CN111484492A

CN111484492A - Substituted pyridino-imidazole compound and application thereof in preparation of medicine for treating malignant tumor diseases

Info

Publication number: CN111484492A
Application number: CN202010245491.1A
Authority: CN
Inventors: 张小雷; 王元相; 刘培庆; 胡文浩; 郑琪瑶; 董辉
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2020-03-31
Filing date: 2020-03-31
Publication date: 2020-08-04
Anticipated expiration: 2040-03-31
Also published as: CN111484492B

Abstract

The invention belongs to the field of medicines, and discloses a substituted pyridoimidazole compound shown as the following and application thereof in preparing a medicine for treating lung cancer. The cell level experiment proves that the compound can inhibit proliferation, migration, invasion and clone formation of lung cancer cells, induce the lung cancer cells to be apoptotic, and inhibit STAT3 phosphorylation level; mouse in vivo experiments show that the compound can inhibit the growth and metastasis of lung cancer cells, specifically inhibit STAT3 signal pathways, inhibit the growth of lung cancer cell transplantable tumors and patient PDX tumors in animal models, lay the foundation for the research and development of new drugs, and have important theoretical significance and wide application rangeAnd (5) landscape.

Description

Substituted pyridino-imidazole compound and application thereof in preparation of medicine for treating malignant tumor diseases

Technical Field

The invention relates to the technical field of medicines, in particular to a substituted pyridoimidazole compound and application thereof in preparing a medicine for treating malignant tumor diseases.

Technical Field

According to the data of the global cancer statistical result in 2018, the lung cancer is still one of the malignant tumors which have the fastest global morbidity and mortality and have the greatest threat to human health and life. 2018 the global lung cancer high incidence area is mainly distributed in North America and eastern Asia, wherein China is one of the major lung cancer incidence countries. By 2018, the incidence and mortality of lung cancer in the country are ranked first, wherein the incidence and mortality of lung cancer in men are the first of all cancers, the incidence of lung cancer in women is the third, the mortality is second to breast cancer, and the number of lung cancer patients in men is obviously higher than that in women. This gender distribution may be associated with risk factors for induction of lung cancer. Statistics show that about 85% of lung cancer development is associated with smoking (both active and passive), with the rest of factors including environmental (e.g., PM2.5), occupational exposure (e.g., asbestos inhalation), radiation, genetic, etc.

The lung cancer mainly originates from bronchial epithelium, glands, bronchioles and alveolar epithelium, the classification of the lung cancer is mainly divided into two categories of small cell lung cancer (SC L C, accounting for about 15%) and non-small cell lung cancer (NSC L C, accounting for about 85%) according to histopathological characteristics at present, wherein the non-small cell lung cancer can be further divided into lung adenocarcinoma (accounting for about 40%), squamous cell lung cancer (accounting for about 30%) and large cell lung cancer (accounting for about 15%).

Signal Transducer and Activator of transcription 3 (STAT 3) is an important nuclear transcription factor widely distributed in the nucleus, cytoplasm and mitochondria, STAT3 protein has 6 domains, each of which plays a different function, from the N-terminal to the C-terminal, (1) an N-terminal domain, stabilizing STAT protein dimer, (2) a coiled coil domain, which mainly regulates the interaction between STAT3 protein and other proteins, (3) a DNA-binding domain (DBD), which directly regulates the interaction between STAT3 protein and DNA, (4) a L inker linker domain, which connects DBD and 493SH 2 domains, (5) an SH2(Src-homology 2) domain, which regulates the binding of STAT 4 protein to receptor and the formation of 3 dimer, (6) a C-terminal transcription Activator domain (Transactivation), which regulates the proliferation of cancer cells, such as breast cancer cells, breast cancer, and the like.

Although STAT3 abnormal activation is found in the development process of diseases such as tumor and tumor drug resistance, the importance of STAT3 is already clear, and the design ideas of the current inhibitor directly targeting STAT3 protein mainly include the following: inhibits STAT3 dimer formation; interfering with binding of STAT3 protein to DNA sequences; inhibits phosphorylation of STAT 3; designing Antisense oligonucleotide (ASO) to bind mRNA and inhibit translation of corresponding protein; STAT 3-related microRNAs, and the like. However, there is no compound targeting STAT3 approved by FDA and on the market, so finding and developing a specific inhibitor targeting STAT3 has been one of the problems to be solved urgently.

Disclosure of Invention

The invention aims to provide a novel compound targeting STAT3 and application thereof in preparing a medicament for treating malignant tumor diseases. The invention discloses a small molecular candidate drug for treating lung cancer, clarifies the action mechanism of the small molecular candidate drug, lays a foundation for the research and development of new drugs, and has important theoretical significance and wide application prospect.

In order to realize the purpose, the invention adopts the technical scheme that:

a substituted pyridino-imidazole compound, wherein the structural formula of the substituted pyridino-imidazole compound is shown as a formula (I):

wherein R is₃、R₅Is mono-, di-or polysubstituted, R₂Is mono-or polysubstituted, R₁Is monosubstituted, R₁、R₂、R₃、R₅The substituents are independently selected from H, halogen, -CF₃、-OH、-CN、NO₂、-NH₂、(CH₃)₃O-CO-、-L-C₁-C₆Alkyl group of-L-C₁-C₆An alkenyl group, -L-substituted or unsubstituted heteroaryl group, or-L-substituted or unsubstituted aryl group, wherein L is O, S, -S (═ O)₂、NH、C(O)、CH₂One or more of-NHC (O) O, -HC (O) or-C (O) NH, R₄Selected from H, C₁-C₃Alkyl of (C)₃-C₆Cycloalkyl groups of (a); x is-CH₂-, -NH- -, - -O- -S- -.

As a more preferred embodiment, R₁、R₂、R₃、R₅The substituents are independently selected from H, halogen, -CF₃、-OH、(CH₃)₃O-CO-、-L-C₁-C₆Wherein L is O, NH, C (O), CH₂One or more of-NHC (O) O, -HC (O) or-C (O) NH, R₄Selected from H, C₁-C₃Alkyl of (C)₃-C₆Cycloalkyl groups of (a); x is one of-O-, -S-.

As a most preferred embodiment, the substituted pyridoimidazoles have the formula:

the invention also protects the application of the substituted pyridino-imidazole compound in preparing a medicament for treating malignant tumor diseases.

As a preferred embodiment, the medicament comprises a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate.

Preferably, the medicament is in the form of injection, tablet, pill, capsule, suspension or emulsion.

As a preferred embodiment, the malignant tumor includes a hematological tumor and a solid tumor; wherein the hematologic tumor comprises lung cancer, leukemia, lymphoma, and the solid tumor comprises lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, stomach cancer, intestinal cancer, head and neck cancer, anal cancer, cancer of the extrahepatic and biliary tract, bladder cancer, bone cancer, brain stem 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, larynx cancer, blood cancer, liver cancer, non-small cell lung cancer, melanoma, prostate cancer, rectal cancer, salivary gland cancer, sarcoma, small intestine cancer, soft tissue sarcoma, uterine sarcoma, testicular cancer, or breast cancer.

More preferably, the solid tumor includes lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, stomach cancer, intestinal cancer, head and neck cancer.

Definitions of terms used in connection with the present invention: the initial definitions provided herein for a group or term apply to that group or term throughout the specification unless otherwise indicated; for terms not specifically defined herein, the meanings that would be given to them by a person skilled in the art are to be given in light of the disclosure and the context.

"substituted" means that a hydrogen atom in a molecule is replaced by a different atom or molecule.

The minimum and maximum carbon atom content of a hydrocarbon group is indicated by a prefix, e.g., the prefix (Ca-Cb) alkyl indicates any alkyl group containing "a" to "b" carbon atoms. Thus, for example, a (C1-C4) alkyl group refers to an alkyl group containing 1-4 carbon atoms.

The alkyl group of C1-C6 refers to alkyl groups of C1, C2, C3, C4, C5 and C6, i.e., straight-chain or branched alkyl groups having 1-6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, hexyl and the like. Alkoxy of C1 to C6 also has the corresponding meaning for the radicals.

The term "pharmaceutically acceptable" means that the carrier, cargo, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients comprising a pharmaceutical dosage form and physiologically compatible with the recipient.

The terms "salt", "acceptable salt" and "pharmaceutically acceptable salt" refer to acid and/or base salts of the above compounds or stereoisomers thereof, with inorganic and/or organic acids and bases, as well as zwitterionic (inner) salts, and also quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. The compound or a stereoisomer thereof may be obtained by appropriately (e.g., equivalently) mixing the above compound or a stereoisomer thereof with a predetermined amount of an acid or a base. These salts may form precipitates in the solution which are collected by filtration, or they may be recovered after evaporation of the solvent, or they may be prepared by reaction in an aqueous medium followed by lyophilization.

Further, the compounds are useful for inhibiting proliferation, growth, metastasis, invasion, infiltration, and clonogenic of lung cancer.

Further, the compounds inhibit proliferation of a STAT3 active protein, pSTAT3Y705 high expressing lung cancer cell.

Further, the compounds inhibit the growth of lung cancer cells.

Further, the compounds inhibit clonogenic lung cancer cells.

Further, the compounds inhibit the invasion of lung cancer cells.

Further, the compounds inhibit metastasis and invasion of lung cancer cells.

Further, the compounds promote apoptosis of lung cancer cells.

Further, the compound can interact with STAT3 protein, and can be applied to preparation of anti-malignant tumor medicines for inhibiting STAT3 signal pathways.

In the application of the invention, the lung cancer cells and the gastric cancer cells are taken as examples, and the W2014 small-molecule compound is found to be capable of selectively inhibiting the proliferation of the lung cancer cells and the gastric cancer cells which highly express a STAT3 active protein, namely p-STAT3 (Y705).

In the application of the invention, the compound W2014S small molecular compound inhibits the clone formation of lung cancer cell lines; wherein the lung cancer cells comprise A549, PC-9 and H460.

In the application of the invention, the compound W2014S small molecular compound inhibits the migration, invasion and infiltration of lung cancer cell lines; wherein the lung cancer cells comprise A549.

In the application of the invention, the compound W2014S small molecular compound inhibits the expression of p-STAT3(Y705) in lung cancer cell strains and the protein expression of downstream target genes; wherein the lung cancer cells comprise A549 and PC-9.

In the application of the invention, the compound W2014S small molecule compound inhibits STAT3 overactivated cell strain to inhibit formation of STAT3 dimerization.

In the application of the invention, the compound W2014S small molecular compound inhibits STAT3 translocation into the nucleus in lung cancer cell strains; wherein the lung cancer cells comprise A549.

In the application of the invention, the compound W2014S small molecule compound inhibits STAT3 transcriptional activity in STAT3 overactivated cells.

In the application of the invention, the compound W2014S small molecule compound can induce lung cancer cell apoptosis; wherein the lung cancer cells comprise A549 and PC-9.

In the application of the invention, the compound W2014S small-molecule compound can inhibit the growth of lung cancer in a nude mouse subcutaneous cell transplantation tumor model.

In the application of the invention, the compound W2014S small molecule compound can inhibit the expression and malignant proliferation of p-STAT3(Y705) in lung cancer in a nude mouse subcutaneous cell transplantation tumor model.

The invention relates to a small molecular compound W2014S obtained by screening, which can be combined with STAT3 protein on the molecular level, can obviously reduce phosphorylation and nuclear translocation of STAT3(Y705) on the cellular level, can selectively inhibit the proliferation of a high-expression p-STAT3(Y705) cell, and simultaneously inhibit the migration, invasion and clone formation of lung cancer; the animal experiment results also show that W2014S can inhibit the growth of lung cancer.

The compound of the invention has the following characteristics and beneficial effects:

the cell level experiment proves that the compound can inhibit proliferation, migration, invasion and clone formation of lung cancer cells, induce the lung cancer cells to be apoptotic, and inhibit STAT3 phosphorylation level; the mouse in vivo experiments show that the compound can inhibit the growth and the metastasis of lung cancer cells, specifically inhibit STAT3 signal pathways, and inhibit the growth of lung cancer cell transplantable tumors and patient PDX tumors in animal models, thereby laying a foundation for the research and development of new drugs, and having important theoretical significance and wide application prospect.

Drawings

FIG. 1 shows the effect of W2014 on the proliferation of different tumor cell lines. FIG. 1A shows the effect of the same concentration of W2014 on the proliferation of different tumor cells; FIG. 1B shows the IC of W2014 in different tumor cells₅₀A value; FIG. (1C) shows clonogenic profiles of different tumor cells with increasing concentration of W2014; figure (1D) shows a statistical plot of clonogenic profiles of different tumor cells with increasing concentration of W2014.

Fig. 2 shows the effect of W2014 on the migration and invasion ability of lung cancer cells. FIG. 2A shows the migration of lung cancer cells with increasing concentration of W2014; FIG. 2B shows a statistical graph of the migration of lung cancer cells with increasing concentration of W2014; FIG. 2C is a graph showing the invasion of lung cancer cells by W2014 with increasing concentration; fig. 2D shows a statistical view of the invasion of lung cancer cells by W2014 with increasing concentration.

Fig. 3 shows the effect of compound W2014S on the proliferation of different lung cancer cell lines. Figure (3A) shows the effect of compound W2014S on the viability of different lung cancer cells with increasing concentration compared to chiral compound W2014R, which has a different configuration; figure (3B) shows the clonogram of different lung cancer cells with increasing concentrations of compounds W2014S and W2014R; figure (3C) shows a statistical plot of clonogenic for different lung cancer cells with increasing concentrations for compounds W2014S and W2014R.

Fig. 4 shows the effect of compound W2014S on the migration and invasion ability of lung cancer cells. Figure (4A) shows the migration profile of lung cancer cells with increasing concentrations of compounds W2014S and W2014R; figure (4B) shows a statistical plot of the migration of lung cancer cells with increasing concentrations of compounds W2014S and W2014R; figure (4C) shows a graph of lung cancer cell invasion with increasing concentrations of compounds W2014S and W2014R; fig. 4D shows a statistical graph of the invasion of lung cancer cells by compounds W2014S and W2014R with increasing concentrations.

FIG. 5 shows that W2014-S and W2014-R bind specifically to STAT3 protein. FIG. 5A shows a molecular simulation docking diagram of W2014-S with W2014-R and STAT 3; FIG. 5B shows SPR results for W2014-S and W2014-R and STAT 3; figure (5C) shows the effect of compounds W2014S and W2014R on the phosphorylation level of STAT3(Y705) in lung cancer cells with increasing concentration; figure (5D) shows the effect of compound W2014S on the phosphorylation level of STAT3(Y705) in lung cancer cells over extended dosing time.

Figure 6 shows the effect of compound W2014S on STAT3 downstream protein expression, nuclear translocation, dimerization and transcription levels. Figure (6A) shows the effect of compound W2014S on protein expression of target genes downstream of STAT3 in lung cancer with increasing concentration; figure (6B) shows the effect of compound W2014S on STAT3 dimer formation with increasing concentration; figure (6C) shows the effect of compound W2014S on STAT3 transcriptional activity with increasing concentration; figure (6D) shows the effect of compound W2014S on p-STAT3(Y705) translocation into the nucleus in lung cancer cells with increasing concentration.

Figure 7 shows that compound W2014S induced apoptosis of lung cancer cells. Figure (7A) shows the apoptosis pattern of lung cancer cells with increasing concentration of compound W2014S; fig (7B) shows a statistical map of apoptosis of lung cancer cells with increasing concentration of compound W2014S.

Figure 8 is a graph showing that compound W2014S inhibited tumor growth in a nude mouse cell tumor-bearing model. FIG. 8A is a graph showing a statistical graph of tumor volumes of nude mice during 21 days of administration to each group of nude mice; FIG. 8B is a graph showing subcutaneous lung cancer tumors obtained from mice sacrificed 21 days after administration to each group of nude mice; FIG. 8C is a weight statistical chart showing subcutaneous lung cancer tumors obtained when mice were sacrificed 21 days after administration to each group of nude mice; FIG. 8D is a figure showing a statistical graph of body weights of nude mice during administration to the nude mice of respective groups for 21 days; FIG. 8E is a graph showing HE staining patterns of liver, spleen, kidney and lung obtained by sacrifice of mice 21 days after administration to each group of nude mice; FIG. 8F shows immunohistochemical profiles of p-STAT3(Y705) and Ki-67 staining of subcutaneous lung cancer tumors obtained from sacrificed mice 21 days after administration to each group of nude mice. FIG. 8G shows the expression of p-STAT3(Y705) and downstream of STAT3 in subcutaneous lung cancer tumors obtained from mice sacrificed 21 days after administration to each group of nude mice.

Figure 9 shows that compound W2014S inhibited tumor growth in a nude mouse PDX model. FIG. 9A is a graph showing a statistical graph of tumor volumes of nude mice during 21 days of administration to each group of nude mice; FIG. 9B is a graph showing subcutaneous lung cancer tumors obtained by sacrifice of mice 21 days after administration to each group of nude mice; FIG. 9C is a weight statistical chart of subcutaneous lung cancer tumors obtained from sacrificed mice 21 days after administration to each group of nude mice; FIG. 9D is a figure showing a statistical graph of body weights of nude mice during administration to the nude mice of respective groups for 21 days; FIG. 9E is a graph showing HE staining patterns of liver, spleen, kidney and lung obtained by sacrifice of mice 21 days after administration to each group of nude mice.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the following detailed descriptions of the technical solutions of the present invention are provided with reference to specific embodiments. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.

Unless otherwise specified, the devices used in the examples and experimental examples are all conventional experimental devices, the materials and reagents used are commercially available, and the experimental methods without specific descriptions are also conventional experimental methods.

Example 1: preparation of W2014

Weighing the compound 1, iron powder (3 equivalents) and ammonium chloride (4 equivalents), dissolving in 20ml ethanol, heating at 80 ℃, cooling to room temperature after complete reaction, filtering the reaction solution through diatomite, washing the diatomite with a proper amount of ethyl acetate, drying an organic phase with anhydrous sodium sulfate, and spin-drying to obtain a purple solid compound 2 with a yield of 78%,¹H NMR(500MHz,CDCl₃)6.85(d,J＝7.8Hz,1H),6.60(d,J＝7.8Hz,1H),4.45(s,2H),3.28(s,2H).ESI-MS:145.0[M+H]+；

weighing the compound 3, p-hydroxybenzaldehyde (1.1 equivalent) and potassium carbonate (2 equivalent) and dissolving in 40ml acetonitrile, stirring at room temperature overnight for reaction, filtering after complete reaction, purifying by organic phase spin-dry column chromatography to obtain the compound 4 with the yield of 90 percent,¹H NMR(400MHz,CDCl₃)9.84(s,1H),7.80–7.73(m,2H),7.41–7.34(m,4H),7.32–7.28(m,1H),6.99(d,J＝8.7Hz,2H),5.44(q,J＝6.4Hz,1H),1.70(d,J＝6.4Hz,3H).ESI-MS:227.2[M+H]+；

weighing intermediate compound 2 and compound 4(1.1 equivalent) and dissolving in 10ml methanol, heating at 90 deg.C for 30min, cooling, adding iodobenzene diacetic acid (1 equivalent) into the dried organic phase and dissolving in 10ml tetrahydrofuran, stirring at room temperature overnight for reaction, performing column chromatography to obtain crude product of compound 5 with yield of 53%,¹H NMR(400MHz,CDCl₃)11.90(s,1H),8.05–7.70(m,3H),7.36(d,J＝7.6Hz,4H),7.28(s,1H),7.05(s,1H),6.87(t,J＝40.5Hz,2H),5.39(d,J＝6.1Hz,1H),1.68(d,J＝5.8Hz,3H).ESI-MS:350.8[M+H]+；

weighing intermediate compound 5, boric acid ester (2 equivalents), catalyst (0.1 equivalent), and sodium carbonate (2 equivalents) dissolved in a mixed solvent of 12ml dioxane and 4ml water, heating at 100 deg.C overnight under protection of nitrogen, and purifying by spin-drying organic phase column chromatography to obtain compoundW2014, yield 71%,¹H NMR(400MHz,DMSO)13.34(s,1H),12.89(s,1H),8.08(d,J＝8.2Hz,3H),7.94(d,J＝8.1Hz,1H),7.45(d,J＝7.6Hz,4H),7.36(t,J＝7.5Hz,3H),7.27(t,J＝7.2Hz,1H),7.07(d,J＝7.9Hz,3H),6.64(s,1H),5.64(d,J＝6.3Hz,1H),4.07(s,3H),3.57(t,J＝5.3Hz,3H),2.65(s,3H),1.59(d,J＝6.2Hz,4H),1.44(s,9H)；¹³C NMR(126MHz,DMSO)159.72,154.43,153.08,151.16,149.34,143.09,135.48,135.32,129.04,128.55,128.01,126.40,126.19,122.61,116.59,114.36,79.32,75.37,28.59,26.24,24.55。

example 2: preparation of W2014R

Weighing triphenylphosphine (1.8 equivalent) and dissolving in 80 ml tetrahydrofuran, under nitrogen protection, slowly adding DIAD (1.8 equivalent) into the reaction under ice bath condition, after 20 minutes, maintaining ice bath condition, sequentially dissolving compound 6(1 equivalent) and p-hydroxybenzaldehyde (1 equivalent) in 20ml tetrahydrofuran, respectively adding into the reaction, stirring at room temperature for reaction, after 10 hours, completely reacting, spin-drying and concentrating the organic phase, purifying the residue by column chromatography to obtain compound 7 with yield of 44%,¹H NMR(400MHz,CDCl₃)9.84(s,1H),7.78–7.74(m,2H),7.39–7.36(m,4H),7.32–7.28(m,1H),6.99(d,J＝8.7Hz,2H),5.44(q,J＝6.4Hz,1H),1.70(d,J＝6.4Hz,3H).ESI-MS:227.2[M+H]+；

weighing intermediate compound 2 and compound 7(1.1 equivalent) and dissolving in 10ml methanol, heating at 90 deg.C for 30min, cooling, adding iodobenzene diacetic acid (1 equivalent) into the dried organic phase and dissolving in 10ml tetrahydrofuran, stirring at room temperature overnight for reaction, performing column chromatography to obtain crude product of compound 8 with yield of 58%,¹H NMR(400MHz,CDCl₃)11.90(s,1H),8.05–7.69(m,3H),7.35(d,J＝7.6Hz,4H),7.28(s,1H),7.06(s,1H),6.87(t,J＝40.5Hz,2H),5.39(d,J＝6.1Hz,1H),1.67(d,J＝5.8Hz,3H).ESI-MS:350.8[M+H]+；

weighing the intermediate compound 8, boric acid ester (2 equivalents), catalyst (0.1 equivalent) and sodium carbonate (2 equivalents) dissolved in a mixed solvent of 12ml of dioxane and 4ml of water,under the protection of nitrogen, heating at 100 ℃ overnight for reaction, performing spin-drying organic phase column chromatography purification to obtain a compound W2014R with the yield of 67 percent,¹H NMR(500MHz,DMSO)8.07(d,J＝7.8Hz,2H),7.92(s,1H),7.44(d,J＝7.4Hz,3H),7.36(t,J＝7.1Hz,2H),7.27(t,J＝7.0Hz,1H),7.07(d,J＝6.1Hz,2H),5.63(d,J＝5.8Hz,1H),4.07(s,2H),3.57(s,2H),2.65(s,2H),1.59(d,J＝5.7Hz,3H),1.44(s,9H)；¹³C NMR(126MHz,DMSO)159.75,154.44,143.08,129.04,128.59,128.00,126.18,122.61,116.59,114.35,79.31,75.38,28.59,26.20,24.53.；ESI-MS:497.0[M+H]+。

example 3: preparation of W2014S

Weighing triphenylphosphine (1.8 equivalent) and dissolving in 80 ml tetrahydrofuran, under nitrogen protection, slowly adding DIAD (1.8 equivalent) into the reaction under ice bath condition, after 20 minutes, maintaining ice bath condition, dissolving compound 9(1 equivalent) and p-hydroxybenzaldehyde (1 equivalent) in 20ml tetrahydrofuran, respectively adding into the reaction, stirring at room temperature for reaction, after 10 hours, completely reacting, spin-drying and concentrating the organic phase, purifying the residue by column chromatography to obtain compound 10 with yield of 48%,¹H NMR(400MHz,CDCl₃)9.84(s,1H),7.79–7.74(m,2H),7.40–7.35(m,4H),7.32–7.28(m,1H),6.99(d,J＝8.7Hz,2H),5.44(q,J＝6.4Hz,1H),1.70(d,J＝6.4Hz,3H).ESI-MS:227.2[M+H]+；

weighing intermediate compound 2 and compound 10(1.1 equivalent) and dissolving in 10ml methanol, heating at 90 deg.C for 30min, cooling, adding iodobenzene diacetic acid (1 equivalent) into the dried organic phase and dissolving in 10ml tetrahydrofuran, stirring at room temperature overnight for reaction, performing column chromatography to obtain crude product of compound 11 with yield of 50%,¹H NMR(400MHz,CDCl₃)11.91(s,1H),8.06–7.70(m,3H),7.38(d,J＝7.6Hz,4H),7.29(s,1H),7.05(s,1H),6.87(t,J＝40.5Hz,2H),5.40(d,J＝6.1Hz,1H),1.69(d,J＝5.8Hz,3H).ESI-MS:350.8[M+H]+；

intermediate compound 11, borate (2 equivalents), catalyst (0.1 equivalent), sodium carbonate (2 equivalents) were weighed out and dissolved in 12ml of dioxane and 4ml of waterThe mixed solvent is heated at 100 ℃ for overnight reaction under the protection of nitrogen, and the compound W2014S is obtained by spin-dry organic phase column chromatography and purification with the yield of 61 percent,¹H NMR(500MHz,DMSO)8.07(d,J＝8.1Hz,2H),7.90(d,J＝5.2Hz,1H),7.44(d,J＝7.8Hz,3H),7.36(t,J＝7.3Hz,2H),7.26(t,J＝7.2Hz,1H),7.07(d,J＝8.1Hz,2H),6.63(s,1H),5.63(d,J＝6.2Hz,1H),4.06(s,2H),3.57(s,2H),2.65(s,2H),1.58(d,J＝6.0Hz,3H),1.43(s,9H).¹³C NMR(126MHz,DMSO)159.75,154.44,151.28,143.08,135.60,129.04,128.60,128.00,126.18,122.59,116.59,114.29,79.31,75.39,62.50,28.59,26.20,25.95,24.52。

example 4: inhibition of proliferation of different cancer cells by W2014

Technical method

(1) Cell culture

Lung cancer cells A549 and H460, gastric cancer cells AGS and MGC-803 are cultured in RPIM-1640 medium containing 10% fetal calf serum in 1% double antibody (penicillin and streptomycin), and breast cancer cells MCF-7 are cultured in DMEM medium containing 10% fetal calf serum in 1% double antibody (penicillin and streptomycin). Cell culture at 37 deg.C with 5% CO₂In a cell culture incubator.

(2) CCK8 method for determining cell viability

The CCK8 method is a method for indirectly measuring the number of living cells by the absorbance of cells.

1) Digesting the cells, counting, inoculating 3000 cells/100 mu L cell suspension into a 96-well plate, and standing and culturing for 24 hours in a cell culture box;

2) after the cells adhere to the wall, adding 50 mu L of compound diluted by the culture medium into each hole, and standing and culturing for 72 hours;

3) performing dark operation, adding 10 mu L volume of Bimake Cell Counting Kit-8(CCK-8) detection reagent into each hole, and performing static culture in an incubator at 37 ℃ for 1-4 hours;

4) when the color is changed into orange, taking out the 96-hole plate, shaking for 5min to ensure uniform color in the hole, and blowing off bubbles in the hole by using a blower;

5) reading the light absorption value at 450nm with microplate reader to make the OD value of control group be between 0.8-1.5, if the OD value is less than 0.8, placing in incubator.

6) And (3) calculating the cell viability:

cell viability [ (+) -a (drug +) -a (blank) ]/[ a (drug-) -a (blank) ] × 100%

A (drug +): absorbance of wells with cells, CCK-8 solution and drug solution;

a (drug-): absorbance of wells with cells, CCK-8 solution, but no drug solution;

a (blank): absorbance of wells with medium and CCK-8 solution without cells;

cell viability: cell proliferation activity or cytotoxic activity.

(3) Clone formation experiments

1) Cells were digested and counted to 5 × 10 per well²Uniformly inoculating each cell in a six-hole plate;

2) standing and culturing for 24 hours, and sequentially adding the cells into complete culture media containing compounds with different concentrations after the cells adhere to the walls;

3) the complete culture medium is replaced every three days to ensure the growth of the cells;

4) culturing for 8-12 days until white cell clone groups are visible in the six-hole plate, removing the culture medium, and washing with normal temperature PBS once;

5) then adding normal temperature 4% paraformaldehyde, and fixing the cells for 15 minutes at room temperature;

6) absorbing the fixing solution, washing with PBS once, adding crystal violet dye in dark, and dyeing for 30min in dark;

7) washing off excessive dye with water, and drying at room temperature;

8) the plates were scanned and the number of cell clones formed per well was counted.

The experimental result is shown in fig. 1, and the graph (1A) shows that the lung cancer cell a549, the gastric cancer cell AGS, MGC-803 and the breast cancer cells MDA-MB-231 and MCF-7 are treated for 72 hours under the condition that the W2014 has a concentration of 5 μ M, and the result shows that the W2014 has the most obvious inhibition effect on the lung cancer cell a549 under the same concentration; FIG. 1B shows the IC of W2014 in various tumor cells₅₀Values for IC50 in A549, H460, AGS, MGC-803 were 1.251. mu.M, 1.152. mu.M, 5.512. mu.M, 4.856. mu.M, respectively; FIG. 1C shows the pair W2014The statistical result of figure (1D) shows that W2014 can more significantly inhibit the clonal formation of lung cancer cells, while the clonal formation of gastric cancer cells AGS and MGC-803 is not obvious, which indicates that W2014 has better selectivity for lung cancer and can more significantly inhibit the growth of lung cancer.

Example 5: w2014 inhibits migration and invasion of lung cancer cells.

Technical method

(1) Cell scratch test

1) Cells were digested and counted to × 10 per well (3-5)⁵Uniformly inoculating each cell in a six-hole plate, and standing and culturing for 24 hours;

2) the cells are grown to a density of about 95-100%, and the diameter of the hole is scratched by a blue gun head;

3) then adding compounds with different concentrations respectively for treatment for 24 h;

4) and observing the scratch position under a quadruple microscope when the medicine is treated for 0h, 12h and 24h respectively, photographing, and counting the cell migration rate.

(2) Transwell cell invasion assay

1) Digesting and counting the cells, and subjecting the cell suspension to (3-5) × 10⁵One cell/well (300. mu. L) was inoculated into the upper chamber of a Transwell chamber and incubated for 12-24 hours;

2) after the cells adhere to the wall, the culture medium in the upper chamber is changed into a blank culture medium containing compounds with different concentrations, and the lower layer is changed into a complete culture medium of 500 mu L;

3) continuously culturing in the incubator for 12-24h, taking out the chamber, and fixing with 4% paraformaldehyde for 15 min;

4) gently scrape the upper cells (uninfected cells) with a cotton swab;

5) washing with PBS twice, and dyeing with crystal violet in dark for 30 min;

6) and washing twice with PBS, washing off unstained crystal violet, drying at room temperature, observing under a tenfold microscope, photographing, and counting.

As shown in fig. 2, the graphs (2A) and (2B) are the scratch graph and the statistical graph of the lung cancer a549 after 24h treatment in W2014, and it can be seen that W2014 can inhibit the migration of a549 in a concentration-dependent manner. Figures (2C) and (2D) show dose-dependent inhibition of lung cancer cell a549 and H460 invasion after 12H treatment with W2014, with corresponding statistical plots of clone numbers.

Example 6: W2014-S can inhibit the multiplication of STAT3 overactivated lung cancer and has better inhibition effect compared with W2014-R.

Technical method

(1) The cell culture method is the same as above;

(2) the CCK8 method is the same as the method for measuring the cell viability;

(3) the experimental procedure for cell monoclonality formation was as above.

The experimental results are shown in fig. 3, and fig. 3A shows the results of IC50 of lung cancer cells a549 and PC-9 treated at different concentrations of W2014-S, W2014-R, respectively, and the results show that IC50 of W2014-S in a549 and PC-9 is lower and the inhibition capability of lung cancer cell proliferation is stronger; FIG. 3C shows the effect of W2014-S, W2014-R at different concentrations on the clonality of lung cancer cells A549 and PC-9, and W2014-S can inhibit the clonality of the cells A549 and PC-9 better and more efficiently than W2014-R at the same concentration.

Example 7: W2014-S inhibits migration and invasion of lung cancer cells.

Technical method

(1) The cell scratching test method is the same as above;

(2) the Transwell chamber cell invasion assay was as above.

As shown in FIG. 4, the scratch patterns and statistical patterns of W2014-S or W2014-R after 48h treatment in FIGS. (4A) and (4B) show that the concentration-dependent inhibition of the migration of A549 and PC-9 by W2014-S is observed. FIGS. 4C and 4D show that after treatment with W2014-S or W2014-R for 12h, the invasion of lung cancer cells A549 and PC-9 is inhibited in a dose-dependent manner, and corresponding clone number statistical graphs are shown.

Example 8: W2014-S is capable of interacting directly with STAT3 protein and down-regulating the phosphorylation level of STAT3 (Y705).

Technical method

(1) Molecular docking experiments

Using Maestro 11.1 software(

Inc.) molecular mock docking of small molecule compounds with STAT3 protein. Respectively utilize

Protein Preparation Wizard and

l igPrep mapping protein and small molecule structures for docking

L igand Docking of protein structure and ligand, wherein STAT3 protein is rigid and micromolecule is flexible in Docking process, XP value is used as Docking parameter for reference

Protein Surface Analyzer analysis of Protein structure based on electronegativity.

(2) SPR surface plasmon resonance technique

Dissolving protein on ice, low-temperature ultrafiltering to make it contain no glycerin and imidazole, using PBS buffer solution to make 100 microgram/M L concentration, using amino coupling mode to anchor it on CM5 chip, respectively dissolving W2014-S and W2014-R in PBS buffer solution filtered by 0.22 microgram filter membrane to prepare 20 microgram mother liquor, using SPR buffer solution containing 0.1% DMSO to make sesquidilution to obtain 10 microgram, 5 microgram, 2.5 microgram, 1.25 microgram, 0.625 microgram and 0.3175 microgram dilution, successively passing compounds with different concentrations through chip from low to high to obtain response signal, using Biacore Insight evaluation software to calculate kinetic and affinity to define binding affinity (K)_D)。

(3) Western blotting (Western Blot)

Treating cells for 24 hours by using W2014-S or W2014-R with different concentrations, absorbing a culture medium, adding a cell lysate RIPA containing a protease inhibitor and a phosphatase inhibitor to lyse the cells, shaking and lysing the cells on a horizontal shaking table for 15 minutes, centrifuging the lysate for 15 minutes at 15000rpm and 4 ℃, taking a supernatant for quantification, adding 5 × loading buffer, boiling and denaturing, separating a protein sample by polyacrylamide gel SDS-PAGE electrophoresis, transferring the protein sample to a PVDF membrane, sealing the PVDF membrane for 1 hour by 5% skim milk, incubating overnight at 4 ℃ by using primary antibodies of pY705-STAT3, STAT3 and β -Actin respectively, incubating for 1 hour at room temperature by using a rabbit secondary antibody and a mouse secondary antibody respectively, and finally detecting the expression level of the protein by using a Tanon developing instrument.

The experimental results are shown in FIG. 5, using the molecular docking software Maestro 11.1 software (

Inc.) STAT3 protein was docked with W2014-S or W2014-R, respectively, and scored using a scoring function, with the results shown in figure (5A), where both W2014-S and W2014-R interacted with the STAT3 SH2 domain and gave XPScore values of-2.568 and-2.165, respectively, for both compounds, indicating that W2014-S is more suitable for binding to the SH2 domain. As shown in FIG. 5B, the affinity of W2014-S, W2014-R and wild-type STAT3 was measured by Biacore 8K, and K was measured_DThe values were 3.64 and 3.39. mu.M, respectively, with no significant difference, indicating that W2014-S/R has a similar affinity for STAT 3. FIG. 5C shows the expression changes of STAT3 phosphorylation of lung cancer cells A549 and PC-9 after 24h treatment at different concentrations of W2014-S, W2014-R, respectively. The result shows that W2014S can obviously inhibit the phosphorylation of STAT3Y705 site, and the concentration gradient is dependent on down-regulation of phosphorylation level. Figure (5D) shows the inhibitory effect of formula (I) W2014S on phosphorylation of STAT3Y705 site at different dosing times, demonstrating that formula (I) W2014S exhibits time-dependent inhibition of p-STAT3(Y705) expression.

Example 9: W2014-S down-regulates the expression of target genes downstream of STAT3, inhibiting nuclear translocation, dimerization, and transcription levels.

Technical method

(1) The Western Blot method (Western Blot) was as above;

(2) immunofluorescence (Immunoflorence)

The lung cancer cell A549 is digested and resuspended, the cell suspension is inoculated into a Confocol dish, and after standing and culturing for 24 hours, W2014-S with different concentrations is added. After the compound is treated for 3 hours, removing the culture medium by suction, adding 4% paraformaldehyde at normal temperature, standing and fixing for 10min at room temperature, removing the paraformaldehyde by suction, naturally volatilizing for 10min by opening a cover, and washing for three times (5 min/time) by normal-temperature PBS; adding 0.3% Triton X-100 at room temperature for membrane rupture for 15min, and washing with PBS for three times (5 min/time); after the PBS was aspirated, the cells were blocked with 10% normal goat serum for 1h, and then 1:100 goat serum diluted pY705-STAT3 primary antibody, and incubating overnight at 4 ℃; and (3) recovering the primary antibody, washing with PBS for three times, adding a fluorescent secondary antibody corresponding to the primary antibody, incubating for 1h in a dark place, washing with PBS for three times in a dark place, adding the ready-to-use DAPI dye, incubating for 10min in a dark place, finally washing with PBS for two times, storing the dish in PBS, and collecting images with a laser confocal microscope.

(3) Co-immunoprecipitation

293T cells were digested and resuspended, seeded in a six well plate at the appropriate density, after standing culturing the cells for 18-24h, HA-STAT3(2500ng) and Flag-STAT3(2500ng) plasmids were co-transfected with L ipo (5. mu. L/well), after 24h transfection the cells were treated with W2014-S at different concentrations for 24h, after the end, the culture was gently washed 1 time with pre-cooled PBS Buffer, IP lysate (1: 100 protease inhibitor and phosphatase inhibitor was added), incubation on ice for 5min, scraping the cells with a cell scraper, transferring to a 1.5m L EP tube, incubation on ice for a further 30min, centrifugation at 15000rpm for 15min at 4 ℃ for 15min, transfer supernatant to a new EP tube, quantification, taking a protein volume of 500ug protein total amount at protein concentration into a new EP tube, adding 15. mu. Anffing-Flag to each tube, centrifugation for 4 min, Gefti for a final centrifugation for a further centrifugation for 50 g, followed by centrifugation for three additional aliquots, washing at 5000. mu. 10 g, centrifugation for 10 g for three additional aliquots, washing, finally, centrifugation at 10 g for 10 g, followed by centrifugation for three additional aliquots.

(4) Dual luciferase reporter genes

293T cells were digested and counted to 2 × 10 per well⁴Inoculating the cells into a 96-well plate, and after standing and culturing for 18-24h, co-transfecting pG L3-STAT 3(50 ng/well) and renilla luciferin reporter gene plasmidTKR L (10 ng/well), after transfection for 24h, adding W2014-S with different concentrations for treatment for 24h, adding I L-6 (100ng/m L) for stimulation for 6h, using a Biyunshan dual-luciferase reporter gene detection kit for detection according to the operation steps, discarding the culture medium, adding 50 mu L reporter gene cell lysate into each well, shaking for 5min, thawing the firefly luciferase detection reagent and the renilla luciferase detection buffer, and reaching the room temperature, placing the renilla luciferase detection substrate (100X) on an ice bath or an ice box for later use, taking a proper amount of renilla luciferase detection buffer according to the amount of 100 mu L required by each well, adding the renilla luciferase detection substrate (100X) according to the ratio of 1:100 to prepare a renilla luciferase detection working solution, after shaking, adding 50 mu L firefly luciferase detection reagent into each well, after shaking for 5min, obtaining R L U2(relat light unit), using the reporter gene lysate as a control solution, adding the firefly luciferase detection reagent for detection for detecting the degree of 857U 84, and obtaining the STAT luciferase detection ratio of 8942U 7375U for detection after shaking for detecting the piyunshut.

The experimental results are shown in figure 6, and in figure (6A), after a549 and PC-9 cells are treated by W2014-S at different concentrations for 24h, the expression of pY705-STAT3 is inhibited in a dose-dependent manner, and meanwhile, the expression amount of downstream genes Bcl-2, Bcl-x L and C-Myc of STAT3 is reduced, so that it can be seen that W2014-S not only inhibits the phosphorylation level of STAT3, but also inhibits the expression of downstream genes of STAT 3. in figure (6B), the expression amount of HA-STAT3 is reduced along with the increase of the concentration of W2014-S, and W2014-S can inhibit the dimerization of STAT 3. in figure (6C), the relative fluorescence intensity is reduced along with the increase of the concentration of W2014-S, and it is presumed that W2014-S can inhibit the transcription activity of 3 in a concentration-dependent manner.

Example 10: W2014-S can cause apoptosis.

Technical method

(1) Flow assay for apoptosis

The Annexin V labeled with FITC is used as a fluorescent probe, and the occurrence of apoptosis can be detected by using a flow cytometer or a fluorescence microscope. Cell membrane damage also occurs during cell necrosis, and necrotic cells bind Annexin V-FITC. The cell membrane of normal and early apoptotic cells is intact, and Propidium Iodide (PI) is a nucleic acid dye that cannot permeate the intact cell membrane, but in cells in the late stages of apoptosis and necrotic cells, PI can permeate the cell membrane to bind to the nucleus and appear red. The use of Annexin v in combination with PI allows differentiation between cells in the early and late stages of apoptosis, as well as dead cells. Necrotic cells can be developed by binding to Annexin V-FITC and PI at the same time, while PI is excluded from live cells (FITC-negative) and early apoptotic cells (FITC-positive). In the absence of macrophages, the final stage of apoptosis is like necrosis in that the cells in the late stage of apoptosis are also stained double-positively by FITC and PI binding.

Tumor cells A549, PC-9 were digested and resuspended at × 10 per well (1-2)⁵Inoculating each cell into a six-well plate, standing and culturing for 24h, adding different concentrations of W2014-S to treat for 72h, digesting the cells with pancreatin without EDTA, re-suspending and washing cell precipitates twice with cold PBS, finally adding 400 mu L1 × Annexin V to re-suspend the cells, adding 5 mu L Annexin V-FITC dye into each sample, uniformly mixing, standing for 15min in a dark place on ice, adding 10 mu L PI dye into each sample, uniformly mixing, standing for 5min in a dark place on ice, detecting by using a flow cytometer, and analyzing and processing data by using FlowJo 7.6.

The experimental results are shown in FIG. 7, and the apoptosis rates of A549 cells and PC-9 cells are increased in a concentration-dependent manner after W2014-S is treated for 72 hours at the concentrations of 0.3 mu M and 1 mu M, which indicates that W2014-S can promote the apoptosis of lung cancer cells.

Example 11: W2014-S can inhibit the growth of lung cancer tumor.

Technical method

(1) Subcutaneous tumor test in nude mice

The lung cancer cells A549 were digested and counted, PBS and matrix Gel were mixed at a ratio of 1:1, and the cells were resuspended to yield 3 × 10⁶100 mu L cells in suspension were placed on ice, and 100 mu L cells were injected into each subcutaneous site on the dorsum and abdomen of immunodeficient nude miceSuspension, the volume of the subcutaneous tumour is about 100mm³The tumor cells were randomly divided into three groups, i.e., a control group, W2014-S5 mg/kg groups, and W2014-S15 mg/kg groups, and were administered to the abdominal cavity, and the tumor volumes were weighed and measured every other day. After three weeks of continuous dosing, mice were sacrificed and tumors and internal organs were removed and weighed for photographing.

(2) Toxicity test in nude mice

The liver, spleen, lung and kidney of each group of mice were taken out, fixed with 4% paraformaldehyde, dehydrated, paraffin-embedded, sliced, stained with hematoxylin and eosin, dehydrated and mounted, observed under an inverted microscope and photographed to detect the influence of W2014-S on the viscera.

(3) Immunohistochemical experiment (IHC)

Fixing the separated tumor with 4% paraformaldehyde, dehydrating, embedding in paraffin, slicing, dewaxing, repairing antigen, removing catalase, sealing antigen locus, incubating Ki67 or p-STAT3 primary antibody, secondary antibody, developing color, staining with hematoxylin, dehydrating, sealing, observing under 20 × microscope, taking pictures, and detecting the expression of p-STAT3 and Ki 67.

(4) Western blotting (Western Blot) method was as described above

Experimental results As shown in FIG. 8, the graph (8A) shows that the W2014S of the formula (I) can significantly inhibit the growth of tumors of nude mice under the conditions of 5mg/kg and 15mg/kg doses, and the inhibition effect is more obvious when the dose is 15mg/kg, which indicates that the W2014S of the formula (I) can significantly inhibit the growth of tumors of lung cancer in a dose-dependent manner, the graph (8B) shows that the final volume size of the tumors after 21 days of administration is large, the graph (8C) shows a tumor weight statistical graph, which indicates that the W2014S of the formula (I) can significantly inhibit the weight of the tumors of lung cancer in a dose-dependent manner, the graph (8D) shows that the weight change of the nude mice during administration, which the W2014S of the formula (I) has no influence on the weight of the nude mice, the graph (8E) shows that the HE staining of livers, spleens, kidneys and lungs of the nude mice taken after administration is high, which 705 and 18C shows that the HE express the malignant lung cancer is a more obviously reduced with the higher dose-expressing the highest expression level of the lung cancer cells of the lung cancer expressed by the brown STAT-2014-18C-19, the brown STAT-18C-18-8C shows that the lung cancer is expressed in a brown STAT-18-1-8C which shows that the tumor expressing a decreased expression of a tumor expressing a malignant tumor expressing a decreased as a malignant tumor expressing a malignant tumor expressing a decreased as a tumor expressing a decreased as a tumor expressing a tumor.

Example 12: W2014-S can inhibit the growth of lung cancer PDX tumor.

Technical method

(1) Nude mouse PDX subcutaneous tumor-bearing test

Carefully remove the tumor tissue with scissors, place it in a petri dish (on an ice box) containing pre-cooled PBS or blank DMEM medium, and cut the tissue with a blade to a size of about 3 × 3mm³The cut tissue sample is transferred to a sterile 50m L centrifuge tube, precooled PBS is added, centrifugation is carried out for 2min at 2000rpm, supernatant is discarded, precooled PBS is added for re-suspension, centrifugation is carried out again, cold PBS is added and the mixture is placed on ice, meanwhile, TROCHAR is put into 75% alcohol for disinfection and preservation, and a tube of cold PBS is prepared for washing TROCHAR.

Injecting 3.5% chloral hydrate into the abdominal cavity of a nude mouse, shaving off two side hairs after anesthesia, carefully cutting a small opening with scissors, sucking a tissue small block with TROCHAR, penetrating the tissue into the subcutaneous part of the nude mouse from the small opening, pushing the tissue into the subcutaneous part, taking care to prevent the tissue block from being taken out when the TROCHAR is pulled out, finally sticking a wound with 502 glue, randomly dividing the nude mouse into 3 groups when the tumor volume reaches about 5mm × 5mm, and injecting 6 compounds W2014-S into the abdominal cavity at doses of 5mg/kg and 15mg/kg respectively when the tumor volume reaches about 5mm 895 mm, treating the control group with the solvent used for dispensing the drug by the abdominal cavity injection (15% castor oil and 85% sterile PBS), continuously administering the drug for three weeks, recording the tumor size and the weight of the nude mouse by using a vernier caliper and a weight scale for three times a.

(2) The toxicity test method in nude mice is the same as above

The experimental results are shown in fig. 9, and fig. 9A shows that formula (I) W2014S can significantly inhibit the growth of tumors in nude mice at 5mg/kg and 15mg/kg doses, which indicates that formula (I) W2014S can significantly inhibit the growth of tumors in lung cancer in a dose-dependent manner; FIG. 9B shows the final volume size of the tumor after 21 days of administration; figure (9C) shows a tumor weight statistic graph illustrating that formula (I) W2014S is able to dose-dependently inhibit the weight of lung cancer tumors; fig. 9D shows the body weight change of nude mice during administration, illustrating that formula (I) W2014S has no effect on the body weight of nude mice; fig. 9E shows HE staining patterns of the liver, spleen, kidney and lung of nude mice taken after administration, which indicates that formula (I) W2014S has no significant toxic effect on the nude mouse organs. In conclusion, formula (I) W2014-S can inhibit PDX tumor activity.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A substituted pyridoimidazole compound is characterized in that the structural formula of the substituted pyridoimidazole compound is shown as the formula (I):

wherein R is₃、R₅Is mono-, di-or polysubstituted, R₂Is mono-or polysubstituted, R₁Is monosubstituted, R₁、R₂、R₃、R₅The substituents are independently selected from H, halogen, -CF₃、-OH、-CN、NO₂、-NH₂、(CH₃)₃O-CO-、-L-C₁-C₆Alkyl group of-L-C₁-C₆Alkenyl, -L-substituted or unsubstituted heteroaryl, or-L-substituted or unsubstitutedSubstituted aryl, wherein L is O, S, -S (═ O)₂、NH、C(O)、CH₂One or more of-NHC (O) O, -HC (O) or-C (O) NH, R₄Selected from H, C₁-C₃Alkyl of (C)₃-C₆Cycloalkyl groups of (a); x is-CH₂-, -NH- -, - -O- -S- -.

2. The substituted pyridoimidazoles according to claim 1, wherein R is₁、R₂、R₃、R₅The substituents are independently selected from H, halogen, -CF₃、-OH、(CH₃)₃O-CO-、-L-C₁-C₆Wherein L is O, NH, C (O), CH₂One or more of-NHC (O) O, -HC (O) or-C (O) NH, R₄Selected from H, C₁-C₃Alkyl of (C)₃-C₆Cycloalkyl groups of (a); x is one of-O-, -S-.

3. The substituted pyridoimidazole compound of claim 1, wherein the substituted pyridoimidazole compound has the structural formula:

4. an application of the substituted pyridoimidazole compound of claim 1 in preparing a medicament for treating malignant tumor diseases.

5. The use of claim 4, wherein the medicament comprises a pharmaceutically acceptable salt or a pharmaceutically acceptable solvate.

6. The use according to claim 4, wherein the medicament is in the form of an injection, tablet, pill, capsule, suspension or emulsion.

7. The use of claim 4, wherein the malignancy comprises a hematological tumor and a solid tumor; wherein the hematologic tumor comprises lung cancer, leukemia, lymphoma, and the solid tumor comprises lung cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, stomach cancer, intestinal cancer, head and neck cancer, anal cancer, cancer of the extrahepatic and biliary tract, bladder cancer, bone cancer, brain stem 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, larynx cancer, blood cancer, liver cancer, non-small cell lung cancer, melanoma, prostate cancer, rectal cancer, salivary gland cancer, sarcoma, small intestine cancer, soft tissue sarcoma, uterine sarcoma, testicular cancer, or breast cancer.

8. A STAT3 inhibitor comprising the substituted pyridoimidazoles of claim 1.