A MET transcription inhibitor and its preparation method and application

Abstract

The invention discloses a MET transcription inhibitor and a preparation method and application thereof. The inhibitor has inhibitory activity on the translation of tyrosine kinase c-Met by inhibiting the transcription of MET gene, and the preparation method is simple, without dangerous reactions and with high reaction yield. The inhibitor is applied in drug preparation, has broad-spectrum anti-tumor activity and has significant anti-proliferation activity on cell lines resistant to c-Met inhibitors.

Description

Technical Field

[0001] The present invention relates to a MET transcription inhibitor, and also relates to a preparation method and application of the inhibitor. Background Art

[0002] c-Met is MET The protein encoded by the proto-oncogene is composed of an extracellular α-subunit and a disulfide-bonded heterodimeric β-subunit. Among them, the β-chain forms the extracellular ligand-binding domain and the intracellular domain, which is responsible for the regulation of kinase activity and effector signal transduction. The c-Met extracellular domain includes: SEMA domain, PSI integrin domain and four immunoglobulin-like IPT domains. The intracellular domain also consists of three domains, namely the membrane-proximal domain, the tyrosine kinase catalytic domain and the C-terminal multifunctional binding region. HGF is mainly secreted by stromal cells and is composed of active heterodimers containing α-chain and β-chain. Mature HGF contains three specific domains and activates the c-Met receptor on adjacent epithelial cells in a paracrine manner. When the SEMA domain of c-Met binds to the α-chain of HGF, the β-chain of HGF can induce the activation of c-Met, thereby activating downstream signal transduction. Abnormal MET Signal transduction can occur through a variety of molecular mechanisms, including MET The HGF / MET signaling pathway is activated by mitochondria (gene amplification, mutation, rearrangement, and overexpression), leading to the persistent activation of the HGF / MET signaling pathway, thereby driving tumorigenesis in multiple cancer types.

[0003] However, as a target for precision tumor therapy, the c-Met target has a difficult problem to solve in terms of drug resistance. Amino acid mutations at Y1230 and D1228 are the most common mutation sites induced by type I inhibitors, while L1195 and F1200 are the most common mutation sites induced by type II inhibitors. Therefore, drug resistance to kinase inhibitors is a serious problem facing clinical applications; new tumor treatment compounds with stronger efficacy and overcoming drug resistance are urgently needed. In the central dogma, nucleic acid molecules are upstream of proteins, and the development of drugs targeting nucleic acid molecules has the potential to overcome the drug resistance of protein-targeted drugs. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a MET transcription inhibitor that targets and stabilizes the G quadruplex (G4) of the MET gene and has good anti-proliferative activity in cell lines resistant to c-Met inhibitors, and also provides a method for preparing the above-mentioned inhibitor and its use in the preparation of drugs for preventing and / or treating cancer or viral infection-related diseases.

[0005] Technical solution: The present invention discloses a MET transcription inhibitor, the structure of which is shown in Formula I: ;

[0006] Wherein, X1 and X2 are independently selected from CH, O, S;

[0007] R is selected from a substituted five-membered aromatic heterocycle, a substituted six-membered aromatic ring or a six-membered aromatic heterocycle, and a substituted cyclic ring, specifically: .

[0008] Wherein, the inhibitor, when X 1 When it is CH, X 2 is O; when X 1 When S, X 2 is CH; the R group is selected from the following: .

[0009] Wherein, the inhibitor, when X 1 is S and X 2 When CH, the R group is selected from the following: .

[0010] Wherein, the compound is selected from any one of the compounds shown in formula (B1) to formula (B12):

[0011] Wherein, the inhibitor also includes pharmaceutically acceptable salts or isomers thereof.

[0012] Among them, the pharmaceutically acceptable salt is an acid addition salt of the compound shown in Formula I, wherein the acid used for salt formation includes inorganic acids and organic acids, the inorganic acid includes hydrochloric acid, sulfuric acid or phosphoric acid, and the organic acid includes acetic acid, trichloroacetic acid, trifluoroacetic acid, propionic acid, butyric acid, maleic acid, p-toluenesulfonic acid, malic acid, methanesulfonic acid, malonic acid, cinnamic acid, citric acid, fumaric acid, camphoric acid, digluconic acid, aspartic acid or tartaric acid.

[0013] The preparation method of the above-mentioned MET transcription inhibitor comprises the following steps: ;

[0014] Dissolve 1,10-phenanthroline-5,6-dione and aldehyde in glacial acetic acid, add ammonium acetate, heat to react, transfer to ice water after the reaction is completed, adjust the pH value to neutral, and purify to obtain a MET transcription inhibitor.

[0015] The present invention also discloses a pharmaceutical composition, comprising the compound shown in formula I or a pharmaceutically acceptable salt or an isomer thereof, and a pharmaceutically acceptable carrier.

[0016] Above MET Transcription inhibitors can also be used to prepare anti-tumor drugs.

[0017] The tumors include solid tumors and blood tumors, specifically lung cancer, liver cancer, pancreatic cancer, gastric cancer, kidney cancer, colon cancer, esophageal cancer, glioblastoma, leukemia or multiple myeloma.

[0018] Wherein, the target for exerting anti-tumor activity is a G quadruplex; the G quadruplex is located at MET Gene promoter region.

[0019] The term "heterocycle" includes saturated and unsaturated polycyclic nitrogen-containing heterocycles, including but not limited to thiophene, furanpyrazine, pyrrole, tetrahydropyrrole, imidazole, pyridine, pyrazine, and the like.

[0020] The term "paracyclic ring" includes aromatic and aliphatic paracyclic rings, including but not limited to quinoline, quinoxaline, benzoheterocyclic ring, benzoalicyclic ring, and the like.

[0021] Principle of the invention: MET Transcriptional inhibitors inhibit MET The transcription of the gene has inhibitory activity on the translation of tyrosine kinase c-Met; the target of the anti-tumor activity is the G quadruplex; the G quadruplex is located at MET Gene promoter region. Since the G-quadruplex is a nucleic acid secondary structure composed of continuously stacked guanines through Hoogsteen hydrogen bonds, MET The G-quadruplex structure in the 5' untranslated region (5'UTR) of the gene plays a crucial regulatory role in the expression of c-Met protein; MET Transcriptional regulators can stabilize MET The G-quadruplex inhibits the translation of c-Met, has a broad spectrum of anti-tumor activity and has significant anti-proliferative activity against cell lines resistant to c-Met inhibitors.

[0022] Considering that the spatial structure of the G quadruplex is one or more planes formed by stacking guanine, the small molecule targeted compound designed by the present invention uses phenanthroline as the parent core for structural optimization and extension. Among them, the phenanthroline parent core of the multi-ring conjugated structure is a planar structure in space and can be embedded in the planar cavity of the G quadruplex; the extension of the side chain mainly plays an important role in improving the drugability and selectivity of the compound.

[0023] c-Met is a proto-oncogene MET The protein encoded by the c-Met protein is highly expressed and is closely related to the occurrence and development of tumors. Compared with traditional c-Met protein targeted drugs, the compound obtained in the present invention is the first MET DNA targeting small molecules by binding and stabilizing MET G-quadruplexes in the DNA promoter region inhibit METGene transcription and inhibition of c-Met protein expression achieve the purpose of anti-tumor. More importantly, the acquired resistance of c-Met inhibitors currently seen in clinical practice makes the effect of c-Met inhibitors limited in the later stages of treatment. MET Transcription inhibitors interfere with DNA transcription upstream of the protein and can effectively overcome the common acquired resistance to c-Met inhibitors in clinical practice.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The MET transcription inhibitor of the present invention inhibits MET (1) It inhibits the transcription of genes and has inhibitory activity on the translation of tyrosine kinase c-Met; (2) The preparation method is simple, has no dangerous reactions, and has a high reaction yield; (3) It is used in drug preparation and has a broad spectrum of anti-tumor activity and significant anti-proliferative activity against cell lines resistant to c-Met inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the structural formula of the MET transcription inhibitor of the present invention;

[0026] Figure 2 This is the result of the thioflavin competition experiment;

[0027] Figure 3 The MET transcription inhibitor inhibits the expression of c-Met protein;

[0028] Figure 4 The effect of the MET transcription inhibitor, preferably compound B6, on inhibiting the expression of c-Met protein, wherein (A) is the effect of compound B6 on the c-Met protein level in A549 cells, (B) is the effect of compound B6 on the c-Met protein level in NCI-H1975 cells, and (C) is the effect of compound B6 on the MET mRNA level in A549 and NCI-H1975 cells;

[0029] Figure 5 The binding ability of compound B6 to MET-G4 is determined by MST, wherein (A) is the binding ability of B6 to MET-G4 determined by MST, and (B) is the binding ability of B6 to mutated MET-G4 determined by MST;

[0030] Figure 6 This is the synthetic route of intermediate compounds M1~M12;

[0031] Figure 7 This is the synthesis route of the MET transcription inhibitors B3-B12 of the present invention. DETAILED DESCRIPTION

[0032] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0033] Synthesis of intermediates

[0034] Intermediate M1: 4-(3-cyanophenyl)thiophene-2-carboxaldehyde (M1): 4-bromothiophene-2-carboxaldehyde (1eq, 2 mmol), 3-cyanophenylboronic acid (1.2 eq, 2.4 mmol), triphenylphosphine palladium (0.05eq, 0.10 mmol), potassium carbonate (2eq, 4mmol) were dissolved in 1,4-dioxane, and the reaction was heated at 90°C overnight. After the reaction was completed, M1 was purified by silica gel column with a yield of 75%. ESI-MS m / z : 214.3 [M+H] + .

[0035] Intermediate M2: 4-(3-methoxyphenyl)thiophene-2-carboxaldehyde (M2): M2 was prepared using 4-bromothiophene-2-carboxaldehyde and 2-(3-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane as raw materials in the same synthetic method as M1, with a yield of 73%. ESI-MS m / z : 219.6 [M+H] + .

[0036] Intermediate M3: 4-(2,3-dihydrobenzo[ b [1,4]dioxin-6-yl)thiophene-2-carboxaldehyde (M3): 4-bromothiophene-2-carboxaldehyde and (2,3-dihydrobenzo[ b M3 was prepared by the same synthesis method as M1 with a yield of 80%. ESI-MS m / z : 247.3[M+H] + .

[0037] Intermediate M4: 4-(1-methyl-1 H -pyrazol-4-yl)thiophene-2-carboxaldehyde (M4): 4-bromothiophene-2-carboxaldehyde and (1-methyl-1 H -pyrazole-4-yl)boronic acid as raw material, and M4 was prepared by the same synthesis method of M1 with a yield of 70%. ESI-MS m / z : 193.4[M+H] + .

[0038] Intermediate M5: 3-(5-formylfuran-2-yl)benzonitrile (M5): M5 was prepared using 5-bromofuran-2-carboxaldehyde and 3-cyanophenylboronic acid as raw materials in the same synthetic method as M1, with a yield of 82%. ESI-MS m / z : 198.2[M+H] + .

[0039] Intermediate M6: 5-(3-methoxyphenyl)furan-2-carboxaldehyde (M6): 5-bromofuran-2-carboxaldehyde and 2-(3-methoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborinane were used as raw materials and M6 was prepared by the same synthetic method as M1 with a yield of 81%. ESI-MS m / z : 203.1[M+H] + .

[0040] Intermediate M7: 5-(2,3-dihydrobenzo[ b [1,4]dioxin-6-yl)furan-2-carboxaldehyde (M7): 5-bromofuran-2-carboxaldehyde and (2,3-dihydrobenzo[ b M7 was prepared by the same synthesis method as M1 with a yield of 84%. ESI-MS m / z : 231.4[M+H] + .

[0041] Intermediate M8: 5-(1-methyl-1 H -pyrazol-4-yl) furan-2-carboxaldehyde (M8): 5-bromofuran-2-carboxaldehyde and (1-methyl-1 H -pyrazol-4-yl)boronic acid as raw material, and M8 was prepared by the same synthesis method of M1 with a yield of 75%. ESI-MS m / z : 177.4[M+H] + .

[0042] Intermediate M9: 5-(4-nitrophenyl)furan-2-carboxaldehyde (M9): M9 was prepared using 5-bromofuran-2-carboxaldehyde and 4-nitrophenylboronic acid as raw materials in the same synthetic method as M1, with a yield of 78%. ESI-MS m / z : 218.3[M+H] + .

[0043] Intermediate M10: 5-(4-hydroxyphenyl)furan-2-carboxaldehyde (M10): M10 was prepared using 5-bromofuran-2-carboxaldehyde and 4-hydroxyphenylboronic acid as raw materials in the same synthetic method as M1, with a yield of 83%. ESI-MS m / z : 189.2[M+H] + .

[0044] Intermediate M11: 5-(3-fluorophenyl)furan-2-carboxaldehyde (M11): 5-bromofuran-2-carboxaldehyde and 2-(3-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolan were used as raw materials and M11 was prepared by the same synthetic method as M1 with a yield of 87%. ESI-MS m / z : 191.2 [M+H] + .

[0045] Intermediate M12: 5-(4-cyanophenyl)furan-2-carboxaldehyde (M12): M12 was prepared using 5-bromofuran-2-carboxaldehyde and 4-cyanophenylboronic acid as raw materials in the same synthetic method as M1, with a yield of 77%. ESI-MS m / z : 198.4 [M+H] + .

[0046] Example 1

[0047] 3-(5-(1 H -imidazo[4,5- f Synthesis of [1,10]phenanthroline-2-yl)thiophene-3-yl)benzonitrile (B1): 1,10-phenanthroline-5,6-dione (1eq, 1 mmol), M1 (1eq, 1mmol) and ammonium acetate (20 eq, 20 mmol) were added to glacial acetic acid, and the reaction was heated under reflux for 6 hours. After the reaction was complete as monitored by TLC, the reaction was poured into ice water, and the pH was adjusted to neutral with ammonia water. The target compound B1 was purified by silica gel column with a yield of 72%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 9.04 (dd, J =4.3, 1.8 Hz, 2H), 8.87 (dd, J = 8.1, 1.8 Hz, 2H), 8.36 (d, J = 1.5 Hz, 1H), 8.29– 8.22 (m, 2H), 8.12 (d, J = 8.0 Hz, 1H), 7.84 (dd, J = 8.1, 4.7 Hz, 3H), 7.70(s, 1H).

[0048] .

[0049] Example 2

[0050] 2-(4-(3-methoxyphenyl)thiophen-2-yl)-1 H -imidazo[4,5- f Synthesis of [1,10]phenanthroline (B2): Using 1,10-phenanthroline-5,6-dione and M2 as raw materials, the target compound B2 was prepared by the same synthetic method as B1 with a yield of 78%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.90 (s, 1H), 9.05 (dd, J = 4.3, 1.7 Hz, 2H),8.86 (dd, J = 8.2, 1.8 Hz, 2H), 8.31 (d, J = 1.5 Hz, 1H), 8.11 (d, J = 1.4 Hz, 1H),7.84 (dd, J = 8.2, 4.3 Hz, 2H), 7.42 (t, J = 7.8 Hz, 1H), 7.38 – 7.33 (m, 2H), 6.96 (ddd, J = 8.1, 2.6, 1.1 Hz, 1H), 3.86 (s, 3H).

[0051] .

[0052] Example 3

[0053] 2-(4-(2,3-dihydrobenzo[ b [1,4]dioxin-6-yl)thiophen-2-yl)-1 H -imidazo[4,5- f Synthesis of ][1,10]phenanthroline (B3): Using 1,10-phenanthroline-5,6-dione and M3 as raw materials, the target compound B3 was prepared by the same synthetic method as B1 with a yield of 70%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 14.10 (s, 1H), 9.05 (dd, J =4.4, 1.8 Hz, 2H), 8.96 – 8.85 (m, 2H), 8.35 (d, J = 1.5 Hz, 1H), 7.95 (d, J= 1.5Hz, 1H), 7.86 (dt, J = 21.0, 6.9 Hz, 2H), 7.29 (d, J = 2.1 Hz, 1H), 7.26 (dd, J =8.3, 2.2 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 4.31 (s, 4H).

[0054] Example 4

[0055] 2-(4-(1-methyl-1 H -pyrazol-4-yl)thiophen-2-yl)-1 H -imidazo[4,5- f Synthesis of ][1,10]phenanthroline (B4): Using 1,10-phenanthroline-5,6-dione and M4 as raw materials, the target compound B4 was prepared by the same synthetic method as B1 with a yield of 63%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.81 (s, 1H), 9.05 (dd, J = 4.3, 1.8 Hz,2H), 8.87 (dd, J = 8.1, 1.7 Hz, 2H), 8.08 (d, J = 16.5 Hz, 2H), 7.84 (q, J = 4.1Hz, 3H), 7.77 – 7.71 (m, 1H), 3.91 (s, 3H).

[0056] Example 5

[0057] 3-(5-(1 H -imidazo[4,5- f Synthesis of [1,10]phenanthroline-2-yl)furan-2-yl)benzonitrile (B5): Using 1,10-phenanthroline-5,6-dione and M5 as raw materials, the target compound B5 was prepared according to the same synthetic method as B1 with a yield of 80%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 14.01 (s, 1H), 9.06 (dd, J= 4.4, 1.7 Hz, 2H),9.02 – 8.90 (m, 2H), 8.42 (t, J = 1.7 Hz, 1H), 8.27 (dt, J = 8.0, 1.4 Hz, 1H),7.84 (dt, J = 7.7, 1.4 Hz, 3H), 7.74 (t, J = 7.8 Hz, 1H), 7.45 (d, J = 3.6 Hz, 1H),7.42 (d, J = 3.6 Hz, 1H).

[0058] Example 6

[0059] 2-(5-(3-methoxyphenyl)furan-2-yl)-1 H -imidazo[4,5- f Synthesis of ][1,10]phenanthroline (B6): Using 1,10-phenanthroline-5,6-dione and M6 as raw materials, the target compound B6 was prepared by the same synthetic method as B1 with a yield of 83%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.92 (s, 1H), 9.06 (dd, J = 4.3, 1.8 Hz, 2H),8.98 – 8.91 (m, 2H), 7.85 (dd, J = 8.5, 4.4 Hz, 2H), 7.55 (ddd, J = 8.5, 5.3, 1.5Hz, 2H), 7.46 (t, J = 7.9 Hz, 1H), 7.37 (d, J = 3.6 Hz, 1H), 7.30 (d, J = 3.5 Hz,1H), 6.99 (dd, J = 8.1, 2.3 Hz, 1H), 3.88 (s, 3H).

[0060] Example 7

[0061] 2-(5-(2,3-dihydrobenzo[ b [1,4]dioxin-6-yl)furan-2-yl)-1 H -imidazo[4,5- fSynthesis of ][1,10]phenanthroline (B7): Using 1,10-phenanthroline-5,6-dione and M7 as raw materials, the target compound B7 was prepared by the same synthetic method as B1 with a yield of 80%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.90 (s, 1H), 9.04 (dd, J =4.4, 1.8 Hz, 2H), 8.94 (dd, J = 8.1, 1.8 Hz, 2H), 7.83 (dd, J = 8.1, 4.3 Hz, 2H),7.49 (d, J = 2.1 Hz, 1H), 7.41 (dd, J = 8.4, 2.1 Hz, 1H), 7.31 (d, J = 3.6 Hz, 1H),7.10 (d, J = 3.6 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 4.32 (s, 4H).

[0062] Example 8

[0063] 2-(5-(1-methyl-1 H -pyrazol-4-yl)furan-2-yl)-1 H -imidazo[4,5- f Synthesis of ][1,10]phenanthroline (B8): Using 1,10-phenanthroline-5,6-dione and M8 as raw materials, the target compound B8 was prepared by the same synthetic method as B1 with a yield of 60%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.98 (s, 1H), 9.03 (dd, J = 4.3, 1.8 Hz,2H), 8.92 (dd, J = 8.1, 1.8 Hz, 2H), 8.13 (s, 1H), 7.88 (s, 1H), 7.83 (dd, J =8.2, 4.3 Hz, 2H), 7.29 (d, J = 3.5 Hz, 1H), 6.78 (d, J= 3.5 Hz, 1H), 3.91 (s, 3H).

[0064] Example 9

[0065] 2-(5-(4-nitrophenyl)furan-2-yl)-1 H -imidazo[4,5- f Synthesis of ][1,10]phenanthroline (B9): Using 1,10-phenanthroline-5,6-dione and M9 as raw materials, the target compound B9 was prepared by the same synthetic method as B1 with a yield of 77%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 12.87 (s, 1H), 9.04 (dd, J = 4.3, 1.7 Hz, 2H),8.91 (dd, J = 8.1, 1.8 Hz, 2H), 8.42 – 8.27 (m, 2H), 8.20 – 8.06 (m, 2H), 7.84(dd, J = 8.1, 4.3 Hz, 2H), 7.56 (d, J = 3.6 Hz, 1H), 7.42 (d, J = 3.7 Hz, 1H).

[0066] Example 10

[0067] 2-(5-(4-Hydroxyphenyl)furan-2-yl)-1 H -imidazo[4,5- f Synthesis of ][1,10]phenanthroline (B10): Using 1,10-phenanthroline-5,6-dione and M10 as raw materials, the target compound B10 was prepared by the same synthetic method as B1 with a yield of 65%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.83 (s, 1H), 9.88 (s, 1H), 9.05 (dd, J = 4.4,1.8 Hz, 2H), 8.95 (dd, J = 8.1, 1.7 Hz, 2H), 7.85 (dd, J = 8.1, 4.3 Hz, 2H), 7.81– 7.71 (m, 2H), 7.32 (d, J= 3.5 Hz, 1H), 7.01 (d, J = 3.5 Hz, 1H), 6.96 – 6.89(m, 2H).

[0068] Embodiment 11

[0069] 2-(5-(3-Fluorophenyl)furan-2-yl)-1 H -imidazo[4,5- f Synthesis of [1,10]phenanthroline (B11): Using 1,10-phenanthroline-5,6-dione and M11 as raw materials, the target compound B11 was prepared by the same synthetic method as B1 with a yield of 85%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 9.04 (dd, J = 4.3, 1.7 Hz, 2H), 8.94 (dd, J = 8.0,1.8 Hz, 2H), 7.91 – 7.74 (m, 4H), 7.56 (td, J = 8.1, 6.0 Hz, 1H), 7.43 – 7.31(m, 2H), 7.22 (td, J = 8.6, 2.6 Hz, 1H).

[0070] Example 12

[0071] 2-(5-(4-cyanophenyl)furan-2-yl)-1 H -imidazo[4,5- f Synthesis of ][1,10]phenanthroline (B12): Using 1,10-phenanthroline-5,6-dione and M12 as raw materials, the target compound B12 was prepared by the same synthetic method as B1 with a yield of 80%. 1 H NMR (400 MHz, DMSO- d 6 ) δ 9.01 (dd, J = 4.3, 1.8 Hz, 2H), 8.91 (dd, J = 8.1,1.7 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 7.92 (d, J = 8.5 Hz, 2H), 7.80 (dd, J = 8.1,4.3 Hz, 2H), 7.44 (d,J = 3.7 Hz, 1H), 7.38 (d, J = 3.6 Hz, 1H).

[0072] Biological evaluation experiment:

[0073] (1) Anti-A549 cell proliferation activity assay:

[0074] The inhibitory effect of the compound on A549 cell proliferation was determined according to the CCK-8 method, and the half inhibitory concentration IC of the compound's inhibitory activity on cell proliferation was obtained. 50 Cells in logarithmic growth phase were seeded in 96-well plates at 3000-5000 cells / well and placed at 37°C with 5% CO 2 The culture plates were incubated for 12-24 hours under the following conditions; 100 μL of the test compound solution with different concentrations was added to the culture plates, and the culture plates were incubated at 37 °C and 5% CO 2 Incubate for 72 hours under incubator conditions; 4 hours before the end of incubation, add 10 μL of CCK-8 solution (5 mg / mL) to each well. After incubation, measure OD with a microplate reader. 450 , inhibition rate = (OD value of control group - OD value of experimental group) / OD value of control group × 100%; after obtaining the data, GraphPad Prism fitted and obtained IC 50 The IC values ​​of the compounds 50 Classification by description:

[0075] "A" stands for IC 50 The measured value is less than or equal to 1 μM;

[0076] "B" stands for IC 50 The measured value is less than or equal to 5 μM and greater than 1 μM;

[0077] "C" stands for IC 50 The measured value was greater than 5 μM;

[0078] Table 1. Antiproliferative activity of compounds against A549

[0079]

[0080] It can be seen from Table 1 that the compounds of the examples of the present invention have potent anti-proliferative activity against pancreatic cancer cell lines.

[0081] (2) Thioflavin competition experiment:

[0082] Thioflavin T (ThT) is a fluorescent dye that is commonly used to detect the formation of amyloid fibers. However, in the study of G-quadruplex (G4), it is also used as a tool to screen small molecule compounds that can stabilize or interfere with the G-quadruplex structure. The fluorescence signal value of G4 treated with the target compound at 480 nm was measured on a black 384-well plate using an ELISA reader (BioTek, USA). The concentration of G4s in the experiment was 1μM and the concentration of the compound was 5μM. After deducting the fluorescence value of the buffer and the compound itself, the final fluorescence signal value is expressed as F / F 0 , where F is the fluorescence value of G4s after incubation with the compound, F 0 is the fluorescence value of G4s itself.

[0083] from Figure 2 It can be seen that the example compounds of the present invention can significantly inhibit the fluorescence level of thioflavin and compete with the binding of thioflavin to G4, indicating that the example compounds can better bind to MET - G4 binding.

[0084] (3) Detection of c-Met protein level:

[0085] Cells in logarithmic growth phase were cultured at 4×10 5 - 5×10 5 Cells / well were seeded in 6-well plates and incubated at 37°C with 5% CO 2 The culture plates were incubated for 12-24 hours under the following conditions; 100 μL of the test compound solution with different concentrations was added to the culture plates, and the culture plates were incubated at 37 °C and 5% CO 2 Incubate for 24 hours under incubator conditions; after incubation, collect cells, centrifuge at 1200 rpm for 3 minutes, wash twice with PBS, resuspend cells in 1 mL PBS, transfer to a 1.5 mL centrifuge tube and centrifuge at 2500 rcf for 5 minutes; carefully discard the supernatant, add cell lysis buffer and protease inhibitors and phosphatase inhibitors, shake for 15 minutes, centrifuge at 4°C 1400 rcf for 15 minutes, collect supernatant to determine protein concentration for subsequent Western-Blotting detection.

[0086] from Figure 3 It can be seen that the compounds B2, B6, B7, B10 and B11 of the present invention can significantly inhibit the expression of c-Met protein.

[0087] (4) Detection of c-Met mRNA level:

[0088] Cells in logarithmic growth phase were cultured at 4×10 5 - 5×10 5Cells / well were seeded in 6-well plates and incubated at 37°C with 5% CO 2 The culture plates were incubated for 12-24 hours under the following conditions; 100 μL of the test compound solution with different concentrations was added to the culture plates, and the culture plates were incubated at 37 °C and 5% CO 2 Incubate for 24 hours under incubator conditions; after incubation, collect cells, centrifuge at 1200 rpm for 3 minutes, wash twice with PBS, resuspend cells in 1 mL PBS, transfer to 1.5 mL centrifuge tubes and centrifuge at 2500 rcf for 5 minutes; carefully discard the supernatant, add 500 μL of RNA-easy reagent and blow until the cells are fully lysed, add 200 μL RNA-free double distilled water to mix, and let stand for 5 minutes; take 650 μL of supernatant and add an equal volume of isopropanol to mix, invert and stand at -20 °C for 30 minutes; centrifuge at 12000 rcf, discard the supernatant, add 500 μL 75% ethanol to wash twice, centrifuge at 8000 rcf for 3 minutes, aspirate the supernatant, and air-dry; add 30 μL RNA-free double distilled water to fully dissolve, and store at -80 °C; the obtained RNA sample was used for subsequent PCR experiments after the concentration was measured.

[0089] from Figure 4 It can be seen from C that the preferred compound B6 in the present embodiment significantly affects MET The levels of mRNA and c-Met protein expression indicate that the compound B6 of the present invention affects MET The transcription level of c-Met protein is affected.

[0090] (5) Microscale thermophoresis (MST) testing of compounds and MET -G4's binding ability:

[0091] Experimental Materials:

[0092] Capillaries:MO-K022 MonolithNT.115 Capillaries Lot:899917;

[0093] Buffer: Folding Buffer: pH 7.5, 50 mM HEPES, 100 mM KCl, 10 mM MgCl 2 , 0.005% Tween-20;

[0094] DNA: Prepare a 20 nM DNA solution in buffer and incubate at 95 oC for 3 minutes and slowly cooled to room temperature. Prepare 25 μM compounds with buffer and dilute 16 concentration gradients starting from 25 μM. Mix the compounds with G4-DNA 1:1 at room temperature and incubate in the dark for 20 minutes. After incubation, use MO-K022 glass capillaries to draw the mixture into the sample rack in sequence and detect it with MO.Affinity software. Data processing was performed using MO.AffinityAnalysisv2.2.4 software (Nano Temper Technologies).

[0095] from Figure 5 It can be seen that the compound B6 of the present invention has MET G4 (Pu23) has a stronger binding ability, with a Kd value of 73.5 nM; MET G4 mutant MET G4 (Pu23 G1T) did not bind. This indicates that compound B6 binds MET- G4 exerts its transcriptional repression activity.

[0096] (6) Broad-spectrum anti-tumor cell activity assay:

[0097] In addition to the above-mentioned non-small cell lung cancer cell lines A549 and NCI-H1975, the inhibitory effect of compound B6 on the proliferation of various cancer cells was determined according to the CCK-8 method, and the half inhibitory concentration IC of the compound's inhibitory activity on cell proliferation was obtained. 50 Cells in logarithmic growth phase were seeded in 96-well plates at 3000-5000 cells / well and placed at 37°C with 5% CO 2 The culture plates were incubated for 12-24 hours under the following conditions; 100 μL of the test compound solution with different concentrations was added to the culture plates, and the culture plates were incubated at 37 °C and 5% CO 2 Incubate for 72 hours under incubator conditions; 4 hours before the end of incubation, add 10 μL of CCK-8 solution (5 mg / mL) to each well.

[0098] After the incubation, OD was measured using a microplate reader. 450 , inhibition rate = (OD value of control group - OD value of experimental group) / OD value of control group × 100%; after obtaining the data, GraphPad Prism fitted and obtained IC 50 The IC values ​​of the compounds 50 Classification by description:

[0099] "A" stands for IC 50 The measured value is less than or equal to 1 μM;

[0100] "B" stands for IC 50The measured value is less than or equal to 5 μM and greater than 1 μM;

[0101] "C" stands for IC 50 The measured value was greater than 5 μM.

[0102] According to the experimental results, it can be concluded that the compounds of the present invention have a strong anti-proliferative activity against pancreatic cancer cell lines:

[0103] Table 2. Antiproliferative activity of compound B6

[0104]

[0105] It can be seen from Table 2 that the compound B6 of the present invention has a broad-spectrum anti-tumor cell activity.

[0106] (7) In vitro activity assay of mutant cell lines:

[0107] The inhibitory effect of the compound on the proliferation of engineered cell lines Ba / F3-TPR-MET-L1195V, Ba / F3-TPR-MET-D1228N and Ba / F3-TPR-MET-F1200I was determined according to the CellTiter Glo® method, and the half inhibitory concentration IC of the compound's inhibitory activity on cell proliferation was obtained 50 The cells in logarithmic growth phase were seeded at 5000 cells / well in a 96-well plate and placed at 37°C with 5% CO 2 The culture plates were incubated for 12-24 hours under the following conditions; 100 μL of the test compound solution with different concentrations was added to the culture plates, and the culture plates were incubated at 37 °C and 5% CO 2 Incubate for 72 hours under incubator conditions. Before measurement, the assay plate was equilibrated to room temperature, 50 µL of CellTiter Glo® reagent was added to each well, the contents were mixed on an orbital shaker for 2 minutes to induce cell lysis, followed by incubation at room temperature for 10 minutes to stabilize the luminescent signal, and the luminescence value (RLU) was recorded on the SpectraMax Paradigm reader.

[0108] The inhibition rate (Inh%) relative to the control wells treated with vehicle (DMSO) was calculated using the following formula: Inh% = 100-(RLU of compound group - RLU of blank group) / (RLU of control group - RLU of blank group)*100%; After the data were obtained, GraphPad Prism was used for fitting to obtain the IC 50 .

[0109] Table 3. Antiproliferative activity of compound B6 of the present invention against various c-Met mutant drug-resistant cells

[0110]

[0111] As shown in Table 3, in the engineered cell lines containing common mutation types of c-Met type I inhibitors (D1228N) and type II inhibitors (L1195V, F1200I), the anti-proliferative activity of compound B6 was significantly better than that of the marketed positive drugs for c-Met, and it has the potential to overcome acquired resistance to c-Met inhibitors.

[0112] Therefore, the MET transcription regulator prepared by the present invention can stabilize the G quadruplex of MET, inhibit the expression of c-Met, has a broad-spectrum anti-tumor activity and has significant anti-proliferation activity on cell lines resistant to c-Met inhibitors.

Claims

1. A MET A transcription inhibitor, characterized in that The structure of the inhibitor is shown in Formula I: ; Wherein, when X1 is CH, X2 is O; when X1 is S, X2 is CH; The R group is selected from the following: .

2. The inhibitor according to claim 1, characterized in that When X1 is S and X2 is CH, the R group is selected from the following: .

3. The inhibitor according to claim 1, characterized in that The inhibitor also includes pharmaceutically acceptable salts thereof.

4. The inhibitor according to claim 3, characterized in that The pharmaceutically acceptable salt is an acid addition salt of the compound shown in formula I, wherein the acid used for salt formation includes an inorganic acid and an organic acid, wherein the inorganic acid includes hydrochloric acid, sulfuric acid or phosphoric acid, and the organic acid includes acetic acid, trichloroacetic acid, trifluoroacetic acid, propionic acid, butyric acid, maleic acid, p-toluenesulfonic acid, malic acid, methanesulfonic acid, malonic acid, cinnamic acid, citric acid, fumaric acid, camphoric acid, digluconic acid, aspartic acid or tartaric acid.

5. A pharmaceutical composition, characterized in that Comprising the compound represented by formula I of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

6. Use of the MET transcription inhibitor according to claim 1 in the preparation of a drug for preventing and / or treating cancer.

7. The use according to claim 6, characterized in that: The cancer is lung cancer, liver cancer, pancreatic cancer, stomach cancer, kidney cancer, colon cancer, esophageal cancer, glioblastoma, leukemia or multiple myeloma.

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