Condensed ring compound as well as preparation method and application thereof

Abstract

The invention provides a fused ring compound and a preparation method and application thereof, the fused ring compound has a structure as shown in a 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 or hematoma such as gastric cancer, breast cancer, lung cancer and lymphoma.

Description

Technical Field

[0001] The present application relates to the field of medicine, and in particular to a condensed ring compound and a preparation method and use thereof. Background Art

[0002] Antibody drug conjugate (ADC) consists of antibodies, bioactive molecules and linkers. The bioactive molecules are covalently coupled to the antibodies through linkers; antibodies (such as monoclonal antibodies) can specifically recognize specific targets on the surface of tumor cells, and then guide ADC to the surface of cancer cells, and allow ADC to enter cancer cells through cellular endocytosis; then the bioactive molecules are released inside the cancer cells, killing cancer cells while minimizing damage to normal tissue cells.

[0003] As an important component of antibody-drug conjugates, the structure of the drug-linker part is closely related to the overall efficacy and safety of ADC drugs. Therefore, the development of new drug-linker structures is of great significance for the development of antibody conjugates with good efficacy and safety. Summary of the invention

[0004] The present application relates to a fused ring compound and a preparation method thereof. The fused ring compound can be used to prepare an antibody-drug conjugate and achieve good efficacy and safety.

[0005] Compound

[0006] 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, which has a structure shown in formula DEL-M', wherein:

[0007] 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;

[0008] L is a structural fragment connecting M' and E;

[0009] E is a structural fragment connecting L and D;

[0010] D is a cytotoxic drug fragment.

[0011] In some embodiments, M' is Lg is a leaving group for a nucleophilic substitution reaction (e.g.,

[0012] Halogen, methylsulfonyl, 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 The alkyl group is substituted; M1 is selected from a single bond and C 1-20 Alkylene, C 2-20 Alkenylene or C 2-20 Alkyne;

[0013] The L, E and D structures are as defined in any of the above.

[0014] 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 aromatic heterocyclic 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 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 Alkyne group.

[0015] In some embodiments, M' is Selected from M1 is selected from a single bond and C 5-8 Alkylene, C 5-8 Alkenylene or C 5-8 Alkyne group.

[0016] In some embodiments, M' is selected from

[0017] In some embodiments, M' is

[0018] In some embodiments, M' is selected from

[0019] And, when M' is When E is or wherein s is selected from an integer of 1-20.

[0020] 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 The alkyl group is substituted; M1 is selected from a single bond and C 1-20 Alkylene, C 2-20 Alkenylene or C 2-20 Alkyne group.

[0021] 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 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 Alkyne group.

[0022] 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 Alkyne group.

[0023] In some embodiments, M is selected from the following structures:

[0024]

[0025] In some embodiments, Lg is selected from halogen, substituted or unsubstituted C 1-6 sulfonyl, 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 as follows: 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.

[0029] In some embodiments, L is selected from a structure consisting of one or more substituted or unsubstituted fragments as follows: 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]

[0032]

[0033] wherein s is selected from an integer of 1-20.

[0034] In some embodiments, L is selected from the following structures:

[0035]

[0036] In some embodiments, L is selected from the following structures:

[0037]

[0038] In some embodiments, E is a single bond, or substituted or unsubstituted -NH-CH2-, -NH-CH2-O-CH2-CO-,

[0039] In some embodiments, E is a single bond, or substituted or unsubstituted -NH-CH2-, -NH-CH2-O-CH2-CO-,

[0040] In some embodiments, E is -NH-CH2-O-CH2-CO-,

[0041] In some embodiments, E is -NH-CH2-O-CH2-CO- or

[0042] In some embodiments, M is selected from the following structures:

[0043]

[0044] L is selected from the following structures:

[0045]

[0046] E is -NH-CH2-O-CH2-CO-,

[0047] In some embodiments, Selected from the following substituted or unsubstituted structures:

[0048]

[0049]

[0050]

[0051] In some embodiments, Select from the following structures:

[0052]

[0053]

[0054] 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.

[0055] The cytotoxic drugs disclosed in the present 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 Only non-hydrogen substituents on N are represented here, and the cytotoxic drug can be attached to the linker in the conjugate via these functional groups.

[0056] In some embodiments, the cytotoxic drug is linked to E in the antibody drug conjugate via -OH, -SH, primary amino, secondary amine or tertiary amine groups on the cytotoxic drug.

[0057] In some embodiments, the cytotoxic drug is selected from the following Formula I and Formula II:

[0058]

[0059] Wherein, R1, R2 are each independently selected from C 1-6 Alkyl and halogen;

[0060] R3 is selected from H and -CO-CH2OH;

[0061] 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;

[0062] R6 is selected from hydrogen or -C 1-4 Alkylene-NR a R b ;

[0063] R7 is selected from C 1-6 Alkyl and -C 1-4 Alkylene-NR a R b ;

[0064] 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.

[0065] In some embodiments, the cytotoxic drug is selected from the following compounds:

[0066]

[0067]

[0068] In some embodiments, the cytotoxic drug is selected from the following compounds:

[0069]

[0070] 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 -OH, -NH2 or a secondary amine group on the cytotoxic drug.

[0071] In some embodiments, D is selected from the following structures:

[0072]

[0073]

[0074] In some embodiments, the compound is selected from A-01 to A-25, B-01 to B-05 shown below:

[0075] A-01:

[0076] A-02:

[0077] A-03:

[0078] A-04:

[0079] A-05:

[0080] A-06:

[0081] A-07:

[0082] A-08:

[0083] A-09:

[0084] A-10:

[0085] A-11:

[0086] A-12:

[0087] A-13:

[0088] A-14:

[0089] A-15:

[0090] A-16:

[0091] A-17:

[0092] A-18:

[0093] A-19:

[0094] A-20:

[0095] A-21:

[0096] A-22:

[0097] A-23:

[0098] A-24: A-25:

[0099] B-01:

[0100] B-02:

[0101] B-03: B-04:

[0102] B-05:

[0103] Conjugate

[0104] The second aspect of the present invention provides a conjugate as shown in formula (II), wherein:

[0105] Ab-[MLED]x

[0106] Formula (II)

[0107] Ab is a targeting moiety; M, L, E and D are as described above;

[0108] x is 1 to 10.

[0109] 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. .

[0110] In some preferred embodiments, Ab is a small molecule ligand, such as a folic acid derivative, a glutamate urea derivative, a somatostatin derivative, an aromatic sulfonamide derivative (such as a carbonic anhydrase IX inhibitor), a polyene connecting two aliphatic indoles, a cyanine dye, or IR-783 or a derivative thereof.

[0111] In some preferred embodiments, Ab is an antibody, such as a monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody or the 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.

[0112] In some embodiments, the conjugate is selected from ADC A-01 to ADC A-25, ADC B-01 to ADCB-05 shown below:

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123] wherein the HA in each conjugate is an antibody or an antigen-binding fragment thereof;

[0124] in, It indicates the specific connection mode between the thiol group in the antibody or its antigen-binding fragment and the M fragment;

[0125] It indicates the specific connection mode between the amino group in the antibody or its antigen-binding fragment and the M fragment.

[0126] 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.

[0127] In some embodiments, Ab-[MLED] x In the antibody drug conjugates shown, x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0128] In some embodiments, the conjugate is selected from:

[0129]

[0130]

[0131] wherein the HA in each conjugate is an antibody or an antigen-binding fragment thereof;

[0132] in, represents the specific connection mode between the thiol group in the antibody or antigen-binding fragment thereof and the M fragment; x is 3-8, for example 3-4 or 7-8.

[0133] Composition

[0134] In another aspect, the present application provides compositions that may include a plurality of ADCs described herein. Each antibody molecule in the composition may be coupled to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 compounds described herein. Thus, the composition is characterized in that the "drug-antibody ratio" (DAR) is in the range of about 1 to about 10. Methods for determining DAR are well known to the skilled person, including methods using reverse phase chromatography or HPLC-MS.

[0135] 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.

[0136] Synthetic intermediates

[0137] On the other hand, the present application provides a compound shown in the following structure:

[0138]

[0139]

[0140]

[0141] Wherein, s, R1, R2, R3, Ra are as described above;

[0142] PG is a 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).

[0143] In some embodiments, the present application provides a compound shown in the following structure:

[0144]

[0145]

[0146]

[0147] Synthesis method

[0148] In another aspect, the present application provides methods for preparing the compounds described herein.

[0149] Method 1

[0150] In some embodiments, the method 1 includes a method for preparing compound B-04a, which includes subjecting compound B-04-4a to a coupling reaction;

[0151] B-04a:

[0152]

[0153] 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.

[0154] In some embodiments, the coupling reaction is performed in the presence of a coupling reagent; preferably, the coupling reagent is cuprous bromide.

[0155] In some embodiments, the method 1 includes a method for preparing compound B-04-4a, which includes deprotecting compound B-04-3a;

[0156]

[0157] Wherein, s, R1, R2, R3, 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.

[0158] In some embodiments, the deprotection is performed under acidic conditions; preferably, the acidic conditions are trifluoroacetic acid.

[0159] In some embodiments, the method 1 includes a method for preparing compound B-04-3a, which includes subjecting compound B-04-2a to a hydrolysis reaction:

[0160]

[0161]

[0162] Wherein, s, R1, R2, R3, 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.

[0163] In some embodiments, the hydrolysis reaction is carried out under alkaline conditions; preferably, the alkaline conditions are sodium salts; further preferably, the sodium salts are sodium carbonate or sodium bicarbonate.

[0164] In some embodiments, the method 1 includes a method for preparing compound B-04-2a, which includes subjecting compound B-04-1a to a coupling reaction:

[0165]

[0166] Wherein, s, R1, R2, PG are as described above; preferably: s is 8, R1 is methyl, R2 is chlorine, and PG is MMT.

[0167] In some embodiments, the hydrolysis reaction is carried out in the presence of a coupling reagent; preferably, the coupling reagent includes one or both of DMAP and triphosgene.

[0168] In some embodiments, the method 1 includes a method for preparing compound B-04-1a, which includes subjecting compound 1-4 to a coupling reaction:

[0169]

[0170] Wherein, R1, R2, and R3 are as described above; preferably, R1 is methyl, R2 is chlorine, and R3 is -C(=O)-CH2-OH.

[0171] In some embodiments, the hydrolysis reaction is performed in the presence of a coupling reagent; preferably, the coupling reagent comprises DIPEA.

[0172] Method 2

[0173] In some embodiments, the method 2 includes a method for preparing compound A-17a, which includes subjecting compound A-17-06a to a coupling reaction;

[0174]

[0175] Wherein, R1, R2, s are as described above; preferably, s is 11, R1 is methyl and R2 is chlorine.

[0176] In some embodiments, the hydrolysis reaction is performed in the presence of a coupling reagent; preferably, the coupling reagent comprises DIPEA.

[0177] In some embodiments, the method 2 includes a method for preparing compound A-17-06a, which includes subjecting compound A-17-05a to a deprotection reaction;

[0178]

[0179] Wherein, R1, R2, PG, and s are as described above; preferably, s is 11, R1 is methyl, R2 is chlorine, and PG is Fmoc.

[0180] 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.

[0181] In some embodiments, the method 2 includes a method for preparing compound A-17-05a, which includes subjecting compound A-17-04a to a coupling reaction;

[0182]

[0183] Wherein, R1, R2, PG, and s are as described above; preferably, s is 11, R1 is methyl, R2 is chlorine, and PG is Fmoc.

[0184] In some technical solutions, the coupling reaction is carried out in the presence of a coupling reagent; preferably, the coupling reagent includes DIPEA.

[0185] In some embodiments, the method 2 includes a method for preparing compound A-17-04a, which includes subjecting compound A-17-03a to a coupling reaction;

[0186]

[0187] Wherein, s and PG are as described above; preferably, s is 11, and PG is Fmoc.

[0188] 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.

[0189] In some embodiments, the method 2 includes a method for preparing compound A-17-03a, which includes subjecting compounds A-17-01a and A-17-02a to a coupling reaction;

[0190]

[0191] Wherein, s and PG are as described above; preferably, s is 11, and PG is Fmoc.

[0192] In some technical solutions, the coupling reaction is carried out in the presence of a coupling reagent; preferably, the coupling reagent includes DIPEA.

[0193] Method 3

[0194] In some embodiments, the method three includes a method for preparing compound B-03a, which includes subjecting compound B-03-8a to a coupling reaction;

[0195]

[0196] Wherein, Ra is as described above; preferably: Ra is isopropyl.

[0197] 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; and diisopropylethylamine is further preferred.

[0198] In some embodiments, the method three includes a method for preparing compound B-03-8a, which includes subjecting compound B-03-7a to a deprotection reaction;

[0199]

[0200]

[0201] Wherein, Ra and PG are as described above; preferably, Ra is isopropyl, and PG is Fmoc.

[0202] 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.

[0203] In some embodiments, the method three includes a method for preparing compound B-03-7a, which includes subjecting compound B-03-6a to a coupling reaction;

[0204]

[0205] Wherein, Ra and PG are as described above; preferably, Ra is isopropyl, and PG is Fmoc.

[0206] 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.

[0207] In some embodiments, the method three includes a method for preparing compound B-03-6a, which includes subjecting compound B-03-5a to a coupling reaction;

[0208]

[0209] Wherein, PG is as described above; preferably, PG is Fmoc.

[0210] In some technical solutions, the coupling reaction is carried out in the presence of a coupling reagent; preferably, the coupling reagent includes diisopropylethylamine.

[0211] In some embodiments, the method three includes a method for preparing compound B-03-5a, which includes subjecting compound B-03-4a to a coupling reaction;

[0212]

[0213] Wherein, PG is as described above; preferably, PG is Fmoc.

[0214] In some technical solutions, the coupling reaction is carried out in the presence of [2-[2-(Fmoc-amino)ethoxy]ethoxy]acetic acid.

[0215] In some embodiments, the method three includes a method for preparing compound B-03-4, which includes subjecting compound B-03-3 to a coupling reaction;

[0216]

[0217] In some technical solutions, the coupling reaction is carried out in the presence of PtO2 and hydrogen.

[0218] Pharmaceutical composition

[0219] In another aspect, the present application provides a pharmaceutical composition, which contains the compound or conjugate described in any one of the above items, and one or more pharmaceutical excipients.

[0220] Pharmaceutical excipients include, for example, pharmaceutically acceptable carriers and / or excipients.

[0221] The compounds or conjugates described herein are usually formulated in a unit injectable form together 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 may be administered by any route appropriate to the individual to be treated.

[0222] The compounds or conjugates described herein, pharmaceutical compositions 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 composition of the present invention should be sterile and stable under production and storage conditions. The preferred dosage form is an injection. This injection can be a sterile injection solution. For example, a sterile injection 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., permeants, preservatives, diluents or any combination thereof) are optionally added, and then filtered and sterilized. In addition, for ease of storage and use, sterile injections can be prepared into sterile lyophilized powders (e.g., by vacuum drying or freeze drying). This sterile lyophilized powder can be dispersed in a suitable carrier, such as sterile pyrogen-free water, before use.

[0223] In addition, the compounds or conjugates described herein can be present in a pharmaceutical composition in a unit dosage form for 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 person will understand that the route and / or mode of administration will vary according to the intended purpose. In some preferred embodiments, the ADC and pharmaceutical compositions described herein are administered by intravenous infusion or injection.

[0224] In certain embodiments, the pharmaceutical composition may also include 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.

[0225] application

[0226] The present application provides the use of the above-mentioned compound or its pharmaceutically acceptable salt, conjugate, composition, or pharmaceutical composition in the preparation of a drug for treating cancer or tumor.

[0227] The present application provides the above-mentioned compound or its pharmaceutically acceptable salt, conjugate, composition, or pharmaceutical composition for use in treating cancer or tumor.

[0228] The present application provides the above-mentioned compound or its pharmaceutically acceptable salt, conjugate, composition, or pharmaceutical composition for use in treating cancer or tumor.

[0229] The present application provides a method for treating cancer or tumors, which comprises administering a therapeutically effective amount of the compound described above or a pharmaceutically acceptable salt, conjugate, composition, or pharmaceutical composition thereof to an individual in need thereof.

[0230] In some embodiments, the compound, or a pharmaceutically acceptable salt, conjugate, composition, or pharmaceutical composition thereof, is sufficient (e.g., in a subject):

[0231] (1) Inhibit the proliferation of cells (such as tumor cells);

[0232] (2) Inhibit tumor growth;

[0233] (3) Induce and / or increase antibody-dependent cellular cytotoxicity activity;

[0234] (4) Inhibition of signal transduction;

[0235] (5) prevention and / or treatment of cancer; or

[0236] (6) Any combination of the above (1) to (5).

[0237] 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.

[0238] definition

[0239] Unless otherwise defined below, the meanings of all technical terms and scientific terms used herein are intended to be the same as those commonly understood by those skilled in the art. Reference to the technology used herein is intended to refer to the technology commonly understood in the art, including those changes in technology that are obvious to those skilled in the art or the replacement of equivalent technology. In addition, laboratory operation steps such as genomics, nucleic acid chemistry, molecular biology, etc. used herein are all conventional steps 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.

[0240] 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, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH regulators 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 commonly understood by those skilled in the art, which can stabilize 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 their degradation products (such as lactalbumin hydrolysate), etc.

[0241] 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.

[0242] The term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, prevent, 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 prevent the disease and its complications in a patient who already has the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount 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 simultaneously, etc. 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 simultaneously, etc.

[0243] The term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, a beneficial or desired clinical outcome includes, but is not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., not worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" may also refer to prolonging survival compared to the expected survival if not receiving treatment.

[0244] 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.

[0245] The terms "cancer" and "tumor" are used interchangeably to refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors, or cells that invade neighboring tissues and may metastasize 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 tumors, especially hematologic malignancies.

[0246] The term "hematologic malignancies" includes lymphomas, leukemias, myelomas or lymphoid malignancies, as well as spleen 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 lymphomas. Hematologic malignancies also include leukemias, such as secondary leukemias or acute lymphocytic leukemias. Hematologic malignancies also include myeloma (e.g., multiple myeloma) and other blood and / or B-cell or T-cell related cancers.

[0247] The term "alkyl" refers to a straight chain or branched hydrocarbon radical with one hydrogen atom removed, for example, "C 1-20 Alkyl", "C 1-10 Alkyl", "C 1-6 Alkyl", "C 1-4 Alkyl", "C 1-3 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, and the like.

[0248] 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.

[0249] 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.

[0250] The term "alkynylene" refers to a divalent group derived from a straight or branched hydrocarbon group containing at least one carbon-carbon triple bond losing 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.

[0251] 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.

[0252] 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.

[0253] The term "aromatic ring system" refers to a monocyclic or polycyclic system comprising at least one aromatic ring (e.g., benzene ring, etc.) or heteroaromatic ring (e.g., 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.

[0254] The term "substituted" refers to the replacement of one or more (e.g., 1, 2, 3, 4, or 5) hydrogens on the specified compound or structural fragment by a substituent, provided that the normal atomic valence of the specified atom in the current situation is not exceeded and the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only when 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 (sub)alkyl, C1-C6 halo (sub)alkyl, C1-C6 alkoxy, C2-C6 (sub)alkenyl, C2-C6 (sub)alkynyl, C3-C8 (sub)cycloalkyl, 3-8 member (sub)heterocyclyl, C6-C 10(sub)aryl and 5-10 membered (sub)heteroaryl, etc.; wherein R' is H or C1-C6 alkyl, C1-C6 haloalkyl, C3-C8 cycloalkyl, 3-8 membered heterocyclic, C6-C 10 Aryl or 5-10 membered heteroaryl.

[0255] If a functional group or structural fragment is described as "substituted or unsubstituted," the functional group or structural fragment can be (1) unsubstituted or (2) substituted.

[0256] 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 10% or greater.

[0257] 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

[0258] 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:

[0259] Figure 1A -C shows the bystander effect test results of anti-human HER3 antibody drug conjugate on HEK293T and HEK293T-h HER3 cells.

[0260] Figure 2 The results of plasma stability test of anti-human HER3 antibody drug conjugate are shown.

[0261] Figure 3A The HPLC spectra of compounds A-07-A and A-07-B prepared in Example 3 are shown.

[0262] Figure 3B The HPLC spectra of compound A-07-A prepared in Example 3 and compound A-05 prepared in Example 9 are shown. DETAILED DESCRIPTION

[0263] 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 is by no means intended to limit 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 creative work are within the scope of protection of the present invention.

[0264] The abbreviations used in this document have the following meanings:

[0265]

[0266]

[0267] The structures of the compounds described in the following examples were determined by nuclear magnetic resonance ( 1 H NMR) or mass spectrometry (MS).

[0268] Nuclear magnetic resonance (NMR) 1 H NMR) was measured using a Bruker 400 MHz nuclear magnetic resonance instrument; the deuterated reagent was hexadeuterated dimethyl sulfoxide (DMSO-d6); and the internal standard substance was tetramethylsilane (TMS).

[0269] The abbreviations in the nuclear magnetic resonance (NMR) spectra used in the Examples are shown below.

[0270] 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.

[0271] Mass spectrometry (MS) was measured using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

[0272] 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-tetraazahexadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1-H-pyrrol-1-yl)hexanamide (A-01)

[0273]

[0274] Compound A-01-1 (0.40 g, 640.59 μmol, its synthesis reference patent application CN 111936169A), exotecan 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 the mixture was reacted at 25° C. for 4 hours. DIPEA was removed under reduced pressure and freeze-dried with water to remove most of the DMF to obtain a crude product, which was purified by preparative high performance liquid chromatography (conditions as follows) to obtain 273 mg of the title compound.

[0275] Chromatographic column: Waters XBridge Prep C18 OBD 45mm×450mm×8.0μm

[0276] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0277] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 30 70 70 5.00 30 70 70 60.00 70 30 70

[0278] The structural characterization data are as follows:

[0279] ESI-MS (m / z): 1034.4 [M+H] + .

[0280] 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)

[0281]

[0282] Step 1: Synthesis of 1-chloro-3-bromo-2-methyl-5-nitrobenzene (1-8-2)

[0283] At 25°C, compound 1-8-1 (5.00 g, 29.14 mmol) was dissolved in n-heptane (25 mL), 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 mixture was kept at 50°C for 2 hours. The reaction was detected 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, extracted with toluene, and the organic phases were combined, 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), and the preparative solution was freeze-dried to obtain 4.88 g of the title compound.

[0284] Chromatographic column: C18 ODS 45mm×450mm×8.0μm

[0285] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0286] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 60 40 60 10 60 40 60 40 100 0 60

[0287] Step 2: Synthesis of 3-chloro-5-bromo-4-methylaniline (1-8-3)

[0288] At 25°C, compound 1-8-2 (4.88 g, 19.48 mmol) was dissolved in ethyl acetate (100 mL), and platinum carbon (2.00 g, 19.48 mmol, content 5%) was added. After hydrogen replacement, the mixture was reacted at 60°C for 4 h under hydrogen protection, and the reaction was monitored by HPLC-MS / MS. The reaction solution was filtered and concentrated to obtain 3.68 g of crude title compound, which was directly used in the next step without further purification.

[0289] Step 3: Synthesis of N-(3-chloro-5-bromo-4-methylphenyl)acetamide (1-8-4)

[0290] At 20°C, compound 1-8-3 (3.63 g, 14.82 mmol) was dissolved in ethyl acetate (70 mL), triethylamine (4.50 g, 44.45 mmol) and acetic anhydride (2.27 g, 22.23 mmol) were added, and the reaction was maintained at 20°C for 20 h, and the reaction was monitored by HPLC-MS. Water was added to the reaction solution, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product, which was slurried with a mixed solvent of ethyl acetate: petroleum ether = 1:5 to obtain 2.86 g of the title compound.

[0291] Step 4: Synthesis of (Z)-4-(5-acetamido-3-chloro-2-methylphenyl)but-3-enoic acid (1-8-5)

[0292] At 20°C, compound 1-8-4 (1.80 g, 6.86 mmol) was dissolved in THF (20 mL) and water (5 mL), and vinylacetic acid (708.31 mg, 8.23 ​​mmol), DIPEA (1.95 g, 15.08 mmol), tri(o-methylphenyl)phosphine (62.60 mg, 0.20 mmol) were added. The reaction system was replaced with nitrogen and heated to 70°C for 5 h. The reaction was monitored by HPLC-MS. 1N sodium hydroxide solution was added to the reaction solution to adjust pH=8, and ethyl acetate was added for extraction. The aqueous phase was adjusted to pH=3 with 1N hydrochloric acid, extracted with ethyl acetate, and 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 directly used in the next step.

[0293] Step 5: Synthesis of 4-(5-acetamido-3-chloro-2-methylphenyl)butyric acid (1-8-6)

[0294] At 20°C, compound 1-8-5 (2.60 g, 9.71 mmol) was dissolved in THF (50 mL), Pd / C (0.52 g, content 10%) was added, the system was replaced with hydrogen and reacted at 40°C for 2 h under the protection of a hydrogen balloon, and 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 directly used in the next step without further purification.

[0295] Step 6: Synthesis of N-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-7)

[0296] Compound 1-8-6 (2.43 g, 9.01 mmol) was dissolved in trifluoroacetic acid (10 mL), cooled to 5°C, trifluoroacetic anhydride (3.78 g, 18.02 mmol, 2.50 mL) was added dropwise, and the reaction was maintained at 5°C for 4 h. The reaction was monitored by HPLC-MS. The reaction solution was added to water, dissolved in 10N sodium hydroxide to adjust pH = 9, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column (ethyl acetate: petroleum ether = 0-20%) to obtain 1.53 g of the title compound.

[0297] 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)

[0298] At 5°C, potassium tert-butoxide (1.50 g, 13.37 mmol) was dissolved in THF (16 mL) and tert-butanol (4 mL), and 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 the reaction was monitored by HPLC-MS. The reaction solution was adjusted to pH = 5 with 1N hydrochloric acid, extracted with ethyl acetate, the combined organic phases were dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the crude product was beaten with methyl tert-butyl ether to obtain 1.20 g of the title compound.

[0299] Step 8: Synthesis of N-(7-amino-3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-8-9)

[0300] At 20°C, compound 1-8-8 (0.50 g, 1.78 mmol) was dissolved in methanol (8 mL) and 2N hydrochloric acid (8 mL), Pd / C (0.15 g, 10% content) was added, the system was replaced with hydrogen and kept at 5°C for 2 h under the protection of a hydrogen balloon, and the reaction was monitored by HPLC-MS / MS. The reaction solution was filtered and concentrated to obtain 0.52 g of the hydrochloride of the title compound, which was directly used in the next step without further purification.

[0301] Step 9: Synthesis of N,N'-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (1-8-10)

[0302] At 20°C, compound 1-8-9 (0.52 g, 1.70 mmol) was dissolved in pyridine (5 mL), acetic anhydride (2 mL) was added, and the reaction was maintained at 20°C for 2 h. The reaction was monitored by HPLC-MS / MS. The reaction solution was added to water, extracted with ethyl acetate, the organic phase was washed with water, combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column (ethyl acetate: petroleum ether = 0-30%) to obtain 0.22 g of the title compound.

[0303] Step 10: Synthesis of N-(8-amino-6-chloro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (1-8-11)

[0304] At 20°C, compound 1-8-10 (450.97 mg, 1.46 mmol) was dissolved in methanol (16 mL), 2N hydrochloric acid (16 mL) was added, and the mixture was heated to 60°C for 2 h, and the reaction was monitored by HPLC-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.

[0305] 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)

[0306] Compound 1-8-11 (230.00 mg, 0.78 mmol) was dissolved in toluene (10 mL), (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, and the mixture was heated to 140°C for 5 h, and the reaction was monitored by HPLC-MS / MS. The reaction solution was concentrated, and the crude product was purified by silica gel column (methanol: dichloromethane = 0-10%) to obtain 150.00 mg of the title compound.

[0307] 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)

[0308] Compound 1-8-12 (40.00 mg, 0.081 mmol) was added to concentrated hydrochloric acid (1 mL), heated to 100 ° C for 5 h, and the reaction was monitored by HPLC-MS. The reaction solution was filtered, and the filtrate was purified by preparative HPLC (conditions as follows), and the preparative solution was freeze-dried to obtain the hydrochloride of the title compound 1-2. The hydrochloride of 1-2 was separated under the following purification conditions to obtain two isomers, which were named 1-2-A (trifluoroacetate 5.00 mg, retention time 9.85 min) and 1-2-B (trifluoroacetate 7.00 mg, retention time 10.62 min) according to the retention time.

[0309] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0310] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0311] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 5 95 28 2 5 95 28 18 50 50 28

[0312] The structural characterization data are as follows:

[0313] 1-2-A:

[0314] 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).

[0315] ESI-MS (m / z): 452.1 [M+H] + .

[0316] 1-2-B:

[0317] 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).

[0318] ESI-MS (m / z): 452.0 [M+H] + .

[0319] 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 ((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 (1-8-13-A and 1-8-13-B)

[0320] 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 HPLC-MS / MS. After the reaction was completed, water was added to the reaction solution, and the mixture was extracted with dichloromethane / methanol (v / v=10 / 1). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by preparative thin layer chromatography (dichloromethane:methanol=20:1) to separate two isomers, which were named 1-8-13-A (15.00 mg, Rf value was 0.3) and 1-8-13-B (12.00 mg, Rf value was 0.35) according to their Rf values.

[0321] 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)

[0322] 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 bottles, and a mixture of tetrabutylammonium fluoride (1M tetrahydrofuran solution) / glacial acetic acid (v / v=13 / 1) (50 μL) was added dropwise, and the reaction was maintained at 25°C for 0.5 hours, and the reaction was monitored by HPLC-MS. After the reaction was completed, the reaction solution was purified by preparative HPLC, and the preparative solution was lyophilized to obtain the title compounds 1-8-A (6.94 mg) and 1-8-B (4.00 mg).

[0323] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0324] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0325] Time [min] Mobile phase A [%] Mobile phase B [%] Flow rate [mL / min]

[0326] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 20 80 28 3 20 80 28 18 90 10 28

[0327] The structural characterization data of 1-8-A are as follows:

[0328] 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).

[0329] ESI-MS (m / z): 510.1 [M+H] + .

[0330] The structural characterization data of 1-8-B are as follows:

[0331] 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).

[0332] ESI-MS (m / z): 510.1 [M+H] + .

[0333] 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-tetraazahexadecane-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-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-07-A and A-07-B)

[0334]

[0335] 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)

[0336] At 25°C, compound A-07-2 (30.00 mg, 0.07 mmol) was dissolved in DMF (0.2 mL), 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, and 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.

[0337] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0338] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0339]

[0340]

[0341] The structural characterization data are as follows:

[0342] ESI-MS (m / z): 691.0 [M+H2O] + .

[0343] 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-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide and 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-tetraazahexadecane-16-yl) 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-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynamide (A-07-A and A-07-B)

[0344] At 25°C, the hydrochloride of 1-2 (30.00 mg, 61.43 μmol) was dissolved in N, N-dimethylformamide (1 mL), and 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 in sequence, and the reaction was maintained at 25°C for 0.5 hours, and the reaction was monitored by HPLC-MS. After the reaction was completed, the reaction solution was purified by preparative HPLC (conditions as follows), and the preparative solution was freeze-dried to obtain the title compound A-07. A-07 was separated under the following purification conditions to obtain two isomers, which were named A-07-A (11.04 mg, retention time 7.5 min) and A-07-B (19.42 mg, retention time 8.0 min) according to the retention time.

[0345] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0346] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0347] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 30 70 28 3 30 70 28 18 90 10 28

[0348] The structural characterization data are as follows:

[0349] A-07-A:

[0350] ESI-MS (m / z): 1107.3 [M+H] + .

[0351] A-07-B:

[0352] ESI-MS (m / z): 1107.3 [M+H] + .

[0353] Example 4 (S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolane[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (2-2)

[0354]

[0355] Step 1: Synthesis of 3-(isopropylamino)-1-(6-nitrobenzo[d][1,3]dioxin-5-yl)propan-1-one (2-2-2)

[0356] 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 formate salt of the title compound.

[0357] The structural characterization data are as follows:

[0358] ESI-MS (m / z): 281.1 [M+H] + .

[0359] Step 2: Synthesis of 1-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-(isopropylamino)propan-1-one (2-2-3)

[0360] The formate of compound 2-2-2 (1.25 g, 4.46 mmol) was added to tetrahydrofuran (20 mL), and 10% palladium carbon (125.00 mg) was added. After hydrogen replacement three times, the mixture was reacted at 25° C. for 16 hours. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to dryness to obtain 895.00 mg of the crude title compound, which was used directly in the next step without purification.

[0361] The structural characterization data are as follows:

[0362] ESI-MS (m / z): 251.1 [M+H] + .

[0363] Step 3: Synthesis of ((S)-7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxolane[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione (2-2)

[0364] 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 removed 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), and 3 drops of 3M hydrochloric acid were added to the prepared solution, followed by freeze drying to obtain 8.70 mg of the hydrochloride of the title compound.

[0365] The structural characterization data are as follows:

[0366] ESI-MS (m / z): 478.2 [M+H] + .

[0367] 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).

[0368] 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)

[0369]

[0370] 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)

[0371] 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 hour and then filtered. The filtrate was purified by preparative HPLC (conditions as follows) to obtain 30.00 mg of the title compound.

[0372] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0373] Mobile phase A: acetonitrile; Mobile phase B: water

[0374] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 40 60 28 8.00 40 60 28 50.00 90 10 28

[0375] The structural characterization data are as follows:

[0376] ESI-MS(m / z):815.9[(M-273) / 2+H] + .

[0377] 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)

[0378] 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 solution, and the reaction was carried out at room temperature for 30 min. The reaction solution was concentrated under reduced pressure and purified by preparative high performance liquid chromatography (conditions as follows) to obtain 20.00 mg of trifluoroacetate salt of the title compound.

[0379] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0380] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% trifluoroacetic acid)

[0381] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 15 85 28 8.00 15 85 28 50.00 60 40 28

[0382] The structural characterization data are as follows: ESI-MS (m / z): 816.0 [M / 2+H] + .

[0383] 1 H 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=8.5 6Hz,2H),7.32(d,J=8.56Hz,2H),7.06(s,1H),5.56(d,J=16.96Hz,1H),5.51(d,J=16.96Hz,1H),5.47(d, J=19.28Hz,1H),5.42(d,J=19.28Hz,1H),5.14(d,J=12.20Hz,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).

[0384] 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]dioxolane[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)

[0385]

[0386] 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)

[0387] 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 (compound B-03-2, 5.00 g, 29.56 mmol) were dissolved in acetonitrile (200 mL), and silver oxide (27.40 g, 118.25 mmol) was added under stirring. After nitrogen replacement, the mixture was reacted at room temperature in the dark for 12 hours. The reaction was monitored by HPLC-MS / MS; the reaction solution was filtered through diatomaceous earth, 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.

[0388] The structural characterization data are as follows:

[0389] ESI-MS (m / z): 503 [M+18] + .

[0390] 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)

[0391] Compound B-03-3 (2.20 g, 4.53 mmol) was dissolved in ethyl acetate and tetrahydrofuran (50 mL each), PtO2 (0.20 g) was added, and then the reaction system was replaced with a hydrogen balloon three times, and the reaction was carried out under a hydrogen atmosphere for 2 hours. The reaction was monitored by HPLC-MS / MS; the reaction solution was directly filtered, the filter cake was washed 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 directly used in the next step.

[0392] The structural characterization data are as follows:

[0393] ESI-MS (m / z): 456.1 [M+1] + .

[0394] Step 3: Synthesis of (2S,3R,4S,5S,6S)-2-(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)

[0395] 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), and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (495.22 mg, 2.00 mmol) was added under stirring, and 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.

[0396] The structural characterization data are as follows:

[0397] ESI-MS (m / z): 823.3 [M+1] + .

[0398] Step 4: Synthesis of (2S,3R,4S,5S,6S)-2-(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)

[0399] Compound B-03-5 (507.00 mg, 616.18 μmol) and diisopropylethylamine (238.91 mg, 1.85 mmol) were dissolved in dichloromethane (20 mL), and then p-nitrophenyl chloroformate (372.60 mg, 1.85 mmol) was dissolved in dichloromethane (1 mL) and slowly added dropwise to the reaction solution. After addition, the mixture was reacted at room temperature for 15 h. 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 496.00 mg of the title compound.

[0400] The structural characterization data are as follows:

[0401] ESI-MS (m / z): 988.5 [M+1] + .

[0402] Step 5: Synthesis of (2S,3R,4S,5S,6S)-2-(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]dioxolane[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)

[0403] 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, and the mixture was stirred for 12 h, and the reaction was monitored by HPLC-MS. Water and ethyl acetate were added and stirred, and the mixture was separated at rest. 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.

[0404] The structural characterization data are as follows:

[0405] ESI-MS (m / z): 1326.2 [M+1] + .

[0406] 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]dioxolane[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)

[0407] Compound B-03-7 (100.00 mg, 0.08 mmol) was dissolved in MeOH (5 mL), 1 drop of dichloromethane was added, and an aqueous solution (1 mL) of lithium hydroxide monohydrate (15.82 mg, 0.377 mmol) was added, and the mixture was stirred for 2 hours. The reaction was monitored by HPLC-MS / MS; 3N hydrochloric acid aqueous solution was added to adjust the pH of the reaction solution to 4, and the solution was concentrated under reduced pressure and purified by preparative HPLC (conditions are as follows), and the preparative solution was freeze-dried to obtain 27.00 mg of the title compound.

[0408] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0409] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0410] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 20 80 28 2.00 20 80 28 18.00 80 20 28

[0411] The structural characterization data are as follows:

[0412] ESI-MS (m / z): 964.2 [M+1] + .

[0413] 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]dioxolane[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)

[0414] 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), and diisopropylethylamine (3.62 mg, 0.03 mmol) was added dropwise under stirring. The mixture was reacted at room temperature for 4 hours, and the reaction was monitored by HPLC-MS. The reaction solution was purified by preparative HPLC (conditions are as follows), and the preparative solution was freeze-dried to obtain 11.70 mg of the title compound.

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

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

[0417] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 20 80 28 2.00 20 80 28 18.00 80 20 28

[0418] The structural characterization data are as follows:

[0419] ESI-MS (m / z): 1214.4 [M+1] + .

[0420] 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)

[0421]

[0422] Step 1: Synthesis of 3-bromo-4-chloro-5-fluoroaniline (1-5-02)

[0423] Compound 1-5-01 (2.00 g, 10.53 mmol) was dissolved in N,N-dimethylformamide (30 mL), and then N-chlorosuccinimide (1.69 g, 12.63 mmol) was slowly added. After the addition was completed, the mixture was reacted at room temperature for 16 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by flash silica gel column (ethyl acetate: petroleum ether = 0-25%) to obtain 0.95 g of the title compound.

[0424] The structural characterization data are as follows:

[0425] 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).

[0426] Step 2: Synthesis of N-(3-bromo-4-chloro-5-fluorophenyl)acetamide (1-5-03)

[0427] Compound 1-5-02 (0.95 g, 4.23 mmol) was dissolved in ethyl acetate (20 mL), and acetic anhydride (648.13 mg, 6.35 mmol) was added under nitrogen protection. After the addition was completed, the temperature was raised to 50°C for 15 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the reaction solution was quenched with methanol (5 mL), it was directly evaporated to dryness under reduced pressure to obtain a crude product, which was purified by rapid silica gel column (ethyl acetate: petroleum ether = 0-40%) to obtain 1.01 g of the title compound.

[0428] The structural characterization data are as follows:

[0429] ESI-MS (m / z): 265.9 [M+H] + .

[0430] Step 3: Synthesis of (E)-4-(5-acetamido-2-chloro-3-fluorophenyl)-3-butenoic acid (1-5-04)

[0431] 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), and then N,N-diisopropylethylamine (1.45 g, 11.26 mmol), tri(o-methylphenyl)phosphine (114.21 mg, 375.24 μmol) and palladium acetate (42.12 mg, 187.62 μmol) were added. After the addition, the reaction system was replaced with nitrogen three times, and the temperature was raised to 100 ° C under a nitrogen atmosphere for 16 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the reaction solution was cooled to room temperature, 1N sodium hydroxide aqueous solution (60 mL) and ethyl acetate (50 mL) were added to shake and layer. 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.

[0432] The structural characterization data are as follows:

[0433] ESI-MS (m / z): 272.0 [M+H] + .

[0434] Step 4: Synthesis of 4-(5-acetamido-2-chloro-3-fluorophenyl)butyric acid (1-5-05)

[0435] The crude product of compound 1-5-04 (1.00 g, 3.68 mmol) was dissolved in tetrahydrofuran (15 mL), and then 10% palladium carbon (0.10 g) was added. After the addition was complete, the reaction system was replaced three times with a hydrogen balloon, and the reaction was carried out under a hydrogen atmosphere for 4 hours. The reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to dryness to obtain 1.00 g of the crude product of the title compound.

[0436] The structural characterization data are as follows:

[0437] ESI-MS (m / z): 274.0 [M+H] + .

[0438] Step 5: Synthesis of N-(4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-06)

[0439] The crude product of compound 1-5-05 (1.00 g, 3.65 mmol) was dissolved in trifluoroacetic acid (5 mL), cooled to 5°C, and trifluoroacetic anhydride (3.84 g, 18.27 mmol, 2.54 mL) was slowly added. After the addition was completed, the reaction was maintained at 5°C for 2 hours, and 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 rapid silica gel column to obtain 0.43 g of the title compound.

[0440] The structural characterization data are as follows:

[0441] ESI-MS (m / z): 256.1 [M+H] + .

[0442] Step 6: Synthesis of N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-07)

[0443] Tetrahydrofuran (16 mL) and tert-butanol (4 mL) were added to the reaction bottle, cooled to 5 ° C in an ice bath, potassium tert-butoxide (415.18 mg, 3.70 mmol) was added, and 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 completed, the reaction was maintained at 5 ° C for 1 hour, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the reaction solution was quenched with saturated ammonium chloride aqueous solution, it was 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 the crude product of the title compound.

[0444] The structural characterization data are as follows:

[0445] ESI-MS (m / z): 285.0 [M+H] + .

[0446] Step 7: Synthesis of N-(7-amino-4-chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1-5-08)

[0447] 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 carbon (40.00 mg) were added. After the addition, the reaction system was replaced with hydrogen three times, and the reaction was carried out at room temperature for 1 hour under a hydrogen atmosphere. 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 of the title compound.

[0448] The structural characterization data are as follows:

[0449] ESI-MS (m / z): 271.0 [M+H] + .

[0450] Step 8: Synthesis of (9H-fluoren-9-ylmethyl)(8-acetamide-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (1-5-09)

[0451] The crude hydrochloride of compound 1-5-08 (0.43 g, 1.19 mmol) was dissolved in 1,4-dioxane (15 mL), and then sodium bicarbonate (400.35 mg, 4.77 mmol), water (5 mL) and 9-fluorenylmethyl-N-succinimidyl carbonate (481.81 mg, 1.43 mmol) were added. After the addition was completed, the reaction was stirred at room temperature for 2 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was poured into water, and then 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 a crude product. The crude product was purified by C18 reverse phase column (acetonitrile: 0.05% formic acid water = 20%-100%) to obtain 301.00 mg of the title compound.

[0452] The structural characterization data are as follows:

[0453] ESI-MS (m / z): 493.2 [M+H] + .

[0454] 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)

[0455] 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 the addition was completed, the temperature was raised to 60°C for reaction for 2 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. The reaction solution was poured into water, and then 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 a crude product. The crude product was purified by rapid silica gel column (ethyl acetate: petroleum ether = 0-50%) to obtain 198.00 mg of the title compound.

[0456] The structural characterization data are as follows:

[0457] ESI-MS (m / z): 451.1 [M+H] + .

[0458] 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)

[0459] (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 reacted for 4 hours. The reaction solution was directly evaporated to dryness under reduced pressure at 140 ° C to obtain a crude product. The crude product was purified by rapid silica gel column (methanol: dichloromethane = 0-5%) to obtain 256.00 mg of the title compound.

[0460] The structural characterization data are as follows:

[0461] ESI-MS (m / z): 678.1 [M+H] + .

[0462] 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)

[0463] Compound 1-5-11 (201.18 mg, 296.67 μmol) was dissolved in N,N-dimethylformamide (4 mL), and then diethylamine (108.49 mg, 1.48 mmol) was added. After the addition was completed, the mixture was reacted at room temperature for 0.5 hours, and the reaction was detected by high performance liquid chromatography-mass spectrometry. After the ethylenediamine was evaporated under reduced pressure, the pH of the reaction solution was adjusted to 2-3 with 1 mol / L hydrochloric acid aqueous solution, and the reaction solution was directly purified by preparative high performance liquid chromatography to obtain the title compounds 1-5-A (44.00 mg) and 1-5-B (43.00 mg).

[0464] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0465] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0466] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 10 90 28 3 10 90 28 18 70 30 28

[0467] 1-5-A (LCMS peak at 6 min, retention time: 1.276 min)

[0468] The structural characterization data are as follows:

[0469] 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).

[0470] ESI-MS (m / z): 456.1 [M+H] + .

[0471] The structural characterization data of 1-5-B (6min LCMS peak later, retention time: 1.300min) are as follows:

[0472] 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).

[0473] ESI-MS (m / z): 456.1 [M+H] + .

[0474] 6min LCMS conditions:

[0475] Chromatographic column: Waters SunFire C18 OBD 4.6mm×50mm×5.0μm

[0476] Mobile phase A: 0.05% acetonitrile; Mobile phase B: water (0.05% formic acid)

[0477] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 90 10 2 4.2 10 90 2 5.7 10 90 2 5.71 90 10 2 6.70 90 10 2

[0478] 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-tetraazahexadecane-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-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexyl-5-amide (1-5-12-A and 1-5-12-B)

[0479] 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), and then 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were added. After addition, the mixture was reacted at room temperature for 1 hour, and the reaction was detected by HPLC-MS. The reaction solution was directly purified by HPLC to obtain the single-configuration title compound 1-5-12-A (51.00 mg).

[0480] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0481] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0482] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 30 70 28 3 30 70 28 18 90 10 28

[0483] The structural characterization data are as follows:

[0484] ESI-MS (m / z): 1111.0 [M+H] + .

[0485] 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), and then 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 μmol) and triethylamine (24.19 mg, 239.10 μmol) were added. After addition, the mixture was reacted at room temperature for 1 hour, and the reaction was detected by HPLC-MS. The reaction solution was directly purified by HPLC to obtain the single-configuration title compound 1-5-12-B (52.00 mg).

[0486] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0487] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0488] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 30 70 28 3 30 70 28 18 90 10 28

[0489] The structural characterization data are as follows:

[0490] ESI-MS (m / z): 1111.0 [M+H] + .

[0491] 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)

[0492] Weigh compound 1-5-12-A (40.00 mg, 35.99 μmol) and dissolve it in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL), then add 4 mol / L hydrochloric acid in ethyl acetate (1 mL), react at room temperature for 0.5 hours, and detect the reaction by HPLC-MS / MS. The reaction solution is directly concentrated under reduced pressure to obtain a crude product, which is purified by HPLC to obtain the title compound 1-11-A (4.75 mg) of a single configuration.

[0493] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0494] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0495] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 15 85 28 3 15 85 28 18 90 10 28

[0496] The structural characterization data are as follows:

[0497] 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).

[0498] ESI-MS (m / z): 514.0 [M+H] + .

[0499] Weigh compound 1-5-12-B (40.00 mg, 35.99 μmol) and dissolve it in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL), then add 4 mol / L hydrochloric acid ethyl acetate (1 mL), react at room temperature for 0.5 hours, and detect the reaction by HPLC-MS / MS chromatography. The reaction solution is directly concentrated under reduced pressure to obtain a crude product, which is purified by HPLC to obtain the title compound 1-11-B (8.24 mg) of a single configuration.

[0500] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0501] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0502] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 15 85 28 3 15 85 28 18 90 10 28

[0503] The structural characterization data are as follows:

[0504] 1H 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).

[0505] ESI-MS (m / z): 514.0 [M+H] + .

[0506] 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-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]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-dodecaoctatridecane-38-amide (A-17-A)

[0507]

[0508] Step 1: Synthesis of (2S)-2-(2,5,8,11,14,17,20,23,26,29,32,35-dodeca-38-octadecane-38-amido)-6-(N-(9H-fluoren-9-ylmethoxycarbonyl)amino)hexanoic acid (A-17-03)

[0509] The hydrochloride of compound A-17-02 (389.68 mg, 962.45 μmol) was dissolved in dichloromethane (8 mL), and DIPEA (518.28 mg, 4.01 mmol, 713.88 μL) and compound A-17-01 (550.00 mg, 802.04 μmol) were added, and the mixture was 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 drained under reduced pressure. The concentrate was purified by preparative high performance liquid chromatography to obtain the title compound A-17-03 (450.00 mg).

[0510] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0511] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0512] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 10 90 28 2 10 90 28 18 80 20 28

[0513] ESI-MS (m / z): 939.3 [M+H] + .

[0514] 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)

[0515] Compound A-17-03 (50.00 mg, 118.09 μmol) was dissolved in DMF (2.5 mL), 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, and the mixture was reacted at 25° C. for 1 hour. The solvent was removed under reduced pressure, and the concentrate was purified by preparative high performance liquid chromatography to obtain the title compound A-17-04 (40.00 mg).

[0516] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0517] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0518] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 10 90 28 2 10 90 28 18 80 20 28

[0519] ESI-MS (m / z): 1344.4 [M+H] + .

[0520] 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 (a-17-05) 1,6,9,12,15-pentaoxy-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)carbamoyl)-38-oxo-2,5,8,11,14,17,20,23,26,29,35-dodecano-39-aza-44-yl)carbamate

[0521] Compound A-17-04 (29.86 mg, 59.50 μmol) was dissolved in DMF (3 mL), 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, and the mixture was reacted at 25° C. for 1 hour. The solvent was removed under reduced pressure, and the concentrate was purified by preparative high performance liquid chromatography to obtain the title compound A-17-05 (50.00 mg).

[0522] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0523] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0524] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 10 90 28 2 10 90 28 18 80 20 28

[0525] ESI-MS (m / z): 1781.6 [M+H] + .

[0526] 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-dodecatrioxa-38-amide (A-17-06)

[0527] 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 removed 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).

[0528] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0529] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0530] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 15 85 28 2 15 85 28 18 80 20 28

[0531] ESI-MS (m / z): 1559.7 [M+H]+.

[0532] 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-ynylamide)-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-dodecanotrioctadecane-38-amide (A-17-A)

[0533] 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, and the mixture was reacted at 25° C. for 2 hours. The solvent was removed under reduced pressure, and the concentrate was purified by preparative high performance liquid chromatography to obtain the title compound A-17-A (5.60 mg).

[0534] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0535] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0536] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0 20 80 28 2 20 80 28 18 80 20 28

[0537] ESI-MS (m / z): 1809.8 [M+H] + .

[0538] 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]indolizine[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxyl-5,8,11,14-tetraazahexadecane-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexane-5-amide (A-05)

[0539]

[0540] 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), cooled to 5-15°C with ice water, added DMTMM (0.98 g, 3.53 mmol), then added dropwise DIPEA (1.14 g, 8.84 mmol), 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, which 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).

[0541] A-05 separation and purification method is as follows:

[0542] Chromatographic column: Waters SunFire Prep C18 OBD (5μm*19mm*150mm)

[0543] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0544] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 30 70 24 2.00 30 70 24 18.00 80 20 24

[0545] A-05 structural characterization data are as follows:

[0546] MS m / z(ESI):1107.3[M+H] +.

[0547] 1 H 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).

[0548] The following HPLC conditions were used to detect the compounds A-07-A and A-07-B prepared in Example 3; as well as the compound A-07-A prepared in Example 3 and the compound A-05 prepared in Example 9:

[0549] Instrument: Agilent 1260 HPLC VWD detector

[0550] Chromatographic column: Waters Xbridge C18 4.6*100mm*3.5μm

[0551] Mobile phase A: 0.01M ammonium phosphate aqueous solution / acetonitrile = 90 / 10; Mobile phase B: acetonitrile

[0552] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 6 22 78 11 22 78

[0553] As attached Figure 3A As shown, the retention times of compound A-07-A and compound A-07-B are 6.6 min and 6.8 min, respectively, indicating that the above HPLC conditions have good separation of isomers;

[0554] As attached Figure 3BAs shown, compound A-07-A prepared in Example 3 and compound A-05 prepared in Example 9 were shown as one peak in HPLC with a retention time of 6.6 min, indicating that compound A-07-A was compound A-05.

[0555] 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)

[0556]

[0557] 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)

[0558] 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]indolizine[1,2-b]quinoline-10,13-dione (2g, 3.65mmol) in DMF (50mL), add DIPEA (1.18g, 9.12mmol, 1.59mL), add acetoxyacetyl chloride (548.12mg, 4.01mmol, 431.59μL) under ice-cooling and stirring, and continue stirring and reacting for 1 hour. Add the reaction solution into 0.1M dilute hydrochloric acid aqueous solution to precipitate solid, and filter. 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 (methanol / dichloromethane = 0% to 5%) and concentrated again to obtain the title compound (1.7 g, 3.077 mmol)

[0559] Its structural characterization data are as follows:

[0560] ESI-MS (m / z): 552.2 [M+1] + .

[0561] 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)

[0562] 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-oxoacetic acid ethyl ester (500 mg,

[0563] 0.905mmol) and DMAP (885.33mg, 7.25mmol) were dissolved in dry dichloromethane (5mL), cooled to 0℃ under nitrogen protection, and triphosgene (268.81mg, 0.905mmol) in dichloromethane solution (5mL) was added dropwise, and the mixture was stirred and reacted for 0.5 hours. (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonyloxy-6-azatrinitroamino)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanamide (1.44g, 1.36mmol) in dichloromethane solution was slowly added dropwise, and the mixture was naturally allowed to react at room temperature for 4 hours. Water was added to quench the reaction, and the organic phases were combined after extraction with dichloromethane for 3 times (100ml x 3), washed with saturated brine, and dried and concentrated. Purification on a silica gel column (MeOH / DCM=0% to 5%) gave the title compound (498 mg, 0.304 mmol)

[0564] Its structural characterization data are as follows:

[0565] ESI-MS (m / z): 1352.8 [M+1] + .

[0566] Step 3: Preparation of 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 (B-04-3)

[0567] 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 0,13,15-Hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indazolidin[1,2-b]quinolin-1-yl)amino)-2-oxoacetic acid ethyl ester (200mg, 0.122mmol) was dissolved in THF (3mL) and MeOH (3mL), and sodium carbonate (25.88mg, 0.224mmol) aqueous solution (1mL) was added dropwise under stirring. After the addition was completed, stirring was continued for 1 hour. Dilute hydrochloric acid was added dropwise to the reaction solution to neutralize the reaction, and the next step was directly carried out after reduced pressure concentration.

[0568] Its structural characterization data are as follows:

[0569] ESI-MS (m / z): 1596.7 [M+1] + .

[0570] 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)

[0571] 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 then separated, and the organic phase was concentrated to obtain a crude product. The crude product 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).

[0572] Its structural characterization data are as follows:

[0573] ESI-MS (m / z): 1323.6 [M+1] + .

[0574] 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)

[0575] 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), and cuprous bromide (9.42 mg, 0.066 mmol) was added and stirred for 2 hours. The reaction solution was directly filtered, and the concentrated crude product was purified by preparative high performance liquid chromatography and freeze-dried to obtain the title compound (42.2 mg, 24.69 μmol).

[0576] Its structural characterization data are as follows:

[0577] ESI-MS (m / z): 1628.7 [M+1] + .

[0578] The preparation high performance liquid chromatography method is as follows:

[0579] Chromatographic column: SunFire Prep C18 OBD 19mm×150mm×5.0μm

[0580] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[0581] Time [min] Mobile phase A[%] Mobile phase B[%] Flow rate [mL / min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[0582] 3. Conjugation of compounds containing cell bioactive molecules and linkers to antibodies

[0583] The sources of antibodies involved in the antibody-drug conjugates prepared in the following examples are as follows: The preparation of 22B6 antibody refers to the description of Examples 2 and 4 of International Patent Application WO2023165475A1, the heavy chain variable region sequence refers to SEQ ID NO: 1 in the application; the heavy chain constant region sequence refers to SEQ ID NO: 63 in the application; the light chain variable region sequence refers to SEQ ID NO: 2 in the application; the light chain constant region sequence refers to SEQ ID NO: 65 in the application;

[0584] The preparation of 47A3 antibody refers to the description of Examples 2 and 4 of International Patent Application WO2023165475A1, the heavy chain variable region sequence refers to SEQ ID NO: 5 in the application; the heavy chain constant region sequence refers to SEQ ID NO: 63 in the application; the light chain variable region sequence refers to SEQ ID NO: 6 in the application; the light chain constant region sequence refers to SEQ ID NO: 65 in the application;

[0585] The preparation of 100H7 antibody refers to the description of Examples 2 and 4 of International Patent Application WO2023165475A1, the heavy chain variable region sequence refers to SEQ ID NO: 7 in the application; the heavy chain constant region sequence refers to SEQ ID NO: 63 in the application; the light chain variable region sequence refers to SEQ ID NO: 8 in the application; the light chain constant region sequence refers to SEQ ID NO: 65 in the application;

[0586] 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.

[0587] The sample coupling preparation is as follows:

[0588] 0.46 ml of 22B6, 47A3, 100H7, and U1-59 antibodies (all adjusted to 11.0 mg / mL) were taken respectively, diluted with 0.1 M edetate disodium solution (pH 7.7), and then adjusted to pH 7.7 with 1 M Na2HPO4 solution, and 10 mM TCEP (tris (2-carboxyethyl) phosphine) solution was added and mixed, and allowed to stand at room temperature for 90 min. 4.0-10 times the amount of the drug linker compound of the present invention dissolved in dimethyl sulfoxide was added to the above solution system, mixed, and allowed to stand at room temperature for 2 h. After completion, the buffer was replaced with a 10 mM histidine buffer solution at pH 6.0 by a NAP-5 gel column (Cytiva), and then sucrose and Tween 20 were added and mixed to obtain an antibody drug conjugate (i.e., ADC), as shown in Table 2.

[0589] The ADC drug adoption / antibody ratio (DAR value) is determined as follows:

[0590] LC-MS determination of ADC sample molecular weight and calculation of drug / antibody ratio DAR value

[0591] ADC samples were subjected to LC-MS molecular weight analysis.

[0592] Chromatographic conditions:

[0593] Liquid chromatography column: Thermo MAbPac RP 3.0*100mm;

[0594] Mobile phase A: 0.1% FA / H2O; Mobile phase B: 0.1% FA / ACN;

[0595] Flow rate: 0.25 ml / min; Sample chamber temperature: 8 °C; Column temperature: 60 °C; Injection volume: 2 μl;

[0596] Time (minutes) 2 20 22 25 26 30 Mobile phase A (volume %) 75 60 5 5 75 75 Mobile phase B (volume %) 25 40 95 95 25 25

[0597] Mass spectrometry conditions:

[0598] Mass spectrometer model: AB Sciex Triple TOF 5600+;

[0599] GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 250; CE 10; Accumulation time 0.5s;

[0600] m / z 600-4000; Time bins to sum 40.

[0601] Table 2 ADC number and DAR

[0602]

[0603] 1. LC-MS determination of the molecular weight of ADC 22B6-A-07-A-1 and calculation of the drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 22B6-A-07-A-1 is shown in Table 3.

[0604]

[0605] Table 3: Measured molecular weight of ADC 22B6-A-07-A-1

[0606]

[0607] Table 4: DAR values ​​for ADC 22B6-A-07-A-1

[0608]

[0609] The calculated drug / antibody ratio of ADC 22B6-A-07-A-1 was DAR=7.99 (see Table 4).

[0610] 2. LC-MS determination of the molecular weight of ADC 22B6-A-07-A-2 and calculation of the drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 22B6-A-07-A-2 is shown in Table 5.

[0611]

[0612] Table 5: Measured molecular weight of ADC 22B6-A-07-A-1

[0613]

[0614] Table 6: DAR values ​​for ADC 22B6-A-07-A-2

[0615]

[0616] The drug / antibody ratio of ADC 22B6-A-07-A-2 was calculated to be DAR=3.56 (see Table 6).

[0617] 3. LC-MS determination of ADC 47A3-A-07-A molecular weight and calculation of drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 47A3-A-07-A is shown in Table 7.

[0618]

[0619] Table 7: ADC 47A3-A-07-A measured molecular weight

[0620]

[0621] Table 8: DAR values ​​for ADC 47A3-A-07-A

[0622]

[0623] The drug / antibody ratio of ADC 47A3-A-07-A was calculated to be DAR=6.27 (see Table 8).

[0624] 4. LC-MS determination of the molecular weight of ADC 100H7-A-07-A and calculation of the drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 100H7-A-07-A is shown in Table 9.

[0625]

[0626] Table 9: ADC 100H7-A-07-A measured molecular weight

[0627]

[0628] Table 10: DAR values ​​for ADC 100H7-A-07-A

[0629]

[0630] The calculated drug / antibody ratio of ADC 100H7-A-07-A was DAR=8.01 (see Table 10).

[0631] 5. LC-MS determination of ADC U1-59-A-07-A molecular weight and calculation of drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC U1-59-A-07-A is shown in Table 11.

[0632]

[0633] Table 11: ADC U1-59-A-07-A measured molecular weight

[0634]

[0635]

[0636] Table 12: DAR values ​​for ADC U1-59-A-07-A

[0637]

[0638] The calculated drug / antibody ratio of ADC U1-59-A-07-A was DAR=8.02 (see Table 12).

[0639] 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 11.

[0640]

[0641] Table 13: Measured molecular weight of ADC 22B6-A-01

[0642]

[0643] Table 14: DAR values ​​for ADC 22B6-A-01

[0644]

[0645]

[0646] The drug / antibody ratio of ADC 22B6-A-01 was calculated to be DAR=7.93 (see Table 12).

[0647] 6. LC-MS determination of ADC U1-59-A-01 molecular weight and calculation of drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC U1-59-A-01 is shown in Table 13.

[0648]

[0649] Table 15: ADC U1-59-A-01 measured molecular weight

[0650]

[0651] Table 16: DAR values ​​for ADC U1-59-A-01

[0652]

[0653] The calculated drug / antibody ratio of ADC U1-59-A-01 is DAR=7.89 (see Table 14).

[0654] 7. LC-MS determination of the molecular weight of ADC 22B6-B-03-1 and calculation of the drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 22B6-B-03-1 is shown in Table 15.

[0655]

[0656] Table 17: Measured molecular weight of ADC 22B6-B-03-1

[0657]

[0658] Table 18: DAR values ​​for ADC 22B6-B-03-1

[0659]

[0660] The drug / antibody ratio of ADC 22B6-B-03-1 was calculated to be DAR=8.0 (see Table 16).

[0661] 8. LC-MS determination of the molecular weight of ADC 22B6-B-03-2 and calculation of the drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 22B6-B-03-2 is shown in Table 17.

[0662]

[0663] Table 19: Measured molecular weight of ADC 22B6-B-03-2

[0664]

[0665]

[0666] Table 20: DAR values ​​for ADC 22B6-B-03-2

[0667]

[0668] The drug / antibody ratio of ADC 22B6-B-03-2 was calculated to be DAR=3.14, see Table 18.

[0669] 9. LC-MS determination of the molecular weight of ADC 22B6-B-01 and calculation of the drug / antibody ratio DAR value. The LC-MS molecular weight analysis of ADC 22B6-B-01 is shown in Table 19.

[0670]

[0671] Table 20: ADC 22B6-B-01 measured molecular weight

[0672]

[0673] Table 21: DAR values ​​for ADC 22B6-B-01

[0674]

[0675]

[0676] The calculated drug / antibody ratio of ADC 22B6-B-01 was DAR=8.07 (see Table 20).

[0677] 10. Preparation Example of ADC 22B6-B-04-1

[0678] Take 0.854ml 22B6 antibody (23.43mg / mL), dilute with 42.68uL 20mM PB+0.1M EDTA (pH 7.60), then adjust the pH to 7.57 with 1M Na2HPO4 solution, add 20mM TCEP (tri(2-carboxyethyl)phosphine, 73.68uL, pH7.60) solution and mix well, and leave at room temperature for 1.5 hours. Then slowly add 12 times the amount of substance dissolved in dimethyl sulfoxide B-04 (164uL, 10mM) solution and mix well, let stand at room temperature overnight, and after completion, use NAP-5 gel column (Cytiva) to replace the buffer with 20mM histidine buffer solution at pH 6.0 to obtain ADC 22B6-B-04-1. The DAR value determined by mass spectrometry is 8.62.

[0679] 11. Preparation Example of ADC 22B6-B-04-2

[0680] Take 2.667ml 22B6 antibody (18.75mg / mL), dilute with 133.35uL 20mM PB+0.1M EDTA (pH 7.60), then adjust the pH to 7.63 with 1M Na2HPO4 solution, add 10mM TCEP (tri(2-carboxyethyl)phosphine, 184.19uL, pH7.60) solution and mix well, and let stand at room temperature for 1.5h. Then slowly add 10 times the amount of substance dissolved in dimethyl sulfoxide B-04 (341.73uL, 10mM) solution and mix well, let stand at room temperature for 2h, and after completion, use NAP-5 gel column (Cytiva) to replace the buffer with 20mM histidine buffer solution of pH 6.0 to obtain ADC 22B6-B-04-2, and the DAR value determined by mass spectrometry is 6.87.

[0681] IV. Detection of Antibody Drug Conjugate Activity

[0682] 1. In vitro cytotoxicity of anti-human HER3 antibody-drug conjugates

[0683] MDA-MB-453, HCC1569, and HEK293T-h HER3 cells were digested with TrypLE (manufacturer Gibco) solution, and an appropriate amount of cells were counted and taken. After diluting with growth medium, 5000 cells or 3000 cells (100 μl) / well were plated on a 96-well plate and cultured at 37°C and 5% CO2 overnight; the next day, ADC was diluted with growth medium, starting at 150 μg / ml, and diluted 2.5 times, and then 100 μl of the diluted ADC was added to the 96-well plate containing cells, and the plate was placed in an incubator. After culturing at 37°C and 5% CO2 for 6 days, 20 μl of CCK8 was added to each well, incubated at 37°C for 2-5 hours, and the OD450nm absorbance value was read by an enzyme reader; the raw data was imported into GraphPrism 6 to calculate IC 50 value.

[0684] Table 22 Anti-human HER3 ADC in vitro cytotoxicity results

[0685]

[0686]

[0687] Note: “ / ” means not detected.

[0688] 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.

[0689] 2. Bystander effect of anti-human HER3 antibody-drug conjugates

[0690] HEK293T and HEK293T-h HER3 cells were digested with 0.25% Trypsin-EDTA (manufacturer Gibco), and appropriate amount of cells were counted and taken respectively. After washing twice with PBS, the cell density was adjusted to 1*10 6 cells / ml, 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 HEK293T-labled: HEK293T=4000:12000 cells / well, HEK293T-labled: HEK293T-hHER3=4000:12000 cells / well, HEK293T-h HER3-labled: HEK293T-h HER3=4000:12000 cells / well, and cultured overnight at 37°C, 5% CO2;

[0691] On the second day, ADC was diluted with growth medium to a concentration of 20 nM, 4 nM, and 0.8 nM, and then 500 μl of the diluted ADC was added to the corresponding 24-well plates containing cells, and the plates were placed in an incubator at 37°C and 5% CO2 for 4 days;

[0692] HEK293T and HEK293T-h HER3 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 min, the cells were detected by flow cytometry (Thermo, model Attune NxT). Figure 1A , 1B As shown in Figure 1C, the ADC formed by the drug linker compound of the present invention coupled with the antibody has basically no killing effect on the negative cells HEK293T, but has significant specific killing effect on HEK293T-HER3. Under the condition that negative cells HEK293T and positive cells HEK293T-HER3 coexist, the ADC has a significant killing effect on the negative cells, indicating that the ADC formed by the drug linker compound of the present invention coupled with the antibody has bystander activity.

[0693] 3. Anti-human HER3 antibody-drug conjugate plasma stability test

[0694] Accurately measure 3.59μl of 20.9mg / ml 22B6-A-07-A-1ADC sample into an EP tube, add 496μl of blank plasma from humans, cynomolgus monkeys and mice respectively, mix well to obtain human, cynomolgus monkey and mouse plasma samples containing 22B6-A-07-A-1ADC sample. Place the sample in a constant temperature incubator and incubate at 37°C. Take out 50μl of the sample at 1, 3, 6, 24, 48, 96 and 168h, add 200μl of 0.5% formic acid-acetonitrile to precipitate the protein, centrifuge at 4°C, 13,000rpm for 10min, take the supernatant, store at -80°C, and use LC-MS / MS to detect the concentration of cytotoxic drugs after all samples are collected. The results are as follows Figure 2 As shown, the cytotoxic drug shedding does not exceed 5%, indicating that the drug linker compound of the present invention is stable in the plasma of different species after being coupled to ADC.

[0695] 4. In vivo efficacy testing of anti-human HER3 antibody-drug conjugate H358 model

[0696] NCI-H358 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. NCI-H358 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and inoculated subcutaneously in female Balb / c-nu mice to establish a non-small cell lung cancer model. When the average tumor volume was about 130-150mm 3 At 14:00, 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 1 mg / kg group, 22B6-B-01 1 mg / kg group, 22B6-A-07-A-1 1 mg / kg group and 3 mg / kg group, 22B6-B-03-1 1 mg / kg group. All were injected through the tail vein, and the drugs were administered 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 mortality was observed and recorded every day. The specific results are shown in Table 23.

[0697] The tumor growth inhibition rate TGI (%) was calculated using the following formula to evaluate the tumor inhibition efficacy:

[0698] TGI (%) = [1-(VT end-VT beginning) / (VC end-VC beginning)]*100%

[0699] VTend: mean tumor volume at the end of the treatment group experiment

[0700] VTstart: mean tumor volume at the start of drug administration in the treatment group

[0701] VC end: mean tumor volume at the end of the negative control group experiment

[0702] VC Start: Mean tumor volume at the beginning of drug administration in the negative control group

[0703] It can be seen from the test results that the ADC of the present invention has a significant tumor growth inhibition effect on the NCI-H358 non-small cell lung cancer transplant model. Compared with the solvent control group, after 3 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, and the TGI of the 22B6-A-07-A-1 1mg / kg group and the 3mg / kg group was 81.22% and 111.53%, respectively, and the TGI of the 22B6-B-03-1 1mg / kg group was 121.45%. There was no animal death and significant animal weight loss in each treatment group on Day32, and no obvious drug toxicity was observed. During the treatment period, the mice tolerated the ADC well.

[0704] Table 23 Human non-small cell lung cancer cell NCI-H358 CDX model

[0705]

[0706] Note: TGI is tumor growth inhibition rate, T / C is relative tumor proliferation rate.

[0707] As shown in the above table, 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.

[0708] 5. In vivo efficacy testing of anti-human HER3 antibody-drug conjugates in the N87 model

[0709] NCI-N87 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. NCI-N87 cells in the exponential growth phase were collected, resuspended in PBS to a suitable concentration, and inoculated subcutaneously in female Balb / c-nu mice to establish a human gastric cancer model. When the average tumor volume was about 120 mm 3 At 14:00, the mice were randomly divided into groups according to the tumor size: vehicle control group, 22B6-A-07-A-1 3mg / kg group, 47A3-A-07-A 3mg / kg group, and 100H7-A-07-A 3mg / kg group. All were injected through the tail vein on Day 0, Day 4, and Day 7, 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 mortality was observed and recorded every day. The specific results are shown in Table 24.

[0710] The test results show that compared with the vehicle control group, after three doses, the data of Day32 showed that the tumor growth inhibition rate (TGI) of the 22B6-A-07-A-13mg / kg group, the 47A3-A-07-A3mg / kg group and the 100H7-A-07-A3mg / kg group was 66.59%, 47.24% and 36.04%, respectively. There was no animal death or significant animal weight loss in each treatment group on Day32, and no obvious drug toxicity was observed. During the treatment period, the mice tolerated the ADC well.

[0711] Table 24 Human gastric cancer cell NCI-N87 CDX model

[0712]

[0713] As shown in the above table, 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.

[0714] 6. In vivo efficacy testing of anti-human HER3 antibody-drug conjugates in the MDA-MB-453 model

[0715] 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 in the exponential growth phase were collected, resuspended to a suitable concentration by adding PBS and a final concentration of 50% matrix gel, and inoculated subcutaneously in female NCG immunodeficient mice to establish a human breast cancer transplant model. When the average tumor volume was about 150mm 3 At the same time, the mice were randomly divided into vehicle control group, 22B6-A-07-A-1 3 mg / kg group and 10 mg / kg group, U1-59-A-01 3 mg / kg group and 10 mg / kg group, hIgG1-B-03-2 3 mg / kg group, and 22B6-B-03-2 3 mg / kg group according to the tumor size. After grouping, the mice were injected into the tail vein and given a single dose on Day 0. After administration, the tumor volume and body weight of the mice were observed and measured regularly. The specific results are shown in Table 25.

[0716] The ADC of the present invention has a significant inhibitory effect on the tumor growth of NCG mice with subcutaneous transplantation of human breast cancer MDA-MB-453. Data on Day32 after a single administration showed that compared with the vehicle control group, the TGI of the 22B6-A-07-A-1 1mg / kg, 3mg / kg and 10mg / kg groups were 118.30% and 200.00%, respectively, among which the tumor of the mice in the 10mg / kg group completely disappeared from Day19 and no growth was seen on Day32; the TGI of the 22B6-B-03-2 3mg / kg group was 148.58%; the TGI of the U1-59-A-01 3mg / kg group and the 10mg / kg group were 76.11% and 180.65%, respectively. During the treatment period, the mice tolerated the ADC well.

[0717] Table 25 Human breast cancer cell MDA-MB-453CDX model

[0718]

[0719] As shown in the above table, 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.

[0720] 9. Anti-human HER3 antibody-drug conjugate endocytosis activity test

[0721] The endocytic activity of anti-human HER3 ADC in MDA-MB-453 and HCC1569 cells was detected using a flow cytometer (Beckman, model Cytoflex).

[0722] 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 4The 96-well plate was placed in a 37°C, CO2 constant temperature incubator for 24 h. The 96-well plate was removed, the culture medium was discarded, and 50 μl of fresh complete culture medium was added to each well; the bispecific antibody and the control antibody were gradiently diluted with complete culture medium, and the final concentrations were 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 to 12 μg / ml with complete culture medium (the final concentration of PHrodo was 3 μg / ml); the gradient diluted antibody to be tested was mixed evenly with the diluted PHrodo reagent in a ratio of 1:1 (30 μl:30 μl), and incubated at room temperature in the dark for 30 min; 50 μl of the mixture of the antibody to be tested and the PHrodo reagent was added to the 96-well plate, and cultured at 37°C and 5% CO2 for 24 h; the 96-well plate was removed, the culture medium was discarded, and sterile PBS was washed once, and 100 μl of Tryps was added to each well. Digest the cells with in-EDTA (0.25%) and then add 100 μl of complete medium to neutralize; disperse the cells in the wells by pipetting and then detect them on FACS. Data processing: export the average fluorescence signal value, then import it into GraphPad Prism 6 software to calculate EC 50 , the results are shown in Table 26.

[0723] Table 26 Anti-human HER3 antibody and ADC endocytosis activity assay results

[0724]

[0725] As shown in the above table, the ADC formed after the drug linker compound of the present invention is coupled with the antibody still has good endocytosis activity.

[0726] 10. In vitro cytotoxicity of anti-human HER3 antibody-drug conjugates

[0727] MDA-MB-453 cells were digested with TrypLE (manufacturer Gibco) solution, and an appropriate amount of cells were counted and taken. After diluting with growth medium, 5000 cells (100 μl / well) were plated on a 96-well plate 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 times. Then 100 μl of the diluted ADC was added to the 96-well plate containing cells, and the plate was placed in an incubator. After culturing at 37°C and 5% CO2 for 6 days, 20 μl of CCK8 was added to each well, incubated at 37°C for 2-5 hours, and the OD450nm absorbance was read by a microplate reader. The raw data was imported into Graph Prism 6 to calculate the IC50 value.

[0728] Table 27 Anti-human HER3 ADC in vitro cytotoxicity results

[0729]

[0730] As shown in Table 27, the ADCs formed by the drug linker compounds of the present invention all exhibited significant cell killing activity.

[0731] 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 within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled substance, metabolite, or 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 Alkyne; L is the 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 aliphatic ring; the saturated heterocyclic aliphatic ring, the unsaturated heterocyclic aliphatic ring and the 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 Alkyne group.

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 Alkyne group.

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 The alkyl group is substituted; M1 is selected from a single bond and C 1-20 Alkylene, C 2-20 Alkenylene or C 2-20 Alkyne; 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 Alkyne; 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 Alkyne group.

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 19, 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 19, 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 19, 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 a 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 to a coupling reaction; B-04a: 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 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 coupling reaction: in, R1, R2, R3 are as described in any one of claims 1-12, 20; preferably: R1 is methyl, R2 is chlorine, and R3 is -C(=O)-CH2-OH.

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