Pyrrolobenzodiazepine-anthracene imide hybrids, methods of making and uses thereof

Abstract

The application discloses a pyrrolobenzodiazepine-anthracene imide hybrid molecule and a preparation method and application thereof, and belongs to the technical field of chemical medicines. In order to solve the problems of few kinds of warheads of an existing antibody drug conjugate, single action mechanism and drug resistance easily generated in long-term use, a pyrrolobenzodiazepine-anthracene imide hybrid molecule shown in formula I and a preparation method and application thereof are provided. Experimental results show that the compound has strong anti-proliferation activity in various tumor cells such as ovarian cancer cells, gastric cancer cells and breast cancer cells, and an antibody drug conjugate thereof has good in-vivo and in-vitro anti-tumor activity and good safety.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical medicine technology, and particularly relates to a pyrrolobenzodiazepine-anthracene imide hybrid molecule as an antitumor drug and an antibody drug conjugate thereof. BACKGROUND

[0002] Antibody-drug conjugates (ADCs) are a new type of tumor targeted therapy drug, which is composed of three parts of monoclonal antibody (Antibody), linker / connector (Linker) and warhead (Warhead / Payload). It combines the high selectivity of antibody and the high activity of cytotoxic drug, and has become a research hotspot of tumor targeted therapy. After decades of development, the research and development of ADCs has achieved many results. As of December 2021, 12 ADCs drugs have been approved for marketing, and more than 150 ADCs have entered clinical trials (see Esnault, C., Schrama, D., Houben, R., Guyétant, S., Desgranges, A., Martin, C., … & Samimi, M. (2022). Antibody-Drug Conjugates as an Emerging Therapy in Oncodermatology. Cancers, 14(3), 778.).

[0003] Warheads, as the key components of ADCs, directly affect the therapeutic effect of ADCs. The warheads of ADCs that have been successfully listed and entered clinical trials are mostly tubulin inhibitors and DNA damage agents, such as calicheamicins, maytansinoids, auristatins, PBD dimers and camptothecins, etc. Although these warheads have achieved good results when applied to ADCs, these warheads have single action mechanism, long-term use is easy to produce drug resistance, and the types of ADC warheads are few, most of the warhead structures are complex, and the synthesis is difficult, time-consuming and laborious. Therefore, it is of great significance to develop new ADC warheads. Using "molecular hybridization technology" to develop hybrid molecules that meet the requirements of warheads is a good strategy to broaden the types of warheads. Hybrid molecules are single-molecule new chemical entities formed by combining two or more active molecules. Compared with traditional warheads, their parent structures are relatively simple, the synthesis difficulty is lower, and it is also easier to design multi-target drugs (see Decker, M. (Ed.). (2017). Design of hybrid molecules for drug development. Elsevier.).

[0004] Pyrrolobenzodiazepines (PBDs) are a class of potent DNA minor groove binders (see Mantaj, J., Jackson, P. J., Rahman, K. M., & Thurston, D. E. (2017). From anthramycin to pyrrolobenzodiazepine (PBD)-containing antibody-drug conjugates (ADCs). Angewandte Chemie International Edition, 56(2), 462-488.). PBD dimers such as SGD-1882, SG3199 and Indolinobenzodiazepine have been used as warheads for ADCs due to their high cytotoxicity, among which ADC drug ADCT-402 with SG3199 as warhead has been launched, which is indicated for relapsed or refractory diffuse large B-cell lymphoma (see Mullard, A (2021). FDA approves ADC Therapeutics' loncastuximab tesirine, ushering in a new cytotoxic payload. Nature reviews Drug discovery, 20(6):414.).In addition to this, PBD monomers and other anticancer small molecules “hybridized” to give hybrid molecules such as PBD-CBI and PBD-BIA are also starting to be used as payloads for ADCs, suggesting that there is good promise in using PBD hybrid molecules as payloads for ADCs (see Giddens, A. C., Lee, H. H., Lu, G. L., Miller, C. K., Guo, J., Phillips, G. D. L.,... & Tercel, M. (2016). Analogues of DNA minor groove cross-linking agents incorporating amino CBI, an amino derivative of the duocarmycins: Synthesis, cytotoxicity, and potential as payloads for antibody-drug conjugates. Bioorganic & Medicinal Chemistry, 24(22), 6075-6081. and Reid, E. E., Archer, K. E., Shizuka, M., McShea, M. A., Maloney, E. K., Ab, O.,... & Miller, M. L. (2019). Design, synthesis and evaluation of novel, potent DNA alkylating agents and their antibody-drug conjugates (ADCs). Bioorganic & Medicinal Chemistry Letters, 29(17), 2455-2458.).

[0005] Naphthalimides are a class of polycyclic amides that are DNA intercalators, which can intercalate into the bases of DNA through non-covalent interaction, thus killing tumor cells (see Brana, M. F., & Ramos, A. (2001). Naphthalimides as anticancer agents: synthesis and biological activity. Current Medicinal Chemistry-Anti-Cancer Agents, 1(3), 237-255.). Mitonafide, Amonafide, Elinafide and Bisnafide have entered the clinical stage (I / II phase), but the compounds have myelosuppressive toxicity, which limits their clinical application (see Kamal, A., Bolla, N. R., Srikanth, P. S., & Srivastava, A. K. (2013). Naphthalimide derivatives with therapeutic characteristics: A patent review. Expert opinion on therapeutic patents, 23(3), 299-317.). After structural optimization, a more active derivative anthracene carbonamide, Azonafide, was developed. VelosBio company uses it as a warhead to develop ADC drugs targeting receptor tyrosine kinase orphan receptor 1 (ROR1), which is currently in the preclinical research stage (see Sami, S. M., Dorr, R. T., Alberts, D. S., & Remers, W. A. (1993). 2-Substituted 1,2-dihydro-3H-dibenz[de,h]isoquinoline-1,3-diones. A new class of antitumor agent. Journal of medicinal chemistry, 36(6), 765-770. and US20180369406).

[0006] SUMMARY

[0007] The present application aims to provide a preparation method and use of pyrrolobenzodiazepine-anthracene carbonamide hybrid molecules, to solve the problems of few types of existing ADC warheads, single mechanism of action, and easy drug resistance after long-term use.

[0008] The present application provides a compound shown in formula I or a tautomer, mesomer, racemate, enantiomer, diastereomer thereof or a pharmaceutically acceptable salt thereof, the structure of formula I is as follows:

[0009]

[0010] Z is selected from l, m, n are independently selected from integers from 1 to 8; l end is connected with N, and m end is connected with O;

[0011] R1 is selected from C1-C8 alkyl, substituted or unsubstituted 6-10 membered aryl; wherein the substituent of the substituted 6-10 membered aryl is selected from C1-C6 alkyl, C1-C6 alkoxy or amino;

[0012] R2 is selected from -H, C1-C8 alkoxy or C1-C8 alkylthiol;

[0013] R3 is selected from C1-C6 alkyl.

[0014] In the above compound, l, m, n are independently selected from integers from 1 to 6. Preferably, in the above compound, l is selected from integers from 1 to 3, m is selected from integers from 2 to 6, and n is selected from integers from 2 to 6. Most preferably, in the above compound, l is 2, m is selected from integers from 3 to 5, and n is selected from integers from 3 to 5.

[0015] In the above compound, R1 is selected from C1-C6 alkyl, substituted or unsubstituted 6-10 membered aryl; wherein the substituent of the substituted 6-10 membered aryl is selected from C1-C4 alkyl, C1-C4 alkoxy or amino.

[0016] Preferably, in the above compound, R1 is selected from C1-C4 alkyl, substituted or unsubstituted 6-membered aryl; wherein the substituent of the substituted 6-membered aryl is selected from C1-C4 alkyl, C1-C4 alkoxy or amino.

[0017] Most preferably, in the above compound, R1 is selected from methyl, p-aminophenyl or p-methoxyphenyl.

[0018] In the above compound, R2 is selected from -H, C1-C6 alkoxy or C1-C6 alkylthiol. Preferably, in the above compound, R2 is selected from -H, C1-C4 alkoxy or C1-C4 alkylthiol. More preferably, in the above compound, R2 is selected from -H, methoxy or methylthiol. Most preferably, in the above compound, R2 is -H.

[0019] In the above compound, R3 is selected from C1-C4 alkyl. Preferably, in the above compound, R3 is methyl.

[0020] The present application also provides some specific compounds of formula I, as follows:

[0021]

[0022]

[0023]

[0024] The present application also provides a class of drug-linker compounds of formula II or its tautomer, meso, racemate, enantiomer, diastereomer or its pharmaceutically acceptable salt, based on the compound of formula I, whose structure is as follows:

[0025]

[0026] wherein, L is selected from o, q are independently selected from an integer from 1 to 10, p is selected from an integer from 1 to 15; R2 is as described above in formula I; R3 is as described above in formula I; Z is as described above in formula I.

[0027] wherein, in the above compound, o, q are independently selected from an integer from 1 to 8, p is selected from an integer from 2 to 10. Preferably, in the above compound, o, q are independently selected from an integer from 1 to 7, p is selected from an integer from 6 to 8. Most preferably, in the above compound, o is 5, q is 2, p is 7.

[0028] wherein, in the above compound, is connected to the benzene para position.

[0029] wherein, in the above compound, Z is l, m are as described above in formula I.

[0030] The present application also provides some specific compounds of formula II, as follows:

[0031]

[0032]

[0033]

[0034] The present application also provides a preparation method of the above-mentioned compound of formula I or formula II, whose synthetic route comprises the following steps:

[0035] a, compound 1 is subjected to Suzuki coupling reaction to obtain compound 2:

[0036]

[0037] b. Compound 2 is reduced by Zn powder / AcOH system to obtain compound 3:

[0038]

[0039] c. Compound 3 is reacted with Alloc-Cl (allyl chloroformate) under basic condition to obtain compound 4:

[0040]

[0041] wherein R1of compound 4 is R1when R1does not contain amino group, and is mono-N-Alloc substituted R1when R1contains amino group;

[0042] d. Compound 4 is deprotected from TBS (tert-butyldimethylsilyl) under acidic condition to obtain compound 5:

[0043]

[0044] e. Compound 5 is subjected to Swern oxidation and ring closure to obtain compound 6:

[0045]

[0046] f. Compound 6 is reacted with TBS-OTf under basic condition to obtain compound 7:

[0047]

[0048] g. Compound 7 is reacted with LiOAc to remove TIPS (triisopropylsilyl) to obtain compound 8:

[0049]

[0050] h. Compound 8 is reacted with diiodoalkane under basic condition to obtain compound 9:

[0051]

[0052] i. Compound 10 is reacted with bromoalkane under basic condition to obtain compound 11:

[0053]

[0054] j. Compound 11 is reacted with compound 12 under basic condition to obtain compound 13:

[0055]

[0056] k. Compound 13 is deprotected from Boc under the action of TFA to obtain compound 14:

[0057]

[0058] I. reacting compound 9 and compound 12 under basic conditions to obtain compound 15:

[0059]

[0060] m. removing TBS from compound 15 under TBAF / AcOH system to obtain compound 16:

[0061]

[0062] n. removing Alloc protecting group from compound 16 under Pd catalyst to obtain product 17:

[0063]

[0064] o. reacting compound 9 and compound 14 under basic conditions to obtain compound 18:

[0065]

[0066] p. removing TBS from compound 18 under TBAF / AcOH system to obtain compound 19:

[0067]

[0068] q. removing Alloc protecting group from compound 19 under Pd catalyst to obtain product 20:

[0069]

[0070] r. when R1 in formula I is amino-substituted phenyl, condensing compound 17 or 20 with linker L-H via amide to obtain formula II:

[0071]

[0072] In the above method, in step a, compound 1, R1-boronic acid, base and Pd catalyst are reacted under a protective atmosphere.

[0073] In the above preparation method, in step a, the molar ratio of compound 1 to R1-boronic acid is 1:(1.0-5.0). Preferably, in step a, the molar ratio of compound 1 to R1-boronic acid is 1:4.0.

[0074] In the above preparation method, in step a, the molar ratio of compound 1 to base is 1:(1.0-8). Preferably, in step a, the molar ratio of compound 1 to base is 1:6.0.

[0075] In the above process, in step a, the molar ratio of compound 1 to palladium catalyst is 1 : 0.02 to 0.05. Preferably, in step a, the molar ratio of compound 1 to palladium catalyst is 1 : 0.05.

[0076] In the above process, in step a, the Pd catalyst is at least one selected from Pd(dppf)Cl2CH2Cl2, PdCl2(PPh3)2, Pd[P(Ph)3]4, Pd(OAc)2. In the above process, in step a, the reaction solvent is at least one selected from acetone, acetonitrile, tetrahydrofuran, toluene, DMF, 1,2-dimethoxyethane, water. In the above process, in step a, the base is at least one selected from potassium phosphate, potassium carbonate, sodium carbonate, sodium hydride, barium hydroxide, cesium carbonate. In the above process, in step a, the reaction temperature is 25°C. In the above process, in step a, the reaction is carried out under nitrogen protection.

[0077] In the above process, in step b, the reaction solvent is selected from methanol or ethanol.

[0078] In the above process, in step c, the base is pyridine.

[0079] In the above process, in step d, the acid is aqueous acetic acid, and the reaction solvent is selected from methanol, tetrahydrofuran, a mixture of methanol: tetrahydrofuran in a volume ratio of 1 : 1.

[0080] In the above process, in step e, compound 5, DMSO, oxalyl chloride and base are reacted under a protective atmosphere.

[0081] In the above process, in step e, the molar ratio of compound 5 to oxalyl chloride is 1 : (1.0-4.0). Preferably, in step e, the molar ratio of compound 5 to oxalyl chloride is 1 : 1.3.

[0082] In the above process, in step e, the molar ratio of compound 5 to DMSO is 1 : (1.0-8.0). Preferably, in step e, the molar ratio of compound 5 to DMSO is 1 : 2.6.

[0083] In the above process, in step e, the molar ratio of compound 5 to base is 1 : (1.0-8.0). Preferably, in step e, the molar ratio of compound 5 to base is 1 : 5.0.

[0084] In the above process, in step e, the base is one or both of diisopropylethylamine and triethylamine. In the above process, in step e, the reaction solvent is at least one selected from dichloromethane, chloroform, tetrahydrofuran. In the above process, in step e, the reaction temperature is -78°C. In the above process, in step e, the reaction is carried out under nitrogen protection.

[0085] In the above preparation method, in step f, the base is 2,6-lutidine.

[0086] In the above preparation method, in step g, the reaction solvent is a mixed solution of N,N-dimethylformamide and water.

[0087] In the above preparation method, in step h, compound 8, diiodoalkane and the base are reacted under a protective atmosphere.

[0088] In the above preparation method, in step h, the molar ratio of compound 8 to diiodoalkane is 1:(1.0-6.0). Preferably, in step h, the molar ratio of compound 8 to diiodoalkane is 1:5.0.

[0089] In the above preparation method, in step h, the molar ratio of compound 8 to the base is 1:(1.0-4.0). Preferably, in step h, the molar ratio of compound 8 to the base is 1:1.3.

[0090] In the above preparation method, in step h, the base is at least one selected from the group consisting of diisopropylethylamine, triethylamine, potassium phosphate, potassium carbonate, sodium carbonate and cesium carbonate. In the above preparation method, in step h, the reaction solvent is at least one selected from the group consisting of N,N-dimethylformamide, N,N-dimethylacetamide, 2-butanone and toluene. In the above preparation method, in step h, the reaction temperature is 75°C. In the above preparation method, in step h, the reaction is carried out under nitrogen protection.

[0091] In the above preparation method, in step i, compound 10, dibromoalkane and the base are reacted under a protective atmosphere.

[0092] In the above preparation method, in step i, the molar ratio of compound 10 to dibromoalkane is 1:(1.0-6.0). Preferably, in step i, the molar ratio of compound 10 to dibromoalkane is 1:4.3.

[0093] In the above preparation method, in step i, the molar ratio of compound 10 to the base is 1:(1.0-4.0). Preferably, in step i, the molar ratio of compound 10 to the base is 1:1.1.

[0094] In the above preparation method, in step i, the base is at least one selected from the group consisting of diisopropylethylamine, triethylamine, potassium phosphate, potassium carbonate, sodium carbonate and cesium carbonate. In the above preparation method, in step i, the reaction solvent is at least one selected from the group consisting of N,N-dimethylformamide, N,N-dimethylacetamide, 2-butanone, dichloromethane and toluene. In the above preparation method, in step i, the reaction temperature is 45°C. In the above preparation method, in step i, the reaction is carried out under nitrogen protection.

[0095] In the above preparation method, in step j, compound 11, compound 12 and the base are reacted under a protective atmosphere.

[0096] In step j of the above preparation method, the molar ratio of compound 11 to compound 12 is 1:(1.0-3.0). Preferably, in step j, the molar ratio of compound 11 to compound 12 is 1:1.0.

[0097] In step j of the above preparation method, the molar ratio of compound 11 to base is 1:(1.0-4.0). Preferably, in step j, the molar ratio of compound 11 to base is 1:1.5.

[0098] In step j of the above preparation method, the base is at least one selected from the group consisting of diisopropylethylamine, triethylamine, potassium phosphate, potassium carbonate, sodium carbonate, and cesium carbonate. In step j of the above preparation method, the reaction solvent is at least one selected from the group consisting of N,N-dimethylformamide, N,N-dimethylacetamide, 2-butanone, and toluene. In step j of the above preparation method, the reaction temperature is 85°C. In step j of the above preparation method, the reaction is carried out under nitrogen protection.

[0099] In step k of the above preparation method, the reaction solvent is at least one selected from the group consisting of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, and N,N-dimethylacetamide.

[0100] In step l of the above preparation method, compound 12, compound 9, and base are reacted under a protective atmosphere.

[0101] In step l of the above preparation method, the molar ratio of compound 12 to compound 9 is 1:(1.0-3.0). Preferably, in step l, the molar ratio of compound 12 to compound 9 is 1:1.1.

[0102] In step l of the above preparation method, the molar ratio of compound 12 to base is 1:(1.0-4.0). Preferably, in step l, the molar ratio of compound 12 to base is 1:1.5.

[0103] In step l of the above preparation method, the base is at least one selected from the group consisting of diisopropylethylamine, triethylamine, potassium phosphate, potassium carbonate, sodium carbonate, and cesium carbonate. In step l of the above preparation method, the reaction solvent is at least one selected from the group consisting of N,N-dimethylformamide, N,N-dimethylacetamide, 2-butanone, and toluene. In step l of the above preparation method, the reaction temperature is 85°C. In step l of the above preparation method, the reaction is carried out under nitrogen protection.

[0104] In step m of the above preparation method, the reaction solvent is at least one selected from the group consisting of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, and N,N-dimethylacetamide.

[0105] In step n of the above method, compound 16, tetrahydropyrrole, and Pd catalyst are reacted under a protective atmosphere.

[0106] In step n of the above preparation method, the molar ratio of compound 16 to tetrahydro pyrrole is 1 : (1.0-4.0). Preferably, in step n, the molar ratio of compound 16 to tetrahydro pyrrole is 1 : 3.5.

[0107] In step n of the above preparation method, the molar ratio of compound 16 to Pd catalyst is 1 : 0.02-0.10. Preferably, in step n, the molar ratio of compound 16 to Pd catalyst is 1 : 0.06.

[0108] In step n of the above preparation method, the Pd catalyst is selected from at least one of Pd(dppf)Cl2CH2Cl2, PdCl2(PPh3)2, Pd[P(Ph)3]4, Pd(OAc)2. In step n of the above preparation method, the reaction solvent is selected from at least one of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide. In step n of the above preparation method, the reaction temperature is 25°C. In step n of the above preparation method, the reaction is carried out under nitrogen protection.

[0109] In step o of the above preparation method, compound 14, compound 9 and base are reacted under a protective atmosphere.

[0110] In step o of the above preparation method, the molar ratio of compound 14 to compound 9 is 1 : (1.0-3.0). Preferably, in step o, the molar ratio of compound 14 to compound 9 is 1 : 1.1.

[0111] In step o of the above preparation method, the molar ratio of compound 14 to base is 1 : (1.0-4.0). Preferably, in step o, the molar ratio of compound 14 to base is 1 : 1.5.

[0112] In step n of the above preparation method, the base is selected from at least one of diisopropylethylamine, triethylamine, potassium phosphate, potassium carbonate, sodium carbonate, cesium carbonate. In step o of the above preparation method, the reaction solvent is selected from at least one of N,N-dimethylformamide, N,N-dimethylacetamide, 2-butanone, toluene. In step o of the above preparation method, the reaction temperature is 85°C. In step o of the above preparation method, the reaction is carried out under nitrogen protection.

[0113] In step p of the above preparation method, the reaction solvent is selected from at least one of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide.

[0114] In step q of the above method, compound 19, tetrahydro pyrrole and Pd catalyst are reacted under a protective atmosphere.

[0115] In the step q of the above preparation method, the molar ratio of compound 19 to tetrahydro-pyrrole is 1:(1.0-4.0). Preferably, in the step q, the molar ratio of compound 19 to tetrahydro-pyrrole is 1:3.5.

[0116] In the step q of the above preparation method, the molar ratio of compound 19 to Pd catalyst is 1:0.02-0.10. Preferably, in the step q, the molar ratio of compound 19 to Pd catalyst is 1:0.06.

[0117] In the step q of the above preparation method, the Pd catalyst is at least one selected from Pd(dppf)Cl2·CH2Cl2, PdCl2(PPh3)2, Pd[P(Ph)3]4, Pd(OAc)2. In the step q of the above preparation method, the reaction solvent is at least one selected from dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide. In the step q of the above preparation method, the reaction temperature is 25℃. In the step q of the above preparation method, the reaction is carried out under nitrogen protection.

[0118] In the step r of the above preparation method, compound 17 or 20, linker L-H and condensing agent are reacted under a protective atmosphere.

[0119] In the step r of the above preparation method, the molar ratio of compound 17 or 20 to linker L-H is 1:(1.0-5.0). Preferably, in the step r, the molar ratio of compound 17 or 20 to linker L-H is 1:3.0.

[0120] In the step r of the above preparation method, the molar ratio of compound 17 or 20 to condensing agent is 1:(1.0-5.0). Preferably, in the step r, the molar ratio of compound 17 or 20 to condensing agent is 1:3.0.

[0121] In the step r of the above preparation method, the reaction solvent is selected from dichloromethane, chloroform, methanol, tetrahydrofuran, N,N-dimethylformamide, a mixture of dichloromethane:methanol in a volume ratio of 20:1 or a mixture of chloroform:methanol in a volume ratio of 20:1. In the step r of the above preparation method, the reaction temperature is 25℃. In the step r of the above preparation method, the reaction is carried out under nitrogen protection.

[0122] The present application also provides a kind of pharmaceutical composition, it is with the compound described in formula I and / or formula II or its tautomer, meso body, racemate, enantiomer, diastereoisomer or its pharmaceutically acceptable salt as active ingredient, preparation by adding pharmaceutically acceptable adjuvant.

[0123] The present application also provides the use of the compound of formula I and / or formula II or a tautomer, mesomer, racemate, enantiomer, diastereomer thereof or a pharmaceutically acceptable salt thereof, the pharmaceutical composition in the preparation of an antitumor drug. Through cell experiments, the compound of the present application exhibits strong anti-proliferative activity in various tumors such as gastric cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer or urothelial carcinoma.

[0124] The present application also provides the use of the compound of formula I and / or formula II or a tautomer, mesomer, racemate, enantiomer, diastereomer thereof or a pharmaceutically acceptable salt thereof, the pharmaceutical composition in the preparation of an antibody drug conjugate.

[0125] Definitions of terms:

[0126] The compounds and derivatives provided in the present application can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstract Service, Columbus, OH) nomenclature system.

[0127] The term "alkyl" is a straight-chain or branched saturated hydrocarbon radical. Examples of C1-C6 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), i-propyl (C3), n-butyl (C4), t-butyl (C4), sec-butyl (C4), i-butyl (C4), n-pentyl (C5), 3-pentyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butyl (C5), t-amyl (C5), and n-hexyl (C6). Unless otherwise indicated, each instance of an alkyl group is independently optionally substituted, i.e., unsubstituted or substituted with one or more substituents. "Substitution" refers to the replacement of a hydrogen atom in a molecule by an atom or molecule of different identity. In some embodiments, the C1-C6 alkyl group is a C1-C6 alkyl group substituted with a halogen (fluorine, chlorine, bromine, iodine). In the case of a C1-C6 alkyl group substituted with a substituent, the number of carbon atoms of the substituent is not counted.

[0128] The term "aryl" refers to a 4n+2 aromatic ring system containing or not containing heteroatoms in the aromatic ring system, wherein the heteroatoms are independently selected from nitrogen, oxygen, and sulfur. Unless otherwise indicated, each instance of an aryl group is independently optionally substituted, i.e., unsubstituted or substituted with one or more substituents.

[0129] The term "alkoxy" refers to the group -OR, wherein R is substituted or unsubstituted alkyl. Examples of C1-C6alkoxy groups include methoxy, ethoxy, n-propyloxy, isopropoxy, n-butyloxy, t-butyloxy, sec-butyloxy, n-pentyloxy, n-hexyloxy, and 1,2-dimethylbutyloxy. In some embodiments, R is alkyl substituted with halo (fluoro, chloro, bromo, iodo). The number of carbon atoms from the C1-C6alkoxy group is not counted when R is substituted with a substituent.

[0130] The term "alkylthio" refers to the group R-S-, wherein R is alkyl as defined above.

[0131] The term "pharmaceutically acceptable" means that which is generally compatible with the other ingredients of a pharmaceutical dosage form and / or the recipient of the form, chemically or toxicologically, and that which is generally compatible with the physiological mechanisms of the recipient.

[0132] The term "pharmaceutically acceptable salt" means an acid or base salt of a compound that is physically and chemically stable, non-toxic, and otherwise biologically acceptable, for use in pharmaceutical applications. Such salts include acid addition salts and base salts. The salts of the present application can be formed by conventional means.

[0133] In certain embodiments of the present application, the present application includes isotopically-labeled compounds, which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. The isotopes of hydrogen, carbon, nitrogen, oxygen, and sulfur, i.e. 2 H, 3 H、 13 C、 14 C、 15 N、 17 O、 18 O、 35 S, can be found in the present application. Compounds of the present application that contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of the present application. Prodrugs

[0134] The mode of administration of the compounds or pharmaceutical compositions of the present application is not particularly limited, and representative modes of administration include, but are not limited to, parenteral (intravenous, intramuscular or subcutaneous), and topical administration. The compositions for parenteral injection can include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof. The dosage forms of the compounds of the present application for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants, if necessary.

[0135] The pharmaceutically acceptable adjuvant of the present application refers to a substance contained in the dosage form in addition to the active ingredient.

[0136] The pharmaceutically acceptable adjuvant of the present application has certain physiological activity, but the addition of the ingredient does not change the dominant position of the above-mentioned pharmaceutical composition in the treatment of diseases, and only plays an auxiliary role, which is only the use of the known activity of the ingredient and the auxiliary treatment method commonly used in the medical field. If the above-mentioned adjuvant is used with the pharmaceutical composition of the present application, it still belongs to the scope of protection of the present application.

[0137] The beneficial effects of the present application:

[0138] The present application provides a kind of novel structure pyrrolobenzodiazepine-anthracene imide hybrid molecule, it is by "molecular hybridization technology", with DNA minor groove binding agent pyrrolobenzodiazepine and DNA intercalator anthracene imide as parent, synthesis after reasonable design is obtained.In vitro anti-tumor cell proliferation experiments show that the compound of the present application in gastric cancer, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer or urothelial carcinoma and other various tumor cells all show very strong in vitro antiproliferative activity, especially compound 20b3 shows superior antitumor activity;And further experiments prove that it can form interstrand crosslinking by inducing DNA, cause DNA damage, eventually lead to cell cycle arrest and apoptosis.In addition, after the compound of the present application is made into antibody drug conjugate, it still has good in vitro and in vivo antitumor activity, and good safety.The present application provides a new type of antibody drug conjugate warhead for clinical use. BRIEF DESCRIPTION OF DRAWINGS

[0139] Figure 1Figure showing the results of compound 20b3 inducing cell cycle arrest and apoptosis in Example 6; wherein, A is the result of cell cycle arrest, the drug concentration is 0.33 nM, 1 nM and 3 nM, and the drug action time is 24 h; B is the result of inducing apoptosis, the drug concentration is 0.33 nM, 1 nM and 3 nM, and the drug action time is 48 h.

[0140] Figure 2 Figure showing the DNA cross-linking experimental scheme of linearized pBR322 plasmid in Example 7; wherein, A represents double-stranded DNA (dsDNA) that is not subjected to drug action and is denatured into two complementary single-stranded DNA (ssDNA) by heat; B represents that after drug action, part of the double-stranded DNA will be cross-linked between the strands, and these cross-linked DNA shows stronger anti-denaturation ability when heated, as double-stranded DNA migration.

[0141] Figure 3 Figure showing the results of compound 20b3 inducing DNA interstrand cross-linking in Example 7; wherein, A is a 1% neutral agarose gel electrophoresis diagram of double-stranded (DS) and single-stranded (SS) DNA, 0 (DS) is linear double-stranded DNA that is not denatured and not added with drugs, 0 (SS) is linear single-stranded DNA that is heat denatured and not added with drugs, and the rest of the samples are heat denatured after being treated with different concentrations of compound 20b3 and oxaliplatin (positive control) before electrophoresis; B is the cross-linking rate of double-stranded DNA after treatment with compound 20b3.

[0142] Figure 4 Figure showing the fluorescence spectrum of compound 20b3 in Example 8; wherein, A is a three-dimensional fluorescence contour plot of compound 20b3, and the darker the color, the stronger the fluorescence intensity; B is the emission spectrum of compound 20b3 at an excitation wavelength of 488 nm.

[0143] Figure 5 Figure showing the distribution results of compound 20b3 in tumor cells in Example 8.

[0144] Figure 6 Figure showing the results of compound 20b3 inducing DNA damage in tumor cells in Example 9.

[0145] Figure 7 Figure showing the structure of T-PBA in Example 11.

[0146] Figure 8 Figure showing the coupling of T-PBA by SDS-PAGE combined with ultraviolet fluorescence analysis in Example 11; wherein, the upper half is Coomassie blue staining, and the lower half is ultraviolet excitation fluorescence diagram.

[0147] Figure 9 Figure showing the results of flow cytometry detecting the targeting of T-PBA in Example 12.

[0148] Figure 10 The graph shows the results of flow cytometry detection of T-PBA internalization efficiency in Example 13.

[0149] Figure 11 This is a diagram showing the distribution of T-PBA in tumor cells in Example 14.

[0150] Figure 12 The diagram shows the results of cell cycle inhibition and apoptosis induction in Example 16; where A represents the cell cycle inhibition results, with drug concentrations of 1.6 nM, 8 nM, and 40 nM, and drug treatment for 24 h; B represents the apoptosis induction results, with drug concentrations of 1.6 nM, 8 nM, and 40 nM, and drug treatment for 48 h.

[0151] Figure 13 This is a diagram showing the results of T-PBA-induced DNA damage in tumor cells in Example 17.

[0152] Figure 14 The graph shows the in vivo antitumor activity results of T-PBA in Example 18; where A represents the antitumor effect of T-PBA in the SKOV3 xenograft model; B represents the antitumor effect of T-PBA in the NCI-N87 xenograft model; and the arrows indicate the time points for drug administration.

[0153] Figure 15 This is a diagram showing the H&E staining results of important organs in mice after T-PBA treatment in Example 18. Detailed Implementation

[0154] This invention is the first to synthesize and evaluate the pyrrolobenzodiazepine-anthraimid hybrid molecule shown in Formula I through pharmacodynamic experiments. In vitro experimental results showed that the hybrid molecules all exhibited good antitumor activity. Further investigation of the mechanism of action of the representative compound 20b3 using flow cytometry, agarose gel electrophoresis, laser confocal microscopy, and Western blotting revealed that the target of this type of hybrid molecule is DNA. It can activate the DNA damage response system by inducing interstrand crosslinking in DNA, leading to Chk2 phosphorylation, which in turn activates the Caspase signaling pathway, promoting PARP and Caspase-3 cleavage, inhibiting DNA damage repair, and ultimately resulting in cell cycle arrest and apoptosis. Subsequently, this type of hybrid molecule was used as a warhead, conjugated with a corresponding linker to prepare a drug-linker as shown in Formula II, and then conjugated with a corresponding antibody to prepare an antibody-drug conjugate. The bioactivity of the conjugate was evaluated, and the resulting antibody-drug conjugate showed good in vitro and in vivo antitumor activity against ovarian cancer and gastric cancer, with good safety profile.

[0155] Example 1: Synthesis of compounds 9a1-9b3

[0156]

[0157] Synthesis of compound 2a:

[0158] Into a 250 mL three-necked round-bottom flask, compound 1 (15.0 g, 21.06 mmol, prepared by the method disclosed in the literature“Tiberghien, A. C.,... & Howard, P. W. (2016). Design and synthesis of tesirine, a clinical antibody-drug conjugate pyrrolobenzodiazepine dimer payload. ACS medicinal chemistry letters, 7(11), 983-987.”) and toluene (90 mL) were added, followed by potassium phosphate (26.8 g, 126.36 mmol) and Pd(dppf)Cl2·CH2Cl2(86.0 mg, 1.05 mmol), and then the system was replaced with nitrogen three times, followed by the addition of methylboronic acid (5.0 g, 84.24 mmol), and then the system was replaced with nitrogen, and then the reaction was carried out at 65 °C for 0.5 h, and the reaction was monitored by TLC (developing agent: petroleum ether / ethyl acetate = 4 / 1, ultraviolet coloration). After cooling to room temperature, water (60 mL) was added for extraction, and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by column chromatography (mobile phase: PE / EA = 10 / 1) to obtain the product 2a (9.85 g, 65.7%) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 7.69 (s, 1H), 6.76 (s, 1H), 5.51 (d, J = 1.9 Hz, 1H), 4.70-4.60 (m, 1H), 3.89 (s, 3H), 2.83-2.72 (m, 1H), 2.54 (d, J = 16.4 Hz, 1H), 1.61 (d, J = 1.6 Hz, 3H), 1.32-1.24 (m, 3H), 1.11 (s, 9H), 1.09 (s, 9H), 0.90 (s, 9H), 0.10 (d, J = 2.4 Hz, 6H). 13 C NMR (101 MHz, CDCl3) δ 156.18, 145.88, 137.69, 127.13, 123.19, 123.06, 116.04, 110.29, 110.24, 59.00, 56.10, 56.02, 36.75, 25.85, 18.21, 17.81, 13.76, 12.79, -5.30. HRMS (ESI, m / z) calcd for C 29 H 51 N2O6Si2[M+H]+ 579.3286, found: 579.3282. [a] D (CHCl3) -64.36°, c = 0.26 g / 100 mL.

[0159] Synthesis of compound 2b:

[0160] Into a 1000 mL three-necked round-bottom flask, compound 1 (32.0 g, 44.93 mmol) was added, then a mixed solvent of toluene, ethanol and water (Toluene / EtOH / H2O = 256 / 128 / 128 mL) was added, followed by 4-aminobenzeneboronic acid pinacol ester (12.8 g, 58.41 mmol), sodium carbonate (21.9 g, 206.67 mmol) and Pd[P(Ph)3]4(1.04 g, 0.90 mmol) in sequence, replaced with nitrogen for three times, then reacted at 25 °C for 24 h, TLC monitored the reaction completion (developing agent: petroleum ether / ethyl acetate = 6 / 1, ultraviolet coloration). The reaction solution was concentrated under reduced pressure to remove excess toluene and ethanol, EA (500 mL) was added for extraction, the organic layer was collected, then the organic layer was washed with saturated sodium chloride (200 mL) again, the organic layer was collected, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, the crude product was purified by column chromatography (mobile phase: PE / EA = 9 / 1-7 / 1) to obtain yellow solid product 2b (13.0 g, 44.2%). 1 H NMR (400 MHz, CDCl3) δ 7.74 (s, 1H), 7.00 (d, J = 8.4 Hz, 2H), 6.81 (s, 1H), 6.58 (d, J = 8.5 Hz, 2H), 6.06 (s, 1H), 4.82-4.72 (m, 1H), 3.94-3.85 (m, 4H), 3.20-3.08 (m, 1H), 2.97 (d, J = 16.9 Hz, 1H), 1.33-1.27 (m, 3H), 1.13 (s, 9H), 1.11 (s, 9H), 0.89 (s, 9H), 0.11 (d, J = 2.9 Hz, 6H). 13 C NMR (101 MHz, CDCl3) δ 156.28, 146.06, 145.80, 137.82, 126.90, 125.90, 125.42, 124.21, 120.45, 116.27, 115.02, 110.34, 110.29, 58.93, 56.17, 56.09, 33.20, 25.91, 25.88, 18.27, 17.84, 12.82, -5.21, -5.25. HRMS (ESI, m / z) calcd for C 34 H 53 N3NaO6Si2[M+Na] +678.3371 found 678.3358.[α] D (CHCl3) -108.28°, c = 0.28 g / 100 mL.

[0161] Synthesis of compound 3a:

[0162] Into a 250 mL single necked round bottom flask, methanol (80 mL), acetic acid (5 mL), water (5 mL) were added in sequence, then zinc powder (41.8 g, 639.78 mmol) was added under ice bath, stirred for 0.5 h, then 2a (10.0 g, 17.29 mmol) in methanol (20 mL) was added dropwise, the reaction was monitored by TLC (eluent: petroleum ether / ethyl acetate = 5 / 1, UV visualization) at 5 °C for 0.5 h. The reaction was removed to room temperature, suction filtered (pad with celite), the filter cake was washed with methanol (75 mL), then the filter cake was washed with EA (75 mL), the filtrate was concentrated under reduced pressure, after the methanol was removed, ethyl acetate (200 mL) was added, then water (60 mL) was added to extract, the organic layer was washed with saturated sodium bicarbonate (200 mL) and saturated sodium chloride (200 mL) in sequence, the organic layer was collected, dried with anhydrous sodium sulfate, suction filtered, concentrated under reduced pressure, to give the product 3a (8.6 g, 90.6%) as yellow oil, this intermediate could not be stored for a long time, and should be used as soon as possible for the next step synthesis. 1 HNMR (400 MHz, CDC13) δ 6.72 (s, 1H), 6.25 (s, 1H), 6.16 (s, 1H), 4.70-4.58 (m, 1H), 3.98-3.87 (m, 1H), 3.80-3.75 (m, 1H), 3.71 (s, 3H), 2.71 (dd, J = 16.4, 10.3 Hz, 1H), 2.57-2.50 (m, 1H), 1.68 (d, J = 1.6 Hz, 3H), 1.10 (s, 9H), 1.08 (s, 9H), 0.89 (s, 9H), 0.06 (d, J = 6.3 Hz, 6H). 13 CNMR (101 MHz, CDC13) δ 165.98, 148.66, 142.98, 140.97, 125.66, 121.04, 113.86, 112.41, 109.16, 62.61, 58.81, 56.64, 36.45, 25.81, 18.19, 17.91, 13.79, 12.89, -5.40. HRMS (ESI, m / z) calcd for C 29 H 53 N2O4Si2[M+H] + 549.3544, found: 549.3545.

[0163] Synthesis of compound 3b:

[0164] Into a 250 mL single necked round bottom flask, methanol (4 mL), acetic acid (0.5 mL) and water (0.5 mL) were added in sequence, then zinc powder (11 g, 169.23 mmol) was added under ice-bath, stirred for 0.5 h, then compound 2b (3.0 g, 4.57 mmol) in methanol (100 mL) was added dropwise, the reaction was carried out at 5 °C for 0.5 h, TLC monitored the reaction was complete (developing agent: petroleum ether / ethyl acetate = 3 / 1, UV coloration). Removed to room temperature, suction filtration (pad diatomite), the filter cake was washed with EA (100 mL), the filtrate was concentrated under reduced pressure (30 °C), after removing methanol, ethyl acetate (150 mL) was added, then water (45 mL) was added to extract, the organic layer was washed with saturated sodium bicarbonate (60 mL) and saturated sodium chloride (60 mL) in sequence, the organic layer was collected, dried over anhydrous sodium sulfate, suction filtration, concentrated under reduced pressure, to obtain the crude compound 3b (2.86 g, 100%), which was directly used in the next step. 1 H NMR (400 MHz, CDCl3) d 7.07 (d, J = 8.1 Hz, 2H), 6.78 (s, 1H), 6.61 (d, J = 8.5 Hz, 2H), 6.28 (s, 1H), 4.74 (s, 1H), 4.01 - 3.84 (m, 3H), 3.71 (s, 3H), 3.13 - 3.02 (m, 1H), 3.00 - 2.91 (m, 1H), 1.31 - 1.23 (m, 3H), 1.12 (s, 9H), 1.10 (s, 9H), 0.86 (s, 9H), 0.06 (s, 6H). 13 C NMR (101 MHz, CDCl3) d 166.40, 148.87, 145.40, 143.05, 141.33, 125.72, 124.94, 123.39, 115.11, 113.72, 111.90, 109.25, 62.57, 58.91, 56.56, 32.85, 25.84, 18.22, 17.93, 12.91, -5.31, -5.33. HRMS (ESI, m / z) calcd for C 34 H 55 N3NaO4Si2[M+Na] + 648.3629, found: 648.3621.

[0165] Synthesis of compound 4a:

[0166] In a 250 mL round bottom flask, compound 3a (12 g, 21.88 mmol) was dissolved in dichloromethane (96 mL), then pyridine (3.9 mL, 3.8 g, 48.14 mmol) was added, and Alloc-Cl (2.6 mL, 2.9 g, 24.07 mmol) was slowly added dropwise into the reaction system at -10 °C under nitrogen protection. After the dropwise addition was completed, the reaction was stirred at -5 °C for 1 h, and TLC monitoring showed that the reaction was complete (developing agent: petroleum ether / ethyl acetate = 4 / 1, ultraviolet coloration). 10% aqueous citric acid solution (96 mL) was added and stirred, and the reaction solution was poured into a separatory funnel and allowed to stand to separate into layers. The organic layer was successively washed with saturated sodium bicarbonate (48 mL) and saturated sodium chloride (48 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the product 4a (13.2 g, 95.1%) as a yellow oil. 1 H NMR (400 MHz, CDC13) δ 8.59 (s, 1H), 7.75 (s, 1H), 6.77 (s, 1H), 6.17 (s, 1H), 6.00-5.89 (m, 1H), 5.33 (dd, J = 17.2, 1.6 Hz, 1H), 5.22 (dd, J = 10.5, 1.4 Hz, 1H), 4.67-4.60 (m, 3H), 3.87-3.77 (m, 2H), 3.75 (s, 3H), 2.72 (dd, J = 16.5, 10.0 Hz, 1H), 2.56 (dd, J = 16.5, 4.1 Hz, 1H), 1.68 (s, 3H), 1.32-1.27 (m, 3H), 1.11 (d, J = 7.4 Hz, 18H), 0.88 (s, 9H), 0.05 (d, J = 8.0 Hz, 6H). 13 CNMR (101 MHz, CDC13) δ 165.98, 148.66, 142.98, 140.97, 125.66, 121.04, 113.86, 112.41, 109.16, 62.61, 58.81, 56.64, 36.45, 25.81, 18.19, 17.91, 13.79, 12.89, -5.40. HRMS (ESI, m / z) calcd for C 33 H 57 N2O6Si2[M+H] + 633.3755, found: 633.3751. [a] D (CHCl3) -88.49°, c = 0.39 g / 100 mL.

[0167] Synthesis of compound 4b:

[0168] Into a 25 mL round-bottom flask, compound 3 (2.86 g, 4.57 mmol) was dissolved in dichloromethane (30 mL), then pyridine (1.62 mL, 1.59 g, 20.11 mmol) was added. The reaction was protected by nitrogen, and Alloc-Cl (1.07 mL, 1.21 g, 10.05 mmol) was added dropwise under ice bath. After the dropwise addition was completed, the reaction was stirred for 1 h under ice bath. TLC monitoring showed that the reaction was completed (eluent: petroleum ether / ethyl acetate = 4 / 1, UV coloration). 10% citric acid aqueous solution (30 mL) was added and stirred. The reaction solution was poured into a separatory funnel and allowed to stand to separate into layers. The organic layer was successively washed with saturated sodium bicarbonate (15 mL) and saturated sodium chloride (15 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (mobile phase: PE / EA = 90 / 10) to obtain the product 4b (2.49 g, 68.8%) as a yellow oil. 1 H NMR (400 MHz, CDC13) δ 8.51 (s, 1H), 7.73 (s, 1H), 7.29 (d, J = 8.3 Hz, 2H), 7.15 (d, J = 8.2 Hz, 2H), 6.80 (d, J = 4.6 Hz, 3H), 5.98 - 5.80 (m, 2H), 5.34 - 5.13 (m, 4H), 4.79 - 4.68 (m, 1H), 4.61 (d, J = 5.8 Hz, 2H), 4.57 (d, J = 5.5 Hz, 2H), 4.05 - 3.79 (m, 2H), 3.71 (s, 3H), 3.12 - 3.03 (m, 1H), 2.96 (dd, J = 15.9, 3.6 Hz, 1H), 1.31 - 1.22 (m, 3H), 1.08 (d, J = 7.4 Hz, 18H), 0.80 (s, 9H), 0.01 (d, J = 7.7 Hz, 6H). 13 C NMR (101 MHz, CDC13) δ 165.90, 153.36, 153.24, 148.49, 146.01, 137.02, 132.51, 132.42, 129.20, 125.27, 124.65, 123.84, 118.75, 118.15, 117.88, 112.50, 65.81, 65.70, 62.44, 59.43, 56.26, 32.63, 25.78, 18.16, 17.90, 12.85, -5.39. HRMS (ESI, m / z) calcd for C 42 H 63 N3NaO8Si2[M+Na] + 816.4051 found, 816.4044. [a] D (CHCl3) -35.34°, c = 0.26 g / 100 mL.

[0169] Synthesis of compound 5a:

[0170] In a 250 mL round-bottom flask, compound 4a (3 g, 4.744 mmol) was dissolved in a mixed solvent of acetic acid, methanol, THF and water (AcOH / MeOH / THF / H2O = 21 / 3 / 3 / 6 mL), stirred at room temperature for 5 h, TLC monitoring reaction completion (developing agent: petroleum ether / ethyl acetate = 2 / 1, ultraviolet coloration). Diluted with ethyl acetate (900 mL), extracted with pure water (300 mL*2) in turn, collected the organic layer, washed with saturated sodium bicarbonate (300 mL*2) and saturated sodium chloride (300 mL*2) in turn, dried with anhydrous sodium sulfate, suction filtered, concentrated under reduced pressure, and the crude product was purified by column chromatography (mobile phase: PE / EA = 4 / 1) to obtain the product 5a (2.06 g, 83.8%) as a yellow oil. 1 H NMR (400 MHz, CDC13) d 8.35 (s, 1H), 7.69 (s, 1H), 6.78 (s, 1H), 6.15 (s, 1H), 6.00-5.90 (m, 1H), 5.34 (dd, J = 17.2, 1.6 Hz, 1H), 5.25-5.21 (m, 1H), 4.77-4.70 (m, 1H), 4.67-4.59 (m, 3H), 3.86-3.80 (m, 2H), 3.77 (s, 3H), 2.88 (dd, J = 16.8, 10.2 Hz, 1H), 2.24-2.15 (m, 1H), 1.70 (s, 3H), 1.29 (d, J = 7.3 Hz, 3H), 1.11 (d, J = 7.5 Hz, 18H). 13 C NMR (101 MHz, CDC13) d 166.99, 153.34, 148.56, 146.02, 132.54, 132.01, 124.99, 122.65, 117.95, 112.64, 66.85, 65.74, 56.39, 37.29, 17.89, 13.69, 12.83. HRMS (ESI, m / z) calcd for C 27 H 43 N2O6Si[M+H] + 519.2890, found: 519.2882. [a] D (CHCl3)-64.02°, c = 0.28 g / 100 mL.

[0171] Synthesis of compound 5b:

[0172] In a 250 mL round-bottom flask, compound 4b (3 g, 4.744 mmol) was dissolved in a mixed solvent of acetic acid, methanol, THF and water (AcOH / MeOH / THF / H2O = 21 / 3 / 3 / 6 mL), stirred at room temperature for 5 h, TLC monitoring reaction completion (developing agent: petroleum ether / ethyl acetate = 2 / 1, ultraviolet coloration). Diluted with ethyl acetate (900 mL), extracted with pure water (300 mL*2) in turn, collected organic layer, washed with saturated sodium bicarbonate (300 mL*2) and saturated sodium chloride (300 mL*2) in turn, dried with anhydrous sodium sulfate, suction filtered, concentrated under reduced pressure, and the crude product was purified by column chromatography (mobile phase: PE / EA = 4 / 1) to obtain the product 5b (2.06 g, 83.8%) as a yellow oil. 1 H NMR (400 MHz, CDCl3) δ 8.34 (s, 1H), 7.69 (s, 1H), 7.34 (d, J = 8.3 Hz, 2H), 7.17 (d, J = 8.2 Hz, 2H), 6.83 (d, J = 17.2 Hz, 3H), 6.03-5.84 (m, 2H), 5.39-5.17 (m, 4H), 4.91-4.80 (m, 1H), 4.66 (d, J = 5.6 Hz, 2H), 4.60 (d, J = 5.7 Hz, 2H), 3.96-3.83 (m, 2H), 3.76 (s, 3H), 3.32-3.21 (m, 1H), 2.67 (dd, J = 15.9, 3.6 Hz, 1H), 1.35-1.27 (m, 3H), 1.12 (d, J = 7.5 Hz, 18H). 13 C NMR (101 MHz, CDCl3) δ 167.51, 153.42, 153.05, 148.82, 146.29, 137.14, 132.40, 132.27, 128.53, 125.39, 124.30, 123.92, 118.75, 118.37, 118.08, 112.36, 65.94, 65.84, 61.90, 56.32, 56.23, 33.55, 17.88, 12.82. HRMS (ESI, m / z) calcd for C 36 H 49 N3NaO8Si[M+Na] + 702.3187, found: 702.3189.[α] D (CHCl3)-65.41°, c = 0.25 g / 100 mL.

[0173] General synthesis method of compounds 6a-6b:

[0174] Into a 100 mL round bottom flask was added anhydrous DCM (25 mL) under nitrogen protection, and stirred at -78 °C, then oxalyl chloride (0.52 mL, 0.78 g, 6.145 mmol) was added, followed by dropwise addition of DMSO (1.0 mL, 1.1 g, 14.1 mmol) and stirring at -78 °C for 10 min. A solution of 5a-5b (5.41 mmol) in DCM (20 mL) was added dropwise, and the reaction solution became turbid. After the dropwise addition was completed, the solution was stirred at -78 °C for 15 min, and then TEA (3.77 mL, 2.75 g, 27.17 mmol) was added dropwise. After the dropwise addition was completed, the solution was allowed to warm to room temperature and stirred for 2 h. TLC monitoring showed that the reaction was complete (developing agent: petroleum ether / ethyl acetate = 2 / 1, ultraviolet coloration). A 5% aqueous citric acid solution (28 mL) was added and stirred for 5 min. The organic layer was collected, and then washed with saturated sodium bicarbonate (14 mL) and water (14 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography to obtain the white foam product 6a-6b.

[0175] Synthesis of compound 6a:

[0176] Compound 5a was synthesized to 6a according to the above method. The crude product was purified by column chromatography (mobile phase: PE / EA = 5 / 1) to obtain the white foam product 6a (2.28 g, 96.9%). 1 H NMR (400 MHz, CDCl3) δ 7.20 (s, 1H), 6.70 (s, 2H), 5.74 (d, J = 9.6 Hz, 2H), 5.22-5.10 (m, 2H), 4.63-4.43 (m, 2H), 3.92-3.82 (m, 4H), 3.82-3.77 (m, 1H), 2.96 (dd, J = 17.0, 10.1 Hz, 1H), 2.59 (d, J = 17.2 Hz, 1H), 1.78 (s, 3H), 1.27-1.20 (m, 3H), 1.08 (dd, J = 7.4, 2.3 Hz, 18H). 13 C NMR (101 MHz, CDCl3) δ 163.00, 150.58, 147.80, 131.82, 127.71, 125.67, 123.25, 121.76, 121.56, 118.39, 111.21, 86.10, 67.02, 59.39, 55.54, 38.75, 17.86, 17.84, 13.69, 12.84. HRMS (ESI, m / z) calcd for C 27 H 41 N2O6Si[M+H] + 517.2734, found: 517.2729. [a] D(CHCl3) 23.36°, c = 0.30 g / 100 mL.

[0177] Synthesis of compound 6b:

[0178] Compound 5b was synthesized as above for 6b. The crude product was purified by column chromatography (mobile phase: PE / EA = 5 / 1) to give the product 6b (2.4 g, 65.4%) as a white foam. 1 H NMR (400 MHz, CDC13) δ 7.43 - 7.33 (m, 3H), 7.28 (d, J = 3.9 Hz, 1H), 7.22 (s, 1H), 7.04 (s, 1H), 6.74 (s, 1H), 6.02 - 5.90 (m, 1H), 5.83 (d, J = 9.7 Hz, 2H), 5.36 (dd, J = 17.2, 1.6 Hz, 1H), 5.26 (dd, J = 10.4, 1.4 Hz, 1H), 5.21 - 5.09 (m, 2H), 4.67 (d, J = 5.7 Hz, 2H), 4.60 (dd, J = 13.2, 5.7 Hz, 1H), 4.46 (dd, J = 13.8, 5.4 Hz, 1H), 3.98 (td, J = 10.0, 3.3 Hz, 1H), 3.85 (s, 3H), 3.33 (ddd, J = 16.7, 10.2, 2.2 Hz, 1H), 3.02 (dd, J = 16.7, 1.8 Hz, 1H), 1.28 - 1.22 (m, 3H), 1.08 (dd, J = 7.4, 2.3 Hz, 18H). 13 C NMR (101 MHz, CDC13) δ 163.60, 156.06, 153.19, 150.71, 148.10, 137.08, 132.39, 131.76, 128.86, 127.84, 125.60, 125.41, 123.09, 122.56, 121.99, 118.83, 118.36, 111.26, 85.96, 67.12, 65.98, 59.54, 55.63, 55.54, 35.06, 17.88, 12.88. HRMS (ESI, m / z) calcd for C 36 H 47 N3NaO8Si[M+Na] + 700.3030, found: 700.3029. [a] D (CHCl3) 93.98°, c = 0.16 g / 100 mL.

[0179] General synthesis method of compounds 7a-7b:

[0180] Into a 100 mL round bottom flask, compound 6a-6b (4.36 mmol) was dissolved in anhydrous DCM (22.5 mL), then 2,6-lutidine (2.0 mL, 1.84 g, 17.17 mmol) was added, and TBS-OTf (3.0 mL, 3.45 g, 13.08 mmol) was added dropwise under ice-bath cooling. After the addition was completed, the reaction was allowed to react at room temperature for 1 h. The reaction was monitored by TLC (eluent: petroleum ether / ethyl acetate = 2 / 1, UV visualization). The reaction solution was added to saturated sodium bicarbonate (15 mL), and the organic layer was collected. The organic layer was washed with water (15 mL), and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was used directly in the next step.

[0181] Synthesis of compound 7a:

[0182] Compound 6a was synthesized to 7a (2.635 g, 95.9%) according to the above method. The obtained crude 7a was a yellow oily liquid, which was used directly in the next step.

[0183] Synthesis of compound 7b:

[0184] Compound 6b was synthesized to 7b (2.646 g, 76.7%) according to the above method. The obtained crude 7b was a yellow oily liquid, which was used directly in the next step.

[0185] General synthesis method of compounds 8a-8b:

[0186] Into a 50 mL round bottom flask, compound 7a-7b (4.18 mmol) was dissolved in a mixed solvent of DMF and water (25 / 0.5 mL), and LiOAc (0.28 g, 4.26 mmol) was added. The reaction was stirred at 25°C for 10 h. The reaction was monitored by TLC (eluent: petroleum ether / ethyl acetate = 2 / 1, UV visualization). The reaction solution was concentrated under reduced pressure (membrane pump, 56°C), and the excess DMF was removed. EA (50 mL) was added to extract, and the organic layer was washed with pure water (25 mL). The water layer was extracted with EA (20 mL), and the organic layers were combined. The organic layer was washed with 5% citric acid (25 mL) and saturated sodium chloride (25 mL), respectively. The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography to obtain white solid 8a-8b.

[0187] Synthesis of compound 8a:

[0188] Compound 7a was synthesized to 8a according to the above method. The crude product was purified by column chromatography (mobile phase: PE / EA = 3 / 1-3 / 2) to obtain white foamed product 8a (1.84 g, 91.35%). 1H NMR (400 MHz, CDC13) δ 7.24 (s, 1H), 6.73 (s, 1H), 6.68 (s, 1H), 5.85 (d, J = 8.9 Hz, 1H), 5.80-5.71 (m, 1H), 5.14-5.04 (m, 2H), 4.60 (dd, J = 13.4, 5.0 Hz, 1H), 4.42 (dd, J = 13.8, 5.3 Hz, 1H), 3.95 (s, 3H), 3.75 (td, J = 12.8 Hz, 3.6 Hz, 1H), 2.91 (dd, J = 17.1, 10.2 Hz, 1H), 2.38 (d, J = 17.3 Hz, 1H), 1.77 (s, 3H), 0.87 (s, 9H), 0.23 (d, J = 17.2 Hz, 6H). 13 C NMR (101 MHz, CDC13) δ 163.43, 155.17, 148.01, 146.37, 132.11, 129.24, 125.51, 123.61, 121.22, 117.35, 116.38, 110.38, 86.89, 66.41, 61.47, 56.26, 39.05, 25.68, 17.89, 13.84, -4.27, -5.39. HRMS (ESI, m / z) calcd for C 24 H 35 N2O6 Si[M + H] + 475.2264, found: 475.2260. [a] D (CHCl3) 47.44°, c = 0.48 g / 100 mL.

[0189] Synthesis of compound 8b:

[0190] Compound 7b was synthesized to 8b by the above method, the crude product was purified by column chromatography (mobile phase: PE / EA = 3 / 1-2 / 1) to give white solid product 8b (2.54 g, 95.6%). 1H NMR (400 MHz, CDC13) δ 7.38 (d, J = 8.5 Hz, 3H), 7.29 (d, J = 8.5 Hz, 2H), 6.76 (s, 1H), 6.71 (s, 1H), 6.03 - 5.91 (m, 2H), 5.84 - 5.70 (m, 1H), 5.37 (dd, J = 17.2, 1.5 Hz, 1H), 5.27 (dd, J = 10.4, 1.4 Hz, 1H), 5.17 - 5.01 (m, 2H), 4.67 (d, J = 5.0 Hz, 2H), 4.61 (dd, J = 14.0, 4.6 Hz, 1H), 4.42 (dd, J = 14.0, 5.2 Hz, 1H), 3.97 - 3.87 (m, 4H), 3.31 (ddd, J = 16.4, 10.3, 2.2 Hz, 1H), 2.81 (dd, J = 16.3, 3.5 Hz, 1H), 0.95 - 0.90 (s, 9H), 0.24 (d, J = 17.4 Hz, 6H). 13 C NMR (101 MHz, CDC13) δ 163.89, 155.17, 153.17, 148.24, 146.45, 137.12, 132.40, 132.04, 129.34, 128.93, 125.55, 125.31, 122.99, 122.67, 118.81, 118.33, 117.46, 116.46, 110.41, 86.66, 66.51, 65.94, 61.46, 56.30, 35.11, 25.69, 17.94, -4.24, -5.29. HRMS (ESI, m / z) calcd for C 30 H 38 N2NaO7Si[M + Na] + 658.2561, found: 658.2554. [a] D (CHCl3) 55.40°, c = 0.33 g / 100 mL.

[0191] General synthetic procedure for compounds 9a1-9b3:

[0192] In a 10 mL round-bottom flask, 8a-8b (0.424 mmol) was dissolved in 2-butanone (2.5 mL), and then potassium carbonate (0.076 g, 0.5483 mmol) and diiodoalkane (2.10 mmol) were added. The reaction was stirred at 75 °C for 16 h under nitrogen protection. TLC was used to monitor the completion of the reaction (eluent: petroleum ether / ethyl acetate = 3 / 1 or 2 / 1, UV coloration). The reaction solution was concentrated under reduced pressure to remove excess 2-butanone. EA (10 mL) was added to extract the product, and the organic layer was washed with saturated sodium chloride (3 mL). The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (mobile phase: PE / EA = 9 / 1-8 / 1) to obtain the yellow oil product 9a1-9b3.

[0193] Synthesis of compound 9a1:

[0194] Compound 8a and 1,3-diiodopropane (0.24 mL, 0.618 g, 2.10 mmol) were used to synthesize 9a1 according to the above method. The crude product was purified by column chromatography (mobile phase: PE / EA = 9 / 1-8 / 1) to obtain the yellow oil product 9a1 (160 mg, 59%). 1 H NMR (400 MHz, CDCl3) δ 7.24 (s, 1H), 6.69 (s, 1H), 6.64 (d, J = 4.7 Hz, 1H), 5.90-5.83 (m, 1H), 5.82-5.73 (m, 1H), 5.15-5.05 (m, 2H), 4.65-4.60 (m, 1H), 4.16-4.01 (m, 2H), 3.93-3.88 (m, 3H), 3.79-3.70 (m, 1H), 3.38 (t, J = 6.6 Hz, 2H), 2.93 (dd, J = 17.0, 10.3 Hz, 1H), 2.43-2.24 (m, 3H), 1.78 (s, 3H), 0.88 (d, J = 3.8 Hz, 9H), 0.25 (d, J = 4.3 Hz, 6H). 13 C NMR (101 MHz, CDCl3) δ 161.03, 152.73, 147.59, 146.82, 129.82, 126.24, 124.07, 121.40, 118.84, 115.19, 112.21, 108.59, 84.70, 66.28, 64.10, 59.20, 53.80, 36.76, 30.16, 23.40, 23.36, 15.62, 11.55, -6.44, -7.56. HRMS (ESI, m / z) calcd for C 27 H 40 IN2O6Si[M+H] + 643.1700, found: 643.1703. [a] D(CHCl3) 90.38°, c = 0.50 g / 100 mL.

[0195] Synthesis of compound 9a2:

[0196] Compound 8a and 1,4-diiodobutane (0.28 mL, 0.65 g, 2.10 mmol) were synthesized to 9a2 as described above, the crude product was purified by column chromatography (mobile phase: PE / EA = 85 / 15-80 / 20) to give the product 9a2 (193.7 mg, 70%) as yellow oil. 1 H NMR (400 MHz, CDC13) δ 7.23 (s, 1H), 6.69 (s, 1H), 6.60 (s, 1H), 5.87 (d, J = 8.9 Hz, 1H), 5.82 - 5.70 (m, 1H), 5.09 (t, J = 12.8 Hz, 2H), 4.62 (dd, J = 13.6, 4.7 Hz, 1H), 4.40 (dd, J = 13.6, 5.2 Hz, 1H), 4.07 - 3.94 (m, 2H), 3.91 (s, 3H), 3.73 (td, J = 9.8, 3.3 Hz, 1H), 3.26 (t, J = 6.7 Hz, 2H), 2.92 (dd, J = 17.0, 10.3 Hz, 1H), 2.38 (d, J = 16.6 Hz, 1H), 2.07 - 2.01 (m, 2H), 1.99 - 1.93 (m, 2H), 1.78 (s, 3H), 0.88 (s, 9H), 0.24 (d, J = 8.7 Hz, 6H). 13 C NMR (101 MHz, CDC13) δ 163.37, 155.04, 150.05, 149.07, 132.15, 128.53, 126.13, 123.65, 121.08, 117.32, 114.18, 110.82, 87.01, 67.84, 66.37, 61.50, 56.21, 39.05, 30.08, 29.82, 25.62, 17.90, 13.84, 6.14, -4.20, -5.26. HRMS (ESI, m / z) calcd for C 28 H 42 IN2O6Si[M+H] + 657.1857, found: 657.1855. [a] D (CHCl3) 90.38°, c = 0.50 g / 100 mL.

[0197] Synthesis of compound 9a3:

[0198] Compound 8a and 1,5-diiodopentane (0.31 mL, 0.68 g, 2.10 mmol) were used to synthesize 9a3 according to the above method. The crude product was purified by column chromatography (mobile phase: PE / EA = 85 / 15-80 / 20) to give the product 9a3 (262.9 mg, 93%) as yellow oil. 1 H NMR (400 MHz, CDC13) δ 7.23 (s, 1H), 6.69 (s, 1H), 6.60 (s, 1H), 5.87 (d, J = 8.9 Hz, 1H), 5.83 - 5.69 (m, 1H), 5.14 - 5.03 (m, 2H), 4.63 (dd, J = 13.7, 5.0 Hz, 1H), 4.40 (dd, J = 13.8, 5.2 Hz, 1H), 4.05 - 3.95 (m, 2H), 3.91 (s, 3H), 3.74 (td, J = 9.6, 3.7 Hz, 1H), 3.21 (t, J = 7.0 Hz, 2H), 2.92 (dd, J = 16.8, 10.4 Hz, 1H), 2.38 (d, J = 16.4 Hz, 1H), 1.93 - 1.85 (m, 4H), 1.78 (s, 3H), 1.63 - 1.56 (m, 2H), 0.88 (s, 9H), 0.24 (d, J = 8.1 Hz, 6H). 13 C NMR (101 MHz, CDC13) δ 163.40, 155.04, 150.15, 149.05, 132.13, 128.55, 125.98, 123.69, 120.99, 117.28, 114.16, 110.81, 87.01, 68.69, 66.33, 61.50, 56.16, 39.05, 33.13, 27.82, 27.02, 25.62, 17.88, 13.79, 6.40, -4.23, -5.28. HRMS (ESI, m / z) calcd for C 29 H 44 IN2O6Si[M+H] + 671.2013, found: 671.2006. [a] D (CHCl3) 26.91°, c = 0.32 g / 100 mL.

[0199] Synthesis of compound 9b1:

[0200] Compound 8b and 1,3-diiodopropane (0.24 mL, 0.618 g, 2.10 mmol) were used to synthesize 9b1 according to the above method. The crude product was purified by column chromatography (mobile phase: PE / EA = 85 / 15-67 / 33) to give the product 9b1 (248.6 mg, 73%) as yellow oil. 1HNMR (400 MHz, CDC13) δ 7.38 (d, J = 8.8 Hz, 3H), 7.29 (d, J = 8.4 Hz, 2H), 6.74 (s, 1H), 6.68 (s, 1H), 6.03 - 5.90 (m, 2H), 5.84 - 5.71 (m, 1H), 5.37 (dd, J = 17.2, 1.5 Hz, 1H), 5.27 (dd, J = 10.4, 1.3 Hz, 1H), 5.18 - 5.04 (m, 2H), 4.68 (d, J = 5.7 Hz, 2H), 4.65 - 4.60 (m, 1H), 4.41 (dd, J = 13.3, 6.0 Hz, 1H), 4.11 - 4.04 (m, 2H), 3.95 - 3.88 (m, 4H), 3.39 (t, J = 6.6 Hz, 2H), 3.35 - 3.28 (m, 1H), 2.81 (d, J = 16.3 Hz, 1H), 2.33 (p, J = 6.2 Hz, 2H), 0.91 (d, J = 3.7 Hz, 9H), 0.26 (d, J = 7.0 Hz, 6H). 13 C NMR (101 MHz, CDC13) δ 163.75, 154.99, 153.14, 150.13, 149.18, 137.19, 132.39, 131.98, 128.76, 128.57, 125.99, 125.48, 122.89, 122.62, 118.74, 118.21, 117.53, 114.50, 110.88, 86.74, 68.55, 66.45, 65.84, 61.48, 56.16, 35.06, 32.38, 25.66, 17.89, 2.23, -4.17, -5.21. HRMS (ESI, m / z) calcd for C 36 H 46 IN3NaO8Si[M+Na] + 826.1997, found: 826.1990.[α] D (CHCl3) 152.33°, c = 0.21 g / 100 mL.

[0201] Synthesis of compound 9b2:

[0202] Compound 8b and 1,4-diiodobutane (0.28 mL, 0.65 g, 2.10 mmol) were synthesized as 9b2 following the above described method, the crude was purified by column chromatography (mobile phase: PE / EA = 85 / 15-67 / 33) to give the product 9b2 (235.6 mg, 68%) as yellow oil. 1H NMR (400 MHz, CDC13) δ 7.38 (d, J = 9.0 Hz, 3H), 7.29 (d, J = 8.3 Hz, 2H), 6.71 (s, 1H), 6.63 (s, 1H), 6.03 - 5.90 (m, 2H), 5.83 - 5.71 (m, 1H), 5.37 (dd, J = 17.2, 1.6 Hz, 1H), 5.27 (dd, J = 10.5, 1.4 Hz, 1H), 5.18 - 5.02 (m, 2H), 4.68 (d, J = 5.8 Hz, 2H), 4.65 - 4.59 (m, 1H), 4.41 (d, J = 11.8 Hz, 1H), 4.07 - 3.97 (m, 2H), 3.92 (s, 4H), 3.40 - 3.30 (m, 1H), 3.27 (t, J = 6.7 Hz, 2H), 2.81 (d, J = 16.1 Hz, 1H), 2.08 - 2.02 (m, 2H), 2.01 - 1.93 (m, 2H), 0.91 (s, 9H), 0.25 (d, J = 11.5 Hz, 6H). 13 C NMR (101 MHz, CDC13) δ 163.80, 155.00, 153.11, 150.30, 149.14, 137.12, 132.38, 132.03, 128.83, 128.57, 125.76, 125.49, 122.96, 122.55, 118.74, 118.25, 117.38, 114.19, 110.83, 86.75, 67.84, 66.43, 65.86, 61.44, 56.10, 35.07, 30.03, 29.77, 25.62, 17.89, 6.08, -4.21, -5.20. HRMS (ESI, m / z) calcd for C 37 H 48 IN3NaO8Si[M+Na] + 840.2153, found: 840.2154.[α] D (CHCl3) 85.53°, c = 0.35 g / 100 mL.

[0203] Synthesis of compound 9b3:

[0204] Compound 8b and 1,5-diiodopentane (0.31 mL, 0.68 g, 2.10 mmol) were synthesized to 9b3 as described above, the crude product was purified by column chromatography (mobile phase: PE / EA = 85 / 15-67 / 33) to give the product 9b3 (317.2 mg, 90%) as yellow oil. 1H NMR (400 MHz, CDC13) δ 7.38 (d, J = 9.3 Hz, 3H), 7.29 (d, J = 8.5 Hz, 2H), 6.71 (s, 1H), 6.63 (s, 1H), 6.04 - 5.90 (m, 2H), 5.83 - 5.71 (m, 1H), 5.37 (dd, J = 17.2, 1.5 Hz, 1H), 5.27 (dd, J = 10.4, 1.3 Hz, 1H), 5.19 - 5.00 (m, 2H), 4.68 (d, J = 5.7 Hz, 2H), 4.65 - 4.59 (m, 1H), 4.42 (d, J = 12.9 Hz, 1H), 4.07 - 3.95 (m, 2H), 3.93 (s, 3H), 3.92 - 3.86 (m, 1H), 3.38 - 3.27 (m, 1H), 3.22 (t, J = 7.0 Hz, 2H), 2.81 (d, J = 15.9 Hz, 1H), 1.97 - 1.82 (m, 4H), 1.64 - 1.60 (m, 2H), 0.92 (s, 9H), 0.25 (d, J = 11.0 Hz, 6H). 13 C NMR (101 MHz, CDC13) δ 163.84, 155.01, 153.13, 150.38, 149.10, 137.16, 132.39, 132.03, 128.80, 128.58, 125.59, 125.48, 122.94, 122.54, 118.74, 118.21, 117.34, 114.09, 110.78, 86.76, 68.68, 66.41, 65.84, 61.48, 56.12, 35.07, 33.08, 27.78, 26.99, 25.63, 17.89, 6.45, -4.22, -5.21. HRMS (ESI, m / z) calcd for C 38 H 50 IN3NaO8Si[M+Na] + 854.2310, found: 854.2313.[α] D (CHCl3) 91.81°, c = 0.27 g / 100 mL.

[0205] Example 2: Synthesis of compound 14

[0206]

[0207] Synthesis of compound 11:

[0208] In a 1000 mL round bottom flask, add N-Boc-piperazine (10, 24 g, 128.94 mmol), then dissolve in 400 mL anhydrous DCM, then add 1,2-dibromoethane (47.0 mL, 102.6 g, 552.0 mmol) and DIEA (23.5 mL, 18.33 g, 141.8 mmol), reflux at 45 °C for 96 h. TLC monitor the reaction is not complete (developing agent: PE / EA = 3 / 1, iodine display), after treatment, cool to room temperature, spin off DCM, add ethyl acetate (600 mL) and stir for 10 min, then suction filter, collect the filtrate, spin dry, concentrate under reduced pressure, and purify the crude product by column chromatography (mobile phase: PE / EA = 9 / 1-17 / 3) to give the light yellow solid product 11 (10.15 g, 27.0%). 1 H NMR (400 MHz, CDC13) δ 3.66 - 3.54 (m, 2H), 3.45 (s, 4H), 2.74 (t, J = 7.1 Hz, 2H), 2.47 (s, 4H), 1.48 - 1.43 (m, 9H). 13 C NMR (101 MHz, CDC13) δ 153.78, 81.36, 60.12, 55.77, 52.88, 28.34, 28.28.

[0209] Synthesis of compound 13:

[0210] In a 50 mL round bottom flask, add 11 (724 mg, 2.479 mmol) and 12 (612.5 mg, 2.479 mmol, prepared by the method disclosed in the literature "Yao, J. H. et. (2010). Bisanthracene bis(dicarboxylic imide)s as soluble and stable nir dyes. Chemistry-A European Journal, 15(37), 9299-9302."), dissolve in 20 mL anhydrous DMF, then add K2CO3 (1.38 g, 9.916 mmol), reflux at 85 °C for 12 h. TLC monitor the reaction is complete (developing agent: PE / EA = 3 / 1, iodine display), cool to room temperature, spin off DMF, add DCM (60 mL) to dissolve, then add water (10 mL) to extract, collect the organic layer, the water layer is extracted with DCM twice (20 mL*2), combine the organic layers, dry the organic layer with anhydrous sodium sulfate, suction filter, concentrate under reduced pressure, and purify the crude product by column chromatography (mobile phase: DCM / EA = 4 / 1-2 / 1) to give the yellow solid product 13 (1.1 g, 96.7%). 1H NMR (400 MHz, Chloroform-d) δ 9.98 (t, J = 8.0 Hz, 1H), 8.83 (d, J = 12.1 Hz, 1H), 8.75 (t, J = 7.1 Hz, 1H), 8.36 (t, J = 8.7 Hz, 1H), 8.12 (t, J = 7.5 Hz, 1H), 7.88 - 7.80 (m, 1H), 7.77 - 7.70 (m, 1H), 7.68 - 7.60 (m, 1H), 4.46 (t, J = 7.1 Hz, 2H), 3.44 (s, 4H), 2.89 - 2.75 (m, 2H), 2.62 (s, 4H), 1.45 (s, 9H). 13 C NMR (101 MHz, DMSO-d6) δ 164.26, 162.62, 153.73, 136.94, 135.50, 133.22, 132.42, 131.78, 131.32, 130.04, 128.35, 127.52, 126.25, 125.68, 125.62, 121.57, 114.13, 78.61, 55.04, 52.56, 37.02, 27.95, 13.98.

[0211] Synthesis of compound 14:

[0212] In a 50 mL round bottom flask, 13 (700 mg, 1.524 mmol) was dissolved in 15 mL chloroform, then trifluoroacetic acid (1.13 mL, 1.737 g, 15.24 mmol) was added, and stirred at 25 °C for 16 h. TLC monitoring reaction complete (developing agent: DCM / MeOH = 10 / 1, ultraviolet coloration), saturated sodium bicarbonate solution (20 mL) was added to adjust pH to 8, then DCM / MeOH = 10 / 1 (10 mL) was added to extract, the organic layer was collected, the water layer was extracted twice with DCM / MeOH = 10 / 1 (10 mL*2), the organic layer was combined, dried with anhydrous sodium sulfate, suction filtered, and concentrated under reduced pressure to obtain yellow solid product 14 (0.5 g, 91.3%), which was directly used in the subsequent reaction without purification.

[0213] Example 3: Synthesis of compounds 17a1-17b3

[0214]

[0215] General synthesis method of compounds 15a1-15b3:

[0216] Into a 25 mL round-bottom flask, 9a1-9b3 (0.336 mmol) was dissolved in anhydrous DMF (5 mL), then potassium carbonate (63.3 mg, 0.458 mmol) and 12 (75.4 mg, 0.305 mmol) were added. The reaction was stirred at 85 °C for 20 h under nitrogen protection. TLC was used to monitor the completion of the reaction (eluent: petroleum ether / ethyl acetate = 3 / 1, UV coloration). The reaction solution was concentrated under reduced pressure, and the excess DMF was removed. EA (15 mL) and pure water (5 mL) were added to extract, and the water layer was extracted with EA (15 mL*2) again. The combined organic layers were washed with saturated sodium chloride (10 mL*2), and the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography to obtain the yellow foam product 15a1-15b3.

[0217] Synthesis of compound 15a1:

[0218] Compound 9a1 and 12 were used to synthesize 15a1 according to the above method. The crude product was purified by column chromatography (mobile phase: PE / EA = 85 / 15-65 / 35) to obtain the yellow foam product 15a1 (83.7 mg, 36%). 1 H NMR (400 MHz, CDCl3) δ 9.97 (d, J = 9.2 Hz, 1H), 8.86 (s, 1H), 8.75 (dd, J = 7.1, 1.3 Hz, 1H), 8.38 (d, J = 8.4 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.84-7.78 (m, 1H), 7.77-7.71 (m, 1H), 7.64 (t, J = 7.5 Hz, 1H), 7.07 (s, 1H), 6.66 (s, 1H), 6.63 (s, 1H), 5.83 (d, J = 8.9 Hz, 1H), 5.74-5.63 (m, 1H), 5.05-4.92 (m, 2H), 4.53 (t, J = 6.7 Hz, 3H), 4.37 (dd, J = 13.5, 4.9 Hz, 1H), 4.24-4.12 (m, 2H), 3.69 (td, J = 9.1, 2.8 Hz, 1H), 3.56 (s, 3H), 2.94-2.83 (m, 1H), 2.42 (t, J = 6.6 Hz, 2H), 2.39-2.32 (m, 1H), 1.76 (s, 3H), 0.79 (s, 9H), 0.22 (s, 3H), 0.19 (s, 3H). 13C NMR (101 MHz, CDC13) δ 165.04, 163.59, 163.34, 154.98, 150.12, 148.81, 136.13, 134.94, 133.41, 133.30, 132.27, 131.90, 131.16, 129.56, 128.65, 128.34, 128.23, 126.64, 126.36, 125.65, 125.30, 123.57, 122.38, 120.85, 117.11, 115.18, 113.91, 110.37, 86.83, 67.42, 66.18, 61.35, 55.56, 38.90, 37.82, 27.83, 25.42, 17.64, 13.70, -4.37, -5.48. HRMS (ESI, m / z) calcd for C 43 H 48 N3O8Si[M+H] + 762.3211, found: 762.3228. [a] D (CHCl3) 76.47°, c = 0.26 g / 100 mL.

[0219] Synthesis of compound 15a2:

[0220] Compound 9a2 and 12 were synthesized as described above for 15a2, the crude was purified by column chromatography (mobile phase: PE / EA = 85 / 15-65 / 35) to give the product 15a2 as a yellow foam (180 mg, 76%). 1H NMR (400 MHz, CDC13) δ 10.02 (d, J = 9.1 Hz, 1H), 8.85 (s, 1H), 8.76 (dd, J = 6.9, 1.2 Hz, 1H), 8.37 (d, J = 8.1 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.86 - 7.81 (m, 1H), 7.78 - 7.72 (m, 1H), 7.68 - 7.62 (m, 1H), 7.22 (s, 1H), 6.71 - 6.62 (m, 2H), 5.90 - 5.82 (m, 1H), 5.81 - 5.70 (m, 1H), 5.15 - 5.03 (m, 2H), 4.66 - 4.58 (m, 1H), 4.45 - 4.31 (m, 3H), 4.16 - 4.04 (m, 2H), 3.91 - 3.85 (m, 3H), 3.79 - 3.70 (m, 1H), 2.98 - 2.86 (m, 1H), 2.42 - 2.33 (m, 1H), 2.07 - 2.00 (m, 4H), 1.77 (s, 3H), 0.88 (d, J = 10.0 Hz, 9H), 0.24 (d, J = 8.3 Hz, 6H). 13 C NMR (101 MHz, CDC13) δ 165.01, 163.59, 163.49, 155.11, 150.37, 149.10, 136.31, 135.09, 133.44, 132.32, 132.11, 131.28, 129.72, 128.68, 128.58, 128.20, 126.66, 126.44, 125.88, 125.38, 123.73, 123.66, 122.36, 121.03, 117.40, 115.10, 114.25, 110.80, 87.02, 68.87, 66.36, 61.51, 56.18, 40.07, 39.06, 26.76, 25.65, 24.84, 17.89, 13.84, -4.21, -5.26. HRMS (ESI, m / z) calcd for C 44 H 49 N3NaO8Si[M+Na] + 798.3187, found: 798.3182. [a] D (CHCl3) 124.95°, c = 0.28 g / 100 mL.

[0221] Synthesis of compound 15a3:

[0222] Compound 9a3 and 12 were synthesized according to the above method, the crude product was purified by column chromatography (mobile phase: PE / EA = 85 / 15-65 / 35) to give yellow foamy product 15a3 (77 mg, 32%). 1 H NMR (400 MHz, CDC13) δ 10.04 (d, J = 9.1 Hz, 1H), 8.87 (s, 1H), 8.78 (dd, J = 7.1, 1.3 Hz, 1H), 8.39 (d, J = 7.8 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 7.88-7.82 (m, 1H), 7.76 (dd, J = 8.4, 7.1 Hz, 1H), 7.69-7.62 (m, 1H), 7.21 (s, 1H), 6.68 (s, 1H), 6.60 (s, 1H), 5.85 (d, J = 9.0 Hz, 1H), 5.79-5.70 (m, 1H), 5.12-5.05 (m, 2H), 4.63-4.58 (m, 1H), 4.44-4.38 (m, 1H), 4.35-4.29 (m, 2H), 4.07-3.98 (m, 2H), 3.87 (s, 3H), 3.76-3.71 (m, 1H), 2.94-2.87 (m, 1H), 2.40-2.33 (m, 1H), 1.99-1.86 (m, 4H), 1.77 (s, 3H), 1.70-1.63 (m, 2H), 0.82 (s, 9H), 0.22-0.18 (m, 6H). 13 C NMR (101 MHz, CDC13) δ 165.08, 163.62, 163.51, 155.10, 150.33, 149.03, 136.30, 135.07, 133.51, 133.45, 132.38, 132.11, 131.31, 129.72, 128.73, 128.56, 128.26, 126.72, 126.46, 125.73, 125.41, 123.73, 122.45, 121.00, 17.36, 115.21, 113.99, 110.74, 87.01, 68.88, 66.35, 61.52, 56.08, 40.42, 39.05, 28.75, 27.95, 25.59, 23.74, 17.84, 13.83, -4.24, -5.28. HRMS (ESI, m / z) calcd for C 45 H 52 N3O8Si[M+H] + 790.3524, found: 790.3524. [a] D (CHCl3) 51.80°, c = 0.26 g / 100 mL.

[0223] Synthesis of compound 15b1:

[0224] Compound 9b1 and 12 were synthesized as described in (25) for 15b1, the crude was purified by column chromatography (mobile phase: PE / EA = 90 / 10-60 / 40) to give the product 15b1 as a yellow foam (126.6 mg, 45%). 1 H NMR (400 MHz, CDC13) δ 9.97 (d, J = 9.2 Hz, 1H), 8.86 (s, 1H), 8.75 (dd, J = 7.1, 1.3 Hz, 1H), 8.39 (d, J = 8.2, 1H), 8.13 (d, J = 8.5 Hz, 1H), 7.81 (ddd, J = 9.2, 6.6, 1.4 Hz, 1H), 7.75 (dd, J = 8.3, 7.0 Hz, 1H), 7.64 (t, J = 7.2 Hz, 1H), 7.40 - 7.34 (m, 3H), 7.29 (s, 1H), 7.09 (s, 1H), 6.72 (s, 1H), 6.65 (s, 1H), 6.02 - 5.88 (m, 2H), 5.75 - 5.63 (m, 1H), 5.40 - 5.34 (m, 1H), 5.27 (dd, J = 10.4, 1.3 Hz, 1H), 5.05 - 4.96 (m, 2H), 4.67 (d, J = 5.7 Hz, 2H), 4.61 - 4.50 (m, 3H), 4.37 (dd, J = 13.7, 4.9 Hz, 1H), 4.27 - 4.15 (m, 2H), 3.85 (td, J = 9.8, 3.6 Hz, 1H), 3.56 (s, 3H), 3.34 - 3.24 (m, 1H), 2.82 - 2.73 (m, 1H), 2.43 (p, J = 6.4 Hz, 2H), 0.82 (s, 9H), 0.24 (s, 3H), 0.20 (s, 3H). 13C NMR (101 MHz, CDC13) δ 165.22, 163.96, 163.79, 155.12, 153.16, 150.55, 149.07, 137.11, 136.33, 135.12, 133.57, 133.48, 132.43, 131.98, 131.33, 129.74, 128.94, 128.82, 128.57, 128.40, 126.79, 126.52, 125.50, 125.47, 123.05, 123.01, 122.54, 122.47, 118.76, 118.31, 118.23, 117.35, 115.34, 114.15, 110.59, 86.76, 67.62, 66.43, 65.91, 61.55, 55.73, 37.97, 35.09, 27.96, 25.60, 17.82, -4.20, -5.24. HRMS (ESI, m / z) calcd for C 52 H 54 N4NaO 10 Si[M+Na] + 945.3507, found: 945.3508. [a] D (CHCl3) 254.38°, c = 0.23 g / 100 mL.

[0225] Synthesis of compound 15b2:

[0226] Compound 9b2 and 12 were synthesized as described in (25) for 15b2, the crude was purified by column chromatography (mobile phase: PE / EA = 90 / 10-60 / 40) to give the product 15b2 as a yellow foam (148.5 mg, 52%). 1H NMR (400 MHz, CDC13) δ 10.03 (d, J = 9.1 Hz, 1H), 8.87 (s, 1H), 8.78 (dd, J = 7.0, 1.3 Hz, 1H), 8.39 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 7.90 - 7.80 (m, 1H), 7.76 (dd, J = 8.4, 7.1 Hz, 1H), 7.70 - 7.63 (m, 1H), 7.42 - 7.34 (m, 3H), 7.29 (d, J = 8.4 Hz, 2H), 6.69 (d, J = 16.0 Hz, 2H), 6.05 - 5.90 (m, 2H), 5.83 - 5.71 (m, 1H), 5.37 (dd, J = 17.2, 1.5 Hz, 1H), 5.27 (dd, J = 10.4, 1.3 Hz, 1H), 5.18 - 5.01 (m, 2H), 4.67 (d, J = 5.7 Hz, 2H), 4.66 - 4.59 (m, 1H), 4.38 (t, J = 7.0 Hz, 3H), 4.10 (d, J = 7.2 Hz, 2H), 3.90 (s, 4H), 3.37 - 3.25 (m, 1H), 2.88 - 2.75 (m, 1H), 2.04 (d, J = 6.1 Hz, 4H), 0.90 (s, 9H), 0.24 (s, 6H). 13 C NMR (101 MHz, CDC13) δ 165.04, 163.90, 163.62, 155.06, 153.11, 150.60, 149.17, 137.11, 136.35, 135.10, 133.47, 132.38, 132.34, 131.98, 131.28, 129.71, 128.83, 128.70, 128.61, 128.24, 126.64, 126.42, 125.52, 125.44, 125.38, 122.97, 122.47, 122.37, 118.75, 118.20, 118.15, 117.43, 115.14, 114.26, 110.78, 86.76, 68.86, 66.41, 65.82, 61.50, 56.07, 40.03, 35.04, 26.69, 25.62, 24.79, 17.87, -4.24, -5.23. HRMS (ESI) calcd for C 53 H 56 N4O 10 Si[M + Na] + 959.3766 found 959.3660.

[0227] Synthesis of compound 15b3:

[0228] Compound 9b3 and 12 were synthesized from 15b3 following the procedure described in (25), the crude was purified by column chromatography (mobile phase: PE / EA = 90 / 10-60 / 40) to give the product 15b3 as a yellow foam (153.6 mg, 53%). 1 H NMR (400 MHz, CDC13) δ 10.03 (d, J = 9.2 Hz, 1H), 8.86 (s, 1H), 8.78 (dd, J = 7.1, 1.3 Hz, 1H), 8.38 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 8.5 Hz, 1H), 7.89 - 7.80 (m, 1H), 7.75 (dd, J = 8.4, 7.0 Hz, 1H), 7.65 (dd, J = 8.3, 6.6 Hz, 1H), 7.42 - 7.34 (m, 3H), 7.30 (s, 1H), 7.24 (s, 1H), 6.73 (s, 1H), 6.64 (s, 1H), 6.03 - 5.89 (m, 2H), 5.80 - 5.68 (m, 1H), 5.41 - 5.32 (m, 1H), 5.27 (dd, J = 10.4, 1.3 Hz, 1H), 5.16 - 4.99 (m, 2H), 4.68 (d, J = 5.7 Hz, 2H), 4.66 - 4.58 (m, 1H), 4.45 - 4.37 (m, 1H), 4.33 (t, J = 7.6 Hz, 2H), 4.11 - 3.99 (m, 2H), 3.94 - 3.83 (m, 4H), 3.36 - 3.24 (m, 1H), 2.88 - 2.74 (m, 1H), 2.02 - 1.94 (m, 2H), 1.91 - 1.84 (m, 2H), 1.72 - 1.64 (m, 2H), 0.86 (s, 9H), 0.22 (d, J = 10.1 Hz, 6H). 13C NMR (101 MHz, CDC13) δ 165.12, 163.98, 163.67, 155.11, 153.17, 150.63, 149.16, 137.15, 136.36, 135.12, 133.55, 133.49, 132.44, 132.41, 132.03, 131.34, 129.75, 128.91, 128.77, 128.65, 128.30, 126.73, 126.48, 125.50, 125.43, 123.04, 122.48, 118.81, 118.72, 118.27, 118.21, 117.47, 115.24, 114.09, 110.79, 86.81, 68.93, 66.45, 65.89, 61.58, 56.20, 40.43, 35.09, 28.75, 27.96, 25.64, 23.74, 17.88, -4.20, -5.18. HRMS (ESI, m / z) calcd for C 54 H 58 N4NaO 10 Si[M+Na] + 973.3820, found: 973.3823. [a] D (CHCl3) 54.69°, c = 0.31 g / 100 mL.

[0229] General synthetic method for compounds 16a1-16b3:

[0230] In a 10 mL round-bottom flask, 15a1-15b3 (0.263 mmol) was dissolved in anhydrous THF (3 mL), then TBAF (1 mol / L in THF, 0.45 mL, 0.447 mmol) and glacial acetic acid (39 μL, 41 mg, 0.684 mmol) were added. The reaction was stirred at 25 °C for 16 h under nitrogen protection. TLC was used to monitor the completion of the reaction (eluent: DCM / MeOH = 20 / 1, UV coloration). The reaction solution was concentrated under reduced pressure to remove excess THF, then DCM (10 mL) was added. The aqueous layer was extracted with DCM (10 mL*2), and the organic layer was washed with saturated sodium chloride (10 mL*2). The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by preparative TLC plate to obtain yellow solid product 16a1-16b3.

[0231] Synthesis of compound 16a1:

[0232] Compound 15a1 was synthesized according to the above method, and the crude product was purified by TLC plate (eluent: DCM / MeOH = 20 / 1) to give yellow solid product 16a1 (163.4 mg, 96%). 1 H NMR (400 MHz, CDC13) δ 9.96 (d, J = 9.2 Hz, 1H), 8.84 (s, 1H), 8.76-8.69 (m, 1H), 8.37 (d, J = 8.3 Hz, 1H), 8.12 (d, J = 8.4 Hz, 1H), 7.87-7.78 (m, 1H), 7.73 (dd, J = 8.3, 7.1 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.05 (s, 1H), 6.79 (s, 1H), 6.73 (s, 1H), 5.74 (d, J = 9.7 Hz, 2H), 5.05 (t, J = 14.4 Hz, 2H), 4.65-4.57 (m, 1H), 4.50 (t, J = 6.8 Hz, 2H), 4.46-4.36 (m, 1H), 4.28-4.18 (m, 2H), 3.85-3.76 (m, 1H), 3.54-3.44 (m, 4H), 3.01-2.90 (m, 1H), 2.65-2.56 (m, 1H), 2.39 (m, 2H), 1.78 (s, 3H). 13 C NMR (101 MHz, CDC13) δ 165.17, 163.84, 162.91, 155.97, 150.15, 148.81, 136.32, 135.13, 133.55, 133.49, 132.40, 131.75, 131.37, 129.72, 128.77, 128.34, 128.03, 126.73, 126.52, 125.46, 125.04, 123.21, 122.48, 121.65, 117.96, 115.31, 114.24, 110.61, 86.23, 67.71, 66.79, 59.51, 55.70, 38.77, 38.00, 27.83, 13.75. HRMS (ESI, m / z) calcd for C 37 H 34 N3O8[M+H] + 648.2346, found: 648.2344.[α] D (CHCl3) 90.91°, c = 0.14 g / 100 mL. Synthesis of compound 16a2:

[0233] Compound 15a2 was synthesized according to the above procedure for 16a2, the crude product was purified by TLC plate (eluent: DCM / MeOH = 20 / 1) to give yellow solid product 16a2 (139.1 mg, 80%). 1 H NMR (400 MHz, CDC13) δ 10.02 (d, J = 9.2 Hz, 1H), 8.86 (s, 1H), 8.77 (d, J = 7.0 Hz, 1H), 8.39 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 7.89 - 7.83 (m, 1H), 7.76 (t, J = 7.7 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.21 (s, 1H), 6.74 (s, 1H), 6.71 (s, 1H), 5.24 - 5.08 (m, 2H), 4.72 - 4.64 (m, 1H), 4.68 (dd, J = 13.3, 5.6 Hz, 1H), 4.54 - 4.42 (m, 1H), 4.36 (t, J = 6.9 Hz, 2H), 4.18 - 4.06 (m, 2H), 3.88 (s, 3H), 3.86 - 3.81 (m, 1H), 3.02 - 2.91 (m, 1H), 2.65 - 2.56 (m, 1H), 2.04 - 1.94 (m, 4H), 1.78 (s, 3H). 13 C NMR (101 MHz, CDC13) δ 165.00, 163.67, 162.96, 155.96, 150.42, 148.86, 136.34, 135.13, 133.49, 133.45, 132.31, 131.80, 131.34, 129.72, 128.65, 128.19, 126.64, 126.45, 125.39, 125.03, 123.30, 123.24, 122.30, 121.68, 118.07, 115.07, 114.07, 110.83, 86.23, 68.91, 66.79, 59.51, 56.11, 40.06, 38.78, 26.74, 24.69, 13.75. HRMS (ESI, m / z) calcd for C 38 H 35 N3NaO8[M+Na] + 684.2322, found: 684.2316.[α] D (CHCl3) 48.00°, c = 0.18 g / 100 mL.

[0234] Synthesis of compound 16a3:

[0235] Compound 15a3 was synthesized according to the above method, the crude product was purified by TLC plate (eluent: DCM / MeOH = 20 / 1) to give yellow solid product 16a3 (136.7 mg, 77%). 1 H NMR (400 MHz, CDC13) δ 10.03 (d, J = 9.1 Hz, 1H), 8.87 (s, 1H), 8.82-8.75 (m, 1H), 8.39 (d, J = 8.3 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 7.90-7.83 (m, 1H), 7.76 (dd, J = 8.4, 7.0 Hz, 1H), 7.65 (t, J = 7.5 Hz, 1H), 7.22 (s, 1H), 6.72 (d, J = 8.9 Hz, 2H), 5.78 (d, J = 9.8 Hz, 2H), 5.20-5.05 (m, 2H), 4.70-4.62 (m, 1H), 4.50 - 4.40 (m, 1H), 4.36-4.27 (m, 2H), 4.14-4.03 (m, 2H), 3.88 (s, 3H), 3.85-3.80 (m, 1H), 3.05-2.91 (m, 1H), 2.64-2.56 (m, 1H), 2.03-1.95 (m, 2H), 1.92-1.85 (m, 2H), 1.79 (s, 3H), 1.69-1.64 (m, 2H). 13 C NMR (101 MHz, CDC13) δ 165.08, 163.78, 162.95, 155.90, 150.38, 148.74, 136.37, 135.14, 133.57, 133.52, 132.37, 131.74, 131.35, 129.72, 128.72, 128.26, 128.11, 126.67, 126.45, 125.43, 124.83, 123.25, 122.37, 121.59, 117.99, 115.18, 113.71, 110.74, 86.20, 68.90, 66.73, 59.53, 56.12, 40.36, 38.74, 28.49, 27.69, 23.48, 13.71. HRMS (ESI, m / z) calcd for C 39 H 37 N3NaO8[M+Na] + 698.2478, found: 698.2478.[α] D (CHCl3) 54.29°, c = 0.23 g / 100 mL.

[0236] Synthesis of compound 16b1:

[0237] Compound 15b1 was synthesized according to the above procedure for 16b1, and the crude product was purified by TLC plate (eluent: DCM / MeOH = 20 / 1) to give yellow solid product 16b1 (157.3 mg, 74%).1H NMR (400 MHz, CDC13) δ 9.95 (dd, J = 9.1, 1.0 Hz, 1H), 8.83 (s, 1H), 8.72 (dd, J = 7.1, 1.3 Hz, 1H), 8.36 (dd, J = 8.5, 1.4 Hz, 1H), 8.12 (d, J = 8.7 Hz, 1H), 7.82 (ddd, J = 9.2, 6.6, 1.4 Hz, 1H), 7.73 (dd, J = 8.4, 7.1 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.36 (d, J = 9.4 Hz, 3H), 7.29 (s, 1H), 7.07 (s, 1H), 6.86 (s, 1H), 6.77 (s, 1H), 6.02 - 5.91 (m, 1H), 5.82 (d, J = 9.6 Hz, 1H), 5.78 - 5.64 (m, 1H), 5.40 - 5.33 (m, 1H), 5.29 - 5.24 (m, 1H), 5.16 - 4.94 (m, 2H), 4.67 (d, J = 5.7 Hz, 2H), 4.65 - 4.58 (m), 4.50 (t, J = 6.8 Hz, 2H), 4.47 - 4.38 (m, 1H), 4.30 - 4.20 (m, 2H), 4,01 - 3.93 (m, 1H), 3.51 (s, 3H), 3.38 - 3.28 (m, 1H), 3.09 - 3.00 (m, 1H), 2.44 - 2.35 (m, 2H). 13 C NMR (101 MHz, CDC13) δ 165.11, 163.86, 163.49, 155.92, 153.26, 150.47, 148.91, 137.17, 136.32, 135.14, 133.48, 132.43, 132.33, 131.73, 131.36, 129.72, 128.72, 128.69, 128.26, 126.69, 126.51, 125.53, 125.43, 124.77, 123.12, 123.11, 122.38, 118.78, 118.68, 118.32, 118.27, 117.97, 115.20, 114.38, 110.62, 86.08, 67.78, 66.84, 65.93, 59.65, 55.75, 38.03, 35..01, 27.85. HRMS (ESI, m / z) calcd for C 46 H 40 N4NaO 10 [M+Na]+ 831.2642, found: 831.2638. [a] D (CHCl3) 121.56°, c = 0.23 g / 100 mL.

[0238] Synthesis of compound 16b2:

[0239] Compound 15b2 was synthesized according to the above method to produce 16b2 (168.7 mg, 78%) as a yellow solid, which was purified by preparative TLC plate (developing solvent: DCM / MeOH = 20 / 1). 1 H NMR (400 MHz, CDC13) δ 9.93 (d, J = 9.1 Hz, 1H), 8.74 (s, 1H), 8.70 (dd, J = 7.1, 1.3 Hz, 1H), 8.30 (dd, J = 8.5, 1.2 Hz, 1H), 8.06 (d, J = 8.3 Hz, 1H), 7.85 - 7.76 (m, 1H), 7.69 (dd, J = 8.4, 7.0 Hz, 1H), 7.65 - 7.57 (m, 1H), 7.35 (d, J = 11.9 Hz, 3H), 7.24 (t, J = 4.3 Hz, 2H), 7.09 (s, 1H), 6.80 (s, 1H), 6.03 - 5.72 (m, 3H), 5.39 - 5.32 (m, 1H), 5.27 - 5.22 (m, 1H), 5.19 - 5.07 (m, 2H), 4.66 (d, J = 5.4 Hz, 3H), 4.56 (s, 1H), 4.50 - 4.41 (m, 1H), 4.35 - 4.27 (m, 2H), 4.12 (t, J = 6.4 Hz, 2H), 4.04 - 3.95 (m, 1H), 3.88 (s, 3H), 3.35 - 3.23 (m, 1H), 3.06 - 2.96 (m, 1H), 2.04 - 1.97 (m, 4H). 13C NMR (101 MHz, CDC13) δ 165.06, 163.76, 163.45, 155.90, 153.16, 150.64, 148.91, 137.06, 136.43, 135.19, 133.55, 133.51, 132.35, 131.71, 131.37, 129.75, 128.73, 128.69, 128.25, 126.64, 126.46, 125.51, 125.40, 124.66, 123.09, 123.06, 122.47, 122.31, 118.76, 118.68, 118.29, 118.11, 115.11, 114.10, 110.81, 86.04, 68.89, 66.85, 65.90, 59.57, 56.14, 40.07, 34.97, 26.69, 24.66. HRMS (ESI, m / z) calcd for C 47 H 42 N4NaO 10 [M+Na] + 845.2799, found: 845.2801.[α] D (CHCl3) 114.63°, c = 0.19 g / 100 mL.

[0240] Synthesis of compound 16b3:

[0241] Compound 15b3 was synthesized to 16b3 following the above procedure, the crude was purified on preparative TLC plate (eluent: DCM / MeOH = 20 / 1) to give the product 16b3 as a yellow solid (138.6 mg, 63%). 1H NMR (400 MHz, CDC13) δ 9.92 (t, J = 7.9 Hz, 1H), 8.76 - 8.65 (m, 2H), 8.32 - 8.23 (m, 1H), 8.04 (t, J = 6.4 Hz, 1H), 7.82 - 7.74 (m, 1H), 7.72 - 7.63 (m, 1H), 7.63 - 7.54 (m, 1H), 7.35 (d, J = 8.4 Hz, 3H), 7.25 - 7.20 (m, 3H), 6.78 (s, 1H), 5.99 - 5.83 (m, 2H), 5.82 - 5.68 (m, 1H), 5.37 - 5.29 (m, 1H), 5.25 - 5.19 (m, 1H), 5.11 (s, 1H), 5.08 (s, 1H), 4.64 (d, J = 5.7 Hz, 3H), 4.47 - 4.37 (m, 1H), 4.30 - 4.21 (m, 2H), 4.09 - 3.93 (m, 3H), 3.87 (s, 3H), 3.33 - 3.22 (m, 1H), 3.04 - 2.93 (m, 1H), 1.99 - 1.92 (m, 2H), 1.90 - 1.81 (m, 2H), 1.68 - 1.59 (m, 2H). 13 C NMR (101 MHz, CDC13) δ 165.06, 163.82, 163.49, 155.82, 153.17, 150.66, 148.84, 137.10, 136.39, 135.16, 133.59, 133.48, 132.36, 131.70, 131.35, 129.72, 128.67, 128.21, 126.64, 126.44, 125.50, 125.41, 124.52, 123.02, 123.01, 122.48, 122.30, 118.71, 118.66, 118.26, 118.00, 117.95, 115.10, 113.83, 110.75, 86.03, 68.93, 66.76, 65.86, 59.63, 56.14, 40.38, 34.96, 28.48, 27.67, 23.49. HRMS (ESI, m / z) calcd for C 48 H 44 N4O 10 [M+Na] + 859.2955, found: 859.2955.[α] D (CHCl3) 142.80°, c = 0.13 g / 100 mL.

[0242] General synthetic method for compounds 17a1-17b3:

[0243] In a 10 mL round-bottom flask, compound 16a1-16b3 (0.155 mmol) was dissolved in anhydrous DCM (3 mL), then tetrahydro-pyrrole (45 μL, 39 mg, 0.548 mmol) was added, and the mixture was stirred under nitrogen protection for 0.5 h at room temperature. TLC monitoring showed that the reaction was complete (eluent: dichloromethane / methanol = 20 / 1, ultraviolet coloration). DCM (10 mL) was added for dilution, and saturated ammonium chloride (4 mL) was added for extraction. The aqueous layer was extracted with DCM (10 mL*2), and the organic layers were combined and washed with saturated sodium chloride (10 mL). The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by preparative TLC plate to obtain yellow solid product 17a1-17b3.

[0244] Synthesis of compound 17a1

[0245] Compound 16a1 was synthesized according to the above method, and the crude product was purified by preparative TLC plate to obtain yellow solid product 17a1 (68.4 mg, 81%). The purity of 17a1 was detected by Waters Alliance e2695 HPLC system (equipped with Waters 2996 diode array detector), XBridge C18 (4.6 mm*150 mm, 5 μm) reversed-phase column, detection conditions: mobile phase A was acetonitrile containing 0.1% formic acid, mobile phase B was water containing 0.1% formic acid, gradient elution (0 min, mobile phase A was 10%; 0-10 min, mobile phase A was increased to 100%; 10-11 min, mobile phase A was kept at 100%; 11-15 min, mobile phase A was 10%), flow rate: 0.6 mL / min, temperature: 50°C, injection volume: 10 μL, detection wavelength: 264 nm. The purity of the compounds synthesized subsequently was detected by this method, and the HPLC purity of 17a1 was 97.05%. 1H NMR (400 MHz, CDC13) δ 9.96 (d, J = 9.1 Hz, 1H), 8.85 (s, 1H), 8.73 (dd, J = 7.1, 1.3 Hz, 1H), 8.37 (dd, J = 8.4, 1.3 Hz, 1H), 8.12 (d, J = 8.4 Hz, 1H), 7.84 - 7.78 (m, 1H), 7.77 - 7.75 (m, 1H), 7.63 (ddd, J = 7.9, 6.7, 1.1 Hz, 1H), 7.28 (s, 1H), 6.79 (s, 1H), 6.71 (d, J = 1.8 Hz, 1H), 4.53 (t, J = 6.7 Hz, 2H), 4.33 - 4.28 (m, 1H), 4.22 - 4.16 (m, 1H), 3.44 (s, 3H), 3.19 - 3.09 (m, 1H), 2.98 - 2.88 (m, 1H), 2.44 (t, J = 6.6 Hz, 2H), 1.82 (s, 3H), 0.88 (t, J = 6.7 Hz, 2H). 13 C NMR (101 MHz, CDC13) δ 165.11, 163.75, 162.50, 160.87, 151.00, 147.77, 140.12, 136.14, 134.95, 133.42, 133.34, 132.30, 131.16, 129.62, 128.69, 128.22, 126.72, 126.39, 125.36, 123.54, 122.47, 121.43, 119.11, 115.27, 111.39, 110.65, 67.57, 55.58, 53.75, 39.20, 38.08, 27.88, 13.63. HRMS (ESI, m / z) calcd for C 33 H 28 N3O5[M+H] + 546.2029, found: 546.2030.[α] D (CHCl3) 279.85°, c = 0.13 g / 100 mL.

[0246] Synthesis of compound 17a2

[0247] Compound 16a2 was synthesized according to the above procedure to give 17a2 as a yellow solid product (60.7 mg, 70%) after purification by preparative TLC plate. 1H NMR (400 MHz, CDC13) δ 10.01 (d, J = 9.1 Hz, 1H), 8.84 (s, 1H), 8.76 (dd, J = 7.1, 1.4 Hz, 1H), 8.37 (d, J = 8.4 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.84 (dd, J = 8.5, 6.7 Hz, 1H), 7.76 - 7.71 (m, 1H), 7.67 - 7.62 (m, 1H), 7.46 (s, 1H), 6.82 (s, 1H), 6.74 (d, J = 1.8 Hz, 1H), 4.38 (t, J = 6.9 Hz, 2H), 4.26 - 4.17 (m, 2H), 4.16 - 4.09 (m, 1H), 3.95 (s, 1H), 3.89 (s, 3H), 3.22 - 3.10 (m, 1H), 3.00 - 2.89 (m, 1H), 2.07 - 2.00 (m, 4H), 1.83 (d, J = 1.7 Hz, 3H). 13 CNMR (101 MHz, CDC13) δ 165.13, 163.73, 162.60, 160.91, 151.13, 147.94, 140.19, 136.38, 135.13, 133.53, 132.41, 132.03, 131.32, 129.73, 128.77, 128.33, 126.74, 126.45, 125.41, 123.59, 122.45, 121.45, 119.20, 115.25, 111.72, 110.81, 68.73, 56.15, 53.80, 40.06, 39.24, 26.66, 24.74, 13.64. HRMS (ESI, m / z) calcd for C 35 H 33 N3NaO6[M+CH3OH+Na] + 614.2267, found: 614.2264. HPLC purity 99.34%. [a] D (CHCl3) 46.90°, c = 0.13 g / 100 mL.

[0248] Synthesis of compound 17a3

[0249] Compound 16a3 was synthesized as described above for 17a3 and the crude product was purified using preparative TLC plate to give yellow solid product 17a3 (48.0 mg, 54%). 1H NMR (400 MHz, CDC13) δ 10.02 (d, J = 9.2 Hz, 1H), 8.84 (s, 1H), 8.76 (d, J = 6.9 Hz, 1H), 8.36 (d, J = 8.4 Hz, 1H), 8.12 (d, J = 8.5 Hz, 1H), 7.84 (ddd, J = 8.9, 6.7, 1.5 Hz, 1H), 7.79 (d, J = 4.0 Hz, 1H), 7.77 - 7.71 (m, 1H), 7.54 (d, J = 6.0 Hz, 1H), 7.47 (s, 1H), 6.79 (s, 1H), 6.74 (d, J = 2.0 Hz, 1H), 4.32 (t, J = 7.6 Hz, 2H), 4.17 - 4.04 (m, 2H), 4.16 - 4.06 (m, 2H), 3.89 (s, 3H), 3.21 - 2.10 (m, 1H), 3.00 - 2.89 (m, 1H), 2.03 - 1.96 (m, 2H), 1.93 - 1.87 (m, 2H), 1.83 (s, 3H), 1.70 - 1.65 (m, 2H). 13 C NMR (101 MHz, CDC13) δ 165.05, 163.62, 162.59, 160.92, 151.16, 147.91, 140.22, 136.24, 135.02, 133.41, 132.33, 131.24, 129.67, 128.68, 128.21, 126.70, 126.39, 125.36, 123.57, 122.41, 121.44, 119.11, 115.18, 112.58, 111.70, 110.67, 68.83, 56.14, 53.79, 40.37, 39.22, 28.71, 27.87, 23.64, 13.63. HRMS (ESI, m / z) calcd for C 35 H 32 N3O5[M+H] + 574.2342, found: 574.2338. HPLC purity 99.26%. [a] D (CHCl3) 43.33°, c = 0.15 g / 100 mL.

[0250] Synthesis of compound 17b1

[0251] Compound 16b1 was synthesized according to the above procedure to synthesize 17b1, the crude product was purified by preparative TLC plate to give yellow solid product 17b1 (73.3 mg, 76%). 1H NMR (400MHz, CDCl3) δ9.98(d,J=9.1Hz,1H),8.87(s,1H),8.74(d,J=7.0Hz,1H),8.38(d, J=8.3Hz,1H),8.14(d,J=8.5Hz,1H),7.84–7.79(m,1H),7.77–7.72(m,1H),7.67–7.62(m, 1H),7.30(d,J=5.7Hz,1H),7.21(d,J=8.3Hz,3H),6.81(s,1H),6.68(d,J=8.1Hz,2H),4.5 5(t,J=6.6Hz,3H),4.36–4.23(m,3H),3.44(s,3H),3.40–3.28(m,2H),2.49–2.41(m,2H). 13 C NMR (101MHz, CDCl) 3 / CDOD3=10 / 1)δ165.38,164.75,164.31,164.23,152.56,151.82,145.22,142.19,1 38.83,136.89,136.69,135.46,133.67,132.51,131.49,129.91,128.87,128.45, 126.59,126.15,126.02,125.54,124.04,123.26,122.42,121.17,119.53,114.25 ,112.27,107.46,67.60,58.65,55.81,38.18,35.83,27.95.HRMS(ESI,m / z)calcd for C 38 H 31 N4O5[M+H] + 623.2294,found:623.2286.HPLC purity 97.38%.[α] D (CHCl3)538.42°, c=0.13g / 100mL.

[0252] Synthesis of compound 17b2

[0253] Compound 16b2 was synthesized into 17b2 according to the above method. The crude product was purified by preparative TLC to give a yellow solid product 17b2 (85.8 mg, 87%). 1H NMR (400 MHz, CDC13) δ 10.03 (d, J = 9.1 Hz, 1H), 8.87 (s, 1H), 8.78 (d, J = 6.8 Hz, 1H), 8.38 (d, J = 8.5 Hz, 1H), 8.14 (d, J = 8.6 Hz, 1H), 7.88 - 7.83 (m, 1H), 7.78 - 7.73 (m, 1H), 7.69-7.63 (m, 1H), 7.49 (s, 1H), 7.33 (s, 1H), 7.21 (d, J = 8.7 Hz, 2H), 6.84 (s, 1H), 6.68 (d, J = 8.3 Hz, 2H), 4.44 - 4.30 (m, 3H), 4.24 - 4.13 (m, 2H), 3.91 (s, 3H), 3.86 - 3.78 (m, 1H), 3.60 - 3.52 (m, 1H), 3.41 - 3.29 (m, 1H), 2.05 - 1.94 (m, 4H). 13 C NMR (101 MHz, CDC13 / CD30D = 10 / 1) δ 164.97, 164.58, 163.78, 163.05, 152.63, 151.88, 145.12, 142.15, 138.85, 136.95, 136.48, 135.25, 133.46, 132.21, 131.28, 129.72, 128.56, 128.05, 126.34, 125.97, 125.82, 125.29, 123.85, 123.03, 122.03, 121.02, 119.31, 114.51, 112.46, 107.30, 68.58, 58.51, 56.17, 40.03, 35.72, 26.64, 24.58. HRMS (ESI, m / z) calcd for C 39 H 33 N4O5[M+H] + 637.2451, found: 637.2449. HPLC purity 95.77%. [a] D (CHCl3) 121.34°, c = 0.16 g / 100 mL.

[0254] Synthesis of compound 17b3

[0255] Compound 16b3 was synthesized according to the above procedure to give 17b3 as a yellow solid (76.6 mg, 76%) after purification of the crude product on a preparative TLC plate. 1H NMR (400 MHz, CDC13) δ 10.04 (d, J = 9.0 Hz, 1H), 8.86 (s, 1H), 8.78 (d, J = 6.9 Hz, 1H), 8.38 (d, J = 8.3 Hz, 1H), 8.14 (d, J = 8.7 Hz, 1H), 7.88 - 7.83 (m, 1H), 7.78 - 7.71 (m, 1H), 7.67 - 7.62 (m, 1H), 7.49 (s, 1H), 7.33 (s, 1H), 7.21 (d, J = 8.8 Hz, 2H), 6.81 (s, 1H), 6.68 (d, J = 8.1 Hz, 2H), 4.41 - 4.27 (m, 3H), 4.16-4.09 (m, 2H), 3.99 - 3.81 (m, 4H), 3.60 - 3.51 (m, 1H), 3.39 - 3.30 (m, 1H), 2.03 - 1.97 (m, 2H), 1.93 - 1.85 (m, 2H), 1.69 - 1.64 (m, 2H). 13 C NMR (101 MHz, CDC13 / CD30D = 10 / 1) δ 164.75, 164.58, 163.59, 163.04, 152.73, 151.97, 145.04, 142.08, 139.01, 137.08, 136.29, 135.10, 133.27, 132.01, 131.17, 129.63, 128.36, 127.83, 126.26, 125.94, 125.79, 125.18, 123.88, 122.93, 121.89, 120.90, 119.15, 114.56, 112.46, 107.07, 68.65, 58.50, 56.18, 40.31, 35.72, 28.57, 27.62, 23.43. HRMS (ESI, m / z) calcd for C 40 H 34 N4O5[M+H] + 651.2607, found: 651.2603. HPLC purity 95.53%. [a] D (CHC13) 166.62°, c = 0.17 g / 100 mL.

[0256] Example 4: Synthesis of compounds 20a1-20b3

[0257]

[0258] General method for synthesis of compounds 18a1-18b3:

[0259] Into a 25 mL round-bottom flask was added 9a1-9b3 (0.336 mmol), dissolved in anhydrous DMF (5 mL), followed by potassium carbonate (116.1 mg, 0.84 mmol) and 14 (108.0 mg, 0.437 mmol), and the reaction was stirred at 85 °C for 20 h under nitrogen protection. The reaction was monitored by TLC (eluent: petroleum ether / ethyl acetate = 2 / 1 and DCM / CH3OH = 10 / 1, UV coloration). The reaction was concentrated under reduced pressure, and the excess DMF was removed. The reaction was extracted with EA (15 mL) and pure water (5 mL), and the water layer was extracted with EA (15 mL*2). The organic layers were combined and washed with saturated sodium chloride (10 mL*2). The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography to give yellow solid product 18a1-18b3. Synthesis of compound 18a1:

[0260] Compound 9a1 and 14 were synthesized according to the above method to give yellow foam product 18a1 (169.3 mg, 57.7%) after column chromatography (mobile phase: DCM / CH3OH = 60 / 1-45 / 1). 1 H NMR (400 MHz, CDC13) δ 9.92 (d, J = 9.1 Hz, 1H), 8.74 (s, 1H), 8.69 (dd, J = 7.0, 1.3 Hz, 1H), 8.29 (d, J = 8.3 Hz, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.80 (ddd, J = 9.3, 6.6, 1.5 Hz, 1H), 7.69 (dd, J = 8.4, 7.0 Hz, 1H), 7.60 (dd, J = 8.2, 6.6 Hz, 1H), 7.29 (s, 1H), 6.69 (s, 1H), 6.62 (s, 1H), 5.87 (d, J = 8.9 Hz, 1H), 5.82 - 5.70 (m, 1H), 5.14 - 5.03 (m, 2H), 4.67 - 4.57 (m, 1H), 4.46 - 4.37 (m, 3H), 4.11 - 3.98 (m, 2H), 3.90 (s, 3H), 3.79 - 3.68 (m, 1H), 3.00 - 2.84 (m, 2H), 2.82 - 2.67 (m, 6H), 2.64 - 2.48 (m, 6H), 2.42 - 2.33 (m, 1H), 2.12 - 2.04 (m, 2H), 1.77 (s, 3H), 0.88 (s, 9H), 0.24 (d, J = 6.2 Hz, 6H). 13C NMR (101 MHz, CDC13) δ 165.04, 163.63, 163.40, 155.04, 150.23, 149.05, 136.27, 135.06, 133.50, 133.40, 132.37, 132.09, 131.26, 129.68, 128.74, 128.56, 128.29, 126.71, 126.43, 125.97, 125.39, 123.67, 122.44, 120.99, 117.31, 115.25, 114.26, 110.80, 87.00, 67.48, 66.32, 61.50, 56.11, 55.68, 54.95, 53.12, 53.04, 39.03, 37.61, 26.19, 25.63, 17.87, 13.78, -4.22, -5.29. HRMS (ESI, m / z) calcd for C 49 H 60 N5O8Si[M+H] + 874.4211, found: 874.4214. [a] D (CHCl3) 72.66°, c = 0.30 g / 100 mL.

[0261] Synthesis of compound 18a2:

[0262] Compound 9a2 and 14 were synthesized as 18a2 following the above described procedure. The crude product was purified by column chromatography (mobile phase: DCM / CH3OH = 60 / 1) to give the product 18a2 as a yellow foam (189.6 mg, 63.6%). 1H NMR (400 MHz, CDC13) δ 10.01 (d, J = 9.0 Hz, 1H), 8.87 (s, 1H), 8.76 (dd, J = 7.0, 1.3 Hz, 1H), 8.39 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 7.85 (ddd, J = 9.1, 6.6, 1.4 Hz, 1H), 7.75 (dd, J = 8.4, 7.1 Hz, 1H), 7.65 (ddd, J = 8.0, 6.7, 1.1 Hz, 1H), 7.21 (s, 1H), 6.68 (s, 1H), 6.58 (s, 1H), 5.86 (d, J = 8.9 Hz, 1H), 5.80 - 5.70 (m, 1H), 5.15 - 4.99 (m, 2H), 4.66 - 4.57 (m, 1H), 4.46 (t, J = 6.9 Hz, 3H), 4.43 - 4.36 (m, 1H), 4.07 - 3.92 (m, 2H), 3.89 - 3.80 (m, 3H), 3.77 - 3.68 (m, 1H), 3.32 - 2.15 (m, 16H), 1.90 - 1.85 (m, 2H), 1.77 (s, 3H), 0.87 (s, 9H), 0.23 (d, J = 10.3 Hz, 6H). 13 C NMR (101 MHz, CDC13) δ 165.05, 163.64, 163.42, 155.05, 150.21, 149.00, 136.28, 135.07, 133.51, 133.41, 132.38, 132.10, 131.27, 129.69, 128.75, 128.53, 128.30, 126.72, 126.44, 125.85, 125.40, 123.68, 122.45, 120.97, 117.28, 115.26, 114.01, 110.73, 87.00, 68.76, 66.31, 61.49, 57.99, 56.08, 55.68, 53.08, 52.96, 39.03, 37.61, 26.85, 25.61, 23.14, 17.86, 13.78, -4.24, -5.30. HRMS (ESI, m / z) calcd for C 50 H 62 N5O8Si[M+H] + 888.4368, found: 888.4363.[α] D (CHCl3) 53.56°, c = 0.26 g / 100 mL.

[0263] Synthesis of compound 18a3:

[0264] Compound 9a3 and 14 were synthesized according to the above method for 18a3, the crude product was purified by column chromatography (mobile phase: DCM / CH3OH = 60 / 1) to give the yellow foamy product 18a3 (202.7 mg, 66.9%). 1 H NMR (400 MHz, CDC13) δ 10.02 (d, J = 9.1 Hz, 1H), 8.88 (s, 1H), 8.77 (dd, J = 7.1, 1.3 Hz, 1H), 8.40 (d, J = 8.2 Hz, 1H), 8.15 (d, J = 8.5 Hz, 1H), 7.90 - 7.81 (m, 1H), 7.76 (dd, J = 8.4, 7.1 Hz, 1H), 7.66 (t, J = 7.5 Hz, 1H), 7.22 (d, J = 2.7 Hz, 1H), 6.68 (s, 1H), 6.58 (s, 1H), 5.86 (d, J = 8.8 Hz, 1H), 5.79 - 5.69 (m, 1H), 5.13 - 5.02 (m, 2H), 4.66 - 4.58 (m, 1H), 4.46 (t, J = 6.9 Hz, 2H), 4.43 - 4.36 (m, 1H), 4.03 - 3.92 (m, 2H), 3.88 (s, 3H), 3.78 - 3.68 (m, 1H), 3.27 - 2.23 (m, 16H), 1.91 - 1.82 (m, 2H), 1.77 (s, 3H), 1.54 - 1.46 (m, 2H), 0.87 (s, 9H), 0.23 (d, J = 9.7 Hz, 6H). 13 C NMR (101 MHz, CDC13) δ 164.97, 163.57, 163.43, 155.05, 150.28, 148.99, 136.20, 135.01, 133.42, 133.34, 132.30, 132.10, 131.21, 129.64, 128.68, 128.55, 128.20, 126.68, 126.40, 125.79, 125.35, 123.68, 122.38, 120.94, 117.26, 115.17, 114.01, 110.74, 87.00, 68.91, 66.30, 61.50, 58.41, 56.12, 55.70, 53.20, 53.09, 39.04, 37.62, 28.79, 26.38, 25.61, 24.02, 17.86, 13.78, -4.25, -5.29. HRMS (ESI, m / z) calcd for C 51 H 64 N5O8Si[M+H] + 902.4524, found: 902.4518.[α] D(CHCl3) 60.46°, c = 0.34 g / 100 mL.

[0265] Synthesis of compound 18b1:

[0266] Compound 9b1 and 14 were synthesized according to the above method for 18b1, the crude product was purified by column chromatography (mobile phase: DCM / CH3OH = 50 / 1-30 / 1) to give yellow foamy product 18b1 (184.5 mg, 52.4%). 1 H NMR (400 MHz, CDC13) δ 10.02 (d, J = 9.2 Hz, 1H), 8.86 (s, 1H), 8.76 (d, J = 7.0 Hz, 1H), 8.38 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 7.88 - 7.81 (m, 1H), 7.75 (t, J = 7.7 Hz, 1H), 7.69 - 7.61 (m, 1H), 7.37 (d, J = 9.5 Hz, 3H), 7.30 (s, 1H), 7.25 (s, 1H), 6.73 (s, 1H), 6.64 (s, 1H), 6.04 - 5.89 (m, 2H), 5.83 - 5.70 (m, 1H), 5.42 - 5.32 (m, 1H), 5.27 (d, J = 10.5 Hz, 1H), 5.19 - 4.97 (m, 2H), 4.71 - 4.57 (m, 3H), 4.53 - 4.32 (m, 3H), 4.14 - 3.97 (m, 2H), 3.96 - 3.84 (m, 4H), 3.38 - 3.25 (m, 1H), 3.05 - 2.30 (m, 13H), 2.10 - 1.98 (m, 2H), 0.91 (s, 9H), 0.25 (d, J = 9.5 Hz, 6H). 13C NMR (101 MHz, CDC13) δ 164.93, 163.86, 163.55, 155.04, 153.14, 150.58, 149.16, 137.22, 136.17, 134.98, 133.36, 133.30, 132.43, 132.25, 132.01, 131.16, 129.62, 128.73, 128.62, 128.14, 126.64, 126.36, 125.58, 125.41, 125.31, 122.93, 122.48, 122.32, 118.73, 118.18, 117.39, 115.11, 114.26, 110.83, 86.79, 67.59, 66.40, 65.79, 61.50, 56.13, 55.77, 54.93, 53.34, 53.21, 37.60, 35.06, 26.36, 25.67, 17.90, -4.20, -5.19. HRMS (ESI, m / z) calcd for C 58 H 67 N6O 10 Si[M+H] + 1035.4688, found: 1035.4680. [a] D (CHCl3) 132.46°, c = 0.23 g / 100 mL.

[0267] Synthesis of compound 18b2:

[0268] Compound 9b2 and 14 were synthesized as 18b2 following the above described procedure. The crude was purified by column chromatography (mobile phase: DCM / CH3OH = 50 / 1-30 / 1) to give the product 18b2 as a yellow foam (189.6 mg, 63.6%). 1H NMR (400 MHz, CDC13) δ 10.00 (d, J = 9.2 Hz, 1H), 8.85 (s, 1H), 8.75 (dd, J = 7.1, 1.3 Hz, 1H), 8.38 (dd, J = 8.5, 1.3 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.85 (ddd, J = 9.2, 6.6, 1.4 Hz, 1H), 7.75 (dd, J = 8.4, 7.1 Hz, 1H), 7.65 (ddd, J = 8.0, 6.6, 1.1 Hz, 1H), 7.38 (t, J = 7.9 Hz, 3H), 7.29 (s, 1H), 7.23 (s, 1H), 6.78 (s, 1H), 6.61 (s, 1H), 6.04 - 5.89 (m, 2H), 5.82 - 5.70 (m, 1H), 5.42 - 5.33 (m, 1H), 5.27 (dd, J = 10.5, 1.3 Hz, 1H), 5.16 - 5.01 (m, 2H), 4.71 - 4.59 (m, 3H), 4.49 - 4.37 (m, 3H), 4.07 - 3.94 (m, 2H), 3.93 - 3.83 (m, 4H), 3.37 - 3.24 (m, 1H), 2.99 - 2.51 (m, 13H), 1.93 - 1.81 (m, 4H), 0.91 (s, 9H), 0.25 (d, J = 12.6 Hz, 6H). 13 C NMR (101 MHz, CDC13) δ 165.09, 163.89, 163.69, 155.04, 153.11, 150.49, 149.12, 137.08, 136.35, 135.12, 133.54, 133.46, 132.40, 132.05, 131.30, 129.73, 128.90, 128.78, 128.64, 128.34, 126.73, 126.46, 125.57, 125.47, 125.42, 123.03, 122.45, 118.78, 118.83, 118.28, 117.39, 115.27, 114.08, 110.79, 86.79, 68.78, 66.41, 65.89, 61.52, 57.95, 56.13, 55.68, 53.03, 52.86, 37.59, 35.09, 26.85, 25.66, 23.06, 17.91, -4.20, -5.18. HRMS (ESI, m / z) calcd for C 59 H 69 N6O 10 Si[M+H] + 1049.4844, found: 1049.4843. [a]D (CHCl3) 9.21°, c = 0.25 g / 100 mL.

[0269] Synthesis of compound 18b3:

[0270] Compound 9b3 and 14 were synthesized as described above for 18b3, the crude was purified by column chromatography (mobile phase: DCM / CH3OH = 50 / 1-30 / 1) to give the product 18b3 as a yellow foam (223.8 mg, 62.7%). 1 H NMR (400 MHz, CDC13) δ 10.01 (d, J = 9.2 Hz, 1H), 8.86 (s, 1H), 8.76 (dd, J = 7.1, 1.3 Hz, 1H), 8.39 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 7.90 - 7.81 (m, 1H), 7.75 (dd, J = 8.4, 7.1 Hz, 1H), 7.65 (dd, J = 8.3, 6.7 Hz, 1H), 7.39 (d, J = 5.8 Hz, 2H), 7.31 - 7.29 (m, 1H), 7.24 (d, J = 1.3 Hz, 1H), 6.75 - 6.68 (m, 1H), 6.62 (s, 1H), 6.03 - 5.90 (m, 2H), 5.78 - 5.71 (m, 1H), 5.41 - 5.33 (m, 1H), 5.29 - 5.25 (m, 1H), 5.15 - 5.01 (m, 2H), 4.71 - 4.59 (m, 3H), 4.50 - 4.37 (m, 3H), 4.05 - 3.94 (m, 2H), 3.93 - 3.85 (m, 4H), 3.37 - 3.27 (m, 1H), 3.01 - 2.38 (m, 13H), 1.92 - 1.83 (m, 2H), 1.74 - 1.64 (m, 2H), 1.55 - 1.48 (m, 2H), 0.91 (s, 9H), 0.25 (d, J = 12.3 Hz, 6H). 13C NMR (101 MHz, CDC13) δ 164.97, 163.89, 163.60, 155.05, 153.16, 150.58, 149.12, 137.22, 136.23, 135.03, 133.37, 132.44, 132.29, 132.04, 131.21, 129.66, 128.78, 128.65, 128.19, 126.67, 126.39, 125.49, 125.43, 125.34, 122.96, 122.48, 122.35, 118.76, 118.76, 118.21, 117.36, 115.13, 114.07, 110.80, 86.80, 68.95, 66.41, 65.82, 61.52, 58.41, 56.16, 55.74, 53.20, 53.10, 37.61, 35.09, 28.80, 26.39, 25.66, 24.03, 17.90, -4.21, -5.17. HRMS (ESI, m / z) calcd for C 60 H 71 N6O 10 Si[M+H] + 1063.5001, found: 1063.4995. [a] D (CHCl3) 124.95°, c = 0.23 g / 100 mL.

[0271] General synthetic method of compounds 19a1-19b3

[0272] In a 10 mL round-bottom flask, 19a1-19b3 (0.263 mmol) was dissolved in anhydrous THF (3 mL), then TBAF (1 mol / L in THF, 0.53 mL, 0.526 mmol) and glacial acetic acid (45 μL, 47 mg, 0.789 mmol) were added. The reaction was stirred at 25 °C for 16 h under nitrogen protection. TLC monitoring showed that the reaction was complete (eluent: DCM / MeOH = 15 / 1, UV coloration). The reaction solution was concentrated under reduced pressure to remove excess THF, then DCM (10 mL) was added. The aqueous layer was extracted with DCM (10 mL*2), and the organic layer was washed with saturated sodium chloride (10 mL*2). The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by preparative TLC plate to obtain yellow solid product 19a1-19b3.

[0273] Synthesis of compound 19a1:

[0274] Compound 18a1 was synthesized according to the above method, and the crude product was purified by TLC plate (eluent: DCM / MeOH = 15 / 1) to give yellow solid product 19a1 (163.6 mg, 81.9%). 1 H NMR (400 MHz, CDC13) δ 10.01 (d, J = 9.2 Hz, 1H), 8.86 (s, 1H), 8.76 (dd, J = 7.1, 1.3 Hz, 1H), 8.44 - 8.35 (m, 1H), 8.14 (d, J = 8.4 Hz, 1H), 7.85 (ddd, J = 9.2, 6.6, 1.4 Hz, 1H), 7.75 (dd, J = 8.4, 7.1 Hz, 1H), 7.69 - 7.62 (m, 1H), 7.21 (s, 1H), 6.70 (s, 2H), 5.76 (d, J = 9.9 Hz, 2H), 5.17 - 5.01 (m, 2H), 4.70 - 4.62 (m, 1H), 4.46 (t, J = 7.1 Hz, 3H), 4.09 - 3.99 (m, 2H), 3.88 (s, 3H), 3.85 - 3.80 (m, 1H), 3.01 - 2.92 (m, 1H), 2.86 - 2.43 (m, 13H), 1.92 - 1.83 (m, 2H), 1.77 (s, 3H). 13 C NMR (101 MHz, CDC13) δ 165.09, 163.67, 162.95, 155.85, 150.35, 148.79, 136.39, 135.16, 133.55, 133.50, 132.41, 131.86, 131.32, 129.73, 128.78, 128.34, 128.27, 126.72, 126.47, 125.43, 125.15, 123.29, 122.43, 121.58, 117.91, 115.25, 114.09, 110.84, 86.12, 67.58, 66.67, 59.64, 56.14, 55.65, 54.72, 53.20, 53.09, 38.88, 37.59, 26.23, 13.73. HRMS (ESI, m / z) calcd for C 43 H 46 N5O8[M+H] + 760.3346, found: 760.3339.[α] D (CHCl3) 85.11°, c = 0.30 g / 100 mL.

[0275] Synthesis of compound 19a2:

[0276] Compound 18a2 was synthesized according to the above method, the crude product was purified by preparative TLC plate (eluent: DCM / MeOH = 15 / 1) to give yellow solid product 19a2 (174.3 mg, 85.7%). 1 H NMR (400 MHz, CDC13) δ 10.00 - 9.89 (m, 1H), 8.81 - 8.66 (m, 2H), 8.37 - 8.26 (m, 1H), 8.13 - 8.02 (m, 1H), 7.88 - 7.56 (m, 3H), 7.34 - 7.27 (m, 1H), 6.84 - 6.69 (m, 2H), 5.91 - 5.71 (m, 2H), 5.20 - 5.05 (m, 2H), 4.74 - 4.60 (m, 1H), 4.52 - 4.36 (m, 3H), 4.15 - 4.05 (m, 2H), 3.96 - 3.82 (m, 4H), 3.05 - 2.92 (m, 1H), 2.84 - 2.43 (m, 13H), 2.08 - 1.99 (m, 2H), 1.82 (s, 1H), 1.78 (s, 2H), 1.31 - 1.22 (m, 2H). 13 C NMR (101 MHz, CDC13) δ 165.08, 163.67, 162.97, 155.82, 150.38, 148.74, 136.37, 135.15, 133.53, 133.46, 132.39, 131.87, 131.32, 129.73, 128.77, 128.32, 128.25, 126.71, 126.46, 125.41, 125.03, 123.29, 122.43, 121.51, 117.80, 115.24, 113.87, 110.75, 86.08, 68.98, 66.63, 59.67, 58.02, 56.12, 55.61, 53.19, 53.07, 38.87, 37.61, 27.08, 23.18, 13.72. HRMS (ESI, m / z) calcd for C 44 H 48 N5O8[M+H] + 774.3503, found: 774.3502.[α] D (CHCl3) 39.15°, c = 0.39 g / 100 mL.

[0277] Synthesis of compound 19a3:

[0278] Compound 18a3 was synthesized according to the above method, the crude product was purified by preparative TLC plate (eluent: DCM / MeOH = 15 / 1) to give yellow solid product 19a3 (131.7 mg, 63.6%).1 H NMR (400 MHz, CDC13) δ 9.93 (d, J = 9.2 Hz, 1H), 8.75 (s, 1H), 8.69 (d, J = 7.0 Hz, 1H), 8.30 (d, J = 8.3 Hz, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.84 - 7.75 (m, 1H), 7.69 (t, J = 7.7 Hz, 1H), 7.61 (t, J = 7.5 Hz, 1H), 7.23 (s, 1H), 6.72 (s, 2H), 5.87 - 5.71 (m, 2H), 5.23 - 5.03 (m, 2H), 4.71 - 4.61 (m, 1H), 4.49 - 4.35 (m, 3H), 3.99 (d, J = 6.7 Hz, 2H), 3.90 (s, 3H), 3.86 - 3.78 (m, 1H), 3.03 - 2.91 (m, 1H), 2.83 - 2.69 (m, 5H), 2.66 - 2.44 (m, 6H), 2.36 (t, J = 7.8 Hz, 2H), 1.89 - 1.80 (m, 2H), 1.78 (s, 3H), 1.60 - 1.52 (m, 2H), 1.48 - 1.41 (m, 2H). 13 C NMR (101 MHz, CDC13) δ 164.99, 163.59, 163.01, 155.75, 150.36, 148.74, 136.31, 135.09, 133.48, 133.42, 132.35, 131.96, 131.28, 129.69, 128.73, 128.33, 128.27, 126.70, 126.44, 125.39, 125.12, 123.30, 122.40, 121.59, 117.73, 115.19, 113.88, 110.78, 86.05, 68.95, 66.57, 59.81, 58.38, 56.15, 55.60, 53.14, 53.09, 38.92, 37.61, 28.79, 26.28, 23.96, 13.75. HRMS (ESI, m / z) calcd for C 45 H 50 N5O8[M+H] + 788.3659, found: 788.3658.[α] D (CHCl3) 20.07°, c = 0.45 g / 100 mL.

[0279] Synthesis of compound 19b1:

[0280] Compound 18b1 was synthesized according to the above procedure for 19b1, and the crude product was purified by TLC plate (eluent: DCM / MeOH = 15 / 1) to give yellow solid product 19b1 (115.7 mg, 47.8%). 1 H NMR (400 MHz, CDC13) δ 9.97 (d, J = 9.1 Hz, 1H), 8.81 (s, 1H), 8.72 (d, J = 7.0 Hz, 1H), 8.34 (d, J = 8.3 Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.79 (dd, J = 9.2, 6.6 Hz, 1H), 7.71 (t, J = 7.7 Hz, 1H), 7.59 (t, J = 7.5 Hz, 1H), 7.40 (s, 1H), 7.34 (d, J = 8.4 Hz, 2H), 7.28 (s, 1H), 7.25 (s, 1H), 6.95 (s, 1H), 6.82 (s, 1H), 6.02 - 5.87 (m, 2H), 5.84 - 5.72 (m, 1H), 5.40 - 5.32 (m, 1H), 5.26 (d, J = 10.4 Hz, 1H), 5.12 (d, J = 11.3 Hz, 2H), 4.66 (d, J = 6.0 Hz, 3H), 4.45 (t, J = 7.3 Hz, 3H), 4.16 - 4.07 (m, 2H), 4.04 - 3.97 (m, 1H), 3.91 (s, 3H), 3.39 - 3.29 (m, 1H), 3.09 - 3.01 (m, 1H), 2.84 - 2.66 (m, 6H), 2.62 - 2.42 (m, 7H), 2.05 - 1.98 (m, 2H). 13 C NMR (101 MHz, CDC13) δ 165.18, 163.73, 163.44, 155.81, 153.10, 150.67, 148.89, 137.02, 136.47, 135.22, 133.60, 133.56, 132.45, 132.37, 131.82, 131.39, 129.76, 128.90, 128.83, 128.40, 126.70, 126.50, 125.57, 125.46, 124.81, 122.95, 122.63, 122.46, 118.77, 118.34, 117.99, 115.30, 114.15, 110.88, 85.94, 67.65, 66.76, 65.92, 59.63, 56.18, 55.70, 54.62, 53.30, 53.14, 37.60, 35.11, 26.21. HRMS (ESI, m / z) calcd for C 52 H 53 N6O 10[M+H] + 921.3823, found: 921.3823. [α] D (CHCl3) 35.68°, c = 0.32 g / 100 mL.

[0281] Synthesis of compound 19b2:

[0282] Compound 18b2 was synthesized according to the above method to give 19b2 (222.9 mg, 90.7%) as a yellow solid. 1 H NMR (400 MHz, CDC13) δ 10.00 (d, J = 9.2 Hz, 1H), 8.85 (d, J = 5.1 Hz, 1H), 8.75 (dt, J = 7.1, 1.8 Hz, 1H), 8.41 - 8.34 (m, 1H), 8.12 (d, J = 8.3 Hz, 1H), 7.87 - 7.78 (m, 1H), 7.73 (t, J = 7.8 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.39 (s, 1H), 7.36 (d, J = 8.2 Hz, 2H), 7.29 (s, 1H), 7.24 (s, 1H), 6.81 (s, 1H), 6.03 - 5.92 (m, 1H), 5.88 - 5.72 (m, 2H), 5.42 - 5.33 (m, 1H), 5.29 - 5.25 (m, 1H), 5.12 (d, J = 11.7 Hz, 2H), 4.71 - 4.61 (m, 3H), 4.45 (t, J = 7.1 Hz, 3H), 4.14 - 3.98 (m, 3H), 3.89 (s, 3H), 3.39 - 3.29 (m, 1H), 2.97 - 2.34 (m, 13H), 1.92 - 1.85 (m, 2H), 1.80 - 1.72 (m, 2H). 13C NMR (101 MHz, CDC13) δ 165.20, 163.76, 163.46, 155.79, 153.10, 150.68, 148.83, 137.00, 136.49, 135.23, 133.61, 133.54, 132.46, 132.37, 131.82, 131.40, 129.78, 128.90, 128.83, 128.41, 126.71, 126.49, 125.55, 125.45, 124.68, 122.86, 122.63, 122.47, 118.74, 118.34, 117.90, 115.30, 113.90, 110.80, 85.92, 69.09, 66.73, 65.92, 59.65, 58.01, 56.16, 55.63, 53.28, 53.12, 37.65, 35.09, 27.11, 23.17. HRMS (ESI, m / z) calcd for C 53 H 55 N6O 10 [M+H] + 935.3980, found: 935.3982. [a] D (CHCl3) 176.00°, c = 0.16 g / 100 mL.

[0283] Synthesis of compound 19b3:

[0284] Compound 18b3 was synthesized according to the above method to give 19b3 (218.5 mg, 87.6%) as a yellow solid product, which was purified by preparative TLC plate (developing solvent: DCM / MeOH = 15 / 1). 1H NMR (400 MHz, CDC13) δ 10.00 (d, J = 9.2 Hz, 1H), 8.85 (d, J = 5.1 Hz, 1H), 8.75 (dt, J = 7.1, 1.8 Hz, 1H), 8.41 - 8.34 (m, 1H), 8.12 (d, J = 8.3 Hz, 1H), 7.87 - 7.78 (m, 1H), 7.73 (t, J = 7.8 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.39 (s, 1H), 7.36 (d, J = 8.2 Hz, 2H), 7.29 (s, 1H), 7.24 (s, 1H), 6.87 - 6.70 (m, 2H), 6.03 - 5.92 (m, 1H), 5.91 - 5.74 (m, 2H), 5.40 - 5.33 (m, 1H), 5.29 - 5.24 (m, 1H), 5.12 (d, J = 11.7 Hz, 2H), 4.71 - 4.61 (m, 3H), 4.45 (t, J = 7.1 Hz, 3H), 4.14 - 3.98 (m, 3H), 4.07 - 3.95 (m, 3H), 3.41 - 3.29 (m, 1H), 3.12 - 03 (m, 1H), 2.95 - 2.35 (m, 13H), 1.91 - 1.82 (m, 2H), 1.69 - 1.58 (m, 2H), 1.50 - 1.43 (m, 2H). 13 C NMR (101 MHz, CDC13) δ 165.07, 163.67, 163.50, 155.75, 153.14, 150.61, 148.83, 137.07, 136.42, 135.18, 133.55, 133.52, 132.41, 131.93, 131.37, 129.75, 128.85, 128.79, 128.47, 128.35, 126.71, 126.49, 125.55, 125.44, 124.83, 123.03, 122.57, 122.42, 118.75, 118.30, 117.84, 115.23, 114.00, 110.85, 85.90, 68.91, 66.70, 65.89, 59.82, 58.19, 56.18, 55.51, 52.97, 52.72, 37.60, 35.11, 28.61, 25.84, 23.82. HRMS (ESI, m / z) calcd for C 54 H 57 N6O 10 [M+H] + 949.4136, found: 949.4133.[α] D (CHCl3) 32.59°, c = 0.38 g / 100 mL.

[0285] General synthesis method of compounds 20a1-20b3:

[0286] In a 10 mL round-bottom flask, compound 19a1-19b3 (0.155 mmol) was dissolved in anhydrous DCM (3 mL), and then tetrahydro-pyrrole (45 μL, 39 mg, 0.548 mmol) was added under nitrogen protection. Pd[P(Ph)3]4 (11.5 mg, 0.01 mmol) was added under nitrogen protection, and the mixture was stirred at room temperature for 0.5 h. TLC monitoring showed that the reaction was complete (eluent: dichloromethane / methanol = 15 / 1, ultraviolet coloration). The reaction mixture was diluted with DCM (10 mL), and then extracted with saturated ammonium chloride (4 mL). The aqueous layer was extracted with DCM (10 mL*2), and the combined organic layers were washed with saturated sodium chloride (10 mL). The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by a preparative TLC plate to obtain yellow solid product 20a1-20b3.

[0287] Synthesis of compound 20a1:

[0288] Compound 19a1 was synthesized according to the above method to synthesize 20a1. The crude product was purified by a preparative TLC plate to obtain yellow solid product 20a1 (70.6

[0289] mg, 69.3%). 1 H NMR (400 MHz, CDCl3) δ 9.95 (d, J = 9.2 Hz, 1H), 8.77 (s, 1H), 8.71 (d, J = 7.1

[0290] Hz, 1H), 8.31 (d, J = 8.4 Hz, 1H), 8.08 (d, J = 8.5 Hz, 1H), 7.81-7.78 (m, 1H), 7.70 (t, J = 7.7 Hz, 1H), 7.61 (t, J = 7.7 Hz, 1H), 7.49 (s, 1H), 6.82 (s, 1H), 6.73 (s, 1H), 4.44 (t, J = 7.3 Hz, 2H), 4.27-4.20 (m, 1H), 4.19-4.06 (m, 2H), 3.91 (d, J = 10.2 Hz, 3H), 3.86-3.78 (m, 1H), 3.22-3.10 (m, 1H), 3.00-2.90 (m, 1H), 2.81-2.69 (m, 5H), 2.57-2.47 (m, 7H), 2.09-2.01 (m, 2H), 1.82 (s, 3H). 13C NMR (101 MHz, CDC13) δ 164.94, 164.92, 163.54, 162.66, 160.85, 151.04, 147.88, 140.21, 136.19, 135.00, 133.32, 132.24, 131.20, 129.63, 128.61, 128.13, 126.63, 126.38, 125.32, 123.54, 122.31, 121.48, 119.25, 115.08, 111.73, 110.84, 67.39, 56.15, 55.67, 54.85, 53.78, 53.48, 53.12, 39.21, 37.57, 26.22, 13.63. HRMS (ESI, m / z) calcd for C 40 H 44 N5O6[M+CH3OH+H] + 690.3292, found: 690.3291. HPLC purity 95.54%. [a] D (CHC13) 123.21 °, c = 0.16 g / 100 mL. Synthesis of compound 20a2:

[0291] Compound 19a2 was synthesized as above for 20a2, the crude product was purified with preparative TLC plate to give yellow solid product 20a2 (80.5 mg, 77.4%). 1 H NMR (400 MHz, CDC13) δ 10.05 - 9.97 (m, 1H), 8.86 (s, 1H), 8.76 (dd, J = 7.1, 1.3 Hz, 1H), 8.38 (dd, J = 8.4, 1.4 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 7.85 (ddd, J = 9.3, 6.6, 1.4 Hz, 1H), 7.75 (dd, J = 8.4, 7.0 Hz, 1H), 7.65 (ddd, J = 8.0, 6.7, 1.1 Hz, 1H), 7.49 (s, 1H), 6.78 (s, 1H), 6.74 (q, J = 1.9 Hz, 1H), 4.46 (t, J = 7.1 Hz, 2H), 4.29 - 4.19 (m, 1H), 4.16 - 3.99 (m, 3H), 3.91 (s, 3H), 3.21 - 3.12 (m, 1H), 2.99 - 2.91 (m, 1H), 2.88 - 2.68 (m, 7H), 2.66 - 2.48 (m, 5H), 1.92 - 1.87 (m, 2H), 1.83 (s, 3H), 1.79 - 1.73 (m, 2H). 13C NMR (101 MHz, CDC13) δ 165.10, 163.69, 162.65, 160.91, 151.10, 147.92, 140.19, 136.33, 135.11, 133.56, 133.47, 132.42, 131.30, 129.72, 128.80, 128.36, 126.76, 126.47, 125.43, 123.59, 122.49, 121.44, 119.19, 115.33, 111.70, 110.66, 68.80, 58.08, 56.16, 55.72, 53.80, 53.27, 53.18, 39.23, 37.68, 26.91, 23.26, 13.63. HRMS (ESI, m / z) calcd for C 41 H 46 N5O6[M+CH3OH+H] + 704.3448, found: 704.3448. HPLC purity 95.28%. [a] D (CHC13) 273.96°, c = 0.15 g / 100 mL.

[0292] Synthesis of compound 20a3:

[0293] Compound 19a3 was synthesized to 20a3 following the above procedure, the crude product was purified with preparative TLC plate to give yellow solid product 20a3 (80.5 mg, 77.4%). 1 H NMR (400 MHz, CDC13) δ 10.02 - 9.90 (m, 1H), 8.84 - 8.66 (m, 2H), 8.39 - 8.27 (m, 1H), 8.14 - 8.02 (m, 1H), 7.80 (s, 1H), 7.75 - 7.66 (m, 1H), 7.66 - 7.58 (m, 1H), 7.49 (d, J = 2.2 Hz, 1H), 6.81 - 6.69 (m, 2H), 4.49 - 4.37 (m, 2H), 4.27 - 4.19 (m, 1H), 4.10 - 3.98 (m, 2H), 3.93 (s, 3H), 3.83 (dd, J = 6.9, 2.9 Hz, 1H), 3.22 - 3.10 (m, 1H), 2.99 - 2.91 (m, 1H), 2.81 - 2.68 (m, 5H), 2.65 - 2.44 (m, 5H), 2.43 - 2.35 (m, 2H), 1.94 - 1.80 (m, 2H), 1.83 (s, 3H), 1.62 - 1.54 (m, 2H), 1.52 - 1.44 (m, 2H). 13C NMR (101 MHz, CDC13) δ 164.85, 163.48, 162.66, 160.83, 151.08, 147.85, 140.19, 136.14, 134.96, 133.27, 133.25, 132.17, 131.15, 129.60, 128.54, 128.05, 126.56, 126.33, 125.27, 123.50, 122.22, 121.48, 119.14, 114.98, 111.67, 110.63, 68.82, 58.33, 56.14, 55.65, 53.77, 53.12, 52.95, 39.19, 37.55, 28.73, 26.21, 23.92, 13.62. HRMS (ESI, m / z) calcd for C 42 H 48 N5O6[M+CH3OH+H] + 718.3605, found: 718.3589. [a] D (CHCl3) 49.24°, c = 0.22 g / 100 mL. Synthesis of compound 20b1:

[0294] Compound 19b1 was synthesized as above for 20b1, the crude product was purified with preparative TLC plate to give yellow solid product 20b1 (74.4 mg, 65.4%). 1 H NMR (400 MHz, DMSO-d6) δ 9.79 (d, J = 9.7 Hz, 1H), 9.14 - 9.03 (m, 1H), 8.62 - 8.46 (m, 2H), 8.21 (d, J = 8.7 Hz, 1H), 7.89 - 7.75 (m, 2H), 7.66 (t, J = 8.0 Hz, 1H), 7.45 - 7.34 (m, 1H), 7.33 - 7.25 (m, 1H), 7.22 - 7.12 (m, 1H), 7.08 (d, J = 8.0 Hz, 1H), 6.64 - 6.48 (m, 2H), 6.45 - 6.32 (m, 1H), 5.34 - 5.09 (m, 2H), 4.62 - 4.49 (m, 1H), 4.27 - 4.07 (m, 3H), 4.00 - 3.85 (m, 2H), 3.73 - 3.61 (m, 3H), 3.22 (s, 2H), 2.64 - 2.47 (m, 7H), 2.44 - 2.27 (m, 5H), 1.94 - 1.80 (m, 2H). 13C NMR (101 MHz, CDC13 / CD30D = 10 / 1) δ 164.92, 164.64, 163.73, 163.12, 151.82, 146.17, 145.12, 142.07, 136.50, 135.29, 133.45, 133.35, 132.22, 131.34, 129.77, 128.58, 128.10, 126.42, 126.06, 125.92, 125.34, 124.05, 123.30, 122.99, 122.08, 120.96, 114.81, 112.56, 109.06, 107.37, 67.26, 58.63, 56.30, 56.27, 55.58, 54.97, 52.92, 37.43, 35.83, 26.14. HRMS (ESI, m / z) calcd for C 45 H 47 N6O6[M+CH3OH+H] + 767.3557, found: 767.3553. HPLC purity 95.53%. [a] D (CHCl3) 226.58°, c = 0.19 g / 100 mL.

[0295] Synthesis of compound 20b2:

[0296] Compound 19b2 was synthesized to 20b2 following the above procedure, the crude was purified with preparative TLC plate to give the product 20b2 (53.7 mg, 46.3%) as a yellow solid. 1H NMR (400 MHz, CDC13) δ 10.05 - 9.95 (m, 1H), 8.85 (s, 1H), 8.76 (d, J = 7.2 Hz, 1H), 8.37 (d, J = 8.3 Hz, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.88 - 7.82 (m, 1H), 7.74 (t, J = 7.7 Hz, 1H), 7.64 (t, J = 7.5 Hz, 1H), 7.52 (d, J = 4.1 Hz, 1H), 7.35 - 7.30 (m, 1H), 7.23 - 7.13 (m, 2H), 6.81 (s, 1H), 6.65 (t, J = 9.2 Hz, 2H), 4.50 - 4.42 (m, 2H), 4.41-4.34 (m, 1H), 4.30 - 4.18 (m, 1H), 4.14 - 3.99 (m, 2H), 3.93 (s, 3H), 3.59 - 3.50 (m, 1H), 3.33 - 3.24 (m, 2H), 2.86 - 2.70 (m, 6H), 2.66 - 2.42 (m, 6H), 1.91 - 1.86 (m, 2H), 1.76 - 1.69 (m, 2H). 13 C NMR (101 MHz, CDC13) δ 165.00, 163.62, 162.66, 161.11, 151.20, 147.96, 146.23, 140.23, 136.27, 135.06, 133.43, 133.38, 132.31, 131.24, 129.68, 128.69, 128.23, 126.63, 126.39, 126.08, 125.98, 125.35, 123.82, 123.42, 122.37, 120.66, 119.05, 115.08, 111.71, 110.72, 68.79, 58.03, 56.23, 56.16, 55.69, 53.78, 53.15, 37.62, 29.31, 26.88, 23.16. HRMS (ESI, m / z) calcd for C 46 H 49 N6O6[M + CH3OH + H] + 781.3714, found: 781.3710. HPLC purity 97.75%. [a] D (CHCl3) 93.79°, c = 0.15 g / 100 mL.

[0297] Synthesis of compound 20b3:

[0298] Compound 19b3 was synthesized according to the above procedure for 20b3, the crude product was purified by preparative TLC plate to give yellow solid product 20b3 (94.5 mg, 80%). 1 H NMR (400 MHz, CDC13) δ 12.30 (s, 1H), 10.00 (d, J = 9.2 Hz, 1H), 9.75 (s, 1H), 8.89 (s, 1H), 8.77 (d, J = 7.1 Hz, 1H), 8.41 (d, J = 8.5 Hz, 1H), 8.16 (d, J = 8.5 Hz, 1H), 7.90 - 7.84 (m, 1H), 7.77 (t, J = 7.7 Hz, 1H), 7.67 (t, J = 7.5 Hz, 1H), 7.49 (s, 1H), 7.33 (s, 1H), 7.22 (d, J = 8.1 Hz, 2H), 6.77 (s, 1H), 6.68 (d, J = 8.2 Hz, 2H), 4.50 - 4.33 (m, 3H), 4.18 - 3.99 (m, 3H), 3.90 (s, 3H), 3.81 (s, 1H), 3.54 (s, 1H), 3.37 (s, 1H), 3.35 - 3.25 (m, 2H), 3.24 - 3.14 (m, 2H), 3.09 - 2.98 (m, 2H), 2.94 - 2.64 (m, 6H), 1.94 - 1.84 (m, 2H), 1.67 - 1.61 (m, 2H), 1.58 - 1.52 (m, 2H). 13 C NMR (101 MHz, CDC13) δ 165.22, 163.82, 162.70, 161.12, 151.07, 147.93, 146.14, 140.25, 136.52, 135.26, 133.69, 133.59, 132.52, 131.46, 129.83, 128.90, 128.50, 126.73, 126.55, 126.15, 126.03, 125.50, 123.79, 123.61, 122.52, 120.79, 119.21, 115.15, 111.76, 110.75, 68.65, 60.40, 56.17, 55.40, 53.81, 52.64, 44.93, 37.50, 35.57, 28.46, 24.38, 23.77. HRMS (ESI, m / z) calcd for C 47 H 51 N6O6[M+CH3OH+H] + 795.3870, found: 795.3878. HPLC purity 98.77%. [a] D (CHCl3) 100.85°, c = 0.15 g / 100 mL.

[0299] Example 5: CCK-8 method for detecting cytotoxicity of hybrid molecules in vitro

[0300] DMEM, RMPI1640, fetal bovine serum were purchased from Gibco BRL company (Invitrogen Corporation, USA). Human ovarian cancer cell line SKOV3, human gastric cancer cell line NCI-N87 and human breast cancer cell line MDA-MB-231 were purchased from ATCC (American type culture collection), and were cultured according to the recommended method in the literature, that is, MDA-MB-231 cells and SKOV3 cells were cultured in DMEM medium containing 10% fetal bovine serum, and NCI-N87 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum. The test compounds used were prepared by the laboratory. The CCK-8 kit was purchased from Japan Tongren Chemical Research Institute.

[0301] Logarithmic growth phase human breast cancer cell line MDA-MB-231, human ovarian cancer cell line SKOV3 and human gastric cancer cell line NCI-N87 were taken, and the cell density was adjusted with DMEM medium containing 10% fetal bovine serum, SKOV3 cell density was 3000 cells per well, NCI-N87 cell density was 5000 cells per well, and MDA-MB-231 density was 4000 cells per well, 100 μL single cell suspension was added per well in a 96-well culture plate, and was cultured at 37°C, 5% CO2.

[0302] After the cells were attached overnight, different concentrations of each test compound were added, the hybrid molecules to be detected in the present application were 17a1-17b3 and 20a1-20b3, and oxaliplatin was selected as a positive control. These compounds were dissolved in DMSO as stock solution and stored in a 4°C refrigerator for use, the stock solution concentration of the hybrid molecules was 10 mM, and the stock solution concentration of oxaliplatin was 100 mM. Eight gradient concentrations were set for the in vitro cytotoxicity study of the present application, the hybrid molecule concentrations were set as 1000 nM, 500 nM, 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, and the concentration of oxaliplatin was set as 500 μM, 50 μM, 10 μM, 2 μM, 0.4 μM, 80 nM, 16 nM, 3.2 nM, which needed to be diluted to a specific concentration in a biosafety cabinet by diluting a certain volume of stock solution with the corresponding medium. Since the present application does not aspirate the culture medium in each well of the 96-well plate in this experiment, but directly adds an equal volume (100 μL) of compound solution for co-incubation with the cells, therefore, the gradient solutions of each compound actually need to be prepared in double amount, of course, only the initial concentration needs to be doubled.

[0303] After the compound is co-incubated with the cells for 72 h, CCK-8 reagent is added to each 96-well plate, 20 μL per well, and attention is paid to not generate bubbles in the wells when adding the drug. After addition, the 96-well plate is incubated at 37 °C in the dark for about 2 h, during which the color gradient of the 96-well plate is observed. Then the 96-well plate is placed in an enzyme-linked immunoassay instrument for detection of the OD value of each well at 450 nm, and the inhibition rate of each well is calculated, and the calculation formula is as follows: Inhibition rate % = (A 对照孔 -A 化合物孔 ) / (A 对照孔 -A 空白孔 ) x 100%, wherein:

[0304] A 对照孔 is the OD value measured for the well containing cells, CCK-8 and culture medium without the compound;

[0305] A 化合物孔 is the OD value measured for the well containing cells, CCK-8, culture medium and the compound;

[0306] A 空白孔 is the OD value measured for the well containing CCK-8 and culture medium without cells and the compound;

[0307] The inhibition rate of each well of each compound is input into Graphpad prism software to calculate the IC 50 value (half-inhibitory concentration, i.e. the compound / drug concentration corresponding to 50% inhibition of cell proliferation) of each compound for each cell. The above experimental operation is repeated more than 3 times, and the in vitro toxicity data of the hybrid molecules are comprehensively analyzed, and the results are shown in Table 1 below:

[0308] Table 1 In vitro cytotoxicity of hybrid molecules (72 h)

[0309]

[0310] P < 0.05, the difference is statistically significant.

[0311] The experimental results show that the hybrid molecules designed and synthesized by us all have good anti-tumor activity (0.17-221.2 nM), which is much higher than that of oxaliplatin (4.1 μM-11.2 μM), and the activity of these hybrid molecules is also higher than that of the PBD-naphthalimide hybrid molecules (0.1 μM-0.1 mM) reported in the literature, except 17a1, 17a2 and 17a3. Among all the tested hybrid molecules, 20b3 shows superior anti-tumor activity (0.17-0.94 nM), and the synthesis yield is also the highest. In addition, 20b3 is located at the p-phenylamino group of the C ring of PBD, which can introduce the linker of ADC, which is conducive to the preparation of subsequent ADC. Therefore, 20b3 is a potential candidate warhead.

[0312] Example 6: 20b3 inhibits tumor cell cycle and induces apoptosis

[0313] SKOV3 cells in logarithmic growth phase were collected, counted, and plated in 6-well plates at a cell density of 500,000 cells per well in a volume of 2 mL per well in a constant temperature cell incubator (37°C, 5% CO2). The candidate compound 20b3 (10 mM in DMSO) was diluted with culture medium to 0.33 nM, 1 nM, and 3 nM for use. After 24 h of cell culture, the culture medium in the 6-well plates was removed, and freshly prepared 20b3 solution was added to each 6-well plate at a volume of 2 mL per well, and an equal volume of culture medium was added to the negative control, and each 6-well plate was returned to the incubator for continued culture for 24 h. Then the cell supernatant was collected into a 15 mL centrifuge tube, and the remaining adherent cells were also collected into the centrifuge tube after trypsinization with 1 mL of trypsin. Centrifugation was performed (1000 g, 5 min), and the supernatant was removed. Then 1 mL of pre-cooled PBS was added, the cells were resuspended, transferred to a 1.5 mL centrifuge tube, centrifuged (4°C, 1000 g, 5 min), the supernatant was removed, and 1 mL of pre-cooled 70% ethanol was added. The cells were mixed gently and uniformly, placed in a 4°C refrigerator for overnight fixation, and then centrifuged again (4°C, 1000 g, 5 min), and the supernatant was removed. Then 0.5 mL of freshly prepared PI staining solution (prepared according to the instructions) was added to each cell sample, and the cells were resuspended slowly and thoroughly. After 30 min of incubation at 37°C in the dark, the sample was transferred to a flow cytometry sample tube, and the inhibitory effect of 20b3 on tumor cell cycle was detected and analyzed using a flow cytometer (Agilent, Novocyte 2070R).

[0314] The cell culture and cell collection in the apoptosis experiment were basically the same as in the cell cycle experiment, except that the plating density in the apoptosis experiment was 300,000 cells per well, the 20b3 action time was 48 h, and trypsin without EDTA was used to digest the cells. The Annexin V-FITC apoptosis detection kit was taken out of the -20°C refrigerator in advance, thawed, and the appropriate amount of staining solution was prepared according to the instructions and stored on ice in the dark for use. Four negative control groups were set up for flow cytometry detection parameter adjustment, namely, no Annexin V-PI staining, Annexin V-PI double staining, Annexin V single staining, and PI single staining. After the staining solution was prepared, 210 μL was added to each cell sample, and the sample was incubated at room temperature in the dark for 15 min, then transferred to a flow cytometry sample tube, and flow cytometry detection was performed within 1 h to analyze the apoptosis induction effect of 20b3 on tumor cells.

[0315] The experimental results of 20b3 inhibition of cell cycle and induction of apoptosis are shown in Figure 1The results show that in the cell cycle experiment, the S phase ratio of the blank control group is 17.96%, and after the action of 0.33 nM, 1 nM and 3 nM 20b3, the S phase ratio of the tumor cells is obviously increased and shows a concentration-dependent effect, which is 19.76%, 26.01% and 50.45% respectively. In the apoptosis experiment, the apoptosis cell (Annexin V + ) ratio of the blank control group is 9.65%, and after the action of 0.33 nM, 1 nM and 3 nM 20b3, the apoptosis cell ratio is obviously increased and shows a concentration-dependent effect, which is 9.71%, 16.11% and 58.68% respectively. The cell cycle and apoptosis experiment results show that 20b3 can dose-dependently inhibit cell mitosis in the S phase, induce cell apoptosis and play an anti-tumor role.

[0316] Example 7: DNA interstrand cross-linking induced by 20b3

[0317] The pBR322 plasmid is cut by restriction enzyme HindIII-HF (star enzyme) to linearize it, and then the DNA is precipitated by EtOH, and the plasmid is recovered by using a genomic DNA extraction kit, the concentration is determined by NanoDrop 2000 and diluted to 100 ng / μL for use. A 20b3 stock solution (1 mM in DMSO) is prepared, and ultra-pure water is used to dilute it to 50, 25, 2, 15, 10 and 5 μM solutions, and an oxaliplatin solution is prepared in the same way to 100, 10 and 1 μM, both of which are stored at -20℃ for use. Take 11 200 μL PCR tubes, numbered 1-11 in turn, and add 1 μL of linearized pBR322 plasmid DNA (100 ng / μL), then add 1 μL of ultra-pure water containing 5% DMSO to tubes 1-2, add 1 μL of 5, 10, 15, 20, 25 and 50 μM 20b3 solution to tubes 3-8 respectively, add 1, 10 and 100 μM oxaliplatin solution to tubes 9-11 respectively, and finally all the PCR tubes are supplemented with TEOA buffer to 10 μL, vortexed, instantaneously separated, and incubated at 37℃ for 2 h, then tube 1 is placed on ice, and tubes 2-11 are heated at 92℃ for 3 min on a PCR instrument and then immediately placed on ice. Then 2 μL of loading buffer (6x) is added to each PCR tube, mixed well, and 6 μL is taken and added to a 1% agarose gel well, electrophoresed at 100 V for 45 min, and then the gel is taken out, observed and adjusted in a Bio-Rad gel imaging system, scanned and imaged, and the gray scale analysis was performed by ImageJ to calculate the cross-linking rate of the DNA of each sample group. The experimental scheme is shown in Figure 2 .

[0318] The experimental results are shown in Figure 3As shown, using ImageJ to analyze the gray scale of each band, the DNA cross-linking rate of 20b3 corresponding to 0 μM, 0.01 μM, 0.1 μM, 0.5 μM, 1 μM, 2.5 μM and 5 μM was calculated as 0%, 1.6%, 20.3%, 64.4%, 81.6%, 95.1% and 98.8%, while the DNA cross-linking rate of oxaliplatin corresponding to 1 μM, 10 μM and 100 μM was 2.3%, 17.3% and 100.0%, which indicated that 20b3 could induce interstrand cross-linking of DNA, and the interstrand cross-linking rate of DNA showed a dose-dependent manner after treatment with different concentrations of 20b3. In addition, it can be seen that the cross-linking rate of 20b3 at 5 μM is basically equivalent to the cross-linking rate of the positive control drug oxaliplatin at 100 μM, indicating that the interstrand cross-linking ability of 20b3 is about 20 times that of oxaliplatin.

[0319] Example 8: Intracellular distribution of 20b3

[0320] A DMSO stock solution of 500 μM 20b3 was prepared, and an appropriate volume was added to a 1 cm quartz cuvette, which was placed at room temperature for 10 min. Then, the three-dimensional scanning mode was selected, and the scanning parameters were set. The sample was scanned in three dimensions, and then the data were processed and plotted using Oringin Pro 2019b software to obtain the three-dimensional fluorescence contour map of 20b3. SKOV3 and MDA-MB-231 cells in the logarithmic growth phase were collected and plated into 35 mm laser confocal culture dishes, with a cell density of 100,000 cells / dish and a volume of 2 mL / dish. The dishes were incubated in an incubator for 24 h. The old culture medium in the culture dishes was aspirated, and a freshly prepared 25 nM 20b3 solution was added to the confocal culture dishes at 2 mL / dish. The dishes were then returned to the incubator for further incubation for 12 h. The old culture medium was aspirated again, and the cells were washed three times with 1 mL of 1×PBS. Hoechst 33342 (diluted 1:100 in PBS, 1 mL) was used to stain the cell nuclei, and the dishes were incubated at 37°C for 0.5 h. The old culture medium was aspirated again, and the cells were washed three times with 1 mL of 1×PBS. Then, 1 mL of PBS was added, and the dishes were observed under the laser confocal microscope by selecting the appropriate excitation light channel.

[0321] In the present study, we used a fluorescence spectrophotometer to measure the three-dimensional fluorescence spectrum data of 20b3. From the detection results, it can be seen that the fluorescence spectrum of 20b3 is a broad band with a peak at 450 nm, and the fluorescence intensity is relatively strong. The fluorescence spectrum of 20b3 is shown in FIG. 6. Figure 4), the maximum excitation wavelength of 20b3 is 468 nm, and the emission spectrum range is 480-560 nm. The emission spectrum has strong fluorescence intensity under the excitation wavelength of 464-550 nm, and the emission light of 480-560 nm mainly presents green light. Therefore, we can select 488 nm as the excitation wavelength of the confocal microscope, and the 20b3 under the microscope presents green fluorescence. On the other hand, we use the DNA fluorescent dye Hoechst 33342 for nuclear staining, which presents blue light under the excitation wavelength of 405 nm, and does not show color under the excitation wavelength of 488 nm. Therefore, combined with the fluorescence characteristics of 20b3, we can observe the distribution of 20b3 in the cell after entering the cell under the confocal microscope. From the observation results of the laser confocal microscope, Figure 5 ), the green fluorescence of 20b3 mainly appears in the nucleus, and there is basically no distribution in the cytoplasm, indicating that 20b3 mainly distributes in the nucleus after entering the tumor cell, and further proving that the target of 20b3 is the DNA in the nucleus.

[0322] Example 9: 20b3 induces DNA damage in tumor cells

[0323] SKOV3 and NCI-N87 cells in logarithmic growth phase were cultured and collected, and were plated in 6-well plates at a cell density of 250,000 cells / well and 500,000 cells / well, respectively, with a volume of 2 mL / dish, and were incubated in an incubator for 24 h. The culture medium in the 6-well plate was aspirated, and freshly prepared 20b3 solution (concentrations were 12.5, 2.5, 0.5 nM, 0.1 nM and 0.02 nM) was added to each 6-well plate at a volume of 2 mL / well, and an equal volume of culture medium was added to the negative control, and each 6-well plate was returned to the incubator for continuous culture for 48 h. Pre-cooled PBS was washed twice, and the cells were scraped into a 1.5 mL centrifuge tube with a cell scraper, centrifuged (4°C, 3500 rpm, 5 min), and the supernatant was discarded. An appropriate amount of RIPA lysis buffer was added, mixed thoroughly, and lysed on ice for 2 h. The supernatant was centrifuged (4°C, 13000 g, 20 min), and the total protein was obtained. An appropriate amount of supernatant was taken, and the total protein concentration was determined using a BCA protein quantification kit. The rest was added with an appropriate amount of 5x SDS-PAGE protein loading buffer, and was heated at 100°C for 5 min. The sample was aliquoted and stored at -20°C for standby.

[0324] Separation gel (10%, 5 ml) was prepared and added to the gap of a glass plate, which was immediately sealed with a saturated mixture of isopropanol and water. After the separation gel solidified, the upper liquid was poured off and dried with filter paper. Concentration gel (5%, 2 mL) was prepared and added to the upper layer of the separation gel, and a comb was inserted. The newly prepared SDS-PAGE gel was placed in an electrophoresis tank, and after the electrophoresis buffer was added, the comb was removed, and the marker and protein sample (30 μg) were sequentially added for electrophoresis. A transfer membrane "sandwich" was assembled, the transfer tank was placed in an ice water bath, the "sandwich" was placed in the tank, transfer buffer was added until the "sandwich" was completely immersed, and transfer was performed. After the transfer was completed, the hybridization membrane was removed, the membrane was washed with an appropriate amount of TBST, and the membrane was transferred to 5% skim milk, and sealed for 2 h at room temperature on a shaker.

[0325] Then the membrane was washed with an appropriate amount of TBST for 3 times, 5 min / time, the membrane was removed, and horseradish peroxidase-labeled secondary antibody diluted with TBST (diluted at a ratio of 1:5000) was added, and incubated at 37°C for 1 h. Then the membrane was washed with an appropriate amount of TBST for 3 times, 5 min / time. Finally, ECL color developing substrates Solution A and Solution B were mixed at a ratio of 1:1, added to the membrane, and detected using a chemiluminescence imager.

[0326] The present application studies the influence of 20b3 on the expression level of DNA damage and apoptosis-related proteins by Western Blot. Figure 6 After 20b3 was used for 48 h, the expression level of γ-H2AX in SKOV3 and NCI-N87 cells was significantly increased compared with the blank control group, and showed a concentration-dependent manner, indicating that 20b3 can induce DNA damage in tumor cells. In addition, it was also found that the expression of p-Chk2 in the cells also increased, indicating that after DNA damage occurred, the DNA repair-related pathway was activated, and Chk2 was phosphorylated. It was further found that the expression levels of PARP and Caspase-3 in the cells decreased, and the expression levels of their cleavage products Cleaved Caspase-3 and Cleaved PARP increased, and showed a concentration-dependent manner. These results show that 20b3 can induce DNA damage, activate the DNA damage response system, phosphorylate Chk2, and then activate the Caspase signaling pathway, promote the cleavage of PARP and Caspase-3, inhibit DNA damage repair, and ultimately lead to cell cycle arrest and apoptosis.

[0327] Example 10: Synthesis of Drug-Linker 21:

[0328]

[0329] In a 25 mL round bottom flask, 2-ethoxy-l-ethoxycarbonyl-l,2-dihydroquinoline (EEDQ) (48.7 mg, 0.20 mmol) and Me-Val-Ala-OH (75 mg, 0.20 mmol) were added successively, dissolved in a mixed solvent of dichloromethane and methanol (DCM / MeOH = 20 / 1, 2 mL), stirred for 0.5 h at room temperature under nitrogen protection, then placed in an ice bath for 5 min, and then a solution of compound 20b3 (50 mg, 0.066 mmol) in DCM / MeOH = 20 / 1, 3 mL) was added, the ice bath was removed, and stirring was continued at room temperature for 16 h. TLC monitoring showed that the reaction was complete (developing agent: dichloromethane / methanol = 15 / 1, UV coloration). After the solvent was removed by concentration under reduced pressure, the crude product was dissolved in a small amount of a mixed solvent of dichloromethane and methanol (DCM / MeOH = 10 / 1), and then purified by preparative TLC plate to obtain yellow solid product 21 (33 mg, 44.7%). 1 H NMR (400 MHz, CDC13) δ 9.92 - 9.82 (m, 1H), 8.70 - 8.60 (m, 2H), 8.24 (d, J = 9.2 Hz, 1H), 8.01 (d, J = 7.8 Hz, 1H), 7.79 - 7.72 (m, 1H), 7.69 - 7.61 (m, 2H), 7.60 - 7.54 (m, 2H), 7.49 (d, J = 8.4 Hz, 1H), 7.41 (s, 1H), 7.30 (s, 1H), 7.27 - 7.10 (m, 3H), 6.66 (s, 2H), 4.39 (d, J = 7.4 Hz, 3H), 3.92 (s, 2H), 3.79 (s, 1H), 3.48 - 3.33 (m, 5H), 2.77 (d, J = 7.6 Hz, 6H), 2.57 (s, 3H), 2.48 - 2.37 (m, 3H), 2.32 - 2.19 (m, 2H), 1.91 - 1.75 (m, 2H), 1.71 - 1.37 (m, 15H), 1.28 - 1.23 (m, 2H), 0.99 - 0.90 (m, 6H). 13C NMR (101 MHz, CDC13) δ 174.07, 173.39, 172.19, 171.78, 170.85, 170.83, 164.94, 163.58, 162.73, 161.36, 153.55, 151.40, 148.59, 147.94, 145.34, 140.32, 137.57, 136.25, 135.05, 134.04, 133.37, 132.26, 131.23, 129.65, 128.64, 128.17, 126.63, 126.38, 125.34, 122.31, 120.10, 118.71, 115.10, 111.68, 110.73, 68.87, 58.33, 56.18, 55.64, 53.13, 52.97, 50.45, 37.56, 37.48, 29.66, 28.76, 28.27, 28.17, 26.37, 26.24, 26.19, 25.27, 25.00, 23.93, 19.23, 18.90, 18.52, 18.19. HRMS (ESI, m / z) calcd for C 65 H 76 N9O 11 [M+CH3OH+H] + 1158.5664, found: 1158.5616. HPLC purity 98.20%. [a] D (CHCl3) 15.45°, c = 0.13 g / 100 mL.

[0330] Example 11: Preparation of antibody conjugate T-PBA and analysis of conjugation

[0331] The ADC conjugation buffer and ADC storage buffer were prepared before the experiment, and stored at 4°C for standby. A suitable amount of Trastuzumab freeze-dried powder was dissolved in water for injection as the antibody stock solution, and the concentration was controlled at about 20 mg / mL, and stored at -80°C for standby. Before use, the antibody stock solution was replaced into the ADC conjugation buffer (14.4 mM NaH2PO4+5.6 mM Na2HPO4+20 mM NaCl+1 mM EDTA, pH 7.40) using AKTA Purifier 100 protein purification system, and then concentrated using 30KD Millipore ultrafiltration concentration tube. During the process, the antibody concentration was determined using Nanaodrop 2000, and controlled at about 10 mg / mL, and stored at -20°C for standby.

[0332] Before the conjugation reaction, the interchain disulfide bond of the antibody was partially reduced with TCEP (1.74 mM in ADC conjugation buffer, prepared immediately before use) at a molar ratio of 1:2 between the antibody and TCEP. The reaction was carried out in a 1.5 mL centrifuge tube, with a volume of 0.8 mL per reaction system, so that the final concentration of the antibody was 5 mg / mL. The reaction centrifuge tube was placed horizontally on a shaker (37°C, 120 rpm) for 1.5 h. After the reduction reaction was completed, freshly prepared DMF solution of drug-linker 21 was directly added to the reaction system in a biological safety cabinet. The molar ratio of drug-linker 21 to antibody was 4:1, and the proportion of DMF in the entire reaction system was 10%. Therefore, 88.9 μL of DMF solution with a concentration of 1.361 mg / mL was required. The conjugation reaction centrifuge tube was placed horizontally on a shaker (25°C, 120 rpm) for reaction. After 2 h of conjugation reaction, centrifugation was performed (4°C, 4000 rpm, 5 min), and the supernatant was transferred to a new 1.5 mL centrifuge tube. The desalting column (HiTrap TM Desalting 5 mL) combined with the AKTA Purifier 100 protein purification system was used to replace the conjugation reaction sample from the ADC conjugation buffer into the ADC storage buffer (14.4 mM NaH2PO4+5.6 mM Na2HPO4+50 mM NaCl, pH 7.40). Concentration was performed using a 30 KD Millipore ultrafiltration concentration tube to obtain a sample concentration of about 10 mg / mL. The sample was aliquoted, labeled as T-PBA, and the concentration and time were recorded. The sample was stored at -80°C for later use.

[0333] After the preparation of the antibody conjugate T-PBA, the conjugation of T-PBA was analyzed by SDS-PAGE combined with ultraviolet fluorescence and ultraviolet double wavelength method.

[0334] In the SDS-PAGE experiment, Trastuzumab and T-PBA were diluted with PBS to the appropriate concentration, and each was divided into two parts, 10 μg each. Then 3 μL of 5x denatured reducing and non-denatured non-reducing protein loading buffer was added to each part, mixed well, and supplemented with UP water to 15 μL. The samples mixed with 5x denatured reducing protein loading buffer were boiled at 100°C for 3 min. All the prepared samples were centrifuged immediately and stored at 4°C for later use.

[0335] After the sample preparation, the newly prepared SDS-PAGE gel (the same method as before) is placed in the electrophoresis tank, the comb is pulled out after adding the electrophoresis buffer, 3 μg or 10 μL of the just prepared sample and Marker are added to each lane, and the electrophoresis is started. The electrophoresis condition is observed, and the electrophoresis is terminated when the bromophenol blue band runs to the bottom of the glass plate. After the electrophoresis is completed, the SDS-PAGE gel on the glass plate is peeled off with a spatula, washed with clean water, and placed flat on the gel loading platform of the Bio-Rad gel scanner, excited with ultraviolet light, and the luminescence image of the SDS-PAGE gel excited by ultraviolet light is collected. Then the SDS-PAGE gel is immersed in the Coomassie brilliant blue staining solution, placed in a shaking bed for light shaking, and stained overnight. After staining, the decolorizing solution is added for immersion, and the shaking bed is used for light shaking for decolorization. After the decolorization is completed, the SDS-PAGE gel is scanned on the gel scanner and the staining results are collected.

[0336] When the coupling of T-PBA is analyzed by the ultraviolet double-wavelength method, the molar absorption coefficients (ε) of the drug-linker 21 and Trastuzumab at 280 nm and 335 nm need to be determined first. The 280 nm is the characteristic absorption wavelength of the antibody, and the 335 nm is the characteristic absorption wavelength of the drug-linker 21. The molar correlation coefficient of the antibody at 280 nm can be queried as 219123, and the antibody has no absorption at 335 nm, so the molar extinction coefficient at this time is 0. The method for determining the molar absorption coefficients (ε) of the drug-linker 21 at the two wavelengths is as follows: the drug-linker 21 is prepared into a 0.025 mg / mL DMF / H2O solution, and then the absorbance value of the solution at 280 nm and 335 nm is determined on the ultraviolet spectrophotometer. The average value is obtained by determining three times in parallel. Then, according to the Lambert-Beer law A = Ecl, the extinction coefficients of the bullet-linker at 280 nm and 335 nm are calculated and and the molar extinction coefficients and

[0337] After the preparation of T-PBA is completed, the absorbance values of T-PBA at 280 nm and 335 nm can be determined, and then the antibody drug coupling ratio (i.e. the coupling rate, DAR) of T-PBA is calculated according to the UV-DAR calculation formula, as follows: wherein, c D is the concentration of the drug-linker 21, c Ab is the concentration of the antibody, is the molar absorption coefficient of the antibody at 280 nm, A 280 is the absorbance value of T-PBA at 280 nm, A 335 is the absorbance value of T-PBA at 335 nm, The molar absorption coefficient of the antibody at 335 nm (no absorption), The molar absorption coefficient of the drug-linker 21 at 335 nm, The molar absorption coefficient of the drug-linker 21 at 280 nm.

[0338] In the present application, we prepared the antibody conjugate T-PBA (Formula 1) based on the thiol coupling method of partially reducing the inter-chain disulfide bond of the antibody. Figure 7 Then we analyzed the conjugation of T-PBA by SDS-PAGE combined with UV fluorescence and UV dual-wavelength method.

[0339] Due to the rigid planar structure and large π bond of the drug-linker 21, T-PBA has strong fluorescence characteristics in addition to the properties of the antibody itself. Therefore, the present application can analyze the conjugation of T-PBA by SDS-PAGE combined with UV detection. The detection results are shown in Figure 1. Figure 8 In the non-reducing electrophoresis in the upper right corner, the molecular weight of the main band of Trastuzumab and T-PBA is about 150 KD, the band is clear, and the purity meets the requirements of subsequent experiments. In the reducing electrophoresis in the upper left corner, Trastuzumab and T-PBA are both reduced into two fragments with molecular weights of 50 KD (heavy chain) and 25 KD (light chain), respectively. The small band slightly above the light chain of T-PBA is due to the increase in molecular weight after the conjugation of the warhead. In the UV excitation fluorescence graph below, the reducing electrophoresis in the lower right corner shows that T-PBA has fluorescence at the position of 150 KD, while Trastuzumab does not show fluorescence at 150 KD, indicating that the antibody has successfully conjugated with the drug-linker 21. In the reducing electrophoresis in the lower left corner, T-PBA shows fluorescence at 50 KD and 25 KD, respectively, and the band at 50 KD is stronger than that at 25 KD, while Trastuzumab does not show fluorescence at these two band positions, indicating that the light chain and heavy chain of the antibody have both conjugated with the drug-linker 21, but mainly conjugated with the heavy chain.

[0340] In the present application, we also analyzed the conjugation of T-PBA by UV dual-wavelength method. We first measured the absorbance of 0.025 mg / mL drug-linker 21 at 280 nm and 335 nm, calculated the average value, and calculated the extinction coefficient and Then we calculated the molar extinction coefficient of the drug-linker 21 and (ε D = M × E D , E unit L / (g·cm), M 40= 1126), the average DAR of T-PBA was calculated to be 2.26, see Table 2. and are 15764 L / (mol-cm) and 19818 L / (mol-cm), respectively. In addition, we also measured the UV-DAR of Trastuzumab is 219123 L / (mol-cm), while Trastuzumab has no absorbance at 335 nm, is 0. After obtaining T-PBA, we measured the absorbance A 280 and A 335 , and put all the data into the UV-DAR calculation formula: The average DAR of T-PBA was calculated to be 2.26, see Table 2.

[0341] Table 2. UV-DAR determination results of T-PBA

[0342]

[0343] Example 12: Targeting of T-PBA

[0344] SKOV3 (HER2 3+), NCI-N87 (HER2 3+) and MDA-MB-231 (HER2 0-1+) cells were cultured, when the cells were in the logarithmic growth phase, 2 mL of trypsin was added to each dish, and the cells were digested for about 4 min, then 2 mL of serum-containing medium was added to terminate the digestion, collected into a 15 mL centrifuge tube, centrifuged (800 rpm, 3 min), and the supernatant was discarded. Then 10 mL of PBS was added to resuspend the cells, and they were again centrifuged (800 rpm, 3 min), and the supernatant was discarded. An appropriate amount of PBS was added to resuspend the cells, counted, and the cell suspension was divided into 1.5 mL centrifuge tubes, 10 6 After the tubes were separated, they were again centrifuged (4°C, 3500 rpm, 3 min), the supernatant was removed, and placed on ice. 100 μL of primary antibody (i.e., Trastuzumab and T-PBA, diluted to 10 μg / mL with pre-cooled PBS just before use, and PBS as a negative control) was added to each tube, the cells were suspended, and incubated on ice for 40 min. Then 500 μL of pre-cooled PBS was added to each tube, the cells were gently mixed and centrifuged (4°C, 3500 rpm, 3 min), the supernatant was removed, and repeated 2-3 times. Then 100 μL of secondary antibody (i.e., goat anti-human IgG / FITC, diluted to an appropriate volume with pre-cooled PBS at a ratio of 1:200 just before use) was added to each tube, the cells were suspended, and incubated on ice for 40 min, and then washed 2-3 times with pre-cooled PBS. Finally, 350 μL of pre-cooled PBS was added to each tube, the cells were gently mixed, transferred to a flow injection tube, and subjected to flow cytometry (Novocyte 2070R) detection and analysis.

[0345] After the above experimental operation and data analysis, the research results are as follows Figure 9 As shown in the figure (red for PBS control group, green for Trastuzumab, and blue for T-PBA). As can be seen from the figure, T-PBA and Trastuzumab both exhibit strong binding ability to SKOV3 and NCI-N87 cells with high expression of HER2, and both have very weak binding ability to MDA-MB-231 cells with low expression of HER2, indicating that after coupling with the small molecule compound (drug-linker 21), the targeting of the antibody to HER2 does not change significantly.

[0346] Example 13: Internalization efficiency of T-PBA

[0347] SKOV3 (HER2 3+) and NCI-N87 (HER2 3+) cells in the logarithmic growth phase were cultured and collected, and each was divided into 9 1.5 mL centrifuge tubes, a total of 18 tubes. Then 100 μL of primary antibody (i.e. Trastuzumab and T-PBA, diluted to 10 μg / mL with pre-cooled PBS before use, and PBS as a negative control) was added to each tube, the cells were suspended, and incubated on ice for 40 min, then washed with 500 μL of pre-cooled PBS for 2-3 times. Subsequently, 100 μL of pre-cooled PBS was added to each tube, the cells were suspended, and incubated in a 37°C incubator for 0 h, 1.5 h and 4 h to allow the antibody to internalize. After incubation to the specified time point, the incubation was stopped, centrifuged (4°C, 3500 rpm, 3 min), and the supernatant was aspirated, then 100 μL of secondary antibody (i.e. goat anti-human IgG / FITC, diluted to an appropriate volume with pre-cooled PBS at a ratio of 1:200 before use) was added to each tube, the cells were suspended, and incubated on ice for 40 min, then washed with 500 μL of pre-cooled PBS for 2-3 times. Finally, 350 μL of pre-cooled PBS was added to each tube, the cells were mixed, transferred to a flow injection tube, and subjected to flow cytometry (Novocyte 2070R) detection and analysis.

[0348] After the above experimental operation and data analysis, the research results are as follows Figure 10The results are shown in the figure (the leftmost yellow line represents the PBS group, the red line represents internalization 0h, the green line represents internalization 1.5h, and the blue line represents internalization 4h). As can be seen from the figure, the degree of internalization of Trastuzumab and T-PBA gradually increases with the increase of incubation time. By calculation, the internalization efficiency of Trastuzumab and T-PBA in SKOV3 cells is 13.74% and 14.65% respectively at 1.5h, and 21.16% and 28.15% respectively at 4h; in NCI-N87 cells, the internalization efficiency of Trastuzumab and T-PBA is 17.69% and 15.52% respectively at 1.5h, and 25.79% and 21.05% respectively at 4h. The internalization efficiency of Trastuzumab and T-PBA in the two cells is similar, indicating that the internalization level of the antibody does not change significantly after coupling with the small molecule compound (drug-linker 21).

[0349] Example 14: Intracellular distribution of T-PBA

[0350] SKOV3 and MDA-MB-231 cells in logarithmic growth phase were cultured and collected, and then plated into 35mm laser confocal culture dishes, with a cell density of 100,000 cells / dish and 2mL / dish. After incubation in an incubator (37℃, 5% CO2) for 24h, the culture medium was removed, and 2mL / dish of T-PBA solution diluted to 1μM with culture medium just before use was added, and then placed back into the incubator for continued incubation for 12h. After removing the culture medium, the cells were washed three times with 1×PBS, 1mL each time. Then 1mL of Lyso-Tracker (Red) labeled lysosomes diluted 20000 times with serum-free and antibiotic-free DMEM culture medium was added, and incubated at 37℃ for 0.5h. After removing the culture medium, the cells were washed three times with 1×PBS, 1mL each time. After washing, 1mL of Hoechst 33342 diluted 100 times with PBS was added for nuclear staining, and incubated at 37℃ for 0.5h. After incubation, the culture medium was removed, and the cells were washed again with 1×PBS three times. Finally, 1mL of PBS was added, and the cells were observed under a laser confocal microscope.

[0351] The present application has proved that the warhead 20b3 of T-PBA exhibits green light under an excitation wavelength of 488nm, therefore in this experiment, we chose to observe the intracellular distribution of T-PBA after internalization under an excitation wavelength of 488nm, and used Hoechst 33342 dye which exhibits blue light under an excitation wavelength of 405nm for nuclear localization, and Lyso-Tracker Red dye which exhibits red light under an excitation wavelength of 561nm for lysosome localization. As can be seen from the experimental results, Figure 11), the distribution of T-PBA in SKOV3 cells with high HER2 expression was significantly higher than that in MDA-MB-231 cells with low HER2 expression, and mainly distributed in lysosomes. The above results show that T-PBA enters tumor cells through HER2-mediated internalization, and is transported to lysosomes for degradation, releasing warhead 20b3 to exert a killing effect.

[0352] Example 15: In vitro cytotoxicity of T-PBA

[0353] The method is basically the same as the in vitro cytotoxicity experiment of hybrid molecules (Example 5), but the gradient concentration of T-PBA is set to 1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, and 0.0128 nM. In addition, the in vitro cytotoxicity of warhead 20b3 and drug-linker 21 is also detected at the same time, and the concentration is set the same as T-PBA.

[0354] The experimental results show that T-PBA has strong killing activity on SKOV3 and NCI-N87 cells with high HER2 expression, and the IC 50 is in the nanomolar level (IC 50 = 5.4-56.1 nM), while for MDA-MB-231 cells with low HER2 expression, the activity of T-PBA is significantly reduced, and the IC 50 is 344.6 nM. In the gradient concentration range, Trastuzumab does not exert an anti-tumor effect, and the IC 50 is greater than 1000 nM (Table 3). In addition, we also detected the in vitro anti-tumor activity of warhead (20b3) and warhead-linker (40) for comparison. Obviously, warhead 20b3 has strong killing activity on the tested cells, and the IC 50 is in the sub-nanomolar level (IC 50 = 0.17-0.94 nM), while the activity of drug-linker 21 is significantly reduced compared to warhead, and the IC 50 is in the nanomolar level (IC 50 = 237.8-341.7), which is reduced by 2-3 orders of magnitude, which is likely due to the blocking of the amino group of the pharmacophore after the warhead is connected to the linker, resulting in a decrease in activity. These experimental results show that the killing effect of warhead 20b3 and drug-linker 21 on different tumor cells does not have obvious specificity, but after being made into ADC, the killing effect of ADC has cell specificity, and the activity is related to the HER2 expression of tumor cells.

[0355] Table 3 In vitro anti-tumor activity of T-PBA, warhead

[0356]

[0357] Example 16: T-PBA inhibits tumor cell cycle and induces apoptosis

[0358] The method was basically the same as the 20b3 tumor cell cycle inhibition experiment and apoptosis induction experiment (Example 6), but the concentration of T-PBA was set to 1.6 nM, 8 nM and 40 nM.

[0359] The results of the tumor cell cycle inhibition experiment show that ( Figure 12 The proportion of cells in S phase in the blank control group was 24.56%. However, after treatment with different concentrations (1.6 nM, 8 nM, and 40 nM) of T-PBA, the proportion of cells in S phase increased significantly in a concentration-dependent manner, reaching 34.46%, 43.09%, and 54.82%, respectively, while the proportions in G0 / G1 and G2 / M phases decreased. The results of the tumor cell apoptosis experiment (Figure x) show that the apoptotic cells (Annexin V) in the blank control group... + The initial proportion of apoptotic cells was 0.14%, but after treatment with different concentrations of T-PBA (1.6 nM, 8 nM, and 40 nM), the proportion of apoptotic cells increased significantly in a dose-dependent manner, reaching 15.42%, 26.86%, and 36.81%, respectively. Cell cycle and apoptosis experiments showed that, like 20b3, T-PBA inhibited cell mitosis in the S phase in a concentration-dependent manner, induced apoptosis, and exerted an anti-tumor effect.

[0360] Example 17: T-PBA-induced DNA damage

[0361] The method was basically the same as the 20b3-induced DNA damage experiment (Example 7), but the concentrations of T-PBA were set to 125 nM, 25 nM, 5 nM, 1 nM and 0.2 nM.

[0362] The experimental results show that ( Figure 13), and compared with the blank control group, the expression level of γ-H2AX in SKOV3 and NCI-N87 cells was significantly increased and showed a concentration-dependent manner after 48 h of T-PBA treatment, indicating that T-PBA can cause DNA damage in SKOV3 and NCI-N87 cells. In addition, the expression of p-Chk2 in cells also increased, indicating that after DNA damage, the DNA damage response system was activated, and Chk2 was activated. In addition, the expression levels of PARP and Caspase-3 in cells decreased, while the expression levels of their cleavage products Cleaved Caspase-3 and Cleaved PARP increased and showed a concentration-dependent manner. These results show that after T-PBA is internalized into cells, it can release the warhead small molecule 20b3 through the lysosomal pathway, and then induce DNA damage, activate the DNA damage response system, phosphorylate Chk2, and then activate the Caspase signaling pathway, promote the cleavage of PARP and Caspase-3, inhibit DNA damage repair, and ultimately lead to cell cycle arrest and apoptosis.

[0363] Example 18: In vivo anti-tumor activity of T-PBA

[0364] The present application constructs a Balb / c nude mouse subcutaneous tumor-bearing model of human ovarian cancer SKOV3 cells and human gastric cancer NCI-N87 cells to evaluate the in vivo anti-tumor activity of T-PBA, and the specific experimental method is as follows:

[0365] 6-8 weeks old Balb / c athymic female nude mice (body weight 18-20 g) were purchased in advance and acclimated to the animal room for 1 week, and SKOV3 cells and NCI-N87 cells were cultured to the required amount during the period. Then collect the logarithmic growth phase cells into a 50 mL centrifuge tube, centrifuge (800 rpm, 3 min), remove the supernatant, and wash the cells with an appropriate amount of serum-free antibiotic-free medium (double-free medium) 3 times. Then add an appropriate amount of double-free medium to resuspend the cells, count, and adjust the SKOV3 cell density to 10 8 × 10 7 live cells / mL, and the NCI-N87 cell density to 2.5 × 10 3 live cells / mL. After the cells are collected, immediately inoculate subcutaneously on the right upper limb of the nude mice, 100 μL per mouse. After inoculation, closely observe the tumor volume, and when the average tumor volume grows to about 200 mm 3 , randomly divide each model into 5 groups, namely the control group (normal saline), the naked antibody group (10 mg / kg), the T-PBA group (10 mg / kg), the T-PBA group (5 mg / kg), and the T-PBA group (1 mg / kg), with 6 nude mice in each group.

[0366] After grouping, tail vein injection was started (the drug solution was prepared in a biological safety cabinet in advance), 100 μL per mouse, once every 3 days, for a total of 4 times. Starting from the first administration, the tumor long diameter and short diameter (unit: mm) were measured every 3-4 days using an electronic vernier caliper, and the tumor volume (mm 3 ) was calculated according to the formula: tumor volume = 1 / 2 x long diameter x short diameter 2 At the same time, the body weight of the nude mice (unit: g) was measured using an electronic balance, and the physiological and living conditions of the nude mice were observed. When the tumor was in a large area of ulceration or the volume reached 2500 mm 3 , the treatment was ended, and the mice were sacrificed. When a significant therapeutic effect was observed, the observation time could be extended. According to the changes in tumor volume and nude mouse body weight, the drug tumor inhibition curve and the nude mouse body weight change curve were plotted using Graphpad Prism software, the tumor inhibition rate at the end of the observation time (the difference between the average tumor volumes of the control and experimental groups, divided by the average tumor volume of the control group, multiplied by 100%) was calculated, and the therapeutic effect of the drug was analyzed and evaluated.

[0367] According to the results of the animal experiment, Figure 14 , in the SKOV3 model, the tumor inhibition rates of the high-, medium-, and low-dose groups were 91.5%, 70.1%, and 57.5%, respectively, showing a dose-dependent effect. In the NCI-N87 model, the tumor inhibition rates of the high-, medium-, and low-dose groups were 97.1%, 88.1%, and 50.2%, respectively, also showing a dose-dependent effect. These results indicate that T-PBA can significantly delay tumor growth at doses of 10 mg / kg and 5 mg / kg, and has good anti-tumor effects. In addition, during the entire treatment period, the transplanted tumor nude mice were in good condition, and their body weight did not decrease significantly, indicating that T-PBA did not have significant toxic side effects at the administered doses.

[0368] At the end of the above experiment, the heart, liver, spleen, lung, and kidney of the control, antibody (10 mg / kg), and T-PBA (10 mg / kg) groups were removed and fixed in 4% paraformaldehyde, and then sent to the Chengdu Rilabio Biotechnology Co., Ltd. for H&E staining. The results are shown in Figure 15 Compared with the control group, no obvious histopathological changes were found in the organs of the antibody and T-PBA administration groups, indicating that T-PBA had no significant toxic side effects at the therapeutic dose and had good safety.

Claims

1. A compound of the formula or a pharmaceutically acceptable salt thereof, characterized in that: The structure is as follows: Formula wherein Z is selected from or ; I, m, n are independently selected from an integer from 1 to 8; wherein I is connected to N at the end and m is connected to O at the end. R1 is selected from C1-C8 alkyl, substituted 6-membered aryl; wherein the substituent of the substituted 6-membered aryl is selected from C1-C6 alkyl, C1-C6 alkoxy or amino; R2 is selected from -H; R3 is selected from C1-C6 alkyl.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: l, m, n are independently selected from integers from 1 to 6.

3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein: l is selected from integers from 1 to 3, m is selected from integers from 2 to 6, and n is selected from integers from 2 to 6.

4. The compound of claim 3, or a pharmaceutically acceptable salt thereof, wherein: l is 2, m is selected from integers from 3 to 5, and n is selected from integers from 3 to 5.

5. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: R1 is selected from C1-C6 alkyl, substituted 6-membered aryl; wherein the substituent of the substituted 6-membered aryl is selected from C1-C4 alkyl, C1-C4 alkoxy or amino.

6. The compound of claim 5, or a pharmaceutically acceptable salt thereof, wherein: R1 is selected from C1-C4 alkyl, substituted 6-membered aryl; wherein the substituent of the substituted 6-membered aryl is selected from C1-C4 alkyl, C1-C4 alkoxy or amino.

7. The compound of claim 6, or a pharmaceutically acceptable salt thereof, wherein: R1 is selected from methyl, p-aminophenyl or p-methoxyphenyl.

8. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: R3 is selected from C1-C4 alkyl.

9. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein: R3 is methyl.

10. A compound, or a pharmaceutically acceptable salt thereof, characterized in that: The compound is selected from: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or .

11. A compound, or a pharmaceutically acceptable salt thereof, characterized in that: The compound is selected from: 。 12. A method of preparing a compound, characterized by: The method comprises the following steps: a. Compound 1 is subjected to Suzuki coupling reaction to obtain compound 2: ； b. Compound 2 is reduced by Zn powder / AcOH system to obtain compound 3: ； c. Compound 3 is reacted with Alloc-Cl under alkaline conditions to obtain compound 4: ； wherein, when R1does not contain an amino group, R 、 is R1, when R1contains an amino group, R 、 is a group in which the amino group in R1is monosubstituted with Alloc; d. Compound 4 is subjected to TBS protecting group removal under acidic conditions to obtain compound 5: ； e. Compound 5 is subjected to Swern oxidation and ring closure to obtain compound 6: ； f. Compound 6 is reacted with TBS-OTf under alkaline conditions to obtain compound 7: ； g. Compound 7 is reacted with LiOAc to remove TIPS to obtain compound 8: ； h. Compound 8 is reacted with diiodoalkane under alkaline conditions to obtain compound 9: ； i. Compound 10 is reacted with bromoalkane under alkaline conditions to obtain compound 11: ； j. Compound 11 is reacted with compound 12 under alkaline conditions to obtain compound 13: ； k. Compound 13 is subjected to Boc removal under the action of TFA to obtain compound 14: ； l. Compound 9 and compound 12 are reacted under alkaline conditions to obtain compound 15: ； m. Compound 15 is subjected to TBS removal under TBAF / AcOH system to obtain compound 16: ； n. Compound 16 is subjected to Alloc protecting group removal under the action of Pd catalyst to obtain product 17: ； o. Compound 9 and compound 14 are reacted under alkaline conditions to obtain compound 18: ； p. Compound 18 is subjected to TBS removal under TBAF / AcOH system to obtain compound 19: ； q. Compound 19 is subjected to Alloc protecting group removal under the action of Pd catalyst to obtain product 20: ； r. Compound 20b3 is subjected to amide condensation with linker Mc-Val-Ala-OH to obtain compound 21: ； wherein l, m, n are independently selected from integers from 1 to 8; R1 is selected from C1-C8 alkyl, substituted 6-membered aryl; wherein the substituent of the substituted 6-membered aryl is selected from C1-C6 alkyl, C1-C6 alkoxy or amino; R2 is selected from -H; R3 is selected from C1-C6 alkyl.

13. The method of claim 12, wherein: Step a, compound 1, , base and Pd catalyst, under a protective atmosphere.

14. The method of claim 13, wherein: At least one of the following is satisfied: Step a, compound 1 with in a molar ratio of 1 : (1.0-5.0); In step a, the molar ratio of compound 1 to base is 1:(1.0-8); Step a, the Pd catalyst is selected from one or more of Pd(dppf)Cl2CH2Cl2, PdCl2(PPh3)2, Pd[P(Ph)3]4, Pd(OAc)2; Step a, the molar ratio of compound 1 to the Pd catalyst is 1:0.02-0.05; Step a, the reaction system further comprises a reaction solvent selected from one or more of acetone, acetonitrile, tetrahydrofuran, toluene, DMF, 1,2-dimethoxyethane, water; Step a, the base is selected from at least one of potassium phosphate, potassium carbonate, sodium carbonate, sodium hydride, barium hydroxide, cesium carbonate; Step a, the reaction temperature is 25°C; Step a, the reaction is carried out under nitrogen protection.

15. The method of claim 13, wherein: At least one of the following is met: Step a, compound 1 with a molar ratio of 1 :4.0; Step a, the molar ratio of compound 1 to the base is 1:6.0; Step a, the molar ratio of compound 1 to the Pd catalyst is 1:0.

05.

16. The method of claim 12, wherein: At least one of the following is met: Step b, the reaction system further comprises a reaction solvent selected from methanol or ethanol; Step c, the base is pyridine; Step d, the acid is aqueous acetic acid, and the reaction system further comprises a reaction solvent selected from methanol, tetrahydrofuran, a mixture of methanol: tetrahydrofuran in a volume ratio of 1:

1.

17. The method of claim 12 wherein: Step e, compound 5, DMSO, oxalyl chloride, and a base are reacted under a protective atmosphere.

18. The method of claim 17, wherein: At least one of the following is met: Step e, the molar ratio of compound 5 to oxalyl chloride is 1:(1.0-4.0); Step e, the molar ratio of compound 5 to DMSO is 1:(1.0-8.0); Step e, the molar ratio of compound 5 to the base is 1:(1.0-8.0); Step e, the base is selected from one or both of diisopropylethylamine and triethylamine; Step e, the reaction system further comprises a reaction solvent selected from at least one of dichloromethane, chloroform, and tetrahydrofuran; Step e, the reaction temperature is -78°C; Step e, the reaction is carried out under nitrogen protection.

19. The method of claim 17, wherein: At least one of the following is met: Step e, the molar ratio of compound 5 to oxalyl chloride is 1:1.3; Step e, the molar ratio of compound 5 to DMSO is 1:2.6; Step e, the molar ratio of compound 5 to the base is 1:5.

0.

20. The method of claim 12 wherein: At least one of the following is met: Step f, the base is 2,6-lutidine; Step g, the reaction system further comprises a reaction solvent which is a mixed solution of N,N-dimethylformamide and H2O.

21. The method of claim 12 wherein: Step h, compound 8, diiodoalkane, and a base are reacted under a protective atmosphere.

22. The method of claim 21 wherein: At least one of the following is met: Step h, the molar ratio of compound 8 to diiodoalkane is 1:(1.0-6.0); Step h, the molar ratio of compound 8 to the base is 1:(1.0-4.0); Step h, the base is selected from at least one of diisopropylethylamine, triethylamine, potassium phosphate, potassium carbonate, sodium carbonate, and cesium carbonate; Step h, the reaction system further comprises a reaction solvent selected from at least one of N,N-dimethylformamide, N,N-dimethylacetamide, 2-butanone, and toluene; Step h, the reaction temperature is 75°C; Step h, the reaction is carried out under nitrogen protection.

23. The method of claim 21 wherein: At least one of the following is met: Step h, the molar ratio of compound 8 to diiodoalkane is 1:5.0; Step h, the molar ratio of compound 8 to base is 1:1.

3.

24. The method of claim 12 wherein: Step i, compound 10, dibromoalkane and base are reacted under a protective atmosphere.

25. The method of claim 24 wherein: At least one of the following is satisfied: Step i, the molar ratio of compound 10 to dibromoalkane is 1:(1.0-6.0); Step i, the molar ratio of compound 10 to base is 1:(1.0-4.0); Step i, the base is at least one selected from diisopropylethylamine, triethylamine, potassium phosphate, potassium carbonate, sodium carbonate, cesium carbonate; Step i, the reaction system further comprises a reaction solvent, and the reaction solvent is at least one selected from N,N-dimethylformamide, N,N-dimethylacetamide, 2-butanone, dichloromethane, toluene; Step i, the reaction temperature is 45°C; Step i, the reaction is carried out under nitrogen protection.

26. The method of claim 24 wherein: At least one of the following is satisfied: Step i, the molar ratio of compound 10 to dibromoalkane is 1:4.3; Step i, the molar ratio of compound 10 to base is 1:1.

1.

27. The method of claim 12 wherein: Step j, compound 11, compound 12 and base are reacted under a protective atmosphere.

28. The method of claim 27 wherein: At least one of the following is satisfied: Step j, the molar ratio of compound 11 to compound 12 is 1:(1.0-3.0); Step j, the molar ratio of compound 11 to base is 1:(1.0-4.0); Step j, the base is at least one selected from diisopropylethylamine, triethylamine, potassium phosphate, potassium carbonate, sodium carbonate, cesium carbonate; Step j, the reaction system further comprises a reaction solvent, and the reaction solvent is at least one selected from N,N-dimethylformamide, N,N-dimethylacetamide, 2-butanone, toluene; Step j, the reaction temperature is 85°C; Step j, the reaction is carried out under nitrogen protection.

29. The method of claim 27, wherein: At least one of the following is satisfied: Step j, the molar ratio of compound 11 to compound 12 is 1:1.0; Step j, the molar ratio of compound 11 to base is 1:1.

5.

30. The method of claim 12 wherein: Step k, the reaction system further comprises a reaction solvent, and the reaction solvent is at least one selected from dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide.

31. The method of claim 12 wherein: Step l, compound 12, compound 9 and base are reacted under a protective atmosphere.

32. The method of claim 31 wherein: At least one of the following is satisfied: Step l, the molar ratio of compound 12 to compound 9 is 1:(1.0-3.0); Step l, the molar ratio of compound 12 to base is 1:(1.0-4.0); Step l, the base is at least one selected from diisopropylethylamine, triethylamine, potassium phosphate, potassium carbonate, sodium carbonate, cesium carbonate; Step l, the reaction system further comprises a reaction solvent, and the reaction solvent is at least one selected from N,N-dimethylformamide, N,N-dimethylacetamide, 2-butanone, toluene; Step l, the reaction temperature is 85°C; Step l, the reaction is carried out under nitrogen protection.

33. The method of claim 31 wherein: At least one of the following is satisfied: Step l, the molar ratio of compound 12 to compound 9 is 1:1.1; Step l, the molar ratio of compound 12 to base is 1:1.

5.

34. The method of claim 12 wherein: Step m, the reaction system further comprises a reaction solvent selected from at least one of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide.

35. The method of claim 12 wherein: Step n, compound 16, tetrahydro-pyrrole and Pd catalyst, under the protection of atmosphere.

36. The method of claim 35 wherein: At least one of the following is met: Step n, the molar ratio of compound 16 to tetrahydro-pyrrole is 1:(1.0-4.0); Step n, the Pd catalyst is selected from at least one of Pd(dppf)Cl2·CH2Cl2, PdCl2(PPh3)2, Pd[P(Ph)3]4, Pd(OAc)2; Step n, the molar ratio of compound 16 to Pd catalyst is 1:0.02-0.10; Step n, the reaction system further comprises a reaction solvent selected from at least one of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide; Step n, the reaction temperature is 25℃; Step n, the reaction is carried out under the protection of nitrogen.

37. The method of claim 35, wherein: At least one of the following is met: Step n, the molar ratio of compound 16 to tetrahydro-pyrrole is 1:3.5; Step n, the molar ratio of compound 16 to Pd catalyst is 1:0.

06.

38. The method of claim 12 wherein: Step o, compound 14, compound 9 and a base, under the protection of atmosphere.

39. The method of claim 38 wherein: At least one of the following is met: Step o, the molar ratio of compound 14 to compound 9 is 1:(1.0-3.0); Step o, the molar ratio of compound 14 to the base is 1:(1.0-4.0); Step o, the base is selected from at least one of diisopropylethylamine, triethylamine, potassium phosphate, potassium carbonate, sodium carbonate, cesium carbonate; Step o, the reaction system further comprises a reaction solvent selected from at least one of N,N-dimethylformamide, N,N-dimethylacetamide, 2-butanone, toluene; Step o, the reaction temperature is 85℃; Step o, the reaction is carried out under the protection of nitrogen.

40. The method of claim 38 wherein: At least one of the following is met: Step o, the molar ratio of compound 14 to compound 9 is 1:1.1; Step o, the molar ratio of compound 14 to the base is 1:1.

5.

41. The method of claim 12 wherein: Step p, the reaction system further comprises a reaction solvent selected from at least one of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide.

42. The method of claim 12 wherein: Step q, compound 19, tetrahydro-pyrrole and Pd catalyst, under the protection of atmosphere.

43. The method of claim 42, wherein: At least one of the following is met: Step q, the molar ratio of compound 19 to tetrahydro-pyrrole is 1:(1.0-4.0); Step q, the Pd catalyst is selected from at least one of Pd(dppf)Cl2·CH2Cl2, PdCl2(PPh3)2, Pd[P(Ph)3]4, Pd(OAc)2; Step q, the molar ratio of compound 19 to Pd catalyst is 1:0.02-0.10; Step q, the reaction system further comprises a reaction solvent selected from at least one of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide; Step q, the reaction temperature is 25℃; Step q, the reaction is carried out under the protection of nitrogen.

44. The method of claim 42, wherein: At least one of the following is met: Step q, the molar ratio of compound 19 to tetrahydropyrrole was 1:3.5; Step q, the molar ratio of compound 19 to Pd catalyst was 1:0.

06.

45. A pharmaceutical composition characterized in that: The compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 11 is used as an active ingredient.

46. Use of the compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, or the pharmaceutical composition according to claim 45, in the preparation of an antitumor drug for gastric cancer, breast cancer, or ovarian cancer.

47. Use of the compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 11 in the preparation of an antibody drug conjugate.

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