Benzimidazo 2-amino-1, 3, 4-thiadiazole carboxylate derivative and preparation method and medicinal application thereof

Abstract

The invention belongs to the technical field of medicines, and relates to a benzimidazo 2-amino-1, 3, 4-thiadiazole carboxylic ester derivative shown as a formula I, a pharmaceutically acceptable salt and a preparation method thereof, a composition containing one or more compounds, and application of the compounds as an STAT3 signaling pathway inhibitor in treatment of STAT3 high-expression cancer diseases, and particularly relates to a preparation method of the benzimidazo 2-amino-1, 3, 4-thiadiazole carboxylic ester derivative shown as the formula I. The invention also relates to a preparation method of the benzimidazo 2-amino-1, 3, 4-thiadiazole carboxylic ester derivative.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to compounds that inhibit human tumor cells and are used in tumor immunotherapy. This research also relates to the synthesis of these compounds and their use as effective drug candidates for the clinical treatment of cancer patients. More specifically, this invention relates to certain benzimidazolo-2-amino-1,3,4-thiadiazole carboxylic acid derivatives that inhibit STAT3-overexpressing tumor cells, and their use in the preparation of drugs for the prevention, relief, and / or treatment of solid tumors. Background Technology

[0002] Signal transducer and activator of transcription 3 (STAT3) is a crucial regulator of immune responses. The Tyr705 domain in the Sarcoma (Src) homology 2 (SH2) domain of the STAT3 protein can be phosphorylated and activated by Janus kinase, which is essential for STAT3 activation. Recent studies have shown that STAT3 is closely related to the occurrence and development of cancer. STAT3 in various tissues and cells can be activated by various cytokines and growth factors, participating in immune responses, regulating cell proliferation and apoptosis, and inducing carcinogenesis in normal tissues. Abnormal activation of STAT3 promotes cancer cell proliferation, angiogenesis, and immune evasion, accelerating cancer progression (Int. J. Mol. Sci., 2018, 19, 159).

[0003] STAT3 is closely related to the survival, proliferation, invasion, metastasis, angiogenesis, and immune evasion of tumor cells (Cell Prolif., 2021, 54:e12974). Aberrant activation of STAT3 often occurs in various solid tumors and hematologic malignancies. Aberrant expression of STAT3 has been observed in breast cancer, ovarian cancer, melanoma, prostate cancer, pancreatic cancer, multiple myeloma, colorectal cancer, and hematopoietic system cancers. Therefore, inhibiting STAT3 can block the occurrence of cancer in its early stages (Eur. J. Med. Chem., 2020 (187), 111922).

[0004] Currently, the development of STAT3 inhibitors mainly focuses on the SH2 domain (Clin. Oncol., 2018, 15(4), 234-248). Several STAT3 inhibitors have entered clinical trials, but only the small molecule inhibitor Napabucasin (BBI-608), which targets the STAT3 signaling pathway, has been approved for marketing (Expert Opin. Investig. Drugs, 2021, 30(3), 245-251). Napabucasin was granted orphan drug designation by the FDA in 2016 for the treatment of tumors at the gastric and esophageal junction and pancreatic cancer. Napabucasin inhibits STAT3 activity by directly targeting and binding to the SH2 domain of STAT3. Clinical trial data on its use as a monotherapy and in combination with chemotherapy drugs for the treatment of tumors have shown that it has good anti-tumor effects and prolongs the overall survival and progression-free survival of cancer patients (Lancet., 2018, 3, 263-27). However, in subsequent clinical trials, the phase III trial results (NCT02178956) of the combination therapy of Napabucasin and paclitaxel for the treatment of tumors at the gastric and esophageal junction did not show a significant improvement in overall survival or progression-free survival. Currently, the search for and research of novel small-molecule inhibitors targeting STAT3 is urgently needed in the field of cancer treatment.

[0005] In addition, TTI-101, which targets the SH2 domain, and AZD9150, an antisense oligonucleotide of STAT3, are currently in clinical trials for the treatment of colorectal cancer (Biom.J., 2021, 9, 1016). However, although STAT3 inhibitors have shown significant inhibitory activity in preclinical studies, they have not yet been used in clinical treatment due to a lack of selectivity and poor bioavailability.

[0006] This patent designs a series of small molecule inhibitors targeting the STAT3 protein signaling pathway. By introducing carboxylic acid ester groups, its physicochemical parameters and pharmacokinetics are improved, with the aim of obtaining novel, highly efficient, low-toxic, physicochemically suitable, and highly specific STAT3-targeting compounds.

[0007] The benzimidazolo-2-amino-1,3,4-thiadiazole carboxylic acid derivatives of the present invention are all novel compounds, and there are no reports on their application in the prevention and treatment of cancer and its complications. Summary of the Invention

[0008] One aspect of the present invention is a compound having the following structure (general formula I) and various intermediates and byproducts involved in its synthesis, including optical isomers, pharmaceutically acceptable salts, or solvates:

[0009]

[0010] In the formula:

[0011] Linker is a straight or branched chain, either substituted or unsubstituted. 1-20 Alkyl, substituted or unsubstituted C 1-20 Cycloalkyl, substituted or unsubstituted C atoms other than carbon atoms 1-20 Heteroalkyl, substituted or unsubstituted aza- or oxa- or thia- or phospha-C 1-20 Cycloalkyl, substituted or unsubstituted compounds containing multiple non-carbon Cs 1-20 Cycloalkyl, substituted or unsubstituted C 1-20 Alkyl, substituted and unsubstituted aryl and heteroaryl groups; wherein the alkyl, cycloalkyl, heteroalkyl, cyclohexaalkyl, and aryl groups may be unsubstituted or substituted with one or more groups selected from the following, including C 1-20 Alkyl, C 1-20 Alkoxy, straight-chain or branched substituted and unsubstituted amino groups, C 1-20 Cyclic amino, halogen, cyano, C 1-20 Phenoxy, trifluoromethyl, trifluoromethoxy, carboxyl, nitro, C 1-20 Ester group, hydroxyl group, C 1-20 amide group, C 1-20 heteroaryl, C 4-24 Triazolyl, trifluoromethylphenyloxy, C 1-20 thioalkyl, C 1-20 sulfonamide, phenyl, morpholino, C 1-20 alkynyl group, C 1-24 Cycloalkoxy, C 1-24 alkeneoxy, hydrazine, C 1-20 Acylhydrazide group, C 5-24 Aromatic heterocyclic, ester group, aromatic cyclic group, fused heterocyclic group, furanyl, pyrazolyl, imidazoleyl, pyridinyl, pyridinyl, pyrimidinyl, pyrroleyl, thiophenyl, pyrazinyl, triazinyl, fused heterocyclic group, benzofuranyl, benzothiophenyl, benzopyrroleyl, benzopyranyl, purinyl, carbazoleyl;

[0012] R is hydrogen, C is... 1-6 Alkyl, C 1-6 Alkoxy-substituted C 1-6 Alkyl, alkyl-substituted C 1-6 Alkyl and C 3-6 Cycloalkyl.

[0013] Preferred

[0014] Linker is a straight or branched chain, either substituted or unsubstituted. 1-15 Alkyl, substituted or unsubstituted C 1-15 Cycloalkyl, substituted or unsubstituted C atoms other than carbon atoms 1-15 Heteroalkyl, substituted or unsubstituted aza- or oxa- or thia- or phospha-C1-15 Cycloalkyl, substituted or unsubstituted compounds containing multiple non-carbon Cs 1-15 Cycloalkyl, substituted or unsubstituted C 1-15 Alkyl, substituted or unsubstituted aryl; wherein the alkyl, cycloalkyl, heteroalkyl, cyclohexaalkyl, and aryl groups may be unsubstituted or substituted by one or more groups selected from the following, including C 1-15 Alkyl, C 1-15 Alkoxy, straight-chain or branched substituted and unsubstituted amino groups, C 1-15 Cyclic amino, halogen, cyano, C 1-15 Phenoxy, trifluoromethyl, trifluoromethoxy, carboxyl, nitro, C 1-15 Ester group, hydroxyl group, C 1-15 amide group, C 1-15 heteroaryl, C 4-12 Triazolyl, trifluoromethylphenyloxy, C 1-15 thioalkyl, C 1-15 sulfonamide, phenyl, morpholino, C 1-15 alkynyl group, C 1-12 Cycloalkoxy, C 1-12 alkeneoxy, hydrazine, C 1-15 Acylhydrazide group, C 5-12 Aromatic heterocyclic, ester group, aromatic cyclic group, fused heterocyclic group, furanyl, pyrazolyl, imidazoleyl, pyridinyl, pyridinyl, pyrimidinyl, pyrroleyl, thiophenyl, pyrazinyl, triazinyl, fused heterocyclic group, benzofuranyl, benzothiophenyl, benzopyrroleyl, benzopyranyl, purinyl, carbazoleyl;

[0015] R is hydrogen, C is... 1-6 Alkyl, alkyl-substituted C 1-6 Alkyl or C 3-6 Cycloalkyl.

[0016] More preferred

[0017] Linker is a straight or branched chain, either substituted or unsubstituted. 1-12 Alkyl, substituted or unsubstituted C 1-12 Cycloalkyl, substituted or unsubstituted C atoms other than carbon atoms 1-12 Heteroalkyl, substituted or unsubstituted aza- or oxa- or thia- or phospha-C 1-12 Cycloalkyl, substituted or unsubstituted compounds containing multiple non-carbon Cs 1-12 Cycloalkyl, substituted and unsubstituted C 1-12 Alkyl, substituted or unsubstituted aryl; wherein the alkyl, cycloalkyl, heteroalkyl, cyclohexaalkyl, and aryl groups may be unsubstituted or substituted by one or more groups selected from the following, including C 1-12 Alkyl, C 1-12Alkoxy, straight-chain or branched substituted and unsubstituted amino groups, C 1-12 Cyclic amino, halogen, cyano, C 1-12 Phenoxy, trifluoromethyl, trifluoromethoxy, carboxyl, nitro, C 1-12 Ester group, hydroxyl group, C 1-12 amide group, C 1-12 heteroaryl, C 4-12 Triazolyl, trifluoromethylphenyloxy, C 1-12 thioalkyl, C 1-12 sulfonamide, phenyl, morpholino, C 1-12 alkynyl group, C 1-12 Cycloalkoxy, C 1-12 alkeneoxy, hydrazine, C 1-12 Acylhydrazide group, C 5-12 Aromatic heterocyclic, ester group, aromatic cyclic group, fused heterocyclic group, furanyl, pyrazolyl, imidazoleyl, pyridinyl, pyridinyl, pyrimidinyl, pyrroleyl, thiophenyl, pyrazinyl, triazinyl, fused heterocyclic group, benzofuranyl, benzothiophenyl, benzopyrroleyl, benzopyranyl, purinyl, carbazoleyl.

[0018] R is hydrogen, C is... 1-6 alkyl.

[0019] More preferred

[0020] Linker is a straight or branched chain, either substituted or unsubstituted. 1-9 Alkyl, substituted or unsubstituted C 1-9 Cycloalkyl, substituted or unsubstituted C atoms other than carbon atoms 1-9 Heteroalkyl, substituted or unsubstituted aza- or oxa- or thia- or phospha-C 1-9 Cycloalkyl, substituted or unsubstituted compounds containing multiple non-carbon Cs 1-9 Cycloalkyl, substituted or unsubstituted C 1-9 Alkyl, substituted or unsubstituted aryl; wherein the alkyl, cycloalkyl, heteroalkyl, cyclohexaalkyl, and aryl groups may be unsubstituted or substituted by one or more groups selected from the following, including C 1-9 Alkyl, C 1-9 Alkoxy, straight-chain or branched substituted and unsubstituted amino groups, C 1-9 Cyclic amino, halogen, cyano, C 1-9 Phenoxy, trifluoromethyl, trifluoromethoxy, carboxyl, nitro, C 1-9 Ester group, hydroxyl group, C 1-9 amide group, C 1-9 heteroaryl, C 4-12 Triazolyl, trifluoromethylphenyloxy, C 1-9 thioalkyl, C 1-9 sulfonamide, phenyl, morpholino, C 1-9 alkynyl group, C1-12 Cycloalkoxy, C 1-12 alkeneoxy, hydrazine, C 1-9 Acylhydrazide group, C 5-12 Aromatic heterocyclic, ester group, aromatic cyclic group, fused heterocyclic group, furanyl, pyrazolyl, imidazoleyl, pyridinyl, pyridinyl, pyrimidinyl, pyrroleyl, thiophenyl, pyrazinyl, triazinyl, fused heterocyclic group, benzofuranyl, benzothiophenyl, benzopyrroleyl, benzopyranyl, purinyl, carbazoleyl;

[0021] R represents hydrogen, methyl, ethyl, or propyl.

[0022] In addition, the present invention also relates to the use of a compound of general formula I or a stereoisomer thereof containing a pharmaceutically effective amount, a pharmaceutically acceptable salt thereof, for the preparation of a medicine for the prevention or treatment of tumor-related diseases and for the preparation of a medicine for the prevention or treatment of tumor-related diseases.

[0023] The most preferred compounds include, but are not limited to,

[0024] Compound 1: Methyl 4-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)benzoate

[0025]

[0026] Compound 2: methyl 2-(4-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)phenylacetate

[0027]

[0028] Compound 3: Ethyl 4-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)butyrate

[0029]

[0030] Compound 4: Ethyl 3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)propionate

[0031]

[0032] Compound 5: Methyl 5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)furan-2-carboxylic acid ester

[0033]

[0034] Compound 6: 1-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)cyclopropane-1-carboxylic acid ethyl ester

[0035]

[0036] Compound 7: Methyl 4-(5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)furan-2-yl)benzoate

[0037]

[0038] Compound 8: 2-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)cyclopropane-1-carboxylic acid ethyl ester

[0039]

[0040] Compound 9: Methyl 5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)valerate

[0041]

[0042] Compound 10: Methyl 5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)furan-3-carboxylic acid ester

[0043]

[0044] Compound 11: Methyl 3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)benzoate

[0045]

[0046] Compound 12: Ethyl 6-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)hexanoate

[0047]

[0048] Compound 13: methyl 6-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)nicotinic acid

[0049]

[0050] Compound 14: ethyl 6-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)pyridinecarboxylate

[0051]

[0052] Compound 15: Ethyl 5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)pyridinecarboxylate

[0053]

[0054] Compound 16: Methyl 9-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)nonanoate

[0055]

[0056] Compound 17: 4'-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)-[1,1'-biphenyl]-4-carboxylic acid methyl ester

[0057]

[0058] Compound 18: (E)-Methyl-3-(4-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)methyl phenylacrylate

[0059]

[0060] Compound 19: (1s, 4s)-4-(5-(1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)cyclohexyl-1-carboxylic acid methyl ester

[0061]

[0062] Compound 20: Methyl 3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)-1H-indole-6-carboxylic acid

[0063]

[0064] Compound 21: 2-(3-(5-((1-cyclopropyl-6-(4-ethylpiperazin-1-yl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)-1H-indole-1-yl)ethyl acetate

[0065]

[0066] Compound 22: methyl 4-(2-(5-(1-cyclopropyl-6-(4-ethylpiperazin-1-yl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)phenoxy)methylbenzoate

[0067]

[0068] Compound 23: ethyl 2-(3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)phenylacetate

[0069]

[0070] Compound 24: 7-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)benzofuran-2-carboxylic acid ethyl ester

[0071]

[0072] Compound 25: Methyl 4-(6-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)-2-pyridylbenzoate

[0073]

[0074] Compound 26: ethyl 4-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)pyridinecarboxylate

[0075]

[0076] Compound 27: methyl 2-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)isonicotinic acid

[0077]

[0078] Compound 28: ethyl 5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)pyrazine-2-carboxylate

[0079]

[0080] Compound 29: (E)-3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)methyl acrylate

[0081]

[0082] On the other hand, the present invention also relates to a complete preparation route and method for compounds with the general formula I structure:

[0083]

[0084] In the above formula, Linker and R are defined as above. The synthetic method of the present invention is described in detail below. In the above reaction, compound 1 is commercially available. Compound 1 can be obtained from compound 4, namely the multi-substituted 5-aminobenzimidazole intermediate, by substitution (a, b), reduction (c), and cyclization (d) according to the literature (J.Comb.Chem.2004,6,811-821).

[0085] In step five, reaction (e), the reagents used are thiocarbonyl diimidazole and triethylamine, with a preferred molar ratio to compound 4 of 1:1.2:2.2. The solvent is an aprotic solvent such as dichloromethane, the temperature is 0°C to 1°C, and the reaction time is 1 hour. In step five, reaction (f), hydrazine hydrate is subsequently added to the reaction solution. The molar ratio to compound 4 is 1:2; the temperature is 80°C; and the reaction time is 1 to 24 hours, preferably 3 hours, to obtain compound 5.

[0086] The reagents used in step seven (g) are esters containing formyl groups with the corresponding R substituents, including methyl 4-formylphenylacetate, methyl p-formylphenylacetate, methyl 5-oxovalerate, methyl 5-formylfuran-2-carboxylate, methyl 4-(5-formyl-2-furanyl)benzoate, methyl 2-formylfuran-4-carboxylate, methyl 4-oxobutyrate, methyl 1-formylcyclopropane-1-carboxylate, ethyl 2-formylcyclopropane-1-carboxylate, methyl meta-formylbenzoate, ethyl 7-oxoheptanoate, methyl 6-formyl-2-pyridinecarboxylate, methyl 9-formylnonanoate, methyl 4'-formyl-[1,1'-biphenyl]-4-carboxylate, and methyl p-formylcinnamate. Methyl trans-4-formylcyclohexanecarboxylate, methyl 3-formylindole-6-carboxylate, ethyl 3-formyl-1-indole-ethyl acetate, methyl 4-[(2-formylphenoxy)methyl]-benzoate, methyl meta-formylphenylacetate, methyl 7-formylbenzofuran-2-carboxylate, methyl 5-formylpyridin-2-carboxylate, methyl 4-formylpyridin-2-carboxylate, methyl 2-formyl sulfoxide, methyl 5-formylpyrazine-2-carboxylate, methyl 4-(6-formylpyridin-2-yl)benzoate, methyl 6-formylnicotinate, etc., are prepared using protic solvents such as ethanol, with 5%–8% CH3COOH as the catalyst, at a temperature of 80°C, and for 1 hour. In step 8 (f), the reagent used was ferric chloride hexahydrate, with a molar ratio of 1:2.5 to compound 5; the temperature was 80℃; the reaction time was 6 to 12 hours; and the compound of formula (I) was obtained by column chromatography purification.

[0087] The above reactions are relatively mild, have short reaction times, stable yields, and are highly versatile. Therefore, they are advantageous for synthesizing compound libraries using methods such as combinatorial chemistry. Such methods for synthesizing compound libraries using combinatorial chemistry also fall within the scope of this invention.

[0088] Those skilled in the art can modify the above steps to improve the yield. They can determine the synthetic route based on basic knowledge in the art, such as selecting reactants, solvents, and temperatures. Such modifications or variations are all within the scope of this invention. Yields can also be improved by using various conventional protecting groups to avoid side reactions. These conventional protection methods can be found, for example, in Protecting Groups in Organic Synthesis (T. Greene, the Fourth Edition, John Wiley & Sons, Inc.).

[0089] The use of the general formula of the compounds mentioned above in the preparation of medicaments for the prevention, relief and / or treatment of symptoms caused by cancer, inflammation, and autoimmune diseases, as long as these benzimidazole compounds and their derivatives are used in cancer and its complications, are all within the scope of compound protection of this invention.

[0090] The cancers mentioned specifically refer to breast cancer, prostate cancer, ovarian cancer, liver cancer, stomach cancer, lung cancer, colon cancer, esophageal cancer, leukemia, human glioma, lymphoma, blood cancer, and melanoma.

[0091] In this invention, the prevention, relief, and / or treatment of cancer or symptoms are selected from drugs that inhibit the activity of STAT3, inhibit the expression of STAT3-related genes, and inhibit the proliferation and metastasis of breast cancer, prostate cancer, ovarian cancer, liver cancer, stomach cancer, lung cancer, colon cancer, esophageal cancer, leukemia, human glioma, lymphoma, hematological malignancy, and melanoma.

[0092] This invention is achieved through the following technical solutions: the series of derivatives are prepared through artificial synthesis; the inhibitory activity of these compounds on the IL-6-STAT3 pathway is evaluated using the HEK-BLUE-IL-6 cell model; the inhibitory activity of these compounds on the HCT116 colorectal cancer cell line is evaluated using the MTT assay; the interaction between these compounds and STAT3 protein is evaluated using the surface plasmon resonance assay; the inhibition of intracellular STAT3 protein phosphorylation by these compounds is evaluated using Western blotting; the in vivo antitumor effect of these compounds on mouse MC38 subcutaneous colon cancer xenografts is evaluated using animal experiments; and the cell cycle disruption and apoptosis effects of these compounds on HCT116 cells are evaluated using flow cytometry.

[0093] The benzimidazolo-2-amino-1,3,4-thiadiazole carboxylate derivative of formula I of the present invention, when formulated into any dosage form, has a pharmaceutical effect of preventing, alleviating, and / or treating cancer. Any pharmaceutical preparation containing benzimidazolo-2-amino-1,3,4-thiadiazole carboxylate derivative of formula I as a component, or prepared solely as a benzimidazolo-2-amino-1,3,4-thiadiazole carboxylate derivative of formula I, is also within the scope of protection of the present invention, provided that its packaging, instructions, or other promotional materials state or indicate that it has an effect of treating cancer and its complications. Attached Figure Description

[0094] Figure 1. Kinetic analysis curves of the interaction between STAT3 and the compounds in the examples

[0095] Figure 2. Immunoblotting assay to detect the inhibition of STAT3Y705 phosphorylation in HCT116 cells by the compounds in the examples

[0096] Figure 3. The compound in this example inhibits the growth of MC38 subcutaneous xenografts.

[0097] Figure 4. The compound in this example can arrest HCT116 cells in the G2 / M phase.

[0098] Figure 5. Example: Compounds induce apoptosis in HCT116 cells Detailed Implementation

[0099] Example

[0100] The following examples illustrate the present invention further. However, the present invention is not limited to these examples.

[0101] Chemical Experiment Section

[0102] Melting point was determined using an RY-2 melting point apparatus manufactured by Tianjin Analytical Instrument Factory; the temperature was not calibrated. Mass spectrometry was performed using a Thermo Finnigan LCQ-Advantage mass spectrometer. Proton and carbon NMR spectra were performed using a Varian Mercury 400MHz, 500MHz, or 700MHz NMR spectrometer with DMSO-d6 as the solvent. Mass spectrometry was performed using a Thermo exactive-orbitrap mass spectrometer.

[0103] Example 1: Methyl 4-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)benzoate

[0104] Step 1: 1-Cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-trifluoromethoxyphenyl)-5-aminobenzimidazole

[0105]

[0106] Compound 1 (1 mmol, 204.1 mg) was dissolved in 10 mL of tetrahydrofuran. N,N-diisopropylethylamine (DIPEA, 1.2 mmol) was added, followed by the slow addition of 2 mL of tetrahydrofuran solution containing cyclopropylamine (1.1 mmol). The reaction was carried out at room temperature. After the reaction was complete, N,N-diisopropylethylamine (DIPEA, 1.2 mmol) was added, followed by the slow addition of N-ethylpiperazine (1 mmol). The reaction was carried out at room temperature. After the reaction was complete, a saturated ammonium chloride solution was added, and the mixture was filtered. The filter cake was the disubstituted product. The disubstituted intermediate (1 mmol) was dissolved in 5 mL of ethanol and 5 mL of tetrahydrofuran. 4.0 eq of 10% Pd-C and 7.5 eq of HCOONH4 were added with stirring, and the mixture was stirred at room temperature for 30 min. The reaction solution was quickly filtered into 5 mL of tetrahydrofuran solution containing 1.2 mmol of p-fluorobenzaldehyde, and 1 mL of acetic acid was added. The mixture was stirred at room temperature for 5 h. HPLC-MS was performed until the reaction was complete. The mixture was concentrated under reduced pressure, the pH was adjusted to neutral with saturated sodium bicarbonate solution, and extracted with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate for 0.5 h. The mixture was filtered, concentrated, prepared into a granulated powder, and purified by column chromatography, eluting with dichloromethane-ethanol to give a brown solid. Melting point: 204-208℃. ESI-MS (m / z): calcd.for C 23 H 27 F3N5O[M+H] + ,446.21,found 446.28.

[0107] Step 2: 1-Cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-trifluoromethoxyphenyl)-5-aminothioureabenzimidazole

[0108]

[0109] 1 mmol of compound 1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-trifluoromethoxyphenyl)-5-aminobenzimidazole was dissolved in 5 mL of anhydrous dichloromethane. 2.2 mmol of triethylamine was added with stirring, followed by slow dropwise addition of 1 mmol of thiocarbonyldiimidazole under ice bath conditions. The reaction was allowed to proceed at room temperature for 1 h. The mixture was then heated to reflux, and 1 mmol of hydrazine hydrate was added. The mixture was refluxed for 3 h. HPLC-MS was used to monitor the reaction until complete. A large amount of white precipitate formed during reflux. The reaction solution was concentrated under reduced pressure, and after crystallization with dichloromethane and petroleum ether, the solution was slurryed, filtered, and the resulting filter cake was a pure white solid. Melting point: 192-194 °C. ESI-MS (m / z): calcd.for C24 H 29 F3N7S[M+H] + ,520.2028,found520.17.

[0110] Step 3: Methyl 4-(5-(1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethyloxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)benzoate

[0111]

[0112] 0.5 mmol of 1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-trifluoromethoxyphenyl)-5-aminothiourea-benzimidazole was dissolved in 20 mL of anhydrous ethanol. Methyl 4-formylbenzoate (0.5 mmol) and acetic acid (0.5 mL) were added, and the mixture was heated under reflux for 2 hours. FeCl3·6H2O was added to the reaction solution, and the mixture was reacted at 85°C for 12 hours. After the reaction was completed by TLC, saturated NaHCO3 solution was added to neutralize the solution. The mixture was filtered, and the filtrate was purified by column chromatography to give methyl 4-(5-(1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethyloxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)benzoate as a yellow solid. Melting point: 168-170°C. ESI-MS(m / z):calcd.for C 33 H 33 F3N7O3S[M+H] + ,664.2318,found 664.2328. 1 H NMR(400MHz,DMSO-d6)δ9.70(s,1H),8.20(s,1H),8.16(d,J=8.6Hz,2H),8.07(d,J =8.2Hz,2H),7.99(d,J=8.3Hz,2H),7.56(d,J=8.3Hz,2H),7.41(s,1H),3.90(s,3H ),3.82(dq,J=7.0,3.4Hz,1H),3.00(t,J=4.7Hz,4H),2.64(s,4H),2.45(q,J=7.1H z,2H),1.18(p,J=7.2,6.2Hz,2H),1.06(t,J=7.1Hz,3H),0.69(p,J=4.8Hz,2H)ppm.

[0113] Example 2 2-(4-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)phenylacetic acid methyl ester

[0114]

[0115] The procedure is the same as in Example 1, except that in step three, methyl 4-formylphenylacetate is used instead of methyl 4-formylbenzoate to obtain methyl 2-(4-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)phenylacetate, a yellow solid. Melting point: 176-178℃. ESI-HRMS (m / z): [M+H] + calcd.for C 34 H 35 F3N7O3S:678.2474,found:678.2465. 1 H NMR(400MHz,DMSO-d6)δ9.57(s,1H),8.32(s,1H),8.22–8.09(m,2H),7.79( d,J=8.2Hz,2H),7.55(d,J=8.3Hz,2H),7.40(d,J=7.7Hz,3H),3.82(dq,J=6. 9,3.5Hz,1H),3.76(d,J=9.2Hz,2H),3.64(s,2H),3.11(s,6H),2.81(s,3H), 1.19(d,J=7.1Hz,3H), 1.15(d,J=6.3Hz,2H), 0.68(p,J=5.3,4.8Hz,2H)ppm.

[0116] Example 3: Ethyl 4-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)butyrate

[0117]

[0118] The procedure is the same as in Example 1, except that in step three, methyl 5-oxovalerate is used instead of methyl p-formylbenzoate to obtain ethyl 4-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)butyrate, a yellow solid. Melting point: 185-186℃. ESI-HRMS (m / z): [M+H] +calcd.for C 31 H 37 F3N7O3S:644.2631,found:644.2618. 1 HNMR(400MHz,DMSO-d6)δ9.25(s,1H),8.30(s,1H),8.13(d,J=8.5Hz,2H),7.54(d,J=8.3Hz ,2H),7.36(s,1H),4.07(q,J=7.1Hz,2H),3.80(tt,J=7.3,3.9Hz,1H),3.60(s,1H),3.09(s, 7H),2.93(t,J=7.5Hz,2H),2.83(s,2H),2.43(q,J=7.6Hz,2H),1.96(dp,J=22.3,7.3Hz,3H ), 1.20 (s, 2H), 1.18 (d, J = 7.3Hz, 3H), 1.14 (d, J = 6.7Hz, 2H), 0.66 (p, J = 5.4, 4.7Hz, 2H) ppm.

[0119] Example 4: Ethyl 3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)propionate

[0120]

[0121] The procedure is the same as in Example 1, except that in step three, methyl 4-oxobutyrate is used instead of methyl p-formylbenzoate to obtain ethyl 3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)propionate, a yellow solid. Melting point: 79-82℃. ESI-HRMS (m / z): [M+H] + calcd.forC 30 H 35 F3N7O3S:630.2469,found:630.2462. 1H NMR (400MHz, DMSO-d6) δ9.26 (s, 1H), 8.33 (s, 1H), 8.13 (d, J = 8.3Hz, 2H), 7.54 ( d,J=8.3Hz,2H),7.36(s,1H),4.09(q,J=7.1Hz,2H),3.80(tt,J=7.2,4.0Hz,1H ),3.15(dd,J=14.7,7.6Hz,10H),2.78(t,J=7.0Hz,2H),1.23(s,3H),1.21(s,2 H), 1.18 (d, J = 7.1Hz, 3H), 1.14 (d, J = 6.7Hz, 2H), 0.66 (q, J = 5.3, 4.2Hz, 2H) ppm.

[0122] Example 5: Methyl 5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)furan-2-carboxylic acid ester

[0123]

[0124] The procedure is the same as in Example 1, except that in step three, methyl 5-formylfuran-2-carboxylate is used instead of methyl p-formylbenzoate to obtain methyl 5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)furan-2-carboxylate, a yellow solid. Melting point: 134-138℃. ESI-HRMS (m / z): [M+H] + calcd.for C 31 H 31 F3N7O4S:654.2032,found:654.2044. 1 H NMR(400MHz,DMSO-d6)δ9.86(s,1H),8.29(s,1H),8.21–8.12(m,2H),7.57(d ,J=8.3Hz,2H),7.49(dd,J=8.1,3.7Hz,1H),7.41(s,1H),7.25(t,J=3.6Hz,1H ),3.87(s,3H),3.83(dt,J=7.0,3.4Hz,1H),3.11(s,8H),2.74(d,J=35.8Hz, 2H),1.33(t,J=7.1Hz,2H),1.19–1.15(m,3H),0.69(q,J=5.1,4.1Hz,2H)ppm.

[0125] Example 6: Ethyl 1-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)cyclopropane-1-carboxylic acid ester

[0126]

[0127] The procedure is the same as in Example 1, except that in step three, methyl 1-formylcyclopropane-1-carboxylate is used instead of methyl p-formylbenzoate to obtain ethyl 1-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)cyclopropane-1-carboxylate, a yellow solid. Melting point: 94-97℃. ESI-HRMS (m / z): [M+H] + calcd.for C 31 H 35 F3N7O3S:642.2469,found:642.2460. 1 H NMR (400MHz, DMSO-d6) δ9.32(s,1H),8.35(s,1H),8.20–8.10(m,2H),7.56(d,J=8.3Hz,2H),7.39(s,1H),4.15(dq,J=23.2,7.1Hz,2H),3.82(tt,J =7.2,3.9Hz,1H),3.20–3.03(m,6H),2.91(s,2H),1.80–1.69(m,4H),1.2 2(s,3H),1.20(d,J=1.9Hz,2H),1.19–1.13(m,3H),0.72–0.65(m,2H)ppm.

[0128] Example 7 Methyl 4-(5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)furan-2-yl)benzoate

[0129]

[0130] The procedure is the same as in Example 1, except that in step three, methyl 4-(5-formyl-2-furanyl)benzoate is used instead of methyl p-formylbenzoate to obtain methyl 4-(5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)furan-2-yl)benzoate, a white solid. Melting point: 204-207℃. ESI-HRMS(m / z): calcd.for C 37 H 35 F3N7O4S:730.2418,found:730.2411. 1 H NMR (400MHz, DMSO-d6) δ9.77 (s, 1H), 8.41 (s, 1H), 8.17 (d, J = 8.4Hz, 2H), 8. 07(d,J=8.2Hz,2H),7.94(d,J=8.1Hz,2H),7.58(d,J=8.3Hz,2H),7.42(d,J= 3.5Hz,2H),7.28(d,J=3.7Hz,1H),3.89(s,3H),3.87–3.80(m,1H),3.23(s, 5H),1.30(s,3H),1.25(s,2H),1.18(d,J=6.9Hz,3H),0.76–0.66(m,2H)ppm.

[0131] Example 8: Ethyl 2-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)cyclopropane-1-carboxylic acid ester

[0132]

[0133] The procedure is the same as in Example 1, except that in step three, ethyl 2-formylcyclopropane-1-carboxylate is used instead of methyl p-formylbenzoate to obtain ethyl 2-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)cyclopropane-1-carboxylate, a yellow solid. Melting point: 110-113℃. ESI-HRMS (m / z): calcd.for C 31 H 35 F3N7O3S:642.2469,found:642.2463. 1H NMR(400MHz,DMSO-d6)δ8.17–8.09(m,3H),7.64(s,1H),7.53(d,J=8.3Hz,2H),7.35(s,1H), 7.01(s,2H),4.12(q,J=7.1Hz,2H),3.78(tt,J=7.5,4.1Hz,1H),2.94(t,J=4.8Hz,4H),2.83 –2.74(m,1H),2.62(s,4H),2.44(q,J=7.1Hz,2H),2.23–2.14(m,1H),1.55(dd,J=8.4,6.4Hz ,2H),1.22(t,J=7.1Hz,3H),1.16–1.10(m,2H),1.06(t,J=7.1Hz,3H),0.72–0.58(m,2H)ppm.

[0134] Example 9: Methyl 5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)valerate

[0135]

[0136] The procedure is the same as in Example 1, except that in step three, methyl 6-oxohexanoate is used instead of methyl p-formylbenzoate to obtain methyl 5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)valerate, a yellow solid. Melting point: 144-148℃. ESI-HRMS (m / z): [M+H] + calcd.for C 31 H 37 F3N7O3S:644.2625,found:644.2621. 1H NMR(400MHz,DMSO-d6)δ9.21(s,1H),8.28(s,1H),8.19–8.07(m,2H),7.54(d,J=8.5Hz ,2H),7.36(s,1H),3.79(tt,J=6.9,3.7Hz,1H),3.59(s,2H),3.33(s,4H),3.06(s,4H) ,2.91(t,J=7.2Hz,2H),2.34(p,J=7.8Hz,2H),1.65(dq,J=23.7,7.7Hz,4H),1.23(s,2 H), 1.18 (t, J = 7.1Hz, 3H), 1.12 (dd, J = 13.8, 7.0Hz, 3H), 0.66 (p, J = 5.2, 4.7Hz, 2H) ppm.

[0137] Example 10 Methyl 5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)furan-3-carboxylic acid ester

[0138]

[0139] The procedure is the same as in Example 1, except that in step three, methyl 2-formylfuran-4-carboxylate is used instead of methyl p-formylbenzoate to obtain methyl 5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)furan-3-carboxylate, a yellow solid. Melting point: 150-154℃. ESI-HRMS (m / z): [M+H] + calcd.for C 31 H 31 F3N7O4S:654.2105,found:654.2100. 1H NMR(400MHz,DMSO-d6)δ9.71(s,1H),8.60(d,J=9.0Hz,1H),8.20(s,1H),8.18–8.13(m,2H ),7.66(s,1H),7.56(d,J=8.3Hz,2H),7.40(s,1H),7.29(d,J=5.7Hz,1H),7.03(s,3H),3.8 3(s,3H),3.80(dd,J=7.0,3.8Hz,1H),2.98(t,J=4.8Hz,4H),2.60(s,4H),2.42(q,J=7.1H z,2H),1.15(dd,J=7.4,5.5Hz,2H),1.05(t,J=7.1Hz,3H),0.68(dt,J=7.0,3.6Hz,2H)ppm.

[0140] Example 11 Methyl 3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)benzoate

[0141]

[0142] The procedure is the same as in Example 1, except that in step three, methyl meta-formylbenzoate is used instead of methyl para-formylbenzoate to obtain methyl 3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)benzoate, a yellow solid. Melting point: 191-192.5℃. ESI-HRMS (m / z): [M+H] + calcd.for C 33 H 33 F3N7O3S:664.2312,found:664.2311. 1H NMR (400MHz, DMSO-d6) δ9.64(s,1H),8.39(d,J=2.1Hz,1H),8.21(d,J=3.0Hz,1H),8.16(d, J=8.7Hz,2H),8.12–8.01(m,2H),7.68(t,J=7.8Hz,1H),7.56(d,J=8.3Hz,2H),7.41(s,1H) ,3.92(s,3H),3.82(tt,J=7.1,3.7Hz,1H),3.00(t,J=4.8Hz,4H),2.61(s,4H),2.47–2.37( m,2H),1.38(dd,J=15.4,8.3Hz,2H),1.05(t,J=7.1Hz,3H),0.69(p,J=5.3,4.8Hz,2H)ppm.

[0143] Example 12 Ethyl 6-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)hexanoate

[0144]

[0145] The procedure is the same as in Example 1, except that in step three, ethyl 7-oxoheptanoate is used instead of methyl p-formylbenzoate to obtain ethyl 6-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzimidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)hexanoate, a white solid. Melting point: 167-168℃. ESI-HRMS (m / z): [M+H] + calcd.for C 33 H 41 F3N7O3S:672.2938,found:672.2936. 1HNMR(400MHz, DMSO-d6)δ9.17(s,1H),8.27–8.09(m,3H),7.55(d,J=8.3Hz,2H),7.38(s,1H),4.06(q,J=7.0 Hz,2H),3.80(tt,J=7.1,3.9Hz,1H),2.97(t,J=4.7Hz,4H),2.89(t,J=7.4Hz,2H),2.66(s,4H),2.47(t,J=7 .4Hz,2H),2.28(dt,J=17.9,7.4Hz,2H),1.68(p,J=7.5Hz,2H),1.57(q,J=7.5Hz,2H),1.38(ddt,J=9.0,6.8 ,3.7Hz,2H),1.19(d,J=7.1Hz,2H),1.16(q,J=5.0,4.3Hz,3H),1.08(t,J=7.1Hz,3H),0.75–0.59(m,2H)ppm.

[0146] Example 13 6-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)nicotinic acid methyl ester

[0147]

[0148] The procedure is the same as in Example 1, except that in step three, methyl 6-formylnicotinate is used instead of methyl p-formylbenzoate to obtain methyl 6-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)nicotinate, a yellow solid. Melting point: 98-99℃. ESI-HRMS (m / z): [M+H] + calcd.for C 32 H 32 F3N8O3S:665.2265,found:665.2262. 1H NMR (400MHz, DMSO-d6) δ9.81 (s, 1H), 9.10 (d, J = 2.2Hz, 1H), 8.43 (dd, J = 8.4, 2.1Hz, 1H), 8.28 (dd,J=8.4,2.0Hz,1H),8.21–8.12(m,3H),7.57(d,J=8.3Hz,2H),7.40(d,J=2.0Hz,1H),7.04 (s,1H),3.93(d,J=2.0Hz,3H),3.87–3.78(m,1H),3.00(t,J=4.7Hz,4H),2.61(s,4H),2.42(q ,J=7.2Hz,2H),1.17(d,J=6.8Hz,2H),1.05(td,J=7.2,2.0Hz,3H),0.69(d,J=4.1Hz,2H)ppm.

[0149] Example 14 Ethyl 6-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)pyridinecarboxylate

[0150]

[0151] The procedure is the same as in Example 1, except that in step three, methyl 6-formyl-2-pyridinecarboxylate is used instead of methyl p-formylbenzoate to obtain ethyl 6-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)pyridinecarboxylate, a yellow solid. Melting point: 231-234℃. ESI-HRMS (m / z): [M+H] + calcd.for C 33 H 34 F3N8O3S:679.2421,found:679.2426. 1H NMR (400MHz, DMSO-d6) δ9.71 (s, 1H), 8.37 (dd, J = 7.8, 1.2 Hz, 2H), 8.16 (dd, J = 8.4, 3.0 Hz, 3H), 8.11 (dd, J=7.7, 1.3Hz, 1H), 7.57 (d, J=8.3Hz, 2H), 7.42 (s, 1H), 4.40 (q, J=7. 1Hz,2H),3.83(tt,J=7.2,3.8Hz,1H),3.34(s,3H),3.10(s,5H),2.70(s,2H),1.36(t, J=7.1Hz,3H),1.26(d,J=6.8Hz,2H),1.20–1.14(m,3H),0.70(p,J=5.3,4.8Hz,2H)ppm.

[0152] Example 15 Ethyl 5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)pyridinecarboxylate

[0153]

[0154] The procedure was the same as in Example 1, except that methyl 5-formylpyridine-2-carboxylate was used in step three to obtain ethyl 5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)pyridinecarboxylate, a yellow solid. Melting point: 127-130℃. ESI-HRMS (m / z): [M+H] + calcd.forC 33 H 34 F3N8O3S:679.2421,found:679.2421. 1 H NMR (400MHz, DMSO-d6) δ9.83 (s, 1H), 9.16 (d, J = 2.2Hz, 1H), 8.40 (dd, J = 8.2, 2.3Hz, 1 H),8.21–8.12(m,4H),7.56(d,J=8.3Hz,2H),7.41(s,1H),4.39(q,J=7.1Hz,2H),3.8 2(tt,J=7.2,3.8Hz,1H),3.00(t,J=4.7Hz,4H),2.60(s,4H),2.42(s,2H),1.37(t,J= 7.1Hz,3H),1.21–1.14(m,2H),1.05(t,J=7.1Hz,3H),0.69(p,J=5.2,4.8Hz,2H)ppm.

[0155] Example 16 Methyl 9-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)nonanoate

[0156]

[0157] The procedure is the same as in Example 1, except that in step three, methyl 9-formylnonanoate is used instead of methyl p-formylbenzoate to obtain methyl 9-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)nonanoate, a yellow solid. Melting point: 144-146℃. ESI-HRMS (m / z): [M+H] + calcd.for C 36 H 47 F3N7O3S:714.3408,found:714.3403. 1 HNMR(400MHz,DMSO-d6)δ9.23(s,1H),8.33(s,1H),8.21–8.06(m,2H),7.54(d,J=8.3Hz,2H),7.36(s,1H ),4.03(q,J=7.1Hz,2H),3.80(tt,J=7.2,3.8Hz,1H),3.33(s,4H),3.11(s,4H),2.89(t,J=7.4Hz,3H),2 .32–2.22(m,2H),1.66(q,J=7.3Hz,2H),1.51(t,J=7.1Hz,2H),1.33(dt,J=14.6,5.3Hz,4H),1.29–1.24 (m,6H),1.22(d,J=10.2Hz,2H),1.17(d,J=7.1Hz,2H),1.14(d,J=6.2Hz,3H),0.66(p,J=4.8Hz,2H)ppm.

[0158] Example 17 4'-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)-[1,1'-biphenyl]-4-carboxylic acid methyl ester

[0159]

[0160] The procedure is the same as in Example 1, except that in step three, methyl 4'-formyl-[1,1'-biphenyl]-4-carboxylate is used instead of methyl p-formylbenzoate to obtain methyl 4'-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)-[1,1'-biphenyl]-4-carboxylate, a yellow solid. Melting point: 234-236℃. ESI-HRMS (m / z): [M+H] + calcd.for C 39 H 37 F3N7O3S:740.2625,found:740.2614. 1 HNMR(400MHz,DMSO-d6)δ9.59(s,1H),8.21(s,1H),8.17–8.12(m,2H),8.07(d,J =8.4Hz,2H),7.92(dt,J=16.4,8.4Hz,6H),7.55(d,J=8.3Hz,2H),7.40(s,1H),3 .89(s,3H),3.80(tt,J=7.1,3.8Hz,1H),2.99(t,J=4.8Hz,4H),2.62(s,4H),2.4 2(s,2H),1.18–1.12(m,2H),1.05(td,J=7.1,3.9Hz,3H),0.72–0.63(m,2H)ppm.

[0161] Example 18 (E)-Methyl-3-(4-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)methyl phenylacrylate

[0162]

[0163] The procedure is the same as in Example 1, except that in step three, methyl p-formylcinnamate is used instead of methyl p-formylbenzoate to obtain (E)-methyl-3-(4-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)phenylacrylate, a yellow solid. Melting point: 146-147℃. ESI-HRMS (m / z): [M+H] + calcd.for C 35 H 35 F3N7O3S:690.2469,found:690.2461. 1H NMR (400MHz, DMSO-d6) δ9.61 (s, 1H), 8.24–8.08 (m, 3H), 7.85 (d, J = 1.6Hz, 4H), 7. 70(d,J=16.0Hz,1H),7.54(d,J=8.3Hz,2H),7.39(s,1H),6.72(d,J=16.1Hz,1H),3 .80(tt,J=7.1,3.9Hz,1H),3.74(s,3H),2.97(t,J=4.8Hz,4H),2.58(s,4H),2.40 (q,J=7.2Hz,2H),1.19–1.10(m,2H),1.03(t,J=7.1Hz,3H),0.73–0.60(m,2H)ppm.

[0164] Example 19 (1s, 4s)-4-(5-(1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)cyclohexyl-1-carboxylic acid methyl ester

[0165]

[0166] The procedure is the same as in Example 1, except that in step three, methyl trans-4-formylcyclohexanecarboxylate is used instead of methyl p-formylbenzoate to obtain (1s,4s)-4-(5-(1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)cyclohexyl-1-carboxylic acid methyl ester, a yellow solid. Melting point: 171-171.5℃. ESI-HRMS (m / z): [M+H] + calcd.for C 33 H 39 F3N7O3S:670.2782,found:670.2777. 1HNMR(400MHz,DMSO-d6)δ9.17(s,1H),8.25–8.08(m,3H),7.55(d,J=8.3Hz,2H),7.38(s,1 H),4.08(q,J=7.1Hz,1H),3.79(tt,J=7.1,3.9Hz,1H),3.63(s,2H),2.96(d,J=4.4Hz,4H), 2.94(s,1H),2.60(s,4H),2.42(p,J=7.3Hz,3H),2.15–2.06(m,2H),2.04–1.96(m,2H),1. 60–1.45(m,4H),1.14(dd,J=7.4,5.5Hz,2H),1.06(t,J=7.2Hz,3H),0.72–0.63(m,2H)ppm.

[0167] Example 20 Methyl 3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)-1H-indole-6-carboxylic acid

[0168]

[0169] The procedure is the same as in Example 20, except that in step three, methyl 3-formylindole-6-carboxylate is used instead of methyl p-formylbenzoate to obtain methyl 3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)-1H-indole-6-carboxylate, a yellow solid. Melting point: 292-295℃. ESI-HRMS (m / z): [M+H] + calcd.for C 35 H 34 F3N8O3S:703.2421,found:703.2418. 1H NMR (400MHz, DMSO-d6) δ8.22(dd,J=8.8,2.9Hz,2H),8.09(d,J=1.5Hz,1H),7.86(s,1H),7.81(dd,J=8.6,1 .6Hz,1H),7.69(s,1H),7.60(d,J=8.3Hz,1H),7.43(s,1H),7.05(s,3H),6.55(d,J=2.9Hz,1H),3.88(s,3H ),3.87–3.83(m,1H),3.08–2.97(m,2H),2.32(s,2H),2.17(tt,J=7.2,3.8Hz,2H),2.07(s,2H),1.19(qd,J =10.0,8.3,5.4Hz,2H),0.88(t,J=7.2Hz,3H),0.77(dd,J=10.1,4.9Hz,1H),0.70(q,J=5.4,4.7Hz,1H)ppm.

[0170] Example 21 2-(3-(5-((1-cyclopropyl-6-(4-ethylpiperazin-1-yl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)-1H-indole-1-yl)ethyl acetate

[0171]

[0172] The procedure is the same as in Example 1, except that in step three, ethyl 3-formyl-1-indole-ethyl acetate is used instead of methyl paraformylbenzoate to obtain ethyl acetate (2-3-(5-((1-cyclopropyl-6-(4-ethylpiperazin-1-yl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)-1H-indole-1-yl) ethyl acetate, a yellow solid. Melting point: 142-146℃. ESI-HRMS (m / z): [M+H] + calcd.for C 37 H 38 F3N8O3S:731.2734found:731.2731. 1HNMR(700MHz,DMSO-d6)δ9.28(s,1H),8.32–8.27(m,1H),8.19–8.16(m,1H),8.15–8.14(m,1H),8.11(d,J=7.8Hz, 1H),8.01(s,1H),7.98(d,J=2.3Hz,1H),7.56–7.52(m,3H),7.29–7.23(m,2H),5.23(d,J=10.4Hz,2H),4.18(q,J=7 .1Hz,2H),3.80(d,J=10.6Hz,1H),3.68(s,3H),3.49(d,J=10.6Hz,2H),3.20(d,J=12.2Hz,2H),2.99(s,2H),2.67 (s,2H),2.48–2.40(m,2H),1.40(t,J=7.1Hz,2H),1.15–1.10(m,3H),0.67(dt,J=7.1,3.6Hz,1H),0.63(s,1H)ppm.

[0173] Example 22 Methyl 4-((2-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)phenoxymethyl)benzoate

[0174]

[0175] The procedure is the same as in Example 1, except that in step three, methyl 4-[(2-formylphenoxy)methyl]-benzoate is used instead of methyl p-formylbenzoate to obtain methyl 4-((4-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)phenoxymethyl)benzoate, a yellow solid. Melting point: 110-113℃. ESI-HRMS (m / z): [M+H] + calcd.for C 40 H 39 F3N7O4S:770.2731,found:770.2723. 1H NMR(400MHz, DMSO-d6)δ9.32(s,1H),8.28–8.13(m,4H),8.01(dd,J=8.3,2.2Hz,2H),7.72–7.64(m, 2H),7.56(d,J=8.3Hz,2H),7.47(td,J=7.9,7.3,1.8Hz,1H),7.37(s,1H),7.31(d,J=8.5Hz,1H),7. 15(t,J=7.6Hz,1H),5.46(s,2H),3.83(s,3H),3.81(dd,J=7.0,3.7Hz,1H),2.94(t,J=4.8Hz,4H),2 .59(s,4H),2.43(s,2H),1.16(dd,J=7.4,5.5Hz,2H),1.06(t,J=7.2Hz,3H),0.73–0.64(m,2H)ppm.

[0176] Example 23 Ethyl 2-(3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)phenylacetate

[0177]

[0178] The procedure is the same as in Example 1, except that in step three, methyl meta-formylphenylacetate is used instead of methyl p-formylbenzoate to obtain ethyl acetate, a yellow solid, consisting of 2-(3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)phenylacetate. Melting point: 121-123℃. ESI-HRMS (m / z): [M+H] + calcd.for C 35 H 37 F3N7O3S:692.2625,found:692.2622. 1HNMR (400MHz, DMSO-d6) δ8.22(s,1H),8.15(d,J=8.5Hz,2H),7.72(dd,J=24.1,16.3Hz,2H),7.56( d,J=8.4Hz,2H),7.46(t,J=7.7Hz,1H),7.42–7.35(m,2H),7.04(s,1H),4.12(q,J=7.1Hz,2H),3.8 2(dd,J=7.3,3.5Hz,1H),3.79(s,2H),3.01(t,J=4.8Hz,4H),2.67(s,4H),2.47(t,J=7.2Hz,2H),1 .21(t,J=7.1Hz,3H),1.16(d,J=6.8Hz,2H),1.07(t,J=7.1Hz,3H),0.68(q,J=5.4,4.1Hz,2H)ppm.

[0179] Example 24 7-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)benzofuran-2-carboxylic acid ethyl ester

[0180]

[0181] The procedure is the same as in Example 1, except that in step three, methyl 7-formylbenzofuran-2-carboxylate is used instead of methyl p-formylbenzoate to obtain ethyl 7-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)benzofuran-2-carboxylate, a yellowish-brown solid. Melting point: 221-224℃. ESI-HRMS (m / z): [M+H] + calcd.for C 36 H 35 F3N7O4S:718.2418,found:718.2414. 1H NMR(400MHz,DMSO-d6)δ9.68(s,1H),8.27–8.19(m,2H),8.18–8.11(m,2H),7.95–7 .87(m,2H),7.58–7.49(m,3H),7.41(s,1H),4.38(q,J=7.1Hz,2H),3.81(tt,J=7.0, 3.8Hz,1H),3.00(t,J=4.8Hz,4H),2.61(s,4H),2.41(d,J=7.4Hz,2H),1.34(t,J=7. 1Hz,3H),1.15(dd,J=7.4,5.5Hz,2H),1.03(t,J=7.1Hz,3H),0.72–0.64(m,2H)ppm.

[0182] Example 25 Methyl 4-(6-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)-2-pyridylbenzoate

[0183]

[0184] The procedure is the same as in Example 1, except that in step three, methyl 4-(6-formylpyridin-2-yl)benzoate is used instead of methyl p-formylbenzoate to obtain methyl 4-(6-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)-2-pyridylbenzoate, a yellow solid. Melting point: 252-256℃. ESI-HRMS (m / z): [M+H] + calcd.for C 38 H 36 F3N8O3S:741.2578,found:741.2578. 1 H NMR(400MHz, DMSO-d6)δ9.66(s,1H),8.33–8.24(m,3H),8.21–8.08(m,7H),7.57(d,J=8.3Hz,2H),7.43(s,1H),3.91(s,3H),3.82(tt ,J=7.1,3.8Hz,1H),3.01(t,J=4.7Hz,4H),2.65(s,4H),2.45(s,2H),1.22–1.13(m,2H),1.06(t,J=7.1Hz,3H),0.73–0.65(m,2H)ppm.

[0185] Example 26 Ethyl 4-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)pyridinecarboxylate

[0186]

[0187] The procedure is the same as in Example 1, except that in step three, methyl 4-formylpyridine-2-carboxylate is used instead of methyl p-formylbenzoate to obtain ethyl 4-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)pyridinecarboxylate, a yellow solid. Melting point: 158-158.5℃. ESI-HRMS (m / z): [M+H] + calcd.for C 33 H 34 F3N8O3S:679.2421,found:679.2418. 1 H NMR(400MHz,DMSO-d6)δ8.83(d,J=5.1Hz,1H),8.41(s,1H),8.23–8.12(m,3H),8.00( d,J=5.1Hz,1H),7.57(d,J=8.2Hz,2H),7.42(s,1H),7.06(s,1H),4.41(q,J=7.2Hz,2 H),3.83(s,1H),3.05(s,3H),2.89(s,1H),2.74(s,4H),2.03(dt,J=14.8,7.2Hz,2H) ,1.38(t,J=7.6Hz,3H),1.17(d,J=6.7Hz,2H),1.09(t,J=7.2Hz,3H),0.70(s,2H)ppm.

[0188] Example 27 2-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)isonicotinic acid ethyl ester

[0189]

[0190] The procedure is the same as in Example 1, except that in step three, methyl 2-formyl sulfoxide is used instead of methyl p-formylbenzoate to obtain ethyl 2-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)isonicotinic acid, a yellow solid. Melting point: 97-98℃. ESI-HRMS (m / z): [M+H] + calcd.for C 32 H 32 F3N8O3S:665.2265,found:665.2258. 1 H NMR(400MHz,DMSO-d6)δ9.73(s,1H),8.81(d,J=5.1Hz,1H),8.51(s,1H),8.21–8.11(m,3H),7 .87(d,J=5.1Hz,1H),7.55(d,J=8.3Hz,2H),7.39(s,1H),4.41(q,J=7.1Hz,1H),3.96(s,1H), 3.81(td,J=7.2,3.7Hz,1H),3.00(s,4H),2.64(d,J=22.5Hz,2H),2.41(d,J=2.8Hz,2H),1.38 (t,J=7.1Hz,2H),1.15(d,J=6.8Hz,3H),1.06(t,J=6.8Hz,3H),0.67(q,J=5.2,4.1Hz,2H)ppm.

[0191] Example 28 Ethyl 5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)pyrazine-2-carboxylic acid ester

[0192]

[0193] The procedure is the same as in Example 1, except that in step three, methyl 5-formylpyrazine-2-carboxylate is used instead of methyl p-formylbenzoate to obtain ethyl 5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)pyrazine-2-carboxylate, a yellow solid. Melting point: 149-152℃. ESI-HRMS (m / z): [M+H] + calcd.for C 32 H 33 F3N9O3S:680.2374,found:680.2369. 1H NMR (400MHz, DMSO-d6) δ10.03(s,1H),9.46(d,J=1.5Hz,1H),9.21(d,J=1.4Hz,1H ),8.16(d,J=8.8Hz,3H),7.57(d,J=8.3Hz,2H),7.41(s,1H),4.43(q,J=7.1Hz,2H) ,3.82(dt,J=6.9,3.2Hz,1H),3.02(s,4H),2.61(s,4H),2.43(s,2H),1.38(t,J=7. 1Hz,3H),1.20–1.14(m,2H),1.05(t,J=7.3Hz,3H),0.70(p,J=5.2,4.7Hz,2H)ppm.

[0194] Example 29 (E)-3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)methyl acrylate

[0195]

[0196] The procedure is the same as in Example 1, except that methyl fumarate is used instead of methyl p-formylbenzoate in step three to obtain (E)-3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2-yl)methyl acrylate, a yellow solid. Melting point: 99-100℃. ESI-HRMS (m / z): [M+H] + calcd.for C 29 H 31 F3N7O3S:614.2156,found:614.2150. 1 H NMR(400MHz,DMSO-d6)δ9.91(s,1H),8.20–8.07(m,4H),7.67(dd,J=15.9,3.5H z,1H),7.56(d,J=8.3Hz,2H),7.39(s,1H),6.50(dd,J=15.9,7.8Hz,1H),3.81( dd,J=7.1,3.6Hz,1H),3.76(s,3H),2.98(s,5H),2.64(d,J=42.9Hz,3H),2.40( s,2H),1.18–1.15(m,2H),1.05(t,J=7.2Hz,3H),0.69(q,J=4.1,3.6Hz,2H)ppm.

[0197] Pharmacologically active part

[0198] Experimental Example 1: Evaluation of the inhibitory effect of the compounds in the examples on the IL-6 / JAK / STAT3 signaling pathway

[0199] 1) Principle: This study utilizes HEK-Blue IL-6 cells from Invivogen, which stably express the IL-6 receptor and a reporter gene containing a STAT3-binding region for secreted embryonic alkaline phosphatase (SEAP). Upon IL-6 stimulation, phosphorylated STAT3 in the cytoplasm forms a dimer, translocates into the nucleus, binds to the SEAP reporter gene, and initiates transcription, ultimately producing SEAP that can be secreted extracellularly. SEAP in the cell culture supernatant reacts with Quanti-Blue; the amount of SEAP produced can be calculated by measuring the product uptake at 655 nm, thus reflecting the activity of the IL-6-STAT3 pathway. Therefore, by treating cells with the drug, reacting the supernatant with Quanti-Blue, and detecting the amount of SEAP produced at 655 nm, the inhibitory effect of the compound on the IL-6-stimulated STAT3 signaling pathway can be evaluated.

[0200] 2) Experimental Method: When the cell density reached 80%, the adherent cells were gently tapped off the plate, resuspended in preheated (37℃) complete culture medium, and counted. A sterile 96-well cell culture plate was used, and the cell suspension (final cell count per well was 5 × 10⁶) was added. 4 The system consisted of 160 μL of complete culture medium, 20 μL of different concentrations of compounds, and 20 μL of IL-6 cytokine (final concentration 1 ng / mL), for a total of 200 μL. The negative control group consisted of 40 μL of complete culture medium and 160 μL of cell suspension; the positive control group consisted of 20 μL of IL-6 cytokine (final concentration 1 ng / mL), 20 μL of complete culture medium, and 160 μL of cell suspension. After incubation at 37°C for 16 h, 20 μL of solution was transferred from each well of a 96-well plate to another 96-well plate, and 180 μL of preheated (37°C) QUANTI-Blue chromogenic solution was added. After incubation at 37°C for 1 h, the cells were detected at 655 nm.

[0201] 3) The results showed that the compounds in the examples inhibited the IL-6 / JAK / STAT3 pathway in a dose-dependent manner, and the experiments were reproducible. The IC50 values ​​of the compounds in the examples are shown below. 50 Listed in Table 1.

[0202] Table 1. Inhibitory activity of the compounds in the examples on the IL-6 / JAK / STAT3 signaling pathway in HEK-Blue cells. (Note: A: IC) 50 ≤5μM, B:5μM>IC50 ≤10μM, C:10μM>IC 50 ≤15μM, D:15μM>IC 50 ≤20μM, E:IC 50 >20μM)

[0203]

[0204]

[0205] Experimental Example 2: MTT assay to determine the inhibitory effect of the compounds in the examples on tumor cells

[0206] 1) Experimental method: HCT116 colorectal cancer cell line in logarithmic growth phase was digested with trypsin and then prepared into a concentration of 2×10⁻⁶ cells / mL. 4 Cell sap was seeded at 100 μL per well in a 96-well plate. The following day, 100 μL of fresh culture medium (DMSO final concentration <0.2%) with different concentrations of drug and solvent control was added to each well, with 3 parallel wells per group. After incubation at 37℃ for 48 h, the supernatant was discarded; 50 μg / mL of DMSO was added to each well. -1 After incubating the MTT solution at 37°C for 1 hour, discard the supernatant. Add DMSO, vortex, and mix well. Measure the absorbance at 570 nm using a microplate reader.

[0207] 2) Results showed that the compounds in the examples inhibited the growth of HCT116 cells in a dose-dependent manner (Table 2), and the experiment was reproducible. The compounds in the examples were determined to have inhibitory activity against the proliferation of HCT116 tumor cells.

[0208] Table 2. Screening results of the compounds in the examples using the MTT assay in HCT116 cells. (Note: A: IC) 50 ≤5μM, B:5μM>IC 50 ≤10μM, C:10μM>IC 50 ≤15μM, D:15μM>IC 50 ≤20μM, E:IC 50 >20μM)

[0209]

[0210] Experimental Example 3: Evaluation of the binding interaction between the compounds in the examples and the STAT3 protein using surface plasmon resonance (SPR) technique.

[0211] 1) Principle: Surface plasmon resonance (SPR) is a biosensing and analysis method based on the principle of physical optics. It utilizes the resonance between light and plasma waves after evanescent waves are generated in different media. It is often used to detect interactions between proteins and ligands, nucleic acids and ligands, and proteins and nucleic acids. This method does not cause any damage to biomolecules and does not require any labeling.

[0212] The procedure involves first bonding a molecule (target molecule) to the surface of the biosensor, then injecting and flowing a solution containing another molecule (analyte) that may interact with the target molecule through the biosensor surface. The intermolecular bonding increases the surface mass of the biosensor, resulting in a proportional increase in the refractive index.

[0213] 2) Experimental methods:

[0214] a) Preparation of experimental materials, reagents, and instruments: PBS buffer, Tween 20, DMSO, EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; 0.4M aqueous solution), NHS (N-hydroxysuccinimide; 0.1M aqueous solution), 1M ethanolamine pH 8.5, 10mM sodium acetate pH 4.0, 50mM NaOH; centrifuge, SPR instrument (GE Healthcare, model: Biacore T200), CM5 chip, etc.

[0215] b) STAT3 protein coupling: i) Washing: Replace with a new CM5 chip, and replace the running buffer with PBS. First, wash the new CM5 chip twice with a mixture of 400 μl of 50 mM NaOH and 4 μl of 0.05% SDS, then wash the CM5 chip once with 400 μl of 50 mM NaOH at a flow rate of 30 μl / min. ii) Activation: Mix equal volumes of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; 0.4 M aqueous solution) and NHS (N-hydroxysuccinimide; 0.1 M aqueous solution) and inject the mixture at a flow rate of 5 μl / min for 10 min to activate the Fc1 and Fc2 channels of the chip. iii) Protein coupling: Dilute the STAT3 protein with 10 mM sodium acetate at pH 4.0 to a final concentration of 5 μg / ml. The Fc2 channel was injected multiple times at a flow rate of 5 μl / min, and the expected protein conjugation amount was approximately 940.7 RU; the Fc1 channel served as a control. iv) Blocking: After conjugation, the Fc1 and Fc2 channels were blocked with ethanolamine solution at a flow rate of 5 μl / min for 10 min.

[0216] c) Running buffer and sample preparation: i) Preparation of running buffer and solvent calibration curve: 0.05% Tween 20 PBS buffer. Specific preparation method: Add 0.5 ml of Tween 20 to 1000 mL of PBS buffer. For later use, refer to this as PBST buffer. For small molecule samples, use PBST buffer containing 5% DMSO. According to the table below, add DMSO to prepare 5% DMSO running buffer and 4.5% and 5.8% solvent calibration stock solutions. Replace the original running buffer in the left tray of the system with PBST buffer containing 5% DMSO and insert the corresponding inlet tubes.

[0217]

[0218]

[0219] Prepare a 5% DMSO concentration calibration curve by mixing 4.5% and 5.8% mother liquor according to the table below.

[0220] Buffer / Vial 1 2 3 4 4.5% DMSO 1500 1000 500 0 5.8% DMSO 0 500 1000 1500

[0221] ii) Small molecule sample preparation. Dilute the 10 mM compound stock solution 20-fold with DMSO-free PBST buffer to obtain 500 μM of the compound in 5% DMSO-containing PBST buffer. Then, using the prepared running buffer (5% DMSO-containing PBST buffer), dilute the compound down 11 times at a concentration gradient of 2-fold, starting from the designed highest concentration of 40 μM, increasing the concentration by 0.

[0222] d)K D Detection. Select LMW kinetics mode and set the injection program. The default detection temperature is 25℃. The flow rate is 30 μL / min; the contact time is 60 s; the dissociation time is 60 s; and the regeneration time using 10 mM NaOH is 30 s.

[0223] 3) Results showed that after SPR assay of the compounds in the examples with STAT3 protein, the compounds in the examples exhibited high binding levels to STAT3 protein and a significant concentration gradient trend, indicating specific binding. This suggests that the compounds in the examples stably bind to STAT3 protein. Figure 1) .

[0224] The binding curve of compound 26 with STAT3 in Example 26 showed a slow dissociation process. Kinetic 1:1 binding mode fitting was performed using a concentration of 9 to determine its K0. D The value was 6.16 μM, indicating high reliability of the fitting results (Table 3).

[0225] Table 3. Kinetic study of STAT3 in Example 26.

[0226]

[0227] Experiment Example 4: Evaluation of the inhibitory effect of the compounds in the examples on intracellular STAT3 protein phosphorylation by Western blotting

[0228] HCT116 cells treated with control group and different concentrations of the compound from the examples for 24 h were collected, washed twice with pre-cooled PBS, and then lysed with an appropriate amount of RIPA lysis buffer (R0010, Solarbio, China), a mixed solution of phosphatase inhibitor and protease inhibitor. After lysis on ice for 30 min, the cells were centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatant was collected, and after protein quantification using BCA, the protein was denatured at 98 °C for 5 min. 20 μg of protein was subjected to 10% SDS-PAGE electrophoresis, transferred to a membrane, and blocked with 5% BSA for 1 h. Primary antibodies were p-STAT3 (Tyr705) (1:1000, #9131, Cell Signaling Technology, the United States) and β-actin (1:2000, 66009-1-Ig, ProteinTech, the United States) incubated overnight at 4 °C. The following day, after warming to 30 minutes, the sample was washed three times with TBST for 5 minutes each time. It was then incubated at room temperature for 1 hour with the corresponding rabbit horseradish peroxidase-labeled secondary antibody, followed by three more TBST washes for 5 minutes each. ECL chemiluminescent substrate reaction solution was added, and the gel was developed in a gel imaging system. The image was saved. After exposure, the p-stat3 bands were washed with stripping buffer for 30 minutes, blocked with 5% skim milk for one hour, and then incubated overnight with the primary antibody stat3 (1:1000, 60199-1-Ig, ProteinTech, the United States). The next day, the subsequent procedures were repeated as described above.

[0229] The results showed that compound 26 of the examples was able to inhibit phosphorylation of tyrosine 705 of STAT3 protein in HCT116 cells in a dose-dependent manner, without affecting the total amount of STAT3. Figure 2) .

[0230] Experimental Example 5: In vivo antitumor effect of the compound in the example against subcutaneous xenografts of colon cancer MC38 in mice.

[0231] The experimental animals were C57BL / 6J mice, 18-20g, SPF grade, male, provided by Spiford (Beijing) Biotechnology Co., Ltd. Under aseptic conditions, MC38 tumor tissue was extracted from tumor-bearing mice, minced, ground evenly, and counted. Each mouse was then divided into doses of 2 × 10⁻⁶.6 Cells were subcutaneously inoculated into the axillary back of mice. The following day, animals were randomly divided into groups according to body weight, and daily administration began. Mice were weighed every three days. After 12 days of administration, mice were euthanized by cervical dislocation, tumor tissue was dissected, weighed, and photographed. The tumor inhibition rate was calculated to evaluate the intensity of the antitumor effect.

[0232] Grouping: There were five groups in total: blank control group, positive control group (stattic, ip 20 mg / kg), high-dose intraperitoneal injection group of compound 26 in example (ip, 20 mg / kg), high-dose intraperitoneal injection group (ip, 5 mg / kg), and oral administration group (po, 20 mg / kg).

[0233] Drug preparation: Weigh out a quantity of the compound, add a small amount of Tween-80 as a solubilizer, and dissolve in sterile physiological saline. The intraperitoneal administration volume per animal / 20g body weight is 0.2mL. Calculation method: Results were statistically analyzed and interpreted according to the "Technical Guidelines for Non-Clinical Evaluation of Cytotoxic Antitumor Drugs".

[0234] Relative tumor proliferation rate T / C (%): T / C% = T / C × 100%. (T: tumor weight in the treatment group; C: tumor weight in the negative control group).

[0235] Tumor proliferation inhibition rate (TGI%): TGI% = (1-T / C) × 100%. (T: tumor weight in the treatment group; C: tumor weight in the negative control group).

[0236] Evaluation criteria: T / C (%) > 40% is invalid; T / C (%) ≤ 40%, and after statistical processing, P < 0.05 is valid.

[0237] Experimental Results: Mice were inoculated with MC38 tumor cells, and drug administration began one day after inoculation. Compound 26 was administered once daily at doses of 20 mg / kg intraperitoneally, 5 mg / kg intraperitoneally, and 100 mg / kg by gavage. Twelve days after administration, the tumors of the treated animals were weighed. Compound 26 inhibited tumor growth (…). Figure 3) The antitumor effects of compound 26 in the MC38 subcutaneous xenograft model are shown in Table 4.

[0238] Table 4. Antitumor effects of the compounds in the examples in the MC38 subcutaneous xenograft model.

[0239] Group Tumor weight (g) TGI Model 1.7207 Static 10mg / kg ip 1.1612 32.5％ Example 26 5mg / kg ip 0.7987 53.6％ Example 26 20mg / kg ip 0.6911 59.8％ Example 26 100mg / kg po 1.2035 30.1％

[0240] Experimental Example 6: Compound Interference with HCT116 Cell Cycle

[0241] HCT116 cells were seeded into 12-well plates at a density of 180,000 cells / well. After adhesion the following day, 0.5, 2.5, and 5 μM of the compound from the example were added to three parallel wells for each group. After 24 hours, cells were collected by trypsin digestion with EDTA-free enzyme and washed once with PBS. 500 μL of DNA staining solution and 5 μL of permeabilization solution were added to each sample, and the mixture was vortexed. After incubation at room temperature in the dark for 30 minutes, the cells were analyzed using a BD flow cytometer at the lowest loading rate.

[0242] The results showed that the compound in the examples could arrest HCT116 cells in the G2 / M phase ( Figure 4) .

[0243] Experimental Example 7: Compounds from the Example Inducing Apoptosis in HCT116 Cells

[0244] HCT116 cells were seeded into 12-well plates at a density of 180,000 cells / well. After adhesion the following day, 0.5, 2.5, 5, and 10 μM of the compound from the example were added to three parallel wells for each group. After 24 hours, the supernatant was collected. Cells were then digested with EDTA-free trypsin and combined with the supernatant. The cells were centrifuged at 1500 rpm for 5 min, washed once with PBS, and resuspended in 1× binding buffer. Then, 5 μL of PI and 5 μL of FITC were added to each sample, stained at room temperature for 5 min, and analyzed using Flowjo v10.8.1 software.

[0245] The results showed that compound 26 in Example 26 induced apoptosis in HCT116 cells in a dose-dependent manner. Figure 5) .

Claims

1. A compound of the following structural formula I ###0001### and pharmaceutically acceptable salts thereof.

2. A compound of the following structural formula I ###0002### and pharmaceutically acceptable salts thereof. Linker is a straight or branched chain C 1-20 alkyl, substituted or unsubstituted C 1-20 cycloalkyl, substituted or unsubstituted C 1-20 heteroalkyl, substituted or unsubstituted C 1-20 cycloalkyl, substituted or unsubstituted C 1-20 cycloheteroalkyl, substituted or substituted C 1-20 alkyl, substituted or unsubstituted C 1-20 alkyl, C 1-20 alkoxy, straight or branched chain substituted or unsubstituted amine, C 1-20 cycloalkyl, halogen, cyano, C 1-20 phenyloxy, trifluoromethyl, trifluoromethyloxy, carboxyl, nitro, C 1-20 ester, hydroxyl, C 1-20 amide, C 1-20 heteroaryl, C 4-24 triazolyl, trifluoromethylphenyloxy, C 1-20 thioalkyl, C 1-20 sulfonamide, phenyl, morpholinyl, C 1-20 alkynyl, C 1-24 cycloalkyloxy, C 1-24 alkenyloxy, hydrazine, C 1-20 hydrazide, C 5-24 aromatic heterocycle, ester, aromatic ring group, fused heterocycle group, furanyl, pyrazolyl, imidazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrrolyl, thienyl, pyrazinyl, triazinyl, fused heterocycle group, benzofuranyl, benzothienyl, benzopyrrolyl, benzopyranyl, purinyl, carbazolyl; R is hydrogen, C 1-6 alkyl, C 1-6 alkoxy-substituted C 1-6 alkyl, alkyl-substituted C 1-6 alkyl or C 3-6 cycloalkyl.

2. The compound of claim 1, and pharmaceutically acceptable salts thereof, characterized in that Linker is a straight or branched chain C 1-15 alkyl, substituted or unsubstituted C 1-15 cycloalkyl, substituted or unsubstituted C 1-15 heteroalkyl, substituted or unsubstituted nitrogen-, oxygen-, sulfur-, or phosphorus- containing C 1-15 cycloalkyl, substituted or unsubstituted C 1-15 cycloheteroalkyl, substituted or unsubstituted C 1-15 alkyl, substituted or unsubstituted aryl; wherein each of the alkyl, cycloalkyl, heteroalkyl, cycloheteroalkyl, aryl groups can be unsubstituted or substituted with one or more groups selected from C 1-15 alkyl, C 1-15 alkoxy, straight or branched chain substituted and unsubstituted amine, C 1-15 cycloalkyl, halogen, cyano, C 1-15 phenyloxy, trifluoromethyl, trifluoromethyloxy, carboxyl, nitro, C 1-15 ester, hydroxyl, C 1-15 amide, C 1-15 heteroaryl, C 4-12 triazolyl, trifluoromethylphenyloxy, C 1-15 thioalkyl, C 1-15 sulfonamide, phenyl, morpholinyl, C 1-15 alkynyl, C 1-12 cycloalkyloxy, C 1-12 alkenyloxy, hydrazine, C 1-15 hydrazide, C 5-12 aromatic heterocycle, ester, aromatic ring group, fused heterocycle group, furanyl, pyrazolyl, imidazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrrolyl, thienyl, pyrazinyl, triazinyl, fused heterocycle group, benzofuranyl, benzothienyl, benzopyrrolyl, benzopyranyl, purinyl, carbazolyl; R is hydrogen, C 1-6 alkyl, alkyl-substituted C 1-6 alkyl or C 3-6 cycloalkyl.

3. A compound of claim 2, characterized in that ###0003### and pharmaceutically acceptable salts thereof. Linker is a straight or branched chain C 1-12 alkyl, substituted or unsubstituted C 1-12 cycloalkyl, substituted or unsubstituted C 1-12 heteroalkyl, substituted or unsubstituted nitrogen or oxygen or sulfur or phosphorus C 1-12 cycloalkyl, substituted or unsubstituted C containing multiple non-carbon atoms 1-12 cycloheteroalkyl, substituted and unsubstituted C 1-12 alkyl, substituted and unsubstituted aryl; wherein the alkyl, cycloalkyl, heteroalkyl, cycloheteroalkyl, aryl can be unsubstituted or substituted with one or more groups selected from C 1-12 alkyl, C 1-12 alkoxy, straight or branched chain substituted and unsubstituted amine, C 1-12 cycloamine, halogen, cyano, C 1-12 phenyloxy, trifluoromethyl, trifluoromethyloxy, carboxyl, nitro, C 1-12 ester, hydroxyl, C 1-12 amide, C 1-12 heteroaryl, C 4-12 triazolyl, trifluoromethylphenyloxy, C 1-12 sulfanyl, C 1-12 sulfonamide, phenyl, morpholinyl, C 1-12 alkynyl, C 1-12 cycloalkyloxy, C 1-12 alkenyloxy, hydrazine, C 1-12 hydrazide, C 5-12 aromatic heterocycle, ester, aromatic ring group, fused heterocycle group, furanyl, pyrazolyl, imidazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrrolyl, thienyl, pyrazinyl, triazinyl, fused heterocycle group, benzofuranyl, benzothienyl, benzopyrrolyl, benzopyranyl, purinyl, carbazolyl; R is hydrogen, C 1-6 alkyl.

4. A compound of claim 3, characterized in that ###0004### and pharmaceutically acceptable salts thereof. Linker is a straight or branched chain C 1-9 alkyl, substituted or unsubstituted C 1-9 cycloalkyl, substituted or unsubstituted C 1-9 heteroalkyl, substituted or unsubstituted C 1-9 cycloalkyl, substituted or unsubstituted C 1-9 cycloheteroalkyl, substituted or unsubstituted C 1-9 alkyl, substituted or unsubstituted C 1-9 alkyl, C 1-9 alkoxy, straight or branched chain substituted and unsubstituted amine, C 1-9 cycloalkyl, halogen, cyano, C 1-9 phenyloxy, trifluoromethyl, trifluoromethyloxy, carboxyl, nitro, C 1-9 ester, hydroxyl, C 1-9 amide, C 1-9 heteroaryl, C 4-12 triazolyl, trifluoromethylphenyloxy, C 1-9 thioalkyl, C 1-9 sulfonamide, phenyl, morpholinyl, C 1-9 alkyne, C 1-12 cycloalkyloxy, C 1-12 alkenyloxy, hydrazine, C 1-9 hydrazide, C 5-12 aromatic heterocycle, ester, aromatic ring group, fused heterocycle group, furanyl, pyrazolyl, imidazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrrolyl, thienyl, pyrazinyl, triazinyl, fused heterocycle group, benzofuranyl, benzothienyl, benzopyrrolyl, benzopyranyl, purinyl, carbazolyl; R is hydrogen, methyl, ethyl, propyl.

5. A compound according to any one of claims 1 to 4, selected from the group consisting of: ###0005### Compound 1: methyl 4-(5-((l-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-lH- benzimidazol-5-yl)amino)-l,3,4-thiadiazol-2-yl)benzoate ###0006### Compound 2: methyl 2-(4-(5-((l-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-lH- benzo[d]imidazol-5-yl)amino)-l,3,4-thiadiazol-2-yl)phenylacetate ###0007### Compound 3: ethyl 4-(5-((l-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-lH- benzimidazol-5-yl)amino)-l,3,4-thiadiazol-2-yl)butanoate ###0008### Compound 4: ethyl 3-(5-((l-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-lH- benzimidazol-5-yl)amino)-l,3,4-thiadiazol-2-yl)propanoate ###0009### Compound 5: methyl 5-(5-((l-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-lH- benzo[d]imidazol-5-yl)amino)-l,3,4-thiadiazol-2-yl)furan-2-carboxylate ###0010### Compound 6: ethyl l-(5-((l-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-lH- benzo[d]imidazol-5-yl)amino)-l,3,4-thiadiazol-2-yl)cyclopropane-l-carboxylate ###0011### Compound 7: methyl 4-(5-(5-((l-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-lH- benzimidazol-5-yl)amino)-l,3,4-thiadiazol-2-yl)furan-2-yl)benzoate ###0012### Compound 8: ethyl 2-(5-((l-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-lH- benzo[d]imidazol-5-yl)amino)-l,3,4-thiadiazol-2-yl)cyclopropane-l-carboxylate ###0013### Compound 9: methyl 5-(5-((l-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-lH- benzimidazol-5-yl)amino)-l,3,4-thiadiazol-2-yl)pentanoate ###0014### Compound 10: methyl 5-(5-((l-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4-(trifluoromethoxy)phenyl)-lH- benzo[d]imidazol-5-yl)amino)-l,3,4-thiadiazol-2-yl)furan-3-carboxylate and pharmaceutically acceptable salts thereof. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ Compound 11: methyl 3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4- (trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2- yl)benzoate Compound 12: ethyl 6-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4- (trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2- yl)hexanoate Compound 13: methyl 6-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4- (trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2- yl)nicotinate Compound 14: ethyl 6-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4- (trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2- yl)picolinate Compound 15: ethyl 5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4- (trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2- yl)picolinate Compound 16: methyl 9-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4- (trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2- yl)nonanoate Compound 17: methyl 4'-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4- (trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2- yl)-[1,1'-biphenyl]-4-carboxylate Compound 18: methyl (E)-3-(4-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2- (4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2- yl)phenyl)acrylate Compound 19: methyl (1s,4s)-4-(5-(1-cyclopropyl-6-(4-ethylpiperazinyl)-2- (4-(trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2- yl)cyclohexyl-1-carboxylate Compound 20: methyl 3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4- (trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2- yl)-1H-indole-6-carboxylate Compound 21: ethyl 2-(3-(5-((1-cyclopropyl-6-(4-ethylpiperazin-1-yl)-2-(4- (trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2- yl)-1H-indol-1-yl)acetate Compound 22: methyl 4-(2-(5-(1-cyclopropyl-6-(4-ethylpiperazin-1-yl)-2-(4- (trifluoromethoxy)phenyl)-1H-benzoimidazol-5-ylamino)-1,3,4-thiadiazol-2-yl)phenoxy) methylbenzoate Compound 23: ethyl 2-(3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4- (trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2- yl)phenylacetate Compound 24: ethyl 7-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4- (trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2- yl)benzofuranyl-2-carboxylate Compound 25: methyl 4-(6-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4- (trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2- yl)-2-pyridinylbenzoate Compound 26: ethyl 4-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4- (trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2- yl)picolinic acid Compound 27: methyl 2-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4- (trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2- yl)isonicotinate Compound 28: ethyl 5-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4- (trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2- yl)pyrazine-2-carboxylate Compound 29: methyl (E)-3-(5-((1-cyclopropyl-6-(4-ethylpiperazinyl)-2-(4- (trifluoromethoxy)phenyl)-1H-benzo[d]imidazol-5-yl)amino)-1,3,4-thiadiazol-2- yl)acrylate 6. A pharmaceutical composition, characterized by, A pharmaceutically effective amount of a compound of any one of claims 1-5, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

7. Use of a compound of any one of claims 1-5, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for inhibiting the activity of STAT3.

8. Use of a compound of any one of claims 1-5, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention or treatment of cancer.

9. Use according to claim 8, characterised in that, wherein the cancer is selected from breast cancer, prostate cancer, ovarian cancer, liver cancer, gastric cancer, lung cancer, colon cancer, esophageal cancer, leukemia, human brain glioma, lymphoma, hematological cancer, or melanoma.

10. A process for the preparation of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, characterized in that, comprising the steps of: a) monosubstitution; b) disubstitution; c) reduction; d) cyclization; e) formation of isocyanate; f) formation of aminothiosemicarbazide group; g) condensation; h) cyclization. wherein: ​ 1) the compound of formula (1) is subjected to a substitution reaction with a primary amine to give a compound of formula (2); 2) the compound of formula (2) is subjected to a substitution reaction with a secondary amine to give a compound of formula (3); 3) the compound of formula (3) is subjected to a reduction with palladium on carbon and ammonium formate and to a cyclization to give a compound of formula (4); 4) the compound of formula (4) is subjected to a reaction with a thio-carbonyl diimidazole to give an isocyanate, which is subjected to a reaction with hydrazine hydrate to give a compound of formula (5); 5) the compound of formula (5) is subjected to a condensation reaction with an aldehyde and to a cyclization in the presence of ferric trichloride to give a compound of formula (I); wherein Linker and R are as defined in any one of claims 1 to 5.

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