Camptothecin derivatives and their conjugates, their preparation methods and pharmaceutical uses

Abstract

Camptothecin derivatives and their conjugates, their preparation methods, and their pharmaceutical uses. Derivatives of camptothecin compounds and drug conjugates containing ligands thereof, pharmaceutical compositions containing such conjugates, and the use of such conjugates in the treatment of cancer.

Description

Technical Field

[0001] This invention relates to novel camptothecin derivatives and their conjugates, methods for their preparation, pharmaceutical compositions containing the same, and their use in the preparation of antitumor drugs. Background Technology

[0002] Antibody-drug conjugates (ADCs) are a relatively new class of anticancer drugs designed to combine the selectivity of monoclonal antibodies with the cell-killing properties of cytotoxic agents. ADCs have attracted considerable interest as a novel therapeutic approach and continue to be developed. ADCs are designed to link monoclonal antibodies or antibody fragments to biologically active cytotoxic agents via linkers, fully utilizing the specificity of antibodies binding to tumor cell surface antigens and the high efficiency of cytotoxic substances, while avoiding the drawbacks of low efficacy of antibodies themselves and excessive toxicity of cytotoxic agents. This means that, compared to traditional chemotherapy drugs, antibody-drug conjugates can more precisely kill tumor cells and reduce toxicity to normal cells. Therefore, the linker in ADC drugs is not only the molecular part that forms a covalent link between the antibody and the small molecule drug, but also a key element with design properties in targeted drug therapy. This involves multiple considerations: the addition of the linker should not induce aggregation; ensure acceptable pharmacokinetic (PK) properties; improve blood circulation stability; and ensure effective release of the active molecule at the target site.

[0003] Camptothecin (CPT) is a pentacyclic quinoline alkaloid, originally isolated from the wood and bark of the native Chinese tree species *Camptotheca acuminata*. Camptothecin exhibits significant antitumor activity by inhibiting topoisomerase I, an enzyme overexpressed in various tumor cell lines and crucial for DNA synthesis. Camptothecin binds to the Topo I-DNA complex, stabilizing it and preventing the rejoining of broken DNA strands, thus inhibiting DNA replication and RNA synthesis. Due to its broad-spectrum antitumor activity and unique mechanism of action, efforts have been made to develop clinical analogues of camptothecin. Currently, only three camptothecin analogues are marketed: irinotecan (approved by the FDA in 1994 by Pfizer), topotecan (approved by the FDA in 2007 by Novartis), and belotecan (approved in South Korea in 2003 by Chong Kun Dang Pharmaceuticals).

[0004] However, camptothecin and most of its derivatives exhibit poor solubility and low activity under physiological conditions, limiting the clinical development of camptothecin analogues. Therefore, formulating camptothecin into an ADC can overcome these limitations. Irinotecan is a prodrug whose active metabolite SN-38 has poor water solubility and a short half-life. Immunomedics uses SN-38 as a toxicant linked to sacituzumab, which targets Trop-2-expressing cancer cells for the treatment of adult patients with metastatic triple-negative breast cancer (TNBC). The humanized lyophilized sacituzumab Govitecan-Hziy injection was approved by the FDA for treatment in April 2022 (US7999083B2).

[0005] The amino group of essanotecan (DX-8951f) contributes to its water solubility, while the rigidity conferred by the cyclohexane ring is thought to benefit the balance between the active lactone form and the inactive hydrolyzed hydroxy acid, thereby enhancing its activity; however, clinical trials did not meet the expected endpoints. Daiichi Sankyo utilized aminohydroxyacetylation to generate DXd, which has 2-4 times lower activity than essanotecan (US20210169852 A). Using an enzyme-cleavable Gly-Gly-Phe-Gly tetrapeptide linker to link DXd, the resulting ADC (Enhertu) conjugated with an anti-HER2 antibody showed great potential against HER2-expressing cancers in a clinical setting. Therefore, Enhertu received accelerated approval from the FDA on December 20, 2019, for adult patients with HER2-positive, unresectable, or metastatic breast cancer who have received two or more prior lines of anti-HER2 therapy in metastatic disease. While the cyclohexane ring of DXd is thought to stabilize the bioactive lactone form, it carries a chiral center, complicating synthetic work and SAR studies. To overcome this challenge, researchers at ImmunoGen designed a new group of camptothecin analogs. The ring was opened, the extra chiral center was eliminated, and a group was introduced at position 7 that could be linked to an antibody. When conjugated with an antibody against human epidermal growth factor receptor (HuEGFR), the resulting ADC was effective in an EGFR-positive HSC-2 tumor xenograft model (US20210077482A1). Researchers at MediBoston used a similar method to derive a functional group at position 9 to link the antibody, introducing a hydrophilic peptide linker, and the resulting ADC showed promising preclinical results (WO 2021173773).

[0006] Although camptothecin analogues possess good antitumor activity, especially after conjugation with humanized antibodies, exhibiting excellent targeting and tumor-killing activity along with a low onset dose, thus reducing toxicity and increasing the therapeutic window, different types of camptothecin derivatives show significant differences in half-life, permeability, and in vitro activity, as well as substantial variations in toxicity to different cancer cells. Therefore, we aim to improve the activity and permeability of camptothecin derivatives against tumor cells (i.e., enhance the bystander effect of camptothecin derivatives) by designing novel structures, thereby increasing the killing effect on tumor cells with low or no antigen expression, in order to achieve certain clinical efficacy. Summary of the Invention

[0007] In one aspect, this invention provides a compound of general formula (A) or a pharmaceutically acceptable salt thereof.

[0008] L-L2-L1-Dr

[0009] (A)

[0010] in:

[0011] Dr is selected from the following structure:

[0012]

[0013] R 1 Selected from hydrogen, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, -NR d R e -(CH2) m -OH, -(CH2) m -NR d R e -OC(=O)NR f -(CH2) m -OH, -OC(=O)NR f -(CH2) m -NR d R e -(CH2) m -C(=O)OH, -(CH2) m -C(=O)-NR d R e -(CH2) m -C(=O)NR f -(CH2) n -OH, -(CH2) m -C(=O)NR f -(CH2) n -NR d R e -NRf -(CH2) m -OH, -NR f -(CH2) m -NR d R e -O-(CH2) m -OH, -O-(CH2) m -NR d R e -NR f C(=O)O-(CH2) m -OH, -NR b C(=O)O-(CH2) m -NR d R e -(CH2) m -NR f C(=O)O-(CH2) n -OH-, -(CH2) m -NR f C(=O)O-(CH2) n -NR d R e -(CH2) m -OC(=O)NR f -(CH2) n -NR d R e -(CH2) m -OC(=O)NR f -(CH2) n -OH, -(CH2) m -NR f C(=O)-(CH2) n -NR d R e -(CH2) m -NR f C(=O)-(CH2) n -OH, -(CH2) m -NR f C(=O)-G-(CH2) n -OH, wherein the alkyl, alkoxy, alkenyl, alkynyl, or cycloalkyl group is optionally further substituted with one or more groups selected from halogen, deuterium, amino, alkyl, or hydroxyl groups;

[0014] R 2 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, alkenyl, and alkynyl, wherein the alkyl, alkoxy, alkenyl, and alkynyl groups are optionally further substituted by one or more groups selected from halogens;

[0015] R 3 Selected from hydrogen, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, alkenyl, alkynyl, CH2=, -NR d R e -(CH2) m -OH, -(CH2) m -NR d R e -OC(=O)NR f -(CH2) m -OH, -OC(=O)NR f -(CH2) m -NR d R e -(CH2) m -C(=O)OH, -(CH2) m -C(=O)-NR d R e -(CH2) m -C(=O)NR f -(CH2) n -OH, -(CH2) m -C(=O)NR f -(CH2) n -NR d R e -NR f -(CH2) m -OH, -NR f -(CH2) m -NR d R e -NR f -C(=O)R d -NR f -C(=O)-(CH2) m -R d -NR f C(=O)O-(CH2) m -R d -O-(CH2) m -OH, -O-(CH2) m -NR d R e -NR f C(=O)O-(CH2) m -OH, -NR f C(=O)NR d -(CH2) m -OH, -NR f C(=O)NR d -(CH2) m-O-(CH2) n -OH, -NR f C(=O)O-(CH2) m -O-(CH2) n -OH, -NR f C(=O)NR d -(CH2) m -O-(CH2) n -NR d R e -NR f C(=O)O-(CH2) m -O-(CH2) n -NR d R e -NR f C(=O)NR d -(CH2) m -NR d R e -NR f C(=O)O-(CH2) m -NR d R e -(CH2) m -NR f C(=O)O-(CH2) n -OH, -(CH2) m -NR f C(=O)O-(CH2) n -NR d R e -(CH2) m -OC(=O)NR f -(CH2) n -NR d R e -(CH2) m -OC(=O)NR f -(CH2) n -OH, -(CH2) m -NR f C(=O)-(CH2) n -NR d R e -(CH2) m -NR f C(=O)-(CH2) n -OH, wherein the alkyl, alkoxy, alkenyl, or alkynyl groups are optionally further substituted with one or more groups selected from halogen, deuterium, amino, alkyl, or hydroxyl groups;

[0016] R 4Selected from hydrogen, halogen, hydroxyl, carboxyl, amino, cyano, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, -NR d R e -(CH2) m -OH, -(CH2) m -NR d R e -OC(=O)NR f -(CH2) m -OH, -OC(=O)NR f -(CH2) m -NR d R e -(CH2) m -C(=O)OH, -(CH2) m -C(=O)-NR d R e -(CH2) m -C(=O)NR f -(CH2) n -OH, -(CH2) m -C(=O)NR f -(CH2) n -NR d R e -NR f -(CH2) m -OH, -NR f -(CH2) m -NR d R e -O-(CH2) m -OH, -O-(CH2) m -NR d R e -NR f C(=O)O-(CH2) m -OH, -NR b C(=O)O-(CH2) m -NR d R e -(CH2) m -NR f C(=O)O-(CH2) n -OH-, -(CH2) m -NR f C(=O)O-(CH2) n -NR d R e -(CH2) m -OC(=O)NR f -(CH2)n -NR d R e -(CH2) m -OC(=O)NR f -(CH2) n -OH, -(CH2) m -NR f C(=O)-(CH2) n -NR d R e -(CH2) m -NR f C(=O)-(CH2) n -OH, -(CH2) m -NR f C(=O)-G-(CH2) n -OH, wherein the alkyl, alkoxy, alkenyl, alkynyl, or cycloalkyl group is optionally further substituted with one or more groups selected from halogen, deuterium, amino, alkyl, or hydroxyl groups; the above-mentioned -(CH2) m -Optionally substituted with one or more deuterium or halogens; G is selected from cycloalkylene, heterocyclic, heteroaryl, and aryl;

[0017] R 5 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, alkenyl, and alkynyl, wherein the alkyl, alkoxy, alkenyl, and alkynyl groups are optionally further substituted by one or more groups selected from halogens;

[0018] R 6 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, alkenyl, and alkynyl, wherein the alkyl, alkoxy, alkenyl, and alkynyl groups are optionally further substituted by one or more groups selected from halogens;

[0019] R 7 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, alkenyl, and alkynyl, wherein the alkyl, alkoxy, alkenyl, and alkynyl groups are optionally further substituted by one or more groups selected from halogens;

[0020] R d and R e Each is independently selected from hydrogen and alkyl groups;

[0021] R f Selected from hydrogen, alkyl, -C(O)R c -S(O)R c -S(O)2R c The alkyl group may optionally be further substituted with a cycloalkyl group; wherein R c Selected from hydrogen, hydroxyl, and alkyl;

[0022] L1 is selected from the bond, -(CH2).m -*、-O-*、-NR a -*、-(CH2) m -O-*、-(CH2) m -NR a -*、-OC(=O)NR b -(CH2) m -O-*、-OC(=O)NR b -(CH2) m -NR a -*、-(CH2) m -C(=O)O-*、-(CH2) m -C(=O)NR a -*、-(CH2) m -C(=O)NR b -(CH2) n -O-*、-(CH2) m -C(=O)NR b -(CH2) n -NR a -*、-NR b -(CH2) m -O-*、-NR b -(CH2) m -NR a -*、-O-(CH2) m -O-*, -O-(CH2) m -NR a -*、-NR b C(=O)O-(CH2) m -O-*、-NR b C(=O)O-(CH2) m -NR a -*、-(CH2) m -NR b C(=O)O-(CH2) n -O-*、-(CH2) m -NR b C(=O)O-(CH2) n -NR a -*、-(CH2) m -OC(=O)NR b -(CH2) n -NR a -*、-(CH2) m -OC(=O)NR b -(CH2) n -O-*、-(CH2) m -NRb C(=O)-(CH2) n -NR a -*、-(CH2) m -NR b C(=O)-(CH2) n -O-*, where * is the connection site with L2;

[0023] R a and R b Each is independently selected from hydrogen, alkyl, -C(O)R c -S(O)R c -S(O)2R c The alkyl group may optionally be further substituted with a cycloalkyl group; wherein R c Selected from hydrogen, hydroxyl, and alkyl;

[0024] L2 is selected from the key, Where * represents the connection site with L1;

[0025] L is

[0026] L3 is an amino acid residue formed by two or more amino acids, and L3 optionally includes one or more structures selected from the following: L6 is selected from one or more structures selected from the following:

[0027]

[0028] Among them, R, R aa R bb Each is independently selected from hydrogen and alkyl groups;

[0029] L4 is

[0030] Z1 is selected from the bond, -(CH2). p -、-(C2H4O) q -、-(CH2) p -C(O)NH-, -(CH2) p -O-(CH2) p -C(O)NH-, -(CH2) p -C(O)-L6-NH-、-(CH2) p -O-(CH2) p -C(O)-L6-NH-, -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)-, -NH-, -O- and -OC(O)NH-;

[0031] m is an integer from 1 to 6;

[0032] n is an integer from 1 to 6;

[0033] s is an integer from 1 to 6;

[0034] t is an integer from 0 to 10;

[0035] s1, s2, s3, and s4 are each independently an integer from 0 to 10; preferably an integer from 0 to 8 or an integer from 0 to 6; even more preferably an integer from 0 to 4, and even more preferably an integer from 0 to 2 or an integer from 1 to 2.

[0036] s5 and s6 are each independent integers from 1 to 6;

[0037] t1 is an integer from 1 to 6;

[0038] t2 is an integer from 0 to 6;

[0039] t3 is an integer from 1 to 6;

[0040] t4 is an integer from 0 to 10;

[0041] t5 is an integer from 0 to 10;

[0042] p is an integer from 1 to 10;

[0043] q is an integer from 1 to 10;

[0044] Q stands for connector unit.

[0045] Z1 is connected to Q.

[0046] In one specific embodiment, according to the invention, a compound of general formula (A) or a pharmaceutically acceptable salt thereof, wherein,

[0047] Dr is selected from the following structure:

[0048]

[0049] L1 is selected from -O-*, -NR a -*、-(CH2) m -O-*、-(CH2) m -NR a -*、-OC(=O)NR b -(CH2) m -O-*、-OC(=O)NR b -(CH2) m -NR a -*、-(CH2) m -C(=O)O-*、-(CH2) m -C(=O)NR a -*、-NR b-(CH2) m -O-*、-NR b -(CH2) m -NR a -*、-O-(CH2) m -O-*、-O-(CH2) m -NR a -*、-NR b C(=O)O-(CH2) m -O-*、-NR b C(=O)O-(CH2) m -NR a -*、-(CH2) m -NR b C(=O)-(CH2) n -NR a -*、-(CH2) m -NR b C(=O)-(CH2) n -O-*, where * is the connection site with L2;

[0050] R a Selected from hydrogen and C1-C6 alkyl groups;

[0051] R b Selected from hydrogen, alkyl, -C(O)R c -S(O)R c -S(O)2R c , where R c Selected from hydrogen, hydroxyl, and C1-C6 alkyl groups;

[0052] m is an integer from 1 to 6; preferably an integer from 1 to 4; more preferably an integer from 1 to 2;

[0053] n is an integer from 1 to 6; preferably an integer from 1 to 4; more preferably an integer from 1 to 2;

[0054] R 1 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl are optionally further substituted with one or more groups selected from halogen; preferably C1-C6 alkyl and C1-C6 alkoxy.

[0055] R 2The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl may optionally be further substituted with one or more groups selected from halogen; halogen is preferred.

[0056] In another specific embodiment, according to the invention, a compound of general formula (A) or a pharmaceutically acceptable salt thereof, wherein,

[0057] Dr is selected from the following structure:

[0058]

[0059] L1 is selected from -O-*, -NR a -*、-(CH2) m -O-*、-(CH2) m -NR a -*、-OC(=O)NR b -(CH2) m -O-*、-OC(=O)NR b -(CH2) m -NR a -*、-(CH2) m -C(=O)O-*、-(CH2) m -C(=O)NR a -*、-NR b -(CH2) m -O-*、-NR b -(CH2) m -NR a -*、-O-(CH2) m -O-*、-O-(CH2) m -NR a -*、-NR b C(=O)O-(CH2) m -O-*、-NR b C(=O)O-(CH2) m -NR a -*、-(CH2) m -NR b C(=O)-(CH2) n -NR a -*、-(CH2) m -NR b C(=O)-(CH2) n -O-*, where * is the connection site with L2;

[0060] Ra Selected from hydrogen and C1-C6 alkyl groups;

[0061] R b Selected from hydrogen, alkyl, -C(O)R c -S(O)R c -S(O)2R c The alkyl group may optionally be further substituted with a cycloalkyl group; wherein R c Selected from hydrogen, hydroxyl, and C1-C6 alkyl groups;

[0062] m is an integer from 1 to 6; preferably an integer from 1 to 4; more preferably an integer from 1 to 2;

[0063] n is an integer from 1 to 6; preferably an integer from 1 to 4; more preferably an integer from 1 to 2;

[0064] R 1 Selected from hydrogen, halogen, hydroxyl, amino, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, -NR d R e -(CH2) m -OH, -(CH2) m -NR d R e -OC(=O)NR f -(CH2) m -OH, -OC(=O)NR f -(CH2) m -NR d R e -(CH2) m -C(=O)OH, -(CH2) m -C(=O)-NR d R e -(CH2) m -C(=O)NR f -(CH2) n -OH, -(CH2) m -C(=O)NR f -(CH2) n -NR d R e -NR f -(CH2) m -OH, -NR f -(CH2) m -NR d R e -O-(CH2) m -OH, -O-(CH2) m -NRd R e -NR f C(=O)O-(CH2) m -OH, -NR b C(=O)O-(CH2) m -NR d R e -(CH2) m -NR f C(=O)O-(CH2) n -OH-, -(CH2) m -NR f C(=O)O-(CH2) n -NR d R e -(CH2) m -OC(=O)NR f -(CH2) n -NR d R e -(CH2) m -OC(=O)NR f -(CH2) n -OH, -(CH2) m -NR f C(=O)-(CH2) n -NR d R e -(CH2) m -NR f C(=O)-(CH2) n -OH, -(CH2) m -NR f C(=O)-G-(CH2) n -OH, wherein the C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, or C3-C6 cycloalkyl may optionally be further substituted with one or more groups selected from halogens, deuterium, amino, C1-C6 alkyl, or hydroxyl; preferably C1-C6 alkyl or C1-C6 alkoxy; the above-mentioned -(CH2) m -Optionally substituted with one or more deuterium or halogens; G is selected from cycloalkylene, heterocyclic, heteroaryl, and aryl;

[0065] R 2 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl may optionally be further substituted with one or more groups selected from halogen; halogen is preferred.

[0066] R d R e R f , m, n are as defined by general formula (A).

[0067] In another specific embodiment, according to the invention, a compound of general formula (A) or a pharmaceutically acceptable salt thereof, wherein,

[0068] Dr is selected from the following structure:

[0069]

[0070] L1 is selected from the bond, -(CH2). m -*、-O-*、-(CH2) m -O-*、-NR a -*、-(CH2) m -NR a -*、-NR b -(CH2) m -O-*、-NR b -(CH2) m -NR a -*、-O-(CH2) m -O-*、-O-(CH2) m -NR a -*、-(CH2) m -OC(=O)NR b -(CH2) n -NR a -*、-(CH2) m -OC(=O)NR b -(CH2) n -O-*、-(CH2) m -NR b C(=O)-(CH2) n -NR a -*、-(CH2) m -NR b C(=O)-(CH2) n -O-*, where * is the connection site with L2;

[0071] R a Selected from hydrogen and C1-C6 alkyl groups;

[0072] R b Selected from hydrogen, alkyl, -C(O)R c -S(O)R c -S(O)2R c The alkyl group may optionally be further substituted with a cycloalkyl group; wherein Rc Selected from hydrogen, hydroxyl, and C1-C6 alkyl groups;

[0073] m is an integer from 1 to 6; preferably an integer from 1 to 4;

[0074] n is an integer from 1 to 6; preferably an integer from 1 to 4;

[0075] R 2 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl may optionally be further substituted with one or more groups selected from halogen; halogen is preferred.

[0076] In another specific embodiment, according to the invention, a compound of general formula (A) or a pharmaceutically acceptable salt thereof, wherein,

[0077] Dr is selected from the following structure:

[0078]

[0079] L1 is selected from -O-*, where * is the connection site with L2;

[0080] R 2 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl may optionally be further substituted with one or more groups selected from halogen; halogen is preferred.

[0081] R 3 Selected from hydrogen, halogen, hydroxyl, amino, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, CH2=, -NR d R e -(CH2) m -OH, -(CH2) m -NR d R e -OC(=O)NR f -(CH2) m -OH, -OC(=O)NR f -(CH2) m -NR d R e -(CH2) m -C(=O)OH, -(CH2) m -C(=O)-NR d Re 、-(CH2) m -C(=O)NR f -(CH2) n -OH、-(CH2) m -C(=O)NR f -(CH2) n -NR d R e 、-NR f -(CH2) m -OH、-NR f -(CH2) m -NR d R e 、-NR f -C(=O)R d 、-NR f -C(=O)-(CH2) m -R d 、-NR f C(=O)O-(CH2) m -R d 、-O-(CH2) m -OH、-O-(CH2) m -NR d R e 、-NR f C(=O)O-(CH2) m -OH、-NR f C(=O)NR d -(CH2) m -OH、-NR f C(=O)NR d -(CH2) m -O-(CH2) n -OH、-NR f C(=O)O-(CH2) m -O-(CH2) n -OH、-NR f C(=O)NR d -(CH2) m -O-(CH2) n -NR d R e 、-NR f C(=O)O-(CH2) m -O-(CH2) n -NR d R e 、-NR f C(=O)NR d -(CH2) m-NR d R e -NR f C(=O)O-(CH2) m -NR d R e -(CH2) m -NR f C(=O)O-(CH2) n -OH, -(CH2) m -NR f C(=O)O-(CH2) n -NR d R e -(CH2) m -OC(=O)NR f -(CH2) n -NR d R e -(CH2) m -OC(=O)NR f -(CH2) n -OH, -(CH2) m -NR f C(=O)-(CH2) n -NR d R e -(CH2) m -NR f C(=O)-(CH2) n -OH, wherein the C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl groups are optionally further substituted with one or more groups selected from halogens, deuterium, amino, C1-C6 alkyl, and hydroxyl groups; preferably hydrogen or hydroxyl.

[0082] R 4 Selected from hydrogen, halogen, hydroxyl, carboxyl, amino, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, -NR d R e -(CH2) m -OH, -(CH2) m -NR d R e -OC(=O)NR f -(CH2) m -OH, -OC(=O)NR f -(CH2) m -NR d R e -(CH2) m -C(=O)OH, -(CH2)m -C(=O)-NR d R e 、-(CH2) m -C(=O)NR f -(CH2) n -OH、-(CH2) m -C(=O)NR f -(CH2) n -NR d R e 、-NR f -(CH2) m -OH、-NR f -(CH2) m -NR d R e 、-O-(CH2) m -OH、-O-(CH2) m -NR d R e 、-NR f C(=O)O-(CH2) m -OH、-NR b C(=O)O-(CH2) m -NR d R e 、-(CH2) m -NR f C(=O)O-(CH2) n -OH-、-(CH2) m -NR f C(=O)O-(CH2) n -NR d R e 、-(CH2) m -OC(=O)NR f -(CH2) n -NR d R e 、-(CH2) m -OC(=O)NR f -(CH2) n -OH、-(CH2) m -NR f C(=O)-(CH2) n -NR d R e 、-(CH2) m -NR f C(=O)-(CH2) n -OH、-(CH2) m -NR fC(=O)-G-(CH2) n -OH, wherein the C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, or C3-C6 cycloalkyl may optionally be further substituted with one or more groups selected from halogens, deuterium, amino, C1-C6 alkyl, or hydroxyl; preferably hydroxyl, C1-C6 alkyl, or C1-C6 alkoxy; the above-mentioned -(CH2) m -Optionally substituted with one or more deuterium or halogens; G is selected from cycloalkylene, heterocyclic, heteroaryl, and aryl;

[0083] R d R e R f , m, n are as defined by general formula (A).

[0084] In another specific embodiment, according to the invention, a compound of general formula (A) or a pharmaceutically acceptable salt thereof, wherein,

[0085] Dr is selected from the following structure:

[0086]

[0087] L1 is selected from -O-*, where * is the connection site with L2;

[0088] R 2 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl may optionally be further substituted with one or more groups selected from halogen; halogen is preferred.

[0089] R 3 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, CH2=, wherein the C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl are optionally further substituted with one or more groups selected from halogen; preferably hydrogen or hydroxyl;

[0090] R 4 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl are optionally further substituted with one or more groups selected from halogen; preferably hydroxyl, C1-C6 alkyl, and C1-C6 alkoxy.

[0091] In another specific embodiment, according to the invention, a compound of general formula (A) or a pharmaceutically acceptable salt thereof, wherein,

[0092] Dr is selected from the following structure:

[0093]

[0094] L1 is selected from -(CH2) m -O-*、-(CH2) m -NR a -*, where * is the connection site with L2;

[0095] m is an integer from 1 to 6; preferably an integer from 1 to 4;

[0096] R a Selected from hydrogen and C1-C6 alkyl groups;

[0097] R 5 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl may optionally be further substituted with one or more groups selected from halogen; halogen is preferred.

[0098] R 7 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl may optionally be further substituted with one or more groups selected from halogen; preferably hydroxyl and amino.

[0099] In another specific embodiment, according to the invention, a compound of general formula (A) or a pharmaceutically acceptable salt thereof, wherein,

[0100] Dr is selected from the following structure:

[0101]

[0102] L1 is selected from -O-*, where * is the connection site with L2;

[0103] R 5 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl may optionally be further substituted with one or more groups selected from halogen; halogen is preferred.

[0104] R6 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl may optionally be further substituted with one or more groups selected from halogen; preferably hydroxyl and amino.

[0105] R 7 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl, wherein the C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, and C2-C6 alkynyl may optionally be further substituted with one or more groups selected from halogen; preferably hydroxyl and amino.

[0106] In a preferred embodiment, the compound of formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein Dr is selected from:

[0107]

[0108] In another preferred embodiment, the compound represented by general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein,

[0109] When L1 is selected from -NR a -*、-(CH2) m -NR a -*、-OC(=O)NR b -(CH2) m -NR a -*、-(CH2) m -C(=O)NR a -*、-(CH2) m -C(=O)NR b -(CH2) n -NR a -*、-NR b -(CH2) m -NR a -*、-O-(CH2) m -NR a -*、-NR b C(=O)O-(CH2) m -NR a -*、-(CH2) m -NR b C(=O)O-(CH2) n -NR a -*、-(CH2) m -OC(=O)NRb -(CH2) n -NR a -* or -(CH2) m -NR b C(=O)-(CH2) n -NR a -*, where * is the connection site with L2;

[0110] L2 is selected from key or Where * represents the connection site with L1;

[0111] R a R b , m, n are as defined by general formula (A).

[0112] In another preferred embodiment, the compound represented by general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein,

[0113] L1 is selected from the following: -O-*, -(CH2). m -O-*、-NR a -*、-OC(=O)NR b -(CH2) m -O-*、-(CH2) m -C(=O)O-*、-(CH2) m -C(=O)NR b -(CH2) n -O-*、-NR b -(CH2) m -O-*、-O-(CH2) m -O-*、-NR b C(=O)O-(CH2) m -O-*、-(CH2) m -NR b C(=O)O-(CH2) n -O-*、-(CH2) m -OC(=O)NR b -(CH2) n -O-* or -(CH2) m -NR b C(=O)-(CH2) n -O-*, where * is the connection site with L2;

[0114] L2 is selected from the key, Where * represents the connection site with L1;

[0115] R a Rb , m, n are as defined by general formula (A).

[0116] In another preferred embodiment, the compound represented by general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein,

[0117] L3 is an amino acid residue formed from two or more amino acids selected from phenylalanine, alanine, glycine, valine, leucine, isoleucine, tryptophan, tyrosine, histidine, lysine, citrulline, serine, threonine, cysteine, glutamic acid, glutamine, aspartic acid, asparagine, methionine, and arginine, and L3 optionally includes one or more structures selected from the following:

[0118]

[0119]

[0120] Preferred

[0121] Among them, R, R aa R bb Each is independently selected from hydrogen and C1-C6 alkyl groups;

[0122] s is an integer from 1 to 6; preferably an integer from 2 to 6; more preferably an integer from 2 to 4;

[0123] s5 and s6 are each independently an integer from 1 to 6; preferably an integer from 2 to 6; more preferably an integer from 2 to 4;

[0124] t is an integer from 0 to 10; preferably an integer from 2 to 8; more preferably an integer from 3 to 7;

[0125] t1 is an integer from 1 to 6; preferably an integer from 1 to 4, even more preferably an integer from 2 to 4, and even more preferably 1 or 2;

[0126] t2 is an integer from 0 to 6; preferably an integer from 1 to 4, and even more preferably 1 or 2;

[0127] t3 is an integer from 1 to 6; preferably an integer from 1 to 4, more preferably 1 or 2;

[0128] t4 is an integer from 0 to 10;

[0129] t5 is an integer from 0 to 10.

[0130] In another embodiment, L3 is an amino acid residue formed from two or more amino acids selected from phenylalanine, alanine, glycine, valine, leucine, isoleucine, tryptophan, tyrosine, histidine, lysine, citrulline, serine, threonine, cysteine, glutamic acid, glutamine, aspartic acid, asparagine, methionine, and arginine, and L3 optionally includes one or more structures selected from the following:

[0131]

[0132] R, R aa R bb s, t, t1-t5, s5-s6 are as described above.

[0133] In another preferred embodiment, the compound represented by general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein L3 is... * indicates the position where L2 is attached, and · indicates the position where carbonyl or methylene is attached;

[0134] Among them, L 1b and L' 1b Each is an amino acid residue formed independently from one or more amino acids selected from phenylalanine, alanine, glycine, valine, leucine, isoleucine, tryptophan, tyrosine, histidine, lysine, citrulline, serine, threonine, cysteine, glutamic acid, glutamine, aspartic acid, asparagine, methionine, and arginine.

[0135] L 1a It can be a key or selected from one or more of the following structures:

[0136]

[0137] Preferred

[0138] R is selected from hydrogen and C1-C6 alkyl groups, with hydrogen being preferred;

[0139] R aa R bb Each is independently selected from C1-C6 alkyl groups;

[0140] s is an integer from 1 to 6; preferably an integer from 2 to 6; more preferably an integer from 2 to 4;

[0141] s5 and s6 are each independently an integer from 1 to 6; preferably an integer from 2 to 6; more preferably an integer from 2 to 4;

[0142] t is an integer from 0 to 10; preferably an integer from 2 to 8; more preferably an integer from 3 to 7;

[0143] t1 is an integer from 1 to 6; preferably an integer from 1 to 4, even more preferably an integer from 2 to 4, and even more preferably 1 or 2;

[0144] t2 is an integer from 0 to 6; preferably an integer from 1 to 4, and even more preferably 1 or 2;

[0145] t3 is an integer from 1 to 6; preferably an integer from 1 to 4, more preferably 1 or 2;

[0146] t4 is an integer from 0 to 10;

[0147] t5 is an integer from 0 to 10.

[0148] In another implementation, L3 is * indicates the position where L2 is attached, and · indicates the position where carbonyl or methylene is attached;

[0149] Among them, L 1b and L' 1b Each is an amino acid residue formed independently from one or more amino acids selected from phenylalanine, alanine, glycine, valine, leucine, isoleucine, tryptophan, tyrosine, histidine, lysine, citrulline, serine, threonine, cysteine, glutamic acid, glutamine, aspartic acid, asparagine, methionine, and arginine.

[0150] L 1a It can be a key or selected from one or more of the following structures:

[0151]

[0152] R, R aa R bb s, t, t1-t5, s5-s6 are as described above.

[0153] In another preferred embodiment, the compound represented by general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein L3 is selected from:

[0154]

[0155]

[0156] in:

[0157] L 1b and L' 1bEach is an amino acid residue formed independently from one or more amino acids selected from phenylalanine, alanine, glycine, valine, leucine, isoleucine, tryptophan, tyrosine, histidine, lysine, citrulline, serine, threonine, cysteine, glutamic acid, glutamine, aspartic acid, asparagine, methionine, and arginine.

[0158] R is selected from hydrogen and C1-C6 alkyl groups, with hydrogen being preferred;

[0159] R aa R bb Each is independently selected from C1-C6 alkyl groups;

[0160] s is an integer from 1 to 6; preferably an integer from 2 to 6; more preferably an integer from 2 to 4;

[0161] s5 and s6 are each independently an integer from 1 to 6; preferably an integer from 2 to 6; more preferably an integer from 2 to 4;

[0162] t is an integer from 0 to 10; preferably an integer from 2 to 8; more preferably an integer from 3 to 7;

[0163] t1 is an integer from 1 to 6; preferably an integer from 1 to 4, even more preferably an integer from 2 to 4, and even more preferably 1 or 2;

[0164] t2 is an integer from 0 to 6; preferably an integer from 1 to 4, and even more preferably 1 or 2;

[0165] t3 is an integer from 1 to 6; preferably an integer from 1 to 4, more preferably 1 or 2;

[0166] t4 is an integer from 0 to 10;

[0167] t5 is an integer from 0 to 10;

[0168] * indicates the location where it connects to L2.

[0169] The position where it is attached to the carbonyl or methylene group.

[0170] In another preferred embodiment, the compound represented by general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein L3 is selected from:

[0171]

[0172]

[0173] in:

[0174] L 1b and L' 1bEach is an amino acid residue formed independently from one or more amino acids selected from phenylalanine, alanine, glycine, valine, leucine, isoleucine, tryptophan, tyrosine, histidine, lysine, citrulline, serine, threonine, cysteine, glutamic acid, glutamine, aspartic acid, asparagine, methionine, and arginine.

[0175] R is selected from hydrogen and C1-C6 alkyl groups, with hydrogen being preferred;

[0176] R aa R bb Each is independently selected from C1-C6 alkyl groups;

[0177] s5 and s6 are each independently an integer from 1 to 6; preferably an integer from 2 to 6; more preferably an integer from 2 to 4;

[0178] t is an integer from 0 to 10; preferably an integer from 2 to 8; more preferably an integer from 3 to 7;

[0179] t1 is an integer from 1 to 6; preferably an integer from 1 to 4, even more preferably an integer from 2 to 4, and even more preferably 1 or 2;

[0180] t2 is an integer from 0 to 6; preferably an integer from 1 to 4, and even more preferably 1 or 2;

[0181] t3 is an integer from 1 to 6; preferably an integer from 1 to 4, more preferably 1 or 2;

[0182] t4 is an integer from 0 to 10;

[0183] t5 is an integer from 0 to 10;

[0184] * indicates the location where it connects to L2.

[0185] The position where it is attached to the carbonyl or methylene group.

[0186] In another preferred embodiment, the compound represented by general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein L 1b and L' 1b Each amino acid residue is independently formed from one or more amino acids selected from glycine, phenylalanine, citrulline, leucine, isoleucine, alanine, valine, asparagine, glutamine, arginine, glutamic acid, and lysine, preferably from two or more amino acids selected from glycine, phenylalanine, citrulline, valine, lysine, glutamine, glutamic acid, leucine, and alanine.

[0187] In another preferred embodiment, the compound represented by general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein L 1band L' 1bEach independently selected from: -Gly-*, -Val-*, -Gly-Phe-Gly-*, -Phe-Gly-*, -Gly-Val-Cit-*, -Val-Cit-*, -Gly-Val-Arg-*, -Val-Arg-*, -Gly-Val-Ala-*, -Val-Ala-*, -Gly-Phe-*, -Phe-Gly-*, -Gly-Gly-Gly-*, Gly-Gly-*, -Gly-Val-Gly-*, -Gly-Ala-Gly-*, -Gly-Phe-Cit-*, -Gly-Phe-Val-*, -Gly-Phe-Ala-*, -Gly-Phe-Lys-*, -Phe-Lys-*, -Gly-Val-*, -Gly-Cit-*, -Gly-Ala-*, -Gly-Gly-Lys-*, Gly-Lys’-*, -Ala-Ala-Ala-*, -Gln-Val-Ala-*, -Gln-Val-Cit-*, -Asp-Val-Ala-*, -Asp-Val-Cit-*, -Lys-Gly-Val-Ala-*, -Lys-Gly-Val-Cit-*, -Lys-Gly-Gly-Val-Ala-*, -Lys-Gly-Gly-Val-Cit-*, Gly-Gly-Phe-Gly-*, -Lys-Gln-Val-Cit-*, -Lys-Gln-Val-Ala-*, -Lys-Glu-Val-Cit-*, -Lys-Glu-Val-ALa-*, -Lys-Asp-Val-Cit-*, -Lys-Asp-Val-Ala-*, Glu-Val-Cit-*, Glu-Val-Ala-*, -Lys-Val-Ala-*, -Lys-Val-Cit-*, -Val-Lys-Gly-*, -Val-Lys*Preferably -Gly-Phe-Gly-*, -Gly-Val-Cit-*, -Val-Cit-*, -Gly-Val-Ala-*, -Val-Ala-*, -Gly-Phe-Gly-*, -Phe-Gly-*, -Gly-Phe-Lys-, -Phe-Lys-*, -Gl n-Val-Ala-*, -Gln-Val-Cit-*, -Asp-Val-Ala-*, -Lys-Gly-Val-Ala-*, -Lys-Gly-Val-Cit-*, -Lys-Gly-Gly-Val-Ala-*, -Lys-Gly-Gly-Val-Cit -*, Gly-Gly-Phe-Gly-*, -Lys-Gln-Val-Cit-*, -Lys-Gln-Val-Ala-*, -Lys-Glu-Val-Cit-*, -Lys-Glu-Val-ALa-*, -Lys-Asp-Val-Cit-*, -Lys-Asp-Val-Ala-*, Glu-Val-Cit-*, Glu-Val-Ala-*, -Lys-Val-Ala-*, -Lys-Val-Cit-*, -Val-Lys-Gly-*, -Val-Lys-*, -Asp-Val-Cit-*; where * indicates the position connected to L2.

[0188] In another preferred embodiment, the compound represented by general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein L3 is selected from:

[0189]

[0190]

[0191]

[0192]

[0193]

[0194] * indicates the location where it connects to L2.

[0195] The position where it is attached to the carbonyl or methylene group.

[0196] In another preferred embodiment, the compound represented by general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein Q is selected from:

[0197] Preferred

[0198] In another preferred embodiment, the compound represented by general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein Z1 is selected from bond, -(CH2). p -、-(CH2) p -C(O)NH-, -(CH2) p -O-(CH2) p -C(O)NH-, -(CH2) p -C(O)-L6-NH-、-(CH2) p -O-(CH2) p -C(O)-L6-NH-, -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)- and -OC(O)NH-; p is an integer from 1 to 10, preferably an integer from 1 to 6; more preferably an integer from 2 to 4; most preferably an integer from 2 to 3;

[0199] s1, s2, s3, and s4 are each independently an integer from 0 to 10; preferably an integer from 0 to 8 or an integer from 0 to 6; even more preferably an integer from 0 to 4, and even more preferably an integer from 0 to 2 or an integer from 1 to 2.

[0200] L6 is selected from

[0201] s is an integer from 1 to 6; preferably an integer from 2 to 6; more preferably an integer from 2 to 4;

[0202] t is an integer from 0 to 10; preferably an integer from 2 to 8; more preferably an integer from 3 to 7;

[0203] L6 is preferred

[0204] In another preferred embodiment, the compound represented by general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein,

[0205] Z1 is selected from the bond, -(CH2). p -、-(CH2) p -C(O)NH-, -(CH2) p -O-(CH2) p -C(O)NH-, -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)- and -OC(O)NH-;

[0206] s1 is an integer from 0 to 6, preferably an integer from 0 to 2;

[0207] s2 is an integer from 0 to 6, preferably an integer from 0 to 2;

[0208] s3 is 0;

[0209] s4 is 0;

[0210] p is an integer from 1 to 10, preferably an integer from 1 to 6; more preferably an integer from 2 to 4; and most preferably an integer from 2 to 3.

[0211] In another preferred embodiment, the compound represented by general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein,

[0212] Z1 is selected from -(CH2) p -C(O)NH-, -(CH2) p -O-(CH2) p -C(O)NH-, -C(O)NH-;

[0213] s1 is an integer from 1 to 6, preferably an integer from 2 to 6;

[0214] s2 is an integer from 1 to 10, preferably an integer from 2 to 10;

[0215] s3 is 0;

[0216] s4 is 0;

[0217] p is an integer from 1 to 10, preferably an integer from 1 to 6; more preferably an integer from 2 to 4; and most preferably an integer from 2 to 3.

[0218] In another preferred embodiment, the compound represented by general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein Q-L4- is selected from:

[0219]

[0220]

[0221] in:

[0222] Z1 is selected from -C(O)NH-, -C(O)O-, -C(O-, -OC(O-)NH-, and -OC(O)NH-, with -C(O)NH- being preferred;

[0223] p is an integer from 1 to 10, preferably an integer from 1 to 6; more preferably an integer from 2 to 4; and most preferably an integer from 2 to 3.

[0224] s1 is an integer from 0 to 6, preferably an integer from 0 to 2;

[0225] s2 is an integer from 1 to 10, preferably an integer from 1 to 8;

[0226] s3 is an integer from 0 to 6, preferably an integer from 0 to 2;

[0227] s4 is an integer from 1 to 6, preferably an integer from 1 to 2;

[0228] s7 is an integer from 0 to 6, preferably an integer from 1 to 2;

[0229] s8 is an integer from 1 to 4, preferably an integer from 1 to 2;

[0230] s9 is an integer from 1 to 10, preferably an integer from 1 to 8;

[0231] s 10 It can be an integer from 1 to 4, preferably an integer from 1 to 2.

[0232] In another preferred embodiment, the compound represented by general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein L is selected from:

[0233]

[0234]

[0235]

[0236] in,

[0237] L 1bAmino acid residues formed from two or more amino acids selected from glycine, phenylalanine, citrulline, leucine, isoleucine, alanine, valine, asparagine, glutamine, arginine, glutamic acid, and lysine; preferably, amino acid residues formed from two or more amino acids selected from glycine, phenylalanine, citrulline, leucine, alanine, valine, glutamine, glutamic acid, and lysine; more preferably, -Gly-*, -Val-*, -Gly-Phe-Gly-*, -Phe-Gly-*, -Gly-Val-Cit-*, -Val-Cit-*, -Gly -Val-Arg-*, -Val-Arg-*, -Gly-Val-Ala-*, -Val-Ala-*, -Gly-Phe-*, -Phe-Gly-*, -Gly-Gly-Gly-*, Gly-Gly-*, -Gly-Val-G ly-*, -Gly-Ala-Gly-*, -Gly-Phe-Cit-*, -Gly-Phe-Val-*, -Gly-Phe-Ala-*, -Gly-Phe-Lys-*, -Phe-Lys-*, -Gly-Val-*, -Gl y-Cit-*, -Gly-Ala-*, -Gly-Gly-Lys-*, Gly-Lys'-*, -Ala-Ala-Ala-*, -Gln-Val-Ala-*, -Gln-Val-Cit-*, -Asp-Val-Ala-*, -Asp-Val-Cit-*, -Lys-Gly-Val-Ala-*, -Lys-Gly-Val-Cit-*, -Lys-Gly-Gly-Val-Ala-*, -Lys-Gly-Gly-Val-Cit-*, Gly-Gl y-Phe-Gly-*, -Lys-Gln-Val-Cit-*, -Lys-Gln-Val-Ala-*, -Lys-Glu-Val-Cit-*, -Lys-Glu-Val-ALa-*, -Lys-Asp-Val-Cit- *, -Lys-Asp-Val-Ala-*, Glu-Val-Cit-*, Glu-Val-Ala-*, -Lys-Val-Ala-*, -Lys-Val-Cit-*, -Val-Lys-Gly-*, -Val-Lys-*,Preferably -Gly-Phe-Gly-*, -Gly-Val-Cit-*, -Val-Cit-*, -Gly-Val-Ala-*, -Val-Ala-*, -Gly-Phe-Gly-*, -Phe-Gly-*, -Gly-Phe-Lys-, -Phe-Lys-*, -Gln-Val-Ala-*, -Gln-Val-Cit-*, -Asp-Val-Ala-*, -Lys-Gly-Val-Ala-*, -Lys-Gly-Val-Cit-*, -Lys-Gly-Gly-Val-Ala-*, -Lys-Gly-Gly-V al-Cit-*, Gly-Gly-Phe-Gly-*, -Lys-Gln-Val-Cit-*, -Lys-Gln-Val-Ala-*, -Lys-Glu-Val-Cit-*, -Lys-Glu-Val-ALa-*, -Lys-Asp-Val-Cit- *, -Lys-Asp-Val-Ala-*, Glu-Val-Cit-*, Glu-Val-Ala-*, -Lys-Val-Ala-*, -Lys-Val-Cit-*, -Val-Lys-Gly-*, -Val-Lys-*, -Asp-Val-Cit-*;,

[0238] * indicates the location where it connects to L2;

[0239] Z1 is selected from -C(O)NH-, -C(O)O-, -C(O-, -OC(O-)NH-, and -OC(O)NH-, with -C(O)NH- being preferred;

[0240] p is an integer from 1 to 10, preferably an integer from 1 to 6;

[0241] s1 is an integer from 0 to 6, preferably an integer from 0 to 2;

[0242] s2 is an integer from 1 to 6, preferably an integer from 1 to 2;

[0243] s3 is an integer from 0 to 6, preferably an integer from 0 to 2;

[0244] s4 is an integer from 1 to 6, preferably an integer from 1 to 2;

[0245] t is an integer from 0 to 10;

[0246] t1 is an integer from 1 to 6; preferably an integer from 1 to 4, even more preferably an integer from 2 to 4, and even more preferably 1 or 2;

[0247] t2 is an integer from 0 to 6; preferably an integer from 1 to 4, and even more preferably 1 or 2;

[0248] t3 is an integer from 1 to 6; preferably an integer from 1 to 4, more preferably 1 or 2.

[0249] In a preferred embodiment, the compound of general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein L 1bIt is an amino acid residue formed from one or more amino acids selected from glycine, phenylalanine, citrulline, leucine, isoleucine, alanine, valine, asparagine, glutamine, arginine, glutamic acid, and lysine; preferably, it is an amino acid residue formed from two or more amino acids selected from glycine, phenylalanine, citrulline, leucine, alanine, valine, glutamine, glutamic acid, and lysine; more preferably, it is an amino acid residue formed from -Gly-*, -Val-*, -Gly-Phe-Gly-*, -Phe-Gly-*, -Gly-Val-Cit-*, -Val-Cit-*, or -Gly -Val-Arg-*, -Val-Arg-*, -Gly-Val-Ala-*, -Val-Ala-*, -Gly-Phe-*, -Phe-Gly-*, -Gly-Gly-Gly-*, Gly-Gly-*, -Gly-Val-G ly-*, -Gly-Ala-Gly-*, -Gly-Phe-Cit-*, -Gly-Phe-Val-*, -Gly-Phe-Ala-*, -Gly-Phe-Lys-*, -Phe-Lys-*, -Gly-Val-*, -Gl y-Cit-*, -Gly-Ala-*, -Gly-Gly-Lys-*, Gly-Lys'-*, -Ala-Ala-Ala-*, -Gln-Val-Ala-*, -Gln-Val-Cit-*, -Asp-Val-Ala-*, -Asp-Val-Cit-*, -Lys-Gly-Val-Ala-*, -Lys-Gly-Val-Cit-*, -Lys-Gly-Gly-Val-Ala-*, -Lys-Gly-Gly-Val-Cit-*, Gly-Gl y-Phe-Gly-*, -Lys-Gln-Val-Cit-*, -Lys-Gln-Val-Ala-*, -Lys-Glu-Val-Cit-*, -Lys-Glu-Val-ALa-*, -Lys-Asp-Val-Cit- *, -Lys-Asp-Val-Ala-*, Glu-Val-Cit-*, Glu-Val-Ala-*, -Lys-Val-Ala-*, -Lys-Val-Cit-*, -Val-Lys-Gly-*, -Val-Lys-*;Preferably -Gly-Phe-Gly-*, -Gly-Val-Cit-*, -Val-Cit-*, -Gly-Val-Ala-*, -Val-Ala-*, -Gly-Phe-Gly-*, -Phe-Gly-*, -Gly-Phe-Lys-, -Phe-Lys-*, -Gln-Val-Ala-*, -Gln-Val-Cit-*, -Asp-Val-Ala-*, -Lys-Gly-Val-Ala-*, -Lys-Gly-Val-Cit-*, -Lys-Gly-Gly-Val-Ala-*, -Lys-Gly-Gly-V al-Cit-*, Gly-Gly-Phe-Gly-*, -Lys-Gln-Val-Cit-*, -Lys-Gln-Val-Ala-*, -Lys-Glu-Val-Cit-*, -Lys-Glu-Val-ALa-*, -Lys-Asp-Val-Cit- *, -Lys-Asp-Val-Ala-*, Glu-Val-Cit-*, Glu-Val-Ala-*, -Lys-Val-Ala-*, -Lys-Val-Cit-*, -Val-Lys-Gly-*, -Val-Lys-*, -Asp-Val-Cit-*. ;

[0250] In another preferred embodiment, the compound represented by general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein L' 1bIt is an amino acid residue formed from one or more amino acids selected from glycine, phenylalanine, citrulline, leucine, isoleucine, alanine, valine, asparagine, glutamine, arginine, glutamic acid, and lysine; preferably, it is an amino acid residue formed from two or more amino acids selected from glycine, phenylalanine, citrulline, leucine, aspartic acid, alanine, valine, glutamine, glutamic acid, and lysine; more preferably, it is an amino acid residue formed from -Gly-*, -Val-*, -Gly-Phe-Gly-*, -Phe-Gly-*, -Gly-Val-Cit-*, -Val-Cit-*, - Gly-Val-Arg-*, -Val-Arg-*, -Gly-Val-Ala-*, -Val-Ala-*, -Gly-Phe-*, -Phe-Gly-*, -Gly-Gly-Gly-*, Gly-Gly-*, -Gly-Va l-Gly-*, -Gly-Ala-Gly-*, -Gly-Phe-Cit-*, -Gly-Phe-Val-*, -Gly-Phe-Ala-*, -Gly-Phe-Lys-*, -Phe-Lys-*, -Gly-Val-*, - Gly-Cit-*, -Gly-Ala-*, -Gly-Gly-Lys-*, Gly-Lys'-*, -Ala-Ala-Ala-*, -Gln-Val-Ala-*, -Gln-Val-Cit-*, -Asp-Val-Ala- *, -Asp-Val-Cit-*, -Lys-Gly-Val-Ala-*, -Lys-Gly-Val-Cit-*, -Lys-Gly-Gly-Val-Ala-*, -Lys-Gly-Gly-Val-Cit-*, Gly-G ly-Phe-Gly-*, -Lys-Gln-Val-Cit-*, -Lys-Gln-Val-Ala-*, -Lys-Glu-Val-Cit-*, -Lys-Glu-Val-ALa-*, -Lys-Asp-Val-Cit -*, -Lys-Asp-Val-Ala-*, Glu-Val-Cit-*, Glu-Val-Ala-*, -Lys-Val-Ala-*, -Lys-Val-Cit-*, -Val-Lys-Gly-*, -Val-Lys-*,Preferably -Gly-Phe-Gly-*, -Gly-Val-Cit-*, -Val-Cit-*, -Gly-Val-Ala-*, -Val-Ala-*, -Gly-Phe-Gly-*, -Phe-Gly-*, -Gly-Phe-Lys-, -Phe-Lys-*, -Gln-Val-Ala-*, -Gln-Val-Cit-*, -Asp-Val-Ala-*, -Lys-Gly-Val-Ala-*, -Lys-Gly-Val-Cit-*, -Lys-Gly-Gly-Val-Ala-*, -Lys-Gly-Gly-V al-Cit-*, Gly-Gly-Phe-Gly-*, -Lys-Gln-Val-Cit-*, -Lys-Gln-Val-Ala-*, -Lys-Glu-Val-Cit-*, -Lys-Glu-Val-ALa-*, -Lys-Asp-Val-Cit- *, -Lys-Asp-Val-Ala-*, Glu-Val-Cit-*, Glu-Val-Ala-*, -Lys-Val-Ala-*, -Lys-Val-Cit-*, -Val-Lys-Gly-*, -Val-Lys-*, -Asp-Val-Cit-*. ,

[0251] In another preferred embodiment, the compound represented by general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein L 1b and L' 1b For -Gly-* or -Val-*.

[0252] In another preferred embodiment, the compound represented by general formula (A) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272] Another aspect of the present invention provides a compound of general formula (I) or a stereoisomer, tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof.

[0273]

[0274] in,

[0275] R 8 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, alkenyl, and alkynyl, wherein the alkyl, alkoxy, alkenyl, and alkynyl groups are optionally further substituted by one or more groups selected from halogens;

[0276] R 9 Selected from hydrogen, halogen, hydroxyl, carboxyl, cyano, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, -NR d R e -(CH2) m -OH, -(CH2) m -NR d R e -OC(=O)NR f -(CH2) m -OH, -OC(=O)NR f -(CH2) m -NR d R e -(CH2) m-C(=O)OH、-(CH2) m -C(=O)-NR d R e 、-(CH2) m -C(=O)NR f -(CH2) n -OH、-(CH2) m -C(=O)NR f -(CH2) n -NR d R e 、-NR f -(CH2) m -OH、-NR f -(CH2) m -NR d R e 、-O-(CH2) m -OH、-O-(CH2) m -NR d R e 、-NR f C(=O)O-(CH2) m -OH、-NR b C(=O)O-(CH2) m -NR d R e 、-(CH2) m -NR f C(=O)O-(CH2) n -OH、-(CH2) m -NR f C(=O)O-(CH2) n -NR d R e 、-(CH2) m -OC(=O)NR f -(CH2) n -NR d R e 、-(CH2) m -OC(=O)NR f -(CH2) n -OH、-(CH2) m -NR f C(=O)-(CH2) n -NR d R e 、-(CH2) m -NR f C(=O)-(CH2) n -OH、-(CH2) m-NR f C(=O)-G-(CH2) n -OH, wherein the alkyl, alkoxy, alkenyl, alkynyl, or cycloalkyl group is optionally further substituted with one or more groups selected from deuterium, halogen, amino, alkyl, or hydroxyl; the above-mentioned -(CH2) m -Optionally replaced by one or more deuterium or halogens;

[0277] G is selected from cycloalkylene, heterocyclic, heteroarylene, and arylene;

[0278] R 10 Selected from hydrogen, halogen, hydroxyl, cyano, alkyl, alkoxy, alkenyl, alkynyl, CH2=, -NR d R e -(CH2) m -OH, -(CH2) m -NR d R e -OC(=O)NR f -(CH2) m -OH, -OC(=O)NR f -(CH2) m -NR d R e -(CH2) m -C(=O)OH, -(CH2) m -C(=O)-NR d R e -(CH2) m -C(=O)NR f -(CH2) n -OH, -(CH2) m -C(=O)NR f -(CH2) n -NR d R e -NR f -(CH2) m -OH, -NR f -(CH2) m -NR d R e -NR f -C(=O)R d -NR f -C(=O)-(CH2) m -R d -NR f C(=O)O-(CH2) m -R d -O-(CH2) m -OH, -O-(CH2)m -NR d R e 、-NR f C(=O)O-(CH2) m -OH、-NR f C(=O)NR d -(CH2) m -OH、-NR f C(=O)NR d -(CH2) m -O-(CH2) n -OH、-NR f C(=O)O-(CH2) m -O-(CH2) n -OH、-NR f C(=O)NR d -(CH2) m -O-(CH2) n -NR d R e 、-NR f C(=O)O-(CH2) m -O-(CH2) n -NR d R e 、-NR f C(=O)NR d -(CH2) m -NR d R e 、-NR f C(=O)O-(CH2) m -NR d R e 、-(CH2) m -NR f C(=O)O-(CH2) n -OH、-(CH2) m -NR f C(=O)O-(CH2) n -NR d R e 、-(CH2) m -OC(=O)NR f -(CH2) n -NR d R e 、-(CH2) m -OC(=O)NR f -(CH2) n -OH、-(CH2) m -NR fC(=O)-(CH2) n -NR d R e -(CH2) m -NR f C(=O)-(CH2) n -OH, wherein the alkyl, alkoxy, alkenyl, or alkynyl groups are optionally further substituted with one or more groups selected from halogens;

[0279] R d and R e Each is independently selected from hydrogen and C1-C6 alkyl groups;

[0280] R f Selected from hydrogen, C1-C6 alkyl, -C(O)R c -S(O)R c -S(O)2R c The C1-C6 alkyl group may optionally be further substituted with a C3-C6 cycloalkyl group; wherein R c Selected from hydrogen, hydroxyl, and C1-C6 alkyl groups;

[0281] m is an integer from 1 to 6;

[0282] n is an integer from 1 to 6.

[0283] In a preferred embodiment, the compound of general formula (I) according to the present invention, or its stereoisomers, tautomers, meso compounds, racemates, enantiomers, diastereomers, mixtures thereof, or pharmaceutically acceptable salts thereof, wherein R 8 The halogen is preferred, with fluorine or chlorine being the most suitable.

[0284] In another preferred embodiment, the compound represented by general formula (I) according to the present invention, or a stereoisomer, tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein R 9 Selected from hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, C3-C6 cycloalkyl, -NR d R e -(CH2) m -OH, -(CH2) m -NR d R e -(CH2) m -NR b C(=O)O-(CH2) n -OH, -(CH2) m -OC(=O)NR f -(CH2) n -OH, -(CH2)m -NR f C(=O)-(CH2) n -OH, -O-(CH2) m -NR d R e -O-(CH2) m -OH, -(CH2) m -NR f C(=O)-G-(CH2) n -OH; the above-mentioned -(CH2) m -Optionally replaced by one or more deuterium or halogens;

[0285] G is selected from cycloalkylene, heterocyclic, heteroarylene, and arylene;

[0286] R d and R e Each is independently selected from hydrogen and C1-C6 alkyl groups, with hydrogen being preferred;

[0287] R f Selected from hydrogen and C1-C6 alkyl groups, with hydrogen being preferred;

[0288] m is an integer from 1 to 6, preferably an integer from 1 to 4;

[0289] n is an integer from 1 to 6, preferably 1 or 2.

[0290] In another preferred embodiment, the compound represented by general formula (I) according to the present invention, or a stereoisomer, tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein R 10 Selected from hydrogen, hydroxyl, amino, and -(CH2). m -OH, -OC(=O)NR f -(CH2) m -OH, -(CH2) m -C(=O)OH, -(CH2) m -C(=O)NR f -(CH2) n -OH, -NR f -(CH2) m -OH, -NR f C(=O)O-(CH2) m -OH, -NR f C(=O)NR d -(CH2) m -OH, -NR f C(=O)NR d -(CH2) m -O-(CH2)n -OH, -NR f C(=O)O-(CH2) m -O-(CH2) n -OH, -NR f C(=O)NR d -(CH2) m -O-(CH2) n -NR d R e -NR f C(=O)O-(CH2) m -O-(CH2) n -NR d R e -NR f C(=O)NR d -(CH2) m -NR d R e -NR f C(=O)O-(CH2) m -NR d R e ;

[0291] R d and R e Each is independently selected from hydrogen and C1-C6 alkyl groups, with hydrogen being preferred;

[0292] R f Selected from hydrogen, C1-C6 alkyl, -C(O)R c -S(O)R c -S(O)2R c , where R c Selected from hydrogen, hydroxyl, and C1-C6 alkyl groups;

[0293] m is an integer from 1 to 6, preferably an integer from 1 to 4, and even more preferably 1 or 2;

[0294] n is an integer from 1 to 6, preferably 1 or 2.

[0295] In another preferred embodiment, the compound represented by general formula (I) according to the present invention, or a stereoisomer, tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein R 9 Selected from hydrogen, C 1-6 Alkyl, C 1-6 Alkoxy, hydroxy, carboxyl, -NR d R e -(CH2) m -OH, -(CH2) m -NRd R e -(CH2) m -NR f C(=O)O-(CH2) n -OH, -(CH2) m -OC(=O)NR f -(CH2) n -OH, -(CH2) m -NR f C(=O)-(CH2) n -OH, -O-(CH2) m -NR d R e -O-(CH2) m -OH;

[0296] R d and R e Each is independently selected from hydrogen and C1-C6 alkyl groups;

[0297] R f Selected from hydrogen and C1-C6 alkyl groups;

[0298] m is an integer from 1 to 6;

[0299] n is an integer from 1 to 4, preferably 1 or 2.

[0300] In another preferred embodiment, the compound represented by general formula (I) according to the present invention, or a stereoisomer, tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein R 10 Selected from hydrogen, hydroxyl, amino, CH2=, -(CH2) m -OH, -OC(=O)NR f -(CH2) m -OH, -(CH2) m -C(=O)OH, -(CH2) m -C(=O)NR f -(CH2) n -OH, -NR f -(CH2) m -OH, -NR f -C(=O)R d -NR f -C(=O)-(CH2) m -R d -NR f C(=O)O-(CH2) m -R d -NR f-C(=O)-(CH2) m -OH, -NR f C(=O)O-(CH2) m -OH;

[0301] R d and R e Each is independently selected from hydrogen and C1-C6 alkyl groups;

[0302] R f Selected from hydrogen, C1-C6 alkyl, -C(O)R c -S(O)R c -S(O)2R c The C1-C6 alkyl group may optionally be further substituted with a C3-C6 cycloalkyl group; wherein R c Selected from hydrogen, hydroxyl, and C1-C6 alkyl groups;

[0303] m is an integer from 1 to 6; preferably an integer from 1 to 4 or from 4 to 6.

[0304] n is an integer from 1 to 4, preferably 1 or 2.

[0305] In another preferred embodiment, the compound represented by general formula (I) according to the present invention, or its stereoisomers, tautomers, meso compounds, racemates, enantiomers, diastereomers, mixtures thereof, or pharmaceutically acceptable salts thereof, are selected from:

[0306] In another aspect, the present invention provides a compound of general formula (II) or a stereoisomer, tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof.

[0307]

[0308] in,

[0309] R 11 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, alkenyl, and alkynyl, wherein the alkyl, alkoxy, alkenyl, and alkynyl groups are optionally further substituted by one or more groups selected from halogens;

[0310] R 12Selected from hydrogen, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, alkenyl, alkynyl, -NR d R e -(CH2) m -OH, -(CH2) m -NR d R e -OC(=O)NR f -(CH2) m -OH, -OC(=O)NR f -(CH2) m -NR d R e -(CH2) m -C(=O)OH, -(CH2) m -C(=O)-NR d R e -(CH2) m -C(=O)NR f -(CH2) n -OH, -(CH2) m -C(=O)NR f -(CH2) n -NR d R e -NR f -(CH2) m -OH, -NR f -(CH2) m -NR d R e -O-(CH2) m -OH, -O-(CH2) m -NR d R e -NR f C(=O)O-(CH2) m -OH, -NR b C(=O)O-(CH2) m -NR a -*、-(CH2) m -NR b C(=O)O-(CH2) n -O-*、-(CH2) m -NR f C(=O)O-(CH2) n -NR d R e -(CH2) m -OC(=O)NR f -(CH2) n -NRd R e -(CH2) m -OC(=O)NR f -(CH2) n -OH, -(CH2) m -NR f C(=O)-(CH2) n -NR d R e -(CH2) m -NR f C(=O)-(CH2) n -OH, wherein the alkyl, alkoxy, alkenyl, or alkynyl groups are optionally further substituted with one or more groups selected from halogens;

[0311] R 13 Selected from hydrogen, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, alkenyl, alkynyl, -NR d R e -(CH2) m -OH, -(CH2) m -NR d R e -OC(=O)NR f -(CH2) m -OH, -OC(=O)NR f -(CH2) m -NR d R e -(CH2) m -C(=O)OH, -(CH2) m -C(=O)-NR d R e -(CH2) m -C(=O)NR f -(CH2) n -OH, -(CH2) m -C(=O)NR f -(CH2) n -NR d R e -NR f -(CH2) m -OH, -NR f -(CH2) m -NR d R e -O-(CH2) m -OH, -O-(CH2) m -NR d R e -NR fC(=O)O-(CH2) m -OH, -NR b C(=O)O-(CH2) m -NR a -*、-(CH2) m -NR b C(=O)O-(CH2) n -O-*、-(CH2) m -NR f C(=O)O-(CH2) n -NR d R e -(CH2) m -OC(=O)NR f -(CH2) n -NR d R e -(CH2) m -OC(=O)NR f -(CH2) n -OH, -(CH2) m -NR f C(=O)-(CH2) n -NR d R e -(CH2) m -NR f C(=O)-(CH2) n -OH, wherein the alkyl, alkoxy, alkenyl, or alkynyl groups are optionally further substituted with one or more groups selected from halogens;

[0312] R d and R e Each is independently selected from hydrogen and C1-C6 alkyl groups;

[0313] R f Selected from hydrogen, C1-C6 alkyl, -C(O)R c -S(O)R c -S(O)2R c , where R c Selected from hydrogen, hydroxyl, and C1-C6 alkyl groups;

[0314] m is an integer from 1 to 6;

[0315] n is an integer from 1 to 6.

[0316] In a preferred embodiment, the compound of general formula (II) according to the present invention, or its stereoisomers, tautomers, meso compounds, racemates, enantiomers, diastereomers, mixtures thereof, or pharmaceutically acceptable salts thereof, wherein R 11Selected from halogens, preferably fluorine or cyano.

[0317] In another preferred embodiment, the compound of general formula (II) according to the present invention, or a stereoisomer, tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein R 12 Selected from hydroxyl, amino, C1-C6 alkyl, -(CH2) m -OH, -(CH2) m -NR d R e ;

[0318] R d and R e Each is independently selected from hydrogen and C1-C6 alkyl groups;

[0319] m is an integer from 1 to 6, preferably an integer from 1 to 4.

[0320] In another preferred embodiment, the compound of general formula (II) according to the present invention, or a stereoisomer, tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein R 13 Selected from hydroxyl and amino groups.

[0321] In another preferred embodiment, the compound of general formula (II) according to the present invention, or a stereoisomer, tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein R 13 Selected from hydroxyl, C 1-6 Alkyl group.

[0322] In another preferred embodiment, the compound represented by general formula (II) according to the present invention, or its stereoisomers, tautomers, meso compounds, racemates, enantiomers, diastereomers, mixtures thereof, or pharmaceutically acceptable salts thereof, are selected from:

[0323]

[0324] Another aspect of the present invention provides a ligand-drug conjugate of general formula (B) or a pharmaceutically acceptable salt thereof.

[0325]

[0326] in:

[0327] Dr is selected from the following structure:

[0328]

[0329] R 1 Selected from hydrogen, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, -NR d R e -(CH2) m -OH, -(CH2) m -NR d R e -OC(=O)NR f -(CH2) m -OH, -OC(=O)NR f -(CH2) m -NR d R e -(CH2) m -C(=O)OH, -(CH2) m -C(=O)-NR d R e -(CH2) m -C(=O)NR f -(CH2) n -OH, -(CH2) m -C(=O)NR f -(CH2) n -NR d R e -NR f -(CH2) m -OH, -NR f -(CH2) m -NR d R e -O-(CH2) m -OH, -O-(CH2) m -NR d R e -NR f C(=O)O-(CH2) m -OH, -NR b C(=O)O-(CH2) m -NR d R e -(CH2) m -NR f C(=O)O-(CH2) n -OH-, -(CH2) m -NR f C(=O)O-(CH2) n -NR d R e -(CH2)m -OC(=O)NR f -(CH2) n -NR d R e -(CH2) m -OC(=O)NR f -(CH2) n -OH, -(CH2) m -NR f C(=O)-(CH2) n -NR d R e -(CH2) m -NR f C(=O)-(CH2) n -OH, -(CH2) m -NR f C(=O)-G-(CH2) n -OH, wherein the alkyl, alkoxy, alkenyl, alkynyl, or cycloalkyl group is optionally further substituted with one or more groups selected from halogen, deuterium, amino, alkyl, or hydroxyl groups; the above-mentioned -(CH2) m -Optionally substituted with one or more deuterium or halogens; G is selected from cycloalkylene, heterocyclic, heteroaryl, and aryl;

[0330] R 2 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, alkenyl, and alkynyl, wherein the alkyl, alkoxy, alkenyl, and alkynyl groups are optionally further substituted by one or more groups selected from halogens;

[0331] R 3 Selected from hydrogen, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, alkenyl, alkynyl, CH2=, -NR d R e -(CH2) m -OH, -(CH2) m -NR d R e -OC(=O)NR f -(CH2) m -OH, -OC(=O)NR f -(CH2) m -NR d R e -(CH2) m -C(=O)OH, -(CH2) m -C(=O)-NR d R e -(CH2) m -C(=O)NR f-(CH2) n -OH、-(CH2) m -C(=O)NR f -(CH2) n -NR d R e 、-NR f -(CH2) m -OH、-NR f -(CH2) m -NR d R e 、-NR f -C(=O)R d 、-NR f -C(=O)-(CH2) m -R d 、-NR f C(=O)O-(CH2) m -R d 、-O-(CH2) m -OH、-O-(CH2) m -NR d R e 、-NR f C(=O)O-(CH2) m -OH、-NR f C(=O)NR d -(CH2) m -OH、-NR f C(=O)NR d -(CH2) m -O-(CH2) n -OH、-NR f C(=O)O-(CH2) m -O-(CH2) n -OH、-NR f C(=O)NR d -(CH2) m -O-(CH2) n -NR d R e 、-NR f C(=O)O-(CH2) m -O-(CH2) n -NR d R e 、-NR f C(=O)NR d -(CH2) m -NR d R e 、-NR fC(=O)O-(CH2) m -NR d R e -(CH2) m -NR f C(=O)O-(CH2) n -OH, -(CH2) m -NR f C(=O)O-(CH2) n -NR d R e -(CH2) m -OC(=O)NR f -(CH2) n -NR d R e -(CH2) m -OC(=O)NR f -(CH2) n -OH, -(CH2) m -NR f C(=O)-(CH2) n -NR d R e -(CH2) m -NR f C(=O)-(CH2) n -OH, wherein the alkyl, alkoxy, alkenyl, or alkynyl groups are optionally further substituted with one or more groups selected from halogen, deuterium, amino, alkyl, or hydroxyl groups;

[0332] R 4 Selected from hydrogen, halogen, hydroxyl, carboxyl, amino, cyano, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, -NR d R e -(CH2) m -OH, -(CH2) m -NR d R e -OC(=O)NR f -(CH2) m -OH, -OC(=O)NR f -(CH2) m -NR d R e -(CH2) m -C(=O)OH, -(CH2) m -C(=O)-NR d R e -(CH2) m -C(=O)NR f -(CH2)n -OH, -(CH2) m -C(=O)NR f -(CH2) n -NR d R e -NR f -(CH2) m -OH, -NR f -(CH2) m -NR d R e -O-(CH2) m -OH, -O-(CH2) m -NR d R e -NR f C(=O)O-(CH2) m -OH, -NR b C(=O)O-(CH2) m -NR d R e -(CH2) m -NR f C(=O)O-(CH2) n -OH-, -(CH2) m -NR f C(=O)O-(CH2) n -NR d R e -(CH2) m -OC(=O)NR f -(CH2) n -NR d R e -(CH2) m -OC(=O)NR f -(CH2) n -OH, -(CH2) m -NR f C(=O)-(CH2) n -NR d R e -(CH2) m -NR f C(=O)-(CH2) n -OH, -(CH2) m -NR f C(=O)-G-(CH2) n -OH, wherein the alkyl, alkoxy, alkenyl, alkynyl, or cycloalkyl group is optionally further substituted with one or more groups selected from halogen, deuterium, amino, alkyl, or hydroxyl groups; the above-mentioned -(CH2)m -Optionally substituted with one or more deuterium or halogens; G is selected from cycloalkylene, heterocyclic, heteroaryl, and aryl;

[0333] R 5 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, alkenyl, and alkynyl, wherein the alkyl, alkoxy, alkenyl, and alkynyl groups are optionally further substituted by one or more groups selected from halogens;

[0334] R 6 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, alkenyl, and alkynyl, wherein the alkyl, alkoxy, alkenyl, and alkynyl groups are optionally further substituted by one or more groups selected from halogens;

[0335] R 7 The group is selected from hydrogen, halogen, hydroxyl, amino, cyano, alkyl, alkoxy, alkenyl, and alkynyl, wherein the alkyl, alkoxy, alkenyl, and alkynyl groups are optionally further substituted by one or more groups selected from halogens;

[0336] R d and R e Each is independently selected from hydrogen and alkyl groups;

[0337] R f Selected from hydrogen, alkyl, -C(O)R c -S(O)R c -S(O)2R c The C1-C6 alkyl group may optionally be further substituted with a C3-C6 cycloalkyl group; wherein R c Selected from hydrogen, hydroxyl, and alkyl;

[0338] L1 is selected from the bond, -(CH2). m -*、-O-*、-NR a -*、-(CH2) m -O-*、-(CH2) m -NR a -*、-OC(=O)NR b -(CH2) m -O-*、-OC(=O)NR b -(CH2) m -NR a -*、-(CH2) m -C(=O)O-*、-(CH2) m -C(=O)NR a -*、-(CH2) m -C(=O)NR b -(CH2) n -O-*、-(CH2) m -C(=O)NRb -(CH2) n -NR a -*、-NR b -(CH2) m -O-*、-NR b -(CH2) m -NR a -*、-O-(CH2) m -O-*、-O-(CH2) m -NR a -*、-NR b C(=O)O-(CH2) m -O-*、-NR b C(=O)O-(CH2) m -NR a -*、-(CH2) m -NR b C(=O)O-(CH2) n -O-*、-(CH2) m -NR b C(=O)O-(CH2) n -NR a -*、-(CH2) m -OC(=O)NR b -(CH2) n -NR a -*、-(CH2) m -OC(=O)NR b -(CH2) n -O-*、-(CH2) m -NR b C(=O)-(CH2) n -NR a -*、-(CH2) m -NR b C(=O)-(CH2) n -O-*, where * is the connection site with L2;

[0339] R a and R b Each is independently selected from hydrogen, alkyl, -C(O)R c -S(O)R c -S(O)2R c The alkyl group may optionally be further substituted with a cycloalkyl group; wherein R c Selected from hydrogen, hydroxyl, and alkyl;

[0340] L2 is selected from the key, Where * represents the connection site with L1;

[0341] L' is

[0342] Q' is selected from:

[0343] Where * indicates the location connected to L4. This is the location where it connects to the Pc.

[0344] L3 is an amino acid residue formed by two or more amino acids, and L3 optionally includes one or more structures selected from the following: L6 is selected from one or more structures selected from the following:

[0345]

[0346] Among them, R, R aa R bb Each is independently selected from hydrogen and alkyl groups;

[0347] L4 is

[0348] Z1 is selected from the bond, -(CH2). p -、-(C2H4O) q -、-(CH2) p -C(O)NH-, -(CH2) p -O-(CH2) p -C(O)NH-, -(CH2) p -C(O)-L6-NH-、-(CH2) p -O-(CH2) p -C(O)-L6-NH-, -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)-, -NH-, -O- and -OC(O)NH-;

[0349] m is an integer from 1 to 6;

[0350] n is an integer from 1 to 6;

[0351] s is an integer from 1 to 6;

[0352] t is an integer from 0 to 10;

[0353] s1, s2, s3, and s4 are each independently an integer from 0 to 10; preferably an integer from 0 to 8 or an integer from 0 to 6; even more preferably an integer from 0 to 4, and even more preferably an integer from 0 to 2 or an integer from 1 to 2.

[0354] s5 and s6 are each independent integers from 1 to 6;

[0355] t1 is an integer from 1 to 6;

[0356] t2 is an integer from 0 to 6;

[0357] t3 is an integer from 1 to 6;

[0358] t4 is an integer from 0 to 10;

[0359] t5 is an integer from 0 to 10;

[0360] p is an integer from 1 to 10;

[0361] q is an integer from 1 to 10;

[0362] v is between 1 and 10, and v can be a decimal or an integer;

[0363] Pc is an antibody or its antigen-binding fragment, or a modified antibody;

[0364] The modified antibody has Pc'-((L5) w -F) x Structure, in which:

[0365] Pc' is an antibody;

[0366] L5 is a connector;

[0367] w is 0 or 1;

[0368] F is a click probe or thiol group or its precursor that can be linked to Q' after a reaction, such as a metal-free click reaction; preferably, F is an azide group.

[0369] x is an integer from 1 to 8.

[0370] Z1 is connected to Q'.

[0371] In a preferred embodiment, the ligand-drug conjugate of general formula (B) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein:

[0372] L3 is selected from:

[0373]

[0374] R is selected from hydrogen and C1-C6 alkyl groups, with hydrogen being preferred;

[0375] R aa R bb Each is independently selected from C1-C6 alkyl groups;

[0376] s is an integer from 1 to 6; preferably an integer from 2 to 6; more preferably an integer from 2 to 4;

[0377] s5 and s6 are each independently an integer from 1 to 6; preferably an integer from 2 to 6; more preferably an integer from 2 to 4;

[0378] t is an integer from 0 to 10; preferably an integer from 2 to 8; more preferably an integer from 3 to 7;

[0379] t1 is an integer from 1 to 6; preferably an integer from 1 to 4, even more preferably an integer from 2 to 4, and even more preferably 1 or 2;

[0380] t2 is an integer from 0 to 6; preferably an integer from 1 to 4, and even more preferably 1 or 2;

[0381] t3 is an integer from 1 to 6; preferably an integer from 1 to 4, more preferably 1 or 2;

[0382] t4 is an integer from 0 to 10;

[0383] t5 is an integer from 0 to 10;

[0384] * indicates the location where it connects to L2;

[0385] The position where it is attached to the carbonyl or methylene group;

[0386] L 1b and L' 1b Each amino acid residue is independently formed from one or more amino acids selected from glycine, phenylalanine, citrulline, leucine, isoleucine, alanine, valine, asparagine, glutamine, arginine, glutamic acid, and lysine, preferably formed from two or more amino acids selected from glycine, phenylalanine, citrulline, valine, lysine, glutamine, glutamic acid, leucine, and alanine.

[0387] Preferably, L 1b and L' 1bEach independently selected from: -Gly-*, -Val-*, -Gly-Phe-Gly-*, -Phe-Gly-*, -Gly-Val-Cit-*, -Val-Cit-*, -Gly-Val-Arg-*, -Val-Arg-*, -Gly-Val-Ala-*, -Val-Ala-*, -Gly-Phe-*, -Phe-Gly-*, -G ly-Gly-Gly-*, Gly-Gly-*, -Gly-Val-Gly-*, -Gly-Ala-Gly-*, -Gly-Phe-Cit-*, -Gly-Ph e-Val-*, -Gly-Phe-Ala-*, -Gly-Phe-Lys-*, -Phe-Lys-*, -Gly-Val-*, -Gly-Cit-*, -Gly -Ala-*, -Gly-Gly-Lys-*, Gly-Lys'-*, -Ala-Ala-Ala-*, -Gln-Val-Ala-*, -Gln-Val-Cit-*, -Asp-Val-Ala-*, -Asp-Val-Cit-*, preferably -Gly-Phe-Gly-*, -Gly-Val-Cit-*, -Val-Cit- *, -Gly-Val-Ala-*, -Val-Ala-*, -Gly-Phe-Gly-*, -Phe-Gly-*, -Gly-Phe-Lys-, -Phe-Lys-*, -Gln-Val-Ala-*, -Gln-Val-Cit-*, -Asp-Val-Ala-*, -Asp-Val-Cit-; where * indicates the position connected to L2.

[0388] In a preferred embodiment, the ligand-drug conjugate of general formula (B) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein:

[0389] L' is

[0390] Q' is selected from:

[0391] Where * indicates the location connected to L4. This is the location where it connects to the Pc.

[0392] L3 is an amino acid residue formed by two or more amino acids selected from glycine, phenylalanine, citrulline, leucine, isoleucine, alanine, valine, asparagine, glutamine, arginine, glutamic acid, and lysine, preferably formed by two or more amino acids selected from glycine, phenylalanine, citrulline, valine, lysine, glutamine, glutamic acid, leucine, and alanine.

[0393] L4 is

[0394] Z1 is selected from the bond, -(CH2). p -、-(CH2) p -C(O)NH-, -(CH2) p -O-(CH2) p -C(O)NH-, -(CH2) p -C(O)-L6-NH-、-(CH2) p -O-(CH2) p -C(O)-L6-NH-, -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)-, -NH-, -O- and -OC(O)NH-;

[0395] s1 is an integer from 1 to 6, preferably an integer from 2 to 6;

[0396] s2 is an integer from 1 to 10, preferably an integer from 2 to 10;

[0397] s3 is 0;

[0398] s4 is 0;

[0399] p is an integer from 1 to 10, preferably an integer from 1 to 6; more preferably an integer from 2 to 4; and most preferably an integer from 2 to 3.

[0400] L6 is selected from

[0401] s is an integer from 1 to 6; preferably an integer from 2 to 6; more preferably an integer from 2 to 4;

[0402] t is an integer from 0 to 10; preferably an integer from 2 to 8; more preferably an integer from 3 to 7;

[0403] L6 is preferred

[0404] In one specific embodiment, the ligand-drug conjugate of general formula (B) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein L5 is...

[0405] Z2 and Z3 are each independently selected from -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)-, -NH-, -O- and -OC(O)NH-; preferably -C(O)NH-;

[0406] r1 is an integer from 1 to 8, preferably an integer from 1 to 6, and more preferably an integer from 1 to 3;

[0407] r2 is an integer from 1 to 6, preferably an integer from 1 to 4, and more preferably an integer from 1 to 2;

[0408] r3 is an integer from 0 to 6, preferably an integer from 0 to 4, and more preferably an integer from 0 to 2;

[0409] r4 is an integer from 0 to 6, preferably an integer from 0 to 4, and more preferably an integer from 0 to 2;

[0410] r5 is an integer from 1 to 6, preferably an integer from 1 to 4, and more preferably an integer from 1 to 2.

[0411] In another specific embodiment, the ligand-drug conjugate of general formula (B) according to the present invention or a pharmaceutically acceptable salt thereof, wherein when w is 0, F is a mercapto group; when w is 1, F is a click probe that can be linked to Q' after a reaction, such as a metal-free click reaction, preferably F is an azide group.

[0412] In another specific embodiment, the ligand-drug conjugate of general formula (B) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein:

[0413] Pc is a modified antibody, and the modified antibody has a specific structure.

[0414] in:

[0415] Pc' is an antibody;

[0416] Z2 and Z3 are each independently selected from -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)-, -NH-, -O- and -OC(O)NH-; preferably -C(O)NH-;

[0417] r1 is an integer from 1 to 8, preferably an integer from 1 to 6, and more preferably an integer from 1 to 3;

[0418] r2 is an integer from 1 to 6, preferably an integer from 1 to 4, and more preferably an integer from 1 to 2;

[0419] r3 is an integer from 0 to 6, preferably an integer from 0 to 4, and more preferably an integer from 0 to 2;

[0420] r4 is an integer from 0 to 6, preferably an integer from 0 to 4, and more preferably an integer from 0 to 2;

[0421] r5 is an integer from 1 to 6, preferably an integer from 1 to 4, and more preferably an integer from 1 to 2.

[0422] In a preferred embodiment, the ligand-drug conjugate of general formula (B) according to the present invention, or a pharmaceutically acceptable salt thereof, wherein: Pc is a modifying antibody, and Pc-Q' is selected from the structure:

[0423]

[0424] Pc' is an antibody;

[0425] Z2 and Z3 are each independently selected from -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)-, -NH-, -O- and -OC(O)NH-; preferably -C(O)NH-;

[0426] r1 is an integer from 1 to 8, preferably an integer from 1 to 6, and more preferably an integer from 1 to 3;

[0427] r2 is an integer from 1 to 6, preferably an integer from 1 to 4, and more preferably an integer from 1 to 2;

[0428] r3 is an integer from 0 to 6, preferably an integer from 0 to 4, and more preferably an integer from 0 to 2;

[0429] r4 is an integer from 0 to 6, preferably an integer from 0 to 4, and more preferably an integer from 0 to 2;

[0430] r5 is an integer from 1 to 6, preferably an integer from 1 to 4, and more preferably an integer from 1 to 2.

[0431] In a preferred embodiment, the ligand-drug conjugate of general formula (B) according to the present invention or a pharmaceutically acceptable salt thereof is selected from:

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446] in:

[0447] v is between 1 and 10, and v can be a decimal or an integer;

[0448] Pc is an antibody or its antigen-binding fragment;

[0449] Pc' is an antibody.

[0450] In some embodiments, the ligand-drug conjugate of formula (B) according to the present invention or a pharmaceutically acceptable salt thereof, wherein the antibody is selected from murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies.

[0451] In other embodiments, the ligand-drug conjugate of general formula (B) according to the present invention or a pharmaceutically acceptable salt thereof, wherein the antibody or its antigen-binding fragment is selected from anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody, anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-ROR1 antibody, anti-CLDN6 antibody, anti-CLDN9 antibody, anti-CLDN18.2 antibody, anti-NaPi-2b antibody, anti-T NF-α antibody, anti-ENPP3 antibody, anti-DLL3 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD28 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD38 antibody, anti-CD44 antibody, anti-CD45 antibody, anti-CD47 antibody, anti-CD48 antibody, anti-CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD98 antibody, anti-CD105 antibody, anti-CEA antibody, anti-EphA2 antibody, anti-MUCI antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-Integrin antibody, anti-PSMA antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-CD79 antibody, anti-TROP-2 antibody, anti-CD79B antibody, anti-Mesothelin antibody, anti-Nectin-4 antibody, anti-TPBG antibody, or their antigen-binding fragments.

[0452] In other embodiments, the ligand-drug conjugate of general formula (B) according to the present invention or a pharmaceutically acceptable salt thereof, wherein the antibody or its antigen-binding fragment is selected from trastuzumab, cetuximab, pertuzumab, nimotuzumab, enoblituzumab, emibetuzumab, Inotuzumab, vitine-pinatuzumab, brentuximab, gemtuzumab, bivatuzumab, morovatuzumab, or an antigen-binding fragment thereof.

[0453] In other embodiments, the ligand-drug conjugate of general formula (B) according to the present invention, or a pharmaceutically acceptable salt thereof, is selected from:

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467] Where v is an integer or decimal from 1 to 10, preferably an integer from 2 to 8.

[0468] The present invention further relates to a pharmaceutical composition comprising a ligand drug conjugate of formula (B) according to the present invention or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

[0469] The present invention also relates to the use of compounds of general formula (A) according to the present invention, or pharmaceutically acceptable salts thereof, in the preparation of ligand-drug conjugates.

[0470] The present invention also relates to the use of compounds of general formula (I) according to the present invention, or their stereoisomers, tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, in the preparation of ligand-drug conjugates.

[0471] The present invention further relates to the use of the ligand drug conjugate or its pharmaceutically acceptable salt or pharmaceutical composition containing the present invention in the preparation of a medicament for treating tumors or cancers, preferably breast cancer, ovarian cancer, soft tissue sarcoma, liposarcoma, lung cancer, non-small cell lung cancer, gastric cancer, melanoma, head and neck cancer, cervical cancer, and prostate cancer.

[0472] The present invention also relates to the use of the ligand drug conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the ligand drug conjugate according to the present invention in a medicament for treating tumors or cancers, preferably breast cancer, ovarian cancer, soft tissue sarcoma, liposarcoma, lung cancer, non-small cell lung cancer, gastric cancer, melanoma, head and neck cancer, cervical cancer, and prostate cancer.

[0473] The present invention also relates to a method for treating tumors or cancer, comprising administering to a subject in need an effective amount of a ligand-drug conjugate according to the present invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the thereof, wherein the cancer is preferably breast cancer, ovarian cancer, soft tissue sarcoma, liposarcoma, lung cancer, non-small cell lung cancer, gastric cancer, melanoma, head and neck cancer, cervical cancer, or prostate cancer.

[0474] The present invention further relates to the use of compounds of general formula (I) or their stereoisomers, tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, or compounds of general formula (II) or their stereoisomers, tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions containing thereof, in the preparation of medicaments for treating tumors or cancers, preferably breast cancer, ovarian cancer, soft tissue sarcoma, liposarcoma, lung cancer, non-small cell lung cancer, gastric cancer, melanoma, head and neck cancer, cervical cancer, and prostate cancer.

[0475] The present invention also relates to the use of compounds of general formula (I) or thereof, stereoisomers, tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, or compounds of general formula (II) or thereof, stereoisomers, tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions containing thereof, in the treatment of tumors or cancers, preferably breast cancer, ovarian cancer, soft tissue sarcoma, liposarcoma, lung cancer, non-small cell lung cancer, gastric cancer, melanoma, head and neck cancer, cervical cancer, and prostate cancer.

[0476] The present invention also relates to a method for treating tumors or cancer, comprising administering to a subject in need an effective amount of a compound of general formula (I) according to the present invention, or a stereoisomer, tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a compound of general formula (II) according to the present invention, or a stereoisomer, tautomer, meso compound, racemic compound, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the thereof, wherein the cancer is preferably breast cancer, ovarian cancer, soft tissue sarcoma, liposarcoma, lung cancer, non-small cell lung cancer, gastric cancer, melanoma, head and neck cancer, cervical cancer, or prostate cancer. Invention Details

[0478] Unless otherwise specified, all technical and scientific terms used herein are consistent with the common understanding of one of ordinary skill in the art to which this disclosure pertains. While this disclosure may be practiced or tested using any methods and materials similar to or equivalent to those described herein, preferred methods and materials are described herein.

[0479] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0480] The term "connector unit (or linker segment)" refers to a chemical structural segment or bond that is connected to a ligand at one end and to a drug at the other end. It can also be connected to other linkers before being linked to a drug.

[0481] The term "ligand-drug conjugate" refers to a ligand linked to a biologically active drug via a stable linker unit. In this disclosure, "ligand-drug conjugate" is preferably an antibody-drug conjugate (ADC), which refers to a monoclonal antibody or antibody fragment linked to a biologically active toxic drug, namely a camptothecin derivative, via a stable linker unit.

[0482] Examples of the three-letter and single-letter codes for amino acids and their structures used in this disclosure are shown in the table below:

[0483]

[0484]

[0485] The term "antibody" refers to immunoglobulin, a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further divide them into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on differences in the constant region. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0486] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conserved backbone regions (FRs). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The CDR amino acid residues in the LCVR and HCVR regions of the antibody or antigen-binding fragments described in this disclosure conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3) in number and position, or conform to the Kabat and Chothia numbering rules (HCDR1).

[0487] The term "antigen-binding fragment" refers to one or more fragments of an antibody that maintain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in "antigen-binding fragments" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bridges on hinge regions; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and VL domains of a single arm of the antibody; (v) single-domain or dAb fragments consisting of a VH domain; and (vi) separate complementarity-determining regions (CDRs) or (vii) combinations of two or more separate CDRs optionally linked by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by synthetic linkers using recombinant methods, thereby enabling the production of a single protein chain (called a single-chain Fv (scFv)) in which the VL and VH regions pair to form a monovalent molecule. Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding moieties can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be different types of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0488] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms (including 1, 2, 3, 4, 5 or 6 carbon atoms). Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc. More preferably, lower alkyl groups containing 1 to 6 carbon atoms are used. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. Alkyl groups can be substituted or unsubstituted. When substituted, the substituents can be substituted at any usable connection point. The substituents are preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0489] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0490] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms (containing 3, 4, 5, 6, 7, or 8 carbon atoms). Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

[0491] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S(O). m The ring consists of heteroatoms (where m is an integer 0, 1, or 2), but excludes ring portions of -OO-, -OS-, or -SS-, with the remaining ring atoms being carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms (1, 2, 3, or 4 heteroatoms); more preferably, the cycloalkyl ring contains 3 to 10 ring atoms (containing 3, 4, 5, 6, 7, 8, 9, or 10 ring atoms). Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups.

[0492] The term "spiroheterocyclic group" refers to a polycyclic heterocyclic group consisting of 5 to 20 member monocyclic rings sharing a single atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O). mThe ring atoms are (where m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a fully conjugated π-electron system. Preferably, it is 6 to 14-membered, more preferably 7 to 10-membered. Spirocyclic groups are classified into monospirocyclic, bispirocyclic, or multispirocyclic groups according to the number of shared spiroatoms between rings, preferably monospirocyclic and bispirocyclic groups. More preferably, it is a 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospirocyclic group. Non-limiting examples of spirocyclic groups include:

[0493]

[0494] The term "fused heterocyclic group" refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with the other rings in the system. One or more rings may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are selected from nitrogen, oxygen, or S(O). m (where m is an integer 0, 1, or 2) heteroatoms, with the remaining ring atoms being carbon. Preferably, they are 6 to 14 members, more preferably 7 to 10 members (7-, 8-, 9-, or 10-membered rings). Depending on the number of rings, they can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclic groups, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic groups. Non-limiting examples of fused heterocyclic groups include:

[0495]

[0496] The term "bridged heterocyclic group" refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two non-directly bonded atoms. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are selected from nitrogen, oxygen, or S(O). m The heteroatom is a carbon atom (where m is an integer 0, 1, or 2). Preferably, it consists of 6 to 14 members, more preferably 7 to 10 members (7-, 8-, 9-, or 10-membered rings). Depending on the number of rings, it can be classified as a bicyclic, tricyclic, tetracyclic, or polycyclic bridged heterocyclic group, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

[0497]

[0498] The heterocyclic ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic group, and non-limiting examples include:

[0499] wait.

[0500] The heterocyclic group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0501] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered (6-, 7-, 8-, 9-, or 10-membered), such as phenyl and naphthyl, with phenyl being more preferred. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, and non-limiting examples include:

[0502]

[0503] The aryl group can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0504] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms (1, 2, 3, or 4 heteroatoms) and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl is preferably 5 to 10-membered (5-, 6-, 7-, 8-, 9-, or 10-membered heteroaryl), more preferably 5- or 6-membered, such as furanyl, thiophene, pyridyl, pyrrole, N-alkylpyrrole, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, and non-limiting examples include:

[0505]

[0506] The heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0507] The term "amino protecting group" is used to protect the amino group by a group that is easily removed, so that the amino group remains unchanged when other parts of the molecule react. Non-limiting examples include 9-fluorenemethoxycarbonyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl, etc. These groups may optionally be replaced by 1 to 3 substituents (one, two, or three substituents) selected from halogens, alkoxy groups, or nitro groups. The amino protecting group is preferably 9-fluorenemethoxycarbonyl.

[0508] The term "halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the alkyl group is as defined above.

[0509] The term “deuterated alkyl” refers to an alkyl group that is replaced by one or more deuterium atoms, wherein the alkyl group is as defined above.

[0510] The term "hydroxyalkyl" refers to an alkyl group that is replaced by one or more hydroxyl groups, wherein the alkyl group is as defined above.

[0511] The term "hydroxyl group" refers to the -OH group.

[0512] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0513] The term "amino" refers to -NH2.

[0514] The term "nitro" refers to -NO2.

[0515] The term "cyano" refers to -CN.

[0516] The term "acylamino" refers to -C(O)N(alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0517] The term "carboxylic acid ester group" refers to -C(O)O (alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0518] This disclosure also includes various deuterated forms of compounds of formula (I). Each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize the deuterated forms of compounds of formula (I) with reference to relevant literature. Commercially available deuterated starting materials can be used in the preparation of the deuterated forms of compounds of formula (I), or they can be synthesized using conventional techniques with deuterating reagents, including but not limited to deuterboranes, trideuterontetrahydrofuran solutions, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane.

[0519] "Optional" or "optionally" means that the event or environment described below may but does not have to occur, and the description includes the possibility that the event or environment may or may not occur. For example, "optionally alkyl-substituted heterocyclic group" means that an alkyl group may but does not have to be present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0520] "Substituted" refers to one or more hydrogen atoms in a group, preferably up to five, more preferably one, two, or three hydrogen atoms, which are independently substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0521] The term "pharmaceutical composition" refers to a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0522] The term "pharmaceutically acceptable salt" or "medicinal salt" refers to a salt of the ligand-drug conjugate of this disclosure, or a salt of the compounds described in this disclosure, which is safe and effective in mammals and has the intended biological activity. The ligand-drug conjugate of this disclosure contains at least one amino group and can therefore form a salt with an acid. Non-limiting examples of medicinal salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0523] The term "carrier" is used in the context of the drugs disclosed herein, referring to a system that can alter the way a drug enters the body and its distribution within the body, control the rate of drug release, and deliver the drug to the target organ. Drug carrier release and targeting systems can reduce drug degradation and loss, decrease side effects, and improve bioavailability. For example, high-molecular-weight surfactants, due to their unique amphiphilic structure, can self-assemble to form various forms of aggregates, preferably such as micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules while also exhibiting good membrane permeability, making them excellent drug carriers.

[0524] The term "excipient" refers to any additive in a pharmaceutical preparation other than the active pharmaceutical ingredient (API). Examples of excipients include binders, fillers, disintegrants, and lubricants in tablets; the base portion in semi-solid preparations such as ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solvents, osmotic pressure regulators, and colorants in liquid preparations.

[0525] Pharmaceutical compositions containing an active ingredient can be in forms suitable for oral administration, such as tablets, sugar lozenges, tablets, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Oral compositions can be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions may contain one or more ingredients selected from: sweeteners, flavoring agents, coloring agents, and preservatives to provide an appealing and palatable pharmaceutical formulation. Tablets contain an active ingredient and non-toxic, pharmaceutically acceptable excipients suitable for tablet preparation for mixing. These excipients can be inert excipients such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating agents and disintegrants such as microcrystalline cellulose, croscarmellose sodium, corn starch, or alginate; binders such as starch, gelatin, polyvinylpyrrolidone, or gum arabic; and lubricants such as magnesium stearate, stearic acid, or talc. These tablets may be uncoated or coated using known techniques that provide sustained release over a longer period of time by masking the taste of the drug or by delaying disintegration and absorption in the gastrointestinal tract. For example, water-soluble taste-masking substances such as hydroxypropyl methylcellulose or hydroxypropyl cellulose may be used, or time-extending substances such as ethylcellulose or cellulose acetate butyrate may be used.

[0526] Oral formulations can also be provided in hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin, or in soft gelatin capsules in which the active ingredient is mixed with a water-soluble carrier such as polyethylene glycol or an oil solvent such as peanut oil, liquid paraffin or olive oil.

[0527] Aqueous suspensions contain active substances and excipients suitable for preparing aqueous suspensions for mixing. Such excipients are suspending agents, such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinylpyrrolidone, and gum arabic; dispersants or wetting agents, which may be naturally occurring phospholipids such as lecithin, or condensation products of olefinic oxygen and fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide and long-chain fatty alcohols, such as heptadecaethyleneoxy cetanol, or condensation products of ethylene oxide and partial esters derived from fatty acids and hexitols, such as polyoxyethylene sorbitan monooleate, or condensation products of ethylene oxide and partial esters derived from fatty acids and hexitol anhydrides, such as polyoxyethylene dehydrated sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives such as ethylparaben or n-propylparaben, one or more colorants, one or more flavoring agents, and one or more sweeteners such as sucrose, saccharin, or aspartame.

[0528] Oil suspensions can be formulated by suspending the active ingredient in vegetable oils such as peanut oil, olive oil, sesame oil, or coconut oil, or mineral oils such as liquid paraffin. Oil suspensions may contain thickeners such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners and flavoring agents mentioned above can be added to provide a palatable formulation. These compositions can be preserved by adding antioxidants such as butylated hydroxyanisole (BHA) or α-tocopherol.

[0529] By adding water, dispersible powders and granules suitable for preparing aqueous suspensions can provide active ingredients and dispersants or wetting agents, suspending agents, or one or more preservatives for mixing. Suitable dispersants or wetting agents and suspending agents are as described above. Other excipients such as sweeteners, flavoring agents, and coloring agents may also be added. These compositions are preserved by adding antioxidants such as ascorbic acid.

[0530] The pharmaceutical compositions of the present invention may also be in the form of an oil-in-water emulsion. The oil phase may be a vegetable oil, such as olive oil or peanut oil, or a mineral oil, such as liquid paraffin, or a mixture thereof. Suitable emulsifiers may be naturally occurring phospholipids, such as soybean lecithin, and esters or metaesters derived from fatty acids and hexitan anhydrides, such as sorbitan monooleate, and condensation products of said metaesters and ethylene oxide, such as poly(ethylene oxide) sorbitan monooleate. The emulsion may also contain sweeteners, flavoring agents, preservatives, and antioxidants. Syrups and elixirs formulated with sweeteners such as glycerin, propylene glycol, sorbitol, or sucrose may be used. Such formulations may also contain moderating agents, preservatives, coloring agents, and antioxidants.

[0531] The pharmaceutical compositions of the present invention can be in the form of sterile injectable aqueous solutions. Acceptable solvents and media that can be used include water, Ringer's solution, and isotonic sodium chloride solution. The sterile injectable formulation can be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then treated with a mixture of water and glycerol to form a microemulsion. The injection solution or microemulsion can be injected into the patient's bloodstream by local large-volume injection. Alternatively, it is preferable to administer the solution and microemulsion in a manner that maintains a constant circulating concentration of the compounds of the present invention. To maintain such a constant concentration, a continuous intravenous delivery device can be used.

[0532] The pharmaceutical compositions of the present invention can be in the form of sterile injectable aqueous or oil suspensions for intramuscular and subcutaneous administration. These suspensions can be formulated using suitable dispersants or wetting agents and suspending agents as described above, according to known techniques. The sterile injectable formulations can also be sterile injectable solutions or suspensions prepared in non-toxic, parenteral-acceptable diluents or solvents, such as solutions prepared in 1,3-butanediol. Furthermore, sterile fixative oils can be conveniently used as solvents or suspension media. For this purpose, any blended fixative oil, including synthetic mono- or diglycerides of glycerol, can be used. Additionally, fatty acids such as oleic acid can also be used to prepare injectable formulations.

[0533] Those skilled in the art will recognize that the dosage of a drug depends on a variety of factors, including, but not limited to, the activity of the specific compound used, the patient's age, weight, health condition, lifestyle, diet, timing of administration, route of administration, rate of excretion, and combination of drugs. Furthermore, optimal treatment modalities, such as treatment patterns, daily dosage of general formula compounds, or types of pharmaceutically acceptable salts, can be validated based on conventional treatment protocols. Attached Figure Description

[0534] Figure 1 The tumor growth curve of the NCI-N87 gastric cancer-bearing mouse model in Test Example 3 was used.

[0535] Figure 2 The tumor growth curve of the human breast cancer JIMT-1 tumor mouse model in Test Example 4 is shown.

[0536] Figure 3A The growth inhibition curve of Her2-negative 468-luc cells in test case 6 is shown.

[0537] Figure 3B The growth inhibition curve of Her2-positive N87 cells in test case 6 is shown.

[0538] Figure 3C To test the growth inhibition curves of N87 cells and 468-luc cells co-incubated in Example 6.

[0539] Figures 4-5 The curve showing the change in body weight of rats after a single dose of 200 mg / kg in Test Example 7.

[0540] Figures 6A-6B The tumor growth curve of the NCI-N87 gastric cancer-bearing mouse model in Test Example 5 was used. Detailed Implementation

[0541] Further examples are provided to illustrate the compounds of the present invention and their preparation, and these examples demonstrate methods for preparing or using said compounds. However, it is to be understood that these examples do not limit the scope of the invention. Variations of the invention now known or further developed are considered to fall within the scope of the invention described and claimed herein.

[0542] The compounds of this invention are prepared using convenient starting materials and common preparation steps. Typical or preferred reaction conditions are provided, such as reaction temperature, time, solvent, pressure, and molar ratio of reactants. However, unless otherwise specified, other reaction conditions may also be adopted. Optimal conditions may vary depending on the specific reactants or solvents used, but in general, the optimal reaction steps and conditions can be determined.

[0543] In addition, some protecting groups may be used in this invention to protect certain functional groups from unnecessary reactions. Suitable protecting groups for various functional groups and their protection or deprotection conditions are well known to those skilled in the art. For example, TW Greene and GMWuts' "Protecting Groups in Organic Preparations" (3rd edition, Wiley, New York, 1999 and cited references in the book) describes in detail a large number of protecting or deprotecting groups.

[0544] The separation and purification of compounds and intermediates are carried out using appropriate methods and procedures depending on specific needs, such as filtration, extraction, distillation, crystallization, column chromatography, preparative thin-layer chromatography, preparative high-performance liquid chromatography, or a combination of the above methods. Specific methods can be found in the examples described in this invention. Of course, other similar separation and purification methods can also be used. Conventional methods (including physical constants and spectroscopic data) can be used for characterization.

[0545] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts were expressed in 10⁻¹⁰. -6 The unit (ppm) is given. NMR measurements were performed using a Brukerdps 400 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), deuterated methanol (CD3OD), and deuterated water (D2O). The internal standard was tetramethylsilane (TMS).

[0546] The determination of small molecules by MS was performed using an LC (Waters 2695) / MS (Quattro Premier xE) mass spectrometer (manufacturer: Waters) (Photodiode Array Detector).

[0547] MS determination of ADC was performed using UPLC-MS, Thermo Fisher Scientific, DionexUltiMate 3000UPLC-Q Exactive MS; High resolution mass spectrometer, Thermo Q EXACTIVE HF-X.

[0548] HIC analysis was performed using an Agilent 1200 high-performance liquid chromatograph with a ThermoMAbPac HIC-Bulty column (5 μm, 4.6 × 100 mm). Gradient elution was performed using a mixed solution of 1.5 M ammonium sulfate and 25 mM phosphate (pH 7.0)-isopropanol (95:5) as mobile phase A and 25 mM phosphate solution (pH 7.0)-isopropanol (80:20) as mobile phase B at a flow rate of 1.0 mL / min.

[0549] SEC was analyzed by ADC using an Agilent 1200 high-performance liquid chromatograph with a Waters BioResolve SEC mAb column (2.5 μm, 7.8 × 300 mm). Isocratic elution was performed using PBS as the mobile phase at a flow rate of 0.5 ml / min.

[0550] ADC analysis of RP was performed using an Agilent 1200 high-performance liquid chromatograph for HIC testing. The chromatographic column was a ThermoMAbPac RP (4 μm, 3 × 100 mm). Gradient elution was performed using 0.1% trifluoroacetic acid solution as mobile phase A and acetonitrile-isopropanol-trifluoroacetic acid (80:20:0.1) as mobile phase B at a flow rate of 1.0 ml / min.

[0551] Preparative high-performance liquid chromatography (HPLC) was performed using an lc6000 HPLC system (manufacturer: Innovation Tongheng). The column was a Daisogel C18 10μm 100A (30mm × 250mm), and the mobile phase was acetonitrile / water; Oriendo, model BRIX-2860. Alternatively, a Phenomenex Luna C18 250 × 50mm × 10μm column was used. The mobile phase was water (0.225% trifluoroacetic acid)-acetonitrile.

[0552] The silica gel plates used for thin-layer chromatography are Qingdao Ocean Chemical GF254 silica gel plates. The silica gel plates used in thin-layer chromatography (TLC) have a diameter of 0.20 mm to 0.25 mm, while the diameter used for preparing products separated and purified by thin-layer chromatography is 0.5 mm.

[0553] Column chromatography typically uses Qingdao Ocean Chemical's 100-200 mesh, 200-300 mesh, and 300-400 mesh silica gel as a carrier.

[0554] The known starting materials of this invention can be synthesized using or according to methods known in the art, or can be purchased from companies such as NetEase, Beijing Ouhe, Sigma, Bailingwei, Yishiming, Shanghai Shuya, Shanghai Inokai, Anaiji Chemical, Shanghai Bide, Haoyuan Pharmaceutical, Yilai Bio, and Duchuang Pharmaceutical.

[0555] Unless otherwise specified in the examples, all reactions can be carried out under a nitrogen atmosphere.

[0556] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0557] The terms "reaction solvent," "organic solvent," or "inert solvent" are used to indicate that the solvent does not participate in the reaction under the described reaction conditions. These include solvents such as benzene, toluene, acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), chloroform, dichloromethane, diethyl ether, methanol, N-methylpyrrolidone (NMP), and pyridine. Unless otherwise specified in the examples, "solution" refers to an aqueous solution.

[0558] The chemical reactions described in this invention are generally carried out under normal pressure. The reaction temperature is between -78°C and 200°C. The reaction time and conditions are, for example, at one atmosphere, between -78°C and 200°C, approximately 1 to 24 hours. If the reaction is carried out overnight, the reaction time is generally 16 hours. Unless otherwise specified in the examples, the reaction temperature is room temperature, between 20°C and 30°C.

[0559] The reaction process in the examples was monitored using thin-layer chromatography (TLC). The developing solvent systems used in the reactions were: A: dichloromethane and methanol system, B: petroleum ether and ethyl acetate system, and C: acetone. The volume ratio of the solvents was adjusted according to the polarity of the compounds.

[0560] The eluent system for column chromatography and the developing solvent system for thin-layer chromatography used to purify the compound include: A: dichloromethane and methanol system, B: petroleum ether and ethyl acetate system. The volume ratio of the solvent is adjusted according to the polarity of the compound. Small amounts of basic or acidic reagents such as triethylamine and trifluoroacetic acid can also be added for adjustment.

[0561] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in the methods of this invention.

[0562] The compounds of the present invention are prepared according to the exemplary procedures provided herein and modifications known to those skilled in the art.

[0563] Preparation Example

[0564] Preparation Example 1: Preparation of (9R)-9-ethyl-5-fluoro-1,9-dihydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-1)

[0565]

[0566] NaNO2 (270 mg, 3.91 mmol) was placed in a 25 mL three-necked flask, dissolved in 6 mL of water, and then 1 mL of glacial acetic acid was added. Subsequently, an aqueous solution of icetane mesylate (Haoyuan Pharmaceutical, 100 mg, 0.188 mmol) in acetic acid (3 mL glacial acetic acid, 3 mL water) was slowly added to the NaNO2 solution, and the reaction was carried out at room temperature for 1.5 h. The reaction solution was filtered to collect the solid, which was dried to obtain a reddish-brown solid. The solid was purified by high-performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50 mm×10 μm; mobile phase: water (0.5% formic acid)-acetonitrile, eluting from 40% to 80%), and lyophilized to obtain 12 mg of a faint red powder, yield: 14%.

[0567] LCMS (ESI): m / z, 437.1 [M+1] + .

[0568] 1 H NMR (400MHz, DMSO-d6) δ7.75(d,J=11.0Hz,1H),7.31(s,1H),6.50(s,1H),6.04-5.83(m,1H),5.48-5.29(m,4H),5.16(d,J=5.6Hz,1H),3.23 (dd,J=13.4,8.6Hz,2H),3.09-2.98(m,1H),2.37(s,3H),2.02(dd,J=16.4,6.8Hz,1H),1.86(dq,J=14.0,7.2Hz,2H),0.88(t,J=7.3Hz,3H).

[0569] Preparation Example 2: Preparation of (9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl(2-hydroxyethyl)carbamate (PY-1AB)

[0570]

[0571] Step 1: Preparation of (9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl(4-nitrophenyl)carbonate (PY-1D)

[0572] Compound PY-1 (40.0 mg, 0.09 mmol, 1.0 eq) and triethylamine (27.3 mg, 0.27 mmol, 3.0 eq) were dissolved in dichloromethane (5 mL). Nitrophenyl p-chloroformate (36.2 mg, 0.18 mmol, 2.0 eq) was added at 0 °C, and the mixture was then heated to 35 °C and stirred for 3 hours. The reaction solution was cooled to room temperature and then concentrated to give a crude yellow solid (60 mg crude product, 49.3% purity, 0.05 mmol, yield 55.6%).

[0573] LCMS: RT=0.909min, MS(ESI)m / z=602.1[M+H] + .

[0574] Step 2: Preparation of (9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl(2-hydroxyethyl)carbamate (PY-1AB)

[0575] Compound PY-1D (0.05 mmol, 1.0 eq) and triethylamine (10.1 mg, 0.1 mmol, 2.0 eq) were dissolved in dichloromethane (5 mL). Ethanolamine (31.4 mg, 0.04 mmol, 1.1 eq) was added at 0 °C, and the mixture was stirred at 25 °C for 3 hours. The reaction solution was concentrated under cold to obtain a crude product, which was purified by reversed-phase chromatography (column: Welch Xtimate C18 150 × 30 mm × 5 μm; mobile phase: [water(FA)-ACN]; gradient: 6%-46% B over 25 min) to give the title compound PY-1AB (2.23 mg, 0.004 mmol, yield 8.00%) as a white solid.

[0576] LCMS: RT=1.197min, MS(ESI)m / z=524.2[M+H] + .

[0577] 1 HNMR (400MHz, DMSO-d) 6)δppm 7.81(d,J=10.8Hz,1H),7.45(t,J=5.6Hz,1H),7.33(s,1H),6.52(s,1H),6.27(s,1H),5.44-5.24(m,4H),4.70(t,J=5.6Hz,1H),3 .48-3.44(m,2H),3.22-3.12(m,4H),2.40(s,3H),2.20-2.17(m,1H),1.91-1.86(m,2H),1.27-1.22(m,1H),0.88(t,J=7.2Hz,3H).

[0578] Preparation Example 3: Preparation of (9S)-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-10,13-dione (PY-2) and (S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1-methylene-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-10,13-dione (PY-2b)

[0579]

[0580] Step 1: Preparation of (E)-N-(3-fluoro-7-(hydroxymethylene)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-22)

[0581] In a 100 mL three-necked flask, N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-21) (1.0 g, 4.25 mmol, 1.0 eq) was dissolved in THF (20 mL). The mixture was cooled to 0 °C in ice water, and t-BuOK (12.75 mmol, 1 M, 12.75 mL, 3 eq) was slowly added. The system temperature was maintained at 0 °C, and the mixture was stirred for 30 minutes. Then, ethyl formate (787 mg, 10.63 mmol, 2.5 eq) was added to the reaction system, and the mixture was stirred for 2 hours. Quenching with ammonium chloride (20 ml), extraction with ethyl acetate, drying with anhydrous sodium sulfate, filtration, and concentration under reduced pressure yielded a yellow solid compound (E)-N-(3-fluoro-7-(hydroxymethylene)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (1.22 g, 95% yield).

[0582] LCMS (ESI): m / z, 264.1 [M+1] + .

[0583] Step 2: Preparation of N-(3-fluoro-7-(hydroxymethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-23)

[0584] Under a nitrogen atmosphere, PtO2 (69.00 mg, 303.90 μmol, 0.2 eq) was added to a methanol (50 mL) solution of compound (E)-N-(3-fluoro-7-(hydroxymethylene)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide PY-22 (400 mg, 1.52 mmol, 1.0 eq). Hydrogen was purged three times, and the reaction was carried out under a hydrogen atmosphere for 24 hours (40 °C). Residual starting material was detected. PtO2 (69.00 mg, 303.90 μmol, 0.2 eq) was added, and the reaction was continued for 24 hours under a hydrogen atmosphere. The mixture was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane 1:20 to 1:5) to give a yellow solid compound N-(3-fluoro-7-(hydroxymethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (324 mg, yield 63%).

[0585] LCMS (ESI): m / z, 266.1 [M+1] + .

[0586] Step 3: Preparation of 8-amino-6-fluoro-2-(hydroxymethyl)-5-methyl-3,4-dihydronaphthyl-1(2H)-one (PY-24)

[0587] Under ice bath conditions, SOCl2 (2.71 mmol, 1 M / L, 2.71 mL, 4.0 eq) was added to an anhydrous MeOH (15 mL) solution of compound N-(3-fluoro-7-(hydroxymethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide PY-23 (180 mg, 0.68 μmol, 1.0 eq). After the addition was complete, nitrogen gas was purged three times, and the mixture was stirred at 50 °C for 60 min. The residue was concentrated under reduced pressure, and purified by preparative high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50mm×10μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 40% to 80%) to give a pale yellow solid compound 8-amino-6-fluoro-2-(hydroxymethyl)-5-methyl-3,4-dihydronaphthyl-1(2H)-one (28.5 mg, 25% yield).

[0588] LCMS (ESI): m / z, 264.1 [M+1] + .

[0589] 1 H NMR (400MHz, DMSO-d) 6 )δ7.43(brs,2H),6.35(d,J=12.0Hz,1H),3.74(dd,J=10.8,4.4Hz,1H),3.60(dd,J=10.8,7.2Hz,1H) ,2.90(dt,J=17.2,4.8Hz,1H),2.75-2.54(m,2H),2.18-2.08(m,1H),1.98(s,3H),1.84-1.79(m,1H).

[0590] Step 4: Preparation of (9S)-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-10,13-dione (PY-2) and (S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1-methylene-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-10,13-dione (PY-2b)

[0591] To a xylene (5 mL) solution of compound 8-amino-6-fluoro-2-(hydroxymethyl)-5-methyl-3,4-dihydronaphthyl-1(2H)-one (22 mg, 98.5 μmol, 1.0 eq), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-3,6,10(4H)-trione (26 mg, 98.50 μmol, 1.0 eq) and PPTS (5 mg, 19.70 μmol, 0.2 eq) were added. After the addition was complete, the mixture was purged with nitrogen three times and stirred at 140 °C for 3 h. The residue was concentrated under reduced pressure and purified by preparative high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50mm×10μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, from 48% to 80%) to give a yellow solid PY-2 (3.42 mg, yield 7%) and a dark brown, somewhat viscous solid PY-2b (5.31 mg, yield 10%).

[0592] PY-2:

[0593] LCMS (ESI): m / z, 451.2 [M+1] + .

[0594] 1H NMR (400MHz, DMSO-d) 6 )δ11.80(s,1H),8.14(s,1H),7.17(d,J=12.4Hz,1H),6.73(s,1H),6.46 (s,1H),6.11(d,J=2.0Hz,1H),5.61(d,J=2.0Hz,1H),5.36(s,1H),4.73( d,J=2.4Hz,1H),2.95(t,J=6.8Hz,2H),2.79(t,J=6.8Hz,2H),2.25–2.03 (m,5H),2.00(d,J=2.0Hz,1H),1.86-1.76(m,2H),0.85(t,J=7.2Hz,3H).

[0595] PY-2b:

[0596] LCMS(ESI): m / z, 433.2 [M+1] + .

[0597] 1 H NMR (400MHz, CDCl3) δ7.67 (d, J = 10.8Hz, 1H), 7.62 (s, 1H), 5.77-5.70 (m, 3H), 5.38-5.30 (m, 3H), 3.77 (s,1H),3.22-3.19(m,2H),2.85-2.82(m,2H),2.41(s,3H),1.96-1.82(m,2H),1.04(t,J=8.0Hz,3H).

[0598] Preparation Example 4: Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxymethyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-4)

[0599]

[0600] Iciticon mesylate (Haoyuan Pharmaceutical) (50 mg, 114.82 μmol) was dissolved in DMF (2 mL). Ethylene oxide (1 M, 1.15 mL) and glacial acetic acid (3.45 mg, 57.41 μmol) were added to the solution. The reaction mixture was stirred at 90 °C for 16 hours. The reaction solution was cooled to room temperature, and 20 mL of water was added to the system. The mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50mm×10μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 40% to 80%). One drop of hydrochloric acid was added to the resulting solution and lyophilized to give a yellow solid compound PY-4 (5.82 mg, yield 21%).

[0601] LCMS: [M+H] + =480.9.

[0602] 1 H NMR (DMSO-d) 6 ,400MHz): δ(ppm)0.72–0.83(t,J=14.7Hz,3H),1.72–1.85(m,2H),2.13-2.22(s,3H),2.43-2.56(m,1H),2.80-3.06(m,2H),3.18 –3.31(s,2H),3.31-3.40(m,1H),3.74-3.82(m,2H),5.08-5.13(s,1H),5.18–5.41(m,4H),7.10-7.14(s,1H),7.15-7.21(s,1H).

[0603] Preparation Example 5: Preparation of 2-hydroxyethyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4:6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (PY-4Car)

[0604]

[0605] Step 1: Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-isocyanate-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-10,13-dione (4Car2)

[0606] At 0°C, three equivalents of triethylamine (14 mg, 0.14 mmol) and one equivalent of triphosgene (14 mg, 0.046 mmol) were added to a solution of ixotecan mesylate (20 mg, 0.046 mmol) in dichloromethane (2 mL), and the mixture was stirred at 0°C for three hours. The solution was concentrated under reduced pressure to obtain 20 mg of crude product, which was then directly used for the next reaction.

[0607] Step 2: Preparation of 2-hydroxyethyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3',4:6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (PY-4Car)

[0608] The compound (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-isocyanate-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4]:6,7]indolazino[1,2-b]quinoline-10,13-dione (4Car2) (20 mg, 0.043 mmol) was dissolved in 1 mL of dichloromethane, and 10 equivalents of ethylene glycol (27 mg, 0.43 mmol) were added dropwise. The mixture was stirred at room temperature for 3 hours. The residue was concentrated under reduced pressure and purified by reversed-phase preparative chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50mm×10μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 40% to 80%) to give 6.5 mg of the compound PY-4Car as a yellow solid.

[0609] LCMS: m / z = 524.2 [M+1] + .

[0610] 1 H NMR (400MHz, DMSO-d) 6)δ8.43(s,1H),8.00(d,J=8.4Hz,1H),7.78(d,J=10.8Hz,1H),7.31(s,1 H),6.55(s,1H),5.43(s,2H),5.24(d,J=7.20Hz,2H),4.81(t,J=5.2Hz,1 H),3.97-4.17(m,2H),3.61(d,J=3.6Hz,2H),3.02-3.18(m,2H),2.30-2 .41(m,3H),2.05-2.28(m,2H),1.75-1.94(m,2H),0.87(t,J=7.2Hz,3H).

[0611] Preparation Example 6: Preparation of 4-hydroxybutyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (PY-4Car2)

[0612]

[0613] Step 1: Preparation of 4-((tert-butyldimethylsilyl)oxy)butyl(4-nitrophenyl)carbonate (PY-4car2-b)

[0614] To a solution of 4-((tert-butyldimethylsilyl)oxy)but-1-ol (1 g, 4.89 mmol, 1 eq) and di(p-nitrobenzene) carbonate (2.98 g, 9.79 mmol, 2 eq) in N,N-dimethylformamide (15 mL), N,N-diisopropylethylamine (2.43 mL, 14.68 mmol, 3 eq) was added, and the mixture was stirred overnight at room temperature. The reaction solution was concentrated, diluted with dichloromethane (10 mL), and then diluted with water. The organic phase was washed three times with 10 mL of saturated brine and washed once with 10 mL of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1:10, dichloromethane / petroleum ether = 40%) to give the product 4-((tert-butyldimethylsilyl)oxy)butyl(4-nitrophenyl)carbonate (PY-4car2-b) (1.63 g, yield = 90.2%).

[0615] LCMS(ESI): m / z, 370 [M+H] + .

[0616] Step 2: Preparation of 4-((tert-butyldimethylsilyl)oxy)butyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (PY-4car2-d)

[0617] To a solution of ixotecan (20 mg, 37.62 μmol, 1 eq, mesylate, Haoyuan Pharmaceutical) and N,N-diisopropylethylamine (6.22 μL, 37.62 μmol, 1 eq) in dichloromethane (6 mL), triethylamine (20.92 μL, 150.48 μmol, 4 eq), compound PY-4car2-b (18.1 mg, 48.92 μmol, 1.3 eq), and 1-hydroxybenzotriazole (2.4 mg, 18.81 μmol, 0.5 eq) were added. The mixture was stirred at room temperature for 48 hours, concentrated, and the residue was analyzed by high-performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model BRIX-2860; column: Welch Xtimate C18). 250×30mm×10μm. Purification was carried out using water (0.225% HCOOH)-acetonitrile as the mobile phase (elution ratio of acetonitrile from 50% to 60%) to give the title compound PY-4car2-d (15mg, yield = 59.8%).

[0618] LCMS(ESI): m / z, 666 [M+H] + .

[0619] Step 3: Preparation of 4-hydroxybutyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (PY-4Car2)

[0620] Under ice bath conditions, triethylamine trihydrofluoride (14.69 μL, 90.12 μmol, 4 eq) was added to a tetrahydrofuran (3 mL) solution of compound PY-4car2-d (15 mg, 22.53 μmol, 1 eq). After stirring for 5 minutes, the mixture was stirred overnight at 25 °C. The residue was purified by reversed-phase preparative chromatography (SHIMADZU LC-20AP, YMC-Triart Prep C18 250×30 mm×10 μm column, water (0.225% HCOOH)-acetonitrile, with acetonitrile elution ratios ranging from 20% to 55%) to give the title compound PY-4Car2 (3.77 mg, yield = 30.34%).

[0621] LCMS(ESI): m / z, 552 [M+H] + .

[0622] 1 H NMR (400MHz, DMSO-d6) δ7.91(d,J=8.8Hz,1H),7.78(d,J=10.8Hz,1H),7.31(s,1H),6. 51(s,1H),5.42(s,2H),5.24(d,J=4.2Hz,2H),4.41(t,J=5.2Hz,1H),4.15-4.03(m,2H ),3.43(q,J=6.2Hz,2H),3.29–3.06(m,3H),2.38(d,J=1.8Hz,3H),2.25-2.08(m,2H), 1.93-1.81(m,2H),1.65(p,J=6.8Hz,2H),1.50(p,J=6.6Hz,2H),0.87(t,J=7.2Hz,3H). 19 F NMR(377MHz,DMSO-d6)δ-111.37.

[0623] Preparation Example 7: Preparation of 3-hydroxypropyl ((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7] indo[1,2-b]quinoline-1-yl)carbamate (PY-4Car3C)

[0624]

[0625]

[0626] Step 1: Preparation of compound 3-((tert-butyldimethylsilyl)oxy)propyl(4-nitrophenyl)carbonate

[0627] At 0°C, di(p-nitrobenzene) carbonate (1.28 g, 852.31 μL, 4.20 mmol, 2 eq) and DIPEA (543.14 mg, 694.55 μL, 4.20 mmol, 2 eq) were added to a DMF (5 mL) solution of compound 3-(tert-butyldimethylsiloxane)propanol (400 mg, 2.10 mmol, 1 eq). The mixture was stirred at room temperature (25°C) for 16 hours, and the reaction solution was poured into 60 mL of water. Extracted three times with ethyl acetate, 20 mL each time, the organic phases were combined and washed with brine (60 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether 1:30 to 1:20) to obtain a light yellow oily compound 3-((tert-butyldimethylsilyl)oxy)propyl(4-nitrophenyl) carbonate (740 mg, Yield: 99.07%).

[0628] 1 H NMR(400MHz,Chloroform-d)δ8.30-8.26(m,2H),7.40-7.35(m,2H),4.41(t,J= 6.4Hz, 2H), 3.76 (t, J = 5.9Hz, 2H), 1.99-1.93 (m, 2H), 0.90 (s, 9H), 0.07 (s, 6H).

[0629] Step 2: Preparation of compound PY-4Car 3C-a

[0630] HOBt (10.17 mg, 99.96%, 6.78 μL, 75.24 μmol, 1 eq) and DIPEA (29.17 mg, 99%, 37.31 μL, 225.73 μmol, 3 eq) were added to a DMF (2 mL) solution of 3-((tert-butyldimethylsilyl)oxy)propyl(4-nitrophenyl) carbonate (32.10 mg, 90.29 μmol, 1 eq) and eczema methanesulfonate (40 mg, 75.24 μmol, 1 eq). After addition, the mixture was stirred at room temperature (25 °C) for 1 hour. The reaction was confirmed to be complete by LC-MS. The reaction solution was prepared by high-performance liquid chromatography (HPLC) (Oriendo BRIX-2860 chromatographic column, Welch Xtimate C18). 150×21.2mm×5μm. Purified by mobile phase water (0.225% HCOOH)-acetonitrile, with water elution ratio from 50% to 80%, and lyophilized to give a light yellow solid compound PY-4Car 3C-a (45mg, Yield: 91.75%).

[0631] LCMS: m / z = 652.3[M+1]+ Rt = 3.098 min.

[0632] Step 3: Preparation of compound PY-4Car 3C

[0633] Triethylamine trihydrofluoride (499.70 mg, 98%, 505.26 μL, 3.04 mmol, 50 eq) was added to a 2 mL THF solution of compound PY-4Car3C-a (45 mg, 60.75 μmol, 1 eq) at room temperature. After addition, the reaction mixture was stirred at 40 °C for 10 hours. LC-MS showed that the reaction was complete. The reaction mixture was purified by high-performance liquid chromatography (Shimadzu LC-20AP, Welch Xtimate C18 250 × 30 mm × 10 μm column, mobile phase water (0.225% HCOOH)-acetonitrile, elution ratio of water from 30% to 60%). The purified solid was lyophilized to give PY-4Car3C (27.40 mg, Yield: 83.90%, Purity: 98.19%).

[0634] LCMS: m / z = 538.2 [M+1] + Rt = 1.508 min.

[0635] 1 H NMR (400MHz, DMSO-d6) δ7.92(d,J=8.8Hz,1H),7.77(d,J=10.9Hz,1H),7.30(s,1H),6. 52(s,1H),5.42(s,2H),5.23(d,J=6.4Hz,3H),4.51(s,1H),4.20-4.15(m,1H),4.13-4 .05(m,1H),3.50(t,J=6.4Hz,2H),3.23(s,1H),3.14-3.07(m,1H),2.37(d,J=1.8Hz,3 H), 2.24-2.09 (m, 2H), 1.92-1.82 (m, 2H), 1.76 (p, J = 6.5Hz, 2H), 0.87 (t, J = 7.3Hz, 3H).

[0636] Preparation Example 8: Preparation of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(2-hydroxyethyl)(methyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-5)

[0637]

[0638] The compound (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxymethyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-4) (25 mg, 52.14 μmol) was dissolved in methanol (2 mL). Paraformaldehyde (7.83 mg, 260.69 μmol) and glacial acetic acid (626.19 μg, 10.43 μmol) were added to the solution, and the reaction system was stirred at 60 °C for two hours. Sodium cyanoborohydride (16.38 mg, 260.69 μmol) was added, and the reaction system was stirred at 30 °C for 16 hours. The reaction solution was cooled to room temperature, and 20 mL of saturated ammonium chloride solution was added to the system. Extraction was performed with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250 × 50 mm × 10 μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 40% to 80%) to obtain a clean product solution. One drop of hydrochloric acid was added to this solution, and the solution was lyophilized to give a yellow solid PY-5 (7.63 mg, yield 29.65%).

[0639] LCMS: [M+H] + =494.8.

[0640] 1 ¹H NMR (deuterated dimethyl sulfoxide, 400 MHz): δ (ppm) 0.65–0.81 (t, J = 7.4 Hz, 3H), 1.73–1.84 (m, 2H), 2.05–2.22 (s, 3H), 2.48–2.57 (m, 1H), 2.57–2.69 (s, 3H), 2.93–3.15 (m, 2H), 3.19–3.31 (m, 1H), 3.34–3.42 (m, 1H), 3.78–3.92 (m, 2H), 5.08–5.44 (m, 4H), 7.12–7.14 (s, 1H), 7.14–7.18 (d, J = 10.4 Hz, 1H).

[0641] Preparation Example 9: Preparation of N-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-1-yl)-N-(2-hydroxyethyl)formamide (PY-6A1)

[0642]

[0643] Acetic anhydride (63.87 mg, 625.65 μmol) was added dropwise to an anhydrous tetrahydrofuran (1 mL) solution of acetic acid (31.31 mg, 521.38 μmol), and the reaction system was reacted at 50 °C for 1 hour. An anhydrous tetrahydrofuran (5 mL) solution of compound (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxymethyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-4) (100 mg, 208.55 μmol) was added to the above reaction system, and the reaction was carried out at 20 °C for 4 hours. 20 mL of water was added to the reaction system, and the mixture was extracted with ethyl acetate (20 mL × 3). After combining the organic phases, the mixture was washed with saturated brine, dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50mm×10μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 40% to 80%), and then lyophilized to give a white solid compound PY-6A1 (4.02 mg, 7.92 μmol, yield 3.80%).

[0644] LCMS: [M+H] + =508.2.

[0645] 1 H NMR (DMSO-d6, 400MHz): δ (ppm) 0.77-0.95 (t, J = 0.9Hz, 3H), 1.75-1.91 (m, 2H), 2.15-2.33 (m, 1H), 2.322.39 (s, 3H), 2.94-3.14 (m, 2H), 3.40 -3.66(m,2H),4.67-5.30(m,3H),5.31-5.57(m,3H),6.46-6.51(s,1H),7.26-7.30(s,1H),7.71-7.82(m,1H),8.11-8.29(d,J=44.6Hz,1H).

[0646] Preparation Example 10: Preparation of N-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12Hbenzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-1-yl)-N-(2-hydroxyethyl)acetamide (PY-6A2)

[0647]

[0648] At 0 °C, three equivalents of triethylamine (12 mg, 0.12 mmol) and one equivalent of acetyl chloride (3.27 mg, 41.70 μmol) were added to a solution of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxymethyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-4) (20 mg, 0.04 mmol) in dichloromethane (1 mL), and the mixture was stirred at 0 °C for two hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50mm×10μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 40% to 80%) to give (3.25 mg, 15%) a white solid compound PY-6A2.

[0649] LCMS (ESI): m / z, 522.2 [M+1] + .

[0650] 1 H NMR (400MHz, DMSO-d) 6 )δ8.13(s,1H)7.75(br d,J=9.6Hz,1H)7.25-7.38(m,1H)6.52(s,1H)5.30-5.53(m,4H)4.04-4.33(m,2H)2.85- 3.19(m,5H)2.29-2.41(m,4H)2.03-2.23(m,5H)1.83-1.95(m,2H)0.87(t,J=7.2Hz,3H).

[0651] Preparation Example 11: Preparation of N-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12Hbenzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-1-yl)-N-(2-hydroxyethyl)methanesulfonamide (PY-6B1)

[0652]

[0653] At 0 °C, three equivalents of triethylamine (12 mg, 0.12 mmol) and one equivalent of methanesulfonyl chloride (5 mg, 0.04 mmol) were added to a solution of (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-((2-hydroxymethyl)amino)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-4) (20 mg, 0.04 mmol) in dichloromethane (1 ml), and the mixture was stirred at 0 °C for two hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50mm×10μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 48% to 80%) to give a white solid compound PY-6B1 (1.3 mg, yield: 6%).

[0654] LCMS (ESI): m / z, 558.2 [M+1] + .

[0655] 1 H NMR (400MHz, DMSO-d) 6 )δ8.22(d,J=13.6Hz,1H),7.70-7.81(m,1H),7.28-7.35(m,1H),6.55(d,J=1.6Hz,1H),5.43(s,3H),4.15-4.37(m,1H ),2.80-3.11(m,4H),2.63-2.70(m,1H),2.27-2.40(m,6H),2.01-2.24(m,2H),1.53-1.95(m,4H),0.80-0.94(m,3H).

[0656] Preparation Example 12: Preparation of (S)-9-ethyl-5-fluoro-9-hydroxy-4-(3-hydroxypropyl)-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-8)

[0657]

[0658] Step 1: Preparation of 1-bromo-3-fluoro-2-methoxy-5-nitrobenzene (PY-82)

[0659] Concentrated sulfuric acid (30 mL) was added to a solution of 2-fluoro-1-methoxy-4-nitrobenzene (PY-81) (50 g, 292 mmol) and NBS (57.2 g, 321 mmol) in acetic acid (500 mL), and the mixture was stirred overnight at 120 °C. The reaction solution was concentrated, allowed to stand to precipitate a milky white solid, filtered, and the filter cake was washed with water and ethanol and dried to give compound PY-82 (58 g, 79% yield).

[0660] LCMS (ESI): m / z, 249.9 [M+H] + .

[0661] 1 H NMR (400MHz, DMSO-d) 6 )δ8.34-8.23(m,2H),4.07(d,J=3.2Hz,3H).

[0662] Step 2: Preparation of 3-bromo-5-fluoro-4-methoxyaniline (PY-83)

[0663] To a solution of compound 1-bromo-3-fluoro-2-methoxy-5-nitrobenzene (PY-82) (50 g, 200 mmol) and iron powder (55.85 g, 1 mol) in ethanol (500 mL), concentrated hydrochloric acid (3.34 mL, 0.55 equiv.) and 83 mL of water were added sequentially, and the mixture was stirred overnight at 80 °C. The reaction solution was filtered through diatomaceous earth, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (PE:EA = 3:1) to give compound PY-83 (40 g, 90% yield).

[0664] LCMS (ESI): m / z, 219.9 [M+H] + .

[0665] 1 H NMR (400MHz, DMSO-d) 6)δ6.59(t,J=2.0Hz,1H),6.41(dd,J=13.2,2.6Hz,1H),5.40(s,2H),3.67(s,3H).

[0666] Step 3: Preparation of N-(3-bromo-5-fluoro-4-methoxyphenyl)acetamide (PY-84)

[0667] Acetyl chloride (4 mL, 1.2 equiv.) was added to a solution of 3-bromo-5-fluoro-4-methoxyaniline (PY-83) (10.0 g, 45.4 mmol) and triethylamine (13.9 mL, 2.2 equiv.) in dichloromethane (500 mL), and the mixture was stirred overnight at 25 °C. The reaction was quenched with 150 mL of saturated ammonium chloride, followed by extraction with 3 × 80 mL of dichloromethane. The organic phase was washed with 80 mL of saturated sodium chloride solution, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (PE:EA = 3:1) to give compound PY-84 (10 g, 84% yield).

[0668] LCMS (ESI): m / z, 264.0 [M+H] + .

[0669] Step 4: Preparation of (Z)-4-(5-acetamido-3-fluoro-2-methoxyphenyl)but-3-enoic acid ethyl ester (PY-85)

[0670] To a solution of N-(3-bromo-5-fluoro-4-methoxyphenyl)acetamide (PY-84) (15 g, 57.23 mmol) and Pd(t-Bu3P)2 (1.46 g, 0.05 equiv) in toluene (200.0 mL), DIPEA (60 mL, 6.0 equiv.) and ethyl butyrate (1.5 equiv., 10.89 mL) were added sequentially, and the mixture was stirred overnight at 13 °C. The reaction mixture was then concentrated directly under reduced pressure, and the residue was purified by silica gel column chromatography (PE:EA = 1:2) to give compound PY-85 (7.9 g, yield 46%).

[0671] LCMS (ESI): m / z, 296.1 [M+H] + .

[0672] Step 5: Preparation of ethyl 4-(5-acetamido-3-fluoro-2-methoxyphenyl)butyrate (PY-86)

[0673] Palladium on carbon (0.1 equiv., 2.88 g) was added to a methanol (100.0 mL) solution of compound (Z)-4-(5-acetamido-3-fluoro-2-methoxyphenyl)but-3-enoic acid ethyl ester (PY-85) (8.0 g, 27.08 mmol), and the mixture was reacted overnight at 50 °C. The reaction mixture was filtered through diatomaceous earth, the filtrate was concentrated under reduced pressure, and dried to obtain the crude product compound PY-86, which was used directly in the next reaction.

[0674] LCMS (ESI): m / z, 298.1 [M+H] + .

[0675] Step 6: Preparation of 4-(5-acetamido-3-fluoro-2-methoxyphenyl)butyric acid (PY-87)

[0676] Lithium hydroxide (3 equiv., 1.93 g) was added to a 1:1:1 mixture of methanol, tetrahydrofuran, and water (120 ml) containing ethyl 4-(5-acetamido-3-fluoro-2-methoxyphenyl)butyrate (PY-86) (8 g, 26.91 mmol) and reacted overnight at room temperature. The reaction mixture was concentrated under reduced pressure, and impurities were extracted with ethyl acetate (2 × 80 ml) to remove the organic phase. The aqueous phase was then adjusted to pH 1 with concentrated hydrochloric acid and extracted with ethyl acetate (3 × 80 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound PY-87 (3.8 g, yield: 70%).

[0677] LCMS (ESI): m / z, 270.1 [M+H] + ;292.1[M+Na] + .

[0678] 1 H NMR (400MHz, DMSO-d) 6 )δ12.07(s,1H),9.99(s,1H),7.50(dd,J=13.6,2.4Hz,1H),7.09-7.03(m,1H),3.77(d,J=1. 2Hz, 3H), 2.63-2.53 (m, 2H), 2.25 (t, J = 7.2Hz, 2H), 2.02 (s, 3H), 1.75 (dq, J = 9.2, 7.2Hz, 2H).

[0679] Step 7: Preparation of N-(3-fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-88)

[0680] Compound 4-(5-acetamido-3-fluoro-2-methoxyphenyl)butyric acid (PY-87) (1 g, 3.71 mmol) was stirred in a solution of PPA (polyphosphoric acid) for 3 hours at 95 °C. The reaction mixture was poured into ice water, washed with water, extracted with ethyl acetate (3 × 30 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250 × 50 mm × 10 μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 45% to 85%), and lyophilized to give compound PY-88 (110 mg, yield 11%).

[0681] LCMS (ESI): m / z, 252.1 [M+H] + ; 274.0 [M+Na] + .

[0682] 1 H NMR (400MHz, DMSO-d) 6 )δ12.05(s,1H),8.37(d,J=14.8Hz,1H),3.81(d,J=1.2Hz,3H),2.95(t,J=6 .0Hz, 2H), 2.66 (dd, J = 7.2, 5.6Hz, 2H), 2.16 (s, 3H), 1.99 (q, J = 6.4Hz, 2H).

[0683] Step 8: Preparation of N-(3-fluoro-4-hydroxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-89)

[0684] 1.3 g of N-(3-fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-88) was dissolved in 20 ml of DCE, and 5 equivalents of AlCl3 were added. The mixture was stirred at 60 °C for 5 hours. The solution was diluted with 100 ml of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 910 mg of a yellow solid, PY-89.

[0685] LCMS (ESI): m / z, 238.0 [M+1] + .

[0686] Step 9: Preparation of 4-acetamido-2-fluoro-5-oxo-5,6,7,8-tetrahydronaphth-1-yltrifluoromethanesulfonate (PY-810)

[0687] The compound N-(3-fluoro-4-hydroxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-89) (0.9 g) was dissolved in DCM (20 mL), and 3 equivalents of triethylamine and 3 equivalents of trifluoromethanesulfonic anhydride were added. The mixture was stirred at room temperature for 3 hours, concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (ethyl acetate / n-hexane 1:5) to obtain 0.9 g of yellow solid compound PY-810.

[0688] LCMS (ESI): m / z, 370.1 [M+1] + .

[0689] Step 10: Preparation of N-(4-(3-(benzyloxy)prop-1-yn-1-yl)-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-811)

[0690] 420 mg of compound 4-acetamido-2-fluoro-5-oxo-5,6,7,8-tetrahydronaphth-1-yltrifluoromethanesulfonate (PY-810) was dissolved in 10 mL of DMF, and 2 equivalents of triethylamine, 3 equivalents of propynyl benzyl ether, 0.2 equivalents of Pd(PPh3)2Cl2, and 0.1 equivalents of CuI were added. The mixture was stirred at 80 °C for 12 hours under a nitrogen atmosphere. The solution was diluted with 100 mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by high-performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50 mm×10 μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 45% to 85%) to obtain 250 mg of the yellow solid compound PY-811.

[0691] LCMS (ESI): m / z, 366.1 [M+1] + .

[0692] Step 11: Preparation of N-(3-fluoro-4-(3-hydroxypropyl)-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-812)

[0693] 250 mg of N-(4-(3-(benzyloxy)prop-1-yn-1-yl)-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-811) was dissolved in 10 mL of MeOH, and 0.1 equivalent of palladium on carbon was added. The mixture was stirred at room temperature for 12 hours under a hydrogen atmosphere. The solution was filtered, and the filtrate was concentrated under reduced pressure to give 150 mg of a yellow solid, PY-812.

[0694] LCMS (ESI): m / z, 280.2 [M+1] +.

[0695] Step 12: Preparation of 8-amino-6-fluoro-5-(3-hydroxypropyl)-3,4-dihydronaphthyl-1(2H)-one (PY-813)

[0696] The compound N-(3-fluoro-4-(3-hydroxypropyl)-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-812) (150 mg) was dissolved in 6N HCl (2 mL) and EtOH (2 mL), stirred at 60 °C for 3 hours, filtered, and concentrated under reduced pressure to obtain 125 mg of yellow solid compound PY-813.

[0697] LCMS (ESI): m / z, 238.1 [M+1] + .

[0698] Step 13: Preparation of (S)-9-ethyl-5-fluoro-9-hydroxy-4-(3-hydroxypropyl)-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-8)

[0699] Compound 8-amino-6-fluoro-5-(3-hydroxypropyl)-3,4-dihydronaphthyl-1(2H)-one (PY-813) (125 mg, 0.53 mmol) and 1.1 equivalents of (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran[3,4-f]indoleazinazino-3,6,10(4H)-trione (Haoyuan Pharmaceutical, 152 mg, 0.58 mmol) were dissolved in 10 mL of xylene, and 0.3 equivalents of PPTS (pyridine p-toluenesulfonate) (40 mg, 0.16 mmol) were added. The mixture was stirred at 120 °C for 12 hours under a nitrogen atmosphere. The residue was concentrated under reduced pressure, and then purified by high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50mm×10μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 45% to 85%). The residue was then lyophilized to give a yellow solid compound PY-8 (42 mg, yield 17%).

[0700] LCMS (ESI): m / z, 456.2 [M+1] + .

[0701] 1 H NMR (400MHz, DMSO-d) 6)δ7.72(d,J=11.2Hz,1H),7.30(s,1H),6.51(s,1H),5.43(s,2H),5.24(s,2H),4.60(t,J=5.2Hz,1H),3.49(q,J=6.0Hz,2H),3.1 6(t,J=6.0Hz,4H),2.87(t,J=8.0Hz,2H),2.08(t,J=6.0Hz,2H),1.88-1.87(m,2H),1.69(p,J=6.4Hz,2H),0.88(t,J=7.2Hz,3H).

[0702] Preparation Example 13: Preparation of (S)-9-ethyl-5-fluoro-9-hydroxy-4-propyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-8B)

[0703]

[0704] Step 1: Preparation of N-(3-bromo-5-fluorophenyl)acetamide (PY-8a)

[0705] 3-Bromo-5-fluoroaniline (50 g, 0.263 mol) was dissolved in dichloromethane (700 mL), and triethylamine (53.2 g, 0.526 mol) was added at room temperature. Acetic anhydride (40.3 g, 0.395 mol) was slowly added dropwise under an ice-water bath. After the addition was complete, the reaction was allowed to proceed at room temperature for 2 hours. Water (400 mL) was added, and the aqueous phase was extracted again with dichloromethane (200 mL). The organic phases were combined and washed successively with saturated sodium chloride solution (300 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was then slurried with n-heptane and filtered to give N-(3-bromo-5-fluorophenyl)acetamide (54 g, yield: 88.4%).

[0706] Step 2: Preparation of (E)-4-(3-acetamido-5-fluorophenyl)but-3-enoic acid tert-butyl ester (PY-8b)

[0707] N-(3-bromo-5-fluorophenyl)acetamide (PY-8a) (54 g, 0.2327 mol) was dissolved in N,N-dimethylformamide (700 mL), and then di(tri-tert-butylphosphine)palladium (5.97 g, 11.64 mmol), tris(o-methylphenyl)phosphine (7.07 g, 23.27 mmol), N-methyldicyclohexylamine (100 g, 0.5119 mol) and tert-butyl 1-buten-4-o-acetic acid (66.1 g, 0.4654 mol) were added sequentially. After purging with nitrogen three times, the reaction was carried out at 100 °C for 16 hours. Extracted with ethyl acetate (600 mL) and water (1 L), the aqueous phase was then extracted with ethyl acetate (300 mL), the organic phases were combined and washed successively with saturated sodium chloride solution (500 mL × 2), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with ethyl acetate / n-heptane (0-30%) as the eluent to give (E)-4-(3-acetamido-5-fluorophenyl)but-3-enoic acid tert-butyl ester (65 g, yield: 95.3%).

[0708] Step 3: Preparation of tert-butyl 4-(3-acetamido-5-fluorophenyl)butyrate (PY-8c)

[0709] (E)-4-(3-acetamido-5-fluorophenyl)but-3-enoic acid tert-butyl ester (PY-8b) (65 g, 0.2218 mol) was dissolved in methanol (1.3 L). After purging with nitrogen once, Pd / C (33 g, 0.3101 mol) was added. After the addition was complete, nitrogen was purged again, followed by two purgings with hydrogen. The reaction was carried out at room temperature for 16 hours. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase chromatography using acetonitrile / purified water (0-90%) as the eluent to obtain 4-(3-acetamido-5-fluorophenyl)butyrate tert-butyl ester (PY-8c) (55 g, yield: 84%).

[0710] Step 4: Preparation of tert-butyl 4-(5-acetamido-2-bromo-3-fluorophenyl)butyrate (PY-8d)

[0711] 4-(3-acetamido-5-fluorophenyl)butyrate tert-butyl ester (PY-8c) (55 g, 0.1864 mol) was dissolved in N,N-dimethylformamide (600 mL). Under ice-water bath, NBS (36.5 g, 0.2051 mol) was added in portions. After the addition was complete, the mixture was stirred at room temperature for 1 h. Then, ethyl acetate (300 mL) and water (400 mL) were added for extraction. The aqueous phase was extracted again with ethyl acetate (100 mL). The organic phases were combined and washed successively with saturated sodium chloride solution (300 mL × 2). The mixture was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography with ethyl acetate / n-heptane (0-30%) as the eluent to give 4-(5-acetamido-2-bromo-3-fluorophenyl)butyrate tert-butyl ester (59 g, yield: 84.6%).

[0712] Step 5: Preparation of 4-(5-acetamido-2-bromo-3-fluorophenyl)butyric acid (PY-8e)

[0713] 4-(5-acetamido-2-bromo-3-fluorophenyl)butyrate tert-butyl ester (PY-8d) (59 g, 0.1577 mol) was dissolved in dichloromethane (300 mL). HCl / dioxane (400 mL, 1.6 mol) was added dropwise under an ice-water bath. After the addition was complete, the mixture was reacted at room temperature for 16 hours. The mixture was filtered, and the filtrate was concentrated under reduced pressure to give 4-(5-acetamido-2-bromo-3-fluorophenyl)butyric acid (42 g, yield: 83.7%).

[0714] Step 6: Preparation of N-(4-bromo-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-8g)

[0715] In a 1L three-necked flask, 4-(5-acetamido-2-bromo-3-fluorophenyl)butyric acid (PY-8e) (42g, 0.1321mol) and Eaton reagent (210g) were added. After purging with nitrogen three times, the mixture was reacted at 85°C for 1 hour. Under an ice-water bath, the reaction solution was slowly added dropwise to water (2L), filtered, and the filter cake was washed clean with water. The filtrate was then concentrated to dryness under reduced pressure and purified by silica gel column chromatography with an eluent system of ethyl acetate / dichloromethane (0-30%) to obtain a crude product (30g). The crude product was then slurried with an ethyl acetate (10mL) / petroleum ether (100mL) system, filtered, and N-(4-bromo-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (20g, yield 50.4%) was obtained.

[0716] LCMS (ESI): m / z, 300.0 [M+H] + .

[0717] 1H NMR (400MHz, CDCl3) δ12.35 (s, 1H), 8.61 (d, J = 11.6Hz, 1H), 3.07 (t, J = 6.0Hz, 2H), 2.69 (dd, J = 7.2Hz, 2H), 2.24 (s, 3H), 2.11 (p, J = 6.4Hz, 2H).

[0718] Step 7: Preparation of N-(4-(3-(benzyloxy)prop-1-yn-1-yl)-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-8h)

[0719] Under a nitrogen atmosphere, N-(4-bromo-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-8g) (1.8g, 6.0mmol) was dissolved in anhydrous DMF (50mL). Then, (prop-2-en-1-oxy)methyl)benzene (4.34mL, 29.99mmol), cuprous iodide (228.4mg, 1.2mmol), bis(triphenylphosphine)palladium dichloride (841.9mg, 1.2mmol), and triethylamine (3.33mL, 23.99mmol) were added sequentially. The resulting mixture was purged with nitrogen three times and then heated to 100°C with stirring for 16 hours. The reaction mixture was concentrated to dryness, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane 1:20 to 1:5) to give compound PY-8h (1.0g, yield 45.6%) as a yellow solid.

[0720] LCMS: m / z = 366.1 [M+1] + Rt = 1.108.

[0721] Step 8: Preparation of N-(3-fluoro-8-oxo-4-propyl-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-8i)

[0722] Compound PY-8h (2.5 g, 6.84 mmol) was dissolved in methanol (20 mL) and tetrahydrofuran (20 mL), and palladium on carbon (4.0 g) was added. The mixture was purged with hydrogen three times and heated to 45 °C with stirring for 4 hours. The reaction solution was filtered and concentrated directly, and the crude product was used directly in the next reaction step.

[0723] LCMS: m / z=264.1[M+1]+; Rt=1.056.

[0724] Step 9: Preparation of 8-amino-6-fluoro-5-propyl-3,4-dihydronaphthyl-1(2H)-one (PY-8j)

[0725] Compound PY-8i (1.91 g, 6.84 mmol) was dissolved in 6 M hydrochloric acid (20 mL) and ethanol (20 mL), and the mixture was heated to 60 °C and stirred for 1 hour. The reaction solution was concentrated to dryness, and the crude product was used directly in the next reaction step.

[0726] Step 10: Preparation of (S)-9-ethyl-5-fluoro-9-hydroxy-4-propyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-8B)

[0727] Under a nitrogen atmosphere, compound PY-8j (960 mg, 1.0 eq) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-3,6,10(4H)-trione (PY-8f) (Haoyuan Pharmaceutical) (1.17 g, 1.1 eq) were dispersed in toluene (25 mL), and p-toluenesulfonic acid pyridine (508 mg, 0.5 eq) was added. The mixture was heated to 120 °C and stirred for 16 hours. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 10:1) to give compound PY-8B (340 mg, yield 18%), a brown solid.

[0728] LCMS: m / z = 449.2 [M+H] + Rt = 3.313 min.

[0729] 1 H NMR (400MHz, DMSO-d) 6) δ7.72(d,J=11.4Hz,1H),7.30(s,1H),6.52(s,1H),5.43(s,2H),5.23(s,2H),3.15(q,J=5.6Hz,4H),2.81(t,J=7.7Hz,2H ), 2.08 (t, J = 5.9Hz, 2H), 1.94-1.78 (m, J = 7.1Hz, 2H), 1.58 (h, J = 7.4Hz, 2H), 0.97 (t, J = 7.3Hz, 3H), 0.88 (t, J = 7.3Hz, 3H).

[0730] Preparation Example 14: Preparation of (S)-9-ethyl-5-fluoro-9-hydroxy-4-(2-hydroxyethoxy)-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-9)

[0731]

[0732] In a 2 mL aqueous solution of compound PY-13 (20 mg), 1 equivalent of tetrabutylammonium bromide (TBAB) (15.3 mg), 0.2 equivalents of palladium chloride catalyst (1.7 mg), 250 mL of ethylene oxide, and 2.5 equivalents of potassium carbonate (16.4 mg) were added. The reaction was carried out at 25 °C for 16 hours. The reaction solution was subjected to preparative high-performance liquid chromatography (SHIMADDZU LC-20AP, Welch Xtimate C18 250×30 mm×10 μm column, water (0.225% HCOOH)-acetonitrile mobile phase, with water elution ratios ranging from 12% to 42%), and lyophilized to obtain crude white solid compound PY-9. This white solid compound was further purified by thin-layer chromatography to obtain 3.03 mg of compound PY-9.

[0733] LCMS (ESI): m / z, 467.2 [M+H] + .

[0734] 1 H NMR (400MHz, DMSO-d6) δ7.83(d,J=12.5Hz,1H),7.29(s,1H),6.51(s,1H),5.43(s,2H),5.25(s,2H),4.93(t,J=5.4Hz,1H),4.14(t,J=4. 9Hz, 2H), 3.73 (q, J = 5.1Hz, 2H), 3.16 (dt, J = 11.1, 6.1Hz, 4H), 2.02 (dt, J = 12.6, 6.6Hz, 2H), 1.86 (h, J = 7.0Hz, 2H), 0.88 (t, J = 7.3Hz, 3H); 19 F NMR (377MHz, DMSO-d6) δ-123.40.

[0735] Preparation Example 15: Preparation of (S)-9-ethyl-5-fluoro-9-hydroxy-4-(hydroxymethyl)-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-10)

[0736]

[0737] Step 1: Preparation of 8-amino-5-bromo-6-fluoro-3,4-dihydronaphthyl-1(2H)-one (PY-10b)

[0738] To a solution of compound PY-8 g (2 g, 6.66 mmol, 1 eq) in ethanol (6 mL), 6 N HCl (6 mL) was added. The reaction was stirred at 80 °C for 3 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, diluted with 20 mL of water, and the pH was adjusted to neutral with sodium bicarbonate solution. The solution was then extracted with dichloromethane (30 mL × 3). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a yellow solid crude compound 8-amino-5-bromo-6-fluoro-3,4-dihydronaphthyl-1(2H)-one (PY-10b) (1.52 g, yield: 88%; purity: 91%).

[0739] LCMS (ESI): m / z, 258 [M+1] + 260 [M+1] + .

[0740] Step 2: Preparation of (S)-4-bromo-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-10d)

[0741] To a 20 mL toluene solution of compound PY-10b (1.52 g, 5.89 mmol, 1 eq), compounds PY-8f (1.55 g, 5.89 mmol, 1 eq) and PPTS (1.48 g, 5.89 mmol, 1 eq) were added. The reaction was carried out under a nitrogen atmosphere at 120 °C and stirred for 16 hours. The residue was purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to give a yellow solid compound PY-10d (1.3 g, yield: 45%; purity: 88%).

[0742] LCMS (ESI): m / z, 485 [M+H] + 487[M+H] + .

[0743] Step 3: Preparation of (S)-9-ethyl-5-fluoro-9-hydroxy-4-(hydroxymethyl)-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-10)

[0744] To a solution of compound PY-10d (1.30 g, 2.68 mmol, 1 eq) in dioxane (5 mL), (tributyltin)methanol (2.84 g, 8.84 mmol, 3.3 eq) and Xphos Pd G2 (210.77 mg, 267.87 μmol, 0.1 eq) were added, respectively. The reaction was carried out under a nitrogen atmosphere at 90 °C with stirring for 16 hours. The mixture was concentrated under reduced pressure, diluted with water and dichloromethane (50 mL / 50 mL), and a solid precipitated. The solid was filtered, and the filter cake was collected separately. The filtrate was extracted with dichloromethane (50 mL × 3), and the organic phases were combined and washed with saturated brine (50 mL). The mixture was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue and filter cake were purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to give a yellow solid compound PY-10 (649 mg, yield: 55.5%, purity: 100%).

[0745] LCMS(ESI): m / z, 437 [M+H] + .

[0746] Preparation Example 16: Preparation of (S)-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-carboxylic acid (PY-10A) and (S)-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-10B)

[0747]

[0748] Step 1: Preparation of N-(3-fluoro-4-formyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-10-1)

[0749] At 0 °C, H₂SO₄ (4.34 g, 44.21 mmol, 5.2 eq) was added to a chloroform (100 mL) solution of N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (2 g, 8.5 mmol, 1.0 eq) and MnO₂ (11.09 g, 127.52 mmol, 15 eq). The reaction was gradually brought back to room temperature and continued for 16 hours. After filtration, the pH was adjusted to neutral with a saturated sodium bicarbonate solution, and the mixture was extracted with dichloromethane (30 mL × 3). The extract was concentrated to give a brownish-yellow crude solid (2.1 g), which was used directly in the next step without purification.

[0750] LCMS (ESI): m / z, 250.1 [M+1] + .

[0751] Step 2: Preparation of 4-acetamido-2-fluoro-5-oxo-5,6,7,8-tetrahydronaphthalene-1-carboxylic acid (PY-10-2)

[0752] At 25°C, dimethyl dibutylene (1.55 g, 22.07 mmol, 1 eq) was added to a methanol / water solution of N-(3-fluoro-4-formyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (1.1 g, 2.21 mmol, 1 eq), sodium chlorite (1.20 g, 13.24 mmol, 6 eq), and sodium dihydrogen phosphate (534 mg, 4.41 mmol, 2 eq) in a 1 / 1 (30 mL) mixture. The reaction was stirred at 50°C for 2 hours. The pH was adjusted to 8 with a saturated sodium bicarbonate aqueous solution, and the starting material was recovered by extraction with dichloromethane (30 mL × 3). The aqueous phase was then adjusted to pH 2 with 2N HCl, extracted with ethyl acetate (30 mL × 3), and concentrated to give compound PY-10-2 (324 mg, yield: 55.36%).

[0753] LCMS (ESI): m / z, 266.0 [M+1] + .

[0754] Step 3: Preparation of 4-amino-2-fluoro-5-oxo-5,6,7,8-tetrahydronaphthalene-1-carboxylic acid (PY-10-3)

[0755] The compound 4-acetamido-2-fluoro-5-oxo-5,6,7,8-tetrahydronaphthalene-1-carboxylic acid (415 mg, 1.67 mmol, 1 eq) was added to a NaOH solution (5 M, 15 mL). The reaction was stirred at 80 °C for 2 hours. After adjusting the pH to 2 with 2N dilute hydrochloric acid, the mixture was extracted with ethyl acetate (30 mL × 3). The organic phase was concentrated to give the compound PY-10-3 (320 mg, yield: 85.6%).

[0756] LCMS (ESI): m / z: 224.0 [M+1] + .

[0757] Step 4: Preparation of (S)-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-4-carboxylic acid (PY-10A) and (S)-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-10,13-dione (PY-10B)

[0758] Compound 4-amino-2-fluoro-5-oxo-5,6,7,8-tetrahydronaphthalene-1-carboxylic acid (364.64 mg, 1.43 mmol, 1 eq) was dissolved in toluene (15 mL) along with indene-3,6,10(4H)-trione (377.42 mg, 1.43 mmol, 1 eq) and PPTS (360.29 mg, 1.43 mmol, 1 eq) from (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran[3,4-f] and stirred at 120 °C for 16 hours. After removing the solvent under reduced pressure, the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol 10:1 to 5:1) to obtain a crude, brown, oily compound. This crude compound was then prepared by high performance liquid chromatography (preparative chromatograph manufacturer: Oriendo, model Lab311-DJ-R2, column: YMC-Triart Prep C18). 250×30mm×10μm. Separation was performed using a mobile phase of water (0.225% HCOOH)-acetonitrile (elution ratio of water ranging from 30% to 60%), followed by lyophilization to give compound PY-10A (18.19 g, yield: 2.82%). LCMS (ESI): m / z: 451.1 [M+1] + ; and compound PY-10B (46 mg, yield: 7.9%), LCMS (ESI): m / z: 407.1 [M+1] + .

[0759] Preparation Example 17: Preparation of (S)-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)methyl(2-hydroxyethyl)carbamate (PY-10Car)

[0760]

[0761] Step 1: Preparation of (S)-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)methyl(4-nitrophenyl)carbonate (PY-10Cara)

[0762] Compound PY-10 (8.0 mg, 0.018 mmol, 1.0 eq), bis(4-nitrobenzene) carbonate (44.6 mg, 0.147 mmol, 8.0 eq), and N,N-diisopropylethylamine (28.4 mg, 0.220 mmol, 12.0 eq) were added to a solution of N,N-dimethylformamide (2 mL) and stirred at 50 °C for 16 h. LC-MS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure and purified using a thin-layer chromatography plate developing solvent system of dichloromethane / methanol (15 / 1) to give a yellow oily compound PY-10Car-a (18 mg, excess weight not calculated in yield).

[0763] LCMS (ESI): m / z, 602.3 [M+H] + .

[0764] Step 2: Preparation of (S)-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)methyl(2-hydroxyethyl)carbamate (PY-10Car)

[0765] Compound PY-10Car-a (18.0 mg, impure), aminoethanol (1.83 mg, 0.030 mmol), and N,N-diisopropylethylamine (11.6 mg, 0.090 mmol) were added to a solution of N,N-dimethylformamide (1 mL) at room temperature and stirred at 25 °C for 0.5 h. LC-MS showed that the reaction was complete. The reaction solution was prepared by high performance liquid chromatography (preparative chromatograph manufacturer: Luna, model: Lab311-ISCO-R4. Column: Phenomenex Luna C18 250×50 mm×10 μm. Mobile phase: water (0.225% formic acid)-acetonitrile, with water elution ratios ranging from 12% to 42%). The eluent was lyophilized to give a white solid product PY-10Car (4.14 mg, 7.56 μmol, two-step yield: 42%).

[0766] LCMS(ESI): m / z, 524.2 [M+H] + .

[0767] 1H NMR (400MHz, DMSO-d) 6 )δ7.79(d,J=11.2Hz,1H),7.32(s,1H),7.19(t,J=5.6Hz,1H),6.54(s,1H),5.44(s,2H),5.27(d,J=4.4Hz,3H),4.63(t,J=5.6H z,1H),3.30–3.23(m,5H),3.18(s,2H),3.04(q,J=6.0Hz,2H),2.08(s,2H),1.86(dt,J=15.2,7.2Hz,2H),0.87(t,J=7.2Hz,3H).

[0768] Preparation Example 18: Preparation of (S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-11)

[0769]

[0770] Step 1: Preparation of 8-amino-6-fluoro-5-methoxy-3,4-dihydronaphthyl-1(2H)-one (PY-111)

[0771] At 100 °C, compound N-(3-fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-88) (100 mg) was stirred in HCl (7 N, 10 mL) for 2 hours. The reaction was quenched by slowly adding saturated NaHCO3 solution (10 mL) and the pH was adjusted to neutral (pH 7). The mixture was extracted with ethyl acetate (3 × 30 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a yellow solid compound PY-111 (66 mg, 79%).

[0772] LCMS(ESI): m / z, 210.1 [M+H] + .

[0773] Step 2: Preparation of (S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-11)

[0774] PPTS (0.67 eq, 16 mg) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-3,6,10(4H)-trione (1 eq, 25 mg) were added to a 10 mL toluene solvent containing 20 mg of compound 8-amino-6-fluoro-5-methoxy-3,4-dihydro-1H-pyrano[3,4-f]indoleazine-3,6,10(4H)-trione and reacted at 140 °C for 16 h. Toluene was removed by vacuum concentration, and 5 mL of DMF was added. The mixture was then purified by high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50 mm×10 μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 45% to 85%) to give a white solid compound PY-11 (8.4 mg, yield 20%).

[0775] LCMS (ESI): m / z, 437.2 [M+H] + .

[0776] 1 H NMR (400MHz, DMSO-d) 6 )δ7.85(d,J=12.5Hz,1H),7.30(s,1H),6.52(s,1H),5.43(s,2H),5.25(s,2H),3.94(s,3H),3.14(dt ,J=11.2,6.0Hz,4H),2.05(q,J=6.4,5.8Hz,2H),1.88(dq,J=14.4,7.2Hz,2H),0.88(t,J=7.2Hz,3H).

[0777] Preparation Example 19: Preparation of (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-10,13-dione and (1R,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-12A and PY-12B)

[0778]

[0779] Step 1: Preparation of (E)-N-(3-fluoro-7-(hydroxyimino)-4-methoxy-8-oxy-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-121)

[0780] At 0 °C, 3 equivalents of potassium tert-butoxide tetrahydrofuran solution and tert-butyl nitrite were added to 10 mL of a tetrahydrofuran solution of compound N-(3-fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-88) (200 mg). The mixture was stirred at 0 °C for 1 hour, diluted with 30 mL of water, and extracted three times with 10 mL of ethyl acetate each time. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50 mm×10 μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 45% to 85%) to give 180 mg of yellow solid compound PY-121.

[0781] LCMS (ESI): m / z, 281.1 [M+1] + .

[0782] Step 2: Preparation of N,N'-(3-fluoro-4-methoxy-8-oxy-5,6,7,8-tetrahydronaphthalene-1,7-diacyl)diacetamide (PY-122)

[0783] At room temperature, 1 mL of acetic anhydride and 2 mL of acetic acid were added to compound (E)-N-(3-fluoro-7-(hydroxyimino)-4-methoxy-8-oxy-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-121) (180 mg), along with zinc powder (35 mg). The mixture was stirred at room temperature for 4 hours. The reaction solution was concentrated under reduced pressure, diluted with 100 mL of water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative high-performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50 mm×10 μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 45% to 85%) to give 150 mg of white solid compound PY-122.

[0784] LCMS (ESI): m / z, 309.1 [M+1] + .

[0785] Step 3: Preparation of N-(8-amino-6-fluoro-5-methoxy-1-oxy-1,2,3,4-tetrahydronaphth-2-yl)acetamide (PY-123)

[0786] The compound N,N'-(3-fluoro-4-methoxy-8-oxy-5,6,7,8-tetrahydronaphthalene-1,7-diacyl)diacetamide (PY-122) (150 mg) was dissolved in 2 mL of 6N HCl and 2 mL of EtOH, and stirred at 60 °C for 3 hours. The reaction solution was concentrated under reduced pressure to obtain 115 mg of crude product, which was directly used in the next step of the reaction.

[0787] LCMS (ESI): m / z, 267.1 [M+1] + .

[0788] Step 4: Preparation of N-(9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-1-yl)acetamide (PY-124)

[0789] To a toluene solution of N-(8-amino-6-fluoro-5-methoxy-1-oxy-1,2,3,4-tetrahydronaphth-2-yl)acetamide (PY-123) (80 mg), 1 equivalent of (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-3,6,10(4H)-trione (Haoyuan Pharmaceutical) (92 mg) and 0.5 equivalents of PPTS (24 mg) were added, and the mixture was stirred at 120 °C for 12 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50 mm×10 μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 45% to 85%) to obtain 55 mg of a yellow solid mixture, PY-124.

[0790] LCMS (ESI): m / z, 494.2 [M+1] + .

[0791] Step 5: Preparation of (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-10,13-dione and (1R,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-12A and PY-12B)

[0792] N-(9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12Hbenzo[de]pyran[3',4:6,7]indolazine[1,2-b]quinoline-1-yl)acetamide (PY-124) (55 mg) was dissolved in 3 mL of 6N hydrochloric acid and stirred at 85 °C for 5 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50 mm×10 μm; mobile phase: water (0.225% HCOOH)-acetonitrile, eluting from 45% to 85%). The residues were lyophilized to give compounds PY-12A (4.55 mg) and PY-12B (6.24 mg), respectively.

[0793] PY-12A (LCMS retention time 0.651 min):

[0794] LCMS (ESI): m / z, 452.2 [M+1] + Rt = 0.651.

[0795] 1 H NMR (400MHz, DMSO-d) 6 )δ7.87(d,J=12.4Hz,1H),7.30(s,1H),6.53(s,1H),5.62(d,J=19.2Hz,1H),5.46–5.29(m,3H),4.38(s ,1H),3.95(s,3H),3.28–3.03(m,4H),2.08(s,2H),1.87(dd,J=9.2,7.2Hz,2H),0.87(t,J=7.2Hz,3H).

[0796] PY-12B (LCMS retention time 0.677 min):

[0797] LCMS (ESI): m / z, 452.2 [M+1] + Rt = 0.677.

[0798] 1 H NMR (400MHz, DMSO-d) 6)δ8.66(s,2H),8.01(d,J=12.4Hz,1H),7.34(s,1H),6.57(s,1H),5.90(d,J=19.2Hz,1H),5.60-5.30(m,3H),5.08(s,1H), 4.00(d,J=1.2Hz,3H), 3.15(t,J=12.8Hz,3H), 2.12(t,J=13.6Hz,1H), 1.97-1.76(m,J=7.2Hz,2H), 0.87(t,J=7.2Hz,3H).

[0799] Preparation Example 20: Preparation of (S)-9-ethyl-5-fluoro-4,9-dihydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4:6,7]indoleazino[1,2-b]quinoline-10,13-dione (PY-13)

[0800]

[0801] Step 1: Preparation of N-(3-fluoro-4-hydroxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-131)

[0802] Three equivalents of AlCl3 were added to a DCM solution of N-(3-fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-88) (200 mg), and the reaction was carried out at 60 °C for 2 hours. The reaction was quenched by slowly adding saturated NaHCO3 solution (10 mL) and the pH was adjusted to 3–4. The mixture was extracted with ethyl acetate (3 × 30 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a yellow solid compound PY-131 (130 mg, 68%).

[0803] LCMS (ESI): m / z, 238.0 [M+H] + , 260.1[M+Na].

[0804] Step 2: Preparation of 8-amino-6-fluoro-5-hydroxy-3,4-dihydronaphthyl-1(2H)-one (PY-132)

[0805] At 100 °C, a solution of N-(3-fluoro-4-hydroxy-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-131) (70 mg) in HCl (12N, 3.5 mL) was stirred for 16 hours. The reaction was quenched by slowly adding saturated NaHCO3 solution (10 mL), the pH was adjusted to 3–4, and the mixture was extracted with ethyl acetate (30 × 3 mL). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. 5 mL of MeCN was added, and the mixture was separated by preparative high-performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250 × 50 mm × 10 μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 45% to 85%) to obtain a yellow solid compound PY-132 (12 mg, 20%).

[0806] LCMS (ESI): m / z, 196.1 [M+H] + .

[0807] Step 3: Preparation of (S)-9-ethyl-5-fluoro-4,9-dihydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4:6,7]indolazido[1,2-b]quinoline-10,13-dione (PY-13)

[0808] To a toluene (10 mL) solution of 15 mg of 8-amino-6-fluoro-5-hydroxy-3,4-dihydronaphthyl-1(2H)-one (PY-132), PPTS (0.67 eq, 12.9 mg) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-3,6,10(4H)-trione (1 eq, 20.2 mg) were added, and the mixture was reacted at 140 °C for 16 h. Toluene was removed under reduced pressure, and 5 mL of DMF was added. The mixture was purified by high-performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250 × 50 mm × 10 μm; mobile phase: water (0.225% HCOOH)-acetonitrile, eluting from 45% to 85%) to give a white solid compound PY-13 (3.2 mg).

[0809] LCMS (ESI): m / z, 423.1 [M+H] + .

[0810] 1 H NMR (400MHz, DMSO-d) 6)δ7.77(d,J=11.9Hz,1H),7.26(s,1H),6.50(s,1H),5.42(s,2H),5.22(s,2H),3.11(t,J=6.1Hz,2H ), 3.03 (t, J = 6.2Hz, 2H), 2.01 (q, J = 6.2Hz, 2H), 1.87 (dq, J = 14.5, 7.1Hz, 2H), 0.87 (t, J = 7.3Hz, 3H).

[0811] Preparation Example 21: Preparation of (S)-4,11-diethyl-8,10-difluoro-4,9-dihydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolazido[1,2-b]quinoline-3,14(4H)-dione (PY-14)

[0812]

[0813] Step 1: Preparation of 1-(2,4-difluoro-3-methoxyphenyl)prop-1-one (PY-14-c)

[0814] At -70°C, n-BuLi (1.07 g, 10.41 mL, 16.65 mmol, 1.2 eq, 1.60 M) was slowly added dropwise to 20 mL of anhydrous THF solvent containing compound 1,3-difluoro-2-methoxybenzene (PY-14-a) (2 g, 13.88 mmol, 1 eq). The mixture was stirred at the same temperature for half an hour. Subsequently, compound N-methoxy-N-methylpropionamide (PY-14-b) (4.55 g, 34.69 mmol, 2.5 eq) was added, and the mixture was stirred at -70°C for another half hour. The cryogenic bath was then removed, and the temperature was raised to 25°C and stirred for 16 hours. LCMS showed product formation. The reaction was quenched at room temperature with 5 mL of saturated ammonium chloride solution, and the organic phase was extracted with 3 × 10 mL of ethyl acetate. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 10:1) to give a pale yellow oily title compound (1.6 g, yield: 54%).

[0815] LCMS (ESI): m / z, 214.9 [M+H] + .

[0816] Step 2: Preparation of 1-(2,4-difluoro-3-methoxy-6-nitrophenyl)prop-1-one (PY-14-d)

[0817] At -40°C, fuming nitric acid (699.74 mg, 95%, 474.82 μL, 10.55 mmol, 0.96 eq) was slowly added dropwise to 22 mL of H₂SO₄ solvent containing 2.20 g (10.99 mmol, 1 eq) of 1-(2,4-difluoro-3-methoxyphenyl)prop-1-one (1.2 g, yield: 34%). The mixture was stirred at this temperature for 1 hour. The reaction mixture was poured into ice water, and the reaction flask was washed with 10 mL of anhydrous ethanol. The organic phase was extracted with 100 mL of DCM, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 10:1). The residue was dried to give a pale yellow oily compound, 1-(2,4-difluoro-3-methoxy-6-nitrophenyl)prop-1-one (1.2 g, yield: 34%).

[0818] LCMS(ESI): m / z, 246.1 [M+H] + .

[0819] 1 H NMR (400MHz, DMSO-d6) δ8.24–8.13(m,1H),4.16(t,J=2.1Hz,3H),2.89–2.81(m,2H),1.14(t,J=7.1Hz,3H).

[0820] Step 3: Preparation of 1-(6-amino-2,4-difluoro-3-methoxyphenyl)prop-1-one (PY-14-e)

[0821] To 10 mL of anhydrous ethanol solvent containing 1-(2,4-difluoro-3-methoxy-6-nitrophenyl)prop-1-one (PY-14-d) (200 mg, 815.73 μmol, 1 eq), iron powder (250.57 mg, 31.88 μL, 4.49 mmol, 5.5 eq), water (339.85 mg, 339.85 μL, 18.86 mmol, 23.12 eq), and concentrated hydrochloric acid (16.36 mg, 37.39 μL, 448.65 μmol, 0.55 eq, 12 M) was added. The reaction was carried out under a nitrogen atmosphere and stirred at 80 °C for 16 hours. After the reaction solution was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 10:1) to give a white solid compound 1-(6-amino-2,4-difluoro-3-methoxyphenyl)prop-1-one (141 mg, yield: 80%).

[0822] LCMS (ESI): m / z, 216.1 [M+H] + .

[0823] Step 4: Preparation of 1-(6-amino-2,4-difluoro-3-hydroxyphenyl)prop-1-one (PY-14-f)

[0824] To 10 mL of DCM solvent containing 300 mg (1.39 mmol, 1 eq) of 1-(6-amino-2,4-difluoro-3-methoxyphenyl)prop-1-one (PY-14-e) and 3 equivalents of AlCl3 (557.61 mg, 4.18 mmol, 3 eq), the reaction was stirred at 70 °C for 4 hours under a nitrogen atmosphere. The reaction was quenched with 100 mL of saturated ammonium chloride solution, and the organic phase was extracted with 2 × 100 mL of DCM. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a yellow crude solid of 1-(6-amino-2,4-difluoro-3-hydroxyphenyl)prop-1-one (165 mg, crude product yield not considered).

[0825] LCMS (ESI): m / z, 202.0 [M+H] + .

[0826] 1 H NMR (400MHz, DMSO-d) 6 )δ9.06(s,1H),6.42(dd,J=13.0,2.1Hz,1H),3.45(s,2H),2.87-2.82(m,2H),1.05(t,J=7.2Hz,3H).

[0827] Step 5: Preparation of (S)-4,11-diethyl-8,10-difluoro-4,9-dihydroxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3,14(4H)-dione (PY-14)

[0828] To 10 mL of toluene solvent containing 1-(6-amino-2,4-difluoro-3-hydroxyphenyl)prop-1-one (PY-14-f) (30 mg, 149.13 μmol, 1 eq), PPTS (25.11 mg, 99.92 μmol, 0.67 eq) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-3,6,10(4H)-trione (Haoyuan Pharmaceutical) (30 mg, 149.13 μmol, 1 eq) were added, and the reaction was stirred at 130 °C for 16 hours under a nitrogen atmosphere. The reactants were purified by preparative high-performance liquid chromatography (SHIMADDZU, LC-20AP; YMC-Triart Prep C18 column, 250×30mm×10μm; mobile phase: water (0.225% HCOOH)-acetonitrile, with water elution rates ranging from 12% to 42%), and lyophilized to give a yellow solid (35.55 mg, 55.65%).

[0829] LCMS (ESI): m / z, 429.1 [M+H] + .

[0830] 1 H NMR (400MHz, DMSO-d) 6 )δ7.84(d,J=11.4Hz,1H),7.26(s,1H),6.52(s,1H),5.43(s,2H),5.31(s,2H),3.22– 3.15 (m, 2H), 1.86 (hept, J = 7.1Hz, 2H), 1.32 (t, J = 7.4Hz, 3H), 0.87 (t, J = 7.3Hz, 3H).

[0831] Preparation Example 22: Preparation of (S)-4-(aminomethyl)-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-16)

[0832]

[0833] Step 1: Preparation of N-(4-((benzylamino)methyl)-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-16d)

[0834] To a solution of N-(3-fluoro-4-formyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (1.22 g, 8.5 mmol, 1 eq) and benzylamine (642.29 μL) in DCM (110 mL), NaBH(OAc)3 (2.06 g, 9.79 mmol, 2 eq) was added. The reaction was stirred at room temperature for 12 hours. The reaction was quenched with water (100 mL), extracted three times with DCM (50 mL), the combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether: 1 / 5-1 / 2) to give a brown oily substance N-(4-((benzylamino)methyl)-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-16d) (499.5 mg, yield: 30%).

[0835] LCMS(ESI): m / z, 341[M+H] + .

[0836] Step 2: Preparation of 8-amino-5-((benzylamino)methyl)-6-fluoro-3,4-dihydronaphthyl-1(2H)-one (PY-16e)

[0837] To compound N-(4-((benzylamino)methyl)-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (499.5 mg, 1.47 mmol, 1 eq), 6N HCl (3 mL) and EtOH (3 mL) were added. The reaction was stirred at 60 °C for 3 hours and concentrated under reduced pressure to obtain a brown oily crude product 8-amino-5-((benzylamino)methyl)-6-fluoro-3,4-dihydronaphth-1(2H)-one (PY-16e) (517.9 mg).

[0838] LCMS(ESI): m / z, 341[M+H] + .

[0839] Step 3: Preparation of (S)-4-((benzylamino)methyl)-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-16g)

[0840] To a toluene (15 mL) solution of compound 8-amino-5-((benzylamino)methyl)-6-fluoro-3,4-dihydronaphthyl-1(2H)-one (517.9 mg, 1.22 mmol, 1 eq) and compound (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran[3,4-f] containing indene-3,6,10(4H)-trione (Haoyuan Pharmaceutical) (319.87 mg, 1.22 mmol, 1 eq), PPTS (305.35 mg, 1.22 mmol, 1 eq) was added, and the reaction was stirred at 120 °C for 12 hours. The reaction was quenched with water (100 mL), followed by extraction three times with DCM (50 mL). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol: 12 / 1–11 / 1) to give a brown oily substance (S)-4-((benzylamino)methyl)-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-16g) (466mg, yield: 73%).

[0841] LCMS (ESI): m / z, 526 [M+H] + .

[0842] Step 4: Preparation of (S)-4-(aminomethyl)-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-16)

[0843] Under a hydrogen atmosphere, Pd / C (100 mg, 939.67 μmol, 2.27 eq) and Pd (…) were added to a MeOH (10 mL) solution of compound (S)-4-((benzylamino)methyl)-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (218 mg, 414.78 μmol, 1 eq). OH)2 (100 mg, 712.1 μmol, 1.72 eq) was reacted with the mixture at room temperature and stirred for 5 hours. After filtration through diatomaceous earth and concentration, a brown solid product (S)-4-(aminomethyl)-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-16) (296.6 mg, yield: 82%) was obtained.

[0844] LCMS (ESI): m / z, 436 [M+H] + .

[0845] 1 H NMR (400MHz, DMSO-d) 6 )δ8.27(s,1H),7.75(d,J=11.2Hz,1H),7.31(s,1H),6.54(s,1H),5.44(s,2H),5.24(s,2H),4.00(s,2H),3.25( t,J=5.8Hz,2H),3.16(t,J=6.2Hz,2H),2.09(t,J=6.2Hz,2H),1.87(hept,J=7.0Hz,2H),0.88(t,J=7.2Hz,3H); 19 F NMR (377MHz, DMSO-d6) δ-113.60.

[0846] Preparation Example 23: Preparation of 2-hydroxyethyl (S)-((9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)methyl)carbamate (PY-16Car)

[0847]

[0848] Step 1: Preparation of 2-((tert-butyldimethylsilyl)oxy)ethyl(4-nitrophenyl)carboxylic acid ester (PY-16Carb)

[0849] At 0 °C, tert-butyldimethylhydroxyethoxysilane (2.0 g, 11.34 mmol, 1.0 eq) was added to a solution of bis(4-nitrobenzene) carbonate (NPC) (5.18 g, 17.01 mmol, 1.5 eq) and N,N-diisopropylethylamine (DIPEA, 4.40 g, 34.03 mmol, 3.0 eq) in tetrahydrofuran (120 mL), and the mixture was stirred at 25 °C for 16 h. The reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography with ethyl acetate / petroleum ether (0-10%) as the eluent to give a yellow oily compound PY-16Carb (2.70 g, 7.91 mmol, yield: 70%).

[0850] 1 H NMR (400MHz, CHCl3-d) δ 8.28 (d, J = 9.2 Hz, 2H), 7.38 (d, J = 9.2 Hz, 2H), 4.36 (dd, J = 5.6, 4.1 Hz, 2H), 3.91 (dd, J = 5.6, 4.1 Hz, 2H), 0.91 (s, 9H), 0.10 (s, 6H).

[0851] Step 2: Preparation of 2-((tert-butyldimethylsilyl)oxy)ethyl(S)-((9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)methyl)carbamate (PY-16Carc)

[0852] At room temperature, compound 2-((tert-butyldimethylsilyl)oxy)ethyl(4-nitrophenyl)carboxylic acid ester (35.75 mg, 0.105 mmol, 1.2 eq), compound (S)-4-(aminomethyl)-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7] indo[1,2-b]quinoline-10,13-dione (38.0 mg, 0.087 mmol, 1.0 eq), and N,N-diisopropylethylamine (33.84 mg, 0.262 mmol, 3.0 eq) were added to a solution of N,N-dimethylformamide (2 mL) and stirred at 25 °C for 1 hour. The reaction solution was used directly for the next step.

[0853] LCMS (ESI): m / z, 638.3 [M+H] + .

[0854] Step 3: Preparation of 2-hydroxyethyl (S)-((9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)methyl)carbamate (PY-16Car)

[0855] MeOH (2 mL) and 4N hydrochloric acid were added to the reaction solution from the previous step, and the mixture was stirred at 25 °C for 1 hour. The reaction solution was then subjected to high performance liquid chromatography (HPLC) (preparative chromatograph manufacturer: Luna, model: Lab311-ISCO-R4; column: Phenomenex Luna C18 250×50 mm×10 μm; mobile phase: water (0.225% formic acid)-acetonitrile; elution ratio of water from 12% to 42%). The eluent was lyophilized to give a white solid product, PY-16Car (20.4 mg, 0.039 mmol, two-step yield: 45%).

[0856] LCMS (ESI): m / z, 524.2 [M+H] + .

[0857] 1 H NMR(400MHz,DMSO-d6)δ7.73(d,J=11.2Hz,1H),7.65(t,J=5.6Hz,1H),7.31(s,1H ),6.52(s,1H),5.43(s,2H),5.24(s,2H),4.71(t,J=5.2Hz,1H),4.43(d,J=5.2Hz, 2H),3.96(t,J=5.2Hz,2H),3.52(q,J=5.2Hz,2H),3.24(t,J=6.0Hz,2H),3.15(t, J=6.4Hz,2H),2.12–2.02(m,2H),1.87(hept,J=7.2Hz,2H),0.88(t,J=7.2Hz,3H).

[0858] Preparation Example 24: Preparation of (S)-4-(2-aminoethoxy)-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolazido[1,2-b]quinoline-10,13-dione (PY-17)

[0859]

[0860]

[0861] Step 1: Preparation of (S)-(2-((9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)oxy)ethyl)tert-butyl carbamate (PY-17-a)

[0862] Potassium carbonate (10.7 mg, 77.26 μmol, 2.0 eq) was added to a DMF solvent containing compound PY-13 (41 mg, 38.63 μmol, 1.0 eq), and the mixture was stirred at 25 °C for half an hour. Subsequently, tert-butyl (2-bromoethyl)carbamate (21.6 mg, 91.6 μmol, 2.5 eq) was added at the same temperature, and the mixture was stirred at 25 °C for 16 hours. The reactants were purified by preparative high-performance liquid chromatography (SHIMADDZU LC-20AP, YMC-Triart Prep C18 250×30 mm×10 μm column, water (0.225% HCOOH)-acetonitrile mobile phase, with water elution ratios ranging from 12% to 42%), and lyophilized to give a gray solid compound PY-17-a (5 mg, yield: 23%).

[0863] LCMS(ESI): m / z, 566.2 [M+H] + .

[0864] Step 2: Preparation of (S)-4-(2-aminoethoxy)-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-17): Under ice-water bath conditions, 0.5 mL of LTFA was slowly added dropwise to 5 mL of DCM solvent containing compound PY-17-a (7 mg, 12.38 μmol, 1.0 eq). The mixture was then naturally heated to 25 °C and stirred for 16 hours. The reactants were purified by preparative high-performance liquid chromatography (SHIMADDZU, LC-20AP; YMC-Triart Prep C18 column, 250×30mm×10μm; mobile phase: water (0.225% HCOOH)-acetonitrile, with water elution ratios ranging from 12% to 42%), and lyophilized to give a white solid compound PY-17 (1.97 mg, yield: 34%).

[0865] LCMS (ESI): m / z, 466.2 [M+H] + .

[0866] 1 H NMR (400MHz, DMSO-d)6 )δ8.06(s,3H),7.90(d,J=12.3Hz,1H),7.30(s,1H),6.52(s,1H),5.44(s,2H),5.27(s,2H),4.27(t,J= 5.1Hz, 2H), 3.17 (t, J = 6.1Hz, 5H), 2.06 (t, J = 6.2Hz, 2H), 1.87 (p, J = 6.8Hz, 2H), 0.88 (t, J = 7.3Hz, 3H).

[0867] Preparation Example 25: Preparation of (S)-9-ethyl-5-fluoro-9-hydroxy-4-(2-hydroxyethyl)-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-18)

[0868]

[0869]

[0870] Step 1: Preparation of N-(4-(2-(benzyloxy)ethyl)-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-18c)

[0871] Under a nitrogen atmosphere, 3.3 equivalents of potassium trifluoroborate (532 mg), 3 equivalents of potassium carbonate, 30 mg of palladium acetate, and 88 mg of S-Phos ligand were added sequentially to 12.5 mL of a mixed solvent of toluene and water (4:1, v / v) containing 200 mg of N-(4-bromo-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-18c) in a nitrogen atmosphere. The reaction was stirred at 100 °C for 16 hours. After concentration under reduced pressure, the residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane 1:20 to 1:8) to give 140 mg of white solid N-(4-(2-(benzyloxy)ethyl)-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-18c) (yield: 59%).

[0872] LCMS (ESI): m / z, 356 [M+1] + .

[0873] 1H NMR (400MHz, DMSO-d6) δ12.26(s,1H),8.29(d,J=13.3Hz,1H),5.10(t,J=5.3Hz,1H),4.50(dd,J=5.4,J =2.2Hz, 2H), 3.10 (t, J = 6.2Hz, 2H), 2.67 (dd, J = 7.3, J = 5.8Hz, 2H), 2.16 (s, 3H), 1.99 (p, J = 6.3Hz, 2H).

[0874] Step 2: Preparation of N-(3-fluoro-4-(2-hydroxyethyl)-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-18d)

[0875] To 10 mL of methanol solvent containing 140 mg of N-(4-(2-(benzyloxy)ethyl)-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide, 0.25 equivalents of palladium on carbon catalyst and 0.25 equivalents of Pd(OH)₂ catalyst were added, and the reaction was carried out at 25 °C for 16 hours under a hydrogen atmosphere. After filtering out the catalyst, the product was concentrated and dried under reduced pressure to obtain a pale white solid crude product, N-(3-fluoro-4-(2-hydroxyethyl)-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-18d) (100 mg, crude product yield negligible).

[0876] LCMS (ESI): m / z, 266 [M+H] + 288[M+Na] + .

[0877] Step 3: Preparation of 8-amino-6-fluoro-5-(2-hydroxyethyl)-3,4-dihydronaphthyl-1(2H)-one (PY-18e)

[0878] 80 mg of N-(3-fluoro-4-(2-hydroxyethyl)-8-oxo-5,6,7,8-tetrahydronaphthyl-1-yl)acetamide was added to 4 mL of 6N HCl and reacted at 40 °C for 3 hours. The reaction solution was cooled to room temperature, diluted with water, and the pH was adjusted to 3-5 with 2N dilute hydrochloric acid. The organic phase was then extracted with ethyl acetate, washed with saturated brine, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to give 30 mg of a yellow solid compound 8-amino-6-fluoro-5-(2-hydroxyethyl)-3,4-dihydronaphthyl-1(2H)-one (PY-18e) (yield: 37%).

[0879] LCMS(ESI): m / z, 224 [M+H] + .

[0880] Step 4: Preparation of (S)-9-ethyl-5-fluoro-9-hydroxy-4-(2-hydroxyethyl)-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-18)

[0881] Under a nitrogen atmosphere, 0.67 equivalents of PPTS (18.8 mg) and 1 equivalent of the nitrogen-indene-3,6,10(4H)-trione (29.4 mg) from compound (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran[3,4-f] were added to 5 ml of toluene solvent (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran[3,4-f] (Haoyuan Pharmaceutical). The reaction was stirred at 130 °C for 16 hours. The solution was prepared by high performance liquid chromatography (SIMADZU, LC-20AP, Synergi Max-RP column). 280×30mm×10μm. Separation was performed using a mobile phase of water (0.225% HCOOH)-acetonitrile (elution ratios of water ranging from 12% to 42%), followed by lyophilization to yield 6.0 mg of a yellow solid (S)-9-ethyl-5-fluoro-9-hydroxy-4-(2-hydroxyethyl)-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-18) (yield: 12%).

[0882] LCMS (ESI): m / z, 451 [M+H] + .

[0883] 1 H NMR (400MHz, DMSO-d) 6 )δ7.71(dd,J=11.3,6.3Hz,1H),7.33–7.28(m,1H),6.54(s,1H),5.43(s,2H),5.23(d,J=11.2Hz,2H),4.88(t,J=5.3Hz,1H),3 .61(d,J=6.5Hz,2H),3.20–3.12(m,4H),3.01(d,J=6.6Hz,2H),2.07(s,2H),1.87(hept,J=7.0Hz,2H),0.88(t,J=7.3Hz,3H). 19 F NMR (377MHz, DMSO-d6) δ-112.5.

[0884] Preparation Example 26: Preparation of (S)-4-(3-aminopropyl)-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-19)

[0885]

[0886] Step 1: Preparation of a mixed intermediate of (S,E)-(3-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolaz[1,2-b]quinoline-4-yl)allyl)tert-butyl carbamate (PY-19c) and (S,E)-(3-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolaz[1,2-b]quinoline-4-yl)propyl-1-en-1-yl)tert-butyl carbamate (PY-19c')

[0887] To 5 mL of toluene solvent containing 20 mg of azido[1,2-b]quinoline-10,13-dione in (S)-4-bromo-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7], 0.1 equivalents of bis(tert-butylphosphine)palladium (2.1 mg), 0.2 equivalents of tri(o-tolyl)phosphine (2.5 mg), 6 equivalents of DIPEA (41 μL), and 2.5 equivalents of allyl carbamate tert-butyl ester (17.8 mg) were added. The reaction mixture was stirred at 120 °C for 16 hours. The reaction solution was concentrated and purified by silica gel column chromatography (eluent: dichloromethane / methanol 20:1 to 10:1). The purified solution was then concentrated under reduced pressure to obtain... 10 mg of compound (S,E)-(3-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinolin-4-yl)allyl)tert-butyl carbamate (PY-19c) and (S,E)-(3-( A mixture of 9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)propyl-1-en-1-yl)tert-butyl carbamate (PY-19c') (yield: 43%; purity: 77%).

[0888] Step 2: Preparation of (S)-(3-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)propyl)tert-butyl carbamate (PY-19d)

[0889] The mixture (S,E)-(3-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolaz[1,2-b]quinoline-4-yl)allyl)tert-butyl carbamate (PY-19c) and (S,E)-(3-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolaz[1,2-b]quinoline-4-yl)prop-1-en-1-yl)tert-butyl carbamate (PY-19c') In 5 mL of methanol solvent containing 30 mg of Pd, 0.25 equivalents of Pd / C (14.2 mg) and 0.25 equivalents of Pd(OH)2 (9.4 mg) were added respectively. The reaction was carried out under a hydrogen atmosphere at 25 °C and stirred for 16 hours. After filtration, the reaction solution was concentrated under reduced pressure to obtain 19.6 mg of crude compound (S)-(3-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)propyl) tert-butyl carbamate (PY-19d) (yield: 65%), which was directly used in the next step of the reaction.

[0890] LCMS (ESI): m / z, 564.3 [M+1] + .

[0891] Step 3: Preparation of (S)-4-(3-aminopropyl)-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-19)

[0892] Under ice-water bath conditions, 0.8 mL of TFA was slowly added to 2.4 mL of dichloromethane solvent containing compound (S)-(3-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)propyl)carbamate (16.3 mg, purity: 82%). The reaction was then naturally heated to 25 °C and stirred for 16 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol 20:1 to 8:1) to obtain a crude product. This crude product was then purified by preparative high-performance liquid chromatography (SHIMADZU LC-20AP, Welch Xtimate C18 250×50mm×10μm column, water (0.225% FA)-ACN mobile phase, with water elution ratios ranging from 12% to 42%). The purified product was lyophilized to give 2.53 mg of compound (S)-4-(3-aminopropyl)-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-19) (yield: 21%).

[0893] LCMS (ESI): m / z, 473.2 [M+H] + .

[0894] 1 H NMR (400MHz, DMSO-d6)δ 1 H NMR (400MHz, DMSO-d6) δ8.39(s,1H),7.75(d,J=11.4Hz,1H),7.30(s,1H),6.53(s,1H),5.43(s,2H),5.25(s,2H),3.16(d, J=6.5Hz,6H),2.88(s,2H),2.82(s,2H),2.08(t,J=6.2Hz,2H),1.87(p,J=7.0Hz,2H),1.76(s,2H),0.88(t,J=7.3Hz,3H); 19 F NMR(377MHz,DMSO-d6)δ-113.12.

[0895] Preparation Example 27: Preparation of (S)-4-(2-aminoethyl)-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indoleazino[1,2-b]quinoline-10,13-dione (PY-20)

[0896]

[0897] Step 1: Preparation of (S)-(2-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)ethyl)carbamate (PY-20a)

[0898] Compound PY-10d (90.91 mg, 66%, 123.63 μmol, 1 eq) was dissolved in dioxane (4 mL) / water (1 mL), and then potassium benzyloxycarbonylaminoethyl trifluoroborate (148.91 mg, 494.53 μmol, 4 eq), tri-o-toluenephosphide (18.81 mg, 61.82 μmol, 0.5 eq) and potassium phosphate (78.73 mg, 30.71 μL, 370.90 μmol, 3 eq) were added. The reaction mixture was stirred at 90 °C for 12 hours under a nitrogen atmosphere. The reaction solution was concentrated under reduced pressure, filtered, and the residue was purified by preparative high-performance liquid chromatography (HPLC) using Oriendo BRIX-2860 (R1, 4, 5, 6) column (Phenomenex Luna C18 250×50mm×10μm, mobile phase: water (0.225% HCOOH)-acetonitrile, with water elution ratios ranging from 30% to 60%) to obtain a white solid product, PY-20a (10 mg, yield: 13.86%).

[0899] LCMS (ESI): m / z, 584.2 [M+H] + .

[0900] Step 2: Preparation of (S)-4-(2-aminoethyl)-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-20)

[0901] Compound PY-20a (10 mg, 17.13 μmol, 1 eq) was dissolved in MeCN (0.5 mL), and TMSI (56 mg, 40 μL, 279.87 μmol, 16.334 eq) was added to the solution. The solution was stirred at 25 °C for 12 hours. The reaction solution was purified by preparative high-performance liquid chromatography (Shimadzu LC-20AP, YMC-Triart Prep C18 250×30 mm×10 μm column, mobile phase: water (0.225% HCOOH)-acetonitrile, with water elution ratios ranging from 3% to 33%) to give a white solid product (5.16 mg, yield: 67%, purity: 97.64%).

[0902] LCMS (ESI): m / z, 450.2 [M+H] + .

[0903] Preparation Example 28: Preparation of (S)-4-ethyl-8-fluoro-4,9-dihydroxy-11-propyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolazido[1,2-b]quinoline-3,14(4H)-dione (PY-21-A)

[0904]

[0905] Step 1: Preparation of 1-(2-amino-4-fluoro-5-methoxyphenyl)but-1-one (PY-21-Ab)

[0906] Under ice-water bath conditions, BCl3 (1 eq, 830.04 mg, 7.08 mmol) was added to 10 mL of anhydrous benzene solvent, followed by a solution of 3-fluoro-4-methoxyaniline (1 eq, 1 g, 7.08 mmol) dissolved in 20 mL of anhydrous benzene solvent. Under a nitrogen atmosphere, nitrile butyronitrile (2 eq, 979.24 mg, 14.17 mmol, 1.23 mL) and AlCl3 (1.1053 eq, 1.04 g, 1.04 mol) were added sequentially, and the reaction was carried out at 100 °C for 16 hours. After cooling in an ice-water bath, 50 mL of 2 M hydrochloric acid was slowly added, and the mixture was stirred at 80 °C for another hour. The reaction was quenched with 100 mL of water under ice-water bath conditions, and the organic phase was extracted with 2 × 100 mL of ethyl acetate. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 5:1) to give a pale yellow solid compound PY-21-Ab (440 mg, yield: 29%).

[0907] LCMS (ESI): m / z, 212.1 [M+H] + .

[0908] Step 2: Preparation of (S)-4-ethyl-8-fluoro-4-hydroxy-9-methoxy-11-propyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolazido[1,2-b]quinoline-3,14(4H)-dione (PY-21-Ad)

[0909] To a 10 mL toluene solution of 1-(2-amino-4-fluoro-5-methoxyphenyl)but-1-one (PY-21-Ab) (1 eq, 62.31 mg, 236.70 μmol), azido-3,6,10(4H)-trione (1 eq, 62.31 mg, 236.70 μmol) and PPTS (1 eq, 59.48 mg, 236.70 μmol, 10 mL) of (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran[3,4-f] were added separately. The reaction was carried out under a nitrogen atmosphere and stirred at 130 °C for 16 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 5:1) to give a yellow solid compound PY-21-Ad (80 mg, yield: 77%).

[0910] LCMS(ESI): m / z, 439.1 [M+H] + .

[0911] Step 3: Preparation of (S)-4-ethyl-8-fluoro-4,9-dihydroxy-11-propyl-1,12-dihydro-14H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3,14(4H)-dione (PY-21-A)

[0912] AlCl3 (18.25 mg, 136.84 μmol, 6 eq) was added to 4 mL of a DCM solution containing compound PY-21-Ad (10 mg, 22.81 μmol, 1 eq). The reaction was stirred at 70 °C for 16 hours. The reaction was quenched with 100 mL of saturated ammonium chloride solution, and the organic phase was extracted with 2 × 100 mL of ethyl acetate. The extract was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (SHIMADDZU, LC-20AP; YMC-Triart Prep C18 column, 250 × 30 mm × 10 μm; mobile phase: water (0.225% HCOOH)-acetonitrile, with water elution from 12% to 42%). The purified solid was lyophilized to give compound PY-21-A (4.3 mg, yield: 44%).

[0913] LCMS(ESI): m / z, 425.2 [M+H] + .

[0914] 1 H NMR(400MHz, DMSO-d6)δ10.90(s,1H),7.91(dd,J=11.9,2.8Hz,1H),7.64–7.57(m,1H),7.26(s,1H),6.49(s,1H),5.43(s,2H),5.27(d, J=3.1Hz,2H),3.06(t,J=7.9Hz,2H),1.86(dq,J=14.1,7.0Hz,2H),1.73(p,J=7.5Hz,2H),1.05(t,J=7.3Hz,3H),0.88(t,J=7.3Hz,3H).

[0915] Preparation Example 29: Preparation of (S)-4-(4-aminobutyl)-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indoleazino[1,2-b]quinoline-10,13-dione (PY-24)

[0916]

[0917] Step 1: Preparation of (S)-(4-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)but-3-en-1-yl)tert-butyl carbamate (PY-24c)

[0918] To 15 mL of toluene solvent containing 200 mg of 200 mg of azido[1,2-b]quinoline-10,13-dione in (S)-4-bromo-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7], 0.1 equivalents of di(tri-tert-butylphosphine)palladium (21 mg), 0.2 equivalents of tri(o-methylphenyl)phosphine (25 mg), 6 equivalents of DIPEA (410 μL), and 2.5 equivalents of tert-butyl but-3-en-1-ylcarbamate (176 mg) were added, and the reaction was stirred at 120 °C for 16 hours. After concentration, the reaction solution was purified by silica gel column chromatography (eluent: dichloromethane / methanol 20:1 to 10:1), and concentrated under reduced pressure to obtain 129 mg of crude product (yield: 54%), which was directly used in the next reaction.

[0919] LCMS (ESI): m / z, 576.3 [M+1] + .

[0920] Step 2: Preparation of (S)-(4-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)butyl)carbamate (PY-24d)

[0921] To 3 mL of methanol containing 6.8 mg of crude (S)-(4-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)but-3-en-1-yl)carbamate, 0.25 equivalents of Pd / C (1.3 mg) and 0.25 equivalents of Pd(OH)₂ (1.7 mg) were added, respectively. Under a specific atmosphere, the reaction was stirred at 25°C for 16 hours. After filtration, 6.8 mg of the crude compound (S)-(4-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)butyl)carbamate (PY-24d) (yield: 55%, purity: 63%) was directly used in the next step of the reaction.

[0922] LCMS (ESI): m / z, 578.2 [M+1] + .

[0923] Step 3: Preparation of (S)-4-(4-aminobutyl)-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-24)

[0924] Under ice-water bath conditions, 0.7 mL of TFA was slowly added to 2.1 mL of dichloromethane solvent containing compound (S)-(4-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)butyl)carbamate (6.8 mg, purity: 63%). The reaction was allowed to proceed naturally to 25 °C and stirred for 16 hours. The product was then prepared by high-performance liquid chromatography (HPLC) (SIMADZU LC-20AP, Welch Xtimate C18 column). 250×50mm×10μm. Purification was carried out using water (0.225% FA)-ACN as the mobile phase (elution ratio of water from 12% to 42%), followed by lyophilization to give 0.35 mg of compound PY-24 (yield: 5.4%).

[0925] LCMS (ESI): m / z, 473.2 [M+H] + .

[0926] 1 H NMR(400MHz,DMSO-d6)δ1H NMR(400MHz,DMSO-d6)δ8.37(s,1H),7.78-7.71(m,1H),7.30(s,1H),6.51(s,1H),5.43(s,2H),5.25(s,2H),3.16( d,J=6.0Hz,6H),2.86(s,2H),2.76(s,2H),2.08(s,2H),1.88(q,J=7.0Hz,2H),1.59(s,2H),0.88(t,J=7.3Hz,3H); 19 F NMR (377MHz, DMSO-d) 6 )δ-112.93.

[0927] Preparation Example 30: Preparation of 2-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-1-yl)acetic acid (PY-25)

[0928]

[0929] Step 1: Preparation of methyl 2-(8-acetamido-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)acetate (PY-251)

[0930] N-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (2 g, 8.5 mmol) was placed in a 100 mL three-necked flask and dissolved in 30 mL of THF under a nitrogen atmosphere. The mixture was cooled to -78 °C, and LDA (10.6 mL, 2 M in THF) was slowly added to the reaction solution. After the addition was complete, the mixture was reacted at -78 °C for 1 h. Then, methyl bromoacetate (1.3 g, 8.5 mmol) was added to the reaction solution, and the mixture was allowed to rise to room temperature for 16 h. The reaction solution was added to 150 mL of water and extracted with ethyl acetate (100 mL × 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (80% PE / 20% EA) to give 800 mg of a pale yellow solid compound, methyl 2-(8-acetamido-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthyl-2-yl)acetate (PY-251), yield: 30.6%.

[0931] Step 2: Preparation of 2-(8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)acetic acid (PY-252)

[0932] 2 mL of NaOH (2 mol / mL) aqueous solution was added to an EtOH (8 mL) solution of methyl 2-(8-acetamido-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)acetate (PY-251) (400 mg, 1.3 mol), and the reaction was carried out at 75 °C for 16 h. The pH of the reaction solution was adjusted to pH 7-8 with 2N hydrochloric acid, concentrated under reduced pressure, extracted with ethyl acetate (30 mL × 3), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 200 mg of crude product, which was used directly in the next step.

[0933] Step 3: Preparation of 2-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-1-yl)acetic acid (PY-25)

[0934] 2-(8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)acetic acid (PY-252) (200 mg, 0.796 mmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-3,6,10(4H)-trione (251 mg, 0.954 mmol) were placed in a 50 mL three-necked flask, toluene (10 mL, 50 v) was added, followed by p-toluenesulfonic acid (27.3 mg, 0.159 mmol) and o-cresol (0.6 mL, 3 v), and the reaction was carried out at 120-125 °C for 16 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50mm×10μm; mobile phase: water (0.225% HCOOH)-acetonitrile, eluting from 45% to 85%) to give 70 mg of yellow powder compound PY-25, with a yield of 18.38%.

[0935] 1 H NMR (400MHz, DMSO) δ12.50 (s, 1H), 7.76 (d, J = 11.1Hz, 1H), 7.31 (s, 1H), 6. 51(s,1H),5.44(s,2H),5.36(s,2H),3.83–3.74(m,1H),3.19-3.05(m,2H) ,2.70-2.64(m,1H),2.58-2.53(m,1H),2.38(s,3H),2.23(d,J=13.6Hz,1H ), 2.06-1.95 (m, 1H), 1.87 (tt, J = 14.1, 7.0Hz, 2H), 0.87 (t, J = 7.3Hz, 3H).

[0936] Preparation Example 31: Preparation of 2-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-1-yl)-N-(2-hydroxyethyl)acetamide and 2-((1R,9S)-9-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazin[1,2-b]quinoline-1-yl)-N-(2-hydroxyethyl)acetamide (PY-25A and PY-25B)

[0937]

[0938] Under ice bath conditions, HATU (17 mg, 45.14 μmol, 1.2 eq), DIEPA (20 mg, 150.5 μmol, 4 eq), and ethanolamine (2.3 mg, 37.62 μmol, 1.0 eq) were added to a DMF (3 mL) solution of compound 2-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4]:6,7]indolazino[1,2-b]quinoline-1-yl)acetic acid (PY-25) (18 mg, 37.62 μmol, 1.0 eq) (PY-25) (3 mL) (18 mg, 37.62 μmol, 1.0 eq) (PY-25) ( ... The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50mm×10μm; mobile phase: water (0.225% HCOOH)-acetonitrile, eluting from 45% to 85%) to obtain white solid compounds PY-25A (4.01 mg, yield 18%) and PY-25B (6.62 mg, yield 30%).

[0939] PY-25A (LCMS retention time 1.73 min):

[0940] LCMS (ESI): m / z, 522.3 [M+1] + .

[0941] 1 H NMR (400MHz, DMSO-d) 6 )δ8.07(t,J=4.0Hz,1H),7.76(d,J=12.0Hz,1H),7.30(s,1H),6.51(s,1H),5.44(s,2H),5.34(s,2H),4.67(t,J=4.0Hz,1H),3.78-3.77(m ,1H),3.44-3.36(m,3H),3.18-3.14(m,4H),2.45-2.44(m,1H),2.39(s,3H),2.13-2.11(m,1H),1.97-1.80(m,3H),0.87(t,J=8.0Hz,3H).

[0942] PY-25B (LCMS retention time 1.83 min):

[0943] LCMS (ESI): m / z, 522.2 [M+1] + .

[0944] 1H NMR (400MHz, DMSO-d) 6 )δ8.46(brs,0.36H,HCOOH),8.08-8.06(m,1H),7.75(d,J=12.0Hz,1H),7.30(s,1H),6.51(s,1H),5.44(s,2H),5.34(s,2H),4.66(s,1H ),3.78-3.76(m,1H),3.38-3.36(m,2H),3.15-3.12(m,,4H),2.48–2.30(m,5H),2.14(s,1H),1.88-1.85(m,3H),0.87(t,J=8.0Hz,3H).

[0945] Preparation Example 32: Preparation of (S)-N-((9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)methyl)-2-hydroxyacetamide (PY-26)

[0946]

[0947] At 0 °C, N,N-diisopropylethylamine (26.71 mg, 206.68 μmol, 3 eq) and HATU (31.43 mg, 82.67 μmol, 1.2 eq) were added to a solution of compound PY-16 (30 mg, 68.89 μmol, 1 eq) and 2-hydroxyacetic acid (6.29 mg, 82.67 μmol, 1.2 eq) in N,N-dimethylformamide (3 mL). After addition, the reaction mixture was reacted at 25 °C for 2 hours. The reaction solution was purified by preparative high-performance liquid chromatography (ISCO, model ISCO-R1; YMC-Triart Prep C18 column 250×50 mm×7 μm; mobile phase: water (0.225% FA)-acetonitrile; acetonitrile elution ratio from 28% to 37%) to give a white solid compound PY-26 (4.37 mg, yield = 12.8%).

[0948] LCMS (ESI): m / z, 494.2 [M+H] + .

[0949] 1H NMR (400MHz, DMSO-d6) δ8.06-8.05(m,1H),7.74(d,J=12Hz,1H),7.31(s,1H),6.54(s,1H),5.43-5.42(m,3H),5.25(s,2H),4.56(d,J= 8.0Hz,2H),3.82(d,J=8.0Hz,2H),3.26-3.23(m,2H),3.15-3.15(m,2H),2.08-2.05(m,2H),1.90-1.83(m,2H),0.87(t,J=7.2Hz,3H). 19 F NMR(377MHz,DMSO-d6)δ-112.11.

[0950] Preparation Example 33: Preparation of (S)-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)methyl(2-hydroxyethyl)carbamate (PY-27)

[0951]

[0952]

[0953] Step 1: Preparation of (S)-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)methyl(4-nitrophenyl)carbonate (PY-27a)

[0954] At room temperature, compound PY-10 (8.0 mg, 0.018 mmol, 1.0 eq), bis(4-nitrobenzene) carbonate (44.6 mg, 0.147 mmol, 8.0 eq), and N,N-diisopropylethylamine (28.4 mg, 0.220 mmol, 12.0 eq) were added to N,N-dimethylformamide (2 mL), and the mixture was stirred at 50 °C for 16 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by thin-layer chromatography using dichloromethane / methanol (15 / 1) as the developing solvent to give a yellow oily compound PY-27a (18 mg).

[0955] LCMS (ESI): m / z, 602.3 [M+H] + .

[0956] Step 2: Preparation of (S)-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)methyl(2-hydroxyethyl)carbamate (PY-27)

[0957] At room temperature, compound PY-27a (18.0 mg, impure), aminoethanol (1.83 mg, 0.030 mmol), and N,N-diisopropylethylamine (11.6 mg, 0.090 mmol) were added to N,N-dimethylformamide (1 mL), and stirred at 25 °C for 0.5 h. The reaction solution was purified by preparative high-performance liquid chromatography (HPLC) (preparative chromatograph manufacturer: Luna, model: Lab311-ISCO-R4; column: Phenomenex Luna C18 250×50 mm×10 μm; mobile phase: water (0.225% formic acid)-acetonitrile; elution ratio of water from 12% to 42%), and lyophilized to give a white solid product PY-27 (4.14 mg, 7.56 μmol, two-step yield: 42%).

[0958] LCMS (ESI): m / z, 524.2 [M+H] + .

[0959] 1 H NMR (400MHz, DMSO-d6) δ7.79(d,J=11.2Hz,1H),7.32(s,1H),7.19(t,J=5.6Hz,1H),6.54(s,1H),5.44(s,2H),5.27(d,J=4.4Hz,3H),4.63 (t,J=5.6Hz,1H),3.30-3.23(m,5H),3.18(s,2H),3.04(q,J=6.0Hz,2H),2.08(s,2H),1.86(dt,J=15.2,7.2Hz,2H),0.87(t,J=7.2Hz,3H).

[0960] Preparation Example 34: Preparation of (S)-4-ethyl-8,10-difluoro-4,9-dihydroxy-11-(4-hydroxybutyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolazido[1,2-b]quinoline-3,14(4H)-dione (PY-29) and (S)-4-ethyl-8,10-difluoro-4-hydroxy-11-(4-hydroxybutyl)-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolazido[1,2-b]quinoline-3,14(4H)-dione (PY-29B)

[0961]

[0962]

[0963] Step 1: Preparation of 2,4-difluoro-3-methoxybenzaldehyde (PY-29a)

[0964] At -78°C, 1.2 equivalents of n-BuLi (9.3 mL, 2.5 M) were added to 20 mL of THF solvent containing 2.8 g of 1,3-difluoro-2-methoxybenzene. The mixture was stirred at this temperature for half an hour. Then, 6 equivalents of DMF (9.15 mL) were added, and stirring continued for another half hour. The low-temperature reaction bath was removed, and the reaction was placed at 15°C for 1 hour. The reaction was quenched with 4 M HCl (6 mL), extracted with ethyl acetate (3 × 50 mL), and the organic layer was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate 20:1 to 10:1) to give 1.8 g of a yellow oily compound PY-29a (yield: 53%).

[0965] LCMS(ESI): m / z, 173[M+1] + .

[0966] 1 H NMR (400MHz, DMSO-d6) δ10.96-9.56(m,1H),7.83-7.43(m,1H),7.30(b,1H),4.22-3.90(m,3H).

[0967] Step 2: Preparation of 2,4-difluoro-3-methoxy-6-nitrobenzaldehyde (PY-29b-1)

[0968] To 20.3 mL of sulfuric acid solvent containing 1.78 g of compound 2,4-difluoro-3-methoxybenzaldehyde, 0.96 equivalents of fuming nitric acid (446 μL) were added, and the reaction was carried out at 25 °C for 1 hour. The reaction solution was poured into ice water, filtered under pressure, washed with water, and dried to give 925 mg of yellow solid compound 2,4-difluoro-3-methoxy-6-nitrobenzaldehyde (PY-29b-1) (yield: 40%).

[0969] 1 H NMR (400MHz, DMSO-d6) δ10.14 (s, 1H), 8.21 (dd, J = 11.1, 2.0Hz, 1H), 4.13 (t, J = 1.9Hz, 3H).

[0970] Step 3: Preparation of 6-amino-2,4-difluoro-3-methoxybenzaldehyde (PY-29c)

[0971] Iron powder (5.5 eq, 1.31 g), water (23.12 eq, 1.77 mL), and hydrochloric acid (0.55 eq, 71 μ L) were added to 30 mL of ethanol solvent containing 2,4-difluoro-3-methoxy-6-nitrobenzaldehyde (925 mg). The reaction was stirred at 80 °C for 16 hours. LC-MS showed that a product was formed. The reaction solution was cooled to room temperature, then filtered under pressure, and the crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate 10:1 to 5:1). After drying, 488 mg of green solid 6-amino-2,4-difluoro-3-methoxybenzaldehyde (PY-29d) (61% yield) was obtained.

[0972] LCMS(ESI): m / z, 187.9 [M+H] + .

[0973] 1 H NMR (400MHz, DMSO-d6) δ 10.04 (s, 1H), 7.48 (s, 2H), 6.44 (dd, J = 13.4, 2.0Hz, 1H), 3.77 (s, 3H).

[0974] Step 4: Preparation of (S)-4-ethyl-8,10-difluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3,14(4H)-dione (PY-29d)

[0975] To 10 mL of toluene solvent containing 150 mg of compound 6-amino-2,4-difluoro-3-methoxybenzaldehyde (PY-29c), 0.67 equivalents of PPTS (134.9 mg) and 1.2 equivalents of compound PY-8f (253.2 mg) were added, respectively. The reaction was carried out under nitrogen atmosphere at 130 °C and stirred for 16 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate 5:1 to 0:1) to give 233 mg of yellow solid compound PY-29d (yield: 67%; purity: 78%).

[0976] LCMS (ESI): m / z, 415 [M+H] + .

[0977] Step 5: Preparation of (S)-11-(4-(benzyloxy)butyl)-4-ethyl-8,10-difluoro-4-hydroxy-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3,14(4H)-dione (PY-29f)

[0978] Compound PY-29d (193 mg, 78% purity) was dissolved in 14.6 mL of glacial acetic acid. Under ice bath conditions, 3.6 mL of concentrated sulfuric acid was slowly added. In another reaction flask, 1.2 equivalents of ferrous sulfate (66.2 mg) and 2.6 mL of deionized water were added. At the same temperature, this ferrous sulfate solution was added to the PY-29d solution. Subsequently, 5 equivalents of 5-(benzyloxy)pentanal (PY-29e) (349 mg) and 54.4 μL of hydrogen peroxide were added sequentially. The reaction was stirred for 45 minutes under ice bath conditions. The reaction solution was poured into ice water, and the organic phase was extracted with ethyl acetate (50 mL × 3). The solution was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol 20:1 to 4:1) to obtain 230 mg of compound PY-29f (64% purity).

[0979] LCMS (ESI): m / z, 577.3 [M+H] + .

[0980] Step 6: Preparation of (S)-4-ethyl-8,10-difluoro-4-hydroxy-11-(4-hydroxybutyl)-9-methoxy-1,12-dihydro-14H-pyrano[3',4':6,7]indolazino[1,2-b]quinoline-3,14(4H)-dione (PY-29B)

[0981] To a 10 mL methanol solution of compound PY-29f (497 mg), 0.25 equivalents of palladium on carbon (78 mg) and 0.25 equivalents of Pd(OH)₂ (51.4 mg) were added, respectively. The reaction was stirred at 30 °C for 16 hours. After filtration, the solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol 20:1 to 10:1) to give 55 mg of compound PY-29B (yield: 38%; purity: 78%).

[0982] LCMS (ESI): m / z, 487.1 [M+H] + .

[0983] Step 7: Preparation of (S)-4-ethyl-8,10-difluoro-4,9-dihydroxy-11-(4-hydroxybutyl)-1,12-dihydro-14H-pyrano[3',4':6,7]indolazido[1,2-b]quinoline-3,14(4H)-dione (PY-29)

[0984] At 90 °C, 6 equivalents of AlCl3 compound (79 mg) were added in portions over 2 hours to 4 mL of DCM solvent containing compound PY-29B (48 mg, 78% purity). The reaction mixture was purified by high performance liquid chromatography (SHIMADDZU LC-20AP, YMC-Triart Prep C18 250×30 mm×10 μm column, mobile phase water (0.225% HCOOH)-acetonitrile, elution ratio of water from 12% to 42%), and lyophilized to give 6.77 mg of white solid compound PY-29 (yield: 14.5%).

[0985] LCMS (ESI): m / z, 473.2 [M+H] + .

[0986] 1 H NMR (400MHz, DMSO-d6) δ8.35(d,J=6.8Hz,1H),7.89(d,J=7.5Hz,2H),7.76(d,J=9.0Hz, 1H),7.71(d,J=7.5Hz,2H),7.55(t,J=6.2Hz,1H),7.42(t,J=7.5Hz,2H),7.33(t,J=7.5H z,2H),4.33–4.20(m,4H),4.11(p,J=7.0Hz,1H),3.67(d,J=6.2Hz,2H),1.95(dt,J=12.6 ,6.3Hz,1H),1.38(d,J=1.9Hz,9H),1.24(d,J=7.2Hz,3H),0.86(dd,J=19.2,6.7Hz,6H); 19 F NMR (377MHz, DMSO-d6) δ-125.50 (d, J=13.5Hz).

[0987] Preparation Example 35: Preparation of (S)-4-ethyl-8,10-difluoro-4,9-dihydroxy-1,12-dihydro-14H-pyran[3',4':6,7]indolazido[1,2-b]quinoline-3,14(4H)-dione (PY-29A)

[0988]

[0989] Step 1: Preparation of (S)-4-ethyl-8,10-difluoro-4,9-dihydroxy-1,12-dihydro-14H-pyran[3',4':6,7]indolazino[1,2-b]quinoline-3,14(4H)-dione (PY-29A)

[0990] AlCl3 (22.2 mg, 166.52 μmol, 3.0 eq) was added to 3 mL of DCM solution containing compound PY-29d (29.5 mg, 55.51 μmol, 1.0 eq, 78% purity), and the mixture was reacted at 70 °C for 2 hours. The reactant was purified by preparative high-performance liquid chromatography (SHIMADDZU LC-20AP, YMC-Triart Prep C18 250 × 30 mm × 10 μm column, mobile phase: water (0.225% HCOOH)-acetonitrile, with water elution ratios ranging from 12% to 42%), and lyophilized to give a white solid compound PY-29A (1.46 mg, yield: 4.9%).

[0991] LCMS(ESI): m / z, 401.1 [M+H] + .

[0992] 1 H NMR (400MHz, DMSO-d6) δ8.54 (s, 1H), 7.77 (d, J = 11.9Hz, 1H), 7.25 (s, 1H), 6.52 (s,1H),5.42(s,2H),5.23(s,2H),1.86(p,J=7.0Hz,2H),0.87(t,J=7.3Hz,3H).

[0993] Preparation Example 36: Preparation of (S)-4-(aminomethyl)-5-chloro-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10h,13h-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-36)

[0994]

[0995]

[0996] Step 1: Preparation of N-(3-bromo-5-chlorophenyl)acetamide (PY-36b)

[0997] 3-Bromo-5-chloroaniline (PY-36a) (5.00 g, 24.2 mmol, 1.0 eq) and triethylamine (4.90 g, 48.4 mmol, 6.74 mL, 2.0 eq) were added to dichloromethane (50.0 mL), and the mixture was purged with nitrogen three times. Acetyl chloride (2.85 g, 36.3 mmol, 2.58 mL, 1.5 eq) was added dropwise at 0 °C, and the mixture was stirred at 25 °C for 2 hours under a nitrogen atmosphere. At 0 °C, 30.0 mL of water was added dropwise to the reaction mixture, causing a precipitate to form. The precipitate was filtered, and the filtrate was concentrated under reduced pressure to give N-(3-bromo-5-chlorobenzene)acetamide (5.90 g, 97% yield) as a white solid.

[0998] 1 H NMR (400MHz, DMSO-d) 6 )δ10.26(s,1H),7.77(d,J=1.2Hz,1H),7.68(d,J=1.2Hz,1H),7.36(d,J=2.0Hz,1H),2.05(s,3H).

[0999] Step 2: Preparation of (E)-4-(3-acetamido-5-chlorophenyl)but-3-enoic acid tert-butyl ester (PY-36c)

[1000] N-(3-bromo-5-chlorophenyl)acetamide (5.00 g, 20.1 mmol, 1.0 eq) was dissolved in N,N-dimethylformamide (30 mL), and tert-butyl 1-buten-4-o-octanoate (4.29 g, 30.2 mmol, 4.89 mL, 1.5 eq), tris(o-methylphenyl)phosphine (306.2 mg, 1.01 mmol, 0.05 eq), triethylamine (4.07 g, 40.2 mmol, 5.60 mL, 2.0 eq), bis(tert-butylphosphine)palladium (103 mg, 201.2 μmol, 0.01 eq), and N-methyldicyclohexylamine (7.86 g, 40.2 mmol, 8.54 mL, 2.0 eq) were added. The reaction mixture was carried out at 100 °C for 4 hours under a nitrogen atmosphere. The reaction solution was cooled to room temperature, water (50.0 mL) was added, and the mixture was extracted twice with ethyl acetate (25.0 mL). The organic phase was washed three times with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography, eluting with petroleum ether / ethyl acetate = 15 / 1-3 / 2, to give (E)-4-(3-acetamido-5-chlorophenyl)but-3-enoic acid tert-butyl ester (5.10 g, 16.5 mmol, yield 82.09%), a white solid.

[1001] LCMS: RT=0.634min, MS(ESI)m / z=254.0[M+H] + .

[1002] Step 3: Preparation of tert-butyl 4-(3-acetamido-5-chlorophenyl)butyrate (PY-36d)

[1003] (E)-4-(3-acetamido-5-chlorophenyl)but-3-enoic acid tert-butyl ester (5.00 g, 16.1 mmol, 1.0 eq) was added to methanol (50.0 mL), and tris(triphenylphosphine)rhodium chloride (I) (1.49 g, 1.61 mmol, 0.1 eq) was added under an argon atmosphere. The reaction mixture was then reacted at 25 °C under hydrogen (30 Psi) for two hours. The reaction mixture was filtered, and the filtrate was concentrated to give crude 4-(3-acetamido-5-chlorophenyl)butyrate tert-butyl ester (3.60 g, 11.5 mmol, yield 71.42%), a white solid.

[1004] 1 HNMR(400MHz,CDCl3)δppm 7.41(s,1H),7.13-7.08(m,2H),6.85(s,1H),2.52(t,J=7.2Hz,2H),2.15(t,J=7.6Hz,1H),2.10(s,1H),1.83-1.81(m,2H),1.38(s,9H).

[1005] Step 4: Preparation of tert-butyl 4-(5-acetamido-2-bromo-3-chlorophenyl)butyrate (PY-36e)

[1006] 100 mg (320.7 μmol, 1.0 eq) of tert-butyl 4-(3-acetamido-5-chlorophenyl)butyrate was dissolved in N,N-dimethylformamide (5.00 mL), and NBS (68.5 mg, 385 μmol, 1.2 eq) was slowly added. The reaction was carried out at 25 °C for 1 hour. Water (10.0 mL) was added dropwise to the reaction solution, and the mixture was extracted twice with ethyl acetate (10.0 mL). The organic phase was washed three times with saturated brine (10.0 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give crude tert-butyl 4-(5-acetamido-2-bromo-3-chlorophenyl)butyrate (120 mg, 96.0%), a white solid.

[1007] LCMS: RT=2.885min, MS(ESI)m / z=336.1[M+H] + .

[1008] Step 5: Preparation of 4-(5-acetamido-2-bromo-3-chlorophenyl)butyric acid (PY-36f)

[1009] 100 mg (256 μmol, 1.0 eq) of tert-butyl 4-(5-acetamido-2-bromo-3-chlorophenyl)butyrate was dissolved in 2.00 mL of dichloromethane. Trifluoroacetic acid (2.00 mL) was slowly added dropwise at 0 °C, and the reaction mixture was reacted at 25 °C for 2 hours. The reaction mixture was directly concentrated under reduced pressure to obtain a solid. Then, 8.00 mL of dichloromethane was added, and the mixture was stirred and filtered to obtain crude 4-(5-acetamido-2-bromo-3-chlorophenyl)butyric acid (60.0 mg, 70.0%), a white solid.

[1010] 1 HNMR(400MHz,CDCl3)δppm 12.09(br,1H),10.19(s,1H),7.87(d,J=2.4Hz,1H),7.37(d,J=2.4Hz,1H),2. 72(t,J=7.6Hz,2H), 2.28(t,J=7.2Hz,2H), 2.04(s,3H), 1.78(t,J=7.6Hz,2H).

[1011] Step 6: Preparation of N-(4-bromo-3-chloro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-36g)

[1012] 4-(5-acetamido-2-bromo-3-chlorophenyl)butyric acid (1.80 g, 5.38 mmol, 1.0 eq) was dissolved in Eaton reagent (113.6 g, 477.3 mmol, 75.00 mL, 88.73 eq) and reacted at 100 °C for 1 hour under a nitrogen atmosphere. The reaction solution was cooled to room temperature and quenched slowly in cold water. The mixture was filtered to obtain a solid. The solid was dissolved in dichloromethane (10.0 mL), concentrated, and slurried with a mixed solvent (petroleum ether / ethyl acetate = 10 / 1). The mixture was filtered to give N-(4-bromo-3-chloro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (1.20 g, 3.79 mmol, yield 70.45%), a yellow solid.

[1013] LCMS: RT=0.961min, MS(ESI)m / z=317.9[M+H] + .

[1014] 1 HNMR (400MHz, CDCl3) δppm 12.11 (s, 1H), 8.86 (s, 1H), 3.03 (t, J = 6.0Hz, 2H), 2.61 (t, J = 6.4Hz, 2H), 2.16 (s, 3H), 2.04 (t, J = 6.4Hz, 2H).

[1015] Step 7: Preparation of 8-amino-5-bromo-6-chloro-3,4-dihydronaphthyl-1(2H)-1-one (PY-36h)

[1016] N-(4-bromo-3-chloro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (900 mg, 2.84 mmol, 1.0 eq) was dissolved in ethanol (15 mL), and hydrochloric acid solution (5 mL, 6 mol / mL) was added at room temperature. The mixture was stirred at 80 °C for 3 hours. The reaction solution was cooled to room temperature and concentrated. The pH of the concentrate was adjusted to 8-9 with saturated sodium bicarbonate solution, and then extracted twice with ethyl acetate (10 mL). The organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product 8-amino-5-bromo-6-chloro-3,4-dihydronaphth-1(2H)-1-one (700 mg, 2.55 mmol, yield 89.8%), a yellow solid.

[1017] LCMS: RT=0.942min, MS(ESI)m / z=275.8[M+H] + .

[1018] Step 8: Preparation of (S)-4-bromo-5-chloro-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10h,13h-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-36i)

[1019] 8-Amino-5-bromo-6-chloro-3,4-dihydronaphthyl-1(2H)-1-one (574 mg, 2.09 mmol, 1.1 eq) and compound PY-8f (500 mg, 1.90 mmol, 1.0 eq) were dissolved in toluene (10 mL), and p-toluenesulfonic acid (36.1 mg, 0.19 mmol, 0.1 eq) was added. The reaction was carried out at 110 °C for 16 hours under a nitrogen atmosphere. The reaction solution was cooled to room temperature, and a solid precipitated out. The solid was filtered and then dried to give crude PY-36i, a yellow solid (850 mg, 1.69 mmol, yield 89.4%).

[1020] LCMS: RT=2.233min, MS(ESI)m / z=503.3[M+H] + .

[1021] Step 9: Preparation of (S)-(5-chloro-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1h,12h-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)methyl)carbamate tert-butyl ester (PY-36j)

[1022] Compound PY-36i (400 mg, 0.80 mmol, 1.0 eq), potassium N-Boc-aminomethyltrifluoroborate (948 mg, 4.00 mmol, 5.0 eq), n-butyl-bis(1-adamantyl)phosphine (375 mg, 0.40 mmol, 0.5 eq), potassium carbonate (221 mg, 1.60 mmol, 2.0 eq), and palladium acetate (53.88 mg, 0.24 mmol, 0.3 eq) were dissolved in 1,4-dioxane (10 mL) and water (2 mL) and stirred at 80 °C for 2 hours under a nitrogen atmosphere. The reaction solution was cooled to room temperature and concentrated. Water (10 mL) was added to the concentrate, and then extracted twice with ethyl acetate (10 mL). The organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by reversed-phase chromatography (column: Ultimate C18 150×40 mm×10 μm; mobile phase: [water (FA)-ACN]; gradient: 26%-66% B, 36 min) to obtain compound PY-36j (54.2 mg, 0.095 mmol, yield 11.87%), a white solid.

[1023] LCMS: RT=2.127min, MS(ESI)m / z=552.5[M+H] + .

[1024] Step 10: Preparation of (S)-4-(aminomethyl)-5-chloro-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10h,13h-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-36)

[1025] Compound PY-36j (52.4 mg, 0.095 mmol, 1.0 eq) was dissolved in dichloromethane (5 mL), and trifluoroacetic acid (2 mL) was added at 0 °C. The reaction mixture was reacted at 25 °C for 2 hours. The reaction solution was concentrated under reduced pressure, and the crude product was purified by reversed-phase chromatography (column: Ultimate C18 150 × 40 mm × 10 μm; mobile phase: [water (TFA)-ACN]; gradient: 0%-38% B, 30 min) to give compound PY-36 (4.55 mg, 0.01 mmol, yield 10.53%), a white solid.

[1026] LCMS: RT=1.566min, MS(ESI)m / z=452.2[M+H] + .

[1027] 1 HNMR (400MHz, DMSO-d) 6)δppm 8.23(s,1H),8.19(s,2H),7.34(s,1H),6.55(s,1H),5.45(s,2H),5.30(s,2H),4.41(s, 2H), 3.20-3.18 (m, 4H), 2.12 (t, J = 4.8Hz, 2H), 1.90-1.86 (m, 2H), 0.88 (t, J = 7.2Hz, 3H).

[1028] Preparation Example 37: Preparation of (S)-5-chloro-9-ethyl-9-hydroxy-4-(hydroxymethyl)-1,2,3,9,12,15-hexahydro-10h,13h-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-37)

[1029]

[1030] Compound PY-36i (200 mg, 0.40 mmol, 1.0 eq) and (tributyltinyl)methanol (192 mg, 0.60 mmol, 1.5 eq) were dissolved in 1,4-dioxane (5 mL). The catalyst chloro(2-dicyclohexylphosphino-2,4,6-triisopropyl-1,1-biphenyl)[2-(2-amino-1,1-biphenyl)]palladium(II) (XPhos Pd G2) (31.4 mg, 0.04 mmol, 0.1 eq) was added. The reaction was carried out at 90 °C for 4 hours under a nitrogen atmosphere. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure, diluted with water, and then extracted twice with ethyl acetate (10 mL). The organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by reversed-phase chromatography (column: Welch Xtimate C18 150×30mm×5μm; mobile phase [water(FA)-ACN]; gradient: 6%-46% B, 25 min) to give compound PY-37 (2.08 mg, 0.005 mmol, yield 1.25%), a white solid.

[1031] LCMS: RT=1.475min, MS(ESI)m / z=453.3[M+H] + .

[1032] 1 HNMR (400MHz, DMSO-d) 6)δppm 8.10(s,1H),7.32(s,1H),6.53(s,1H),5.44(s,2H),5.28(s,2H),5.19(t,J=5.2Hz,1H),4.83(d,J=5.2Hz,2H ), 3.17 (t, J = 7.6 Hz, 2H), 2.09 (t, J = 7.6 Hz, 2H), 1.89-1.84 (m, 2H), 1.15-1.14 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H).

[1033] Preparation Example 38: Preparation of (S)-9-ethyl-9-hydroxy-4-(hydroxymethyl)-1,2,3,9,12,15-hexahydro-10h,13h-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-37A)

[1034]

[1035] Compound PY-36i (200 mg, 0.40 mmol, 1.0 eq) and (tributyltinyl)methanol (192 mg, 0.60 mmol, 1.5 eq) were dissolved in 1,4-dioxane (5 mL). The catalyst chloro(2-dicyclohexylphosphino-2,4,6-triisopropyl-1,1-biphenyl)[2-(2-amino-1,1-biphenyl)]palladium(II) (XPhos Pd G2) (31.4 mg, 0.04 mmol, 0.1 eq) was added. The reaction was carried out at 90 °C for 4 hours under a nitrogen atmosphere. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure, diluted with water, and then extracted twice with ethyl acetate (10 mL). The organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by reversed-phase chromatography (column: Welch Xtimate C18 150×30mm×5μm; mobile phase: [water(FA)-ACN]; gradient: 6%-46% B, 25 min) to give compound PY-37A (2.23 mg, 0.005 mmol, yield 1.25%), a white solid.

[1036] LCMS: RT=1.233min, MS(ESI)m / z=419.4[M+H] + .

[1037] 1 HNMR (400MHz, DMSO-d) 6)δppm 7.99(d,J=8.8Hz 1H),7.90(d,J=8.8Hz1H),7.32(s,1H),6.51(s,1H),5.44(s,2H),5.33(t,J=4.4Hz,1H),5.27(s,2H),4.73(d,J=5.2Hz,2H) ,3.19-3.16(m,1H),3.10-3.07(m,1H),2.08(t,J=3.2Hz,2H),1.90-1.86(m,2H),1.15-1.13(m,2H),0.89(t,J=7.2Hz,3H).

[1038] Preparation Example 39: Preparation of (S)-5-chloro-9-ethyl-4,9-dihydroxy-1,2,3,9,12,15-hexahydro-10h,13h-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-38)

[1039]

[1040] Step 1: Preparation of (S)-5-chloro-9-ethyl-9-hydroxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)-1,2,3,9,12,15-hexahydro-10h,13h-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-38a)

[1041] Compound PY-36i (400 mg, 0.80 mmol, 1.0 eq), bis(pinacolborate) borate (305 mg, 1.20 mmol, 1.5 eq), potassium acetate (235 mg, 2.40 mmol, 3.0 eq), and 1,1-bis(diphenylphosphine)ferrocene palladium chloride (176 mg, 0.24 mmol, 0.3 eq) were dissolved in 1,4-dioxane (10 mL). The mixture was stirred at 90 °C for 5 hours under a nitrogen atmosphere. The reaction solution was cooled to room temperature and concentrated. Water (10 mL) was added to the concentrate, and the extract was extracted twice with ethyl acetate (10 mL). The organic phase was washed once with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by preparative thin-layer chromatography (PE / EA = 1 / 4) to give compound PY-38a (70.2 mg, 0.13 mmol, yield 16.25%) as a yellow solid.

[1042] LCMS: RT=2.391min, MS(ESI)m / z=549.3[M+H] + .

[1043] Step 2: Preparation of (S)-5-chloro-9-ethyl-4,9-dihydroxy-1,2,3,9,12,15-hexahydro-10h,13h-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-38)

[1044] Compound PY-38a (70.2 mg, 0.13 mmol, 1.0 eq) was dissolved in tetrahydrofuran (0.2 mL) and water (0.2 mL). Sodium perborate tetrahydrate (44.15 mg, 0.39 mmol, 3.0 eq) was added at 0 °C, and the reaction was carried out at 25 °C for 1 h. The reaction solution was quenched with saturated ammonium chloride solution (0.2 mL) and then purified by reversed-phase chromatography (column: Ultimate C18 150 × 40 mm × 10 μm; mobile phase: [water (FA)-ACN]; gradient: 10%-50% B, 36 min) to give compound PY-38 (0.82 mg, 0.002 mmol, yield 1.53%) as a white solid.

[1045] LCMS: RT=1.491min, MS(ESI)m / z=439.3[M+H] + .

[1046] 1 HNMR (400MHz, DMSO-d) 6 )δppm 8.36(s,0.16H),8.03(s,1H),7.23(s,1H),6.49(s,1H),5.42(s,2H),5.21(s,2H),3.10-3.07(m,2H) ,3.02(t,J=5.6Hz,2H),2.01(t,J=6.4Hz,2H),1.88-1.85(m,2H),1.24(s,1H),0.88(t,J=7.6Hz,3H).

[1047] Preparation Example 40: Preparation of PY-41A and PY-41B

[1048]

[1049] Step 1: Preparation of compounds PY-41A-a and PY-41A-b

[1050] The compound N-(8-amino-5-bromo-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)acetamide (PY-Uc) (preparation method see Preparation Example 44) (140 mg, 444.25 μmol, 1 eq) was dissolved in toluene (10 mL), and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-3,6,10(4H)-trione (PY-8f) (116.95 mg, 444.25 μmol, 1 eq) and PPTS (111.64 mg, 444.25 μmol, 1 eq) were added. The reaction solution was stirred at 125 °C for 12 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by high-performance liquid chromatography (HPLC) (Shimadzu LC-20AP preparative chromatograph; YMC-TriartPrep C18 column, 250×50mm×7μm; mobile phase: water (0.225% HCOOH)-acetonitrile, with water elution ratios ranging from 21% to 51%), yielding two brown isomer solids, PY-41-a (54 mg, yield: 22.41%) and PY-41-b (54 mg, yield: 22.41%).

[1051] LCMS (ESI): m / z, 542.2 [M+H] + .

[1052] Step 2: Preparation of compound PY-41A

[1053] Compound PY-41A-a (15 mg, 27.66 μmol, 1 eq) was dissolved in dioxane (2 mL), and hydroxymethyltributyltinane (26.64 mg, 82.97 μmol, 3 eq) and XPhos Pd G2 (4.35 mg, 5.53 μmol, 0.2 eq) were added. The reaction solution was purged with nitrogen three times, and the mixture was stirred at 90 °C for 12 hours under a nitrogen atmosphere. The reaction solution was filtered, concentrated under reduced pressure, and the residue was purified by preparative high-performance liquid chromatography (HPLC) using Oriendo BRIX-2860 (R1, 4, 5, 6) column (Phenomenex Luna C18 250×50mm×10μm, mobile phase: water (0.225% HCOOH)-acetonitrile, with water elution from 14% to 24%) to obtain a white solid PY-41A (1.10 mg, yield: 8.06%).

[1054] LCMS(ESI): m / z, 494.2 [M+H] + .

[1055] Step 3: Preparation of compound PY-41B

[1056] Compound PY-41A-b (15 mg, 27.66 μmol, 1 eq) was dissolved in dioxane (2 mL), and hydroxymethyltributyltinane (26.64 mg, 82.97 μmol, 3 eq) and XPhos Pd G2 (4.35 mg, 5.53 μmol, 0.2 eq) were added. The reaction mixture was purged with nitrogen three times, and stirred at 90 °C for 12 hours under a nitrogen atmosphere. The reaction solution was filtered, concentrated under reduced pressure, and the residue was purified by high-performance liquid chromatography (HPLC) using Oriendo BRIX-2860 (R1, 4, 5, 6) preparative chromatograms. The column was a Phenomenex Luna C18 250×50mm×10μm. The mobile phase was water (0.225% HCOOH)-acetonitrile, with water elution ratios ranging from 12% to 42%. The result was a white solid product, PY-41B (1.02 mg, yield: 7.47%).

[1057] LCMS(ESI): m / z, 494.1 [M+H] + .

[1058] Preparation Example 41: Preparation of ((9S)-9-ethyl-5-fluoro-9-hydroxy-4-(hydroxymethyl)-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (PY-42)

[1059]

[1060] Compound PY-UA (preparation method as shown in Preparation Example 44) (10.53 mg, 95%, 22.15 μmol, 1 eq) was dissolved in DCM (5 mL), and TEA (4.48 mg, 6.16 μL, 44.30 μmol, 2 eq) was added. Ethyl chloroformate (4.81 mg, 4.24 μL, 44.30 μmol, 2 eq) was added at 0 °C. The reaction solution was stirred at 0 °C for 8 hours. The reaction solution was diluted with DCM (10 mL), washed with water (10 mL), dried the organic phase, filtered, and purified by high performance liquid chromatography (HPLC) (preparative chromatograph manufacturer: Oriendo, model BRIX-2860 (R1,4,5,6). The column was a Welch Xtimate C18 250×30 mm×10 μm. Mobile phase: water (0.225% HCOOH)-acetonitrile, with water elution ratios ranging from 20% to 50%), yielding a white solid product PY-42 (1.12 mg, yield: 9.66%, Purity: 90%).

[1061] LCMS (ESI): m / z, 524.2 [M+H] +.

[1062] Preparation Example 42: Preparation of N-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-(hydroxymethyl)-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-2-hydroxyacetamide (PY-43)

[1063]

[1064] Step 1: Preparation of 2-((tert-butyldimethylsilyl)oxy)-N-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-(hydroxymethyl)-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)acetamide (PY-43-b)

[1065] Compound PY-UA (5 mg, 100%, 11.08 μmol, 1 eq) was dissolved in DMF (1 mL), and DIPEA (2.86 mg, 3.66 μL, 22.15 μmol, 2 eq) and 2,5-dioxopyrrolidone-1-yl-2-((tert-butyldimethylsilyl)oxy)acetate (PY-43-a) (3.82 mg, 13.29 μmol, 1.2 eq) were added. The reaction mixture was stirred at 20 °C for 12 hours. The reaction solution was purified by preparative high-performance liquid chromatography (Preparative chromatograph manufacturer: Oriendo, model BRIX-2860 (R1,4,5,6). The chromatographic column was GS-120-10-C18AP. Mobile phase: water (0.225% HCOOH)-acetonitrile, with water elution ratios ranging from 30% to 60%) to give a white solid product (2 mg, yield: 28.95%).

[1066] LCMS (ESI): m / z, 624.2 [M+H] + .

[1067] Step 2: Preparation of N-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-(hydroxymethyl)-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)-2-hydroxyacetamide (PY-43)

[1068] Compound PY-43-b (2 mg, 3.21 μmol, 1 eq) was dissolved in THF (1 mL), and HCl (116.91 μg, 500 μL, 3.21 μmol, 1 eq) was added. The reaction mixture was stirred at 15 °C for 2 hours. The reaction mixture was purified by preparative high-performance liquid chromatography (Preparative chromatograph manufacturer: Oriendo, model BRIX-2860 (R1,4,5,6). The chromatographic column was Welch Xtimate C18 250×30 mm×10 μm. Mobile phase: water (0.225% HCOOH)-acetonitrile, with water elution ratios ranging from 12% to 42%), yielding a yellow solid product PY-43 (1.10 mg, yield: 67.33%, purity: 100%).

[1069] LCMS (ESI): m / z, 510.2 [M+H] + .

[1070] Preparation Example 43: Preparation of (S)-4-amino-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-A)

[1071]

[1072]

[1073] Step 1: Preparation of 6,8-difluoro-5-nitro-1,2,3,4-tetrahydronaphthyl-1-one (PY-Ab)

[1074] To a 6 mL sulfuric acid solution of 1.0 g of 6,8-difluoro-3,4-dihydronaphthyl-1(2H)-one (PY-Aa), 0.96 equivalents of potassium nitrate (546 mg) were added, and the reaction was stirred in an ice bath for 2 hours. The reaction solution was poured into ice water, quenched with 50 mL of water, and extracted with ethyl acetate (60 mL × 3). The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 800 mg of the crude product compound PY-Ab (41% purity).

[1075] 1 H NMR (400MHz, DMSO-d6) δ7.20–7.07(m,1H),2.96(q,J=6.0Hz,2H),2.61(ddd,J=27.9,7.3,5.8Hz,2H),2.12–2.00(m,2H).

[1076] Step 2: Preparation of 6,8-difluoro-5-amino-1,2,3,4-tetrahydronaphthyl-1-one (PY-Ac)

[1077] To 9 mL of a mixture of ethanol and water (8:1 v / v) containing 800 mg of 6,8-difluoro-5-nitro-1,2,3,4-tetrahydronaphthyl-1-one, 894 mg of iron powder and 321 mg of ammonium chloride were added, and the reaction was stirred at 80 °C for 2 hours. After filtering off the iron powder, the reaction solution was concentrated directly, and the residue was purified by silica gel column chromatography (eluting ethyl acetate / petroleum ether at a 1:1 ratio) to give 176 mg of 6,8-difluoro-5-amino-1,2,3,4-tetrahydronaphthyl-1-one (PY-Ac) (yield: 45%).

[1078] LCMS(ESI): m / z, 198.1 [M+H] + .

[1079] 1 H NMR (400MHz, DMSO-d6) δ7.03 (t, J = 11.4Hz, 1H), 5.02 (s, 2H), 2.70 (t, J = 6.2Hz, 2H), 2.08 (s, 1H), 2.00 (p, J = 6.4Hz, 2H).

[1080] Step 3: Preparation of N-(2,4-difluoro-5-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-Ad)

[1081] To a 25 mL solution of 6,8-difluoro-5-amino-1,2,3,4-tetrahydronaphthyl-1-one (600 mg) in dichloromethane, 1.2 equivalents of acetic anhydride (343 μL) and 1.2 equivalents of triethylamine (508 μL) were added, respectively. The reaction was stirred at 60 °C for 36 hours. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol 20:1) to give 483 mg of N-(2,4-difluoro-5-oxo-5,6,7,8-tetrahydronaphthyl-1-yl)acetamide (PY-Ad) (yield: 66%, purity: 95%).

[1082] LCMS(ESI): m / z, 240.1 [M+H] + .

[1083] Step 4: Preparation of N-(4-amino-2-fluoro-5-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-Ae)

[1084] In a sealed tube, 35 mL of ammonia was added to 30 mL of DMSO solution containing 1.43 g of N-(2,4-difluoro-5-oxo-5,6,7,8-tetrahydronaphthyl-1-yl)acetamide (PY-Ad), and the mixture was stirred at 100 °C for 16 hours. The reaction was quenched by adding 50 mL of water, extracted with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol 20:1) to give 1.01 g of N-(4-amino-2-fluoro-5-oxo-5,6,7,8-tetrahydronaphthyl-1-yl)acetamide (PY-Ae) (yield: 72%, purity: 94%).

[1085] LCMS(ESI): m / z, 237.2 [M+H] + .

[1086] Step 5: Preparation of (S)-N-(9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-4-yl)acetamide (PY-Ag)

[1087] In a 50 mL toluene solution of N-(4-amino-2-fluoro-5-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (1.01 g), 1.2 equivalents of compound PY-8f (1.3 g) and 0.67 equivalents of PPTS (691 mg) were added, respectively. The reaction was stirred at 130 °C for 16 hours. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (eluent: dichloromethane / methanol 10:1) to give 584 mg of compound PY-Ag (yield: 30%, purity: >99%).

[1088] LCMS(ESI): m / z, 464.0 [M+H] + .

[1089] 1 H NMR(400MHz, DMSO-d6)δ9.82(s,1H),7.80(d,J=11.0Hz,1H),7.31(s,1H),6.52(s,1H),5.44(s,2H),5.25(s,2H),3.16 (t,J=6.1Hz,2H),2.98(t,J=6.0Hz,2H),2.14(s,3H),2.06–1.99(m,2H),1.87(p,J=7.1Hz,2H),0.88(t,J=7.3Hz,3H).

[1090] Step 6: Preparation of (S)-4-amino-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (PY-A)

[1091] To a 6 mL ethanol solution of compound PY-Ag (78 mg), 6 mL of 12 N hydrochloric acid was added, and the reaction was stirred at 60 °C for 16 hours. The solution was purified by preparative high-performance liquid chromatography (SHIMADDZU LC-20AP, YMC-Triart Prep C18 250×30 mm×10 μm column, mobile phase water (0.225% HCOOH)-acetonitrile, with water elution ratios ranging from 21% to 51%), and then lyophilized to obtain 43.9 mg of compound PY-A (yield: 61%, purity: 98%).

[1092] LCMS (ESI): m / z, 422.1 [M+H] + .

[1093] 1 H NMR(400MHz,DMSO-d6)δ7.63(d,J=12.5Hz,1H),7.20(s,1H),6.47(s,1H),5.76(s,2H),5.41(s,2H),5.18(s,2H),3.0 6(t,J=6.1Hz,2H),2.84(t,J=6.1Hz,2H),2.02(t,J=6.2Hz,2H),1.86(dq,J=14.4,7.0Hz,2H),0.87(t,J=7.3Hz,3H); 19 F NMR (377MHz, DMSO-d6) δ-125.24.

[1094] Preparation Example 44: Preparation of PY-UA and PY-UB

[1095]

[1096] Step 1: Preparation of (E)-N-(4-bromo-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-Ua)

[1097] At 0 °C and under a nitrogen atmosphere, potassium tert-butoxide (3.36 g, 29.99 mL, 29.99 mmol, 3 eq, 1 M in THF) and tert-butyl nitrite (3.09 g, 3.60 mL, 29.99 mmol, 3 eq) were added dropwise to an anhydrous THF (150 mL) solution of N-(4-bromo-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (PY-8 g) (3 g, 10 mmol, 1 eq). After the addition was complete, the reaction was allowed to proceed at 0-10 °C for 1.5 hours. 200 mL of water was added to the reaction solution at 0 °C, followed by extraction with ethyl acetate (200 mL × 3). The organic phases were combined and washed with saturated brine (100 mL). The mixture was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a yellow solid crude compound PY-Ua (3.20 g, crude product, yield: 48.63%, purity: 50%).

[1098] LCMS (ESI): m / z, 329.0 [M+1] + .

[1099] Step 2: Preparation of compound N,N'-(4-bromo-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (PY-Ub)

[1100] To a solution of compound PY-Ua (3.20 g, 50%, 4.86 mmol, 1 eq) in acetic acid (40 mL) and acetic anhydride (20 mL), zinc powder (4.77 g, 668.47 μL, 72.92 mmol, 15 eq) was added. After the addition was complete, the mixture was reacted at 15-20 °C for 16 hours. The reaction solution was concentrated to remove acetic acid, and the residue was poured into a mixture of ethyl acetate (50 mL) and water (50 mL). The mixture was stirred for 10 minutes, filtered through diatomaceous earth, and the filtrate was separated in a separatory funnel. The aqueous phase was extracted twice with ethyl acetate (50 mL × 2). The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol = 10:1) to give a yellow solid compound PY-Ub (1.15 g, yield: 55.63%, purity: 84%).

[1101] LCMS (ESI): m / z, 357 [M+H] + 359[M+H] + .

[1102] Step 3: Preparation of N-(8-amino-5-bromo-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphth-2-yl)acetamide (PY-Uc)

[1103] Compound PY-Ub (1.15 g, 84%, 2.70 mmol, 1 eq) was dissolved in ethanol (15 mL), and then HCl (3.28 g, 15 mL, 90 mmol, 33.2776 eq, 6 M) was added. The reaction mixture was stirred at 60 °C for 2 hours. Most of the solvent was concentrated from the reaction mixture, neutralized with sodium bicarbonate (30 mL), and then extracted with DCM (20 mL × 2). The organic phase was dried, filtered, concentrated under reduced pressure, and used directly in the next step. The resulting product was a brown solid (700 mg, yield: 68.99%).

[1104] LCMS (ESI): m / z, 315.0 [M+H] + .

[1105] Step 4: Preparation of N-((9S)-4-bromo-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)acetamide (PY-Ud)

[1106] Compound PY-Uc (700 mg, 2.22 mmol, 1 eq) was dissolved in toluene (50 mL). (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indoleazine-3,6,10(4H)-trione (PY-8f) (877.11 mg, 3.33 mmol, 1.5 eq) and PPTS (558.20 mg, 2.22 mmol, 1 eq) were added to this solution. The solution was stirred at 125 °C for 12 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by high-performance liquid chromatography (HPLC) to give a brown solid product (1 g, yield: 83.01%, purity: 75%).

[1107] LCMS (ESI): m / z, 542.1 [M+H] + .

[1108] Step 5: Preparation of N-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-(hydroxymethyl)-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)acetamide (PY-Ue)

[1109] Compound PY-Ud (300 mg, 75%, 414.85 μmol, 1 eq) was dissolved in a solution of dioxane (10 mL), and (tributyltin)methanol (399.62 mg, 1.24 mmol, 3 eq) and XPhos Pd G2 (65.28 mg, 82.97 μmol, 0.2 eq) were added. The reaction mixture was purged with nitrogen and stirred at 90 °C for 12 hours under a nitrogen atmosphere. The reaction mixture was concentrated under reduced pressure, and the residue was purified by high-performance liquid chromatography to give a white solid product (120 mg, yield: 58.6%).

[1110] LCMS (ESI): m / z, 494.1 [M+H] + .

[1111] Step 6: Preparation of compounds PY-UA and PY-UB

[1112] Compound PY-Ue (50 mg, 101.32 μmol, 1 eq) was dissolved in HCl (6 N, 6 mL), and DIPEA was added. The reaction mixture was stirred at 85 °C for 6 hours. The reaction mixture was purified by preparative high-performance liquid chromatography (Preparative chromatograph manufacturer: Oriendo, model R-120g. Column: Phenomenex Luna C18 250 × 50 mm × 10 μm. Mobile phase: water (0.225% HCOOH)-acetonitrile, elution ratio of water from 10% to 20%), yielding two white isomer solids: PY-UA (62 mg, yield: crude product yield negligible) and PY-UB (16 mg, yield: 34.98%).

[1113] LCMS (ESI): m / z, 452.2 [M+H] + .

[1114] Preparation Example 45: Preparation of (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxanepentano[4,5-g]pyrano[3',4':6,7]indolazano[1,2-b]quinoline-8,11(7H)-dione (PY-Y)

[1115]

[1116] Step 1: Preparation of 6-aminobenzo[d][1,3]dioxane-5-carboxaldehyde (PY-Y2)

[1117] Iron powder (3.94 g, 70.47 mmol, 5.5 eq) and ammonium chloride (3.77 g, 70.47 mmol, 5.5 eq) were added to ethanol (30.0 mL) and water (3.00 mL), followed by the addition of 6-nitrobenzene[d][1,3]oxacyclopentan-5-carboxaldehyde (2.50 g, 12.81 mmol, 1 eq). The reaction mixture was reacted at 80 °C for 4 hours. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was dissolved in water and dichloromethane (100 mL). The organic phase was washed with saturated sodium bicarbonate (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (SiO2, DCM:MeOH = 1:0, Rf...). (P) =0.39), yielding 6-aminobenzo[d][1,3]dioxane-5-carboxaldehyde (1.27 g, yield: 59.83%).

[1118] LCMS: RT=0.996min, MS(ESI)m / z=166.1[M+H] + .

[1119] Step 2: Preparation of (S)-7-ethyl-7-hydroxy-10,13-dihydro-11h-[1,3]dioxanepentano[4,5-g]pyrano[3',4':6,7]indolazano[1,2-b]quinoline-8,11(7H)-dione (PY-Y3)

[1120] Compound PY-8f (2.00 g, 7.60 mmol, 1 eq) and 6-aminobenzo[d][1,3]dioxane-5-carboxaldehyde (1.25 g, 7.60 mmol, 1 eq) were dissolved in toluene (160 mL), and then p-toluenesulfonic acid monohydrate (144.52 mg, 759.75 μmol, 0.1 eq) was added. The reaction mixture was stirred at 125 °C for 16 hours, cooled to 25 °C, filtered, and the filter cake was washed with 10 mL of tetrahydrofuran and dried under reduced pressure to give compound PY-Y3 (1.51 g, yield: 50.65%).

[1121] LCMS: RT=1.271min, MS(ESI)m / z=393.2[M+H] + .

[1122] Step 3: Preparation of (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxanepentano[4,5-g]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-8,11(7H)-dione (PY-Y)

[1123] Compound PY-Y3 (400 mg, 795.19 μmol, 1 eq) and ferrous sulfate heptahydrate (353.72 mg, 1.27 mmol, 1.6 eq) were dissolved in 10 mL of water. 98% sulfuric acid (4.59 g, 46.83 mmol, 2.50 mL, 58.89 eq) was added with stirring. After stirring thoroughly for 10 minutes, 4-hydroxybutyraldehyde (343.3 mg, 3.90 mmol, 5 eq) was added dropwise at 0 °C. Then, a solution of hydrogen peroxide (1.26 g, 11.10 mmol, 1.07 mL, 30% purity, 13.96 eq) in 20 mL of water was added dropwise. The reaction solution was stirred at 0-5 °C for 1 hour. The reaction solution was poured into ice water, the pH was adjusted to 8.0 with saturated sodium bicarbonate solution, extracted with ethyl acetate (50 mL × 5), concentrated under reduced pressure, and the residue was purified by high performance liquid chromatography (Xtimate C18 150 × 40 mm × 10 μm; mobile phase: [water (0.1% FA) - acetonitrile]; gradient: 10% - 44% acetonitrile, 36 min) to obtain compound PY-Y (99.5 mg, 99.6% purity).

[1124] LCMS: RT=1.890min, MS(ESI)m / z=451.3[M+H] + .

[1125] 1 HNMR (400MHz, DMSO-d) 6 )δppm 7.63(s,1H),7.50(s,1H),7.24(s,1H),6.50(s,1H),6.29(s,2H),5.42(s,2H),5.25(s,2H),4.68(t,J=5.2Hz,1H),3.49(t, J=5.6Hz,2H),3.15(t,J=6.8Hz,2H),2.53(t,J=1.6Hz,1H),2.33(t,J=1.6Hz,1H),1.81-1.88(m,2H),0.88(t,J=7.2Hz,3H).

[1126] Preparation Example 46: N 2 -(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl)-N 6 Preparation of -(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoyl)-L-lysine (A)

[1127]

[1128] Route 1:

[1129]

[1130] Step 1: N 2 -(benzyloxy)carbonyl)-N 6 Preparation of -(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoyl)-L-lysine (Ac)

[1131] At room temperature, (3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-one (Aa) (1.27 g, 7.13 mmol) and ((benzyloxy)carbonyl)-L-lysine (Ab) (2 g, 7.13 mmol) were dissolved in methanol (30 mL), followed by the addition of triethylamine (1.44 g, 14.27 mmol). The reaction mixture was stirred at 70 °C for 16 hours. The reaction mixture was concentrated under reduced pressure, and the residue was separated by high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250 × 50 mm × 10 μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 45% to 85%) to give a white solid compound Ac (2.5 g, yield 76.43%).

[1132] LCMS: [M+H] + =459.1.

[1133] Step 2: N 6 Preparation of -(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoyl)-L-lysine (Ad)

[1134] At room temperature, compound N 2 -(benzyloxy)carbonyl)-N 6 -(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoyl)-L-lysine (Ac) (2.5 g, 5.45 mmol) was dissolved in methanol (40 mL), and then 10% wet palladium on carbon (250 mg) was added to the reaction solution. The reaction solution was stirred at room temperature for 6 hours under a hydrogen atmosphere. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with water, and the filtrate was lyophilized to give a white solid compound Ad (1.6 g, yield: 88.96%).

[1135] LCMS: [M+H] + =325.1.

[1136] 1 H NMR (400MHz, DMSO-d) 6)δ4.16-4.19(d,J=9.4Hz,1H),3.93-3.97(t,J=9.4Hz,1H),3.72–3.50(m,5H) ,3.10-3.20(m,2H),1.68-1.79(m,2H),1.40-1.55(m,2H),1.20-1.35(m,2H).

[1137] Step 3: N 2 -(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl)-N 6 Preparation of -(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoyl)-L-lysine (A)

[1138] At room temperature, compound N 6 -(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoyl)-L-lysine (Ad) (1.6 g, 4.93 mmol) was dissolved in N,N-dimethylformamide (30 mL), and then 2,5-dioxopyrrolidone-1-yl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate (Ae) (1.52 g, 4.93 mmol) and DIEA (1.27 g, 9.87 mmol) were added to the solution. The reaction mixture was stirred at 70 °C for 16 hours under a nitrogen atmosphere. The reaction solution was filtered and separated by high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50mm×10μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 45% to 85%) to obtain a white solid linker A (400 mg, yield: 15.67%).

[1139] LCMS: [M+H] + =517.9.

[1140] 1 H NMR (400MHz, DMSO-d6) δ7.93(d,J=7.6Hz,1H),7.60(t,J=7.6Hz,1H),6.97(s,2H),5.32(m,1H),4.59–4.30(m,2H),4.17–3.99(m,1H),3.96-3. 91(m,1H),3.89-3.81(m,1H),3.57-3.5(m,1H),3.48-3.39(m,2H),3.3 4(s,4H),3.12–2.89(m,2H),2.05(t,J=6.4Hz,2H),1.82–0.95(m,13H).

[1141] Route 2:

[1142]

[1143] Step 1: N 2 -(tert-Butoxycarbonyl)-N 6 Preparation of -(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoyl)-L-lysine (Ag)

[1144] (tert-Butyloxycarbonyl)-L-lysine (Af) (5 g, 0.02 mol) and (3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-one (Aa) (3.6 g, 0.02 mol) were dissolved in MeOH (80 mL), and TEA (4.1 g, 0.04 mol) was added. The reaction mixture was reacted at 70 °C for 16 hours. The reaction solution was concentrated, and MTBE (50 mL × 3) was added and concentrated under reduced pressure. Then, it was slurryed with petroleum ether to obtain 8 g of crude product, which was a white foamy substance (the product was an acidic compound that formed a salt with TEA). The yield of the crude product was negligible.

[1145] LCMS (ESI): m / z, 425.2 [M+H] + 447.2 [M+Na] + .

[1146] 1 H NMR (400MHz, DMSO) δ7.50 (brs, 1H), 5.61 (d, J = 4.0Hz, 1H), 4.27 (s, 1H), 4.13 (s, 1H), 4.10-3.85 (m, 1H),3.85-3.66(m,4H),3.28-3.15(m,2H),1.80-1.55(m,2H),1.55-1.48(m,2H),1.50-1.21(m,11H.

[1147] Step 2: N 6 Preparation of -(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoyl)-L-lysine (Ad)

[1148] N 2 -(tert-Butoxycarbonyl)-N 6 -(2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanoyl)-L-lysine (Ag) (7g) was dissolved in DCM (35mL), and TFA (35mL) was added. The reaction mixture was reacted at 10-15℃ for 16 hours. The reaction solution was concentrated, and DCM (50mL×3) was added and concentrated under reduced pressure. Then, water was added to dissolve the solution and lyophilized to obtain 8g of crude yellow viscous substance, 100%.

[1149] 1 H NMR (400MHz, DMSO) δ9.26(s,1H),8.26(d,J=25.1Hz,3H),7.69(dd,J=37.2,31.4Hz,1H),3.99(d,J=3.7Hz,1H),3.94–3.84(m,2H),3.58(dd,J=13 .3,3.7Hz,1H),3.51–3.45(m,2H),3.40-3.34(m,1H),3.09(qt,J=15.7, 7.9Hz, 2H), 1.84–1.67 (m, 2H), 1.60–1.27 (m, 4H), 1.18 (t, J = 7.3Hz, 5H).

[1150] The remaining steps are the same as in route one, resulting in connector A.

[1151] Preparation Example 47: Preparation of antibody tag 2-(2-aminoethoxy)-N-(3-azidopropyl)acetamide hydrochloride (TS-1)

[1152]

[1153] Step 1: Preparation of tert-butyl carbamate (2-(2-(3-azidopropyl)amino)-2-oxoethoxy)carbamate (TS-13)

[1154] To a solution of 2-(2-(tert-butoxycarbonyl)amino)ethoxy)acetic acid (TS-12) (Haoyuan Pharmaceutical, 450.0 mg, 2.0 mmol, 1.0 eq) in dichloromethane (4.5 mL), 3-azidopropylamine (205.4 mg, 2.0 mmol, 1.0 eq), DIEA (662.6 mg, 5.1 mmol, 2.5 eq), and T3P (2.3 g, 3.7 mmol, 1.8 eq) were added sequentially. After stirring at room temperature for 16 hours, the mixture was diluted with 30 mL of dichloromethane and washed with 20 mL of water. The aqueous phase was extracted once with 30 mL of dichloromethane, and the organic phases were combined and washed with 20 mL of saturated brine. The organic phase was dried, filtered, and concentrated. The residue was purified by rapid silica gel column chromatography (eluent: ethyl acetate / n-hexane 1:20 to 1:1) to give a colorless oily compound TS-13, 492 mg (yield 79%).

[1155] LCMS(ESI): m / z, 302[M+H] + .

[1156] 1H NMR (400MHz, Chloroform-d) δ6.84(s,1H),4.88(s,1H),3.95(s,2H),3.56(t,J=5.2Hz,2H),3.41–3.31(m,6H),1.81(q,J=6.7Hz,2H),1.44(s,9H).

[1157] Step 2: Preparation of 2-(2-aminoethoxy)-N-(3-azidopropyl)acetamide hydrochloride (TS-1)

[1158] In a three-necked flask, tert-butyl(2-(2-(((3-azidopropyl)amino)-2-oxoethoxy)ethyl)carbamate (TS-13) (482.0 mg, 1.6 mmol, 1.0 eq) and 5 mL of methanol were added. A 1.6 mL solution of 6 M HCl in 1,4-dioxane was slowly added dropwise at 0 °C, with the temperature not exceeding 5 °C. After stirring at room temperature for 16 hours, the reaction mixture was concentrated. The residue was purified by high-performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250 × 50 mm × 10 μm; mobile phase: water (0.1% HCl)-acetonitrile, with water elution ratios ranging from 55% to 85%), and lyophilized to give 94.8 mg of a colorless oily compound TS-1 (yield 24.7%).

[1159] LCMS(ESI): m / z, 202[M+H] + .

[1160] 1 H NMR(400MHz,DMSO-d6)δppm 1.67-1.72(m,J=6.85Hz,2H),2.97–3.04(m,J=5.30Hz,2H),3.15–3.20(q,J=6.71Hz,2H),3 .34-3.37(t,J=6.82Hz,2H),3.62-3.64(t,J=5.00Hz,2H),3.91(s,2H),8.21-8.30(m,3H).

[1161] Example 1: Preparation of compound LY-1

[1162]

[1163]

[1164] Step 1: Preparation of methyl-L-phenylalanylglycine tert-butyl ester (LY-1c)

[1165] ((benzyloxy)carbonyl)-L-phenylalanine (LY-1a) (3.0 g, 10.0 mmol) was dissolved in 50 mL of DMF, and glycine tert-butyl hydrochloride (1.9 g, 10.1 mmol), HOBt (0.68 g, 5.0 mmol), EDCI (2.3 g, 12.0 mmol), and DIPEA (3.87 g, 30.0 mmol) were added sequentially. The mixture was stirred at room temperature for 18 h under a nitrogen atmosphere. After dilution with water (80 mL), the mixture was extracted twice with DCM (100 mL), and the organic phases were combined. The mixture was washed with saturated brine, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane 1:20 to 1:5) to give compound LY-1c (3.5 g, 85% yield) as a colorless oil.

[1166] Step 2: Preparation of L-phenylalanylglycine tert-butyl ester (LY-1d)

[1167] Methyl-L-phenylalanine tert-butyl ester (LY-1c) (3.0 g, 7.3 mmol) was dissolved in 30 mL of dichloromethane, and 10 mL of trifluoroacetic acid was added at 0 °C. The mixture was stirred at room temperature for 2 h under a nitrogen atmosphere. The solution was then concentrated under reduced pressure. The product, a pale yellow solid, was used directly in the next step without purification.

[1168] Step 3: Preparation of ((9H-fluorene-9-yl)methoxy)carbonyl)glycyl-L-phenylalanylglycine (LY-1f)

[1169] L-phenylalanylglycine tert-butyl ester (LY-1d) (1.39 g, 5 mmol) and LY-1e (1.49 g, 5 mmol) were dissolved in 50 mL of DMF, and HOBt (0.68 g, 5.0 mmol), EDCI (1.9 g, 10.0 mmol), and DIPEA (3.87 g, 30.0 mmol) were added sequentially. The mixture was stirred at room temperature for 18 h under a nitrogen atmosphere. The pH was adjusted to 1-2 with 30 mL of dilute hydrochloric acid, and the mixture was extracted twice with 100 mL of DCM. The organic phases were combined, washed with saturated brine, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane 1:20 to 1:5) to give compound LY-1f (3.1 g, yield 84%, two steps), a white solid.

[1170] LCMS (ESI): m / z, 502.4 [M+H] + .

[1171] Step 4: Preparation of (9H-fluorene-9-yl)methyl-(2-(1-(2-(4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)carbamate (LY-1g)

[1172] The compound ((9H-fluorene-9-yl)methoxy)carbonyl)glycyl-L-phenylalanylglycine (2.5 g, 5 mmol) was dissolved in 20 mL of DMF, and HATU (2.3 g, 6.0 mmol), DIPEA (1.9 g, 15.0 mmol), and 4-aminobenzyl alcohol (0.74 g, 6 mmol) were added sequentially. The mixture was stirred at room temperature for 10 h under a nitrogen atmosphere. The solution was diluted with 50 mL of water, extracted twice with 100 mL of DCM, and the organic phases were combined, washed with saturated brine, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane 1:20 to 1:5) to give compound LY-1 g (2.8 g, 92% yield) as a white solid.

[1173] LCMS (ESI): m / z, 607.2 [M+H] + .

[1174] Step 5: Preparation of (9H-fluorene-9-yl)methyl-(2-(1-(2-(4-(4-(nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)carbamate (LY-1h)

[1175] (9H-fluorene-9-yl)methyl-(2-(1-(2-(4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)carbamate (LY-1g) (2.0g, 3.3mmol) and bis(4-nitrophenyl) carbonate (2g, 6.6mmol) were dissolved in DMF (20mL), and DIPEA (0.43g, 3.3mmol) was added. The mixture was stirred at room temperature for 2h under a nitrogen atmosphere. The solution was diluted with water (30mL), extracted twice with DCM, and the organic phases were combined, washed with saturated brine, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate / n-hexane 1:20 to 1:5) to give compound LY-1h (0.92g, yield 36%) as a white solid.

[1176] Step 6: Preparation of compounds (LY-1i) and (LY-1j)

[1177] (9H-fluorene-9-yl)methyl-(2-(1-(2-(4-(4-(nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)carbamate (LY-1h) (397 mg, 0.54 mmol) and icettecan mesylate (300 mg, 0.56 mmol) were dissolved in DMF (2 mL). HOBt (73 mg, 0.54 mmol), pyridine (425 mg, 5.38 mmol), and DIPEA (208 mg, 1.61 mmol) were added, and the mixture was stirred overnight at room temperature to obtain LY-1i. NMM (109 mg, 1.08 mmol) was added, and the mixture was stirred at room temperature for 8 h. The reaction solution was separated by preparative HPLC (column: Phenomenex LunaC18 250×50mm×10μm; mobile phase: water (0.225% HCOOH)-acetonitrile, eluting from 45% to 85%) to obtain compound LY-1j (123 mg, yield 21%), a white solid.

[1178] LCMS (ESI): m / z, 1068.5 [M+H] + .

[1179] Step 7: Preparation of compound (LY-1)

[1180] To a solution of linker A (11.2 mg, 0.022 mmol, 1.2 eq) in anhydrous tetrahydrofuran (7 mL), HATU (10.3 mg, 0.027 mmol, 1.5 eq), DIPEA (7.0 mg, 0.054 mmol, 3.0 eq), and LY-1j (15 mg, 0.018 mmol, 1 eq) were added sequentially. The reaction mixture was stirred at 60 °C for 2 hours. The reaction mixture was purified by high performance liquid chromatography (HPLC) (column: Phenomenex Luna C18 250 × 50 mm × 10 μm; mobile phase: water (0.225% HCOOH)-acetonitrile, eluting from 45% to 85%), and then lyophilized to give a yellow solid, LY-1 (9.4 mg, yield 38%).

[1181] LCMS (ESI): m / z, 1345.6 [M+H] + 673.5 [1 / 2M+H] + .

[1182] 1H NMR (400MHz, DMSO-d6) δ9.87 (s, 1H), 8.40 (s, 1H), 8.10 (dd, J = 20.8, 8.0Hz, 3H) ,7.94(d,J=7.2Hz,1H),7.78(d,J=10.8Hz,1H),7.66–7.53(m,3H),7.37(d,J=8. 2Hz,2H),7.31(s,1H),7.27–7.22(m,3H),7.18(t,J=4.4Hz,1H),6.98(d,J=2.2 Hz,2H),6.53(s,1H),5.44(s,2H),5.35(s,1H),5.29(s,3H),5.08(s,2H),4.50( dd,J=13.6,8.4Hz,5H),4.18–4.07(m,1H),3.96(s,1H),3.93–3.83(m,3H),3.7 2(s,1H),3.64–3.51(m,2H),3.46(s,2H),3.23(s,4H),3.05(dt,J=15.2,7.2Hz, 5H),2.90–2.77(m,2H),2.38(d,J=1.9Hz,3H),2.25–2.01(m,5H),1.92–1.82(m ,2H),1.58(s,2H),1.49–1.33(m,6H),1.26–1.10(m,4H),0.87(t,J=7.3Hz,3H).

[1183] Example 2: Preparation of compound LY-2

[1184]

[1185] Step 1: Preparation of ((9H-fluorene-9-yl)methoxy)carbonyl)glycyl-L-pentanoyl-L-alanine tert-butyl ester (LY-2b)

[1186] (((9H-fluorene-9-yl)methoxy)carbonyl)glycyl-L-valine (LY-2a) (8 g, 20.2 mmol) and tert-butyl glycine (3.66 g, 20.2 mmol) were added to a 250 mL three-necked flask. DMF (80 mL) was added, and DIEA (7.8 g, 60.6 mmol) was added dropwise under a nitrogen atmosphere. After the addition was complete, the mixture was stirred for 5 minutes. Then, a DMF (30 mL) solution of HATU (9.2 g, 24.2 mmol) was added dropwise. After the addition was complete, the mixture was stirred at room temperature for 1 hour. The reaction solution was slowly poured into water (800 mL), stirred for 30 minutes, filtered, and the solid was dissolved in DCM (200 mL). The solid was washed once with water, and the organic phase was dried over anhydrous sodium sulfate. The solution was filtered, concentrated, and 9.5 g of a yellow oil was obtained, with a yield of 90%.

[1187] 1 H NMR (400MHz, DMSO) δ8.32(d,J=6.7Hz,1H),7.88(d,J=7.5Hz,2H),7.70(d,J=7.3Hz,3H),7.51(t,J=6.0Hz,1H),7.41(t,J=7.4Hz,2H),7 .32(t,J=7.4Hz,2H),4.19(m,5H),3.66(d,J=5.1Hz,2H),1.95(m,1H),1.37(s,9H),1.23(d,J=7.2Hz,3H),0.85(dd,J=18.7,6.8Hz,6H).

[1188] Step 2: Preparation of ((9H-fluorene-9-yl)methoxy)carbonyl)glycyl-L-valine-L-alanine (LY-2c)

[1189] ((9H-fluorene-9-yl)methoxy)carbonyl)glycyl-L-pentanoyl-L-alanine tert-butyl ester (LY-2b) (6 g, 11.47 mmol) was dissolved in DCM (30 mL). Under nitrogen atmosphere, TFA (20 mL) was added dropwise, and the reaction was allowed to proceed at room temperature for 2 hours after the addition was complete. The reaction solution was concentrated to a small volume, and the pH was adjusted to 7-8 with saturated sodium bicarbonate solution. Impurities were extracted with DCM (100 mL), and the pH was adjusted to 1-2 with 1N HCl. Extraction was performed with EA (200 mL × 2). The organic phase was washed with water (100 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 4.8 g of white solid, yield: 89%.

[1190] 1 H NMR (400MHz, DMSO) δ7.88(d,J=7.5Hz,2H),7.70(d,J=7.4Hz,2H),7.41(t,J=7.4Hz,2H),7.32(t,J=7. 3Hz, 2H), 4.21 (m, 5H), 3.65 (s, 2H), 1.92 (m, 1H), 1.26 (d, J = 7.3Hz, 3H), 0.84 (dd, J = 20.8, 6.7Hz, 6H).

[1191] Step 3: Preparation of (9H-fluorene-9-yl)methyl(2-((S)-1-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-2-oxoethyl)carbamate (LY-2d)

[1192] ((9H-fluorene-9-yl)methoxy)carbonyl)glycyl-L-valine-L-alanine (LY-2c) (2.5 g, 5.35 mmol) and aminobenzyl alcohol (645 mg, 5.24 mmol) were added to a reaction flask and dissolved in DMF (25 mL). Under a nitrogen atmosphere, 2,6-dimethylpyridine (1.72 g, 16 mmol) was added, and the mixture was stirred for 5 minutes. Then, a solution of HATU (2.44 g, 6.4 mmol) in DMF (8 mL) was added dropwise, and the mixture was allowed to react at room temperature for 1 hour. The reaction solution was then added to water (200 mL), extracted with EA (200 mL × 2), and the organic phase was washed with dilute hydrochloric acid and sodium bicarbonate. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 2.4 g of a nearly white solid, yield: 80%.

[1193] 1 H NMR (400MHz, DMSO) δ7.90(d,J=7.5Hz,2H),7.70(d,J=7.5Hz,2H),7.55(d,J=8.3Hz,2H),7.43(t,J=7.4Hz,2H),7.33(t,J=7.4Hz,2H),7.24(d,J=8.3H z,2H),4.48–4.35(m,3H),4.25(dd,J=21.4,6.6Hz,4H),3.70(d,J=2.4Hz,2 H), 2.00–1.95 (m, 1H), 1.33 (d, J = 7.1Hz, 3H), 0.87 (dd, J = 19.4, 6.8Hz, 6H).

[1194] Step 4: Preparation of (9H-fluorene-9-yl)methyl(2-((S)-3-methyl-1-((S)-1-((4-(nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropane-2-yl)amino)-1-oxobutane-2-yl)amino)-2-oxoethyl)carbamate (LY-2e)

[1195] (9H-fluorene-9-yl)methyl(2-((S)-1-((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-2-oxoethyl)carbamate (LY-2d) (1 g, 1.74 mmol) and di(4-nitrophenyl)) carbonate (0.8 g, 2.62 mmol) were dissolved in DMF (10 mL). Under a nitrogen atmosphere, DIEA (563 mg, 4.37 mmol) was added dropwise, and the reaction was allowed to proceed at room temperature for 2 hours after the addition was complete. The reaction solution was added to water (50 mL), extracted with EA (100 mL × 2), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (PE:EA = 10:1) to give 1 g of near-white solid, yield: 78%.

[1196] 1 H NMR (400MHz, DMSO) δ9.96 (s, 1H), 8.35–8.23 (m, 3H), 7.87 (m, 3H), 7.62 (m, 7H), 7.41 (t, J = 6.8Hz, 4H), 7.32 (t, J = 7.0Hz, 2H),5.23(s,2H),4.45–4.33(m,1H),4.22(m,4H),3.70(s,2H),1.99(m,1H),1.33(m,3H),0.86(dd,J=19.9,6.8Hz,6H).

[1197] Step 5: Preparation of (9H-fluorene-9-yl)methyl(2-((S)-1-((S)-1-((4-(((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4:6,7]indolazino[1,2-b]quinoline-1-yl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)amino)-2-oxoethyl)carbamate (LY-2f)

[1198] (9H-fluorene-9-yl)methyl(2-((S)-3-methyl-1-((S)-1-((4-(nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropane-2-yl)amino)-1-oxobutane-2-yl)amino)-2-oxoethyl)carbamate (LY-2e) (415 mg, 0.54 mmol) and icettecon mesylate (300 mg, 0.56 mmol) were dissolved in DMF (2 mL), and HOBt (73 mg, 0.54 mmol), pyridine (425 mg, 5.38 mmol), and DIPEA (208 mg, 1.61 mmol) were added. The mixture was stirred overnight at room temperature to obtain LY-2f. The reaction solution was used directly for the next step.

[1199] Step 6: Preparation of compound (LY-2g)

[1200] The reaction solution from step 5 was added to piperidine (92 mg, 1.08 mmol) and stirred at room temperature for 8 h. The reaction solution was purified by high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50 mm×10 μm; mobile phase: water (0.225% HCOOH)-acetonitrile, eluting from 45% to 85%) to give compound LY-2 g (81.0 mg, yield 18.5%), a white solid, which was used directly in the next step.

[1201] LCMS (ESI): m / z, 834.4 [M+Na] + 812.5 [M+H] + .

[1202] Step 7: Preparation of compound (LY-2)

[1203] To a solution of linker A (3.9 mg, 0.0074 mmol, 1.2 eq) in anhydrous tetrahydrofuran (2 mL), HATU (2.83 mg, 0.0074 mmol, 1.2 eq), DIPEA (2.4 mg, 0.019 mmol, 3.0 eq), and compound LY-2 (5 mg, 0.0062 mmol, 1 eq) were added sequentially. The reaction mixture was stirred at 60 °C for 2 hours. The reaction mixture was separated by high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250 × 50 mm × 10 μm; mobile phase: water (0.225% HCOOH)-acetonitrile, eluting from 45% to 85%), and lyophilized to give a yellow solid compound LY-2 (2.0 mg, yield 24.6%).

[1204] LCMS(ESI): m / z, 1333.5[M+Na]+ 1311.6 [M+H] + 656.4 [1 / 2M+H] + .

[1205] 1 H NMR (400MHz, DMSO-d) 6 )δ9.91(s,1H),8.25–8.14(m,2H),8.06(d,J=8.8Hz,1H),7.93(d,J=7.6Hz,1H),7.75(dd,J=21.2,9.6Hz,2H),7.65–7.55(m,3H),7.36(d,J =8.2Hz,2H),7.31(s,1H),7.00(s,2H),6.52(s,1H),5.45(s,2H),5.29(s,3H),5.07(s,2H),4.37(t,J=7.0Hz,2H),4.23–4.10(m,3H),3.97 (d,J=3.6Hz,1H),3.90(s,1H),3.73(d,J=5.6Hz,2H),3.59–3.53(m,1H),3.46(s,2H),3.22(s,1H),3.04(t,J=7.6Hz,3H),2.38(s,3H),2.2 5–2.04(m,5H),2.01–1.80(m,4H),1.60(s,2H),1.52–1.35(m,7H),1.30(d,J=7.2Hz,3H),1.19(dt,J=15.4,9.0Hz,4H),0.92–0.75(m,9H).

[1206] Example 3: Preparation of compound LY-3

[1207]

[1208] Step 1: Preparation of tert-butyl (2-(S)-1-(S)-1-(4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-2-oxoethyl)carbamate (LY-3b)

[1209] At room temperature, (S)-2-((S)-2-amino-3-methylbutamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (Haoyuan Biotechnology, LY-3a) (300 mg, 0.79 mmol) was dissolved in N,N-dimethylformamide (10 mL), followed by the addition of Boc-protected glycine (138.5 mg, 0.79 mmol), EDCI (227.7 mg, 1.19 mmol), HOBT (53.4 mg, 0.40 mmol), and triethylamine (240 mg, 2.37 mmol). The reaction mixture was stirred at room temperature for 16 hours. The reaction solution was filtered and separated by high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50mm×10μm; mobile phase: water (0.225% HCOOH)-acetonitrile, eluting from 45% to 85%) to give a white solid compound LY-3b (300 mg, yield 69.04%).

[1210] LCMS: [M+H] + =537.3.

[1211] 1 H NMR (400MHz, DMSO-d6) δ9.84(s,1H),8.32–8.04(d,J=7.6Hz,1H),7.58-7.64(d,J=8.6Hz,1H),7.49-7.54(d,J=8. 8Hz,2H),7.17-7.22(d,J=8.8Hz,2H),7.00-7.08(t,J=7.6Hz,1H),5.90-6.00(t,J=5.6Hz,1H),5.37(s,2H),5.06( t,J=6.0Hz,1H),4.37-4.42(d,J=5.6Hz,2H),4.30–4.36(m,1H),4.19-4.26(m,1H),3.52-3.58(m,2H),2.85-3.05 (m,3H),2.05-2.07(m,1H),1.85-2.05(m,1H),1.74–1.44(m,2H),1.33(s,9H),0.75–0.90(dd,J=17.6,6.8Hz,6H).

[1212] Step 2: Preparation of tert-butyl carbamate (LY-3c)

[1213] At room temperature, (2-(S)-1-(S)-1-(4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-2-oxoethyl)aminomethyltert-butyric acid (LY-3b) (250 mg, 0.466 mmol) was dissolved in N,N-dimethylformamide (10 mL), and 4-nitrocarbonate (383.45 mg, 0.932 mmol) and DIEA (180.3 mg, 1.4 mmol) were added. The reaction mixture was stirred at room temperature for 16 hours. The reaction solution was filtered and separated by high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50mm×10μm; mobile phase: water (0.225% HCOOH)-acetonitrile, eluting from 45% to 85%) to give a yellow solid compound LY-3c (200 mg, yield 61.18%).

[1214] LCMS: [M+H] + =702.3.

[1215] Step 3: Preparation of compound (LY-3d)

[1216] Compound (LY-3c) (200 mg, 0.285 mmol) was dissolved in N,N-dimethylformamide (10 mL) at room temperature, followed by the addition of icetane mesylate (148.9 mg, 0.342 mmol), HOBT (19.26 mg, 0.143 mmol), and DIEA (110.3 mg, 0.855 mmol). The reaction mixture was stirred at room temperature for 8 hours. The reaction mixture was filtered and separated by high-performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50 mm×10 μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 45% to 85%) to obtain a white solid compound LY-3d (200 mg, yield 70.31%).

[1217] LCMS: [M+H] + =998.60.

[1218] Step 4: Preparation of compound (LY-3e)

[1219] Compound LY-3d (200 mg, 0.2 mmol) was added to a 10% trifluoroacetic acid / dichloromethane solution (15 mL) at 0 °C, and the reaction mixture was stirred at 0 °C for 4 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by preparative HPLC (mobile phase: acetonitrile / water; acidic system: 0.1% HCl) to give a yellow solid compound LY-3e (120 mg, yield: 66.69%).

[1220] LCMS: [M+H] + =898.3.

[1221] Step 5: Preparation of compound (LY-3)

[1222] At room temperature, compound LY-3e (120 mg, 0.133 mmol), linker A (83 mg, 0.160 mmol), and HATU (61 mg, 0.160 mmol) were dissolved in N,N-dimethylformamide (6 mL), and then DIEA (51.72 mg, 0.4 mmol) was added. The reaction mixture was stirred at 60 °C for 1 hour. The reaction mixture was filtered and then separated by high performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50 mm×10 μm; mobile phase: water (1% formic acid)-acetonitrile, eluting from 45% to 85%) to give a yellow solid compound LY-3 (74.4 mg, yield 39.84%).

[1223] LCMS: [M / 2+2H] + =699.4.

[1224] 1H NMR (400MHz, DMSO-d6) δ9.92 (s, 1H), 8.18-8.08 (m, 2H), 8.02 (d, J = 8.5Hz, 1H), 7 .90(d,J=6.0Hz,1H),7.73(d,J=10.9Hz,1H),7.68(d,J=9.0Hz,1H),7.57(m,3H), 7.32(d,J=7.2Hz,2H),7.27(s,1H),6.96(s,2H),5.97(s,1H),5.41(s,2H),5.24 (m,3H),5.04(s,2H),4.33(m,1H),4.19(m,1H),4.13–4.06(m,1H),3.94(d,J=4Hz 1H),3.86(m,1H),3.70(m,3H),3.31-3.17(m,9H),3.13-2.83(m,7H),2.33(m,3H),2.22–1.02(m,25H),0.97–0.67(m,10H).

[1225] Example 4: Preparation of compound LY-5

[1226]

[1227] Step 1: N 6 -(tert-Butoxycarbonyl)-N 2 Preparation of -(6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoyl)-L-lysine (LY-5a)

[1228] Under a nitrogen atmosphere, N2 is added to the reaction flask. 6 -(tert-Butoxycarbonyl)-L-lysine (300 mg, 1.0 eq), 2,5-dioxopyrrolidone-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoate (Ae) (375 mg, 1.0 eq), and DIPEA (315 mg, 2 eq) were added, followed by the addition of 10 mL of anhydrous DMF. After purging with nitrogen three times, the reaction was carried out at room temperature for 16 h. The mixture was then separated by high-performance liquid chromatography (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250×50 mm×10 μm; mobile phase: water (0.225% trifluoroacetic acid)-acetonitrile, eluting from 45% to 85%) to obtain 400 mg of colorless colloidal compound LY-5a.

[1229] LCMS(ESI): m / z, 340.2[M-100+1] + 462.3 [M+Na] + .

[1230] Step 2: Preparation of compound (LY-5b)

[1231] Under a nitrogen atmosphere, compounds LY-5a (6.33 mg, 1.3 eq), HATU (5 mg, 1.2 eq), LY-2g (9 mg, 1.0 eq), and DIPEA (4.3 mg, 3 eq) were added to a reaction flask, followed by 3 mL of DMF. The reaction was carried out at room temperature for 2 h under a nitrogen atmosphere. The reaction solution was separated by preparative high-performance liquid chromatography (HPLC) (column: Phenomenex Luna C18 250×50 mm×10 μm; mobile phase: water (0.225% HCOOH)-acetonitrile, eluting from 45% to 85%) to obtain 2 mg of compound LY-5b as a yellow solid.

[1232] LCMS(ESI): m / z, 1255.5[M+Na] + , 1233.8[M+H] + .

[1233] Step 3: Preparation of compound (LY-5)

[1234] Compound LY-5b (2 mg, 1.0 eq) was added to a reaction flask, followed by 3 mL of DCM and 0.3 mL of TFA. The reaction mixture was reacted at 15 °C for 1 hour. The reaction solution was concentrated under reduced pressure, and 5 mL of DCM was added. The solution was then concentrated under reduced pressure. The residue was separated by preparative HPLC (Oriendo, BRIX-2860; column: Phenomenex Luna C18 250 × 50 mm × 10 μm; mobile phase: water (0.225% HCOOH)-acetonitrile, eluting from 45% to 85%) to give 0.61 mg of compound LY-5b as a white solid.

[1235] LCMS(ESI): m / z, 1155.6[M+Na] + 1133.6 [M+H] + .

[1236] 1 H NMR (400MHz, DMSO-d) 6 )δ 1 H NMR (400MHz, DMSO-d) 6)δ9.92(s,1H),8.22(d,J=6.8Hz,1H),8.16(s,1H),8.01(dd,J=39.6,8.2Hz,3H),7.79(d,J=10.4Hz,2H),7.59(d,J=8.2Hz,4H),7.36(d,J=8.2H z,2H),7.31(s,1H),7.00(s,2H),6.53(s,1H),5.45(s,2H),5.29(s,2H) ,5.08(s,2H),4.38(t,J=7.0Hz,2H),4.20(d,J=7.7Hz,2H),3.74(d,J=5 .7Hz,2H),2.78-2.72(m,3H),2.68-2.65(m,2H),2.38(s,3H),2.34-2.3 2(m,3H),2.20(d,J=10.4Hz,2H),2.10(t,J=7.2Hz,2H),1.98(q,J=6.8H z,2H),1.88(dd,J=15.6,7.2Hz,3H),1.52-1.48(m,4H),1.31(d,J=7.2Hz,3H),1.18(t,J=7.8Hz,2H),0.89-0.86(m,4H),0.82(d,J=6.8Hz,3H).

[1237] Example 5: Preparation of compound LY-6

[1238]

[1239]

[1240] Step 1: Preparation of ((1R,8S,9S)bicyclo[6.1.0]non-4-yn-9-yl)methyl(4-nitrophenyl)carboxylic acid ester (LY-61)

[1241] ((1R,8S,9S)bicyclo[6.1.0]non-4-yn-9-yl)methanol (BCN) (500 mg, 3.33 mmol) and DIE...

Claims

1. A compound of Formula (A) or a pharmaceutically acceptable salt thereof, L-L2-L1-Dr (A) wherein: Dr is selected from the following structures: R 1 selected from hydrogen, C1-C6alkyl; L1is selected from -NR b C(=O)O-(CH2) 4-6 -O-*, wherein * is the point of attachment to L2; or Dr is selected from the following structures: L1is selected from -(CH2) m -O-*, -(CH2) m -NR a -*, -O-(CH2) m -NR a -*, wherein * is the point of attachment to L2; R a selected from hydrogen, C1-C6alkyl; R b selected from hydrogen, C1-C6alkyl; m is an integer from 1 to 6; R 2 selected from hydrogen, halogen; L2is selected from a bond, wherein * is the site of attachment to L1; L is L3 is an amino acid residue formed from two or more amino acids, and L3 optionally comprises one or more structures selected from the following, L6 is selected from one or more structures: wherein R, R aa , R bb are each independently selected from hydrogen and Ci-C6alkyl; L4 is Z1 is selected from the bond, -(CH2). p -、-(C2H4O) q -、-(CH2) p -C(O)NH-, -(CH2) p -O-(CH2) p -C(O)NH-, -(CH2) p -C(O)-L6-NH-、-(CH2) p -O-(CH2) p -C(O)-L6-NH-, -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)-, -NH-, -O- and -OC(O)NH-; s is an integer from 1 to 6; t is an integer from 0 to 10; s1, s2, s3, s4 are each independently an integer from 0 to 10; s5, s6 are each independently an integer from 1 to 6; t1 is an integer from 1 to 6; t2 is an integer from 0 to 6; t3 is an integer from 1 to 6; t4 is an integer from 0 to 10; t5 is an integer from 0 to 10; p is an integer from 1 to 10; q is an integer from 1 to 10; Q is a linker unit.

2. The compound of Formula (A) or a pharmaceutically acceptable salt thereof according to claim 1, wherein, Dr is selected from the following structures: L1is selected from -NR b C(=O)O-(CH2)4-O-*, wherein * is the point of attachment to L2.

3. The compound of Formula (A) or a pharmaceutically acceptable salt thereof according to claim 1, wherein, Dr is selected from the following structures: L1is selected from -(CH2) m -O- *, -(CH2) m -NR a - *, wherein * is the point of attachment to L2; m is an integer from 1 to 4.

4. The compound of claim 1, represented by the general formula (A) or a pharmaceutically acceptable salt thereof, wherein, Dr is selected from:

5. The compound of Formula (A) or a pharmaceutically acceptable salt thereof according to any one of claims 1, 3-4, wherein, when L1is selected from -(CH2) m -NR a -*, -0-(CH2) m -NR a -*, wherein * is the point of attachment to L2; L2is selected from a bond or wherein * is the site of attachment to L1; R a , m are as defined in any one of claims 1, 3 to 4.

6. The compound of Formula (A) or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein, when L1is selected from -(CH2) m -O-*, -NR b C(=O)O-(CH2) 4-6 -O-*, where * is the point of attachment to L2; L2is selected from a bond, wherein * is the site of attachment to L1; R b , m are as defined in any one of claims 1 to 4.

7. The compound of Formula (A) or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein, L3 is an amino acid residue formed from two or more amino acids selected from phenylalanine, alanine, glycine, valine, leucine, isoleucine, tryptophan, tyrosine, histidine, lysine, citrulline, serine, threonine, cysteine, glutamic acid, glutamine, aspartic acid, asparagine, methionine, arginine, and L3 optionally comprises one or more structures selected from the following: wherein R, R aa , R bb are each independently selected from hydrogen and Ci-C6alkyl; s is an integer from 2 to 6; s5, s6 are each independently an integer from 2 to 6; t is an integer from 0 to 10; t1 is an integer from 1 to 4; t2 is an integer from 1 to 4; t3 is an integer from 1 to 4; t4 is an integer from 0 to 10; t5 is an integer from 0 to 10.

8. The compound of Formula (A) or a pharmaceutically acceptable salt thereof according to claim 7, wherein, L3 is * is the position of attachment to L2, • is the position of attachment to a carbonyl or methylene group; wherein L 1b and L' 1b each independently is an amino acid residue formed from one or more amino acids selected from phenylalanine, alanine, glycine, valine, leucine, isoleucine, tryptophan, tyrosine, histidine, lysine, citrulline, serine, threonine, cysteine, glutamic acid, glutamine, aspartic acid, asparagine, methionine, arginine; L 1a is a bond or one or more structures selected from the group consisting of: wherein R is selected from hydrogen and C1-C6 alkyl; R aa , R bb each independently is selected from the group consisting of C1-C6alkyl.

9. The compound of Formula (A) or a pharmaceutically acceptable salt thereof according to claim 8, wherein, L3 is selected from: wherein: * is the position of attachment to L2, for the position of attachment to the carbonyl or methylene group.

10. The compound of Formula (A) or a pharmaceutically acceptable salt thereof according to claim 9, wherein, s is an integer from 2 to 4; s5, s6 are each independently an integer from 2 to 4; t1 is 1 or 2; t2 is 1 or 2; t3 is 1 or 2.

11. The compound of any one of claims 8-10, or a pharmaceutically acceptable salt thereof, wherein L 1b and L' 1b each independently is an amino acid residue formed from two or more amino acids selected from the group consisting of glycine, phenylalanine, citrulline, valine, lysine, glutamine, glutamic acid, aspartic acid, leucine, alanine.

12. The compound of Formula (A) or a pharmaceutically acceptable salt thereof according to any one of claims 8 to 10, wherein, L 1b and L' 1b each independently is selected from the group consisting of -Gly-*, -Val-*, -Gly-Phe-Gly-*, -Phe-Gly-*, -Gly-Val-Cit-*, -Val-Cit-*, -Gly-Val-Arg-*, -Val-Arg-*, -Gly-Val-Ala-*, -Val-Ala-*, -Gly-Phe-*, -Phe-Gly-*, -Gly-Gly-Gly-*, -Gly-Gly-*, -Gly-Val-Gly-*, -Gly-Ala-Gly-*, -Gly-Phe-Cit-*, -Gly-Phe-Val-*, -Gly-Phe-Ala-*, -Gly-Phe-Lys-*, -Phe-Lys-*, -Gly-Val-*, -Gly-Cit-*, -Gly-Ala-*, -Gly-Gly-Lys-*, -Gly-Lys-*, -Ala-Ala-Ala-*, -Gln-Val-Ala-*, -Gln-Val-Cit-*, -Asp-Val-Ala-*, -Asp-Val-Cit-*, -Lys-Gly-Val-Ala-*, -Lys-Gly-Val-Cit-*, -Lys-Gly-Gly-Val-Ala-*, -Lys-Gly-Gly-Val-Cit-*, -Gly-Gly-Phe-Gly-*, -Lys-Gln-Val-Cit-*, -Lys-Gln-Val-Ala-*, -Lys-Glu-Val-Cit-*, -Lys-Glu-Val-Ala-*, -Lys-Asp-Val-Cit-*, -Lys-Asp-Val-Ala-*, -Glu-Val-Cit-*, -Glu-Val-Ala-*, -Lys-Val-Ala-*, -Lys-Val-Cit-*, -Val-Lys-Gly-*, -Val-Lys-*, wherein * is the position of attachment to L2.

13. The compound of Formula (A) or a pharmaceutically acceptable salt thereof according to claim 7, wherein, L3 is selected from: R aa and R bb each independently is selected from the group consisting of C1-C6alkyl; * is the position of attachment to L2, for the position of attachment to the carbonyl or methylene group.

14. The compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein Q is selected from:

15. The compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein Z1 is selected from the bond, -(CH2). p -、-(CH2) p -C(O)NH-, -(CH2) p -O-(CH2) p -C(O)NH-, -(CH2) p -C(O)-L6-NH-、-(CH2) p -O-(CH2) p -C(O)-L6-NH-, -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)- and -OC(O)NH-; p is an integer from 1 to 10; s1, s2, s3, s4 are each independently an integer from 0 to 6; L6is selected from s is an integer from 2 to 6; t is an integer from 0 to 10.

16. The compound of claim 15, or a pharmaceutically acceptable salt thereof, wherein p is an integer from 1 to 6; s1, s2, s3, s4 are each independently an integer from 0 to 4; L6 is 17. The compound of Formula (A) or a pharmaceutically acceptable salt thereof according to claim 15, wherein, Z1 is selected from the bond, -(CH2). p -、-(CH2) p -C(O)NH-, -(CH2) p -O-(CH2) p -C(O)NH-, -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)- and -OC(O)NH-; s1 is an integer from 0 to 6; s2 is an integer from 0 to 6; s3 is 0; s4 is 0; p is an integer from 1 to 10.

18. The compound of Formula (A) or pharmaceutically acceptable salt thereof according to claim 17, wherein, s1 is an integer from 0 to 2; s2 is an integer from 0 to 2; p is an integer from 1 to 6.

19. The compound according to any one of claims 1 to 4, represented by general formula (A), or a pharmaceutically acceptable salt thereof, wherein, Z1is selected from -(CH2) p -C(O)NH-, -(CH2) p -O-(CH2) p -C(O)NH-, -C(O)NH-; s1 is an integer from 1 to 6; s2 is an integer from 1 to 10; s3 is 0; s4 is 0; p is an integer from 1 to 10.

20. The compound of Formula (A) or pharmaceutically acceptable salt thereof according to claim 19, wherein, s1 is an integer from 2 to 6; s2 is an integer from 2 to 10; p is an integer from 1 to 6.

21. The compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein Q-L4- is selected from: wherein: Z1 is selected from -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)- and -OC(O)NH-; p is an integer from 1 to 10; s1 is an integer from 0 to 6; s2 is an integer from 1 to 10; s3 is an integer from 0 to 6; s4 is an integer from 1 to 6; s7 is an integer from 0 to 6; s8 is an integer from 1 to 4; s9 is an integer from 1 to 10; s 10 is an integer from 1 to 4.

22. The compound according to claim 21, represented by general formula (A), or a pharmaceutically acceptable salt thereof, wherein, p is an integer from 1 to 6; s1 is an integer from 0 to 2; s2 is an integer from 1 to 8; s3 is an integer from 0 to 2; s4 is an integer from 1 to 2; s7 is an integer from 1 to 2; s8 is an integer from 1 to 2; s9 is an integer from 1 to 8; s 10 is an integer from 1 to 2.

23. A compound represented by one of the following, or a pharmaceutically acceptable salt thereof:

24. A compound represented by general formula (I), or a stereoisomer, a tautomer, an entgegen and an enanti isomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein, R 8 selected from hydrogen, halogen; R 9 selected from hydrogen, C 1-6 alkyl; R 10 selected from -NR f C(=O)O-(CH2) 4-6 -OH; R f selected from hydrogen, C1-C6alkyl.

25. The compound according to claim 24, represented by general formula (I), or a stereoisomer, a tautomer, an entgegen and an enanti isomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein, R 9 selected from hydrogen, C 1-6 alkyl; R 10 selected from -NR f C(=O)O-(CH2)4-OH.

26. The compound according to any one of claims 24 to 25, represented by general formula (I), or a stereoisomer, a tautomer, an entgegen and an enanti isomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein R 8 selected from halogen.

27. A compound represented by the following, or a stereoisomer, a tautomer, an entgegen and an enanti isomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof:

28. A Ligand Drug Conjugate represented by general formula (B), or a pharmaceutically acceptable salt thereof, wherein: Dr is selected from the following structures: R 1 selected from hydrogen, Ci-C6alkyl; L1is selected from -NR b C(=O)O-(CH2) 4-6 -O-*, wherein * is the point of attachment to L2; or Dr is selected from the following structures: L1is selected from -(CH2) m -O-*, -(CH2) m -NR a -*, -O-(CH2) m -NR a -*, wherein * is the point of attachment to L2; R a selected from hydrogen, C1-C6alkyl; R b selected from hydrogen, Ci-C6alkyl; m is an integer from 1 to 6; R 2 selected from hydrogen, halogen; L2is selected from a bond, wherein * is the site of attachment to L1; L' is Q' is selected from: wherein * is the position of attachment to L4, is the position of attachment to Pc; L3 is an amino acid residue formed by two or more amino acids, and L3 optionally comprises one or more structures selected from the following, L6 is selected from one or more structures: wherein R, R aa , R bb are each independently selected from hydrogen and Ci-C6alkyl; L4 is Z1is selected from the group consisting of a bond, -(CH2) p - -(C2H4O) q - -(CH2) p - C(O)NH-, -(CH2) p - O-(CH2) p - C(O)NH-, -(CH2) p - C(O)-L6-NH-, -(CH2) p - O-(CH2) p - C(O)-L6-NH-, -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)-, -NH-, -O-, and -OC(O)NH-; s is an integer from 1 to 6; t is an integer from 0 to 10; s1, s2, s3, s4 are each independently an integer from 0 to 10; s5, s6 are each independently an integer from 1 to 6; t1 is an integer from 1 to 6; t2 is an integer from 0 to 6; t3 is an integer from 1 to 6; t4 is an integer from 0 to 10; t5 is an integer from 0 to 10; p is an integer from 1 to 10; q is an integer from 1 to 10; v is 1 to 10, v is a decimal or an integer; or, the ligand drug conjugate of general formula (B) is one of the following: Pc is an antibody or an antigen binding fragment thereof, or is a modified antibody; The modified antibody has the structure Pc'-((L5) w -F) x structure, wherein: Pc' is an antibody; L5 is a linker; w is 0 or 1; F is a click probe or a thiol or a precursor thereof that is capable of being linked to Q' after reaction; x is an integer from 1 to 8.

29. The ligand drug conjugate of Formula (B) or a pharmaceutically acceptable salt thereof according to claim 28, wherein, The reaction is a metal-free click reaction.

30. The Ligand Drug Conjugate of Formula (B) or a pharmaceutically acceptable salt thereof according to claim 28 or 29, wherein: When w is 0, F is a thiol; when w is 1, F is a click probe that is capable of being linked to Q' after reaction.

31. The ligand drug conjugate of general formula (B) according to claim 30, or a pharmaceutically acceptable salt thereof, wherein the click probe that is capable of being linked to Q' after reaction is an azido group.

32. The ligand drug conjugate of general formula (B) according to claim 28 or 29, or a pharmaceutically acceptable salt thereof, wherein: L3 is selected from: R is selected from hydrogen and C1-C6 alkyl; R aa , R bb each independently is selected from the group consisting of C1-C6alkyl; s is an integer from 2 to 6; s5, s6 are each independently an integer from 2 to 6; t is an integer from 0 to 10; t1 is an integer from 1 to 4; t2 is an integer from 1 to 4; t3 is an integer from 1 to 4; t4 is an integer from 0 to 10; t5 is an integer from 0 to 10; * is the position of attachment to L2; for the position of attachment to a carbonyl or methylene group; L 1b and L' 1b each independently is an amino acid residue formed from one or more amino acids selected from glycine, phenylalanine, citrulline, leucine, isoleucine, alanine, valine, asparagine, glutamine, arginine, glutamic acid, lysine.

33. The ligand drug conjugate of general formula (B) according to claim 32, or a pharmaceutically acceptable salt thereof, wherein, s is an integer from 2 to 4; s5, s6 are each independently an integer from 2 to 4; t1 is 1 or 2; t2 is 1 or 2; t3 is 1 or 2.

34. The ligand drug conjugate of general formula (B) according to claim 32, or a pharmaceutically acceptable salt thereof, wherein, L 1b and L' 1b each independently is an amino acid residue formed from two or more amino acids selected from the group consisting of glycine, phenylalanine, citrulline, valine, lysine, glutamine, glutamic acid, leucine, alanine.

35. The ligand drug conjugate of general formula (B) according to claim 32, or a pharmaceutically acceptable salt thereof, wherein, L 1b and L' 1b each independently is selected from the group consisting of -Gly-*, -Val-*, -Gly-Phe-Gly-*, -Phe-Gly-*, -Gly-Val-Cit-*, -Val-Cit-*, -Gly-Val-Arg-*, -Val-Arg-*, -Gly-Val-Ala-*, -Val-Ala-*, -Gly-Phe-*, -Phe-Gly-*, -Gly-Gly-Gly-*, -Gly-Gly-*, -Gly-Val-Gly-*, -Gly-Ala-Gly-*, -Gly-Phe-Cit-*, -Gly-Phe-Val-*, -Gly-Phe-Ala-*, -Gly-Phe-Lys-*, -Phe-Lys-*, -Gly-Val-*, -Gly-Cit-*, -Gly-Ala-*, -Gly-Gly-Lys-*, -Gly-Lys-*, -Ala-Ala-Ala-*, -Gln-Val-Ala-*, -Gln-Val-Cit-*, -Asp-Val-Ala-*, -Asp-Val-Cit-*, -Lys-Gly-Val-Ala-*, -Lys-Gly-Val-Cit-*, -Lys-Gly-Gly-Val-Ala-*, -Lys-Gly-Gly-Val-Cit-*, -Gly-Gly-Phe-Gly-*, -Lys-Gln-Val-Cit-*, -Lys-Gln-Val-Ala-*, -Lys-Glu-Val-Cit-*, -Lys-Glu-Val-Ala-*, -Lys-Asp-Val-Cit-*, -Lys-Asp-Val-Ala-*, -Glu-Val-Cit-*, -Glu-Val-Ala-*, -Lys-Val-Ala-*, -Lys-Val-Cit-*, -Val-Lys-Gly-*, -Val-Lys-*, wherein * is the position of attachment to L2.

36. The ligand drug conjugate of general formula (B) according to claim 28 or 29, or a pharmaceutically acceptable salt thereof, wherein: L' is Q' is selected from: wherein * is the position of attachment to L4, is the position of attachment to Pc; L3 is an amino acid residue formed from two or more amino acids selected from glycine, phenylalanine, citrulline, leucine, isoleucine, alanine, valine, asparagine, glutamine, arginine, glutamic acid, lysine; L4 is Z1is selected from the group consisting of a bond, -(CH2) p - -(CH2) p - -(CH2) p - -(CH2) p - -(CH2) p - -(CH2) p - -(CH2) p - -(CH2) - C(O)-L6-NH-, -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)-, -NH-, -O-, and -OC(O)NH-; s1 is an integer from 1 to 6; s2 is an integer from 1 to 10; s3 is 0; s4 is 0; p is an integer from 1 to 10; L6is selected from s is an integer from 2 to 6; t is an integer from 0 to 10.

37. The ligand drug conjugate of Formula (B) or a pharmaceutically acceptable salt thereof according to claim 36, wherein, s1 is an integer from 2 to 6; s2 is an integer from 2 to 10; p is an integer from 1 to 6; L6 is 38. The ligand drug conjugate of Formula (B) or a pharmaceutically acceptable salt thereof according to claim 28, wherein, L5 is Z2 and Z3 are each independently selected from -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)-, -NH-, -O-, and -OC(O)NH-; r1 is an integer from 1 to 8; r2 is an integer from 1 to 6; r3 is an integer from 0 to 6; r4 is an integer from 0 to 6; r5 is an integer from 1 to 6.

39. The ligand drug conjugate of general formula (B) according to claim 28, or a pharmaceutically acceptable salt thereof, wherein: Pc is a modified antibody, the modified antibody having the structure: wherein: Pc' is an antibody; Z2and Z3are each independently selected from the group consisting of -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)-, -NH-, -O-, and -OC(O)NH-; r1is an integer from 1 to 8; r2is an integer from 1 to 6; r3is an integer from 0 to 6; r4is an integer from 0 to 6; r5is an integer from 1 to 6.

40. The ligand drug conjugate of Formula (B) or a pharmaceutically acceptable salt thereof according to claim 28, wherein: Pc is a modified antibody, Pc-Q' is selected from the group consisting of structures: Pc' is an antibody; Z2and Z3are each independently selected from the group consisting of -C(O)NH-, -C(O)O-, -C(O)-, -OC(O)-, -NH-, -O-, and -OC(O)NH-; r1is an integer from 1 to 8; r2is an integer from 1 to 6; r3is an integer from 0 to 6; r4is an integer from 0 to 6; r5is an integer from 1 to 6.

41. The Ligand Drug Conjugate of Formula (B) or a pharmaceutically acceptable salt thereof according to any one of claims 38-40, wherein, r1is an integer from 1 to 6; r2is an integer from 1 to 4; r3is an integer from 0 to 4; r4is an integer from 0 to 4; r5is an integer from 1 to 4.

42. The Ligand Drug Conjugate of Formula (B) or a pharmaceutically acceptable salt thereof according to claim 41, wherein, r1is an integer from 1 to 3; r2is an integer from 1 to 2; r3is an integer from 0 to 2; r4is an integer from 0 to 2; r5is an integer from 1 to 2.

43. The Ligand Drug Conjugate of Formula (B) or a pharmaceutically acceptable salt thereof according to any one of claims 28, 38-40, selected from the group consisting of: wherein: v is 1 to 10, v is a decimal or an integer; Pc is an antibody or an antigen binding fragment thereof; Pc' is an antibody.

44. The Ligand Drug Conjugate of Formula (B) or a pharmaceutically acceptable salt thereof according to claim 43, wherein the antibody is selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

45. The Ligand Drug Conjugate of Formula (B) or a pharmaceutically acceptable salt thereof according to claim 44, wherein the antibody or antigen-binding fragment thereof is selected from an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-c-Met antibody, an anti-HER3 (ErbB3) antibody, an anti-HER4 (ErbB4) antibody, an anti-ROR1 antibody, an anti-CLDN6 antibody, an anti-CLDN9 antibody, an anti-CLDN18.2 antibody, an anti-NaPi-2b antibody, an anti-TNF-a antibody, an anti-ENPP3 antibody, an anti-DLL3 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD28 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD37 antibody, an anti-CD38 antibody, an anti-CD44 antibody, an anti-CD45 antibody, an anti-CD47 antibody, an anti-CD48 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD73 antibody, an anti-CD98 antibody, an anti-CD105 antibody, an anti-CEA antibody, an anti-EphA2 antibody, an anti-MUC1 antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-Integrin antibody, an anti-PSMA antibody, an anti-Tenascin-C antibody, an anti-SLC44A4 antibody, an anti-CD79 antibody, an anti-TROP-2 antibody, an anti-CD79B antibody, an anti-Mesothelin antibody, an anti-Nectin-4 antibody, an anti-TPBG antibody, or an antigen-binding fragment thereof.

46. The Ligand Drug Conjugate of Formula (B) or a pharmaceutically acceptable salt thereof according to claim 44, wherein the antibody or antigen-binding fragment thereof is selected from Trastuzumab, Cetuximab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, or an antigen-binding fragment thereof.

47. The Ligand Drug Conjugate of Formula (B) or a pharmaceutically acceptable salt thereof according to claim 43, selected from: wherein v is an integer or decimal number from 1 to 10.

48. The Ligand Drug Conjugate of Formula (B) or a pharmaceutically acceptable salt thereof according to claim 47, wherein v is an integer or decimal number from 2 to 8.

49. A pharmaceutical composition comprising the Ligand Drug Conjugate of Formula (B) or a pharmaceutically acceptable salt thereof according to any one of claims 28 to 48, and one or more pharmaceutically acceptable carriers or excipients. ​ 50. Use of a compound according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof or a compound according to any one of claims 24 to 27 or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a Ligand-Drug Conjugate.

51. Use of a Ligand-Drug Conjugate according to any one of claims 28 to 48 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 49 for the manufacture of a medicament for the treatment of a tumor or cancer.

52. Use according to claim 51, wherein the cancer is preferably breast cancer, ovarian cancer, soft tissue sarcoma, liposarcoma, lung cancer, gastric cancer, melanoma, head and neck cancer, cervical cancer, prostate cancer.

53. Use of a compound according to any one of claims 24 to 27 or a stereoisomer, tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a tumor or cancer.

54. The use of claim 53, wherein, the cancer is preferably breast cancer, ovarian cancer, soft tissue sarcoma, liposarcoma, lung cancer, gastric cancer, melanoma, head and neck cancer, cervical cancer, prostate cancer.

55. The use of claim 52 or 54, wherein, the cancer is selected from non-small cell lung cancer.

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