Camptothecin derivative for treating or preventing cancer, and antibody-drug conjugate thereof

Abstract

A camptothecin derivative for treating or preventing cancer, and a drug-linker conjugate and antibody-drug conjugate thereof, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope marker thereof. The present invention also relates to a pharmaceutical composition containing the camptothecin derivative or the drug-linker conjugate or antibody-drug conjugate thereof, and a preparation method therefor and a use thereof.

Description

Camptothecin derivatives and antibody-drug conjugates thereof for treating or preventing cancer Technical Field

[0001] In summary, the present invention relates to camptothecin derivatives and their drug-linker conjugates and antibody-drug conjugates for treating or preventing cancer, or pharmaceutically acceptable salts or esters, solvates, tautomers, stereoisomers, prodrugs, or isotope-labeled forms thereof. The present invention also relates to pharmaceutical compositions comprising the camptothecin derivatives, their drug-linker conjugates, or antibody-drug conjugates, as well as methods for their preparation and uses.

[0002] Background of the Invention

[0003] Antibody-Drug Conjugate (ADC) is a highly promising targeted drug, consisting mainly of three parts: an antibody (Ab) responsible for selectively recognizing antigens on the surface of tumor cells, a small molecule cytotoxic drug (Payload) responsible for killing tumor cells, and a linker (Linker) that connects the Payload to the Ab. The antibody acts as a carrier to transport the small molecule cytotoxic drug to the target cells.

[0004] By binding antibodies to tumor cell surface antigens, ADCs enter the cell via endocytosis. The small molecule cytotoxic drug then dissociates from the ADC and exerts its biological function. Thus, ADCs combine the potent killing effects of traditional small molecule cytotoxic drugs with the tumor-targeting properties of antibodies. Currently, there are many ADC drugs used to treat liquid tumors and solid tumors, such as POLIVY (Polatuzumab vedotin-piiq, CD79b-MMAE), which is approved by the FDA for the treatment of relapsed or refractory diffuse large B-cell lymphoma (R / RDLBCL); PADCEV (Enfortumab vedotin-ejfv, Nectin4-MMAE), which is approved by the FDA for the treatment of locally advanced or metastatic urothelial carcinoma (UC); Enhertu (Fam-trastuzumab deruxtecan-nxki, Her2-Dxd), used to treat breast cancer, etc.; and Trodelvy (sacituzumab govitecan-hziy, TROP2-SN38), used to treat patients with metastatic triple-negative breast cancer.

[0005] Camptothecin (CPT) is a small molecule, plant-based anticancer drug that acts as a DNA topoisomerase I inhibitor, showing promising results against gastrointestinal and head and neck cancers. Several camptothecin derivatives, such as irinotecan and exatecan, have been developed and are clinically used to treat tumors, including colorectal, pancreatic, and lung cancers. Camptothecin derivatives have also been used in ADC development, such as Enhertu, which uses Dxd as a payload, and Trodelvy, which uses SN-38 as a payload. However, many solid tumors still respond poorly to camptothecin drugs (Joshua Z Drago, Shanu Modi, Sarat Chandarlapaty et al. Unlocking the potential of antibody-drug conjugates for cancer therapy. Nat Rev Clin Oncol. 2021 Jun; 18(6): 327-344. doi: 10.1038 / s41571-021-00470-8. Epub 2021 Feb 8.). Tumor cells can develop resistance to camptothecins and their ADCs through various mechanisms, such as reduced cellular accumulation of the drug (GL Beretta, L Gatti, P Perego et al. Camptothecin resistance in cancer: insights into the molecular mechanisms of a DNA-damaging drug. Curr Med Chem. 2013; 20(12): 1541-65. doi: 10.2174 / 0929867311320120006.), which is related to the fact that SN38 is a substrate of P-glycoprotein (Michael Tagen, Yanli Zhuang, Fan Zhang et al. P-glycoprotein, but not multidrug resistance protein 4, plays a role in the systemic clearance of irinotecan and SN-38 in mice. Drug Metab Lett. 2010 Dec; 4(4): 195-201.).

[0006] Therefore, there is still a need to develop new camptothecin derivatives with good cytotoxicity and high-efficiency, low-toxicity ADC drugs. In particular, the camptothecin derivatives and ADC drugs of the present invention have good therapeutic efficacy. Moreover, the camptothecin derivatives and ADC drugs of the present invention have good tolerability, low toxic side effects, and a wide therapeutic window. The ADC drugs of the present invention also have good tumor tissue targeting and a strong bystander effect.

[0007] Summary of the Invention

[0008] SUMMARY OF THE INVENTION

[0009] In a first aspect, the present application provides an antibody-drug conjugate of formula (I) or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof.

[0010] In a second aspect, the present application provides a compound of formula (II) or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof.

[0011] In a third aspect, the present application provides a drug-linker conjugate of formula (III) or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof.

[0012] In a fourth aspect, the present application provides a pharmaceutical composition comprising a substance of the invention as defined herein and a pharmaceutically acceptable carrier, diluent or excipient.

[0013] In a fifth aspect, the present application provides a substance of the invention as defined herein for use in the treatment or prevention of cancer.

[0014] In a sixth aspect, the present application provides a method for preventing or treating cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of a substance of the present invention as defined herein.

[0015] In a seventh aspect, the present application provides use of a substance of the present invention as defined herein in the preparation of a medicament for treating or preventing cancer.

[0016] In an eighth aspect, the present application provides a kit for treating or preventing cancer, the kit comprising a substance of the present invention as defined herein and other co-drugs. The co-drugs may have the same or different efficacy as the substance of the present invention.

[0017] In a ninth aspect, the present application provides a method of preparing a substance of the invention as defined herein.

[0018] The substances of the present invention, including the camptothecin derivatives and drug-linker conjugates and antibody-drug conjugates of the present invention, have high cytotoxicity and can be used to treat or prevent tumors such as cancer, including but not limited to solid tumors, in particular lung cancer (such as small cell lung cancer, non-small cell lung cancer), liver cancer, digestive system cancer (such as intestinal cancer, gastric cancer, cardia cancer, esophageal cancer, appendix cancer, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer), bladder cancer, melanoma, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, basal cell carcinoma, bile duct cancer, squamous cell carcinoma, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, basal cell carcinoma, bile duct cancer, squamous cell carcinoma, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, basal cell carcinoma, bile duct cancer, squamous cell carcinoma, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, basal cell carcinoma, bile duct cancer, squamous cell carcinoma, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, basal cell carcinoma, cholangiocarcinoma ... Cell carcinoma, thyroid cancer, brain cancer, head and neck cancer, peritoneal cancer, renal cancer, urothelial carcinoma, testicular cancer, central nervous system tumors (e.g., gliomas, glioblastomas such as glioblastoma multiforme, gliomas or sarcomas), choriocarcinoma, oral epidermoid carcinoma; or hematological malignancies, in particular leukemias (e.g., acute or chronic myeloid leukemia, acute or chronic myeloid leukemia), lymphomas (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, acute B-cell lymphoma, follicular lymphoma), multiple myeloma. Preferably, the cancer is ovarian cancer, colon cancer, lung adenocarcinoma or oral epidermoid carcinoma. The substances of the present invention have stronger tumoricidal activity than Dxd and SN38.

[0019] Detailed Description of the Invention

[0020] In one aspect, the present invention provides an antibody drug conjugate of formula (I):

[0021] or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein:

[0022] Ab is an antibody or an antigen-binding fragment thereof;

[0023] L1 is each independently a linker unit;

[0024] L2 is each independently a linking unit selected from the following formula: -(CR m R n ) t -(L M ) 0-1 -(CH2CH2O) s -(CR m R n ) t -CO-,-(CR m R n ) t -L M -(CR m R n ) t -(L M ) 0-1 -LN -L M -(CH2O) s -(CR m R n ) t -CO-,-(CR m R n ) t -L M -(CR m R n ) t -N(R p )-(CR m R n ) t -N(R p )-(CR m R n ) t -CO-, and -(CR m R n ) t -L M -CH(R p )-CO-, wherein the -CO- end of L2 is connected to L3, and the other end of L2 is connected to L1, and

[0025] in,

[0026] R m and R n are each independently H or C 1-6 alkyl,

[0027] L M are each independently -NH-CO- or -CO-NH-,

[0028] L N Each independently is -(CH2CH2O) m -(CH2) t -、-(CH2) t -(OCH2CH2) m -, -(3-8 membered heteroarylene)-(CH2) t -(OCH2CH2) m or -(OCH2CH2) m -(CH2) t -(3-8 membered heteroarylene)-,

[0029] R p Each independently is -CO-(CH2CH2O) m -CH3 or -(CH2) t -L M -(CH2CH2O) m-CH3,

[0030] s and t are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, and

[0031] m is each independently 2, 3, 4, 5, 6, 7 or 8;

[0032] L3 is independently an amino acid residue, a short peptide chain consisting of 2-10 amino acid residues, or -NH-(CH2) p -CO-;

[0033] L4 are each independently a valence bond or a spacer unit;

[0034] D is each independently a small molecule drug moiety derived from a compound of formula (II);

[0035] in,

[0036] R1 is: H; halogen; CN; OH; SH; C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, said alkyl, alkenyl and alkynyl groups each being optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino; -(CH2) p -(C 3-8 Cycloalkyl) or -(CH2) p -(C 3-8 Heterocycloalkyl), said cycloalkyl and heterocycloalkyl are each optionally substituted by one or more independently selected from halogen, CN, OH, SH, amino, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl, C 1-6 Cyanoalkyl, and C 1-6 The substituent of aminoalkyl is substituted; -(CH2) p -(C 1-6 alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ; -(CH2) p -CH=NR a ;-NH-CO-R a ;-NH-CO-(CH2) p -CH(OH)-R a ;-NH-(CH2) p -(CH=CH)-R a ; or -COOH;

[0037] R2 and R3 are each independently halogen, cyano, NO2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, -NH-CO-(C 1-6 hydroxyalkyl), or NR a R b , or R2 and R3 together with the atoms to which they are attached form a 4- to 8-membered heterocyclic group;

[0038] R4 is H or C 3-8 Cycloalkyl, preferably H;

[0039] R a and R b Each independently is H, C 1-6 Alkyl, -CO-(C 1-6 alkyl), -SO-(C 1-6 alkyl), -SO2-(C 1-6 alkyl), or -(CH2) q -(C 3-8 cycloalkyl), said alkyl and cycloalkyl are each optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino, and said cycloalkyl is further optionally substituted with one or more substituents independently selected from C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl, C 1-6 Cyanoalkyl and C 1-6 The aminoalkyl group is substituted with a substituent; and

[0040] p and q are each independently 0, 1, 2, 3, 4, 5 or 6;

[0041] wherein the point of attachment of D to L4 is on R1 or R3 or through the hydroxyl oxygen shown in formula (II); and

[0042] n is an integer of 0 to 10, preferably an integer of 0 to 8.

[0043] In another aspect, the present application provides an antibody drug conjugate of formula (I)

[0044] or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein:

[0045] Ab is an antibody or an antigen-binding fragment thereof;

[0046] L1 is each independently a linker unit;

[0047] L2 is each independently of the formula -(CH2CH2O) x -(CR m R n ) y -CO- linking unit, wherein the -CO- end is connected to L3 and the other end is connected to L1, and wherein R m and R n are each independently H or C 1-6 Alkyl, x is an integer from 0 to 5, y is an integer from 1 to 5, and x+y≤6;

[0048] L3 is independently an amino acid residue, a short peptide chain consisting of 2-10 amino acid residues, or -NH-(CH2) p -CO-;

[0049] L4 are each independently a valence bond or a spacer unit;

[0050] D is each independently a small molecule drug moiety derived from a compound of formula (II);

[0051] in,

[0052] R1 is: H; halogen; CN; OH; SH; C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, said alkyl, alkenyl and alkynyl groups each optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino; -(CH2) p -(C 3-8 Cycloalkyl) or -(CH2) p -(C 3-8 Heterocycloalkyl), said cycloalkyl and heterocycloalkyl are each optionally substituted by one or more independently selected from halogen, CN, OH, SH, amino, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl, C 1-6 Cyanoalkyl, and C 1-6 The substituent of aminoalkyl is substituted; -(CH2) p -(C 1-6 alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ; -(CH2) p -CH=NR a ;-NH-CO-R a ;-NH-CO-(CH2) p-CH(OH)-R a ;-NH-(CH2) p -(CH=CH)-R a ; or -COOH;

[0053] R2 and R3 are each independently halogen, cyano, NO2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, -NH-CO-(C 1-6 hydroxyalkyl), or NR a R b , or R2 and R3 together with the atoms to which they are attached form a 4- to 8-membered heterocyclic group;

[0054] R4 is H or C 3-8 Cycloalkyl, preferably H;

[0055] R a and R b Each independently is H, C 1-6 Alkyl, -CO-(C 1-6 alkyl), -SO-(C 1-6 alkyl), -SO2-(C 1-6 alkyl), or -(CH2) q -(C 3-8 cycloalkyl), said alkyl and cycloalkyl are each optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino, and said cycloalkyl is further optionally substituted with one or more substituents independently selected from C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl, C 1-6 Cyanoalkyl and C 1-6 The aminoalkyl group is substituted with a substituent; and

[0056] p and q are each independently 0, 1, 2, 3, 4, 5 or 6;

[0057] wherein the point of attachment of D to L4 is on R1 or R3 or through the hydroxyl oxygen shown in formula (II); and

[0058] n is an integer of 0 to 10, preferably an integer of 0 to 8.

[0059] In another aspect, the present invention provides an antibody drug conjugate of formula (I):

[0060] or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein:

[0061] Ab is an antibody or an antigen-binding fragment thereof;

[0062] L1 is each independently a linker unit;

[0063] L2 is each independently of the formula -(CH2CH2O) x -(CR m R n ) y -CO- linking unit, wherein the -CO- end is connected to L3 and the other end is connected to L1, and wherein R m and R n are each independently H or C 1-6 Alkyl, x is an integer from 0 to 5, y is an integer from 1 to 5, and x+y≤6;

[0064] L3 is independently an amino acid residue, a short peptide chain consisting of 2-10 amino acid residues, or -NH-(CH2) p -CO-;

[0065] L4 are each independently a valence bond or a spacer unit;

[0066] D is each independently a small molecule drug moiety derived from a compound of formula (II);

[0067] in,

[0068] R1 is: H; halogen; CN; OH; SH; C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, said alkyl, alkenyl and alkynyl groups each being optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino; -(CH2) p -(C 3-8 cycloalkyl), which is optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino; -(CH2) p -(C 1-6 alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ;-CH=NR a ;-NH-CO-R a ;-NH-CO-(CH2) p -CH(OH)-R a ; or -COOH;

[0069] R2 and R3 are each independently halogen, cyano, NO2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, or NR a R b , or R2 and R3 together with the atoms to which they are attached form a 4- to 8-membered heterocyclic group;

[0070] R4 is H or C 3-8 Cycloalkyl, preferably H;

[0071] R a and R b Each independently is H, C 1-6 Alkyl, -SO-(C 1-6 alkyl), -SO2-(C 1-6 alkyl), or -(CH2) q -(C 3-8 cycloalkyl), said alkyl and cycloalkyl being each optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino;

[0072] p and q are each independently 0, 1, 2, 3, 4, 5 or 6;

[0073] wherein the point of attachment of D to L4 is on R1 or R3 or through the hydroxyl oxygen shown in formula (II); and

[0074] n is an integer of 0 to 10, preferably an integer of 0 to 8.

[0075] In another aspect, the present invention provides a camptothecin derivative of formula (II):

[0076] or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein

[0077] R1 is: H; halogen; CN; OH; SH; C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, said alkyl, alkenyl and alkynyl groups each optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino; -(CH2) p -(C 3-8 Cycloalkyl) or -(CH2) p -(C 3-8 Heterocycloalkyl), said cycloalkyl and heterocycloalkyl are each optionally substituted by one or more independently selected from halogen, CN, OH, SH, amino, C 1-6 Alkyl, C 1-6Halogenated alkyl, C 1-6 Hydroxyalkyl, C 1-6 Cyanoalkyl, and C 1-6 The substituent of aminoalkyl is substituted; -(CH2) p -(C 1-6 alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ; -(CH2) p -CH=NR a ;-NH-CO-R a ;-NH-CO-(CH2) p -CH(OH)-R a ;-NH-(CH2) p -(CH=CH)-R a ; or -COOH;

[0078] R2 and R3 are each independently halogen, cyano, NO2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, -NH-CO-(C 1-6 hydroxyalkyl), or NR a R b , or R2 and R3 together with the atoms to which they are attached form a 4- to 8-membered heterocyclic group;

[0079] R4 is H or C 3-8 Cycloalkyl, preferably H;

[0080] R a and R b Each independently is H, C 1-6 Alkyl, -CO-(C 1-6 Alkyl), -SO-(C 1-6 alkyl), -SO2-(C 1-6 alkyl), or -(CH2) q -(C 3-8 cycloalkyl), said alkyl and cycloalkyl are each optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino, and said cycloalkyl is further optionally substituted with one or more substituents independently selected from C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl, C 1-6 Cyanoalkyl and C 1-6 The aminoalkyl group is substituted with a substituent; and

[0081] p and q are each independently 0, 1, 2, 3, 4, 5 or 6.

[0082] In another aspect, the present invention provides a compound of formula (II):

[0083] or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein,

[0084] R1 is: H; halogen; CN; OH; SH; C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, said alkyl, alkenyl and alkynyl groups each being optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino; -(CH2) p -(C 3-8 cycloalkyl), which is optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino; -(CH2) p -(C 1-6 alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ;-CH=NR a ;-NH-CO-R a ;-NH-CO-(CH2) p -CH(OH)-R a ; or -COOH;

[0085] R2 and R3 are each independently halogen, cyano, NO2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, or NR a R b , or R2 and R3 together with the atoms to which they are attached form a 4- to 8-membered heterocyclic group;

[0086] R4 is H or C 3-8 Cycloalkyl, preferably H;

[0087] R a and R b Each independently is H, C 1-6 Alkyl, -SO-(C 1-6 alkyl), -SO2-(C 1-6 alkyl), or -(CH2) q -(C3-8 cycloalkyl), said alkyl and cycloalkyl being each optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino; and

[0088] p and q are each independently 0, 1, 2, 3, 4, 5 or 6.

[0089] In another aspect, the present invention provides a drug-linker conjugate of formula (III): Lg-L1-L2-L3-L4-D (III)

[0090] or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotopically labeled thereof, wherein L1, L2, L3, L4 and D are as defined herein; and wherein

[0091] When L1 is When Lg and L1 form

[0092] When L1 is not When Lg is a leaving group. Preferably, Lg is a halogen, a sulfonyl group (e.g., methanesulfonyl, p-toluenesulfonyl), a sulfonyloxy group (e.g., methanesulfonyloxy, CF3SO3-, p-toluenesulfonyloxy), a tertiary amine group (e.g., Me3N + or Et3N + ) or a diazonium salt group. More preferably, Lg is F, Cl, Br, or a methylsulfonyl group. Particularly preferably, Lg is a methylsulfonyl group.

[0093] In some embodiments, R1 is: H; halogen; CN; OH; SH; C 1-6 Alkyl; C 1-6 Haloalkyl; C 1-6 Hydroxyalkyl; -(CH2) p -(C 3-8 Cycloalkyl) or -(CH2) p -(C 3-8 Heterocycloalkyl), said cycloalkyl and heterocycloalkyl are each optionally substituted by one or more independently selected from OH, SH, C 1-6 Alkyl and C 1-6 Substituents of hydroxyalkyl; -(CH2) p -(C 1-6 alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ; -(CH2) p -CH=NR a ;-NH-CO-R a;-NH-CO-(CH2) p -CH(OH)-R a ;-NH-(CH2) p -(CH=CH)-R a ; or -COOH. Further, R a and R b Each independently is H, C 1-6 Alkyl, C 1-6 Hydroxyalkyl, C 1-6 aminoalkyl, -SO2-(C 1-6 alkyl), or -(CH2) q -(optionally replaced by one or more independently selected from hydroxyl and C 1-6 The substituents of hydroxyalkyl are substituted with C 3-8 cycloalkyl).

[0094] In other embodiments, R1 is H; C 1-6 Alkyl; C 1-6 Haloalkyl; C 1-6 Hydroxyalkyl; -(CH2) p -(C 3-8 cycloalkyl); -(CH2) p -(C 3-8 Hydroxycycloalkyl); -(CH2) p -(C 3-8 Heterocycloalkyl), said heterocycloalkyl being optionally substituted by one or more independently selected from OH, C 1-6 Alkyl and C 1-6 The substituents of the hydroxyalkyl group are preferably selected from OH and C 1-6 The substituents of the hydroxyalkyl group are more preferably selected from C 1-6 Substituents of hydroxyalkyl; -(CH2) p -(C 1-6 alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ; -(CH2) p -CH=NR a ;-NH-CO-R a ;-NH-CO-(CH2) p -CH(OH)-R a ;-NH-(CH2) p -(CH=CH)-R a ; or -COOH. Further, R a and R b Each independently is H, C 1-6 Alkyl, C1-6 Hydroxyalkyl, C 1-6 aminoalkyl, -SO2-(C 1-6 alkyl), -(CH2) q -(C 3-8 Cycloalkyl), -(CH2) q -(C 3-8 Hydroxycycloalkyl), -(CH2) q -(C substituted by one or more amino groups 3-8 Cycloalkyl), -(CH2) q -(by one or more C 1-6 Hydroxyalkyl substituted C 3-8 cycloalkyl).

[0095] In other embodiments, R1 is H; C 1-6 Alkyl; C 1-6 Haloalkyl; C 1-6 Hydroxyalkyl; -(CH2) p -(C 3-8 Heterocycloalkyl), said heterocycloalkyl being optionally substituted by one or more independently selected from OH and C 1-6 The substituents of the hydroxyalkyl group are more preferably selected from C 1-6 Substituents of hydroxyalkyl; -(CH2) p -(C 1-6 alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NH2; -(CH2) p -NH(C 1-6 alkyl); -(CH2) p -NH(C 1-6 Hydroxyalkyl); -(CH2) p -NH(C 1-6 aminoalkyl); -(CH2) p -NH-(CH2) q -(optionally replaced by one or more C 1-6 Hydroxyalkyl substituted C 3-8 cycloalkyl); -(CH2) p -N(C 1-6 Alkyl)(-SO2-C 1-6 alkyl); -(CH2) p -CH=N(C 1-6 alkyl); -NH-CO-(C 1-6 Hydroxyalkyl); -NH-CO-(C 3-8 Hydroxycycloalkyl); -NH-CO-(CH2) p -CH(OH)-(C1-6 Alkyl); -NH-CO-(CH2) p -CH(OH)-(C 3-8 Cycloalkyl); -NH-(CH2) p -(CH=CH)-(C 1-6 alkyl); or -COOH.

[0096] In other embodiments, R1 is H; C 1-6 Alkyl; C 1-6 Haloalkyl; C 1-6 Hydroxyalkyl; -(CH2) p -(C 3-8 Heterocycloalkyl), the heterocycloalkyl group is optionally substituted by one or more C 1-6 Hydroxyalkyl substituted; -(CH2) p -(C 1-6 alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NH2; -(CH2) p -NH(C 1-6 alkyl); -(CH2) p -NH(C 1-6 Hydroxyalkyl); -(CH2) p -NH(C 1-6 aminoalkyl); -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), the cycloalkyl group is optionally substituted by one or more selected from C 1-6 Substituents of hydroxyalkyl; -(CH2) p -N(C 1-6 Alkyl)(-SO2-C 1-6 alkyl); -CH=N(C 1-6 alkyl); -NH-CO-(C 1-6 Hydroxyalkyl); -NH-CO-(C 3-8 Hydroxycycloalkyl); -NH-CO-(CH2) p -CH(OH)-(C 1-6 Alkyl); -NH-CO-(CH2) p -CH(OH)-(C 3-8 Cycloalkyl); -NH-(CH2) p -(CH=CH)-(C 1-6 or -COOH.

[0097] In other embodiments, R1 is H; C 1-6 Alkyl; C1-6 Haloalkyl; C 1-6 Hydroxyalkyl; -(CH2) p -(C 3-8 Heterocycloalkyl), the heterocycloalkyl group is optionally substituted by one or more C 1-6 Hydroxyalkyl substituted; -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NH2; -(CH2) p -NH(C 1-6 alkyl); -(CH2) p -NH(C 1-6 Hydroxyalkyl); -(CH2) p -NH(C 1-6 aminoalkyl); -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), the cycloalkyl group is optionally substituted by one or more selected from C 1-6 Substituents of hydroxyalkyl; -(CH2) p -N(C 1-6 Alkyl)(-SO2-C 1-6 alkyl); -CH=N(C 1-6 alkyl); -NH-CO-(C 1-6 Hydroxyalkyl); -NH-CO-(C 3-8 Hydroxycycloalkyl); -NH-CO-(CH2) p -CH(OH)-(C 3-8 Cycloalkyl); -NH-(CH2) p -(CH=CH)-(C 1-6 or -COOH.

[0098] In some embodiments, R1 is H; C 1-6 Alkyl; -(CH2) p -(optionally replaced by one or more C 1-6 Hydroxyalkyl substituted C 3-8 heterocycloalkyl); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -(NH2);-(CH2) p -NH(C 1-6 alkyl); -(CH2) p -NH(C 1-6 Hydroxyalkyl); -(CH2) p -NH-(CH2) q -(optionally replaced by one or more C 1-6Hydroxyalkyl substituted C 3-8 Cycloalkyl); -NH-CO-(CH2) p -CH(OH)-(C 3-8 cycloalkyl); or -NH-(CH2) p -(CH=CH)-(C 1-6 alkyl).

[0099] In some embodiments, R1 is H, C 1-6 Alkyl, -(CH2) p -(optionally replaced by one or more C 1-6 Hydroxyalkyl substituted C 3-8 Nitrogen-containing heterocycloalkyl), -(CH2) p -(C 1-6 alkylthio), -NH2, -(CH2) p -NH(C 1-6 Alkyl), -NH(C 1-6 Hydroxyalkyl), -(CH2) p -NH-(CH2) q -(optionally replaced by one or more C 1-6 Hydroxyalkyl substituted C 3-8 Cycloalkyl), -NH-CO-CH(OH)-(C 3-8 cycloalkyl), or -NH-(CH=CH)-(C 1-6 alkyl).

[0100] In a further embodiment, R1 is H, C 1-6 Alkyl, optionally substituted by one or more C 1-6 Hydroxyalkyl substituted C 3-8 Nitrogen-containing heterocycloalkyl, -CH2-(C 1-6 Alkylthio), -NH2, -CH2-NH(C 1-6 Alkyl), -NH(C 1-6 Hydroxyalkyl), -NH-(optionally replaced by one or more C 1-6 Hydroxyalkyl substituted C 3-8 Cycloalkyl), -CH2-NH-CH2-(C 3-8 Cycloalkyl), -NH-CO-CH(OH)-(C 3-8 cycloalkyl), or -NH-(CH=CH)-(C 1-6 alkyl).

[0101] In some embodiments, R1 is C 1-6 Alkyl, -(CH2) p -(optionally replaced by one or more C 1-6 Hydroxyalkyl substituted C 3-8 Nitrogen-containing heterocycloalkyl), -(CH2)p -(C 1-6 alkylthio), -(CH2) p -NH(C 1-6 alkyl), -(CH2) p -NH-(CH2) q -(optionally replaced by one or more C 1-6 Hydroxyalkyl substituted C 3-8 cycloalkyl), or -(CH2) p -NH(C 1-6 Hydroxyalkyl), or -NH-(CH2) p -(CH=CH)-(C 1-6 Preferably, R1 is C 1-6 Alkyl, -(CH2) p -(optionally replaced by a C 1-6 Hydroxyalkyl substituted C 3-8 Nitrogen-containing heterocycloalkyl), -CH2-(C 1-6 Alkylthio), -NH(C 1-6 Alkyl), -NH(C 1-6 Hydroxyalkyl), -(CH2) p -NH-(CH2) q -(optionally replaced by a C 1-6 Hydroxyalkyl substituted C 3-8 cycloalkyl), or -NH-(CH=CH)-(C 1-6 alkyl).

[0102] In some embodiments, R1 is C 1-6 Alkyl, -(CH2) p -(optionally replaced by a C 1-6 Hydroxyalkyl substituted C 3-8 Nitrogen-containing heterocycloalkyl), -(CH2) p -(C 1-6 alkylthio), -NH2, -(CH2) p -NH(C 1-6 alkyl), -(CH2) p -NH(C 1-6 Hydroxyalkyl), -(CH2) p -NH-(CH2) q -(optionally replaced by a C 1-6 Hydroxyalkyl substituted C 3-8 Cycloalkyl), -NH-CO-(C 1-6 Hydroxyalkyl), -NH-CO-(CH2) p -CH(OH)-(C 3-8 cycloalkyl), or -NH-(CH=CH)-(C 1-6Preferably, R1 is C 1-6 Alkyl, a C 1-6 Hydroxyalkyl substituted C 3-8 Nitrogen-containing heterocycloalkyl, -NH2, -(CH2) p -NH(C 1-6 alkyl), -(CH2) p -NH(C 1-6 Hydroxyalkyl), -NH-(with a C 1-6 Hydroxyalkyl substituted C 3-8 Cycloalkyl), -NH-CO-(C 1-6 Hydroxyalkyl), or -NH-(CH=CH)-(C 1-6 alkyl).

[0103] In some embodiments, R1 is selected from: H; methyl, ethyl, propyl, butyl, pentyl, hexyl; chloromethyl, fluoromethyl, bromomethyl, chloroethyl, fluoroethyl, bromoethyl, chloropropyl, fluoropropyl, bromopropyl, chlorobutyl, fluorobutyl, bromobutyl; hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl; -(piperidinyl)-CH2OH; -CH2-(methylthio), -CH2-(ethylthio), -CH2-(propylthio), -CH2-(butylthio), -CH2-(pentylthio) ), -CH2-(hexylthio); -NH2, -NHMe, -NHEt, -NHPr, -NHBu, -NH(pentyl), -NH(hexyl); -NH(hydroxymethyl), -NH(hydroxyethyl), -NH(hydroxypropyl), -NH(hydroxybutyl), -NH(hydroxypentyl), -NH(hydroxyhexyl); -NH(aminomethyl), -NH(aminoethyl), -NH(aminopropyl), -NH(aminobutyl), -NH(aminopentyl), -NH(aminohexyl); -CH2-NHMe, -CH2-NHEt, -CH2-NHPr, -CH2-NHBu, -CH2-NH(pentyl), -CH2-NH(hexyl); -CH2-NH-CH2-(cyclopropyl), -CH2-NH-CH2-(cyclobutyl), -CH2-NH-CH2-(cyclopentyl), -CH2-NH-CH2-(cyclohexyl); -(CH2)2-N(Pr)(-SO2Me); -CH=NMe, -CH=NEt, -CH=NPr, -CH=NBu, -CH=N(pentyl), -CH=N(hexyl); -NH-CO-hydroxymethyl, -NH-CO-hydroxyethyl, -NH-CO-hydroxypropyl, -NH-CO-hydroxybutyl, -NH-CO-hydroxypentyl, -NH-CO-hydroxyhexyl; -NH-CO-(hydroxycyclohexyl); -NH-CO-(hydroxycyclobutyl), -NH-CO-(hydroxycyclopentyl), -NH-CO-(hydroxycyclohexyl); -NH-CO-CH(OH)-(cyclopropyl), -NH-CO-CH(OH)-(cyclobutyl), -NH-CO-CH(OH)-(cyclopentyl), -NH-CO-CH(OH)-(cyclohexyl); -NH-CO-CH2-CH(OH)(Me), -NH-CO-CH2-CH(OH)(Et), -NH-CO-CH2-CH(OH)(Pr), -NH-CO-CH2-CH(OH)(Bu), -NH-CO-CH2-CH(OH)(pentyl), -NH-CO-CH2-CH(OH)(hexyl); -NH-(CH2)-(CH=CH)-(CH2OH); or -COOH.

[0104] In some embodiments, R1 is H; pentyl; chloromethyl; hydroxybutyl; -(piperidinyl)-CH2OH; -CH2-(propylthio); -NH2; -NH(pentyl); -CH2-NH(pentyl); -NH(hydroxybutyl); -NH(aminobutyl); -CH2-NH-CH2-(cyclopentyl); -(CH2)2-N(Pr)(-SO2Me); -CH=N(pentyl); -NH-CO-(hydroxymethyl); -NH-CO-(hydroxycyclohexyl); -NH-CO-CH(OH)-(cyclopropyl); -NH-CO-CH2-CH(OH)(Me); -NH-(CH2)-(CH=CH)-(CH2OH); or -COOH.

[0105] In some embodiments, R1 is: H; halogen; CN; OH; SH; C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6Alkynyl, said alkyl, alkenyl and alkynyl groups each being optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino; -(CH2) p -(C 3-8 cycloalkyl), which is optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino; -(CH2) p -(C 1-6 alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ;-CH=NR a ;-NH-CO-R a ;-NH-CO-(CH2) p -CH(OH)-R a ; or -COOH; wherein R a and R b Each independently is H, C 1-6 Alkyl, -SO-(C 1-6 alkyl), -SO2-(C 1-6 alkyl), or -(CH2) q -(C 3-8 cycloalkyl), said alkyl and cycloalkyl being each optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino.

[0106] In some embodiments, R1 is: H; halogen; CN; OH; SH; C 1-6 Alkyl; C 1-6 Haloalkyl; C 1-6 Hydroxyalkyl; -(CH2) p -(C 3-8 cycloalkyl); -(CH2) p -(C 3-8 Hydroxycycloalkyl); -(CH2) p -(C 1-6 alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ;-CH=NR a ;-NH-CO-R a ;-NH-CO-(CH2) p -CH(OH)-R a ; or -COOH.

[0107] In a further embodiment, R1 is H; C 1-6 Alkyl; C 1-6 Haloalkyl; C 1-6 Hydroxyalkyl; -(CH2) p -(C 1-6 alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ;-CH=NR a ;-NH-CO-R a ;-NH-CO-(CH2) p -CH(OH)-R a ; or -COOH; preferably wherein R a and R b Each independently is H, C 1-6 Alkyl, C 1-6 Hydroxyalkyl, C 1-6 aminoalkyl, -SO2-(C 1-6 alkyl), -(CH2) q -(C 3-8 cycloalkyl), or -(CH2) q -(C 3-8 hydroxycycloalkyl).

[0108] In a further embodiment, R1 is H; C 1-6 Alkyl; C 1-6 Haloalkyl; C 1-6 Hydroxyalkyl; -(CH2) p -(C 1-6 alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NH2; -(CH2) p -NH(C 1-6 alkyl); -(CH2) p -NH(C 1-6 Hydroxyalkyl); -(CH2) p -NH(C 1-6 aminoalkyl); -(CH2) p -NH-(CH2) q -(C 3-8 cycloalkyl); -(CH2) p -N(C 1-6 Alkyl)(-SO2-C 1-6 alkyl); -CH=N(C 1-6 alkyl); -NH-CO-(C 1-6Hydroxyalkyl); -NH-CO-(C 3-8 Hydroxycycloalkyl); -NH-CO-(CH2) p -CH(OH)-(C 1-6 Alkyl); -NH-CO-(CH2) p -CH(OH)-(C 3-8 cycloalkyl); or -COOH.

[0109] In some embodiments, R1 is H, C 1-6 Alkyl, -(CH2) p -(C 1-6 alkylthio), -(CH2) p -(NH2), -(CH2) p -NH(C 1-6 alkyl), -(CH2) p -NH(C 1-6 Hydroxyalkyl), -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), or -NH-CO-(CH2) p -CH(OH)-(C 3-8 cycloalkyl).

[0110] In a further embodiment, R1 is H, C 1-6 Alkyl, -(CH2) p -(C 1-6 alkylthio), -NH2, -(CH2) p -NH(C 1-6 Alkyl), -NH(C 1-6 Hydroxyalkyl), -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), or -NH-CO-CH(OH)-(C 3-8 cycloalkyl).

[0111] In a further embodiment, R1 is H, C 1-6 Alkyl, -CH2-(C 1-6 Alkylthio), -NH2, -CH2-NH(C 1-6 Alkyl), -NH(C 1-6 Hydroxyalkyl), -CH2-NH-CH2-(C 3-8 Cycloalkyl), or -NH-CO-CH(OH)-(C 3-8 cycloalkyl).

[0112] In some embodiments, R1 is C 1-6Alkyl, -(CH2) p -(C 1-6 alkylthio), -(CH2) p -NH(C 1-6 alkyl), or -(CH2) p -NH(C 1-6 Preferably, R1 is C 1-6 Alkyl, -CH2-(C 1-6 Alkylthio), -NH(C 1-6 alkyl), or -NH(C 1-6 hydroxyalkyl).

[0113] In some embodiments, R1 is C 1-6 Alkyl, -(CH2) p -(C 1-6 alkylthio), -NH2, -(CH2) p -NH(C 1-6 alkyl), -(CH2) p -NH(C 1-6 Hydroxyalkyl), -NH-CO-(C 1-6 Hydroxyalkyl), or -NH-CO-(CH2) p -CH(OH)-(C 3-8 Preferably, R1 is C 1-6 Alkyl, -NH2, -(CH2) p -NH(C 1-6 alkyl), -(CH2) p -NH(C 1-6 Hydroxyalkyl), or -NH-CO-(C 1-6 hydroxyalkyl).

[0114] In some embodiments, R1 is: C 1-6 Alkyl; -(CH2) p -(C 3-8 Heterocycloalkyl), said heterocycloalkyl being optionally substituted by one or more independently selected from OH, C 1-6 Alkyl or C 1-6 Substituents of hydroxyalkyl; -(CH2) p -NR a R b , where R a and R b Each independently is H, C 1-6 Alkyl, C 1-6 Hydroxyalkyl, or -(CH2) q -(optionally replaced by one or more independently selected from hydroxyl and C 1-6 The substituents of hydroxyalkyl are substituted with C 3-8cycloalkyl); or -NH-(CH2) p -(CH=CH)-R a , where R a C 1-6 hydroxyalkyl, and p is 0, 1, 2, 3 or 4. Preferably, R1 is: C 1-6 Alkyl; -(CH2) p -(optionally replaced by a C 1-6 Hydroxyalkyl substituted C 3-8 heterocycloalkyl); -(CH2) p -NR a R b , where R a is H, and R b It is C 1-6 Hydroxyalkyl, or -(CH2) q -(optionally replaced by a C 1-6 Hydroxyalkyl substituted C 3-8 cycloalkyl); or -NH-(CH2) p -(CH=CH)-R a , where R a C 1-6 hydroxyalkyl, and p is 0, 1, 2, 3 or 4.

[0115] In other embodiments, R1 is C 1-6 Alkyl; -(CH2) p -(C 3-8 Heterocycloalkyl), the heterocycloalkyl group is optionally substituted by one or more C 1-6 Hydroxyalkyl substituted; -(CH2) p -NH(C 1-6 Hydroxyalkyl); -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), the cycloalkyl group is optionally substituted by one or more selected from C 1-6 Substituted with hydroxyalkyl; or -NH-(CH2) p -(CH=CH)-(C 1-6 hydroxyalkyl).

[0116] In other embodiments, R1 is C 1-6 Alkyl; -(CH2) p -(C 3-8 Heterocycloalkyl), the heterocycloalkyl group is optionally substituted by one or more C 1-6 Hydroxyalkyl substituted; -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NH2; -(CH2)p -NH(C 1-6 alkyl); -(CH2) p -NH(C 1-6 Hydroxyalkyl); -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), the cycloalkyl group is optionally substituted by one or more selected from C 1-6 Substituents of hydroxyalkyl; -NH-CO-(C 1-6 hydroxyalkyl); or -NH-CO-(CH2) p -CH(OH)-(C 3-8 cycloalkyl).

[0117] In other embodiments, R1 is C 1-6 Alkyl; -(CH2) p -(C 3-8 Heterocycloalkyl), the heterocycloalkyl group is optionally substituted by one or more C 1-6 Hydroxyalkyl substituted; -(CH2) p -NH2; -(CH2) p -NH(C 1-6 alkyl); -(CH2) p -NH(C 1-6 Hydroxyalkyl); -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), the cycloalkyl group is optionally substituted by one or more selected from C 1-6 Substituents of hydroxyalkyl; -NH-CO-(C 1-6 hydroxyalkyl); or -NH-CO-(CH2) p -CH(OH)-(C 3-8 cycloalkyl).

[0118] In other embodiments, R1 is C 1-6 Alkyl; -(CH2) p -(C 3-8 Heterocycloalkyl), the heterocycloalkyl group is optionally substituted by one or more C 1-6 Hydroxyalkyl substituted; -(CH2) p -NH(C 1-6 Hydroxyalkyl); -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), the cycloalkyl group is optionally substituted by one or more selected from C 1-6 Substituted with a hydroxyalkyl group; or -NH-CO-(CH2) p -CH(OH)-(C3-8 cycloalkyl).

[0119] In other embodiments, R1 is C 1-6 Alkyl; -(CH2) p -(C 3-8 Heterocycloalkyl), the heterocycloalkyl group is optionally substituted by one or more C 1-6 Hydroxyalkyl substituted; -(CH2) p -NH(C 1-6 hydroxyalkyl); or -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), the cycloalkyl group is optionally substituted by one or more selected from C 1-6 The substituent of the hydroxyalkyl group is substituted.

[0120] In further embodiments, p is 0, 1, 2, 3, or 4. In some further embodiments, p is 0. In other further embodiments, p is 1. In other further embodiments, p is 2. In other further embodiments, p is 3. In other further embodiments, p is 4.

[0121] In some embodiments, R1 is selected from: H; methyl, ethyl, propyl, butyl, pentyl, hexyl; chloromethyl, fluoromethyl, bromomethyl, chloroethyl, fluoroethyl, bromoethyl, chloropropyl, fluoropropyl, bromopropyl, chlorobutyl, fluorobutyl, bromobutyl; hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl; -CH2-(methylthio), -CH2-(ethylthio), -CH2-(propylthio), -CH2-(butylthio), -CH2-(pentylthio), -CH2-(hexylthio); -NH2, -NHMe, -NHEt, -NHPr, -NHBu, -NH(pentyl), -NH(hexyl); -NH(hydroxymethyl), -NH(hydroxyethyl), -NH(hydroxypropyl), -NH(hydroxybutyl), -NH(hydroxypentyl), -NH(hydroxyhexyl); -NH(aminomethyl), -NH(aminoethyl ... NH(aminopropyl), -NH(aminobutyl), -NH(aminopentyl), -NH(aminohexyl); -CH2-NHMe, -CH2-NHEt, -CH2-NHPr, -CH2-NHBu, -CH2-NH(pentyl), -CH2-NH(hexyl); -CH2-NH-CH2-(cyclopropyl), -CH2-NH-CH2-(cyclobutyl), -CH2-NH-CH2-(cyclopentyl), -CH2-NH-CH2-(cyclohexyl); -(CH2)2-N(Pr)(-SO2Me); -CH=NMe, -CH=NEt, -CH=NPr, -CH=NBu, -CH=N(pentyl), -CH=N(hexyl); -NH-CO-hydroxymethyl, -NH-CO-hydroxyethyl, -NH-CO-hydroxypropyl, -NH-CO-hydroxybutyl, -NH-CO- -NH-CO-hydroxypentyl, -NH-CO-hydroxyhexyl; -NH-CO-(hydroxycyclopropyl), -NH-CO-(hydroxycyclobutyl), -NH-CO-(hydroxycyclopentyl), -NH-CO-(hydroxycyclohexyl); -NH-CO-CH(OH)-(cyclopropyl), -NH-CO-CH(OH)-(cyclobutyl), -NH-CO-CH(OH)-(cyclopentyl), -NH-CO-CH(OH)-(cyclohexyl); -NH-CO-CH2-CH(OH)(Me), -NH-CO-CH2-CH(OH)(Et), -NH-CO-CH2-CH(OH)(Pr), -NH-CO-CH2-CH(OH)(Bu), -NH-CO-CH2-CH(OH)(pentyl), -NH-CO-CH2-CH(OH)(hexyl); or -COOH.

[0122] In some embodiments, R1 is H; pentyl; chloromethyl; hydroxybutyl; -CH2-(propylthio); -NH2; -NH(pentyl); -CH2-NH(pentyl); -NH(hydroxybutyl); -NH(aminobutyl); -CH2-NH-CH2-(cyclopentyl); -(CH2)2-N(Pr)(-SO2Me); -CH=N(pentyl); -NH-CO-(hydroxymethyl); -NH-CO-(hydroxycyclohexyl); -NH-CO-CH(OH)-(cyclopropyl); -NH-CO-CH2-CH(OH)(Me); or -COOH.

[0123] In some embodiments, R1 is -NH(hydroxymethyl), -NH(hydroxyethyl), -NH(hydroxypropyl), -NH(hydroxybutyl), -NH(hydroxypentyl), -NH(hydroxyhexyl); -(azacyclopentane)-(CH2OH), -(azacyclohexane)-(CH2OH), -(azacycloheptane)-(CH2OH); or -NH-cyclopentane-(CH2OH), -NH-cyclohexane-(CH2OH), -NH-cycloheptane-(CH2OH). Preferably, R1 is -NH(hydroxybutyl), -(azacyclohexane)-(CH2OH), or -NH-cyclohexane-(CH2OH).

[0124] In some embodiments, R1 is: pentyl; -NH(hydroxyethyl), -NH(hydroxypropyl), -NH(hydroxybutyl), -NH(hydroxypentyl); -(azacyclopentane)-(CH2OH); -NH-cyclohexane-(CH2OH); or -NH-(CH2)-(CH=CH)-(CH2OH).

[0125] In other embodiments, R1 is: pentyl, -CH2-(propylthio), -NH2, -NH(pentyl), -NH(hydroxybutyl), -CH2-NH(pentyl), -NH-CO-hydroxymethyl, -NH-CO-CH(OH)-(cyclopropyl), -(azacyclohexane)-(hydroxymethyl), or -NH-(cyclohexane)-(hydroxymethyl).

[0126] In other embodiments, R1 is pentyl, -NH2, -NH(hydroxybutyl), -CH2-NH(pentyl), -NH-CO-hydroxymethyl, -NH-CO-CH(OH)-(cyclopropyl), -(azacyclohexane)-(hydroxymethyl), or -NH-(cyclohexane)-(hydroxymethyl).

[0127] In other embodiments, R1 is pentyl, -NH(hydroxybutyl), -NH-CO-CH(OH)-(cyclopropyl), -(azacyclohexane)-(hydroxymethyl), or -NH-(cyclohexane)-(hydroxymethyl).

[0128] In other embodiments, R1 is pentyl, -NH(hydroxybutyl), -(azacyclohexane)-(hydroxymethyl), or -NH-(cyclohexane)-(hydroxymethyl).

[0129] In some embodiments, R2 is halogen, such as fluorine, chlorine, bromine, or iodine. In further embodiments, R2 is fluorine.

[0130] In some embodiments, R3 is C 1-6 Alkyl, C 1-6 Alkoxy, halogen, cyano, -NH2, -NH-CO-(C 1-6 Preferably, R3 is C 1-6 Alkyl, halogen, -NH2, -NH-CO-(C 1-6 In some embodiments, R is methyl, ethyl, propyl, butyl, fluorine, chlorine, bromine, -NH, -NH-CO-(CHOH), or NO. In some embodiments, R is methyl, ethyl, propyl, butyl, fluorine, chlorine, bromine, -NH, -NH-CO-(CHOH), or NO. In some embodiments, R is methyl, bromine, -NH, -NH-CO-(CHOH), or NO.

[0131] In some embodiments, R3 is halogen, cyano, NO2, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, or NR a R b , where R a and R b Each independently is H, C 1-6 Alkyl, -SO-(C 1-6 alkyl), -SO2-(C 1-6 alkyl), or -(CH2) q -(C 3-8 cycloalkyl), said alkyl and cycloalkyl being each optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino.

[0132] In some embodiments, R3 is C 1-6 Alkyl, C 1-6 In a further embodiment, R3 is C 1-6 Alkyl, halogen, -NH2 or NO2. In particular, R3 is methyl, ethyl, propyl, butyl, fluorine, chlorine, bromine, -NH2 or NO2. More particularly, R3 is methyl, bromine, -NH2 or NO2.

[0133] In some embodiments, R3 is C 1-6Alkyl or NR a R b , where R a and R b Each independently is H or -CO-(C 1-6 alkyl), wherein the alkyl is optionally substituted by one or more OH groups. Further, R3 is C 1-6 Alkyl, -NH2 or -NH-CO-(CH2OH). Further, R3 is C 1-6 Alkyl or -NH-CO-(CH2OH).

[0134] In some embodiments, R3 is C 1-6 Alkyl groups, such as methyl.

[0135] In some embodiments, R2 and R3 together with the atoms to which they are attached form a 4- to 8-membered heterocyclic group containing N, O or S as heteroatom, such as dioxole, for example in the compounds of Examples 6 or 7.

[0136] In some embodiments, D is linked to L4 through a nitrogen or oxygen atom in R1 or R3 or through a hydroxy oxygen atom as shown in formula (II), for example, through an amide bond, an aminomethyl ether bond, or a carbamate bond.

[0137] In some embodiments, D is a small molecule drug moiety obtained by losing an atom (e.g., a hydrogen atom) or a group of atoms from a compound of formula (II), such as losing a hydrogen atom attached to a nitrogen or oxygen atom in R1 or R3 or losing a hydrogen atom in a hydroxyl group shown in formula (II). In particular, D is a moiety selected from the following:

[0138] In some embodiments, each L1 is independently:

[0139] Preferred More preferably Among them, position 1 is connected to Ab, and position 2 is connected to L2.

[0140] In some embodiments, each L2 is independently a linking unit selected from the following formula:

[0141] -(CH2) t -(L M ) 0-1 -(CH2CH2O) s -(CH2) t -CO-, -(CH2) t -L M -(CH2)t -(L M ) 0-1 -L N -L M -(CH2O) s -(CH2) t -CO-, -(CH2) t -L M -(CH2) t -N(R p )-(CH2) t -N(R p )-(CH2) t -CO-, and (CH2) t -L M -CH(R p )-CO-,

[0142] The -CO- end of L2 is connected to L3, and the other end of L2 is connected to L1.

[0143] In some embodiments, each L2 is independently -(CR m R n ) t -(L M ) 0-1 -(CH2CH2O) s -(CR m R n ) t -CO-, for example -(CR m R n ) t -(CH2CH2O) s -(CR m R n ) t -CO- or -(CR m R n ) t -L M -(CH2CH2O) s -(CR m R n ) t -CO-, wherein the -CO- end of L2 is connected to L3, and the other end of L2 is connected to L1. In a further embodiment, each L2 is independently -(CH2) t -(L M ) 0-1 -(CH2CH2O) s -(CH2) t -CO-, for example -(CH2) t -(CH2CH2O) s -(CH2)t -CO- or -(CH2) t -L M -(CH2CH2O) s -(CH2) t -CO-, wherein the -CO- end of L2 is connected to L3, and the other end of L2 is connected to L1. In a further embodiment, each L2 is independently -(CH2) t -(CH2CH2O) s -(CH2) t -CO-, -(CH2) t -CO-NH-(CH2CH2O) s -(CH2) t -CO-, or -(CH2) t -NH-CO-(CH2CH2O) s -(CH2) t -CO-, preferably -(CH2) t -(CH2CH2O) s -(CH2) t -CO-, or -(CH2) t -CO-NH-(CH2CH2O) s -(CH2) t -CO-, wherein the -CO- end of L2 is connected to L3, and the other end of L2 is connected to L1.

[0144] In some embodiments, each L2 is independently

[0145] -(CR m R n ) t -L M -(CR m R n ) t -(L M ) 0-1 -L N -L M -(CH2O) s -(CR m R n ) t -CO-, e.g.

[0146] -(CR m R n ) t -L M -(CR m R n ) t -L N -L M -(CH2O)s -(CR m R n ) t -CO-, or

[0147] -(CR m R n ) t -L M -(CR m R n ) t -L M -L N -L M -(CH2O) s -(CR m R n ) t -CO-, wherein the -CO- end of L2 is connected to L3, and the other end of L2 is connected to L1. In a further embodiment, L2 are each independently

[0148] -(CH2) t -L M -(CH2) t -L N -L M -(CH2O) s -(CH2) t -CO-, or

[0149] -(CH2) t -L M -(CH2) t -L M -L N -L M -(CH2O) s -(CH2) t -CO-, wherein the -CO- end of L2 is connected to L3, and the other end of L2 is connected to L1. In a further embodiment, L2 are each independently

[0150] -(CH2) t -CO-NH-(CH2) t -(3-8 membered heteroarylene)-(CH2) t -(OCH2CH2) m -NH-CO-(CH2O)-(CH2) t -CO-(preferably -(CH2) t -CO-NH-(CH2) t -(Triazolylene)-(CH2) t -(OCH2CH2) m -NH-CO-(CH2O)-(CH2)t -CO-), -(CH2) t -CO-NH-(CH2) t -NH-CO-(CH2CH2O) m -(CH2) t -NH-CO-(CH2O)-(CH2) t -CO-, or -(CH2) t -CO-NH-(CH2) t -NH-CO-(CH2) t -(OCH2CH2) m -NH-CO-(CH2O)-(CH2) t -CO-, wherein the -CO- end of L2 is connected to L3, and the other end of L2 is connected to L1.

[0151] In some embodiments, each L2 is independently

[0152] -(CR m R n ) t -L M -(CR m R n ) t -N(R p )-(CR m R n ) t -N(R p )-(CR m R n ) t -CO-, e.g.

[0153] -(CH2) t -L M -(CH2) t -N(R p )-(CH2) t -N(R p )-(CH2) t -CO-, wherein the -CO- end of L2 is connected to L3, and the other end of L2 is connected to L1. In a further embodiment, each L2 is independently -(CH2) t -NH-CO-(CH2) t -N(R p )-(CH2) t -N(R p )-(CH2) t -CO-, or

[0154] -(CH2) t -CO-NH-(CH2)t -N(R p )-(CH2) t -N(R p )-(CH2) t -CO-, preferably

[0155] -(CH2) t -NH-CO-(CH2) t -N(R p )-(CH2) t -N(R p )-(CH2) t -CO-, wherein the -CO- end of L2 is connected to L3, and the other end of L2 is connected to L1. In a further embodiment, R p Each independently is -CO-(CH2CH2O) m -CH3, -(CH2) t -NH-CO-(CH2CH2O) m -CH3, or -(CH2) t -CO-NH-(CH2CH2O) m -CH3, preferably -CO-(CH2CH2O) m -CH3.

[0156] In some embodiments, each L2 is independently -(CR m R n ) t -L M -CH(R p )-CO-, for example -(CH2) t -L M -CH(R p )-CO-, also for example -(CH2) t -CO-NH-CH(R p )-CO- or -(CH2) t -NH-CO-CH(R p )-CO-, wherein the -CO- end of L2 is connected to L3, and the other end of L2 is connected to L1. In a further embodiment, R p Each independently is -CO-(CH2CH2O) m -CH3, -(CH2) t -NH-CO-(CH2CH2O) m -CH3, or -(CH2) t -CO-NH-(CH2CH2O) m -CH3, preferably -(CH2) t -NH-CO-(CH2CH2O)m -CH3.

[0157] Preferably, each L2 is independently: -(CH2) t -(CH2CH2O) s -(CH2) t -CO-, -(CH2) t -CO-NH-(CH2CH2O) s -(CH2) t -CO-, -(CH2) t -NH-CO-(CH2CH2O) s -(CH2) t -CO-, -(CH2) t -CO-NH-(CH2) t -(3-8 membered heteroarylene)-(CH2) t -(OCH2CH2) m -NH-CO-(CH2O)-(CH2) t -CO-, -(CH2) t -CO-NH-(CH2) t -NH-CO-(CH2CH2O) m -(CH2) t -NH-CO-(CH2O)-(CH2) t -CO-, -(CH2) t -CO-NH-(CH2) t -NH-CO-(CH2) t -(OCH2CH2) m -NH-CO-(CH2O)-(CH2) t -CO-, -(CH2) t -NH-CO-(CH2) t -N(R p )-(CH2) t -N(R p )-(CH2) t -CO-, -(CH2) t -CO-NH-(CH2) t -N(R p )-(CH2) t -N(R p )-(CH2) t -CO-, or -(CH2) t -CO-NH-CH(R p )-CO- or -(CH2) t -NH-CO-CH(R p)-CO-, wherein the -CO- end of L2 is connected to L3, and the other end of L2 is connected to L1.

[0158] Understandably, as L M The structural units (e.g., -NH-CO- or -CO-NH-) are arranged in the structural formula of L2 from left to right. Similarly, as L N The structural units are also arranged from left to right in the structural formula of L2.

[0159] In some embodiments, each s is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8. In further embodiments, each s is independently 0. In some embodiments, each s is independently 3. In other embodiments, each s is independently 8.

[0160] In some embodiments, each t is independently 0, 1, 2, 3, 4, 5, 6, 7, or 8. In further embodiments, each s is independently 0, 1, 2, 3, 4, 5, or 6, preferably each s is independently 0, 1, 2, 3, 4, or 5.

[0161] In some embodiments, each m is independently 3, 4, 5, 6, 7, or 8. In further embodiments, each m is independently 3. In other embodiments, each m is independently 8.

[0162] In some embodiments, each L2 is independently of the formula -(CH2CH2O) x -(CH2) y-CO- linking unit, wherein the -CO- end is connected to L3 and the other end is connected to L1, and wherein x is an integer from 0 to 5, y is an integer from 1 to 5, and x+y≤6; preferably, L2 is -(CH2)-CO-, -(CH2)2-CO-, -(CH2)3-CO-, -(CH2)4-CO-, -(CH2)5-CO-, -(CH2)6-CO-, -(CH2CH2O)-(CH2)-CO-, -(CH2CH2O)-(CH2)2-CO-, -(CH2CH2O)-(CH2)3-CO- More preferably, L2 is each independently -(CH2)5-CO- or -(CH2CH2O)3-(CH2)2-CO-.

[0163] In other embodiments, each L2 is independently:

[0164] ◆-(CH2)5-CO-;

[0165] ◆-(CH2CH2O)3-(CH2)2-CO-;

[0166] ◆-(CH2)5-CO-NH-(CH2CH2O)8-(CH2)2-CO-;

[0167] ◆-(CH2)3-CO-NH-(CH2)-(triazolylene)-(CH2)2-(OCH2CH2)8-NH-CO-(CH2)-O-(CH2)-CO-;

[0168] ◆-(CH2)3-CO-NH-(CH2)2-NH-CO-(CH2CH2O)8-(CH2)2-NH-CO-(CH2)-O-(CH2)-CO-;

[0169] ◆-(CH2)3-CO-NH-(CH2)2-NH-CO-(CH2)2-(OCH2CH2)8-NH-CO-(CH2)-O-(CH2)-CO-;

[0170] ◆ ◆ ◆

[0171] The -CO- end of L2 is connected to L3 and the other end of L2 is connected to L1, or position 1 is connected to L1 and position 2 is connected to L3.

[0172] In some embodiments, L3 is independently an amino acid residue, a short peptide chain consisting of 2-6 amino acid residues, or -NH-(CH2) p -CO-, wherein p is 0, 1, 2, 3, 4, 5 or 6. In particular, the amino acid is a natural or unnatural amino acid, preferably selected from glycine (Gly), alanine (Ala), valine (Val), phenylalanine (Phe), citrulline (Cit), lysine (Lys) and asparagine (Asn). In some embodiments, L3 is Gly-Gly-Phe-Gly, Val-Cit, Val-Ala, Phe-Lys, Val-Lys, Lys, Ala-Ala-Ala, Ala-Ala-Asn, or -NH-(CH2)2-CO-. In particular, L3 is Gly-Gly-Phe-Gly, Val-Cit, Val-Ala, Phe-Lys, Val-Lys, Ala-Ala-Ala, Ala-Ala-Asn, or -NH-(CH2)2-CO-. In some embodiments, L3 is Gly-Gly-Phe-Gly, Val-Cit, Val-Ala, Phe-Lys, Lys or -NH-(CH2)2-CO-. In particular, L3 is Gly-Gly-Phe-Gly, Val-Ala, Phe-Lys or Lys.

[0173] In some embodiments, each L4 is independently:

[0174] Valence bond, Bit 1 is connected to L3 and bit 2 is connected to D.

[0175] In further embodiments, L4 are each independently a bond, Bit 1 is connected to L3 and bit 2 is connected to D.

[0176] In some embodiments, each L4 is independently Bit 1 is connected to L3 and bit 2 is connected to D.

[0177] In some embodiments, each -L1-L2-L3-L4- moiety is independently selected from:

[0178] Among them, position 1 is connected to Ab and position 2 is connected to D.

[0179] In some embodiments, each -L1-L2-L3-L4- moiety is independently selected from:

[0180] Among them, position 1 is connected to Ab and position 2 is connected to D.

[0181] In some embodiments, each -L1-L2-L3-L4-D moiety is independently selected from:

[0182] Among them, position 1 represents the site of connection with Ab.

[0183] In some embodiments, Ab is an antibody or antigen-binding fragment thereof that binds to a tumor cell surface antigen; for example, an anti-Her2 antibody or antigen-binding fragment thereof and / or an anti-FRα antibody or antigen-binding fragment thereof. In some embodiments, Ab is fatuinumab or an antigen-binding fragment thereof and / or trastuzumab or an antigen-binding fragment thereof.

[0184] In some embodiments, when L1 is When Lg and L1 form

[0185] In other embodiments, when L1 is not When Lg is a leaving group. The leaving group can easily leave when reacting with the antibody or its antigen-binding fragment. In some embodiments, Lg is a halogen, a sulfonyl group (e.g., a methanesulfonyl group, a p-toluenesulfonyl group), a sulfonyloxy group (e.g., a methanesulfonyloxy group, a CF3SO3- group, a p-toluenesulfonyloxy group), a tertiary amine group (e.g., a Me3N + or Et3N + ) or a diazonium salt group. Preferably, Lg is F, Cl, Br, or a methylsulfonyl group. More preferably, Lg is a methylsulfonyl group.

[0186] In some embodiments, the antibody drug conjugate of formula (I) of the present invention is selected from:

[0187] wherein, in Fa-ADCs, mAb represents fatuinumab (Fa); in Tra-ADCs, mAb represents trastuzumab (Tra); and n is an integer from 0 to 8, such as 0, 1, 2, 3, 4, 5, 6, 7 or 8.

[0188] In some embodiments, the antibody drug conjugates of the present invention have an average DAR selected from any value within the range of 1.0-10.0, preferably an average DAR selected from any value within the range of 2.0-8.0, and more preferably an average DAR selected from any value within the range of 6.0-8.0.

[0189] In some embodiments, the compound of formula (II) of the present invention is selected from:

[0190] In some embodiments, the drug-linker conjugate of formula (III) of the present invention is selected from:

[0191] In another aspect, the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate of the present invention or its pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label, or the compound of the present invention or its pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label, or the drug-linker conjugate of the present invention or its pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label, and a pharmaceutically acceptable carrier, diluent or excipient.

[0192] In another aspect, the present invention provides an antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label thereof, or a compound of the present invention or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label thereof, or a drug-linker conjugate of the present invention or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label thereof, for use in treating or preventing cancer.

[0193] In another aspect, the present invention provides a method for preventing or treating cancer in a patient, comprising administering to the patient a therapeutically effective amount of an antibody-drug conjugate of the present invention, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label thereof, or a compound of the present invention, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label thereof, or a drug-linker conjugate of the present invention, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label thereof.

[0194] In another aspect, the present invention provides use of the antibody-drug conjugate of the present invention, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, or the compound of the present invention, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, or the drug-linker conjugate of the present invention, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, in the preparation of a medicament for treating or preventing cancer.

[0195] In some embodiments, the cancer is a solid tumor, particularly lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), liver cancer, digestive system cancer (e.g., intestinal cancer, stomach cancer, cardia cancer, esophageal cancer, appendix cancer, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer), bladder cancer, melanoma, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, basal cell carcinoma, bile duct cancer, squamous cell carcinoma, thyroid cancer, brain cancer, head and neck cancer, peritoneal cancer, kidney cancer, urothelial cancer, testicular cancer, central nervous system cancer, Preferably, the cancer is a glioma (e.g., a glioblastoma such as glioblastoma multiforme, a glioma or a sarcoma), a choriocarcinoma, an oral epidermoid carcinoma; or a hematological malignancy, in particular a leukemia (e.g., acute or chronic myeloid leukemia, acute or chronic myeloid leukemia), a lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, acute B-cell lymphoma, follicular lymphoma), multiple myeloma; preferably, the cancer is ovarian cancer, colon cancer, lung adenocarcinoma or oral epidermoid carcinoma.

[0196] The terms and scientific and technical terms used in this application generally have the meanings commonly understood by those skilled in the art, unless otherwise specified. In particular, the terms used in this application have the following meanings.

[0197] As used herein, the terms “a,” “an,” “the” and the entity itself are to be construed to cover both the singular and the plural, unless otherwise indicated or clearly contradicted by context.

[0198] A hyphen ("-") that is not between two letters or symbols indicates the point of attachment of a substituent. For example, -NRa R b Indicates that the group is attached to the rest of the molecule through a nitrogen atom, -(CH2) p -NR a R b Indicates that the group is represented by -(CH2) p - is connected to the rest of the molecule. When the attachment point of the substituent is obvious to those skilled in the art (such as for halogen, CN, OH, NH2, etc.), the "-" can also be omitted.

[0199] The term "halogen" or "halo" refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I), preferably fluorine, chlorine or bromine.

[0200] The term "alkyl" alone or as part of another group refers to a fully saturated straight or branched chain hydrocarbon group consisting of carbon and hydrogen atoms. Preferably, the alkyl group has 1 to 6 carbon atoms (C 1-6 Alkyl), 1-4 carbon atoms (C 1-4 Alkyl) or 1-3 carbon atoms (C 1-3 Examples of the alkyl group include, but are not limited to, methyl (Me), ethyl (Et), propyl (Pr) (including n-propyl and isopropyl), butyl (Bu) (including n-butyl, isobutyl, sec-butyl and tert-butyl), pentyl (including n-pentyl, isopentyl, neopentyl, etc.), hexyl, heptyl, octyl, etc.

[0201] The term "alkenyl" alone or as part of another group refers to a straight or branched hydrocarbon group consisting of carbon and hydrogen atoms containing at least one double bond. Preferably, the alkenyl group has 2 to 6 carbon atoms (C 2-6 alkenyl), 2-4 carbon atoms (C 2-4 alkenyl) or 2-3 carbon atoms (C 2-3 Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, allyl, butenyl, isobutenyl, pentenyl, isopentenyl, and hexenyl.

[0202] The term "alkynyl" alone or as part of another group refers to a straight-chain or branched hydrocarbon group consisting of carbon and hydrogen atoms and containing at least one triple bond. Preferably, the alkynyl group has 2 to 6 carbon atoms (C 2-6 Alkynyl), 2-4 carbon atoms (C 2-4 Alkynyl) or 2-3 carbon atoms (C 2-3 Representative examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, propargyl, butynyl, isobutynyl, pentynyl, isopentenyl, hexynyl, and the like.

[0203] The terms "alkoxy" and "alkyl-O-" are used interchangeably to denote an alkyl group as defined above attached through an oxygen atom. Preferably, the alkoxy group has 1 to 6 carbon atoms (C 1-6 Alkoxy), 1-4 carbon atoms (C 1-4 Alkoxy) or 1-3 carbon atoms (C 1-3 Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy, isopropoxy), butoxy (including n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, etc.), pentoxy (including n-pentoxy, isopentoxy, neopentoxy, etc.), hexyloxy, heptyloxy, octyloxy, etc.

[0204] The terms "alkylthio" and "alkyl-S-" are used interchangeably to denote an alkyl group as defined above attached through a sulfur atom. Preferably, the alkylthio group has 1 to 6 carbon atoms (C 1-6 Alkylthio), 1-4 carbon atoms (C 1-4 Alkylthio) or 1-3 carbon atoms (C 1-3 Examples of alkylthio groups include, but are not limited to, methylthio, ethylthio, propylthio (including n-propylthio, isopropylthio), butylthio (including n-butylthio, sec-butylthio, isobutylthio, tert-butylthio, etc.), pentylthio (including n-pentylthio, isopentylthio, neopentylthio, etc.), hexylthio, heptylthio, octylthio, etc.

[0205] The term "haloalkyl" refers to an alkyl group as defined herein that is substituted with one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) halogens. It will be understood that when there are more than one halogen substituent, the halogen substituents may be the same or different and may be located on the same or different carbon atoms. Preferably, the haloalkyl group is C 1-6 Halogenated alkyl, C 1-4 Haloalkyl or C 1-3 Examples of haloalkyl groups include, but are not limited to, fluoromethyl, chloromethyl, difluoromethyl, dichloromethyl, fluorochloromethyl, trifluoromethyl, trichloromethyl, dichlorofluoromethyl, difluoroethyl, trifluoroethyl, trichloroethyl, difluorochloroethyl, difluoropropyl, and trifluoropropyl.

[0206] The term "hydroxyalkyl" refers to an alkyl group as defined herein that is substituted with one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) hydroxy groups. It will be understood that when there are more than one hydroxy substituent, the hydroxy substituents may be located on the same or different carbon atoms. Preferably, the hydroxyalkyl group is C 1-6 Hydroxyalkyl, C 1-4 Hydroxyalkyl or C 1-3 Examples of hydroxyalkyl groups include, but are not limited to, hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, and the like.

[0207] The term "cyanoalkyl" refers to an alkyl group as defined herein which is substituted with one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) cyano groups. It will be understood that when there are more than one cyano substituent, the cyano substituents may be located on the same or different carbon atoms. Preferably, the cyanoalkyl group is C 1-6 Cyanoalkyl, C 1-4 Cyanoalkyl or C 1-3 Examples of cyanoalkyl groups include, but are not limited to, cyanomethyl, cyanoethyl, cyanopropyl, and the like.

[0208] The term "aminoalkyl" refers to an alkyl group as defined herein which is substituted with one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) amino groups. It will be understood that when there are more than one amino substituent, the amino substituents may be located on the same or different carbon atoms. Preferably, the aminoalkyl group is C 1-6 aminoalkyl, C 1-4 aminoalkyl or C 1-3 Aminoalkyl. Examples of aminoalkyl groups include, but are not limited to, aminomethyl, aminoethyl, aminopropyl, and the like.

[0209] The term "cycloalkyl" alone or as part of another group means a fully or partially saturated non-aromatic monocyclic or bicyclic hydrocarbon group consisting of carbon and hydrogen atoms, preferably fully saturated. Preferably, the cycloalkyl group has 3 to 8 ring carbon atoms (C 3-8 Cycloalkyl) or 3-6 ring carbon atoms (C 3-6 Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, and the like. When a cycloalkyl group is substituted with one or more hydroxyl groups, it may be referred to as a "hydroxycycloalkyl group," including C 3-8 Hydroxycycloalkyl or C 3-6 Hydroxycycloalkyl.

[0210] The term "heterocyclic group" refers to a non-aromatic monocyclic or bicyclic group containing one or more, for example 1, 2, 3 or 4, heteroatoms independently selected from N, O or S, with the remaining ring members being carbon, which is fully saturated (i.e., heterocycloalkyl) or partially saturated (e.g., heterocycloalkenyl containing one or more double bonds, heterocycloalkynyl containing one or more triple bonds, etc.). The carbon members of the heterocyclic group may optionally be replaced by -CO-, and any N and S heteroatoms may optionally be oxidized (e.g., in NO, SO, SO2) and any N heteroatoms may optionally be quaternized (e.g., in [NR] + Cl - 、[NR] + OH -Preferably, the heterocyclic group is a 3-8 membered heterocyclic group, a 4-8 membered heterocyclic group, a 4-6 membered heterocyclic group, or a 4-5 membered heterocyclic group. A nitrogen-containing heterocyclic group refers to a heterocyclic group containing one or more nitrogen heteroatoms and the remaining ring members are carbon, such as pyrrole, dihydropyrrole, pyrrolidine, pyridine, dihydropyridine, tetrahydropyridine, piperidine, etc. Examples of heterocyclic groups include, but are not limited to, aziridinyl, oxiranyl, azetidinyl, oxetanyl, thietanyl, dihydropyrrolyl, pyrrolidinyl, pyrrolidonyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothiophenyl, tetrahydrothiophenyl, tetrahydrothiophenyl 1-oxide, tetrahydrothiophenyl 1,1-dioxide, dioxolanyl, dioxolenyl, dihydropyrazolyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, imidazolonyl, dihydroimidazolonyl, tetrahydroimidazolonyl, dihydrooxazolyl, oxazolidinyl, dihydroisoxazolyl, isoxazolidinyl, dihydrothiazolyl, thiazolidinyl, dihydroisothiazolyl, isothiazolidinyl, dihydropyrazolyl, The heterocyclic group may be attached to the rest of the molecule via a carbon atom or a heteroatom, as long as it is chemically feasible. The heterocyclic group may also be optionally fused to other cyclic groups which may be aromatic or non-aromatic and which may optionally contain one or more heteroatoms selected from N, O or S, for example in middle.

[0211] The term "aryl" refers to a monovalent carbon ring having one or more rings, preferably 1 or 2 rings, wherein at least one ring is aromatic and the other rings (if present) may be aromatic or non-aromatic. 6-10 aryl), more preferably 6 ring carbon atoms (i.e., C6 aryl or phenyl). Representative examples of aryl include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indenyl, dihydroindenyl or tetrahydronaphthyl, etc.

[0212] The term "heteroaryl" refers to a monocyclic or bicyclic cyclic group having one or more, preferably 1, 2, 3, 4, 5 or 6, heteroatoms independently selected from N, O or S, and the remaining ring members being carbon, wherein at least one ring is aromatic and the other rings (if present) may be aromatic or non-aromatic. Any N and S heteroatoms of the heteroaryl group may optionally be oxidized (e.g., in NO, SO, SO2) and any N heteroatoms may optionally be quaternized (e.g., in [NR] + Cl - 、[NR] + OH - ). Heteroaryl is preferably a 3-8 membered, more preferably a 5-8 membered heteroaryl (e.g., a monocyclic, e.g., nitrogen-containing). Representative examples of heteroaryl include, but are not limited to, pyrrolyl, furyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, isothiazolyl, thiazolyl, isothiazolyl, triazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyranyl, thiopyranyl, oxazinyl, oxadiazinyl, indolyl, isoindolyl, azaindolyl (e.g., 7-azaindolyl, 6-azaindolyl, 5-azaindolyl, 4-azaindolyl), benzofuranyl, isobenzofuranyl, benzothienyl, benzothiazolyl , indazolyl, benzimidazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, benzopyranyl, cinnolinyl, quinazolinyl, quinoxalinyl, benzoxazinyl, benzotriazolyl, purinyl, indolizinyl, pyrrolopyridinyl, pyrrolopyrimidinyl, pyrrolopyrazinyl, imidazopyridinyl, imidazopyridinyl, pyrazolopyridinyl, pyrazolopyridazinyl, pyrazolopyrimidinyl, pyrazolopyrazinyl, pyridopyrimidinyl, pyrimidopyrimidinyl, pyrazinopyrazinyl, phthalazine, naphthyridinyl, and the like. Heteroaryl groups can be attached to the rest of the compound via a carbon atom or a heteroatom, as chemically feasible.

[0213] The terms "heteroarylene" and "heteroarylene" are used interchangeably to refer to a divalent heteroaryl group as defined herein.

[0214] The term "subunit" or "subunit" refers to a divalent group derived by removing two hydrogen atoms from a molecule.

[0215] The term "CN" refers to a cyano group.

[0216] The term "OH" refers to a hydroxyl group.

[0217] The term "SH" refers to a thiol group.

[0218] The term "NH2" refers to an amino group.

[0219] The term "-CO-" or "-C(=O)-" represents a carbonyl group.

[0220] The term "-SO-" represents a sulfinyl group.

[0221] The term "-SO2-" represents a sulfonyl group.

[0222] The term "-COOH" represents a carboxyl group.

[0223] The term "NO2" means a nitro group.

[0224] The term "leaving group" refers to an atom or functional group that is easily separated from a molecule in a chemical reaction. Examples include, but are not limited to, halogens such as F, Cl, or Br; sulfonyl groups such as methanesulfonyl or p-toluenesulfonyl; sulfonyloxy groups such as alkylsulfonyloxy (e.g., methanesulfonyloxy), trifluoromethylsulfonyloxy, or arylsulfonyloxy (e.g., p-toluenesulfonyloxy); tertiary amine groups (e.g., Me3N + or Et3N + ); or diazonium base.

[0225] The expression "optional," "optionally," or "optionally" means that the subsequently described event may or may not occur, and that the expression includes instances where the event occurs as well as instances where the event does not occur. For example, "optionally substituted with" includes instances where the event is not substituted as well as instances where the event is substituted. "Optional substituent" indicates that the substituent may or may not be present.

[0226] When any variable occurs more than one time in a structural formula, it is independently defined at each occurrence. For example, the phrase "optionally substituted with one or more substituents independently selected from..." means substitution with one or more independently selected substituents, each of which may be the same or different. Combinations of substituents and / or variables are permissible so long as stable compounds result.

[0227] The term "comprise" or "include" refers to the inclusion of the elements, integers or steps described, but does not exclude any other elements, integers or steps. In this article, when the term "comprise" or "include" is used, unless otherwise indicated, the situation of combinations of the elements, integers or steps described is also covered.

[0228] The term "substance of the present invention" includes the antibody drug conjugate of formula (I) of the present invention, the small molecule camptothecin derivative of formula (II) of the present invention, the drug linker conjugate of formula (III) of the present invention, or a salt or ester, solvate (e.g., hydrate), tautomer, stereoisomer, prodrug, metabolite or isotope label (e.g., deuterated) thereof. In some embodiments, "substance of the present invention" specifically refers to the compound or conjugate of the Examples, or a salt or ester, solvate (e.g., hydrate), tautomer, stereoisomer, prodrug, metabolite or isotope label (e.g., deuterated) thereof.

[0229] The term "pharmaceutically acceptable" refers to substances or compositions that do not produce adverse, allergic or other undesirable reactions when administered to animals, such as humans.

[0230] The term "pharmaceutically acceptable salts" refers to those salts that retain the biological effectiveness of the substances of the invention and are not biologically or otherwise undesirable. The substances of the invention may be in the form of salts, preferably pharmaceutically acceptable salts, including acid addition salts and base addition salts. Acid addition salts can be formed with inorganic or organic acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, and perchloric acid, and organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, glutaric acid, adipic acid, succinic acid, fumaric acid, maleic acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, pamoic acid, phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, ethanedisulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, xylenesulfonic acid, isethionic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, salicylic acid, oleic acid, nicotinic acid, palmitic acid, stearic acid, furoic acid, hippuric acid, orotic acid, and pamoic acid. Base addition salts can be formed with organic or inorganic bases, including but not limited to alkali metal salts such as lithium, sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts; organic base salts such as ammonium salts formed with organic bases containing N groups. Salts can be synthesized from the parent compound by conventional methods.

[0231] Pharmaceutically acceptable salts are preferred. However, other salts may also be useful, for example in isolation or purification steps, which may be employed during the preparation and are therefore encompassed within the scope of this application.

[0232] As used herein, the term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds with one or more (e.g., one, two, three, or four) asymmetric centers, racemic mixtures, single enantiomers, diastereomeric mixtures, and individual diastereomers may occur. Specific individual molecules may also exist as geometric isomers (cis / trans).

[0233] The term "tautomer" refers to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions via reorganization of some of the bonding electrons.

[0234] Solid lines, solid wedges, or dashed wedges may be used herein to depict valence bonds in the materials of the present invention. Solid lines attached to asymmetric carbon atoms are intended to indicate that all possible stereoisomers at that carbon atom are included (e.g., specific enantiomers, racemic mixtures, etc.). Solid or dashed wedges attached to asymmetric carbon atoms are intended to indicate the presence of the stereoisomers shown. When in a racemic mixture, solid and dashed wedges are used to define relative stereochemistry, not absolute stereochemistry. Unless otherwise indicated, the materials of the present invention may exist as stereoisomers, including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotational isomers, conformational isomers, atropisomers, and mixtures thereof. The materials of the present invention may exhibit more than one type of isomerism and consist of mixtures thereof (e.g., racemic mixtures and diastereoisomer pairs).

[0235] The present invention also includes prodrugs of the substances of the present invention. The term "prodrug" refers to a chemically modified active or inactive compound that, after administration to an individual, undergoes physiological action (e.g., hydrolysis, necrolysis, etc.) in the body and is converted into the substance of the present invention. Therefore, in this application, the term "administering" includes administering a prodrug of the substance of the present invention to treat various diseases or conditions, wherein the prodrug is converted into the substance of the present invention in the body. The techniques for making and using prodrugs are well known to those skilled in the art.

[0236] The present invention also includes all pharmaceutically acceptable isotopically labeled substances which are identical to the substances of the present invention but in which one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number prevalent in nature. Examples of isotopes suitable for incorporation into the substances of the present invention include, but are not limited to, isotopes of hydrogen (e.g. 2 H. 3 H); isotopes of carbon (e.g. 11 C. 13 C and 14 C); isotopes of chlorine (e.g. 36 Cl); isotopes of fluorine (e.g. 18 F); isotopes of iodine (such as 123 I and 125 I); isotopes of nitrogen (e.g. 13 N and 15 N); oxygen isotopes (e.g. 15 O. 17 O and 18 O); isotopes of phosphorus (such as 32 P); and sulfur isotopes (e.g. 35 S).

[0237] The present invention also includes metabolites of the substances of the present invention, i.e., substances formed in vivo after administration of the substances of the present invention. These can be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic hydrolysis, and the like. Thus, the present invention includes metabolites of the substances of the present invention, including compounds produced by contacting the substances of the present invention with a mammal for a period of time sufficient to produce a metabolic product thereof.

[0238] Some of the substances of the present invention can exist in unsolvated and solvated forms, including hydrates. The term "solvate" refers to a complex of a solvent molecule and a substance of the present invention. Examples of solvents that form solvates include water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. The term "hydrate" refers to a complex in which the solvent molecule is water. Methods of solvation are well known in the art.

[0239] The term "subject" or "patient" refers to an animal, preferably a mammal. Examples of subjects include, but are not limited to, primates (e.g., humans and non-human primates such as monkeys), horses, cows, sheep, cats, dogs, rabbits, rabbits, and rodents (e.g., mice and rats). In some embodiments, the subject is a human, including a child, adolescent, or adult.

[0240] The term "treating" refers to (i) treating or preventing a particular disease, condition, or disorder, (ii) reducing, ameliorating, or eliminating one or more symptoms of a particular disease, condition, or disorder, and optionally (iii) preventing or delaying the onset of one or more symptoms of a particular disease, condition, or disorder as described herein. In some embodiments, "treating" refers to improving at least one physical parameter, which may not be noticeable to the patient. In other embodiments, "treating" refers to modulating a disease or condition physically (e.g., stabilizing a noticeable symptom) or physiologically (e.g., stabilizing a physical parameter), or both.

[0241] The term "prevention" refers to the administration of one or more pharmaceutical substances, particularly compounds of the present invention and / or pharmaceutically acceptable salts thereof, to an individual with a predisposition to the disease or condition in question, in order to prevent the individual from developing the disease.

[0242] The terms "inhibit" and "alleviate" and the like refer to a reduction or suppression of a particular condition, symptom or disorder or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0243] The term "effective amount" refers to an amount effective to achieve the desired therapeutic or prophylactic effect at the required dosage and for the required period of time. It can be determined by the attending physician or veterinary practitioner and will vary with factors such as the compound, the disease state being treated, the severity of the disease being treated, the age and relative health of the individual, the route and form of administration, and the judgment of the attending physician or veterinary practitioner. Generally, a "prophylactically effective amount" will be less than a "therapeutically effective amount."

[0244] The term "preparation" or "pharmaceutical composition" refers to a composition suitable for administration to an animal, preferably a mammal (including a human) comprising at least one active ingredient and at least one inactive ingredient, such as a pharmaceutically acceptable excipient. The preparation of the present invention can be any preparation suitable in the art, such as a tablet, capsule, liquid preparation, etc.

[0245] The term "pharmaceutically acceptable excipient" refers to an ingredient in a pharmaceutical formulation other than the active ingredient that is non-toxic to a subject. Examples of pharmaceutically acceptable carriers include, but are not limited to, binders, disintegrants, lubricants, solvents, dispersion media, buffers, excipients, antioxidants, preservatives, or flavoring agents.

[0246] When referring to a chemical reaction, "treating," "contacting," and "reacting" refer to the addition or mixing of two or more reagents under appropriate conditions to produce the indicated and / or desired product. It should be understood that the reaction that produces the indicated and / or desired product may not necessarily result directly from the combination of the two reagents initially added, that is, there may be one or more intermediates generated in the mixture that ultimately lead to the formation of the indicated and / or desired product.

[0247] The term "about" when used in conjunction with a numerical value means a range of ±20%, preferably ±10%, more preferably ±5% of the numerical value.

[0248] Antibody Drug Conjugates

[0249] The term "antibody-drug conjugate" or "ADC" as used herein refers to a substance obtained by coupling a small molecule drug portion (Payload) to an antibody or its antigen-binding fragment portion (Ab, which is responsible for targeting function and sometimes also has biological activity) via a linker.

[0250] Methods for coupling linkers to antibodies or antigen-binding fragments thereof are well known to those skilled in the art and include non-site-specific and site-specific coupling, such as, but not limited to, lysine coupling, cysteine ​​coupling, Thiomab technology, introduction of unnatural amino acids, and enzyme-catalyzed methods. For example, with maleimide linkers, cysteine ​​residues on the antibody serve as attachment points for the payload. Reduction of interchain disulfide bonds on the antibody generates eight free sulfhydryl groups, which react with maleimide via Michael addition to attach the payload, resulting in an ADC product with a DAR value approaching 8.

[0251] Antibody drug conjugates can be characterized by the drug-antibody ratio (DAR). The term "drug-antibody ratio" or "DAR" as used herein refers to the quantitative ratio of the small molecule drug portion (D) coupled to the antibody or its antigen-binding fragment (Ab) to the antibody or its antigen-binding fragment portion (Ab). The average drug-antibody ratio (average DAR) represents the ratio of the molar concentrations of the total drug molecules and antibody molecules in the assay system. The DAR can range from 1 to 20, but higher loadings are also possible, depending on the number of attachment sites on the antibody. Methods for determining the DAR are known in the art, such as reversed-phase high performance liquid chromatography (RP-HPLC), size exclusion chromatography (SEC-HPLC), mass spectrometry, and hydrophobic chromatography (HIC-HPLC).

[0252] In some embodiments, the antibody drug conjugates of the present invention have an average DAR selected from any value within the following ranges: 1.0-20.0, 1.0-18.0, 1.0-16.0, 1.0-10.0, 2.0-14.0, 3.0-12.0, 4.0-10.0, 5.0-9.0, 6.0-8.0, or 2.0-8.0. In some embodiments, the antibody drug conjugates of the present invention have an average DAR selected from any value within the following ranges: 2±0.4, 4±0.4, 6±0.4, or 8±0.4. In some embodiments, the antibody drug conjugates of the present invention have an average DAR of about 2.0, 4.0, 6.0, 8.0, 10.0, or 12.0.

[0253] In some embodiments, the average DAR value of the antibody drug conjugate of the invention is about 1.0-20.0, such as about 1.0-18.0, 1.0-16.0, 2.0-14.0, 3.0-12.0, 4.0-10.0, 5.0-9.0, 6.0-8.0, 1.0-8.0, 2.0-6.0, such as about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4 , 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7. 9.8, 9.9, 10.0, 12.0, and 16.0, and ranges having any two of these values ​​as endpoints.

[0254] Antibodies or antigen-binding fragments thereof

[0255] The terms "complete antibody", "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules having the structure of natural immunoglobulin molecules. In the case of conventional four-chain IgG antibodies, full-length antibodies comprise two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of heavy chain antibodies having only heavy chains and lacking light chains, full-length antibodies comprise two heavy chains (H) interconnected by disulfide bonds.

[0256] The term "antibody fragment" includes a portion of an intact antibody. Preferably, the antibody fragment is an antigen-binding fragment.

[0257] The term "antigen-binding fragment" refers to a molecule, other than an intact antibody, that comprises a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies such as VHH; diabodies or fragments thereof; or camelid antibodies.

[0258] The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. Those skilled in the art will appreciate that any macromolecule, including essentially all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. In some embodiments, the antigen is a tumor cell surface antigen, such as Her2 or FRα.

[0259] The term "complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms a structurally determined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact points"). CDR is primarily responsible for binding to the antigen epitope. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia (Chothia et al. 1989, Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948, 1997), Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics (ImmunoGeneTics), and the like. database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and North's CDR definitions based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Concepts", Journal of Molecular Biology, 406, 228-256, 2011).

[0260] The term "IgG form antibody" refers to the IgG form of the antibody's heavy chain constant region. For example, an IgG4 form antibody means that its heavy chain constant region is derived from IgG4, or an IgG1 form antibody means that its heavy chain constant region is derived from IgG1.

[0261] The antibodies or antigen-binding fragments thereof suitable for use in the present application can bind to any antigen suitable for use in antibodies, preferably tumor cell surface antigens, such as Her2 or FRα.

[0262] In some embodiments, the antibodies or antigen-binding fragments thereof suitable for the present application are antibodies or antigen-binding fragments thereof that bind to Her2, particularly human Her2. Any Her2 antibody or antigen-binding fragment thereof known to those skilled in the art is suitable for the present invention, including but not limited to disclosed antibodies or commercially available antibodies, such as trastuzumab.

[0263] In some embodiments, the antibodies or antigen-binding fragments thereof suitable for use in the present application are antibodies or antigen-binding fragments thereof that bind to FRα, particularly human FRα. Any FRα antibody or antigen-binding fragment thereof known to those skilled in the art is suitable for use in the present invention, including but not limited to disclosed antibodies or commercially available antibodies, such as fatuin.

[0264] In some embodiments, the antibody suitable for the present application is an antibody in the form of IgG1, an antibody in the form of IgG2, an antibody in the form of IgG3, or an antibody in the form of IgG4. Preferably, the antibody suitable for the ADC of the present application is an antibody in the form of IgG1 or an antigen-binding fragment thereof.

[0265] In some embodiments, antibodies suitable for use in the present invention are monoclonal antibodies. In some embodiments, antibodies suitable for use in the present invention are humanized. In some embodiments, antibodies suitable for use in the present invention are human antibodies. In some embodiments, antibodies suitable for use in the present invention are chimeric antibodies.

[0266] In one embodiment, antibodies suitable for use in the present application are full-length antibodies.

[0267] In one embodiment, the antigen-binding fragment of the antibody suitable for use in the present application is an antibody fragment selected from the following: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody), linear antibody, half antibody, or multispecific antibody such as bispecific antibody, trispecific antibody, tetraspecific antibody. In some embodiments, the antigen-binding fragment of the ADC suitable for use in the present application includes but is not limited to VHH, Fv molecule, scFv molecule, Fab molecule, and F(ab')2 molecule.

[0268] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof suitable for use in the present application is trastuzumab or fatuinumab or an antigen-binding fragment thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises one or more CDRs (preferably 3 CDRs, i.e., HCDR1, HCDR2, and HCDR3; or LCDR1, LCDR2, and LCDR3, more preferably 6 CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) of trastuzumab or fatuinumab or an antigen-binding fragment thereof, or comprises VH and / or VL of trastuzumab or fatuinumab or an antigen-binding fragment thereof, or comprises the heavy chain and / or light chain of the antibody.

[0269] In some embodiments, the anti-Her2 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region CDR1, CDR2 and CDR3, and the light chain variable region CDR1, CDR2 and CDR3 from trastuzumab. In some embodiments, the anti-Her2 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region and light chain variable region from trastuzumab. In some embodiments, the anti-Her2 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain and light chain variable region from trastuzumab.

[0270] In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region CDR1, CDR2, and CDR3, and the light chain variable region CDR1, CDR2, and CDR3 from fatuinumab. In some embodiments, the anti-FRα antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region and light chain variable region from fatuinumab. In some embodiments, the anti-Her2 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain and light chain from fatuinumab.

[0271] Linker

[0272] The term "linker" as used herein is a connecting portion used to connect a small molecule cytotoxic drug (Payload) to an antibody or its antigen-binding fragment (Ab), and can be represented as a -L1-L2-L3-L4- portion in this application. Linkers generally include cleavable linkers and non-cleavable linkers. Cleavable linkers utilize the difference in conditions between the blood circulation and tumor cells (such as pH, proteolysis or glutathione concentration) or utilize the specific action of antibodies in lysosomes (such as lysosomal proteolytic enzymes) to break, releasing the payload and exerting the drug's active effect. Non-cleavable linkers rely on the degradation of antibodies in lysosomes to release the drug and produce drug activity.

[0273] The linkers that can be used in the present invention include cleavable linkers and non-cleavable linkers, such as acid-sensitive linkers (e.g., hydrazone bond linkers), glutathione-sensitive linkers (e.g., disulfide bond linkers), enzyme-cleavable linkers (e.g., short peptide linkers, β-glucuronide linkers), amide bond linkers, thioether linkers, etc. In particular, the linkers that can be used in the present application include but are not limited to those given above, especially those used in the examples.

[0274] The term "Linker Unit" is a component (L1) of the Linker (-L1-L2-L3-L4- portion) that serves to attach the Linker or a Linker that is part of a Drug-Linker Conjugate to an antibody or antigen-binding fragment thereof. Examples include, but are not limited to

[0275] Among them, position 1 is connected to Ab, and position 2 is connected to L2.

[0276] The term "linker unit" is a component (L2) of the linker (-L1-L2-L3-L4- moiety) that serves to connect the linker unit (L1) to the amino acid residue, short peptide chain or -NH-(CH2) p -CO- L3 part connection.

[0277] The component L3 of the linker of the present application is an amino acid residue, a short peptide chain consisting of 2-10 (preferably 2-6) amino acid residues, or -NH-(CH2) p -CO-, for example, -NH-(CH2)2-CO-. The amino acid can be either natural or non-natural, such as glycine (Gly), alanine (Ala), valine (Val), phenylalanine (Phe), citrulline (Cit), lysine (Lys) and asparagine (Asn). L3 can be directly linked to the small molecule drug moiety or linked to the small molecule drug moiety through a spacer unit.

[0278] The term "spacer unit" is a component (L4) of the linker (-L1-L2-L3-L4- moiety). When present, its function is to connect the linker to the small molecule drug moiety (D), or to provide an additional structural moiety to facilitate the release of the small molecule drug moiety from the ADC. Examples include, but are not limited to

[0279] Bit 1 is connected to L3 and bit 2 is connected to D.

[0280] Small molecule drug part

[0281] The terms "small molecule drug moiety," "small molecule cytotoxic drug moiety," and "payload" are used interchangeably herein to refer to the component of an antibody-drug conjugate or drug-linker conjugate responsible for killing tumor cells. Following administration, the ADC undergoes cleavage between or within target cells, releasing the small molecule drug or its derivative, which then exerts its biological activity. For the avoidance of doubt, it should be noted that "drug" does not refer solely to "pharmaceuticals" approved by pharmaceutical regulatory authorities; it also encompasses any compound with potential biological activity in clinical practice, R&D, or academic research.

[0282] In the present invention, the small molecule drug portion is derived from a camptothecin derivative of formula (II), in particular, a camptothecin derivative from the Examples herein. It is a portion obtained by losing an atom (e.g., a hydrogen atom) or a group of atoms from a camptothecin derivative of formula (II). For example, the small molecule drug portion is a portion obtained by losing a hydrogen atom connected to a nitrogen or oxygen atom in R1 or R3 or by losing a hydrogen atom from a hydroxyl group shown in formula (II). The hydroxyl group shown in formula (II) refers to the hydroxyl group indicated by the arrow in the following structure:

[0283] The small molecule drug moiety of the present invention can be linked to the linker via any chemically permitted bonding means, including but not limited to amide bonds, aminomethyl ether bonds, and carbamate bonds.

[0284] Efficacy and beneficial effects

[0285] The substances of the present invention, including the antibody drug conjugate of formula (I), the camptothecin derivative of formula (II) and the drug linker conjugate of formula (III), or pharmaceutically acceptable salts or esters, solvates, tautomers, stereoisomers, or isotope-labeled substances thereof, have high cytotoxic activity and can be used to treat or prevent tumors such as cancer, including but not limited to solid tumors, in particular lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), liver cancer, digestive system cancer (e.g., intestinal cancer, gastric cancer, cardia cancer, esophageal cancer, appendix adenocarcinoma, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer), bladder cancer, melanoma, breast cancer, ovarian cancer, cervical cancer, Endometrial cancer, prostate cancer, basal cell carcinoma, bile duct cancer, squamous cell carcinoma, thyroid cancer, brain cancer, head and neck cancer, peritoneal cancer, renal cancer, urothelial carcinoma, testicular cancer, central nervous system tumors (e.g., gliomas, glioblastomas such as glioblastoma multiforme, gliomas or sarcomas), choriocarcinoma, oral epidermoid carcinoma; or hematological malignancies, in particular leukemias (e.g., acute or chronic myeloid leukemia, acute or chronic myeloid leukemia), lymphomas (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, acute B-cell lymphoma, follicular lymphoma), multiple myeloma. Preferably, the substances of the present invention can be used to treat and / or prevent ovarian cancer, colon cancer, lung adenocarcinoma or oral epidermoid carcinoma. Compared with known camptothecin derivatives such as SN-38 and DXD, the substances of the present invention have stronger tumoricidal activity and / or lower side effects.

[0286] The substance of the present invention has good tolerance and does not cause obvious behavioral abnormalities or weight fluctuations (such as weight loss) while effectively killing tumor cells.

[0287] The substance of the present invention has good solubility and physicochemical stability and can be easily formulated into a pharmaceutical preparation suitable for administration.

[0288] The ADCs of the present invention have an appropriate drug-to-antibody ratio, and the distribution of the drug-to-antibody ratio is uniform.

[0289] The ADCs of the present invention are stable in the blood circulation, reducing drug molecule shedding in non-target tissues and alleviating off-target toxicity. Furthermore, the ADCs of the present invention exhibit excellent tumor tissue targeting and are enriched in the tumor microenvironment, increasing the concentration ratio of the active drug molecule within the tumor and in the blood, reducing the mechanism-related toxicity of the conjugate, and exhibiting a good therapeutic index. The small molecule drugs released from the ADCs of the present invention can also exert a bystander effect, further killing tumor cells surrounding the target tumor cells that do not express or express low levels of the antigen.

[0290] The ADCs of the present invention have better therapeutic or preventive efficacy and / or lower side effects than corresponding small molecule camptothecin derivatives, and have a wider therapeutic window.

[0291] Pharmaceutical compositions and administration

[0292] The substances of the present invention can be administered in the form of pharmaceutical compositions by any suitable route, such as, but not limited to, oral administration (e.g., in the form of tablets, capsules, liquid preparations such as solutions, emulsions or suspensions), inhalation (e.g., in the form of sprays), rectal administration (e.g., in the form of suppositories), or parenteral administration (e.g., in the form of injections, such as intravenous, intramuscular, subcutaneous, intraperitoneal, intracranial, etc.). Parenteral administration, such as intravenous injection, is particularly preferred.

[0293] The technology of making the substance of the present invention into a pharmaceutical composition is well known in the art. For example, the substance of the present invention can be processed into the form of a pharmaceutical composition using one or more pharmaceutically acceptable carriers, diluents or excipients, such as tablets, capsules, liquid preparations (such as injections, infusions, syrups, emulsions, suspensions, etc.), powder injections, dispersions, sprays, suppositories, liposomes, etc. Pharmaceutically acceptable carriers, diluents or excipients are well known in the art, such as fillers, disintegrants, solvents, solubilizers, stabilizers, wetting agents, emulsifiers, preservatives, sweeteners, colorants, flavoring agents, salts for changing osmotic pressure, buffers, masking agents or antioxidants, etc.

[0294] The dosage can vary over a wide range and must, of course, be adjusted according to the individual needs in each specific case. The determination of the appropriate dosage can be determined by the attending physician based on the type of disease being treated and its severity, the individual's health and medical history, the specific compound being administered, the route of administration, and co-medications, as appropriate. The daily dosage for a 70 kg adult is typically from about 0.01 mg to about 1000 mg of the substance of the present invention or a corresponding amount of a pharmaceutically acceptable salt or ester. The dosage of the substance of the present invention may exceed this dosage range as needed. The daily dosage may be administered as a single dose or in divided doses.

[0295] The substance of the present invention can be used alone or in combination with one or more other activating agents or therapy, and the other activating agents or therapy can have the same or different pharmacological efficacy as the substance of the present invention. The substance of the present invention can be used simultaneously, respectively or sequentially by the same or different routes of administration with other shared activating agents. They can be included in the same pharmaceutical composition (fixed composition), or can be a combination product of separate forms, such as medicine box forms. They can be prepared and / or supplied by the same or different manufacturers. When the substance of the present invention is used in combination with other activating agents, the dosage of the activating agent used in combination will certainly change according to factors such as shared medicine, disease to be treated, individual general health, physician or veterinarian's judgment. In order to help the patient's compliance, the application's medicine box can generally include instructions for use.

[0296] General preparation method

[0297] The substances of the present invention can be prepared by a variety of methods, including the methods described in the following processes, the methods given in the examples, or similar methods. For each reaction step, appropriate reaction conditions are known to those skilled in the art or can be easily determined. The starting materials are generally commercially available or can be easily prepared using methods known in the art or methods described herein. The variables in the general formula have the meanings defined herein, unless otherwise stated.

[0298] In the preparation of compounds of the present invention, the protection of groups (e.g., amino protecting groups, hydroxy protecting groups) may be desired, which can be easily determined by those skilled in the art. For a general description of protecting groups and their uses, see TW Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

[0299] If necessary, the starting materials and intermediates in the synthetic reaction schemes can be separated and purified by conventional techniques, including but not limited to filtration, distillation, crystallization, chromatography, etc. The materials can be characterized by conventional methods including physical constants and spectral data.

[0300] For illustrative purposes only, the following scheme provides an exemplary approach to synthesizing the materials of the present invention. It will be appreciated by those skilled in the art that other synthetic approaches are also available, and the compounds prepared by the methods described below may be further modified according to the content of this application, utilizing conventional compounds well known to those skilled in the art.

[0301] Process: Synthesis of camptothecin derivatives

[0302] Wherein, each variable is as defined herein.

[0303] This reaction can be achieved by The camptothecin derivatives can be synthesized by quinoline synthesis, for example, in the presence of an acid, such as p-toluenesulfonic acid, glacial acetic acid or other Lewis acid, to obtain a reaction product. The resulting product can then be further modified, for example, by substitution reactions, condensation reactions (forming amide bonds), Buchwald-Hartwig coupling reactions or amino deprotection to produce other camptothecin derivatives. The reaction conditions are readily determined by those skilled in the art of organic synthesis.

[0304] The synthesis of drug-linker conjugates is within the capabilities of those skilled in the art of organic synthesis. For example, drug-linker conjugates can be prepared by Williamson ether synthesis, condensation reaction, and the like.

[0305] Methods for conjugating antibodies to drug-linker conjugates are known in the field of ADCs, such as lysine conjugation, cysteine ​​conjugation, Thiomab technology, unnatural amino acid technology, enzyme catalysis technology, etc. In particular, the antibody can be first reduced, and then the antibody and drug-linker conjugate can be conjugated, and the resulting antibody-drug conjugate can be optionally purified. BRIEF DESCRIPTION OF THE DRAWINGS

[0306] Figures 1a to 1d show the killing efficacy of ADCs of the present invention on Capan-1 cells; RLU stands for relative light units (Example B).

[0307] Figures 2a to 2d show the killing efficacy of ADCs of the present invention on NCI-N87 cells; RLU represents relative light units (Example B).

[0308] Figures 3a to 3e show that the ADCs of the present invention have a bystander effect (Example C).

[0309] Figure 4 shows that ADCs of the present invention inhibited the growth of human ovarian cancer cell SKOV-3 tumors in a mouse model (Example D.2).

[0310] FIG5 shows that the ADCs of the present invention did not cause weight loss and were well tolerated in the SKOV-3 immunodeficient mouse model (Example D.2).

[0311] Figures 6a and 6b respectively show the tumor growth curve (6a) and body weight change curve (6b) after administration of ADCs of the present invention in a JIMT-1 tumor-bearing mouse model (Example D.3).

[0312] Figures 7a and 7b respectively show the tumor growth curve (7a) and body weight change curve (7b) after administration of ADCs of the present invention in a Capan-1 tumor-bearing mouse model (Example D.3).

[0313] Figures 8a and 8b respectively show the tumor growth curve (8a) and body weight change rate curve (8b) after administration of ADCs of the present invention in the NCI-N87 tumor-bearing mouse model (Example D.3).

[0314] Figures 9a and 9b respectively show the tumor growth curve (9a) and body weight change rate curve (9b) after administration of ADCs of the present invention in a JIMT-1 tumor-bearing mouse model (Example D.4).

[0315] Figures 10a and 10b respectively show the tumor growth curve (10a) and body weight change rate curve (10b) after administration of ADCs of the present invention in a Capan-1 tumor-bearing mouse model (Example D.4).

[0316] Figures 11a and 11b respectively show the tumor growth curve (11a) and body weight change rate curve (11b) after administration of ADCs of the present invention in the NCI-N87 tumor-bearing mouse model (Example D.4).

[0317] Figures 12a and 12b respectively show the tumor growth curve (12a) and body weight change curve (12b) after administration of ADCs of the present invention in a Calu-3 tumor-bearing mouse model (Example D.5).

[0318] Figures 13a and 13b respectively show the tumor growth curve (13a) and body weight change rate curve (13b) after administration of ADCs of the present invention in an OE-19 tumor-bearing mouse model (Example D.5).

[0319] Figures 14a and 14b respectively show the tumor growth curve (14a) and body weight change rate curve (14b) after administration of ADCs of the present invention in a Capan-1 tumor-bearing mouse model (Example D.6). Example

[0320] The following examples are provided to further illustrate the present invention. It should be understood that they are only for a better understanding of the present invention and are not intended to limit the scope of the present invention in any way.

[0321] In this application, when a chemical name and structural formula are inconsistent, the structural formula shall prevail unless the context indicates that the chemical name, rather than the structural formula, is correct. For simplicity, not all hydrogen atoms are explicitly labeled in some of the compound structural formulas presented in this application. When there are vacant valences in a compound, this indicates the presence of unlabeled hydrogen atoms.

[0322] Unless otherwise specified, the experimental materials and reagents used in the examples can be obtained from commercial channels, or can be easily prepared using methods known to those skilled in the art.

[0323] Abbreviations

[0324] Abbreviations used herein (eg, for chemical groups and compounds) generally have their meanings commonly understood in the art unless otherwise indicated. For example, the following abbreviations are used herein: acetyl (Ac), boron trichloride (BCl3), tert-butyloxycarbonyl (Boc), deuterated chloroform (CDCl3), N,N'-dicyclohexylcarbodiimide (DCC), 1,2-dichloroethane (DCE), dichloromethane (DCM), N,N-diisopropylethylamine (DIPEA / DPEA), 4-dimethylaminopyridine (DMAP), N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), deuterated dimethyl sulfoxide (DMSO-d6), ethyl acetate (EA), 6-(maleimido)hexanoic acid succinimidyl ester (EMCS), triethylamine (Et3N), 9-fluorenylmethoxycarbonyl (Fmoc), glycine (Gly or G), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), lysine (Lys or K), molar / liter (M), m-chloroperbenzoic acid (mCPBA), acetonitrile (MeCN), N-hydroxysuccinimide (NHS), trifluoromethanesulfonate (OTf), tris(dibenzylideneindeneacetone)dipalladium(0) [Pd2(dba)3], 1,1-bis(diphenylphosphino)ferrocenepalladium dichloride [Pd(dppf)Cl2], petroleum ether (PE), pentafluorophenol (PFP-OH), phenylalanine (Phe or F), pyridinium p-toluenesulfonate (PPTS), preparative thin layer chromatography (Prep-TLC), pyridine (Py), trifluoroacetic acid (TFA), trifluoromethanesulfonic anhydride (Tf2O), thin layer chromatography (TLC), p-toluenesulfonyl (Ts), p-toluenesulfonic acid (TsOH), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos), and sodium triacetoxyborohydride (STAB).

[0325] Basic information on testing and preparation

[0326] Liquid chromatography-mass spectrometry (LCMS)

[0327] Test instrument model: Agilent 1260II G6125B

[0328] Test method: Electrospray ionization source positive / negative ion mode (ESI ± )

[0329] Chromatographic column model: YMC-TRIART C18 (4.6×50mm×5μm)

[0330] Mobile phase: A (10 mM NH4HCO3)–B (MeCN): 0–1.5 min (5% B–100% B), 1.5–2.5 min (100% B–100% B), 2.5–2.6 min (100% B–5% B), 2.6–3.75 min (5% B–5% B); flow rate: 2.0 mL / min;

[0331] Column temperature: 40°C

[0332] LCMS data were generated using the following conditions: Chlorine isotopes are reported as 35 Cl, bromine isotopes are reported as 79 Br.

[0333] Nuclear magnetic resonance spectroscopy ( 1 H NMR)

[0334] Test instrument: Bruker AVANCE NEO 400 NMR

[0335] Test method: The solvent is DMSO-d6 (+D2O) or CDCl3, and the internal standard is tetramethylsilane (TMS)

[0336] Signal multiplicities are indicated by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; dt, doublet of triplets; tt, triplet of triplets; td, triplet of doublets; ddd, doublet of doublets; br, broad; hept, septet; m, multiplet. All observed coupling constants, J, are reported in Hertz. Exchangeable protons, including but not limited to active hydrogens such as hydroxyl, carboxyl, and amino groups, are not always observed.

[0337] Medium and high voltage reverse preparation

[0338] Preparation instrument: SHIMADZU LC-20AP

[0339] Chromatographic column: SHIMADZU C18 (50×250mm×10μm)

[0340] Acid mobile phase: 0.05% TFA aqueous solution-MeCN; Alkaline mobile phase: 0.1% NH4HCO3 aqueous solution-MeCN; Neutral mobile phase: H2O-MeCN;

[0341] Flow rate: 15 mL / min; Detection wavelength: 214 nm, 254 nm

[0342] Medium and low pressure reverse preparation

[0343] Preparation instrument: ISOLERA PRIME

[0344] Chromatographic column: Spherical C18 (40-750μm, )

[0345] Alkaline mobile phase: 0.1% NH4HCO3 aqueous solution-MeCN; Acid mobile phase: 0.05% TFA aqueous solution-MeCN;

[0346] Flow rate: 25 mL / min; Detection wavelength: 214 nm, 254 nm

[0347] Example 1: Synthesis of (S)-4-ethyl-4-hydroxy-8-fluoro-9-methyl-11-(chloromethyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (STI-A-4)

[0348] Step 1: Under nitrogen and an ice bath, add 160 mL of DCE and a 1M solution of boron trichloride in DCM (32.0 mL, 32.0 mmol) to a reaction flask. Then, add compound 1a (5.0 g, 40.0 mmol) and stir at 0°C for 10 minutes. Chloroacetonitrile (5.4 mL, 47.0 mmol) and aluminum trichloride (7.0 g, 52.0 mmol) are added under an ice bath, and the reaction system is slowly warmed to room temperature. Stir at room temperature for 10 minutes, then reflux and stir for 39 hours. After the reaction, add 80 mL of ice water, followed by 40 mL of 4M HCl, and stir at room temperature for 30 minutes. Extract with 80 mL of dichloromethane, dry the organic phase over anhydrous sodium sulfate, and concentrate under reduced pressure. The residue is subjected to medium- and high-pressure reverse elution (neutral mobile phase) and lyophilized to yield 1.0 g of compound 1b as a yellow solid powder in a 12.4% yield. LCMS (254 nm) purity 97.2%, Rt = 1.789 min; MS calculated value: 201.0; MS found value: 202.0 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ7.68 (d, J = 8.7 Hz, 1H), 7.30 (s, 2H), 6.53 (d, J = 12.5 Hz, 1H), 4.97 (s, 2H), 2.08 (s, J = 8.6 Hz, 3H).

[0349] Step 2: Dissolve compound 1b (500 mg, 2.48 mmol) and compound 1c (490 mg, 1.86 mmol) in 30 mL of anhydrous toluene under nitrogen atmosphere. Reflux for 30 minutes with a water separator. Add p-toluenesulfonic acid (48 mg, 0.25 mmol) and continue stirring at reflux for 3.5 hours. After completion, the reaction system was cooled to room temperature, filtered, washed with acetone, and the filter cake dried under vacuum at room temperature to yield 719 mg of compound STI-A-4 as a yellow solid in a 90.2% yield. LCMS (254 nm) revealed a purity of 96.8%, Rt = 1.711 min; MS calculated: 428.1; MS found: 429.2 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ8.36(d,J=8.1Hz,1H),7.94(d,J=10.7Hz,1H),7.32(s,1H),5.4 4(d,J=5.1Hz,4H),5.35(s,2H),2.54(s,3H),1.92–1.78(m,2H),0.88(t,J=7.3Hz,3H).

[0350] Example 2: Synthesis of (S,E)-4-ethyl-4-hydroxy-8-fluoro-9-methyl-11-((neopentylimino)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (STI-A-6)

[0351] Compound STI-A-4 (500 mg, 1.17 mmol) from Example 1 and silver p-toluenesulfonate (390 mg, 1.40 mmol) were dissolved in 30 mL of DMSO and stirred at room temperature overnight under nitrogen. The mixture was then heated and stirred at 90°C for 3 hours. After completion of the reaction, as monitored by LCMS, the mixture was cooled to room temperature, p-toluenesulfonic acid (21 mg, 0.12 mmol) and compound 2a (234 mg, 2.69 mmol) were added, and the mixture was stirred at room temperature for 3.5 hours. After completion of the reaction, as monitored by LCMS, the mixture was prepared by reverse chromatographic separation at medium and low pressure (alkaline mobile phase) and lyophilized to afford 152 mg of the title compound (STI-A-6) as a yellow solid in a 27.3% yield. LCMS (254 nm) revealed a purity of 94.8%, Rt = 2.234 min; MS calculated: 477.2; MS found: 478.1 [M+H] + . 1H NMR(400MHz, DMSO-d6)δ9.29(s,1H),8.56(d,J=8.1Hz,1H),7.78(d,J=10.6Hz,1H),7.22(s,1H),6.45(s,1H) ,5.34(s,2H),5.17(s,2H),3.56(s,2H),2.42(s,3H),1.87–1.76(m,2H),1.00(s,9H),0.83(t,J=7.4Hz,3H).

[0352] Example 3: (S)-4-ethyl-4-hydroxy-8-fluoro-9-methyl-11-((neopentylamino)methyl)-1,12-dihydro-14H-pyrano[3-( ...

[0353] Synthesis of [3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (STI-A)

[0354] Compound STI-A-6 (152 mg, 0.32 mmol) from Example 2 and 10% Pd / C (51 mg, 0.05 mmol) were suspended in 10 mL of methanol and stirred at room temperature under hydrogen atmosphere (1 atm) for 4 hours. After completion of the reaction, monitored by LCMS, the system was filtered, and the filtrate was subjected to low-pressure reverse chromatography (alkaline mobile phase) and lyophilized to yield 37 mg of the title compound (STI-A) as a yellow solid in a 24.2% yield. LCMS (254 nm) revealed a purity of 99.0%, Rt = 2.147 min; MS calculated: 479.2; MS found: 480.3 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ8.36(d,J=8.5Hz,1H),7.85(d,J=10.8Hz,1H),7.31(s,1H),6.51(s,1H),5 .43(s,2H),5.38(s,2H),4.30(s,2H),2.49(s,3H),2.43(s,2H),1.93–1.81(m,2H),0.88(s,12H).

[0355] Example 4: Synthesis of (S)-4-ethyl-4-hydroxy-8-fluoro-9-methyl-11-(((cyclopentylmethyl)amino)methyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (STI-A2)

[0356] Step 1: Compound STI-A-4 (150 mg, 0.35 mmol) from Example 1 and silver p-toluenesulfonate (117 mg, 0.42 mmol) were dissolved in 10 mL of DMSO and stirred at room temperature overnight under nitrogen. The mixture was then heated and stirred at 90°C for 3 hours. After completion of the reaction, as monitored by LCMS, the mixture was cooled to room temperature, p-toluenesulfonic acid (3 mg, 0.02 mmol) and compound 4a (73 mg, 0.74 mmol) were added, and the mixture was stirred at room temperature for 1 hour. After completion of the reaction, as monitored by LCMS, the mixture was prepared by reverse transpiration at medium and low pressure (alkaline mobile phase) and lyophilized to afford 16 mg of compound 4b as a yellow solid in a 10.4% yield. LCMS (254 nm) revealed a purity of 95.5%, Rt = 2.084 min; MS calculated: 489.2; MS found: 490.2 [M+H] + . 1 H NMR(400MHz, DMSO-d6)δ9.45(s,1H),8.73(d,J=8.1Hz,1H),7.94(d,J=10.9Hz,1H),7.32(s,1H),6.52(s,1H),5.42(s,2H),5 .33(s,2H),2.52(s,3H),1.89–1.78(m,4H),1.72–1.62(m,2H),1.63–1.48(m,3H),1.49–1.37(m,4H),0.88(t,J=7.3Hz,3H).

[0357] Step 2: Compound 4b (10 mg, 0.02 mmol) and 10% Pd / C (3 mg, 0.003 mmol) were suspended in 3 mL of methanol and stirred at room temperature under hydrogen atmosphere (1 atm) for 6 hours. After completion of the reaction, the system was filtered, and the filtrate was subjected to low-pressure reverse preparative chromatography (alkaline mobile phase) and lyophilized to afford 3 mg of the title compound (STI-A2) as a yellow solid in a 24.2% yield. LCMS (254 nm) revealed a purity of 95.7%, Rt = 1.959 min; MS calculated: 491.2; MS found: 492.3 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ8.32(d,J=8.5Hz,1H),7.88(d,J=10.7Hz,1H),7.57(s,1H),5.32(s,2H),4.80–4.61(m,2H),4.35(s,2H),4.19(d,J =13.5Hz,1H),2.65(d,J=7.5Hz,2H),2.47(s,3H),2.09–2.01(m,2H),1.72(d,J=14.2Hz,3H),1.52(d,J=22.0Hz,6H),0.86(d,J=7.1Hz,3H).

[0358] Example 5: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-11-carboxylic acid (STI-F-01)

[0359] Compound STI-A-4 (250 mg, 0.58 mmol) from Example 1 and silver p-toluenesulfonate (200 mg, 0.70 mmol) were dissolved in 20 mL of DMSO and stirred at room temperature overnight under nitrogen. The mixture was then heated and stirred at 90°C for 3 hours. After LCMS monitoring of the aldehyde formation, a portion of the reaction solution was subjected to low-pressure reverse transpiration (alkaline mobile phase), air-oxidized, and lyophilized to yield 50 mg of the title compound (STI-F-01) as a white solid. LCMS, Rt = 1.25 min; MS calculated: 424.1; MS found: 425.2 [M+H] + . 1H NMR(400MHz,DMSO-d6)δ8.85(d,J=8.5Hz,1H),7.84(d,J=11.0Hz,1H),7.29(s,1H),6.50 (s,1H),5.42(s,2H),5.27(s,2H),2.47(s,3H),1.92–1.80(m,2H),0.88(t,J=7.4Hz,3H).

[0360] Example 6: (S)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (STI-E05), and

[0361] Example 7: Synthesis of (S)-7-ethyl-7-hydroxy-14-(4-hydroxybutyl)-10,13-dihydro-11H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (STI-E)

[0362] Step 1: Under nitrogen, compound 6a (1 g, 7.24 mmol) was added to a reaction flask, followed by dissolution in 15 mL of DMF. Cesium carbonate (3.54 g, 10.86 mmol) and dibromomethane (1.89 g, 10.86 mmol) were added. The mixture was heated to 110°C and stirred for 2.5 hours. The reaction was stopped after completion as monitored by TLC (PE:DCM = 1:1, product Rf = 0.7). Prep-TLC purification afforded 972 mg of compound 6b in an 89.4% yield.

[0363] Step 2: Compound 6b (972 mg, 6.48 mmol) was added to a reaction flask, followed by 20 mL of nitric acid. The mixture was allowed to react at room temperature under nitrogen for 0.5 h. After completion of the reaction, as determined by TLC (PE:EA = 5:1, product Rf = 0.5), ice water was added to the reaction system, the mixture was allowed to stand, and then filtered. The filter cake was dried under vacuum at room temperature to afford 1.053 g of compound 6c as a yellow solid, with a yield of 83.3%. 1 H NMR (400MHz, CDCl3) δ10.22(s,1H),7.45(s,1H),7.26(s,1H),6.14(s,2H).

[0364] Step 3: Dissolve compound 6c (7.0 g, 35.87 mmol) in 350 mL of 50% aqueous ethanol and heat at 100°C. Dissolve FeSO4·7H2O (70 g, 251.09 mmol) in 350 mL of water, heat at 100°C for 2 minutes, and add to the reaction system. Slowly add 91.0 mL of concentrated aqueous ammonia. Heat at 100°C for 15 minutes. After completion, the reaction was cooled to room temperature, filtered, and cooled in an ice bath to precipitate 4.5 g of yellow needles (compound 6d) in a 76.0% yield. LCMS (254 nm) revealed a purity of 96.61%, Rt = 1.40 min; MS calculated: 165.0; MS found: 166.1 [M+H] + . 1 H NMR (400MHz, CDCl3) δ9.61 (d, J = 0.6Hz, 1H), 6.82 (s, 1H), 6.27 (s, 2H), 6.14 (s, 1H), 5.93 (s, 2H).

[0365] Step 4: Compound 6d (300 mg, 1.82 mmol) and compound 1c (360 mg, 1.37 mmol) were suspended in 4 mL of toluene and 4 mL of acetic acid was added. The mixture was heated at 80°C overnight under nitrogen. After completion of the reaction, the mixture was cooled to room temperature, filtered, and washed with acetone to obtain 45 mg of compound STI-E05 as a pale yellow solid in a 45.9% yield. LCMS (254 nm) revealed a purity of 99.62%, Rt = 1.49 min; MS calculated: 392.1; MS found: 393.2 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ8.46(s,1H),7.51(s,2H),7.25(s,1H),6.50(s,1H),6.28(dd, J=1.4,0.7Hz,2H),5.41(s,2H),5.21(s,2H),1.92–1.80(m,2H),0.87(t,J=7.3Hz,3H).

[0366] Step 5: Compound STI-E05 (100 mg, 0.26 mmol), n-butanol (58 mg, 0.78 mmol), terpyridyl ruthenium chloride hexahydrate (1.6 mg, 0.0026 mmol) and 4,5,6,7-tetrafluoro-1-hydroxy-1λ 3 1-Benzo[d][1,2]iodoxol-3(1H)-one (175 mg, 0.52 mmol) was added to the reaction flask and suspended in 5 mL of hexafluoroisopropanol (HFIP). Argon was bubbled through the reaction mixture for 1 hour. The mixture was then heated to 70°C under argon protection and irradiated with a 23W energy-saving lamp. The mixture was stirred for 65 hours. LCMS analysis indicated approximately 20% conversion, and the reaction was stopped. Reverse chromatography (alkaline mobile phase) was performed at medium-low pressure and lyophilized to yield 20 mg of compound STI-E as a brownish-yellow solid. LCMS (254 nm) purity was 94.36%, Rt = 1.75 min; MS calculated: 464.2; MS found: 465.2 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ7.55(s,1H),7.50(s,1H),7.30(s,1H),6.51(s,1H),6.36(t,J=6.2Hz,1H),6.29(s,2H),5.42(s, 2H), 5.26 (s, 2H), 3.92–3.82 (m, 2H), 3.79 (d, J = 6.7Hz, 2H), 1.92–1.81 (m, 4H), 1.38–1.26 (m, 2H), 0.84 (t, J = 6.8Hz, 3H).

[0367] Example 8: Synthesis of (S)-4-ethyl-4-hydroxy-8-fluoro-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (STI-F-8)

[0368] Step 1: NaSO (22.7 g, 159.81 mmol) and chloral hydrate (3.0 g, 17.58 mmol) were added to a three-necked flask. 67 mL of water was then added, followed by compound 1a (2.0 g, 15.98 mmol), hydroxylamine hydrochloride (4.4 g, 63.92 mmol), and 25 mL of 1 M hydrochloric acid. The mixture was stirred and heated at 80°C overnight under nitrogen. After completion of the reaction, the mixture was filtered and the filter cake was dried at 50°C to obtain 2.9 g of compound 8a as a yellow solid in a 92.5% yield. LCMS (254 nm) revealed a purity of 97.76%, Rt = 1.522 min; MS calculated: 196.1; MS found: 197.1 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ12.21(s,1H),10.28(s,1H),7.64(s,1H),7.61(dd,J=12.3,2 .0Hz, 1H), 7.36 (dd, J = 8.3, 2.0Hz, 1H), 7.22 (t, J = 8.6Hz, 1H), 2.19 (d, J = 1.9Hz, 3H).

[0369] Step 2: Dissolve compound 8a (2.9 g, 14.79 mmol) in 10 mL of concentrated sulfuric acid and heat with stirring at 80°C for 3 hours. LCMS monitoring indicates completion of the reaction. Cool the reaction system to room temperature, add 200 mL of ice water to the reaction system under an ice bath, filter, and wash the filter cake with water until the filtrate is neutral. Dry the filter cake at 50°C to obtain 2.07 g of compound 8b as an orange solid, with a yield of 78.2%. 1 H NMR (400MHz, DMSO-d6) δ11.06 (s, 1H), 7.51 (d, J = 7.7Hz, 1H), 6.69 (d, J = 9.8Hz, 1H), 2.16 (d, J = 2.2Hz, 3H).

[0370] Step 3: Dissolve compound 8b (2.07 g, 11.6 mmol) and KOH (1.3 g, 23.2 mmol) in 20 mL of water. Slowly add 590 mg of H₂O₂ dropwise under ice-cooling. Stir at room temperature for 3 hours. After the reaction, adjust the pH to 4-6 with hydrochloric acid under ice-cooling. Filter, wash, and dry the filter cake at 50°C to obtain 660 mg of compound 8c as a yellow solid. LCMS (254 nm) purity: 94.31%, Rt = 1.065 min; MS calculated: 169.1; MS found: 170.1 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ7.61 (d, J = 9.2 Hz, 1H), 6.49 (d, J = 12.3 Hz, 1H), 2.07 (s, 3H).

[0371] Step 4: Add 10 mL of tetrahydrofuran to a reaction tube, under nitrogen protection, and cool to 0°C in an ice bath. LiAlH4 (352 mg, 9.28 mmol) was then added, followed by a slow dropwise addition of a solution of compound 8c (100 mg, 0.6 mmol) in tetrahydrofuran (10 mL). The mixture was allowed to return to room temperature and allowed to react overnight. After completion of the reaction, the tube was cooled to below 10°C in an ice bath, and the reaction was quenched by the slow addition of water. The reaction was extracted with ethyl acetate, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and spin-dried to afford 510 mg of compound 8d as a brown solid in an 82.9% yield. LCMS (254 nm) revealed a purity of 88.5%, Rt = 1.375 min; MS calculated: 155.1; MS found: 156.1 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ6.90 (d, J = 9.0 Hz, 1H), 6.36 (d, J = 12.2 Hz, 1H), 4.94 (d, J = 5.5 Hz, 3H), 4.32 (d, J = 4.7 Hz, 2H), 2.05 (s, 3H).

[0372] Step 5: Dissolve compound 8d (510 mg, 3.29 mmol) in 10 mL of dichloromethane, add MnO2 (1.5 g, 17.11 mmol), and stir overnight at room temperature. After the reaction, filter, wash, and spin dry. Purify by column chromatography to obtain 464 mg of compound 8e as a pale yellow solid in a 92.2% yield. LCMS (254 nm) purity: 84.82%, Rt = 1.787 min; MS calculated: 153.1; MS found: 154.1 [M+H] + . 1H NMR (400MHz, CDCl3) δ9.69 (s, 1H), 7.21 (d, J = 8.4Hz, 1H), 6.23 (d, J = 11.6Hz, 1H), 6.17–5.88 (m, 2H), 2.11 (d, J = 1.8Hz, 3H).

[0373] Step 6: Compound 8e (466 mg, 3.04 mmol) and compound 1c (600 mg, 2.28 mmol) were dissolved in 28 mL of toluene under nitrogen atmosphere. The mixture was heated to 130°C and stirred at reflux for 30 minutes. TsOH (131 mg, 0.76 mmol) was then added and stirred continuously. The reaction was monitored by LCMS. After completion of the reaction, the mixture was cooled to room temperature, filtered, and washed with acetone. The filtrate was concentrated and subjected to low-medium pressure reverse transpiration (alkaline mobile phase). The target fraction was collected and lyophilized to obtain 350 mg of the title compound (STI-F-8) as a pale yellow solid in a 40.4% yield. LCMS (254 nm) revealed a purity of 94.07%, Rt = 1.589 min; MS calculated: 380.1; MS found: 381.1 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.61(s,1H),8.05(d,J=8.5Hz,1H),7.88(d,J=10.9Hz,1H),7.32(s,1H) ,6.53(s,1H),5.42(s,2H),5.25(s,2H),2.48(s,3H),1.92–1.80(m,2H),0.88(t,J=7.4Hz,3H).

[0374] Example 9: (S)-N-(4-ethyl-4-hydroxy-8-fluoro-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)-2-hydroxyacetamide (STI-F2), and

[0375] Example 10: Synthesis of (S)-4-ethyl-4-hydroxy-8-fluoro-9-methyl-11-amino-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (STI-F-03c-1)

[0376] Step 1: At room temperature, 3.0 mL of acetic anhydride and 2.4 mL of pyridine were mixed, and compound STI-F-8 (300 mg, 0.79 mmol) was added. The mixture was heated and stirred at 40°C, and monitored by LCMS. After completion of the reaction, saturated sodium chloride was added, and the mixture was extracted with acetonitrile. The organic phase was concentrated, and the target fraction was collected by reverse preparative reaction at medium and low pressure (alkaline mobile phase). The fraction was lyophilized to obtain 117 mg of compound 9a as a yellow-brown solid, with a yield of 35.1%. LCMS (254 nm) showed a purity of 97.16%, Rt = 1.698 min; MS calculated: 422.1; MS found: 423.2 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.23(s,1H),7.77–7.64(m,2H),7.12(s,1H),5.60(d,J=17.2Hz,1H),5.33(d,J=17.2Hz,1H),5.19(t,J=1. 5Hz, 2H), 2.46 (t, J = 1.4Hz, 3H), 2.21 (dd, J = 13.9, 7.5Hz, 1H), 2.15 (s, 3H), 2.08 (dd, J = 13.9, 7.5Hz, 1H), 0.91 (t, J = 7.5Hz, 3H).

[0377] Step 2: Compound 9a (480 mg, 1.14 mmol) was dissolved in 5 mL of acetic acid. 30% aqueous hydrogen peroxide (6 mL) was added and stirred at 70°C for 3.5 hours. The mixture was extracted with DCM, dried over anhydrous sodium sulfate, filtered, and spun down to dryness. Dissolve the mixture in 24 mL of DMF, cool to 0°C in an ice bath, and slowly add 1.5 mL of oxalyl chloride dropwise. Stir the mixture back to room temperature and monitor with LCMS. After completion, the reaction was quenched with water, extracted with DCM, and spun down to dryness. The target fraction was collected and lyophilized to afford 420 mg of compound 9b as a pink solid in an 81.0% yield. LCMS (254 nm) revealed a purity of 97.37%, Rt = 2.014 min; MS calculated: 456.1; MS found: 457.0 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.24(d,J=8.0Hz,1H),7.98(d,J=10.6Hz,1H),7.05(s,1H),5.50(d,J=4.2 Hz,2H),5.28(d,J=4.7Hz,2H),2.54(s,3H),2.22(s,3H),2.18–2.12(m,2H),0.91(t,J=7.4Hz,3H).

[0378] Step 3: Compound 9b (240 mg, 0.52 mmol), t-BuOK (128 mg, 1.04 mmol), Pd(OAc)2 (16 mg, 0.07 mmol), Xantphos (30 mg, 0.05 mmol), and benzophenone imine (104 mg, 0.58 mmol) were added to a reaction tube. The atmosphere was purged with nitrogen three times, and 16 mL of ultra-dry toluene was added. The mixture was heated at 100°C with stirring for 6 hours. The reaction mixture was spin-dried and dissolved in acetonitrile. The reaction mixture was then subjected to reverse preparative reaction at medium-low pressure (alkaline mobile phase). The desired fractions were collected and lyophilized to obtain the coupling product. 90 mg of the coupling product was added to 15 mL of THF and 2 mL of 6 M hydrochloric acid. The mixture was stirred at room temperature overnight. The reaction mixture was concentrated and subjected to reverse preparative reaction at medium-low pressure (alkaline mobile phase). The desired fractions were collected and lyophilized to obtain 50 mg of compound 9c as a light yellow solid. The total yield for the two steps was 21.7%. LCMS (254 nm) purity 90.36%, Rt = 1.730 min; MS calculated value: 437.1; MS found value: 438.2 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.25(d,J=8.3Hz,1H),7.58(d,J=11.4Hz,1H),7.38(s,2H),6.87(s,1H),5 .46(s,2H),5.08–4.97(m,2H),2.43(s,3H),2.20(s,3H),2.15–2.07(m,2H),0.90(t,J=7.4Hz,3H).

[0379] Step 4: Compound 9c (50 mg, 0.011 mmol) and DMAP (1 mg, cat) were added to a reaction flask under nitrogen protection. Ultra-dry DMF (1.5 mL) was then added, followed by DIPEA (29 mg, 0.023 mmol) and acetoxyacetyl chloride (31 mg, 0.023 mmol). The reaction was carried out at room temperature for 2 hours, then heated to 55°C for 3 hours and stirred at room temperature overnight. LCMS showed that the reaction was approximately 50%. The reaction was stopped and the system was concentrated to dryness under reduced pressure. Methanol (4 mL) and LiOH (50 mg) were added and stirred at room temperature for approximately 30 min. The pH was adjusted to 2-3 with acetic acid. The system was concentrated to dryness and prepared by reverse phase high pressure with medium-low pressure alkaline method. The mixture was lyophilized to obtain compound STI-F2 as a light yellow solid, approximately 7.8 mg. The acetyl group of the remaining compound 9c in the reaction system was hydrolyzed with LiOH to obtain compound STI-F-03c-1 as a light yellow solid, approximately 8.3 mg.

[0380] Compound STI-F2: LCMS (254 nm) purity 97.89%, Rt = 1.51 min; MS calculated value: 453.1; MS found value: 454.2 [M+H]+ . 1H NMR (400MHz, DMSO-d6) δ10.65(s,1H),8.13(d,J=8.0Hz,1H),7.90(d,J=10.8Hz,1H),7.32(s,1H) ,5.81(br,1H),5.43(s,2H),5.12(s,2H),4.30(s,2H),1.92–1.81(m,2H),0.88(t,J=7.6Hz,3H).

[0381] Compound STI-F-03c-1: LCMS (254 nm) purity 97.67%, Rt = 1.60 min; MS calculated value: 395.1; MS found value: 396.2 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.76-8.58 (m, 2H), 8.38 (d, J = 8.0Hz, 1H),7.60(d,J=10.84Hz,1H),7.46-7.42(m,1H),6.61(br,1H),5.44(s, 2H), 5.05 (s, 2H), 2.44 (s, 3H), 1.93–1.78 (m, 2H), 0.87 (t, J = 7.2Hz, 3H).

[0382] Example 11: Synthesis of (S)-4-ethyl-4-hydroxy-8-fluoro-9-amino-11-pentyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (STI-G1)

[0383] Step 1: Under nitrogen and an ice bath, compound 11a (500 mg, 3.18 mmol) was dissolved in 30 mL of DCM. Hexanoyl chloride (0.67 mL, 4.77 mmol) and triethylamine (0.88 mL, 6.36 mmol) were added to the reaction flask with stirring at 0°C. The reaction was continued at this temperature for 30 minutes. TLC (PE:EA = 30:1, product Rf = 0.5) confirmed the completion of the reaction. The mixture was extracted twice with 50 mL of dichloromethane and 50 mL of water. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude brown product was purified by silica gel column chromatography (PE:EA = 10:1) to obtain 650 mg of compound 11b as a colorless liquid in an 80.9% yield. 1H NMR (400MHz, DMSO-d6) δ8.25(t,J=8.9Hz,1H),7.54(dd,J=12.1,2.4Hz,1H),7.27(dt,J=9.1,1.8Hz,1 H), 2.62 (t, J = 7.4Hz, 2H), 1.64 (dd, J = 10.0, 4.8Hz, 2H), 1.33 (q, J = 3.7Hz, 4H), 0.89 (t, J = 6.9Hz, 3H).

[0384] Step 2: Compound 11b (500 mg, 1.96 mmol) was dissolved in 4 mL of nitrobenzene. Under nitrogen, aluminum trichloride (290 mg, 2.16 mmol) was added and the mixture was allowed to react at 150°C for 5 hours. After completion of the reaction, as determined by TLC (PE:EA = 30:1, product Rf = 0.5), the reaction system was cooled to room temperature and added to 50 mL of 1N HCl in ice-cold water. The mixture was stirred at room temperature for 30 minutes. Extraction was then performed with 50 mL of ethyl acetate. The aqueous phase was then extracted twice with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting brown crude product was purified by silica gel column chromatography (PE:EA = 200:1 to remove the nitrobenzene solvent and PE:EA = 20:1 to remove the product). After spin drying, 300 mg of compound 11c was obtained as a yellow solid in a 60% yield. 1 H NMR (400MHz, DMSO-d6) δ12.66(s,1H),8.54(d,J=9.0Hz,1H),7.04(d,J=13.0Hz,1H),3. 06(t,J=7.2Hz,2H),1.60(dd,J=8.5,5.9Hz,2H),1.33–1.28(m,4H),0.89–0.85(m,3H).

[0385] Step 3: Compound 11c (500 mg, 1.96 mmol) was dissolved in 20 mL of dichloromethane. Pyridine (0.63 mL, 7.84 mmol) and Tf2O (0.99 mL, 5.88 mmol) were added to the reaction system under nitrogen in an ice bath. The reaction was allowed to react at 0°C for 30 minutes. After completion of the reaction, as determined by TLC (PE:EA = 20:1, product Rf = 0.5), the reaction system was diluted with 20 mL of dichloromethane and extracted with water. The aqueous phase was then extracted twice with 10 mL of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting brown crude product was purified by silica gel column chromatography (PE:EA = 20:1) and dried to afford 650 mg of compound 11d as a yellow solid in an 85.7% yield. 1H NMR (400MHz, CDCl3) δ8.55(d,J=7.9Hz,1H),7.36(d,J=9.9Hz,1H),2.96(t,J=7.3 Hz, 2H), 1.75 (dd, J = 9.1, 5.6Hz, 2H), 1.36 (q, J = 3.9Hz, 4H), 0.93 (d, J = 6.8Hz, 3H).

[0386] Step 4: Compound 11d (1.12 g, 2.90 mmol) was dissolved in 28 mL of 1,4-dioxane. Under nitrogen, tert-butyl carbamate (680 mg, 5.80 mmol), Xantphos (336 mg, 0.58 mmol), Pd2(dba)3 (266 mg, 0.29 mmol), and cesium carbonate (1.89 g, 5.80 mmol) were added to the reaction system. The nitrogen atmosphere was replaced three times, and the reaction was allowed to react at 80°C for 1 hour. After completion, as determined by TLC (PE:EA = 10:1, product Rf = 0.6), the reaction was cooled to room temperature and diluted with 20 mL of ethyl acetate. The mixture was extracted with water, and the aqueous phase was extracted twice with 10 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting crude brown product was purified by silica gel column chromatography (PE:EA = 50:1) to yield 440 mg of a yellow liquid after spin drying. The yellow liquid was dissolved in 12 mL of 50% TFA / DCM solution and allowed to react at room temperature for 30 minutes. After completion of the reaction, the mixture was diluted with 20 mL of dichloromethane and extracted with water. The aqueous phase was then extracted twice with 10 mL of dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting brown crude product was purified by silica gel column chromatography (PE:EA = 5:1) and dried to dryness to afford 260 mg of compound 11e as a light yellow solid in a 35.3% yield. LCMS (254 nm) Rt = 2.108 min; MS Calcd: 254.1; MS Found: 253.1 [MH] - . 1 H NMR(400MHz,DMSO-d6)δ8.64(d,J=8.7Hz,1H),8.53–7.43(m,2H),6.67(d,J=14.4Hz,1H),3 .01(t,J=7.3Hz,2H),1.59(t,J=7.2Hz,2H),1.31(dq,J=7.6,3.4Hz,4H),0.92–0.83(m,3H).

[0387] Step 5: Compound 11e (200 mg, 0.79 mmol) was dissolved in 12 mL of a 3:1 ethanol:water mixture. Iron powder (176 mg, 3.16 mmol) and ammonium chloride (44 mg, 0.79 mmol) were added to the reaction system under nitrogen. The nitrogen atmosphere was replaced three times, and the reaction was refluxed at 80°C for 1 hour. After completion, the reaction was cooled to room temperature, the solvent was evaporated, and the crude product was purified by silica gel column chromatography (PE:EA=2:1). After evaporation, 160 mg of compound 11f was obtained as a brown solid in a 90% yield. LCMS (254 nm) Rt = 2.189 min; MS calcd: 224.1; MS found: 225.2 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ7.25(d,J=10.1Hz,1H),6.70(s,2H),6.45(d,J=13.5Hz,1H),4.39(s,2H) ,2.78(t,J=7.4Hz,2H), 1.57(t,J=7.3Hz,2H), 1.30(tt,J=5.9,3.2Hz,4H), 0.87(t,J=6.8Hz,3H).

[0388] Step 6: Dissolve compound 11f (20 mg, 0.09 mmol) and compound 1c (24 mg, 0.09 mmol) in 1 mL of anhydrous toluene under nitrogen atmosphere. Reflux with a water separator for 30 minutes. Add p-toluenesulfonic acid (4 mg, 0.023 mmol) and continue stirring at reflux for 3.5 hours. After completion of the reaction, as determined by TLC (DCM:MeOH = 30:1, product Rf = 0.4) and LCMS, cool the reaction system to room temperature, filter, and wash with acetone. The crude product is purified by preparative plate to yield the title compound (STI-G1) as a yellow solid (5 mg). LCMS (254 nm) Rt = 1.839 min; MS calcd: 451.2; MS found: 452.3 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ7.74(d,J=12.3Hz,1H),7.30(d,J=9.5Hz,1H),7.19(s,1H),6.46(s,1H),6.06(s,2H),5.40(s,2H),5.21(s,2H),3.0 0(t,J=8.1Hz,2H),2.05–1.94(m,2H),1.91–1.79(m,2H),1.73–1.62(m,2H),1.48–1.40(m,2H),0.90(t,J=6.0Hz,3H),0.87(t,J=5.9Hz,3H).

[0389] Example 12: Synthesis of (S)-4-ethyl-4-hydroxy-8-fluoro-9-nitro-11-pentyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (STI-G03)

[0390] Compound 11e (20 mg, 0.078 mmol) and compound 1c (20 mg, 0.078 mmol) were dissolved in 1 mL of anhydrous toluene under nitrogen atmosphere. The mixture was refluxed for 30 minutes with a water separator. p-Toluenesulfonic acid (3.5 mg, 0.02 mmol) was added, and stirring at reflux continued for 3.5 hours. After completion of the reaction, the reaction system was cooled to room temperature, filtered, and washed with acetone. The crude product was purified using a preparative plate to obtain the title compound as a yellow solid (8 mg, 21% yield). LCMS (254 nm) revealed a purity of 93.78% (Rt = 2.01 min); MS calculated: 481.2; MS found: 482.2 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ9.05(d,J=7.9Hz,1H),8.26(d,J=12.2Hz,1H),7.38(s,1H),6.57(s,1H),5.45(s,2H),5.35(s,2H),3 .30–3.25(m,2H),1.92–1.82(m,2H),1.72(t,J=7.7Hz,2H),1.49–1.44(m,2H),1.38(t,J=7.1Hz,2H),0.88(d,J=8.1Hz,6H).

[0391] Example 13: Synthesis of (S)-9-bromo-4-ethyl-8-fluoro-4-hydroxy-11-propylthiomethyl-1,12-dihydro-14H-pyrano[3,4]:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (STI-G2-06c)

[0392] Step 1: Under nitrogen and an ice bath, add 70 mL of DCE and a 1M solution of boron trichloride in DCM (12.5 mL, 12.5 mmol) to a reaction flask. Then, add compound 13a (3.0 g, 15.6 mmol) and stir at 0°C for 10 minutes. Chloroacetonitrile (1416 mg, 18.7 mmol) and aluminum trichloride (2706 mg, 20.3 mmol) are then added under ice bath, and the reaction system is slowly warmed to room temperature. Stir at room temperature for 10 minutes, then reflux and stir for 39 hours. After the reaction, add 100 mL of ice water, followed by 40 mL of 2M HCl, and stir at room temperature for 1 hour. Extract with 100 mL of dichloromethane, dry the organic phase over anhydrous sodium sulfate, and concentrate under reduced pressure. The residue is then subjected to neutral medium-pressure reverse elution (alkaline mobile phase) and lyophilized to yield 1.1 g of compound 13b as a yellow solid powder in a 26.2% yield. LCMS (254 nm) purity 100%, Rt = 1.93 min; MS calculated value: 264.9; MS found value: 263.9 [MH] - . 1 H NMR (400MHz, DMSO-d6) δ 8.03 (d, J = 7.9 Hz, 1H), 7.56 (s, 2H), 6.74 (d, J = 11.6 Hz, 1H), 5.05 (s, 2H).

[0393] Step 2: Compound 13b (250 mg, 0.94 mmol) was dissolved in 5 mL of dichloromethane under nitrogen protection. Propanethiol (94 μL, 1.03 mmol) and triethylamine (261 μL, 1.88 mmol) were added and stirred at room temperature overnight. After completion of the reaction, the mixture was washed with water, dried, and purified by Pre-TLC to obtain 218 mg of compound 13c as a light yellow solid with a yield of 75.7%. 1 H NMR(400MHz,DMSO-d6)δ8.08(d,J=8.0Hz,1H),7.53(s,2H),6.71(d,J=11.5Hz,1H) ,3.90(s,2H),2.47(d,J=7.3Hz,2H),1.54(h,J=7.3Hz,2H),0.91(t,J=7.3Hz,3H).

[0394] Step 3: Dissolve compound 13c (187 mg, 0.61 mmol) and compound 1c (121 mg, 0.46 mmol) in 10 mL of anhydrous toluene under nitrogen atmosphere. Reflux the mixture for 30 minutes. Add p-toluenesulfonic acid (11 mg, 0.06 mmol) and continue stirring at reflux for 4 hours. After completion of the reaction, as determined by LCMS, cool the reaction system to room temperature, filter, wash with acetone, and purify by Pre-TLC (DCM:MeOH = 15:1) to obtain 230 mg of compound STI-G2-06c as a yellow solid in a 94.0% yield. Rt = 2.09 min; MS calcd: 532.1; MS found: 533.1 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.79(d,J=7.5Hz,1H),8.13(d,J=9.8Hz,1H),7.33(s,1H),6.54(s,1H),5.44(s,2H),5.33( s,2H),4.45(s,2H),2.05–1.95(m,2H),1.92–1.81(m,2H),1.63–1.52(m,2H),0.93–0.88(m,3H),0.87–0.82(m,3H).

[0395] Example 14: Synthesis of (S)-9-amino-4-ethyl-8-fluoro-4-hydroxy-11-propylthiomethyl-1,12-dihydro-14H-pyrano[3,4:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (STI-G2)

[0396] Step 1: Under nitrogen, compound STI-G2-06c (53 mg, 0.10 mmol) from Example 13 and tert-butyl carbamate (23 mg, 0.20 mmol) were dissolved in 5 mL of anhydrous toluene. Cesium carbonate (65 mg, 0.20 mmol), Xantphos (5.8 mg, 0.01 mmol), and palladium acetate (2.2 mg, 0.01 mmol) were added under nitrogen. The mixture was stirred until uniform, heated to 100°C, and reacted for 2.0 hours. After completion, as determined by TLC (DCM:MeOH = 15:1, product Rf = 0.5), the reaction mixture was concentrated to dryness under reduced pressure, extracted with ethyl acetate and water, and the organic phase was concentrated to afford 40 mg of compound 14a as a yellow solid in a 70.5% yield. LCMS (254 nm) revealed a purity of 98.42%, Rt = 2.07 min; calculated: 569.2; found: 570.3 [M+H] + .

[0397] Step 2: Compound 14a (65 mg, 0.11 mmol) was dissolved in 2.0 mL of DCM, and 0.6 mL of TFA was added. The mixture was stirred at room temperature under nitrogen for 0.5 h. The reaction was terminated and concentrated to dryness under reduced pressure. Reverse chromatography (acidic mobile phase) was performed using a medium- and high-pressure preparative column (with an acidic mobile phase), and lyophilized to afford 10 mg of compound STI-G2 as a reddish-brown solid in an 18.7% yield. LCMS (254 nm) revealed a purity of 98.48%, Rt = 1.75 min; MS calculated: 469.2; MS found: 470.2 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ7.76(d,J=12.3Hz,1H),7.35(d,J=9.6Hz,1H),7.20(s,1H),6.48(s,1H),6.16(s,2H),5.41(s,2H),5.24 (s,2H),4.19(s,2H),2.58(t,J=7.2Hz,2H),1.91–1.80(m,2H),1.65–1.56(m,2H),0.92(t,J=7.3Hz,3H),0.87(t,J=7.3Hz,3H).

[0398] Example 15: Synthesis of (S)-10-cyclopropyl-4-ethyl-8-fluoro-4-hydroxy-11-(4-hydroxybutyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (STI-D)

[0399] Step 1: Compound 15a (10.0 g, 80.0 mmol) was dissolved in 100 mL of DCM at room temperature. 100 mL of methanol was added, and Br2 (32.0 g, 200.0 mmol) was added dropwise. The mixture was allowed to react at room temperature for two hours. After completion of the reaction, the mixture was concentrated to dryness under reduced pressure. 1 M aqueous sodium thiosulfate (100 mL) and 100 mL of ethyl acetate were added, and the pH was adjusted to 7-8 with saturated NaHCO3. The aqueous phase was washed with EA (100 mL x 2). The combined organic phases were washed sequentially with 100 mL of 1 M aqueous sodium thiosulfate and saturated brine, dried over anhydrous Na2SO4, concentrated to dryness under reduced pressure, and purified by column chromatography (PE) to afford 20.0 g of compound 15b as a pale purple solid in an 89.3% yield. 1 H NMR (400MHz, DMSO-d6) δ7.44 (d, J = 8.8 Hz, 1H), 5.16 (s, 2H), 2.21 (d, J = 2.4 Hz, 3H).

[0400] Step 2: TsOH (40.0 g, 232.6 mmol) was added to MeCN (240.0 mL), cooled to 10°C, and compound 15b (20.0 g, 70.8 mmol) was added. NaNO2 (9.8 g, 141.6 mmol) and KI (29.4 g, 141.6 mmol) were dissolved in water (60.0 mL) and added dropwise to the reaction system. The system was allowed to return to room temperature and allowed to react for 1 hour before stopping the reaction. The system was poured into 200 mL of water, and the pH was adjusted to approximately 9 with saturated NaHCO3. A 1 M aqueous sodium thiosulfate solution was then added until the system did not fade. The product was extracted with EA (200.0 mL x 3). The organic phases were combined, dried over anhydrous Na2SO4, concentrated to dryness under reduced pressure, and purified by column chromatography (PE) to afford 18.6 g of compound 15c as a white solid in a 67.1% yield. 1 H NMR (400MHz, DMSO-d6) δ7.79 (d, J = 9.2 Hz, 1H), 2.26 (d, J = 2.4 Hz, 3H).

[0401] Step 3: Compound 15c (5040 mg, 12.8 mmol) was added to a reaction flask under nitrogen atmosphere. Toluene was added and the temperature was cooled to approximately -30°C. A 2M solution of iPrMgCl in tetrahydrofuran (9.6 mL, 19.2 mmol) was added dropwise. The mixture was kept at -30°C for 1.5 hours. DMF (3083 mg, 42.2 mmol) was then added dropwise. The mixture was allowed to return to room temperature and react for 1.5 hours. After completion of the reaction, as determined by TLC (PE:EA = 20:1, product Rf = 0.3), the reaction was quenched by the addition of 50.0 mL of saturated aqueous NH4Cl. The mixture was extracted with EA (50.0 mL x 3). The organic phases were combined, dried over anhydrous Na2SO4, concentrated to dryness under reduced pressure, and purified by column chromatography (PE-PE:EA = 100:1) to afford 3.5 g of compound 15d as a pale yellow solid in a yield of 79.8%. 1 H NMR (400MHz, DMSO-d6) δ10.14 (s, 1H), 7.32 (d, J = 12.6 Hz, 1H), 2.29 (d, J = 2.8 Hz, 3H).

[0402] Step 4: Compound 15d (2959 mg, 10.0 mmol) was added to a three-necked flask under nitrogen protection. DCE (30.0 mL) was added to dissolve the mixture, and ethylene glycol (3724 mg, 60.0 mmol), triethyl orthoformate (1630 mg, 11.0 mmol) and TsOH (86 mg, 0.5 mmol) were added in sequence. The mixture was refluxed for 3 hours. After completion of the reaction, the reaction was stopped and the system was naturally cooled to room temperature. 50 mL of DCM was added to dilute the reaction solution, and the mixture was extracted with 80 mL of saturated aqueous Na2CO3 solution. The organic phase was washed with 80 mL of saturated brine, dried over anhydrous Na2SO4, and concentrated to dryness under reduced pressure to obtain 3.3 g of compound 15e as a light yellow solid with a yield of 97.7%. 1 H NMR (400MHz, DMSO-d6) δ7.33 (d, J = 8.8Hz, 1H), 6.42 (s, 1H), 4.35-4.31 (m, 2H), 4.09-4.06 (m, 2H), 2.33 (d, J = 2.4Hz, 3H).

[0403] Step 5: Compound 15e (3.3 g, 10.0 mmol) was added to a reaction flask under nitrogen protection, toluene (20.0 mL) was added, and then benzophenone imine (1450 mg, 8.0 mmol), t-BuOK (2356 mg, 21.0 mmol), Pd(OAc)2 (225 mg, 1.0 mmol) and Xantphos (579 mg, 1.0 mmol) were added to the reaction flask in sequence. The temperature was raised to 100°C and the reaction was allowed to react for 1 hour. The reaction was then stopped and the system was filtered through a celite pad. The filter cake was washed with EA, and the filtrate was concentrated to dryness under reduced pressure and purified by column chromatography (PE:EA=400:1) to obtain 730 mg of compound 15f as a light yellow solid with a yield of 16.6%.

[0404] Step 6: Compound 15f (730 mg, 1.67 mmol) was dissolved in 4.0 mL of THF and cooled to 0-5°C. Concentrated hydrochloric acid (11.0 mL, 116.8 mmol) was added dropwise. The mixture was kept at 0-5°C for 10 minutes before stopping the reaction. The pH was adjusted to neutral with solid NaHCO₃. EA (20.0 mL) and water (20.0 mL) were added for extraction. The aqueous phase was washed with EA (20.0 mL x 2). The combined organic phases were dried over anhydrous Na₂SO₄, concentrated to dryness under reduced pressure, and purified by pre-TLC to afford 130 mg of compound 15g as a yellow solid in a 33.7% yield. LCMS (254 nm) revealed a purity of 92.14%, Rt = 2.024 min; MS calcd: 231.0; MS found: 230.0 [MH] - .1 H NMR (400MHz, DMSO-d6) δ10.39 (s, 1H), 6.51 (br, 2H) 6.31 (d, J = 11.2Hz, 1H), 2.25 (d, J = 2.4Hz, 3H).

[0405] Step 7: Compound 15g (130 mg, 0.56 mmol) was added to a reaction flask under nitrogen atmosphere. After dissolving in THF, the temperature was lowered to 0-5°C. The THF solution of compound 15h was then added dropwise to the reaction mixture and allowed to react at 0-5°C for 1 hour. The reaction was stopped and quenched by adding 20 mL of saturated aqueous NH4Cl. The mixture was extracted with EA, dried over anhydrous Na2SO4, and concentrated to dryness under reduced pressure. Purification by pre-TLC (PE:EA = 3:1) afforded 130 mg of compound 15i as a brown oil in a 54.6% yield. LCMS (254 nm) revealed a purity of 98.60%, Rt = 2.174 min; MS calculated: 425.1; found: 424.1 [MH] - . 1 H NMR (400MHz, DMSO-d6) δ7.26-7.17(m,2H),6.93-6.84(m,2H),6.44(d,J=12.0Hz,1H),5.79(d,J=3.8Hz,1H),5.66(s,2H),5.16(dt ,J=8.7,4.2Hz,1H),4.34(s,2H),3.74(s,3H),3.36(t,J=6.2Hz,2H),2.11(d,J=2.2Hz,3H),1.87-1.74(m,1H),1.55-1.46(m,5H).

[0406] Step 8: Compound 15i (130 mg, 0.31 mmol) was dissolved in 4.0 mL of ethyl acetate, and 2-iodobenzoic acid (IBX) (95 mg, 0.34 mmol) was added. The mixture was heated to reflux for 4 hours, then the reaction was stopped and the system was allowed to return to room temperature. The mixture was filtered through a pad of Celite, and the filter cake was washed with ethyl acetate. The filtrate was concentrated to dryness under reduced pressure and purified by Pre-TLC (PE:EA = 5:2) to obtain 130 mg of compound 15j as a brown oil in a 100.0% yield. LCMS (214 nm) purity: 90.82%, Rt = 2.226 min; MS calcd: 423.1; found: 422.1 [MH] - . 1H NMR (400MHz, DMSO-d6) δ7.26-7.21(m,2H),6.91-6.87(m,2H),6.52(d,J=12.0Hz,1H),5.35(br,2H),4.36(s,2H),3. 74(s,3H),3.39(t,J=6.4Hz,2H),2.77(t,J=7.2Hz,2H),2.10(d,J=2.4Hz,3H),1.68-1.63(m,2H),1.61-1.55(m,2H).

[0407] Step 9: Compound 15j (130 mg, 0.31 mmol) was dissolved in 1 mL of DCM and cooled to 0-5°C. A 50% trifluoroacetic acid solution in dichloromethane (3.0 mL) was added dropwise. The mixture was kept at 0-5°C for 20 minutes before stopping the reaction. The mixture was concentrated to dryness under reduced pressure and redissolved in DCM. The pH was adjusted to approximately 7 with saturated NaHCO₃. Extraction was performed, and the aqueous phase was washed with DCM (20.0 mL). The combined organic phases were dried over anhydrous Na₂SO₄, concentrated to dryness under reduced pressure, and purified by pre-TLC (PE:EA = 5:2) to afford 80 mg of compound 15k in an 86.0% yield. LCMS (254 nm) revealed a purity of 94.67%, Rt = 1.763 min; MS calculated: 303.0; found: 302.0 [MH] - . 1 H NMR(400MHz, DMSO-d6)δ6.51(d,J=12.0Hz,1H),5.35(s,2H),4.34(s,1H),3.40(t,J=6.4H z, 2H), 2.77 (t, J = 7.2Hz, 2H), 2.11 (d, J = 2.0Hz, 3H), 1.67-1.60 (m, 2H), 1.50-1.53 ​​(m, 2H).

[0408] Step 10: Compound 15k (40 mg, 0.13 mmol) was dissolved in 1,4-dioxane (4.0 mL), and cyclopropylboronic acid (67 mg, 0.78 mmol), K3PO4 (165 mg, 0.78 mmol) and Pd(dppf)Cl2 (22 mg, 0.03 mmol) were added in sequence. The mixture was protected by nitrogen and heated to 90°C for 1.5 hours. The reaction was stopped after LCMS monitoring until the starting material was completely consumed. The system was naturally cooled to room temperature, filtered through celite, and the filter cake was washed with ethyl acetate. The filtrate was concentrated to dryness under reduced pressure and purified by Pre-TLC (DCM:MeOH = 20:1) to obtain 30 mg of compound 15l as a white solid in a yield of 85.8%.

[0409] Step 11: Compound 151 (21 mg, 0.08 mmol), compound 1c (40 mg, 0.13 mmol), and TsOH·H2O (40 mg, 0.13 mmol) were added to a reaction flask. Toluene was added and the mixture was heated to reflux under nitrogen for 1 hour. The reaction was then stopped and the system was concentrated to dryness under reduced pressure. The mixture was then lyophilized using medium- and high-pressure reverse-phase separation (acidic mobile phase) to afford 6.5 mg of compound STI-D as a pale yellow solid. LCMS (254 nm) revealed a purity of 98.45%, Rt = 1.990 min; MS calculated: 492.2; found: 493.1 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ6.72 (s, 1H), 6.29 (s, 1H), 6.20 (d, J = 11.2Hz, 1H), 6.13 (s,1H),4.56(dd,J=24.4,16.0Hz,2H),4.16-3.92(m,4H),2.59-2.54(m,2H),2 .13(d,J=2.0Hz,3H),2.12-2.08(m,1H)1.95-1.85(m,2H),1.84-1.61(m,4H),0 .99-0.88(m,2H),0.75(t,J=7.2Hz,3H),0.54-0.48(m,1H),0.32-0.28(m,1H).

[0410] Intermediate compound 15h was prepared as follows:

[0411] Magnesium particles (97 mg, 4.03 mmol) were added to a three-necked flask under nitrogen protection. 4.0 mL of THF and 1,2-dibromoethane (2 drops, cat.) were added and heated until initiation. 15h-1 (914 mg, 3.36 mmol) was dissolved in 2.0 mL of THF and added to the reaction system. The mixture was reacted at 50°C for 30 min to obtain a THF solution of 15h.

[0412] Example 16: Synthesis of 2-cyclopropyl-N-((S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)-2-hydroxyacetamide (STI-F)

[0413] Step 1: Compound 16a (23 mg, 0.2 mmol), compound 9b from Example 9 (46 mg, 0.1 mmol), Xantphos (6 mg, 0.01 mmol), palladium acetate (2.2 mg, 0.01 mmol), and cesium carbonate (65 mg, 0.20 mmol) were added to a reaction flask under nitrogen atmosphere. 3 mL of anhydrous toluene was added and stirred. The mixture was heated to 100°C and reacted for 1.0 hour. After completion of the reaction, as determined by TLC (DCM:MeOH = 20:1, product Rf = 0.5), the mixture was concentrated to dryness under reduced pressure and purified by column chromatography (DCM:MeOH = 100:1) to afford 17 mg of compound 16b as a yellow solid in a 31.8% yield. LCMS (254 nm) revealed a purity of 99.42%. Rt = 1.89 min; Calcd: 533.2; Found: 534.2 [M+H] + .

[0414] Step 2: Compound 16b (17 mg, 0.032 mmol) was added to DCM (1.0 mL) under nitrogen protection. STAB (28 mg, 0.140 mmol) was added in four portions and reacted at room temperature for 1.5 hours. After completion of the reaction, 10 mL of DCM and 10 mL of water were added to the system for extraction. The aqueous phase was then washed with DCM (10 mL x 2) and the mixture was stirred for 1 h. The organic phases were washed, combined, and concentrated to dryness under reduced pressure. Methanol (3 mL) and lithium hydroxide (20 mg) were added and the mixture was allowed to react at room temperature for 10 minutes. The pH was adjusted to 3-4 with acetic acid. A mixture of dichloromethane and methanol (DCM:MeOH = 10:1, 10 mL) and water (10 mL) were added for extraction. The aqueous phase was washed again with a mixture of dichloromethane and methanol (DCM:MeOH = 10:1, 10 mL x 2). The organic phases were combined and concentrated to dryness under reduced pressure. Reverse phase preparative reaction (acidic mobile phase) was performed under medium-low pressure, and lyophilized to afford 6 mg of compound STI-F as a light yellow solid in a 37.9% yield. LCMS (254 nm) purity was 98.86%, Rt = 1.60 min; MS calculated value: 493.2; MS found: 494.2 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ10.60(s,1H),8.07(d,J=8.0Hz,1H),7.92(d,J=10.8Hz,1H),7.33(s,1H),6.52(s,1H),5.91(br,1H),5.42(s, 2H), 5.11 (s, 2H), 3.95 (d, J = 6.8Hz, 1H), 2.50 (s, 3H), 1.92-1.79 (m, 2H), 1.34-1.27 (m, 1H), 0.88 (t, J = 7.2Hz, 3H), 0.62-0.49 (m, 4H).

[0415] Intermediate compound 16a was prepared as follows:

[0416] Compound 16a-1 (250 mg, 2.19 mmol) was dissolved in THF (5 mL), and triethylamine (0.61 mL, 4.38 mmol) was added. The mixture was cooled to -20°C under nitrogen and a solution of isopropyl chloroacetate in tetrahydrofuran (299 mg, 2.19 mmol) was added dropwise. After stirring at this constant temperature for 30 minutes, concentrated aqueous ammonia (1279 mg, 10.95 mmol) was added to the system and allowed to react at room temperature for 2 hours to terminate the reaction. The system was concentrated to dryness under reduced pressure and extracted with saturated aqueous sodium bicarbonate and EA. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Pre-TLC (PE:EA = 2:1) gave 92 mg of compound 16a as a white solid in a 37.2% yield. 1 H NMR (400MHz, DMSO-d6) δ7.91(s,1H),7.69(s,1H),2.86-2.80(m,1H),1.12-1.08(m,2H),0.97-0.94(m,2H).

[0417] Example 17: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-11-(4-hydroxybutyl)amino-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (STI-F6)

[0418] Step 1: Compound 9b (91 mg, 0.20 mmol) from Example 9, 17a (52 mg, 0.40 mmol), Xantphos (23 mg, 0.04 mmol), palladium acetate (5 mg, 0.02 mmol), and cesium carbonate (195 mg, 0.60 mmol) were added to a reaction flask and dissolved in 6 mL of anhydrous toluene. Under nitrogen, the mixture was heated to 100°C and reacted for 5 hours. The mixture was concentrated to dryness and purified by silica gel column chromatography (DCM:MeOH = 100:1) to afford 20 mg of compound 17b as a beige solid in an 18.2% yield. LCMS (254 nm) revealed a purity of 97.1%, Rt = 1.89 min; MS calcd: 551.2; MS found: 552.3 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.30(d,J=8.1Hz,1H),7.56(d,J=11.1Hz,1H),7.37(t,J=5.8Hz,1H),6.87(s,1H),5.46(s,2H),5.50-5.35(m,2H),4 .11-4.03(m,2H),3.67-3.61(m,2H),2.43(s,3H),2.20(s,3H),2.20-2 .03(m,2H),2.00(s,3H),1.75(h,J=4.3Hz,4H),0.90(t,J=7.4Hz,3H).

[0419] Step 2: Compound 17b (35 mg, 0.06 mmol) was dissolved in a mixture of 2 mL of methanol and 1 mL of dichloromethane. LiOH (35 mg, 1.46 mmol) was added, and the mixture was stirred at room temperature under nitrogen for 80 minutes. The pH was adjusted to 3-4 with glacial acetic acid. After concentration, the mixture was subjected to medium-low pressure reverse preparative reaction (acidic mobile phase) and lyophilized to afford 15 mg of compound STI-F6 as a pale yellow solid in a 50.6% yield. LCMS (254 nm) revealed a purity of 98.6%, Rt = 1.59 min; MS calculated: 467.2; MS found: 468.3 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.43(d,J=7.7Hz,1H),7.57(d,J=10.3Hz,1H),7.45(s,1H),6.61(s,1H),5.46(s,2H),5.44(s,2H ),3.74(q,J=6.7Hz,2H),3.48(t,J=6.4Hz,2H),2.43(s,3H),1.96–1.73(m,4H),1.66–1.54(m,2H),0.87(t,J=7.3Hz,3H).

[0420] Intermediate compound 17a was prepared as follows:

[0421] Step 1: Dissolve compound 17a-1 (3.0 g, 15.9 mmol) in 15 mL of pyridine, add 15 mL of acetic anhydride, and heat at 40°C under nitrogen for 1 hour. Extract with water and EA under ice-cooling. Wash the organic phase with 2 M hydrochloric acid to a pH ≤ 2, then with saturated brine, dry over anhydrous sodium sulfate, and rotary evaporation to obtain 3.56 g of compound 17a-2 as a clear oil (97.1% yield). 1 H NMR (400MHz, DMSO-d6) δ6.81(t,J=5.8Hz,1H),3.98(t,J=6.6Hz,2H),2.92(q,J=6.6Hz,2H),1.99(s,3H),1.59–1.49(m,2H),1.45–1.33(m,11H).

[0422] Step 2: Dissolve compound 17a-2 (92 mg, 0.4 mmol) in 0.5 mL of dichloromethane, add 0.5 mL of trifluoroacetic acid, and react at room temperature under nitrogen for 20 minutes. The crude product of compound 17a is obtained after spin drying and can be used directly in the next step.

[0423] Example 18: Synthesis of (S)-11-((4-aminobutyl)amino)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (STI-F7)

[0424] Step 1: Compound 9b from Example 9 (91 mg, 0.2 mmol), Xantphos (23 mg, 0.04 mmol), palladium acetate (5 mg, 0.02 mmol), and cesium carbonate (130 mg, 0.4 mmol) were added to a reaction flask under nitrogen. 18a (75 mg, 0.4 mmol) was dissolved in 6 mL of anhydrous toluene and added to the system. The mixture was stirred evenly, heated to 100°C, and reacted for 1.0 hour. After completion of the reaction, as determined by TLC (DCM:MeOH = 30:1, product Rf = 0.3), the system was concentrated to dryness under reduced pressure and purified by column chromatography (DCM:MeOH = 100:1) to afford 100 mg of compound 18b as a brown solid in a 54.8% yield. 1H NMR (400MHz, DMSO-d6) δ8.31(d,J=8.4Hz,1H),7.57(d,J=11.2Hz,1H),7.40(br, 1H),6.87(s,1H),6.83(t,J=5.6Hz,1H),5.46(s,2H),5.53(d,J=4.8Hz,2H),3.6 0(q,J=6.4Hz,2H),2.97(q,J=6.4Hz,2H),2.44(s,3H),2.20(s,3H),2.12-2.07( m,2H),1.70-1.63(m,2H),1.56-1.49(m,2H),1.33(s,9H),0.90(t,J=7.2Hz,3H).

[0425] Step 2: Compound 18b (100 mg, 0.16 mmol) was added to DCM (1.5 mL), followed by trifluoroacetic acid (0.5 mL). The reaction was allowed to react at room temperature for 20 minutes. The reaction was terminated and the system was concentrated to dryness under reduced pressure. Methanol (2.0 mL) and LiOH (100 mg) were added and stirred at room temperature for another 20 minutes. The reaction was terminated. The pH was adjusted to 4-5 with acetic acid. After concentration, the product was subjected to low-medium pressure reverse preparative reaction (acidic mobile phase) and lyophilized to 60 mg of compound STI-F7 as a pale yellow solid in an 83.3% yield. LCMS (254 nm) revealed a purity of 100.0%, Rt = 1.789 min; MS calculated: 466.2; MS found: 467.2 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.39(d,J=8.0Hz,1H),8.06(br,1H),7.73(br,3H),7.61(d,J=10.8Hz,1H),7.36(s,1H),6.56(br,1H),5.46(s,2 H), 5.43 (s, 2H), 3.60 (q, J = 6.4Hz, 2H), 2.97 (q, J = 6.4Hz, 2H), 2.44 (s, 3H), 1.91-1.81 (m, 2H), 1.79-1.67 (m, 4H), 0.87 (t, J = 7.2Hz, 3H).

[0426] Example 19: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-11-pentylamino-1,12-dihydro-14H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione (STI-F13)

[0427] Step 1: Compound 9b from Example 9 (91 mg, 0.20 mmol), n-pentylamine (35 mg, 0.40 mmol), Xantphos (23 mg, 0.04 mmol), palladium acetate (5 mg, 0.02 mmol), and cesium carbonate (130 mg, 0.40 mmol) were added to a reaction flask, dissolved in 6 mL of anhydrous toluene, and heated to 100°C for 5 hours under nitrogen. The mixture was concentrated to dryness and purified by silica gel column chromatography (DCM:MeOH = 200:1 to 100:1) to obtain 30 mg of compound 19a as a light red solid in a yield of 29.6%.

[0428] Step 2: Compound 19a (23 mg, 0.05 mmol) was dissolved in a mixture of 2 mL of methanol and 1 mL of dichloromethane. LiOH (23 mg, 0.96 mmol) was added, and the mixture was stirred at room temperature for 80 minutes under nitrogen atmosphere. The pH was adjusted to 3-4 with glacial acetic acid. After concentration, the mixture was subjected to medium-low pressure reverse preparative reaction (acidic mobile phase) and lyophilized to afford 12 mg of compound STI-F13 as a pale yellow solid in a 56.9% yield. LCMS (254 nm) revealed a purity of 98.5%, Rt = 1.97 min; MS calculated: 465.2; MS found: 466.3 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.41(d,J=7.7Hz,1H),7.55(d,J=10.3Hz,1H),7.42(s,1H),6.60(s,1H),5.50-5.37(m,4H),3.72-3.67(m, 2H), 2.42 (s, 3H), 1.87 (dq, J = 14.1, 7.1Hz, 2H), 1.74 (q, J = 7.4Hz, 2H), 1.41 (td, J = 12.8, 7.1Hz, 4H), 0.90 (dt, J = 18.2, 7.2Hz, 6H).

[0429] Example 20: Synthesis of (S)-N-(2-(9-amino-4-ethyl-8-fluoro-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-N-isopropylmethanesulfonamide (STI-G4)

[0430] Step 1: Compound 20a (2.0 g, 10.0 mmol) was added to a mixture of nitric acid (1.2 mL, 28.0 mmol) and sulfuric acid (8.0 mL) and allowed to react at room temperature for 2 hours. TLC (PE:EA = 10:1, product Rf = 0.7) confirmed the completion of the reaction. The mixture was poured into ice water and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting crude brown product was purified by column chromatography (PE:EA = 50:1) to afford 2.2 g of compound 20b as a yellow solid in an 89.1% yield. 1 H NMR (400MHz, DMSO-d6) δ10.15 (s, 1H), 8.32 (d, J = 8.4Hz, 1H), 7.26 (d, J = 6.8Hz, 1H).

[0431] Step 2: Compound 20b (247 mg, 1.0 mmol), iron powder (173 mg, 3.1 mmol) and glacial acetic acid (1.0 mL, 18.0 mmol) were added to a mixed solvent of ethanol (5.0 mL) and water (1.0 mL). The reaction was incubated at 80°C for 1 hour. The reaction was confirmed to be complete by TLC (PE:EA = 10:1, product Rf = 0.5). The system was naturally cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (PE:EA = 50:1) to obtain 190 mg of compound 20c as a yellow solid in a yield of 81.5%. 1 H NMR (400MHz, DMSO-d6) δ9.75 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.44 (s, 2H), 6.68 (d, J = 11.6 Hz, 1H).

[0432] Step 3: Dissolve compound 20c (233 mg, 1.0 mmol) and compound 1c (121 mg, 1.0 mmol) in 5 mL of anhydrous toluene under nitrogen atmosphere. Reflux the mixture for 30 minutes. Add p-toluenesulfonic acid (11 mg, 0.06 mmol) and continue stirring at reflux for 1 hour. After completion of the reaction as determined by LCMS, cool the reaction system to room temperature, concentrate to dryness under reduced pressure, and slurry with acetone to afford 260 mg of compound 20d as a yellow solid in a 58.6% yield. 1 H NMR (400MHz, DMSO-d6) δ8.69(s,1H),8.66(d,J=8.0Hz,1H),8.12(d,J=10.0Hz,1H),7.35( s,1H),6.53(br,1H),5.43(s,2H),5.29(s,2H),1.92-1.81(m,2H),0.88(t,J=6.4Hz,3H).

[0433] Step 4: Compound 20d (222 mg, 0.5 mmol) and ferrous sulfate heptahydrate (52 mg, 0.19 mmol) were added sequentially to 8 mL of water. The temperature was lowered to 0-5°C. Glacial acetic acid (0.52 mL, 9.2 mmol) and sulfuric acid (3.6 mL) were added under nitrogen. Tert-butyl peroxide (0.34 mL, 3.5 mmol) was added dropwise to the mixture. The mixture was allowed to react at room temperature for 3 hours. The mixture was poured into ice water and extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (DCM:MeOH = 100:1) to obtain 130 mg of compound 20e as a yellow-brown solid in a 56.8% yield. 1 H NMR(400MHz,DMSO-d6)δ8.65(d,J=7.6Hz,1H),8.10(d,J=10.0Hz,1H),7.33(s,1H),6.53 (s,1H),5.43(s,2H),5.29(s,2H),2.79(s,3H),1.91-1.81(m,2H),0.88(t,J=7.6Hz,3H).

[0434] Step 5: Compound 20e (92 mg, 0.2 mmol) was added to 2 mL of DMSO. Under nitrogen, hydrochloric acid (0.12 mL, 1.4 mmol) and isopropylamine (0.1 mL, 1.2 mmol) were added to the reaction system and reacted at 140°C for 1 hour. After completion of the reaction, the system was concentrated to dryness under reduced pressure and subjected to medium- and high-pressure reverse transpiration (acidic mobile phase). The reaction mixture was lyophilized to afford 35 mg of compound 20f as a yellow solid in a 33.0% yield.

[0435] Step 6: Compound 20f (30 mg, 0.057 mmol) was added to 2 mL of DCM and the temperature was lowered to 0-5°C. Methanesulfonyl chloride (8 mg, 0.068 mmol) and triethylamine (7 mg, 0.068 mmol) were added to the reaction system under nitrogen and allowed to react at room temperature for 1 hour. After completion of the reaction, as determined by LCMS, the organic layer was washed sequentially with 1 M aqueous hydrochloric acid and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield 30 mg of the compound (20 g) of crude product, which was used directly in the next step.

[0436] Step 7: Dissolve 20 g of the crude compound from the previous step (30 mg, 0.049 mmol) and tert-butyl carbamate (12 mg, 0.01 mmol) in 3 mL of anhydrous toluene. Under nitrogen, add cesium carbonate (33 mg, 0.01 mmol), Xantphos (1 mg, 0.001 mmol), and palladium acetate (0.2 mg, 0.001 mmol). Stir thoroughly, raise the temperature to 100°C, and react for 2.0 hours. After completion of the reaction, as determined by LCMS, concentrate the system to dryness under reduced pressure, extract with dichloromethane and water, concentrate the organic phase, and add 25% trifluoroacetic acid in dichloromethane (2 mL) to the residue. Stir at room temperature for 30 minutes. After completion of the reaction, as determined by LCMS, concentrate the system to dryness under reduced pressure. High-pressure reverse transpiration (acidic mobile phase) was used, and lyophilized to afford 8 mg of compound STI-G4 as a yellow solid in a 30.0% yield.

[0437] Similarly, the following example compounds were synthesized:

[0438] Example P1: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-11-((2-hydroxyethyl)amino)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indole[1,2-b]quinoline-3,14(4H)-dione (STI-F5)

[0439] Step 1: Dissolve compound F5-01b (1.22 g, 20.00 mmol) and triethylamine (4.17 mL, 30.00 mmol) in 20 mL of dichloromethane. Slowly add a solution of tert-butyldimethylsilyl trifluoromethanesulfonate (6.34 g, 24.00 mmol) in 10 mL of dichloromethane dropwise over 3 minutes. Under nitrogen, react at room temperature for 1.5 hours. Extract with 30 mL of water and twice with dichloromethane. Combine the organic phases, dry over anhydrous sodium sulfate, and rotary evaporate the solvent to obtain 3.5 g of compound F5-02b as a colorless oil in 100% yield. 1 H NMR (400MHz, DMSO-d6) δ3.57 (t, J = 5.8 Hz, 2H), 2.67 (t, J = 5.8 Hz, 2H), 0.87 (s, 9H), 0.04 (s, 6H).

[0440] Step 2: (S)-11-chloro-4-ethyl-8-fluoro-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indol[1,2-b]quinolin-4-yl acetate (184 mg, 0.40 mmol), F5-02b (140 mg, 0.80 mmol), Xantphos (23 mg, 0.04 mmol), palladium acetate (9 mg, 0.04 mmol), and cesium carbonate (391 mg, 0.80 mmol) were added to a reaction flask, dissolved in 10 mL of anhydrous toluene, and heated to 100°C for 1.5 hours under nitrogen. The mixture was concentrated to dryness and purified by silica gel column chromatography (DCM:MeOH = 200:1) to obtain 127 mg of compound F5-03b as a light yellow solid in a 52.9% yield. 1 H NMR (400MHz, DMSO-d6) δ8.23(d,J=8.2Hz,1H),7.56(d,J=11.1Hz,1H),7.52(t,J=6.5Hz,1H),6.86(s,1H),5.46(s,2H),5.43(s,2H),3.83(t,J= 5.6Hz,2H),3.70(t,J=5.5Hz,2H),2.43(s,3H),2.20(s,3H),2.16–2.08 (m,2H),0.89(t,J=7.6Hz,3H),0.67(s,9H),-0.11(s,3H),-0.13(s,3H).

[0441] Step 3: F5-03b (127 mg, 0.21 mmol) was dissolved in 1 mL of dichloromethane, and a 4 M HCl solution in 1,4-dioxane was added. The mixture was stirred at room temperature for 40 minutes under nitrogen protection. After the reaction was completed by LCMS, the reaction solution was dried and the crude F5-04b was used directly in the next reaction.

[0442] Step 4: Dissolve the crude F5-04b obtained in the previous step in 12 mL of methanol, add lithium hydroxide (150 mg), and stir at room temperature under nitrogen for 1 hour. Adjust the pH to 6-7 with glacial acetic acid. Perform a high-pressure reverse-phase preparative reaction (mobile phase: acetonitrile / 0.05% formic acid in water) and lyophilize to obtain 16 mg of compound STI-F5 as a pale yellow solid in a 56.9% yield. LCMS (254 nm) purity: 99.7%, Rt = 1.45 min; MS calculated: 439.2; MS found: 440.1 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ8.27(d,J=8.1Hz,1H),7.57(d,J=11.1Hz,1H),7.37(t,J=6.1Hz,1H),7.20(s,1H),6.46(s,1H),5.4 3(s,2H),5.40(s,2H),5.00(s,1H),3.69(d,J=2.9Hz,4H),2.43(s,3H),1.94–1.76(m,J=7.2Hz,2H),0.87(t,J=7.3Hz,3H).

[0443] Example P2: Synthesis of (S,E)-4-ethyl-8-fluoro-4-hydroxy-11-((4-hydroxy-2-en-1-yl)amino)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indole[1,2-b]quinoline-3,14(4H)-dione (STI-F8)

[0444] Step 1: Dissolve compound F8-01 (1.2 g, 13.60 mmol) in 40 mL of tetrahydrofuran, cool to 0°C, and under nitrogen. Add phthalimide (1.00 g, 6.80 mmol) and a solution of diethyl azodicarboxylate (3.57 g, 13.60 mmol) in 60 mL of toluene. React at room temperature for 2 hours. Rotary evaporate the solvent and purify on a silica gel column (PE:EA = 5:1) to obtain 500 mg of compound F8-02 as a white solid. 1 H NMR (400MHz, DMSO-d6) δ7.93–7.80(m,4H),5.74–5.60(m,2H),4.71(t,J=5.5Hz,1H),4.21–4.15(m,2H),3.90(m,2H).

[0445] Step 2: Dissolve F8-02 (759 mg, 3.5 mmol) and triethylamine (1062 mg, 10.5 mmol) in 8 mL of dichloromethane. Slowly add a solution of tert-butyldimethylsilyl chloride (633 mg, 4.2 mmol) in 2 mL of dichloromethane dropwise under nitrogen. Stir at room temperature overnight. Rotary evaporate the solvent and purify on a silica gel column (PE:EA = 10:1) to obtain 536 mg of compound F8-02c as a colorless oil. 1 H NMR (400MHz, DMSO-d6) δ7.93–7.80(m,4H),5.77–5.61(m,2H),4.21–4.15(m,2H),4.10(dd,J=2.9,1.5Hz,2H),0.83(s,9H),0.00(s,6H).

[0446] Step 3: Dissolve F8-02C (536 mg, 1.62 mmol) in 8 mL of anhydrous ethanol, add 80% hydrazine hydrate (112 mg, 1.78 mmol), and reflux for 6 hours. Filter, spin-dry the filtrate, add cold dichloromethane, filter again, and spin-dry the filtrate to obtain 250 mg of compound F8-01b as a yellow oil.

[0447] Step 4: (S)-11-chloro-4-ethyl-8-fluoro-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indol[1,2-b]quinolin-4-yl acetate (184 mg, 0.40 mmol), Xantphos (23 mg, 0.04 mmol), palladium acetate (9 mg, 0.04 mmol), and cesium carbonate (391 mg, 0.80 mmol) were added to a reaction flask. F8-01b (161 mg, 0.80 mmol) was added and dissolved in 10 mL of anhydrous toluene. The mixture was heated to 100°C and reacted for 1 hour under nitrogen. The mixture was concentrated to dryness and purified by silica gel column chromatography (DCM:MeOH = 200:1 to 150:1) to obtain 100 mg of compound F8-02b as a brown solid in a 39.9% yield. MS calculated value: 621.2; MS found value: 622.3 [M+H] + .

[0448] Step 5: F8-02b (100 mg, 0.16 mmol) was suspended in 3 mL of dichloromethane and a mixture of 4 M HCl in 1,4-dioxane (5 mL). The mixture was stirred at room temperature for 1 hour under nitrogen atmosphere, then dried by evaporation. The crude product was used directly in the next reaction. LCMS (254 nm) revealed a purity of 85.0%, Rt = 1.67 min; MS calculated: 507.2; MS found: 508.2 [M+H] + .

[0449] Step 6: The crude product from the previous step was dissolved in 8 mL of methanol, and 38 mg of lithium hydroxide was added. The mixture was reacted under nitrogen atmosphere at room temperature for 1 hour. The pH was adjusted to 5 with glacial acetic acid under ice-cooling. High pressure reverse phase preparative chromatography (mobile phase: acetonitrile / 0.05% aqueous formic acid) was used, and lyophilized to afford 35 mg of compound STI-F8 as a pale yellow solid. LCMS (254 nm) purity: 97.5%, Rt = 1.50 min; MS calculated: 465.2; MS found: 466.2 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ8.34(d,J=8.1Hz,1H),7.77(t,J=6.3Hz,1H),7.61(d,J =11.1Hz,1H),7.25(s,1H),6.51(s,1H),5.94(dt,J=15.8,4.5Hz,1H),5.71(dt, J=15.7,5.1Hz,1H),5.45(s,2H),5.37(s,2H),4.75(s,1H),4.28(t,J=5.8Hz,2H ), 3.97 (d, J = 4.9Hz, 2H), 2.47 (s, 3H), 1.97–1.85 (m, 2H), 0.93 (t, J = 7.3Hz, 3H).

[0450] Example P3: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-11-(4-(hydroxymethyl)piperidin-1-yl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indole[1,2-b]quinoline-3,14(4H)-dione (STI-F14)

[0451] Step 1: Dissolve compound F14-01 (1.00 g, 4.60 mmol) in 20.0 mL of dichloromethane, add pyridine (790 mg, 9.20 mmol) and acetyl chloride (630 mg, 7.40 mmol), and react at room temperature under nitrogen overnight. Add 1 M aqueous hydrochloric acid under ice to adjust the pH to 1-2. Extract with ethyl acetate, wash with saturated sodium bicarbonate and saturated brine, dry over anhydrous sodium sulfate, filter, and spin-dry the filtrate to afford 1143 mg of compound F14-02 as a light yellow oil (96.6% yield). 1 H NMR(400MHz,DMSO-d6)δ3.94(d,J=13.0Hz,2H),3.86(d,J=6.5Hz,2H),2.69(s,2H),2.01 (s,3H),1.81–1.71(m,1H),1.61(dd,J=12.7,3.5Hz,2H),1.39(s,9H),1.12–0.97(m,2H).

[0452] Step 2: Dissolve F14-02 (680 mg, 2.64 mmol) in 5 mL of dichloromethane, add 3 mL of trifluoroacetic acid, protect with nitrogen, stir at room temperature for 1 hour, spin dry the reaction solution, and use the crude F14-03 directly in the next reaction.

[0453] Step 3: (S)-11-chloro-4-ethyl-8-fluoro-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indol[1,2-b]quinolin-4-yl acetate (184 mg, 0.40 mmol), Xantphos (23 mg, 0.04 mmol), palladium acetate (9 mg, 0.04 mmol) and cesium carbonate (391 mg, 0.80 mmol) were added to a reaction flask, dissolved in 10 mL of anhydrous toluene, and a 1 mL tert-butanol solution of F14-03 (217 mg, 0.80 mmol) was added. The mixture was protected by nitrogen and heated to 100 ° C for 1 hour. The system was concentrated to dryness and purified by silica gel column chromatography (DCM:MeOH = 300:1 to 100:1) to obtain 120 mg of compound F14-04 as a pale yellow solid in a 52.0% yield. LCMS (254 nm) purity: 82.5%, Rt = 2.04 min; MS calculated: 577.2; MS found: 578.3 [M+H] + .

[0454] Step 4: Dissolve F14-04 (80 mg, 0.14 mmol) in 5 mL of methanol, add lithium hydroxide (33 mg, 1.40 mmol), and stir at room temperature under nitrogen for 40 minutes. Adjust the pH to 5-6 with glacial acetic acid. Perform high-pressure reverse-phase preparative chromatography (mobile phase: acetonitrile / 0.05% trifluoroacetic acid in water) and lyophilize to obtain 15 mg of compound STI-F14 as a pale yellow solid in a 21.7% yield. LCMS (254 nm) purity: 99.3%, Rt = 1.67 min; MS calculated: 493.2; MS found: 494.3 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ7.91(d,J=8.4Hz,1H),7.74(d,J=10.8Hz,1H),7.29(s,1H),6.58(d,J=63.2Hz,2H),5.46(s,2H),5.42(s,2H),3.69–3.64( m,2H),3.42(d,J=6.2Hz,2H),3.25(t,J=11.5Hz,2H),2.47(s,3H),1.93– 1.78(m,4H),1.74–1.65(m,1H),1.58–1.45(m,2H),0.87(t,J=7.3Hz,3H).

[0455] Example P4: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-11-((1r,4S)-4-(hydroxymethyl)cyclohexyl)amino-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indole[1,2-b]quinoline-3,14(4H)-dione (STI-F15)

[0456] Step 1: Dissolve compound F15-01 (1.50 g, 11.62 mmol) and di-tert-butyl dicarbonate (3.04 g, 13.94 mmol) in 20 mL of tetrahydrofuran under nitrogen atmosphere and react at room temperature overnight. Rotary evaporate the solvent, extract with ethyl acetate and water, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and spin-dry the filtrate to obtain 2.74 g of compound F15-02 as a white solid (100% yield). 1 H NMR (400MHz, DMSO-d6) δ6.66(d,J=8.0Hz,1H),4.36(t,J=5.3Hz,1H),3.18(t,J=5.8Hz,2H),3.15–3. 06(m,1H),1.80–1.67(m,4H),1.37(s,9H),1.29–1.18(m,1H),1.17–1.02(m,2H),0.94–0.80(m,2H).

[0457] Step 2: Dissolve compound F15-02 (1.10 g, 4.80 mmol) in 2.5 mL of pyridine, add 5 mL of acetic anhydride, and heat at 40°C under nitrogen for 4 hours. Add 1 M aqueous hydrochloric acid to adjust the pH to 1-2 under ice-cooling. Extract with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and spin-dry the filtrate to afford 979 mg of compound F15-03 as a white solid, in a 75% yield.

[0458] Step 3: Dissolve F15-03 (740 mg, 2.73 mmol) in 5 mL of dichloromethane, add 3 mL of trifluoroacetic acid, protect with nitrogen, stir at room temperature for 2 hours, spin dry the reaction solution, and use the crude F15-04 directly in the next reaction. 1 H NMR (400MHz, DMSO-d6) δ3.83 (d, J=6.4Hz, 2H), 3.01–2.87 (m, 1H), 2.01 (s, 3H), 1.98–1. 89(m,2H),1.80–1.71(m,2H),1.60–1.47(m,1H),1.35–1.21(m,2H),1.11–0.96(m,2H).

[0459] Step 4: (S)-11-chloro-4-ethyl-8-fluoro-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indol[1,2-b]quinolin-4-yl acetate (184 mg, 0.40 mmol), Xantphos (23 mg, 0.04 mmol), palladium acetate (9 mg, 0.04 mmol) and cesium carbonate (391 mg, 0.80 mmol) were added to a reaction flask, dissolved in 10 mL of anhydrous toluene, and a 1 mL tert-butanol solution of F15-04 (114 mg, 0.80 mmol) was added. The mixture was protected by nitrogen and heated to 100 ° C for 1.5 hours. The system was concentrated to dryness and purified by silica gel column chromatography (DCM:MeOH = 300:1 to 100:1) to obtain 135 mg of compound F15-05 as a pale yellow solid in a 57.1% yield. LCMS (254 nm) purity: 79.8%, Rt = 1.97 min; MS calculated: 591.2; MS found: 592.3 [M+H] + .

[0460] Step 5: Dissolve F15-05 (135 mg, 0.23 mmol) in 10 mL of methanol, add lithium hydroxide (55 mg, 2.30 mmol), and stir at room temperature under nitrogen for 1 hour. Adjust the pH to 4-5 with glacial acetic acid. Perform high-pressure reverse-phase preparative chromatography (mobile phase: acetonitrile / 0.05% trifluoroacetic acid in water) and lyophilize to obtain 10 mg of compound STI-F15 as a pale yellow solid in 8.6% yield. LCMS (254 nm) purity: 95.8%, Rt = 1.62 min; MS calculated: 507.2; MS found: 508.3 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.50(d,J=7.9Hz,1H),7.59(d,J=10.5Hz,1H),7.41(s,1H),6.56(s,1H),5.43(s,2H),5.40(s,2H),3.77(s,1H),3.31(d, J=6.0Hz,2H),2.45(s,3H),2.11–1.97(m,2H),1.94–1.79(m,4H),1.59( q,J=11.8Hz,2H),1.42(s,1H),1.22–1.10(m,2H),0.87(t,J=7.3Hz,3H).

[0461] Example P5: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-11-((1r,4S)-4-(hydroxymethyl)cyclohexyl)amino-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7]indole[1,2-b]quinoline-3,14(4H)-dione (STI-F17)

[0462] Step 1: Dissolve compound F17-01 (1.00 g, 4.92 mmol) in 5 mL of pyridine, add 5 mL of acetic anhydride, and heat at 40°C under nitrogen for 4 hours. Add 4 M aqueous hydrochloric acid under ice to adjust the pH to 1-2. Extract with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and spin-dry the filtrate to obtain 1.12 g of compound F17-02 as a clear oil (92.9% yield). 1 H NMR (400MHz, CDCl3) δ4.56 (s, 1H), 4.06 (t, J = 6.6Hz, 2H), 3.12 (q, J = 6.7Hz, 2H), 2.0 5(s,3H),1.64(h,J=6.2Hz,2H),1.55–1.49(m,2H),1.45(s,9H),1.42–1.34(m,2H).

[0463] Step 2: Dissolve F17-02 (1.12 g, 4.57 mmol) in 9 mL of dichloromethane, add 3 mL of trifluoroacetic acid, protect with nitrogen, stir at room temperature for 3 hours, spin dry the reaction solution, and use the crude F17-03 directly in the next reaction.

[0464] Step 3: (S)-11-chloro-4-ethyl-8-fluoro-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indol[1,2-b]quinolin-4-yl acetate (184 mg, 0.40 mmol), Xantphos (46 mg, 0.08 mmol), palladium acetate (9 mg, 0.04 mmol) and cesium carbonate (521 mg, 1.60 mmol) were added to a reaction flask, dissolved in 10 mL of anhydrous toluene, and a solution of F17-03 (114 mg, 0.80 mmol) in 2 mL of 1,4-dioxane was added. The mixture was protected by nitrogen and heated to 100 ° C for 100 minutes. The system was concentrated to dryness and purified by silica gel column chromatography (DCM:MeOH=300:1-200:1) to obtain 300 mg of crude compound F17-04 as a brown solid.

[0465] Step 4: Dissolve F17-04 (140 mg, 0.25 mmol) in 11 mL of methanol, add lithium hydroxide (70 mg, 2.92 mmol), and stir at room temperature under nitrogen for 1 hour. Adjust the pH to 6 with glacial acetic acid. High-pressure reverse-phase preparative chromatography (mobile phase: methanol / 0.05% aqueous formic acid) is performed, and lyophilization is performed to obtain 15 mg of compound STI-F17 as a pale yellow solid in a 12.5% ​​yield. LCMS (254 nm) purity: 99.7%, Rt = 1.55 min; MS calculated: 481.2; MS found: 482.1 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.26(d,J=8.1Hz,1H),7.54(d,J=11.1Hz,1H),7.31(t,J=5.9Hz,1H),7.20(s,1H),6.47(s,1H),5.40(s,2H),5.36(s,2H),4 .43(t,J=5.5Hz,1H),3.60(q,J=6.7Hz,4H),2.42(s,3H),1.93–1.76(m,J =7.2Hz,2H),1.94–1.79(m,4H),1.56–1.39(m,4H),0.87(t,J=7.3Hz,3H).

[0466] Example P6: Synthesis of (S)-7-ethyl-7-hydroxy-14-(4-hydroxybutyl)amino)-10,13-dihydro-11H-[1,3]dioxo[4,5-g]pyrano[3',4':6,7]indole[1,2-b]quinoline-8,11(7H)-dione (STI-F18)

[0467] Step 1: Compound STI-E-05 (1.0 g, 2.6 mmol) was added to 6 mL of pyridine, followed by 6 mL of acetic anhydride. The mixture was heated and stirred at 40°C under nitrogen atmosphere and monitored by LCMS. After completion of the reaction, the mixture was concentrated under reduced pressure and slurried in acetone to afford 360 mg of compound F9-01 as a yellow solid, with a yield of 33.0%.

[0468] Step 2: Compound F9-01 (360 mg, 0.83 mmol) was dissolved in 12 mL of acetic acid, and 30% aqueous hydrogen peroxide (4 mL) was added. Under nitrogen, the mixture was heated and stirred at 70°C for 3.0 hours. The reaction was complete as monitored by LCMS. The mixture was then concentrated under reduced pressure, and the residue was added to ice-water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. The residue was dissolved in 16 mL of dimethylformamide (DMF), cooled to 0°C in an ice bath, and then 1.1 mL of oxalyl chloride was slowly added dropwise. The mixture was returned to room temperature and stirred under reduced pressure. The reaction was monitored by LCMS. After completion, the reaction was quenched with water, filtered, and the filter cake was washed with deionized water and dried under reduced pressure to obtain 300 mg of compound F9-02 as a yellow solid in a 77.3% yield. LCMS (254 nm) purity was 92.13%, Rt = 1.83 min; MS calculated: 468.1; MS found: 469.1 [M+H] + .

[0469] Step 3: Compound F9-02 (140 mg, 0.30 mmol), F18-S2 (122 mg, 0.60 mmol), Xantphos (35 mg, 0.06 mmol), palladium acetate (7 mg, 0.03 mmol), and cesium carbonate (392 mg, 0.60 mmol) were added to a reaction flask. The mixture was heated to 100°C with 6 mL of anhydrous toluene and 6 mL of 1,4-dioxane under nitrogen for 2 hours. The mixture was concentrated to dryness and purified by silica gel column chromatography (DCM:MeOH = 100:1) to afford 50 mg of compound F18-01 as a red solid in a 26.2% yield. LCMS (254 nm) revealed a purity of 78.92%, Rt = 2.22 min; MS calcd: 635.3; MS found: 636.3 [M+H] + .

[0470] Step 4: Compound F18-01 (50 mg, 0.08 mmol) was dissolved in 1 mL of dichloromethane and a 4 M solution of dioxane hydrochloride was added. The mixture was allowed to react at room temperature for 1 hour. After completion of the reaction, the mixture was concentrated to dryness under reduced pressure. 3 mL of methanol and lithium hydroxide (50 mg, 2.08 mmol) were added to the residue and stirred at room temperature for 20 minutes. The pH was adjusted to 3-4 with glacial acetic acid. The mixture was concentrated and subjected to high pressure reverse transpiration (mobile phase: acetonitrile / 0.05% trifluoroacetic acid in water). The mixture was lyophilized to afford 5 mg of compound STI-F18 as a pale yellow solid in a 13.3% yield. LCMS (254 nm) revealed a purity of 97.86%, Rt = 1.38 min; MS calculated: 479.2; MS found: 480.2 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ7.94(s,1H),7.67(brs,1H),7.47(s,1H),7.29(s,1H),6.63(d,J=13.0Hz,2H),6.31(d,1H),5.46(s,2H),5.43 (s,2H),3.70(q,J=7.0Hz,2H),3.48(d,J=6.7Hz,2H),1.92–1.82(m,2H),1.81–1.71(m,2H),1.61–1.54(m,2H),0.88(t,J=7.3Hz,3H).

[0471] Intermediate compound F18-S2 was prepared as follows:

[0472] Step 1: Dissolve 4-(N-tert-Butyloxycarbonylamino)-1-butanol (3785 mg, 20.0 mmol) in 20 mL of dichloromethane, add triethylamine (4.17 mL, 30.0 mmol), and slowly add dropwise a solution of tert-butyldimethylsilyl chloride (3617 mg, 24.00 mmol) in 10 mL of dichloromethane under nitrogen protection. React at room temperature overnight, extract with saturated brine, extract twice with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, and rotary evaporate the solvent to obtain 4.49 g of compound F18-S1 as a colorless oil with a yield of 74.1%. 1 H NMR (400MHz, DMSO-d6) δ6.74(t,J=5.7Hz,1H),3.54(t,J=5.6Hz,2H),2.88(q,J=6.2Hz,2H),1.40–1.38(m,4H),1.35(s,9H),0.87(s,9H),0.04(s,6H).

[0473] Step 2: Dissolve compound F18-S1 (712 mg, 2.35 mmol) in 5 mL of dichloromethane under nitrogen. Cool to 0-5°C, then add trimethylsilyl iodide (604 mg, 2.82 mmol). Continue to react at this temperature for 3 hours. After completion of the reaction, as determined by TLC (DCM:MeOH = 10:1, product Rf = 0.2), methanol (0.1 mL) was added to the reaction system, which was then concentrated to dryness under reduced pressure. Purification by column chromatography (DCM:MeOH = 50:1) afforded 400 mg of compound F18-S2 as a pale yellow solid in an 83.8% yield. 1 H NMR (400MHz, DMSO-d6) δ7.57(s,2H),3.55(t,J=5.9Hz,2H),2.76(t,J=7.5Hz,2H),1.62–1.39(m,4H),0.87(s,9H),0.04(s,6H).

[0474] Example P7: Synthesis of (S)-7-ethyl-7-hydroxy-14-(4-hydroxybutyl)amino)-10,13-dihydro-11H-[1,3]dioxo[4,5-g]pyrano[3',4':6,7]indole[1,2-b]quinoline-8,11(7H)-dione (STI-G5)

[0475] Step 1: Compound G5-01 (1.4 g, 4.32 mmol) was dissolved in 40 mL of ethanol / water (3:1). Iron powder (965 mg, 17.28 mmol) and ammonium chloride (231 mg, 4.32 mmol) were added. After replacing the nitrogen atmosphere, the mixture was reacted at 80°C for 1 hour. TLC confirmed complete conversion of the starting material. The reaction mixture was filtered while hot and then dried by spin drying. The crude product was purified by column chromatography to obtain 1.0 g of compound G5-02 as a brown oil in a 71.4% yield. LCMS (254 nm), Rt = 2.31 min; MS calcd: 324.2; MS found: 323.2 [MH] - .

[0476] Step 2: Compound G5-02 (1.0 g, 3.09 mmol) and 4-dimethylaminopyridine (38 mg, 0.309 mmol) were dissolved in 30 mL of N,N-dimethylformamide. After nitrogen was replaced, N,N-diisopropylethylamine (1.2 g, 9.25 mmol) and acetoxyacetyl chloride (842 mg, 6.18 mmol) were added and reacted at room temperature for 2 hours. TLC confirmed complete conversion of the starting material. The reaction was quenched by adding ethyl acetate and water, followed by extraction three times. The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and spin-dried to dryness. The crude product was redissolved in 15 mL of methanol, and 1.0 g of lithium hydroxide was added and reacted at room temperature for 1 hour. TLC confirmed complete conversion of the starting material. The reaction was quenched by adding ethyl acetate and water, followed by extraction three times. The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and spin-dried to obtain 750 mg of compound G5-03 as a dark yellow oil in a 63.4% yield. The crude product of G5-03 was used directly in the next step. LCMS (254 nm), Rt = 2.21 min; MS calculated value: 382.2; MS found value: 381.2 [MH] - .

[0477] Step 3: Dissolve G5-03 (750 mg, 1.96 mmol) in 10 mL of 50% trifluoroacetic acid in dichloromethane and stir at room temperature for 1 hour. After completion of the reaction by TLC, add dichloromethane and water to quench the reaction mixture, extract three times, and combine the organic layers, wash with saturated brine, dry over anhydrous sodium sulfate, and spin dry. The crude product was purified by column chromatography to obtain 450 mg of compound G5-04 as a black oil in an 81.4% yield. LCMS (254 nm), Rt = 1.70 min; MS calculated: 282.1; MS found: 283.3 [M+H] + .

[0478] Step 4: Dissolve G5-04 (450 mg, 1.60 mmol) in 15 mL of toluene, add (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indol-3,6,10(4H)-one (461 mg, 1.75 mmol), and stir at 130°C for 30 minutes under nitrogen. Then, add p-toluenesulfonic acid (69 mg, 0.40 mmol) and continue the reaction at 130°C for 2 hours. Solid precipitated during the reaction. After completion of the reaction, the reaction solution was evaporated to dryness. The crude product was slurried with acetone to obtain 520 mg of compound STI-G5 as a gray solid, with a yield of 63.8%. LCMS (254 nm), Rt = 1.73 min; MS calculated: 509.2; MS found: 510.2 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ9.73–9.64(m,1H),8.95(d,J=8.5Hz,1H),8.01(d,J=12.0Hz,1H),7.29(s,1H),6.51(s,1H),5.98(s,1H),5.43(s, 2H), 5.27 (s, 2H), 4.15 (s, 2H), 3.11 (d, J = 8.1Hz, 2H), 1.95–1.79 (m, 2H), 1.73 (t, J = 7.8Hz, 2H), 1.48–1.34 (m, 4H), 0.88 (d, J = 6.7Hz, 6H).

[0479] Synthesis of drug-linker conjugates

[0480] Example 23: Synthesis of Drug-Linker Conjugate STI-A3

[0481] Step 1: Compound STI-A (60 mg, 0.13 mmol) from Example 3, Boc-Glycine (46 mg, 0.26 mmol), and HATU (99 mg, 0.26 mmol) were added to a reaction flask. The atmosphere was purged with nitrogen three times, and the mixture was dissolved in 1.5 mL of DMF. Triethylamine (72 μL, 0.13 mmol) was added and stirred at room temperature for 3.5 hours. The reaction was stopped when approximately 30% conversion was observed by LCMS. Reverse chromatography with medium- and high-pressure preparative chromatography (alkaline mobile phase) and lyophilization afforded 9 mg of compound 23a as a yellow solid. 45 mg of starting material was recovered, for a yield of 53.6%. LCMS (254 nm) revealed a purity of 98.32%, Rt = 1.98 min; MS calculated: 636.3; MS found: 635.4 [MH] - . 1 H NMR (400MHz, DMSO-d6) δ8.23(d,J=8.0Hz,1H),7.83(d,J=10.6Hz,1H),7.36(s,1H),5.54–4.92(m,6H),3.64(d,2H), 3.55(t,J=13.2Hz,2H),2.51(s,3H),1.87(qt,J=13.9,6.8Hz,2H),1.21(s,9H),1.06(s,9H),0.89(t,J=7.3Hz,3H).

[0482] Step 2: Compound 23a (6 mg, 0.01 mmol) was dissolved in 1 mL of DCM, 0.3 mL of TFA was added, and the mixture was stirred at room temperature under nitrogen for 0.5 h. The reaction was stopped after LCMS monitoring, and the mixture was concentrated to dryness under reduced pressure. Triethylamine (14 μL, 0.10 mmol), compound 23b (4 mg, 0.01 mmol), and HATU (8 mg, 0.02 mmol) were added to the reaction flask, the atmosphere was purged with nitrogen three times, and 1.0 mL of DMF was added to dissolve the mixture. The mixture was stirred at room temperature for 1 h. The reaction was confirmed to be complete by LCMS. Reverse chromatography with medium- and high-pressure preparative chromatography (alkaline mobile phase) was performed, and the mixture was lyophilized to obtain compound 23c as a light yellow solid (3 mg, 30.0% yield). LCMS (254 nm) purity was 94.25%, Rt = 1.92 min; MS calculated value: 897.4; MS found value: 898.5 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ8.29(d,J=8.2Hz,1H),7.94(t,J=5.3Hz,1H),7.91–7.86(m,1H),7.84(d,J=8.5Hz,1H),7.65 (t,J=5.8Hz,1H),7.35(s,1H),7.14(q,J=8.1Hz,5H),6.72(s,1H),6.34(s,1H),5.51–5.02(m,6H),4.47(td,J=9.0,4 .6Hz,1H),4.08(s,1H),3.85(s,1H),3.72–3.52(m,4H),3.51(d,J=6.0Hz,2H),2.95(dd,J=14.0,4.6Hz,1H),2.69(dd ,J=13.5,9.4Hz,1H),2.54(s,3H),1.88(p,J=6.9Hz,2H),1.36(s,9H),1.05(d,J=10.3Hz,9H),0.89(t,J=7.4Hz,3H).

[0483] Step 3: Compound 23c (16 mg, 0.018 mmol) was dissolved in 1 mL of DCM, and 0.3 mL of TFA was added. The mixture was stirred at room temperature under nitrogen for 0.5 h. LCMS confirmed the reaction was complete, and the reaction was stopped and concentrated to dryness under reduced pressure. Triethylamine (13 μL, 0.09 mmol) and compound 23d (11 mg, 0.036 mmol) were added, and the atmosphere was purged with nitrogen three times. 1.0 mL of DMF was added to dissolve the mixture, and the mixture was stirred at room temperature for 1.5 h. LCMS confirmed the reaction was complete. Reverse chromatography (basic mobile phase) was performed using a medium-pressure reverse transpiration chromatography system, and the resulting product, drug-linker conjugate STI-A3, was lyophilized to give 11 mg of a white solid in a 62.3% yield. LCMS (254 nm) revealed a purity of 96.87%, Rt = 1.81 min; MS calculated: 990.4; MS found: 991.1 [M+H] + . 1H NMR(400MHz,DMSO-d6)δ8.28(d,1H),7.87–7.81(m,3H),7.35(s,1H),7.22(s,2H),7.21–7.13( m,6H),6.94(s,1H),6.34–6.29(m,1H),5.42–5.30(m,6H),4.47–4.45(m,1H),3.69–3.64(m,4H ),3.57(d,J=6.1Hz,2H),3.49(d,J=6.1Hz,2H),3.37(t,J=7.1Hz,2H),2.94(s,1H),2.54(s,3H ),2.21–2.16(m,2H),1.90–1.85(m,2H),1.50–1.46(m,6H),1.05(s,9H),0.92(t,J=7.8Hz,3H).

[0484] Intermediate compound 23b was prepared as follows:

[0485] Compound 23b-1 (1.0 g, 4.31 mmol), 23b-2 (930 mg, 4.31 mmol), and HATU (2.458 g, 6.47 mmol) were added to a reaction flask. The atmosphere was purged with nitrogen three times, and the mixture was dissolved in 20 mL of DCM. Triethylamine (2.4 mL, 17.24 mmol) was added and stirred at room temperature for 7.0 hours. The reaction was stopped after LCMS monitoring. The mixture was concentrated under reduced pressure, extracted with EA and water, and slurried with PE and DCM. Filtered, the solid was dissolved in methanol, cooled in an ice bath, and LiOH (310 mg, 12.93 mmol) was added. The mixture was stirred at 0°C for 30 minutes, then brought to room temperature and stirred for 3 hours. The reaction was complete after LCMS monitoring. The mixture was then dried by spun dry, the pH adjusted to 2-3 with 1 M hydrochloric acid, extracted with EA and water, dried over anhydrous sodium sulfate, and spun dry to afford 694 mg of compound 23b as a white solid in a 53.6% yield. LCMS (214 nm) purity 97.25%, Rt = 1.25 min; MS calculated value: 379.2; MS found value: 378.2 [MH] - . 1H NMR (400MHz, DMSO-d6) δ12.70(s,1H),8.15(d,J=8.1Hz,1H),7.93(t,J=5.7H z,1H),7.28(t,J=7.4Hz,2H),7.21(d,J=7.5Hz,3H),6.99(t,J=6.0Hz,1H),4 .42(td,J=8.5,5.0Hz,1H),3.69(qd,J=16.8,5.6Hz,2H),3.55(d,J=6.0Hz,2 H), 3.05 (dd, J=13.8, 5.1Hz, 1H), 2.87 (dd, J=13.8, 9.0Hz, 1H), 1.38 (s, 9H).

[0486] Intermediate compound 23d was prepared as follows:

[0487] Compound 23d-1 (500 mg, 2.37 mmol) and compound 23d-2 (273 mg, 2.37 mmol) were dissolved in acetonitrile under an ice bath. DCC (489 mg, 2.37 mmol) was added and stirred at 0°C under nitrogen for 2 hours before returning to room temperature and reacting overnight. TLC (PE:EA = 1:1, product Rf = 0.5) monitored the reaction completion and stopped the reaction. The filtrate was filtered, and the filtrate was subjected to silica gel column chromatography (PE:EA = 4:1 to 1:1). The filtrate was concentrated under reduced pressure to dryness to afford compound 23d as a white solid (548 mg, 75.1% yield). 1 H NMR (400MHz, DMSO-d6) δ7.00 (s, 2H), 3.39 (t, J = 7.0Hz, 2H), 2.81 (s, 4H), 2.65 (t, J=7.3Hz,2H),1.62(p,J=7.4Hz,2H),1.51(h,J=7.2Hz,2H),1.31(q,J=8.0Hz,2H).

[0488] Example 24: Synthesis of Drug-Linker Conjugate STI-F3

[0489] Step 1: Compound 9b (1046 mg, 2.3 mmol) from Example 9 was dissolved in a mixed solvent of 60 mL of dichloromethane and 60 mL of methanol. Lithium hydroxide (166 mg, 6.9 mmol) was added and the mixture was reacted at room temperature for 1 hour. The mixture was concentrated to dryness under reduced pressure, the pH was adjusted to 3-4 with 1 M hydrochloric acid, and the mixture was extracted four times with dichloromethane. The organic phase was concentrated to obtain compound 24a as a brownish-red solid (approximately 1.0 g). LCMS (254 nm) purity was 93.42%, Rt = 1.88 min; MS calculated value: 414.1; MS found value: 415.0 [M+H] + .1 H NMR (400MHz, DMSO-d6) δ8.22(d,J=8.0Hz,1H),7.98(d,J=10.6Hz,1H),7.31(s,1H),6.56(s, 1H),5.44(s,2H),5.25(s,2H),2.54(s,3H),1.87(hept,J=7.0Hz,2H),0.88(t,J=7.3Hz,3H).

[0490] Step 2: Compound 24a (200 mg, 0.48 mmol) and Boc-glycinamide (126 mg, 0.72 mmol) were dissolved in 6 mL of anhydrous toluene. Potassium tert-butoxide (108 mg, 0.96 mmol), Xantphos (28 mg, 0.048 mmol), and palladium acetate (11 mg, 0.048 mmol) were added under nitrogen. The mixture was stirred and heated to 100°C for 3.5 hours. After completion of the reaction, as determined by TLC (DCM:MeOH = 15:1, product Rf = 0.2), the mixture was concentrated to dryness under reduced pressure and extracted with 1 M hydrochloric acid and dichloromethane. The organic phase was concentrated and purified on a silica gel column to afford 39 mg of compound 24b as a red solid. 40 mg of starting material 24a was recovered for a yield of 67.0%. 1 H NMR (400MHz, DMSO-d6) δ10.80(s,1H),8.17(d,J=8.1Hz,1H),7.85(d,J=10.7Hz,1H),7.30(s,2H),6.52(s,1H),5. 42(s,2H),5.03(s,2H),4.04(d,J=6.0Hz,2H),2.50(s,3H),1.91–1.81(m,2H),1.44(s,9H),0.88(t,J=7.3Hz,3H).

[0491] Step 3: Compound 24b (39 mg, 0.07 mmol) was dissolved in 1 mL of dichloromethane. 0.3 mL of trifluoroacetic acid was added under nitrogen and stirred at room temperature for 30 minutes. After completion of the reaction, the system was concentrated to dryness under reduced pressure. The crude product was dissolved in 1 mL of anhydrous DMF and nitrogen was added. Triethylamine (1 mL, 0.71 mmol), 23b (27 mg, 0.07 mmol), and HATU (41 mg, 0.11 mmol) were added and reacted at room temperature for 1.5 hours. LCMS confirmed the reaction was complete. The DMF was then dried and extracted three times with ethyl acetate and water. The product was washed with saturated brine, dried over anhydrous sodium sulfate, and dried to afford 50 mg of compound 24c as a light pink product in an 87.2% yield. LCMS (254 nm) revealed a purity of 85.45%, Rt = 1.71 min; MS calculated: 813.3; MS found: 814.1 [M+H]+ . 1 H NMR (400MHz, DMSO-d6) δ10.80(s,1H),8.56(t,J=5.8Hz,1H),8.53(dd,J=8.4,1.4Hz,1H),8.24(s,1H ),8.21(d,J=8.6Hz,1H),7.91(d,J=10.7Hz,2H),7.32(s,1H),7.29–7.23(m,5H),6.52(s,1H),5.42(s ,2H),5.10(s,2H),4.66–4.58(m,1H),4.22(d,J=6.4Hz,2H),3.78(dd,J=16.6,5.7Hz,1H),3.61(dd, J=17.4,4.7Hz,1H),3.53(d,J=5.7Hz,2H),2.52(s,3H),1.89–1.82(m,2H),1.36(s,9H),0.87(s,3H).

[0492] Step 4: Compound 24c (45 mg, 0.055 mmol) was dissolved in 1.0 mL of DCM, and 0.4 mL of TFA was added. The mixture was stirred at room temperature under nitrogen for 0.5 h. LCMS confirmed the reaction was complete, and the reaction was stopped and concentrated to dryness under reduced pressure. Triethylamine (76 μL, 0.550 mmol) and compound 23d (34 mg, 0.110 mmol) were added, and the atmosphere was purged with nitrogen three times. 1.0 mL of DMF was added to dissolve the mixture, and the mixture was stirred at room temperature for 2.5 h. LCMS confirmed the reaction was complete. Reverse chromatography (basic mobile phase) was performed using a medium-pressure preparative chromatography system to obtain 11 mg of the drug-linker conjugate STI-F3 as a yellow solid. LCMS (254 nm) indicated a purity of 100%, Rt = 1.64 min; MS calculated: 906.3; MS found: 907.0 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ10.79(s,1H),8.53(t,J=5.7Hz,1H),8.20(dd,J=8.5,2.7Hz,2H),8.06(t,J=5.8Hz,1H) ,8.01(t,J=5.4Hz,1H),7.90(d,J=10.8Hz,1H),7.32(s,1H),7.31–7.21(m,5H),6.97(s,2H),6.52(s,1H),5.42 (s,2H),5.09(s,2H),4.60(td,J=8.9,4.3Hz,1H),4.24(d,J=7.9Hz,2H),3.77(dd,J=16.7,5.8Hz,2H),3.71–3. 55(m,6H),2.51(s,3H),2.09(t,J=7.5Hz,2H),1.94–1.78(m,2H),1.46(t,J=8.0Hz,6H),0.88(t,J=7.4Hz,3H).

[0493] Example 25: Synthesis of Drug-Linker Conjugate STI-F4

[0494] Step 1: Compound 25a (4.3 g, 12.2 mmol) and lead tetraacetate (6.8 g, 14.7 mmol) were added to a reaction flask. Under nitrogen, tetrahydrofuran (120.0 mL) and toluene (40.0 mL) were added to dissolve the substrate. Pyridine (1.16 mL, 14.7 mmol) was then added and the temperature was raised to 85°C for 3 hours to terminate the reaction. The mixture was filtered through a pad of Celite and extracted with EA and water. The organic phase was dried over anhydrous Na2SO4, concentrated to dryness under reduced pressure, and purified by column chromatography (DCM:MeOH = 50:1) to afford 4.4 g of compound 25b as a white solid in a 98.0% yield. LCMS (254 nm) revealed a purity of 97.2%, Rt = 1.789 min; MS calcd: 368.1; MS found: 386.1 [M+NH4]. + .

[0495] Step 2: Compound STI-F2 from Example 9 (60 mg, 0.13 mmol), 25b (150 mg, 0.39 mmol), and pyridinium p-toluenesulfonate (2 mg, 0.01 mmol) were added to a reaction flask. DCE (6.0 mL) was added, and the mixture was heated to 80°C under nitrogen atmosphere and allowed to react overnight. The mixture was concentrated to dryness under reduced pressure and purified by Pre-TLC (DCM:MeOH = 15:1) to afford 40 mg of compound 25c as a yellow solid in a 34.1% yield. LCMS (254 nm) revealed a purity of 93.2%, Rt = 1.811 min; MS calcd: 761.2; MS found: 762.3 [M+H] + .

[0496] Step 3: Compound 25c (40 mg, 0.053 mmol) was dissolved in DMF (0.8 mL), and piperidine (0.2 mL) was added. The mixture was reacted at room temperature for 20 min. The mixture was concentrated to dryness, diluted with water, and lyophilized. 23b (21 mg, 0.056 mmol), HATU (30 mg, 0.08 mmol), and DMF (1.0 mL) were added thereto under nitrogen protection. Triethylamine (16 mg, 0.159 mmol) was added and the mixture was reacted at room temperature for 1 h. The reaction was stopped and the mixture was concentrated to dryness. EA and saturated brine were added for extraction. The aqueous phase was washed with EA (10.0 mL × 5). The organic phases were combined, dried over anhydrous Na2SO4, and concentrated to dryness under reduced pressure to afford 30 mg of compound 25d as a light yellow solid in a yield of 62.9%. LCMS (254 nm) purity 97.2%, Rt = 1.662 min; MS calculated value: 900.3; MS found value: 901.4 [M+H] + .

[0497] Step 4: Compound 25d (30 mg, 0.033 mmol) was dissolved in 1.0 mL of DCM under nitrogen atmosphere. 0.3 mL of trifluoroacetic acid was added and the mixture was allowed to react at room temperature for 20 minutes. The reaction was stopped by LCMS monitoring until the reaction was complete. The system was concentrated to dryness under reduced pressure, and 1.0 mL of DMF was added. Under nitrogen atmosphere, triethylamine (22 mg, 0.22 mmol) and 23d (10 mg, 0.033 mmol) were added sequentially. The mixture was allowed to react at room temperature for 20 minutes. The reaction was stopped and the system was concentrated to dryness under reduced pressure. The product was then prepared by medium- and high-pressure reverse phase chromatography (acidic mobile phase) and lyophilized to obtain 6 mg of the drug-linker conjugate STI-F4 as a yellow oil in a 27.9% yield. LCMS (254 nm) revealed a purity of 98.65%, Rt = 1.591 min; MS calculated: 993.4; MS found: 994.4 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ10.74(s,1H),8.75(t,J=6.4Hz,1H),8.37(t,J=6.0Hz,1H),8.15(d,J=8.0Hz,1H),8.07(t,J=6.0Hz,1H), 8.02(t,J=5.6Hz,1H),7.90(d,J=10.8Hz,1H),7.32(s,1H),7.27–7.14(m,6H),6.98(s,2H),6.52(s,1H),5.42(s,2H),5.15(s,2H) ,4.84–4.77(m,2H),4.52–4.47(m,1H),4.38(s,2H),3.80(t,J=6.0Hz,2H),3.73–3.57(m,6H),3.04(dd,J=14.0,4.4Hz,1H),2.82 –2.79(m,1H),2.50(s,3H),2.09(t,J=7.6Hz,2H),1.92–1.80(m,2H),1.48–1.41(m,4H),1.20–1.32(m,2H),0.88(t,J=7.2Hz,3H).

[0498] Example 26: Synthesis of Drug-Linker Conjugate STI-G

[0499] Step 1: Compound 11f (200 mg, 0.89 mmol) and Fmoc-Gly-OH (530 mg, 1.78 mmol) were dissolved in 5 mL of tetrahydrofuran. Et3N (360 mg, 3.56 mmol), HOBt (240 mg, 1.78 mmol), and EDCI (340 mg, 1.78 mmol) were added to the reaction mixture. The reaction mixture was stirred at room temperature. After completion of the reaction, the mixture was diluted with 10 mL of ethyl acetate and extracted with water. The aqueous phase was then extracted twice with 10 mL of ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting brown crude product was purified by silica gel column chromatography (PE:EA = 2:1) and dried to dryness to afford 300 mg of compound 26a as a pink solid in a 67% yield. LCMS (254 nm) Rt = 2.112 min; MS Calcd: 503.2; MS Found: 504.2 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ9.45(s,1H),8.07(d,J=8.8Hz,1H),7.90(d,J=7.6Hz,2H),7.73(d,J=7.5Hz,1H),7.61(s,1H),7.42(t,J=7.5Hz,2H),7.34(d,J= 7.1Hz,3H),6.57(d,J=12.9Hz,1H),4.34–4.18(m,3H),3.82(d,J=6.1Hz,2H),2. 81(t,J=7.4Hz,2H), 1.58(t,J=7.3Hz,2H), 1.35–1.19(m,6H), 0.90–0.82(m,3H).

[0500] Step 2: Compound 26a (200 mg, 0.40 mmol) and compound 1c (105 mg, 0.40 mmol) were dissolved in 4 mL of anhydrous toluene under nitrogen atmosphere. The mixture was refluxed for 30 minutes with a water separator. p-Toluenesulfonic acid (18 mg, 0.1 mmol) was added, and the mixture was stirred at reflux for 3.5 hours. TLC (DCM:MeOH = 10:1, product Rf = 0.5) and LCMS analysis revealed that the reaction mixture was cooled to room temperature, dried, slurried with acetone, and filtered to yield 200 mg of compound 26b as a yellow solid in a 70% yield. LCMS (254 nm) revealed a purity of 96.96%, Rt = 2.084 min; MS calculated: 730.3; MS found: 731.3 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ10.21(s,1H),9.00(d,J=8.4Hz,1H),8.01(d,J=12.0Hz,1H),7.90(d,J=7.6Hz ,2H),7.75(d,J=7.8Hz,2H),7.49–7.41(m,2H),7.37–7.28(m,3H),7.11(d,J=7.9Hz,1H),5.43(s,2H), 5.28(s,2H),4.37–4.24(m,3H),4.02(d,J=6.0Hz,2H),3.11(t,J=8.0Hz,2H),2.28(s,1H),1.86(p,J= 7.1Hz, 2H), 1.72 (t, J = 7.7Hz, 2H), 1.42 (d, J = 7.4Hz, 2H), 1.34 (d, J = 7.1Hz, 2H), 0.86 (d, J = 7.6Hz, 6H).

[0501] Step 3: Compound 26b (50 mg, 0.068 mmol) was dissolved in 2 mL of 20% piperidine / DMF solution and stirred at room temperature for 30 minutes. After completion, the reaction solution was purified directly by silica gel column chromatography (DCM:MeOH = 10:1, product Rf = 0.2) and LCMS. The mixture was then spin-dried to give 20 mg of compound 26c as a white solid in a 57.8% yield. LCMS (254 nm) Rt = 1.670 min; MS calcd: 508.2; MS found: 509.3 [M+H] + .

[0502] Step 4: Compound 26c (20 mg, 0.039 mmol) was dissolved in 1 mL of a 9:1 DCM / DMF mixture, and 23b (30 mg, 0.078 mmol), HATU (30 mg, 0.078 mmol), and DIPEA (25 mg, 0.195 mmol) were added. The reaction was stirred at room temperature for 30 minutes. After completion of the reaction, 1 mL of trifluoroacetic acid was added directly to the reaction solution. The reaction was stirred at room temperature for 4 hours. After completion of the reaction, the reaction solution was spin-dried to dryness. The crude product was subjected to medium- and high-pressure reverse chromatographic separation (acidic mobile phase) and lyophilized to obtain 10 mg of compound 26d as a yellow solid in a 33.3% yield. LCMS (254 nm) Rt = 1.616 min; MS calcd: 769.3; MS found: 770.4 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ10.23(s,1H),8.99(d,J=8.4Hz,1H),8.58(t,J=6.0Hz,1H),8.50(t,J=5.6Hz,1H),8.36(d,J=8.4Hz,1H),8.02( d,J=12.1Hz,1H),7.98(s,2H),7.28(t,J=7.7Hz,5H),7.18(t,J=6.9Hz,1H),6.52(s,1H),5.43(s,2H),5.29(s,2H),4.65(td,J=9.2,4.3 Hz,1H),4.12(d,J=5.7Hz,2H),3.89(dd,J=16.8,5.8Hz,1H),3.71(dd,J=16.8,5.3Hz,1H),3.57(s,2H),3.12(q,J=5.7,4.9Hz,3H),2.81 (dd,J=13.8,9.8Hz,1H),1.86(p,J=7.2Hz,2H),1.74(t,J=7.7Hz,2H),1.41(ddd,J=21.6,11.3,5.2Hz,4H),0.87(td,J=7.1,3.8Hz,6H).

[0503] Step 5: Compound 26d (20 mg, 0.026 mmol) was dissolved in 0.2 mL of DMF, and 23d (16 mg, 0.052 mmol) and DIPEA (14 mg, 0.104 mmol) were added. The reaction was stirred at room temperature for 30 minutes. After completion of the reaction, the reaction solution was directly subjected to medium- and high-pressure reverse elution (acidic mobile phase) and lyophilized to obtain 6 mg of the drug-linker conjugate STI-G as a white solid in a 23.9% yield. LCMS (254 nm) revealed a purity of 99.08%, Rt = 1.693 min; MS calculated: 962.4; MS found: 963.1 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ10.19 (s, 1H), 9.00 (dd, J = 8.5, 3.4Hz, 1H), 8.45 (dt, J=9.2,5.8Hz,3H),8.14(d,J=8.3Hz,1H),8.07(t,J=5.7Hz,1H),8.04–7.98( m,2H),7.31–7.24(m,5H),7.21–7.15(m,1H),6.98(s,1H),6.53(d,J=7.8Hz, 1H),5.41(d,J=14.1Hz,2H),5.31(d,J=11.2Hz,2H),4.62–4.50(m,2H),4.11 (d,J=5.8Hz,2H),3.74(d,J=6.2Hz,1H),3.67(d,J=5.7Hz,2H),3.60(d,J=11 .3Hz,1H),3.11(dd,J=13.1,5.2Hz,3H),2.84(dd,J=13.8,9.8Hz,1H),2.10( q,J=6.1,4.7Hz,2H),2.06–1.93(m,2H),1.86(p,J=7.0Hz,2H),1.77–1.70(m ,2H),1.50(d,J=7.0Hz,4H),1.45(dt,J=7.7,3.8Hz,4H),0.89–0.84(m,6H).

[0504] Example 27: Synthesis of Drug-Linker Conjugate STI-G3

[0505] Step 1: Under nitrogen, compound STI-G2-06c (176 mg, 0.33 mmol) from Example 13 and Boc-glycinamide (115 mg, 0.66 mmol) were dissolved in 5 mL of anhydrous toluene. Cesium carbonate (215 mg, 0.66 mmol), Xantphos (19 mg, 0.033 mmol), and palladium acetate (7 mg, 0.033 mmol) were added, stirred, and the mixture was heated to 100°C for 4.0 hours. After completion of the reaction, the mixture was concentrated to dryness under reduced pressure and dissolved in methanol. Reverse chromatography (acidic mobile phase) was performed at medium and low pressure to obtain 45 mg of compound 27a as a brown solid in a 21.7% yield. LCMS (254 nm) revealed a purity of 96.8%, Rt = 1.86 min; calculated value: 626.2; found: 627.3 [M+H] + . 1H NMR(400MHz,DMSO-d6)δ10.14(s,1H),9.04(d,J=8.9Hz,1H),8.07(s,1H), 8.04(d,J=11.9Hz,1H),7.30(s,1H),6.52(s,1H),5.43(s,2H),5.31(s,2H) ,4.66(s,2H),4.30(s,2H),2.59(t,J=7.2Hz,2H),1.87(hept,J=7.1Hz,2H) ,1.59(q,J=7.2Hz,2H),1.42(s,9H),0.93–0.89(m,3H),0.89–0.86(m,3H).

[0506] Step 2: Compound 27a (45 mg, 0.072 mmol) was dissolved in 2 mL of DCM, 0.6 mL of TFA was added, and the mixture was stirred at room temperature under nitrogen for 1 hour. The reaction was stopped after LCMS monitoring, and the mixture was concentrated to dryness under reduced pressure. Triethylamine (73 mg, 0.72 mmol), compound 23b (27 mg, 0.072 mmol), and HATU (41 mg, 0.108 mmol) were added to the reaction flask, the atmosphere was purged with nitrogen three times, and 1.0 mL of DMF was added to dissolve the mixture. The mixture was stirred at room temperature for 1.5 hours. The reaction was confirmed to be complete by LCMS. Reverse chromatography (acidic mobile phase) was performed with medium- and high-pressure preparative chromatography, and the mixture was lyophilized to obtain 20 mg of compound 27b as a yellow solid in a 31.4% yield. LCMS (254 nm) revealed a purity of 98.2%, Rt = 1.73 min; MS calculated: 887.3; MS found: 888.3 [M+H] + . 1 H NMR (400MHz, MeOD) δ8.94(d,J=8.3Hz,1H),7.65(d,J=11.8Hz,1H),7.42(s,1H),7.19(dd,J=4.5,2.7Hz,6H),7.10 (d,J=6.3Hz,2H),5.47(d,J=16.3Hz,1H),5.26(d,J=16.2Hz,1H),5.12(d,J=4.3Hz,1H),4.85(s,1H),4.65–4.60(m ,2H),4.49(s,2H),4.13(s,2H),4.05(d,J=11.3Hz,2H),3.80(s,1H),3.74(s,1H),2.93(dd,J=14.0,9.5Hz,2H),2. 52(t,J=7.3Hz,2H),1.92–1.82(m,2H),1.56(p,J=7.4Hz,2H),1.33(s,11H),0.94–0.88(m,3H),0.88–0.83(m,3H).

[0507] Step 3: Compound 27b (17 mg, 0.019 mmol) was dissolved in 1.0 mL of DCM, and 0.4 mL of TFA was added. The mixture was stirred at room temperature for 0.5 h. LCMS confirmed the reaction was complete, and the reaction was stopped and concentrated to dryness under reduced pressure. Triethylamine (26 μL, 0.190 mmol) and compound 23d (12 mg, 0.038 mmol) were added, and the atmosphere was purged with nitrogen three times. 1.0 mL of DMF was added to dissolve the mixture, and the mixture was stirred at room temperature for 1 h. LCMS confirmed the reaction was complete. Reverse chromatography (acidic mobile phase) was performed using a medium- and high-pressure preparative chromatography to obtain 11 mg of the drug-linker conjugate STI-G3 as a yellow solid. LCMS (254 nm) indicated a purity of 100%, Rt = 1.65 min; MS calculated: 980.4; MS found: 981.3 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ10.33(s,1H),8.98(d,J=8.5Hz,1H),8.10(q,J=7.8Hz,2H),8.05(d,J=11.6Hz,1H),7.95(s,1H),7. 33–7.23(m,5H),7.20(s,2H),7.01(s,2H),6.67(s,1H),6.54(d,J=2.2Hz,1H),6.40(d,J=2.4Hz,1H),5.39(s,2H),4.52(d,J =23.6Hz,2H),4.32–4.25(m,1H),3.89(t,J=12.3Hz,2H),3.83–3.73(m,2H),3.63 (t,J=12.4Hz,2H),3.14(d,J=11.8Hz,1H),2.94(d,J=12.9Hz,1H),2.89(s,2H),2 .73(d,J=2.3Hz,2H),2.10(d,J=7.7Hz,2H),2.00(t,J=7.4Hz,2H),1.84(q,J=7.2 Hz, 2H), 1.57 (dd, J = 7.2, 3.7Hz, 2H), 1.18 (t, J = 8.0Hz, 4H), 0.87 (t, J = 7.3Hz, 6H).

[0508] Example 28: Synthesis of Drug-Linker Conjugate STI-F1002

[0509] Similar to the synthesis of STI-F4, the drug-linker conjugate STI-F1002 was prepared as shown in the following scheme:

[0510] Step 1: Compound STI-F6 (40 mg, 0.09 mmol), 25b (40 mg, 0.10 mmol), and pyridinium p-toluenesulfonate (13 mg, 0.5 mmol) were added to a reaction flask. 1,2-Dichloroethane (2.0 mL) was added under nitrogen. The reaction was heated to 80°C overnight. The mixture was concentrated to dryness under reduced pressure and purified by Prep-TLC (DCM:MeOH = 15:1) to afford 46 mg of compound 28a as a yellow solid in a 69.7% yield. LCMS (254 nm) revealed a purity of 87.90%, Rt = 1.90 min; Calcd: 775.3; Found: 776.2 [M+H] + .

[0511] Step 2: Compound 28a (46 mg, 0.06 mmol) was dissolved in DMF (1.8 mL), piperidine (0.4 mL) was added, and the mixture was reacted at room temperature for 20 minutes. The system was concentrated to dryness, and to the residue were added (tert-butyloxycarbonyl)glycyl-L-phenylalanine (23b) (27 mg, 0.07 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (34 mg, 0.09 mmol). mol) and N,N-dimethylformamide (1.0 mL) were added under nitrogen protection, followed by the addition of triethylamine (16 mg, 0.159 mmol). The reaction was allowed to proceed at room temperature for 1 hour. The reaction was terminated, concentrated to dryness, and extracted with dichloromethane and saturated brine. The aqueous phase was washed with dichloromethane (10.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain 86 mg of crude compound 28b as a yellow oily liquid, with a yield of 62.9%. LCMS (254 nm) revealed a purity of 93.41%, Rt = 1.54 min; calculated value: 914.4; found value: 915.4 [M+H] + .

[0512] Step 3: Compound 28b (86 mg, 0.10 mmol) was dissolved in dichloromethane (1.8 mL) under nitrogen atmosphere. Trifluoroacetic acid (0.1 mL) was added and the mixture was allowed to react at room temperature for 1 hour. The reaction was stopped after monitoring by LCMS until the reaction was complete. The system was concentrated to dryness under reduced pressure and added to N,N-dimethylformamide (2.0 mL). Under nitrogen atmosphere, triethylamine (111 mg, 1.10 mmol) and 6-(maleimido)hexanoic acid succinimidyl ester (EMCS, 52 mg, 0.17 mmol) were added sequentially. The mixture was allowed to react at room temperature for approximately 1 hour. The reaction was stopped and the system was concentrated to dryness under reduced pressure. The mixture was directly subjected to high pressure reverse phase preparation (mobile phase: acetonitrile / 0.05% trifluoroacetic acid in water). After lyophilization, 12 mg of compound STI-F1002 was obtained as a yellow solid in a yield of 12.6%. LCMS (254 nm) purity 98.5%, Rt = 1.58 min; MS calculated value: 1007.4; MS found value: 1008.4 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ10.05(s,1H),8.50(t,J=6.6Hz,1H),8.41(d,J=5.9Hz,1H),8.29(t,J=5.9Hz,1H),8.11(d,J=8.0Hz,1H),8.07(t,J=5.8Hz,1H ),8.01(t,J=5.8Hz,1H),7.59(d,J=10.5Hz,1H),7.27–7.16(m,5H),7.00( s,1H),6.99(s,2H),6.61(br,1H),5.49(s,2H),5.43(s,2H),4.54–4.44(m, 1H),3.76–3.56(m,10H),3.39–3.33(m,4H),3.04(d,J=4.8Hz,1H),3.01(d,J=4.7Hz,1H),2.44(s,3H),2.13(t,J =7.4Hz,2H),1.91–1.80(m,2H),1.69–1.60(m,2H),1.50–1.41(m,4H),1.26–1.14(m,4H),0.87(t,J=7.3Hz,3H).

[0513] Example 29: Synthesis of Drug-Linker Conjugate STI-F1001

[0514] Step 1: Compound STI-F (80 mg, 0.16 mmol), 25b (61 mg, 0.16 mmol), and pyridinium p-toluenesulfonate (4 mg, 0.01 mmol) were added to a reaction flask. 1,2-Dichloroethane (5.0 mL) was added under nitrogen. The reaction was heated to 75°C for 48 hours. The mixture was then concentrated to dryness under reduced pressure and purified by Prep-TLC (DCM:MeOH = 15:1) to afford 40 mg of compound F1001-01 as an orange-yellow solid in a 31.3% yield. LCMS (254 nm) revealed a purity of 92.16%, Rt = 1.905 min; Calcd: 801.3; Found: 802.1 [M+H] + .

[0515] Step 2: Compound F1001-01 (40 mg, 0.05 mmol) was dissolved in N,N-dimethylformamide (0.8 mL), and piperidine (0.2 mL) was added. The mixture was reacted at room temperature for 20 minutes, and the system was concentrated to dryness. To the crude product were added (tert-butyloxycarbonyl)glycyl-L-phenylalanine (23b) (27 mg, 0.07 mmol), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (34 mg, 0.09 mmol) and N,N-dimethylformamide (1.0 mL). Triethylamine (15 mg, 0.15 mmol) was added under nitrogen protection, and the mixture was reacted at room temperature for 1 hour. The system was concentrated to dryness, extracted with dichloromethane and saturated brine, and the aqueous phase was further extracted with dichloromethane (10.0 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure to yield 46 mg of crude compound F1001-02. LCMS (254 nm) purity: 72.13%, Rt = 1.708 min; calculated: 940.4; found: 941.2 [M+H] + .

[0516] Step 3: Compound F1001-02 (46 mg, 0.05 mmol) was dissolved in dichloromethane (1.8 mL) under nitrogen atmosphere. Trifluoroacetic acid (0.1 mL) was added and the mixture was allowed to react at room temperature for 1 hour. The reaction was stopped by LCMS monitoring until the reaction was complete. The system was concentrated to dryness under reduced pressure and added to N,N-dimethylformamide (2.0 mL). Under nitrogen atmosphere, triethylamine (0.07 mL, 0.50 mmol) and 6-(maleimido)hexanoic acid succinimidyl ester (EMCS, 52 mg, 0.17 mmol) were added sequentially. The mixture was allowed to react at room temperature for approximately 1 hour. The reaction was stopped and the system was concentrated to dryness under reduced pressure. The mixture was purified by high-pressure reverse phase chromatography (mobile phase: acetonitrile / 0.05% trifluoroacetic acid in water) and lyophilized to obtain 12 mg of compound STI-F1001 as a yellow solid in a 23.7% yield. LCMS (254 nm) purity 96.79%, Rt = 1.58 min; MS calculated value: 1033.4; MS found value: 1034.1 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ10.69(s,1H),8.72(t,J=6.6Hz,1H),8.35(t,J=5.9Hz,1H),8.12(dd,J=8.3,4.3Hz,2H),8.06(t,J=5.9Hz,1H),8.00(t,J=5.8Hz ,1H),7.91(d,J=10.7Hz,1H),7.33(s,1H),7.26–7.11(m,5H),6.98(s,2H), 6.53(br,1H),5.41(s,2H),5.14(s,2H),4.86–4.71(m,2H),4.48(td,J=8.9, 4.6Hz,1H),3.83(d,J=7.5Hz,1H),3.77(d,J=6.4Hz,2H),3.75–3.53(m,6H) ,3.02(dd,J=14.0,4.4Hz,1H),2.77(dd,J=13.9,9.7Hz,1H),2.50(s,3H),2. 09(t,J=7.6Hz,2H),1.92–1.81(m,2H),1.50–1.41(m,4H),1.31(q,J=6.4Hz, 1H), 1.18 (dd, J=15.4, 7.8Hz, 2H), 0.88 (t, J=7.3Hz, 3H), 0.65–0.56 (m, 4H).

[0517] Similarly, a drug-linker conjugate was prepared as follows:

[0518] Example 32: Synthesis of Drug-Linker Conjugate STI-F1006

[0519] Step 1: Dissolve F1006-S1 (500 mg, 1.13 mmol) in N,N-dimethylformamide (10.0 mL) under nitrogen. Add EMCS (382 mg, 1.24 mmol) and N,N-diisopropylethylamine (219 mg, 1.70 mmol) and react at room temperature for 2 hours. Quench the reaction with water and directly prepare the product using reverse phase chromatography (mobile phase: acetonitrile / 0.05% trifluoroacetic acid in water). After lyophilization, 587 mg of compound F1006-S2 was obtained as a pale yellow oil in an 80.7% yield. LCMS (254 nm) revealed a purity of 98.8%, Rt = 1.38 min; MS calculated: 634.3; MS found: 635.2 [M+H] + .

[0520] Step 2: Dissolve F1006-S2 (578 mg, 0.93 mmol) and N,N'-dicyclohexylcarbodiimide (210 mg, 1.02 mmol) in tetrahydrofuran (12.0 mL) under nitrogen. Add pentafluorophenol (188 mg, 1.02 mmol) and allow to react overnight at room temperature. After completion of the reaction, as monitored by LCMS, filter the system and concentrate the filtrate to dryness under reduced pressure to obtain 587 mg of crude compound F1006-S3 as a pale yellow oily liquid, with a yield of 80.6%. LCMS (254 nm) purity: 80.0%, Rt = 1.85 min; MS calculated: 800.3; MS found: 801.3 [M+H] + .

[0521] Step 3: Compound 28b (100 mg, 0.11 mmol) was dissolved in dichloromethane (1.8 mL) under nitrogen protection. Trifluoroacetic acid (0.2 mL) was added and the mixture was allowed to react at 0-5°C for 3 hours. The reaction was stopped after monitoring by LCMS until the reaction was complete. The system was concentrated to dryness under reduced pressure and added to N,N-dimethylformamide (2.0 mL). The mixture was cooled to 0-5°C under nitrogen protection. N,N-diisopropylethylamine (111 mg, 1.10 mmol) and F1006-S3 (80 mg, 0.10 mmol) were added sequentially. The reaction was allowed to react for about 0.5 hours. The reaction was stopped and the system was concentrated to dryness under reduced pressure. The mixture was directly prepared by high-pressure reverse phase chromatography (mobile phase: acetonitrile / 0.05% trifluoroacetic acid in water). After lyophilization, 16 mg of compound STI-F1006 was obtained as a yellow solid in a yield of 10.2%. LCMS (254 nm) purity 99.8%, Rt = 1.59 min; MS calculated value: 1430.7; MS found value: 1429.5 [MH] - . 1H NMR (400MHz, DMSO-d6) δ8.52(t,J=6.7Hz,1H),8.30(d,J=7.5Hz,2H),8.17(d,J=5.7Hz,1H),8.07(t,J=5.7Hz,1H),8.11(d,J=8.0Hz,1H),8.01(t,J= 5.8Hz,1H),7.82(t,J=5.7Hz,1H),7.58(d,J=11.0Hz,1H),7.38(t,J=5.6H z,1H),7.23–7.18(m,5H),6.99(s,2H),6.48(s,1H),5.44(s,2H),5.41(s,2 H),4.51–4.45(m,1H),3.77–3.68(m,6H),3.66–3.57(m,8H),3.50(s,30H) ,3.19–3.16(m,4H),3.05(d,J=4.8Hz,1H),3.01(d,J=3.6Hz,1H),2.43(s, 3H),2.38(t,J=6.5Hz,2H),2.03(t,J=7.4Hz,2H),1.88–1.81(m,2H),1.66 –1.64(m,2H),1.50–1.43(m,4H),1.24–1.18(m,4H),0.87(t,J=7.3Hz,3H).

[0522] Example 33: Synthesis of Drug-Linker Conjugate STI-F1010

[0523] Step 1: Compound F1007-01 (1.64 g, 4.00 mmol) was added to 50 mL of dichloromethane under an ice bath. N,N-diisopropylethylamine (1551 mg, 12.00 mmol), glycine tert-butyl ester (H-Gly-OtBu, 525 mg, 4.00 mmol), and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (1349 mg, 4.20 mmol) were also added. The mixture was allowed to react under nitrogen atmosphere at room temperature for 1 hour. After completion of the reaction, as monitored by LCMS, the mixture was filtered and washed with dichloromethane several times. The filter cake was added to 8 mL of dichloromethane, followed by 4 mL of trifluoroacetic acid. The mixture was allowed to react at room temperature for 3 hours. After completion of the reaction as monitored by LCMS, the mixture was spin-dried, slurried in dichloromethane / acetonitrile, filtered, and washed with acetonitrile to obtain 1280 mg of compound F1010-01 as a white solid in a 68.5% yield. LCMS (254 nm) revealed a purity of 100%, Rt = 1.695 min; MS calculated: 467.2; MS found: 468.3 [M+H] + .

[0524] Step 2: Compound F1010-01 (545 mg, 1.17 mmol) was dissolved in tetrahydrofuran (15.0 mL) and toluene (5.0 mL). Lead tetraacetate (620 mg, 1.40 mmol) and pyridine (111 mg, 1.40 mmol) were added under nitrogen. The mixture was heated to 85°C and allowed to react for 3 hours, after which the reaction was stopped. The mixture was filtered through a pad of Celite and the filtrate was concentrated to dryness under reduced pressure. Purification by column chromatography (DCM:MeOH = 100:1) afforded 400 mg of compound F1010-02 as a white solid in a 71.0% yield. LCMS (254 nm) revealed a purity of 89.0%, Rt = 1.811 min; MS calcd: 481.2; MS found: 499.7 [M+NH4] + .

[0525] Step 3: Compound STI-F6 (160 mg, 0.34 mmol) from Example 17, F1010-02 (197 mg, 0.41 mmol), and pyridinium p-toluenesulfonate (PPTS, 10 mg) were added to a reaction flask. DMF (5.0 mL) was added and the mixture was heated to 95°C under nitrogen for approximately 3 hours. The reaction was then terminated. The mixture was concentrated to dryness under reduced pressure and purified by column chromatography (DCM:MeOH = 50:1) to afford 110 mg of compound F1010-03 as an orange-red solid in a 25.5% yield. LCMS (254 nm) revealed a purity of 84.0%, Rt = 1.920 min; MS calcd: 888.4; MS found: 889.2 [M+H] + .

[0526] Step 4: Compound F1010-03 (55 mg, 0.062 mmol) was dissolved in DMF (0.5 mL), and piperidine (0.05 mL) was added. The mixture was reacted at room temperature for 20 minutes. The solution was concentrated to dryness, and the residue was washed with petroleum ether and dried under reduced pressure. EMCS (29 mg, 0.093 mmol) and DMF (1.0 mL) were added under nitrogen. Triethylamine (19 mg, 0.19 mmol) was added and the mixture was reacted at room temperature for 1 hour. The reaction was stopped and the mixture was concentrated to dryness. The mixture was then lyophilized using medium- and high-pressure reverse-phase preparative chromatography (mobile phase: acetonitrile / 0.05% aqueous formic acid) to give 16 mg of compound STI-F1010 as a light yellow solid in a 30.2% yield. LCMS (254 nm) revealed a purity of 96.38%, Rt = 1.663 min; MS calculated: 859.4; MS found: 860.4 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ8.79(t,J=6.4Hz,1H),8.59(t,J=6.6Hz,1H),8.42(d,J=7.8Hz,1H),8.00(d,J=7.2Hz,1H),7.76(t,J=8.7Hz, 1H),7.59(d,J=10.5Hz,1H),7.41(s,1H),7.00(s,2H),6.57(br,1H),5.47(s,2H),5.44(s,2H),4.58–4.56(m,2H),4.23(q,J=7.1Hz,1 H),4.14–4.09(m,1H),3.71(d,J=6.8Hz,2H),3.45–3.43(m,2H),3.35(t,J=7.0Hz,2H),2.44(s,3H),2.16–2.05(m,2H),1.88–1.84(m, 1H),1.77–1.72(m,2H),1.67–1.61(m,2H),1.47–1.42(m,4H),1.24–1.12(m,7H),0.88(t,J=6.0Hz,3H),0.79(dd,J=11.3,6.7Hz,6H).

[0527] Example 34: Synthesis of Drug-Linker Conjugate STI-F1011

[0528] Compound F1010-03 (55 mg, 0.062 mmol) from Example 33 was dissolved in DMF (0.5 mL), and piperidine (0.05 mL) was added. The reaction was carried out at room temperature for 10 minutes. The system was concentrated to dryness, and the residue was washed with petroleum ether and dried under reduced pressure. HATU (57 mg, 0.19 mmol), F1006-S2 (29 mg, 0.093 mmol) from Example 32, and DMF (1.0 mL) were added thereto. Under nitrogen protection, triethylamine (19 mg, 0.19 mmol) was added, and the reaction was carried out at room temperature for 0.5 hour. The reaction was stopped, and the system was concentrated to dryness. The reaction was carried out using medium- and high-pressure reverse phase chromatography (mobile phase: acetonitrile / 0.05% formic acid aqueous solution). After lyophilization, 28 mg of compound STI-F1011 was obtained as a light yellow sticky solid with a yield of 35.0%. LCMS (254 nm) purity 96.07%, Rt = 1.597 min; MS calculated value: 1282.6; MS found value: 1283.6 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ8.60–8.56(m,2H),8.49–8.45(m,1H),8.03(d,J=7.3Hz,1H),7.90–7.78(m,2H),7.60(d,J=10.3,1H),7.49(s,1H ),7.00(s,2H),6.53(br,1H),5.51(s,2H),5.45(s,2H),4.62–4.52(m,2H),4.23(t,J=7.2Hz,1H),4.15(dd,J=8.7,6.6Hz,1H),3.75–3.73 (m,2H),3.60–3.52(m,4H),3.51–3.46(m,30H),3.39–3.36(m,4H),3.16(q,J=5.9Hz,2H),2.45(s,3H),2.03(t,J=7.4Hz,2H),1.88–1.84 (m,1H),1.78–1.73(m,2H),1.68–1.62(m,2H),1.50–1.42(m,4H),1.23–1.12(m,7H),0.87(t,J=7.4Hz,3H),0.79(dd,J=11.5,6.8Hz,6H).

[0529] Example 35: Synthesis of Drug-Linker Conjugate STI-F1012

[0530] Compound F1010-03 (70 mg, 0.078 mmol) in Example 33 was dissolved in DMF (0.5 mL), and piperidine (0.05 mL) was added. The mixture was reacted at room temperature for 10 minutes. The system was concentrated to dryness, and the residue was washed with petroleum ether and dried under reduced pressure. HATU (87 mg, 0.23 mmol), 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (25 mg, 0.094 mmol), and DMF (2.0 mL) were added thereto. Under nitrogen protection, triethylamine (23 mg, 0.23 mmol) was added, and the mixture was reacted at room temperature for 0.5 hour. The reaction was stopped, and the system was concentrated to dryness. The mixture was then subjected to medium- and high-pressure reverse phase preparation (mobile phase: acetonitrile / 0.05% trifluoroacetic acid aqueous solution) and lyophilized to obtain 30 mg of compound STI-F1012 as a light yellow solid in a yield of 41.9%. LCMS (254 nm) purity 98.69%, Rt = 1.646 min; MS calculated value: 916.4; MS found value: 917.3 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ9.11(s,2H),8.60(t,J=6.6Hz,1H),8.45(d,J=7.7Hz,1H),8.06(d,J=7.2Hz,1H),7.89(d,J=8.7Hz,1H),7. 58(dd,J=10.3,2.3Hz,1H),7.41(s,1H),6.65(br,1H),5.49(s,2H),5.45(s,2H),4.64–4.51(m,2H),4.24(q,J=7.1Hz,1H),4.16(d d,J=8.7,6.7Hz,1H),3.76–3.69(m,4H),3.45–3.42(m,1H),3.40(s,3H),2.54(d,J=7.3Hz,2H),2.44(s,3H),2.16–2.05(m,2H),1. 89–1.83(m,2H),1.81–1.76(m,4H),1.67–1.61(m,2H),1.21(d,J=7.1Hz,3H),0.87(t,J=7.4Hz,3H),0.81(dd,J=11.8,6.7Hz,6H).

[0531] Example 36: Synthesis of Drug-Linker Conjugate STI-F1013

[0532] Step 1: F1013-01 (2.0 g, 4.27 mmol) was dissolved in dichloromethane (20.0 mL) under nitrogen protection. Pentafluorophenol (943 mg, 5.12 mmol) and DCC (1.05 g, 5.12 mmol) were added and reacted at room temperature for 1 hour. After completion of the reaction monitored by TLC, the reaction solution was filtered, and the filter cake was rinsed with dichloromethane (5 mL*3). The filtrate was concentrated to dryness at room temperature and used directly in the next step. The above product was dissolved in acetone / water (5 mL / 15 mL), and sodium bicarbonate (1.07 g, 12.8 mmol) and glycine (320 mg, 4.27 mmol) were added sequentially. The mixture was stirred at room temperature for 2 hours. After completion of the reaction, 1 M hydrochloric acid was added to adjust the pH to 3-4. The mixture was extracted with ethyl acetate (10 mL x 2), washed with saturated brine (10 mL x 2), dried over anhydrous sodium sulfate, concentrated at 45°C, and directly subjected to medium-low pressure reverse phase preparation (mobile phase: acetonitrile / 0.05% trifluoroacetic acid aqueous solution). After lyophilization, 2.0 g of compound F1013-03 was obtained as a white solid in a 90.9% yield. LCMS (254 nm) purity was 95.6%, Rt = 2.16 min; MS calculated value: 525.2; MS found value: 526.3 [M+H] + .

[0533] Step 2: F1013-03 (2.0 g, 3.80 mmol) was dissolved in tetrahydrofuran (40 mL) and toluene (10 mL) under nitrogen. Pb(OAc)4 (2.03 g, 4.57 mmol) and pyridine (361 mg, 4.57 mmol) were added and the mixture was allowed to react at 85°C for 1 hour. After completion of the reaction, as monitored by LCMS, the reaction solution was concentrated to dryness under reduced pressure at 45°C. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 2 / 3) to afford 1.80 g of compound F1013-04 as a pale yellow solid in an 87.8% yield. LCMS (254 nm) revealed a purity of 92.1%, Rt = 2.36 min; MS calculated: 539.2; MS found: 557.3 [M+NH4]. + .

[0534] Step 3: Compound F1013-04 (260 mg, 0.43 mmol) was dissolved in DMF (5 mL) under nitrogen. STI-F6 (170 mg, 0.36 mmol) and PPTS (17 mg) from Example 17 were added sequentially. The mixture was allowed to react at 95°C for 2 hours. The reaction mixture was concentrated to dryness under reduced pressure at 45°C. The crude product was purified by column chromatography (dichloromethane / methanol = 50 / 3) to afford 70 mg of compound F1013-05 as a black solid in a 14.2% yield. LCMS (254 nm) revealed a purity of 70.1%, Rt = 2.05 min; MS calculated: 946.4; MS found: 947.5 [M+H] + .

[0535] Step 4: Compound F1013-05 (70 mg, 0.07 mmol) was dissolved in DMF (0.7 mL), piperidine (70 μL) was added, and the mixture was stirred under nitrogen for 0.5 h. The reaction was completed after monitoring by LCMS. The reaction mixture was concentrated to dryness at 45°C and then slurried with petroleum ether (2 mL x 3). The mixture was concentrated again and used directly in the next step. F1013-06 (64 mg, 0.07 mmol) was dissolved in DMF (2 mL), and HATU (42 mg, 0.11 mmol) and N,N-diisopropylethylamine (19 mg, 0.148 mmol) were added sequentially. The mixture was stirred under nitrogen for 5 minutes, and then the concentrate was added. The mixture was stirred at room temperature under nitrogen for 1 hour. After completion of the reaction, LCMS was used to monitor the reaction. The mixture was concentrated to dryness at 45°C and the crude product was purified by column chromatography (dichloromethane / methanol = 50 / 7) to obtain 60 mg of compound F1013-07 as a yellow solid in a 52.1% yield. LCMS (254 nm) revealed a purity of 93.0%, Rt = 1.78 min; MS calculated: 1565.1; MS found: 1566.2 [M+H] + .

[0536] Step 5: Compound F1013-07 (60 mg, 0.03 mmol) was dissolved in 10% TFA / DCM (2 mL) under nitrogen atmosphere. Stir on ice for 1 hour. LCMS monitored the reaction for completion. The reaction solution was adjusted to pH 7-8 with triethylamine, dried under nitrogen, and then subjected to high-pressure reverse-phase preparative (mobile phase: acetonitrile / 0.05% trifluoroacetic acid in water). After lyophilization, 6.2 mg of compound STI-F1013 was obtained as a yellow solid in an 11.8% yield. LCMS (254 nm) revealed a purity of 99.0%, Rt = 1.51 min; MS calculated: 1465.6; MS found: 1466.8 [M+H] + . 1 H NMR(400MHz,MeOD)δ8.95(s,2H),8.32(d,J=7.6Hz,1H),7.96(s,1H),7.70(s,1H),7.56(d,J=10.0Hz,1H),5.66-5.58( m,2H),4.77(q,J1=10.4Hz,J2=26.4Hz,2H),4.56(t,J=4.8Hz,2H),4.45-4.41(m,3H),4.03(s,2H),3.98(d,J=2.0Hz,2 H),3.89-3.83(m,4H),3.64-3.57(m,28H),3.52-3.49(m,2H),3.37(s,3H),3.37-3.31(m,6H),2.97(t,J=7.2Hz,2H),2 .60(t,J=7.2Hz,2H),2.52(s,3H),2.45(t,J=7.2Hz,2H),2.03-1.68(m,12H),1.51-1.47(m,2H),1.02(t,J=7.2Hz,3H).

[0537] Intermediate compound F1013-06 was prepared as follows:

[0538] Step 1: Dissolve 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (200 mg, 0.74 mmol) in dichloromethane (2 mL). HATU (425 mg, 1.12 mmol) and N,N-diisopropylethylamine (193 mg, 1.49 mmol) were added sequentially. The mixture was stirred under nitrogen for 10 minutes. Propargylamine (49 mg, 0.89 mmol) was added and stirring continued for 1 hour. LCMS monitored the reaction for completion. The reaction solution was concentrated to dryness under reduced pressure at room temperature. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 1 / 3) to afford 210 mg of compound F1005-02 as a yellow solid in a 92.5% yield. LCMS (254 nm) revealed a purity of 92.3%, Rt = 2.20 min; MS calculated: 305.0; MS found: 306.1 [M+H] + .

[0539] Step 2: Compound F1005-02 (100 mg, 0.32 mmol) was dissolved in a mixture of dimethyl sulfoxide (1 mL) and water (0.1 mL). F1005-03 (144 mg, 0.32 mmol) and CuBr (94 mg, 0.65 mmol) were added sequentially. The mixture was stirred under nitrogen for 1 hour. LCMS monitored the reaction for completion. The reaction mixture was preparatively purified by low-pressure reverse phase chromatography (mobile phase: acetonitrile / 0.05% trifluoroacetic acid in water). After lyophilization, 350 mg of compound F1013-06 was obtained as a green oil. LCMS (254 nm) revealed a purity of 85.2%, Rt = 1.95 min; MS calculated: 859.3; MS found: 860.4 [M+H] + .

[0540] Example 37: Synthesis of Drug-Linker Conjugate STI-F1014

[0541] Step 1: Dissolve ((9H-fluoren-9-yl)methoxy)carbonyl)glycyl-L-phenylalanine (Fmoc-GGF-OH, 400 mg, 0.80 mmol) in DMF (5.4 mL). Add piperidine (0.6 mL) and react at room temperature for 30 minutes. The mixture is concentrated to dryness, slurried with petroleum ether, and redissolved in DMF (8.0 mL). Under nitrogen, cool to 0-5°C, add F1006-03 (832 mg, 1.04 mmol) and DIPEA (154 mg, 1.20 mmol). React at 0-5°C for approximately 1 hour. LCMS monitors the reaction for completion, and stop the reaction. The mixture is concentrated to dryness under reduced pressure and purified by reverse phase purification using an acid method to obtain 380 mg of compound F1006-S3 as a colorless oil in a 40.8% yield. LCMS (254 nm) purity 95.96%, Rt = 1.648 min; MS calculated value: 895.4; MS found value: 896.5 [M+H] + .

[0542] Step 2: Compound F1014-01 (180 mg, 0.22 mmol) was dissolved in DMF (2.7 mL), piperidine (0.3 mL) was added, and the reaction was carried out at room temperature for 30 minutes. The system was concentrated to dryness, slurried with acetone, filtered, and the filter cake was redissolved in DMF (3.0 mL). F1006-S3 (63 mg, 0.07 mmol) and HATU (125 mg, 0.33 mmol) were added thereto. Under nitrogen protection, DIPEA (43 mg, 0.33 mmol) was added and the reaction was carried out at room temperature for 0.5 hour. The reaction was completed after LCMS monitoring. The reaction was stopped and the system was concentrated to dryness. The mixture was prepared by medium- and high-pressure reverse phase (mobile phase: acetonitrile / 0.05% trifluoroacetic acid aqueous solution). After lyophilization, 14 mg of compound STI-F1014 was obtained as a light yellow solid with a yield of 4.4%. LCMS (254 nm) purity 95.8%, Rt = 1.654 min; MS calculated value: 1456.7; MS found value: 1457.8 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ8.52(t,J=6.7Hz,1H),8.37(t,J=6.0Hz,1H),8.19–8.12(m,2H),8.02(t,J=5.7Hz,1H),7.93(t,J=8.6Hz,1H),7.80–7 .75(m,2H),7.28–7.24(m,6H),7.00(s,2H),6.51(br,1H),5.48(s,2H) ,5.42(s,2H),4.61–4.60(m,2H),4.51(q,J=8.3,7.8Hz,1H),3.77–3.6 9(m,6H),3.61–3.59(m,6H),3.49–3.47(m,28H),3.28–3.22(m,6H),3. 18–3.16(m,2H),3.09–3.08(m,1H),3.06–3.05(m,1H),2.49(s,3H),2. 39(t,J=6.6Hz,2H),2.03(t,J=7.3Hz,2H),1.88–1.82(m,2H),1.58–1. 52(m,1H),1.48–1.42(m,4H),1.25–1.17(m,6H),0.88(t,J=6.0Hz,3H).

[0543] The intermediate compound F1014-01 was prepared as follows:

[0544] Procedure: Compound STI-F14 (170 mg, 0.34 mmol), 25b (151 mg, 0.41 mmol), and pyridinium p-toluenesulfonate (20 mg) were added to a reaction flask. DMF (10.0 mL) was added and the mixture was heated to 95°C under nitrogen for approximately 3 hours. The reaction was then terminated. The mixture was concentrated to dryness under reduced pressure and purified by column chromatography (DCM:MeOH = 50:1) to afford 180 mg of compound F1014-01 as a yellow solid in a 63.8% yield. LCMS (254 nm) revealed a purity of 51.39%, Rt = 1.992 min; MS calcd: 801.3; MS found: 802.3 [M+H] + .

[0545] Example 38: Synthesis of Drug-Linker Conjugate STI-F1015

[0546] Step 1: Compound STI-F15 (390 mg, 0.77 mmol), 25b (339 mg, 0.92 mmol), and pyridinium p-toluenesulfonate (20 mg) were added to a reaction flask. DMF (12.0 mL) was added and the mixture was heated to 95°C under nitrogen for approximately 3 hours. The reaction was then terminated. The mixture was concentrated to dryness under reduced pressure and purified by column chromatography (DCM:MeOH = 50:1) to afford 180 mg of compound F1015-01 as a yellow solid in an 18.5% yield. LCMS (254 nm) revealed a purity of 47%, Rt = 1.891 min; MS calcd: 815.3; MS found: 816.4 [M+H] + .

[0547] Step 2: Compound F1015-01 (47%, 180 mg, 0.11 mmol) was dissolved in DMF (2.7 mL), piperidine (0.3 mL) was added, and the reaction was carried out at room temperature for 30 minutes. The system was concentrated to dryness, slurried with acetone, filtered, and the filter cake and HATU (125 mg, 0.30 mmol) were added to the reaction flask. Under nitrogen protection, DMF (3.0 mL), F1006-S3 (107 mg, 0.12 mmol) of Example 37 and DIPEA (43 mg, 0.30 mmol) were added and the reaction was carried out at room temperature for 0.5 hours. The reaction was completed after LCMS monitoring. The reaction was stopped and the system was concentrated to dryness. The mixture was prepared by medium- and high-pressure reverse phase chromatography (acid mobile phase) and lyophilized to obtain 15 mg of compound STI-F1015 as a light yellow solid with a yield of 9.3%. LCMS (254 nm) purity 97.56%, Rt = 1.655 min; MS calculated value: 1470.7; MS found value: 1469.6 [MH] - . 1H NMR (400MHz, DMSO-d6) δ8.52-8.47(m,2H),8.31(t,J=5.7Hz,1H),8.17(t,J =5.7Hz,1H),8.12(d,J=8.1Hz,1H),8.02(t,J=5.8Hz,1H),7.81(t,J=5.7Hz, 1H),7.60(d,J=10.7Hz,1H),7.37(s,1H),7.28–7.20(m,5H),7.00(s,2H),6 .54(br,1H),5.42(s,2H),5.38(s,2H),4.57–4.55(m,2H),4.53–4.48(m,1H) ,3.78–3.69(m,7H),3.63–3.58(m,7H),3.49–3.47(m,28H),3.29(d,J=6.1H z,2H),3.19–3.14(m,2H),3.09–3.08(m,1H),3.06–3.05(m,1H),2.45(s,3H) ,2.39(t,J=6.6Hz,2H),2.07–1.97(t,J=7.3Hz,4H),1.88–1.82(m,2H),1.6 4–1.54(m,2H),1.50–1.42(m,5H),1.21–1.15(m,6H),0.88(t,J=6.0Hz,3H).

[0548] Example 39: Synthesis of Drug-Linker Conjugate STI-F1018

[0549] Step 1: F1005-01 (65 mg, 0.24 mmol) was dissolved in dichloromethane (2.0 mL) under nitrogen protection, and NHS (34 mg, 0.29 mmol) and DCC (60 mg, 0.29 mmol) were added. The reaction was carried out in an ice bath for 1 hour. After the reaction was completed by TLC monitoring, the reaction solution was filtered, and the filter cake was rinsed with DCM (5 mL*3). The filtrate was concentrated to dryness at room temperature and used directly in the next step. The above crude product and F1018-01 were mixed. A 145 mg, 0.24 mmol compound (145 mg, 0.24 mmol) was dissolved in DMF (2 mL), and DIEA (130 μL, 0.72 mmol) was added. The mixture was stirred in an ice bath for 2 hours. After completion of the reaction, acetic acid was added to quench the reaction until the pH became acidic. The reaction was concentrated at 45°C using an oil pump. The crude product was purified by medium-low pressure reverse phase chromatography (mobile phase: acetonitrile / 0.05% trifluoroacetic acid aqueous solution) and lyophilized to obtain 200 mg of compound F1018-02 as a clear oil in a 96.9% yield. LCMS (254 nm) revealed a purity of 92.1%, Rt = 1.95 min; MS calculated: 849.3; MS found: 850.5 [M+H] + .

[0550] Step 2: F1018-02 (53 mg, 0.06 mmol) was dissolved in dichloromethane (2.0 mL) under nitrogen protection, and NHS (9.0 mg, 0.07 mmol) and DCC (16 mg, 0.07 mmol) were added. The mixture was reacted in an ice bath for 1 hour. After the reaction was completed by TLC monitoring, the reaction solution was filtered, and the filter cake was rinsed with DCM (5 mL*3). The filtrate was concentrated to dryness at room temperature and used directly in the next step. The above concentrate was dissolved in DMF (2.0 To a 5% HCl (100 mL) solution of F1016-06 (43 mg, 0.05 mmol) and DIEA (49 mg, 0.37 mmol) was added. The mixture was allowed to react on ice for 2 hours. LCMS confirmed the reaction was complete. The mixture was concentrated at 45°C and purified by column chromatography (dichloromethane / methanol = 10 / 3) to yield 46 mg of crude compound. This crude compound was dissolved in 10% TFA / DCM (2 mL) under nitrogen atmosphere and stirred on ice for 1 hour. LCMS monitored the reaction for completion. The reaction solution was adjusted to pH 7-8 with triethylamine, dried under nitrogen, and then subjected to high-pressure reverse-phase preparative (mobile phase: acetonitrile / 0.05% trifluoroacetic acid in water). After lyophilization, 5.69 mg of compound STI-F1018 was obtained as a yellow solid. LCMS (254 nm) revealed a purity of 93.5%, Rt = 1.57 min; MS calculated: 1602.7; MS found: 1603.6 [M+H] + . 1 H NMR(400MHz, DMSO-d6)δ8.95(s,2H),8.32(t,J=6.0Hz,1H),8.36-8.25(m,2H),8.10(d,J=8.0Hz,1H),7.99( t,J=6.0Hz,1H),7.87(s,2H),7.61-7.58(m,3H),7.31(d,J=2.0Hz,1H),7.21-7.08(m,5H),6.53(brs,1H),5 .41(d,J=16.8Hz,4H),4.59-4.54(m,3H),4.24-4.23(m,1H),3.88-3.79(m,3H),3.67-3.59(m,2H),3.57(t, J=6.8Hz,3H),3.47-3.42(m,34H),3.28-3.23(m,4H),3.16-3.12(m,6H),2.86-2.74(m,4H),2.55(t,J=7.2Hz 2H),2.32-2.22(m,5H),1.88-1.78(m,4H),1.77-1.62(m,5H),1.57-1.50(m,3H),1.34-1.27(m,2H),0.86(t,J=7.6Hz,3H).

[0551] The intermediate compound F1018-01 was prepared as follows:

[0552] Step 1: F1018-01-01 (541 mg, 1.0 mmol) was dissolved in dichloromethane (10.0 mL) under nitrogen atmosphere. HATU (380 mg, 1.0 mmol) and DIEA (258 mg, 2.0 mmol) were added and stirred at room temperature for 5 minutes. F1018-01-02 (318 mg, 1.0 mmol, HCl salt) was added. After completion of the reaction, the reaction solution was concentrated at 45°C to dryness. The crude product was purified by medium-low pressure reverse phase chromatography (mobile phase: acetonitrile / 0.05% trifluoroacetic acid in water) and lyophilized to obtain 680 mg of compound F1018-01-03 as a clear oil in an 84.4% yield. LCMS (254 nm) revealed a purity of 94.5%, Rt = 1.89 min; MS calculated: 805.4; MS found: 806.5 [M+H] + .

[0553] Step 2: F1018-01-03 (200 mg, 0.24 mmol) was dissolved in 4 M HCl / EA (4.0 mL) and reacted at room temperature for 1.0 hour. After the reaction was completed, the reaction solution was concentrated at 45°C and part of the EA was removed with petroleum ether (5 mL*3) to obtain 174 mg of crude product F1018-01-04, which was directly used in the next step.

[0554] Step 3: F1018-01-04 (174 mg, crude) was dissolved in DMF, and F1018-01-05 (143 mg, 1.24 mmol) and DIEA (160 mg, 1.24 mmol) were added. The mixture was stirred at room temperature for 0.5 h. LCMS confirmed the reaction was complete. Acetic acid was added to quench the reaction until the pH reached acidity. The mixture was concentrated at 45°C using an oil pump. The mixture was then purified by medium-low pressure reverse phase chromatography (mobile phase: acetonitrile / 0.05% trifluoroacetic acid in water) and lyophilized to afford 180 mg of compound F1018-01-06 as a clear oil in an 88.4% yield. LCMS (254 nm) revealed a purity of 93.1%, Rt = 1.95 min; MS calculated: 821.3; MS found: 822.5 [M+H] + .

[0555] Step 4: Dissolve compound F1018-01-06 (180 mg, 0.22 mmol) in DMF (2.0 mL) and add 200 μL of piperidine. Stir the mixture under nitrogen for 0.5 hours. LCMS monitors the reaction for completion. Concentrate the reaction mixture to dryness at 45°C using a pump concentrate. Add petroleum ether (2 mL x 3). Discard the supernatant to obtain 145 mg of compound F1018-01, which was used directly in the next step.

[0556] Intermediate compound F1016-06 was prepared as follows:

[0557] Step 1: F1016-01 (1.0 g, 2.58 mmol) was dissolved in dichloromethane (20.0 mL) under nitrogen protection, and NHS (357 mg, 3.1 mmol) and DCC (640 mg, 3.1 mmol) were added. The mixture was reacted in an ice bath for 1 hour. After the reaction was completed by TLC monitoring, the reaction solution was filtered, and the filter cake was rinsed with DCM (5 mL*3). The filtrate was concentrated to dryness at room temperature, and the crude product was used directly in the next step. The above crude product was dissolved in DMF / H2O=1:1 (20 mL), and NH2-Lys(Boc )-OH (635 mg, 2.58 mmol) and NaHCO₃ (665 mg, 7.75 mmol) were reacted in an ice bath for 2 hours. LCMS confirmed the completion of the reaction. 1 M hydrochloric acid was added to adjust the pH to 1-2, and the mixture was extracted with ethyl acetate (10 mL*3), washed with saturated brine (10 mL*2), dried over anhydrous Na₂SO₄, and concentrated. The crude product was purified by medium-low pressure reverse phase chromatography (mobile phase: acetonitrile / 0.05% trifluoroacetic acid aqueous solution) and lyophilized to obtain 900 mg of compound F1016-02 as a white solid in a 56.7% yield. LCMS (254 nm) revealed a purity of 95.1%, Rt = 2.12 min; MS calculated: 615.2; MS found: 616.3 [M+H] + .

[0558] Step 2: F1016-02 (900 mg, 1.46 mmol) was dissolved in tetrahydrofuran (10.0 mL) under nitrogen protection, and NHS (168 mg, 1.46 mmol) and DCC (300 mg, 1.46 mmol) were added. The mixture was reacted in an ice bath for 1 hour. After the reaction was completed by TLC monitoring, the reaction solution was filtered and the filter cake was rinsed with THF (5 mL*3). The mother liquor was concentrated to dryness at room temperature and used directly in the next step, 1.1 g, crude product; the above concentrate was dissolved in DMF / H2O / acetone = 1:1:1 (10 mL). Glycine (110 mg, 1.46 mmol) and NaHCO₃ (369 mg, 4.38 mmol) were added and reacted in an ice bath for 2 hours. LCMS confirmed the reaction was complete. 1N hydrochloric acid was added to adjust the pH to 1-2, and the mixture was extracted with ethyl acetate (10 mL x 3), washed with saturated brine (10 mL x 2), dried over anhydrous Na₂SO₄, and concentrated. The crude product was purified by medium-low pressure reverse phase chromatography (mobile phase: acetonitrile / 0.05% trifluoroacetic acid aqueous solution) and lyophilized to obtain 800 mg of compound F1016-03 as a white solid in an 81.5% yield. LCMS (254 nm) revealed a purity of 96.1%, Rt = 2.02 min; MS calculated: 672.3; MS found: 673.4 [M+H]+ .

[0559] Step 3: F1016-03 (800 mg, 1.19 mmol) was dissolved in 16 mL of tetrahydrofuran and 4 mL of toluene. Pb(OAc)4 (633 mg, 1.42 mmol) and pyridine (113 mg, 1.42 mmol) were added. The mixture was stirred at 85°C under nitrogen for 2.5 hours. LCMS analysis showed that the starting material was completely consumed. The mixture was concentrated to dryness at 45°C. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 2 / 3) to obtain 460 mg of compound F1016-04 as a white solid in a 56.3% yield. LCMS (254 nm) showed a purity of 93.2%, Rt = 1.85 min; MS calculated: 686.3; MS found: 704.1 [M+NH4]. + . 1 H NMR (400MHz, DMSO-d6) δ8.91(t,J=6.8Hz,1H),8.10(d,J=8.0Hz,1H),7.87(d,J=7.2Hz,2H),7.63-7.58(m,3H),7.42-7.38(m,2H),7.32-7.26 (m,7H),6.72(t,J=4.8Hz,1H),5.11-5.06(m,2H),4.31-4.13(m,5H),3 .03-2.73(m,4H),1.98(s,3H),1.63-1.49(m,2H),1.34-1.18(m,13H).

[0560] Step 4: Compound F1016-04 (176 mg, 0.25 mmol) was dissolved in DMF (5 mL) under nitrogen atmosphere. STI-F6 (120 mg, 0.25 mmol) and PPTS (12 mg, cat. 1) were added sequentially. The mixture was reacted at 95°C for 2 hours. The reaction solution was concentrated to dryness under reduced pressure at 45°C. The crude product was purified by column chromatography (dichloromethane / methanol = 50 / 1) to obtain 50 mg of compound F1016-05 as a yellow solid with a purity of 50%; MS calculated value: 1093.5; MS found value: 1094.6 [M+H] + .

[0561] Step 5: Dissolve compound F1016-05 (60 mg, 0.05 mmol) in DMF (2.0 mL) and add 200 μL of piperidine. Stir the mixture under nitrogen for 0.5 hours. LCMS monitors the reaction for completion. Concentrate the reaction mixture to dryness at 45°C using an oil pump. Add petroleum ether (2 mL x 3). Discard the supernatant and concentrate again to yield 43 mg of compound F1016-06, which was used directly in the next step.

[0562] Example 40: Synthesis of Drug-Linker Conjugate STI-F1019

[0563] Compound F1013-05 (30 mg, 0.03 mmol) from Example 36 was dissolved in DMF (2.0 mL). 200 μL of piperidine was added and the mixture was stirred under nitrogen for 0.5 hours. LCMS monitored the reaction for completion. The reaction mixture was concentrated to dryness at 45°C using an oil pump. Petroleum ether (2 mL x 3) was added to the mixture, and the supernatant was discarded and concentrated again to yield 24 mg of the crude Fmoc-free product.

[0564] F1018-02 (32 mg, 0.03 mmol) in Example 39 was dissolved in DMF (2.0 mL) under nitrogen protection. HATU (22 mg, 0.05 mmol) and DIEA (15 mg, 0.11 mmol) were added and stirred for 5 minutes. The above-mentioned crude product of Fmoc removal was added and the reaction was continued for 1 hour. After the completion of the reaction monitored by LCMS, the reaction solution was concentrated to dryness at 45°C. The crude product was purified by column chromatography (dichloromethane / methanol = 20 / 3) to obtain a condensation product.

[0565] The condensation product was dissolved in 10% TFA / DCM (2 mL) and stirred in an ice bath under nitrogen for 1 hour. LCMS monitored the reaction for completion. The reaction solution was quenched with triethylamine to a pH of 7-8, dried under nitrogen, and purified by high-pressure reverse-phase chromatography (mobile phase: acetonitrile / 0.05% trifluoroacetic acid in water). After lyophilization, 2.87 mg of compound STI-F1019 was obtained as a white solid. LCMS (254 nm) revealed a purity of 99.6%, Rt = 1.51 min; MS calculated: 1455.6; MS found: 1456.7 [M+H]+. 1H NMR (400MHz, DMSO-d6) δ9.11(s,2H),8.78(t,J=6.4Hz,1H),8.41-8.39(d,J=7.6Hz,1H),8.11(d,J=8.0Hz,1H),8.03(t,J =4.2Hz,1H),7.88(s,2H),7.65-7.59(m,3H),7.39(s,1H),6.57(brs,1H),5.48(d,J=38Hz,4H),4.59-4.57(m,2H),4.29- 4.26(m,1H),4.01-3.90(m,3H),3.70-3.59(m,7H),3.45-3.30(m,30H),3.32-3.16(m,4H),3.09-3.07(m,3H),2.77-2.73 (m,2H),2.63-2.53(m,4H),2.44(s,3H),2.32-2.22(m,5H),1.89-1.64(m,12H),1.30-1.23(m,2H),0.87(t,J=7.2Hz,3H).

[0566] Example 41: Synthesis of Drug-Linker Conjugate STI-G1001

[0567] Step 1: Compound STI-G5 (140 mg, 0.275 mmol) was dissolved in 2.8 mL of N,N-dimethylacetamide, and 25b (152 mg, 0.42 mmol) and PPTS (7 mg, 0.0275 mmol) were added. The mixture was reacted at 80°C under nitrogen for 4 hours. After completion, the reaction mixture was dried by rotary evaporation, slurried with acetone, filtered, and the filtrate was dried by rotary evaporation and purified by column chromatography to obtain approximately 100 mg of crude compound G1001-01 as a yellow solid in approximately 40% yield. LCMS (254 nm) Rt = 1.918 min; MS calcd: 817.3; MS found: 816.3 [MH] - .

[0568] Step 2: Compound G1001-01 (100 mg, 0.122 mmol) was placed in a reaction flask, and 2 mL of a 10% piperidine / dichloromethane solution was added. The reaction was allowed to react at room temperature for 30 minutes. After completion, the reaction mixture was spin-dried, and the crude product was slurried with acetone and filtered. The filter cake yielded G1001-02, approximately 50 mg, as a yellow solid, with a yield of approximately 68%. LCMS (254 nm) Rt = 1.539 min; MS Calcd: 595.24; MS Found: 596.30 [M+H] + .

[0569] Step 3: Compound G1001-02 (50 mg, 0.084 mmol) was placed in a reaction flask, and F1006-S3 (75 mg, 0.084 mmol) from Example 37 was added. Dissolved in 1 mL of DMF, followed by DIPEA (30 μL, 0.168 mmol) and HATU (60 mg, 0.168 mmol). The mixture was allowed to react at room temperature for 30 minutes. After completion of the reaction, the reaction solution was spin-dried and subjected to high-pressure preparative separation (mobile phase: acetonitrile / 0.05% formic acid in water). After lyophilization, approximately 15 mg of compound STI-G1001 was obtained as a yellow solid. LCMS (254 nm) revealed a purity of 97.5%, Rt = 1.620 min; MS calculated: 1472.68; MS found: 737.50 [M / 2+H]. + . 1 H NMR (400MHz, DMSO-d6) δ9.78 (s, 1H), 9.70 (s, 1H), 8.95 (d, J = 8.4Hz, 1H), 8.87 (d,J=8.4Hz,1H),8.72(t,J=6.8Hz,1H),8.33(t,J=5.6Hz,1H),8.18-8.11(m, 2H),8.04-7.99(m,2H),7.81-7.99(m,1H),7.30(s,2H),7.22(s,2H),6.99(s, 1H),6.52(brs,1H),5.43(s,2H),5.29(s,2H),4.74(d,J=6.8Hz,1H),4.51-4. 47(m,1H),4.22(s,2H),4.15(s,2H),3.80-3.76(m,2H),3.72-3.67(m,2H),3. 64-3.57(m,2H),3.49(s,34H),3.24-3.15(m,2H),3.14-3.10(m,2H),3.05-3. 01(m,2H),2.38(t,J=6.4Hz,2H),2.04-1.99(m,2H),1.90-1.81(m,2H),1.73( s,2H),1.47-1.44(m,4H),1.40-1.36(m,2H),1.23(s,4H),0.91-0.87(m,6H).

[0570] Example 42: Synthesis of Drug-Linker Conjugate STI-F1031

[0571] Step 1: Compound F1031-01 (2.88 g, 12.0 mmol) was dissolved in acetonitrile (48.0 mL). Sodium carbonate (1272 mg, 12.0 mmol) was added dropwise under nitrogen. A solution of tert-butyl bromoacetate (585 mg, 3.0 mmol) in acetonitrile was added dropwise. The mixture was allowed to react at room temperature for 24 hours. The reaction was terminated, filtered, and the filtrate was concentrated under reduced pressure. The mixture was extracted with ethyl acetate and water. The organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (PE:EA = 1:1) to afford 1.1 g of compound F1031-02 as a yellow solid in a 25.9% yield. LCMS (254 nm) revealed a purity of 93.2%, Rt = 1.816 min; MS calculated: 354.2; MS found: 355.3 [M+H] + .

[0572] Step 2: Compound F1031-02 (1200 mg, 3.39 mmol) was dissolved in acetonitrile (20.0 mL). Sodium carbonate (1030 mg, 10.0 mmol) was added. Under nitrogen, a solution of benzyl bromoacetate (1214 mg, 0.5.3 mmol) in acetonitrile was added dropwise. The mixture was allowed to react at room temperature for 1 hour. The reaction was terminated, filtered, and the filtrate was concentrated under reduced pressure. Purification by column chromatography (PE:EA = 30:1) afforded 1.5 g of compound F1031-01b-1 as a yellow oil in an 88.2% yield. LCMS (254 nm) revealed a purity of 93.2%, Rt = 2.421 min; MS calculated: 502.2; MS found: 503.6 [M+H] + .

[0573] Step 3: Compound F1031-01b-1 (1.0 g, 2.0 mmol) was dissolved in methanol (20.0 mL), and 10% palladium on carbon (100 mg) was added. The atmosphere was replaced with hydrogen, and the reaction was allowed to react at room temperature overnight. LCMS monitored the reaction completion, and the reaction was stopped, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 445 mg of compound F1031-02b as a colorless oily liquid, with a yield of 95.9%. LCMS (254 nm) showed a purity of 91.0%, Rt = 1.910 min; MS calculated: 232.1; MS found: 233.2 [M+H] + .

[0574] Step 4: 4,7,10,13,16,19,22,25-Octoxahexacosaic acid (412 mg, 1.0 mmol) and pentafluorophenol (221 mg, 1.2 mmol) were added to tetrahydrofuran (5.0 mL) under nitrogen protection. The temperature was lowered to 0-5°C. N,N'-dicyclohexylcarbodiimide (247 mg, 1.2 mmol) was added and the mixture was reacted at room temperature for 4 hours. The mixture was filtered and the filtrate was concentrated to dryness under reduced pressure. Compound F1031-02b (126 mg, 0.5 mmol) and DMF (5.0 mL) were added under nitrogen protection. DIPEA (258 mg, 2.0 mmol) was added and the mixture was reacted at room temperature overnight. The reaction was monitored to be complete by LCMS. The system was concentrated to dryness under reduced pressure. The crude product (F1031-03b) was used directly in the next step.

[0575] Step 5: The crude product from the previous step was redissolved in DMF (5.0 mL), and F1031-S3, HATU (570 mg, 1.5 mmol), and DIPEA (387 mg, 3.0 mmol) were added. The reaction was allowed to proceed at room temperature under nitrogen for 1 hour. LCMS monitored the reaction for completion. The reaction was stopped, concentrated to dryness, and lyophilized using reverse phase prep (mobile phase: acetonitrile / 0.05% trifluoroacetic acid in water) to obtain 240 mg of compound F1031-04b as a colorless oil in a 40.2% yield. LCMS (254 nm) revealed a purity of 94.1%, Rt = 1.526 min; MS calculated: 1184.7; MS found: 1183.35 [MH] - .

[0576] Step 6: Compound F1031-04b (100 mg, 0.084 mmol) was dissolved in 25% trifluoroacetic acid in dichloromethane and stirred at room temperature for approximately 2 hours. The reaction was complete as monitored by LCMS. The product was then concentrated to dryness under reduced pressure to afford 130 mg of compound F1031-08 as a colorless oily liquid in a 100.0% yield. LCMS (254 nm) purity: 95.1%, Rt = 1.513 min; MS calculated: 1128.6; MS found: 1127.7 [MH] - .

[0577] Step 7: Compound F1010-03 (40 mg, 0.045 mmol) in Example 33 was dissolved in DMF (0.9 mL), piperidine (0.1 mL) was added, and the reaction was carried out at room temperature for 20 minutes. The system was concentrated to dryness, and the residue was washed with PE and dried under reduced pressure. DMF (2.0 mL), HATU (34 mg, 0.09 mmol), F1031-08 (56 mg, 0.05 mmol) and triethylamine (23 mg, 0.23 mmol) were added to the residue. The mixture was protected by nitrogen and reacted at room temperature for 1.0 hour. The reaction was stopped and the system was concentrated to dryness. The mixture was prepared by medium- and high-pressure reverse phase (mobile phase: acetonitrile / 0.05% trifluoroacetic acid aqueous solution) and lyophilized to obtain 17 mg of compound STI-F1031 as a light yellow solid with a yield of 21.3%. LCMS (254 nm) purity 98.58%, Rt = 1.623 min; MS calculated value: 1776.9; MS found value: 1776.0 [MH] - . 1 H NMR (400MHz, DMSO-d6) δ8.63–8.56(m,1H),8.48–8.35(m,2H),8.15(t,J=6.8 Hz,1H),8.10–8.04(m,1H),8.00(d,J=6.5Hz,1H),7.60(d,J=10.6,1H),7.37 (s,1H),6.98(s,2H),6.55(br,1H),5.48(s,2H),5.43(s,2H),4.72–4.69(m, 1H),4.56–4.46(m,2H),4.25–4.18(m,3H),4.12(t,J=4.6Hz,1H),4.08–3.96( m,2H),3.70–3.65(m,3H),3.62–3.58(m,4H),3.51–3.46(m,63H),3.23(s,6H) ),3.05–2.99(m,4H),2.61–2.56(m,4H),2.44(s,3H),2.04–1.97(m,2H),1.88 –1.83(m,2H),1.74(q,J=7.4Hz,2H),1.64(q,J=6.8Hz,2H),1.51–1.42(m,2H ), 1.21 (d, J = 7.1Hz, 3H), 0.87 (t, J = 7.2Hz, 3H), 0.82 (dd, J = 11.2, 4.9Hz, 6H).

[0578] Intermediate compound F1031-S3 was prepared as follows:

[0579] Step 1: Add tert-butyl N-(5-aminopentyl)carbamate (1414 mg, 7.0 mmol) to saturated sodium bicarbonate (35.0 mL), cool to 0-5°C, add F1031-S1 (1085 mg, 7.0 mmol), return to room temperature, and react for 15 minutes. Tetrahydrofuran (55.0 mL) is then added and allowed to react for another 30 minutes at room temperature to terminate the reaction. 50 mL of water and EA are added, and the mixture is extracted. The organic phase is dried over anhydrous sodium sulfate and purified by column chromatography (DCM:EA = 20:1) to afford 1.1 g of compound F1031-S2 as a white solid in a 55.7% yield. LCMS (254 nm) purity: 93.2%, Rt = 1.723 min; MS calcd: 282.2; MS found: 283.1 [M+H] + .

[0580] Step 2: Compound F1031-S2 (423 mg, 1.5 mmol) was dissolved in 25% trifluoroacetic acid in dichloromethane and stirred at room temperature for about 2 hours. The reaction was completed as monitored by LCMS. The mixture was concentrated to dryness under reduced pressure to obtain F1031-S3, which was used directly in the next step.

[0581] Example 43: Synthesis of Drug-Linker Conjugate STI-F1034

[0582] Step 1: Compound STI-F14 (276 mg, 0.56 mmol), compound F1010-02 from Example 33 (269 mg, 0.56 mmol), and PPTS (20 mg) were added to a reaction flask. DMF (10 mL) was added and the mixture was heated to 90°C under nitrogen for 5 hours. The reaction was then terminated. The mixture was concentrated to dryness under reduced pressure and purified by column chromatography (DCM:MeOH = 100:1 to 60:1) to obtain 154 mg of compound F1033-01 as a pale yellow solid in a 30.0% yield. LCMS (254 nm) revealed a purity of 76.8%, Rt = 2.043 min; MS calcd: 914.4; MS found: 915.4 [M+H] + .

[0583] Step 2: Compound F1033-01 (154 mg, 0.17 mmol) was dissolved in 3 mL of DMF, and 300 μL of piperidine was added. The reaction was stirred under nitrogen for 0.5 hours. LCMS monitored the reaction for completion. The reaction solution was concentrated to dryness at 45°C, then slurried with petroleum ether and filtered to obtain 100 mg of compound F1033-02 as a pale yellow solid in an 85.0% yield. LCMS (254 nm) revealed a purity of 98.7%, Rt = 1.692 min; MS calculated: 692.3; MS found: 693.4 [M+H] + .1 H NMR (400MHz, DMSO-d6) δ8.74(t,J=6.6Hz,1H),8.13(s,1H),7.91(d,J=8.4Hz,1H),7.76(d,J=10.7Hz,1 H),7.27(s,1H),6.51(s,1H),5.46(s,2H),5.43(s,2H),4.67–4.55(m,2H),4.37–4.29(m,1H),3.63(d,J =11.2Hz,2H),3.44–3.37(m,4H),3.07–3.01(m,1H),2.48(s,3H),1.98–1.88(m,2H),1.89–1.79(m,4H) ,1.60–1.46(m,2H),1.26(d,J=7.0Hz,3H),1.24–1.14(m,2H),0.90–0.86(m,6H),0.79(d,J=6.8Hz,3H).

[0584] Step 3: Compound F1033-02 (67 mg, 0.097 mmol), F1031-08 (120 mg, 0.106 mmol) from Example 42, and HATU (74 mg, 0.194 mmol) were dissolved in 3 mL of DMF. DIPEA (82 μL, 0.485 mmol) was added, and the mixture was reacted under nitrogen for 1 hour at room temperature. The mixture was concentrated to dryness and lyophilized using a medium- and high-pressure reverse-phase preparative method (mobile phase: acetonitrile / 0.05% trifluoroacetic acid in water) to afford 12.5 mg of compound STI-F1034 as a yellow oil in a 6.9% yield. LCMS (254 nm) revealed a purity of 100%, Rt = 1.684 min; MS calculated: 1802.9; MS found: 1803.7 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ8.47(s,1H),8.25–8.17(m,1H),8.13–7.99(m,2H),7.92(d,J=8.5Hz,1H),7.77(d,J=10.8Hz ,1H),7.28(s,1H),6.99(s,2H),6.50(s,1H),5.48(s,2H),5.42(s,2H),4.66–4.50(m,7H),4.32–4.19(m,2H),3.67– 3.44(m,66H),3.23(s,6H),3.09–2.93(m,3H),2.70–2.57(m,4H),2.48(s,3H),2.44–2.36(m,4H),2.36–2.30(m,1H) ,2.05–1.94(m,2H),1.93–1.78(m,5H),1.44–1.34(m,2H),1.31–1.21(m,5H),1.23–1.14(m,2H),0.93–0.80(m,9H).

[0585] Example 44: Synthesis of Drug-Linker Conjugate STI-F1035

[0586] Step 1: 4,7,10,13,16,19,22,25-octaoxahexaconic acid (412 mg, 1.0 mmol) and pentafluorophenol (221 mg, 1.2 mmol) were added to tetrahydrofuran (5.0 mL) under nitrogen protection, cooled to 0-5°C, and N,N'-dicyclohexylcarbodiimide (247 mg, 1.2 mmol) was added. The mixture was reacted at room temperature for 4 hours, filtered, and the filtrate was concentrated to dryness under reduced pressure. Compound F1031-02b (100 mg, 0.43 mmol) and DMF (3.0 mL) were added thereto under nitrogen protection, and DIPEA (111 mg The reaction was continued overnight at room temperature. LCMS monitored the reaction to be complete. The system was then concentrated to dryness under reduced pressure and the residue was redissolved in DMF (5.0 mL). F1035-S4, HATU (327 mg, 0.86 mmol), and DIPEA (166 mg, 1.29 mmol) were added under nitrogen protection and allowed to react at room temperature for 1 hour. LCMS monitored the reaction to be complete. The reaction was stopped and the system was concentrated to dryness. Reverse phase preparation (mobile phase: acetonitrile / 0.05% trifluoroacetic acid aqueous solution) was performed and lyophilized to obtain 179 mg of compound F1035-01 as a colorless oily liquid in a 33.5% yield. LCMS (254 nm) purity was 98.0%, Rt = 1.537 min; MS calculated value: 1241.6; MS found value: 1240.6 [MH] - .

[0587] Step 2: Compound F1035-01 (59 mg, 0.05 mmol) was dissolved in 25% trifluoroacetic acid in dichloromethane and stirred at room temperature for about 5 hours. The reaction was completed as monitored by LCMS. The mixture was concentrated to dryness under reduced pressure to obtain the deprotected product of F1035-01. Compound F1010-03 (40 mg, 0.045 mmol) in Example 33 was dissolved in DMF (0.9 mL), and piperidine (0.1 mL) was added. The mixture was reacted at room temperature for 20 minutes. The system was concentrated to dryness, and the residue was washed with petroleum ether and dried under reduced pressure. The deprotected product of F1035-01, DMF (2.0 mL), HATU (34 mg, 0.09 mmol), and triethylamine (23 mg, 0.23 mmol) were added to the residue. The mixture was reacted at room temperature for 0.5 hours under nitrogen protection. The reaction was stopped, and the system was concentrated to dryness. The mixture was then subjected to medium- and high-pressure reverse phase preparation (mobile phase: acetonitrile / 0.05% trifluoroacetic acid aqueous solution). After lyophilization, 20 mg of compound STI-F1035 was obtained as a yellow solid in a yield of 24.2%. LCMS (254 nm) purity 95.23%, Rt = 1.633 min; MS calculated value: 1833.9; MS found value: 1833.0 [MH] - . 1 H NMR(400MHz, DMSO-d6)δ9.10(s,2H),8.60(t,J=6.0Hz,1H),8.45–8.40(m,2H),8.16(t,J=6.7Hz,1H),8.08(d,J=8.5Hz,1H),7.91–7.80(m,1H),7. 58(d,J=10.5Hz,1H),7.41(s,1H),6.58(br,1H),5.47(s,2H),5.44(s,2H ),4.56–4.49(m,2H),4.26–4.18(m,3H),4.13–4.11(m,1H),4.05–3.95(m, 2H),3.73–3.68(m,2H),3.61–3.56(m,4H),3.51–3.46(m,65H),3.40(s,6 H),3.23(s,3H),2.63–2.55(m,4H),2.43(s,3H),2.41–2.39(m,2H),2.01 –1.91(m,2H),1.90–1.83(m,2H),1.76–1.73(m,2H),1.68–1.60(m,2H),1 .23(d,J=4.6Hz,3H), 0.87(t,J=7.4Hz,3H), 0.82(dd,J=12.7,6.5Hz,6H).

[0588] Intermediate compound F1035-S4 was prepared as follows:

[0589] Step 1: F1035-S1 (303 mg, 1.5 mmol), N-Boc-4-pentyn-1-amine (330 mg, 1.8 mmol), cuprous iodide (30 mg, 0.3 mmol) and bistriphenylphosphine palladium dichloride (35 mg, 0.3 mmol) were added to a reaction flask under nitrogen protection. DMF (2.0 mL) and triethylamine (223 mg, 2.25 mmol) were added, and the temperature was raised to 95°C for 5 hours. The reaction was stopped, and water and EA were added to the system for extraction. The EA layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. The mixture was purified by column chromatography (PE:EA=100:1 to PE:EA=10:1) to obtain 330 mg of compound F1035-S2 as a yellow solid with a yield of 71.6%. LCMS (254 nm) purity 95.0%, Rt = 1.968 min; MS calculated value: 307.1; MS found value: 308.6 [M+H] + .

[0590] Step 2: Dissolve F1035-S2 (300 mg, 0.98 mmol) in dichloromethane under nitrogen. Add m-chloroperbenzoic acid (507 mg, 2.94 mmol) and stir at room temperature overnight. Stop the reaction after monitoring the reaction completion by LCMS. The system was concentrated to dryness under reduced pressure and purified by column chromatography (PE:EA = 20:1 to PE:EA = 2:1) to obtain 260 mg of compound F1035-S3 as a yellow solid in a 78.3% yield. LCMS (254 nm) revealed a purity of 96.0%, Rt = 1.532 min; MS calculated: 339.1; MS found: 338.2 [MH] - .

[0591] Step 3: Compound F1035-S3 (146 mg, 0.43 mmol) was dissolved in 25% trifluoroacetic acid in dichloromethane and stirred at room temperature for about 2 hours. The reaction was completed as monitored by LCMS. The mixture was concentrated to dryness under reduced pressure to obtain F1035-S4, which was used directly in the next reaction.

[0592] Example 45: Synthesis of Drug-Linker Conjugate STI-F1043

[0593] Step 1: F1043-01 (1.0 g, 2.13 mmol) was dissolved in 4 M HCl / EA (10.0 mL) and reacted at room temperature for 1.5 hours. After the reaction was completed as monitored by TLC, the reaction solution was concentrated at 45 ° C to obtain a crude de-Boc product; F1043-02 (220 mg, crude product) and the above de-Boc product (178 mg, 0.48 mml) were dissolved in THF / H2O (2 mL:2 mL), and NaHCO3 (203 mg, 2.42 mmol) was added. The mixture was stirred in an ice bath for 2 hours. After the reaction was completed as monitored by LCMS, 1 M The pH was adjusted to 3-4 with HCl, extracted with ethyl acetate (10 mL x 2), dried with saturated brine (10 mL x 2), dried over anhydrous sodium sulfate, concentrated at 45°C, and directly subjected to medium-low pressure reverse phase preparation (mobile phase: acetonitrile / 0.05% trifluoroacetic acid aqueous solution). Lyophilization afforded 277 mg of compound F1043-03 as a clear oil, with a yield of 74.9%. LCMS (254 nm) purity was 93.6%, Rt = 1.96 min; MS calculated: 762.3; MS found: 763.5 [M+H] + .

[0594] Step 2: F1043-03 (100 mg, 0.13 mmol) was dissolved in dichloromethane (20.0 mL) under nitrogen protection. NHS (18 mg, 0.15 mmol) and DCC (32 mg, 0.15 mmol) were added and the mixture was reacted in an ice bath for 1 hour. After the reaction was completed as monitored by TLC, the reaction solution was filtered and the filter cake was rinsed with DCM (5 mL*3). The filtrate was concentrated at room temperature. The resulting crude product and F1033-02 (90 mg, 0.13 mmol) were dissolved in DMF (4 mL). DIEA (33 mg, 0.26 mmol) was added and stirred in an ice bath for 2 hours. After the reaction was completed as monitored by LCMS, the mixture was concentrated at 45°C using an oil pump. The crude product was purified by column chromatography (dichloromethane / methanol = 25 / 3) to obtain 110 mg of compound F1043-04 as a yellow solid with a yield of 58.9%. LCMS (254 nm) purity 91.2%, Rt = 1.85 min; MS calculated value: 1436.7; MS found value: 1437.9 [M+H] + .

[0595] Step 3: Compound F1043-04 (110 mg, 0.07 mmol) was dissolved in DMF (2.0 mL), and 200 μL of piperidine was added. The reaction was stirred under nitrogen for 0.5 hours. LCMS monitored the reaction for completion. The reaction mixture was concentrated to dryness at 45°C, then slurried with petroleum ether and filtered. The filter cake and EMCS (16 mg, 0.07 mmol) were dissolved in DMF (2 mL), and DIEA (19 mg, 0.15 mmol) was added under nitrogen. The mixture was stirred at room temperature for 2 hours. LCMS monitored the reaction for completion. The crude product was concentrated to dryness at 45°C, and purified by high-pressure reverse phase chromatography (mobile phase: acetonitrile / 0.05% trifluoroacetic acid in water). After lyophilization, 7.18 mg of compound STI-F1043 was obtained as a yellow solid in a 6.6% yield. LCMS (254 nm) purity 99.0%, Rt = 1.71 min; MS calculated value: 1407.7; MS found value: 1408.6 [M+H] + . 1 H NMR (400MHz, DMSO).δ8.81(s,1H),8.64(t,J=6.4Hz,1H),8.06(d,J=6.8Hz,1H),7.95-7.88(m,2H),7.80-7.72(m,2H),7.64( d,J=8.8Hz,1H),7.26(s,1H),6.99(s,2H),5.43(d,J=9.6Hz,4H),4.64-4.55(m,2H),4.29-4.15(m,3H),3.58-3.55(m,22H), 3.49-3.43(m,10H),3.41-3.33(m,5H),3.23(s,4H),3.00-2.98(m,2H),2.47(s,4H ), 2.28 (t, J = 6.4Hz, 2H), 1.89-1.80 (m, 4H), 1.65-1.13 (m, 20H), 0.89-0.81 (m, 9H).

[0596] Intermediate compound F1043-02 was prepared as follows:

[0597] F1043-02a (200 mg, 0.48 mmol) was dissolved in dichloromethane (20.0 mL) under nitrogen protection. NHS (67 mg, 0.58 mmol) and DCC (120 mg, 0.58 mmol) were added and reacted in an ice bath for 2 h. After the completion of the reaction monitored by TLC, the reaction solution was filtered, washed with dichloromethane, and the filtrate was concentrated to dryness at room temperature and used directly in the next step.

[0598] Example 46: Preparation of Antibodies

[0599] Farletuzumab is an anti-folate receptor α (FolRα, FRα) antibody, and its sequence information is as follows:

[0600] Fatuinomab heavy chain sequence:

[0601] Fatuinomab light chain sequence:

[0602] Trastuzumab is an anti-Her2 antibody, and its sequence information is as follows:

[0603] Trastuzumab light chain sequence:

[0604] Trastuzumab heavy chain sequence:

[0605] The isotype is Anti-HEL Human IgG1 antibody, and its sequence information is as follows:

[0606] The light chain sequence of Anti-HEL Human IgG1 monoclonal antibody:

[0607] The heavy chain sequence of Anti-HEL Human IgG1 monoclonal antibody:

[0608] After constructing the above sequence vector, sequencing confirmed that the constructed sequence was consistent with the above sequence. The protein was expressed in Expi293F cells (Thermofisher Catalog No.: A1452) and purified using Protein A (Cytiva, Catalog No.: 17549801).

[0609] Example 47: Preparation and Detection of Antibody Drug Conjugates

[0610] Antibody-drug conjugates (ADCs) were synthesized using the camptothecin derivatives of the present invention, and their DAR values ​​were detected.

[0611] Specifically, the purified antibody (SEC-HPLC purity greater than 95%) is added to 10mM PBS buffer, and 500mM EDTA is added to the reaction system to obtain an EDTA concentration of 2mM, and the antibody concentration is about 1-5mg / ml. 5-8 equivalents of TCEP (TCEP / mAb) are added, stirred evenly, and reacted at 37°C and 600 rpm for 2 hours. After the reaction is completed, the reaction solution is cooled to 4°C, and a drug-linker conjugate solution dissolved in DMSO (10-16eq, drug / mAb) is added, mixed evenly, and reacted at 4°C and 600 rpm for 1 hour. After the reaction is completed, the reaction solution is added to a 30K ultrafiltration tube, concentrated to an appropriate volume, added to a balanced desalting column, and centrifuged to obtain a purified conjugated sample. The concentration, purity, and DAR value of the sample are detected. The DAR value can be detected and calculated using the following method:

[0612] 1. RP-HPLC detection method

[0613] Add 75uL 8M guanidine hydrochloride and 5uL 1M Tris to a centrifuge tube, add an appropriate amount of sample (40ug) (converted to volume according to the concentration), and finally add water to a final volume of 100μL. Add 2uL 1M DTT, then vortex to mix, and incubate at 30℃ for 30min. After incubation, return to room temperature, centrifuge at 13000rpm for 5min, and transfer the supernatant to a liquid injection bottle. Use Vanquish high-performance liquid chromatography, and the chromatographic column is selected from BioResolveTM RP mAb, Polyphenyl, A 2.7 μm, 2.1 x 100 mm column (Part No. 186008945, Serial No. 01293033515811) was used, with a column temperature of 80°C; a DAD detector wavelength of 280 nm; a flow rate of 1 ml / min; and an injection volume of 20 μg. The heavy and light chains were then identified by comparing the naked antibody with the sample. The sample spectrum was then integrated to calculate the DAR value.

[0614] Mobile phase configuration:

[0615] Reversed phase column mobile phase A: 0.05% TFA + 0.05% FA in H2O

[0616] Reversed phase column mobile phase B: 0.05% TFA + 0.05% FA in ACN

[0617] Data analysis: Based on the positions of the light and heavy chains, the spectra of the test samples were integrated and the DAR value was calculated.

[0618] The calculation formula is as follows:

[0619] Sum of LC peak areas = LC peak area + LC+1 peak area

[0620] Total HC peak area = HC peak area + HC+1 peak area + HC+2 peak area + HC+3 peak area

[0621] LC DAR = Σ(number of connected drugs * peak area percentage) / total LC peak area

[0622] HC DAR = Σ(number of connected drugs * peak area percentage) / total HC peak area

[0623] DAR=LC DAR+HC DAR

[0624] 2.SEC-HPLC detection method

[0625] The sample was diluted to 1 mg / mL with ultrapure water, the injection volume was 20 uL, and a high performance liquid chromatography Vanquish column was used. SEC 3.5um, 7.8*150mm Column (Part No.186007639, Serial No.01783201716107); column temperature 25°C; DAD detector wavelength 280nm; flow rate: 0.5ml / min.

[0626] 3. Mass spectrometry detection method

[0627] About 25 μg of protein was diluted to 0.5 μg / μL with 50 mM NH4HCO3, 0.5 μL PNGaseF and 1 μL 1MDDT were added, and the mixture was incubated at 37°C for 60 minutes. 0.5 μg was injected for mass spectrometry analysis. A Waters Xevo G2-XS Q-TOF was used, and the chromatographic column was ACQUITY UPLC BEH C4, 1.7 μm, 2.1 x 50 mm, (186004495), column temperature 80°C; DAD detector wavelength 280 nm; flow rate: 0.3 ml / min; DAR value was calculated based on the response signal of the sample mass spectrometer.

[0628] The antibody drug conjugate of the present invention was synthesized by the above method and its DAR value was measured. The results are shown in Table 1.

[0629] Table 1. Prepared ADCs and their characterization

[0630] Note:

[0631] 1) In Fa-ADCs, mAb represents fatuinumab (Fa); in Tra-ADCs, mAb represents trastuzumab (Tra); in Iso-ADCs, mAb represents Anti-HEL Human IgG1 antibody, and n represents an integer from 0 to 8, e.g., 0, 1, 2, 3, 4, 5, 6, 7, or 8.

[0632] 2) The drug-linker conjugate MC-GGFC-DXD in the ADC reference was purchased from Shanghai Haoyuan Biotechnology Co., Ltd.

[0633] Biology section

[0634] Example A. Camptothecin derivative toxin killing experiment

[0635] Tumor cells (human ovarian cancer cell line SK-OV-3; human colon cancer cell line HCT116; human lung adenocarcinoma cell line A549) in logarithmic phase were digested and the cell density was adjusted to approximately 3×10 4 Cells / mL. The cells were plated into a 96-well plate with a white bottom at about 100 μL / well. The camptothecin derivative toxin of the embodiment of the present application was gradiently diluted with the corresponding cell culture medium and mixed with the cells, and cultured in a 37°C, 5% CO2 incubator for 3 days. The growth inhibition of tumor cells by camptothecin derivative toxin was evaluated by Cell Titer Turbo 2.0 Luminescent Cell Viability Assay (Damas life, RA-GL11). The relative light unit (RLU) value was read on the cell culture plate using an enzyme reader. The experimental data was analyzed using Graphpad Prism 8.0 software to calculate the IC 50 The above experiment was repeated using the following compounds as positive controls:

[0636] The results are shown in Table 2. It can be seen that the compounds of the Examples of the present application exhibited good tumor cell killing activity. In particular, the tumor cell killing activities of the compounds of Examples 3 (STI-A), 4 (STI-A2), 6 (STI-E-05), 8 (STI-F-8), 10 (STI-F-03C-1), 13 (STI-G2-06C), and 16 (STI-F) were comparable to those of SN-38 and Dxd, and the tumor cell killing activities of the compounds of Examples 11 (STI-G1), 14 (STI-G2), 17 (STI-F6), and 19 (STI-F13) were even significantly better than those of SN-38 and Dxd.

[0637] Table 2 Cytotoxicity of camptothecin derivatives against tumor cells

[0638] NT means not tested.

[0639] Example A': Camptothecin derivative toxin killing experiment

[0640] The camptothecin derivatives of the examples were serially diluted with DMSO and corresponding cell culture medium and then added to a white-bottomed 96-well plate. Tumor cells (human ovarian cancer cell line SK-OV-3; human colon cancer cell line HCT116; human lung adenocarcinoma cell line A549; human lung adenocarcinoma cell line NCI-H441; human oral epidermoid carcinoma cell line KB) in logarithmic phase were digested and the cell density was adjusted to approximately 3.33×10 4 / mL. Cells were plated at 90 μL / well into a white-bottomed 96-well plate with sample added and cultured in a 37°C, 5% CO2 incubator for 3 days. The growth inhibition of tumor cells by small molecule compounds was evaluated using the Cell Titer Turbo 2.0 Luminescent Cell Viability Assay (Damas life, RA-GL11). Relative light units (RLU) were read on the cell culture plate using a microplate reader. Graphpad Prism 8.0 software was used to analyze the experimental data and calculate the IC 50 The results are shown in Table 3.

[0641] Table 2 Cytotoxicity of camptothecin derivatives against tumor cells

[0642] NT means not tested.

[0643] Example B. ADC Killing Experiment

[0644] After digestion of tumor cells in logarithmic phase growth, the cell density was adjusted to approximately 1-10x10 4 / mL. The cells were plated into a 96-well plate with a white bottom at 50 μL / well and cultured overnight in a 37°C, 5% CO2 incubator. The ADCs or reference ADCs of the present application were diluted with the cell matrix corresponding to the cell culture and mixed with the cells, and cultured in a 37°C, 5% CO2 incubator for 5-7 days. The growth inhibition of tumor cells by ADC was evaluated by Cell Titer Turbo 2.0 Luminescent Cell Viability Assay (Damas life, RA-GL11). The relative light unit (RLU) value was read on the cell culture plate using an enzyme-linked microplate reader. The experimental data were analyzed using Graphpad Prism 8.0 software to calculate the IC50 As shown in Table 3, the killing activity of Fa-ADC1 against oral epidermoid KB tumor cells was significantly better than that of the positive control Fa-ADC reference.

[0645] Table 3. Cytotoxicity of ADCs against human oral epidermoid KB tumor cells

[0646] N / A means not applicable

[0647] In addition, in vitro drug killing experiments were performed using the Her2 endogenously expressing tumor cell lines NCI-N87 (gastric cancer cell line, Nanjing Kebai, CBP60491) and Capan-1 (pancreatic cancer cell line, Nanjing Kebai, CBP60543).

[0648] NCI-N87 and Capan-1 cells were obtained by digestion with trypsin (Gibco, 25200072). NCI-N87 cells were resuspended in RPMI-1640 medium containing 10% fetal bovine serum (FBS, Gibco, 10091-148) and the cell concentration was adjusted to 2×10 4 Cells / mL were added to a 96-well microplate with a white bottom and a transparent well (Perkin Elmer, 6005181) at a concentration of 100 μL per well. Capan-1 was resuspended in RPMI-1640 medium containing 20% ​​FBS and the cell concentration was adjusted to 4×10 4 Cells / mL were added to a white 96-well microplate at a density of 4,000 cells / mL at 100 μL per well. 200 μL of DPBS was added to the periphery of each well of the 96-well microplate. The 96-well plate was placed in a 37°C, CO2 incubator and cultured for 1 day.

[0649] Add the ADCs of the examples or reference ADCs serially diluted in RPMI-1640 medium containing 10% FBS. For NCI-N87 cells, start with a concentration of 600 nM (6*), perform a 5-fold serial dilution, and add 20 μL per well to the 96-well plate from the previous step. The total volume per well is 120 μL. For Capan-1 cells, start with a concentration of 3000 nM (6*), perform a 5-fold serial dilution, and add 20 μL per well to the 96-well plate from the previous step. The total volume per well is 120 μL. Place the 96-well plate in a 37°C, CO2 incubator for 4-5 days.

[0650] On day 5, remove the 96-well plate containing Capan-1 and Cell Titer Turbo 2.0 (Adama Life Sciences, RA-GL11) reagent from the incubator and allow them to return to room temperature. Then, add 60 μL per well of the 96-well plate and incubate at room temperature for 10 minutes. After 10 minutes, read the fluorescence signal on a Microplate Reader (BMG, CLARIOstar Plus).

[0651] On day 6, remove the 96-well plate containing NCI-N87 and Cell Titer Turbo 2.0 reagent from the incubator and allow them to return to room temperature. Then, add 60 μL per well of the 96-well plate and incubate at room temperature for 10 minutes. After 10 minutes, read the fluorescence signal on a microplate reader.

[0652] As shown in Figures 1a to 1d (killing experiments of ADC drugs on Capan-1) and Figures 2a to 2d (killing experiments of ADC drugs on NCI-N87), the ADC drugs of the present invention exhibited in vitro killing activity on both NCI-N87 and Capan-1.

[0653] Example C. Bystander Effect of ADCs

[0654] After digestion of antigen-positive cells (folate receptor α-positive cells KB) and antigen-negative cells (293T cells) in logarithmic phase growth, the cells were adjusted to an appropriate cell density. Antigen-positive cells and antigen-negative cells were added to 96-well white low-transparency plates in different proportions and placed in a 37°C, 5% CO2 incubator for cell culture overnight. The next day, the ADC was gradiently diluted with the corresponding cell culture medium and added to the 96-well plate with cells at 100 μL / well, and cultured in a 37°C, 5% CO2 incubator for 5-7 days. The growth inhibition of tumor cells by ADC was evaluated by Cell Titer Turbo 2.0 Luminescent Cell Viability Assay (Adamas life, RA-GL11-A). The relative light unit (RLU) value was read on the cell culture plate using a microplate reader. The ADCs of the embodiments of the present application have a strong bystander effect.

[0655] CHOK1-EGFP cells (a Chinese hamster ovary cell substrain, target-negative, laboratory-generated) and OE19 cells (human esophageal carcinoma cells, target-positive, Kebai CBP60495) were obtained by trypsinization, resuspended and counted, and OE19 cells were seeded into 96-well plates at a density of 0-8000 cells / well, while CHOK1-EFGP cells were seeded into 96-well plates at a density of 500 cells / well. The ADCs of the examples or reference ADCs were added to the 96-well plates at a density of 1 μg / mL. The 96-well plates were placed in an Incucyte detector and incubated for 7 days. The green fluorescence signal was monitored in real time. After 7 days, data was collected and plotted to evaluate the bystander effect of the ADCs. The data are shown in Figures 3a to 3e. The results showed that when target-negative CHOK1-EGFP cells were added alone, the ADC drug had no killing effect. As the number of co-cultured target-positive cells OE19 increased, the killing effect of the ADC drug on CHOK1-EGFP increased, indicating that the ADC drug had a bystander effect.

[0656] Example D. Evaluation of the efficacy of ADCs in mouse tumor models

[0657] Example D.1. Evaluation of therapeutic efficacy in the human lung cancer cell NCI-H441 immunodeficient mouse (SCID Beige) model

[0658] The NCI-H441 human lung adenocarcinoma model was established using human immunodeficient mice (SCID Beige, CB17.Cg-PrkdcscidLystbg-J / Crl, Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., strain code: 405) and the anti-tumor efficacy of the ADCs of the present application examples in this mouse model was tested. NCI-H441 cells were diluted with serum-free medium (RPMI 1640), and then the corresponding volume of Matrigel (Cat. No.: 354234, BD) was added and mixed with the cell solution (RPMI 1640 medium: Matrigel = 7:3). After that, the cells were plated at a rate of 2.5×10 7 200 μL / mouse was inoculated subcutaneously in the right flank of SCID Beige mice. 3 Mice were divided into groups of 5 and injected with the test sample or an equal volume of PBS vehicle control at a dose of 10 mL / kg via the tail vein. Doses ranged from 1 to 5 mg / kg. Following administration, the mice were housed normally, their survival was observed, and their body weight and tumor volume were recorded twice weekly.

[0659] Fa-ADC1 significantly inhibited the growth of NCI-H441 tumors compared with vehicle control, IgG1-Dxd, and Fa-ADC reference.

[0660] Example D.2. Evaluation of the efficacy of ADCs in the human ovarian cancer cell SKOV-3 immunodeficient mouse (Balb / c Nude) model

[0661] The SKOV-3 human ovarian cancer animal model was established using immunodeficient mice (Balb / c Nude, BALB / cNj-Foxn1nu / Gpt, Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., strain code: D000521) and the in vivo efficacy of the ADCs of the present application examples was tested. SKOV-3 cells were diluted with serum-free medium (RPMI 1640), and then the corresponding volume of Matrigel (Cat. No.: 354234, BD, RPMI 1640 medium: Matrigel = 7:3) was added and mixed with the cell solution. After that, the cells were plated at 2.5×10 7 200 μL / mouse was inoculated subcutaneously in the right flank of immunodeficient mice. 3 When , 5 mice were divided into groups per group, and the test sample or an equal volume of PBS vehicle control was injected into the tail vein according to the dosage volume of 10mL / kg. The drug was administered twice, with an interval of 9 days between the first and second administrations (doses were 1 and 5mg / kg, respectively). After administration, the mice were raised normally, the survival status of the mice was observed, and the weight and tumor volume of the mice were recorded twice a week. The results are shown in Figures 4 and 5. As shown in Figure 4, Fa-ADC1 significantly inhibited the growth of SK-OV-3 tumors compared with the vehicle control, IgG1-Dxd (Iso-ADC reference) and Fa-ADC reference. During the experimental observation period, the body weight of tumor-bearing mice did not decrease, indicating that the mice had good tolerance under this dosage condition (see Figure 5).

[0662] In addition, the anti-tumor efficacy of different ADC molecules was evaluated in NCI-N87 (human gastric cancer cells), Capan-1 (human pancreatic cancer cells), JIMT-1 (human breast cancer cells), Calu-3 (human lung adenocarcinoma cells) and OE19 (human esophageal cancer cells) tumor cell models using the Her2 antibody trastuzumab.

[0663] Example D.3 Efficacy evaluation of Her2-ADCs in Balb / c Nude mouse JIMT-1, Capan-1, and NCI-N87 tumor models

[0664] JIMT-1, Capan-1, ...

Claims

1. Antibody drug conjugate of formula (I): or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein: Ab is an antibody or an antigen-binding fragment thereof; L1 are each independently a linker unit; L2 is each independently a linking unit selected from the following formula: -(CR m R n ) t -(L M ) 0-1 -(CH2CH2O) s -(CR m R n ) t -CO-,-(CR m R n ) t -L M -(CR m R n ) t -(L M ) 0-1 -L N -L M -(CH2O) s -(CR m R n ) t -CO-,-(CR m R n ) t -L M -(CR m R n ) t -N(R p )-(CR m R n ) t -N(R p )-(CR m R n ) t -CO-, and -(CR m R n ) t -L M -CH(R p )-CO-, wherein the -CO- end of L2 is connected to L3, and the other end of L2 is connected to L1, and in, R m and R n Each independently is H or C 1-6 alkyl, L M are each independently -NH-CO- or -CO-NH-, L N Each independently is -(CH2CH2O) m -(CH2) t -、-(CH2) t -(OCH2CH2) m -、-(3-8 membered heteroarylene)-(CH2) t -(OCH2CH2) m or -(OCH2CH2) m -(CH2) t -(3-8 membered heteroarylene)-, R p Each independently is -CO-(CH2CH2O) m -CH3 or -(CH2) t -L M -(CH2CH2O) m -CH3, and s and t are each independently 0, 1, 2, 3, 4, 5, 6, 7 or 8, and m is each independently 2, 3, 4, 5, 6, 7 or 8; L3 is independently an amino acid residue, a short peptide chain consisting of 2-10 amino acid residues, or -NH-(CH2) p -CO-; L4 are each independently a valence bond or a spacer unit; D is each independently a small molecule drug moiety derived from a compound of formula (II); in, R1 is: H; halogen; CN; OH; SH; C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, wherein the alkyl, alkenyl and alkynyl groups are each optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino; -(CH2) p -(C 3-8 Cycloalkyl) or -(CH2) p -(C 3-8 wherein the cycloalkyl and heterocycloalkyl groups are each optionally substituted by one or more independently selected from halogen, CN, OH, SH, amino, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Cyanoalkyl, and C 1-6 Substituents of aminoalkyl are substituted; -(CH2) p -(C 1-6 Alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ; -(CH2) p -CH=NR a ;-NH-CO-R a ; -NH-CO-(CH2) p -CH(OH)-R a ; -NH-(CH2) p -(CH=CH)-R a ; or -COOH; R2 and R3 are each independently halogen, cyano, NO2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, -NH-CO-(C 1-6 Hydroxyalkyl), or NR a R b , or R2 and R3 together with the atoms to which they are attached form a 4- to 8-membered heterocyclic group; R4 is H or C 3-8 Cycloalkyl, preferably H; R a and R b Each independently is H, C 1-6 Alkyl, -CO-(C 1-6 Alkyl), -SO-(C 1-6 Alkyl), -SO2-(C 1-6 alkyl), or -(CH2) q -(C 3-8 cycloalkyl), said alkyl and cycloalkyl are each optionally substituted by one or more substituents independently selected from halogen, CN, OH, SH and amino, and said cycloalkyl is further optionally substituted by one or more substituents independently selected from C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Cyanoalkyl and C 1-6 The aminoalkyl group is substituted with a substituent; and p and q are each independently 0, 1, 2, 3, 4, 5 or 6; wherein the point of attachment of D to L4 is at R1 or R3 or through the hydroxyl oxygen as shown in formula (II); and n is an integer of 0-10, preferably an integer of 0-8.

2. The antibody-drug conjugate of formula (I) according to claim 1: or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein: Ab is an antibody or an antigen-binding fragment thereof; L1 are each independently a linker unit; L2 is each independently of the formula -(CH2CH2O) x -(CR m R n ) y -CO- linking unit, wherein the -CO- end is connected to L3 and the other end is connected to L1, and wherein R m and R n Each independently is H or C 1-6 Alkyl, x is an integer from 0 to 5, y is an integer from 1 to 5, and x+y≤6; L3 is independently an amino acid residue, a short peptide chain consisting of 2-10 amino acid residues, or -NH-(CH2) p -CO-; L4 are each independently a valence bond or a spacer unit; D is each independently a small molecule drug moiety derived from a compound of formula (II); in, R1 is: H; halogen; CN; OH; SH; C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, wherein the alkyl, alkenyl and alkynyl groups are each optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino; -(CH2) p -(C 3-8 Cycloalkyl) or -(CH2) p -(C 3-8 wherein the cycloalkyl and heterocycloalkyl groups are each optionally substituted by one or more independently selected from halogen, CN, OH, SH, amino, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Cyanoalkyl, and C 1-6 Substituents of aminoalkyl are substituted; -(CH2) p -(C 1-6 Alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ; -(CH2) p -CH=NR a ;-NH-CO-R a ; -NH-CO-(CH2) p -CH(OH)-R a ; -NH-(CH2) p -(CH=CH)-R a ;or -COOH; R2 and R3 are each independently halogen, cyano, NO2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, -NH-CO-(C 1-6 Hydroxyalkyl), or NR a R b , or R2 and R3 together with the atoms to which they are attached form a 4- to 8-membered heterocyclic group; R4 is H or C 3-8 Cycloalkyl, preferably H; R a and R b Each independently is H, C 1-6 Alkyl, -CO-(C 1-6 Alkyl), -SO-(C 1-6 Alkyl), -SO2-(C 1-6 alkyl), or -(CH2) q -(C 3-8 cycloalkyl), said alkyl and cycloalkyl are each optionally substituted by one or more substituents independently selected from halogen, CN, OH, SH and amino, and said cycloalkyl is further optionally substituted by one or more substituents independently selected from C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Cyanoalkyl and C 1-6 The aminoalkyl group is substituted with a substituent; and p and q are each independently 0, 1, 2, 3, 4, 5 or 6; wherein the point of attachment of D to L4 is at R1 or R3 or through the hydroxyl oxygen as shown in formula (II); and n is an integer of 0-10, preferably an integer of 0-8.

3. The antibody drug conjugate according to claim 1 or 2, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein R1 is: H; halogen; CN; OH; SH; C 1-6 Alkyl; C 1-6 Haloalkyl; C 1-6 Hydroxyalkyl; -(CH2) p -(C 3-8 Cycloalkyl) or -(CH2) p -(C 3-8 Heterocycloalkyl), wherein the cycloalkyl and heterocycloalkyl are each optionally substituted by one or more independently selected from OH, SH, C 1-6 Alkyl and C 1-6 Substituents of hydroxyalkyl; -(CH2) p -(C 1-6 Alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ; -(CH2) p -CH=NR a ;-NH-CO-R a ; -NH-CO-(CH2) p -CH(OH)-R a ; -NH-(CH2) p -(CH=CH)-R a ; or -COOH; wherein R a and R b Each independently is H, C 1-6 Alkyl, C 1-6 Hydroxyalkyl, C 1-6 Aminoalkyl, -SO2-(C 1-6 alkyl), or -(CH2) q -(optionally represented by one or more independently selected from hydroxyl and C 1-6 The substituents of hydroxyalkyl are substituted with C 3-8 Cycloalkyl); Preferably, R1 is H; C 1-6 Alkyl; C 1-6 Haloalkyl; C1-6 Hydroxyalkyl; -(CH2) p -(C 3-8 Heterocycloalkyl) which is optionally substituted by one or more C 1-6 Hydroxyalkyl substituted; -(CH2) p -(C 1-6 Alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NH2; -(CH2) p -NH(C 1-6 alkyl); -(CH2) p -NH(C 1-6 Hydroxyalkyl); -(CH2) p -NH(C 1-6 aminoalkyl); -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), the cycloalkyl group is optionally substituted by one or more selected from C 1-6 Substituents of hydroxyalkyl; -(CH2) p -N(C 1-6 Alkyl)(-SO2-C 1-6 alkyl); -CH=N(C 1-6 Alkyl); -NH-CO-(C 1-6 Hydroxyalkyl); -NH-CO-(C 3-8 Hydroxycycloalkyl); -NH-CO-(CH2) p -CH(OH)-(C 1-6 Alkyl); -NH-CO-(CH2) p -CH(OH)-(C 3-8 Cycloalkyl); -NH-(CH2) p -(CH=CH)-(C 1-6 Hydroxyalkyl); or -COOH; or R1 is: H; halogen; CN; OH; SH; C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, wherein the alkyl, alkenyl and alkynyl groups are each optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino; -(CH2) p -(C 3-8 Cycloalkyl), the cycloalkyl being optionally substituted by one or more substituents independently selected from halogen, CN, OH, SH and amino; -(CH2) p -(C 1-6 Alkoxy); -(CH2)p -(C 1-6 alkylthio); -(CH2) p -NR a R b ; -CH=NR a ;-NH-CO-R a ; -NH-CO-(CH2) p -CH(OH)-R a ; or -COOH; wherein R a and R b Each independently is H, C 1-6 Alkyl, -SO-(C 1-6 Alkyl), -SO2-(C 1-6 alkyl), or -(CH2) q -(C 3-8 cycloalkyl), said alkyl and cycloalkyl each being optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino; Preferably, R1 is: H; halogen; CN; OH; SH; C 1-6 Alkyl; C 1-6 Haloalkyl; C 1-6 Hydroxyalkyl; -(CH2) p -(C 3-8 Cycloalkyl); -(CH2) p -(C 3-8 Hydroxycycloalkyl); -(CH2) p -(C 1-6 Alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ; -CH=NR a ;-NH-CO-R a ; -NH-CO-(CH2) p -CH(OH)-R a ; or -COOH; More preferably, R1 is H; C 1-6 Alkyl; C 1-6 Haloalkyl; C 1-6 Hydroxyalkyl; -(CH2) p -(C 1-6 Alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ; -CH=NRa ;-NH-CO-R a ; -NH-CO-(CH2) p -CH(OH)-R a ; or -COOH; preferably wherein R a and R b Each independently is H, C 1-6 Alkyl, C 1-6 Hydroxyalkyl, C 1-6 Aminoalkyl, -SO2-(C 1-6 Alkyl), -(CH2) q -(C 3-8 Cycloalkyl), or -(CH2) q -(C 3-8 Hydroxycycloalkyl); More preferably, R1 is H; C 1-6 Alkyl; C 1-6 Haloalkyl; C 1-6 Hydroxyalkyl; -(CH2) p -(C 1-6 Alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NH2; -(CH2) p -NH(C 1-6 alkyl); -(CH2) p -NH(C 1-6 Hydroxyalkyl); -(CH2) p -NH(C 1-6 aminoalkyl); -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl); -(CH2) p -N(C 1-6 Alkyl)(-SO2-C 1-6 alkyl); -CH=N(C 1-6 Alkyl); -NH-CO-(C 1-6 Hydroxyalkyl); -NH-CO-(C 3-8 Hydroxycycloalkyl); -NH-CO-(CH2) p -CH(OH)-(C 1-6 Alkyl); -NH-CO-(CH2) p -CH(OH)-(C 3-8 cycloalkyl); or -COOH.

4. The antibody-drug conjugate according to claim 1 or 2, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein R1 is H, C1-6 Alkyl, -(CH2) p -(C 1-6 Alkylthio), -(CH2) p -(NH2), -(CH2) p -NH(C 1-6 Alkyl), -(CH2) p -NH(C 1-6 Hydroxyalkyl), -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), or -NH-CO-(CH2) p -CH(OH)-(C 3-8 Cycloalkyl); Preferably, R1 is H, C 1-6 Alkyl, -(CH2) p -(C 1-6 -(alkylthio), -NH2, -(CH2) p -NH(C 1-6 Alkyl), -NH(C 1-6 Hydroxyalkyl), -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), or -NH-CO-CH(OH)-(C 3-8 Cycloalkyl); More preferably, R1 is H, C 1-6 Alkyl, -CH2-(C 1-6 Alkylthio), -NH2, -CH2-NH(C 1-6 Alkyl), -NH(C 1-6 Hydroxyalkyl), -CH2-NH-CH2-(C 3-8 Cycloalkyl), or -NH-CO-CH(OH)-(C 3-8 cycloalkyl).

5. The antibody-drug conjugate according to claim 1 or 2, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein R1 is C 1-6 Alkyl, -(CH2) p -(C 1-6 Alkylthio), -(CH2) p -NH(C 1-6 alkyl), or -(CH2) p -NH(C 1-6 Preferably, R1 is C 1-6 Alkyl, -CH2-(C 1-6 Alkylthio), -NH(C 1-6alkyl), or -NH(C 1-6 hydroxyalkyl).

6. The antibody-drug conjugate according to claim 1 or 2, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein R1 is C 1-6 Alkyl, -(CH2) p -(C 1-6 -(alkylthio), -NH2, -(CH2) p -NH(C 1-6 Alkyl), -(CH2) p -NH(C 1-6 Hydroxyalkyl), -NH-CO-(C 1-6 Hydroxyalkyl), or -NH-CO-(CH2) p -CH(OH)-(C 3-8 Preferably, R1 is C 1-6 Alkyl, -NH2, -(CH2) p -NH(C 1-6 Alkyl), -(CH2) p -NH(C 1-6 Hydroxyalkyl), or -NH-CO-(C 1-6 hydroxyalkyl).

7. The antibody drug conjugate according to claim 1 or 2, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein R1 is C 1-6 Alkyl; -(CH2) p -(C 3-8 Heterocycloalkyl) which is optionally substituted by one or more C 1-6 Hydroxyalkyl substituted; -(CH2) p -NH(C 1-6 Hydroxyalkyl); -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), the cycloalkyl group is optionally substituted by one or more selected from C 1-6 Substituents of hydroxyalkyl; or -NH-(CH2) p -(CH=CH)-(C 1-6 Hydroxyalkyl); Or, R1 is C 1-6 Alkyl; -(CH2) p -(C 3-8 Heterocycloalkyl) which is optionally substituted by one or more C 1-6 hydroxyl Alkyl substituted; -(CH2) p -(C1-6 alkylthio); -(CH2) p -NH2; -(CH2) p -NH(C 1-6 alkyl); -(CH2) p -NH(C 1-6 Hydroxyalkyl); -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), the cycloalkyl group is optionally substituted by one or more selected from C 1-6 The substituents of hydroxyalkyl are substituted; -NH-CO-(C 1-6 Hydroxyalkyl); or -NH-CO-(CH2) p -CH(OH)-(C 3-8 Cycloalkyl); Or, R1 is C 1-6 Alkyl; -(CH2) p -(C 3-8 Heterocycloalkyl) which is optionally substituted by one or more C 1-6 Hydroxyalkyl substituted; -(CH2) p -NH2; -(CH2) p -NH(C 1-6 alkyl); -(CH2) p -NH(C 1-6 Hydroxyalkyl); -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), the cycloalkyl group is optionally substituted by one or more selected from C 1-6 The substituents of hydroxyalkyl are substituted; -NH-CO-(C 1-6 Hydroxyalkyl); or -NH-CO-(CH2) p -CH(OH)-(C 3-8 Cycloalkyl); Or, R1 is C 1-6 Alkyl; -(CH2) p -(C 3-8 Heterocycloalkyl) which is optionally substituted by one or more C 1-6 Hydroxyalkyl substituted; -(CH2) p -NH(C 1-6 Hydroxyalkyl); -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), the cycloalkyl group is optionally substituted by one or more selected from C 1-6 Substitution of hydroxyalkyl; or -NH-CO-(CH2) p-CH(OH)-(C 3-8 Cycloalkyl); Or, R1 is C 1-6 Alkyl; -(CH2) p -(C 3-8 Heterocycloalkyl) which is optionally substituted by one or more C 1-6 Hydroxyalkyl substituted; -(CH2) p -NH(C 1-6 hydroxyalkyl); or -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), the cycloalkyl group is optionally substituted by one or more selected from C 1-6 The substituents of the hydroxyalkyl group are substituted.

8. The antibody drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein R2 is halogen, preferably fluorine.

9. The antibody drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein R3 is C 1-6 Alkyl, C 1-6 Alkoxy, halogen, cyano, -NH2, -NH-CO-(C 1-6 Preferably, R3 is C 1-6 Alkyl, halogen, -NH2, -NH-CO-(C 1-6 Hydroxyalkyl), or NO2; Alternatively, R3 is halogen, cyano, NO2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, or NR a R b , where R a and R b Each independently is H, C 1-6 Alkyl, -SO-(C 1-6 Alkyl), -SO2-(C 1-6 alkyl), or -(CH2) q -(C 3-8 cycloalkyl), wherein the alkyl and cycloalkyl are each optionally substituted by one or more substituents independently selected from halogen, CN, OH, SH and amino; preferably, R3 is C 1-6 Alkyl, C 1-6 Alkoxy, halogen, cyano, -NH2 or NO2; more preferably C 1-6Alkyl, halogen, -NH2 or NO2; Or, R3 is C 1-6 Alkyl or NR a R b , where R a and R b Each is independently H or -CO-(C 1-6 Alkyl), the alkyl group is optionally substituted with one or more OH; Or, R3 is C 1-6 Alkyl, -NH2 or -NH-CO-(CH2OH), preferably C 1-6 Alkyl or -NH-CO-(CH2OH); Or R3 is C 1-6 alkyl.

10. The antibody drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein R2 and R3 together with the atoms to which they are attached form a 4- to 8-membered heterocyclic group containing N, O or S as a heteroatom, such as dioxole.

11. An antibody drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label thereof, wherein D is connected to L4 through a nitrogen or oxygen atom in R1 or R3 or through a hydroxyl oxygen atom as shown in formula (II), for example, through an amide bond, an aminomethyl ether bond or a carbamate bond.

12. The antibody drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein D is a moiety selected from the following:

13. The antibody drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein each L1 is independently: Best More preferably in, Position 1 is connected to Ab, and position 2 is connected to L2.

14. The antibody drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label thereof, wherein each L2 is independently a linker selected from the following formula: -(CH2)t -(L M ) 0-1 -(CH2CH2O) s -(CH2) t -CO-, -(CH2) t -L M -(CH2) t -(L M ) 0-1 -L N -L M -(CH2O) s -(CH2) t -CO-, -(CH2) t -L M -(CH2) t -N(R p )-(CH2) t -N(R p )-(CH2) t -CO-, and (CH2) t -L M -CH(R p )-CO-, where The -CO- terminal of L2 is connected to L3, and the other terminal of L2 is connected to L1.

15. The antibody drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label thereof, wherein each L2 is independently a linker selected from the following formula: -(CH2) t -(CH2CH2O) s -(CH2) t -CO-, -(CH2) t -CO-NH-(CH2CH2O) s -(CH2) t -CO-, -(CH2) t -NH-CO-(CH2CH2O) s -(CH2) t -CO-, -(CH2) t -CO-NH-(CH2) t -(3-8 membered heteroarylene)-(CH2) t -(OCH2CH2) m -NH-CO-(CH2O)-(CH2) t -CO-, -(CH2) t -CO-NH-(CH2) t -NH-CO-(CH2CH2O)m -(CH2) t -NH-CO-(CH2O)-(CH2) t -CO-, -(CH2) t -CO-NH-(CH2) t -NH-CO-(CH2) t -(OCH2CH2) m -NH-CO-(CH2O)-(CH2) t -CO-, -(CH2) t -NH-CO-(CH2) t -N(R p )-(CH2) t -N(R p )-(CH2) t -CO-, -(CH2) t -CO-NH-(CH2) t -N(R p )-(CH2) t -N(R p )-(CH2) t -CO-, or -(CH2) t -CO-NH-CH(R p )-CO- or -(CH2) t -NH-CO-CH(R p )-CO-, The -CO- end of L2 is connected to L3, and the other end of L2 is connected to L1.

16. The antibody drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein L3 is independently an amino acid residue, a short peptide chain consisting of 2-6 amino acid residues, or -NH-(CH2) p -CO-, wherein p is 0, 1, 2, 3, 4, 5 or 6; preferably, the amino acid is selected from glycine (Gly), alanine (Ala), valine (Val), phenylalanine (Phe), citrulline (Cit), lysine (Lys) and asparagine (Asn); for example, L3 is Gly-Gly-Phe-Gly, Val-Ala, Phe-Lys or Lys.

17. The antibody drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein L4 is each independently a valence bond, Preferably, a valence bond, More preferably Bit 1 is connected to L3 and bit 2 is connected to D.

18. The antibody drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein: L1 is independently: Best More preferably Among them, position 1 is connected to Ab, and position 2 is connected to L2; L2 is each independently of the formula -(CH2CH2O) x -(CH2) y -CO- connecting unit, wherein the -CO- end is connected to L3 and the other end is connected to L1, and wherein x is an integer from 0 to 5, y is an integer from 1 to 5, and x+y≤6; preferably, L2 is -(CH2)-CO-, -(CH2)2-CO-, -(CH2)3-CO-, -(CH2)4-CO-, -(CH2)5-CO-, -(CH2)6-CO-, -(CH2CH2O)-(CH2)-CO-, -(CH2CH2O)-(CH2)2-CO-, -(CH2CH2O)-(CH2)3-CO-, -(CH2CH2O)-(CH2)4-CO-, - (CH2CH2O)-(CH2)5-CO-, -(CH2CH2O)2-(CH2)-CO-, -(CH2CH2O)2-(CH2)2-CO-, -(CH2CH2O)2-(CH2)2-CO-, -(CH2CH2O)2-(CH2)3-CO-, -(CH2CH2O)2-(CH2)4-CO-, -(CH2CH2O)3-(CH2)-CO-, -(CH2CH2O)3-(CH2)2-CO-, -(CH2CH2O)3-(CH2)2-CO-, -(CH2CH2O)3-(CH2)3-CO-; More preferably, L2 is each independently -(CH2)5-CO- or -(CH2CH2O)3-(CH2)2-CO-; L3 is independently an amino acid residue, a short peptide chain consisting of 2-6 amino acid residues, or -NH-(CH2) p -CO-, wherein p is 0, 1, 2, 3, 4, 5 or 6; preferably, the amino acid is selected from glycine (Gly), alanine (Ala), valine (Val), phenylalanine (Phe), citrulline (Cit), lysine (Lys) and asparagine (Asn); for example, L3 is Gly-Gly-Phe-Gly, Val-Cit, Val-Ala, Phe-Lys, Val-Lys, Ala-Ala-Ala, Ala-Ala-Asn, or -NH-(CH2)2-CO-; and L4 is each independently a valence bond, Bit 1 is connected to L3 and bit 2 is connected to D.

19. The antibody drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label thereof, wherein the -L1-L2-L3-L4- moieties are each independently selected from: in, Position 1 is connected to Ab and position 2 is connected to D.

20. The antibody drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label thereof, wherein the -L1-L2-L3-L4-D moieties are each independently selected from: in, Position 1 indicates the site of attachment to Ab.

21. The antibody-drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein Ab is an antibody or an antigen-binding fragment thereof that binds to a tumor cell surface antigen; for example, an anti-Her2 antibody or an antigen-binding fragment thereof and / or an anti-FRα antibody or an antigen-binding fragment thereof; in particular, facilitation monoclonal antibody or an antigen-binding fragment thereof and / or trastuzumab or an antigen-binding fragment thereof.

22. The antibody drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein the antibody drug conjugate is selected from: in, In Fa-ADCs, mAb represents fatutumomab; In Tra-ADCs, mAb represents trastuzumab, and n is 0, 1, 2, 3, 4, 5, 6, 7 or 8.

23. The antibody drug conjugate according to any one of the preceding claims, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, having an average DAR selected from any value within the range of 1.0-10.0, preferably an average DAR selected from any value within the range of 2.0-8.0, more preferably an average DAR selected from any value within the range of 6.0-8.

0.

24. Compounds of formula (II): or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, in, R1 is: H; halogen; CN; OH; SH; C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, wherein the alkyl, alkenyl and alkynyl groups are each optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino; -(CH2) p -(C 3-8 Cycloalkyl) or -(CH2) p -(C 3-8 wherein the cycloalkyl and heterocycloalkyl groups are each optionally substituted by one or more independently selected from halogen, CN, OH, SH, amino, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Cyanoalkyl, and C 1-6 Substituents of aminoalkyl are substituted; -(CH2) p -(C 1-6 Alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ; -(CH2) p -CH=NR a ;-NH-CO-R a ; -NH-CO-(CH2) p -CH(OH)-R a ; -NH-(CH2) p -(CH=CH)-R a ; or -COOH; R2 and R3 are each independently halogen, cyano, NO2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, -NH-CO-(C 1-6 Hydroxyalkyl), or NR a R b , or R2 and R3 together with the atoms to which they are attached form a 4- to 8-membered heterocyclic group; R4 is H or C 3-8 Cycloalkyl, preferably H; R a and R b Each independently is H, C 1-6 Alkyl, -CO-(C 1-6 Alkyl), -SO-(C 1-6 Alkyl), -SO2-(C 1-6 alkyl), or -(CH2) q -(C 3-8 cycloalkyl), said alkyl and cycloalkyl are each optionally substituted by one or more substituents independently selected from halogen, CN, OH, SH and amino, and said cycloalkyl is further optionally substituted by one or more substituents independently selected from C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Cyanoalkyl and C 1-6 The aminoalkyl group is substituted with a substituent; and p and q are each independently 0, 1, 2, 3, 4, 5 or 6.

25. The compound according to claim 24, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein: R1 is: H; halogen; CN; OH; SH; C 1-6 Alkyl; C 1-6 Haloalkyl; C 1-6 Hydroxyalkyl; -(CH2) p -(C 3-8 Cycloalkyl) or -(CH2) p -(C 3-8 Heterocycloalkyl), wherein the cycloalkyl and heterocycloalkyl are each optionally substituted by one or more independently selected from OH, SH, C 1-6 Alkyl and C 1-6 Substituents of hydroxyalkyl; -(CH2) p -(C 1-6 Alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ; -(CH2) p -CH=NR a ;-NH-CO-R a ; -NH-CO-(CH2) p -CH(OH)-R a ; -NH-(CH2) p -(CH=CH)-R a ; or -COOH; wherein R a and R b Each independently is H, C 1-6 Alkyl, C 1-6 Hydroxyalkyl, C 1-6 Aminoalkyl, -SO2-(C 1-6 alkyl), or -(CH2) q -(optionally represented by one or more independently selected from hydroxyl and C 1-6 The substituents of hydroxyalkyl are substituted with C 3-8 Cycloalkyl); Preferably, R1 is H; C 1-6 Alkyl; C 1-6 Haloalkyl; C 1-6 Hydroxyalkyl; -(CH2) p -(C 3-8 Heterocycloalkyl) which is optionally substituted by one or more C 1-6 Hydroxyalkyl substituted; -(CH2) p -(C 1-6 Alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NH2; -(CH2) p -NH(C 1-6 alkyl); -(CH2) p -NH(C 1-6 Hydroxyalkyl); -(CH2) p -NH(C 1-6 aminoalkyl); -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), the cycloalkyl group is optionally substituted by one or more selected from C 1-6 The substituents of hydroxyalkyl are Generation; -(CH2) p -N(C 1-6 Alkyl)(-SO2-C 1-6 alkyl); -CH=N(C 1-6 Alkyl); -NH-CO-(C 1-6 Hydroxyalkyl); -NH-CO-(C 3-8 Hydroxycycloalkyl); -NH-CO-(CH2) p -CH(OH)-(C 1-6 Alkyl); -NH-CO-(CH2) p -CH(OH)-(C 3-8 Cycloalkyl); -NH-(CH2) p -(CH=CH)-(C 1-6 Hydroxyalkyl); or -COOH; or R1 is: H; halogen; CN; OH; SH; C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, wherein the alkyl, alkenyl and alkynyl groups are each optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino; -(CH2) p -(C 3-8 Cycloalkyl), the cycloalkyl being optionally substituted by one or more substituents independently selected from halogen, CN, OH, SH and amino; -(CH2) p -(C 1-6 Alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ; -CH=NR a ;-NH-CO-R a ; -NH-CO-(CH2) p -CH(OH)-R a ; or -COOH; wherein R a and R b Each independently is H, C 1-6 Alkyl, -SO-(C 1-6 Alkyl), -SO2-(C 1-6 alkyl), or -(CH2) q -(C 3-8 cycloalkyl), said alkyl and cycloalkyl each being optionally substituted with one or more substituents independently selected from halogen, CN, OH, SH and amino; Preferably, R1 is: H; halogen; CN; OH; SH; C 1-6 Alkyl; C 1-6 Haloalkyl; C 1-6 Hydroxyalkyl; -(CH2) p -(C 3-8 Cycloalkyl); -(CH2) p -(C 3-8 Hydroxycycloalkyl); -(CH2) p -(C 1-6 Alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ; -CH=NR a ;-NH-CO-R a ; -NH-CO-(CH2) p -CH(OH)-R a ; or -COOH; More preferably, R1 is H; C 1-6 Alkyl; C 1-6 Haloalkyl; C 1-6 Hydroxyalkyl; -(CH2) p -(C 1-6 Alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NR a R b ; -CH=NR a ;-NH-CO-R a ; -NH-CO-(CH2) p -CH(OH)-R a ; or -COOH; preferably wherein R a and R b Each independently is H, C 1-6 Alkyl, C 1-6 Hydroxyalkyl, C 1-6 Aminoalkyl, -SO2-(C 1-6 Alkyl), -(CH2) q -(C 3-8 Cycloalkyl), or -(CH2) q -(C 3-8 Hydroxycycloalkyl); More preferably, R1 is H; C 1-6 Alkyl; C 1-6 Haloalkyl; C 1-6 Hydroxyalkyl; -(CH2) p -(C 1-6 Alkoxy); -(CH2) p -(C 1-6 alkylthio); -(CH2) p -NH2; -(CH2) p -NH(C 1-6 alkyl); -(CH2) p -NH(C 1-6 Hydroxyalkyl); -(CH2) p -NH(C 1-6 aminoalkyl); -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl); -(CH2) p -N(C 1-6 Alkyl)(-SO2-C 1-6 alkyl); -CH=N(C 1-6 Alkyl); -NH-CO-(C 1-6 Hydroxyalkyl); -NH-CO-(C 3-8 Hydroxycycloalkyl); -NH-CO-(CH2) p -CH(OH)-(C 1-6 Alkyl); -NH-CO-(CH2) p -CH(OH)-(C 3-8 cycloalkyl); or -COOH.

26. The compound according to claim 24, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein R1 is H, C 1-6 Alkyl, -(CH2) p -(C 1-6 Alkylthio), -(CH2) p-(NH2), -(CH2) p -NH(C 1-6 Alkyl), -(CH2) p -NH(C 1-6 Hydroxyalkyl), -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), or -NH-CO-(CH2) p -CH(OH)-(C 3-8 Cycloalkyl); Preferably, R1 is H, C 1-6 Alkyl, -(CH2) p -(C 1-6 -(alkylthio), -NH2, -(CH2) p -NH(C 1-6 Alkyl), -NH(C 1-6 Hydroxyalkyl), -(CH2) p -NH-(CH2) q -(C 3-8 Cycloalkyl), or -NH-CO-CH(OH)-(C 3-8 Cycloalkyl); More preferably, R1 is H, C 1-6 Alkyl, -CH2-(C 1-6 Alkylthio), -NH2, -CH2-NH(C 1-6 Alkyl), -NH(C 1-6 Hydroxyalkyl), -CH2-NH-CH2-(C 3-8 Cycloalkyl), or -NH-CO-CH(OH)-(C 3-8 cycloalkyl).

27. The compound according to claim 24, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein R1 is C 1-6 Alkyl, -(CH2) p -(C 1-6 Alkylthio), -(CH2) p -NH(C 1-6 alkyl), or -(CH2) p -NH(C 1-6 Preferably, R1 is C 1-6 Alkyl, -CH2-(C 1-6 Alkylthio), -NH(C 1-6 alkyl), or -NH(C 1-6 hydroxyalkyl).

28. The compound according to any one of claims 24 to 27, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein R2 is halogen, preferably fluorine.

29. The compound according to any one of claims 24 to 28, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein R3 is C 1-6 Alkyl, C 1-6 Alkoxy, halogen, cyano, -NH2, -NH-CO-(C 1-6 Preferably, R3 is C 1-6 Alkyl, halogen, -NH2, -NH-CO-(C 1-6 Hydroxyalkyl), or NO2; Alternatively, R3 is halogen, cyano, NO2, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, or NR a R b , where R a and R b Each independently is H, C 1-6 Alkyl, -SO-(C 1-6 Alkyl), -SO2-(C 1-6 alkyl), or -(CH2) q -(C 3-8 cycloalkyl), wherein the alkyl and cycloalkyl are each optionally substituted by one or more substituents independently selected from halogen, CN, OH, SH and amino; preferably, R3 is C 1-6 Alkyl, C 1-6 Alkoxy, halogen, cyano, -NH2 or NO2; more preferably C 1-6 Alkyl, halogen, -NH2 or NO2.

30. A compound according to any one of claims 24 to 27, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein R2 and R3 together with the atoms to which they are attached form a 4-8 membered heterocyclic group containing oxygen, nitrogen or sulfur atoms as heteroatoms, such as dioxole.

31. The compound according to claim 24, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, wherein the compound is selected from:

32. Drug-linker conjugate of formula (III): Lg-L1-L2-L3-L4-D (III) or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, in, L1, L2, L3, L4 and D are as defined in any one of claims 1 to 20; and wherein When L1 is When Lg and L1 form When L1 is not When Lg is a leaving group; preferably, Lg is a halogen, a sulfonyl group (e.g., methanesulfonyl, p-toluenesulfonyl), a sulfonyloxy group (e.g., methanesulfonyloxy, CF3SO3-, p-toluenesulfonyloxy), a tertiary amine group (e.g., Me3N + or Et3N + ) or a diazonium salt group; more preferably, Lg is F, Cl, Br, methylsulfonyl; particularly preferably, Lg is methylsulfonyl.

33. The drug-linker conjugate according to claim 32, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope-labeled substance thereof, selected from:

34. A pharmaceutical composition comprising an antibody drug conjugate of any one of claims 1-23 or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label thereof, or a compound of any one of claims 21-31 or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label thereof, or a drug linker conjugate of any one of claims 32-33 or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

35. An antibody drug conjugate or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label of any one of claims 1 to 23, or a compound or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label of any one of claims 21 to 31, or a drug linker conjugate or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label of any one of claims 32 to 33 for use in treating or preventing cancer.

36. A method for preventing or treating cancer in a patient, the method comprising administering to the patient a therapeutically effective amount of an antibody drug conjugate of any one of claims 1-23, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label thereof, or a compound of any one of claims 21-31, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label thereof, or a drug linker conjugate of any one of claims 32-33, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label thereof.

37. Use of an antibody drug conjugate or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label of any one of claims 1 to 23, or a compound or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label of any one of claims 21 to 31, or a drug linker conjugate or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label of any one of claims 32 to 33 in the preparation of a medicament for treating or preventing cancer.

38. The antibody drug conjugate or compound or drug linker conjugate according to claim 35, or a pharmaceutically acceptable salt or ester, solvate, tautomer, stereoisomer, prodrug or isotope label thereof, or a method according to claim 36, or a use according to claim 37, wherein the cancer is a solid tumor, in particular lung cancer (e.g. small cell lung cancer, non-small cell lung cancer), liver cancer, digestive system cancer (e.g. intestinal cancer, gastric cancer, cardia cancer, esophageal cancer, appendix adenocarcinoma, colon cancer, rectal cancer, colorectal cancer, pancreatic cancer), bladder cancer, melanoma, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, basal cell carcinoma, bile duct cancer , squamous cell carcinoma, thyroid cancer, brain cancer, head and neck cancer, peritoneal cancer, renal cancer, urothelial carcinoma, testicular cancer, central nervous system tumors (e.g. glioma, glioblastoma such as glioblastoma multiforme, glioma or sarcoma), choriocarcinoma, oral epidermoid carcinoma; or hematological malignancies, in particular leukemias (e.g. acute or chronic myeloid leukemia, acute or chronic myeloid leukemia), lymphomas (e.g. Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, acute B-cell lymphoma, follicular lymphoma), multiple myeloma; preferably, the cancer is ovarian cancer, colon cancer, lung adenocarcinoma or oral epidermoid carcinoma.

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