1H),1.82–1.70(m,2H).

[0625] Example 3.2: Synthesis of D2

[0626] Step 1:

[0627] Compounds DL001 (40.0 mg, 0.053 mmol) and D1-1 (12.8 mg, 0.11 mmol) were dissolved in a mixed solution of acetonitrile (2 mL) and water (2 mL), and then TEA (16.0 mg, 0.16 mmol) was added. The reaction solution was stirred at room temperature for 2 h. The reaction solution was purified by preparative high performance liquid chromatography (acetonitrile:water containing 0.1% FA = 10%-90%) to obtain the target compound D2 (10.4 mg).

[0628] LCMS(ESI)[M+H] + =789.28.

[0629] 1 H NMR(400MHz,DMSO-d6)δ10.03(s,1H),8.60(d,J=30.7Hz,2H),8.20(s,1H),7.70–7 .30(m,5H),7.16–6.94(m,2H),5.13–5.05(m,1H),4.48–4.27(m,4H),3.82–3.67(m ,2H),3.65–3.51(m,2H),3.38–3.33(m,6H),2.96(s,1H),2.94–2.85(m,1H),2.83( s,2H),2.64–2.52(m,4H),2.47–2.35(m,2H),2.05–1.95(m,1H),1.84–1.61(m,4H).

[0630] Example 3.3: Synthesis of D3

[0631] Step 1:

[0632] Compound DL001-1 (38.56 mg, 0.063 mmol), 6-(2-(methylthio)pyrimidin-5-yl)hex-5-ethynic acid (16.37 mg, 0.069 mmol), and HATU (35.93 mg, 0.095 mmol) were dissolved in DMF (2 mL). DIPEA (24.43 mg, 0.19 mmol) was added with stirring at room temperature. After the addition was complete, the reaction mixture was stirred at room temperature for 1 h, and the reaction was monitored by LCMS. The reaction solution was directly purified by preparative high-performance liquid chromatography (acetonitrile:water containing 0.1% FA = 20%-90%) to obtain the target compound D3 (12.2 mg).

[0633] LCMS(ESI)[M+H] + =716.23.

[0634] 1 H NMR (400MHz, DMSO-d6) δ10.96(s,1H),8.76(s,1H),8.64(d,J=15.9Hz,2H),7.68(d,J=7.8Hz ,1H),7.65–7.61(m,1H),7.50(s,1H),7.45–7.40(m,1H),7.20–7.12(m,2H),6.79(t,J=6.1H z,1H),5.09(dd,J=13.3,5.1Hz,1H),4.49–4.27(m,4H),3.36–3.32(m,4H),2.99–2.84(m,3H ),2.80(s,1H),2.63–2.51(m,5H),2.47–2.30(m,4H),2.03–1.95(m,1H),1.81–1.64(m,4H).

[0635] Example 3.4: Synthesis of D4

[0636] Step 1:

[0637] Compounds DL004 (20.0 mg, 0.026 mmol) and D1-1 (15.75 mg, 0.13 mmol) were dissolved in a mixed solution of acetonitrile (2 mL) and water (2 mL), followed by the addition of triethylamine (7.89 mg, 0.078 mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction solution was then purified directly by preparative high performance liquid chromatography (acetonitrile:water containing 0.1% FA = 10%-90%) to obtain the target compound D4 (10.05 mg).

[0638] LCMS(ESI)[M+H] + =819.28.

[0639] 1 H NMR (400MHz, DMSO-d6) δ10.98(s,1H),9.09(d,J=8.0Hz,2H),8.86(s,1H),7.69(d,J=7.8Hz,1H),7.64(dd,J =7.4,1.7Hz,1H),7.51(s,1H),7.44(dd,J=7.9,1.4Hz,1H),7.21–7.13(m,2H),6.95–6.85(m,1H),5.10(dd, J=13.3,5.1Hz,1H),4.90–4.60(m,1H),4.51–4.25(m,4H),3.55–3.42(m,2H),3.40(d,J=3.7Hz,3H),3.38–3 .34(m,3H),2.97–2.84(m,1H),2.64–2.52(m,7H),2.45–2.31(m,2H),2.04–1.94(m,1H),1.86–1.64(m,4H).

[0640] Example 3.5: Synthesis of D5

[0641] Step 1:

[0642] Compounds DL005 (20.0 mg, 0.021 mmol) and D1-1 (12.72 mg, 0.11 mmol) were dissolved in a mixed solution of acetonitrile (2 mL) and water (2 mL), and then triethylamine (6.37 mg, 0.063 mmol) was added. The reaction solution was stirred at room temperature for 2 h. The reaction solution was directly purified by preparative high performance liquid chromatography (acetonitrile:water containing 0.1% FA = 10%-90%) to obtain the target compound D5 (7.31 mg).

[0643] LCMS(ESI)[M+H] + =995.25.

[0644] 1H NMR (400MHz, DMSO-d6) δ10.97(s,1H),10.11–9.13(m,1H),8.67–8.52(m,2H),8.17(s,1H),7.70–7. 56(m,2H),7.52(s,1H),7.44(d,J=8.1Hz,1H),7.34–6.95(m,3H),5.10(dd,J=13.3,5.2Hz,3H),4.4 7–4.28(m,4H),4.22(dd,J=7.8,3.1Hz,1H),3.84–3.70(m,3H),3.69–3.44(m,10H),3.21–3.12(m,3 H),3.02–2.83(m,3H),2.65–2.51(m,5H),2.47–2.32(m,2H),2.04–1.95(m,1H),1.83–1.64(m,4H).

[0645] Example 4: Preparation of Antibody

[0646] Example 4.1 Preparation of anti-CD123 antibody

[0647] Example 4.1.1 Preparation of CD123 mAb-01

[0648] The sequence of CD123 mAb-01 was obtained by linking the CD123 scFv sequence disclosed in US10906979B2 with the human IgG1 framework sequence.

[0649] The heavy chain sequence of the CD123 mAb-01 monoclonal antibody is shown in SEQ ID NO:1:

[0650] The light chain sequence of the CD123 mAb-01 monoclonal antibody is shown in SEQ ID NO:2:

[0651] Using techniques well-known to those skilled in the art, the amino acid sequences of the CDR region, VH, and VL of CD123 mAb-01 were obtained through analysis, and the results are as follows:

[0652] The amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO:3.

[0653] The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO:4.

[0654] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0655] The heavy and light chain nucleic acid sequences of CD123mAb-01 were cloned into the pcDNA3 plasmid. The plasmid was transfected into HEK293 cells to express the antibody. The supernatant was collected and purified by affinity chromatography using a protein A column. The purified antibody was found to have a purity of over 95% by SEC-HPLC.

[0656] Example 4.1.2 Preparation of G4723A

[0657] The sequence of G4723A was obtained from WO2020 / 092533A2.

[0658] The heavy chain sequence of G4723A is shown in SEQ ID NO:26:

[0659] The light chain sequence of G4723A is shown in SEQ ID NO:27:

[0660] Using techniques well-known to those skilled in the art, the amino acid sequences of the obtained CDR region, VH, and VL were analyzed, and the results are as follows:

[0661] The amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO:28.

[0662] The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO:29.

[0663] The heavy and light chain nucleic acid sequences of G4723A were cloned into the pcDNA3 plasmid, and the plasmid was transfected into HEK293 cells to express antibodies. The supernatant was collected and purified by affinity chromatography using a protein A column. The purified antibodies were found to have a purity of over 95% by SEC-HPLC.

[0664] Example 4.1.3 Preparation of h12F1

[0665] The sequence of the h12F1 monoclonal antibody was obtained from US20230399411A1.

[0666] The heavy chain sequence of h12F1 is shown in SEQ ID NO:30:

[0667] The light chain sequence of h12F1 is shown in SEQ ID NO:31:

[0668] Using techniques well-known to those skilled in the art, the amino acid sequences of the obtained CDR region, VH, and VL were analyzed, and the results are as follows:

[0669] The amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO:32.

[0670] The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO:33.

[0671] The nucleic acid sequences of the h12F1 heavy and light chains were cloned into the pcDNA3 plasmid, and the plasmid was transfected into HEK293 cells to express antibodies. The supernatant was collected and purified by affinity chromatography using a protein A column. The purified antibodies were found to have a purity of over 95% by SEC-HPLC.

[0672] Example 4.2 Preparation of anti-CD33 antibody

[0673] Example 4.2.1 Preparation of Gemtuzumab monoclonal antibody

[0674] The sequence of Gemtuzumab monoclonal antibody (CD33 mAb-1) was obtained from WO2022 / 254377A1.

[0675] The heavy chain sequence of Gemtuzumab monoclonal antibody is shown in SEQ ID NO:34:

[0676] The light chain sequence of Gemtuzumab monoclonal antibody is shown in SEQ ID NO:35:

[0677] Using techniques well-known to those skilled in the art, the amino acid sequences of the CDR region, VH, and VL were obtained through analysis, with the following results:

[0678] The amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO:36.

[0679] The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO:37.

[0680] The amino acid sequences of the three CDR regions of its heavy chain variable region are as follows:

[0681] The amino acid sequences of the three CDR regions of its light chain variable region are as follows:

[0682] The Fab sequence of Gemtuzumab was ligated into human IgG1 Fc to construct the monoclonal antibody CD33 mAb-02.

[0683] The heavy chain sequence of the CD33 mAb-2 monoclonal antibody is shown in SEQ ID NO:66:

[0684] The light chain sequence of the CD33 mAb-2 monoclonal antibody is identical to that of SEQ ID NO:35.

[0685] Example 4.2.2 Preparation of Lintuzumab monoclonal antibody

[0686] The sequence of the lintuzumab monoclonal antibody was obtained from US5530101.

[0687] The heavy chain sequence of Lintuzumab monoclonal antibody is shown in SEQ ID NO:58:

[0688] The light chain sequence of Lintuzumab monoclonal antibody is shown in SEQ ID NO:59:

[0689] Using techniques well-known to those skilled in the art, the amino acid sequences of the CDR region, VH, and VL of Lintuzumab were obtained through analysis, with the following results:

[0690] The amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO:60.

[0691] The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO:61.

[0692] Example 4.2.3 Preparation of YLAb-36 monoclonal antibody

[0693] The heavy chain sequence of YLAb-36 monoclonal antibody is shown in SEQ ID NO:62:

[0694] The light chain sequence of the YLAb-36 monoclonal antibody is shown in SEQ ID NO:63:

[0695] Using techniques well-known to those skilled in the art, the amino acid sequences of the CDR region, VH, and VL of YLAb-36 were obtained through analysis, with the following results:

[0696] The amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO:64.

[0697] The amino acid sequence of the light chain variable region VL is shown in SEQ ID NO:65.

[0698] The nucleic acid sequences of the heavy and light chains of each monoclonal antibody were cloned into the pcDNA3 plasmid, and the plasmid was transfected into HEK293 cells to express the antibody. The supernatant was collected and purified by affinity filtration through a protein A column. The purity of the purified antibodies was higher than 95% as determined by SEC-HPLC.

[0699] Example 5: Preparation of ADC

[0700] Example 5.1 Preparation of anti-HER2-ADC

[0701] Example 5.1.1 Preparation of HER2-ADC-001

[0702] Take 0.8 mL of trastuzumab (20.4 mg / mL), dilute with 0.008 mL of 20 mM PB + 100 mM disodium edetate solution (pH 7.6), adjust the pH to 7.6 with 0.5 M Na2HPO4 solution, add 20 mM TCEP (tris(2-carboxyethyl)phosphine, 0.0303 mL, 0.606 μmol, 5.5 times the molar amount of antibody substance) solution and mix well. Incubate at 25 °C for 90 min. Add DL001 (1.006 mg, 12 times the molar amount of antibody substance) in dimethyl sulfoxide (0.1345 mL) solution to the above solution, mix well, incubate at 25 °C for 2 h. After completion, use a centrifuge ultrafiltration tube (Merck, Amicon Ultra-15) to replace the buffer solution with 20 mM His-HCl, pH 5.9 buffer solution. The conjugate product HER2-ADC-001 of DL001 and trastuzumab was obtained. The DAR value was determined to be 8.0 by mass spectrometry.

[0703] In the table, LC represents the antibody light chain; HC represents the antibody heavy chain; DAR1 represents a conjugate containing one drug linker conjugate (either light or heavy chain); DAR2 represents a conjugate containing two drug linkers conjugate (either light or heavy chain); and DAR3 represents a conjugate containing three drug linkers conjugate (either light or heavy chain). LC, HC, DAR1, DAR2, and DAR3 will be explained as above in the following text.

[0704] Trastuzumab light chain conjugated with 1 toxin molecule (LC+DAR1 ratio of 100%) and heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio of 100%) were measured, and the antibody-drug conjugation ratio (DAR value) of HER2-ADC-001 was calculated to be 8.0.

[0705] Example 5.1.2 Preparation of HER2-ADC-002

[0706] Using the same method as in Example 5.1.1, DL001 was replaced with DL008 to obtain the conjugate product HER2-ADC-002 of DL008 and trastuzumab. The DAR value was determined to be 7.9 by mass spectrometry.

[0707] Trastuzumab was measured to have 0 and 1 toxin molecules conjugated to the light chain (LC+DAR0 ratio of 3%, LC+DAR1 ratio of 97%) and 3 toxin molecules conjugated to the heavy chain (HC+DAR3 ratio of 100%). Based on this, the antibody-drug conjugation ratio (DAR value) of HER2-ADC-002 was calculated to be 7.9.

[0708] Example 5.2 Preparation of anti-CD123 ADC

[0709] Example 5.2.1 Preparation of CD123-ADC-001

[0710] Take 2.2 mL of CD123 mAb-01 antibody (6.39 mg / mL), dilute with 0.022 mL of 20 mM PB + 100 mM disodium edetate solution (pH 7.6), and adjust the pH to 7.2 with 0.5 M Na2HPO4 solution. Add 20 mM TCEP (tris(2-carboxyethyl)phosphine, 0.0290 mL, 0.580 μmol, 6.0 times the molar amount of antibody) solution to the above solution and mix well. Incubate at 25 °C for 90 min. Then add DL001 (0.808 mg, 11 times the molar amount of antibody) in dimethyl sulfoxide (0.1080 mL), mix well, and incubate at 25 °C for 2 h. After completion, use a centrifuge ultrafiltration tube (Merck, Amicon Ultra-15) to replace the buffer solution. Obtain the conjugate product CD123-ADC-001 of DL001 and CD123 antibody. The DAR value determined by mass spectrometry was 8.1.

[0711] The antibody-drug conjugation ratio (DAR value) of CD123 mAb-01 monoclonal antibody was determined to be 8.1 based on the following results: light chain conjugated with 1 toxin molecule (LC+DAR1 ratio of 100%), heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio of 93%), and heavy chain conjugated with 4 toxin molecules (HC+DAR4 ratio of 7%).

[0712] Example 5.2.2 Preparation of CD123-ADC-002

[0713] Using the same method as in Example 5.2.1, DL001 was replaced with DL002 to obtain the conjugate product CD123-ADC-002 of DL002 and CD123 monoclonal antibody. The DAR value was determined to be 8.1 by mass spectrometry.

[0714] The antibody-drug conjugation ratio (DAR value) of CD123 mAb-01 monoclonal antibody was determined to be 8.1 based on the following results: light chain conjugated with 1 toxin molecule (LC+DAR1 ratio of 100%), heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio of 94%), and heavy chain conjugated with 4 toxin molecules (HC+DAR4 ratio of 6%).

[0715] Example 5.2.3 Preparation of CD123-ADC-003

[0716] Using the same method as in Example 5.2.1, CD123 mAb-01 was replaced with G4723A to obtain the conjugate product CD123-ADC-003 of DL001 and G4723A monoclonal antibody, with a DAR value of 8.1 determined by mass spectrometry.

[0717] The antibody-drug conjugation ratio (DAR value) of CD123-ADC-003 was calculated to be 8.1 based on the following measurements: light chain conjugation of 0 toxin molecules (LC ratio of 0%), light chain conjugation of 1 toxin molecule (LC+DAR1 ratio of 100%), heavy chain conjugation of 3 toxin molecules (HC+DAR3 ratio of 94%), and heavy chain conjugation of 4 toxin molecules (HC+DAR4 ratio of 6%).

[0718] Example 5.2.4 Preparation of CD123-ADC-004

[0719] Using the same method as in Example 5.2.1, CD123 mAb-01 was replaced with h12F1 to obtain the conjugate product CD123-ADC-004 of DL001 and h12F1 monoclonal antibody, and the DAR value was determined to be 8.0 by mass spectrometry.

[0720] The antibody-drug conjugation ratio (DAR value) of CD123-ADC-004 was calculated to be 8.0 based on the following results: light chain conjugation of 0 toxin molecules (LC ratio of 1%), light chain conjugation of 1 toxin molecule (LC+DAR1 ratio of 99%), heavy chain conjugation of 2 toxin molecules (HC+DAR2 ratio of 4%), heavy chain conjugation of 3 toxin molecules (HC+DAR3 ratio of 92%), and heavy chain conjugation of 4 toxin molecules (HC+DAR4 ratio of 4%).

[0721] Example 5.2.5 Preparation of CD123-ADC-005

[0722] Using the same method as in Example 5.2.1, DL001 was replaced with DL003 to obtain the conjugate product CD123-ADC-005 of DL003 and CD123 monoclonal antibody. The DAR value was determined to be 8.1 by mass spectrometry.

[0723] The antibody-drug conjugation ratio (DAR value) of CD123 mAb-01 monoclonal antibody was determined to be 8.1 based on the following results: light chain conjugated with 1 toxin molecule (LC+DAR1 ratio of 100%), heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio of 94%), and heavy chain conjugated with 4 toxin molecules (HC+DAR4 ratio of 6%).

[0724] Example 5.2.6 Preparation of CD123-ADC-006

[0725] Using the same method as in Example 5.2.1, DL003 was used instead of DL001, with the amount of DL003 added being 5 times the molar amount of the antibody material, to obtain the conjugate product CD123-ADC-006 of DL003 and CD123 monoclonal antibody. The DAR value was determined by mass spectrometry to be 4.3.

[0726] The antibody-drug conjugation ratio (DAR value) of CD123 mAb-01 monoclonal antibody was determined as follows: light chain conjugated with 0 toxin molecules (LC ratio 48%), light chain conjugated with 1 toxin molecule (LC+DAR1 ratio 52%), heavy chain conjugated with 0 toxin molecules (HC ratio 16%), heavy chain conjugated with 1 toxin molecule (HC+DAR1 ratio 30%), heavy chain conjugated with 2 toxin molecules (HC+DAR2 ratio 30%), and heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio 23%). Based on these results, the antibody-drug conjugation ratio (DAR value) of CD123-ADC-006 was calculated to be 4.3.

[0727] Example 5.2.7 Preparation of CD123-ADC-007

[0728] Using the same method as in Example 5.2.1, DL001 was replaced with DL004 to obtain the conjugate product CD123-ADC-007 of DL004 and CD123 monoclonal antibody. The DAR value was determined to be 8.1 by mass spectrometry.

[0729] The antibody-drug conjugation ratio (DAR value) of CD123 mAb-01 monoclonal antibody was determined to be 8.1 based on the following results: light chain conjugated with 1 toxin molecule (LC+DAR1 ratio of 100%), heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio of 97%), and heavy chain conjugated with 4 toxin molecules (HC+DAR4 ratio of 3%).

[0730] Example 5.2.8 Preparation of CD123-ADC-008

[0731] Using the same method as in Example 5.2.1, DL001 was replaced with DL005 to obtain the conjugate product CD123-ADC-008 of DL005 and CD123 monoclonal antibody. The DAR value was determined to be 7.2 by mass spectrometry.

[0732] The antibody-drug conjugation ratio (DAR value) of CD123 mAb-01 monoclonal antibody was determined as follows: light chain conjugated with 0 toxin molecules (LC ratio 20%), light chain conjugated with 1 toxin molecule (LC+DAR1 ratio 80%), heavy chain conjugated with 1 toxin molecule (HC+DAR1 ratio 7%), heavy chain conjugated with 2 toxin molecules (HC+DAR2 ratio 6%), and heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio 86%). Based on these results, the antibody-drug conjugation ratio (DAR value) of CD123-ADC-008 was calculated to be 7.2.

[0733] Example 5.2.9 Preparation of CD123-ADC-009

[0734] Using the same method as in Example 5.2.1, DL001 was replaced with DL006 to obtain the conjugate product CD123-ADC-009 of DL006 and CD123 monoclonal antibody. The DAR value was determined to be 8.1 by mass spectrometry.

[0735] The antibody-drug conjugation ratio (DAR value) of CD123 mAb-01 monoclonal antibody was determined to be 8.1 based on the following results: light chain conjugated with 1 toxin molecule (LC+DAR1 ratio of 100%), heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio of 96%), and heavy chain conjugated with 4 toxin molecules (HC+DAR4 ratio of 4%).

[0736] Example 5.2.10 Preparation of CD123-ADC-010

[0737] Using the same method as in Example 5.2.1, DL001 was replaced with DL007 to obtain the conjugate product CD123-ADC-010 of DL007 and CD123 monoclonal antibody. The DAR value was determined to be 8.0 by mass spectrometry.

[0738] The antibody-drug conjugation ratio (DAR value) of CD123 mAb-01 monoclonal antibody was determined to be 8.0 based on the light chain conjugation of one toxin molecule (LC+DAR1 ratio of 100%) and the heavy chain conjugation of three toxin molecules (HC+DAR3 ratio of 100%).

[0739] Example 5.2.11 Preparation of CD123-ADC-011

[0740] Using the same method as in Example 5.2.1, DL001 was replaced with DL012 to obtain the conjugate product CD123-ADC-011 of DL012 and CD123 monoclonal antibody. The DAR value was determined to be 8.0 by mass spectrometry.

[0741] The antibody-drug conjugation ratio (DAR value) of CD123 mAb-01 monoclonal antibody was determined as follows: light chain conjugated with 0 toxin molecules (LC ratio of 3%), light chain conjugated with 1 toxin molecule (LC+DAR1 ratio of 94%), light chain conjugated with 2 toxin molecules (LC+DAR2 ratio of 3%), heavy chain conjugated with 2 toxin molecules (HC+DAR2 ratio of 3%), heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio of 92%), and heavy chain conjugated with 4 toxin molecules (HC+DAR4 ratio of 5%). Based on these results, the antibody-drug conjugation ratio (DAR value) of CD123-ADC-011 was calculated to be 8.0.

[0742] Example 5.2.12 Preparation of CD123-ADC-012

[0743] Using the same method as in Example 5.2.1, DL001 was replaced with DL010 to obtain the conjugate product CD123-ADC-012 of DL010 and CD123 monoclonal antibody. The DAR value was determined to be 8.2 by mass spectrometry.

[0744] The antibody-drug conjugation ratio (DAR value) of CD123 mAb-01 monoclonal antibody was determined as follows: light chain conjugated with 0 toxin molecules (LC ratio of 2%), light chain conjugated with 1 toxin molecule (LC+DAR1 ratio of 96%), light chain conjugated with 2 toxin molecules (LC+DAR2 ratio of 2%), heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio of 91%), and heavy chain conjugated with 4 toxin molecules (HC+DAR4 ratio of 9%). Based on these results, the antibody-drug conjugation ratio (DAR value) of CD123-ADC-012 was calculated to be 8.2.

[0745] Example 5.3 Preparation of anti-CD33 ADC

[0746] Example 5.3.1 Preparation of CD33-ADC-001

[0747] Take 2.53 mL of Gemtuzumab antibody (5.93 mg / mL), dilute with 0.025 mL of 20 mM PB + 100 mM disodium edetate solution (pH 7.6), and adjust the pH to 7.5 with 0.5 M Na2HPO4 solution. Add 20 mM TCEP (tris(2-carboxyethyl)phosphine, 0.0928 mL, 1.856 μmol, 18 times the molar amount of antibody) solution to the above solution and mix well. Incubate at 25 °C for 120 min. Then add DL001 (0.864 mg, 11 times the molar amount of antibody) in dimethyl sulfoxide (0.077 mL), mix well, and incubate at 25 °C for 2 h. After completion, use a centrifuge ultrafiltration tube (Merck, Amicon Ultra-15) to replace the buffer solution. The conjugate product CD33-ADC-001 of DL001 and Gemtuzumab antibody is obtained. The DAR value was determined to be 8.0 by mass spectrometry.

[0748] The antibody-drug conjugation ratio (DAR value) of CD33-ADC-001 was calculated to be 8.0 based on the following results: Gemtuzumab light chain conjugated with 0 toxin molecules (LC ratio of 5%), light chain conjugated with 1 toxin molecule (LC+DAR1 ratio of 95%), heavy chain conjugated with 2 toxin molecules (HC+DAR2 ratio of 1%), heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio of 96%), and heavy chain conjugated with 4 toxin molecules (HC+DAR4 ratio of 3%).

[0749] Example 5.3.2 Preparation of CD33-ADC-002

[0750] Using the same method as in Example 5.3.1, DL001 was replaced with DL003 to obtain the conjugate product CD33-ADC-002 of DL003 and Gemtuzumab monoclonal antibody, and the DAR value was determined to be 7.7 by mass spectrometry.

[0751] The antibody-drug conjugation ratio (DAR value) of Gemtuzumab monoclonal antibody was determined as follows: light chain conjugated with 0 toxin molecules (LC ratio of 9%), light chain conjugated with 1 toxin molecule (LC+DAR1 ratio of 91%), heavy chain conjugated with 2 toxin molecules (HC+DAR2 ratio of 9%), heavy chain conjugated with 3 toxin molecules (HC+DAR3 ratio of 88%), and heavy chain conjugated with 4 toxin molecules (HC+DAR4 ratio of 3%). Based on these results, the antibody-drug conjugation ratio (DAR value) of CD33-ADC-002 was calculated to be 7.7.

[0752] Comparative Example 1: Preparation of Control ADC-001

[0753] The ADC-001 was synthesized according to Scheme 7 of patent CN118055779.

[0754] Example 6: Determination of ADC Affinity

[0755] Example 6.1 Determination of anti-CD123 ADC affinity

[0756] The affinity of anti-CD123 monoclonal antibody and anti-CD123 ADC for the target was determined using human acute myeloid leukemia cells MV4-11 (from Procell, catalog number CL-0572) and MOLM-13 (from Procell, catalog number CL-0681) expressing CD123.

[0757] Tumor cell lines were cultured at 37°C and 5% CO2. Cells in the logarithmic growth phase were harvested and counted to ensure cell viability was above 90%. FACS buffer was used to prepare a density of 1×10⁻⁶ cells / mL. 6Add 100 μL of cell suspension per well to a 96-well plate. Centrifuge the cell suspension at 1000 rpm for 5 min, discard the supernatant, and set aside. Dilute the ADC sample 4-fold serially to a total of 10 concentrations, with the highest working concentration being 10 μg / mL. Add 100 μL of ADC working sample solution, mix well, and incubate at 4°C for 30 min. After incubation, centrifuge, wash, and resuspend the cells in fluorescently labeled secondary antibody solution, then incubate at 4°C in the dark for 30 min. After incubation, centrifuge, wash, and resuspend the cells in FACS buffer, then analyze using flow cytometry.

[0758] The specific results are shown in Table 1 below. The results indicate that the affinity of the anti-CD123 antibody did not change significantly before and after conjugation. Therefore, the ADC has excellent specificity for stable binding to the CD123 antigen.

[0759] Table 1. Affinity assay results of anti-CD123 monoclonal antibody and anti-CD123 ADC on cells.

[0760] Example 6.2 Determination of anti-CD33 ADC affinity

[0761] The cell affinity of anti-CD33 monoclonal antibodies and anti-CD33 ADCs for their targets was determined using CD33-expressing human acute myeloid leukemia cells MV4-11 (from Procell, catalog number CL-0572) and MOLM-13 (from Procell, CL-0681).

[0762] Tumor cell lines were cultured at 37°C and 5% CO2. Cells in the logarithmic growth phase were harvested and counted to ensure cell viability was above 90%. FACS buffer was used to prepare a density of 1×10⁻⁶ cells / mL. 6 Add 100 μL of cell suspension per well to a 96-well plate. Centrifuge the cell suspension at 1000 rpm for 5 min, discard the supernatant, and set aside. Dilute the ADC sample 4-fold serially to a total of 10 concentrations, with the highest working concentration being 10 μg / mL. Add 100 μL of ADC working sample solution, mix well, and incubate at 4°C for 30 min. After incubation, centrifuge, wash, and resuspend the cells in fluorescently labeled secondary antibody solution, then incubate at 4°C in the dark for 30 min. After incubation, centrifuge, wash, and resuspend the cells in FACS buffer, then analyze using flow cytometry.

[0763] The specific results are shown in Table 2 below. The results indicate that the affinity of the anti-CD33 antibody did not change significantly before and after conjugation. Therefore, the ADC has excellent specificity for stable binding to the CD33 antigen.

[0764] Table 2. Affinity assay results of anti-CD33 monoclonal antibody and anti-CD33 ADC on cells.

[0765] Example 7: Determination of ADC cell killing activity

[0766] Example 7.1 Test of the inhibitory activity of anti-HER2ADC on the proliferation of NCI-N87 tumor cells

[0767] N87 cells (source: Pronos, catalog number CL-0211) were resuscitated with 1640 + 10% FBS and seeded at a density of 5000 cells / well, 100 μl / well in 96-well plates, and incubated overnight at 37°C. The next day, the target ADC molecules were diluted with 1640 + 10% FBS, with the highest concentration of the target ADC molecules being 200 nM-2000 nM, and 4-fold diluted for 10 spots. 100 μl of the diluted drug was added to the cell culture wells. After incubation at 37°C for 4 days, 50 μl of CellTiter Turbo 2.0 Luminescent Cell Vitality Assay reagent (from Adamas Life, catalog number RA-GL11-A) was added to each well, and the fluorescence value was detected after incubation for 5 minutes.

[0768] The test results show that the anti-HER2ADC molecules of this application have significant inhibitory activity on the proliferation of NCI-N87 tumor cell lines. The test results are shown in Table 3.

[0769] Table 3 Results of the inhibitory activity test of anti-HER2ADC on NCI-N87 tumor cell proliferation.

[0770] Example 7.2 Determination of anti-CD123 ADC cell killing activity

[0771] The cytotoxic activity against CD123 ADCs was measured using human acute myeloid leukemia cells expressing CD123, including MV4-11 (from Procell, catalog number CL-0572), MOLM13 (Kangyuan Bochuang, catalog number KC-0323), OCI-AML-3 (Kangyuan Bochuang, catalog number KC-1736), PL-21 (Kangyuan Bochuang, catalog number KC-2086), and EOL1 cells (Kangyuan Bochuang, catalog number KC-0598).

[0772] Cells were cultured at 37°C and 5% CO2. Cells in the logarithmic growth phase were harvested and counted to ensure cell viability was above 90%. Cell density was adjusted: MV4-11 cells were seeded at 5000 cells / well, and MOLM13, OCI-AML-3, PL-21, and EOL1 cells at 3000 cells / well in 96-well plates. The culture plates were incubated overnight.

[0773] The following day, the ADC molecules used to test MV4-11, MOLM13, OCI-AML-3, and PL-21 cells were diluted 20-fold (20×) to their final concentration in serum-free medium, with a maximum working concentration of 20 nM, using a 4-fold serial dilution, for a total of 9 concentration points. The ADC molecules used to test EOL1 cells were diluted 10-fold (10×) to their final concentration in serum-free medium, with a maximum concentration of 100 nM, using a 4-fold serial dilution, for a total of 9 concentration points.

[0774] Add the diluted analyte sample to the cell culture wells, and then incubate the 96-well plate in an incubator for 3 days. After incubation, equilibrate the 96-well plate to room temperature for 30 minutes. Add 50 μL of CellTiter Turbo 2.0 Luminescent Cell Viability Assay reagent (from Adamas Life, catalog number RA-GL11-A) to each well, incubate for 10 minutes, and then measure the chemiluminescence value.

[0775] The specific results are shown in Tables 4, 5, and 6. The results indicate that the anti-CD123 ADCs all have a strong anti-AML cell proliferation effect and good targeting.

[0776] Table 4 Results of anti-CD123 ADC cell killing activity assay

[0777] Table 5 Results of anti-CD123 ADC cell killing activity assay

[0778] Table 6 Results of anti-CD123 ADC cell killing activity assay

[0779] Example 7.3 Determination of anti-CD33 ADC cell killing activity

[0780] The cytotoxic activity against CD33ADC was determined using MV4-11 human acute myeloid leukemia cells expressing CD33 (from Procell, catalog number CL-0572).

[0781] MV4-11 cells were cultured at 37°C and 5% CO2. Cells in the logarithmic growth phase were harvested and counted to ensure cell viability was above 90%. Cell density was adjusted to 5000 cells / well with 142.5 μL of cell suspension / well in 96-well plates. The plates were incubated overnight. The next day, the ADC molecule was diluted 20-fold (20×) with serum-free medium to the final concentration. The highest working concentration of MV4-11 cells was 20 nM, with 4-fold serial dilutions, resulting in a total of 9 concentration points. 7.5 μL of the diluted analyte sample (20×) was added to each well, and the 96-well plates were incubated for another 3 days. After incubation, the 96-well plates were equilibrated at room temperature for 30 min. Add 50 μL of CellTiter Turbo 2.0 Luminescent Cell Vitality Assay reagent (from Adamas Life, catalog number RA-GL11-A) to each well, incubate for 10 minutes, and then detect the chemiluminescence value.

[0782] The specific results are shown in Table 7. The results indicate that the anti-CD33 ADC exhibits a strong anti-AML cell proliferation effect.

[0783] Table 7 Results of anti-CD33 ADC cell killing activity assay

[0784] Example 8. In vivo activity assay of antibody-drug conjugates (ADCs)

[0785] Example 8.1 Efficacy test of antibody-drug conjugate (ADC) on BT-474 xenograft tumors

[0786] 1. Experimental Materials

[0787] Test compound: HER2-ADC-001, with physiological saline as a negative control.

[0788] Experimental cells: BT-474 cells.

[0789] Laboratory animals: BALB / c nude mice, female, 6-8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. or other qualified suppliers.

[0790] 2. Experimental Design

[0791] 2.1. Cell Treatment

[0792] The culture conditions for BT-474x cells (ATCC-HTB-20) were as follows: ATCC Hybri-Care Medium + 10% FBS, incubated at 37°C in a 5% CO2 incubator. Cells were passaged twice a week using trypsin containing EDTA. After washing twice with PBS, the cells were centrifuged and resuspended in pre-chilled PBS. Cells were counted using a cell counter, and the cell suspension was adjusted to an appropriate concentration before seeding.

[0793] 2.2. Tumor cell transplantation

[0794] BALB / c nude mice were acclimatized to the laboratory environment for 3-5 days. Three days before cell inoculation, 0.36 mg estrogen extended-release tablets were subcutaneously injected into the left posterior back of each mouse. One week after estrogen tablet injection, assisted urination was initiated three times per week; if necessary, assisted urination was initiated daily. 0.2 mL (10 x 10) 6 BT-474x cells were subcutaneously inoculated into the right back near the upper limb of each mouse using PBS plus matrigel (1:1). The average tumor volume reached approximately 150-200 mm. 3 Dosing will begin in groups at that time.

[0795] 2.3. Animal drug administration and detection

[0796] The tumor-bearing nude mice enrolled in the group were administered medication according to the following regimen:

[0797] Table 8. Dosing Regimen

[0798] 2.4. Tumor volume and body weight measurement

[0799] The day of the first dose is recorded as day 0. A total of three doses are administered, and tumor diameter and body weight are measured periodically. Tumor volume, relative tumor proliferation rate, and relative tumor inhibition rate are calculated, and tumor growth curves are plotted. The calculation formulas are as follows:

[0800] The formula for calculating tumor volume (V) is: V = 1 / 2 × L_length × L_short. 2 Where L_long and L_short represent the long and short diameters of the tumor, respectively.

[0801] Relative tumor growth inhibition rate (TGI) (%) = [(1 - (average tumor volume of the treatment group on a certain day - average tumor volume of the treatment group when the treatment group was divided into subgroups)) / (average tumor volume of the solvent control group on a certain day - average tumor volume of the solvent control group when the treatment group was divided into subgroups)] × 100%.

[0802] Relative tumor proliferation rate (T / C%): The calculation formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the solvent control group). RTV, relative tumor volume, is calculated as RTV = Vt / V0, where V0 is the average tumor volume measured at the time of drug administration (i.e., D0), and Vt is the average tumor volume at a certain measurement. TRTV and CRTV are based on data from the same day.

[0803] 3. Experimental Results

[0804] Test results show that the antibody-drug conjugate disclosed herein exhibits a relatively significant antitumor effect. During the administration period, no significant weight loss or drug toxicity was observed in any of the animal groups. Specific results are shown in Table 9.

[0805] Table 9. Tumor volume data of the BT-474 xenograft model

[0806] Example 8.2 Efficacy test of antibody-drug conjugate (ADC) against MV4-11-Luc acute myeloid leukemia systemic xenografts

[0807] 1. Experimental Materials

[0808] Test compound: CD123-ADC-001, with physiological saline as a negative control.

[0809] Experimental cells: MV4-11-Luc cells.

[0810] Experimental animals: NPG mice, female, 6-10 weeks old, purchased from Beijing Vitonda Biotechnology Co., Ltd.

[0811] 2. Experimental Design

[0812] 2.1. Cell Treatment

[0813] The culture conditions for MV4-11-Luc cells were: IMDM + 10% FBS, incubated at 37°C in a 5% CO2 incubator. 0.5 μg / mL puromycin was added to the medium weekly for 72 hours, followed by medium replacement. Cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and used for inoculation of mouse systemic tumors.

[0814] 2.2. Tumor cell transplantation

[0815] Laboratory mice were injected with 1*10 via the tail vein 7 MV-4-11-luc cells were observed regularly, and tumor growth was monitored using the IVIS Lumina III mouse bioluminescence in vivo imaging system (PerkinElmer, USA). The average bioluminescence value reached 5 x 10⁻⁶.6 Grouping is performed at photons / s.

[0816] 2.3. Animal drug administration and detection

[0817] The mice enrolled were administered the drugs according to the following regimen:

[0818] Table 10. Dosing Regimen

[0819] 2.4. Tumor volume and body weight measurement

[0820] The day of the first administration was recorded as day 0. A total of two administrations were administered. Whole-body bioluminescence imaging of mice was performed periodically, and luminescence values ​​were recorded. The relative tumor proliferation rate and relative tumor inhibition rate were calculated based on the luminescence values, and tumor growth curves were plotted. The calculation formulas are as follows:

[0821] Relative tumor growth inhibition rate (TGI) (%) = [(1 - (average luminescence value of the treatment group on a certain day - average luminescence value of the treatment group when administered in groups)) / (average luminescence value of the solvent control group on a certain day - average luminescence value of the solvent control group when administered in groups)] × 100%.

[0822] Relative tumor proliferation rate T / C (%): The calculation formula is as follows: T / C% = T RTV / C RTV ×100% (T) RTV Treatment group RTV; C RTV : Solvent control group RTV). RTV, relative tumor volume, is calculated using the formula RTV = V t / V0, where V0 is the average tumor luminescence value measured during group administration (i.e., D0), V t T represents the average tumor luminescence value during a single measurement. RTV With C RTV Take data from the same day.

[0823] 3. Experimental Results

[0824] Test results show that the antibody-drug conjugate disclosed herein exhibits a relatively significant antitumor effect. During the administration period, no significant weight loss or drug toxicity was observed in any of the animal groups. Specific results are shown in Table 11.

[0825] Table 11. Bioluminescence values ​​of tumors in the MV-4-11 system xenograft model

[0826] Example 8.3 Efficacy test of antibody-drug conjugate (ADC) against MV4-11-Luc acute myeloid leukemia systemic xenografts

[0827] 1. Experimental Materials

[0828] Test compounds: CD123-ADC-005, CD123-ADC-007, CD123-ADC-008, CD123-ADC-009, CD123-ADC-010, and physiological saline as a negative control.

[0829] Experimental cells: MV4-11-Luc cells.

[0830] Experimental animals: NPG mice, female, 6-10 weeks old, purchased from Beijing Vitonda Biotechnology Co., Ltd.

[0831] 2. Experimental Design

[0832] 2.1. Cell Treatment

[0833] The culture conditions for MV4-11-Luc cells were: IMDM + 10% FBS, incubated at 37°C in a 5% CO2 incubator. 0.5 μg / mL puromycin was added to the medium weekly for 72 hours, followed by medium replacement. Cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and used for inoculation of mouse systemic tumors.

[0834] 2.2. Tumor cell transplantation

[0835] Laboratory mice were injected with 1*10 via the tail vein 7 MV-4-11-luc cells were observed regularly, and tumor growth was monitored using the IVIS Lumina III mouse bioluminescence in vivo imaging system (PerkinElmer, USA). The average bioluminescence value reached 5 x 10⁻⁶. 6 Grouping is performed at photons / s.

[0836] 2.3. Animal drug administration and detection

[0837] The mice enrolled were administered the drugs according to the following regimen:

[0838] Table 12. Dosing Regimen

[0839] 2.4. Tumor volume and body weight measurement

[0840] The day of the first administration was recorded as day 0. A total of two administrations were administered. Whole-body bioluminescence imaging of mice was performed periodically, and luminescence values ​​were recorded. The relative tumor proliferation rate and relative tumor inhibition rate were calculated based on the luminescence values, and tumor growth curves were plotted. The calculation formulas are as follows:

[0841] Relative tumor growth inhibition rate (TGI) (%) = [(1 - (average luminescence value of the treatment group on a certain day - average luminescence value of the treatment group when administered in groups)) / (average luminescence value of the solvent control group on a certain day - average luminescence value of the solvent control group when administered in groups)] × 100%.

[0842] Relative tumor proliferation rate T / C (%): The calculation formula is as follows: T / C% = T RTV / C RTV ×100% (T) RTV Treatment group RTV; C RTV : Solvent control group RTV). RTV, relative tumor volume, is calculated using the formula RTV = V t / V0, where V0 is the average tumor luminescence value measured during group administration (i.e., D0), V t T represents the average tumor luminescence value during a single measurement. RTV With C RTV Take data from the same day.

[0843] 3. Experimental Results

[0844] Test results show that the antibody-drug conjugate disclosed herein exhibits a relatively significant antitumor effect. During the administration period, no significant weight loss or drug toxicity was observed in any of the animal groups. Specific results are shown in Table 13.

[0845] Table 13. Bioluminescence values ​​of tumors in the MV-4-11 systemic xenograft model

[0846] Example 8.4 Efficacy test of antibody-drug conjugate (ADC) against OCI-AML-3 cell xenografts

[0847] 1. Experimental Materials

[0848] Test compound: CD123-ADC-008, control ADC-001, and physiological saline as a negative control.

[0849] Experimental cells: OCI-AML-3 cells.

[0850] Experimental animals: BALB / c Nude mice, female, 6-8 weeks old, purchased from Beijing Vitonda Biotechnology Co., Ltd.

[0851] 2. Experimental Design

[0852] 2.1. Cell Treatment

[0853] The culture conditions for OCI-AML-3 cells were as follows: Human leukemia OCI-AML-3 cells (Nanjing Kebai-CBP60817) were cultured in suspension in vitro under the following conditions: RPMI-1640 + 20% FBS, incubated at 37℃ in a 5% CO2 incubator. Routine passage was performed twice a week. When the cell saturation reached 80%-90% and the required number was achieved, the cells were harvested, counted, and seeded.

[0854] 2.2. Tumor cell transplantation

[0855] BALB / c nude mice were acclimatized to the laboratory environment for 3-5 days. Three days before cell inoculation, 0.2 mL (10 x 10⁻⁶) of the solution was added. 6 OCI-AML-3 cells were subcutaneously inoculated into the right posterior back near the upper limb of each mouse using PBS plus matrigel (1:1). The average tumor volume reached approximately 150-200 mm. 3 Dosing will begin in groups at that time.

[0856] 2.3. Animal drug administration and detection

[0857] The mice enrolled were administered the drugs according to the following regimen:

[0858] Table 14. Dosing Regimen

[0859] 2.4. Tumor volume and body weight measurement

[0860] The day of the first dose is recorded as day 0. A total of three doses are administered, and tumor diameter and body weight are measured periodically. Tumor volume, relative tumor proliferation rate, and relative tumor inhibition rate are calculated, and tumor growth curves are plotted. The calculation formulas are as follows:

[0861] The formula for calculating tumor volume (V) is: V = 1 / 2 × L_length × L_short. 2 Where L_long and L_short represent the long and short diameters of the tumor, respectively.

[0862] Relative tumor growth inhibition rate (TGI) (%) = [(1 - (average tumor volume of the treatment group on a certain day - average tumor volume of the treatment group when the treatment group was divided into subgroups)) / (average tumor volume of the solvent control group on a certain day - average tumor volume of the solvent control group when the treatment group was divided into subgroups)] × 100%.

[0863] Relative tumor proliferation rate (T / C%): The calculation formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the solvent control group). RTV, relative tumor volume, is calculated as RTV = Vt / V0, where V0 is the average tumor volume measured at the time of drug administration (i.e., D0), and Vt is the average tumor volume at a certain measurement. TRTV and CRTV are based on data from the same day.

[0864] 3. Experimental Results

[0865] Test results show that the antibody-drug conjugate disclosed herein exhibits a relatively significant antitumor effect. During the administration period, no significant weight loss or drug toxicity was observed in any of the animal groups. Specific results are shown in Table 15.

[0866] Table 15. Tumor data of the OCI-AML-3 xenograft model

[0867] Example 8.5 Efficacy test of antibody-drug conjugate (ADC) against MV4-11-Luc acute myeloid leukemia systemic xenografts

[0868] 1. Experimental Materials

[0869] Test compound: CD33-ADC-001, with physiological saline as a negative control.

[0870] Experimental cells: MV4-11-Luc cells.

[0871] Experimental animals: NPG mice, female, 6-10 weeks old, purchased from Beijing Vitonda Biotechnology Co., Ltd.

[0872] 2. Experimental Design

[0873] 2.1. Cell Treatment

[0874] The culture conditions for MV4-11-Luc cells were: IMDM + 10% FBS, incubated at 37°C in a 5% CO2 incubator. 0.5 μg / mL puromycin was added to the medium weekly for 72 hours, followed by medium replacement. Cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and used for inoculation of mouse systemic tumors.

[0875] 2.2. Tumor cell transplantation

[0876] Laboratory mice were injected with 1*10 via the tail vein 7MV-4-11-luc cells were observed regularly, and tumor growth was monitored using the IVIS Lumina III mouse bioluminescence in vivo imaging system (PerkinElmer, USA). The average bioluminescence value reached 5 x 10⁻⁶. 6 Grouping is performed at photons / s.

[0877] 2.3. Animal drug administration and detection

[0878] The mice enrolled were administered the drugs according to the following regimen:

[0879] Table 16. Dosing Regimen

[0880] 2.4. Tumor volume and body weight measurement

[0881] The day of the first administration was recorded as day 0. A total of two administrations were administered. Whole-body bioluminescence imaging of mice was performed periodically, and luminescence values ​​were recorded. The relative tumor proliferation rate and relative tumor inhibition rate were calculated based on the luminescence values, and tumor growth curves were plotted. The calculation formulas are as follows:

[0882] Relative tumor growth inhibition rate (TGI) (%) = [(1 - (average luminescence value of the treatment group on a certain day - average luminescence value of the treatment group when administered in groups)) / (average luminescence value of the solvent control group on a certain day - average luminescence value of the solvent control group when administered in groups)] × 100%.

[0883] Relative tumor proliferation rate T / C (%): The calculation formula is as follows: T / C% = T RTV / C RTV ×100% (T) RTV Treatment group RTV; C RTV : Solvent control group RTV). RTV, relative tumor volume, is calculated using the formula RTV = V t / V0, where V0 is the average tumor luminescence value measured during group administration (i.e., D0), V t T represents the average tumor luminescence value during a single measurement. RTV With C RTV Take data from the same day.

[0884] 3. Experimental Results

[0885] Test results show that the antibody-drug conjugate disclosed herein exhibits a relatively significant antitumor effect. During the administration period, no significant weight loss or drug toxicity was observed in any of the animal groups. Specific results are shown in Table 17.

[0886] Table 17. Bioluminescence values ​​of tumors in the MV-4-11 system xenograft model

[0887] Unless otherwise specified, all terms used in this application have the meanings commonly understood by those skilled in the art.

[0888] The embodiments described in this application are for illustrative purposes only and are not intended to limit the scope of protection of this application. Those skilled in the art can make various other substitutions, changes and improvements within the scope of this application. Therefore, this application is not limited to the above embodiments, but is only defined by the claims.

Claims

A drug linker conjugate as shown in Formula I, or a pharmaceutically acceptable salt thereof. in, Lg is the leaving group that reacts with antibodies or their antigen-binding fragments; X is selected from direct bond, -NR m -or -C(O)-; R m Selected from hydrogen or C 1-4 Alkyl, the C 1-4 The alkyl group is optionally substituted by one or more substituents selected from hydroxyl, phosphoric acid, sulfonic acid and sugar groups; A is selected from -NR 1 (CH2) m -、-NR 1 (CH2) m Q(CH2) p -、-(O)C(CH2) m -、-(O)C(CH2) m Q(CH2) p -、-S(O)2(CH2) m -、-S(O)2(CH2) m Q(CH2) p -、-S(O)(CH2) m -or -S(O)(CH2) m Q(CH2) p -; Q is selected independently from -O-, -S-, or -NR. 2 -; R 1 Independently selected from hydrogen, sulfonyl, or C 1-4 Alkyl, the C 1-4 The alkyl group is optionally substituted by one or more substituents selected from hydroxyl, sulfonic acid, phosphate and sugar groups; R 2 Selected from hydrogen or C 1-4 alkyl; m and p are each independently selected from any integer between 1 and 5. The drug linker conjugate as claimed in claim 1, or a pharmaceutically acceptable salt thereof, is characterized in that, It meets one or more of the following conditions: (1) Lg is selected from halogen, sulfone, and tertiary amine salt (Me3N) + Et3N + ), diazonium salts, -OMs, MeSO2-, MeS-, CF3SO3-, p-toluenesulfonyl, Or a substituted phenoxy group, wherein the substituent is selected from halogens and / or nitro groups; Preferably, Lg is selected from fluorine, chlorine, bromine, iodine, -OMs, MeSO2-, MeS-, CF3SO3- or p-toluenesulfonyl; More preferably, Lg is MeSO2-; (2)R m Selected from hydrogen, C 1-4 Alkyl, hydroxyl substituted C 1-4 alkyl and phosphate substituted C 1-4 C substituted with alkyl or sulfonic acid groups 1-4 alkyl or sugar-substituted C 1-4 alkyl; Preferably, R m Selected from C 1-4 Alkyl, hydroxyl substituted C 1-4 alkyl, n is selected from 1, 2, 3, and 4, and position 1 is connected to the corresponding nitrogen atom; Preferably, R m Selected from methyl, -CH2CH2OH or Position 1 is connected to the corresponding nitrogen atom; (3) Q is -O-; (4)R 1 Independently selected from hydrogen, sulfonyl, C 1-4 Alkyl, hydroxyl substituted C 1-4 alkyl and phosphate substituted C 1-4 C substituted with alkyl or sulfonic acid groups 1-4 alkyl or sugar-substituted C 1-4 alkyl; Preferably, R 1 Independently selected from sulfonyl, C 1-4 Alkyl, hydroxyl substituted C 1-4 alkyl, n is selected from 1, 2, 3, and 4, and position 1 is connected to the corresponding nitrogen atom; Preferably, R 1 Independently selected from methyl, methanesulfonyl, -CH2CH2OH, Position 1 is connected to the corresponding nitrogen atom; (5)R 2 It can be hydrogen or methyl; (6) m is selected from 1, 2 or 3; (7) p is selected from 1, 2 or 3. The drug linker conjugate or a pharmaceutically acceptable salt thereof as described in claim 1 or 2 is characterized in that, It meets one or more of the following conditions: (1) X is selected from direct bonds, -N(CH3)-, Or -C(O)-, with position 1 connected to the corresponding carbon atom and position 2 connected to A; (2)A is selected from -NR 1 (CH2) m -, -NR 1 (CH2) m O(CH2) p -, -NR 1 (CH2) m S(CH2) p -, -NR 1 (CH2) m NR 2 (CH2) p -, -(O)C(CH2) m -, -(O)C(CH2) m O(CH2) p -, -(O)C(CH2) m S(CH2) p -, -(O)C(CH2) m NR 2 (CH2) p -, -S(O)2(CH2) m -, -S(O)2(CH2) m O(CH2) p -, -S(O)2(CH2) m S(CH2) p -, -S(O)2(CH2) m NR 2 (CH2) p -, -S(O)(CH2) m -, -S(O)(CH2) m O(CH2) p -, -S(O)(CH2) m S(CH2) p - or -S(O)(CH2) m NR 2 (CH2) p -; Preferably, A is selected from -NR 1 (CH2) m -、-NR 1 (CH2) m O(CH2) p -、-(O)C(CH2) m -、-S(O)2(CH2) m -or -S(O)(CH2) m -; Preferably, A is selected from -NR 1 (CH2)3-、-NR 1 (CH2)2O(CH2)2-, -(O)C(CH2)2-, -(O)C(CH2)3-, -S(O)2(CH2)3- or -S(O)(CH2)3-. The drug linker conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1-3 is characterized in that, Selected from 1 is connected to A. The drug linker conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1-4 is characterized in that, Select from the following structure, with one bit connected to X; The drug linker conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1-5 is characterized in that, The drug linker conjugate or its pharmaceutically acceptable salt has the structures shown in Formula I-1, Formula I-2 and Formula I-3: in, A is selected from -S(O)2(CH2) m -、-S(O)2(CH2) m Q(CH2) p -、-S(O)(CH2) m -、-S(O)(CH2) m Q(CH2) p -、-NR 1 (CH2) m -or-NR 1 (CH2) m Q(CH2) p -; Q, R 1 Lg, m, and p are as defined in any one of claims 1-5; Where A is selected from -NR 1 (CH2) m -or-NR 1 (CH2) m Q(CH2) p -; R 1 Q, Lg, m and p are as defined in any one of claims 1-5; Wherein, A is selected from -(O)C(CH2). m -、-(O)C(CH2) m Q(CH2) p -; R m Q, Lg, m and p are as defined in any one of claims 1-5. The drug linker conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1-6 is characterized in that, The drug linker conjugate is selected from: An antibody-drug conjugate as shown in Formula II, or a pharmaceutically acceptable salt thereof. in, Tb is an antibody or its antigen-binding fragment; q is any integer between 1 and 12; X and A are as defined in any one of claims 1-5. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 8 is characterized in that, Tb satisfies one or more of the following conditions: (1) The Tb is an antibody or its antigen-binding fragment that has the activity of binding to the surface antigens of solid or blood tumor cells; (2) The Tb is an anti-Her2 antibody or its antigen-binding fragment, or an anti-Trop-2 antibody or its antigen-binding fragment; (3) The Tb is an anti-Her2 antibody or its antigen-binding fragment, such as anbenitamab, coprelotamab, disitamab, gancotamab, margetuximab, pertuzumab, timigutuzumab, zanidatamab, Trastuzumab, Pertuzumab or its antigen-binding fragment; preferably, Tb is Trastuzumab or Pertuzumab; for example, Tb is Trastuzumab; (4) The Tb is an anti-CD123 or CD33 antibody or its antigen-binding fragment; (5) The Tb is an antibody against CD123 or its antigen-binding fragment, such as Pivekimab, Talacotuzumab, h12F1, G4723A, mAb-01 or its antigen-binding fragment. (6) The Tb is an anti-CD33 antibody or its antigen-binding fragment, such as Gemtuzumab, Vadastuximab, Lintuzumab, YLAb-36 or its antigen-binding fragment. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 8 or 9 is characterized in that, Choose from any of the following options: (a) The anti-CD123 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:3 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:4, wherein the CDRs are defined according to the Chothia, AbM, Kabat, IMGT, Contact scheme or any combination thereof; Optionally, the anti-CD123 antibody or its antigen-binding fragment comprises CDR-H1 shown in SEQ ID NO:7, CDR-H2 shown in SEQ ID NO:12, CDR-H3 shown in SEQ ID NO:15, CDR-L1 shown in SEQ ID NO:18, CDR-L2 shown in SEQ ID NO:21, and CDR-L3 shown in SEQ ID NO:24, as defined in the Kabat scheme. Preferably, the anti-CD123 antibody or its antigen-binding fragment comprises: the heavy chain variable region (VH) of SEQ ID NO:3 and the light chain variable region (VL) of SEQ ID NO:4; More preferably, the anti-CD123 antibody or its antigen-binding fragment comprises: the heavy chain of SEQ ID NO:1 and the light chain of SEQ ID NO:2; (b) The anti-CD123 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:28 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:29, wherein the CDRs are defined according to the Chothia, AbM, Kabat, IMGT, Contact scheme or any combination thereof; Preferably, the anti-CD123 antibody or its antigen-binding fragment comprises: the heavy chain variable region (VH) of SEQ ID NO:28 and the light chain variable region (VL) of SEQ ID NO:29; More preferably, the anti-CD123 antibody or its antigen-binding fragment comprises: the heavy chain shown in SEQ ID NO:26 and the light chain shown in SEQ ID NO:27; or (c) The anti-CD123 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:32 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:33, wherein the CDRs are defined according to the Chothia, AbM, Kabat, IMGT, Contact scheme or any combination thereof; Preferably, the anti-CD123 antibody or its antigen-binding fragment comprises: the heavy chain variable region (VH) of SEQ ID NO:32 and the light chain variable region (VL) of SEQ ID NO:33; More preferably, the anti-CD123 antibody or its antigen-binding fragment comprises: the heavy chain shown in SEQ ID NO:30 and the light chain shown in SEQ ID NO:

31. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 8-10 is characterized in that, Choose from any of the following options: (a) The anti-CD33 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:36 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:37, wherein the CDRs are defined according to the Chothia, AbM, Kabat, IMGT, Contact scheme or any combination thereof; Optionally, the anti-CD33 antibody or its antigen-binding fragment comprises CDR-H1 shown in SEQ ID NO:40, CDR-H2 shown in SEQ ID NO:45, CDR-H3 shown in SEQ ID NO:48, CDR-L1 shown in SEQ ID NO:51, CDR-L2 shown in SEQ ID NO:54, and CDR-L3 shown in SEQ ID NO:57, as defined in the Kabat scheme. Preferably, the anti-CD33 antibody or its antigen-binding fragment comprises: the heavy chain variable region (VH) of SEQ ID NO:36 and the light chain variable region (VL) of SEQ ID NO:37; More preferably, the anti-CD33 antibody or its antigen-binding fragment comprises: the heavy chain of SEQ ID NO:34 and the light chain of SEQ ID NO:35; More preferably, the anti-CD33 antibody or its antigen-binding fragment comprises: the heavy chain of SEQ ID NO:66 and the light chain of SEQ ID NO:35; (b) The anti-CD33 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:60 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:61, wherein the CDRs are defined according to the Chothia, AbM, Kabat, IMGT, Contact scheme or any combination thereof; Preferably, the anti-CD33 antibody or its antigen-binding fragment comprises: the heavy chain variable region (VH) of SEQ ID NO:60 and the light chain variable region (VL) of SEQ ID NO:61; More preferably, the anti-CD33 antibody or its antigen-binding fragment comprises: the heavy chain shown in SEQ ID NO:58 and the light chain shown in SEQ ID NO:59; or (c) The anti-CD33 antibody or its antigen-binding fragment comprises: three HCDRs of the heavy chain variable region (VH) sequence of SEQ ID NO:64 and three LCDRs of the light chain variable region (VL) sequence of SEQ ID NO:65, wherein the CDRs are defined according to the Chothia, ABM, Kabat, IMGT, Contact scheme or any combination thereof; Preferably, the anti-CD33 antibody or its antigen-binding fragment comprises: the heavy chain variable region (VH) of SEQ ID NO:64 and the light chain variable region (VL) of SEQ ID NO:65; More preferably, the anti-CD33 antibody or its antigen-binding fragment comprises: the heavy chain shown in SEQ ID NO:62 and the light chain shown in SEQ ID NO:

63. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 8-11 is characterized in that, The antibody-drug conjugate or its pharmaceutically acceptable salt has the structures shown in Formula II-1, Formula II-2 and Formula II-3. in, A is selected from -S(O)2(CH2) m -、-S(O)2(CH2) m Q(CH2) p -、-S(O)(CH2) m -、-S(O)(CH2) m Q(CH2) P -、-NR 1 (CH2) m -or-NR 1 (CH2) m Q(CH2) p -; Q, R 1 m and p are as defined in any one of claims 1-5, and Tb and q are as defined in any one of claims 8-11; Where A is selected from -NR 1 (CH2) m -or-NR 1 (CH2) m Q(CH2) p -; Q, R 1 m and p are as defined in any one of claims 1-5, and Tb and q are as defined in any one of claims 8-11; Wherein, A is selected from -(O)C(CH2). m -or -(O)C(CH2) m Q(CH2) p -; Q, R m m and p are defined as in any one of claims 1-5, and Tb and q are defined as in any one of claims 8-11. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 8-12 is characterized in that, The antibody-drug conjugate is selected from: A bioactive compound as shown in Formula III, or a pharmaceutically acceptable salt thereof. in, X and A are as defined in any one of claims 1-5. The bioactive compound or a pharmaceutically acceptable salt thereof as claimed in claim 14 is characterized in that, The bioactive compounds are selected from: A compound as shown in Formula IV or a pharmaceutically acceptable salt thereof. in, A is selected from -NR 1 (CH2) m -、-NR 1 (CH2) m Q(CH2) p -、-(O)C(CH2) m -、-(O)C(CH2) m Q(CH2) p -、-S(O)2(CH2) m -、-S(O)2(CH2) m Q(CH2) p -、-S(O)(CH2) m -or -S(O)(CH2) m Q(CH2) p -; Q is selected independently from -O-, -S-, or -NR. 2 -; R 1 Independently selected from hydrogen, sulfonyl, or C 1-4 Alkyl, the C 1-4 The alkyl group is optionally substituted by one or more substituents selected from hydroxyl, sulfonic acid, phosphate and sugar groups; R 2 Selected from hydrogen or C 1-4 alkyl; m and p are each independently selected from any integer between 1 and 5; B is either hydrogen or a hydroxyl group; And R 1 It is not methyl. The compound of claim 16 or a pharmaceutically acceptable salt thereof, characterized in that, The compound is selected from: A non-splittable connector as shown in formula V, in, X is defined as in any one of claims 1-5, wherein position 1 is linked to an antibody or its antigen-binding fragment, and position 2 is linked to a bioactive molecular fragment. The non-splittable connector as described in claim 18 is characterized in that, The non-spliable linkers are selected from: Position 1 is linked to the antibody or its antigen-binding fragment, and position 2 is linked to the bioactive molecule fragment. A method for preparing an antibody-drug conjugate of Formula II, the method comprising: The antibody or its antigen-binding fragment Tb is coupled with the drug linker conjugate shown in Formula I. Preferably, the method includes the step of coupling Tb with the drug linker conjugate shown in Formula I in a solvent to form a CS bond; The molar ratio of Tb to the drug linker conjugate is 1:(1-20), such as 1:(2-16), 1:(2-14), 1:(2-12), or 1:(2-10); The coupling reaction is carried out in water and / or an organic solvent; The organic solvent is selected from one or more of N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone, and nitrile compounds (e.g., acetonitrile); Wherein, the antibody-drug conjugate represented by Formula II is defined as any one of claims 8-13, and the drug linker conjugate represented by Formula I is defined as any one of claims 1-7. A group of antibody-drug conjugates comprising any one of claims 8-13, or a pharmaceutically acceptable salt thereof, or a combination thereof, wherein the antibody-drug conjugate has one, two or more q values; Preferably, the average DAR of the antibody-drug conjugate group is selected from an integer or decimal number from 1 to 16, for example, selected from 1.5-2.5, 3.5-4.5, 5.5-6.5 and 7.5-8.5; More preferably, the average DAR of the antibody-drug conjugate group is selected from about 2.0, 4.0, 6.0 and 8.

0. A pharmaceutical composition comprising an antibody-drug conjugate as described in any one of claims 8-13 or a pharmaceutically acceptable salt thereof; or the group of antibody-drug conjugates as described in claim 21 and optionally one or more pharmaceutical excipients. The use of the drug conjugate or pharmaceutically acceptable salt thereof as described in any one of claims 1-7; or the antibody-drug conjugate or pharmaceutically acceptable salt thereof as described in any one of claims 8-13; or the bioactive compound or pharmaceutically acceptable salt thereof as described in claim 14 or 15; or the compound or pharmaceutically acceptable salt thereof as described in claim 16 or 17; or the antibody-drug conjugate group as described in claim 21; or the use of the pharmaceutical composition as described in claim 22 in the preparation of a medicament for the treatment and / or prevention of diseases (e.g., cancer) associated with abnormal cellular activity; Preferably, the cancer is a solid tumor and / or a hematologic malignancy; Preferably, the cancer is a hematologic malignancy; Preferably, the cancer is a cancer related to the targets of HER2, CD123 and / or CD33; Preferably, the cancer is selected from acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, multiple myeloma, plasmacytoid dendritic cell tumor, non-Hodgkin lymphoma and / or Hodgkin lymphoma.

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