Disulfide-containing compounds, methods of making, and pharmaceutical compositions and uses thereof

Abstract

The application discloses a kind of disulfide bond derivatives, and preparation method and pharmaceutical composition and purposes thereof. Specifically, it is related to the disulfide bond derivative shown in general formula I, its pharmaceutically acceptable salt, its stereoisomer and preparation method, composition containing one or more these compounds, and the purposes of the compound in treating cancer related to PD-1 / PD-L1 signal pathway and or IDO1. It belongs to the field of medicinal chemistry.

Description

Technical Field

[0001] The present invention discloses a class of disulfide bond-containing compounds, their preparation methods, pharmaceutical compositions, and uses. Specifically, the present invention relates to disulfide bond-containing compounds represented by general formula I, their pharmaceutically acceptable salts, their stereoisomers, and methods for their preparation, compositions containing one or more of these compounds, and the use of these compounds in treating cancers associated with the PD-1 / PD-L1 signaling pathway and / or IDO1. This invention belongs to the field of medicinal chemistry. Background Art

[0002] As research into tumor immunity deepens, it has been discovered that the tumor microenvironment can protect tumor cells from recognition and killing by the immune system, and that immune escape by tumor cells plays a crucial role in tumor development and progression. In 2013, Science magazine listed tumor immunotherapy as one of its top ten breakthroughs, once again placing immunotherapy at the forefront of the field of tumor treatment. The activation or inhibition of immune cells is regulated by both positive and negative signals. Programmed death 1 (PD-1) / PD-1 ligand (PD-L1) is a negative immune regulatory signal that suppresses the immune activity of tumor-specific CD8+ T cells and mediates immune escape.

[0003] Tumor cells' ability to evade the immune system is achieved by binding the programmed death ligand (PD-L1) produced on their surface to the PD-1 protein on T cells. The tumor microenvironment in the body induces infiltrating T cells to overexpress the PD-1 molecule, and tumor cells overexpress the PD-1 ligands PD-L1 and PD-L2, leading to sustained activation of the PD-1 pathway in the tumor microenvironment. This suppresses T cell function, preventing them from detecting tumors and, consequently, preventing them from sending signals to the immune system to attack tumors and kill tumor cells. PD-1 antibodies are antibody proteins that target PD-1 or PD-L1, preventing the binding of the former two proteins, blocking this pathway and partially restoring T cell function, allowing these cells to continue killing tumor cells.

[0004] PD1 / PDL1-based immunotherapy is a new generation of immunotherapy that is attracting much attention. It aims to use the body's own immune system to fight tumors and induce apoptosis by blocking the PD-1 / PD-L1 signaling pathway. It has the potential to treat various types of tumors. Recently, a series of surprising research results have confirmed that PD1 / PD-L1 inhibitory antibodies have strong anti-tumor activity against various tumors, which is particularly eye-catching. On September 4, 2014, Merck Merck's Keytruda (pembrolizumab) became the first FDA-approved PD-1 mAb for the treatment of patients with advanced or unresectable melanoma who have failed other drug treatments. Currently, Merck is investigating the potential of Keytruda in more than 30 different types of cancer, including various hematological cancers, lung cancer, breast cancer, bladder cancer, gastric cancer, head and neck cancer. On December 22, 2014, the pharmaceutical giant Bristol-Myers Squibb did not disappoint, took the lead and obtained the accelerated approval from the U.S. Food and Drug Administration (FDA) for its anti-cancer immunotherapy drug nivolumab, marketed under the trade name Opdivo, for the treatment of patients with unresectable or metastatic melanoma who have progressed on or are intolerant to other drugs. It is the second PD-1 inhibitor to be marketed in the United States after Merck's Keytruda. On March 4, 2015, the FDA approved nivolumab for the treatment of metastatic squamous non-small cell lung cancer that has progressed during or following platinum-based chemotherapy or chemotherapy. According to the data of Merck's Keytruda (pembrolizumab) treatment of solid tumors in a phase Ib KEYNOTE-028 study, Keytruda treatment achieved a 28% overall response rate (ORR) in 25 patients with pleural mesothelioma (PM), and 48% of patients had stable disease, and the disease control rate was 76%. Patients with advanced Hodgkin's lymphoma (HL) who had no response to any currently approved drugs achieved complete remission after treatment with Merck's Keytruda and Bristol-Myers' Opdvio. At the 2015 AACR Annual Meeting, Leisha A. Emens, MD, PhD, Associate Professor of Medical Oncology at the Kimmel Cancer Center, reported that Roche's MPDL3280A, a monoclonal antibody with anti-PD-L1 effect, showed a sustained effect in advanced triple-negative breast cancer.

[0005] Indoleamine 2,3-dioxygenase 1 (IDO1) is an intracellular heme-containing enzyme widely distributed in many tissues and cells of humans and animals. It catalyzes the catabolism of tryptophan along the kynurenine pathway (KP), resulting in decreased tryptophan levels and increased kynurenine levels. Kynurenine directly inhibits the proliferation and function of natural killer (NK) and T cells. High IDO1 expression in tumor tissues predicts resistance to chemotherapy and radiotherapy, tumor-mediated immune suppression, and particularly resistance to immune checkpoint therapy. Epacadostat, developed by Incyte, is an oral, potent, and selective small molecule IDO1 inhibitor. Phase I / II clinical trial data in combination with the PD-1 monoclonal antibody Keytruda for the treatment of advanced melanoma showed encouraging results. However, in 2018, Merck announced the early termination of a Phase III clinical trial, citing the failure of epacadostat and Keytruda to improve progression-free survival compared to Keytruda alone.

[0006] Although tumor immunotherapy is considered a revolution in cancer treatment after targeted therapy, monoclonal antibodies have inherent drawbacks: they are easily degraded by proteases, making them unstable in the body and inappropriate for oral administration; they are prone to immune cross-reactivity; product quality is difficult to control, requiring high manufacturing technology; large-scale preparation and purification are difficult, resulting in high production costs; and they are inconvenient to use, requiring only injection or intravenous drip. Therefore, small molecule inhibitors of the PD1 / PD-L1 interaction are a better option for tumor immunotherapy.

[0007] Based on the different mechanisms of action of PD-L1 and IDO1 as targets in tumor immunotherapy, the present invention rationally designed and synthesized a series of PD-L1 / IDO1 dual-target small molecule inhibitor prodrugs that specifically target tumor cells and the tumor microenvironment. Its structure consists of three parts, one end is a PD-L1 small molecule inhibitor structural group, the other end is an IDO1 small molecule inhibitor structural group, and the middle is a connecting chain (such as a disulfide bond or certain peptides) that can only be broken in the tumor microenvironment or tumor cells. The prodrug only releases PD-L1 small molecule inhibitors and IDO1 small molecule inhibitors in tumor tissues or tumor cells, so it can effectively reduce the target side effects of the drug. Given that PD-L1 and IDO1 have different mechanisms of tumor immune escape, and studies have shown that the high expression of IDO1 in tumor tissues is one of the reasons for resistance to tumor immunotherapy, the dual inhibition of PD-L1 and IDO1 not only solves the problem of drug resistance, but also improves the patient response rate and expands the beneficiary population. Its synergistic effect also makes the treatment effect better, which is an ideal combination for tumor immunotherapy. Dual inhibitors of IDO1 and PD-L1 instead of combination therapy may improve progression-free survival and overall survival and enhance the efficacy of immunotherapy. Summary of the Invention

[0008] The technical problem solved by the present invention is to provide a disulfide bond-containing compound having a structural formula I that can simultaneously inhibit PD-L1 and IDO1 and can specifically target tumor tissues, as well as a stereoisomer or a pharmaceutically acceptable salt thereof, a preparation method, a pharmaceutical composition, and its use in the preparation of a drug for preventing or treating tumors related to the PD1 / PD-L1 signaling pathway and / or IDO1.

[0009] In order to solve the technical problems of the present invention, the present invention provides the following technical solutions:

[0010] The first aspect of the technical solution of the present invention is to provide a class of disulfide bond-containing compounds as shown in general formula I and their stereoisomers and pharmaceutically acceptable salts thereof

[0011]

[0012] In the formula

[0013] X is selected from:

[0014] wherein R1 is selected from R2 is selected from methyl, fluorine, chlorine, bromine, and cyano; R3 is selected from 2-cyanopyridine-4-methylene, 5-cyanopyridine-3-methylene, 5-methylsulfonylpyridine-3-methylene, and 5-carbamoylpyridine-3-methylene; R4 is selected from hydrogen, methyl, fluorine, chlorine, and bromine; R5 is selected from hydrogen, methyl, hydroxymethyl, 2-hydroxyethyl, acetoxymethyl, 2-acetoxyethyl, and isopropyl; R6 is selected from hydrogen, methyl, ethyl, and isopropyl; and R7 is selected from hydrogen and methyl;

[0015] Y is selected from carbon, oxygen, and NH;

[0016] W is selected from fluorine, chlorine, bromine, methyl, trifluoromethyl;

[0017] n is selected from: 0, 1, 2, 3, 4, 5;

[0018] Z is selected from ---NH2, ---NH-(CH2) m -NH-SO2NH2,---NH-(CH2) m -NH-S(NH)ONH2,---NH-(CH2) m -NH-S(NCN)ONH2,---NH-(CH2) m -NH-SO2CH3,---NH-(CH2) m -SO2NH2,---NH-(CH2) m -S(NH)ONH2,---NH-(CH2) m -S(NCN)ONH2,---NH-(CH2) m-SO2CH3,---NH-(CH2) m -S(NH)OCH3,---NH-(CH2) m -S(NCN)OCH3,---NH-(CH2) m -NH-S(NH)OCH3,---NH-(CH2) m -NH-S(NCN)OCH3,---NH(CO)NH-(CH2) m -NH-SO2NH2,---NH(CO)NH-(CH2) m -NH-S(NH)ONH2,---NH(CO)NH-(CH2) m -NH-S(NCN)ONH2,---NH(CO)NH-(CH2) m -NH-SO2CH3,---NH(CO)NH-(CH2) m -NH-S(NH)OCH3,---NH(CO)NH-(CH2) m -NH-S(NCN)OCH3,---NH(CO)NH-(CH2) m -SO2CH3,---NH(CO)NH-(CH2) m -S(NH)OCH3,---NH(CO)NH-(CH2) m -S(NCN)OCH3,---NH(CO)NH-(CH2) n -SO2NH2,---NH(CO)NH-(CH2) m -S(NH)ONH2,---NH(CO)NH-(CH2) m -S(NCN)ONH2,

[0019]

[0020] Among them, m=2, 3, 4, 5.

[0021] The preferred disulfide bond-containing compound and its stereoisomers and pharmaceutically acceptable salts are characterized in that the compound is as shown in formula (IA):

[0022]

[0023] In the formula

[0024] R1 is selected from R2 is selected from methyl, fluorine, chlorine, bromine, and cyano; R3 is selected from 2-cyanopyridine-4-methylene, 5-cyanopyridine-3-methylene, 5-methylsulfonylpyridine-3-methylene, and 5-carbamoylpyridine-3-methylene; R4 is selected from hydrogen, methyl, fluorine, chlorine, and bromine; R5 is selected from hydrogen, methyl, hydroxymethyl, 2-hydroxyethyl, acetoxymethyl, 2-acetoxyethyl, and isopropyl; and R6 is selected from hydrogen, methyl, ethyl, and isopropyl;

[0025] Y is selected from carbon, oxygen, and NH;

[0026] W is selected from fluorine, chlorine, bromine, methyl, trifluoromethyl;

[0027] n is selected from: 0, 1, 2, 3, 4, 5;

[0028] Z is selected from ---NH2, ---NH-(CH2) m -NH-SO2NH2,---NH-(CH2) m -NH-S(NH)ONH2,---NH-(CH2) m -NH-S(NCN)ONH2,---NH-(CH2) m -NH-SO2CH3,---NH-(CH2) m -SO2NH2,---NH-(CH2) m -S(NH)ONH2,---NH-(CH2) m -S(NCN)ONH2,---NH-(CH2) m -SO2CH3,---NH-(CH2) m -S(NH)OCH3,---NH-(CH2) m -S(NCN)OCH3,---NH-(CH2) m -NH-S(NH)OCH3,---NH-(CH2) m -NH-S(NCN)OCH3,---NH(CO)NH-(CH2) m -NH-SO2NH2,---NH(CO)NH-(CH2) m -NH-S(NH)ONH2,---NH(CO)NH-(CH2) m -NH-S(NCN)ONH2,---NH(CO)NH-(CH2) m -NH-SO2CH3,---NH(CO)NH-(CH2) m -NH-S(NH)OCH3,---NH(CO)NH-(CH2) m-NH-S(NCN)OCH3,---NH(CO)NH-(CH2) m -SO2CH3,---NH(CO)NH-(CH2) m -S(NH)OCH3,---NH(CO)NH-(CH2) m -S(NCN)OCH3,---NH(CO)NH-(CH2) n -SO2NH2,---NH(CO)NH-(CH2) m -S(NH)ONH2,---NH(CO)NH-(CH2) m -S(NCN)ONH2,

[0029]

[0030] Among them, m=2, 3, 4, 5.

[0031] The preferred disulfide bond-containing compound and its stereoisomers and pharmaceutically acceptable salts thereof are characterized in that the compound is as shown in formula (IA1):

[0032]

[0033] In the formula

[0034] R1 is selected from R2 is selected from methyl, fluorine, chlorine, bromine, and cyano; R3 is selected from 2-cyanopyridine-4-methylene, 5-cyanopyridine-3-methylene, 5-methylsulfonylpyridine-3-methylene, and 5-carbamoylpyridine-3-methylene; R4 is selected from hydrogen, methyl, fluorine, chlorine, and bromine; R5 is selected from hydrogen, methyl, hydroxymethyl, 2-hydroxyethyl, acetoxymethyl, 2-acetoxyethyl, and isopropyl; and R6 is selected from hydrogen, methyl, ethyl, and isopropyl;

[0035] Y is selected from carbon, oxygen, and NH.

[0036] The preferred disulfide bond-containing compound and its stereoisomers and pharmaceutically acceptable salts are characterized in that the compound is as shown in formula (IB):

[0037]

[0038] In the formula

[0039] R1 is selected from R2 is selected from methyl, fluorine, chlorine, bromine, and cyano; R3 is selected from 2-cyanopyridine-4-methylene, 5-cyanopyridine-3-methylene, 5-methylsulfonylpyridine-3-methylene, and 5-carbamoylpyridine-3-methylene; R4 is selected from hydrogen, methyl, fluorine, chlorine, and bromine; R6 is selected from hydrogen, methyl, ethyl, and isopropyl; and R7 is selected from hydrogen and methyl;

[0040] Y is selected from carbon, oxygen, and NH;

[0041] W is selected from fluorine, chlorine, bromine, methyl, trifluoromethyl;

[0042] n is selected from: 0, 1, 2, 3, 4, 5;

[0043] Z is selected from ---NH2, ---NH-(CH2) m -NH-SO2NH2,---NH-(CH2) m -NH-S(NH)ONH2,---NH-(CH2) m -NH-S(NCN)ONH2,---NH-(CH2) m -NH-SO2CH3,---NH-(CH2) m -SO2NH2,---NH-(CH2) m -S(NH)ONH2,---NH-(CH2) m -S(NCN)ONH2,---NH-(CH2) m -SO2CH3,---NH-(CH2) m -S(NH)OCH3,---NH-(CH2) m -S(NCN)OCH3,---NH-(CH2) m -NH-S(NH)OCH3,---NH-(CH2) m -NH-S(NCN)OCH3,---NH(CO)NH-(CH2) m -NH-SO2NH2,---NH(CO)NH-(CH2) m -NH-S(NH)ONH2,---NH(CO)NH-(CH2) m -NH-S(NCN)ONH2,---NH(CO)NH-(CH2) m -NH-SO2CH3,---NH(CO)NH-(CH2) m -NH-S(NH)OCH3,---NH(CO)NH-(CH2) m -NH-S(NCN)OCH3,---NH(CO)NH-(CH2) m -SO2CH3,---NH(CO)NH-(CH2) m -S(NH)OCH3,---NH(CO)NH-(CH2) m -S(NCN)OCH3,---NH(CO)NH-(CH2) n-SO2NH2,---NH(CO)NH-(CH2) m -S(NH)ONH2,---NH(CO)NH-(CH2) m -S(NCN)ONH2,

[0044]

[0045] Among them, m=2, 3, 4, 5.

[0046] The preferred disulfide bond-containing compound and its stereoisomers and pharmaceutically acceptable salts thereof are characterized in that the compound is as shown in formula (IB1):

[0047]

[0048] In the formula

[0049] R1 is selected from R2 is selected from methyl, fluorine, chlorine, bromine, and cyano; R3 is selected from 2-cyanopyridine-4-methylene, 5-cyanopyridine-3-methylene, 5-methylsulfonylpyridine-3-methylene, and 5-carbamoylpyridine-3-methylene; R4 is selected from hydrogen, methyl, fluorine, chlorine, and bromine; R6 is selected from hydrogen, methyl, ethyl, and isopropyl; and R7 is selected from hydrogen and methyl;

[0050] Y is selected from carbon, oxygen, and NH.

[0051] The preferred disulfide bond-containing compound and its stereoisomers and pharmaceutically acceptable salts are characterized in that the compound is as shown in formula (IC):

[0052]

[0053] In the formula

[0054] R1 is selected from R2 is selected from methyl, fluorine, chlorine, bromine, and cyano; R3 is selected from 2-cyanopyridine-4-methylene, 5-cyanopyridine-3-methylene, 5-methylsulfonylpyridine-3-methylene, and 5-carbamoylpyridine-3-methylene; R4 is selected from hydrogen, methyl, fluorine, chlorine, and bromine; and R6 is selected from hydrogen, methyl, ethyl, and isopropyl;

[0055] Y is selected from carbon, oxygen, and NH;

[0056] W is selected from fluorine, chlorine, bromine, methyl, trifluoromethyl;

[0057] n is selected from: 0, 1, 2, 3, 4, 5;

[0058] Z is selected from ---NH2, ---NH-(CH2) m -NH-SO2NH2,---NH-(CH2)m -NH-S(NH)ONH2,---NH-(CH2) m -NH-S(NCN)ONH2,---NH-(CH2) m -NH-SO2CH3,---NH-(CH2) m -SO2NH2,---NH-(CH2) m -S(NH)ONH2,---NH-(CH2) m -S(NCN)ONH2,---NH-(CH2) m -SO2CH3,---NH-(CH2) m -S(NH)OCH3,---NH-(CH2) m -S(NCN)OCH3,---NH-(CH2) m -NH-S(NH)OCH3,---NH-(CH2) m -NH-S(NCN)OCH3,---NH(CO)NH-(CH2) m -NH-SO2NH2,---NH(CO)NH-(CH2) m -NH-S(NH)ONH2,---NH(CO)NH-(CH2) m -NH-S(NCN)ONH2,---NH(CO)NH-(CH2) m -NH-SO2CH3,---NH(CO)NH-(CH2) m -NH-S(NH)OCH3,---NH(CO)NH-(CH2) m -NH-S(NCN)OCH3,---NH(CO)NH-(CH2) m -SO2CH3,---NH(CO)NH-(CH2) m -S(NH)OCH3,---NH(CO)NH-(CH2) m -S(NCN)OCH3,---NH(CO)NH-(CH2) n -SO2NH2,---NH(CO)NH-(CH2) m -S(NH)ONH2,---NH(CO)NH-(CH2) m -S(NCN)ONH2,

[0059]

[0060] 其中,m=2、3、4、5。

[0061] Preferred disulfide-containing compounds, stereoisomers thereof, and pharmaceutically acceptable salts thereof are characterized in that the compounds are of the formula (IC1):

[0062]

[0063] wherein

[0064] R1is selected from R2is selected from methyl, fluoro, chloro, bromo, cyano, R3is selected from 2-cyanopyridine-4-ylmethyl, 5-cyanopyridine-3-ylmethyl, 5-methanesulfonylpyridine-3-ylmethyl, 5-carbamoylpyridine-3-ylmethyl, R4is selected from hydrogen, methyl, fluoro, chloro, bromo, R6is selected from hydrogen, methyl, ethyl, isopropyl;

[0065] Y is selected from carbon, oxygen, NH.

[0066] Most preferred disulfide-containing compounds, stereoisomers thereof, and pharmaceutically acceptable salts thereof are selected from, but not limited to, the following compounds:

[0067] Compound 1 : (S,Z)-1 -(4-amino-1,2,5-oxadiazol-3-yl)-1 -(3-bromo-4-fluoroanilino)- 16-(4-((2-bromo-(1,1 '-biphenyl)-3-yl)methoxy)-5-chloro-2-(2-cyanopyridin-4- ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxa-8,9-dithia-2,5,12-triazahexadec- 1 -ene-17-oic acid ethyl ester

[0068]

[0069] Compound 2: (S,Z)-1 -(4-amino-1,2,5-oxadiazol-3-yl)-1 -(3-bromo-4-fluoroanilino)- 16-(4-((2-chloro-(1,1 '-biphenyl)-3-yl)methoxy)-5-chloro-2-(5-cyanopyridin-3- ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxa-8,9-dithia-2,5,12-triazahexadec- 1 -ene-17-oic acid ethyl ester

[0070]

[0071] Compound 3: (S,Z)-1 -(4-amino-1,2,5-oxadiazol-3-yl)-1 -(3-bromo-4-fluoroanilino)- 16-(4-((2-chloro-(1,1 '-biphenyl)-3-yl)methoxy)-5-chloro-2-(5-cyanopyridin-3- ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxa-8,9-dithia-2,5,12-triazahexadec- 1 -ene-17-oic acid

[0072]

[0073] Compound 4: (S,R,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4- fluorophenylamino)-15-methyl-16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5- chloro-2-(2-cyanopyridin-4-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxa-8,9- disulfur-2,5,12-triazahexadeca-1-ene-17-oic acid ethyl ester

[0074]

[0075] Compound 5: (S,R,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4- fluorophenylamino)-15-methyl-16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5- chloro-2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxa-8,9- disulfur-2,5,12-triazahexadeca-1-ene-17-oic acid ethyl ester

[0076]

[0077] Compound 6: (S,R,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4- fluorophenylamino)-15-methyl-16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5- chloro-2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxa-8,9- disulfur-2,5,12-triazahexadeca-1-ene-17-oic acid

[0078]

[0079] Compound 7: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4- fluorophenylamino)-16-(4-((2-chloro-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-methyl- 2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxa-8,9-disulfur- 2,5,12-triazahexadeca-1-ene-17-oic acid methyl ester

[0080]

[0081] Compound 8: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-chloro-3-(1,4-benzodioxane-6-yl)benzyloxy)-5-methyl-2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid

[0082]

[0083] Compound 9: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-methyl-3-(1,4-benzodioxane-6-yl)benzyloxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid

[0084]

[0085] Compound 10: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid ethyl ester

[0086]

[0087] Compound 11: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid

[0088]

[0089] Compound 12: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(5-methylsulfonylpyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid ethyl ester

[0090]

[0091] Compound 13: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-bromo-3-(1,4-benzodioxane-6-yl)benzyloxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid ethyl ester

[0092]

[0093] Compound 14: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-methyl-16-(4-((2-bromo-3-(1,4-benzodioxane-6-yl)benzyloxy)-5-chloro-2-(2-cyanopyridin-4-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid ethyl ester

[0094]

[0095] Compound 15: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-methyl-16-(4-((2-bromo-3-(1,4-benzodioxane-6-yl)benzyloxy)-5-chloro-2-(2-cyanopyridin-4-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid

[0096]

[0097] Compound 16: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-14-(4-((2-bromo-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzyl)-15-methyl-4,13-dioxo-3,12-dioxo-8,9-dithio-2,5,14-triazahexadecene-1-en-16-oic acid ethyl ester

[0098]

[0099] Compound 17: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-14-(4-((2-bromo-3-(1,4-benzodioxane-6-yl)benzyloxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzyl)-15-methyl-4,13-dioxo-3,12-dioxo-8,9-dithio-2,5,14-triazahexadecene-1-en-16-oic acid

[0100]

[0101] Compound 18: (S,Z)-15-(acetoxymethyl)-14-(4-((2-bromo-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzyl)-1-(3-bromo-4-fluoroanilino)-4,13-dioxo-1-(4-(2-(sulfamoylamino)ethylamino)-1,2,5-oxadiazol-3-yl)-3,12-dioxo-8,9-dithio-2,5,14-triazahexadecene-1-en-16-oic acid ethyl ester

[0102]

[0103] Compound 19: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-14-(4-((2-bromo-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzyl)-15-(acetoxymethyl)--4,13-dioxo-3,12-dioxo-8,9-dithio-2,5,14-triazahexadecene-1-en-16-oic acid ethyl ester

[0104]

[0105] Compound 20: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4- fluorani- lido)-14-(4-((2-bromo-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chloro-2-(5- cyano pyridin-3-ylmethoxy)benzyl)-15-hydroxymethyl-4,13-dioxo-3,12-dioxa-8,9- disulfur-2,5,14-triazahexadec-1-ene-16-oic acid

[0106]

[0107] Compound 21: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4- fluorani- lido)-14-(4-((2-bromo-3-phenylbenzyloxy)-5-chloro-2-(5-cyano pyridin-3- ylmethoxy)benzyl)-15-hydroxymethyl-4,13-dioxo-3,12-dioxa-8,9-disulfur-2,5,14- triazahexadec-1-ene-16-oic acid

[0108] Compound 22: (2S,4S)-4-((((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4- fluorani- lido)-4-oxo-3-oxa-8,9-disulfur-2,5-diazundec-1-en-11-yl)carbamoyl)oxy)-1-(4- ((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-((2-cyanopyridin-4-yl)methoxy) benzyl)pyrrolidine-2-carboxylic acid ethyl ester

[0109]

[0110] Compound 23: (2S,4S)-4-((((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4- fluorani- lido)-4-oxo-3-oxa-8,9-disulfur-2,5-diazundec-1-en-11-yl)carbamoyl)oxy)-1-(4- ((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-((5-cyanopyridin-3-yl)methoxy) benzyl)pyrrolidine-2-carboxylic acid ethyl ester

[0111]

[0112] Compound 24: (2S,4S)-4-((((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4-oxo-3-oxo-8,9-dithio-2,5-diazaundec-1-en-11-yl)carbamoyl)oxy)-1-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-((5-cyanopyridin-3-yl)methoxy)benzyl)pyrrolidine-2-carboxylic acid

[0113]

[0114] Compound 25: (2S,4S)-4-((((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4-oxo-3-oxo-8,9-dithio-2,5-diazaundec-1-en-11-yl)carbamoyl)oxy)-1-(4-((2-chloro-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chloro-2-((5-cyanopyridin-3-yl)methoxy)benzyl)pyrrolidine-2-carboxylic acid ethyl ester

[0115] Compound 26: (2S,4S)-4-((((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4-oxo-3-oxo-8,9-dithio-2,5-diazaundec-1-en-11-yl)carbamoyl)oxy)-1-(4-((2-chloro-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chloro-2-((5-cyanopyridin-3-yl)methoxy)benzyl)pyrrolidine-2-carboxylic acid

[0116] The above-mentioned disulfide bond-containing compound, stereoisomers thereof, and pharmaceutically acceptable salts thereof are characterized in that the pharmaceutically acceptable salts include salts formed by combining with inorganic acids, organic acids, alkali metal ions, alkaline earth metal ions, or organic bases that can provide physiologically acceptable cations, as well as ammonium salts.

[0117] Furthermore, the inorganic acid is selected from hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid; the organic acid is selected from methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, citric acid, maleic acid tartaric acid, fumaric acid, citric acid or lactic acid; the alkali metal ion is selected from lithium ion, sodium ion, potassium ion; the alkaline earth metal ion is selected from calcium ion, magnesium ion; the organic base capable of providing physiologically acceptable cations is selected from methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris(2-hydroxyethyl)amine.

[0118] The second aspect of the technical solution of the present invention is to provide a method for preparing the compound described in the first aspect:

[0119] To prepare the compounds of the present application according to Formula I, the preparation of the compounds of Formula I will be divided into several routes according to the structure of Formula I:

[0120] Route 1:

[0121]

[0122] (a) The chlorooxime compound 1 is reacted with the isocyanate compound 2 in the presence of a base to give compound 3.

[0123] (b) Compound 3 is subjected to a nucleophilic substitution reaction with an aniline derivative 4 in the presence of a base to give compound 5.

[0124] (c) Compound 5 is reacted with 2-mercaptoethylamine to give the amine compound 6.

[0125] (d) The amine compound 6 is subjected to a nucleophilic substitution reaction with the ester compound 7 to give the amide 8.

[0126] (e) Compound 8 is subjected to a deprotection reaction under acidic conditions to give the amine compound 9.

[0127] (f) The amine compound 9 is condensed with the aldehyde compound 10 and subjected to a hydrogenation reduction to give the target compound IB.

[0128] Route 2:

[0129]

[0130] (a) The N-hydroxyl amidine compound 11 is reacted with the isocyanate compound 2 in the presence of a base to give compound 5.

[0131] (c) The amine compound 6 is subjected to a nucleophilic substitution reaction with the ester compound 12 to give the amide 13.

[0132] (e) Compound 13 is subjected to a deprotection reaction under acidic conditions to give the target compound IB.

[0133] Route 3:

[0134]

[0135] (a) The N-hydroxyl amidine compound 11 is reacted with the isocyanate compound 2 in the presence of a base to give compound 5.

[0136] (b) Compound 5 is reacted with 2-mercaptoethanol to give the alcohol compound 14.

[0137] (c) The alcohol compound 14 is subjected to a nucleophilic substitution reaction with the ester compound 15 to give the target compound IA.

[0138] Route 4:

[0139]

[0140] (a) Amine compound 16 is used as a raw material and reacted with tetrahydrothiophene-2-one in an alkaline environment, or dehydrated with 4-mercaptobutyric acid to form an amide, or reacted with 4-mercaptobutyryl chloride to obtain a thiol-containing compound 17.

[0141] (b) Compound 17 reacts with compound 5 containing a disulfide bond to obtain the target compound IA.

[0142] Route 5:

[0143]

[0144] (a) N-hydroxyamidine compound 11 is reacted with isocyanate compound 2 under alkaline conditions to obtain compound 5. (b) Compound 5 is reacted with thiol compound 18 to obtain compound 19.

[0145] (c) Compound 19 undergoes nucleophilic substitution reaction with compound 20 under alkaline conditions to obtain the target compound IC.

[0146] The definitions of R1, R2, R3, R4, R5, R6, R7, W, Y, Z, and n are the same as those in any one of claims 1-7.

[0147] In addition, the starting materials and intermediates in the above reactions are readily available, and each step can be readily synthesized using conventional methods in organic synthesis, based on published literature or readily known to those skilled in the art. The compound of Formula I may exist as a solvate or an unsolvate, and different solvates may be obtained by crystallization using different solvents. Pharmaceutically acceptable salts of Formula I include various acid addition salts, such as those with the following inorganic or organic acids: hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, citric acid, maleic acid, tartaric acid, fumaric acid, citric acid, and lactic acid. Pharmaceutically acceptable salts of Formula I also include various alkali metal salts (lithium, sodium, potassium salts), alkaline earth metal salts (calcium, magnesium salts), and ammonium salts, as well as salts of organic bases that provide physiologically acceptable cations, such as salts of methylamine, dimethylamine, trimethylamine, piperidine, morpholine, and tris(2-hydroxyethyl)amine. All of these salts within the scope of the present invention can be prepared using conventional methods. During the preparation of the compound of general formula I, its solvate and salt thereof, polycrystals or cocrystals may appear under different crystallization conditions.

[0148] The third aspect of the technical solution of the present invention is to provide a pharmaceutical composition, which comprises as active ingredients the disulfide bond-containing compound and its stereoisomers and pharmaceutically acceptable salts described in the first aspect of the present invention and a pharmaceutically acceptable carrier or excipient.

[0149] The present invention also relates to pharmaceutical compositions containing the compounds of the present invention as active ingredients. The pharmaceutical compositions can be prepared according to methods known in the art. The compounds of the present invention can be combined with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants to form any dosage form suitable for human or animal use. The compounds of the present invention are typically present in pharmaceutical compositions in an amount of 0.1-95% by weight.

[0150] The compound of the present invention or the pharmaceutical composition containing the same can be administered in unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0151] The dosage form can be a liquid dosage form, a solid dosage form, or a semisolid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o / w, w / o, and multiple emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), and sprays; semisolid dosage forms can be ointments, gels, pastes, and the like.

[0152] The compound of the present invention can be prepared into common preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

[0153] In order to prepare the compound of the present invention into tablets, various excipients well known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and glidants. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropyl alcohol, etc.; binders can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, etc.; lubricants and glidants can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0154] The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0155] To prepare the dosing unit as a capsule, the active ingredient compound of the present invention can be mixed with a diluent and a glidant, and the mixture can be directly placed in a hard or soft capsule. Alternatively, the active ingredient compound of the present invention can be first mixed with a diluent, a binder, and a disintegrant to form granules or pellets, which are then placed in a hard or soft capsule. The diluents, binders, wetting agents, disintegrants, and glidants used to prepare tablets of the compound of the present invention can also be used to prepare capsules of the compound of the present invention.

[0156] To prepare the compounds of the present invention as injections, water, ethanol, isopropanol, propylene glycol, or mixtures thereof can be used as solvents, and appropriate amounts of solubilizers, cosolvents, pH adjusters, and osmotic pressure regulators commonly used in the art can be added. Examples of solubilizers or cosolvents include poloxamer, lecithin, and hydroxypropyl-β-cyclodextrin; pH adjusters include phosphates, acetates, hydrochloric acid, and sodium hydroxide; and osmotic pressure regulators include sodium chloride, mannitol, glucose, phosphates, and acetates. For lyophilized powder injections, mannitol, glucose, and the like can also be added as support agents.

[0157] Furthermore, if necessary, colorants, preservatives, perfumes, flavorings or other additives may be added to the pharmaceutical preparations.

[0158] To achieve the purpose of medication and enhance the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known method of administration.

[0159] The dosage of the pharmaceutical composition of the compound of the present invention can vary widely depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the route of administration, and the dosage form. Generally speaking, a suitable daily dosage range of the compound of the present invention is 0.001-150 mg / kg body weight, preferably 0.01-100 mg / kg body weight. The above dosage can be administered in a single dosage unit or divided into several dosage units, depending on the clinical experience of the physician and the dosage regimen including the use of other therapeutic means.

[0160] The compound or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs. When the compound of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0161] The fourth aspect of the technical solution of the present invention is to provide the use of disulfide bond-containing compounds, stereoisomers thereof, and pharmaceutically acceptable salts thereof in the preparation of drugs for preventing and / or treating diseases related to the PD-1 / PD-L1 signaling pathway and / or IDO1.

[0162] The disease associated with the PD-1 / PD-L1 signaling pathway and / or IDO1 is selected from cancer and infectious diseases. The cancer is selected from skin cancer, lung cancer, urinary tract tumors, blood tumors, breast cancer, glioma, digestive system tumors, reproductive system tumors, lymphoma, nervous system tumors, brain tumors, and head and neck cancer. The infectious disease is selected from bacterial infection and viral infection.

[0163] Beneficial technical effects:

[0164] Based on the different mechanisms of action of PD-L1 and IDO1 as targets in tumor immunotherapy, this invention rationally designed and synthesized a series of PD-L1 / IDO1 dual-target small molecule inhibitor prodrugs that specifically target tumor cells and the tumor microenvironment. These prodrugs release the PD-L1 and IDO1 small molecule inhibitors only within tumor tissue or cells, effectively reducing the drug's on-target side effects. Given that PD-L1 and IDO1 contribute to tumor immune escape through different mechanisms, and that studies have shown that high IDO1 expression in tumor tissue is one of the causes of resistance to tumor immunotherapy, dual inhibition of PD-L1 and IDO1 not only addresses drug resistance but also improves patient response rates, expanding the population that benefits. Their synergistic effects also enhance therapeutic efficacy, making them an ideal combination for tumor immunotherapy. Dual IDO1 and PD-L1 inhibitors, instead of combined therapy, may improve progression-free survival and overall survival, enhancing the effectiveness of immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0165] Figure 1 : Tumor weight (compared with the blank control group, *P<0.05, *** P < 0.001)

[0166] Figure 2 Antitumor activity of ZC-102 in a CT26 xenograft mouse model. (A) Images of CT26 cell xenograft mouse tumors after different treatments. (B) Tumor weights of each group after treatment. (C) Body weights of each group after treatment.

[0167] Figure 3 A) Images of spleens from mice xenografted with CT26 cells after different treatments. (B) Spleen weight and spleen index. (C) Percentage of regulatory T cells (CD4+CD25+Foxp3+) in the spleen. DETAILED DESCRIPTION

[0168] The invention will be further described below with reference to the following examples, but the scope of the invention is not limited thereto.

[0169] Measurement instruments: Nuclear magnetic resonance spectroscopy was performed using a Vaariaan Mercury 300 nuclear magnetic resonance instrument. Mass spectrometry was performed using ZAD-2F and VG300 mass spectrometers.

[0170] Example 1: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(2-cyanopyridin-4-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid ethyl ester (Compound 1)

[0171]

[0172] Compound 1

[0173] 1. Synthesis of (pyridin-2-yl)(aminoethyl) disulfide hydrochloride

[0174]

[0175] 2,2'-Disulfide dipyridine (13.58 g, 61.64 mmol) was added to 100 ml of methanol, and a methanol solution (100 ml) of mercaptoethylamine hydrochloride (4.67 g, 41.11 mmol) was added under Ar protection. After the addition was complete, the mixture was reacted at room temperature for 12 h. 500 ml of diethyl ether was added to the reaction solution, and the precipitated solid was filtered to obtain 7.60 g of a yellow solid. 1H NMR (300MHz, CD3OD): δ8.65(s,1H,-ArH), 8.24(d,J=6.5Hz,1H,-ArH), 8.12(d,J=7.2Hz,1H,-ArH), 7.67(s,1H,-ArH), 3.23(m,4H,-CH2-,-CH2-).

[0176] 2. Synthesis of (isocyanatoethyl)(pyridin-2-yl) disulfide

[0177]

[0178] The intermediate (pyridin-2-yl)(aminoethyl) disulfide hydrochloride (4 g, 17.96 mmol) and 1,8-bis(dimethylaminonaphthalene) (12.36 g, 53.88 mmol) were added to 120 ml of ultra-dry DCM. Diphosgene (1.08 ml, 8.98 mmol) was added under ice bath conditions. The mixture was moved to room temperature and stirred for 2 h. After evaporation, the next step of the reaction was directly carried out.

[0179] 3. Synthesis of (Z)-4-amino-N-(3-bromo-4-fluorophenyl)-N'-(2-(pyridine-2-disulfanyl)ethylamine formyloxy)-1,2,5-oxadiazole-3-amidine

[0180]

[0181] The crude intermediate (isocyanatoethyl)(pyridin-2-yl) disulfide (3 g, 17.96 mmol) was dissolved in 160 mL of DCM, followed by the addition of the intermediate 4-amino-1,2,5-oxadiazole-3-chloroformaldehyde oxime (1.5 g, 8.98 mmol) and DIPEA (5.93 mL, 35.90 mmol). The mixture was stirred under Argon protection for 12 h. 140 mL of 1N aqueous hydrochloric acid was added to the reaction solution, and the mixture was extracted with dichloromethane (3 × 200 mL). The mixture was washed with 1N aqueous hydrochloric acid (2 × 150 mL) and saturated aqueous sodium chloride (200 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to approximately 100 mL. The precipitated solid was filtered and dried to yield 1.4 g (3.74 mmol) of a white solid. It was dissolved in 13.6 ml of anhydrous ethanol, and 3-bromo-4-fluoroaniline (0.72 g, 3.76 mmol) was added. Under stirring in an ice-water bath, an aqueous solution (10.8 ml) of NaHCO3 (0.72 g, 8.5 mmol) was added. The temperature was raised to 60°C and the reaction was carried out for 2 h. The ethanol was evaporated under reduced pressure, and the product was extracted with ethyl acetate (3×150 ml), washed with saturated aqueous NaCl solution (1×60 ml), dried over anhydrous Na2SO4, concentrated, and separated by column chromatography to obtain 0.78 g of (Z)-4-amino-N-(3-bromo-4-fluorophenyl)-N'-(2-(pyridine-2-disulfanyl)ethylaminecarbonyloxy)-1,2,5-oxadiazole-3-amidine.

[0182] Alternatively, the crude intermediate (isocyanatoethyl)(pyridin-2-yl) disulfide (3 g, 17.96 mmol) was dissolved in 160 mL of DCM, followed by the addition of the intermediate (Z)-4-amino-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-amidine (2.84 g, 8.98 mmol) and DIPEA (5.93 mL, 35.90 mmol). The mixture was stirred under Ar protection for 12 h. 140 mL of 1N aqueous hydrochloric acid was added to the reaction solution, and the mixture was extracted with dichloromethane (3 × 200 mL). The mixture was washed with 1N aqueous hydrochloric acid (2 × 150 mL) and saturated aqueous sodium chloride (200 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to approximately 100 mL. The precipitated solid was filtered to obtain 2.28 g of a white solid.

[0183] 1H NMR (400MHz, DMSO-d6): δ9.78(s,1H,-NH-),8.45(d,J=5.0Hz,1H,-ArH),7.87– 7.74(m,2H,-ArH),7.72(t,J=5.8Hz,1H,-ArH),7.32(dd,J=6.1,2.6Hz,1H,ArH) ,7.29–7.21(m,2H,-ArH),6.98–6.89(m,1H,-NH-),6.43(s,2H,-NH2),3.42(dd ,J=12.8,6.4Hz,2H,-CH2-),2.98(t,J=6.7Hz,2H,-CH2-).HRMS(ESI)m / z:[M+H] + calculated for C 17 H 16 O3N7BrFS2,527.99180; found,527.99121,Δ-2.65ppm.

[0184] 4. Synthesis of 2-(2-cyanopyridin-4-methyleneoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzaldehyde

[0185]

[0186] The intermediate 2-hydroxy-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzaldehyde (1.50 g, 3.59 mmol) was added to 25 ml of DMF, followed by KCO (992 mg, 7.18 mmol) and 4-(bromomethyl)picolinecarbonitrile (1.06 g, 5.39 mmol). After addition, the mixture was stirred at room temperature for 4 h. Water (80 ml) was added to the reaction mixture, and a solid precipitated. The solid was filtered and the filter cake was purified by column chromatography to yield 1.38 g of a white solid.

[0187] 5. N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(2-cyanopyridin-4-methoxy)benzyl)-N-(tert-butoxycarbonyl)-O-(p-nitrophenoxycarbonyl)-L-serine ethyl ester

[0188]

[0189] Dissolve N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(2-cyanopyridin-4-methoxy)benzyl)-L-serine ethyl ester hydrochloride (11 g, 15.98 mmol) and sodium bicarbonate (3.36 g, 39.95 mmol) in a mixture of methanol (100 ml) and water (100 ml). Add di-tert-butyl dicarbonate (3.49 g, 15.98 mmol). After addition, react at room temperature for 8 h. Add 150 ml of water to the reaction mixture, extract with ethyl acetate (2 × 150 ml), wash with saturated brine (2 × 150 ml), dry over anhydrous sodium sulfate, and evaporate to dryness to obtain 14.46 g of a white solid. The product was dissolved in ultra-dry dichloromethane (100 ml), and p-nitrophenyl chloroformate (4.78 g, 24 mmol) and Et3N (3.33 ml, 23.89 mmol) were added. After the addition was complete, the mixture was reacted at room temperature for 6 h, evaporated to dryness, and purified by column chromatography to obtain 11 g of a yellow solid.

[0190] 6. Synthesis of tert-butyl (Z)-(1-(4-amino-1,2,5-oxadiazol-3-yl)-1-((3-bromo-4-fluorophenyl)amino)-4-oxo-3-oxo-8,9-dithio-2,5-diazaundec-1-en-11-yl)carbamate

[0191]

[0192] The intermediate (Z)-4-amino-N-(3-bromo-4-fluorophenyl)-N'-(2-(pyridine-2-disulfanyl)ethylamine formyloxy)-1,2,5-oxadiazole-3-amidine (3 g, 5.69 mmol) was dissolved in a mixture of dichloromethane (100 mL) and methanol (100 mL). 2-tert-Butyloxycarbonylaminoethanethiol (1.31 g, 7.40 mmol) was added and stirred under Ar protection for 1 hour. 200 mL of 0.5N aqueous hydrochloric acid was added to the reaction solution, and the mixture was extracted with dichloromethane (2 × 150 mL). The product was washed with saturated aqueous NaHCO₃ (200 mL), dried over anhydrous sodium sulfate, evaporated to dryness, and purified by silica gel column chromatography to obtain 1.59 g of a yellow oil. 1H NMR (400MHz, Acetone-d6): δ8.94(s,1H,-NH-),7.45(dd,J=6.0,2.6Hz,1H,-Ar H),7.21(t,J=8.6Hz,1H,-NH-),7.16–7.05(m,2H,-ArH),6.19(s,1H,-NH-),6. 01(s,2H,-NH2),3.57(dd,J=12.7,6.4Hz,2H,-CH2-),3.39(dd,J=13.1,6.4Hz, 2H,-CH2-),2.94(dd,J=8.3,5.3Hz,2H,-CH2-),2.91–2.81(2H,-CH2-,covered by the peak of water),1.40(s,9H,-Boc).HRMS(ESI)m / z:[M+H] + calculated for C 19 H 26 O5N7BrFS2,594.05988; found,594.05817.

[0193] 7. Synthesis of (Z)-4-amino-N'-(2-((2-aminoethyl)disulfanyl)ethylcarbamoyloxy)-N-(3-bromo-4-fluorophenyl)-1,2,5-oxadiazole-3-amidine-trifluoroacetate

[0194]

[0195] The intermediate (Z)-(1-(4-amino-1,2,5-oxadiazol-3-yl)-1-((3-bromo-4-fluorophenyl)amino)-4-oxo-3-oxo-8,9-disulfide-2,5-diazaundec-1-en-11-yl)carbamic acid tert-butyl ester (797 mg, 1.34 mmol) was dissolved in 35 ml of dichloromethane, and trifluoroacetic acid (3.5 ml) was added. After the addition was complete, the reaction was carried out under argon protection for 1 hour, and the next reaction was directly carried out after evaporation.

[0196] 8. Synthesis of (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(tert-butyloxycarboxamido)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid ethyl ester

[0197]

[0198] The resulting dark yellow oil, (Z)-4-amino-N'-(2-((2-aminoethyl)disulfanyl)ethylaminoformyloxy)-N-(3-bromo-4-fluorophenyl)-1,2,5-oxadiazole-3-amidine trifluoroacetate, was dissolved in DMF (35 ml). Et3N (559 μl, 4.02 mmol) and (S)-ethyl 2-tert-butoxyformylamino-3-(4-nitrophenoxyformyloxy)propionate (534 mg, 1.34 mmol) were added sequentially. The mixture was stirred under Ar protection for 2 h. 150 ml of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 150 ml). The product was washed with saturated brine (2 × 200 ml), dried over anhydrous sodium sulfate, evaporated to dryness, and purified by silica gel column chromatography to obtain 783 mg of a light yellow oil. 1 H NMR (400MHz, Acetone-d6): δ8.92(s,1H,-NH-),7.46(dd,J=6.0,2.4Hz,1H,-ArH),7.22(t,J=8.6Hz,1H,-NH-),7.16–7.05(m, 2H,-ArH),6.59(s,1H,-NH-),6.19(d,J=7.5Hz,1H,-NH-),6.00(s,2H,-NH2-),4.45–4.37(m,1H,-CH-),4.33(d,J=4.5Hz,2H,- CH2-),4.16(dd,J=7.0,3.2Hz,2H,-CH2-),3.56(dd,J=12.4,6.7Hz,2H,-CH2-),3.45(dd,J=12.5,6.2Hz,2H,-CH2-),2.94(dd ,J=8.1,5.5Hz,2H,-CH2-),2.88(t,J=6.8Hz,2H,-CH2-),1.41(s,9H,-Boc),1.24(t,J=7.1Hz,3H,-CH3).HRMS(ESI)m / z:[M+H] + calculated for C 25 H 35 O9N8BrFS2,753.11303; found,753.10962,Δ-4.53ppm.

[0199] 9. Synthesis of (S,Z)-16-amino-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid ethyl ester trifluoroacetate

[0200]

[0201] The intermediate (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(tert-butoxycarbonyl)-4,13-dioxo-3,14-dioxo-8,9-disulfide-2,5,12-triazahepta-1-ene-17-oic acid ethyl ester (407 mg, 0.54 mmol) was dissolved in 40 ml of ultra-dry dichloromethane, and trifluoroformic acid (4 ml) was added. After the addition was complete, the reaction was carried out under argon protection for 1 h. After evaporation, the next step was directly carried out.

[0202] 10. Synthesis of (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(2-cyanopyridin-4-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid ethyl ester

[0203]

[0204] The obtained yellow oil (S,Z)-16-amino-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4,13-dioxo-3,14-dioxo-8,9-disulfide-2,5,12-triazaheptadecan-1-en-17-oic acid ethyl ester trifluoroacetate was dissolved in 30 ml of DMF, and 2-(2-cyanopyridin-4-methyleneoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzaldehyde (432 mg, 0.81 mmol) and glacial acetic acid (195 mg, 3.24 mmol) were added. The mixture was stirred at room temperature for 1 h, sodium cyanoborohydride (204 mg, 3.24 mmol) was added, and the reaction was continued for 3 h. 150 ml of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (3×150 ml), washed with water (200 ml) and saturated brine (2×200 ml), dried over anhydrous sodium sulfate, evaporated to dryness, and purified by Silica gel column chromatography Compound 1 was isolated and purified to obtain 783 mg of a white solid. 1H NMR (400MHz, Acetone-d6): δ8.91(s,1H,-NH-),8.74(d,J=5.2Hz,1H,-ArH),8.08(s,1H,-NH-),7.84(d,J=4.9Hz,1H,-ArH),7.67(d,J=7.0Hz,1H,-ArH), 7.54–7.38(m,9H,-ArH),7.34(dd,J=10.7,4.2Hz,1H,-ArH),7.20(t,J=8.6H z,1H,-NH-),7.10(dd,J=13.9,8.7Hz,2H,-ArH),6.97(s,1H,-ArH),6.50(s, 1H,-NH-),6.00(s,2H,-NH2),5.42(s,2H,-CH2-),5.29(s,2H,-CH2-),4.30( dd,J=10.9,5.0Hz,1H,-CH2-),4.19(dd,J=10.8,5.5Hz,1H,-CH2-),4.16–4. 07(m,2H,-CH2-),3.88(m,2H,-CH2-),3.60–3.50(m,3H,-CH2-,-CH-),3.43( dd,J=12.6,6.2Hz,2H),2.92(t,J=6.9Hz,2H,-CH2-),2.89-2.84(2H,partly covered by the peak of water),1.21(dd,J=9.0,5.1Hz,3H,-CH3).HRMS(ESI)m / z:[M+H] + 1169.08813.

[0205] Alternatively, the obtained dark yellow oil (Z)-4-amino-N'-(2-((2-aminoethyl)disulfanyl)ethylaminoformyloxy)-N-(3-bromo-4-fluorophenyl)-1,2,5-oxadiazole-3-amidine-trifluoroacetate (1.34 mmol) was dissolved in DMF (35 ml), and Et3N (559 μl, 4.02 mmol) and N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(2-cyanopyridin-4-methoxy)benzyl)-N-(tert-butoxyformyl)-O-(p-nitrophenoxyformyl)-L-serine ethyl ester (1.23 g, 1.34 mmol) were added in sequence, and the mixture was stirred under Ar protection for 2 h. To the reaction solution was added 150 ml of water, extracted with ethyl acetate (3 × 150 ml), washed with saturated brine (2 × 200 ml), dried over anhydrous sodium sulfate, evaporated to dryness, and purified by silica gel column chromatography to obtain 950 mg of a white solid.

[0206] Example 2: (S,R,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-15-methyl-16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(2-cyanopyridin-4-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid ethyl ester (Compound 4)

[0207]

[0208] (S,R)-2-tert-butoxycarbonylamino-3-(4-nitrophenoxycarbonyloxy)butyric acid ethyl ester was used instead of (S)-2-tert-butoxycarbonylamino-3-(4-nitrophenoxycarbonyloxy)propionic acid ethyl ester. The same operation as in Example 1 was carried out to obtain a white solid with a yield of 24.30%. 1 H NMR (400MHz, Acetone): δ8.91(s,1H,-NH-),8.74(d,J=4.9Hz,1H,-ArH),8.08(s,1H,-ArH),7.83(d,J=4.9Hz,1H,-ArH),7.68(dd,J=7.6,1.1Hz,1H ,-ArH),7.53–7.32(m,9H,-ArH,-ArH),7.19(t,J=8.6Hz,1H,-NH-),7.15–7.02(m,2H,-ArH),6.97(s,1H,-ArH),6.41(t,J=5.5Hz,1H,-NH-),6.01( s,2H,-NH2),5.41(s,2H,-CH2-),5.29(s,2H,-CH2-),5.09(dd,J=6.2,4.4Hz,1H,-CH-),4.20–4.02(m,2H,-CH2-),3.92(d,J=13.5Hz,1H,-CH2-),3 .75(d,J=13.5Hz,1H,-CH2-),3.55(dd,J=12.5,6.2Hz,2H,-CH2-),3.40(dd,J=9.7,5.1Hz,3H,-CH2-,-CH-),2.98–2.79(4H,-CH2-,-CH2-,covered by the peak of water),2.30(s,1H,-NH-),1.27(d,J=6.4Hz,3H,-CH3),1.20(t,J=7.1Hz,3H,-CH3).HRMS(ESI)m / z:[M+H] + 1183.10291.

[0209] Example 3: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)

[0210] -16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid ethyl ester (Compound 10)

[0211]

[0212] 2-(5-cyanopyridin-3-methyleneoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzaldehyde was substituted for 2-(2-cyanopyridin-4-methyleneoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzaldehyde. The same procedures as in Example 1 were followed to obtain a white solid in a yield of 33.4%. HRMS (ESI) m / z: [M+H] + 1169.09113.

[0213] Example 4: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(5-methylsulfonylpyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid ethyl ester (Compound 12)

[0214]

[0215] 2-(5-methanesulfonylpyridin-3-methyleneoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzaldehyde was used instead of 2-(2-cyanopyridin-4-methyleneoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzaldehyde. The same procedure as in Example 1 was followed to obtain a white solid in a yield of 32.1%. HRMS (ESI) m / z: [M+H] + 1222.07021.

[0216] Example 5: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-chloro-3-(1,4-benzodioxane-6-yl)benzyloxy)-5-methyl-2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid methyl ester (Compound 7)

[0217]

[0218] 2-(5-cyanopyridin-3-methyleneoxy)-4-(2-chloro-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-methylbenzaldehyde was used in place of 2-(2-cyanopyridin-4-methyleneoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzaldehyde, and (S)-2-tert-butoxycarbonylamino-3-(4-nitrophenoxycarbonyloxy)propionic acid ethyl ester was used in place of (S)-2-tert-butoxycarbonylamino-3-(4-nitrophenoxycarbonyloxy)propionic acid ethyl ester. The same procedures as in Example 1 were followed to give a white solid in a yield of 34.23%. HRMS (ESI) m / z: [M+H] + 1149.18041.

[0219] Example 6: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-chloro-3-(1,4-benzodioxane-6-yl)benzyloxy)-5-methyl-2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid (Compound 8)

[0220]

[0221] (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-chloro-3-(1,4-benzodioxane-6-yl)benzyloxy)-5-methyl-2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid methyl ester was dissolved in an acetone solution containing lithium hydroxide, stirred at room temperature until completely hydrolyzed, poured into ice water, and allowed to settle. Filtration afforded a white solid (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-chloro-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-methyl-2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid). The yield was 85.5%. HRMS (ESI) m / z: [M+H] + 1135.16723.

[0222] Example 7: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)- 16-(4-((2-bromo-3-(1,4benzodioxan-6-yl)benzyloxy)-5-chloro-2-(5-cyanopyridin-3- ylmethoxy)benzamido)-4,13-dioxo-3,14-dioxo-8,9-dithia-2,5,12-triazahexadeca-1- en-17-oic acid ethyl ester (Compound 13)

[0223]

[0224] Using 2-(5-cyanopyridin-3-ylmethoxy)-4-(2-bromo-3-(1,4benzodioxan-6-yl)benzyloxy)- 5-chlorobenzaldehyde instead of 2-(2-cyanopyridin-4-ylmethoxy)-4-(2-bromo-3- phenylbenzyloxy)-5-chlorobenzaldehyde, the procedure was the same as Example 1 to give a white solid in 29.5% yield. HRMS (ESI) m / z: [M+H] + 1227.08641.

[0225] Example 8: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)- 16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(5-cyanopyridin-3- ylmethoxy)benzamido)-4,13-dioxo-3,14-dioxo-8,9-dithia-2,5,12-triazahexadeca-1- en-17-oic acid (Compound 11)

[0226]

[0227] (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-bromo- (1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzamido)- 4,13-dioxo-3,14-dioxo-8,9-dithia-2,5,12-triazahexadeca-1-en-17-oic acid ethyl ester was dissolved in acetone solution containing lithium hydroxide, stirred at room temperature until complete hydrolysis, poured into ice water, and the white solid (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)- 16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(5-cyanopyridin-3- ylmethoxy)benzamido)-4,13-dioxo-3,14-dioxo-8,9-dithia-2,5,12-triazahexadeca-1- en-17-oic acid was obtained by settling and filtration. Yield 79.9%. HRMS (ESI) m / z: [M+H]+ 1141.05563.

[0228] Example 9: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-methyl-16-(4-((2-bromo-3-(1,4-benzodioxane-6-yl)benzyloxy)-5-chloro-2-(2-cyanopyridin-4-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid ethyl ester (Compound 14)

[0229]

[0230] 2-(2-cyanopyridin-4-methyleneoxy)-4-(2-bromo-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chlorobenzaldehyde was substituted for 2-(2-cyanopyridin-4-methyleneoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzaldehyde, and (S)-2-methyl-2-tert-butoxycarbonylamino-3-(4-nitrophenoxycarbonyloxy)propanoic acid ethyl ester was substituted for (S)-2-tert-butoxycarbonylamino-3-(4-nitrophenoxycarbonyloxy)propanoic acid ethyl ester. The same procedures as in Example 1 were followed to give a white solid in a yield of 23.6%. HRMS (ESI) m / z: [M+H] + 1241.10531.

[0231] Example 10: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-14-(4-((2-bromo-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzyl)-15-methyl-4,13-dioxo-3,12-dioxo-8,9-dithio-2,5,14-triazahexadecene-1-en-16-oic acid ethyl ester (Compound 16)

[0232]

[0233] 1. Synthesis of (pyridin-2-yl)(aminoethyl) disulfide hydrochloride

[0234]

[0235] 2,2'-Disulfide dipyridine (13.58 g, 61.64 mmol) was added to 100 ml of methanol, and a methanol solution (100 ml) of mercaptoethylamine hydrochloride (4.67 g, 41.11 mmol) was added under Ar protection. After the addition was complete, the mixture was reacted at room temperature for 12 h. 500 ml of diethyl ether was added to the reaction solution, and the precipitated solid was filtered to obtain 7.60 g of a yellow solid.1 H NMR (300MHz, CD3OD): δ8.65(s,1H,-ArH), 8.24(d,J=6.5Hz,1H,-ArH), 8.12(d,J=7.2Hz,1H,-ArH), 7.67(s,1H,-ArH), 3.23(m,4H,-CH2-,-CH2-).

[0236] 2. Synthesis of (isocyanatoethyl)(pyridin-2-yl) disulfide

[0237]

[0238] The intermediate (pyridin-2-yl)(aminoethyl) disulfide hydrochloride (4 g, 17.96 mmol) and 1,8-bis(dimethylaminonaphthalene) (12.36 g, 53.88 mmol) were added to 120 ml of ultra-dry DCM. Diphosgene (1.08 ml, 8.98 mmol) was added under ice bath conditions. The mixture was moved to room temperature and stirred for 2 h. After evaporation, the next step of the reaction was directly carried out.

[0239] 3. Synthesis of (Z)-4-amino-N-(3-bromo-4-fluorophenyl)-N'-(2-(pyridine-2-disulfanyl)ethylamine formyloxy)-1,2,5-oxadiazole-3-amidine

[0240]

[0241] The crude intermediate (isocyanatoethyl)(pyridin-2-yl) disulfide (3 g, 17.96 mmol) was dissolved in 160 mL of DCM, followed by the addition of the intermediate 4-amino-1,2,5-oxadiazole-3-chloroformaldehyde oxime (1.5 g, 8.98 mmol) and DIPEA (5.93 mL, 35.90 mmol). The mixture was stirred under Argon protection for 12 h. 140 mL of 1N aqueous hydrochloric acid was added to the reaction solution, and the mixture was extracted with dichloromethane (3 × 200 mL). The mixture was washed with 1N aqueous hydrochloric acid (2 × 150 mL) and saturated aqueous sodium chloride (200 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to approximately 100 mL. The precipitated solid was filtered and dried to yield 1.4 g (3.74 mmol) of a white solid. It was dissolved in 13.6 ml of anhydrous ethanol, and 3-bromo-4-fluoroaniline (0.72 g, 3.76 mmol) was added. Under stirring in an ice-water bath, an aqueous solution (10.8 ml) of NaHCO3 (0.72 g, 8.5 mmol) was added. The temperature was raised to 60°C and the reaction was carried out for 2 h. The ethanol was evaporated under reduced pressure, and the product was extracted with ethyl acetate (3×150 ml), washed with saturated aqueous NaCl solution (1×60 ml), dried over anhydrous Na2SO4, concentrated, and separated by column chromatography to obtain 0.78 g of (Z)-4-amino-N-(3-bromo-4-fluorophenyl)-N'-(2-(pyridine-2-disulfanyl)ethylaminecarbonyloxy)-1,2,5-oxadiazole-3-amidine.

[0242] Alternatively, the crude intermediate (isocyanatoethyl)(pyridin-2-yl) disulfide (3 g, 17.96 mmol) was dissolved in 160 mL of DCM, followed by the addition of the intermediate (Z)-4-amino-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-amidine (2.84 g, 8.98 mmol) and DIPEA (5.93 mL, 35.90 mmol). The mixture was stirred under Ar protection for 12 h. 140 mL of 1N aqueous hydrochloric acid was added to the reaction solution, and the mixture was extracted with dichloromethane (3 × 200 mL). The mixture was washed with 1N aqueous hydrochloric acid (2 × 150 mL) and saturated aqueous sodium chloride (200 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to approximately 100 mL. The precipitated solid was filtered to obtain 2.28 g of a white solid.

[0243] 1H NMR (400MHz, DMSO-d6): δ9.78(s,1H,-NH-),8.45(d,J=5.0Hz,1H,-ArH),7.87– 7.74(m,2H,-ArH),7.72(t,J=5.8Hz,1H,-ArH),7.32(dd,J=6.1,2.6Hz,1H,ArH) ,7.29–7.21(m,2H,-ArH),6.98–6.89(m,1H,-NH-),6.43(s,2H,-NH2),3.42(dd ,J=12.8,6.4Hz,2H,-CH2-),2.98(t,J=6.7Hz,2H,-CH2-).HRMS(ESI)m / z:[M+H] + 527.99121.

[0244] 4. Synthesis of (Z)-4-amino-N'-(2-((2-hydroxyethyl)disulfanyl)ethylcarbamoyloxy)-N-(3-bromo-4-fluorophenyl)-1,2,5-oxadiazole-3-amidine

[0245]

[0246] Dissolve 528 mg (1 mmol) of (Z)-4-amino-N-(3-bromo-4-fluorophenyl)-N'-(2-(pyridin-2-disulfanyl)ethylaminecarboxyloxy)-1,2,5-oxadiazole-3-amidine in 15 ml of anhydrous THF, add 94 mg of mercaptoethanol (1.2 mmol), and react at room temperature for 24 hours. Evaporate the solvent under reduced pressure, dissolve the product in 50 ml of ethyl acetate, wash with water, then with saturated brine, dry over anhydrous sodium sulfate, concentrate, and separate by column chromatography to obtain 480 mg of a white solid. LCMS: m / z 496.8445 [M+H] + NMR data: 1 H NMR (400MHz, DMSO-d6) δ9.70 (s, 1H, -NH-), 7.54 (t, J = 5.6Hz, 1H, -NH-), 7.31 (dd, J=6.0,2.5Hz,1H,-PhH),7.25(t,J=8.7Hz,1H,-PhH),6.96–6.87(m,1H,-PhH),6. 40(s,2H,-NH2),4.87(t,J=6.0Hz,1H,-OH),3.65–3.60(m,2H,-CH2-),3.41(q,J= 6.4Hz,2H,-CH2-),2.85(dd,J=12.5,5.6Hz,2H,-CH2-),2.82–2.73(m,2H,-CH2-).

[0247] 5. Synthesis of (S)-N-(4-((2-bromo-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chloro-2-(5-cyanopyridin-3-methyleneoxy)benzyl)-N-(4-nitrophenoxycarbonyl)alanine ethyl ester

[0248] (S)-N-(4-((2-bromo-3-(1,4-benzodioxane-6-yl)benzyloxy)-5-chloro-2-(5-cyanopyridin-3-methyleneoxy)benzyl)alanine ethyl ester (16.6 g, 24 mmol) was dissolved in ultra-dry dichloromethane (100 ml), and p-nitrophenyl chloroformate (4.78 g, 24 mmol) and Et3N (3.33 ml, 23.89 mmol) were added. After the addition was complete, the mixture was reacted at room temperature for 10 h, evaporated to dryness, and 150 ml of ethyl acetate was added to dissolve the mixture. The mixture was washed with saturated sodium bicarbonate aqueous solution, water, and saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 16 g of a yellow solid.

[0249] 6. (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-14-(4-((2-bromo-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzyl)-15-methyl-4,13-dioxo-3,12-dioxo-8,9-dithio-2,5,14-triazahexadecene-1-en-16-oic acid ethyl ester

[0250] Dissolve (S)-N-(4-((2-bromo-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chloro-2-(5-cyanopyridin-3-methyleneoxy)benzyl)-N-(4-nitrophenoxycarbonyl)alanine ethyl ester (858 mg, 1 mmol) in 10 ml Cesium carbonate (652 mg, 2 mmol) and (Z)-4-amino-N'-(2-((2-hydroxyethyl)disulfanyl)ethylcarbamoyloxy)-N-(3-bromo-4-fluorophenyl)-1,2,5-oxadiazole-3-amidine (743 mg, 1.5 mmol) were added to DMF and reacted at room temperature until complete. The mixture was then poured into saturated ammonium chloride water and iced water. The mixture was extracted three times with 100 ml of ethyl acetate. The organic phases were combined, washed with water and saturated brine, dried over anhydrous sodium sulfate, evaporated to dryness, and separated by column chromatography to obtain a white solid in a yield of 61.7%. HRMS (ESI) m / z: [M+H] + 1212.07131.

[0251] Example 11: (S,Z)-15-(acetoxymethyl)-14-(4-((2-bromo-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzyl)-1-(3-bromo-4-fluoroanilino)-4,13-dioxo-1-(4-(2-(sulfamoylamino)ethylamino)-1,2,5-oxadiazol-3-yl)-3,12-dioxo-8,9-dithio-2,5,14-triazahexadecene-1-en-16-oic acid ethyl ester (Compound 18)

[0252]

[0253] (S)-N-(4-((2-bromo-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chloro-2-(5-cyanopyridin-3-methyleneoxy)benzyl)-N-(4-nitrophenoxycarbonyl)-O-acetylserine ethyl ester was used to replace (S)-N-(4-((2-bromo-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chloro-2-(5-cyanopyridin-3-methyleneoxy)benzyl)-N-(4-nitrophenoxycarbonyl)-O-acetylserine ethyl ester, and (Z)-N'- (Z)-4-amino-N'-(2-((2-hydroxyethyl)disulfanyl)ethylaminoformyloxy)-N-(3-bromo-4-fluorophenyl)-1,2,5-oxadiazole-3-amidine was replaced with (2-((2-hydroxyethyl)disulfanyl)ethylaminoformyloxy)-N-(3-bromo-4-fluorophenyl)-1,2,5-oxadiazole-3-amidine. The same procedures as in Example 10 were followed to give a white solid in a yield of 34.8%. HRMS (ESI) m / z: [M+H] + 1392.09033.

[0254] Example 12: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-14-(4-((2-bromo-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzyl)-15-(acetoxymethyl)-4,13-dioxo-3,12-dioxo-8,9-dithio-2,5,14-triazahexadecene-1-en-16-oic acid ethyl ester (Compound 19)

[0255]

[0256] (S)-N-(4-((2-bromo-3-(1,4-benzodioxin-6-yl)benzyloxy)-5-chloro-2-(5-cyanopyridin-3-methyleneoxy)benzyl)-N-(4-nitrophenoxycarbonyl)-O-acetylserine ethyl ester was substituted with (S)-N-(4-((2-bromo-3-(1,4-benzodioxin-6-yl)benzyloxy)-5-chloro-2-(5-cyanopyridin-3-methyleneoxy)benzyl)-N-(4-nitrophenoxycarbonyl)-O-acetylserine ethyl ester. The same procedures as in Example 10 were followed to give a white solid in a yield of 57.2%. HRMS (ESI) m / z: [M+H] + 1270.08451.

[0257] Example 13: (2S,4S)-4-((((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4-oxo-3-oxo-8,9-dithio-2,5-diazaundec-1-en-11-yl)carbamoyl)oxy)-1-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-((2-cyanopyridin-4-yl)methoxy)benzyl)pyrrolidine-2-carboxylic acid ethyl ester (Compound 22)

[0258]

[0259] 1. Synthesis of (pyridin-2-yl)(aminoethyl) disulfide hydrochloride

[0260]

[0261] 2,2'-Disulfide dipyridine (13.58 g, 61.64 mmol) was added to 100 ml of methanol, and a methanol solution (100 ml) of mercaptoethylamine hydrochloride (4.67 g, 41.11 mmol) was added under Ar protection. After the addition was complete, the mixture was reacted at room temperature for 12 h. 500 ml of diethyl ether was added to the reaction solution, and the precipitated solid was filtered to obtain 7.60 g of a yellow solid. 1 H NMR (300MHz, CD3OD): δ8.65(s,1H,-ArH), 8.24(d,J=6.5Hz,1H,-ArH), 8.12(d,J=7.2Hz,1H,-ArH), 7.67(s,1H,-ArH), 3.23(m,4H,-CH2-,-CH2-).

[0262] 2. Synthesis of (isocyanatoethyl)(pyridin-2-yl) disulfide

[0263]

[0264] The intermediate (pyridin-2-yl)(aminoethyl) disulfide hydrochloride (4 g, 17.96 mmol) and 1,8-bis(dimethylaminonaphthalene) (12.36 g, 53.88 mmol) were added to 120 ml of ultra-dry DCM. Diphosgene (1.08 ml, 8.98 mmol) was added under ice bath conditions. The mixture was moved to room temperature and stirred for 2 h. After evaporation, the next step of the reaction was directly carried out.

[0265] 3. Synthesis of (Z)-4-amino-N-(3-bromo-4-fluorophenyl)-N'-(2-(pyridine-2-disulfanyl)ethylamine formyloxy)-1,2,5-oxadiazole-3-amidine

[0266]

[0267] The crude intermediate (isocyanatoethyl)(pyridin-2-yl) disulfide (3 g, 17.96 mmol) was dissolved in 160 mL of DCM, followed by the addition of the intermediate 4-amino-1,2,5-oxadiazole-3-chloroformaldehyde oxime (1.5 g, 8.98 mmol) and DIPEA (5.93 mL, 35.90 mmol). The mixture was stirred under Argon protection for 12 h. 140 mL of 1N aqueous hydrochloric acid was added to the reaction solution, and the mixture was extracted with dichloromethane (3 × 200 mL). The mixture was washed with 1N aqueous hydrochloric acid (2 × 150 mL) and saturated aqueous sodium chloride (200 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to approximately 100 mL. The precipitated solid was filtered and dried to yield 1.4 g (3.74 mmol) of a white solid. It was dissolved in 13.6 ml of anhydrous ethanol, and 3-bromo-4-fluoroaniline (0.72 g, 3.76 mmol) was added. Under stirring in an ice-water bath, an aqueous solution (10.8 ml) of NaHCO3 (0.72 g, 8.5 mmol) was added. The temperature was raised to 60°C and the reaction was carried out for 2 h. The ethanol was evaporated under reduced pressure, and the product was extracted with ethyl acetate (3×150 ml), washed with saturated aqueous NaCl solution (1×60 ml), dried over anhydrous Na2SO4, concentrated, and separated by column chromatography to obtain 0.78 g of (Z)-4-amino-N-(3-bromo-4-fluorophenyl)-N'-(2-(pyridine-2-disulfanyl)ethylaminecarbonyloxy)-1,2,5-oxadiazole-3-amidine.

[0268] Alternatively, the crude intermediate (isocyanatoethyl)(pyridin-2-yl) disulfide (3 g, 17.96 mmol) was dissolved in 160 mL of DCM, followed by the addition of the intermediate (Z)-4-amino-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-amidine (2.84 g, 8.98 mmol) and DIPEA (5.93 mL, 35.90 mmol). The mixture was stirred under Ar protection for 12 h. 140 mL of 1N aqueous hydrochloric acid was added to the reaction solution, and the mixture was extracted with dichloromethane (3 × 200 mL). The mixture was washed with 1N aqueous hydrochloric acid (2 × 150 mL) and saturated aqueous sodium chloride (200 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to approximately 100 mL. The precipitated solid was filtered to obtain 2.28 g of a white solid.

[0269] 1 H NMR (400MHz, DMSO-d6): δ9.78(s,1H,-NH-),8.45(d,J=5.0Hz,1H,-ArH),7.87– 7.74(m,2H,-ArH),7.72(t,J=5.8Hz,1H,-ArH),7.32(dd,J=6.1,2.6Hz,1H,ArH) ,7.29–7.21(m,2H,-ArH),6.98–6.89(m,1H,-NH-),6.43(s,2H,-NH2),3.42(dd ,J=12.8,6.4Hz,2H,-CH2-),2.98(t,J=6.7Hz,2H,-CH2-).HRMS(ESI)m / z:[M+H] + 527.99121.

[0270] 4. Synthesis of 2-(2-cyanopyridin-4-methyleneoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzaldehyde

[0271]

[0272] The intermediate 2-hydroxy-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzaldehyde (1.50 g, 3.59 mmol) was added to 25 ml of DMF, followed by KCO (992 mg, 7.18 mmol) and 4-(bromomethyl)picolinecarbonitrile (1.06 g, 5.39 mmol). After addition, the mixture was stirred at room temperature for 4 h. Water (80 ml) was added to the reaction mixture, and a solid precipitated. The solid was filtered and the filter cake was purified by column chromatography to yield 1.38 g of a white solid.

[0273] 5. (S,S)-N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(2-cyanopyridin-4-methoxy)benzyl)-O-(p-nitrophenoxycarbonyl)-4-hydroxyproline ethyl ester

[0274]

[0275] Dissolve (S,S)-N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(2-cyanopyridin-4-methoxy)benzyl)-4-hydroxyproline ethyl ester (10.8 g, 15.98 mmol) in ultra-dry dichloromethane (100 ml). Add p-nitrophenyl chloroformate (4.78 g, 24 mmol) and Et3N (3.33 ml, 23.89 mmol). After addition, react at room temperature for 6 h, evaporate to dryness, and purify by column chromatography to obtain 11 g of a yellow solid. HRMS (ESI) m / z: [M+H] + 841.11897.

[0276] 6. Synthesis of tert-butyl (Z)-(1-(4-amino-1,2,5-oxadiazol-3-yl)-1-((3-bromo-4-fluorophenyl)amino)-4-oxo-3-oxo-8,9-dithio-2,5-diazaundec-1-en-11-yl)carbamate

[0277]

[0278] The intermediate (Z)-4-amino-N-(3-bromo-4-fluorophenyl)-N'-(2-(pyridine-2-disulfanyl)ethylamine formyloxy)-1,2,5-oxadiazole-3-amidine (3 g, 5.69 mmol) was dissolved in a mixture of dichloromethane (100 mL) and methanol (100 mL). 2-tert-Butyloxycarbonylaminoethanethiol (1.31 g, 7.40 mmol) was added and stirred under Ar protection for 1 hour. 200 mL of 0.5N aqueous hydrochloric acid was added to the reaction solution, and the mixture was extracted with dichloromethane (2 × 150 mL). The product was washed with saturated aqueous NaHCO₃ (200 mL), dried over anhydrous sodium sulfate, evaporated to dryness, and purified by silica gel column chromatography to obtain 1.59 g of a yellow oil. 1H NMR (400MHz, Acetone-d6): δ8.94(s,1H,-NH-),7.45(dd,J=6.0,2.6Hz,1H,-Ar H),7.21(t,J=8.6Hz,1H,-NH-),7.16–7.05(m,2H,-ArH),6.19(s,1H,-NH-),6. 01(s,2H,-NH2),3.57(dd,J=12.7,6.4Hz,2H,-CH2-),3.39(dd,J=13.1,6.4Hz, 2H,-CH2-),2.94(dd,J=8.3,5.3Hz,2H,-CH2-),2.91–2.81(2H,-CH2-,covered by the peak of water),1.40(s,9H,-Boc).HRMS(ESI)m / z:[M+H] + 594.05817.

[0279] 7. Synthesis of (Z)-4-amino-N'-(2-((2-aminoethyl)disulfanyl)ethylcarbamoyloxy)-N-(3-bromo-4-fluorophenyl)-1,2,5-oxadiazole-3-amidine-trifluoroacetate

[0280]

[0281] The intermediate (Z)-(1-(4-amino-1,2,5-oxadiazol-3-yl)-1-((3-bromo-4-fluorophenyl)amino)-4-oxo-3-oxo-8,9-disulfide-2,5-diazaundec-1-en-11-yl)carbamic acid tert-butyl ester (797 mg, 1.34 mmol) was dissolved in 35 ml of dichloromethane, and trifluoroacetic acid (3.5 ml) was added. After the addition was complete, the reaction was carried out under argon protection for 1 hour, and the next reaction was directly carried out after evaporation.

[0282] 8. Synthesis of tert-butyl (2R,4R)-4-(((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4-oxo-3-oxo-8,9-dithio-2,5-diazaundec-1-en-11-yl)carbamoyloxy)pyrrolidine-1-carboxylate-2-carboxylic acid ethyl ester

[0283]

[0284] The resulting (Z)-4-amino-N'-(2-((2-aminoethyl)disulfanyl)ethylcarbamoyloxy)-N-(3-bromo-4-fluorophenyl)-1,2,5-oxadiazole-3-amidine trifluoroacetate was dissolved in DMF (35 ml). Et3N (559 μl, 4.02 mmol) and (S,S)-N-tert-butoxycarbonyl-4-(4-nitrophenoxycarbonyloxy)proline ethyl ester (568 mg, 1.34 mmol) were added sequentially. The mixture was stirred under Ar protection for 2 h. 150 ml of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 150 ml). The mixture was washed with saturated brine (2 × 200 ml), dried over anhydrous sodium sulfate, evaporated to dryness, and purified by silica gel column chromatography to obtain 897 mg of a light yellow oil. HRMS (ESI) m / z: [M+H] + 779.11982.9. Synthesis of (2R,4R)-4-(((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4-oxo-3-oxo-8,9-dithio-2,5-diazaundec-1-en-11-yl)carbamoyloxy)proline ethyl ester trifluoroacetate

[0285]

[0286] The intermediate (2R,4R)-4-(((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4-oxo-3-oxo-8,9-disulfide-2,5-diazaundec-1-en-11-yl)carbamoyloxy)pyrrolidine-1-carboxylic acid tert-butyl ester-2-ethyl carboxylate (420 mg, 0.54 mmol) was dissolved in 40 ml of ultra-dry dichloromethane, and trifluoroformic acid (4 ml) was added. After the addition was complete, the reaction was carried out under argon protection for 1 h. After evaporation to dryness, the next reaction was directly carried out.

[0287] 10. Synthesis of ethyl (2S,4S)-4-((((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4-oxo-3-oxo-8,9-dithio-2,5-diazaundec-1-en-11-yl)carbamoyl)oxy)-1-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-((2-cyanopyridin-4-yl)methoxy)benzyl)pyrrolidine-2-carboxylate

[0288] The obtained yellow oil (2R,4R)-4-(((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4-oxo-3-oxo-8,9-disulfide-2,5-diazaundec-1-en-11-yl)carbamoyloxy)proline ethyl ester trifluoroacetate was dissolved in 30 ml of DMF, and 2-(2-cyanopyridin-4-methyleneoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzaldehyde (432 mg, 0.81 mmol) and glacial acetic acid (195 mg, 3.24 mmol) were added. The mixture was stirred at room temperature for 1 h, sodium cyanoborohydride (204 mg, 3.24 mmol) was added, and the reaction was continued for 3 h. 150 ml of water was added to the reaction solution, extracted with ethyl acetate (3×150 ml), washed with water (200 ml) and saturated brine (2×200 ml), dried over anhydrous sodium sulfate, and evaporated to dryness. Silica gel column chromatography A white solid was obtained by separation and purification, with a yield of 15.88%. 1 H NMR (400MHz, DMSO-d6): δ9.71(s,1H,-NH-),8.76(d,J=5.0Hz,1H,-ArH),8.16(s,1H,-ArH),7.81(d,J=5.0Hz,1H,-ArH),7.62(d,J=7.6Hz,1H,-ArH H),7.57–7.29(m,11H,-NH-,-ArH,-ArH),7.24(t,J=8.7Hz,1H,-NH-),6.98(s,1H,-ArH),6.96–6.90(m,1H,-ArH),6.41(s,2H,-NH2),5.36(q,J=14 .5Hz,2H,-CH2-),5.27(s,2H,-CH2-),5.03(s,1H,-CH-),4.03–3.92(m,2H,-CH2-),3.78(q,J=13.5Hz,2H,-CH2-),3.50(t,J=6.8Hz,1H,-CH-),3.4 5–3.37(m,2H,-CH2-),3.33–3.21(m,3H,-CH2-,-CH2-),2.86(t,J=6.6Hz,2H,-CH2-),2.78(t,J=6.3Hz,2H,-CH2-),2.55–2.49(1H,partly covered by the peak of solvent),2.28–2.15(m,1H,-CH2-),2.12–2.01(m,1H,-CH2-),1.11(t,J=7.1Hz,3H,-CH3).HRMS(ESI)m / z:[M+H] + 1195.10461ppm.

[0289] The obtained dark yellow oil (Z)-4-amino-N'-(2-((2-aminoethyl)disulfanyl)ethylaminoformyloxy)-N-(3-bromo-4-fluorophenyl)-1,2,5-oxadiazole-3-amidine-trifluoroacetate (1.34 mmol) was dissolved in DMF (35 ml), and Et3N (559 μl, 4.02 mmol) and (S,S)-N-(4-(2-bromo-3-phenylbenzyloxy)-5-chloro-2-(2-cyanopyridin-4-methoxy)benzyl)-O-(p-nitrophenoxyformyl)-4-hydroxyproline ethyl ester (1.13 g, 1.34 mmol) were added in sequence, and the mixture was stirred under Ar protection for 2 h. To the reaction solution was added 150 ml of water, extracted with ethyl acetate (3 × 150 ml), washed with saturated brine (2 × 200 ml), dried over anhydrous sodium sulfate, evaporated to dryness, and purified by silica gel column chromatography to obtain 910 mg of a white solid.

[0290] Example 14: (2S,4S)-4-((((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4-oxo-3-oxo-8,9-dithio-2,5-diazaundec-1-en-11-yl)carbamoyl)oxy)-1-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-((5-cyanopyridin-3-yl)methoxy)benzyl)pyrrolidine-2-carboxylic acid ethyl ester (Compound 23)

[0291]

[0292] 2-(5-cyanopyridin-3-methyleneoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzaldehyde was substituted for 2-(2-cyanopyridin-4-methyleneoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzaldehyde. The same procedures as in Example 13 were followed to obtain a white solid in a yield of 31.4%. HRMS (ESI) m / z: [M+H] + 1195.09231.

[0293] Example 15: (2S,4S)-4-((((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4-oxo-3-oxo-8,9-dithio-2,5-diazaundec-1-en-11-yl)carbamoyl)oxy)-1-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-((5-cyanopyridin-3-yl)methoxy)benzyl)pyrrolidine-2-carboxylic acid (Compound 24)

[0294]

[0295] Ethyl (2S,4S)-4-((((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4-oxo-3-oxo-8,9-disulfide-2,5-diazaundec-1-en-11-yl)carbamoyl)oxy)-1-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-((5-cyanopyridin-3-yl)methoxy)benzyl)pyrrolidine-2-carboxylate was dissolved in an acetone solution containing lithium hydroxide and stirred at room temperature until completely hydrolyzed. The mixture was poured into ice water and filtered to obtain a white solid. The yield was 87.2%. HRMS (ESI) m / z: [M+H] + 1167.06621.

[0296] Example 16: (2S,4S)-4-((((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4-oxo-3-oxo-8,9-dithio-2,5-diazaundec-1-en-11-yl)carbamoyl)oxy)-1-(4-((2-chloro-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chloro-2-((5-cyanopyridin-3-yl)methoxy)benzyl)pyrrolidine-2-carboxylic acid ethyl ester (Compound 25)

[0297]

[0298] 2-(5-cyanopyridin-3-methyleneoxy)-4-(2-chloro-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chlorobenzaldehyde was substituted with 2-(5-cyanopyridin-3-methyleneoxy)-4-(2-chloro-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chlorobenzaldehyde. The same procedures as in Example 12 were followed to give a white solid in a yield of 29.94%. HRMS (ESI) m / z: [M+H] + 1209.15541.

[0299] Example 17: (2S,4S)-4-((((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4-oxo-3-oxo-8,9-dithio-2,5-diazaundec-1-en-11-yl)carbamoyl)oxy)-1-(4-((2-chloro-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chloro-2-((5-cyanopyridin-3-yl)methoxy)benzyl)pyrrolidine-2-carboxylic acid (Compound 26)

[0300]

[0301] Ethyl (2S,4S)-4-((((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4-oxo-3-oxo-8,9-dithio-2,5-diazaundec-1-en-11-yl)carbamoyl)oxy)-1-(4-((2-chloro-3-(1,4-benzodioxan-6-yl)benzyloxy)-5-chloro-2-((5-cyanopyridin-3-yl)methoxy)benzyl)pyrrolidine-2-carboxylate was dissolved in an acetone solution containing lithium hydroxide, stirred at room temperature until completely hydrolyzed, poured into ice water, and filtered to obtain a white solid. The yield was 79.2%. HRMS (ESI) m / z: [M+H] + 1181.12325.

[0302] Pharmacological activity

[0303] 1. Evaluation of inhibitory activity on the interaction between PD-1 and PD-L1:

[0304] The in vitro enzymatic level detection method uses the PD-1 / PD-L1 binding assay kit produced by Cisbio.

[0305] Screening principles and methods for PD-1 / PD-L1 small molecule inhibitors

[0306] 1) Principle: The PD-1 protein carries a HIS tag, and the PD-1 ligand PD-L1 carries an hFc tag. Eu-labeled anti-hFc antibody and XL665-labeled anti-HIS antibody are respectively bound to the two tagged proteins. After laser excitation, energy can be transferred from the donor Eu to the acceptor XL665, causing XL665 to emit light. When an inhibitor (compound or antibody) is added, the binding of PD-1 and PD-L1 is blocked, making the distance between Eu and XL665 greater, preventing energy transfer and XL665 from emitting light.

[0307] 2) Experimental Methods: For specific methods, please refer to the PD-1 / PD-L1 kit (Cat. No. 64ICP01PEG) from Cisbio. Briefly, a 96-well white ELISA plate (Cisbio, Cat. No. 66PL96100) was used. 2 μl of diluent or target compound diluted with diluent was added to each well. 4 μl of PD-1 protein and 4 μl of PD-L1 protein were then added to each well. The plates were incubated at room temperature for 15 minutes, and 10 μl of anti-Tag1-Eu3 was added to each well. + The mixture of anti-Tag2-XL665 was incubated at room temperature for 2-6 hours and then the fluorescence signals at 665nm and 620nm were detected using a Tecan Spark instrument. HTRF ratio = (665nm / 620nm)*10 4Each compound was tested at 6-10 concentrations, and IC was calculated using Graphpad software. 50 .

[0308] 3) The screening results are shown in Table 1: A represents IC 50 Value less than 10 -7 M, B stands for IC 50 Value is 10 -6 to 10 -7 M stands for IC 50 Value greater than 10 -6 M.

[0309] Table 1. Screening results of inhibitory activity evaluation of some compounds on the interaction between PD-1 and PD-L1

[0310] Compound <![CDATA[IC 50 (M)]]> Compound IC 50 (M) 1 B 16 C 4 B 18 C 10 B 19 C 12 B 22 B 7 B 23 B 8 A 24 A 13 B 25 B 11 A 26 A 14 B

[0311] 2. Determination of tumor cell survival rate by MTT assay

[0312] Experimental methods:

[0313] Take cells in the logarithmic growth phase and digest them with trypsin to prepare a cell suspension, which was then inoculated into a 96-well plate. The next day, fresh culture medium containing different concentrations of compounds and corresponding solvent controls was added. Each compound was divided into 4-6 dose groups, and each group had at least three parallel wells. After continuing to culture at 37°C for 96 hours, the supernatant was discarded, and freshly prepared serum-free culture medium containing 0.5 mg / mL MTT was added to each well. The culture was continued for 4 hours, and the culture supernatant was discarded. DMSO was added to each well to dissolve the MTT formazan precipitate. After oscillation and mixing, 450 nm was used as the reference wavelength, and the OD value was measured at a wavelength of 570 nm. The tumor cells treated with the solvent control were used as the control group. The inhibition rate of the drug on the tumor cells was calculated, and the IC was calculated according to the median effect equation. 50 .

[0314] Inhibition rate = (average OD value of the control group - average OD value of the drug group) / average OD value of the control group × 100%. Experimental results:

[0315] Table 2 MTT screening results of some compounds

[0316]

[0317]

[0318] Table 3 MTT screening results of some compounds

[0319]

[0320] Tumor cell lines: B16F10-melanoma, HepG2-liver cancer, MGC803-gastric cancer, Mia-PaCa2-pancreatic cancer, UO31-renal cancer, NCI-H1975-lung cancer, MDA-MB-231-breast cancer, PC3-prostate cancer, MC38-intestinal cancer, A2780-ovarian cancer, BIU-87-bladder cancer, U87MG-brain cancer.

[0321] 3. In vivo pharmacodynamic studies of the compounds in the examples

[0322] 1) Tumor inhibition rate of oral administration of ZC-102 (Compound 1) in a mouse melanoma B16F10 subcutaneous transplant tumor model

[0323] Purpose of the experiment

[0324] The in vivo antitumor efficacy of the dual-target inhibitor ZC-102 against mouse melanoma B16F10 was evaluated in a mouse subcutaneous xenograft tumor model.

[0325] Experimental Materials

[0326] ZC-102 should be stored at room temperature and in the dark.

[0327] Positive drug: cyclophosphamide (CTX), Androstenone, specification 0.2g, batch number: 7B153A.

[0328] Animals: C57 mice, weighing 16-18 g, SPF grade, female, were provided by Spavor.

[0329] Experimental methods

[0330] step:

[0331] The passaged B16F10 tumor cells were homogenized with a homogenizer, washed twice with sterile saline, and counted. The cell concentration was adjusted to 9×10 6 / ml, 0.2ml of the cell suspension was inoculated into the right axilla of C57 mice. The day after inoculation, the animals were randomly divided into 10 groups. Each group was weighed and dosed. The test compound was administered once daily for 19 consecutive doses, and the animals were weighed. The animals were then sacrificed, and tumor tissue was removed and weighed. The tumor inhibition rate was calculated and used to evaluate the anti-tumor effect.

[0332] Grouping:

[0333] Blank control group, cyclophosphamide 80 mg (injection), PD-L1 monoclonal antibody 10 mg (injection), ZC-102 compound 37 mg (oral).

[0334] Preparation:

[0335] The test compound, ZC-102, was accurately weighed, ground, and dissolved in 0.5% CMCNa to a concentration of 1.85 mg / ml. Each animal was orally administered 0.4 ml / 20 g (37 mg / kg) of the solution, based on body weight. Prepare immediately before use.

[0336] Calculation method:

[0337] Relative tumor growth rate T / C (%): T / C%=T / C×100% (T: tumor weight of treatment group; C: tumor weight of negative control group).

[0338] Tumor growth inhibition rate (TGI) (%): TGI = (1-T / C) × 100. (T: tumor weight of treatment group; C: tumor weight of negative control group).

[0339] CON and control stand for control group, antibody stands for antibody, CTX stands for cyclophosphamide, IDO stands for (Z)-4-amino-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-1,2,5-oxadiazole-3-amidine, and 008 stands for N-[2-(2-cyanopyridin-4-methyleneoxy)-4-(2-bromo-3-phenylbenzyloxy)-5-chlorobenzyl]serine ethyl ester.

[0340] Experimental results

[0341] Efficacy

[0342] Tumor weight

[0343] The tumor weights of the treated animals were weighed 19 days after administration. The effects of ZC-102 on B16F10 mice were shown in Figure 1 and Table 4.

[0344] Table 4. In vivo antitumor efficacy of the dual-target inhibitor ZC-102 compound on B16F10 mice

[0345]

[0346] 1.TTEST, * P<0.05, *** P<0.001

[0347] NA: Not applicable

[0348] 2) In vivo antitumor efficacy of ZC102 injection and oral administration on CT26 colon cancer mice, mouse body weight and spleen weight, index, and regulatory T cell analysis

[0349] like Figure 2 A and Figure 2As shown in Figure B, in the mouse CT26 xenograft model, daily intraperitoneal injection of 4 mg / kg of zc102 produced significant in vivo anti-tumor activity with a TGI of 54.9%, while daily oral administration of 20 mg / kg of the PD-L1 inhibitor 008 and 100 mg / kg of IDO alone produced TGIs of 40.6% and 32.1%, respectively. The above results indicate that the dual-target inhibitor zc102 has stronger in vivo tumor inhibition activity than single-agent 008 and IDO. In addition, compared with the blank control group, there was no significant change in the body weight of mice in the zc102-treated group ( Figure 2 C).

[0350] like Figure 3 A and Figure 3 As shown in B, zc102 treatment did not affect the spleen weight and spleen index of mice. Figure 3 As shown in Figure C, after zc102 treatment, the number of regulatory T cells (CD4+CD25+Foxp3+) in the spleen of mice showed a downward trend, which indirectly indicates that the dual-target compound zc102 can inhibit the generation of regulatory T cells by inhibiting the activity of IDO1.

Claims

1. Any one of the disulfide bond-containing compounds and stereoisomers or pharmaceutically acceptable salts thereof, wherein the compound is selected from: Compound 1: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(2-cyanopyridin-4-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid ethyl ester Compound 2: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-chloro-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid ethyl ester Compound 3: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-chloro-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid Compound 4: (S,R,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-15-methyl-16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(2-cyanopyridin-4-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid ethyl ester Compound 5: (S,R,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-15-methyl-16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid ethyl ester Compound 6: (S,R,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-15-methyl-16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid Compound 10: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid ethyl ester Compound 11: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-16-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzylamino)-4,13-dioxo-3,14-dioxo-8,9-dithio-2,5,12-triazaheptadecan-1-en-17-oic acid Compound 21: (S,Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-14-(4-((2-bromo-3-phenylbenzyloxy)-5-chloro-2-(5-cyanopyridin-3-ylmethoxy)benzyl)-15-hydroxymethyl-4,13-dioxo-3,12-dioxo-8,9-dithio-2,5,14-triazahexadecene-1-en-16-oic acid Compound 22: (2S,4S)-4-((((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4-oxo-3-oxo-8,9-dithio-2,5-diazaundec-1-en-11-yl)carbamoyl)oxy)-1-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-((2-cyanopyridin-4-yl)methoxy)benzyl)pyrrolidine-2-carboxylic acid ethyl ester Compound 23: (2S,4S)-4-((((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4-oxo-3-oxo-8,9-dithio-2,5-diazaundec-1-en-11-yl)carbamoyl)oxy)-1-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-((5-cyanopyridin-3-yl)methoxy)benzyl)pyrrolidine-2-carboxylic acid ethyl ester Compound 24: (2S,4S)-4-((((Z)-1-(4-amino-1,2,5-oxadiazol-3-yl)-1-(3-bromo-4-fluoroanilino)-4-oxo-3-oxo-8,9-dithio-2,5-diazaundec-1-en-11-yl)carbamoyl)oxy)-1-(4-((2-bromo-(1,1'-biphenyl)-3-yl)methoxy)-5-chloro-2-((5-cyanopyridin-3-yl)methoxy)benzyl)pyrrolidine-2-carboxylic acid 2. The disulfide bond-containing compound according to any one of claim 1, its stereoisomers, or pharmaceutically acceptable salts thereof, characterized in that: The pharmaceutically acceptable salts include salts formed by combining with inorganic acids, organic acids, alkali metal ions, alkaline earth metal ions or organic bases that can provide physiologically acceptable cations, as well as ammonium salts.

3. The disulfide bond-containing compound according to any one of claim 2, its stereoisomers, or pharmaceutically acceptable salts thereof, characterized in that: The inorganic acid is selected from hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid; the organic acid is selected from methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, citric acid, maleic acid tartaric acid, fumaric acid, citric acid or lactic acid; the alkali metal ion is selected from lithium ion, sodium ion, potassium ion; the alkaline earth metal ion is selected from calcium ion and magnesium ion; the organic base capable of providing physiologically acceptable cations is selected from methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris(2-hydroxyethyl)amine.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises as an active ingredient the disulfide bond-containing compound according to any one of claims 1 and its stereoisomers or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier or excipient.

5. Use of the disulfide bond-containing compound according to any one of claim 1, its stereoisomers, or pharmaceutically acceptable salts thereof in the preparation of a medicament for preventing and / or treating diseases related to the PD-1 / PD-L1 signaling pathway and / or IDO1.

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