A class of n-benzyl-[1,1'-biphenyl]-3-carboxamide compounds, and preparation method and application thereof

Abstract

The application relates to the field of pharmaceutical chemistry, and relates to a class of N-benzyl-[1,1'-biphenyl]-3-carboxamide compounds and a preparation method and application thereof, in particular to a class of compounds, which are compounds shown in the formula (I) or pharmaceutically acceptable salts thereof, and application of the compounds and pharmaceutical compositions containing the compounds in preparation of medicines for preventing or treating tumors and diseases related to viral infections and preparation of inhibitors of interaction between lysine methyltransferase p300 and cancer-promoting transcription factor ZEB1. It is proved by in-vitro experiments that the compounds have obvious blocking effect on the interaction between lysine acetyltransferase p300 and cancer-promoting transcription factor ZEB1, the compounds can inhibit the proliferation of various tumor cells, including pancreatic cancer, liver cancer, lung cancer, gastric cancer, kidney cancer, colon cancer, esophageal cancer, glioblastoma, leukemia, multiple myeloma and various solid tumors and hematological tumors.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a class of N-benzyl-[1,1'-biphenyl]-3-carboxamide compounds, their preparation methods, and applications. Background Technology

[0002] p300 is a multifunctional protein with lysine acetyltransferase (KAT) activity, catalyzing the acetylation of histone and non-histone lysine residues. It plays a central role in the regulation of signal transduction, autophagy, gene transcription, protein-protein interactions, cellular stress responses, DNA damage repair, protein stability, and intracellular protein localization. The oncogenic transcription factor ZEB1 is a key regulator in the epithelial-mesenchymal transition (EMT) process, and its abnormally high expression is closely related to tumor growth, invasion, metastasis, stemness maintenance, and treatment resistance.

[0003] Studies have shown a direct and functional interaction between p300 and ZEB1 (p300-ZEB1 PPI). This PPI is crucial for the stability and transcriptional activity of ZEB1. By binding to ZEB1, p300 catalyzes acetylation of the ZEB1 protein, stabilizing it, and forms a transcriptional activation complex, jointly driving a series of gene expression programs that promote tumor malignant progression. However, current therapeutic strategies targeting the p300-ZEB1 signaling axis mainly focus on indirect pathways, such as inhibitors of p300's KAT or BRD domains. These strategies have significant limitations due to their lack of specificity and indirect mechanisms of action.

[0004] To date, no small molecule compounds have been reported that can directly and specifically block the p300-ZEB1 PPI. Therefore, there is an urgent need in the field to develop an inhibitor that can directly target the p300-ZEB1 protein-binding interface, thereby efficiently and specifically disrupting this interaction, providing a novel precision treatment strategy for the treatment of malignant tumors (especially those driven by the p300-ZEB1 signaling axis). Summary of the Invention

[0005] Objective: The objective of this invention is to provide a class of N-benzyl-[1,1'-biphenyl]-3-carboxamide compounds or pharmaceutically acceptable salts thereof that exert potent antitumor activity by blocking p300-ZEB1 PPI, thus providing novel compounds for the development of p300-ZEB1 signaling axis-driven tumor drugs. Another objective of this invention is to provide a method for preparing and using the aforementioned N-benzyl-[1,1'-biphenyl]-3-carboxamide compounds or pharmaceutically acceptable salts thereof.

[0006] Technical solution:

[0007] In a first aspect, the present invention discloses a compound, which is a compound of formula (I), or a pharmaceutically acceptable salt thereof:

[0008] ;

[0009] R1 is selected from -OH or -CH3;

[0010] R2 is selected from -CN, -CF3 or -NO2 with para or meta substitution;

[0011] R3 is selected from H, -OH, -OCH3, -F, -Cl, and -Br;

[0012] R4 is selected from H, -OH, -OCH3, -F, -Cl, and -Br.

[0013] In some embodiments, R1 is selected from -OH or -CH3; R2 is selected from para- or meta-substituted -CN or -CF3; R3 is selected from H, -OH, -OCH3, -F, -Cl; and R4 is selected from H, -OH, -OCH3, -F, -Cl.

[0014] In some embodiments, when R1 is -OH and R2 is para-substituted -CN, R3 is selected from H and R4 is selected from -OH;

[0015] When R1 is -OH, R3 is -F and R4 is -Cl, R2 is selected from meta-substituted -CN or -CF3;

[0016] When R1 is -OH, R3 is -OCH3 and R4 is -OCH3, R2 is selected from meta-substituted -CN;

[0017] When R1 is -CH3, R3 is hydrogen, and R4 is -OCH3, R2 is selected from para- or meta-substituted -CN.

[0018] In some embodiments, the compound represented by formula (I) is selected from:

[0019] A1: 4'-Cyano-2-hydroxy-N-(4-hydroxybenzyl)-[1,1'-biphenyl]-3-carboxamide

[0020] ;

[0021] A2: 3'-Cyano-N-(4-Methoxybenzyl)-2-methyl-[1,1'-biphenyl]-3-carboxamide

[0022] ;

[0023] A3: 3'-Cyano-N-(3,4-Dimethoxybenzyl)-2-hydroxy-[1,1'-biphenyl]-3-carboxamide

[0024] ;

[0025] A4: 4'-Cyano-N-(4-Methoxybenzyl)-2-methyl-[1,1'-Biphenyl]-3-carboxamide

[0026] ;

[0027] A5: N-(3-chloro-4-fluorobenzyl)-3'-cyano-2-hydroxy-[1,1'-biphenyl]-3-carboxamide

[0028] ;

[0029] A6: N-(3-chloro-4-fluorobenzyl)-2-hydroxy-3'-trifluoromethyl-[1,1'-biphenyl]-3-carboxamide

[0030] .

[0031] In some embodiments, the pharmaceutically acceptable salt is a salt formed by the compound of formula (I) and an alkaline substance, wherein the alkaline substance forming the salt includes inorganic bases and organic bases, wherein the inorganic bases include hydroxides, carbonates or bicarbonates of sodium, potassium, lithium, calcium, magnesium, and zinc, and the organic bases include ammonia, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, histidine, choline, meglumine, or tromethamine.

[0032] In a second aspect, the present invention also provides a method for preparing the compound represented by formula (I), comprising:

[0033] S1. Compound A and compound B undergo a condensation reaction to obtain intermediate C;

[0034] S2, intermediate C and compound D undergo a coupling reaction to obtain the compound shown in formula (I);

[0035] The chemical structural formulas of compound A, compound B, intermediate C, and compound D are as follows:

[0036] , , , .

[0037] In this embodiment, the synthesis route is as follows:

[0038] .

[0039] In some embodiments, in step S1, DCC (N,N'-dicyclohexylcarbodiimide) and NHS (N-hydroxysuccinimide) are used as combined condensing agents during the reaction process;

[0040] In some embodiments, in step S2, tetra-(triphenylphosphine)palladium is used as a catalyst during the reaction.

[0041] Furthermore, in this embodiment, the reaction solvent used in steps S1 and S2 is 1,4-dioxane.

[0042] Thirdly, the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0043] Furthermore, the dosage form of the pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhaler, cream, ointment, suppository, or patch.

[0044] The dosage forms of the pharmaceutical compositions of the present invention can be prepared by those skilled in the art according to conventional methods in the pharmaceutical field. For example, the active ingredient can be mixed with one or more carriers (also called excipients) and then formulated into the desired dosage form, including tablets, capsules, granules, and aerosols; it can also be formulated into intravenous injection or intravenous lyophilized formulations according to conventional methods for the production of injectables.

[0045] Fourthly, the present invention also provides the use of the said compounds and pharmaceutical compositions in the preparation of medicaments for the prevention and / or treatment of tumors and viral infection-related diseases.

[0046] Furthermore, the tumors and virus-related diseases mentioned are diseases related to abnormalities in the interaction signaling axis between lysine methyltransferase p300 and the oncogenic transcription factor ZEB1.

[0047] More specifically, the tumor-related diseases are selected from: liver cancer, pancreatic cancer, colon cancer, lung cancer, stomach cancer, kidney cancer, esophageal cancer, breast cancer, glioblastoma, leukemia, and multiple myeloma.

[0048] The present invention also provides the use of the compound and pharmaceutical composition in the preparation of an inhibitor of the interaction between lysine methyltransferase p300 and the oncogenic transcription factor ZEB1.

[0049] Beneficial Effects: Compared with existing technologies, this invention provides for the first time a small molecule compound that can directly target the surface of the p300 protein (i.e., the TAZ2 domain, which is also the binding pocket of ZEB1) and specifically block its binding to ZEB1. This compound does not directly inhibit the KAT catalytic activity of p300, avoiding the severe toxic side effects caused by traditional p300 inhibitors due to their broad impact on epigenetic status, thus exhibiting higher targeting and safety. Since the p300-ZEB1 signaling axis plays a crucial role in the progression and drug resistance relapse of various malignant tumors (such as liver cancer, lung cancer, colorectal cancer, pancreatic cancer, etc.), the compound of this invention, by blocking the interaction between p300 and ZEB1, leads to the degradation of ZEB1, providing broad-spectrum therapeutic potential for treating these highly malignant cancers. Attached Figure Description

[0050] Figure 1 This is a schematic diagram illustrating the effects of compounds A1 to A6 on the transcriptional level of ZEB1.

[0051] Figure 2 This is a schematic diagram showing the effects of compounds A1 to A6 on the expression level of ZEB1 protein in HT1080 cells.

[0052] Figure 3 This is a schematic diagram illustrating the effect of compound A1 on ubiquitination of the ZEB1 protein.

[0053] Figure 4 This is a schematic diagram showing the affinity detection results of compounds A1 and A2 for the p300 TAZ2 domain protein.

[0054] Figure 5 This is a schematic diagram showing the effect of compound A1 on the binding ability of p300-ZEB1.

[0055] Figure 6 A schematic diagram of key amino acid residues in the p300 TAZ2 domain protein pocket where compound A1 binds;

[0056] Figure 7 This is a schematic diagram showing the affinity test results of compound A1 for wild-type or mutant p300 TAZ2 domain proteins.

[0057] Figure 8 This is a schematic diagram illustrating the effect of compound A1 on the in vitro proliferation of cancer cells.

[0058] Figure 9 This diagram illustrates the in vivo anti-hepatocellular carcinoma activity of compound A1 and its synergistic effect when used in combination with PD-L1 antibody.

[0059] Figure 10 A schematic diagram illustrating how compound A1 improves the intratumoral suppressive immune microenvironment and enhances antitumor immune effects.

[0060] Figure 11 This is a schematic diagram showing the effects of compound A1 on the morphology of vital organs and tissues at doses of 30 and 60 mg / kg. Detailed Implementation

[0061] To make the objectives of this invention more easily understood, the specific embodiments of this invention are described in detail below with reference to specific examples. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0062] This invention also provides a method for preparing the above-described compounds. The preparation of the compounds of general formula (I) of this invention can be accomplished by the following exemplary methods and examples, but these methods and examples should not be considered in any way as limiting the scope of this invention. The compounds described in this invention can also be synthesized by synthetic techniques known to those skilled in the art, or by combining synthetic methods known in the art with the methods described in this invention. The products obtained from each reaction step are obtained using separation techniques known in the art, including but not limited to extraction, filtration, distillation, crystallization, and chromatographic separation. The starting materials and chemical reagents required for synthesis can be conventionally synthesized according to literature (such as those provided by SciFmder) or purchased.

[0063] I. Synthesis of intermediates

[0064] Intermediate 1:

[0065] 3-Bromo-2-hydroxy-N-(4-hydroxybenzyl)benzamide (1):

[0066]

[0067] 3-Bromo-2-hydroxybenzoic acid (868 mg, 4.0 mmol) was dissolved in 20 mL of 1,4-dioxane solution. N-hydroxysuccinimide (NHS, 460 mg, 4.0 mmol) and N,N'-dicyclohexylcarbodiimide (DCC, 824 mg, 4.0 mmol) were added. The mixture was stirred at room temperature for 12 h under argon protection and then filtered. 4-hydroxybenzylamine (590 mg, 4.8 mmol) was added to the filtrate, and the mixture was heated at 60 °C for 3 h under argon protection. After the reaction was complete as monitored by TLC, the solvent was removed by vacuum concentration. The crude product was purified by rapid column chromatography to give intermediate 1. A white solid (1027 mg, 80% yield). ESI-MS m / z: 322.2 [M+H] + .

[0068] Intermediate 2:

[0069] 3-Bromo-N-(4-methoxybenzyl)-2-methylbenzamide (2):

[0070]

[0071] 3-Bromo-2-hydroxybenzoic acid (217 mg, 1.0 mmol) was dissolved in 10 mL of N,N-dimethylformamide (DMF) solution, followed by the sequential addition of 4-methoxybenzylamine (164 mg, 1.2 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 573 mg, 3.0 mmol), 1-hydroxybenzotriazole (HOBT, 203 mg, 1.5 mmol), and triethylamine (TEA, 505 mg, 5.0 mmol). The mixture was reacted at room temperature for 3 hours under argon protection. After the reaction was completed as monitored by TLC, the mixture was extracted with ethyl acetate, and the organic phases were combined. The crude product was purified by rapid column chromatography to give intermediate 2. A white solid (200 mg, 60% yield). ESI-MS m / z: 334.3 [M+H] + .

[0072] Intermediate 3:

[0073] 3-Bromo-N-(3,4-Dimethoxybenzyl)-2-hydroxybenzamide (3):

[0074]

[0075] Intermediate 3 was prepared from 3-bromo-2-hydroxybenzoic acid and 3,4-dimethoxybenzylamine via the same route as intermediate 1. It was a white solid in 85% yield, ESI-MS m / z: 366.5 [M+H]. + .

[0076] Intermediate 4:

[0077] 3-Bromo-N-(3-chloro-4-fluorobenzyl)-2-hydroxybenzamide (4):

[0078]

[0079] Intermediate 4 was prepared from 3-bromo-2-hydroxybenzoic acid and 3-chloro-4-fluorobenzylamine via the same route as intermediate 1. It was a white solid in 71% yield, ESI-MS m / z: 358.1 [M+H]. + .

[0080] II. Synthesis of the final product

[0081] Example 1

[0082] Synthesis of 4'-cyano-2-hydroxy-N-(4-hydroxybenzyl)-[1,1'-biphenyl]-3-carboxamide (A1):

[0083]

[0084] Intermediate 1 (1.0 mmol), p-cyanobenzoic acid (1.2 mmol), potassium carbonate (3.0 mmol), and tetra-(triphenylphosphine)palladium (0.06 mmol) were added sequentially to 20 mL of 1,4-dioxane solution. The mixture was heated at 100 °C overnight under argon protection. After the reaction was complete, the reaction mixture was filtered through a diatomaceous earth short column, and the crude product was purified by rapid column chromatography to give compound A1 as a white solid (62% yield). 1 H NMR (400 MHz, DMSO-d6) δ 13.85 (s, 1H), 9.56 (t, J= 5.9 Hz, 1H), 9.36 (s, 1H), 7.99 (dd, J = 8.1, 1.6 Hz, 1H), 7.89 (d, J = 8.4Hz, 2H), 7.78 (d, J = 8.5 Hz, 2H), 7.56 (dd, J = 7.6, 1.5 Hz, 1H), 7.17 (d, J= 8.5 Hz, 2H), 7.02 (t, J = 7.8 Hz, 1H), 6.75 (d, J = 8.5 Hz, 2H), 4.43 (d, J= 5.8 Hz, 2H).

[0085] Example 2

[0086] Synthesis of 3'-cyano-N-(4-methoxybenzyl)-2-methyl-[1,1'-biphenyl]-3-carboxamide (A2):

[0087]

[0088] Starting from intermediates 2- and 3-cyanobenzonic acid, the target product A2 was prepared according to the preparation method of compound A1. It was a white solid (65% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.82 (t, J = 6.1 Hz, 1H), 7.87 (td, J = 4.3, 1.6 Hz, 1H), 7.79 (q, J = 1.4 Hz, 1H), 7.67 (d, J = 5.3Hz, 2H), 7.39 – 7.23 (m, 5H), 6.90 (d, J = 8.7 Hz, 2H), 4.38 (d, J = 6.0 Hz, 2H), 3.73 (s, 3H), 2.15 (s, 3H).

[0089] Example 3

[0090] Synthesis of 3'-cyano-N-(3,4-dimethoxybenzyl)-2-hydroxy-[1,1'-biphenyl]-3-carboxamide (A3):

[0091]

[0092] Starting from intermediate 3 and 3-cyanobenzonic acid, the target product A3 was prepared by referring to the preparation method of compound A1. It was a white solid (80% yield). 1 H NMR (300 MHz, DMSO-d6) δ 13.75 (s, 1H), 9.61 – 9.53 (m, 1H), 8.03 – 7.96 (m, 2H), 7.92 (dt, J = 8.0, 1.4 Hz, 1H), 7.85 – 7.79 (m,1H), 7.65 (d, J = 7.8 Hz, 1H), 7.62 – 7.56 (m, 1H), 7.06 – 6.96 (m, 2H), 6.94– 6.84 (m, 2H), 4.46 (d, J = 5.7 Hz, 2H), 3.74 (d, J = 5.7 Hz, 6H).

[0093] Example 4

[0094] Synthesis of 4'-cyano-N-(4-methoxybenzyl)-2-methyl-[1,1'-biphenyl]-3-carboxamide (A4):

[0095]

[0096] Starting from intermediates 2- and 4-cyanobenzonic acid, the target product A4 was prepared according to the preparation method of compound A1. It was a white solid (70% yield). 1H NMR (400 MHz, DMSO-d6) δ 8.82 (t, J = 6.2 Hz, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.5 Hz, 2H), 7.35 (dt, J = 4.8, 2.9Hz, 2H), 7.30 – 7.22 (m, 3H), 6.90 (d, J = 8.8 Hz, 2H), 4.38 (d, J = 6.2 Hz, 2H), 3.73 (s, 3H), 2.15 (s, 3H).

[0097] Example 5

[0098] Synthesis of N-(3-chloro-4-fluorobenzyl)-3'-cyano-2-hydroxy-[1,1'-biphenyl]-3-carboxamide (A5):

[0099]

[0100] Starting from intermediate 4 and 3-cyanobenzonic acid, the target product A5 was prepared by referring to the preparation method of compound A1. White solid (yield 66%). 1 H NMR (300 MHz, DMSO-d6) δ 13.57 (s, 1H), 9.66 (t, J =5.9 Hz, 1H), 8.04 – 7.94 (m, 2H), 7.92 (dt, J = 7.9, 1.5 Hz, 1H), 7.82 (dt, J= 7.7, 1.4 Hz, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.59 (td, J = 7.6, 2.0 Hz, 2H), 7.45 – 7.34 (m, 2H), 7.05 (t, J = 7.8 Hz, 1H), 4.53 (d, J = 5.8 Hz, 2H).

[0101] Example 6

[0102] Synthesis of N-(3-chloro-4-fluorobenzyl)-3'-trifluoromethyl-2-hydroxy-[1,1'-biphenyl]-3-carboxamide (A6):

[0103]

[0104] Starting from intermediate 4 and 3-cyanobenzonic acid, the target product A6 was prepared by referring to the preparation method of compound A1. It was a white solid (72% yield). 1H NMR (400 MHz, DMSO-d6) δ 13.55 (s, 1H), 9.66 (t, J =6.0 Hz, 1H), 7.98 (dd, J = 8.1, 1.6 Hz, 1H), 7.91 – 7.84 (m, 2H), 7.75 – 7.64(m, 2H), 7.59 (ddd, J = 9.2, 7.3, 1.7 Hz, 2H), 7.39 (pd, J = 7.0, 5.3, 2.9Hz, 2H), 7.05 (t, J = 7.8 Hz, 1H), 4.53 (d, J = 5.8 Hz, 2H).

[0105] III. Biological Evaluation Experiment

[0106] (1) Detection of ZEB1 transcription and protein levels

[0107] HT1080 tumor cells in logarithmic growth phase were used at a rate of 4 × 10⁻⁶ 5 ~ 5×10 5 Cells were seeded in 6-well plates and cultured at 37°C with 5% CO2 for 12–24 hours. 100 μL of different test compound solutions (final concentration 5 μM) were added to the culture plates, and the plates were incubated at 37°C with 5% CO2 for 72 hours. After incubation, cells were collected, centrifuged at 1200 rpm for 3 minutes, washed twice with PBS, resuspended in 1 mL PBS, transferred to 1.5 mL centrifuge tubes, centrifuged at 2500 rcf for 5 minutes, and the supernatant was carefully discarded. TRIZOL lysis buffer was added to extract total RNA for subsequent reverse transcription and quantitative PCR (RT-qPCR) detection. Alternatively, RIPA cell lysis buffer (containing protease inhibitors and phosphatase inhibitors) was added, shaken for 15 minutes, centrifuged at 1400 rcf at 4°C for 15 minutes, and the supernatant was collected to determine protein concentration for subsequent Western blotting.

[0108] Figure 1 This is a schematic diagram illustrating the effects of compounds A1 to A6 on the transcriptional level of ZEB1. Figure 1 The results showed that compounds A1 to A6 had no effect on the transcriptional level of ZEB1.

[0109] Figure 2 This is a schematic diagram showing the effects of compounds A1 to A6 on the expression level of ZEB1 protein in HT1080 cells. Figure 2 The results showed that both compounds A1 and A2 could downregulate ZEB1 protein expression, with compound A1 being more significant. Figure 3 The results showed that compound A1 induced ubiquitination and subsequent degradation of the ZEB1 protein.

[0110] (2) Microscale thermophoresis (MST) was used to test the binding ability of the compound to p300 and the effect of the compound on the p300-ZEB1 interaction.

[0111] Purified p300 TAZ2 domain protein (the region where p300 binds to ZEB1) was labeled with a fluorescent dye under light-protected conditions. After removing free dye using a desalting column, the concentration and fluorescence intensity of the labeled protein were measured. The labeled protein was diluted to an appropriate concentration and mixed with an equal volume fraction of the compound solution to maintain a gradient in compound concentration while keeping the protein concentration constant. The mixed sample was incubated at room temperature for 15 minutes and then added to an MST capillary tube.

[0112] Figure 4 This is a schematic diagram showing the affinity detection results of compounds A1 and A2 for the p300 TAZ2 domain protein. Figure 5 This is a schematic diagram showing the effect of compound A1 on the binding ability of p300-ZEB1.

[0113] Figure 4 The results show that compounds A1 and A2 in the embodiments of the present invention have good binding ability to the p300 TAZ2 domain protein. Compound A1 binds to the K domain of p300 TAZ2. d The value was 1.2 ± 0.32 μM, indicating that compound A2 binds to the K of p300 TAZ2. d The value was 8.6 ± 2.37 μM, indicating that compound A1 had a higher binding affinity for p300 TAZ2 than compound A2 (K). d A higher value indicates lower affinity. Figure 5 The results show that compound A1 enables the K-type binding of p300 TAZ2 to ZEB1 ZF3 (this region is the binding region between ZEB1 and p300). d The value increased from 2.6 ± 0.44 μM to 145.4 ± 39.85 μM (i.e., a 56-fold decrease in affinity), indicating that compound A1 binds with high affinity to p300 TAZ2 and occupies the binding pocket, thereby interfering with the latter's binding to ZEB1. It should be noted that compound A1 hardly binds to ZEB1 (data not shown).

[0114] Figure 6 A schematic diagram of key amino acid residues in the p300 TAZ2 domain protein pocket where compound A1 binds; Figure 7 This is a schematic diagram showing the affinity test results of compound A1 for wild-type or mutant p300 TAZ2 domain proteins.

[0115] Figure 6The results showed that compound A1 binds to amino acid residues I1781 and Q1784 in the p300 TAZ2 domain protein pocket via stable hydrogen bonds, and binds to amino acid residues L1785 and L1788 via weaker π-alkyl hydrophobic interactions. Figure 7 The results showed that compound A1 binds to the K domain of the wild-type (WT) p300 TAZ2 domain protein. d The value was 1.5 ± 0.35 μM, and the K binding of the I1781T mutant protein was... d The value was 25.8 ± 5.62 μM, and the K binding of the Q1784A mutant protein was... d The value was 45.9 ± 8.23 ​​μM, representing the K binding of the I1781T / Q1784A double mutant protein. d The value was 153.4 ± 20.35 μM (the affinity was 100 times lower than that of the wild type).

[0116] (3) Detection of the in vitro antiproliferative activity of the compound

[0117] Logarithmic growth phase cells were fed at 4 × 10 5 ~5×10 5 Cells / wells were seeded into 6-well plates and incubated at 37°C with 5% CO2 for 12–24 hours. 100 μL of serially diluted test compound solutions of different concentrations were added to the culture plates, and the plates were incubated at 37°C with 5% CO2 for 72 hours to detect the antiproliferative activity of the compounds.

[0118] Figure 8 The results showed that compound A1 could significantly inhibit the in vitro proliferation (IC50) of liver cancer and colorectal cancer cells. 50 The IC50 values ​​were 5.0 ± 0.9 μM and 8.1 ± 1.5 μM, respectively, for the in vitro proliferation of p300 gene-silenced cells (p300-KD, where the compound loses its target). 50 The concentrations were 15.3 ± 3.2 μM and 24.1 ± 4.8 μM, respectively, indicating that p300 gene silencing reduced the sensitivity of compound A1 to inhibiting cell proliferation.

[0119] Table 1: IC50 of compounds A1-A6 against liver cancer and colorectal cancer cells 50 value

[0120]

[0121] As shown in Table 1, the synthesized compounds A1-A6 can inhibit the in vitro proliferation of liver cancer and colorectal cancer cells. In particular, compound A1 can significantly inhibit the in vitro proliferation of liver cancer and colorectal cancer cells, and has the potential value for developing tumor-related drugs.

[0122] (4) Evaluation of the in vivo antitumor activity of the compound and preliminary assessment of potential toxic side effects

[0123] The C57 / BL6 mice underwent abdominal preparation the day before surgery and were fasted for 12 hours. An incision was made along the midline of the abdomen of the anesthetized mice to expose the liver. The largest lobe of the liver was gently pulled out of the incision using a sterile cotton swab. An injection needle was inserted obliquely into the liver parenchyma to a depth of approximately 3 mm, and a solution containing 1×10⁻⁶ g / L of [unspecified substance] was slowly injected. 6 100 μL of a suspension of mouse Hep53.4 tumor cells was used. Immediately after needle removal, the puncture site was gently pressed with a cotton swab to stop bleeding. After confirming no active bleeding, the liver was gently returned to the peritoneal cavity, and the cavity was closed layer by layer by suture. The general condition of the mice and the wound were observed daily post-surgery. After 3 days of feeding, the mice were randomly divided into groups and intraperitoneal drug administration was initiated. Two dosage groups were established: 30 mg / kg and 60 mg / kg. PD-L1 antibody and control IgG were administered on days 7 and 10 after tumor inoculation, respectively, at a dose of 50 μg / mouse. The mice's condition and weight were monitored daily during the experiment. Two weeks later, the mice were sacrificed, and liver tissue was harvested. The maximum long diameter of the tumor was measured, the liver-to-body ratio was calculated, and flow cytometry analysis was performed. Histological sections of vital organs were collected and stained.

[0124] Figure 9 This diagram illustrates the in vivo anti-hepatocellular carcinoma activity of compound A1 and its synergistic effect when used in combination with PD-L1 antibody. Figure 10 A schematic diagram illustrating how compound A1 improves the intratumoral suppressive immune microenvironment and enhances antitumor immune effects. Figure 9 The results show that compound A1 in the embodiments of the present invention has significant in vivo anti-hepatocellular carcinoma activity (in a dose-dependent manner) and has a synergistic effect when used in combination with PD-L1 antibody. Figure 10 The results showed that compound A1 significantly increased activated cytotoxic CD8. + The reduction of T cell infiltration and regulatory T cell infiltration indicates that compound A1 can effectively improve the intratumoral suppressive immune microenvironment and enhance antitumor immune effects.

[0125] Figure 11 This is a schematic diagram showing the effects of compound A1 on the morphology of vital organs and tissues at doses of 30 and 60 mg / kg. Figure 11 The results showed that compound A1 had no significant effect on the morphology of vital organs such as the heart, spleen, lungs, and kidneys in tumor-bearing mice at doses of 30 and 60 mg / kg.

[0126] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A compound, characterized in that, The compound represented by formula (I), or a pharmaceutically acceptable salt thereof: ； R1 is selected from -OH or -CH3; R2 is selected from -CN, -CF3 or -NO2, which are para- or meta-substituted; R3 is selected from H, -OH, -OCH3, -F, -Cl, and -Br; R4 is selected from H, -OH, -OCH3, -F, -Cl, and -Br.

2. The compound according to claim 1, characterized in that, R1 is selected from -OH or -CH3; R2 is selected from -CN or -CF3, which are para or meta substitutions; R3 is selected from H, -OH, -OCH3, -F, -Cl; R4 is selected from H, -OH, -OCH3, -F, and -Cl.

3. The compound according to claim 1, characterized in that, When R1 is -OH and R2 is para-substituted -CN, R3 is selected from H and R4 is selected from -OH; When R1 is -OH, R3 is -F and R4 is -Cl, R2 is selected from meta-substituted -CN or -CF3; When R1 is -OH, R3 is -OCH3 and R4 is -OCH3, R2 is selected from meta-substituted -CN; When R1 is -CH3, R3 is hydrogen, and R4 is -OCH3, R2 is selected from para- or meta-substituted -CN.

4. The compound according to claim 1, characterized in that, The compound represented by formula (Ι) is selected from: A1: 4'-Cyano-2-hydroxy-N-(4-hydroxybenzyl)-[1,1'-biphenyl]-3-carboxamide ； A2: 3'-Cyano-N-(4-Methoxybenzyl)-2-methyl-[1,1'-biphenyl]-3-carboxamide ； A3: 3'-Cyano-N-(3,4-Dimethoxybenzyl)-2-hydroxy-[1,1'-biphenyl]-3-carboxamide ； A4: 4'-Cyano-N-(4-Methoxybenzyl)-2-methyl-[1,1'-Biphenyl]-3-carboxamide ； A5: N-(3-chloro-4-fluorobenzyl)-3'-cyano-2-hydroxy-[1,1'-biphenyl]-3-carboxamide ； A6: N-(3-chloro-4-fluorobenzyl)-2-hydroxy-3'-trifluoromethyl-[1,1'-biphenyl]-3-carboxamide 。 5. The compound according to claim 1, characterized in that, The pharmaceutically acceptable salt is a salt formed by the compound represented by formula (I) and an alkaline substance. The alkaline substance forming the salt includes inorganic bases and organic bases. The inorganic bases include hydroxides, carbonates, or bicarbonates of sodium, potassium, lithium, calcium, magnesium, and zinc. The organic bases include ammonia, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, histidine, choline, meglumine, or tromethamine.

6. The method for preparing the compound according to claim 1, characterized in that, The preparation method of the compound shown in formula (I) includes: S1. Compound A and compound B undergo a condensation reaction to obtain intermediate C; S2, intermediate C and compound D undergo a coupling reaction to obtain the compound shown in formula (I); The chemical structural formulas of compound A, compound B, intermediate C, and compound D are as follows: 、 、 、 。 7. The method for preparing the compound according to claim 6, characterized in that, In step S1, N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide are used as a combined condensing agent in the reaction process; And / or, in step S2, tetra-(triphenylphosphine)palladium is used as a catalyst during the reaction.

8. A pharmaceutical composition, characterized in that, It includes a therapeutically effective amount of the compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

9. The use of the compound of any one of claims 1-5, or the pharmaceutical composition of claim 8, in the preparation of medicaments for the prevention and / or treatment of tumors and viral infection-related diseases.

10. The use of the compound of any one of claims 1-5 or the pharmaceutical composition of claim 8 in the preparation of an inhibitor of the interaction between lysine methyltransferase p300 and the oncogenic transcription factor ZEB1.

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