Immunomodulators

Abstract

The present invention discloses an immunomodulatory agent, specifically a compound represented by Formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof. The compounds provided by the present invention can effectively bind to STING, exhibit good STING protein agonist activity, and exhibit good inhibitory effects against various tumors. Therefore, the compounds of the present invention can be used as STING agonists to treat various STING-related diseases. The compounds of the present invention have excellent application prospects in the manufacture of pharmaceuticals for treating diseases associated with STING activity (especially pharmaceuticals for treating inflammatory diseases, allergic diseases, autoimmune diseases, infectious diseases, cancer, or precancerous syndromes) and in the manufacture of immune adjuvants, providing new options for clinical screening and / or manufacturing of pharmaceuticals for diseases associated with STING activity. TIFF2022518853000113.tif6778

Description

[Technical field]

[0001] The present invention relates to immunomodulatory agents, in particular to compounds that activate STING and their use as immunomodulators in the manufacture of medicaments. [Background technology]

[0002] The human immune system can generally be divided into the "innate immune system" and the "adaptive immune system". The innate immune system plays an important role in resisting infection, inhibiting tumor growth, and the pathogenesis of autoimmune diseases, mainly by recognizing pathogenic microorganisms and cancer cell components through pattern recognition receptors, initiating downstream signal pathways, and finally inducing the expression of cytokines, thereby killing pathogenic microorganisms and cancer cell components. Meanwhile, the adaptive immune system promotes the production of antibodies and specific T lymphocytes.

[0003] STING (Stimulator of interferon genes, TMEM173, MITA, etc.) is a key node molecule in the response to DNA invasion into cells, and plays a key role in the process of recognizing the signal of cytoplasmic DNA receptors and inducing interferon production under the stimulation of cytoplasmic DNA. After the host cell's DNA recognition receptor recognizes exogenous or endogenous "non-self" DNA, it transmits the signal to the node molecule STING, which then rapidly dimerizes and translocates from the endoplasmic reticulum to perinuclear bodies. Activation of STING causes upregulation of IRF 3 and NKκB pathways, which leads to the induction of interferon-β and other cytokines.

[0004] Compounds that induce human interferon can be used to treat a variety of diseases, including allergic and other inflammatory diseases, allergic rhinitis and asthma, infectious diseases, neurodegenerative diseases, precancerous syndromes, and cancer, and can also be used as adjuvants in immune compositions or vaccines. Thus, the discovery of novel compounds that can activate STING is an effective way to treat diseases associated with the type 1 IFN pathway, including inflammatory, allergic, and autoimmune diseases, infectious diseases, cancer, and precancerous syndromes. Summary of the Invention [Problem to be solved by the invention]

[0005] To solve the above problems, the present invention provides an immunomodulatory agent. [Means for solving the problem]

[0006] The present invention provides a compound represented by the following formula (I), or a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof: [ka] (wherein L is selected from a C2-C6 alkylene group, a C2-C6 alkenylene group, and a C2-C6 alkynylene group; R 1 , R 3 , R 1’ , and R 3’ are each independently selected from hydrogen, halogen, a C1-C6 alkyl group, a cyano group, a nitro group, a hydroxyl group, an amino group, a C1-C6 alkoxy group, a C1-C6 alkylamino group, and a C1-C6 alkyl group substituted with a halogen, R 2 , and R 2’ are each independently hydrogen, -C(O)NR a R b , -NR a C(O)R b , -C(O)R a , -C(O)OR a, a halogen, a C1-C6 alkyl group, a cyano group, a nitro group, a hydroxy group, an amino group, a C1-C6 alkoxy group, a C1-C6 alkylamino group, or a C1-C6 alkyl group substituted with a halogen, R 4 , and R 4’ are each independently selected from hydrogen and a C1 to C6 alkyl group; X and X' are each independently selected from -CH2-, -NH-, -O-, and -S-; Ring A and ring A' each independently represent 0 to 4 R c selected from 5-6 membered aromatic heterocycles optionally substituted with And, when X and X' are selected from -CH2-, -NH-, and -O-, at least one of the A ring and the A' ring is selected from 0 to 4 R c a sulfur-containing 5-6 membered aromatic heterocycle or an oxygen-containing 5-6 membered aromatic heterocycle, optionally substituted by R 5 , and R 5’ Each independently represents 0 to 2 R d C1-C6 alkyl groups optionally substituted with R a , and R b are each independently selected from hydrogen and a C1 to C6 alkyl group; R c are each independently halogen, -CN, -OR a , a C1-C6 alkyl group, or a C1-C6 alkyl group substituted with halogen; R d are each independently a halogen, -OR a , -NR a R b , 0 to 4 R e a 3- to 6-membered cycloalkyl group optionally substituted with 0 to 4 R e a 3-6 membered heterocycloalkyl group optionally substituted with 0-4 R e a 7-10 membered cycloalkyl group optionally substituted with 0-4 R e a 7-10 membered heterocycloalkyl group optionally substituted with 0-4 Re a 7-10 membered spirocycloalkyl group optionally substituted with 0-4 R e a 7-10 membered spiroheterocycloalkyl group optionally substituted with 0-4 R e a 5-10 membered bridged cycloalkyl group optionally substituted with 0-4 R e a 5-10 membered bridged heterocycloalkyl group optionally substituted with e a 7-10 membered fused cycloalkyl group optionally substituted with 0-4 R e a 7- to 10-membered fused heterocycloalkyl group optionally substituted with R e are each independently a halogen, a C1-C6 alkyl group, a 3- to 6-membered cycloalkyl group, or -C(O)R f Selected from R f are independently -OR a , a C1 to C6 alkyl group, and a 3 to 6 membered cycloalkyl group.)

[0007] Further, the compound has the structure shown in formula (I): [ka] (wherein L is selected from a C2-C6 alkylene group, a C2-C6 alkenylene group, and a C2-C6 alkynylene group; R 1 , R 3 , R 1’ , and R 3’ are each independently selected from hydrogen, halogen, and a C1 to C6 alkyl group; R 2 , and R 2’ are each independently hydrogen, -C(O)NR a R b Selected from R 4 , and R 4’ are each independently selected from hydrogen and a C1 to C6 alkyl group; X and X' are each independently selected from -CH2-, -NH-, -O-, and -S-; Ring A and ring A' each independently represent 0 to 4 R c selected from 5-6 membered aromatic heterocycles optionally substituted with And, when X and X' are selected from -CH2-, -NH-, and -O-, at least one of the A ring and the A' ring has 0 to 4 R c a sulfur-containing 5-6 membered aromatic heterocycle or an oxygen-containing 5-6 membered aromatic heterocycle, optionally substituted by R 5 , and R 5’ Each independently represents 0 to 2 R d C1-C6 alkyl groups optionally substituted with R a , and R b are each independently selected from hydrogen and a C1 to C6 alkyl group; R c are each independently halogen, -CN, -OR a , a C1-C6 alkyl group, or a C1-C6 alkyl group substituted with halogen; R d are each independently a halogen, -OR a , -NR a R b , 0 to 4 R e a 3- to 6-membered cycloalkyl group optionally substituted with 0 to 4 R e a 3- to 6-membered heterocycloalkyl group optionally substituted with R e are each independently a halogen, a C1-C6 alkyl group, a 3- to 6-membered cycloalkyl group, or -C(O)R f Selected from R f are independently -OR a , a C1 to C6 alkyl group, and a 3 to 6 membered cycloalkyl group.)

[0008] In addition, R 1 , R 3 , R 1’ , and R 3’ are each independently hydrogen; R2 , and R 2’ are each independently -C(O)NR a R b Selected from R 4 , and R 4’ are each independently hydrogen; X and X' are each independently selected from -O- and -S-; Ring A and ring A' each independently represent 0 to 3 R c a 5-membered aromatic heterocycle optionally substituted with And when X and X' are -O-, at least one of the A ring and the A' ring is 0 to 3 R c a sulfur-containing 5-membered aromatic heterocycle optionally substituted with R 5 , and R 5’ are each independently 0 to 1 R d C1-C6 alkyl groups optionally substituted with R c are each independently selected from a halogen, a C1-C6 alkyl group, and a C1-C6 alkyl group substituted with a halogen, R d Each independently represents 0 to 2 R e a 3-6 membered cycloalkyl group optionally substituted with 0-2 R e a 3- to 6-membered heterocycloalkyl group optionally substituted with R e are each independently a C1 to C6 alkyl group, a 3 to 6 membered cycloalkyl group, or -C(O)R f Selected from R f are independently -OR a , a C1 to C6 alkyl group, and a 3 to 6 membered cycloalkyl group.

[0009] Furthermore, at least one of X and X' is -S-; Ring A and ring A' each independently represent 0 to 2 R c Optionally replaced with [ka] Selected from R 5 , and R 5’ are each independently a C1 to C6 alkyl group, [ka] Selected from R c’ is selected from a C1-C6 alkyl group and a C1-C6 alkyl group substituted with halogen, R c is selected from halogen, a C1-C6 alkyl group, and a C1-C6 alkyl group substituted with halogen, R e’ are each independently a C1 to C6 alkyl group, a 3 to 6 membered cycloalkyl group, or -C(O)R f Selected from R f are independently -OR a , a C1-C6 alkyl group, and a 3- to 6-membered cycloalkyl group; R a is selected from hydrogen and C1 to C6 alkyl groups.

[0010] Further, X and X' are -O-, At least one of the rings A and A' is 0 to 2 R c Optionally replaced with [ka] Selected from R 5 , and R 5’ are each independently a C1 to C6 alkyl group, [ka] Selected from R c is selected from halogen, a C1-C6 alkyl group, and a C1-C6 alkyl group substituted with halogen, R e’are each independently a C1 to C6 alkyl group, a 3 to 6 membered cycloalkyl group, or -C(O)R f Selected from R f are independently -OR a , a C1-C6 alkyl group, and a 3- to 6-membered cycloalkyl group; R a is selected from hydrogen and C1 to C6 alkyl groups.

[0011] Furthermore, the compound has the structure shown in formula (II): [ka] (wherein X and X' are each independently selected from -O- and -S-; Ring A and ring A' each independently represent 0 to 2 R c Optionally replaced with [ka] Selected from And when either X or X' is -O-, at least one of the ring A and the ring A' is 0 to 2 R c Optionally replaced with [ka] Selected from R c’ is selected from a C1-C2 alkyl group and a C1-C2 alkyl group substituted with halogen, R c is selected from halogen, a C1-C2 alkyl group, and a C1-C2 alkyl group substituted with halogen, Preferably, the halogen is F, R 5 , and R 5’ each independently represents a C1 to C3 alkyl group, [ka] Selected from R e’is a C1-C3 alkyl group, a 3-6 membered cycloalkyl group, -C(O)R f Selected from R f -OR a’ , a C1-C3 alkyl group, and a 3- or 4-membered cycloalkyl group; R a’ is selected from C1 to C3 alkyl groups.

[0012] Further, X is selected from -O- and -S-; X' is -O-; R 5 is selected from C1 to C3 alkyl groups, R 5’ teeth, [ka] Selected from R e’ is a C1-C3 alkyl group, a 3-6 membered cycloalkyl group, -C(O)R f Selected from R f -OR a’ , a C1-C3 alkyl group, and a 3- or 4-membered cycloalkyl group; R a’ is selected from C1 to C3 alkyl groups.

[0013] Furthermore, the 0 to 2 R c Optionally replaced with [ka] and The 0 to 2 R c Optionally replaced with [ka] It is.

[0014] Furthermore, the compound is as follows: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0015] Furthermore, in the compound, R 5 , and R 5’ Each independently represents 0 to 2 R d C1-C6 alkyl groups optionally substituted with R d are each independently a halogen, -OH, or 0 to 4 R e a 3- to 6-membered cycloalkyl group optionally substituted with 0 to 4 R e a 3-6 membered heterocycloalkyl group optionally substituted with 0-4 R e a 7-10 membered cycloalkyl group optionally substituted with 0-4 R e a 7-10 membered heterocycloalkyl group optionally substituted with 0-4 R e a 7-10 membered spirocycloalkyl group optionally substituted with 0-4 R e a 7-10 membered spiroheterocycloalkyl group optionally substituted with 0-4 R e a 5-10 membered bridged cycloalkyl group optionally substituted with 0-4 R ea 5-10 membered bridged heterocycloalkyl group optionally substituted with R e are each independently a halogen, a C1-C6 alkyl group, a 3- to 6-membered cycloalkyl group, or -C(O)R f Selected from R f are independently -OR a , a C1 to C6 alkyl group, and a 3 to 6 membered cycloalkyl group.

[0016] Furthermore, the compound is represented by the following formula (III): [ka] (However, R 1 , R 3 , R 1’ , and R 3’ are each independently selected from hydrogen and halogen; Ring A and ring A' each independently represent 0 to 2 R c Optionally replaced with [ka] Selected from R c’ is selected from a C1-C2 alkyl group and a C1-C2 alkyl group substituted with halogen, R c is selected from halogen, a C1-C2 alkyl group, and a C1-C2 alkyl group substituted with halogen, R 5’ is 0 to 2 R d C1-C3 alkyl groups optionally substituted with R d each independently represents -OH, 0 to 2 R e a 3-6 membered cycloalkyl group optionally substituted with 0-2 R e a 3-6 membered heterocycloalkyl group optionally substituted with 0-2 R e a 7-10 membered cycloalkyl group optionally substituted with 0-2 R ea 7-10 membered heterocycloalkyl group optionally substituted with 0-2 R e a 7-10 membered cycloalkyl group optionally substituted with 0-2 R e a 7-10 membered spiroheterocycloalkyl group optionally substituted with 0-2 R e a 5-10 membered bridged cycloalkyl group optionally substituted with 0-2 R e a 5-10 membered bridged heterocycloalkyl group optionally substituted with e a 7-10 membered fused cycloalkyl group optionally substituted with 0-2 R e a 7- to 10-membered fused heterocycloalkyl group optionally substituted with R e are each independently selected from a halogen and a C1 to C3 alkyl group.

[0017] Further, ring A and ring A' are each independently [ka] Selected from R 5’ is 0 to 2 R d C1-C3 alkyl groups optionally substituted with R d are each independently -OH, [ka] Selected from.

[0018] Furthermore, the compound is as follows: [ka] [ka]

[0019] In some embodiments of the present invention, for the compound represented by formula (I), the A ring and the A′ ring each independently contain 0 to 3 R c and when X and X' are -O-, at least one of the A ring and the A' ring is selected from sulfur and nitrogen-containing 5-membered aromatic heterocycles.

[0020] In some embodiments of the present invention, for the compound represented by formula (I), the A ring and the A′ ring each independently preferably have 0 to 3 R c Pyrrole substituted with 0-3 R c pyrazole substituted with 0-3 R c Imidazole substituted with 0-3 R c Thiazole substituted with 0-3 R c isothiazole substituted with 0-3 R a oxazole substituted with 0-3 R c and when X and X' are -O-, at least one of the A ring and the A' ring is substituted with 0 to 3 R c Thiazole substituted with 0-3 R c is selected from isothiazoles substituted with

[0021] The present invention further provides the use of the above compound, or a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for activating STING.

[0022] The present invention further provides the use of the above compound, or a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for treating a disease associated with the activity of STING.

[0023] Furthermore, the disease associated with STING activity is one or more of diseases associated with inflammation, autoimmune diseases, infectious diseases, cancer, and precancerous syndromes.

[0024] The invention further provides the use of a compound as described above, or a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for treating inflammation, an autoimmune disease, an infectious disease, cancer or a precancerous syndrome.

[0025] The present invention further provides the use of the above compound, or a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof, in the manufacture of an immune adjuvant.

[0026] The present invention further provides a pharmaceutical preparation produced by adding pharma-ceutical acceptable additives to the above-mentioned compound, or a stereoisomer thereof, or a pharma-ceutical acceptable salt thereof as an active ingredient.

[0027] A disease associated with STING activity as defined in the present invention is a disease in which STING plays an important role in the pathogenesis of the disease.

[0028] Diseases associated with STING activity include inflammation, allergic and autoimmune diseases, infectious diseases, cancer, and precancerous syndromes.

[0029] A "cancer" or "malignancy" refers to any one of a variety of diseases characterized by uncontrolled abnormal cell growth, the ability of affected cells to spread locally or via the bloodstream and lymphatic system to other locations in the body (i.e., metastasis), and any one of a number of characteristic structural and / or molecular features. A "cancer cell" is a cell that undergoes multiple stages of tumor development at early, intermediate, or late stages. Cancers include sarcoma, breast cancer, lung cancer, brain cancer, bone cancer, liver cancer, kidney cancer, colon cancer, and prostate cancer. In some embodiments, the compounds of formula (I) are used to treat a cancer selected from colon cancer, brain cancer, breast cancer, fibrosarcoma, and squamous cell carcinoma. In some embodiments, the cancer is selected from melanoma, breast cancer, colon cancer, lung cancer, and ovarian cancer. In some embodiments, the cancer being treated is a metastatic cancer.

[0030] Inflammatory diseases include a variety of disorders characterized by histopathological inflammation. Examples of inflammatory diseases include acne vulgaris, asthma, celiac disease, chronic prostatitis, glomerulonephritis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, vasculitis, airway inflammation caused by house dust mites, and interstitial cystitis. There is a large overlap between inflammatory diseases and autoimmune diseases. Some embodiments of the present invention relate to treating asthma, an inflammatory disease. In general, the immune system is involved in inflammatory diseases, as manifested in allergic reactions and some myopathies, and many disorders of the immune system cause abnormal inflammation.

[0031] The compounds and derivatives provided herein may be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) naming systems.

[0032] For the definition of terms used in the present invention, unless otherwise specified, the definition of a group or term first described in this specification can be applied to all of the groups or terms. For terms that are not specifically defined in this specification, the meaning that a person skilled in the art can give them based on the disclosed content and context is given.

[0033] A "substitution" refers to the replacement of a hydrogen atom in a molecule with another atom or molecule.

[0034] The minimum and maximum carbon atom content in the hydrocarbon group is indicated by a prefix, e.g., the prefix (C a ~C b ) alkyl group refers to any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, a (C1-C6) alkyl group refers to an alkyl group containing 1 to 6 carbon atoms.

[0035] In the present invention, an "alkyl group" refers to a saturated hydrocarbon chain having a specified number of member atoms. For example, a C1-C6 alkyl group refers to an alkyl group having from 1 to 6 carbon atoms. The alkyl group may be linear or branched. Representative examples of branched alkyl groups include groups having one, two, or three branches. The alkyl group may be optionally substituted with one or more substituents as defined herein. Alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, and tert-butyl), pentyl (n-pentyl, isopentyl, and isopentyl), and hexyl. The alkyl group can be part of another group, such as, for example, a C1-C6 alkoxy group.

[0036] C in the present invention a ~C b An alkoxy group is a group obtained by combining an alkyl group containing from "a" to "b" carbon atoms with the corresponding oxygen atom.

[0037] In the present invention, the "C2-C6 alkylene group" is a divalent saturated aliphatic hydrocarbon group having 2 to 6 carbon atoms. The alkylene group includes branched and straight-chain hydrocarbon groups. Examples of the "(C2-C6) alkylene group" include ethylene, propylene, 2-methylpropylene, dimethylethylene, and pentylene.

[0038] Similarly, a "C2-C6 alkenylene group" is a divalent aliphatic hydrocarbon group containing one or more carbon-carbon double bonds and containing 2 to 6 carbon atoms. The alkenylene group includes branched and straight chain groups. The carbon-carbon double bonds in the alkenylene group include cis double bonds and trans double bonds.

[0039] Similarly, a "C2-C6 alkynylene group" is a divalent aliphatic hydrocarbon group containing one or more carbon-carbon triple bonds, containing from 2 to 6 carbon atoms. Alkynylene groups include branched and straight chain groups.

[0040] In the present invention, -C(O)NR a R b , -NR a C(O)R b , -C(O)R a , -C(O)OR a , -C(O)R f In the formula, -C(O)- is a carbonyl group in which two atoms, carbon and oxygen, are connected by a double bond. [ka] Shows.

[0041] In the present invention, "halogen" refers to a halogen radical such as fluorine, chlorine, bromine or iodine.

[0042] The immune adjuvant in the present invention, i.e., the immune regulator, is a substance that has the function of enhancing and regulating immunity in terms of anti-infection, anti-virus, anti-tumor, anti-allergy, asthma, etc., and is clinically mainly used for the adjunctive treatment of diseases related to inflammation, autoimmune diseases, infectious diseases, cancer, and precancerous syndrome. The term "pharmaceutical acceptable" means that a certain carrier, vehicle, diluent, additive, and / or formed salt is generally chemically or physically compatible with other ingredients constituting a pharmaceutical dosage form and physiologically compatible with a receptor.

[0043] The terms "salt" and "pharmaceutically acceptable salt" refer to acid salts and / or base salts formed by the above compounds or their stereoisomers with inorganic and / or organic acids and bases, including zwitterionic salts (internal salts) and further including quaternary ammonium salts such as alkylammonium salts. These salts can be obtained directly from the final isolation and purification of the compounds, or by appropriately mixing the above compounds or their stereoisomers with a certain amount (e.g., an equivalent amount) of an acid or base. These salts can form precipitates in solution and be collected by filtration or recovered after evaporation of the solvent, or can be prepared by reaction in an aqueous medium followed by lyophilization. Salts according to the present invention include hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, or trifluoroacetate salts of the compounds.

[0044] In some embodiments, one or more compounds of the present invention can be used in combination with each other. The compounds of the present invention can be used in combination with any other active agent to prepare a medicament or pharmaceutical composition for regulating cell function or treating disease. When a group of compounds is used, these compounds can be administered to a subject simultaneously, separately, or sequentially.

[0045] The compounds provided by the present invention can effectively bind to STING, have good STING protein agonist function, and show good inhibitory effects on various tumors, and can also activate immune memory mechanisms in mice to inhibit tumor restimulation. Therefore, the compounds of the present invention can be used as STING agonists to treat various related diseases. The compounds of the present invention have very good application prospects in the manufacture of medicines for treating diseases related to STING activity (especially medicines for treating inflammatory diseases, allergic diseases, autoimmune diseases, infectious diseases, cancer or precancerous syndromes) and the manufacture of immune adjuvants, providing new options in the clinical screening and / or manufacture of medicines for diseases related to STING activity.

[0046] Based on the above content of the present invention, it is apparent that various other modifications, substitutions or alterations can be made in accordance with common technical knowledge and commonly used means in this field without departing from the basic technical idea of ​​the present invention.

[0047] The present invention will be described in more detail below through specific embodiments in the form of examples, but it should not be understood that the scope of the subject matter of the present invention is limited to the following specific examples. All techniques implemented based on the content of the present invention belong to the scope of the present invention. [Brief description of the drawings]

[0048] [Figure 1] FIG. 1 is a graph showing the inhibitory effect of the compounds prepared in the examples of the present invention on the CT26 tumor model. [Diagram 2] FIG. 2 is a graph showing the inhibitory effect of compounds prepared in the examples of the present invention on a CT26 tumor restimulation model, in which the curves of Example 2 (4.5 mg / kg), Example 2 (1.5 mg / kg), Example 5 (1.5 mg / kg), Example 15 (3 mg / kg), Example 15 (1 mg / kg), Example 17 (1.5 mg / kg), and Example 29 (1 mg / kg) are overlapping. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] The raw materials, equipment used in the specific embodiments of the present invention are all known products and were obtained from commercially available sources.

[0050] The structure of the compound is identified by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The NMR shifts (δ) are 10 -6 The unit of measurement was ppm. NMR measurements were performed using nuclear magnetic instruments (Bruker AvanceIII 400 and Bruker Avance 300), the measurement solvents were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), and the internal standard was tetramethylsilane (TMS).

[0051] For LC-MS measurements, a Shimadzu liquid chromatograph mass spectrometer (Shimadzu LC-MS 2020 (ESI)) is used.

[0052] For the HPLC measurements, a Shimadzu high pressure liquid chromatograph (Shimadzu LC-20A) is used.

[0053] For reversed phase preparative chromatography, a Gilson GX-281 reversed phase preparative chromatogram apparatus is used.

[0054] For thin-layer chromatography silica gel plate, Yantai Yellow Sea HSGF254 or Qingdao GF254 silica gel plate is used, and the specification of product separation and purification by thin-layer chromatography is 0.4mm~0.5mm.

[0055] Column chromatography generally uses 200-300 mesh silica gel manufactured by Yantai Huanghai as a carrier.

[0056] The known starting materials in the present invention can be synthesized using or according to methods well known in the art, or can be purchased from companies such as Ananji Chemical, Chengdu Hualong Chemical, Shaoyuan Chemical Technology, J&K Scientific, etc.

[0057] By hydrogen atmosphere it is meant that the reaction flask is connected to a hydrogen balloon of approximately 1 L volume.

[0058] The hydrogenation reaction usually involves three cycles of evacuation and refilling with hydrogen.

[0059] In the examples, unless otherwise noted, reactions are carried out under a nitrogen atmosphere.

[0060] In the examples, unless otherwise specified, solutions refer to aqueous solutions.

[0061] In the examples, unless otherwise stated, the reaction temperature is room temperature.

[0062] In the examples, M is in moles per liter unless otherwise specified.

[0063] Room temperature is the optimum reaction temperature, between 20°C and 30°C.

[0064] Overnight is defined as 12±1 hours.

[0065] PE is petroleum ether, EA is ethyl acetate, DCM is dichloromethane, MeOH is methanol, DMF is N,N-dimethylformamide, DMSO is dimethylsulfoxide, DMAP is 4-dimethylaminopyridine, DIPEA is diisopropylethylamine, Boc is tert-butoxycarbonyl group, TFA is trifluoroacetic acid, DBU is 1,8-diazabicycloundec-7-ene, HATU is O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate.

[0066] Below is the preparation of intermediate compounds. 1. Synthesis of intermediate compound: 4-chloro-3-methoxy-5-nitrobenzamide [ka]

[0067] Methyl 4-chloro-3-methoxy-5-nitrobenzoate (18.5 g, 75.3 mmol) was added to a single-neck flask containing aqueous ammonia (200 mL) and stirred for 3 hours at 60° C. The reaction solution was concentrated to 100 mL, cooled, filtered, and the solid was washed with ice water and dried to give 4-chloro-3-methoxy-5-nitrobenzamide (12.5 g, 54.1 mmol) as a brown solid.

[0068] MS(ESI)m / z=231[M+H] + , 1 HNMR (400MHz, DMSO-d6): δ8.29 (s, 1H), 8.04 (d, 1H), 7.87 (d, 1H), 7.78 (s, 1H), 4.01 (s, 3H). 2. Intermediate compound: Synthesis of 4-chloro-3-hydroxy-5-nitrobenzamide [ka]

[0069] In an ice bath, 4-chloro-3-methoxy-5-nitrobenzamide (7.5 g, 32.5 mmol) was dispersed in dried DCM (90 mL), and boron tribromide (120 mL, 1 M) was slowly added dropwise to it. After the addition was completed, the ice bath was removed, and the reaction was allowed to proceed overnight at room temperature under nitrogen gas protection. After the reaction was completed, the reaction solution was poured into ice water, vigorously stirred for 30 minutes, filtered, and the filter cake was washed with water and dried to obtain 4-chloro-3-hydroxy-5-nitrobenzamide (6.00 g, 27.7 mmol, 85.3% yield) as a pale yellow solid.

[0070] MS(ESI)m / z=217[M+H] +, 1 H NMR (400MHz, DMSO-d6): δ11.73 (s, 1H), 8.21 (s, 1H), 7.92 (s, 1H), 7.80 (s, 1H), 7.66 (s, 1H). 3. Intermediate compound: Synthesis of 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate [ka]

[0071] 1-Ethyl-3-methyl-1H-pyrazole-5-carboxylic acid (4.00 g, 25.9 mmol) was dispersed in dry DCM (80 mL) and oxalyl chloride (3.9 g, 31.1 mmol) and catalytic amount of DMF were added dropwise to it in an ice bath. After reacting at room temperature for 1 h, volatile materials were removed by rotary evaporation under reduced pressure. After adding DCM (20 mL) to the crude product and further removing the solvent by rotary evaporation, 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl chloride (4.46 g, 100% yield) was obtained and used directly in the next reaction.

[0072] At 0°C, 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl chloride (4.46g, 25.9mmol) was dissolved in dried acetone (20mL) and added dropwise to a solution of potassium thiocyanate (5.0g, 51.5mmol) in acetone (100mL), stirred at room temperature for 3 hours, filtered to remove inorganic salts, and the crude product obtained after concentrating the filtrate was purified on a silica gel column (eluent: ethyl acetate / petroleum ether (v / v) = 1 / 15) to obtain 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (4.0g, 20.4mmol, 78.7%) as a transparent brown-yellow liquid.

[0073] MS(ESI)m / z=196[M+H] + . 4. Intermediate compound: Synthesis of 4-ethyl-2-methylthiazole-5-carbonyl isothiocyanate [ka]

[0074] Step 1: Synthesis of 4-ethyl-2-methylthiazole-5-carbonyl chloride

[0075] 4-Ethyl-2-methylthiazole-5-carboxylic acid (2 g, 11.7 mmol) was dispersed in dry DCM (40 mL) and oxalyl chloride (1.9 g, 15.1 mmol) and catalytic amount of DMF were added dropwise to it in an ice bath. After reacting at room temperature for 1 h, volatile materials were removed by rotary evaporation under reduced pressure. After adding DCM (20 mL) to the crude product and removing the solvent by rotary evaporation, 4-ethyl-2-methylthiazole-5-carbonyl chloride (2.2 g, 100% yield) was obtained and used directly in the next reaction.

[0076] Step 2: Synthesis of 4-ethyl-2-methylthiazole-5-carbonyl isothiocyanate

[0077] At 0°C, 4-ethyl-2-methylthiazole-5-carbonyl chloride (2.2g, 11.7mmol) was dissolved in dried acetone (10mL) and added dropwise to a solution of potassium thiocyanate (2.3g, 23.4mmol) in acetone (50mL), stirred at room temperature for 3 hours, filtered to remove inorganic salts, and the crude product obtained after concentrating the filtrate was purified on a silica gel column (eluent: ethyl acetate / petroleum ether, v / v=1 / 15) to obtain 4-ethyl-2-methylthiazole-5-carbonyl isothiocyanate (2.15g, 10.2mmol, yield: 87%) as a transparent brown-yellow liquid.

[0078] MS(ESI)m / z=213[M+H] + . 5. Intermediate compound: Synthesis of 4-chloro-3-(3-morpholino)-5-nitrobenzamide [ka]

[0079] To a solution of 3-morpholinopropan-1-ol (1.0 g, 9.79 mmol) in dichloromethane (20 mL), TEA (2.7 mL, 19.58 mmol) and paratoluenesulfonyl chloride (2.0 g, 10.77 mmol) were added, and the mixture was reacted at room temperature for 2 hours. Water and dichloromethane were added for extraction, and the organic phases were washed with water and saturated saline, respectively, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4-morpholinopropyl 4-methylbenzenesulfonate, which was used as it was in the next reaction.

[0080] Potassium carbonate (1.08 g, 7.8 mmol), potassium iodide (0.12 g, 0.72 mmol) and 4-morpholinopropyl 4-methylbenzenesulfonate (1.17 g, 3.9 mmol) were added to a solution of 4-chloro-3-hydroxy-5-nitrobenzamide (845 mg, 3.9 mmol) in DMF (10 mL), and the mixture was reacted at 75° C. for 24 hours. After the reaction was completed, the mixture was filtered to remove inorganic salts, and the filtrate was added to water and extracted with ethyl acetate. The organic phase was washed with water and saturated saline in that order, and then dried and concentrated to obtain 4-chloro-3-(3-morpholino)-5-nitrobenzamide (1.0 g, yield: 75.5%). 6. Intermediate compound: Synthesis of tert-butyl 4-(3-(2-chloro-5-(methoxycarbonyl)-3-nitrophenoxy)propyl)piperazinyl-1-carboxylate [ka]

[0081] Step 1: Synthesis of methyl 4-chloro-3-hydroxy-5-nitrobenzoate

[0082] Methyl 4-chloro-3-methoxy-5-nitrobenzoate (10g, 40.7mmol) was dispersed in anhydrous dichloromethane (100mL), and boron tribromide (40.8g, 162.8mmol) was slowly added dropwise in an ice bath. After the addition was completed, the mixture was slowly warmed to room temperature and reacted overnight with stirring. After the reaction was completed, methanol was slowly added dropwise in an ice bath to quench the reaction, and then it was dried by rotary evaporation. Methanol (100mL) and concentrated sulfuric acid (0.2mL) were added thereto, and the reaction solution was heated to 75°C and stirred overnight. After cooling, the mixture was concentrated under reduced pressure to remove the solvent, and 150mL water was added, and after ultrasonic dispersion, it was filtered, the solid was washed with water, and the solid was dried, and then methyl 4-chloro-3-hydroxy-5-nitrobenzoate (8.89g, 38.4mmol) was obtained.

[0083] Step 2: Synthesis of tert-butyl 4-(3-(2-chloro-5-(methoxycarbonyl)-3-nitrophenoxy)propyl)piperazinyl-1-carboxylate

[0084] Methyl 4-chloro-3-hydroxy-5-nitrobenzoate (10 g, 47.2 mmol), tert-butyl 4-(3-hydroxypropyl)piperazinyl-1-carboxylate (10.6 g, 47.2 mmol) and triphenylphosphine (20.4 g, 77.7 mmol) were dissolved in anhydrous THF (200 mL) under nitrogen gas protection. DIAD (15.8 g, 77.7 mmol) was added dropwise to the above reaction solution in an ice bath, and the reaction solution was warmed to room temperature and stirred for 16 hours. The reaction solution was added to water, extracted with ethyl acetate, and the organic phase was washed with saturated saline, dried over anhydrous magnesium sulfate, and then concentrated. The obtained crude product was purified with a silica gel column (eluent: petroleum ether / ethyl acetate = 2 / 1) to obtain the target compound (13 g, yield: 61%) as a pale yellow solid. 7. Intermediate compound: Synthesis of tert-butyl 4-(3-(2-chloro-5-(methoxycarbonyl)-3-nitrophenoxy)propyl)piperazinyl-1-carboxylate [ka]

[0085] Methyl 4-chloro-3-hydroxy-5-nitrobenzoate (36.8 g, 159.3 mmol) was dissolved in DMF (300 mL), and 4-morpholinopropyl 4-methylbenzenesulfonate (74 g, 247.5 mmol) and K2CO3 (44.5 g, 318.6 mmol) were added. The reaction was stirred at 75°C for 16 hours, filtered to remove inorganic salts, and the DMF was distilled under reduced pressure to remove DMF. The crude product was dissolved in ethyl acetate, washed with water and saturated saline, respectively, dried over anhydrous magnesium sulfate, filtered, and concentrated to the remaining ethyl acetate of 30 mL, and filtered to obtain the target compound (48.7 g, yield: 86%) as a yellow solid. 8. Intermediate compound: Synthesis of methyl 4-fluoro-3-(methylthio)-5-nitrobenzoate [ka]

[0086] Step 1: Synthesis of 3-bromo-4-fluoro-5-nitrobenzoic acid

[0087] 4-Fluoro-3-nitrobenzoic acid (50g, 270mmol) was dispersed in concentrated sulfuric acid (200ml), and NBS (47.5g, 270mmol) was added. The mixture was heated to 75°C and stirred overnight. After cooling to room temperature, the mixture was slowly added to ice water and stirred. A pale yellow solid precipitated. The solid was filtered and dried to obtain the target compound (66g).

[0088] Step 2: Synthesis of methyl 3-bromo-4-fluoro-5-nitrobenzoate

[0089] In an ice bath, thionyl chloride (44.5 g, 373.5 mmol) was added dropwise to a solution of 3-bromo-4-fluoro-5-nitrobenzoic acid (66 g, 249 mmol) in methanol (400 ml). The mixture was heated to 75° C. and stirred overnight. After concentrating until about 100 ml of solvent remained, the solid was subsequently precipitated after cooling, filtered and dried to obtain methyl 3-bromo-4-fluoro-5-nitrobenzoate (56 g).

[0090] Step 3: Synthesis of methyl 4-fluoro-3-(methylthio)-5-nitrobenzoate

[0091] To a solution of methyl 3-bromo-4-fluoro-5-nitrobenzoate (80 mg, 0.14 mmol) in toluene () and tert-butanol (mL), methyl thioacetate (11 mg, 0.14 mmol), Pd2(dba)3 (13 mg, 0.014 mmol), xantphos (8 mg, 0.014 mmol), and K3PO4 (36 mg, 0.166 mmol) were added in that order, and the reaction was completed at 110 ° C overnight under nitrogen gas protection. The reaction solution was cooled to room temperature, concentrated, and the crude product was purified and separated using a normal phase column (eluent: PE / EA = 3 / 1, v / v) to obtain the target compound (g, yield: 45%). 9. Intermediate compound: Synthesis of methyl 4-fluoro-3-((3-morpholinopropyl)thio)-5-nitrobenzoate [ka]

[0092] Step 1: Synthesis of 3-morpholinopropyl mercaptan acetate

[0093] To a solution of 4-morpholinopropyl 4-methylbenzenesulfonate (9 g, 30.1 mmol) in DMF (100 mL), potassium thioacetate (6.86 g, 60.2 mmol) and potassium carbonate (12.5 g, 90.3 mmol) were added and stirred at room temperature overnight. The mixture was filtered to remove inorganic salts, and the solvent was removed by rotary evaporation. The crude product was purified on a silica gel column (eluent: dichloromethane / methanol = 20 / 1) to obtain the target compound (5 g, yield: 81%).

[0094] Step 2: Synthesis of methyl 4-fluoro-3-((3-morpholinopropyl)thio)-5-nitrobenzoate

[0095] To a solution of 3-bromo-4-fluoro-5-nitrobenzoic acid methyl (3.4g, 12.3mmol) in toluene (50mL) and tert-butanol (5mL), 3-morpholinopropyl mercaptan acetate (3g, 14.8mmol), Pd2(dba)3 (1.13g, 1.23mmol), xantphos (0.72g, 1.23mmol), and K3PO4 (7.83g, 36.9mmol) were added in sequence, and the reaction was completed at 110°C overnight under nitrogen gas protection. The reaction solution was cooled to room temperature, and the crude product obtained by concentrating the reaction solution was purified and separated using a normal phase column (eluent: PE / EA = 1 / 1, v / v) to obtain the target compound (1.8g, yield: 41%). 10. Intermediate compound: Synthesis of 1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate [ka]

[0096] Step 1: Synthesis of 1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carbonyl chloride

[0097] 1-Ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxylic acid (2 g, 11.7 mmol) was dispersed in dried DCM (40 mL) and oxalyl chloride (1.9 g, 15.1 mmol) and catalytic amount of DMF were added dropwise to it in an ice bath. After reacting at room temperature for 1 h, volatile materials were removed by rotary evaporation under reduced pressure. After adding DCM (20 mL) to the crude product and removing the solvent by rotary evaporation, 4-ethyl-2-methylthiazole-5-carbonyl chloride (2.2 g, 100% yield) was obtained and used directly in the next reaction.

[0098] Step 2: Synthesis of 1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate

[0099] At 0 ° C., 1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carbonyl chloride (2.2 g, 11.7 mmol) was dissolved in dried acetone (10 mL), dropped into a solution of potassium thiocyanate (2.3 g, 23.4 mmol) in acetone (50 mL), and stirred at room temperature for 3 hours. The reaction system was filtered to remove inorganic salts, and the crude product obtained after concentrating the filtrate was purified on a silica gel column (eluent: ethyl acetate / petroleum ether, v / v = 1 / 15) to obtain 4-ethyl-2-methylthiazole-5-carbonyl isothiocyanate (2.15 g, 10.2 mmol, yield: 87%) as a transparent brown-yellow liquid.

[0100] MS(ESI)m / z=214[M+H] +

[0101] The preparation of the compounds according to the present invention will now be described. Example 1 [ka]

[0102] Step 1: Synthesis of (trans)-tert-butyl (4-(4-carbamoyl-2-methoxy-6-nitrophenyl)amino) n-but-2-enyl)carbamate

[0103] At room temperature, 4-chloro-3-methoxy-5-nitrobenzamide (500 mg, 2.17 mmol) and (4-aminobut-2-en-1-yl)carbamate tert-butyl (966 mg, 4.34 mmol) were dissolved in dimethyl sulfoxide (10 mL), and triethylamine (1.1 g, 10.84 mmol) was added dropwise to the reaction solution. The reaction mixture was heated to 115° C. and stirred at the same temperature overnight. After cooling to room temperature, water was added to dilute the mixture, and a large amount of orange solid precipitated. The mixture was filtered, and the filter cake was washed with water (15 mL×3) and dried with a rotary evaporator equipped with a vacuum pump to obtain (trans)-tert-butyl (4-(4-carbamoyl-2-methoxy-6-nitrophenyl)amino)n-but-2-enyl)carbamate (compound 1b) (450 mg, yield: 57%) as an orange solid.

[0104] MS(ESI)m / z=381[M+H] + .

[0105] Step 2: Synthesis of (trans)-tert-butyl (4-((2-amino-4-carbamoyl-6-methoxyphenyl)amino) n-but-2-enyl)carbamate

[0106] Compound 1b (450 mg, 1.18 mmol) was dissolved in methanol (15 mL), and the solution was cooled to 0°C, followed by the addition of aqueous ammonia (1.62 mL, 11.80 mmol) and an aqueous solution (6 mL) of sodium dithionite (1.22 g, 7.01 mmol). The reaction mixture was stirred at 0°C for 1 hour, and the color of the reaction solution changed from orange-red to white. Methanol was removed from the reaction solution by rotary evaporation, and then the solution was diluted with water and extracted with ethyl acetate (20 mL x 4). The separated organic phase was washed with saturated saline (20 mL x 2), dried over anhydrous sodium sulfate, and then spin-dried to obtain (trans)-tert-butyl (4-((2-amino-4-carbamoyl-6-methoxyphenyl)amino) n-but-2-enyl) carbamate (compound 1c) (260 mg, yield: 58%) as a white solid.

[0107] MS(ESI)m / z=351[M+H] + .

[0108] Step 3: Synthesis of (trans)-tert-butyl (4-(5-carbamoyl-2-(1-ethyl-3-methyl-1-hydro-pyrazole-5-carboxamido)-7-methoxy-1H-benzimidazolyl) n-but-2-enyl)carbamate

[0109] Compound 1c (260 mg, 0.742 mmol) and compound 1-ethyl-3-methyl-1H-pyrazole-5-formyl isothiocyanate (145 mg, 0.74 mmol) were dissolved in N,N-dimethylformamide (5 mL) and stirred at room temperature for 1 hour. Then, HATU (338 mg, 0.89 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (147 mg, 0.96 mmol) were added to the reaction solution, followed by stirring at room temperature for 1 hour. The mixture was diluted with water, filtered, and the resulting white solid was collected and washed with water (5 mL x 3), and then separated on a reverse phase column (eluent: acetonitrile / water = 1 / 3, v / v) to obtain (trans)-tert-butyl (4-(5-carbamoyl-2-(1-ethyl-3-methyl-1-hydro-pyrazole-5-carboxamide)-7-methoxy-1H-benzimidazolyl) n-but-2-enyl)carbamate (compound 1d) (313 mg, yield: 82%) as a white solid.

[0110] MS(ESI)m / z=512[M+H] + 1 H NMR(400MHz,DMSO-d6)δ12.86(s,1H),8.02(s,1H),7.67(s,1H),7.48-7.32(m,2H),7.00-6.87(m,1H),6.65(s,1H),5.8 5-5.50(m,2H),4.93(d,2H),4.61(q,2H),3.98(s,3H),3.51(m,2H),2.55(m,2H),2.18(s,3H),1.35(t,3H),1.32(s,9H).

[0111] Step 4: Synthesis of (trans)-1-(4-amino n-but-2-enyl)-2-(1-ethyl-3-methyl-1-hydro-pyrazole-5-carboxamide)-7-methoxy-1-hydro-benzimidazolyl-5-carboxamide

[0112] Compound 1d (290 mg, 0.57 mmol) was dissolved in methanol (10 mL), and hydrochloric acid (6N aqueous solution, 3 mL, 18 mmol) was added dropwise. The reaction solution was stirred at room temperature for 30 minutes, then heated to 40° C. and stirred for 3 hours. The crude product obtained by spin-drying the reaction solution was separated and purified using a reversed-phase column (eluent: water / acetonitrile=50 / 50, v / v), and compound (trans)-1-(4-amino n-but-2-enyl)-2-(1-ethyl-3-methyl-1-hydro-pyrazole-5-carboxamide)-7-methoxy-1-hydro-benzimidazolyl-5-carboxamide (compound 1e) (230 mg, yield: 98%) was obtained as a pale yellow solid.

[0113] MS(ESI)m / z=412.0[M+H] + .

[0114] Step 5: Synthesis of (E)-1-(4-((4-carbamoyl-2-(3-morpholino)-6-nitrophenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benz[d]imidazolyl-5-carboxamide

[0115] Compound 1e (150 mg, 0.37 mmol), 4-chloro-3-(3-morpholino)-5-nitrobenzamide (127 mg, 0.37 mmol) and DIPEA (220 mg, 1.85 mmol) were mixed and dissolved in n-butanol (5 mL). The reaction mixture was heated to 120° C. and stirred at the same temperature for 48 hours. After cooling to room temperature, water was added to dilute the mixture and extracted with ethyl acetate (20 mL×3). The organic phases were combined and washed with saturated saline (15 mL×2), dried over anhydrous sodium sulfate, and then collected. The solvent was removed by evaporation, and the resulting crude product was separated by reverse phase column chromatography (eluent: acetonitrile / water = 30 / 70, v / v) to give (E)-1-(4-((4-carbamoyl-2-(3-morpholino)-6-nitrophenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benz[d]imidazolyl-5-carboxamide (Compound 1f) (130 mg, yield: 48.9%).

[0116] MS(ESI)m / z=719.3[M+H] + .

[0117] Step 6: Synthesis of (E)-1-(4-((2-amino-4-carbamoyl-6-(3-morpholino)phenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benz[d]imidazolyl-5-carboxamide

[0118] Aqueous ammonia (0.35 mL) was added dropwise to a solution of compound 1f (130 mg, 0.18 mmol) in methanol (5 mL) in an ice bath, and the reaction mixture was stirred at 0° C. for 5 minutes, after which an aqueous solution (2 mL) of sodium dithionite (154 mg, 0.28 mmol) was slowly added. The reaction mixture was slowly warmed to room temperature and continued to be stirred for 3 hours. The mixture was diluted with water, filtered, and the filtrate was spin-dried to obtain a crude product, which was separated by reverse phase column chromatography (eluent: acetonitrile / water = 50 / 50, v / v) to obtain (E)-1-(4-((2-amino-4-carbamoyl-6-(3-morpholino)phenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benz[d]imidazolyl-5-carboxamide 1g (91 mg, yield: 74%) as a white solid.

[0119] MS(ESI)m / z=688.3[M+H] + .

[0120] Step 7: Synthesis of (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(3-morpholinomethyl)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide

[0121] Compound 1g (91 mg, 0.13 mmol) and 4-ethyl-2-methylthiazole-5-carbonyl isothiocyanate (28 mg, 0.13 mmol) were mixed and dissolved in DMF (3 mL), stirred at room temperature for 0.5 hours, and then HATU (49 mg, 0.13 mmol) and DIPEA (34 mg, 0.26 mmol) were added in order, and stirring was continued at room temperature for 2 hours. The reaction solution was purified by reverse phase HPLC to obtain compound 1 (21 mg, 19% yield) as a white solid.

[0122] MS(ESI)m / z=867.4[M+H] + 1 H NMR(400M,DMSO-d6)δ12.80-12.92(m,1H),9.64-9.73(m,1H),7.93-8.01(m,1H),7.64-7.67(m,2H),7.28-7 .43(m,3H),6.52-6.57(m,2H),5.76-5.82(m,2H),4.85-4.94(m,4H),4.51-4.58(m,2H),3.99-4.05(m,3H),3 .90-3.97(m,4H),3.71(s,3H),3.24-3.32(m,4H),3.11-3.17(m,4H),2.92-3.06(m,4H),2.65-2.70(m,4H), 2.54-2.55(m,4H),2.31-2.36(m,4H),2.11(s,3H),1.86-1.95(m,2H),1.26-1.33(m,3H),1.14-1.21(m,3H). Example 2 [ka]

[0123] Step 1: Synthesis of methyl (E)-4-((4-((tert-butoxycarbonyl)amino)but-2-en-1-yl)amino)-3-methoxy-5-nitrobenzoate

[0124] Methyl 4-chloro-3-methoxy-5-nitrobenzoate (30 g, 122 mmol) was dispersed in n-butanol (500 mL), and tert-butyl (E)-(4-aminobut-2-en-1-yl)carbamate (22.8 g, 122 mmol) and DIPEA (78.9 g, 609 mmol) were added. The reaction solution was heated to 120° C. and stirred for 18 hours. The crude product obtained by distillation under reduced pressure was dissolved in ethyl acetate, and an appropriate amount of dilute hydrochloric acid (0.5 M) was added in an ice bath to adjust the pH to neutral. The separated organic phase was washed with water and saturated saline, respectively, dried over anhydrous magnesium sulfate, and then spin-dried to obtain 2b (48.3 g, yield: 99%).

[0125] MS(ESI)m / z=396.3[M+H]

[0126] Step 2: Synthesis of methyl (E)-3-amino-4-((4-((tert-butoxycarbonyl)amino)but-2-en-1-yl)amino)-5-methoxybenzoate

[0127] In an ice bath, ammonia water (120 mL) was added to a solution of 2b (48.3 g, 122 mmol) in methanol (400 mL), and after 10 minutes, an aqueous solution of sodium dithionite (106.3 g, 611 mmol) was added, and the mixture was slowly warmed to room temperature and reacted for 2 hours. The mixture was filtered to remove inorganic salts, and ethyl acetate was added for extraction. The organic phase was washed with saturated saline, dried over anhydrous magnesium sulfate, and then spin-dried to obtain a crude product, which was purified with a silica gel column (eluent: petroleum ether / ethyl acetate = 1 / 1) to obtain compound 2c (28.6 g, yield: 64%).

[0128] MS(ESI)m / z=366.3[M+H]

[0129] Step 3: Synthesis of methyl (E)-1-(4-((tert-butoxycarbonyl)amino)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-methoxy-1H-methyl-benz[d]imidazolyl-5-carboxylate

[0130] In an ice bath, 4-ethyl-2-methylthiazole-5-carbonyl isothiocyanate (8.7 g, 41.1 mmol) was added to a solution of 2c (15 g, 41.1 mmol) in DMF (200 mL). After reacting for 0.5 hours, DIPEA (15.9 g, 123.3 mmol) and HATU (18.8 g, 49.3 mmol) were added, and the mixture was then warmed to room temperature and reacted for 12 hours. The reaction solution was slowly poured into water, and a yellow solid precipitated. The solid was filtered and dried to obtain 2d (14.4 g, yield: 64%).

[0131] MS(ESI)m / z=544.3[M+H]

[0132] Step 4: Synthesis of methyl (E)-1-(4-aminobut-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-methoxy-1H-benz[d]imidazolyl-5-carboxylate

[0133] TFA (6 mL) was added to 2d (6 g, 11 mmol) in DCM (200 mL) in an ice bath, and then the mixture was allowed to warm to room temperature and react for 2 hours. The solvent was removed by rotary evaporation under reduced pressure, ethyl acetate was added, and the mixture was spin-dried under reduced pressure to remove free TFA, yielding compound 2e (4.9 g, yield: 99%).

[0134] MS(ESI)m / z=444.3[M+H]

[0135] Step 5: Synthesis of methyl (E)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-methoxy-1-(4-((4-(methoxycarbonyl)-2-(3-morpholinopropoxy)-6-nitrophenyl)amino)but-2-en-1-yl)-1H-benz[d]imidazolyl-5-carboxylate

[0136] 2e (4.9 g, 11 mmol) was dispersed in n-butanol (100 mL), and 4-chloro-3-(3-morpholinopropoxy)-5-nitrobenzoic acid methyl ester (4.0 g, 11 mmol) and DIPEA (7.2 g, 55 mmol) were added. The reaction mixture was heated to 120° C. and stirred for 18 hours. The crude product obtained by distillation under reduced pressure was dissolved in ethyl acetate, washed with water and saturated saline, dried over anhydrous magnesium sulfate, spin-dried, and purified with a silica gel column (eluent: petroleum ether / ethyl acetate=1 / 2) to obtain 2f (8.4 g, yield: 99%).

[0137] MS(ESI)m / z=766.3[M+H]

[0138] Step 6: Synthesis of methyl (E)-1-(4-((2-amino-4-(methoxycarbonyl)-6-(3-morpholinopropoxy)phenyl)amino)but-2-en-1-yl)-2-(4-ethyl)-2-methylthiazole-5-carboxamido)-7-methoxy-1H-benz[d]imidazolyl-5-carboxylate

[0139] In an ice bath, ammonia water (11 mL) was added to a solution of 2f (8.4 g, 11 mmol) in methanol (50 mL), and after 10 minutes, an aqueous solution of sodium dithionite (9.6 g, 55 mmol) was added, and the mixture was slowly warmed to room temperature and reacted for 2 hours. The mixture was filtered to remove inorganic salts, and ethyl acetate was added for extraction. The organic phase was washed with saturated saline, dried over anhydrous magnesium sulfate, and then spin-dried to obtain a crude product, which was purified with a silica gel column (eluent: petroleum ether / ethyl acetate = 1 / 1) to obtain compound 2g (5.1 g, yield: 63%).

[0140] MS(ESI)m / z=736.3[M+H]

[0141] Step 7: Synthesis of methyl (E)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-1-(4-(2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-5-(methoxycarbonyl)-7-(3-morpholinomethyl)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-methoxy-1H-benz[d]imidazolyl-5-carboxylate

[0142] In an ice bath, 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (1.4 g, 6.94 mmol) was added to a solution of 2g (5.1 g, 6.94 mmol) in DMF (100 mL). After reacting for 0.5 hours, DIPEA (2.7 g, 20.82 mmol) and HATU (3.2 g, 8.33 mmol) were added, and the mixture was then warmed to room temperature and reacted for 12 hours. The reaction solution was slowly poured into water, and a yellow solid precipitated. The solid was filtered and dried to obtain 2h (4.8 g, yield: 77%).

[0143] MS(ESI)m / z=897.3[M+H]

[0144] Step 8: Synthesis of (E)-1-(4-(5-carboxy-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-morpholinomethyl)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-methoxy-1H-benz[d]imidazolyl-5-carboxylic acid

[0145] 2h (4.8 g, 5.38 mmol) was dissolved in a mixture (20 mL) of methanol, tetrahydrofuran and water (volume ratio: 1 / 1 / 1), and lithium hydroxide (2.3 g, 53.8 mmol) was added. The mixture was heated to 75°C and reacted overnight with stirring. The organic solvent was removed by rotary evaporation, cooled to room temperature, and adjusted with dilute hydrochloric acid (1 M) in an ice bath until no solid precipitated, filtered, and the solid was dried to obtain compound 2i (4.65 g, yield: 99%).

[0146] MS(ESI)m / z=869.3[M+H]

[0147] Step 9: Synthesis of (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-morpholino)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-methoxy-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide

[0148] 2i (4.65 g, 5.38 mmol) was dissolved in DMF (100 mL), and HATU (4.9 g, 12.86 mmol) and DIPEA (4.2 g, 32.14 mmol) were added. After 0.5 hours, ammonium bicarbonate (1.3 g, 16.07 mmol) was added and stirred at room temperature for 2 hours. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 2 (2.5 g, yield: 54%).

[0149] MS(ESI)m / z=867.3[M+H] 1 H NMR(400M,DMSO-d6)δ12.79-12.96(m,1H),11.30-11.46(m,1H),7.92-8.14(m,2H),7.63-7.69(m,2H),7 .33-7.45(m,3H),7.26-7.32(m,1H),6.53-6.59(m,1H),5.81-5.92(m,1H),5.68-5.79(m,1H),4.51-4.59 (m,2H),3.97-4.04(m,2H),3.85-3.93(m,2H),3.81(s,3H),3.72-3.79(m,2H),3.02-3.24(m,6H),2.85-2 .98(m,2H),2.58(s,3H),2.13(s,3H),1.90-2.01(m,2H),1.30(t,J=7.08Hz,3H),1.17(t,J=7.44Hz,3H). Example 3 [ka]

[0150] Step 1: Synthesis of (E)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-1-(4-(2-(4-ethyl-2-methylthiazole-5-carboxamido)-5-(methoxycarbonyl)-7-methyl ester-(3-morpholinomethyl)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-methoxy-1H-benz[d]imidazolyl-5-carboxylate methyl

[0151] In an ice bath, 4-ethyl-2-methylthiazole-5-carbonyl isothiocyanate (0.18 g, 0.83 mmol) was added to a solution of 2g (0.5 g, 0.69 mmol) in DMF (5 mL). After reacting for 0.5 hours, DIPEA (0.27 g, 2.08 mmol) and HATU (0.32 g, 0.83 mmol) were added, and the mixture was then warmed to room temperature and reacted for 12 hours. The reaction solution was slowly poured into water, and a yellow solid precipitated. The solid was filtered and dried to obtain 3a (623 mg, yield: 99%).

[0152] MS(ESI)m / z=914.3.3[M+H]

[0153] Step 2: Synthesis of (E)-1-(4-(5-carboxy-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-(3-morpholinomethyl)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-methoxy-1H-benz[d]imidazolyl-5-carboxylic acid

[0154] 3a (623 mg, 0.68 mmol) was dissolved in a mixture of methanol, tetrahydrofuran and water (volume ratio: 1 / 1 / 1), and lithium hydroxide hydrate (0.17 g, 4.08 mmol) was added. The mixture was heated to 75°C and reacted overnight with stirring. The organic solvent was removed by rotary evaporation, cooled to room temperature, and adjusted with dilute hydrochloric acid (1 M) in an ice bath until no solid precipitated, filtered, and the solid was dried to obtain compound 3b (610 mg, yield: 99%).

[0155] MS(ESI)m / z=886.3[M+H]

[0156] Step 3: Synthesis of (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(4-ethyl-2-methylthiazole-5-carboxamide)-7-(3-morpholinomethyl)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-methoxy-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide

[0157] 3b (610 mg, 0.68 mmol) was dissolved in DMF (10 mL), and HATU (4.9 g, 1.36 mmol) and DIPEA (0.53 g, 4.08 mmol) were added. After 0.5 hours, ammonium bicarbonate (0.16 g, 2.04 mmol) was added and stirred at room temperature for 2 hours. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 3 (206 mg, 34% yield).

[0158] MS(ESI)m / z=884.3[M+H] 1 H NMR(400M,DMSO-d6)δ12.80-12.91(m,1H),11.10-11.23(m,1H),7.91-8. 12(m,2H),7.60-7.69(m,2H),7.26-7.48(m,4H),5.71-5.95(m,2H),3.98- 4.11(m,2H),3.85-3.95(m,2H),3.71-3.83(m,5H),3.04-3.29(m,8H),2. 88-3.01 (m, 2H), 2.56-2.60 (m, 4H), 1.95-2.04 (m, 2H), 1.12-1.24 (m, 6H). Example 4 [ka]

[0159] Step 1: Synthesis of methyl (E)-4-((4-((tert-butoxycarbonyl)amino)but-2-en-1-yl)amino)-3-(3-morpholinopropoxy)-5-nitrobenzoate

[0160] Methyl 4-chloro-3-(3-morpholinopropoxy)-5-nitrobenzoate (6.8 g, 19 mmol) was dispersed in n-butanol (100 mL), and tert-butyl (E)-(4-aminobut-2-en-1-yl)carbamate (3.5 g, 19 mmol) and DIPEA (12.3 g, 95 mmol) were added. The reaction solution was heated to 120° C. and stirred for 18 hours. The crude product obtained by distillation under reduced pressure was dissolved in ethyl acetate, and an appropriate amount of dilute hydrochloric acid (0.5 M) was added in an ice bath to neutralize the pH. The separated organic phase was washed with water and saturated saline, respectively, dried over anhydrous magnesium sulfate, and then spin-dried to obtain 4b (7.3 g, yield: 76%).

[0161] MS(ESI)m / z=509.3[M+H]

[0162] Step 2: Synthesis of methyl (E)-3-amino-4-((4-((tert-butoxycarbonyl)amino)but-2-en-1-yl)amino)-5-(3-morpholinopropoxy)benzoate

[0163] In an ice bath, ammonia water (15 mL) was added to a solution of 4b (7.3 g, 14.4 mmol) in methanol (100 mL), and after 10 minutes, an aqueous solution of sodium dithionite (12.5 g, 71.9 mmol) was added, and the mixture was slowly warmed to room temperature and reacted for 2 hours. The mixture was filtered to remove inorganic salts, and ethyl acetate was added for extraction. The organic phase was washed with saturated saline, dried over anhydrous magnesium sulfate, and then spin-dried to obtain a crude product, which was purified with a silica gel column (eluent: petroleum ether / ethyl acetate = 1 / 2) to obtain compound 4c (4.5 g, yield: 65%).

[0164] MS(ESI)m / z=479.3[M+H]

[0165] Step 3: Synthesis of methyl (E)-1-(4-((tert-butoxycarbonyl)amino)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-(3-)morpholinomethyl)-1H-benz[d]imidazolyl-5-carboxylate

[0166] In an ice bath, 4-ethyl-2-methylthiazole-5-carbonyl isothiocyanate (0.96 g, 4.52 mmol) was added to a solution of 4c (1.8 g, 3.76 mmol) in DMF (30 mL). After reacting for 0.5 hours, DIPEA (1.46 g, 11.3 mmol) and HATU (1.72 g, 4.52 mmol) were added, and the mixture was then warmed to room temperature and reacted for 12 hours. The reaction solution was slowly poured into water, and a yellow solid precipitated. The solid was filtered and dried to obtain 4d (1.9 g, yield: 77%).

[0167] MS(ESI)m / z=657.3[M+H]

[0168] Step 4: Synthesis of methyl (E)-1-(4-aminobut-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benz[d])imidazolyl-5-carboxylate

[0169] In an ice bath, TFA (3 mL) was added to 4d (1.9 g, 2.9 mmol) in DCM (40 mL), and the mixture was then warmed to room temperature and reacted for 2 hours. The solvent was removed by rotary evaporation under reduced pressure, ethyl acetate was added, and the mixture was spin-dried under reduced pressure to remove free TFA, yielding compound 4e (1.6 g, yield: 99%).

[0170] MS(ESI)m / z=557.3[M+H]

[0171] Step 5: Synthesis of methyl (E)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-1-(4-((4-(methoxycarbonyl)-2-(methylthio)-6-nitrophenyl)amino)but-2-ketoen-1-yl)-7-(3-morpholinomethyl)-1H-benz[d]imidazolyl-5-carboxylate

[0172] (E)-1-(4-aminobut-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benz[d]]imidazolyl-5-carboxylate methyl (556 mg, 1 mmol) was added to a DMF (10 mL) solution of DIPEA (387 mg, 3 mmol) and 4-fluoro-3-methylthio-5-nitrobenzoic acid methyl (245 mg, 1 mmol), and the mixture was reacted at room temperature for 3 hours. The reaction solution was added to water, extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure to obtain compound 4f (616 mg, yield: 79%).

[0173] MS(ESI)m / z=782.3[M+H]

[0174] Step 6: Synthesis of (E)-1-(4-((2-amino-4-(methoxycarbonyl)-6-(methylthio)phenyl)amino)but-2-en-1-yl)-2-(4-ethyl-2)methyl ester-methylthiazole-5-carboxamido)-7-(3-morpholinomethyl)-1H-benz[d]imidazolyl-5-carboxylate methyl

[0175] In an ice bath, ammonia water (1 mL) was added to a solution of 4f (616 mg, 0.79 mmol) in methanol (5 mL), and after 10 minutes, an aqueous solution of sodium dithionite (686 mg, 3.94 mmol) was added, and the mixture was slowly warmed to room temperature and reacted for 2 hours. The mixture was filtered to remove inorganic salts, and ethyl acetate was added for extraction. The organic phase was washed with saturated saline, dried over anhydrous magnesium sulfate, and then spin-dried to obtain a crude product, which was purified with a silica gel column (eluent: petroleum ether / ethyl acetate = 1 / 1) to obtain compound 4g (435 mg, yield: 73%).

[0176] MS(ESI)m / z=752.3[M+H]

[0177] Step 7: Synthesis of (E)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-1-(4-(2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-5-(methoxycarbonyl)-7-(methylthio)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(3-morpholinomethyl)-1H-benz[d]imidazolyl-5-carboxylate

[0178] In an ice bath, 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (46 mg, 0.232 mmol) was added to a solution of 4g (145 mg, 0.193 mmol) in DMF (5 mL). After reacting for 0.5 hours, DIPEA (75 mg, 0.579 mmol) and HATU (89 mg, 0.232 mmol) were added, and the mixture was then warmed to room temperature and reacted for 12 hours. The reaction solution was slowly poured into water, and a yellow solid precipitated. The solid was filtered and dried to obtain 4h (150 mg, yield: 85%).

[0179] MS(ESI)m / z=913.3[M+H]

[0180] Step 8: Synthesis of (E)-1-(4-(5-carboxy-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(methylthio)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-5-carboxylic acid

[0181] 4h (150 mg, 0.16 mmol) was dissolved in a mixture of methanol, tetrahydrofuran and water (volume ratio: 1 / 1 / 1), and lithium hydroxide (69 mg, 1.64 mmol) was added. The mixture was heated to 75°C and reacted overnight with stirring. The organic solvent was removed by rotary evaporation, cooled to room temperature, and adjusted with dilute hydrochloric acid (1 M) in an ice bath until no solid precipitated, filtered, and the solid was dried to obtain compound 4i (126 mg, yield: 86%).

[0182] MS(ESI)m / z=885.3[M+H]

[0183] Step 9: Synthesis of (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(methylthio)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(3-morpholinomethyl)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide

[0184] 4i (126 mg, 0.143 mmol) was dissolved in DMF (5 mL), and HATU (131 mg, 0.342 mmol) and DIPEA (110 mg, 0.855 mmol) were added. After 0.5 hours, ammonium bicarbonate (34 mg, 0.428 mmol) was added and stirred at room temperature for 2 hours. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 4 (54 mg, yield: 43%).

[0185] MS(ESI)m / z=883.3[M+H] 1 H NMR(400M,DMSO-d6)δ12.79-12.88(m,1H),11.10-11.12(m,1H),7.95-8.12(m,2H),7.85-7.89(m,1H),7.62-7.6 8(m,2H),7.33-7.46(m,3H),6.52(s,1H),5.71-5.78(m,2H),5.11-5.18(m,2H),4.85-4.94(m,2H),4.47-4.55(m, 2H),4.05-4.10(m,4H),3.87-3.91(m,4H),3.71-3.81(m,4H),3.22-3.29(m,2H),3.08-3.18(m,8H),2.89-3.01( m,2H),2.57(s,3H),2.46(s,3H),2.10(s,3H),2.00-2.07(m,2H),1.28(t,J=7.08Hz,3H),1.17(t,J=7.48Hz,3H). Example 5 [ka]

[0186] Compound 5 was prepared by the same synthetic method as compound 4. Compound 5 (67 mg) was obtained by the same steps and experimental methods as compound 4, except that the ring-closing materials used in step 3 and step 7 were replaced, i.e., 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate was used for ring-closing in step 3, and 4-ethyl-2-methylthiazole-5-carbonyl isothiocyanate was used for ring-closing in step 7.

[0187] MS(ESI)m / z=883.3[M+H] 1H NMR(400M,DMSO-d6)δ12.90-13.04(m,1H),10.92-11.05(m,1H),7.93-8.14(m,2H),7.83-7.87(m,1H),7.62-7.70(m ,2H),7.34-7.48(m,2H),7.27-7.32(m,1H),6.51-6.67(m,1H),5.66-5.83(m,4H),5.06-5.12(m,2H),4.89-4.96(m, 2H),4.50-4.59(m,2H),3.99-4.05(m,2H),3.86-3.94(m,2H),3.70-3.80(m,2H),3.19-3.27(m,2H),3.06-3.15(m,4 H),2.88-3.00(m,2H),2.49(s,3H),2.12(s,3H),1.95-2.03(m,2H),1.30(t,J=7.08Hz,3H),1.17(t,J=7.48Hz,3H). Example 6 [ka]

[0188] Compound 6 was prepared by the same synthesis method as compound 2. The synthesis method was the same as that of compound 2, except that methyl 4-fluoro-3-((3-morpholinopropyl)thio)-5-nitrobenzoate was used instead of methyl 4-chloro-3-(3-morpholinopropoxy)-5-nitrobenzoate used in the synthesis of compound 2.

[0189] MS(ESI)m / z=883.3[M+H] 1H NMR(400M,DMSO-d6)δ12.73-12.84(m,1H),10.59-10.82(m,1H),7.98-8.14(m,2H),7.91-7.98(m,1H),7.75-7.83(m,1 H),7.59-7.67(m,1H),7.31-7.49(m,3H),6.51-6.59(m,1H),5.87-5.96(m,1H),5.69-5.78(m,1H),5.18-5.29(m,2H),4 .83-4.90(m,4H),4.51-4.58(m,4H),3.88-3.97(m,2H),3.81(s,3H),3.67-3.79(m,2H),3.27-3.37(m,2H),3.04-3.15( m,4H),2.91-3.03(m,4H),2.55(s,3H),2.11(s,3H),1.77-1.88(m,2H),1.29(t,J=7.04Hz,3H),1.14(t,J=7.48Hz,3H). Example 7 [ka]

[0190] Step 1: Synthesis of methyl (E)-1-(4-((2-(3-(4-(tert-butoxycarbonyl)piperazin-1-yl)propoxy)-4-(methoxycarbonyl)-6-nitrophenyl)amino)but-2-ketoen-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy-1H-benz[d]imidazolyl-5-carboxylate

[0191] (E)-1-(4-aminobut-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benz[d]]imidazolyl-5-carboxylate methyl (0.65g, 1.4mmol) was dispersed in n-butanol (15mL), and 4-(3-(2-chloro-5-(methoxycarbonyl)-3-nitrophenoxy)propyl)piperazinyl-1-carboxylate tert-butyl (630mg, 1.4mmol) and DIPEA (1.8g, 14mmol) were added. The reaction solution was heated to 120°C and stirred for 18 hours. The crude product obtained by distillation under reduced pressure was dissolved in ethyl acetate, washed with water and saturated saline, respectively, dried over anhydrous magnesium sulfate, spin-dried, and purified on a silica gel column (eluent: petroleum ether / ethyl acetate=1 / 2) to obtain 7a (312 mg, yield 26%).

[0192] MS(ESI)m / z=865.2[M+H] +

[0193] Step 2: Synthesis of methyl (E)-1-(4-((2-amino-6-(3-(4-(tert-butoxycarbonyl)piperazin-1-yl)propoxy)-4-(methoxycarbonyl)phenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy-1H-benz[d]imidazolyl-5-carboxylate

[0194] In an ice bath, ammonia water (0.5 mL) was added to a solution of 7a (285 mg, 0.36 mmol) in methanol (10 mL), and after 10 minutes, an aqueous solution of sodium dithionite (311 mg, 1.8 mmol) was added, and the temperature was slowly raised to room temperature and reacted for 2 hours. The mixture was filtered to remove inorganic salts, and ethyl acetate was added for extraction. The organic phase was washed with saturated saline, dried over anhydrous magnesium sulfate, and then spin-dried to obtain compound 7b (296 mg, purity 90%, yield: 96%) as a crude product.

[0195] MS(ESI)m / z=835.0[M+H] +

[0196] Step 3: Synthesis of (E)-7-(3-(4-(tert-butoxycarbonyl)piperazin-1-yl)propoxy)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-1-(4-(2)-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy-5-(methoxycarbonyl)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-ylmethyl)-1H-benz[d]imidazolyl-5-carboxylate methyl

[0197] In an ice bath, 4-ethyl-2-methylthiazole-5-carbonyl isothiocyanate (66 mg, 0.34 mmol) was added to a solution of 7b (296 mg, 0.34 mmol) in DMF (3 mL). After reacting for 0.5 hours, HATU (155 mg, 0.41 mmol) and DIPEA (88 mg, 0.68 mmol) were added, and the mixture was then warmed to room temperature and reacted for 12 hours. The reaction solution was slowly poured into water, and a yellow solid precipitated. The solid was filtered and dried to obtain 7c (346 mg, yield: 97%).

[0198] MS(ESI)m / z=996.4[M+H] + Step 4: Synthesis of (E)-7-(3-(4-(tert-butoxycarbonyl)piperazin-1-yl)propoxy)-1-(4-(5-carboxy-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-1H-benz[d]imidazolyl-5-carboxylic acid

[0199] 7c (340 mg, 0.34 mmol) was dissolved in a mixed solution (5.5 mL) of methanol, tetrahydrofuran and water (volume ratio: 1 / 1 / 0.5), and lithium hydroxide (142 mg, 3.4 mmol) was added. The mixture was heated to 75°C and reacted overnight with stirring. The organic solvent was removed by rotary evaporation, the crude product was dissolved in water, extracted with ethyl acetate, and the aqueous phase obtained by phase separation was adjusted with dilute hydrochloric acid (1 M) in an ice bath until no solid precipitated, filtered, and the solid was dried to obtain compound 7d (220 mg, yield: 67%).

[0200] MS(ESI)m / z=968.4[M+H] +

[0201] Step 5: Synthesis of (E)-4-(3-((5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-tert-butyl ester methoxy-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-1H-benz[d]imidazolyl-7-yl)oxy)propyl)piperazinyl-1-carboxylate tert-butyl

[0202] 7d (158 mg, 0.16 mmol) was dissolved in DMF (2 mL), and HATU (182 mg, 0.48 mmol) and DIPEA (103 mg, 0.8 mmol) were added. After 0.5 hours, ammonium bicarbonate (64.5 mg, 0.82 mmol) was added and stirred at room temperature for 2 hours. Water was added to the reaction solution to precipitate a solid, which was then filtered, washed with pure water, and dried to obtain compound 7e (140 mg, yield: 90%).

[0203] MS(ESI)m / z=966.5[M+H] +

[0204] Step 6: Synthesis of (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(3-(piperazin-1-yl)propoxy)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide

[0205] In an ice bath, TFA (2 mL) was added to a solution of compound 7e (140 mg, 0.14 mmol) in DCM (5 mL), and the mixture was allowed to warm slowly to room temperature and react for 1 hour. The solvent was then removed by rotary evaporation. Free TFA was removed under reduced pressure with an oil pump to give compound 7f (130 mg, 80% purity).

[0206] MS(ESI)m / z=866.4[M+H]

[0207] Step 7: Synthesis of (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(3-(4-methylpiperazin-1-yl)propoxy)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide

[0208] A 30% formaldehyde solution (2 drops) was added to a methanol (1 mL) solution of compound 7f (10 mg, 0.011 mmol), and after 0.5 hours, acetic acid was added to adjust the pH to about 4. Sodium borohydride (1.3 mg) was added and reacted for 3 hours, after which water was added to quench the reaction. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 7 (8.5 mg, yield: 87%).

[0209] 1 H NMR (400MHz, DMSO- d6+D2O)δ7.59(s,1H),7.58(s,1H),7.31(s,1H),7.26(s,1H),6.46(s,1H),5.87-5.6 7(m,2H),4.99-4.78(m,4H),4.53-4.38(m,2H),4.10(t,J=6.1Hz,2H),3.70(s,3H) ,3.48(s,8H),3.17(dt,J=13.6,5.6Hz,2H),3.04(dd,J=15.0,7.7Hz,2H),2.86(s, 3H),2.07(s,3H),2.06-1.97(m,2H),1.21(t,J=7.1Hz,3H),1.09(t,J=7.5Hz,3H). MS(ESI)m / z=880.4[M+H] + . Example 8 [ka]

[0210] Acetaldehyde (18 mg) was added to a methanol solution of compound 7f (60 mg, 0.07 mmol), and after 0.5 hours, acetic acid was added to adjust the pH to about 4. Sodium cyanoborohydride (13 mg, 0.21 mmol) was added and reacted for 3 hours, after which water was added to quench the reaction. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 8 (31 mg, yield 49.5%).

[0211] 1 H NMR(400MHz,MeOD)δ7.67(s,1H),7.63(s,1H),7.38(s,2H),6.76(s,1H),6.07-5.96(m,1H),5 .93-5.79(m,1H),5.19(d,J=5.5Hz,2H),5.07(d,J=5.1Hz,2H),4.68(q,J=7.1Hz,2H),4.21(t, J=5.9Hz,2H),3.87(s,7H),3.64(s,4H),3.51-3.40(m,3H),3.40-3.34(m,2H),3.29-3.22(m,2 H),2.85(s,3H),2.28(s,3H),2.27-2.17(m,2H),1.44(t,J=7.2Hz,6H),1.33(t,J=7.6Hz,3H). MS(ESI)m / z=894.41[M+H] + . Example 9 [ka]

[0212] Acetone (3 mL) was added to a methanol solution of compound 7f (50 mg, 0.058 mmol), and acetic acid was added to adjust the pH to about 4. The mixture was heated to 50°C and reacted for 4 hours. The reaction solution was then cooled to 0°C, and sodium cyanoborohydride (11 mg, 0.17 mmol) was added, and the mixture was reacted overnight at room temperature. Water was added to quench the reaction. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 9 (23 mg, yield: 43%).

[0213] 1 H NMR (400MHz, DMSO -d6 +D2O)δ7.61(s,1H),7.57(s,1H),7.33(s,1H),7.26(s,1H),6.48(s,1H),5.87-5.62( m,2H),4.90(s,4H),4.45(q,J=6.7Hz,2H),4.13(t,J=5.7Hz,2H),3.69(s,3H),3.67- 3.32(m,10H),3.32-3.23(m,2H),3.04(q,J=7.3Hz,2H),2.58(s,3H),2.18-2.09(m,2 H),2.07(s,3H),1.29(d,J=6.6Hz,6H),1.21(t,J=7.1Hz,3H),1.10(t,J=7.5Hz,3H). MS(ESI)m / z=908.43[M+H] + . Example 10 [ka]

[0214] 1-ethoxy-1-trimethylsiloxycyclopropane (48 mg, 0.277 mmol) was added to a methanol solution of compound 7f (60 mg, 0.055 mmol), and acetic acid was added to adjust the pH to about 4. Sodium borohydride acetate (47 mg, 0.22 mmol) was then added, and the mixture was heated to 50° C. and reacted overnight. When LCMS showed that all the intermediates had been converted to imine, the mixture was cooled to room temperature and sodium cyanoborohydride (10 mg, 0.16 mmol) was added. Water was added to quench the reaction. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 10 (27.7 mg, yield: 55%).

[0215] 1 H NMR(400MHz,DMSO-d6+D2O)δ7.57(s,2H),7.23(s,1H),7.21(s,1H),6.46(s,1H) ,5.83-5.63(m,2H),4.85(d,J=10.9Hz,4H),4.45(dd,J=13.3,6.5Hz,2H),3.64( s,4H),3.27-2.91(m,10H),2.87(s,3H),2.05(s,3H),2.01(s,1H),1.85(s,2H), 1.21(t,J=7.1Hz,3H),1.09(t,J=7.5Hz,3H),0.55(d,J=6.5Hz,2H),0.43(s,2H). MS(ESI)m / z=906.41[M+H] + . Example 11 [ka]

[0216] To a solution of 7f (10 mg, 0.011 mmol) in DMF (1 mL), TEA (5.5 mg, 0.055 mmol) and acetic anhydride (3.3 mg, 0.033 mmol) were added and reacted at room temperature for 2 hours. The reaction solution was purified by preparative HPLC to obtain compound 11 (6.5 mg, yield: 62%).

[0217] 1H NMR(400MHz,DMSO-d6+D2O)δ7.61(d,J=1.6Hz,2H),7.27(s,2H),6.48(s,1H),5 .84-5.58(m,2H),4.87(d,J=4.9Hz,4H),4.47(dd,J=13.8,6.8Hz,2H),4.01(s,2 H),3.68(s,3H),3.33(s,4H),3.17-3.04(m,5H),2.87(s,4H),2.54(s,3H),2.07 (s,3H),2.02(s,3H),1.97(s,2H),1.23(t,J=7.1Hz,3H),1.12(t,J=7.5Hz,3H). MS(ESI)m / z=908.39[M+H] + .

[0218] Compounds 12 to 23 in Examples 12 to 23 were also prepared according to the synthesis method of Example 7. Example 12 [ka]

[0219] (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-(piperazin-1-yl)propoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-methoxy-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide (40 mg, 0.044 mmol) in methanol (2 mL) was added with 30% formaldehyde solution (2 drops), and after 0.5 hours, acetic acid was added to adjust the pH to about 4, and sodium borohydride (5.2 mg) was added and reacted for 3 hours, after which water was added to quench the reaction. The reaction mixture was concentrated and then purified by preparative HPLC to obtain compound 12 (29 mg, yield: 75%).

[0220] 1H NMR(400MHz,DMSO)δ12.79(s,1H),11.85(s,2H),8.03(s,1H),7.97(s,1H),7.65(s,1H),7.62(s,1H),7.36(s ,2H),7.34(s,2H),6.49(s,1H),5.97-5.84(m,1H),5.79-5.65(m,1H),4.96(d,J=3.9Hz,2H),4.86(d,J=5.3Hz ,2H),4.49(q,J=7.0Hz,3H),4.15-4.08(m,4H),3.83(s,8H),3.64(s,5H),3.44(s,4H),3.22(s,2H),3.09(q, J=7.5Hz,2H), 2.86(d,J=20.8Hz,3H),2.55(s,3H),2.09(s,5H),1.25(t,J=7.1Hz,3H),1.14(t,J=7.5Hz,3H). MS(ESI)m / z=880.4[M+H] + . Example 13 [ka]

[0221] (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-(piperazin-1-yl)propoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-methoxy-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide (50 mg, 0.058 mmol) was added to a methanol solution, and acetaldehyde (18 mg) was added. After 0.5 hours, acetic acid was added to adjust the pH to about 4, and sodium cyanoborohydride (13 mg, 0.21 mmol) was added and reacted for 3 hours, after which water was added to quench the reaction. The reaction mixture was concentrated and then purified by preparative HPLC to obtain compound 13 (30 mg, yield: 58%).

[0222] 1H NMR(400MHz,DMSO)δ12.80(s,1H),11.80(s,1H),8.03(s,1H),7.96(s,1H),7.65(s,1H),7.63(s,1H) ,7.36(s,3H),7.34(s,1H),6.50(s,1H),5.94-5.89(m,1H),5.78-5.70(m,1H),4.96(d,J=3.8Hz,2H), 4.86(d,J=5.3Hz,2H),4.50(q,J=6.9Hz,2H),4.09(t,J=5.5Hz,2H),3.68(s,4H),3.38(s,4H),3.20( s,4H),3.09(q,J=7.5Hz,2H),2.54(s,3H),2.09(s,5H),1.26(q,J=6.9Hz,6H),1.14(t,J=7.5Hz,3H). MS(ESI)m / z=894.41[M+H] + . Example 14 [ka]

[0223] (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-(piperazin-1-yl)propoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-methoxy-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide (50 mg, 0.058 mmol) was added to a methanol solution, and the pH was adjusted to about 4 with acetic acid, heated to 50 ° C., and reacted for 4 hours. The reaction solution was cooled to 0 ° C., and then sodium cyanoborohydride (11 mg, 0.17 mmol) was added, and the reaction was allowed to proceed overnight at room temperature. The reaction was quenched by adding water. The reaction mixture was concentrated and then purified by preparative HPLC to obtain compound 14 (23 mg, yield: 43.7%).

[0224] 1H NMR(400MHz,DMSO)δ12.80(s,1H),11.86(s,1H),8.03(s,1H),7.96(s,1H),7.65(s,1H),7.63(s,1H),7.36( s,3H),7.34(s,1H),6.50(s,1H),5.99-5.87(m,1H),5.79-5.70(m,1H),4.96(d,J=4.2Hz,2H),4.86(d,J=5.5 Hz,2H),4.50(q,J=6.9Hz,2H),4.10(t,J=5.5Hz,2H),3.83(s,3H),3.71-3.37(m,8H),3.22(s,2H),3.09(q, J=7.4Hz,2H),2.54(s,3H),2.09(s,5H),1.30(d,J=6.5Hz,5H),1.25(t,J=7.1Hz,3H),1.14(t,J=7.5Hz,3H). MS(ESI)m / z=908.43[M+H] + . Example 15 [ka]

[0225] (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-(piperazin-1-yl)propoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-methoxy-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide (50 mg, 0.058 mmol) ) was added to a methanol solution of 1-ethoxy-1-trimethylsiloxycyclopropane (48 mg, 0.277 mmol), and the pH was adjusted to about 4 with acetic acid. Sodium borohydride acetate (47 mg, 0.22 mmol) was then added, and the mixture was heated to 50°C and reacted overnight. When LCMS showed that all of the product had been converted to the intermediate imine, the mixture was cooled to room temperature and sodium cyanoborohydride (10 mg, 0.16 mmol) was added. Water was added to quench the reaction. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 15 (9 mg, 17% yield).

[0226] 1 H NMR(400MHz,DMSO)δ12.82(s,1H),8.03(s,1H),7.96(s,1H),7.66(s,1H),7.64(s,1H),7.45-7.26(m, 3H),6.52(s,1H),5.98-5.84(m,1H),5.79-5.68(m,1H),4.95(s,2H),4.85(d,J=5.4Hz,2H),4.52(dd,J =14.2,7.2Hz,2H),4.03(s,1H),3.79(s,2H),3.66-3.14(m,6H),3.10(dd,J=15.0,7.5Hz,2H),2.53(s, 2H),2.11(s,2H),1.99(s,1H),1.27(t,J=7.1Hz,2H),1.15(t,J=7.5Hz,2H),0.99(s,2H),0.74(s,2H). MS(ESI)m / z=906.41[M+H] + . Example 16 [ka]

[0227] Compound (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(methylthio)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(3-(piperazin-1-yl)propoxy)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide (20 mg, 0.023 mmol) was added to a methanol solution of 30% formaldehyde solution (2 drops), and after 0.5 hours, acetic acid was added to adjust the pH to about 4, and sodium borohydride (2.6 mg, 0.069 mmol) was added and reacted for 3 hours, after which water was added to quench the reaction. The reaction mixture was concentrated and then purified by preparative HPLC to obtain compound 16 (10 mg, yield: 48%).

[0228] MS(ESI)m / z=896.37[M+H] + . Example 17 [ka]

[0229] (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(methylthio)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(3-(piperazin-1-yl)propoxy)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide (60 mg, 0.07 mmol) was added to a methanol solution, and acetaldehyde (18 mg) was added. After 0.5 hours, acetic acid was added to adjust the pH to about 4, and sodium cyanoborohydride (13 mg, 0.21 mmol) was added and reacted for 3 hours, after which water was added to quench the reaction. The reaction mixture was concentrated and then purified by preparative HPLC to obtain compound 17 (26 mg, yield: 41%).

[0230] 1 H NMR(400MHz,DMSO)δ12.80(s,1H),11.77(s,1H),8.06(s,1H),7.98(s,1H),7.85(s,1H),7.63(s,2H),7.40( s,1H),7.36(s,2H),6.51(s,1H),5.92-5.79(m,1H),5.79-5.68(m,1H),5.15(d,J=2.6Hz,2H),4.91(d,J=3.9 Hz,2H),4.51(dd,J=14.1,7.0Hz,4H),4.14(t,J=5.3Hz,2H),3.70(s,4H),3.39(s,5H),3.20(s,4H),3.09(q, J=7.4Hz,2H),2.53(s,3H),2.45(s,3H),2.13(s,2H),2.09(s,3H),1.33-1.19(m,7H),1.14(t,J=7.5Hz,3H). MS(ESI)m / z=910.39[M+H] + . Example 18 [ka]

[0231] ((E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(methylthio)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(3-(piperazin-1-yl)propoxy)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide ( Acetone (3 mL) was added to a methanol solution of 1,2-dichlorophenyl ether (60 mg, 0.07 mmol), and the pH was adjusted to about 4 with acetic acid. The mixture was heated to 50°C and reacted for 4 hours. The reaction solution was then cooled to 0°C, and sodium cyanoborohydride (11 mg, 0.17 mmol) was added. The mixture was reacted overnight at room temperature, and water was added to quench the reaction. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 18 (17 mg, 26%).

[0232] 1 H NMR(400MHz,DMSO)δ12.81(s,1H),12.12(s,1H),11.84(s,1H),8.06(s,1H),7.98(s,1H),7.85(s,1H),7.63(s ,2H),7.41(s,1H),7.37(s,2H),6.52(s,1H),5.91-5.67(m,2H),5.15(d,J=2.5Hz,2H),4.91(d,J=3.5Hz,2H),4 .51(q,J=7.0Hz,2H),4.16(t,J=5.6Hz,2H),3.81-3.39(m,10H),3.24(s,2H),3.09(q,J=7.4Hz,2H),2.54(s,3 H),2.45(s,4H),2.14(s,2H),2.09(s,3H),1.30(d,J=6.5Hz,6H),1.26(t,J=7.1Hz,3H),1.14(t,J=7.5Hz,3H). MS(ESI)m / z=924.40[M+H] + . Example 19 [ka]

[0233] (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(methylthio)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(3-(piperazin-1-yl)propoxy)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide (60 mg, 0.07 mmol) ) was added to a methanol solution of 1-ethoxy-1-trimethylsiloxycyclopropane (48 mg, 0.277 mmol), and the pH was adjusted to about 4 with acetic acid. Sodium borohydride acetate (47 mg, 0.22 mmol) was then added, and the mixture was heated to 50°C and reacted overnight. When LCMS showed that all the imine had been converted to the intermediate, the mixture was cooled to room temperature and sodium cyanoborohydride (10 mg, 0.16 mmol) was added. The reaction was quenched by adding water. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 19 (16 mg, yield: 25%).

[0234] MS(ESI)m / z=922.39[M+H] + . Example 20 [ka]

[0235] (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-(piperazin-1-yl)propoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(methylthio)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide (10 mg, 0.011 mmol) was added to a methanol solution of 30% formaldehyde solution (2 drops), and after 0.5 hours, acetic acid was added to adjust the pH to about 4, and sodium borohydride (1.3 mg, 0.033 mmol) was added and reacted for 3 hours, after which water was added to quench the reaction. The reaction mixture was concentrated and then purified by preparative HPLC to obtain compound 20 (5 mg, yield: 51%).

[0236] MS(ESI)m / z=896.37[M+H] + . Example 21 [ka]

[0237] (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-(piperazin-1-yl)propoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(methylthio)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide (60 mg, 0.07 mmol) was added to a methanol solution, and acetaldehyde (18 mg) was added. After 0.5 hours, acetic acid was added to adjust the pH to about 4, and sodium cyanoborohydride (13 mg, 0.21 mmol) was added and reacted for 3 hours, after which water was added to quench the reaction. The reaction mixture was concentrated and then purified by preparative HPLC to obtain compound 21 (8 mg, yield: 12%).

[0238] 1H NMR(400M,MeOD-d4)δ7.83-7.90(m,1H),7.74-7.79(m,1H),7.68-7.74(m,1H),7.34-7 .45(m,1H),6.75-6.83(m,1H),5.82-6.02(m,2H),5.18-5.34(m,4H),4.67-4.79(m,2H) ,4.17-4.28(m,2H),3.54-3.99(m,8H),3.36-3.47(m,4H),3.26-3.30(m,2H),2.89(s,3 H),2.58(s,3H),2.32(s,3H),2.17-2.27(m,2H),1.42-1.53(m,6H),1.32-1.39(m,3H). MS(ESI)m / z=910.39[M+H] + . Example 22 [ka]

[0239] (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-(piperazin-1-yl)propoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(methylthio)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide (60 mg, 0.07 mmol) was added to a methanol solution, and the pH was adjusted to about 4 with acetic acid, heated to 50 ° C., and reacted for 4 hours. The reaction solution was cooled to 0 ° C., and then sodium cyanoborohydride (11 mg, 0.17 mmol) was added, and the reaction was allowed to proceed overnight at room temperature. Water was added to quench the reaction. The reaction mixture was concentrated and then purified by preparative HPLC to obtain compound 22 (17 mg, 26%).

[0240] 1H NMR (400M, MeOD-d4) δ7.85-7.90(m,1H),7.74-7.79(m,1H),7.70-7.74(m,1H),7.37-7 .41(m,1H),6.78-6.83(m,1H),5.82-6.01(m,2H),5.18-5.34(m,4H),4.68-4.78(m,2H) ,4.19-4.26(m,2H),3.66-3.87(m,8H),3.40-3.47(m,2H),3.28-3.32(m,2H),2.89(s,3 H),2.58(s,3H),2.32(s,3H),2.18-2.28(m,2H),1.44-1.53(m,9H),1.31-1.40(m,3H). MS(ESI)m / z=924.40[M+H] + . Example 23 [ka]

[0241] (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-(piperazin-1-yl)propoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(methylthio)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazide 1-ethoxy-1-trimethylsiloxycyclopropane (39 mg, 0.225 mmol) was added to a methanol solution of zole-5-carboxamide (40 mg, 0.045 mmol), and acetic acid was added to adjust the pH to about 4. Sodium borohydride acetate (47 mg, 0.22 mmol) was then added, and the mixture was heated to 50° C. and reacted overnight, and water was added to quench the reaction. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 23 (10 mg, yield: 24%).

[0242] MS(ESI)m / z=922.39[M+H] + .

[0243] (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(3-(piperazin-1-yl)propoxy)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide was added to a DMF solution with TEA and the corresponding acid chloride, and the mixture was reacted at room temperature for 2 hours. The reaction solution was purified by preparative HPLC to obtain compounds 24, 25, 26, 27, and 28. The structures and properties of the compounds are shown in Table 1 below.

[0244] [Table 1]

[0245] (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-(piperazin-1-yl)propoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-methoxy-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide was added to a DMF solution of TEA and the corresponding acid chloride, and the mixture was reacted at room temperature for 2 hours. The reaction solution was purified by preparative HPLC to obtain compounds 29, 30, 31, 32, 33, and 34. The structures and properties of the compounds are shown in Table 2 below.

[0246] [Table 2-1] [Table 2-2]

[0247] (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(methylthio)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(3-(piperazin-1-yl)propoxy)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide in DMF solution was added with TEA and the corresponding acid chloride, and the mixture was reacted at room temperature for 2 hours. The reaction solution was purified by preparative HPLC to obtain compounds 35, 36, 37, 38, 39, and 40. The structures and properties of the compounds are shown in Table 3 below.

[0248] [Table 3-1] [Table 3-2]

[0249] (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-(piperazin-1-yl)propoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(methylthio)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide was added to a DMF solution and reacted at room temperature for 2 hours. The reaction solution was purified by preparative HPLC to obtain compounds 41, 42, 43, 44, 45, and 46. The structures and properties of the compounds are shown in Table 4 below:

[0250] [Table 4-1] [Table 4-2] Example 47 [ka] Step 1: Synthesis of methyl (E)-methyl-2-(4-ethyl-2-methylthiazole-5-carboxamido)-1-(4-(((2-methoxy-4-(methoxycarbonyl)-6-nitrophenyl)amino)but-2-en-1-yl)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-5-carboxylate

[0251] 4e (2.1 g, 3.8 mmol) was dispersed in n-butanol (20 mL), and 4-chloro-3-(3-morpholinopropoxy)-5-nitrobenzoic acid methyl ester (0.93 g, 3.8 mmol) and DIPEA (2.5 g, 19 mmol) were added. The reaction mixture was heated to 120° C. and stirred for 18 hours. The crude product obtained by distillation under reduced pressure was dissolved in ethyl acetate, washed with water and saturated saline, dried over anhydrous magnesium sulfate, spin-dried, and purified with a silica gel column (eluent: petroleum ether / ethyl acetate=1 / 2) to obtain 47a (2.0 g, yield: 70%).

[0252] MS(ESI)m / z=766.2[M+H] Step 2: Synthesis of methyl (E)-methyl-1-(4-(((2-amino-6-methoxy-4-(methoxycarbonyl)phenyl)amino)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-5-carboxylate

[0253] In an ice bath, ammonia water (1 mL) was added to a solution of 47a (600 mg, 0.78 mmol) in methanol (5 mL), and after 10 minutes, an aqueous solution of sodium dithionite (679 mg, 3.9 mmol) was added, and the mixture was slowly warmed to room temperature and reacted for 2 hours. The mixture was filtered to remove inorganic salts, and extracted with ethyl acetate. The organic phase was washed with saturated saline, dried over anhydrous magnesium sulfate, and then spin-dried to obtain compound 47b (459 mg, yield: 80%).

[0254] MS(ESI)m / z=736.3[M+H] Step 3: Synthesis of methyl (E)-methyl-2-(4-ethyl-2-methylthiazole-5-carboxamido)-1-(4-(2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy-5-(methoxycarbonyl)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-5-carboxylate

[0255] In an ice bath, 1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (58 mg, 0.27 mmol) was added to a solution of 47b (200 mg, 0.27 mmol) in DMF (5 mL). After reacting for 0.5 hours, DIPEA (70 mg, 0.54 mmol) and HATU (102 mg, 0.27 mmol) were added, and the mixture was then warmed to room temperature and reacted for 12 hours. The reaction solution was slowly poured into water, and a yellow solid precipitated. The solid was filtered and dried to obtain 47c (185 mg, yield: 75%).

[0256] MS(ESI)m / z=915.3[M+H] Step 4: Synthesis of (E)-1-(4-(5-carboxy-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-5-carboxylic acid

[0257] 47c (185 mg, 0.2 mmol) was dissolved in a mixture (5 mL) of methanol, tetrahydrofuran and water (volume ratio: 1 / 1 / 1), and lithium hydroxide (42 mg, 1.0 mmol) was added. The mixture was heated to 75°C and reacted overnight with stirring. The organic solvent was removed by rotary evaporation, cooled to room temperature, and adjusted with dilute hydrochloric acid (1 M) in an ice bath until no solid precipitated, filtered, and the solid was dried to obtain compound 47d (141 mg, yield: 80%).

[0258] MS(ESI)m / z=887.3[M+H] Step 5: Synthesis of (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H)-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide

[0259] 47d (141 mg, 0.16 mmol) was dissolved in DMF (5 mL), and HATU (152 mg, 0.4 mmol) and DIPEA (82 mg, 0.64 mmol) were added. After 0.5 hours, ammonium bicarbonate (38 mg, 0.48 mmol) was added and stirred at room temperature for 2 hours. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 47 (30 mg, yield: 43%).

[0260] MS(ESI)m / z=885.4[M+H] 1 H NMR (400M, DMSO- d6 )δ12.81-12.98(m,2H),9.69-9.77(m,1H),7.93-8.02(m,2H),7.61-7.69(m,2H),7.37-7.43(m,2H), 7.26-7.34(m,2H),5.71-5.90(m,4H),4.80-4.94(m,4H),4.46-4.54(m,2H),3.90-4.00(m,4H),3.75( s,3H),3.58-3.64(m,4H),3.21-3.26(m,2H),3.05-3.20(m,6H),2.93-3.00(m,2H),2.64-2.70(m,2H) ),2.31-2.36(m,2H),2.12(s,3H),1.80-1.94(m,4H),1.29(t,J=7.04Hz,3H),1.16(t,J=7.48Hz,3H). Example 48 [ka] Step 1: Synthesis of methyl (E)-methyl-2-(4-ethyl-2-methylthiazole-5-carboxamido)-1-(4-(2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-5-(methoxycarbonyl)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-methoxy-1H-benz[d]imidazolyl-5-carboxylate

[0261] In an ice bath, 1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (0.15 g, 0.69 mmol) was added to a solution of 2g (0.51 g, 0.69 mmol) in DMF (5 mL). After reacting for 0.5 hours, DIPEA (0.27 g, 2.07 mmol) and HATU (0.32 g, 0.83 mmol) were added, and the mixture was then warmed to room temperature and reacted for 12 hours. The reaction solution was slowly poured into water, and a yellow solid precipitated. The solid was filtered and dried to obtain 48a (0.48 g, yield: 77%).

[0262] MS(ESI)m / z=915.3[M+H]

[0263] Step 2: Synthesis of (E)-1-(4-(5-carboxy-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-methoxy-1H-benz[d]imidazolyl-5-carboxylic acid

[0264] 48a (0.48 g, 0.52 mmol) was dissolved in a mixture (20 mL) of methanol, tetrahydrofuran and water (volume ratio: 1 / 1 / 1), and lithium hydroxide hydrate (0.22 g, 5.2 mmol) was added. The mixture was heated to 75°C and reacted overnight with stirring. The organic solvent was removed by rotary evaporation, cooled to room temperature, and adjusted with dilute hydrochloric acid (1 M) in an ice bath until no solid precipitated, filtered, and the solid was dried to obtain compound 48b (0.45 g, yield: 97%).

[0265] MS(ESI)m / z=886.3[M+H]

[0266] Step 3: Synthesis of (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamide))-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-methoxy-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide

[0267] 48b (0.45 g, 0.51 mmol) was dissolved in DMF (5 mL), and HATU (0.46 g, 1.22 mmol) and DIPEA (0.33 g, 2.55 mmol) were added. After 0.5 hours, ammonium bicarbonate (0.2 g, 2.55 mmol) was added and stirred at room temperature for 2 hours. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 48 (0.26 g, yield: 57%).

[0268] MS(ESI)m / z=884.4[M+H] 1 H NMR (400M, DMSO- d6 )δ12.82-12.97(m,2H),9.55-9.85(m,1H),7.92-8.02(m,2H),7.61-7.67(m,2H),7.35-7.45(m,2H) ,7.28-7.35(m,3H),5.77-5.89(m,2H),4.86-4.93(m,4H),4.44-4.52(m,2H),4.03-4.07(m,2H),3. 91-3.98(m,2H),3.73(s,3H),3.59-3.67(m,4H),3.26-3.32(m,4H),3.12-3.18(m,4H),2.95-3.04( m,2H),2.53(s,3H),2.09(s,3H),1.86-1.99(m,2H),1.26(t,J=7.04Hz,3H),1.17(t,J=7.48Hz,3H). Example 49 [ka]

[0269] Step 1: Synthesis of methyl (E)-methyl-2-(4-ethyl-2-methylthiazole-5-carboxamido)-1-(4-(2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-5-(methoxycarbonyl)-7-(methylthio)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl 5-carboxylate

[0270] In an ice bath, 1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (49 mg, 0.23 mmol) was added to a solution of 4g (145 mg, 0.19 mmol) in DMF (5 mL). After reacting for 0.5 hours, DIPEA (75 mg, 0.58 mmol) and HATU (89 mg, 0.23 mmol) were added, and the mixture was then warmed to room temperature and reacted for 12 hours. The reaction solution was slowly poured into water, and a yellow solid precipitated. The solid was filtered and dried to obtain 49a (116 mg, yield: 64%).

[0271] MS(ESI)m / z=931.3[M+H]

[0272] Step 2: Synthesis of (E)-1-(4-(5-carboxy-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-7-(methylthio)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-5-carboxylic acid

[0273] 49a (116 mg, 0.12 mmol) was dissolved in a mixture of methanol, tetrahydrofuran and water (volume ratio: 1 / 1 / 1), and lithium hydroxide hydrate (54 mg, 12.4 mmol) was added. The mixture was heated to 75°C and reacted overnight with stirring. The organic solvent was removed by rotary evaporation, cooled to room temperature, and adjusted with dilute hydrochloric acid (1 M) in an ice bath until no solid precipitated, filtered, and the solid was dried to obtain compound 49b (102 mg, yield: 90%).

[0274] MS(ESI)m / z=903.3[M+H] Step 3: Synthesis of (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamide))-7-(methylthio)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide

[0275] 49b (102 mg, 0.11 mmol) was dissolved in DMF (5 mL), and HATU (103 mg, 0.27 mmol) and DIPEA (88 mg, 0.68 mmol) were added. After 0.5 hours, ammonium bicarbonate (27 mg, 0.34 mmol) was added and stirred at room temperature for 2 hours. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 49 (46 mg, yield: 45%).

[0276] MS(ESI)m / z=901.3[M+H] 1 H NMR (400M, DMSO- d6)δ12.77-13.08(m,1H),10.91-10.99(m,1H),7.94-8.14(m,2H),7.83-7.90(m,1H),7.61-7.68(m,2H),7 .31-7.48(m,3H),5.72-5.84(m,2H),5.09-5.17(m,2H),4.87-4.91(m,2H),4.41-4.49(m,2H),4.03-4.13 (m,2H),3.86-3.97(m,2H),3.69-3.81(m,2H),3.22-3.33(m,2H),3.07-3.20(m,4H),2.91-3.04(m,2H),2 .56(s,3H),2.47(s,3H),2.08(s,3H),1.99-2.07(m,2H),1.25(t,J=7.04Hz,3H),1.16(t,J=7.48Hz,3H). Example 50 [ka]

[0277] Step 1: Synthesis of methyl (E)-methyl-2-(4-ethyl-2-methylthiazole-5-carboxamido)-1-(4-(2-(4-ethyl-2-methylthiazole-5-carboxamido)-5-(methoxycarbonyl)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(methylthio)-1H-benz[d]imidazolyl-5-carboxylate

[0278] In an ice bath, 4-ethyl-2-methylthiazole-5-carbonyl isothiocyanate (49 mg, 0.23 mmol) was added to a solution of 4g (145 mg, 0.19 mmol) in DMF (5 mL). After reacting for 0.5 hours, DIPEA (75 mg, 0.58 mmol) and HATU (89 mg, 0.23 mmol) were added, and the mixture was then warmed to room temperature and reacted for 12 hours. The reaction solution was slowly poured into water, and a yellow solid precipitated. The solid was filtered and dried to obtain 50a (116 mg, yield: 64%).

[0279] MS(ESI)m / z=930.3[M+H] Step 2: Synthesis of (E)-1-(4-(5-carboxy-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-(methylthio)-1H-benz[d]imidazolyl-5-carboxylic acid

[0280] 50a (116 mg, 0.12 mmol) was dissolved in a mixture of methanol, tetrahydrofuran and water (volume ratio: 1 / 1 / 1), and lithium hydroxide hydrate (54 mg, 12.4 mmol) was added. The mixture was heated to 75°C and reacted overnight with stirring. The organic solvent was removed by rotary evaporation, cooled to room temperature, and adjusted with dilute hydrochloric acid (1 M) in an ice bath until no solid precipitated, filtered, and the solid was dried to obtain compound 50b (103 mg, yield: 91%).

[0281] MS(ESI)m / z=902.3[M+H]

[0282] Step 3: Synthesis of (E)-1-(4-(5-carboxy-2-(4-ethyl-2-methylthiazole-5-carboxamide)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamide)-7-(methylthio)-1H-benz[d]imidazolyl-5-carboxamide

[0283] 50b (103 mg, 0.11 mmol) was dissolved in DMF (5 mL), and HATU (103 mg, 0.27 mmol) and DIPEA (88 mg, 0.68 mmol) were added. After 0.5 hours, ammonium bicarbonate (27 mg, 0.34 mmol) was added and stirred at room temperature for 2 hours. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 50 (36 mg, 35% yield).

[0284] MS(ESI)m / z=900.3[M+H] 1 H NMR (400M, DMSO-d6 )δ12.79-12.99(m,1H),10.99-11.08(m,1H),7.89-8.16(m,2H),7.83-7.87(m,1H),7.63 -7.67(m,2H),7.29-7.48(m,3H),5.75-5.81(m,2H),5.08-5.12(m,4H),4.88-4.92(m,4H) ,4.01-4.10(m,2H),3.86-3.95(m,2H),3.70-3.81(m,2H),3.21-3.30(m,2H),3.07-3.17( m,6H),2.90-3.01(m,2H),2.56(s,3H),1.97-2.09(m,2H),1.17(dt,J=7.44,3.00Hz,6H). Example 51 [ka]

[0285] Step 1: Synthesis of methyl (E)-methyl-1-(4-((tert-butoxycarbonyl)amino)but-2-en-1-yl)-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-5-carboxylate

[0286] In an ice bath, 1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (0.34 g, 1.55 mmol) was added to a solution of 4c (0.74 g, 1.55 mmol) in DMF (5 mL). After reacting for 0.5 hours, DIPEA (0.6 g, 4.65 mmol) and HATU (0.71 g, 1.86 mmol) were added, and the mixture was then warmed to room temperature and reacted for 12 hours. The reaction solution was slowly poured into water, and a yellow solid precipitated. The solid was filtered and dried to obtain 51a (0.88 g, yield: 86%).

[0287] MS(ESI)m / z=658.3[M+H]

[0288] Step 2: Synthesis of methyl (E)-methyl-1-(4-aminobut-2-en-1-yl)-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-5-carboxylate

[0289] In an ice bath, TFA (3 mL) was added to 51a (0.88 g, 1.34 mmol) in DCM (40 mL), and the mixture was allowed to warm to room temperature and react for 2 hours. The solvent was removed by rotary evaporation under reduced pressure, ethyl acetate was added, and the mixture was spin-dried under reduced pressure to remove free TFA, yielding compound 51b (0.66 g, yield: 88%).

[0290] MS(ESI)m / z=558.3[M+H]

[0291] Step 3: Synthesis of methyl (E)-methyl-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-1-(4-((4-(methoxycarbonyl)-2-(methylthio)-6-nitrophenyl)amino)but-2-en-1-yl)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-5-carboxylate

[0292] DIPEA (387 mg, 3 mmol) and methyl 4-fluoro-3-methylthio-5-nitrobenzoate (245 mg, 1 mmol) were added to a solution of 51b (557 mg, 1 mmol) in DMF (10 mL), and the mixture was allowed to react at room temperature for 3 hours. The reaction solution was added to water, extracted with ethyl acetate, washed with saturated saline, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation under reduced pressure to obtain compound 51c (451 mg, yield: 58%).

[0293] MS(ESI)m / z=783.3[M+H]

[0294] Step 4: Synthesis of (E)-1-(4-((2-amino-4-(methoxycarbonyl)-6-(methylthio)phenyl)amino)but-2-en-1-yl)-2-(4-ethyl-2)methyl ester-methylthiazole-5-carboxamido)-7-(3-morpholinomethyl)-1H-benz[d]imidazolyl-5-carboxylate

[0295] In an ice bath, ammonia water (1 mL) was added to a solution of 51c (451 mg, 0.58 mmol) in methanol (5 mL), and after 10 minutes, an aqueous solution of sodium dithionite (502 mg, 2.88 mmol) was added, and the mixture was slowly warmed to room temperature and reacted for 2 hours. The mixture was filtered to remove inorganic salts, and ethyl acetate was added for extraction. The organic phase was washed with saturated saline, dried over anhydrous magnesium sulfate, and then spin-dried to obtain a crude product, which was purified with a silica gel column (eluent: petroleum ether / ethyl acetate = 1 / 1) to obtain compound 51d (417 mg, yield: 94%).

[0296] MS(ESI)m / z=753.3[M+H]

[0297] Step 5: Synthesis of methyl (E)-methyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1-(4-(2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-5-(methoxycarbonyl)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(methylthio)-1H-benz[d]imidazolyl-5-carboxylate

[0298] In an ice bath, 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (46 mg, 0.232 mmol) was added to a solution of 51d (139 mg, 0.193 mmol) in DMF (5 mL). After reacting for 0.5 hours, DIPEA (75 mg, 0.579 mmol) and HATU (89 mg, 0.232 mmol) were added, and the mixture was then warmed to room temperature and reacted for 12 hours. The reaction solution was slowly poured into water, and a yellow solid precipitated. The solid was filtered and dried to obtain 51e (120 mg, yield: 71%).

[0299] MS(ESI)m / z=914.3[M+H]

[0300] Step 6: Synthesis of (E)-1-(4-(5-carboxy-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(methylthio)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-5-carboxylic acid

[0301] 51e (120 mg, 0.13 mmol) was dissolved in a mixture of methanol, tetrahydrofuran and water (volume ratio: 1 / 1 / 1), and lithium hydroxide (55 mg, 1.31 mmol) was added. The mixture was heated to 75°C and reacted overnight with stirring. The organic solvent was removed by rotary evaporation, cooled to room temperature, and adjusted with dilute hydrochloric acid (1 M) in an ice bath until no solid precipitated, filtered, and the solid was dried to obtain compound 51f (112 mg, yield: 96%).

[0302] MS(ESI)m / z=886.3[M+H]

[0303] Step 7: Synthesis of (E)-1-(4-(5-(5-carbamoyl-2-)(1-styrene-3-methyl-1H-pyrazole-5-carboxamide)-7-(methylthio)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-2-(1-styrene-4-fluoro-3-methyl-1H-pyrazole-5-anthrylamide)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-5-carboxamide

[0304] 51f (112 mg, 0.126 mmol) was dissolved in DMF (5 mL), and HATU (116 mg, 0.304 mmol) and DIPEA (98 mg, 0.759 mmol) were added. After 0.5 hours, ammonium bicarbonate (30 mg, 0.380 mmol) was added and stirred at room temperature for 2 hours. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 51 (51 mg, yield: 46%).

[0305] MS(ESI)m / z=884.3[M+H] 1 H NMR(400M,DMSO-d6)δ12.76-13.12(m,1H),11.07-11.17(m,1H),7.95-8.13(m,2H),7.85-7.90(m,1H) ,7.61-7.70(m,2H),7.31-7.46(m,3H),6.51(s,1H),5.71-5.76(m,2H),5.09-5.16(m,2H),4.89-4.96( m,2H),4.42-4.47(m,4H),4.00-4.07(m,2H),3.85-3.94(m,2H),3.70-3.81(m,2H),3.18-3.28(m,2H), 3.04-3.13(m,2H),2.87-3.00(m,2H),2.45(s,3H),2.10(s,6H),1.95-2.04(m,2H),1.23-1.32(m,6H). Example 52 [ka]

[0306] Step 1: Synthesis of methyl (E)-methyl-2-(4-ethyl-2-methylthiazole-5-carboxamido)-1-(4-(2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-5-(methoxycarbonyl)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(methylthio)-1H-benz[d]imidazolyl 5-carboxylate

[0307] In an ice bath, 4-ethyl-2-methylthiazole-5-carbonyl isothiocyanate (49 mg, 0.232 mmol) was added to a solution of 51d (139 mg, 0.193 mmol) in DMF (5 mL). After reacting for 0.5 hours, DIPEA (75 mg, 0.579 mmol) and HATU (89 mg, 0.232 mmol) were added, and the mixture was then warmed to room temperature and reacted for 12 hours. The reaction solution was slowly poured into water, and a yellow solid precipitated. The solid was filtered and dried to obtain 52a (118 mg, yield: 68%).

[0308] MS(ESI)m / z=931.3[M+H]

[0309] Step 2: Synthesis of (E)-1-(4-(5-carboxy-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-(methylthio)-1H-benz[d]imidazolyl-5-carboxylic acid

[0310] 52a (118 mg, 0.13 mmol) was dissolved in a mixture of methanol, tetrahydrofuran and water (volume ratio: 1 / 1 / 1), and lithium hydroxide (55 mg, 1.31 mmol) was added. The mixture was heated to 75°C and reacted overnight with stirring. The organic solvent was removed by rotary evaporation, cooled to room temperature, and adjusted with dilute hydrochloric acid (1 M) in an ice bath until no solid precipitated, filtered, and the solid was dried to obtain compound 52b (110 mg, yield: 96%).

[0311] MS(ESI)m / z=903.3[M+H]

[0312] Step 3: Synthesis of (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamide))-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(methylthio)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide

[0313] 52b (110 mg, 0.122 mmol) was dissolved in DMF (5 mL), and HATU (112 mg, 0.293 mmol) and DIPEA (95 mg, 0.732 mmol) were added. After 0.5 hours, ammonium bicarbonate (29 mg, 0.366 mmol) was added and stirred at room temperature for 2 hours. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 52 (32 mg, yield: 29%).

[0314] MS(ESI)m / z=901.3[M+H] 1 H NMR(400M,DMSO-d6)δ12.93-12.98(m,1H),10.88-10.96(m,1H),7.94-8.13(m,2H),7.84-7.88(m,1H),7.6 3-7.68(m,2H),7.28-7.46(m,3H),5.70-5.83(m,2H),5.06-5.12(m,2H),4.90-4.96(m,2H),4.45-4.52(m, 4H),3.98-4.06(m,2H),3.86-3.94(m,2H),3.68-3.80(m,2H),3.19-3.27(m,2H),3.05-3.16(m,4H),2.88- 3.00(m,2H),2.49(s,6H),2.10(s,3H),1.93-2.03(m,2H),1.28(t,J=7.04Hz,3H),1.16(t,J=7.48Hz,3H). Example 53 [ka]

[0315] Step 1: Synthesis of methyl (E)-methyl-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-1-(4-(2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-5-(methoxycarbonyl)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-1-yl)-2-en-1-yl)-7-(methylthio))-1H-benz[d]imidazolyl-5-carboxylate

[0316] In an ice bath, 1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (49 mg, 0.232 mmol) was added to a solution of 51d (139 mg, 0.193 mmol) in DMF (5 mL). After reacting for 0.5 hours, DIPEA (75 mg, 0.579 mmol) and HATU (89 mg, 0.232 mmol) were added, and the mixture was then warmed to room temperature and reacted for 12 hours. The reaction solution was slowly poured into water, and a yellow solid precipitated. The solid was filtered and dried to obtain 53a (108 mg, yield: 62%).

[0317] MS(ESI)m / z=932.3[M+H]

[0318] Step 2: Synthesis of (E)-1-(4-(5-carboxy-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamido)-7-(methylthio)-1H-benz[d]imidazolyl-5-carboxylic acid

[0319] 53a (108 mg, 0.12 mmol) was dissolved in a mixture of methanol, tetrahydrofuran and water (volume ratio: 1 / 1 / 1), and lithium hydroxide (49 mg, 1.16 mmol) was added. The mixture was heated to 75°C and reacted overnight with stirring. The organic solvent was removed by rotary evaporation, cooled to room temperature, and adjusted with dilute hydrochloric acid (1 M) in an ice bath until no solid precipitated, filtered, and the solid was dried to obtain compound 53b (100 mg, yield: 95%).

[0320] MS(ESI)m / z=904.3[M+H]

[0321] Step 3: Synthesis of (E)-1-(4-(5-carbamoyl-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-2-(1-ethyl-4-fluoro-3-methyl-1H-pyrazole-5-carboxamide)-7-(methylthio)-1H-benz[d]imidazolyl-5-carboxamide

[0322] 53b (100 mg, 0.111 mmol) was dissolved in DMF (5 mL), and HATU (102 mg, 0.266 mmol) and DIPEA (88 mg, 0.665 mmol) were added. After 0.5 hours, ammonium bicarbonate (27 mg, 0.333 mmol) was added and stirred at room temperature for 2 hours. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 53 (41 mg, yield: 41%).

[0323] MS(ESI)m / z=902.3[M+H] 1H NMR(400M,DMSO-d6)δ12.89-13.08(m,1H),10.47-10.57(m,1H),7.92-8.13(m,2H),7.85-7.89(m,1H) ,7.61-7.69(m,2H),7.30-7.48(m,3H),5.74-5.79(m,2H),5.09-5.16(m,2H),4.89-4.95(m,2H),4.41- 4.49(m,6H),4.03-4.07(m,2H),3.89-3.95(m,2H),3.66-3.75(m,2H),3.21-3.30(m,2H),3.08-3.17(m ,2H),2.92-3.02(m,2H),2.45(s,3H),2.10(s,3H),2.08(s,3H),1.91-2.03(m,2H),1.20-1.30(m,6H). Example 54 [ka]

[0324] Step 1: Synthesis of methyl (E)-methyl-1-(4-((tert-butoxycarbonyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-5-carboxylate

[0325] In an ice bath, 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (1.35 g, 6.90 mmol) was added to a solution of 4c (3.0 g, 6.27 mmol) in DMF (30 mL). After reacting for 0.5 hours, DIPEA (1.62 g, 12.54 mmol) and HATU (2.62 g, 6.9 mmol) were added, and the mixture was then warmed to room temperature and reacted for 3 hours. The reaction solution was slowly poured into water, and a yellow solid precipitated. The solid was filtered and dried to obtain 54a (3.21 g, yield: 80%).

[0326] MS(ESI)m / z=640.3[M+H]

[0327] Step 2: Synthesis of methyl (E)-methyl-1-(4-aminobut-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-5-carboxylate

[0328] In an ice bath, TFA (5 mL) was added to 54a (3.20 g, 4.0 mmol) in DCM (30 mL), and then the mixture was allowed to warm to room temperature and react for 2 h. The solvent was removed by rotary evaporation under reduced pressure, and ethyl acetate was added, followed by spin drying under reduced pressure to remove free TFA, to give compound 54b (4.2 g), which was used directly in the next step.

[0329] MS(ESI)m / z=540.3[M+H]

[0330] Step 3: Synthesis of methyl (E)-methyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1-(4-((4-(methoxycarbonyl)-2-(methylthio)-6-nitrophenyl)amino)but-2-en-1-yl)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-5-carboxylate

[0331] DIPEA (143 mg, 1.1 mmol) and methyl 4-fluoro-3-methylthio-5-nitrobenzoate (91 mg, 0.37 mmol) were added to a solution of 54b (296 mg, crude product, 0.37 mmol) in DMF (10 mL), and the mixture was reacted at room temperature for 3 hours. After the reaction was completed, the mixture was added to water, filtered, washed with water and dried to obtain 54c (120 mg, yield: 42%) as a yellow solid.

[0332] MS(ESI)m / z=765.3[M+H]

[0333] Step 4: Synthesis of methyl (E)-methyl-1-(4-(((2-amino-4-(methoxycarbonyl)-6-(methylthio)phenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-5-carboxylate

[0334] In an ice bath, ammonia water (1 mL) was added to a solution of 54c (120 mg, 0.16 mmol) in methanol (5 mL), and after 10 minutes, an aqueous solution of sodium dithionite (140 mg, 0.8 mmol) was added, and the mixture was slowly warmed to room temperature and reacted for 2 hours. The mixture was filtered to remove inorganic salts, and extracted with ethyl acetate. The organic phase was washed with saturated saline, dried over anhydrous magnesium sulfate, and then spin-dried to obtain a crude product, which was purified with a silica gel column (eluent: dichloromethane / methanol = 10 / 1, v / v) to obtain compound 54d (55 mg, yield: 48%).

[0335] MS(ESI)m / z=735.3[M+H]

[0336] Step 5: Synthesis of (E)-methyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1-(4-(2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-5-(methoxycarbonyl)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(methylthio)-1H-benz[d]imidazolyl 5-carboxylate methyl

[0337] In an ice bath, 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (16 mg, 0.08 mmol) was added to a solution of 54d (55 mg, 0.074 mmol) in DMF (5 mL). After reacting for 0.5 hours, DIPEA (20 mg, 0.15 mmol) and HATU (34 mg, 0.09 mmol) were added, and the mixture was then warmed to room temperature and reacted for 3.5 hours. The reaction solution was slowly added to water, and a whitish solid precipitated. The solid was filtered and dried to obtain 54e (50 mg, yield: 75%).

[0338] MS(ESI)m / z=897.0[M+H]

[0339] Step 6: Synthesis of (E)-1-(4-(5-carboxy-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-(methylthio)-1H-benz[d]imidazolyl-5-carboxylic acid

[0340] 54e (50 mg, 0.056 mmol) was dissolved in a mixed solution (3.5 mL) of methanol, tetrahydrofuran and water (volume ratio: 2 / 1 / 0.5), and lithium hydroxide (12 mg, 0.28 mmol) was added. The mixture was heated to 75°C and reacted overnight with stirring. The organic solvent was removed by rotary evaporation, cooled to room temperature, and adjusted with dilute hydrochloric acid (1 M) in an ice bath until no solid precipitated, filtered, and the solid was dried to obtain compound 54f (38 mg, yield: 78%).

[0341] MS(ESI)m / z=867.0[M+H]

[0342] Step 7: Synthesis of (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(methylthio)-1H-benz[d]imidazolyl-5-carboxamide

[0343] 54f (38 mg, 0.043 mmol) was dissolved in DMF (5 mL), and HATU (49 mg, 0.129 mmol) and DIPEA (30 mg, 0.215 mmol) were added. After 0.5 hours, ammonium bicarbonate (16 mg, 0.131 mmol) was added and stirred at room temperature for 2 hours. The reaction solution was concentrated and then purified by preparative HPLC to obtain compound 54 (18 mg, yield: 47%) as a white solid.

[0344] MS(ESI)m / z=866.3[M+H] 1 H NMR(400MHz,DMSO)δ12.98(s,2H),9.79(s,1H),8.06(s,1H),7.95(s,1H),7.87(d,J=1.3Hz,1H),7.66(s,1H),7.63 (d,J=1.3Hz,1H),7.43(s,1H),7.37(s,1H),7.29(s,1H),6.52(s,1H),6.51(s,1H),5.78-5.61(m,2H),5.12(s,2H) ,4.90(s,2H),4.52(p,J=7.3Hz,4H),4.00(t,J=5.6Hz,2H),3.94(d,J=12.4Hz,2H),3.26(d,J=11.8Hz,3H),3.11(s ,3H),2.97(s,3H),2.43(d,J=8.4Hz,3H),2.12(s,3H),2.10(s,3H),1.96-1.81(m,2H),1.28(td,J=7.1,4.1Hz,6H). Example 55 [ka]

[0345] Step 1: Synthesis of (E)-1-(4-((2-(3-(((tert-butyldimethylsilyl)oxy)propoxy)-4-carbamoyl-6-nitrophenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benz[d]imidazolyl-5-carboxamide

[0346] Compound 1e (3.0 g, 7.3 mmol), 3-(3-((tert-butyldimethylsilyl)oxy)propoxy)-4-chloro-5-nitrobenzamide (2.84 g, 7.3 mmol) and DIPEA (4.7 g, 36.5 mmol) were mixed and dissolved in n-butanol (60 mL), the reaction mixture was heated to 120 ° C. and stirred at the same temperature for 48 hours, cooled to room temperature, diluted with water, extracted with ethyl acetate (30 mL × 3), the organic phase was combined, washed with saturated saline, dried with anhydrous sodium sulfate, filtered, and the crude product obtained by removing the solvent by rotary evaporation was separated by reverse phase column chromatography (eluent: acetonitrile / water = 50 / 50, v / v) to obtain compound 55a (4.2 g, yield: 75%).

[0347] MS(ESI)m / z=764.3[M+H] + .

[0348] Step 2: Synthesis of (E)-1-(4-((2-amino-6-(3-((tert-butyldimethylsilyl)oxy)propoxy)-4-carbamoylphenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benz[d]imidazolyl-5-carboxamide

[0349] In an ice bath, ammonia water (5.5 mL) was added dropwise to a solution of compound 55a (4.2 g, 5.5 mmol) in methanol (40 mL), and the reaction mixture was stirred at 0° C. for 10 min, followed by slow addition of an aqueous solution (10 mL) of sodium dithionite (4.8 g, 27.5 mmol). The reaction mixture was slowly warmed to room temperature and continued to stir for 3 h. The inorganic salts were removed by filtration, and the filtrate was spin-dried to obtain a crude product, which was separated by reverse phase column chromatography (eluent: acetonitrile / water=60 / 40, v / v) to obtain compound 55b (2.8 g, yield: 69%) as a white solid.

[0350] MS(ESI)m / z=734.3[M+H] + .

[0351] Step 3: Synthesis of (E)-N-(7-(3-((tert-butyldimethylsilyl)oxy)propoxy)-5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide

[0352] In an ice bath, 4-ethyl-2-methylthiazole-5-carbonyl isothiocyanate (805 mg, 3.8 mmol) was added to compound 55b (2.8 g, 3.8 mmol) in DMF (40 mL), and after 20 minutes, HATU (1.59 g, 4.18 mmol) and DIPEA (980 mg, 7.6 mmol) were added in sequence, and the mixture was stirred at room temperature for 2 hours. After the reaction was completed, the mixture was poured into water, and a solid precipitated. The solid was collected by filtration and dried to obtain compound 55c (2.5 g, yield: 71%).

[0353] MS(ESI)m / z=912.4[M+H] +

[0354] Step 4: Synthesis of (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7)-methoxy-1H-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(3-hydroxypropoxy)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide

[0355] Compound 55c (2.5 g, 2.7 mmol) in THF (10 mL) was added with tetrabutylammonium fluoride (8.1 mL, 1 M tetrahydrofuran solution) and reacted at room temperature for 4 hours with stirring. The solvent was removed by rotary evaporation, and the crude product was separated by reverse phase column chromatography (eluent: acetonitrile / water = 40 / 60, v / v) to obtain compound 55 (1.6 g, yield: 74%).

[0356] MS(ESI)m / z=798.3[M+H] + Example 56 [ka]

[0357] In an ice bath, triethylamine (36 mg, 0.36 mmol) and methanesulfonyl chloride (14 mg, 0.13 mmol) were added to a solution of compound 55 (100 mg, 0.12 mmol) in tetrahydrofuran (5 mL). The mixture was warmed to room temperature and reacted for 2 hours, at which point LCMS showed the reaction was complete. Potassium carbonate (50 mg, 0.36 mmol) and 8-oxa-3-azabicyclo[3.2.1]octane (68 mg, 0.6 mmol) were added to the reaction mixture, which was warmed to 50° C. and reacted for 2 hours, after which the solvent was removed by rotary evaporation. The crude product was purified by preparative HPLC to give compound 56 (15 mg).

[0358] According to the preparation method of compound 56, compounds 57, 58, 59, 60, 61, 62, 63, and 64 were obtained from compound 55 and the corresponding amine as raw materials. The structures and properties of the compounds are shown in Table 5 below.

[0359] [Table 5-1] [Table 5-2] Example 65 [ka]

[0360] Step 1: Synthesis of 4-amino-2-fluoro-5-methoxybenzamide

[0361] At room temperature, 10% palladium carbon (1.5 g) was added to a solution of 2-fluoro-5-methoxy-4-nitrobenzamide (10 g, 46.7 mmol) in methanol (100 mL), and the hydrogenation reaction was carried out at room temperature for 5 hours. The palladium carbon was removed by filtration, and the filtrate was spin-dried to obtain compound 65a (7.7 g, yield: 89%).

[0362] MS(ESI)m / z=185[M+H] +

[0363] Step 2: Synthesis of tert-butyl (E)-(4-((4-carbamoyl-5-fluoro-2-methoxyphenyl)amino)but-2-en-1-yl)carbamate

[0364] Compound 65a (7.5 g, 40.7 mmol) was dissolved in DMF (150 mL), and potassium carbonate (8.4 g, 61 mmol) and (E)-(4-bromobut-2-en-1-yl)carbamate tert-butyl (10.1 g, 40.7 mmol) were added in that order. The mixture was heated to 60°C and reacted for 8 hours. After cooling, the mixture was filtered to remove inorganic salts, the filtrate was spin-dried, dissolved in ethyl acetate, washed with water and saturated saline, and the organic phase was spin-dried. The mixture was then purified with a silica gel column (petroleum ether / ethyl acetate = 1 / 2) to obtain compound 65b (5.2 g, yield 36%).

[0365] MS(ESI)m / z=354[M+H] +

[0366] Step 3: Synthesis of tert-butyl (E)-(4-((4-carbamoyl-5-fluoro-2-methoxy-6-nitrophenyl)amino)but-2-en-1-yl)carbamate

[0367] Compound 65b (5.2 g, 14.7 mmol) was dissolved in acetic anhydride (30 mL) and cooled to 0° C. in an ice bath. Nitric acid (1.7 g, 17.6 mmol, 65% purity) was then added dropwise to the reaction solution, and the mixture was reacted for 2 hours while maintaining the temperature at 0° C. The reaction solution was added to water, extracted with ethyl acetate, washed with saturated saline, and the organic phase was concentrated. The mixture was then purified with a silica gel column (petroleum ether / ethyl acetate=1 / 1) to obtain compound 65c (2 g, yield 34%).

[0368] MS(ESI)m / z=399[M+H] +

[0369] Step 4: Synthesis of tert-butyl (E)-(4-((4-carbamoyl-5-fluoro-2-methoxy-6-aminophenyl)amino)but-2-en-1-yl)carbamate

[0370] In an ice bath, ammonia water (5 mL) was added to a solution of 65c (2 g, 5 mmol) in methanol (20 mL), and after 10 minutes, an aqueous solution of sodium dithionite (4.3 g, 25 mmol) was added and reacted for 2 hours. The mixture was filtered to remove inorganic salts, and ethyl acetate was added for extraction. The organic phase was washed with saturated saline, dried over anhydrous magnesium sulfate, and then spin-dried to obtain a crude product, which was purified with a silica gel column (eluent: dichloromethane / methanol = 50 / 1-30 / 1) to obtain compound 65d (1.1 g, yield: 64%).

[0371] MS(ESI)m / z=369[M+H] +

[0372] Step 5: Synthesis of tert-butyl (E)-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-4-fluoro-7-methoxy-1H-benz[d]imidazolyl-1-yl)-2-en-1-yl)carbamate

[0373] In an ice bath, 1-ethyl-3-methyl-1H-pyrazole-5-carbonyl isothiocyanate (565 mg, 2.9 mmol) was added to a solution of 65d (1.1 g, 2.9 mmol) in DMF (20 mL). After reacting for 0.5 hours, DIPEA (748 mg, 5.8 mmol) and HATU (1.3 g, 3.48 mmol) were added, and the mixture was then warmed to room temperature and reacted for 12 hours. The reaction solution was slowly poured into water, and a yellow solid precipitated. The solid was filtered and dried to obtain 65e (1.1 g, yield: 75%).

[0374] MS(ESI)m / z=530[M+H] +

[0375] Step 6: Synthesis of (E)-1-(4-aminobut-2-en-1-yl)-2-(4-ethyl-2-methylthiazole-5-carboxamido)-7-methoxy-1H-benz[d]imidazolyl-5-carboxylic acid methyl

[0376] In an ice bath, TFA (5 mL) was added to 65e (1.1 g, 2.1 mmol) in DCM (10 mL), and then the mixture was allowed to warm to room temperature and react for 2 hours. The solvent was removed by rotary evaporation under reduced pressure, and ethyl acetate was added, followed by spin drying under reduced pressure to remove free TFA, yielding compound 65f (1.5 g, containing trifluoroacetic acid).

[0377] MS(ESI)m / z=430[M+H] +

[0378] Step 7: Synthesis of (E)-1-(4-aminobut-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-4-fluoro-7-methoxy-1H-benz[d]imidazolyl-5-carboxamide

[0379] 65f (1.5g, 2.1mmol, 57% purity) was dispersed in n-butanol (10mL), and 4-chloro-3-(3-morpholinopropoxy)-5-nitrobenzamide (720mg, 2.1mmol) and DIPEA (1.35g, 10.5mmol) were added. The reaction mixture was heated to 120°C and stirred for 18 hours. The crude product obtained by distillation under reduced pressure was dissolved in ethyl acetate, washed with water and saturated saline, dried over anhydrous sodium sulfate, spin-dried, and purified with a silica gel column (eluent: dichloromethane / methanol = 30 / 1) to obtain 65g (770mg, yield: 49%).

[0380] MS(ESI)m / z=737.[M+H] +

[0381] Step 8: Synthesis of (E)-1-(4-(((2-amino-4-carbamoyl-6-(3-morpholinopropoxy)phenyl)amino)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-4-fluoro-7-methoxy-1H-benz[d]imidazolyl-5-carboxamide

[0382] In an ice bath, ammonia water (1 mL) was added to a solution of 65g (500 mg, 0.68 mmol) in methanol (10 mL), and after 10 minutes, an aqueous solution of sodium dithionite (591 mg, 3.4 mmol) was added, and the mixture was slowly warmed to room temperature and reacted for 2 hours. The mixture was filtered to remove inorganic salts, and the filtrate was concentrated to obtain the crude product, which was then purified by reverse phase column chromatography (eluent: acetonitrile / water = 60 / 40) to obtain compound 65h (200 mg, yield: 41%).

[0383] MS(ESI)m / z=707[M+H] +

[0384] Step 9: Synthesis of (E)-N-(5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-4-fluoro-7-methoxy-1H)-benz[d]imidazolyl-1-yl)but-2-en-1-yl)-7-(3-morpholinopropoxy)-1H-benz[d]imidazolyl-2-yl)-4-ethyl-2-methylthiazole-5-carboxamide

[0385] In an ice bath, 4-ethyl-2-methylthiazole-5-carbonyl isothiocyanate (30 mg, 0.14 mmol) was added to a solution of 65h (100 mg, 0.14 mmol) in DMF (3 mL). After reacting for 0.5 hours, DIPEA (54 mg, 0.42 mmol) and HATU (53 mg, 0.14 mmol) were added, and the mixture was then warmed to room temperature and reacted for 2 hours. The reaction solution was purified by preparative HPLC to obtain 65 (20 mg, yield: 16%).

[0386] MS(ESI)m / z=885[M+H] +

[0387] The beneficial effects of the present invention will now be described through experimental examples. Experimental Example 1: Measurement of the binding affinity of the compound according to the present invention to STING protein

[0388] (1) Experimental method The binding affinity of compounds to STING protein was measured by protein thermal shift assay (TSA). 100ug / ml STING protein was mixed with different concentrations of compounds and 5X SYPRO orange dye in a buffer solution of 20mM Hepes, 150mM NaCl, 1mM MgCl2, 1mM DTT, pH=7.5, and the protein melting curve was measured using a qPCR instrument. The Tm values ​​were fitted with Protein Thermal Shift Software 1.3 software, and the Tm difference of the protein with and without different concentrations of compounds was calculated, and the binding affinity of the compound with and without compound was measured. ΔThe dissociation constant Kd was obtained by fitting according to the change in Tm. Compound 3 reported in the literature (Nature. 2018 Dec, 564 (7736): 439-443) was used as a positive reference compound. The test results are shown in Table 6, and the Kd value of each compound is classified according to the following description. "+" indicates a Kd value greater than 10 μM. "++" indicates a Kd value less than 10 μM and greater than 1 μM. "+++" indicates a Kd value less than 1 μM. A lower Kd indicates a stronger binding affinity of the compound to the STING protein.

[0389] (2) Experimental results [Table 6]

[0390] The above experimental results show that the compounds of the present invention have good binding ability with STING protein, which is comparable to the in vitro affinity of the reference compounds, and therefore the compounds of the present invention can be used as effective STING protein regulators. Experimental Example 2. Measurement of the agonist function of the compound according to the present invention against STING protein

[0391] (1) Experimental method In this experiment, the function of sting agonists was evaluated by detecting the changes in IFN-β and CXCL10 (IP10) cytokines produced by THP1 cells (Shanghai Cell Bank), a human peripheral blood monocytic cell line, upon stimulation with compounds. On the first day of the experiment, ELISA plates were coated according to the instructions of the IFN-β (R&D, #DY814-05) and IP10 (BD, #550926) ELISA test kits. Compounds were dissolved in DMSO to make stock solutions, and then diluted to 2X working concentrations with medium, added to a 96-well plate at 100 μL per well, and THP1 cells in the logarithmic growth phase were counted and 2×10 6The cells were diluted to a concentration of 100 μL / mL and added to the 96-well plate containing the above compounds at 100 μL per well, mixed well, and cultured in an incubator at 37°C and 5% CO2 for 18 hours. The next day, 100 μL of the above cell culture supernatant was taken per well and detected according to the IFN-β and IP10 ELISA test kits, respectively, the OD450 values ​​were read and converted to the concentrations of IFN-β and IP10 according to the standard curves, and the dose-response curves were fitted by GraphPad 5.0 to obtain the EC 50 The value was calculated. EC 50 is the concentration for 50% of maximal effect (EC50), which is the drug concentration that induces an effect in 50% of individuals. As a positive reference compound, compound 3 reported in the literature (Nature. 2018 Dec; 564 (7736): 439-443) was used.

[0392] (2) Experimental results The experimental results are shown in Table 7 below. The EC 50 Values ​​are classified according to the following descriptions: "+" stands for EC 50 Values ​​above 1 μM are indicated. "++" stands for EC 50 Values ​​below 1 μM and above 100 nM are indicated. "+++" stands for EC 50 Values ​​less than 100 nM and greater than 10 nM are indicated. "++++" stands for EC 50 Values ​​below 10 nM are indicated.

[0393] [Table 7]

[0394] The above experimental data show that the compounds of the present invention have good activity in stimulating THP1 cells to produce IFN-β and CXCL10 (IP10) cytokines, and have excellent STING protein agonist function. In particular, compared with the reference compounds, the compounds of the present invention, such as compounds 2, 4, 5, 15, 19, 23, 35, 40, 49, 50, 51, 52, and 53, have the same or higher activity in stimulating immune cells to produce cytokines. Experimental Example 3. IFN-β induction experiment in mice

[0395] (1) Experimental method 5 × 10 on the back of Balb / C mice 5 CT26 cells were inoculated subcutaneously. After inoculation, the tumor volume was 200-300 mm 3 Tumor-bearing mice were selected from the 100-mL tumor-bearing mice in the 100-mL tumor-bearing mice range and were randomly divided into 21 groups, 3 mice per group, according to the random method of tumor volume and segment. Different doses of the drug (compound of the present invention) were injected through the tail vein. However, the control group was administered with the control solvent (5% DMSO, 40% PEG400 prepared in saline). Three hours after administration, blood was collected from the mouse orbital vein, and EDTA·2K was used as an anticoagulant. The collected mouse whole blood was centrifuged at 8000g for 5 minutes, and the upper layer of plasma was collected. Three hours after administration, mouse tumor tissues were collected, weighed, and homogenized by adding 9 times the volume of PBS. The homogenate was centrifuged at 10000 rpm for 15 minutes, and the supernatant was collected and prepared for detection. Each was detected according to the IFN-β test kit (R&D, #DY814-05), and the OD450 value was read and converted to the concentration of IFN-β according to the calibration curve.

[0396] (2) Experimental results The experimental results show that tail vein administration in mice can induce the release of IFN-β downstream of the STING pathway in blood and tumors. After administration of the compound of the present invention, the IFN-β level in the tumor is more than three times higher than that in plasma, indicating that the cytokines are relatively concentrated in tumor tissues and less cytokines in the blood, suggesting a higher therapeutic window. Example 4. Tumor-inhibiting effect of the compound according to the present invention (CT26 tumor model)

[0397] (1) Experimental method After resting the Balb / c mice for 1 week, each mouse was cultured with 5 × 10 5 CT26 cells were inoculated subcutaneously into the back of the mice, with 100 μL of cell suspension containing 10 cells. The average tumor volume was 120 mm. 3 When the tumors grew to about 100% in size, they were randomly assigned to groups of 5 mice per group according to the volume size of the tumor. On days 1, 4, and 8 after grouping, the mice in each group were intravenously administered the drug via the tail vein. However, the control group was administered a control solvent (5% DMSO, 40% PEG400 prepared in saline). On the day of grouping, three times a week after the first administration, and before euthanasia, the long and short diameters of the tumors were measured with a Vernier caliper, the tumor volume was calculated, and a tumor growth curve was drawn based on the tumor volume. The tumor volume was calculated according to the following formula: V = 1 / 2 × long diameter × short diameter × short diameter.

[0398] (2) Experimental results The experimental results are shown in Figure 1, and the compound prepared in the embodiment of the present invention can effectively inhibit tumor growth after 20 days of administration, and can show a dose-dependent tumor growth inhibition effect. Example 5. Tumor Inhibitory Effect of the Compound of the Present Invention (H22 Tumor Model)

[0399] (1) Experimental method After the Kunming mice were allowed to rest for 1 week, each mouse was then injected with 5 × 10 6 H22 cells were inoculated subcutaneously into the back of the mice, with 100 μL of cell suspension containing 10 cells per mouse. The average tumor volume was 200 mm. 3When the tumors grew to a certain extent, they were randomly divided into groups, 7 mice per group, according to the volume size of the tumor. On the 1st, 4th, and 8th days after grouping, the mice in the high, medium, and low dose groups were intravenously administered (the compound of the present invention) via the tail vein, respectively, or on the 1st, 8th, and 15th days after grouping, the mice in the high dose group were intravenously administered via the tail vein. However, the control group was administered a control solvent (5% DMSO, 40% PEG400 prepared in saline). On the day of grouping, 3 times a week after the first administration, before euthanasia, the long and short diameters of the tumors were measured with a Vernier caliper, the tumor volume was calculated, and the tumor growth curve was drawn based on the tumor volume. The tumor volume was calculated according to the following formula: V=1 / 2×long diameter×short diameter×short diameter.

[0400] (2) Experimental results The compound of the present invention can effectively inhibit tumor growth after 30 days of administration, and can also show a dose-dependent tumor growth inhibitory effect. Example 6. Tumor-inhibiting effect of the compound according to the present invention (tumor restimulation model)

[0401] (1) Experimental method The mice in each group with tumor regression in the CT26 antitumor effect model of Experimental Example 4 were continuously observed. Thirty days after the last administration, each mouse was administered 5×10 5 CT26 cells were then subcutaneously inoculated into the back of each mouse, with 100uL of cell suspension containing 100 cells. Mice that were not treated with drugs were used as a control group and were inoculated with CT26 cells in the same manner. Tumors were observed and tumor growth was recorded.

[0402] (2) Experimental results The experimental results are shown in Figure 2, in which the control group was able to grow CT26 tumors after inoculation, whereas mice whose tumors had completely regressed after treatment with the compounds of the present invention were unable to grow tumors again after inoculation, indicating that these compounds activated the immune memory mechanisms in the mice, rendering them immune to the reinoculated tumor cells.

[0403] Thus, the present invention discloses compounds of formula (I), which can effectively bind to STING, have good STING protein agonist function, show good inhibitory effects on various tumors, and can also activate immune memory mechanisms in mice to inhibit tumor restimulation. Therefore, the compounds of the present invention can be used as STING agonists to treat various related diseases. The compounds of the present invention have very good application prospects in the manufacture of medicines for treating diseases related to STING activity (especially medicines for treating inflammatory diseases, allergic diseases, autoimmune diseases, infectious diseases, cancer or precancerous syndromes) and the manufacture of immune adjuvants, providing new options in clinical screening and / or manufacturing of medicines for diseases related to STING activity in clinical practice.

Claims

【Request 1】 【Chemical 7-1】 【Chemistry 7-2】 【Chemistry 7-3】 【Chemistry 7-4】 【Chemistry 7-5】 、 or a pharma- ceutically acceptable salt thereof.

2. A pharmaceutical composition comprising as an active ingredient the compound described in claim 1 or a pharma- ceutically acceptable salt thereof, for use in treating a disease associated with STING activity, wherein the disease associated with STING activity is any one of diseases associated with inflammation, autoimmune disease, infectious disease, cancer, precancerous syndrome, or a combination thereof.

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