WO2021088265A1 - 咪唑并吡啶类化合物、包含该化合物的药物组合物及其制备方法和用途 - Google Patents
咪唑并吡啶类化合物、包含该化合物的药物组合物及其制备方法和用途 Download PDFInfo
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- WO2021088265A1 WO2021088265A1 PCT/CN2020/075706 CN2020075706W WO2021088265A1 WO 2021088265 A1 WO2021088265 A1 WO 2021088265A1 CN 2020075706 W CN2020075706 W CN 2020075706W WO 2021088265 A1 WO2021088265 A1 WO 2021088265A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
- C07D471/04—Ortho-condensed systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
Definitions
- the present invention relates to the fields of medicinal chemistry and pharmacotherapy, in particular to imidazopyridine compounds, pharmaceutical compositions containing the compounds, and preparation methods and uses thereof.
- STAT Signal Transduction and Activator of Transcription
- cytokines and growth factor receptors After being activated by different cytokines and growth factor receptors, it undergoes phosphorylation and dimerization, translocates from the cytoplasm to the nucleus, and binds to DNA to regulate the transcription and expression of corresponding target genes.
- STAT3 As an important member of the STAT family, STAT3 is responsible for regulating a series of important physiological processes such as cell growth, proliferation, differentiation and apoptosis.
- STAT3 can also interact with other tumor-related molecules on the cell surface to cross-link, activate, and amplify tumor-related effects, which greatly promotes the occurrence, development and metastasis of tumors. Therefore, inhibiting the STAT3 signaling pathway can inhibit multiple tumor targets from exerting their effects.
- STAT3 inhibitors can also act on EGFR (Epidermal grovth factor reptor). When the inhibitor is used in combination with EGFR inhibitors, it can delay the emergence of EGFR drug-acquired drug resistance and prolong its clinical effect. The service life has important clinical significance.
- the purpose of the present invention is to overcome the problems of low efficacy, low selectivity, and poor effectiveness of the specific inhibitors of STAT3 in the prior art, and provide an imidazo[1,2-a]pyridine compound.
- the second object of the present invention is to provide a method for preparing the above-mentioned imidazo[1,2-a]pyridine compounds.
- the third object of the present invention is to provide applications of the above-mentioned imidazo[1,2-a]pyridine compounds.
- n 0, 1 or 2;
- R 1 , R 2 , R 3 , and R 4 are each independently selected from hydrogen, halogen, cyano, nitro, amino, hydroxyl, trifluoromethyl, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C1-6 alkylamino, substituted or unsubstituted C3-8 cycloalkyl, substituted or unsubstituted C3-8 cycloalkoxy, substituted or unsubstituted Substituted C3-8 cycloalkylamino, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted 3-membered containing 1-2 heteroatoms selected from N and O To 8-membered heterocyclic group, -COR a , -CO 2 R a , -CONR a R b , -NR a C(O
- R a and R b are each independently hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, aryl, heteroaryl;
- R 5 is hydrogen, halogen, cyano, nitro, amino, hydroxyl, trifluoromethyl, substituted or unsubstituted C1-8 alkyl, substituted or unsubstituted C1-8 alkoxy, substituted or unsubstituted C1-C8 alkylamino, substituted or unsubstituted C3-8 cycloalkyl, substituted or substituted C3-C8 cycloalkoxy, substituted or unsubstituted C1-8 alkylamino, substituted or unsubstituted aryl Group or heteroaryl group, the aryl or heteroaryl group is furan, thiophene, pyrrole, oxazole, thiazole, imidazole, pyrazole, benzofuran, benzothiophene, benzoxazole, benzothiazole, phenyl , Pyridine, pyridazine, pyrimidine, pyrazine, quinoline
- R 6 is hydrogen, halogen, cyano, nitro, amino, hydroxy, trifluoro C1-C3 alkyl, C1-C3 alkoxy, C1-C3 alkylamino.
- R 1 , R 2 , R 3 , and R 4 are each independently selected from H, halogen, cyano, nitro, amino, hydroxy, trifluoromethyl, C 1-6 alkyl, C 1-6 hetero Alkyl alkoxy, C 1-6 alkylamino, C 3-8 cycloalkyl, C 3-8 cycloalkoxy, C 3-8 cycloalkylamino, aryl, heteroaryl, containing selected from A 3- to 8-membered heterocyclic group with 1-2 heteroatoms in N and O;
- R 5 is selected from H, halogen, cyano, nitro, amino, hydroxyl, trifluoromethyl, C 1-8 alkyl, C 1-8 alkoxy, C 1-8 alkylamino, C 3-8 Cycloalkyl, C 3-8 cycloalkoxy, C 1-8 alkylamino, aryl, heteroaryl;
- R 6 represents hydrogen, halogen, cyano, nitro, amino, hydroxyl, trifluoromethyl, trifluoroethyl, trifluoropropyl, C 1-3 heteroalkyl.
- R 1 , R 2 , R 3 , and R 4 are each independently selected from: halo, phenyl, C 1-3 alkyl, C 1-3 alkoxy, nitrogen-containing five- or six-membered Heterocyclic group; any hydrogen of R 1 , R 2 , R 3 , and R 4 can be substituted by C 1-3 alkyl or C 1-3 alkoxy; R 5 is aryl or heteroaryl; any of R 5 One hydrogen may be substituted by the following substituents: halo, C 1-3 alkyl, C 1-3 alkoxy, nitro, trifluoromethyl, cyano, methylsulfonyl; R 6 is hydrogen, C 1 -3 alkyl or C 1-3 alkoxy.
- the aryl and heteroaryl groups in R5 include but are not limited to: furan, thiophene, pyrrole, oxazole, thiazole, imidazole, pyrazole, benzofuran, benzothiophene, benzoxazole, benzothiazole, phenyl , Pyridine, pyridazine, pyrimidine, pyrazine, quinoline, naphthyl.
- the "pharmaceutically acceptable salt” of the present invention can be synthesized from the parent compound containing acid or base by conventional chemical methods. Generally, the preparation method of such a salt is: in water or an organic solvent or a mixture of both, It is prepared by reacting these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid.
- “Pharmaceutically acceptable salts” include, but are not limited to: inorganic acid salts, such as hydrochloride, hydrobromide, nitrate, sulfate, phosphate, etc.; organic acid salts, such as formate, acetate, Propionate, benzoate, maleate, fumaric acid, succinate, tartrate, citrate, etc.; alkyl sulfonate, such as methanesulfonate, ethylsulfonate, etc.; Aryl sulfonate, such as benzene sulfonate, p-toluene sulfonate, etc.
- the above-mentioned imidazo[1,2-a]pyridine compounds of the present invention specifically include 24 compounds of formula I-1 to formula I-24;
- the present invention also provides a method for preparing the above-mentioned compound, which is prepared by two routes, and the first route (reaction process) includes the following steps:
- the second route (reaction process) of the above-mentioned compound preparation method includes the following steps:
- the intermediate undergoes condensation reaction at room temperature to obtain the target product
- the present invention has discovered through a large number of experimental studies that the above-mentioned compounds can specifically inhibit STAT3.
- the present invention also provides the application of the above-mentioned compounds in the preparation of drugs for preventing and/or treating tumor growth and metastasis in vivo and in vitro;
- the tumors of the present invention include but are not limited to: acute lymphocytic leukemia, acute myeloid leukemia, adrenal cortical cancer, AIDS-related cancer, AIDS-related lymphoma, anal cancer, extrahepatic duct cancer, bladder cancer, bone cancer, brainstem glioma, brain tumor, bronchial adenoma, Burkitt’s lymphoma, carcinoid tumor, unknown primary Cancer, central nervous system lymphoma, cervical cancer, childhood cancer, germ cell tumor, eye cancer, stomach cancer, kidney cancer, laryngeal cancer, blood cancer, liver cancer, non-small cell lung cancer, melanoma, prostate cancer, rectal cancer, salivary glands Cancer, sarcoma, small bowel cancer, soft tissue sarcoma, uterine sarcoma, test
- the compound of the present invention or its pharmaceutically acceptable salts and solvates can significantly inhibit the proliferation, migration and invasion of various tumor cells in vitro. Therefore, the present invention also provides the compound in the preparation of inhibiting tumor cell proliferation, Application of drugs for migration and invasion.
- the present invention also provides the application of the above-mentioned compound or its pharmaceutically acceptable salts and solvates in the preparation of drugs for promoting tumor cell apoptosis.
- the tumor cells are breast cancer cells, lung cancer cells, gastric adenocarcinoma and/or gastric cancer cells.
- the present invention has studied the mechanism of the compound's inhibition of STAT3 through a series of experiments.
- the results show that the compound of the present invention can significantly inhibit the dimerization of STAT3 and the binding of STAT3 to DNA, and inhibit the tyrosine phosphorylation level of STAT3, and can inhibit the downstream of STAT3.
- the expression of target genes BCL-XL, C-myc and Mcl-1 is concentration-dependent. Therefore, the present invention also provides the application of the compound in the preparation of drugs that inhibit the tyrosine phosphorylation level of STAT3.
- the present invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt or solvate thereof in combination with an EGFR inhibitor; when the inhibitor is used in combination with an EGFR inhibitor , It can delay the emergence of EGFR drug-acquired drug resistance and prolong its clinical service life, which has important clinical significance.
- the present invention has the following technical effects:
- the present invention provides a class of imidazo[1,2-a]pyridine compounds and their pharmaceutically acceptable salts or pharmaceutically acceptable solvates.
- the compounds disclosed in the present invention have high selectivity for inhibiting STAT3 protein, It has the characteristics of strong drug effect, good druggability, safety, etc., and has a good application prospect in the preparation of drugs for diseases related to abnormal proliferation, morphological changes, and hyperkinesia of STAT3 high expression cells, as well as diseases related to angiogenesis or cancer metastasis. Especially suitable for the treatment and prevention of tumor growth and metastasis drugs.
- Fig. 1 Photo of six-well plate in the experiment of inhibitory effect of compound I-1 on the survival of breast cancer, lung cancer and gastric cancer cells;
- Fig. 2 Histogram of the number of colonies in each well in the experiment of compound I-1 on the survival of breast cancer, lung cancer and gastric cancer cells ;
- Figure 4 The inhibitory effect of compound I-1 on breast cancer cell invasion. Transwell experimental results; Figure 4(a) and Figure 4(b) are the invasion microscopic images; Figure 4(c) and Figure 4(d) are the invasion Cell histogram;
- FIG. 5 The Annexin V-FITC/PI double staining experiment results of the ability of compound I-1 to promote the apoptosis of breast cancer and gastric cancer cells; the upper 3 pictures study the breast cancer cells; the upper 3 pictures study the right Gastric cancer cells; I-1-1 ⁇ m represents the addition of 1 ⁇ m drug I-1; W1010-3 ⁇ m represents the addition of drug 3 ⁇ m;
- Figure 7 shows the experimental results of the fluorescent confocal method for the inhibitory activity of compound I-1 on the nuclear translocation of breast cancer and lung cancer cells p-STAT3;
- Figure 8 The experimental results of the fluorescent confocal method for the inhibitory activity of compound I-1 on STAT3 dimerization
- FIG. 9 shows the experimental results of gel migration (EMSA) method for the inhibitory activity of compound I-1 on breast cancer cell STAT3 binding to DNA;
- Figure 10 shows the experimental results of the dual luciferase reporter gene method for the effect of compound I-1 on the transcription activity of STAT3;
- Fig. 11 The relationship between the concentration of compound I-1 and the growth and proliferation of mouse model breast cancer (HCC70); Fig. 11(a) is the effect on tumor size; Fig. 11(b) is the effect on tumor weight;
- Figure 12 Morphology of the main organs of the mouse after the compound I-1 acts on the mouse model
- Figure 13 The relationship between the concentration of compound I-1 and the growth and proliferation in a mouse model of human tumor xenograft (PDX);
- Figure 13(a) shows the effect on tumor size;
- Figure 13(b) shows the effect on tumor weight;
- Figure 14 The morphological map of the main organs of the mouse after the compound I-1 acts on the mouse model of human tumor xenotransplantation (PDX).
- the mouse model used in Figures 11 and 12 is a nude mouse; the mouse model used in Figures 13 and 14 is a NOD-SCID mouse, and different models are used to show the organs twice to verify its influence on the organs.
- the equipment used in this embodiment, comparative example and experimental example are all conventional experimental equipment.
- the materials and reagents used are all commercially available without special instructions, and the experimental methods without special instructions are also conventional experimental methods.
- Step 2 Preparation of ethyl 2-(2-phenylimidazole[1,2-a]pyridin-3-yl)acetate
- DIPEA N,N-diisopropylethylamine
- Step 2 Preparation of ethyl 2-(7-chloro-2-phenylimidazole[1,2-a]pyridin-3-yl)acetate
- Step 3 Preparation of ethyl 2-(7-chloro-2-phenylimidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 4 Preparation of 2-(7-chloro-2-phenylimidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxybenzene[b]thiophen-6-yl) Acetamide (I-2)
- Step 2 Preparation of ethyl 2-(7-bromo-2-phenylimidazole[1,2-a]pyridin-3-yl) ethyl acetate
- Step 3 Preparation of ethyl 2-(7-bromo-2-phenylimidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 4 Preparation of 2-(7-bromo-2-phenylimidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxybenzene[b]thiophen-6-yl) Acetamide (I-3)
- Step 2 Preparation of ethyl 2-(7-methyl-2-phenylimidazole[1,2-a]pyridin-3-yl) ethyl acetate
- Step 3 Preparation of ethyl 2-(7-methyl-2-phenylimidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 4 Preparation of 2-(7-methyl-2-phenylimidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxybenzene[b]thiophen-6-yl )Acetamide (I-4)
- Step 2 Preparation of ethyl 2-(7-methoxy-2-phenylimidazole[1,2-a]pyridin-3-yl) ethyl acetate
- Step 3 Preparation of ethyl 2-(7-methoxy-2-phenylimidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 4 Preparation of 2-(7-methoxy-2-phenylimidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxybenzene[b]thiophene-6- Base) acetamide (I-5)
- Step 2 Preparation of ethyl 2-(8-methoxy-2-phenylimidazole[1,2-a]pyridin-3-yl) ethyl acetate
- Step 3 Preparation of ethyl 2-(8-methoxy-2-phenylimidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 4 Preparation of 2-(8-methoxy-2-phenylimidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxybenzene[b]thiophene-6- Yl)acetamide (I-6)
- Step 2 Preparation of ethyl 2-(6-methoxy-2-phenylimidazole[1,2-a]pyridin-3-yl)acetate
- Step 3 Preparation of ethyl 2-(6-methoxy-2-phenylimidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 4 Preparation of 2-(6-methoxy-2-phenylimidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxybenzene[b]thiophene-6- Yl)acetamide (I-7)
- Step 2 Preparation of ethyl 2-(2-(4-fluorophenyl-7-methoxyimidazole[1,2-a]pyridin-3-yl)acetate
- Step 3 Preparation of ethyl 2-(2-(4-fluorophenyl)-7-methoxyimidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 4 Preparation of 2-(2-(4-fluorophenyl)-7-methoxyimidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxybenzene[b ]Thien-6-yl)acetamide (I-8)
- Step 1 in 1 obtain a brown solid.
- Step 2 Preparation of ethyl 2-(2-(4-trifluoromethylphenyl-7-methoxyimidazole[1,2-a]pyridin-3-yl)acetate
- Step 3 Preparation of ethyl 2-(2-(4-trifluoromethylphenyl)-7-methoxyimidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 4 Preparation of 2-(2-(4-trifluoromethylphenyl)-7-methoxyimidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxy) Benzene[b]thiophen-6-yl)acetamide (I-9)
- Step 1 A white solid is obtained.
- Step 2 Preparation of ethyl 2-(2-(4-cyanophenyl-7-methoxyimidazole[1,2-a]pyridin-3-yl)acetate
- Step 3 Preparation of ethyl 2-(2-(4-cyanophenyl)-7-methoxyimidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 4 Preparation of 2-(2-(4-cyanophenyl)-7-methoxyimidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxybenzene[ b)Thien-6-yl)acetamide (I-10)
- step 1 a white solid is obtained.
- Step 2 Preparation of ethyl 2-(2-(4-nitrophenyl-7-methoxyimidazole[1,2-a]pyridin-3-yl)acetate
- Step 3 Preparation of ethyl 2-(2-(4-nitrophenyl)-7-methoxyimidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 4 Preparation of 2-(2-(4-nitrophenyl)-7-methoxyimidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxybenzene[ b)Thien-6-yl)acetamide (I-11)
- Step 1 A white solid is obtained.
- Step 2 Preparation of ethyl 2-(2-(4-methylthiophenyl-7-methoxyimidazole[1,2-a]pyridin-3-yl)acetate
- Step 3 Preparation of ethyl 2-(2-(4-methylthiophenyl)-7-methoxyimidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 4 Preparation of 2-(2-(4-methylthiophenyl)-7-methoxyimidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxybenzene) [b]Thien-6-yl)acetamide (I-13)
- Step 2 Preparation of ethyl 2-(7-methoxy-2-(thiophen-2-yl)imidazole[1,2-a]pyridin-3-yl)acetate
- Step 3 Preparation of ethyl 2-(7-methoxy-2-(thiophen-2-yl)imidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 4 Preparation of 2-(7-methoxy-2-(thiophen-2-yl)imidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxybenzene[b ]Thien-6-yl)acetamide (I-13)
- Step 1 Preparation of 7-methoxy-2-(naphthyl-2-yl)imidazole [1,2-a]pyridine
- Step 2 Preparation of ethyl 2-(7-methoxy-2-(naphthyl-2-yl)imidazole[1,2-a]pyridin-3-yl)acetate
- Step 3 Preparation of ethyl 2-(7-methoxy-2-(naphthyl-2-yl)imidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 4 Preparation of 2-(7-methoxy-2-(naphthyl-2-yl)imidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxybenzene[ b)Thien-6-yl)acetamide (I-14)
- Step 2 Preparation of ethyl 2-(6-bromo-2-phenylimidazole[1,2-a]pyridin-3-yl)acetate
- Step 3 Preparation of ethyl 2-(6-bromo-2-phenylimidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 4 Preparation of 2-(6-bromo-2-phenylimidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxybenzene[b]thiophen-6-yl) Acetamide (I-15)
- Step 2 Preparation of ethyl 2-(2-(4-cyanophenyl-6-methylimidazole[1,2-a]pyridin-3-yl)acetate
- Step 3 Preparation of ethyl 2-(2-(4-cyanophenyl)-6-methylimidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 4 Preparation of 2-(2-(4-cyanophenyl)-6-methylimidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxybenzene[b ]Thien-6-yl)acetamide (I-17)
- step 1 a white solid is obtained.
- Step 2 Preparation of ethyl 2-(2-(4-chloro-phenyl-6-methylimidazole[1,2-a]pyridin-3-yl)acetate
- Step 3 Preparation of ethyl 2-(2-(4-chloro-phenyl)-6-methylimidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 4 Preparation of 2-(2-(4-chloro-phenyl)-6-methylimidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxybenzene[b ]Thien-6-yl)acetamide (I-18)
- the preparation method is the same as step 1 in Example 1 to obtain a white solid.
- Step 2 Preparation of ethyl 2-(2-(4-methylphenylimidazole[1,2-a]pyridin-3-yl)acetate
- Step 3 Preparation of ethyl 2-(2-(4-methylphenyl)imidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 2 Preparation of ethyl 2-(2-(4-methylthiophenylimidazole[1,2-a]pyridin-3-yl)acetate
- Step 3 Preparation of ethyl 2-(2-(4-methylthiophenyl)imidazole[1,2-a]pyridin-3-yl)acetic acid
- the preparation method is the same as step 3 in Example 1 to obtain a white solid.
- Step 4 Preparation of 2-(2-(4-methylthiophenyl)imidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxybenzene[b]thiophene-6 -Yl)acetamide (I-20)
- the preparation method is the same as step 1 in Example 1 to obtain a white solid.
- Step 2 Preparation of ethyl 2-(2-(4-nitrophenylimidazole[1,2-a]pyridin-3-yl)acetate
- Step 3 Preparation of ethyl 2-(2-(4-nitrophenyl)imidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 1 Preparation of ethyl 2-(2,7-diphenylimidazole[1,2-a]pyridin-3-yl)acetate
- Step 2 Preparation of ethyl 2-(2,7-diphenylimidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 3 Preparation of 2-((2,7-diphenyl)imidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxybenzene[b]thiophen-6-yl )Acetamide (I-22)
- Step 1 Preparation of ethyl 2-(7-(4-methoxyphenyl)-2-phenylimidazole[1,2-a]pyridin-3-yl)acetate
- Step 2 Preparation of ethyl 2-(7-(4-methoxyphenyl)-2-phenylimidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 3 Preparation of 2-(7-(4-methoxyphenyl)-2-phenylimidazole[1,2-a]pyridin-3-yl)-N-(1,1-dioxybenzene[ b)Thien-6-yl)acetamide (I-23)
- Step 1 Preparation of ethyl 2-(7-(1-methyl-1H-pyrazol-4-yl)-2-phenylimidazole[1,2-a]pyridin-3-yl) ethyl acetate
- Step 2 Preparation of ethyl 2-(7-(1-methyl-1H-pyrazol-4-yl)-2-phenylimidazole[1,2-a]pyridin-3-yl)acetic acid
- Step 3 Preparation of 2-(7-(1-methyl-1H-pyrazol-4-yl)-2-phenylimidazole[1,2-a]pyridin-3-yl)-N-(1,1 -Dioxybenzene[b]thiophen-6-yl)acetamide (I-24)
- the compound of general formula inhibits the proliferation, survival, migration and invasion of breast cancer and gastric cancer cells and promotes their apoptosis
- the compound inhibits the proliferation of breast cancer, lung cancer, gastric adenocarcinoma cells and gastric cancer cells
- the cell viability assay was used to test the compound's inhibitory effect on cell viability.
- the experimental method is as follows: (1) Take the logarithmic growth phase cells, according to, experimental well: drug + cell + medium + CCK8, positive control well: drug solvent (SH4 -54) + cells + medium + CCK8, blank wells: medium + CCK8, measure the absorbance at 450 nm with a microplate reader. The general OD value is between 0.5-1.5, and the typical value is between 0.8-1.5.
- Table 1 shows the drug concentration (IC 50 ) when the cell viability is inhibited by half.
- compounds I-1, I-3, I-4, I-5, and I-9 can also significantly inhibit the proliferation of breast cancer cells and human gastric adenocarcinoma cells in vitro; although compound I-8 is effective against breast cancer cells There is no obvious inhibitory effect, but it can significantly inhibit the proliferation of gastric adenocarcinoma cells in vitro.
- the specific operations are: (1) Plating: Take logarithmic growth phase MDA-MB-231, MDA-MB-468, HCC70, A549, MGC-803 and AGS cells, discard the medium, wash with PBS, and digest with trypsin , The culture medium terminates the digestion, after centrifugation, the culture medium is resuspended to form a single cell suspension, the counting plate is counted, and 250-500 cells/ml cell suspension is prepared, and the cells are inoculated into a six-well plate at 2ml/well, and placed Incubate for 24h in a 37°C, 5% CO 2 incubator.
- Compound I-1 inhibits the migration of breast cancer and gastric cancer cells
- the cell scratch test method was used to test the inhibitory effect of compound I-1 on the migration of breast cancer and gastric cancer cells.
- the experimental method is as follows: (1) Plating: Take the logarithmic growth phase (the number of cells is about 80%-90%) MDA-MB -231, MDA-MB-468, HCC70, MGC-803 and AGS cells, discard the culture medium, wash with PBS, trypsinize, terminate the digestion of the culture medium, resuspend the culture medium to form a single cell suspension after centrifugation, and count on a counting plate After making 500,000-1,000,000 cells/ml cell suspension, inoculate the cells in a six-well plate at 2ml/well, and place them in a 37°C, 5% CO 2 incubator for 24 hours.
- Figure 3(a) shows the effect of compound I-1 on breast cancer cells
- Figure 3(a) shows the effect of compound I-1 on MDA-MB-468, MDA-MB-231, The effect of HCC70, after 96h, the results show that when the concentration of compound I-1 is getting higher and higher, the inhibitory effect on the migration of MDA-MB-468, MDA-MB-231 and HCC70 is more obvious
- Figure 3(b) is compound I, respectively The effect of -1 on MGC-803 and AGS, after 24 hours, the results showed that when the concentration of compound I-1 became higher and higher, the inhibitory effect on the migration of MGC-803 and AGS was more obvious.
- the method of Transwell experiment was used to test the inhibitory effect of compound I-1 on the invasion of breast cancer cells.
- the experimental method is as follows: (1) Transwell chamber pre-incubation: remove the nest used in a new 24-well plate, and add a blank medium (without serum) , No double antibody) soak in the incubator at 37°C for 1h.
- Compound I-1 promotes apoptosis of breast cancer and gastric cancer cells
- the Annexin V-FITC/PI double staining method was used to detect the ability of compound I-1 to promote the apoptosis of breast cancer and gastric cancer cells.
- the experimental method is as follows: (1) Plating: Take logarithmic growth phase MDA-MB-231 and MGC-803 cells , Discard the culture medium, wash once with PBS, trypsin digestion, stop the digestion of the culture medium, resuspend the culture medium to form a single cell suspension after centrifugation, count on a counting plate, and mix the cells into 500,000-800,000/ml cell suspension. Inoculate 2ml/well in a six-well plate and place it in a 37°C, 5% CO 2 incubator for 24 hours.
- Compound I-1 inhibits the phosphorylation level of STAT3 in cancer cells and the expression of its downstream target genes.
- the experimental method is as follows: (1) Protein sample extraction: Take MDA-MB- that has been treated with different concentrations of compound I-1 for the corresponding time.
- MDA-MB-468 and HCC70 cells placed on ice, discarded the culture medium, washed once with pre-cooled PBS, discarded PBS, added RIPA lysis buffer (containing protease and phosphatase inhibitor), lysed by shaking on ice for 15 min, Scrape the cells, place the cell culture plate at an angle for 5 minutes, transfer the cell lysate to a 1.5 mL centrifuge tube, vortex for 20 seconds, and let stand on ice for 5 minutes, centrifuge at 15000 rpm, 4°C for 15 minutes. Pipet the supernatant into a new 1.5mL centrifuge tube, and quantify the BCA.
- RIPA lysis buffer containing protease and phosphatase inhibitor
- Compound I-1 inhibits the nuclear translocation of cancer cells p-STAT3.
- the experimental method is as follows: (1) Plating: Take logarithmic growth phase MDA-MB-231 and MGC-803 cells, discard the culture medium, and wash once with PBS. Trypsin digestion, the culture medium terminates the digestion, after centrifugation, the culture medium is resuspended to form a single cell suspension, and the appropriate amount of cells is suspended evenly and dropped into the confocal dish. Let it stand for 30 minutes, and then transfer it to a 37°C, 5% CO 2 incubator. In, culture for 24h.
- Fluorescence confocal method was used to determine the inhibitory activity of compound I-1 on STAT3 dimerization.
- the experimental method is as follows: (1) Plating: Take the logarithmic growth phase (HEK-293T cells, discard the medium, wash once with PBS, trypsin After digestion, the culture medium terminates the digestion. After centrifugation, the culture medium is resuspended into a single cell suspension. The appropriate amount of cells is suspended evenly and dropped into a confocal dish. Let it stand for 30 minutes, then transfer it to a 37°C, 5% CO2 incubator, and cultivate 24h. (2) Transfection: Transfect HA-STAT3 and Flag-STAT3 plasmids into HEK-293T cells after 24h.
- Incubate the primary antibody discard the goat serum, add HA-tag and Flag-tag primary antibodies diluted with goat serum, and place in a humid box at 4°C overnight.
- Incubate the secondary antibody recover the primary antibody and place it in a shaker Wash the bed with PBS 3 times, 5min/time. Then add the fluorescent secondary antibody diluted with goat serum, and incubate for 1h at room temperature in the dark.
- Nucleus staining Discard the secondary antibody and wash 3 times with PBS on a shaker, 5min/time. Add DAPI and incubate for 10 min in the dark. After that, DAPI is discarded and placed on a shaker to wash 3 times with PBS, 5 min/time.
- Take pictures Take pictures with a laser scanning ultra-high resolution microscope (FV3000).
- Compound I-1 inhibits the binding of STAT3 to DNA in cancer cells
- the gel migration (EMSA) method was used to determine the inhibitory activity of compound I-1 on the binding of STAT3 to DNA in breast cancer cells.
- the experimental methods are as follows: (1) Nucleoprotein extraction: Biyuntian cell nuclear protein and cytoplasmic protein extraction kit ( P0027) Extract nucleoprotein. The details are as follows: Take MDA-MB-231 cells that have been treated with different concentrations of compound I-1 for 3 hours, place them on ice, discard the medium, wash once with pre-cooled PBS, discard PBS, scrape the cells with a cell scraper, and use Pipette down the cells. Collect the cells by centrifugation and try their best to aspirate the supernatant, leaving the cell pellet.
- EMSA with EMSA glue (TBE buffer (5x), 1mL; ddH2O, 5mL; acrylamide/bisacrylamide (30%, w/v), 1mL; glycerol, 250 ⁇ L; 10% ammonium persulfate, 100 ⁇ L; TEMED, 10 ⁇ L ); EMSA binding reaction (negative control: Nuclease-Free Water+EMSA/Gel-Shift binding buffer (5X)+STAT3 or STAT5 probe; sample reaction: Nuclease-Free Water+EMSA/Gel-Shift binding buffer (5X )+8 ⁇ g nucleoprotein+STAT3 or STAT5 probe; add various reagents in the above order, mix well before adding STAT3 or STAT5 probe, and place at room temperature for 10 minutes, then add probe, mix well, and place at room temperature for 20 minutes.
- EMSA glue TBE buffer (5x)
- ddH2O 5mL
- acrylamide/bisacrylamide 30%
- electrophoresis analysis (use 0.5xTBE as the electrophoresis solution, 100V, on ice, pre-electrophoresis for 30min. Then add the sample mixed with the loading buffer to the loading wells, and add 10 ⁇ L of diluted 1x to the excess loading wells Loading buffer (blue), used to observe the progress of electrophoresis. 100V on ice, 60-70min. Cut out a nylon membrane of the same size as the gel, soak it in 0.5XTBE for 10 minutes, transfer the membrane on ice, 380mA, 70min. Take out the membrane, UV cross-link for 15 minutes, then soak the membrane in the blocking solution (dissolved at 37°C), and seal at room temperature for 1 hour.
- compound I-1 can inhibit the binding of STAT3 to DNA in MDA-MB-231 cells, but does not affect the binding of STAT5 to DNA, that is, compound I-1 has a specific inhibitory effect on STAT3.
- the dual luciferase reporter gene method was used to determine the effect of compound I-1 on the transcriptional activity of STAT3.
- the experimental method is as follows: (1) Plating: Take HEK-293T cells in logarithmic growth phase, discard the culture medium, wash with PBS, and pancreas. After enzymatic digestion, the culture medium terminates the digestion. After centrifugation, the culture medium is resuspended into a single cell suspension, and 20,000 cells/well are inoculated in a 96-well plate. (2) Transfection: After 24 hours, transfect 50ng pGL3-STAT3+50ng STAT3C+40ng Renilla luciferase reporter gene plasmid TK-RL per well with lipo2000.
- Adding medicine adding different concentrations of compound I-1 after 24 hours of transfection, and treating for 24 hours.
- Renilla luciferase detection buffer For each sample, take an appropriate amount of Renilla luciferase detection buffer, add Renilla luciferase detection substrate (100X) according to 1:100 to prepare Renilla luciferase detection working solution. After the shaking, add 50 ⁇ L of firefly luciferase detection reagent to each well, beat it with a gun, and measure RLU1 (relative light unit). Take the reporter gene cell lysate as a blank control. After completing the above-mentioned determination of firefly luciferase step, add 100 ⁇ L of Renilla luciferase detection working solution, mix well with a pipette, and determine RLU2 (relative light unit). The ratio of RLU1/RLU2 was used to compare the differences in STAT3 transcription activity between different samples.
- Compound I-1 inhibits tumor growth and proliferation in animal models
- the establishment of a tumor-bearing mouse model Take the cancer cells in the logarithmic growth phase for centrifugal digestion, wash them with sterile PBS for 3 times, then count the cells, adjust the cell concentration to about 2*10 ⁇ 7 cells/ml, and then in the mouse 100 ⁇ L of cell suspension was injected subcutaneously on the ventral dorsal side.
- Experimental grouping After the tumor-bearing mouse model is established (about 1-3 weeks), the mice are randomly divided into a control group and an administration group, with about 6-10 mice in each group.
- Drug intervention After the model is established, drug intervention is started.
- the control group was intraperitoneally injected with 15% castor oil-containing PBS (medicine solvent group), and the administration group was intraperitoneally injected with the drug, the administration volume was 100 ⁇ L/head. It lasted for 3-4 weeks, during which the body weight and tumor volume of the mice were measured every day, and the behavioral status of the mice was monitored. Sample collection: After 3-4 weeks of administration, the mice were sacrificed by cervical vertebrae. The tumors were taken out, weighed and measured, and blood, heart, spleen, liver and other organs and tissues were taken for further pharmacology and toxicology the study.
- the specimens can be derived from tissue biopsy and surgical specimens for radical tumor resection. They are collected after the tumor is isolated.
- the fresh tumor tissues are completely immersed in a serum-free and anti-biological medium at 0°C. Cut the tumor tissue into 2*2*2mm tissue pieces with sterile tissue scissors, and wash them with culture medium three times. Under anesthesia, a small opening of about 3 mm was cut on the skin on both sides of the abdomen and back of the mouse under anesthesia, and a small pocket space was separated.
- the tumor tissue was planted under the skin and the wound was sutured. Drop 100 x double antibody solution on the wound to prevent infection.
- Each type of tumor was planted in 5 mice (F1), and the status of planted tumors was observed at least once a week. Observations include the presence or absence of tumor growth and the measurement of tumor volume.
- the transplanted tumor begins to grow into a size of 1-2cm3. Take out the tumor on the ventral and dorsal side of F1 mice, cut the tumor into 2*2*2mm tissue pieces with sterile tissue scissors, and soak in serum-free RPMI1640 medium for washing. The procedure is the same as the above, the tissue block is planted under the skin on both sides of the abdomen and back of the mouse, and 5 mice of each tumor are planted (F2). After tumor-bearing, the tumor to be transplanted grows to a size of 1-2cm3.
- F3 The tumor to be transplanted grows to a size of about 100mm3.
- F3 is randomly divided into control group and administration group. After grouping, the control group is intraperitoneally injected with 15% castor oil-containing PBS (medicine solvent), and the administration group is intraperitoneally injected with drugs and administration
- the volume is about 100 ⁇ L/mouse, during which the tumor volume and body weight are measured every day. After three to four weeks of continuous administration, the mice were killed by cervical dislocation. The tumor was taken out, weighed and volume, and blood, heart, spleen, liver and other organs and tissues were taken for further pharmacological and toxicological studies.
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Abstract
涉及药物化学和药物治疗学技术领域，具体公开了一类咪唑并[1,2-a]吡啶类化合物及其药学上可接受的盐或药学上可接受的溶剂合物，此外还公开了其制备方法、包含该化合物的药物组合物和用途，还公开了化合物具备抑制STAT3蛋白选择性高、药效强、成药性好、安全等特点，在制备STAT3高表达的细胞异常增殖、形态变化以及运动功能亢进等相关的疾病，以及与血管新生或癌症转移相关的疾病的药物中应用前景好，尤其适用于预防和/或治疗肿瘤生长与转移的药物。
Description
本发明涉及药物化学和药物治疗学领域，具体涉及咪唑并吡啶类化合物、包含该化合物的药物组合物及其制备方法和用途。
信号传导与转录激活因子(STAT)是一类双功能蛋白，具有信号转导和转录激活双重功能。其被不同的细胞因子和生长因子受体激活后，发生磷酸化、二聚化，从细胞质转位到细胞核内，与DNA结合后进而调控相应的靶基因的转录和表达。作为STAT家族的重要成员，STAT3负责调控细胞的生长、增值、分化以及凋亡等一系列重要生理过程。然而，研究发现STAT3持续性激活及异常高表达，能够诱导细胞增殖、侵袭、迁移，抑制细胞凋亡，促进血管生成，在肿瘤的发生发展转移过程中发挥重要的作用。因此，靶向STAT3抑制剂的研发已成为抗肿瘤药物研究前沿中的热点。
另外，STAT3还可以与细胞表面其他肿瘤相关分子相互作用，从而交联、激活、放大肿瘤的相关效应，极大程度上促进了肿瘤的发生发展与转移。因此抑制STAT3信号通路可以抑制多个肿瘤靶点发挥效应。其中令人关注的是，STAT3抑制剂同时可以作用于EGFR(Epidermal grovth factor reptor)发挥效应，当该抑制剂与EGFR抑制剂联合用药时，可以延缓EGFR药物获得性耐药的产生、延长其临床使用寿命，具有重要的临床意义。
综上所述，靶向STAT3的特异性抑制剂研究已经成为研发的热点，但现今为止已研发的药物，都存在药效作用小、选择性低、成药性差等不同程度、不同方面的缺点，限制了STAT3抑制剂的临床应用与后期开发。而STAT3作为一个具有前景的肿瘤治疗靶点，迫切需要开发新的一类化合物，能特异性的抑制STAT3。
发明内容
本发明的目的在于克服现有技术中的STAT3的特异性抑制剂药效作用小、选择性低、成效性差等问题，提供了一种咪唑并[1,2-a]吡啶类化合物。
本发明的第二目的是提供上述咪唑并[1,2-a]吡啶类化合物的制备方法。
本发明的第三个目的是提供上述咪唑并[1,2-a]吡啶类化合物的应用。
本发明的目的是通过以下技术方案予以实现的：
一种咪唑并[1,2-a]吡啶类化合物，所述化合物的结构通式如式(I)所示：
其中，n为0、1或2；
R
1、R
2、R
3、R
4各自独立地选自氢、卤素、氰基、硝基、氨基、羟基、三氟甲基、取代或非取代的C1-6烷基、取代或非取代的C1-6烷氧基、取代或非取代的C1-6烷基氨基，取代或非取代的C3-8的环烷基、取代或非取代的C3-8的环烷氧基、取代或非取代的C3-8的环烷基氨基、取代或非取代的芳基、取代或非取代的杂芳基、取代或非取代的含有选自N和O中的1-2个杂原子的3元至8元杂环基、-COR
a、-CO
2R
a、-CONR
aR
b、-NR
aC(O)R
b、-NR
aSO
2R
b、-SR
a、-SOR
a、-SO
2R
a、-SO
2NR
aR
b、-OC(O)R
a、-OC(O)NR
aR
b，所述取代是指至少1个位点被以下取代基取代：卤素、氰基、氨基、硝基、羟基、三氟甲基、C1-3烷基、C1-3烷氧基、C1-3烷基氨基；
其中所述R
a和R
b各自独立地为氢、C1-C6烷基、C3-C6环烷基，芳基，杂芳基；
R
5为氢、卤素、氰基、硝基、氨基、羟基、三氟甲基、取代或非取代的C1-8烷基、取代或非取代的C1-8烷氧基、取代或非取代的C1-C8烷基氨基、取代或非取代的C3-8的环烷基、取代或取代的C3-C8环烷氧基、取代或非取代的C1-8烷基氨基、取代或非取代的芳基或杂芳基，所述芳基或杂芳基为呋喃、噻吩、吡咯、恶唑、噻唑、咪唑、吡唑、苯并呋喃、苯并噻吩、苯并恶唑、苯并噻唑、苯基、吡啶、哒嗪、嘧啶、吡嗪、喹啉或萘基，所述取代是指至少1个位点被以下取代基取代：卤素、氰基、氨基、硝基、羟基、三氟甲基、甲硫基、C1-3烷基、C1-3烷氧基、C1-3烷基氨基；
R
6为氢、卤素、氰基、硝基、氨基、羟基、三氟C1-C3烷基、C1-C3烷氧基、C1-C3烷基氨基。
进一步地，R
1、R
2、R
3、R
4分别独立的选自H、卤素、氰基、硝基、氨基、羟基、三氟甲基、C
1-6烷基、C
1-6杂烷基烷氧基、C
1-6烷基氨基、C
3-8环烷基、C
3-8环烷氧基、C
3-8环烷基氨基、芳基、杂芳基、含有选自N和O中的1-2个杂原子的3元至8元杂环基；
R
5选自H、卤素、氰基、硝基、氨基、羟基、三氟甲基、C
1-8烷基、C
1-8烷氧基、C
1-8烷基氨基、C
3-8环烷基、C
3-8环烷氧基、C
1-8烷基氨基、芳基、杂芳基；
R
6代表氢、卤素、氰基、硝基、氨基、羟基、三氟甲基、三氟乙基、三氟丙基、C
1-3杂烷基。
更优选地，R
1、R
2、R
3、R
4分别独立的选自：卤基、苯基、C
1-3烷基、C
1-3烷氧基、含氮 的五元或六元杂环基；R
1、R
2、R
3、R
4中任意一个氢可C
1-3烷基、C
1-3烷氧基取代；R
5为芳基、杂芳基；R
5中任意一个氢可被以下取代基取代：卤基、C
1-3烷基、C
1-3烷氧基、硝基、三氟甲基、氰基、甲基磺酰基；R
6为氢、C
1-3烷基或C
1-3烷氧基。
进一步地，R
5上的氢取代发生在对位。
其中R5中的芳基、杂芳基包括但不限于：呋喃、噻吩、吡咯、恶唑、噻唑、咪唑、吡唑、苯并呋喃、苯并噻吩、苯并恶唑、苯并噻唑、苯基、吡啶、哒嗪、嘧啶、吡嗪、喹啉、萘基。
本发明的“药学上可以接受的盐”可由含有酸根或碱基的母体化合物通过常规化学方法合成，一般情况下，这样的盐的制备方法为：在水或有机溶剂或者两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。“药学上可以接受的盐”包括但不限于：无机酸盐，如盐酸盐、氢溴酸盐、硝酸盐、硫酸盐、磷酸盐等；有机酸盐，如甲酸盐、乙酸盐、丙酸盐、苯甲酸盐、马来酸盐、富马酸、琥珀酸盐、酒石酸盐、柠檬酸盐等；烷基磺酸盐，如甲基磺酸盐、乙基磺酸盐等；芳基磺酸盐，如苯磺酸盐、对甲苯磺酸盐等。
作为最优选的技术方案，本发明上述咪唑并[1,2-a]吡啶类化合物具体有式I-1到式I-24这24种化合物；
本发明还提供上述化合物的制备方法，其由两种路径制备得到，其第一种路径(反应过程)包括以下步骤：
S1.由式(1)和式(2)混于溶剂中，反应生成中间体(3)；
S2.中间体(3)在铑催化剂的催化作用下，生成中间体(4)；
S3.中间体(4)室温下酯水解成酸(5)，后再经过室温缩合反应，即得目标产物；
上述化合物的制备方法的第二种路径(反应过程)包括以下步骤：
S1.式(6)化合物经过铑催化剂的催化，生成中间体(7)；
S2.中间体经过SUZUKI偶联反应，生成中间体(8)；
S3.中间体经过室温缩合反应得目标产物；
本发明通过大量的实验研究发现，上述化合物能够特异性抑制STAT3。
本发明还提供上述化合物在制备预防和/或治疗肿瘤体内和体外生长与转移的药物的应用；本发明所述的肿瘤包括但不限于：急性淋巴细胞白血病、急性髓细胞白血病、肾上腺皮质癌、艾滋病相关癌症、艾滋病相关淋巴癌、肛门癌、肝胆外管癌、膀胱癌、骨癌、脑干胶质瘤、脑瘤、支气管腺瘤、伯基特氏淋巴瘤、类癌瘤、未知原发癌、中枢神经系统淋巴癌、子宫颈癌、儿童癌症、生殖细胞瘤、眼癌、胃癌、肾癌、喉癌、血癌、肝癌、非小细胞肺癌、黑素瘤、前列腺瘤、直肠癌、唾液腺癌、肉瘤、小肠癌、软组织肉瘤、子宫肉瘤、睾丸癌、以及乳腺癌等癌症。
通过实验研究发现，本发明的化合物或其药学上可以接受的盐、溶剂合物能够显著抑制体外多种肿瘤细胞增殖、迁移、侵袭，因此，本发明还提供上述化合物在制备抑制肿瘤细胞增殖、迁移、侵袭的药物的应用。
另外，研究还发现化合物I-1能够促进肿瘤细胞凋亡，因此本发明还提供上述化合物或其药学上可以接受的盐、溶剂合物在制备促进肿瘤细胞凋亡的药物的应用。
更优选的，所述肿瘤细胞为乳腺癌细胞、肺癌细胞、胃腺癌和/或胃癌细胞。
本发明通过一系列实验研究了化合物对STAT3抑制的机制，结果表明本发明的化合物能够显著抑制STAT3二聚化以及STAT3与DNA的结合，并抑制STAT3的酪氨酸磷酸化水平，能够抑制STAT3下游靶基因BCL-XL、C-myc和Mcl-1的表达，且具有浓度依赖性。因此本发明还提供化合物在制备抑制STAT3的酪氨酸磷酸化水平的药物的应用。
通过体内小鼠实验，考察化合物对体内肿瘤的影响，结果显著化合物本身对小鼠的脏器没有毒性，且化合物能够显著减小体内肿瘤的体积和重量，即明显抑制了肿瘤的生长增殖。
本发明还提供一种药物组合物，所述药物组合物包括所述化合物或其药学上可以接受的 盐、溶剂合物和EGFR抑制剂进行联用；当该抑制剂与EGFR抑制剂联合用药时，可以延缓EGFR药物获得性耐药的产生、延长其临床使用寿命，具有重要的临床意义。
与现有技术相比，本发明具有以下技术效果：
本发明提供了一类咪唑并[1,2-a]吡啶类化合物及其药学上可接受的盐，或药学上可接受的溶剂合物，本发明公开的化合物具备抑制STAT3蛋白选择性高、药效强、成药性好、安全等特点，在制备STAT3高表达的细胞异常增殖、形态变化以及运动功能亢进等相关的疾病，以及与血管新生或癌症转移相关的疾病的药物中应用前景好，尤其适用于治疗和预防肿瘤生长与转移的药物。
图1化合物I-1对乳腺癌、肺癌和胃癌细胞生存的抑制作用实验六孔板照片；图2化合物I-1对乳腺癌、肺癌和胃癌细胞生存的抑制作用实验中各孔集落数柱状图；
图3化合物I-1对乳腺癌和胃癌细胞迁移的抑制作用划痕实验结果图；
图4化合物I-1对乳腺癌细胞侵袭的抑制作用Transwell实验结果图；图4(a)和图4(b)为侵袭显微视野图；图4(c)和图4(d)为侵袭细胞柱状图；
图5化合物I-1促进乳腺癌和胃癌细胞凋亡的能力的Annexin V-FITC/PI双染法实验结果图；上面3幅图研究的是对乳腺癌细胞；上面3幅图研究的是对胃癌细胞；I-1-1μm代表加入1μm药物I-1；W1010-3μm代表加入药物3μm；
图6化合物I-1对乳腺癌细胞中STAT3的磷酸化水平及其下游靶基因表达的影响WesternBlot实验结果图；
图7化合物I-1对乳腺癌和肺癌细胞p-STAT3核转位的抑制活性的荧光共聚焦的方法的实验结果图；
图8化合物I-1对STAT3二聚化的抑制活性的荧光共聚焦方法实验结果图；
图9化合物I-1对乳腺癌细胞STAT3与DNA结合的抑制活性的凝胶迁移(EMSA)方法实验结果图；
图10化合物I-1对STAT3的转录活性的影响的双萤光素酶报告基因的方法实验结果图；
图11化合物I-1浓度与小鼠模型乳腺癌(HCC70)的生长增殖关系图；图11(a)为对肿瘤大小的影响；图11(b)为对肿瘤重量的影响；
图12化合物I-1作用小鼠模型之后，小鼠主要脏器形态图；
图13化合物I-1浓度与人源肿瘤异种移植(PDX)小鼠模型中的生长增殖关系图；图13(a)为对肿瘤大小的影响；图13(b)为对肿瘤重量的影响；
图14化合物I-1作用人源肿瘤异种移植(PDX)小鼠模型之后，小鼠主要脏器形态图。
其中，图11和12使用的小鼠模型是裸鼠；图13和14使用的小鼠模型是NOD-SCID小鼠，采用不同模型两次显示脏器，以验证其对脏器影响。
为使本发明的目的、技术方案和优点更加清楚，下面结合具体实施例和对比例将对本发明的技术方案进行详细的描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式，都属于本发明所保护的范围。
除特殊说明，本实施例、对比例以及实验例中所用的设备均为常规实验设备，所用的材料、试剂无特殊说明均为市售得到，无特殊说明的实验方法也为常规实验方法。
实施例1
N-(1,1-二氧基苯[b]噻吩-6-基)-2-(2-苯基咪唑[1,2-a]吡啶-3-基)乙酰胺(I-1)的结构如下所示
步骤1：制备2-苯咪唑[1,2-a]吡啶
步骤2：制备2-(2-苯基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备2-(2-苯基咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备(1,1-二氧基苯[b]噻吩-6-基)-2-(2-苯基咪唑[1,2-a]吡啶-3-基)乙酰胺(I-1)
唑(HOAt),以及156毫克的N,N-二异丙基乙胺(DIPEA)，在室温下搅拌4小时，反应完全，用乙酸乙酯以及水萃取后，有机相用无水硫酸钠干燥，将溶剂旋干后柱层析(二氯甲烷：甲醇＝20:1，V/V)得淡黄色固体82毫克，产率为：49％。
1H NMR(400MHz,DMSO)δ10.99(s,1H),8.46(d,J＝6.7Hz,1H),8.17(s,1H),7.84–7.76(m,3H),7.65(d,J＝9.0Hz,1H),7.59(dd,J＝11.5,7.6Hz,2H),7.50(t,J＝7.4Hz,2H),7.39(t,J＝7.2Hz,1H),7.34–7.27(m,2H),6.98(t,J＝6.7Hz,1H),4.36(s,2H)；
13C NMR(101MHz,DMSO)δ168.6,144.5,143.5,141.8,137.7,134.8，133.2,130.5，129.1,128.3,128.0,127.0，126.1,125.5,125.1,123.8,117.1,115.1,112.4,111.9,32.3.HRMS calcd for C
23H
17N
3O
3S(M
+):416.1063,found:416.1062.
实施例2
2-(7-氯-2-苯基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-2)的结构如下所示
步骤1：制备7-氯-2-苯基咪唑[1,2-a]吡啶
步骤2：制备乙基2-(7-氯-2-苯基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备乙基2-(7-氯-2-苯基咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备2-(7-氯-2-苯基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-2)
实施例3
2-(7-溴-2-苯基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-3)的结构如下所示
步骤1：制备7-溴-2-苯基咪唑[1,2-a]吡啶
步骤2：制备乙基2-(7-溴-2-苯基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备乙基2-(7-溴-2-苯基咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备2-(7-溴-2-苯基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-3)
实施例4
2-(7-甲基-2-苯基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-4)的结构如下所示
步骤1：制备7-甲基-2-苯基咪唑[1,2-a]吡啶
步骤2：制备乙基2-(7-甲基-2-苯基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备乙基2-(7-甲基-2-苯基咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备2-(7-甲基-2-苯基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-4)
实施例5
2-(7-甲氧基-2-苯基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-5)的结构如下所示
步骤1：制备7-甲氧基-2-苯基咪唑[1,2-a]吡啶
步骤2：制备乙基2-(7-甲氧基-2-苯基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备乙基2-(7-甲氧基-2-苯基咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备2-(7-甲氧基-2-苯基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-5)
实施例6
2-(8-甲氧基-2-苯基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-6)的结构如下所示
步骤1：制备8-甲氧基-2-苯基咪唑[1,2-a]吡啶
步骤2：制备乙基2-(8-甲氧基-2-苯基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备乙基2-(8-甲氧基-2-苯基咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备2-(8-甲氧基-2-苯基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-6)
实施例7
2-(6-甲氧基-2-苯基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-7)的结构如下所示
步骤1：制备6-甲氧基-2-苯基咪唑[1,2-a]吡啶
步骤2：制备乙基2-(6-甲氧基-2-苯基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备乙基2-(6-甲氧基-2-苯基咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备2-(6-甲氧基-2-苯基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-7)
实施例8
2-(2-(4-氟苯基)-7-甲氧基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-8)的结构如下所示
步骤1：制备2-(4-氟苯基)-7-甲氧基咪唑[1,2-a]吡啶
步骤2：制备乙基2-(2-(4-氟苯基-7-甲氧基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
1H NMR(400MHz,CDCl
3)δ7.98(dd,J＝14.3,6.8Hz,1H),7.90–7.77(m,2H),7.17(dt,J＝17.3,8.7Hz,2H),6.99(dd,J＝11.2,9.2Hz,1H),6.61(dd,J＝10.4,3.0Hz,1H),4.24(q,2H),3.90(d,J＝30.0Hz,5H),1.30(t,J＝6.9Hz,3H).EI-MS:m/z(M+H
+):328.12.
步骤3：制备乙基2-(2-(4-氟苯基)-7-甲氧基咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备2-(2-(4-氟苯基)-7-甲氧基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-8)
实施例9
2-(2-(4-三氟甲基苯基)-7-甲氧基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-9)的结构如下所示
步骤1：制备2-(4-三氟甲基苯基)-7-甲氧基咪唑[1,2-a]吡啶
步骤2：制备乙基2-(2-(4-三氟甲基苯基-7-甲氧基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备乙基2-(2-(4-三氟甲基苯基)-7-甲氧基咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备2-(2-(4-三氟甲基苯基)-7-甲氧基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-9)
实施例10
2-(2-(4-氰基苯基)-7-甲氧基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-10)的结构如下所示
步骤1：制备2-(4-氰基苯基)-7-甲氧基咪唑[1,2-a]吡啶
步骤2：制备乙基2-(2-(4-氰基苯基-7-甲氧基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备乙基2-(2-(4-氰苯基)-7-甲氧基咪唑[1,2-a]吡啶-3-基)乙酸
1H NMR(400MHz,MeOD)δ8.65(s,1H),7.97(d,J＝26.4Hz,4H),7.26(d,J＝34.0Hz,2H),4.15(d,J＝48.7Hz,5H).EI-MS:m/z(M+H
+):307.10.
步骤4：制备2-(2-(4-氰基苯基)-7-甲氧基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-10)
实施例11
2-(2-(4-硝基苯基)-7-甲氧基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-11)的结构如下所示
步骤1：制备2-(4-硝基苯基)-7-甲氧基咪唑[1,2-a]吡啶
步骤2：制备乙基2-(2-(4-硝基苯基-7-甲氧基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备乙基2-(2-(4-硝基苯基)-7-甲氧基咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备2-(2-(4-硝基苯基)-7-甲氧基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-11)
实施例12
2-(2-(4-甲硫苯基)-7-甲氧基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-12)的结构如下所示
步骤1：制备2-(4-甲硫基苯基)-7-甲氧基咪唑[1,2-a]吡啶
步骤2：制备乙基2-(2-(4-甲硫基苯基-7-甲氧基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备乙基2-(2-(4-甲硫基苯基)-7-甲氧基咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备2-(2-(4-甲硫基苯基)-7-甲氧基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-13)
实施例13
2-(2-(7-甲氧基-2-(噻吩-2-基)咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-13)的结构如下所示
步骤1：制备7-甲氧基-2-(噻吩-2-基)咪唑[1,2-a]吡啶
步骤2：制备乙基2-(7-甲氧基-2-(噻吩-2-基)咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备乙基2-(7-甲氧基-2-(噻吩-2-基)咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备2-(7-甲氧基-2-(噻吩-2-基)咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-13)
实施例14
2-(2-(7-甲氧基-2-(萘基-2-基)咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-14)的结构如下所示
步骤1：制备7-甲氧基-2-(萘基-2-基)咪唑[1,2-a]吡啶
步骤2：制备乙基2-(7-甲氧基-2-(萘基-2-基)咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备乙基2-(7-甲氧基-2-(萘基-2-基)咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备2-(7-甲氧基-2-(萘基-2-基)咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻 吩-6-基)乙酰胺(I-14)
实施例15
2-(6-溴-2-苯基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-15)的结构如下所示
步骤1：制备6-溴-2-苯基咪唑[1,2-a]吡啶
步骤2：制备乙基2-(6-溴-2-苯基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备乙基2-(6-溴-2-苯基咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备2-(6-溴-2-苯基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-15)
实施例16
2-(2-(4-甲基)-6-甲基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-16)的结构如下所示
实施例17
2-(2-(4-氰基苯基)-6-甲基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-11)的结构如下所示
步骤1：制备2-(4-氰基苯基)-6-甲基咪唑[1,2-a]吡啶
步骤2：制备乙基2-(2-(4-氰基苯基-6-甲基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备乙基2-(2-(4-氰基苯基)-6-甲基咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备2-(2-(4-氰基苯基)-6-甲基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-17)
实施例18
2-(2-(4-氯-苯基)-6-甲基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-18)的结构如下所示
步骤1：制备2-(4-氯-苯基)-6-甲基咪唑[1,2-a]吡啶
步骤2：制备乙基2-(2-(4-氯-苯基-6-甲基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备乙基2-(2-(4-氯-苯基)-6-甲基咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备2-(2-(4-氯-苯基)-6-甲基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-18)
实施例19
2-(2-(4-甲基苯基)咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-19)的结构如下所示
步骤1：制备2-(4-甲基苯基)咪唑[1,2-a]吡啶
步骤2：制备乙基2-(2-(4-甲基苯基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备乙基2-(2-(4-甲基苯基)咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备2-(2-(4-甲基苯基)咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-19)
实施例20
2-(2-(4-甲硫基苯基)咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-20)的结构如下所示
步骤1：制备2-(4-甲硫基苯基)咪唑[1,2-a]吡啶
步骤2：制备乙基2-(2-(4-甲硫基苯基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备乙基2-(2-(4-甲硫基苯基)咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备2-(2-(4-甲硫基苯基)咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-20)
实施例21
2-(2-(4-硝基苯基)咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-21)的结构如下所示
步骤1：制备2-(4-硝基苯基)咪唑[1,2-a]吡啶
步骤2：制备乙基2-(2-(4-硝基苯基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤3：制备乙基2-(2-(4-硝基苯基)咪唑[1,2-a]吡啶-3-基)乙酸
步骤4：制备2-(2-(4-硝基苯基)咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-21)
实施例22
2-((2,7-二苯基)咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-22)的结构如下所示
步骤1：制备乙基2-(2，7-二苯基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤2：制备乙基2-(2，7-二苯基咪唑[1,2-a]吡啶-3-基)乙酸
步骤3：制备2-((2,7-二苯基)咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-22)
实施例23
2-(7-(4-甲氧基苯基)-2-苯基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-23)的结构如下所示
步骤1：制备乙基2-(7-(4-甲氧基苯基)-2-苯基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤2：制备乙基2-(7-(4-甲氧基苯基)-2-苯基咪唑[1,2-a]吡啶-3-基)乙酸
步骤3：制备2-(7-(4-甲氧基苯基)-2-苯基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-23)
实施例24
2-(7-(1-甲基-1H-吡唑-4-基)-2-苯基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-24)的结构如下所示
步骤1：制备乙基2-(7-(1-甲基-1H-吡唑-4-基)-2-苯基咪唑[1,2-a]吡啶-3-基)乙酸乙酯
步骤2：制备乙基2-(7-(1-甲基-1H-吡唑-4-基)-2-苯基咪唑[1,2-a]吡啶-3-基)乙酸
步骤3：制备2-(7-(1-甲基-1H-吡唑-4-基)-2-苯基咪唑[1,2-a]吡啶-3-基)-N-(1,1-二氧基苯[b]噻吩-6-基)乙酰胺(I-24)
生物活性测试部分：
一.通式化合物抑制乳腺癌和胃癌细胞增殖、生存、迁移和侵袭并促进其凋亡
1.化合物抑制乳腺癌、肺癌、胃腺癌细胞和胃癌细胞增殖
采用细胞活力测定的方法测试化合物对细胞活性抑制作用，实验方法如下：(1)取对数生长期细胞，按照，实验孔：药物+细胞+培养基+CCK8，阳性对照孔：药物溶剂(SH4-54)+细胞+培养基+CCK8，空白孔：培养基+CCK8，用酶标仪测量在450nm处的吸光度。一般的OD值在0.5-1.5之间，典型的在0.8-1.5之间。按以下公式计算细胞活性：细胞活力 (％)＝(实验孔OD值-空白孔OD值)/(阴性对照孔OD值-空白孔OD值)*100％。其中，表1为细胞活力被抑制一半时药物的浓度(IC
50)。
表1
从表1结果中可以看出，化合物I-1到化合物I-24均能够抑制肺癌细胞和胃癌细胞体外增殖；这里，以STAT的抑制剂SH4-54为阳性独照，显示化合物I-10、I-12、I-20、I-21抑制肺癌细胞和胃癌细胞体外增殖的效果与抑制剂SH4-54的效果相当，其他化合物抑制肺癌细胞和胃癌细胞体外增殖的效果基本优于抑制剂SH4-54。
除此之外，化合物I-1、I-3、I-4、I-5、I-9还能够显著抑制乳腺癌细胞和人胃腺癌细胞的体外增殖；化合物I-8虽然对乳腺癌细胞没有明显的抑制作用，但是能够显著抑制胃腺癌细胞的体外增殖。
2.下面以化合物I-1为例，说明通式化合物抑制乳腺癌、肺癌和胃癌细胞生存的抑制作用：采用细胞平板克隆实验的方法测试化合物I-1对乳腺癌、肺癌和胃癌细胞生存的抑制作 用，具体操作为：(1)铺板：取对数生长期MDA-MB-231、MDA-MB-468、HCC70、A549、MGC-803和AGS细胞，弃培养基，PBS清洗，胰酶消化，培养基终止消化，离心后用培养基重悬成单细胞悬液，计数板计数，配成250-500个/ml细胞悬液后，将细胞按2ml/孔接种于六孔板，置于37℃、5％CO
2培养箱培养24h。(2)加药：24h后加入不同浓度I-1，并设置空白组(不做处理，Blank)，对照组(DMSO,C)和实验孔(加入药物)，每种药物及浓度均设3个复孔。置于37℃、5％CO2培养箱培养10-15天。(3)固定染色：10-15天后，肉眼可见细胞克隆集落，弃培养基，PBS洗一次，加入4％多聚甲醛，约1ml/孔，固定15min，之后弃多聚甲醛，PBS洗2次，加入结晶紫，约500ul/孔，避光染色30min，之后弃结晶紫，PBS洗至背景干净，室温晾干。(4)拍照并统计每孔集落数，用GraphPad Prism软件进行统计学分析。
结果如图1、图2所示，当化合物I-1的浓度分别为0.03μm、0.1μm、0.3μm和1μm时，肺癌细胞、胃腺癌细胞和胃癌细胞的克隆集落越来越少，说明化合物I-1对肺癌细胞、胃腺癌细胞和胃癌细胞的生存抑制越来越明显，且呈浓度依赖性。另外，当化合物I-1的浓度分别为0.1μm、0.3μm和1μm时，三种乳腺癌系细胞(MDA-MB-231、MDA-MB-468、HCC70)的克隆集落也越来越少，说明化合物I-1对三种乳腺癌系细胞(MDA-MB-231、MDA-MB-468、HCC70)的生存抑制越来越明显，且呈浓度依赖性。
3.化合物I-1抑制乳腺癌和胃癌细胞迁移
采用细胞划痕实验的方法测试化合物I-1对乳腺癌和胃癌细胞迁移的抑制作用，实验方法如下：(1)铺板：取对数生长期(细胞数量约80％-90％)MDA-MB-231、MDA-MB-468、HCC70、MGC-803和AGS细胞，弃培养基，PBS清洗，胰酶消化，培养基终止消化，离心后用培养基重悬成单细胞悬液，计数板计数，配成50万-100万个/ml细胞悬液后，将细胞按2ml/孔接种于六孔板，置于37℃、5％CO
2培养箱培养24h。(2)划痕并加药：24h后细胞密度大于90％时，用枪头比着直尺划痕，之后弃培养基，用PBS洗2次，加入新鲜培养基，显微镜下拍照，加入不同浓度化合物I-1，设置对照组(加入药物溶剂,标记为0组)和实验孔(加入不同浓度的化合物I-1)。置于37℃、5％CO
2培养箱培养24-96h。(3)拍照统计：24-96h后，弃培养基，PBS洗2次，显微镜下拍照。
测试结果如图3所示，图3(a)为化合物I-1对乳腺癌细胞的影响，其中图3(a)分别为化合物I-1对MDA-MB-468、MDA-MB-231、HCC70的影响，在96h后结果表明化合物I-1浓度越来越高时，对MDA-MB-468、MDA-MB-231、HCC70迁移的抑制效果越明显；图3(b)分别为化合物I-1对MGC-803和AGS的影响，在24h后结果表明化合物I-1浓度越来越高时，对MGC-803和AGS迁移的抑制效果越明显。
4.化合物I-1抑制乳腺癌细胞侵袭
采用Transwell实验的方法测试化合物I-1对乳腺癌细胞侵袭的抑制作用，实验方法如下：(1)Transwell小室预孵育：取出所用嵌套至一新24孔板中，加入空白培养基(无血清，无双抗)浸泡，37℃培养箱中静置1h。(2)铺板：取对数生长期MDA-MB-231和HCC70细胞，弃培养基，PBS清洗，胰酶消化，培养基终止消化，离心后用2％FBS培养基重悬成单细胞悬液，计数板计数，配成10万-30万个/ml细胞悬液后，将细胞按300μL/孔接种于transwell小室上层，下层加入500μL/孔的2％FBS培养基，室温静置30min，之后置于37℃、5％CO
2培养箱培养24h。(3)加药：24h后，吸去小室上下层培养基，下层加入500μL的20％FBS培养基，上层加入300μL含不同浓度的化合物I-1的2％FBS培养基，并设置对照组和实验组。置于37℃、5％CO2培养箱培养12h。(4)固定并染色：12h后弃上下层培养基，PBS洗一次，用棉签刮去小室上层细胞，PBS洗上层2-3次，洗去残留细胞，之后用4％多聚甲醛固定15min，PBS洗2次，加入结晶紫避光染色30min。PBS洗去残留结晶紫，室温晾干小室。(5)拍照统计：晾干后，显微镜下随机选取5个视野拍照，用Image J进行统计。
结果如图4所示，从图4(a)、图4(b)和图4(c)中可以看出，化合物I-1浓度越来越高时，对乳腺癌细胞系中的MDA-MB-231和HCC70的抑制效果越来越明显，即化合物I-1可抑制乳腺癌细胞的侵袭，且随浓度升高抑制越强，并具有统计学差异。
5.化合物I-1促进乳腺癌和胃癌细胞凋亡
采用Annexin V-FITC/PI双染法检测化合物I-1促进乳腺癌和胃癌细胞凋亡的能力，实验方法如下：(1)铺板：取对数生长期MDA-MB-231和MGC-803细胞，弃培养基，PBS洗一次，胰酶消化，培养基终止消化，离心后用培养基重悬成单细胞悬液，计数板计数，配成50-80万/ml细胞悬液后，将细胞按2ml/孔接种于六孔板，置于37℃、5％CO
2培养箱培养24h。(2)加药：加入不同浓度的化合物I-1于细胞中，处理48h。(3)检测：采用贝博Annexin V-FITC细胞凋亡检测试剂盒(BB-4101-1)进行检测。弃培养基，PBS清洗，用不含EDTA的胰酶消化，收集并离心，用冷PBS洗2次，吸尽PBS。用400μL 1ⅹAnnexin V结合液悬浮细胞，在悬浮液中加入5μL Annexin V-FITC染色液，轻轻混匀后2冰上避光孵育15min，加入10μL PI染色液后轻轻混匀于冰上避光孵育5min。立即用流式细胞仪检测。之后用FlowJo 7.6软件处理。
结果如图5所示，由图5可知，化合物I-1可促进乳腺癌和胃癌细胞的凋亡，尤其是早期凋亡，且具有浓度依赖性。
二.下面以化合物I-1为例，说明本发明通式化合物抑制癌细胞生长的机制
1.化合物I-1抑制癌细胞STAT3的磷酸化水平及其下游靶基因的表达，实验方法如下： (1)蛋白样品提取：取已经被不同浓度化合物I-1处理相应时间的MDA-MB-231、MDA-MB-468和HCC70细胞，置于冰上，弃培养基，预冷PBS洗一次，弃PBS，加入RIPA裂解液(含蛋白酶和磷酸酶抑制剂)，冰上震荡裂解15min，用细胞刮刮取细胞，斜置细胞培养板5min，将细胞裂解液转移至一1.5mL离心管中，涡旋20s，冰上静置5min，15000rpm，4℃离心15min。吸取上清至一新1.5mL离心管中，BCA定量。确定浓度后稀释成蛋白总量相等的蛋白样品，加入5ⅹLoading buffer，100℃变性5min，-80℃暂存。(2)Western Blot：将上述蛋白样品按每孔等蛋白量加入凝胶中，电泳，电泳完后，冰上转膜。转膜完成后，将膜浸泡至5％BSA中，置于摇床上室温封闭1h。之后将膜裁剪，转移至pY705-STAT3、pS727-STAT3、STAT3、β-actin、BCL-XL、C-myc和Mcl-1一抗中4℃过夜孵育。一抗孵育结束后，用TBST洗3次，10min/次。之后将膜转移至鼠二抗或兔二抗中室温孵育1h。二抗孵育完后，用TBST洗3次，10min/次。现配显影液(A液：B液＝1：1)，使用化学发光显影仪进行显影。
通过蛋白质免疫印迹法，分别检测不同癌细胞中STAT3的705酪氨酸磷酸化水平、727丝氨酸磷酸化水平、STAT3下游靶基因BCL-XL、C-myc和Mcl-1的表达；同时以β-actin为内参，结果入图6，由图6可知，化合物I-1可在短时间内(3h)显著抑制STAT3的705酪氨酸磷酸化水平，具有浓度依赖性，但不影响727丝氨酸的磷酸化水平。同时，它还能抑制STAT3下游靶基因BCL-XL、C-myc和Mcl-1的表达，具有浓度依赖性。
2.化合物I-1抑制癌细胞p-STAT3的核转位，实验方法如下：(1)铺板：取对数生长期MDA-MB-231和MGC-803细胞，弃培养基，PBS洗一次，胰酶消化，培养基终止消化，离心后用培养基重悬成单细胞悬液，将适量细胞悬空均匀地滴入confocal皿中，静置30min，平稳转移至37℃、5％CO
2培养箱中，培养24h。(2)加药：24h后向confocal皿中加入不同浓度W1078，处理12-18h，加入100ng/mL IL-6刺激30min。(3)固定：6h后，弃培养基，PBS洗一遍，加入4％多聚甲醛固定15min。(4)透膜：弃多聚甲醛，置于摇床PBS洗3次，5min/次，加入0.3％Triton-X100透膜10min，之后置于摇床PBS洗3次，5min/次。(5)封闭：加入山羊血清，置于摇床室温封闭1h。(6)孵一抗：弃山羊血清，加入用山羊血清稀释的pY705-STAT3一抗，置于湿盒内，4℃过夜。(7)孵二抗：回收一抗，置于摇床PBS洗3次，5min/次。之后加入用山羊血清稀释的荧光二抗，室温避光孵育1h。(8)染核：弃二抗，置于摇床PBS洗3次，5min/次。加入DAPI避光孵育10min。之后弃DAPI，置于摇床PBS洗3次，5min/次。(9)拍照：用激光扫描超高分辨率显微镜(FV3000)进行拍照。
结果如图7所示，由图7可知，化合物I-1能显著抑制MDA-MB-231和A549细胞中pY705-STAT3的核转位，具有浓度依赖性。
3.化合物I-1抑制STAT3二聚化
采用荧光共聚焦的方法测定化合物I-1对STAT3二聚化的抑制活性，实验方法如下：(1)铺板：取对数生长期(HEK-293T细胞，弃培养基，PBS洗一次，胰酶消化，培养基终止消化，离心后用培养基重悬成单细胞悬液，将适量细胞悬空均匀地滴入confocal皿中，静置30min，平稳转移至37℃、5％CO2培养箱中，培养24h。(2)转染：24h后向HEK-293T细胞中转染HA-STAT3和Flag-STAT3质粒(3)加药：24h后向HEK-293T中加入不同浓度的化合物I-1，处理24h，之后加入100ng/mL IL-6处理1h。(4)固定：弃培养基，PBS洗一遍，加入4％多聚甲醛固定15min。(5)透膜：弃多聚甲醛，置于摇床PBS洗3次，5min/次，加入0.3％Triton-X100透膜10min，之后置于摇床PBS洗3次，5min/次。(6)封闭：加入山羊血清，置于摇床室温封闭1h。(7)孵一抗：弃山羊血清，加入用山羊血清稀释的HA-tag和Flag-tag一抗，置于湿盒内，4℃过夜。(8)孵二抗：回收一抗，置于摇床PBS洗3次，5min/次。之后加入用山羊血清稀释的荧光二抗，室温避光孵育1h。(9)染核：弃二抗，置于摇床PBS洗3次，5min/次。加入DAPI避光孵育10min。之后弃DAPI，置于摇床PBS洗3次，5min/次。(10)拍照：用激光扫描超高分辨率显微镜(FV3000)进行拍照。
结果如图8所示，图中黄色部分明显减少，即HA-STAT3与Flag-STAT3的共定位减少，说明化合物I-1抑制了STAT3二聚体的形成，即化合物I-1抑制了STAT3二聚化。
4.化合物I-1抑制癌细胞STAT3与DNA结合
采用凝胶迁移(EMSA)方法测定化合物I-1对乳腺癌细胞STAT3与DNA结合的抑制活性，实验方法如下：(1)核蛋白提取：采用碧云天细胞核蛋白与细胞浆蛋白抽提试剂盒(P0027)提取核蛋白。具体如下：取已经被不同浓度的化合物I-1处理3h的MDA-MB-231细胞，置于冰上，弃培养基，预冷PBS洗一次，弃PBS，用细胞刮子刮下细胞，并用移液器吹打下细胞。离心收集细胞，尽最大努力吸尽上清，留下细胞沉淀。每20μL细胞沉淀加入200μL含1％PMSF的细胞浆蛋白抽提试剂A。最高速剧烈Vortex 5秒，把细胞沉淀完全悬浮并分散开。冰浴10-15分钟。加入细胞浆蛋白抽提试剂B 10μL。最高速剧烈Vortex 5秒，冰浴1分钟。最高速剧烈Vortex 5秒，4℃ 12,000-16,000g离心5min。完全吸尽上清，加入50μL含1％PMSF的细胞核蛋白抽提试剂。最高速剧烈Vortex 15-30秒，把细胞沉淀完全悬浮并分散开。然后放回冰浴中，每隔1-2min再高速剧烈Vortex 15-30秒，共30min。4℃ 12,000-16,000g离心10min。立即吸取上清至一预冷的塑料管中，即为抽提得到的细胞核蛋白。BCA定量，-80℃暂存。(2)EMSA：配EMSA胶(TBE buffer(5ⅹ),1mL；ddH2O,5mL；acrylamide/bisacrylamide(30％,w/v),1mL；甘油，250μL；10％过硫酸铵，100μL；TEMED,10μL)；EMSA结合反应(阴性对照：Nuclease-Free Water+EMSA/Gel-Shift结合缓冲液(5X)+STAT3或STAT5探针；样品反应：Nuclease-Free Water+EMSA/Gel-Shift结合缓冲液(5X)+8μg细胞核蛋白+STAT3或STAT5探 针；按上述顺序依次加入各种试剂，在加入STAT3或STAT5探针前先混匀，并室温放置10min，之后加入探针，混匀，室温放置20min。)；电泳分析(用0.5ⅹTBE作为电泳液，100V，冰上，预电泳30min。然后把混合上样缓冲液的样品加入到上样孔内，在多余的上样孔内加入10μL稀释好的1ⅹ上样缓冲液(蓝色)，用于观察电泳进行情况。冰上100V，60-70min。剪取与胶大小一致的尼龙膜，用0.5XTBE浸泡10分钟，冰上转膜，380mA，70min。将膜取出，紫外交联15min，然后把膜浸泡在封闭液(37℃溶解)中，室温封闭1h。取50μL Streptavidin-HRP Conjugate加入到15ml封闭液中(1:300稀释)，混匀备用。封闭结束后，去除用于尼龙膜封闭的封闭液，加入上一步中配制的15ml含有Streptavidin-HRP Conjugate的封闭液。在水平摇床上缓慢摇动15分钟。之后弃封闭液，加入洗脱液(37℃溶解，ddH
2O稀释至1ⅹ)，洗4次，10min/次。现配化学发光工作液(A:B＝1:1)，在尼龙膜表面加入工作液，使工作液完全覆盖尼龙膜，室温2min，用化学发光显影仪进行显影。
结果如图9所示，化合物I-1腺癌细胞STAT3与DNA结合选择性抑制的凝胶迁移(EMSA)实验结果图，显示药物对STAT3DNA结合活性的抑制，也同时通过对STAT5DNA结合观察，其对STAT5DNA结合活性影响不大，证明其一定的选择性。
因此，化合物I-1可抑制MDA-MB-231细胞中STAT3与DNA的结合，但并不影响STAT5与DNA的结合，即化合物I-1对STAT3具有特异性的抑制作用。
5.化合物I-1抑制STAT3转录活性
采用双萤光素酶报告基因的方法测定化合物I-1对STAT3的转录活性的影响，实验方法如下：(1)铺板：取对数生长期HEK-293T细胞，弃培养基，PBS清洗，胰酶消化，培养基终止消化，离心后用培养基重悬成单细胞悬液，按20000个/孔接种于96孔板中。(2)转染：24h后，用lipo2000每孔转染50ng pGL3-STAT3+50ng STAT3C+40ng海肾荧光素酶报告基因质粒TK-RL。(3)加药：转染24h后加入不同浓度的化合物I-1，处理24h。(4)检测：采用的碧云天双萤光素酶报告基因检测试剂盒(RG028)进行检测。弃培养基，每孔加入50μL报告基因细胞裂解液，震荡5min。融解萤火虫萤光素酶检测试剂和海肾萤光素酶检测缓冲液，并达到室温。海肾萤光素酶检测底物(100X)置于冰浴或冰盒上备用。按照每个样品需100μL的量，取适量海肾萤光素酶检测缓冲液，按照1:100加入海肾萤光素酶检测底物(100X)配制成海肾萤光素酶检测工作液。震荡结束后，每孔加入50μL萤火虫萤光素酶检测试剂，用枪打匀后测定RLU1(relative light unit)。以报告基因细胞裂解液为空白对照。在完成上述测定萤火虫萤光素酶步骤后，加入100μL海肾萤光素酶检测工作液，用移液器打匀后测定RLU2(relative light unit)。用RLU1/RLU2的比值来比较不同样品间STAT3转录活性的差异。
结果如图10所示，化合物I-1可抑制STAT3的转录活性，且具有浓度依赖性。
6.化合物I-1在动物模型中抑制肿瘤的生长、增殖
a.在荷瘤小鼠模型中的药效、药理及毒理研究
荷瘤小鼠模型的建立：取对数生长期的癌细胞离心消化，用灭菌PBS清洗3次后进行细胞计数，将细胞浓度调至约2*10^7个/ml，然后在小鼠的腹背侧皮下注射细胞悬液100μL。实验分组：待荷瘤小鼠模型建立后(约1-3周)，将小鼠随机分为对照组和给药组，每组约6-10只。药物干预：模型建立后，开始给予药物干预。对照组腹腔注射含15％蓖麻油的PBS(药物溶剂组)，给药组腹腔注射药物，给药体积100μL/只。持续3-4周，期间每天测量小鼠体重和肿瘤体积，并对小鼠的行为学状态进行监测。样本收集：给药3-4周后脱颈椎处死小鼠，将瘤体取出，称量重量并测量体积，并取血液、心脏、脾脏、肝脏等脏器和组织做进一步的药理学和毒理学研究。
b.在人源肿瘤异种移植(PDX)小鼠模型中的药效、药理学研究。
病人肿瘤标本的收集：标本可来源于组织活检和肿瘤根治的手术标本，在肿瘤离体后收集，将新鲜肿瘤组织完全浸泡于0℃无血清无双抗的培养基。用消毒组织剪将肿瘤组织剪成2*2*2mm组织块，并用培养基清洗三次。在麻醉状态下的小鼠的腹背两侧皮肤各划开一个约3mm的小口，并分离出一个小口袋式空间，将肿瘤组织块种植于皮下，缝合伤口。伤口处滴100×双抗溶液以防感染。每种肿瘤都种植于5只小鼠(F1)，每周至少一次观察种植肿瘤情况。观察内容包括有无肿瘤生长以及测量肿瘤体积。约在12-16周后，移植肿瘤开始长成1-2cm3大小。取出F1小鼠腹背侧瘤体，用消毒组织剪将瘤体剪成2*2*2mm组织块，浸泡于无血清无双抗RPMI1640培养基清洗。步骤同上，将组织块种植于小鼠腹背两侧皮下，每种肿瘤种植5只小鼠(F2)。荷瘤后待移植肿瘤长成1-2cm3大小，取出F2小鼠腹背侧瘤体，剪成2*2*2mm组织块，随后将组织块种植于小鼠腹背两侧皮下，每种肿瘤种植若干只小鼠(F3)。待移植肿瘤长成约100mm3大小，将F3随机分组，分为对照组和给药组，分组后对照组腹腔注射含15％蓖麻油的PBS(药物溶剂)，给药组腹腔注射药物，给药约体积100μL/只，期间每天对肿瘤体积和体重进行测量。连续给药三至四周后，小鼠颈椎脱臼处死，将瘤体取出，称量重量和体积，并取血液、心脏、脾脏、肝脏等脏器和组织做进一步的药理学和毒理学研究。
结果如图11-14所示，从图11可以看出，化合物I-1可以显著降低小鼠体内肿瘤(乳腺癌)的体积和重量，说明化合物I-1可以抑制小鼠体内肿瘤的生长和增殖；图12可以看出，化合物I-1对小鼠主要脏器形态等指标基本没有影响；图13和图14可以看出，I-1在人源肿瘤异种移植(PDX)小鼠模型中显著地抑制乳腺癌的生长增殖(且浓度越高，抑制效果越明显)，图15可以看出，I-1对动物主要脏器形态等指标基本没有影响。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，对于本领域的普通技术人员来说，在上述说明及思路的基础上还可以做出其它不同形式的变化或变动，这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。
Claims (10)
- 咪唑并[1,2-a]吡啶类化合物，其特征在于，所述化合物的结构通式如式(I)所示：其中，n为0、1或2；
R 1、R 2、R 3、R 4各自独立地选自氢、卤素、氰基、硝基、氨基、羟基、三氟甲基、取代或非取代的C1-6烷基、取代或非取代的C1-6烷氧基、取代或非取代的C1-6烷基氨基，取代或非取代的C3-8的环烷基、取代或非取代的C3-8的环烷氧基、取代或非取代的C3-8的环烷基氨基、取代或非取代的芳基、取代或非取代的杂芳基、取代或非取代的含有选自N和O中的1-2个杂原子的3元至8元杂环基、-COR a、-CO 2R a、-CONR aR b、-NR aC(O)R b、-NR aSO 2R b、-SR a、-SOR a、-SO 2R a、-SO 2NR aR b、-OC(O)R a、-OC(O)NR aR b，所述取代是指至少1个位点被以下取代基取代：卤素、氰基、氨基、硝基、羟基、三氟甲基、C1-3烷基、C1-3烷氧基、C1-3烷基氨基；其中所述R a和R b各自独立地为氢、C1-C6烷基、C3-C6环烷基，芳基，杂芳基；R 5为氢、卤素、氰基、硝基、氨基、羟基、三氟甲基、取代或非取代的C1-8烷基、取代或非取代的C1-8烷氧基、取代或非取代的C1-C8烷基氨基、取代或非取代的C3-8的环烷基、取代或取代的C3-C8环烷氧基、取代或非取代的C1-8烷基氨基、取代或非取代的芳基或杂芳基，所述芳基或杂芳基为呋喃、噻吩、吡咯、恶唑、噻唑、咪唑、吡唑、苯并呋喃、苯并噻吩、苯并恶唑、苯并噻唑、苯基、吡啶、哒嗪、嘧啶、吡嗪、喹啉或萘基，所述取代是指至少1个位点被以下取代基取代：卤素、氰基、氨基、硝基、羟基、三氟甲基、甲硫基、C1-3烷基、C1-3烷氧基、C1-3烷基氨基；R 6为氢、卤素、氰基、硝基、氨基、羟基、三氟C1-C3烷基、C1-C3烷氧基、C1-C3烷基氨基。 - 根据权利要求1所述咪唑并[1,2-a]吡啶类化合物，其特征在于，R 1、R 2、R 3、R 4分别独立的选自H、卤素、氰基、硝基、氨基、羟基、三氟甲基、C1-6烷基、C1-6杂烷基烷氧基、C1-6烷基氨基、C3-8环烷基、C3-8环烷氧基、C3-8环烷基氨基、芳基、杂芳基、含有选自N和O中的1-2个杂原子的3元至8元杂环基；R 5选自H、卤素、氰基、硝基、氨基、羟基、三氟甲基、C 1-8烷基、C 1-8烷氧基、C 1-8烷基氨基、C 3-8环烷基、C 3-8环烷氧基、C 1-8烷基氨基、芳基、杂芳基；R 6代表氢、卤素、氰基、硝基、氨基、羟基、三氟甲基、三氟乙基、三氟丙基、C 1-3杂烷基。
- 根据权利要求1所述咪唑并[1,2-a]吡啶类化合物，其特征在于，R 1、R 2、R 3、R 4分别独立的选自：卤基、苯基、C1-3烷基、C1-3烷氧基、含氮的五元或六元杂环基；R 1、R 2、R 3、R 4中任意一个氢可C1-3烷基、C 1-3烷氧基取代；R 5为芳基、杂芳基；；R 5中任意一个氢可被以下取代基取代：卤基、C1-3烷基、C1-3烷氧基、硝基、三氟甲基、氰基、甲基磺酰基；R 6为氢、C1-3烷基或C1-3烷氧基。
- 根据权利要求3所述咪唑并[1,2-a]吡啶类化合物，其特征在于，R 5中的氢取代发生在对位。
- 权利要求1所述咪唑并[1,2-a]吡啶类化合物的制备方法，其特征在于，包括以下步骤：S1.化合物混于溶剂中，加热反应生成
S2.步骤S1产物在铑催化剂的催化作用下，生成
S3.室温下酯水解成相应的酸，后再经过室温缩合反应，即得目标产物；
其中，为2-溴苯乙酮或2-溴-4’-硝基苯乙酮；R 1、R 2、R 3、R 4、R 5、R 6的定义同权利要求1。
- 权利要求1所述咪唑并[1,2-a]吡啶类化合物的制备方法，其特征在于，包括以下步骤：S1.化合物经过铑催化剂的催化，得到
S2.经过偶联反应，生成
S3.经过室温缩合反应得目标产物。
- 一种STAT3特异性抑制剂，其特征在于，所述STAT3特异性抑制剂为权利要求1至4任一项所述咪唑并[1,2-a]吡啶类化合物、其药学上可接受的盐或药学上可接受的溶剂合物。
- 权利要求1至4任一项所述化合物在制备预防和/或治疗抑制肿瘤的药物中的应用。
- 根据权利要求8所述的应用，其特征在于，所述药物的剂型为注射剂、片剂、丸剂、胶囊剂、悬浮剂或乳剂。
- 一种药物组合物，其特征在于，所述药物组合物包括权利要求1至4任一项所述化合物和EGFR抑制剂。
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