Fused ring compound, preparation method therefor, and use thereof

Abstract

A fused ring compound, a preparation method therefor, and use thereof. The fused ring compound has a structure represented by the formula M'-L-E-D. The compound can be used for preparing a conjugate, and the conjugate has an excellent targeted-killing effect on solid tumors, such as gastric cancer, breast cancer, lung cancer, and lymphoma, or hematologic tumors.

Description

Condensed ring compound and its preparation method and use

[0001] This application is based on and claims priority to CN application No. 202311408874.6, filed on October 27, 2023, and PCT application No. PCT / CN2023 / 127189, filed on October 27, 2023. The disclosed contents of these applications are hereby incorporated into this application as a whole. Technical Field

[0002] The present application relates to the field of medicine, and specifically to a fused ring compound and its preparation method and use. Background Art

[0003] Antibody drug conjugates (ADCs) consist of an antibody, a bioactive molecule, and a linker. The bioactive molecule is covalently coupled to the antibody via the linker. The antibody (e.g., a monoclonal antibody) can specifically recognize a target on the surface of tumor cells, guiding the ADC to the cancer cell surface and allowing it to enter the cancer cell through endocytosis. The bioactive molecule is then released inside the cancer cell, killing the cancer cell while minimizing damage to normal tissue cells.

[0004] As an important component of antibody-drug conjugates, the structure of the drug-linker portion is closely related to the overall efficacy and safety of ADC drugs. Therefore, the development of novel drug-linker structures is of great significance for the development of antibody conjugates with good efficacy and safety.

[0005] Summary of the Invention

[0006] The present application relates to a fused ring compound and a preparation method thereof. The fused ring compound can be used to prepare antibody-drug conjugates and achieve good efficacy and safety.

[0007] Compound

[0008] In one aspect, the present application provides a compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled compound, metabolite, and prodrug thereof, having a structure shown in formula DEL-M', wherein:

[0009] M' is Lg is a leaving group of a nucleophilic substitution reaction, or is a hydroxyl group (-OH), a thiol group (-SH) or an amino group (-NH2), and ring A is a 5-6 membered saturated aliphatic heterocyclic ring, or a 5-20 membered aromatic ring system; or, M' is Ring A' is a 5-6 membered unsaturated heterocyclic ring; the saturated heterocyclic ring, unsaturated heterocyclic ring and aromatic ring system are optionally substituted by one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl group substitution; M1 is selected from single bond, C 1-20 Alkylene, C 2-20 Alkenylene or C 2-20 Alkynylidene;

[0010] L is the structural fragment connecting M' and E;

[0011] E is a structural fragment connecting L and D;

[0012] D is a cytotoxic drug fragment.

[0013] In some embodiments, M' is Lg is a leaving group for nucleophilic substitution reaction (e.g., halogen, methanesulfonyl, fluorophenol or ), or is hydroxyl (-OH), thiol (-SH) or amino (-NH2), Ring A is a 5-6 membered saturated aliphatic heterocyclic ring, or a 5-20 membered aromatic ring system; or, M' is Ring A' is a 5-6 membered unsaturated heterocyclic ring; the saturated heterocyclic ring, unsaturated heterocyclic ring and aromatic ring system are optionally substituted by one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl group substituted; M1 is selected from single bond and C 1-20 Alkylene, C 2-20 Alkenylene or C 2-20 Alkynylidene;

[0014] The L, E and D structures are as defined in any of the above.

[0015] In some embodiments, M' is Lg is a methylsulfonyl group; Ring A is a 5-membered aliphatic heterocyclic ring, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more 6-membered heteroaromatic rings to a benzene ring via a single bond, or M' is Ring A' is a 5-6 membered unsaturated heterocyclic ring; the saturated heterocyclic ring, unsaturated heterocyclic ring and aromatic ring system are optionally substituted by one or more selected from oxy (=O), halogen and C 1-4 Alkyl group substituted; M1 is selected from single bond and C 3-10 Alkylene, C 3-10 Alkenylene or C 3-10 Alkynylidene.

[0016] In some embodiments, M' is Selected from M1 is selected from a single bond and C 5-8Alkylene, C 5-8 Alkenylene or C 5-8 Alkynylidene.

[0017] In some embodiments, M' is selected from

[0018] In some embodiments, M' is

[0019] In some embodiments, M' is selected from

[0020] And, when M' is When E is or wherein s is selected from an integer of 1-20.

[0021] In some embodiments, M is Wherein ring A is a 5-6 membered alicyclic heterocyclic ring, or a 5-20 membered aromatic ring system, wherein the alicyclic heterocyclic ring and the aromatic ring system are optionally substituted by one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl group substituted; M1 is selected from single bond and C 1-20 Alkylene, C 2-20 Alkenylene or C 2- 20 Alkynylidene.

[0022] In some embodiments, M is wherein Ring A is a 5-membered alicyclic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more 6-membered heteroaromatic rings to a benzene ring via a single bond, wherein the alicyclic heterocycle is optionally substituted with one or more selected from oxy (=O), halogen and C 1-4 Alkyl group substituted; M1 is selected from single bond and C 3-10 Alkylene, C 3-10 Alkenylene or C 3-10 Alkynylidene.

[0023] In some embodiments, M is wherein ring A is selected from M1 is selected from a single bond and C 5-8 Alkylene, C 5-8 Alkenylene or C 5-8 Alkynylidene.

[0024] In some embodiments, M is selected from the following structures:

[0025] In some embodiments, Lg is selected from halogen, substituted or unsubstituted C 1-6sulfonyl, halogenated phenoxy, hydroxyl (-OH), mercapto (-SH) or amino (-NH2).

[0026] In some embodiments, Lg is selected from halogen, substituted or unsubstituted methylsulfonyl, halophenoxy, hydroxyl (-OH), thiol (-SH), or amino (-NH2).

[0027] In some embodiments, Lg is selected from methylsulfonyl or pentafluorophenoxy.

[0028] In some embodiments, L is selected from a structure consisting of one or more substituted or unsubstituted fragments of: C 1-6 Alkylene, -N(R')-, Carbonyl, -O-, Val, Cit, Phe, Lys, Lys(COCH2CH2(OCH2CH2) s OCH3), D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys , Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Gly, Gly-Gly-Phe-Gly (GGFG,SEQ ID NO: 13), Gly-Gly-Gly-Gly-Gly (GGGGGG, SEQ ID NO: 14), Where R' represents hydrogen, C 1-6 Alkyl or alkyl containing -(CH2CH2O)r-; r is selected from an integer of 1-10; s is selected from an integer of 1-20.

[0029] In some embodiments, L is selected from a structure consisting of one or more substituted or unsubstituted fragments of: C 1-6 Alkylene, -NH-, Phe, Lys, Lys(COCH2CH2(OCH2CH2) s OCH3), Gly, Gly-Gly-Phe-Gly, wherein s is selected from an integer of 1-20.

[0030] In some embodiments, L is selected from the following substituted or unsubstituted structures:

[0031] wherein s is selected from an integer of 1-20.

[0032] In some embodiments, L is selected from the following structures:

[0033] In some embodiments, L is selected from the following structures:

[0034] In some embodiments, E is a single bond, or substituted or unsubstituted -NH-CH2-, -NH-CH2-O-CH2-CO-,

[0035] In some embodiments, E is a single bond, or substituted or unsubstituted -NH-CH2-, -NH-CH2-O-CH2-CO-,

[0036] In some embodiments, E is -NH-CH2-O-CH2-CO-,

[0037] In some embodiments, E is -NH-CH2-O-CH2-CO- or

[0038] In some embodiments, M is selected from the following structures:

[0039] L is selected from the following structures:

[0040] E is -NH-CH2-O-CH2-CO-,

[0041] In some embodiments, Selected from the following substituted or unsubstituted structures:

[0042] In some embodiments, Selected from the following structures:

[0043] In some embodiments, the cytotoxic drug is selected from microtubule inhibitors, DNA intercalators, DNA topoisomerase inhibitors and RNA polymerase inhibitors. In some embodiments, the microtubule inhibitor is an auristatin compound or a maytansine compound. In some embodiments, the DNA intercalator is a pyrrolobenzodiazepine (PBD). In some embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, multicarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide). In some embodiments, the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester or analog thereof.

[0044] The cytotoxic drugs disclosed in this application generally contain a variety of functional groups, such as hydroxyl (-OH), carboxyl (-COOH), sulfhydryl (-SH), primary amino (-NH2), secondary amine (-NR A H) or tertiary amine (-NR B R C ), where R A 、R B 、R C These represent only non-hydrogen substituents on N, through which the cytotoxic drug can be attached to the linker in the conjugate.

[0045] In some embodiments, the cytotoxic drug is linked to E in the antibody drug conjugate through -OH, -SH, primary amino, secondary amine, or tertiary amine groups on the cytotoxic drug.

[0046] In some embodiments, the cytotoxic drug is selected from the following Formula I and Formula II:

[0047] Wherein, R1, R2 are each independently selected from C 1-6 Alkyl and halogen;

[0048] R3 is selected from H and -CO-CH2OH;

[0049] R4 and R5 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring;

[0050] R6 is selected from hydrogen or -C 1-4 Alkylene-NR a R b ;

[0051] R7 is selected from C1-6 Alkyl and -C 1-4 Alkylene-NR a R b ;

[0052] where R a 、R b Each occurrence is independently selected from H, C 1-6 Alkyl, -SO2-C 1-6 Alkyl and -CO-C 1-6 alkyl.

[0053] In some embodiments, the cytotoxic drug is selected from the following compounds:

[0054] In some embodiments, the cytotoxic drug is selected from the following compounds:

[0055] The corresponding fragment of the cytotoxic drug obtained after the cytotoxic drug is connected to the linker is D in formula (I) of the present application. In some embodiments, D is a monovalent structure obtained by losing one H from the -OH, -NH2 or secondary amine group on the cytotoxic drug.

[0056] In some embodiments, D is selected from the following structures:

[0057] In some embodiments, the compound is selected from A-01 to A-25, B-01 to B-05 shown below:

[0058] A-01:

[0059] A-02:

[0060] A-03:

[0061] A-04:

[0062] A-05:

[0063] A-06:

[0064] A-07:

[0065] A-08:

[0066] A-09:

[0067] A-10:

[0068] A-11:

[0069] A-12:

[0070] A-13:

[0071] A-14:

[0072] A-15:

[0073] A-16:

[0074] A-17:

[0075] A-18:

[0076] A-19:

[0077] A-20:

[0078] A-21:[[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​The second aspect of the present invention provides a conjugate as shown in formula (II), wherein:

[0090] Ab-[MLED]x

[0091] Formula (II)

[0092] Ab is a targeting moiety; M, L, E and D are as described above;

[0093] x is 1 to 10.

[0094] In some preferred embodiments, the target of Ab is selected from epidermal growth factor, Trop-2, CD37, HER2, CD70, EGFRvIII, Mesothelin, Folate receoptor1, Mucin 1, CD138, CD20, CD19, CD30, SLTRK6, Nectin 4, Tissue factor, Mucin16, Endothelinreceoptor, STEAP1, SLC39A6, Guanylylcyclase C, PSMA, CCD79b, CD22, Sodium phosphate cotransporter 2B, GPNMB, Trophoblast glycoprotein, AGS-16, EGFR, CD33, CD66e, CD74, CD56, PD-L1, TACSTD2, DR5, E16, 0772P, MPF, Napi3b, Sema 5b, PSCA hlg, ETBR, MSG783, STEAP2, TrpM4, CRIPTO, CD21, CD79b, FcRH2, NCA, MDP, IL20Rα, Brevican, EphB2R, ASLG659, PSCA, GEDA, BAFF-R, CD79a, CXCR5, HLA-DOB, P2X5, CD72, LY 64. FcRH1, IRTA2, TENB2, integrin α5β6, α4β7, FGF2, FGFR2, Her3, CA6, DLL3, DLL4, P-cadherin, EpCAM, pCAD, CD223, LYPD3, LY6E, EFNA4, ROR1, SLITRK6, 5T4, ENPP3, Claudin18.2. BMPR1B, Tyro7, c-Met, ApoE, CD1 lc, CD40, CD45(PTPRC), CD49D(ITGA4), CD80, CSF1R, CTSD, GZMB, Ly86, MS4A7, PIK3AP1, PIK3CD, CCR5, IFNG, IL10RA1, I L-6, ACTA2, COL7A1, LOX, LRRC15, MCPT8, MMP10, NOG, SERPINEl, STAT1, TGFBR1, CTSS, PGF, VEGFA, C1QA, C1QB, ANGPTL4, EGLN, EGLN3, BNIP3, AIF1, CCL5, CXCL10, CXCL11, IFI6, PLOD2, KISS1R, STC2, DDIT4, PFKFB3, PGK1, PDK1, AKR1C1, AKR1C 2. CADM1, CDH11, COL6A3, CTGF, HMOX1, KRT33A, LUM, WNT5A, IGFBP3, MMP14, CDCP1, PDGFRA, TCF4, TGF, TGFB1, TGFB2, CDl lb, ADGRE1, EMR2, TNFRSF21, UPK1B, TNFSF9, MMP16, MFI2, IGF-1R, RNF43, NaPi2b and BCMA. .

[0095] In some preferred embodiments, Ab is a small molecule ligand, such as a folic acid derivative, a glutamate urea derivative, a somatostatin derivative, an arylsulfonamide derivative (e.g., a carbonic anhydrase IX inhibitor), a polyene linked to two aliphatic indoles, a cyanine dye, or IR-783 or its derivatives.

[0096] In some preferred embodiments, Ab is an antibody, such as a monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof includes Fab, Fab', F(ab')2, Fd, Fv, dAb, a complementarity determining region fragment, a single-chain antibody (e.g., scFv), a non-human antibody, a humanized antibody, a chimeric antibody, a fully human antibody, a probody, a bispecific antibody or a multispecific antibody.

[0097] In some embodiments, the conjugate is selected from ADC A-01 to ADC A-25, ADC B-01 to ADC B-05 shown below:

[0098] ADC A-01

[0099] wherein the HA in each conjugate is an antibody or an antigen-binding fragment thereof;

[0100] in, Indicates the specific connection mode between the thiol group in the antibody or its antigen-binding fragment and the M fragment;

[0101] Indicates the specific connection method between the amino group in the antibody or its antigen-binding fragment and the M fragment.

[0102] In some embodiments, Ab-[MLED] x In the antibody drug conjugates shown, x is 1-10, e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10 or 9-10.

[0103] In some embodiments, Ab-[MLED] x The antibody drug conjugates shown have x of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0104] In some embodiments, the conjugate is selected from:

[0105] wherein the HA in each conjugate is an antibody or an antigen-binding fragment thereof;

[0106] in, Indicates the specific connection mode between the thiol group in the antibody or its antigen-binding fragment and the M fragment;

[0107] x is 3-8, for example, 3-4 or 7-8.

[0108] Composition

[0109] In another aspect, the present application provides compositions that may comprise a plurality of ADCs as described herein. Each antibody molecule in the composition may be coupled to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 compounds of the invention. Thus, the composition is characterized by a "drug-antibody ratio" (DAR) in the range of about 1 to about 10. Methods for determining DAR are well known to those skilled in the art and include methods using reverse phase chromatography or HPLC-MS.

[0110] In some embodiments, the DAR value (drug-antibody coupling ratio) of the antibody drug conjugate is 1-10, for example: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4- 10, 5-6, 5-7, 5-8, 5-9, 5-10, 6-7, 6-8, 6-9, 6-10, 7-8, 7-9, 7-10, 8-9, 8-10, or 9-10, preferably 3-9, for example, 3.0-3.5, 3.0-4.0, 3.0-4.5, 3.0-5.0, 3.0-5.5, 3.0-6.0, 3.5-4.0, 3.5-4.5, 3.5-5.0, 3.5-5.5, 3.5 ~6.0,3.5~6.5,3.5~7.0,3.5~7.5,3.5~8.0,4.0~4.5,4.0~5.0,4.0~5.5,4.0~6.0,4.0~6.5,4.0~7.0,4.0~7.5,4.0~8.0,4.5~5.0,4.5~5.5,4.5~6.0,4.5~6.5,4.5~7.0,4.5~7.5,4.5~8.0,5.0~5 .5, 5.0~6.0, 5.0~6.5, 5.0~7.0, 5.0~7.5, 5.0~8.0, 5.5~6.0, 5.5~6.5, 5.5~7.0, 5.5~7.5, 5.5~8.0, 6.0~6.5, 6.0~7.0, 6.0~7.5, 6.0~8.5, 6.5~7.0, 6.5~7.5, 6.5~8.5, 7.0~7.5, 7.0~9.0 or 7.5~9.0.

[0111] synthetic intermediates

[0112] On the other hand, the present application provides a compound shown in the following structure:

[0113] Wherein, s, R1, R2, R3, and Ra are as described above;

[0114] PG is an amino protecting group; for example, PG is 9-fluorenylmethoxycarboxylate (Fmoc), tert-butoxycarboxylate (Boc), benzyloxycarboxylate (Cbz), acetate (Ac), dichloroacetate (CAc), benzoate (Bz), tert-butylcarboxylate (Pv), TMS, TES, TBDMS, TIPS, TBDPS, methoxymethyl ether (MOM), 2-methoxyethoxymethyl ether (MEM), benzyloxymethyl ether (BOM) or p-methoxybenzyloxymethyl ether (PMBOM), trityl (Tr), p-methoxytrityl (MMT), dimethoxytrityl (DMT). In some embodiments, PG is Fmoc or MMT.

[0115] In some embodiments, the present application provides a compound shown in the following structure:

[0116] Synthesis method

[0117] In another aspect, the present application provides methods for preparing the compounds described herein.

[0118] Method 1: Preparation of Compound B-04a

[0119] In some embodiments, the method comprises coupling compound B-04-4a and compound B-04-5 to obtain compound B-04a;

[0120] B-04a:

[0121] wherein s, R1, R2, and R3 are as described above; preferably, s is 8, R1 is methyl, R2 is chlorine, and R3 is -C(=O)-CH2-OH.

[0122] In some embodiments, the coupling reaction is carried out in the presence of a coupling reagent; preferably, the coupling reagent is cuprous bromide.

[0123] In some embodiments, the method comprises deprotecting compound B-04-3a to obtain compound B-04-4a;

[0124] wherein s, R1, R2, R3, and PG are as described above; preferably, s is 8, R1 is methyl, R2 is chlorine, R3 is -C(=O)-CH2-OH, and PG is MMT.

[0125] In some embodiments, the deprotection is performed under acidic conditions; preferably, the acidic conditions are trifluoroacetic acid.

[0126] In some embodiments, the method comprises hydrolyzing compound B-04-2a to obtain compound B-04-3a:

[0127] wherein s, R1, R2, R3, and PG are as described above; preferably, s is 8, R1 is methyl, R2 is chlorine, R3 is -C(=O)-CH2-OH, and PG is MMT.

[0128] In some embodiments, the hydrolysis reaction is carried out in the presence of a base; preferably, the base is a sodium salt; further preferably, the sodium salt is sodium carbonate or sodium bicarbonate.

[0129] In some embodiments, the method comprises coupling compound B-04-1a and compound B-04-6a to obtain compound B-04-2a:

[0130] wherein s, R1, R2, and PG are as described above; preferably, s is 8, R1 is methyl, R2 is chlorine, and PG is MMT.

[0131] In some embodiments, the coupling reaction is carried out in the presence of a coupling reagent; preferably, the coupling reagent includes one or both of DMAP and triphosgene.

[0132] In some embodiments, the method comprises subjecting compound 1-4 to a hydrolysis reaction:

[0133] Wherein, R1, R2, and R3 are as described above; preferably, R1 is methyl, R2 is chlorine, and R3 is -C(=O)-CH2-OH.

[0134] In some embodiments, the hydrolysis reaction is carried out under alkaline conditions; preferably, the base is DIPEA.

[0135] Method 2: Preparation of Compound A-17a

[0136] In some embodiments, the method comprises coupling compound A-17-06a and compound A-07-1 to obtain compound A-17a;

[0137] wherein R1, R2, and s are as described above; preferably, s is 11, R1 is methyl, and R2 is chlorine.

[0138] In some embodiments, the coupling reaction is performed in the presence of a coupling reagent; preferably, the coupling reagent comprises DIPEA.

[0139] In some embodiments, the method comprises subjecting compound A-17-05a to a deprotection reaction to obtain compound A-17-06a;

[0140] wherein R1, R2, PG, and s are as described above; preferably, s is 11, R1 is methyl, R2 is chlorine, and PG is Fmoc.

[0141] In some technical solutions, the deprotection reaction is carried out in the presence of an alkaline reagent; preferably, the alkaline reagent is diethylamine or triethylamine.

[0142] In some embodiments, the method comprises coupling compound A-17-04a and compound 1-5-A-1 to obtain compound A-17-05a;

[0143] wherein R1, R2, PG, and s are as described above; preferably, s is 11, R1 is methyl, R2 is chlorine, and PG is Fmoc.

[0144] In some technical solutions, the coupling reaction is carried out in the presence of a coupling reagent; preferably, the coupling reagent includes DIPEA.

[0145] In some embodiments, the method comprises subjecting Compound A-17-03a and Compound A-07-2 to a coupling reaction;

[0146] Wherein, s and PG are as described above; preferably, s is 11, and PG is Fmoc.

[0147] In some technical solutions, the coupling reaction is carried out in the presence of a coupling reagent; preferably, the coupling reagent includes HATU and DIPEA.

[0148] In some embodiments, the method comprises coupling compound A-17-01a and A-17-02a to obtain compound A-17-03a;

[0149] Wherein, s and PG are as described above; preferably, s is 11, and PG is Fmoc.

[0150] In some technical solutions, the coupling reaction is carried out in the presence of a coupling reagent; preferably, the coupling reagent includes DIPEA.

[0151] Method 3: Preparation of Compound B-03a

[0152] In some embodiments, the method comprises coupling compound B-03-8a and compound A-07-1 to obtain compound B-03a;

[0153] Wherein, Ra is as described above; preferably, Ra is isopropyl.

[0154] In some technical solutions, the coupling reaction is carried out in the presence of a coupling reagent; preferably, the coupling reagent includes an amine reagent; more preferably, diisopropylethylamine.

[0155] In some embodiments, the method comprises subjecting compound B-03-7a to a deprotection reaction to obtain compound B-03-8a;

[0156] Wherein, Ra and PG are as described above; preferably, Ra is isopropyl, and PG is Fmoc.

[0157] In some technical solutions, the deprotection reaction is carried out in the presence of an alkaline reagent; preferably, the alkaline reagent includes a lithium reagent; more preferably, it is lithium hydroxide monohydrate.

[0158] In some embodiments, the method comprises subjecting compound B-03-6a and compound 2-2 to a coupling reaction;

[0159] Wherein, Ra and PG are as described above; preferably, Ra is isopropyl, and PG is Fmoc.

[0160] In some technical solutions, the coupling reaction is carried out in the presence of a coupling reagent; preferably, the coupling reagent includes 1-hydroxybenzotriazole and diisopropylethylamine.

[0161] In some embodiments, the method comprises coupling compound B-03-5a and p-nitrophenyl chloroformate to obtain compound B-03-6a;

[0162] Wherein, PG is as described above; preferably, PG is Fmoc.

[0163] In some technical solutions, the coupling reaction is carried out in the presence of a coupling reagent; preferably, the coupling reagent includes diisopropylethylamine.

[0164] In some embodiments, the method comprises coupling compound B-03-4a and compound B-03-9a to obtain compound B-03-5a;

[0165] Wherein, PG is as described above; preferably, PG is Fmoc.

[0166] In some technical solutions, the coupling reaction is carried out in the presence of [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid.

[0167] In some embodiments, the method comprises subjecting compound B-03-3 to a reduction reaction to obtain compound B-03-4;

[0168] In some technical solutions, the coupling reaction is carried out in the presence of PtO2 and hydrogen.

[0169] Pharmaceutical composition

[0170] In another aspect, the present application provides a pharmaceutical composition comprising the compound or conjugate described in any one of the foregoing items, and one or more pharmaceutical excipients.

[0171] Pharmaceutical excipients include, for example, pharmaceutically acceptable carriers and / or excipients.

[0172] The compounds or conjugates described herein are typically formulated in a unit injectable form with a pharmaceutically acceptable parenteral vehicle for parenteral use, such as bolus injection, intravenous injection, intratumoral injection, etc. Optionally, the antibody drug conjugate having the desired purity is mixed with a pharmaceutically acceptable diluent, carrier, excipient or stabilizer in the form of a lyophilized agent or solution (Remington's Pharmaceutical Sciences (1980) 16 th The antibody drug conjugates described herein, or pharmaceutical compositions containing the same, can be administered by any route appropriate to the individual to be treated.

[0173] The compounds or conjugates, pharmaceutical compositions described herein can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under production and storage conditions. The preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by the following method: the necessary dose of the antibody of the present invention is incorporated into a suitable solvent and other required ingredients (including but not limited to pH regulators, surfactants, adjuvants, ionic strength enhancers, etc., penetrants, preservatives, diluents or any combination thereof) are optionally added, followed by filtration sterilization. In addition, for ease of storage and use, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze drying). Such sterile lyophilized powders can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

[0174] In addition, the compounds or conjugates described herein can be present in a pharmaceutical composition in a unit dosage form for ease of administration by any suitable method known in the art, including but not limited to oral, oral, sublingual, ophthalmic, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powders, ointments or drops) or intranasal routes. However, for many therapeutic uses, the preferred route of administration / mode is parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). The skilled artisan will appreciate that the route and / or mode of administration will vary depending on the intended purpose. In some preferred embodiments, the ADC and pharmaceutical compositions described herein are administered by intravenous infusion or injection.

[0175] In certain embodiments, the pharmaceutical composition may further comprise other pharmaceutically active agents. In certain embodiments, the other pharmaceutically active agents are drugs with anti-tumor activity. In certain embodiments, the other pharmaceutically active agents are selected from EGFR inhibitors, HER2 inhibitors, HER3 inhibitors, HER4 inhibitors, IGFR-1 inhibitors, mTOR inhibitors, PI3 kinase inhibitors, c-met or VEGF inhibitors, chemotherapeutic drugs or any combination thereof. In certain embodiments, the compounds or conjugates described herein and other pharmaceutically active agents are provided as separate components or as mixed components. Therefore, the compounds or conjugates described herein and other pharmaceutically active agents can be administered simultaneously, separately or sequentially.

[0176] application

[0177] The present application provides the use of the aforementioned compound or its pharmaceutically acceptable salt, conjugate, composition, or pharmaceutical composition in the preparation of a drug for treating cancer or tumor.

[0178] The present application provides the aforementioned compound or a pharmaceutically acceptable salt, conjugate, composition, or pharmaceutical composition thereof, which is used to treat cancer or tumors.

[0179] The present application provides the aforementioned compound or a pharmaceutically acceptable salt, conjugate, composition, or pharmaceutical composition thereof, which is used to treat cancer or tumors.

[0180] The present application provides a method for treating cancer or tumors, which comprises administering a therapeutically effective amount of the aforementioned compound or a pharmaceutically acceptable salt, conjugate, composition, or pharmaceutical composition thereof to an individual in need thereof.

[0181] In some embodiments, the compound, or a pharmaceutically acceptable salt, conjugate, composition, or pharmaceutical composition thereof, is sufficient (e.g., in a subject):

[0182] (1) Inhibit cell (such as tumor cell) proliferation;

[0183] (2) inhibit tumor growth;

[0184] (3) induce and / or increase antibody-dependent cellular cytotoxicity activity;

[0185] (4) inhibit signal transduction;

[0186] (5) prevention and / or treatment of cancer; or

[0187] (6) Any combination of (1) to (5) above.

[0188] In certain embodiments, the cancer or tumor is selected from breast cancer, gastric cancer, lung cancer (e.g., non-small cell lung cancer), colorectal cancer, pancreatic cancer, head and neck squamous cell carcinoma, melanoma, ovarian cancer, prostate cancer, liver cancer, kidney cancer, bladder cancer, lymphoma, or any combination thereof.

[0189] definition

[0190] Unless otherwise defined below, the meanings of all technical and scientific terms used herein are intended to be the same as those generally understood by those skilled in the art. Reference to the techniques used herein is intended to refer to techniques generally understood in the art, including variations of those techniques that are obvious to those skilled in the art or replacements with equivalent techniques. Furthermore, laboratory procedures such as genomics, nucleic acid chemistry, and molecular biology used herein are conventional procedures widely used in the corresponding fields. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

[0191] The term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein), or degradation products thereof (such as lactalbumin hydrolysate), etc.

[0192] The terms "comprises," "comprising," "having," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0193] The term "effective amount" refers to an amount sufficient to achieve or at least partially achieve the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently. The therapeutically effective amount of an ADC may vary depending on the severity of the disease to be treated, the general state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently.

[0194] The term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization (i.e., no longer worsening) of the disease state, delaying or slowing the progression of the disease, improvement or alleviation of the disease state, and relief of symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival if not receiving treatment.

[0195] The term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (such as a human) suffers from a tumor, or has a risk of suffering from the above-mentioned disease.

[0196] The terms "cancer" and "tumor" are used interchangeably to refer to a broad category of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors, or cells that invade neighboring tissues and may spread to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer also includes blood cancers, particularly hematologic malignancies.

[0197] The term "hematologic malignancies" includes lymphomas, leukemias, myelomas or lymphoid malignancies, as well as splenic cancer and lymph node tumors. Exemplary lymphomas include B-cell lymphomas and T-cell lymphomas. B-cell lymphomas include, for example, Hodgkin's lymphoma. T-cell lymphomas include, for example, cutaneous T-cell lymphoma. Hematologic malignancies also include leukemias, such as secondary leukemias or acute lymphoblastic leukemia. Hematologic malignancies also include myeloma (e.g., multiple myeloma) and other blood and / or B-cell or T-cell related cancers.

[0198] The term "alkyl" refers to a group obtained by removing one hydrogen atom from a straight-chain or branched hydrocarbon group, for example, "C 1-20 Alkyl", "C 1- 10 Alkyl", "C 1-6 Alkyl", "C 1-4 Alkyl", "C 1-3 alkyl”, etc., specific examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, etc.

[0199] The term "alkylene" refers to a group obtained by removing two hydrogen atoms from a straight or branched hydrocarbon group, for example, "C 1-20 Alkylene", "C 1-10 Alkylene", "C 3-10 Alkylene", "C 5-8 Alkylene", "C 1-6 Alkylene", "C 1-4 Alkylene", "C 1-3 Specific examples include, but are not limited to, methylene, ethylene, 1,3-propylene, 1,4-butylene, 1,5-pentylene or 1,6-hexylene.

[0200] The term "alkenylene" refers to a divalent group derived from a straight or branched hydrocarbon group containing at least one carbon-carbon double bond losing two hydrogen atoms, including, for example, "C 2-20 Alkenylene", "C 3-10 Alkenylene", "C 5-8 Examples include, but are not limited to, vinylene, 1-propenylene, 2-propenylene, 1-butenylene, 2-butenylene, 1,3-butadienylene, 1-pentenylene, 2-pentenylene, 3-pentenylene, 1,3-pentadienylene, 1,4-pentadienylene, 1-hexenylene, 2-hexenylene, 3-hexenylene, 1,4-hexadienylene, and the like.

[0201] The term "alkynylene" refers to a divalent group derived from a straight or branched hydrocarbon group containing at least one carbon-carbon triple bond that loses two hydrogen atoms. 2-20 Alkynylidene", "C 3-10 Alkynylidene", "C 5-8Examples include, but are not limited to, ethynylene, 1-propynylene, 2-propynylene, 1-butynylene, 2-butynylene, 1,3-butadiynylene, 1-pentynylene, 2-pentynylene, 3-pentynylene, 1,3-pentadiynylene, 1,4-pentadiynylene, 1-hexynylene, 2-hexynylene, 3-hexynylene, 1,4-hexadiynylene, and the like.

[0202] The term "aliphatic heterocycle" refers to a saturated or partially saturated cyclic structure containing at least one ring member selected from N, O, and S. Specific examples include, but are not limited to, 5-6 membered aliphatic heterocycles, 5-6 membered nitrogen-containing aliphatic heterocycles, 5-6 membered oxygen-containing aliphatic heterocycles, and the like, such as tetrahydrofuran, pyrrolidine, piperidine, tetrahydropyran, and the like.

[0203] The term "heteroaromatic ring" refers to an aromatic ring structure containing at least one ring member selected from N, O and S. Specific examples include, but are not limited to, 5-6 membered aromatic heterocycles, 5-6 membered nitrogen-containing aromatic heterocycles, 5-6 membered oxygen-containing aromatic heterocycles, and the like, such as furan, thiophene, pyrrole, thiazole, isothiazole, thiadiazole, oxazole, isoxazole, oxadiazole, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, pyridine, pyrimidine, pyridazine, pyrazine, 1,2,3-triazine, 1,3,5-triazine, 1,2,4,5-tetrazine, and the like.

[0204] The term "aromatic ring system" refers to a monocyclic or polycyclic ring system comprising at least one aromatic ring (e.g., a benzene ring, etc.) or heteroaromatic ring (e.g., a pyrimidine ring, etc.), two or more aromatic rings and / or heteroaromatic rings may form a fused ring or be connected by a single bond (e.g., dipyrimidinylphenyl, etc.), and the aromatic ring system may be divalent or higher valent (e.g., trivalent or tetravalent), for example, a 5-20 membered aromatic ring system.

[0205] The term "substituted" refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogen atoms on a designated compound or structural fragment with a substituent, provided that the normal valence of the designated atom in the present context is not exceeded and the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form stable compounds. For example, the substituents are each independently composed of one or more of the following structures: -O-, -S-, -NR'-, halogen, -CN, -OH, -NH2, -NO2, -CN, =O, C1-C6 alkylene, C1-C6 haloalkylene, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkylene, 3-8 membered heterocyclyl, C6-C 10(Ethyl)aryl and 5-10 membered (Ethyl)heteroaryl, etc.; wherein R' is H or C1-C6 alkyl, C1-C6 haloalkyl, C3-C8 cycloalkyl, 3-8 membered heterocyclic group, C6-C 10 aryl or 5-10 membered heteroaryl.

[0206] If a functional group or structural moiety is described as "substituted or unsubstituted," the functional group or structural moiety may be (1) unsubstituted or (2) substituted.

[0207] As used herein, the terms "about" or "approximately" when used in conjunction with a numerical variable generally mean that the value of the variable is within the range of experimental error (e.g., within a 95% confidence interval for the mean) or within a range of 10% or greater.

[0208] It should be noted that if there is a discrepancy between a described structure and the name of that structure, the described structure will be given greater weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0209] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0210] Figure 1A-C shows the bystander effect assay results of anti-human HER3 antibody drug conjugates on HEK293T and HEK293T-h HER3 cells.

[0211] FIG2 shows the plasma stability test results of the anti-human HER3 antibody drug conjugate.

[0212] FIG3A shows the HPLC spectra of compounds A-07-A and A-07-B prepared in Example 3.

[0213] FIG3B shows the HPLC spectra of compound A-07-A prepared in Example 3 and compound A-05 prepared in Example 9. DETAILED DESCRIPTION

[0214] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and in no way constitutes any limitation on the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0215] Sequence Information Table

[0216] The abbreviations used in this document have the following meanings:

[0217] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0218] Nuclear magnetic resonance (NMR) 1 H NMR measurements were performed using a Bruker 400 MHz nuclear magnetic resonance instrument; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard was tetramethylsilane (TMS).

[0219] The abbreviations used in the nuclear magnetic resonance (NMR) spectra in the examples are shown below.

[0220] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: hertz, DMSO-d6: deuterated dimethyl sulfoxide. δ values ​​are expressed in ppm.

[0221] Mass spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0222] Example 1 N-((S)-10-benzyl-1-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1-H-pyrrol-1-yl)hexanamide (A-01)

[0223] Compound A-01-1 (0.40 g, 640.59 μmol, its synthesis is described in patent application CN 111936169A) and isotecan mesylate (0.37 g, 704.65 μmol) were dissolved in DMF (8 mL). HATU (0.32 g, 832.77 μmol) and DIPEA (0.25 g, 1.92 mmol) were added and reacted at 25°C for 4 hours. DIPEA was removed under reduced pressure, and the mixture was freeze-dried with water to remove most of the DMF to obtain a crude product. The crude product was purified by preparative HPLC (conditions as follows) to obtain 273 mg of the title compound.

[0224] Column: Waters XBridge Prep C18 OBD 45 mm × 450 mm × 8.0 μm

[0225] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0226] The structural characterization data are as follows:

[0227] ESI-MS (m / z): 1034.4 [M+H] + .

[0228] Example 2 N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (1-8-A and 1-8-B)

[0229] Step 1: Synthesis of 1-chloro-3-bromo-2-methyl-5-nitrobenzene (1-8-2)

[0230] Compound 1-8-1 (5.00 g, 29.14 mmol) was dissolved in n-heptane (25 mL) at 25°C, concentrated sulfuric acid (25 mL) was added, and the mixture was heated to 50°C. NBS (6.22 g, 34.97 mmol) was added in batches at 50°C, and the reaction was maintained at 50°C for 2 hours. The reaction was monitored by thin-layer chromatography (ethyl acetate: petroleum ether = 1:10). The reaction solution was cooled to room temperature and then added dropwise to ice water. The mixture was extracted with toluene, and the organic phases were combined and washed with sodium sulfite solution, water, and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by preparative high-performance liquid chromatography (conditions as follows). The preparative solution was freeze-dried to obtain 4.88 g of the title compound.

[0231] Chromatographic column: C18 ODS 45mm×450mm×8.0μm

[0232] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0233] Step 2: Synthesis of 3-chloro-5-bromo-4-methylaniline (1-8-3)

[0234] Compound 1-8-2 (4.88 g, 19.48 mmol) was dissolved in ethyl acetate (100 mL) at 25°C, and platinum on carbon (2.00 g, 19.48 mmol, 5% content) was added. After hydrogen replacement, the mixture was reacted at 60°C under hydrogen protection for 4 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was filtered and concentrated to obtain 3.68 g of the crude title compound, which was used directly in the next reaction without further purification.

[0235] Step 3: Synthesis of N-(3-chloro-5-bromo-4-methylphenyl)acetamide (1-8-4)

[0236] Compound 1-8-3 (3.63 g, 14.82 mmol) was dissolved in ethyl acetate (70 mL) at 20°C, and triethylamine (4.50 g, 44.45 mmol) and acetic anhydride (2.27 g, 22.23 mmol) were added. The reaction was maintained at 20°C for 20 h, and the reaction was monitored by high-performance liquid chromatography-mass spectrometry. Water was added to the reaction solution, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was slurried in a mixed solvent of ethyl acetate: petroleum ether = 1:5 to obtain 2.86 g of the title compound.

[0237] Step 4: Synthesis of (Z)-4-(5-acetamido-3-chloro-2-methylphenyl)but-3-enoic acid (1-8-5)

[0238] At 20°C, compound 1-8-4 (1.80 g, 6.86 mmol) was dissolved in THF (20 mL) and water (5 mL). Vinyl acetic acid (708.31 mg, 8.23 ​​mmol), DIPEA (1.95 g, 15.08 mmol), and tris(o-methylphenyl)phosphine (62.60 mg, 0.20 mmol) were added. The reaction system was purged with nitrogen and heated to 70°C for 5 h. The reaction was monitored by HPLC-MS / MS. 1N sodium hydroxide solution was added to the reaction solution to adjust the pH to 8, and ethyl acetate was added for extraction. The aqueous phase was adjusted to pH 3 with 1N hydrochloric acid and extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 0.82 g of the title compound, which was used directly in the next step.

[0239] Step 5: Synthesis of 4-(5-acetamido-3-chloro-2-methylphenyl)butyric acid (1-8-6)

[0240] Compound 1-8-5 (2.60 g, 9.71 mmol) was dissolved in THF (50 mL) at 20°C, and Pd / C (0.52 g, 10%) was added. The system was replaced with hydrogen and reacted under a hydrogen balloon at 40°C for 2 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was filtered and the filtrate was concentrated to obtain 2.43 g of the title compound, which was used directly in the next reaction without further purification.

[0241] Step 6: Synthesis of N-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-7)

[0242] Compound 1-8-6 (2.43 g, 9.01 mmol) was dissolved in trifluoroacetic acid (10 mL), cooled to 5°C, and trifluoroacetic anhydride (3.78 g, 18.02 mmol, 2.50 mL) was added dropwise. The reaction temperature was maintained at 5°C for 4 h, and the reaction was monitored by high performance liquid chromatography-mass spectrometry. The reaction solution was added to water, dissolved in 10N sodium hydroxide and adjusted to pH = 9, and extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified on a silica gel column (ethyl acetate:petroleum ether = 0-20%) to obtain 1.53 g of the title compound.

[0243] Step 7: Synthesis of (Z)-N-(3-chloro-7-(hydroxyimino)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-8)

[0244] At 5°C, potassium tert-butoxide (1.50 g, 13.37 mmol) was dissolved in THF (16 mL) and tert-butanol (4 mL). A THF solution (16 mL) of compound 1-8-7 (1.53 g, 6.08 mmol) was added dropwise. After 10 minutes, amyl nitrite (1.14 g, 9.73 mmol) was added dropwise. The reaction was maintained at 5°C for 1 hour and monitored by HPLC-MS / MS. The reaction solution was adjusted to pH 5 with 1N hydrochloric acid and extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was slurried with methyl tert-butyl ether to obtain 1.20 g of the title compound.

[0245] Step 8: Synthesis of N-(7-amino-3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-9)

[0246] Compound 1-8-8 (0.50 g, 1.78 mmol) was dissolved in methanol (8 mL) and 2N hydrochloric acid (8 mL) at 20°C. Pd / C (0.15 g, 10%) was added. The system was replaced with hydrogen and maintained at 5°C under a hydrogen balloon for 2 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was filtered and concentrated to obtain 0.52 g of the hydrochloride salt of the title compound, which was used directly in the next reaction without further purification.

[0247] Step 9: Synthesis of N,N'-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (1-8-10)

[0248] Compound 1-8-9 (0.52 g, 1.70 mmol) was dissolved in pyridine (5 mL) at 20°C, and acetic anhydride (2 mL) was added. The reaction was maintained at 20°C for 2 h, and the reaction was monitored by HPLC-MS / MS. The reaction solution was added to water and extracted with ethyl acetate. The organic phases were washed with water, combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified on a silica gel column (ethyl acetate:petroleum ether = 0-30%) to obtain 0.22 g of the title compound.

[0249] Step 10: Synthesis of N-(8-amino-6-chloro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (1-8-11)

[0250] Compound 1-8-10 (450.97 mg, 1.46 mmol) was dissolved in methanol (16 mL) at 20°C, 2N hydrochloric acid (16 mL) was added, and the mixture was heated to 60°C for 2 h. The reaction was monitored by HPLC-MS / MS. Saturated sodium bicarbonate solution was added to the cooled reaction solution to adjust the pH to 8, and the mixture was extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 230.00 mg of the title compound, which was used directly in the next step without further purification.

[0251] Step 11: Synthesis of N-((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1-8-12)

[0252] Compound 1-8-11 (230.00 mg, 0.78 mmol) was dissolved in toluene (10 mL), and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (230.00 mg, 0.87 mmol) and p-toluenesulfonic acid (26.73 mg, 0.16 mmol) were added. The mixture was heated to 140°C for 5 h and monitored by HPLC-MS / MS. The reaction solution was concentrated and the crude product was purified on a silica gel column (methanol:dichloromethane = 0-10%) to obtain 150.00 mg of the title compound.

[0253] Step 12: Synthesis of (9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (1-2)

[0254] Compound 1-8-12 (40.00 mg, 0.081 mmol) was added to concentrated hydrochloric acid (1 mL) and heated to 100°C for 5 h. The reaction was monitored by HPLC-MS / MS. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (conditions as follows). The preparative solution was freeze-dried to obtain the hydrochloride salt of the title compound 1-2. The hydrochloride salt of 1-2 was separated under the following purification conditions to yield two isomers, designated 1-2-A (trifluoroacetate salt 5.00 mg, retention time 9.85 min) and 1-2-B (trifluoroacetate salt 7.00 mg, retention time 10.62 min) based on their retention times.

[0255] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0256] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0257] The structural characterization data are as follows:

[0258] 1-2-A:

[0259] 1 H NMR(400MHz,DMSO-d6)δ8.42(s,3H),8.27(s,1H),7.36(s,1H),6.59(s,1H),5.78-5.63(m,1H),5.50-5.36(m,3H) ,5.10-5.06(m,1H),3.20-3.04(m,2H),2.56(s,3H),2.26-2.13(m,2H),1.93-1.79(m,2H),0.88(t,J=7.2Hz,3H).

[0260] ESI-MS (m / z): 452.1 [M+H] + .

[0261] 1-2-B:

[0262] 1 H NMR(400MHz,DMSO-d6)δ8.42(s,3H),8.27(s,1H),7.36(s,1H),6.58(s,1H),5.78-5.63(m,1H),5.50-5.36(m,3H) ,5.10-5.06(m,1H),3.20-3.04(m,2H),2.55(s,3H),2.26-2.13(m,2H),1.93-1.79(m,2H),0.88(t,J=7.2Hz,3H).

[0263] ESI-MS (m / z): 452.0 [M+H] + .

[0264] Step 13: 2-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide and 2-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide Synthesis of (1-8-13-A and 1-8-13-B)-N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide

[0265] At 25 ° C, the hydrochloride of compound 1-2 (40.00 mg, 81.91 μmol) was dissolved in N, N-dimethylformamide (1 mL), and 2-((tert-butyldiphenylsilyl)oxy)acetic acid (30.91 mg, 98.29 μmol), HATU (62.25 mg, 163.81 μmol) and N, N-diisopropylethylamine (42.34 mg, 327.63 μmol) were added in sequence. The reaction was maintained at 25 ° C for 0.5 hour and the reaction was monitored by high performance liquid chromatography-mass spectrometry. After the reaction is completed, water is added to the reaction solution, and the mixture is extracted with dichloromethane / methanol (v / v=10 / 1). The organic phases are combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product is purified by preparative thin layer chromatography (dichloromethane:methanol=20:1) to separate and obtain two isomers. The two isomers are named 1-8-13-A (15.00 mg, Rf value 0.3) and 1-8-13-B (12.00 mg, Rf value 0.35) based on the Rf value.

[0266] Step 14: Synthesis of N-((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N-((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (1-8-A and 1-8-B)

[0267] At 25°C, 1-8-13-A (15.00 mg) and 1-8-13-B (12.00 mg) were dissolved in tetrahydrofuran (1 mL) in two reaction flasks. A mixture of tetrabutylammonium fluoride (1 M solution in tetrahydrofuran) and glacial acetic acid (v / v = 13 / 1) (50 μL) was added dropwise. The reaction was maintained at 25°C for 0.5 hours and monitored by HPLC-MS / MS. After completion of the reaction, the reaction solutions were purified by preparative HPLC, and the preparative solutions were lyophilized to give the title compounds 1-8-A (6.94 mg) and 1-8-B (4.00 mg), respectively.

[0268] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0269] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0270] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min]

[0271] The structural characterization data of 1-8-A are as follows:

[0272] 1H NMR (400MHz, DMSO-d6) δ8.43(d,J=8.8Hz,1H),8.16(s,1H),7.31(s,1H),6.55(s,1H),5.65-5.36(m,4H),5.21(q,J=19.0H z,2H),3.95(d,J=5.7Hz,2H),3.26-3.11(m,2H),2.53(s,3H),2.30-2.08(m,2H),1.94-1.79(m,2H),0.87(t,J=7.3Hz,3H).

[0273] ESI-MS (m / z): 510.1 [M+H] + .

[0274] The structural characterization data of 1-8-B are as follows:

[0275] 1H NMR (400MHz, DMSO-d6) δ8.45(d,J=8.9Hz,1H),8.15(s,1H),7.31(s,1H),6.54(s,1H),5.64-5.35(m,4H),5.19(q,J=19.0H z,2H),3.97(d,J=5.2Hz,2H),3.27-3.10(m,2H),2.51(s,3H),2.27-2.10(m,2H),1.93-1.80(m,2H),0.88(t,J=7.3Hz,3H).

[0276] ESI-MS (m / z): 510.1 [M+H] + .

[0277] Example 3 N-((10S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4';6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide and N-((1 0S)-10-benzyl-1-(((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4';6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-07-A and A-07-B)

[0278] Step 1: Synthesis of (S)-10-benzyl-23-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15,18-pentaoxo-3-oxa-5,8,11,14,17-pentaazatricosane-22-ynecarboxylic acid (A-07-3)

[0279] Compound A-07-2 (30.00 mg, 0.07 mmol) was dissolved in DMF (0.2 mL) at 25°C, and 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoate (A-07-1, 28.00 mg, 0.08 mmol) was added. The mixture was reacted at 30°C for 1 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was directly purified by preparative HPLC (conditions as follows) and the preparative solution was freeze-dried to obtain 20.00 mg of the title compound.

[0280] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0281] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0282] The structural characterization data are as follows:

[0283] ESI-MS (m / z): 691.0 [M+H2O] + .

[0284] Step 2: N-((10S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4';6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide and N-((10 Synthesis of S)-10-benzyl-1-(((1R,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4';6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-07-A and A-07-B)

[0285] At 25°C, the hydrochloride salt of 1-2 (30.00 mg, 61.43 μmol) was dissolved in N,N-dimethylformamide (1 mL). A-07-3 (49.66 mg, 73.72 μmol), HATU (35.01 mg, 92.14 μmol), and N,N-diisopropylethylamine (23.82 mg, 184.29 μmol) were added sequentially. The mixture was maintained at 25°C for 0.5 hours and monitored by HPLC-MS / MS. After completion, the reaction solution was purified by preparative HPLC (conditions listed below) and freeze-dried to obtain the title compound A-07. A-07 was separated under the following purification conditions to yield two isomers, designated A-07-A (11.04 mg, retention time 7.5 min) and A-07-B (19.42 mg, retention time 8.0 min) based on their retention times.

[0286] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0287] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0288] The structural characterization data are as follows:

[0289] A-07-A:

[0290] ESI-MS (m / z): 1107.3 [M+H] + .

[0291] A-07-B:

[0292] ESI-MS (m / z): 1107.3 [M+H] + .

[0293] Example 4 (S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (2-2)

[0294] Step 1: Synthesis of 3-(isopropylamino)-1-(6-nitrobenzo[d][1,3]dioxin-5-yl)propan-1-one (2-2-2)

[0295] Compound 2-2-1 (1.90 g, 8.08 mmol) was slowly added to nitric acid (8 mL) at 0°C and the temperature was slowly raised to 25°C for 1 hour. The reaction solution was directly purified by reverse phase column chromatography (acetonitrile / 0.5% formic acid aqueous solution) to obtain 1.50 g of the formate salt of the title compound.

[0296] The structural characterization data are as follows:

[0297] ESI-MS (m / z): 281.1 [M+H] + .

[0298] Step 2: Synthesis of 1-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-(isopropylamino)propan-1-one (2-2-3)

[0299] The formate salt of compound 2-2-2 (1.25 g, 4.46 mmol) was added to tetrahydrofuran (20 mL), followed by 10% palladium on carbon (125.00 mg). The mixture was replaced with hydrogen three times and allowed to react at 25°C for 16 hours. The reaction mixture was filtered through celite, and the filtrate was concentrated to dryness under reduced pressure to obtain 895.00 mg of the crude title compound, which was used directly in the next reaction without purification.

[0300] The structural characterization data are as follows:

[0301] ESI-MS (m / z): 251.1 [M+H] + .

[0302] Step 3: Synthesis of ((S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (2-2)

[0303] Compound 2-2-3 (23.00 mg, 0.09 mmol) and (4S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (21.00 mg, 0.09 mmol) were dissolved in toluene (4 mL), p-toluenesulfonic acid (1.40 mg, 0.009 mmol) was added, and the mixture was reacted at 140°C for 12 hours. The solvent was evaporated under reduced pressure to obtain a crude product of the title compound, which was purified by HPLC (mobile phase A: acetonitrile, mobile phase B: 0.05% formic acid aqueous solution). 3 drops of 3M hydrochloric acid were added to the prepared solution, followed by freeze-drying to obtain 8.70 mg of the hydrochloride salt of the title compound.

[0304] The structural characterization data are as follows:

[0305] ESI-MS (m / z): 478.2 [M+H] + .

[0306] 1 H-NMR (400MHz, DMSO-d6): δ9.38(brs,2H),7.85(s,1H),7.52(s,1H),7.25(s,1H),6. 30(d,J=2.0Hz,2H),5.43(s,2H),5.30(s,2H),3.57-3.45(m,2H),3.40-3.25(m,1H), 3.22-3.06(m,2H),1.95-1.77(m,2H),1.27(d,J=6.4Hz,6H),0.87(t,J=7.6Hz,3H).

[0307] Example 5 Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropyl-N-methylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (B-01)

[0308] Step 1: Synthesis of (S)-4-ethyl-11-(2-(N-isopropyl-N-methylsulfonylamino)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl(4-((S)-2-(4-(((4-methoxyphenyl)diphenylmethyl)amino)butyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxy-3,9-diazapentatriacontamido)benzyl)carbonate (B-01-2)

[0309] At room temperature, compound B-01-1 (413.40 mg, 0.251 mmol, its synthesis reference patent CN111295389B) was dissolved in dimethyl sulfoxide and water (2.0 mL: 0.5 mL), and cuprous bromide (72.95 mg, 0.503 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)-hex-5-ynamide (95.10 mg, 0.302 mmol) were added. The reaction was stirred for 1 h and then filtered. The filtrate was purified by preparative high performance liquid chromatography (conditions as follows) to obtain 30.00 mg of the title compound.

[0310] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0311] Mobile phase A: acetonitrile; mobile phase B: water

[0312] The structural characterization data are as follows:

[0313] ESI-MS(m / z):815.9[(M-273) / 2+H] + .

[0314] Step 2: Synthesis of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonaoxa-3,9-diazapentatriacontamido)benzyl((S)-4-ethyl-11-(2-(N-isopropyl-N-methylsulfonamido)ethyl)-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)carbonate (B-01)

[0315] Compound B-01-2 (30.00 mg, 0.02 mmol) was dissolved in dichloromethane (1.0 mL). Trifluoroacetic acid (0.2 mL) was added to the reaction mixture and allowed to react at room temperature for 30 min. The reaction mixture was concentrated under reduced pressure and purified by preparative HPLC (conditions as follows) to obtain 20.00 mg of the trifluoroacetate salt of the title compound.

[0316] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0317] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0318] The structural characterization data are as follows: ESI-MS (m / z): 816.0 [M / 2+H] + .

[0319] 1H NMR (400MHz, DMSO-d6) δ10.18(s,1H),9.10(s,2H),8.38(t,J=5.56Hz,1H) ,8.32(d,J=8.40Hz,1H),8.22-8.20(m,2H),8.09(t,J=5.68Hz,1H),7.91-7 .87(m,2H),7.82-7.78(m,1H),7.69(brs,3H),7.61(d,J=8.56Hz,2H),7.3 2(d,J=8.56Hz,2H),7.06(s,1H),5.56(d,J=16.96Hz,1H),5.51(d,J=16.96 Hz,1H),5.47(d,J=19.28Hz,1H),5.42(d,J=19.28Hz,1H),5.14(d,J=12.2 0Hz,1H),5.07(d,J=12.16Hz,1H),4.48(t,J=5.24Hz,2H),4.46-4.43(m,1H ),4.29(d,J=5.60Hz,2H),4.08-3.95(m,5H),3.79(t,J=5.28Hz,2H),3.51 -3.43(m,32H),3.40(s,3H),3.39-3.35(m,2H),3.30-3.26(m,2H),3.00(s, 3H),2.82-2.74(m,2H),2.56(t,J=7.08Hz,2H),2.29(t,J=7.36Hz,2H),2.23-2.13(m,2H),1.82(p,J=7.24Hz,2H),1.78-1 .63(m,2H),1.61-1.49(m,2H),1.42-1.27(m,2H),1.15(d,J=6.80Hz,3H),1.13(d,J=6.76Hz,3H),0.90(t,J=7.32Hz,3H).

[0320] Example 6 (2S,3S,4S,5R,6S)-6-(4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-(2-(2-(2-(2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)ethoxy)ethoxy)acetamido))phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (B-03)

[0321] Step 1: Synthesis of (2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (B-03-3)

[0322] Compound (2R,3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetic acid triester (B-03-1, 12.32 g, 31.02 mmol) and 4-hydroxy-3-nitrobenzyl alcohol (B-03-2, 5.00 g, 29.56 mmol) were dissolved in acetonitrile (200 mL). Silver oxide (27.40 g, 118.25 mmol) was added with stirring. After nitrogen substitution, the mixture was allowed to react at room temperature in the dark for 12 hours. The reaction was monitored by HPLC-MS / MS. The reaction solution was filtered through celite, and the filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 1:3) to obtain 12.80 g of the title compound.

[0323] The structural characterization data are as follows:

[0324] ESI-MS (m / z): 503 [M+18] + .

[0325] Step 2: Synthesis of (2S,3R,4S,5S,6S)-2-(2-amino-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (B-03-4)

[0326] Compound B-03-3 (2.20 g, 4.53 mmol) was dissolved in ethyl acetate and tetrahydrofuran (50 mL each), and PtO2 (0.20 g) was added. The reaction system was then purged with a hydrogen balloon three times and allowed to react under a hydrogen atmosphere for 2 hours. The reaction was monitored by HPLC-MS / MS. The reaction mixture was filtered, the filter cake was rinsed with ethyl acetate, and the filtrate was evaporated to dryness under reduced pressure to obtain 2.02 g of the crude title compound, which was used directly in the next reaction.

[0327] The structural characterization data are as follows:

[0328] ESI-MS (m / z): 456.1 [M+1] + .

[0329] Step 3: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(2-(2-(2-(9H-fluoren-9-ylmethoxycarbonyl)amino)ethoxy)ethoxy)acetamido)-4-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (B-03-5)

[0330] Compound B-03-4 (456.00 mg, 1.00 mmol) and [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid (385.91 mg, 1.00 mmol) were dissolved in dichloromethane (10 mL). 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (495.22 mg, 2.00 mmol) was added with stirring. The mixture was stirred for 2 hours. The reaction was monitored by HPLC-MS / MS. The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography (methanol:dichloromethane = 1:20) to obtain 507.00 mg of the title compound.

[0331] The structural characterization data are as follows:

[0332] ESI-MS (m / z): 823.3 [M+1] + .

[0333] Step 4: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(2-(2-(2-(9H-fluoren-9-ylmethoxycarbonyl)amino)ethoxy)ethoxy)acetamido)-4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (B-03-6)

[0334] Compound B-03-5 (507.00 mg, 616.18 μmol) and diisopropylethylamine (238.91 mg, 1.85 mmol) were dissolved in dichloromethane (20 mL). p-Nitrophenyl chloroformate (372.60 mg, 1.85 mmol) was then dissolved in dichloromethane (1 mL) and slowly added dropwise to the reaction mixture. The mixture was allowed to react at room temperature for 15 h. The reaction was monitored by HPLC-MS / MS. The reaction mixture was concentrated under reduced pressure and purified by silica gel column chromatography (methanol:dichloromethane = 1:20) to obtain 496.00 mg of the title compound.

[0335] The structural characterization data are as follows:

[0336] ESI-MS (m / z): 988.5 [M+1] + .

[0337] Step 5: Synthesis of (2S,3R,4S,5S,6S)-2-(2-(2-(2-(2-(9H-fluoren-9-ylmethoxycarbonyl)amino)ethoxy)ethoxy)acetamido)-4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxy-8,10,11,13-tetrahydro-7H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate (B-03-7)

[0338] Compound B-03-6 (165.51 mg, 0.17 mmol), compound 2-2 (40.00 mg, 0.084 mmol) and 1-hydroxybenzotriazole (33.96 mg, 0.25 mmol) were dissolved in DMF (4 mL), and diisopropylethylamine (32.48 mg, 0.25 mmol) was added dropwise. The mixture was stirred for 12 h and monitored by HPLC-MS / MS. Water and ethyl acetate were added and stirred, and the mixture was allowed to stand for separation. The organic phase was washed with saturated brine and dried, and concentrated under reduced pressure to obtain 100.00 mg of the crude title compound, which was directly used for the next step.

[0339] The structural characterization data are as follows:

[0340] ESI-MS (m / z): 1326.2 [M+1] + .

[0341] Step 6: Synthesis of (2S,3S,4S,5R,6S)-6-(2-(2-(2-(aminoethoxy)ethoxy)acetamido)-4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxy-8,10,11,13-tetrahydro-7H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (B-03-8)

[0342] Compound B-03-7 (100.00 mg, 0.08 mmol) was dissolved in MeOH (5 mL), and 1 drop of dichloromethane was added dropwise. An aqueous solution of lithium hydroxide monohydrate (15.82 mg, 0.377 mmol) (1 mL) was then added dropwise. The mixture was stirred and reacted for 2 hours. The reaction was monitored by HPLC-MS / MS. 3N aqueous hydrochloric acid was added dropwise to adjust the pH of the reaction solution to 4. The solution was concentrated under reduced pressure and purified by preparative HPLC (conditions as follows). The preparative solution was freeze-dried to obtain 27.00 mg of the title compound.

[0343] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0344] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0345] The structural characterization data are as follows:

[0346] ESI-MS (m / z): 964.2 [M+1] + .

[0347] Step 7: Synthesis of (2S,3S,4S,5R,6S)-6-(4-((((2-((S)-7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolano[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(isopropyl)carbamoyl)oxy)methyl)-2-(2-(2-(2-(2-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)ethoxy)ethoxy)acetamido))phenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (B-03)

[0348] Compound B-03-8 (27.00 mg, 0.03 mmol) and 2,5-dioxopyrrolidin-1-yl 6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynoate (A-07-1), 11.26 mg, 0.03 mmol) were dissolved in DMF (1 mL). Diisopropylethylamine (3.62 mg, 0.03 mmol) was added dropwise with stirring. The mixture was allowed to react at room temperature for 4 hours. The reaction was monitored by HPLC-MS / MS. The reaction solution was purified by preparative HPLC (conditions as follows) and the preparative solution was freeze-dried to obtain 11.70 mg of the title compound.

[0349] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0350] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0351] The structural characterization data are as follows:

[0352] ESI-MS (m / z): 1214.4 [M+1] + .

[0353] Example 7 N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (1-11-A and 1-11-B)

[0354] Step 1: Synthesis of 3-bromo-4-chloro-5-fluoroaniline (1-5-02)

[0355] Compound 1-5-01 (2.00 g, 10.53 mmol) was dissolved in N,N-dimethylformamide (30 mL), and N-chlorosuccinimide (1.69 g, 12.63 mmol) was slowly added. The reaction was allowed to react at room temperature for 16 hours, and the reaction was monitored by HPLC-MS / MS. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was then purified on a flash silica gel column (ethyl acetate:petroleum ether = 0-25%) to obtain 0.95 g of the title compound.

[0356] The structural characterization data are as follows:

[0357] 1 H NMR (400MHz, DMSO-d6) δ6.77 (dd, J=2.5, 1.4Hz, 1H), 6.51 (dd, J=11.7, 2.5Hz, 1H), 5.84 (s, 2H).

[0358] Step 2: Synthesis of N-(3-bromo-4-chloro-5-fluorophenyl)acetamide (1-5-03)

[0359] Compound 1-5-02 (0.95 g, 4.23 mmol) was dissolved in ethyl acetate (20 mL). Acetic anhydride (648.13 mg, 6.35 mmol) was added under nitrogen. After addition, the temperature was raised to 50°C and the reaction mixture was reacted for 15 hours. The reaction was monitored by HPLC-MS / MS. The reaction solution was quenched with methanol (5 mL) and evaporated to dryness under reduced pressure to obtain a crude product. The crude product was purified on a flash silica gel column (ethyl acetate:petroleum ether = 0-40%) to obtain 1.01 g of the title compound.

[0360] The structural characterization data are as follows:

[0361] ESI-MS (m / z): 265.9 [M+H] + .

[0362] Step 3: Synthesis of (E)-4-(5-acetamido-2-chloro-3-fluorophenyl)-3-butenoic acid (1-5-04)

[0363] Compound 1-5-03 and 3-butenoic acid (387.65 mg, 4.50 mmol) were dissolved in a mixed solvent of 1,4-dioxane (24 mL) and water (8 mL). N,N-diisopropylethylamine (1.45 g, 11.26 mmol), tris(o-methylphenyl)phosphine (114.21 mg, 375.24 μmol), and palladium acetate (42.12 mg, 187.62 μmol) were then added. After the addition, the reaction system was purged with nitrogen three times and heated to 100°C under a nitrogen atmosphere for 16 hours. The reaction was monitored by high-performance liquid chromatography-mass spectrometry. After the reaction solution was cooled to room temperature, 1N aqueous sodium hydroxide solution (60 mL) and ethyl acetate (50 mL) were added and the layers were shaken. After separating the lower aqueous phase, the pH was adjusted to about 3 with a 4 mol / L hydrochloric acid aqueous solution, then extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain 1.00 g of a crude product of the title compound.

[0364] The structural characterization data are as follows:

[0365] ESI-MS (m / z): 272.0 [M+H] + .

[0366] Step 4: Synthesis of 4-(5-acetamido-2-chloro-3-fluorophenyl)butyric acid (1-5-05)

[0367] The crude product of compound 1-5-04 (1.00 g, 3.68 mmol) was dissolved in tetrahydrofuran (15 mL), and 10% palladium on carbon (0.10 g) was added. After the addition was complete, the reaction system was purged with a hydrogen balloon three times and allowed to react under a hydrogen atmosphere for 4 hours. The reaction was monitored by HPLC-MS / MS. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 1.00 g of the crude product of the title compound.

[0368] The structural characterization data are as follows:

[0369] ESI-MS (m / z): 274.0 [M+H] + .

[0370] Step 5: Synthesis of N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-06)

[0371] The crude product of compound 1-5-05 (1.00 g, 3.65 mmol) was dissolved in trifluoroacetic acid (5 mL). After cooling to 5°C, trifluoroacetic anhydride (3.84 g, 18.27 mmol, 2.54 mL) was slowly added. After the addition was complete, the reaction was maintained at 5°C for 2 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was slowly poured into water, then extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. The filtrate was evaporated to dryness under reduced pressure to obtain a crude product, which was purified by flash silica gel column to obtain 0.43 g of the title compound.

[0372] The structural characterization data are as follows:

[0373] ESI-MS (m / z): 256.1 [M+H] + .

[0374] Step 6: Synthesis of N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-07)

[0375] Tetrahydrofuran (16 mL) and tert-butanol (4 mL) were added to a reaction flask. After cooling to 5°C in an ice bath, potassium tert-butoxide (415.18 mg, 3.70 mmol) was added. Compound 1-5-06 (0.43 mg, 1.68 mmol) was dissolved in tetrahydrofuran (1 mL) and slowly added dropwise to the reaction solution. After 10 minutes, isoamyl nitrite (315.24 mg, 2.69 mmol) was added. After the addition was complete, the reaction was maintained at 5°C for 1 hour. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was quenched with saturated aqueous ammonium chloride solution and extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure to obtain 455.00 mg of a crude product of the title compound.

[0376] The structural characterization data are as follows:

[0377] ESI-MS (m / z): 285.0 [M+H] + .

[0378] Step 7: Synthesis of N-(7-amino-4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-08)

[0379] The crude product of compound 1-5-07 (0.40 g, 1.41 mmol) was dissolved in methanol (10 mL), and then 3 mol / L aqueous hydrochloric acid solution (1 mL) and 10% palladium on carbon (40.00 mg) were added. After the addition, the reaction system was replaced with hydrogen three times and reacted at room temperature under a hydrogen atmosphere for 1 hour. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 0.43 g of the crude hydrochloride salt of the title compound.

[0380] The structural characterization data are as follows:

[0381] ESI-MS (m / z): 271.0 [M+H] + .

[0382] Step 8: Synthesis of (9H-fluoren-9-ylmethyl)(8-acetamido-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (1-5-09)

[0383] The crude hydrochloride of compound 1-5-08 (0.43 g, 1.19 mmol) was dissolved in 1,4-dioxane (15 mL), followed by the addition of sodium bicarbonate (400.35 mg, 4.77 mmol), water (5 mL), and 9-fluorenylmethyl-N-succinimidyl carbonate (481.81 mg, 1.43 mmol). The mixture was stirred at room temperature for 2 hours and monitored by HPLC-MS / MS. The reaction mixture was poured into water and extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified on a C18 reverse-phase column (acetonitrile: 0.05% formic acid in water = 20%-100%) to obtain 301.00 mg of the title compound.

[0384] The structural characterization data are as follows:

[0385] ESI-MS (m / z): 493.2 [M+H] + .

[0386] Step 9: Synthesis of (9H-fluoren-9-ylmethyl)(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (1-5-10)

[0387] Compound 1-5-09 (300.00 mg, 608.61 μmol) was dissolved in dioxane (5 mL) and 12 mol / L concentrated hydrochloric acid (1 mL) was added. After addition, the temperature was raised to 60°C and the reaction mixture was reacted for 2 hours. The reaction was monitored by HPLC-MS / MS. The reaction solution was poured into water and extracted with ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified on a flash silica gel column (ethyl acetate:petroleum ether = 0-50%) to obtain 198.00 mg of the title compound.

[0388] The structural characterization data are as follows:

[0389] ESI-MS (m / z): 451.1 [M+H] + .

[0390] Step 10: Synthesis of (9H-fluoren-9-ylmethyl)((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (1-5-11)

[0391] (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (138.72 mg, 526.96 μmol) and compound 1-5-10 (198.00 mg, 439.13 μmol) were added to toluene (10 mL), and then p-toluenesulfonic acid (75.53 mg, 439.13 μmol) was added. After the addition was completed, the temperature was raised to 140 ° C. and the reaction was reacted for 4 hours. The reaction solution was directly evaporated to dryness under reduced pressure at 140 ° C. to obtain a crude product, which was purified by flash silica gel column (methanol: dichloromethane = 0-5%) to obtain 256.00 mg of the title compound.

[0392] The structural characterization data are as follows:

[0393] ESI-MS (m / z): 678.1 [M+H] + .

[0394] Step 11: Synthesis of (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione and (1R,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (1-5-A and 1-5-B)

[0395] Compound 1-5-11 (201.18 mg, 296.67 μmol) was dissolved in N,N-dimethylformamide (4 mL), and diethylamine (108.49 mg, 1.48 mmol) was added. After the addition was complete, the mixture was reacted at room temperature for 0.5 hours, and the reaction was monitored by HPLC-MS / MS. The ethylenediamine was evaporated under reduced pressure, and the pH of the reaction solution was adjusted to 2-3 with 1 mol / L aqueous hydrochloric acid. The reaction solution was then directly purified by preparative HPLC to obtain the title compounds 1-5-A (44.00 mg) and 1-5-B (43.00 mg).

[0396] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0397] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0398] 1-5-A (LCMS peak at the front at 6 min, retention time: 1.276 min)

[0399] The structural characterization data are as follows:

[0400] 1 H NMR (400MHz, DMSO-d6) δ8.00(d,J=10.3Hz,1H),7.33(s,1H),6.54(s,1H),5.62(d,J=19.3Hz,1H),5.44(s,2H),5.38(d,J=19.3 Hz,1H),4.43-4.38(m,1H),3.28-3.10(m,2H),2.22-2.12(m,1H),2.12-2.02(m,1H),1.93-1.80(m,2H),0.87(t,J=7.3Hz,3H).

[0401] ESI-MS (m / z): 456.1 [M+H] + .

[0402] The structural characterization data of 1-5-B (6 min LCMS peak later, retention time: 1.300 min) are as follows:

[0403] 1 H NMR (400MHz, DMSO-d6) δ7.98(d,J=10.3Hz,1H),7.32(s,1H),5.61(d,J=19.4Hz,1H),5.44(s,2H),5.32(d,J=19 .4Hz,1H),4.44-4.36(m,1H),3.33-3.25(m,1H),3.22-3.11(m,1H),2.23-2.13(m,1H),2.11-2.03(m,1H),1.96 -1.82(m,2H),0.89(t,J=7.3Hz,3H).

[0404] ESI-MS (m / z): 456.1 [M+H] + .

[0405] 6 min LCMS conditions:

[0406] Column: Waters SunFire C18 OBD 4.6 mm × 50 mm × 5.0 μm

[0407] Mobile phase A: 0.05% acetonitrile; Mobile phase B: water (0.05% formic acid)

[0408] Step 12: N-((S)-10-benzyl-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide and N-((S)- Synthesis of 10-benzyl-1-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-amide (1-5-12-A and 1-5-12-B)

[0409] The single-configuration compound 1-5-A (36.00 mg, 79.70 μmol) and compound A-07-3 (64.43 mg, 95.64 μmol) were dissolved in N,N-dimethylformamide (2 mL). 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were then added. The mixture was reacted at room temperature for 1 hour. The reaction was monitored by HPLC-MS / MS. The reaction solution was directly purified by HPLC to obtain the single-configuration title compound 1-5-12-A (51.00 mg).

[0410] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0411] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0412] The structural characterization data are as follows:

[0413] ESI-MS (m / z): 1111.0 [M+H] + .

[0414] The single-configuration compound 1-5-B (36.00 mg, 79.70 μmol) and compound A-07-3 (64.43 mg, 95.64 μmol) were dissolved in N,N-dimethylformamide (2 mL). 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were then added. The mixture was reacted at room temperature for 1 hour. The reaction was monitored by HPLC-MS / MS. The reaction solution was directly purified by HPLC to obtain the single-configuration title compound 1-5-12-B (52.00 mg).

[0415] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0416] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0417] The structural characterization data are as follows:

[0418] ESI-MS (m / z): 1111.0 [M+H] + .

[0419] Step 13: Synthesis of N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide (1-11-A and 1-11-B)

[0420] Compound 1-5-12-A (40.00 mg, 35.99 μmol) was weighed and dissolved in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL). A 4 mol / L hydrochloric acid solution in ethyl acetate (1 mL) was then added. The mixture was allowed to react at room temperature for 0.5 hours. The reaction was monitored by HPLC-MS / MS. The reaction solution was directly concentrated to dryness to obtain a crude product, which was purified by HPLC to obtain the title compound 1-11-A (4.75 mg) in a single configuration.

[0421] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0422] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0423] The structural characterization data are as follows:

[0424] 1 H NMR (400MHz, DMSO-d6) δ8.50(d,J=8.9Hz,1H),8.05(d,J=10.3Hz,1H),7.33(s,1H),6.55(s,1H),5.67-5.60(m,1H),5.49(t,J=5.8Hz, 1H),5.43(s,2H),5.21(s,2H),3.96(d,J=5.8Hz,2H),3.32-3.22(m,2H),2.28-2.15(m,2H),1.93-1.80(m,2H),0.87(t,J=7.3Hz,3H).

[0425] ESI-MS (m / z): 514.0 [M+H] + .

[0426] Compound 1-5-12-B (40.00 mg, 35.99 μmol) was weighed and dissolved in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL). 4 mol / L hydrochloric acid and ethyl acetate (1 mL) were then added. The mixture was allowed to react at room temperature for 0.5 hours. The reaction was monitored by HPLC-MS / MS. The reaction solution was directly concentrated under reduced pressure to dryness to obtain a crude product, which was purified by HPLC to obtain the title compound 1-11-B (8.24 mg) in a single configuration.

[0427] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0428] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0429] The structural characterization data are as follows:

[0430] 1 H NMR (400MHz, DMSO-d6) δ8.52(d,J=9.0Hz,1H),8.05(d,J=10.3Hz,1H),7.34(s,1H),6.55(s,1H),5.68-5.58(m,1H),5.53(t,J=5.8Hz,1H),5.43 (d,J=2.9Hz,2H),5.20(d,J=7.3Hz,2H),3.97(d,J=5.7Hz,2H),3.31-3.21(m,2H),2.26-2.15(m,2H),1.92-1.82(m,2H),0.87(t,J=7.3Hz,3H).

[0431] ESI-MS (m / z): 514.0 [M+H] + .

[0432] Example 8 N-((10S,19S)-23-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)-10-benzyl-1-(((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[ 3',4:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3-oxa-5,8,11,14,17-pentaazatricosan-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodeca-triacontane-38-carboxamide or N -((10S,19S)-23-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)-10-benzyl-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4: (6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3-oxa-5,8,11,14,17-pentaazatricosan-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxatriacontane-38-carboxamide (A-17-A)

[0433] Step 1: Synthesis of (2S)-2-(2,5,8,11,14,17,20,23,26,29,32,35-dodeca-38-octadecanecarboxamide)-6-(N-(9H-fluoren-9-ylmethoxycarbonyl)amino)hexanoic acid (A-17-03)

[0434] The hydrochloride salt of compound A-17-02 (389.68 mg, 962.45 μmol) was dissolved in dichloromethane (8 mL). DIPEA (518.28 mg, 4.01 mmol, 713.88 μL) and compound A-17-01 (550.00 mg, 802.04 μmol) were added and reacted at 25°C for 1.5 hours. The pH of the reaction solution was adjusted to neutral with dilute hydrochloric acid, and the solvent was removed under reduced pressure. The concentrate was purified by preparative HPLC to obtain the title compound A-17-03 (450.00 mg).

[0435] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0436] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0437] ESI-MS (m / z): 939.3 [M+H] + .

[0438] Step 2: Synthesis of 2-((2-((2S)-2-(2-(2-((2S)-2-(2,5,8,11,14,17,20,23,26,29,32,35-dodeca-triacontane-38-amido)-6-(N-(9H-fluoren-9-ylmethoxycarbonyl)amino)hexanamido)acetamido)acetamido)-3-phenylpropionamido)acetamido)methoxy)acetic acid (A-17-04)

[0439] Compound A-17-03 (50.00 mg, 118.09 μmol) was dissolved in DMF (2.5 mL), and HATU (49.39 mg, 129.89 μmol), compound A-07-2 (133.07 mg, 141.70 μmol), and DIPEA (45.78 mg, 354.26 μmol, 63.06 μL) were added. The mixture was reacted at 25°C for 1 hour. The solvent was evaporated under reduced pressure, and the concentrate was purified by preparative HPLC to obtain the title compound A-17-04 (40.00 mg).

[0440] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0441] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0442] ESI-MS (m / z): 1344.4 [M+H] + .

[0443] Step 3: (9H-fluoren-9-ylmethyl)((40S)-40-(((10S)-10-benzyl-1-(((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2- Synthesis of [b]quinolin-1-yl)amino]-1,6,9,12,15-pentaoxy-3-oxa-5,8,11,14-tetraazahexadec-16-yl)carbamoyl)-38-oxo-2,5,8,11,14,17,20,23,26,29,35-dodeca-39-aza-44-yl)carbamate (A-17-05)

[0444] Compound A-17-04 (29.86 mg, 59.50 μmol) was dissolved in DMF (3 mL), and HATU (27.15 mg, 71.40 μmol), compound 1-5-A (80.00 mg, 59.50 μmol), and DIPEA (38.45 mg, 297.51 μmol, 52.96 μL) were added. The mixture was reacted at 25°C for 1 hour. The solvent was evaporated under reduced pressure, and the concentrate was purified by preparative HPLC to obtain the title compound A-17-05 (50.00 mg).

[0445] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0446] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0447] ESI-MS (m / z): 1781.6 [M+H] + .

[0448] Step 4: Synthesis of N-((10S,19S)-23-amino-10-benzyl-1-(((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3-oxa-5,8,11,14,17-pentaazatricosan-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoctatriacontane-38-amide (A-17-06)

[0449] Compound A-17-05 (20.00 mg, 11.22 μmol) was dissolved in DMF (2.5 mL) and diethylamine (0.5 mL) and reacted at 25°C for 2 hours. The solvent was evaporated under reduced pressure and the concentrate was purified by preparative HPLC to obtain the formate salt of the title compound A-17-06 (10.00 mg).

[0450] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0451] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0452] ESI-MS (m / z): 1559.7 [M+H] +.

[0453] Step 5: N-((10S,19S)-23-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)-10-benzyl-1-(((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3 ',4:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3-oxa-5,8,11,14,17-pentaazatricosan-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoctatrioxane-38-amide or N-( (10S,19S)-23-(6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamido)-10-benzyl-1-(((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7 Synthesis of (1,6,9,12,15,18-hexaoxo-3-oxa-5,8,11,14,17-pentaazatricosan-19-yl)-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoctatriacontane-38-carboxamide (A-17-A)

[0454] The formate salt of compound A-17-06 (7.00 mg, 4.49 μmol) and compound A-07-1 (3.28 mg, 8.97 μmol) were dissolved in DMF (1 mL), and DIPEA (1.74 mg, 13.46 μmol, 2.40 μL) was added. The mixture was reacted at 25°C for 2 hours. The solvent was evaporated under reduced pressure, and the concentrate was purified by preparative HPLC to obtain the title compound A-17-A (5.60 mg).

[0455] Column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0456] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0457] ESI-MS (m / z): 1809.8 [M+H] + .

[0458] Example 9: N-((S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizin[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxyl-5,8,11,14-tetraazahexadec-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-05)

[0459] Under nitrogen protection, 2,5-dioxopyrrolidin-1-yl-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynoate (A-07-1, 0.66 g, 1.80 mmol) and A-07-2 (0.75 g, 1.77 mmol) were added to DMF (19 mL), heated to 35 ° C for 16 hours, and (1S, 9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2 ,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (1-4 (i.e., 1-2-A), 1.00 g, 1.77 mmol), ice water was cooled to 5-15°C, DMTMM (0.98 g, 3.53 mmol) was added, and then DIPEA (1.14 g, 8.84 mmol) was added dropwise. The reaction mixture was reacted at 25°C for 16 hours. The reaction solution was poured into a mixture of DCM (600 mL), IPA (60 mL), and water (100 mL) and stirred for 10 minutes. The DCM phase was separated, washed with brine (100 ml), and concentrated to obtain a crude product. The crude product was purified by preparative high performance liquid chromatography and freeze-dried to obtain 0.98 g of compound A-05 (i.e., A-07-A in Example 3).

[0460] A-05 separation and purification method is as follows:

[0461] Column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0462] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0463] A-05 structural characterization data are as follows:

[0464] MS m / z(ESI):1107.3[M+H] + .

[0465] 1H NMR (400MHz, DMSO) δ9.10 (s, 2H), 8.66-8.63 (m, 1H), 8.51 (d, J = 8.8Hz, 1H), 8.34-8.31 (m, 1H), 8.21-8.19 (m, 1H), 8.17-8.09 (m, 2 H),8.08-8.04(m,1H),7.30(s,1H),7.26-7.15(m,5H),6.55(s,1H),5.56-5.55(m,1H),5.48-5.35(m,2H),5.25-5.10(m,2H),4.6 4(d,J=6.4Hz,2H),4.45-4.44(m,1H),4.06-3.98(m,2H),3.77-3.52(m,6H),3.41(s,3H),3.25-3.12(m,2H),3.03-3.00(m,1H),2 .83-2.72(m,1H),2.58-2.56(m,2H),2.48(s,3H),2.33-2.30(m,2H),2.21-2.13(m,2H),1.91-1.76(m,4H),0.87(t,J=7.2Hz,3H).

[0466] The following HPLC conditions were used to detect compounds A-07-A and A-07-B prepared in Example 3; as well as compound A-07-A prepared in Example 3 and compound A-05 prepared in Example 9:

[0467] Instrument: Agilent 1260 HPLC with VWD detector

[0468] Chromatographic column: Waters Xbridge C18 4.6*100mm*3.5μm

[0469] Mobile phase A: 0.01M ammonium phosphate aqueous solution / acetonitrile = 90 / 10; Mobile phase B: acetonitrile

[0470] As shown in FIG3A , the retention times of compound A-07-A and compound A-07-B were 6.6 min and 6.8 min, respectively, indicating that the above HPLC conditions have good separation of isomers;

[0471] As shown in FIG3B , compound A-07-A prepared in Example 3 and compound A-05 prepared in Example 9 showed one peak in HPLC with a retention time of 6.6 min, indicating that compound A-07-A was compound A-05.

[0472] Example 10: 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl((1S,9R)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-04)

[0473] Step 1: Preparation of ethyl 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoacetate (B-04-1)

[0474] Dissolve (1S,9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (2 g, 3.65 mmol) in DMF (50 mL). Add DIPEA (1.18 g, 9.12 mmol, 1.59 mL) dropwise. Add acetoxyacetyl chloride (548.12 mg, 4.01 mmol, 431.59 μL) dropwise with ice-cooling and stirring. Continue stirring for 1 hour. Add the reaction solution to 0.1 M dilute hydrochloric acid to precipitate a solid, which is then filtered. The filter cake was dissolved in dichloromethane and methanol, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by silica gel column chromatography (methanol / dichloromethane = 0% to 5%) and concentrated again to obtain the title compound (1.7 g, 3.077 mmol).

[0475] Its structural characterization data are as follows:

[0476] ESI-MS (m / z): 552.2 [M+1] + .

[0477] Step 2: Preparation of ethyl 2-(((1S,9S)-9-(((4-((S)-35-azido-2-(4-(4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl)oxy)carbonyl)oxy-5-chloro-9-ethyl-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indazolidin-1-yl)amino)-2-oxoacetate (B-04-2)

[0478] Ethyl 2-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoacetate (500 mg, 0.905 mmol) and DMAP (885.33 mg, 7.25 mmol) were dissolved in dry dichloromethane (5 mL), cooled to 0°C under nitrogen, and a dichloromethane solution (5 mL) of triphosgene (268.81 mg, 0.905 mmol) was added dropwise. The mixture was stirred and heated for 0.5 h. A solution of (S)-2-(3,2-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatriacontamide)-N-(4-hydroxymethylphenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (1.44 g, 1.36 mmol) in dichloromethane was slowly added dropwise and allowed to react at room temperature for 4 hours. The reaction was quenched with water and extracted three times with dichloromethane (100 ml x 3). The organic phases were combined, washed with saturated brine, dried, and concentrated. Purification on a silica gel column (MeOH / DCM = 0% to 5%) afforded the title compound (498 mg, 0.304 mmol).

[0479] Its structural characterization data are as follows:

[0480] ESI-MS (m / z): 1352.8 [M+1] + .

[0481] Step 3: Preparation of 4-((S)-35-azido-2-(4-((4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-04-3)

[0482] 2-(((1S,9S)-9-(((4-((S)-35-azido-2-(4-(4-methoxyphenyl)diphenylmethyl)amino)butyl)-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl)oxy)carbonyl)oxy-5-chloro-9-ethyl-4-methyl-10,13-dioxo-2,3,9,1 Ethyl 2-oxoacetate (200 mg, 0.122 mmol) (0,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indazolidin[1,2-b]quinolin-1-yl)amino) was dissolved in THF (3 mL) and MeOH (3 mL). A 1 mL aqueous solution of sodium carbonate (25.88 mg, 0.224 mmol) was added dropwise with stirring. Stirring was continued for 1 hour after the addition was complete. Dilute hydrochloric acid was added dropwise to the reaction mixture to neutralize the reaction. After concentration under reduced pressure, the mixture was directly transferred to the next step.

[0483] Its structural characterization data are as follows:

[0484] ESI-MS (m / z): 1596.7 [M+1] + .

[0485] Step 4: Preparation of 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-04-4)

[0486] 4-((S)-35-Azido-2-(4-((4-methoxyphenyl)benzhydryl)amino)butyl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (190 mg, 119.04 μmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (0.5 mL) was added and the reaction was continued for 1 hour. Saturated sodium bicarbonate aqueous solution was added dropwise to the reaction solution for neutralization, and the organic phase was concentrated to obtain a crude product, which was purified by reverse phase column chromatography (acetonitrile / 1% formic acid aqueous solution = 0% to 50%) and freeze-dried to obtain the title compound (95 mg, 69.35 μmol).

[0487] Its structural characterization data are as follows:

[0488] ESI-MS (m / z): 1323.6 [M+1] + .

[0489] Step 5: Preparation of 4-((S)-2-(4-aminobutyl)-35-(4-((6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-ynamido)methyl)-1H-1,2,3-triazol-1-yl)-4,8-dioxo-6,12,15,18,24,27,30,33-nonyloxy-3,9-diazapentaazatriamido)benzyl((1S,9R)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-9-yl)carbonate (B-04)

[0490] 4-((S)-2-(4-aminobutyl)-35-azido-4,8-dioxo-6,12,15,18,21,24,27,30,33-nonyloxy-3,9-diazapentabenzotriamido)benzyl((1S,9S)-5-chloro-9-ethyl-1-(2-hydroxyacetamido)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[ [3',4':6,7] indolizino[1,2-b]quinolin-9-yl) carbonate (90 mg, 0.066 mmol) and 6-(2-(methylsulfonyl)pyrimidin-5-yl)-N-(prop-2-yn-1-yl)hex-5-ynamide (24.07 mg, 0.079 mmol) were dissolved in DMSO (2 mL) and water (0.2 mL). Cuprous bromide (9.42 mg, 0.066 mmol) was added and stirring continued for 2 hours. The reaction mixture was directly filtered and the concentrated crude product was purified by preparative HPLC and freeze-dried to obtain the title compound (42.2 mg, 24.69 μmol).

[0491] Its structural characterization data are as follows:

[0492] ESI-MS (m / z): 1628.7 [M+1] + .

[0493] The preparative high performance liquid chromatography method is as follows:

[0494] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0495] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0496] Conjugation of compounds containing cell-active molecules and linkers to antibodies

[0497] The sources of the antibodies involved in the antibody-drug conjugates prepared in the following examples are as follows: The preparation of the 22B6 antibody is described in Examples 2 and 4 of International Patent Application WO2023165475A1, the heavy chain variable region sequence is SEQ ID NO: 1 (see SEQ ID NO: 1 in that application); the heavy chain constant region sequence is SEQ ID NO: 2 (see SEQ ID NO: 63 in that application); the light chain variable region sequence is SEQ ID NO: 3 (see SEQ ID NO: 2 in that application); and the light chain constant region sequence is SEQ ID NO: 4 (see SEQ ID NO: 65 in that application);

[0498] The preparation of the 47A3 antibody is described in Examples 2 and 4 of International Patent Application WO2023165475A1. The heavy chain variable region sequence is SEQ ID NO: 5 (see SEQ ID NO: 5 in that application); the heavy chain constant region sequence is SEQ ID NO: 6 (see SEQ ID NO: 63 in that application); the light chain variable region sequence is SEQ ID NO: 7 (see SEQ ID NO: 6 in that application); and the light chain constant region sequence is SEQ ID NO: 8 (see SEQ ID NO: 65 in that application).

[0499] The preparation of the 100H7 antibody is described in Examples 2 and 4 of International Patent Application WO2023165475A1. The heavy chain variable region sequence is SEQ ID NO: 9 (see SEQ ID NO: 7 in that application); the heavy chain constant region sequence is SEQ ID NO: 10 (see SEQ ID NO: 63 in that application); the light chain variable region sequence is SEQ ID NO: 11 (see SEQ ID NO: 8 in that application); and the light chain constant region sequence is SEQ ID NO: 12 (see SEQ ID NO: 65 in that application).

[0500] The U1-59 control antibody was derived from patent application CN200680049887. After codon optimization, the antibody heavy and light chain nucleotide sequences were synthesized and cloned into the pTT5 vector and expressed and purified.

[0501] The coupling preparation of the sample is as follows:

[0502] 0.46 ml of each of the 22B6, 47A3, 100H7, and U1-59 antibodies (all adjusted to 11.0 mg / mL) was diluted with a 0.1 M disodium edetate solution (pH 7.7). The pH was then adjusted to 7.7 with a 1 M Na2HPO4 solution. A 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution was added, mixed, and allowed to stand at room temperature for 90 minutes. A 4.0-10-fold amount of the drug linker compound of the present invention dissolved in dimethyl sulfoxide was added to the above solution, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 10 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva). Sucrose and Tween 20 were then added and mixed to obtain the antibody-drug conjugate (ADC), as shown in Table 1.

[0503] The ADC drug-to-antibody ratio (DAR value) is determined as follows:

[0504] LC-MS determination of ADC sample molecular weight and calculation of drug / antibody ratio DAR value

[0505] ADC samples were subjected to LC-MS molecular weight analysis.

[0506] Chromatographic determination conditions:

[0507] Liquid chromatography column: Thermo MAbPac RP 3.0*100mm;

[0508] Mobile phase A: 0.1% FA / H2O; Mobile phase B: 0.1% FA / ACN;

[0509] Flow rate: 0.25 ml / min; sample chamber temperature: 8°C; column temperature: 60°C; injection volume: 2 μl;

[0510] Mass spectrometry conditions:

[0511] Mass spectrometer model: AB Sciex Triple TOF 5600+;

[0512] GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 250; CE 10; Accumulation time 0.5s;

[0513] m / z 600-4000; Time bins to sum 40.

[0514] Table 1 ADC number and DAR

[0515] 1. Determine the molecular weight of ADC 22B6-A-07-A-1 by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC 22B6-A-07-A-1 is shown in Table 2.

[0516] Table 2: Measured molecular weight of ADC 22B6-A-07-A-1

[0517] Table 3: DAR values ​​for ADC 22B6-A-07-A-1

[0518] The calculated drug / antibody ratio of ADC 22B6-A-07-A-1 was DAR = 7.99 (see Table 3).

[0519] 2. Determine the molecular weight of ADC 22B6-A-07-A-2 by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC 22B6-A-07-A-2 is shown in Table 4.

[0520] Table 4: Measured molecular weight of ADC 22B6-A-07-A-1

[0521] Table 5: DAR values ​​for ADC 22B6-A-07-A-2

[0522] The calculated drug / antibody ratio of ADC 22B6-A-07-A-2 was DAR=3.56 (see Table 5).

[0523] 3. Determine the molecular weight of ADC 47A3-A-07-A by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC 47A3-A-07-A is shown in Table 6.

[0524] Table 6: Measured molecular weight of ADC 47A3-A-07-A

[0525] Table 7: DAR values ​​for ADC 47A3-A-07-A

[0526] The calculated drug / antibody ratio of ADC 47A3-A-07-A was DAR = 6.27 (see Table 7).

[0527] 4. Determine the molecular weight of ADC 100H7-A-07-A by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC 100H7-A-07-A is shown in Table 8.

[0528] Table 8: Measured molecular weight of ADC 100H7-A-07-A

[0529] Table 9: DAR values ​​for ADC 100H7-A-07-A

[0530] The drug / antibody ratio of ADC 100H7-A-07-A was calculated to be DAR = 8.01 (see Table 9).

[0531] 5. Determine the molecular weight of ADC U1-59-A-07-A by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC U1-59-A-07-A is shown in Table 10.

[0532] Table 10: Measured molecular weight of ADC U1-59-A-07-A

[0533] Table 11: DAR values ​​for ADC U1-59-A-07-A

[0534] The calculated drug / antibody ratio of ADC U1-59-A-07-A was DAR=8.02 (see Table 11).

[0535] 6. LC-MS determination of the molecular weight of ADC 22B6-A-01 and calculation of the drug / antibody ratio (DAR) value. The LC-MS molecular weight analysis of ADC 22B6-A-01 is shown in Table 12.

[0536] Table 12: Measured molecular weight of ADC 22B6-A-01

[0537] Table 13: DAR values ​​for ADC 22B6-A-01

[0538] The calculated drug / antibody ratio of ADC 22B6-A-01 was DAR = 7.93 (see Table 13).

[0539] 7. Determine the molecular weight of ADC U1-59-A-01 by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC U1-59-A-01 is shown in Table 14.

[0540] Table 14: Measured molecular weight of ADC U1-59-A-01

[0541] Table 15: DAR values ​​for ADC U1-59-A-01

[0542] The calculated drug / antibody ratio of ADC U1-59-A-01 was DAR = 7.89 (see Table 15).

[0543] 8. Determine the molecular weight of ADC 22B6-B-03-1 by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC 22B6-B-03-1 is shown in Table 16.

[0544] Table 16: Measured molecular weight of ADC 22B6-B-03-1

[0545] Table 17: DAR values ​​for ADC 22B6-B-03-1

[0546] The drug / antibody ratio of ADC 22B6-B-03-1 was calculated to be DAR=8.0 (see Table 17).

[0547] 9. Determine the molecular weight of ADC 22B6-B-03-2 by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC 22B6-B-03-2 is shown in Table 18.

[0548] Table 18: Measured molecular weight of ADC 22B6-B-03-2

[0549] Table 19: DAR values ​​for ADC 22B6-B-03-2

[0550] The drug / antibody ratio of ADC 22B6-B-03-2 was calculated to be DAR = 3.14, see Table 19.

[0551] 10. Determine the molecular weight of ADC 22B6-B-01 by LC-MS and calculate the drug / antibody ratio (DAR). The LC-MS molecular weight analysis of ADC 22B6-B-01 is shown in Table 20.

[0552] Table 20: Measured molecular weight of ADC 22B6-B-01

[0553] Table 21: DAR values ​​for ADC 22B6-B-01

[0554] The calculated drug / antibody ratio of ADC 22B6-B-01 was DAR = 8.07 (see Table 21).

[0555] 11. Preparation Example of ADC 22B6-B-04-1

[0556] 0.854 ml of 22B6 antibody (23.43 mg / mL) was diluted with 42.68 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.57 with 1 M Na₂HPO₄ solution. 20 mM TCEP (tris(2-carboxyethyl)phosphine, pH 7.60, 73.68 μL) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 12-fold amount of B-04 dissolved in dimethyl sulfoxide (164 μL, 10 mM) was then slowly added, mixed, and allowed to stand at room temperature overnight. After completion, the buffer was exchanged with a 20 mM histidine buffer at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain ADC 22B6-B-04-1. The DAR value was determined by mass spectrometry to be 8.62.

[0557] 12. Preparation Example of ADC 22B6-B-04-2

[0558] 2.667 ml of 22B6 antibody (18.75 mg / mL) was diluted with 133.35 μL of 20 mM PB + 0.1 M EDTA (pH 7.60). The pH was then adjusted to 7.63 with 1 M Na₂HPO₄ solution. 10 mM TCEP (tris(2-carboxyethyl)phosphine, 184.19 μL, pH 7.60) was added, mixed, and allowed to stand at room temperature for 1.5 hours. A 10-fold amount of B-04 dissolved in dimethyl sulfoxide (341.73 μL, 10 mM) was then slowly added, mixed, and allowed to stand at room temperature for 2 hours. After completion, the buffer was exchanged with a 20 mM histidine buffer solution at pH 6.0 using a NAP-5 gel column (Cytiva) to obtain ADC 22B6-B-04-2. The DAR value was 6.87 as determined by mass spectrometry.

[0559] Detecting the activity of antibody-drug conjugates

[0560] 1. In vitro cytotoxicity of anti-human HER3 antibody-drug conjugates

[0561] MDA-MB-453, HCC1569, and HEK293T-h HER3 cells were digested with TrypLE (Gibco) solution, and an appropriate amount of cells was counted and taken. After dilution with growth medium, 5000 cells or 3000 cells (100 μl) / well were plated into 96-well plates and cultured overnight at 37°C and 5% CO2. The next day, ADC was diluted with growth medium, starting at 150 μg / ml, and diluted 2.5-fold. Then, 100 μl of the diluted ADC was added to the 96-well plate containing cells. The plate was placed in an incubator and cultured at 37°C and 5% CO2 for 6 days. After that, 20 μl of CCK8 was added to each well and incubated at 37°C for 2-5 hours. The OD450nm absorbance value was read by a microplate reader. The raw data was imported into Graph Prism 6 to calculate IC 50 value.

[0562] Table 22 Anti-human HER3 ADC in vitro cytotoxicity results Note: “ / ” means not tested.

[0563] As shown in Table 22, the ADCs formed by coupling the drug-linker compounds of the present invention to antibodies (e.g., 22B6-A-01, 22B6-A-07-A-1) have target-specific killing activity.

[0564] 2. Bystander Effect of Anti-Human HER3 Antibody-Drug Conjugates

[0565] HEK293T and HEK293T-h HER3 cells were digested with 0.25% Trypsin-EDTA (Gibco), and appropriate amount of cells were taken. After washing twice with PBS, the cell density was adjusted to 1×10 6 cells / ml, and then added Cell Trace Violet at a final concentration of 5 μM for labeling, mixed and placed in an incubator for incubation for 20 min, then washed with growth medium and counted, and plated into 24-well plates (500 μl / well) according to the following formulas: HEK293T-labled: HEK293T = 4000: 12000 cells / well, HEK293T-labled: HEK293T-h HER3 = 4000: 12000 cells / well, HEK293T-h HER3-labled: HEK293T-h HER3 = 4000: 12000 cells / well, and cultured overnight at 37°C, 5% CO2;

[0566] On the second day, ADC was diluted with growth medium to concentrations of 20 nM, 4 nM, and 0.8 nM. 500 μl of the diluted ADC was then added to the corresponding 24-well plates containing cells. The plates were placed in an incubator at 37°C and 5% CO2 for 4 days.

[0567] HEK293T and HEK293T-hHER3 cells were digested with 0.25% Trypsin-EDTA, and growth medium containing PI at a final concentration of 3 μM was added and mixed. After incubation at room temperature for 15 minutes, the cells were analyzed by flow cytometry (Thermo, model Attune NxT). The results, as shown in Figures 1A, 1B, and 1C, show that the ADC formed by the drug-linker compound of the present invention coupled with the antibody had essentially no killing effect on negative HEK293T cells, but exhibited significant specific killing effect on HEK293T-HER3 cells. In the presence of both negative HEK293T cells and positive HEK293T-HER3 cells, the ADC exhibited a significant killing effect on negative cells, indicating that the ADC formed by the drug-linker compound of the present invention coupled with the antibody has bystander activity.

[0568] 3. Plasma stability test of anti-human HER3 antibody-drug conjugates

[0569] 3.59 μl of 20.9 mg / ml 22B6-A-07-A-1 ADC sample was precisely measured and transferred to an EP tube. 496 μl of blank plasma from human, cynomolgus macaque, and mouse was added and mixed thoroughly to obtain human, cynomolgus macaque, and mouse plasma samples containing the 22B6-A-07-A-1 ADC sample. The samples were incubated at 37°C. 50 μl of sample was removed at 1, 3, 6, 24, 48, 96, and 168 hours. Protein was precipitated with 200 μl of 0.5% formic acid-acetonitrile. The supernatant was centrifuged at 13,000 rpm for 10 minutes at 4°C and stored at -80°C. After all samples were collected, LC-MS / MS was used to analyze the cytotoxic drug concentration. As shown in Figure 2, cytotoxic drug shedding did not exceed 5%, demonstrating that the drug-linker compound of the present invention, after conjugation to the ADC, was stable in plasma from various species.

[0570] 4. In vivo efficacy testing of anti-human HER3 antibody-drug conjugate H358 model

[0571] NCI-H358 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. Exponentially growing NCI-H358 cells were harvested, resuspended in PBS to an appropriate concentration, and inoculated subcutaneously into female Balb / c-nu mice to establish a non-small cell lung cancer model. The tumors were grown until the average volume reached approximately 130-150 mm. 3 At the time of treatment, the mice were randomly divided into 6 groups according to the tumor size, namely: vehicle control group (i.e. negative control group), 22B6-A-01 1mg / kg group, 22B6-B-01 1mg / kg group, 22B6-A-07-A-1 1mg / kg group and 3mg / kg group, 22B6-B-03-1 1mg / kg group. All the drugs were injected through the tail vein on Day 0, Day 1, Day 4, and Day 10, for a total of 3 times. After administration, the tumor diameter was measured with a vernier caliper twice a week, and the tumor volume was calculated according to the following formula: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. Animal deaths were observed and recorded every day. The specific results are shown in Table 23.

[0572] The tumor growth inhibition rate (TGI) was calculated using the following formula to evaluate the tumor inhibition efficacy:

[0573] TGI (%) = [1 - (VT end - VT start) / (VC end - VC start)] * 100%

[0574] VTend: mean tumor volume at the end of the treatment group experiment

[0575] VT start: mean tumor volume at the start of drug administration in the treatment group

[0576] VC end: mean tumor volume of negative control group at the end of the experiment

[0577] VC Start: Mean tumor volume at the beginning of drug administration in the negative control group

[0578] The experimental results show that the ADC of the present invention has a significant tumor growth inhibitory effect on the NCI-H358 non-small cell lung cancer xenograft model. Compared with the solvent control group, after three doses, the Day32 data showed that the tumor growth inhibition rate (TGI) of the 22B6-A-01 1mg / kg group and the 22B6-B-01 1mg / kg group was 79.80% and 64.68%, respectively. The TGI of the 22B6-A-07-A-1 1mg / kg group and the 3mg / kg group was 81.22% and 111.53%, respectively. The TGI of the 22B6-B-03-1 1mg / kg group was 121.45%. There was no animal death or significant animal weight loss in each treatment group on Day32, and no obvious drug toxicity was observed. The mice tolerated the ADC well during the treatment period.

[0579] Table 23 Human non-small cell lung cancer cell NCI-H358 CDX model Note: TGI is tumor growth inhibition rate, T / C is relative tumor proliferation rate.

[0580] As shown in the table above, the ADCs formed by coupling the drug linker compounds of the present invention with various antibodies all have excellent killing activity against tumor cells and good safety.

[0581] 5. In vivo efficacy testing of anti-human HER3 antibody-drug conjugates in the N87 model

[0582] NCI-N87 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. NCI-N87 cells were harvested during the exponential growth phase, resuspended in PBS to an appropriate concentration, and inoculated subcutaneously into female Balb / c-nu mice to establish a human gastric cancer model. The tumors were grown until the average volume was approximately 120 mm. 3 At 4 hr, the animals were randomly divided into the following groups according to tumor size: vehicle control group, 22B6-A-07-A-1 3 mg / kg group, 47A3-A-07-A 3 mg / kg group, and 100H7-A-07-A 3 mg / kg group. All animals were injected via the tail vein on Day 0, Day 4, and Day 7, for a total of three doses. Tumor diameters were measured with a vernier caliper twice weekly after administration, and tumor volume was calculated using the following formula: V = 0.5a × b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. Animal deaths were observed and recorded every day. The specific results are shown in Table 24.

[0583] The experimental results show that compared with the vehicle control group, after three doses, the tumor growth inhibition rates (TGI) in the 22B6-A-07-A-1 3mg / kg group, the 47A3-A-07-A 3mg / kg group, and the 100H7-A-07-A 3mg / kg group were 66.59%, 47.24%, and 36.04%, respectively, as shown in the Day 32 data. No animal deaths or significant weight loss were observed in any of the treatment groups on Day 32, and no obvious drug toxicity was observed. The mice tolerated the ADC well during treatment.

[0584] Table 24 Human gastric cancer cell NCI-N87 CDX model

[0585] As shown in the table above, the ADCs formed by coupling the drug linker compounds of the present invention with various antibodies all have excellent killing activity against tumor cells and good safety.

[0586] 6. In vivo efficacy testing of anti-human HER3 antibody-drug conjugates in the MDA-MB-453 model

[0587] Human breast cancer MDA-MB-453 cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum at 37°C and 5% CO2. MDA-MB-453 cells were harvested during the exponential growth phase, resuspended in PBS and a final concentration of 50% Matrigel to an appropriate concentration, and inoculated subcutaneously into female NCG immunodeficient mice to establish a human breast cancer xenograft model. When the average tumor volume was approximately 150 mm, the cells were grown. 3 At 4 pm, mice were randomly divided into groups based on tumor size: a vehicle control group, 22B6-A-07-A-1 3 mg / kg and 10 mg / kg groups, U1-59-A-01 3 mg / kg and 10 mg / kg groups, hIgG1-B-03-2 3 mg / kg group, and 22B6-B-03-2 3 mg / kg group. After grouping, mice were injected via the tail vein, with a single dose on Day 0. Following administration, tumor volume and body weight were measured regularly. The results are shown in Table 25.

[0588] The ADCs of the present invention significantly inhibited tumor growth in NCG mice bearing subcutaneous transplants of human breast cancer MDA-MB-453. Data from Day 32 after a single dose showed that compared with the vehicle control group, the 22B6-A-07-A-1 1 mg / kg, 3 mg / kg, and 10 mg / kg groups had a TGI of 118.30% and 200.00%, respectively. The 10 mg / kg group showed complete tumor regression from Day 19, with no growth observed by Day 32. The 22B6-B-03-2 3 mg / kg group had a TGI of 148.58%. The U1-59-A-01 3 mg / kg and 10 mg / kg groups had TGIs of 76.11% and 180.65%, respectively. The mice tolerated the ADCs well during treatment.

[0589] Table 25 Human breast cancer cell MDA-MB-453CDX model

[0590] As shown in the table above, the ADCs formed by coupling the drug linker compounds of the present invention with various antibodies all have excellent killing activity against tumor cells and good safety.

[0591] 7. Anti-human HER3 antibody-drug conjugate endocytosis activity test

[0592] The endocytic activity of anti-human HER3 ADC in MDA-MB-453 and HCC1569 cells was detected using a flow cytometer (Beckman, model Cytoflex).

[0593] The adherent cells were digested with Trypsin-EDTA (0.25%, Shanghai Yuanpei) solution and counted. The cell density was adjusted to 1×10 5 100 μl of cell suspension was added to each well of a 96-well plate (the number of cells was 1×10 4 / well), and the 96-well plate was placed in a 37°C, CO2 constant temperature incubator for 24 hours. The 96-well plate was removed, the culture medium was aspirated, and 50 μl of fresh complete culture medium was added to each well; the bispecific antibody and the control antibody were serially diluted with complete culture medium to a final concentration of 0.55, 1.64, 4.94, 14.81, 44.44, 133.33, 400, and 1200 ng / ml, for a total of 8 concentration points; the pHrodo reagent (Thermo) was diluted with complete culture medium to 12 μg / ml (the final concentration of pHrodo was 3 μg / ml); the serially diluted test antibody was mixed with the diluted Mix the pHrodo reagent in a 1:1 ratio (30 μl:30 μl) and incubate at room temperature in the dark for 30 minutes. Add 50 μl of the test antibody and pHrodo reagent mixture to a 96-well plate and incubate at 37°C, 5% CO2 for 24 hours. Remove the 96-well plate, discard the culture medium, wash once with sterile PBS, add 100 μl of Trypsin-EDTA (0.25%) to each well to digest the cells, and then add 100 μl of complete culture medium to neutralize them. After pipetting and dispersing the cells in the wells, analyze them on a FACS machine. Data processing: Export the average fluorescence signal value and then import it into GraphPad Prism 6 software to calculate the EC. 50 , the results are shown in Table 26.

[0594] Table 26 Anti-human HER3 antibody and ADC endocytosis activity assay results

[0595] As shown in the table above, the ADC formed after the drug linker compound of the present invention is coupled to the antibody still has good endocytosis activity.

[0596] 8. In vitro cytotoxicity of anti-human HER3 antibody-drug conjugates

[0597] MDA-MB-453 cells were digested with TrypLE (Gibco) solution, counted, and an appropriate amount of cells was obtained. After dilution with growth medium, 5,000 cells (100 μl / well) were plated onto a 96-well plate and incubated overnight at 37°C, 5% CO2. The next day, the ADC was diluted with growth medium, starting at 150 μg / ml and serially diluted 2.5-fold. 100 μl of the diluted ADC was then added to the 96-well plate containing the cells. The plate was placed in an incubator and incubated at 37°C, 5% CO2 for 6 days. After that, 20 μl of CCK8 was added to each well and incubated at 37°C for 2-5 hours. The absorbance at OD450 nm was read using a microplate reader. The raw data were imported into Graph Prism 6 to calculate the IC50 value.

[0598] Table 27 Anti-human HER3 ADC in vitro cytotoxicity results

[0599] As shown in Table 27, the ADCs formed from the drug-linker compounds of the present invention all exhibited significant cell-killing activity.

[0600] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are all within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite and prodrug thereof, wherein the compound has a structure as shown in the following formula (I): M'-LED Formula (I) in: M' is Lg is a leaving group of a nucleophilic substitution reaction, or is a hydroxyl group (-OH), a thiol group (-SH) or an amino group (-NH2), and ring A is a 5-6 membered saturated aliphatic heterocyclic ring, or a 5-20 membered aromatic ring system; or, M' is Ring A' is a 5-6 membered unsaturated heterocyclic ring; the saturated heterocyclic ring, unsaturated heterocyclic ring and aromatic ring system are optionally substituted by one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1-6 Alkyl radical substitution; M1 is selected from a single bond, C 1-20 Alkylene, C 2-20 Alkenylene or C 2-20 Alkynylidene; L is a structural fragment connecting M' and E; E is a structural fragment connecting L and D; D is a cytotoxic drug fragment.

2. The compound of claim 1 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite, and prodrug thereof, wherein: M' is Lg is a methylsulfonyl group; Ring A is a 5-membered saturated aliphatic heterocyclic ring, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more 6-membered heteroaromatic rings to a benzene ring via a single bond, or M' is Ring A' is a 5-6 membered unsaturated heterocyclic ring; the saturated heterocyclic ring, unsaturated heterocyclic ring and aromatic ring system are optionally substituted by one or more selected from oxy (=O), halogen and C 1-4 Alkyl radical substitution; M1 is selected from a single bond, C 3-10 Alkylene, C 3-10 Alkenylene or C 3-10 Alkynylidene.

3. The compound of claim 1 or 2, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite, and prodrug thereof, wherein: M' is in Selected from M1 is selected from a single bond and C 5-8 Alkylene, C 5-8 Alkenylene or C 5-8 Alkynylidene.

4. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite and prodrug thereof, wherein: M'Selected 5. The compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite, and prodrug thereof, wherein: M is Ring A is a 5-6 membered alicyclic heterocyclic ring or a 5-20 membered aromatic ring system, wherein the alicyclic heterocyclic ring and the aromatic ring system are optionally substituted by one or more selected from oxy (=O), halogen, cyano, amino, carboxyl, thiol and C 1- 6 alkyl groups; M1 is selected from a single bond and C 1-20 Alkylene, C 2-20 Alkenylene or C 2-20 Alkynylidene; Preferably, M is Wherein ring A is a 5-membered alicyclic heterocycle, a 6-membered heteroaromatic ring, or a polycyclic ring formed by connecting one or more 6-membered aromatic heterocycles to a benzene ring via a single bond, wherein the alicyclic heterocycle is optionally substituted with one or more selected from oxy (=O), halogen and C 1-4 The alkyl group is substituted; M1 is selected from a single bond and C 3-10 Alkylene, C 3-10 Alkenylene or C 3-10 Alkynylidene; Preferably, M is wherein ring A is selected from M1 is selected from a single bond and C 5-8 Alkylene, C 5-8 Alkenylene or C 5-8 Alkynylidene.

6. The compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite and prodrug thereof, wherein L is selected from a structure consisting of one or more substituted or unsubstituted fragments of the following: C 1-6 Alkylene, -N(R')-, Carbonyl, -O-, Val, Cit, Phe, Lys, Lys(COCH2CH2(OCH2CH2) s OCH3), D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-G ly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Val-Lys-Gly, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly, Where R' represents hydrogen, C 1-6 Alkyl or alkyl containing -(CH2CH2O)r-; r is selected from an integer of 1-10; s is selected from an integer of 1-20; Preferably, L is selected from the structure consisting of one or more substituted or unsubstituted fragments as follows: C 1-6 Alkylene, -NH-, Phe, Lys, Lys(COCH2CH2(OCH2CH2) s OCH3), Gly, Gly-Gly-Phe-Gly, Wherein s is selected from an integer from 1 to 20; Preferably, L is selected from the following structures: wherein s is selected from an integer of 1-20.

7. The compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite and prodrug thereof, wherein E is a single bond, or a substituted or unsubstituted -NH-CH2-, -NH-CH2-O-CH2-CO-, 8. A compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite or prodrug thereof, Selected from the following substituted or unsubstituted structures:

9. The compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite and prodrug thereof, wherein the cytotoxic drug is selected from a microtubule inhibitor, a DNA intercalator, a DNA topoisomerase inhibitor and an RNA polymerase inhibitor; preferably, the microtubule inhibitor is an auristatin compound or a maytansine compound; preferably, the DNA intercalator is a pyrrolobenzodiazepine (PBD); preferably, the DNA The topoisomerase inhibitor is a topoisomerase I inhibitor (e.g., camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, or rubitecan) or a topoisomerase II inhibitor (e.g., doxorubicin, PNU-159682, duocarmycin, daunorubicin, mitoxantrone, podophyllotoxin, or etoposide); preferably, the RNA polymerase inhibitor is α-amanitin or a pharmaceutically acceptable salt, ester or analog thereof; Preferably, the cytotoxic drug is selected from the compounds represented by Formula I and Formula II: in, R1, R2 are each independently selected from C 1-6 Alkyl and halogen; R3 is selected from H and -CO-CH2OH; R4 and R5 are each independently selected from H, halogen and hydroxyl; or R4 and R5 are connected to the connected carbon atom to form a 5-6 membered oxygen-containing heterocyclic ring; R6 is selected from hydrogen or -C 1-4 Alkylene-NR a R b ; R7 is selected from C 1-6 Alkyl and -C 1-4 Alkylene-NR a R b ; Where R a , R b Each occurrence is independently selected from H, C 1-6 Alkyl, -SO2-C 1-6 Alkyl and -CO-C 1-6 alkyl.

10. The compound of any one of claims 1 to 9 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite and prodrug thereof, wherein the cytotoxic drug is selected from the following compounds:

11. The compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope label, metabolite and prodrug thereof, wherein D is a monovalent structure obtained by losing one H from -OH, -NH2 or a secondary amine group on the cytotoxic drug; Preferably, D is selected from the following structures:

12. The compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite and prodrug thereof, wherein the compound has the following structure:

13. A pharmaceutical composition comprising the compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutical excipients.

14. Use of the compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 13, in the preparation of a medicament for treating cancer or tumor.

15. The use of claim 14, wherein the cancer is selected from a solid tumor or a hematological malignancy; for example, selected from a solid tumor or a hematological malignancy, such as colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.

16. The compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 13, for use in treating cancer or tumors.

17. A compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 13, for use in treating solid tumors or hematological malignancies; for example, selected from solid tumors or hematological malignancies, such as colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, specifically lung adenocarcinoma), or lymphoma.

18. A method for treating cancer, comprising administering to an individual in need thereof a therapeutically effective amount of the compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 13.

19. The method of claim 18, wherein the cancer or tumor is selected from a solid tumor or a hematological malignancy, such as colon cancer, gastric cancer, breast cancer, lung cancer (e.g., non-small cell lung cancer, particularly lung adenocarcinoma), or lymphoma.

20. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite, and prodrug thereof, wherein the compound has the following structure: in, s, R1, R2, R3, Ra are as described in any one of claims 1-12; PG is an amino protecting group; for example, PG is 9-fluorenylmethoxycarboxylate (Fmoc), tert-butoxycarboxylate (Boc), benzyloxycarboxylate (Cbz), acetate (Ac), dichloroacetate (CAc), benzoate (Bz), tert-butylcarboxylate (Pv), TMS, TES, TBDMS, TIPS, TBDPS, methoxymethyl ether (MOM), 2-methoxyethoxymethyl ether (MEM), benzyloxymethyl ether (BOM) or p-methoxybenzyloxymethyl ether (PMBOM), trityl (Tr), p-methoxytrityl (MMT), dimethoxytrityl (DMT).

21. The compound of claim 20 or its pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite and prodrug, wherein the compound structure is as follows:

22. A method for preparing compound B-04a, the method comprising subjecting compound B-04-4a and compound B-04-5 to a coupling reaction; in, s, R1, R2, R3 are as described in any one of claims 1-12; preferably, s is 8, R1 is methyl, R2 is chlorine, and R3 is -C(=O)-CH2-OH.

23. A method for preparing compound B-04-4a, the method comprising deprotecting compound B-04-3a; in, s, R1, R2, R3, PG are as described in any one of claims 1-12, 20; preferably, s is 8, R1 is methyl, R2 is chlorine, R3 is -C(=O)-CH2-OH, and PG is MMT.

24. A method for preparing compound B-04-3a, the method comprising subjecting compound B-04-2a to a hydrolysis reaction: in, s, R1, R2, R3, PG are as described in any one of claims 1-12, 20; preferably: s is 8, R1 is methyl, R2 is chlorine, R3 is -C(=O)-CH2-OH, and PG is MMT.

25. A method for preparing compound B-04-2a, the method comprising subjecting compound B-04-1a and compound B-04-6a to a coupling reaction: in, s, R1, R2, PG are as described in any one of claims 1-12, 20; preferably, s is 8, R1 is methyl, R2 is chlorine, and PG is MMT.

26. A method for preparing compound B-04-1a, the method comprising subjecting compound 1-4 to a hydrolysis reaction: in, R1, R2, and R3 are as described in any one of claims 1-12 and 20; preferably, R1 is methyl, R2 is chlorine, and R3 is -C(=O)-CH2-OH.

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