Small-molecule inhibitor for specifically targeting ARHGAP9, preparation method of small-molecule inhibitor and application of small-molecule inhibitor in tumor resistance

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a small-molecule inhibitor of specific targeting ARHGAP9, a preparation method and application in tumor resistance. The invention firstly provides a novel small molecular compound N-(4-fluorobenzyl)-6-(3-phenylpiperazine-1-yl) picolinamide, the novel small molecular compound targets ARHGAP9 high-expression tumors, it is proved in anti-tumor experiments that the novel small molecular compound can effectively activate immune cells and has a good aging and dose-effect relationship, and the novel small molecular compound can be used for preparing ARHGAP9 high-expression tumors. The positive effect of resisting the growth of various transplanted tumors is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a small molecule inhibitor specifically targeting ARHGAP9, a preparation method thereof, and use thereof in anti-tumor treatment. Background Art

[0002] Cancer poses a serious threat to human health. The discovery of new targets and the development of interventional drugs are cornerstones of clinical cancer treatment. Immune checkpoints refer to inhibitory signaling pathways within the immune system, a core mechanism by which tumor cells evade immune control. Inhibiting immune checkpoints can effectively enhance the activity of immune cells in killing tumor cells.

[0003] ARHGAP9 (RhoGTPase activating protein 9) is one of the 47 currently known members of the Rho-GAPs subfamily. However, current reports on the function of ARHGAP9 in cancer are conflicting. Some studies have reported that ARHGAP9 is a pro-oncogene, including one that promotes the proliferation and metastasis of colon cancer cells (Sun et al. Tissue cell. 2022). ARHGAP9 is highly expressed in ovarian cancer and negatively correlated with patient prognosis, suggesting it could serve as a prognostic marker for ovarian cancer (Shen et al. Transl Cancer Res. 2021;10: 4440-4453). However, there are also contradictory reports suggesting that ARHGAP9 is a tumor suppressor gene. These include bioinformatics analysis showing that patients with breast and bladder cancers with high ARHGAP9 expression have longer survival and better prognosis than those with low ARHGAP9 expression (Chen et al. Oncol Lett. 2019;18:6017-6025). ARHGAP9 inhibits liver cancer cell proliferation and migration (Zhang et al. Cell Death Dis. 2018;9:916). ARHGAP9 knockout promotes lung cancer metastasis (Song et al. Genomics. 2023). Therefore, the role of ARHGAP9 in cancer development and progression remains controversial. Furthermore, no ARHGAP9 inhibitors have been reported, leaving a significant gap in this field.

[0004] In summary, there is an urgent need to develop new small molecule drugs that can effectively inhibit ARHGAP9 to fill the gap in the industry and assist in clinical and scientific research. Summary of the Invention

[0005] The purpose of the present invention is to provide a novel small molecule drug that can effectively inhibit ARHGAP9, and its preparation method and use, to partially solve or alleviate the above-mentioned deficiencies in the prior art. The present invention specifically adopts the following technical solutions.

[0006] On the one hand, the present invention provides a novel small molecule compound and its application in the field of tumor treatment.

[0007] A novel small molecule compound targeting ARHGAP9 high-expressing tumors, wherein the novel small molecule compound is N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)pyridine amide; the chemical formula is C 23 H 23 FN4O; the structural formula is shown in the following formula (I): Formula (I).

[0008] The compound of formula (I) has a pyridineamide parent structure, connected to fluorobenzyl and phenylpiperazine substituents; the structure is shown below: Formula (II); Wherein R1 represents fluorobenzyl; R2 represents phenylpiperazine.

[0009] Furthermore, the novel small molecule compound has the function of competitively binding to ARHGAP9 with the ARHGAP9 functional ligand RAC2.

[0010] A small molecule compound or a pharmaceutically acceptable salt thereof is used in the preparation of an anti-tumor drug targeting ARHGAP9, wherein the small molecule compound is N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)pyridinecarboxamide; the structural formula is shown in the compound of formula (I).

[0011] Furthermore, the types of tumors include solid tumors and hematological tumors.

[0012] Furthermore, the solid tumor or blood tumor is a solid tumor or blood tumor mediated by high expression of ARHGAP9, including: (1) Patients with solid tumors whose tumor tissue or lymph node pathological sections were evaluated for ARHGAP9 expression, and whose CPS (combined positive score of tumor cells and immune cells) was greater than 1% or IC (positive score of immune cells) was greater than 1% or TC (positive proportion of tumor cells) was greater than 25%.

[0013] (2) Patients with hematologic tumors (such as leukemia, myeloma, lymphoma, etc.) whose bone marrow puncture or peripheral blood flow cytometry tests show CPS (combined positive score of tumor cells and immune cells) > 1% or IC (positive score of immune cells) > 1% or TC (positive proportion of tumor cells) > 25%.

[0014] Furthermore, the pharmaceutically acceptable salt forms include salts formed between the compound of formula (I) and inorganic or organic acids.

[0015] Furthermore, the inorganic acid includes typical inorganic acids such as hydrochloric acid, sulfuric acid, bisulfate, phosphoric acid, dihydrogen phosphate, monohydrogen phosphate, hydrobromic acid, nitric acid, hydroiodic acid, carbonic acid, bicarbonate or aminosulfonic acid.

[0016] Furthermore, the organic acid includes typical organic acids such as methanesulfonic acid, citric acid, tartaric acid, maleic acid, fumaric acid, acetic acid, propionic acid, butyric acid, lactic acid, malic acid, succinic acid, malonic acid, benzoic acid, phenylacetic acid, phenylpropionic acid, p-toluenesulfonic acid, methanesulfonic acid, adipic acid, ascorbic acid, glutaric acid, propionic acid, oxalic acid, glutamic acid, aspartic acid, arginine, pamoic acid, camphorsulfonic acid, stearic acid, picric acid, boric acid, salicylic acid, nicotinic acid, fumaric acid, edetic acid, gluconic acid, gentisic acid, oleic acid, pamoic acid, sorbic acid or lauric acid.

[0017] The term "pharmaceutically acceptable" as used herein refers to compounds, raw materials, compositions and / or preparations that can be prepared into pharmaceutically acceptable dosage forms with the compound of formula (I) of the present invention within a reasonable medical dosage range, and that have no excessive toxicity, irritation, allergic reaction or side effects upon contact with patients, and are effective for their intended use.

[0018] Furthermore, the dosage form of the anti-tumor drug includes a dosage form for gastrointestinal administration and / or an injection dosage form.

[0019] The "gastrointestinal dosage forms" described in the present invention include common tablets, capsules, solutions, suspensions, etc.

[0020] The "injection dosage form" mentioned in the present invention includes common intravenous injections, intramuscular injections or subcutaneous injections, etc.

[0021] An anti-tumor agent comprising immune cells and a small molecule compound; the small molecule compound is N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)pyridineamide; the structural formula is shown in the compound of formula (I).

[0022] Preferably, the immune cells include T cells and NK cells.

[0023] Furthermore, the small molecule compound is configured as an in vitro immune activator of the immune cells.

[0024] Another aspect of the present invention provides a method for synthesizing the novel small molecule compound.

[0025] A method for synthesizing a novel small molecule compound, N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)pyridineamide, comprises the following steps: S01: In the presence of triethanolamine and acetonitrile, ethyl α-bromophenylacetate reacts with N-Boc-ethylenediamine to generate ethyl 2-[(2-{(tert-butoxycarbonyl)amino}ethyl)amino]-2-phenylacetate; S02: the ethyl 2-[(2-{(tert-butoxycarbonyl)amino}ethyl)amino]-2-phenylacetate is first reacted in the presence of trifluoroacetic acid and dichloromethane, and then in the presence of triethylamine and ethanol to produce 3-phenylpiperazin-2-one; S03: The 3-phenylpiperazine-2-one reacts with Boc anhydride and sodium bicarbonate in the presence of a H2O / THF mixed solvent to generate 1-tert-butyloxycarbonyl-3-oxo-2-phenylpiperazine; S04: Under the action of borane-tetrahydrofuran, the amide carbonyl group of the 1-tert-butoxycarbonyl-3-oxo-2-phenylpiperazine is reduced to generate 1-tert-butoxycarbonyl-2-phenylpiperazine; S05: The 1-tert-butoxycarbonyl-2-phenylpiperazine reacts with 6-chloro-2-pyridinecarboxylic acid methyl ester under the action of cesium carbonate to generate 6-[4-(tert-butoxycarbonyl)-3-phenylpiperazin-1-yl]picolinic acid methyl ester; S06: Under the action of lithium hydroxide, the ester group of the methyl 6-[4-(tert-butoxycarbonyl)-3-phenylpiperazin-1-yl]picolinate is reduced to a carboxylic acid to generate 6-[4-(tert-butoxycarbonyl)-3-phenylpiperazin-1-yl]picolinic acid; S07: The 6-[4-(tert-butoxycarbonyl)-3-phenylpiperazin-1-yl]picolinic acid reacts with p-fluorobenzylamine to generate an amide compound, N-(4-fluorobenzyl)-6-(3-phenylpiperazin-4-tert-butoxycarbonyl-1-yl)picolinamide; S08: The N-(4-fluorobenzyl)-6-(3-phenylpiperazine-4-tert-butoxycarbonyl-1-yl)picolinamide is subjected to the action of hydrochloric acid to remove the Boc substituent to generate N-(4-fluorobenzyl)-6-(3-phenylpiperazine-1-yl)picolinamide.

[0026] Furthermore, 1.0 equivalent of ethyl α-bromophenylacetate and 1.2 equivalents of N-Boc-ethylenediamine were added to triethylamine and acetonitrile solution under a nitrogen atmosphere, and reacted at 60° C. to generate ethyl 2-[(2-{(tert-butoxycarbonyl)amino}ethyl)amino]-2-phenylacetate.

[0027] Further, 1.0 equivalent of ethyl 2-[(2-{(tert-butoxycarbonyl)amino}ethyl)amino]-2-phenylacetate was added to 2.0 equivalents of trifluoroacetic acid and dichloromethane under a nitrogen atmosphere, and the mixture was reacted at room temperature to obtain a first reaction solution, and then saturated sodium carbonate was added to adjust the pH of the first reaction solution to alkaline; the organic phase was then extracted and concentrated; triethylamine and ethanol solution were then added under a nitrogen atmosphere to obtain a second reaction solution, and the second reaction solution was placed in an 80° C. oil bath to react; after completion of the reaction, the reaction was quenched with water, and then the organic phase was extracted, washed, concentrated, and purified to obtain 3-phenylpiperazine-2-one.

[0028] Furthermore, 1.0 equivalent of the 3-phenylpiperazine-2-one, 1.2 equivalents of Boc anhydride and 1.2 equivalents of sodium bicarbonate were added to a mixed solvent of H2O and THF to obtain a third reaction solution, and the third reaction solution was placed in a 60°C water bath for reaction; after the reaction was completed, the organic phase was extracted, washed, concentrated, and purified to obtain 1-tert-butoxycarbonyl-3-oxo-2-phenylpiperazine.

[0029] Furthermore, 1.0 equivalents of 1-tert-butoxycarbonyl-3-oxo-2-phenylpiperazine were added to a tetrahydrofuran solution under a nitrogen atmosphere to obtain a fourth reaction liquid, the fourth reaction liquid was placed in a 0°C reactor, and then 1.5 equivalents of BH3-THF solution were added to obtain a fifth reaction liquid, and the temperature of the fifth reaction liquid was raised to 70°C for reaction; after completion of the reaction, the reaction was quenched with water, and then the organic phase was extracted, washed, concentrated, and purified to obtain 1-tert-butoxycarbonyl-2-phenylpiperazine.

[0030] Further, 1.0 equivalents of the 1-tert-butoxycarbonyl-2-phenylpiperazine, 1.2 equivalents of methyl 6-chloro-2-pyridinecarboxylate, 15 mol% of 2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl, 5 mol% of trisdibenzylideneacetone dipalladium and 3 equivalents of cesium carbonate were added to a 1,4-dioxane solution under a nitrogen atmosphere to obtain a sixth reaction solution; the sixth reaction solution was heated to 100° C. in an oil bath for reaction; after the reaction was completed, the reaction product was concentrated and purified to obtain methyl 6-[4-(tert-butoxycarbonyl)-3-phenylpiperazin-1-yl]picolinate.

[0031] Furthermore, 1.0 equivalent of methyl 6-[4-(tert-butoxycarbonyl)-3-phenylpiperazin-1-yl]picolinate was dissolved in a mixed solvent of THF and water, and 2.0 equivalents of LiOH were added, and the mixture was reacted at room temperature. After the reaction was completed, saturated brine was added, and the organic phase was subsequently extracted, concentrated, and purified to obtain 6-[4-(tert-butoxycarbonyl)-3-phenylpiperazin-1-yl]picolinic acid.

[0032] Further, 1.0 equivalents of the 6-[4-(tert-butoxycarbonyl)-3-phenylpiperazin-1-yl]picolinic acid and 1.2 equivalents of p-fluorobenzylamine were added to a mixed solution of N,N'-dimethylformamide and dichloromethane under a nitrogen atmosphere to obtain a seventh reaction solution; then 1.2 equivalents of 1-hydroxybenzotriazole and 1.2 equivalents of triethylamine were slowly added to obtain an eighth reaction solution, and the mixture was reacted at room temperature, and then the eighth reaction solution was cooled to 0°C; then 1.5 equivalents of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to react at 0°C, and then the reaction system was heated to room temperature and the reaction continued; after the reaction was completed, the reaction solution was concentrated, and the organic phases were extracted, combined, washed, concentrated, and purified to obtain N-(4-fluorobenzyl)-6-(3-phenylpiperazin-4-tert-butoxycarbonyl-1-yl)picolinamide.

[0033] Furthermore, 1.0 equivalent of the N-(4-fluorobenzyl)-6-(3-phenylpiperazine-4-tert-butoxycarbonyl-1-yl)picolinamide was dissolved in HCl in 1,4-dioxane at room temperature to obtain a ninth reaction solution. After the reaction was completed, a saturated sodium carbonate solution was added to adjust the pH value of the ninth reaction solution to alkaline. The organic phase was then extracted and concentrated to obtain the N-(4-fluorobenzyl)-6-(3-phenylpiperazine-1-yl)picolinamide.

[0034] Another aspect of the present invention is to provide various combinations of the novel small molecule compounds.

[0035] The novel small molecule compound N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)pyridine amide provided by the present invention can be used in combination with one or more tumor treating drugs, including but not limited to the following drugs.

[0036] (1) Chemotherapy drugs: such as cyclophosphamide, cisplatin, oxaliplatin, carboplatin, busulfan, thiotepa, mitomycin, methotrexate, pemetrexed, fluorouracil, gemcitabine, cytarabine, doxorubicin, epirubicin, irinotecan, topotecan, etoposide, taxanes, vinca alkaloids, eribulin, and asparaginase.

[0037] (2) Small molecule targeted drugs: EGFR inhibitors: gefitinib, erlotinib, icotinib, osimertinib, ametinib, vumetinib, etc.; ALK inhibitors: crizotinib, alectinib, lorlatinib, etc.; MEK inhibitors: trametinib, etc.; HER2 inhibitors: lapatinib, cilotinib, neratinib, tucatinib, etc.; PARP inhibitors: olaparib, niraparib, fluzoparib, etc.; mTOR inhibitors: everolimus, etc.; HDAC inhibitors: cedamide, etc.; BCR-ABL inhibitors: imatinib, dasatinib, nilotinib, ponatinib, etc.; MET inhibitors: Saivotinib, Capmatinib, etc.; RET inhibitors: Pratinib, BRAF inhibitors: Dabrafenib, Vemurafenib, Encorafenib, etc., CDK4 / 6 inhibitors: Guabecib, Abemaciclib, etc., NTRK inhibitors: Larotrectinib, Entrectinib, etc.; BTK inhibitors: Ibrutinib, Zanubrutinib, etc.; JAK inhibitors: Ruxolitinib, etc.; PI3K inhibitors: Alpelisib, etc., Anti-vascular multikinase inhibitors: Anlotinib, Apatinib, Lenvatinib, Axitinib, Sunitinib, Cabozantinib, Regorafenib, Sorafenib, etc.; PDGFR / c-Kit inhibitors: Imatinib, Nilotinib, Afatinib, etc.; Protease inhibitors: Bortezomib, Ixazomib, etc.; FGFR2 inhibitors: Pemigatinib, etc.; IDH1 inhibitors: Ivosidenib, etc.; Nuclear transport protein inhibitors: Selinexor, etc.

[0038] (3) Antibody targeted drugs: Anti-HER2: trastuzumab, pertuzumab, ZW25, KN026, etc.; Anti-EGFR: cetuximab, nimotuzumab, etc.; Anti-VEGF: bevacizumab, ramucirumab, human endostatin, etc.; Anti-CD20: rituximab, etc., Anti-CD38: daratumumab, etc.; Anti-CD19-CD3: blinatumomab, etc.; Anti-EGFR-MET: JNJ-372, etc.; Anti-DLL4-VEGF: navicixizumab, etc.

[0039] (4) Immune checkpoint inhibitors (Anti-PD1: nivolumab, pembrolizumab, toripalizumab, sintilimab, tislelizumab, carrelizumab, etc., Anti-PDL1: atezolizumab, durvalumab, sugemalimab, avelumab, etc., Anti-CTLA4: ipilimumab, etc., Anti-LAG3: relalizumab, etc., Anti-TIGIT: MK-7684A, etc., Anti-PD1 / CLTA4: KN046, XmAb2 0717, Cadonilimab, etc., Anti-PD-1 / TIM-3: RO-7121661, etc., Anti-PD1 / CD27: CDX-527, etc., Anti-PDL1 / TGFB: JS201, etc., Anti-PD1 / PDL1: Reozalimab, IB1318, etc., Anti-PD-1 / VEGF: Ivonescimab, etc., Anti-PD-1 / HER2: Fidasimtamab, etc.).

[0040] (5) Immunomodulators: such as thalidomide, lenalidomide, thymosin, retinoic acid, etc.

[0041] (6) Antibody-drug conjugates: venetoclax, U3-1402, trastuzumab emtansine, etc. (7) Hormones: tamoxifen, toremifene, letrozole, anastrozole, enzalutamide, bicalutamide, abiraterone, etc.

[0042] (8) Cell therapy: CAR-T, CAR-NK, etc.

[0043] Beneficial technical effects: The present invention provides a novel small molecule compound, N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)pyridineamide, that targets tumors with high ARHGAP9 expression. This novel small molecule compound, with fluorobenzyl and phenylpiperazine attached to the pyridineamide ring structure, competitively binds to ARHGAP9's functional ligand, RAC2, thereby inhibiting ARHGAP9. Animal experiments have confirmed that this novel small molecule compound exhibits significant anti-tumor immune effects while also possessing good biosafety.

[0044] Currently, the role of ARHGAP9 in the development and progression of cancer is still controversial. Although there are reports that knocking down ARHGAP9 expression can inhibit the progression of certain specific cancers (Sun et al. Int J Clin Exp Pathol. 2017, 10:11979-11985; Wang et al. J Cell Biochem. 2018, 119:7747-7756), there are also reports that ARHGAP9 knockout can promote lung cancer metastasis (Song et al. Genomics. 2023). The present invention found that this new small molecule compound can effectively activate NK cells and CD8 + T cells play a positive role in resisting the growth of multiple transplanted tumors. They can be used as new small molecule inhibitors of ARHGAP9 and potential drugs targeting tumors with high ARHGAP9 expression, and have dual value in scientific research and clinical practice.

[0045] The present invention also provides a method for synthesizing the novel small molecule compound. The synthesis method has a clear pathway and the obtained final product has stable properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the embodiments or the description of the prior art. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the various elements or parts are not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without inventive work.

[0047] Figure 1 Schematic diagram of the synthesis process of N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)picolinamide in one embodiment of the present invention; Figure 2 This is a hydrogen spectrum of N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)picolinamide synthesized in one of the examples of the present invention; Figure 3 This is a carbon spectrum of N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)picolinamide synthesized in one of the embodiments of the present invention; Figure 4 This is a mass spectrum of N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)picolinamide synthesized in one of the examples of the present invention; Figure 5This is a verification of the specific binding of N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)picolinamide to ARHGAP9 in one of the embodiments of the present invention; Figure 6 This is the result of in vitro activation of T cells and NK cells by N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)picolinamide in one of the embodiments of the present invention; Figure 7 This is the result of N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)picolinamide enhancing anti-tumor immunity in mice in one of the examples of the present invention; Figure 8 This is an in vivo safety evaluation of N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)picolinamide in one of the examples of the present invention. DETAILED DESCRIPTION

[0048] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0049] As used herein, "and / or" includes any and all combinations of one or more of the associated listed items.

[0050] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0051] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0052] In this specification, certain embodiments may be disclosed in a format that is within a range. It should be understood that this description of "within a range" is merely for convenience and brevity and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within this range. For example, the description of a range of 1-6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. Regardless of the breadth of the range, the above rules apply.

[0053] Example 1 This example provides an example of a synthesis method of N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)pyridinecarboxamide (hereinafter referred to as P801-0710). The synthesis flow chart is shown in FIG. Figure 1 .

[0054] Compound 3: In the presence of triethanolamine and acetonitrile, ethyl α-bromophenylacetate (1) reacts with N-Boc-ethylenediamine (2) to generate compound (3) ethyl 2-[(2-{(tert-butoxycarbonyl)amino}ethyl)amino]-2-phenylacetate.

[0055] Specifically, 1.0 equivalent (equiv) of ethyl α-bromophenylacetate (1) and 1.2 equiv of N-Boc-ethylenediamine (2) were added with dry triethylamine and acetonitrile solution under nitrogen atmosphere and reacted at 60°C for 12 hours to generate compound (3).

[0056] Compound 4: Compound (3) was first reacted at room temperature for 2 h under the action of trifluoroacetic acid and dichloromethane, and then reacted at 80°C under the action of triethylamine and ethanol solution to generate compound (4) 3-phenylpiperazine-2-one.

[0057] Specifically, 1.0 equiv of compound (3) was added to 2.0 equiv of trifluoroacetic acid and dichloromethane under a nitrogen atmosphere and reacted at room temperature for 2 hours. A saturated sodium carbonate solution was added until the pH value of the reaction solution was alkaline. The solution was then extracted three times with dichloromethane and the organic phase was concentrated. Subsequently, triethylamine and ethanol solution were added under a nitrogen atmosphere, and the reaction solution was placed in an 80°C oil bath for reaction. After the reaction was completed, the solution was quenched with water, extracted three times with dichloromethane, and then the organic phases were combined, washed with saturated brine, concentrated, and finally purified by silica gel column chromatography to obtain compound (4).

[0058] Compound 5: Compound (4) was reacted with Boc anhydride and sodium bicarbonate in a H2O / THF (volume ratio 1:1) mixed solvent at 60°C for 12 h to generate compound (5) 1-tert-butyloxycarbonyl-3-oxo-2-phenylpiperazine.

[0059] Specifically, 1.0 equiv of compound (4), 1.2 equiv of Boc anhydride, and 1.2 equiv of sodium bicarbonate were weighed into a dry round-bottom flask, followed by the addition of a H2O / THF (volume ratio 1:1) mixed solvent. The reaction solution was placed in a 60°C water bath for 12 hours. After the reaction was complete, the mixture was extracted three times with dichloromethane, and the organic phases were combined, washed with saturated brine, concentrated, and purified by silica gel column chromatography to obtain compound (5).

[0060] Compound 6: Under the action of borane-tetrahydrofuran, the amide carbonyl group is reduced to generate compound (6) 1-tert-butyloxycarbonyl-2-phenylpiperazine.

[0061] Specifically, 1.0 equiv of compound (5) was weighed into a dry round-bottom flask, and dry tetrahydrofuran solution was added under a nitrogen atmosphere. The reaction solution was placed in a low-temperature reactor at 0°C, and then 1.5 equiv of BH3-THF solution was added to the reaction. The reaction temperature was raised to 70°C. After the reaction was completed, the reaction was quenched with water, extracted three times with dichloromethane, and then the organic phases were combined, washed with saturated brine, concentrated, and finally purified by silica gel column chromatography to obtain compound (6).

[0062] Compound 8: Compound (6) reacts with compound (7) 6-chloro-2-pyridinecarboxylic acid methyl ester in the presence of cesium carbonate to generate compound (8) 6-[4-(tert-butyloxycarbonyl)-3-phenylpiperazin-1-yl]picolinic acid methyl ester.

[0063] Specifically, 1.0 equiv of compound (6), 1.2 equiv of compound (7), 15 mol% of 2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl (Ruphos), 5 mol% of tris(dibenzylideneacetone)dipalladium, and 3 equiv of cesium carbonate were weighed into a dry round-bottom flask, and 1,4-dioxane solution was added under a nitrogen atmosphere to obtain a mixed reaction solution. The mixed reaction solution was heated to 100°C in an oil bath for 12 hours. After the reaction was completed, the reaction product was concentrated and then purified by silica gel column chromatography to obtain compound (8).

[0064] Compound 9: Under the action of lithium hydroxide, the ester group of compound (8) is reduced to a carboxylic acid to generate compound (9) 6-[4-(tert-butyloxycarbonyl)-3-phenylpiperazin-1-yl]picolinic acid.

[0065] Specifically, 1.0 equiv of compound 8 was dissolved in a mixed solvent of THF and water, and 2.0 equiv of LiOH was added. The mixture was reacted at room temperature for 8 h. After the reaction was completed, saturated brine was added, followed by extraction with dichloromethane three times. The organic phase was concentrated and finally purified by silica gel column chromatography to obtain compound (9).

[0066] Compound 11: Compound (9) and compound (10) react with fluorobenzylamine to form amide compound (11) N-(4-fluorobenzyl)-6-(3-phenylpiperazine-4-tert-butoxycarbonyl-1-yl)pyridineamide.

[0067] Specifically, 1 equiv of compound (9) and 1.2 equiv of p-fluorobenzylamine were weighed into a dry round-bottom flask and added to a mixed solution of N,N'-dimethylformamide and dichloromethane (volume ratio 4:1) under a nitrogen atmosphere. 1.2 equiv of 1-hydroxybenzotriazole and 1.2 equiv of triethylamine were then slowly added to the reaction solution. After reacting at room temperature for 30 minutes, the reaction solution was cooled to 0°C, and then 1.5 equiv of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC-HCl) was added. After reacting at 0°C for 30 minutes, the reaction system was heated to room temperature and the reaction was continued. After the reaction was completed, the reaction solution was concentrated, extracted with dichloromethane three times, and the organic phases were combined and washed with 1M hydrochloric acid, saturated sodium bicarbonate solution and water in sequence. The organic phase was concentrated and finally purified by silica gel column chromatography to obtain compound (11).

[0068] P801-0710: Compound (11) is treated with hydrochloric acid to remove the Boc substituent to generate P801-0710.

[0069] Specifically, 1.0 equiv of compound (11) was dissolved in 4N HCl in 1,4-dioxane at room temperature. After the reaction was complete, saturated sodium carbonate solution was added until the pH of the reaction solution became alkaline. The product was then extracted three times with dichloromethane, the organic phase was concentrated, and finally purified by silica gel column chromatography to obtain P801-0710. The characterization data of P801-0710 are as follows, and the structural characterization diagram is shown in Figure 2-Figure 4 : 1 H NMR (600 MHz, DMSO-d6) δ = 9.03 (s, 1H), 7.66 (t, J = 7.9 Hz, 1H), 7.50– 7.45 (m, 2H), 7.34 (t, J = 7.5 Hz, 4H), 7.29 (t, J= 7.1 Hz, 2H), 7.13 (dd, J =9.8, 8.0 Hz, 2H), 7.05 (d, J = 8.6 Hz, 1H), 4.53 – 4.40 (m, 3H), 4.27 (d, J =12.3 Hz, 1H), 3.74 (dd, J = 10.5, 2.9 Hz, 1H), 3.07 (d, J = 9.4 Hz, 1H), 2.90 –2.78 (m, 3H), 2.73 – 2.65 (m, 1H).

[0070] 13 C{ 1 H} NMR (151 MHz, DMSO-d6) δ .8, 45.8, 45.3, 42.0.

[0071] HRMS (ESI+) m / z: ([M]+H + ) Calculated value: C 23 H 24 FN4O + = 391.1929, measured value: 391.1930.

[0072] Example 2 Validation of the binding of P801-0710 to ARHGAP9.

[0073] The binding mode of compound P801-0710 with ARHGAP9 was predicted by molecular docking. The docking score of compound P801-0710 at the RAC2 and ARHGAP9 binding site was -12.2 kcal / mol. P801-0710 forms hydrogen bond interactions with the side chain of the glutamic acid (E640) residue of ARHGAP9, and also forms hydrophobic interactions with three amino acid residues: phenylalanine (F637), glutamine (Q61), and lysine (K16). Figure 5A). Surface plasmon resonance (SPR) technology was used to detect the affinity of P801-0710 to ARH. The results showed that P801-0710 exhibited a concentration-dependent binding reaction with ARHGAP9 protein in vitro, and P801-0710 exhibited high binding strength with ARHGAP9 (KD value was 3.60e-7 M) ( Figure 5 B). The above results prove that P801-0710 can specifically bind to ARHGAP9 and is a potential specific inhibitor of ARHGAP9. Figure 5 The concentrations of analyte P801-0710 in B, from low to high, are: 0.04882813 μM, 0.09765625 μM, 0.1953125 μM, 0.390625 μM, 0.78125 μM and 1.5625 μM.

[0074] Example 3 P801-0710 Validation of in vitro activated immune cells.

[0075] Jurkat cells and NK92MI cells were used to investigate the in vitro activation effect of P801-0710 on T cells and NK cells. Different drug concentration groups (P801-0710 concentrations were set at 0μM, 5μM, 10μM, and 20μM) were co-cultured with Jurkat cells and NK92MI cells, respectively. After 24 hours of culture, flow cytometry was used to detect the activation of T cells and NK cells by P801-0710 in vitro. The results are shown in Figure 2. Figure 6 .

[0076] Figure 6 A is the detection of compound P801-0710 to enhance the release of cytokines by T cells: IFN-γ in Jurkat cells after administration of P801-0710 + T cells and TNF-α + T cell expression increased with drug concentration, indicating that P801-0710 could significantly stimulate Jurkat cells to release cytokines IFNγ and TNFα, showing a good dose-effect relationship; this proved that P801-0710 could effectively activate T cells. Figure 6 B is qPCR detection of the expression of NK cell activation markers (IFNγ, TNFα) in NK92MI cells after administration of P801-0710. The results showed that P801-0710 can increase the expression of NK cell activation markers (IFNγ, TNFα), showing a good dose-effect relationship, proving that P801-0710 can effectively activate NK cells.

[0077] Based on this, this embodiment also provides an anti-tumor agent comprising immune cells (T cells and / or NK cells) and P801-0710, wherein the immune cells and P801-0710 are independently prepared. When used for anti-tumor treatment or research, P801-0710 is added to an immune cell culture medium to culture the immune cells, thereby effectively activating the immune cells.

[0078] Example 4 Antitumor immune effect of P801-0710 in a mouse ectopic tumor model.

[0079] To further verify the immune activation effect of P801-0710, C57 mice were subcutaneously inoculated with lymphoma E.G7 cells (1×10 5 The results showed that daily injection of P801-0710 (10 mg / kg) significantly inhibited the growth of transplanted tumors and significantly reduced the tumor volume compared with the control group ( Figure 7 A, B), but had no significant effect on the body weight of mice ( Figure 7 C). Subsequently, the infiltrating lymphocytes in the transplanted tumor tissue were separated using Percoll separation medium, and CD8 + The results showed that P801-0710 could effectively increase the expression of CD8 T and NK cells and immune activation markers TNFα and IFNγ in transplanted tumor tissues. + The ratio of T cells and NK cells ( Figure 7 D), and significantly increased tumor infiltration of TNFα + CD8 + T cells, IFNγ + CD8 + T cell ratio and TNFα + NK cells, IFNγ + NK cell ratio ( Figure 7 E). The above results show that P801-0710 can effectively increase the expression of CD8 + The number of T cells and NK cells was significantly increased, and tumor infiltrating CD8 + T and NK cells released cytokines TNFα and IFNγ, proving that P801-0710 can significantly enhance the anti-tumor immunity of mice.

[0080] Example 5 In vivo safety evaluation of P801-0710.

[0081] No abnormalities in behavior, diet, excretion, etc. were found in mice treated with P801-0710 during the administration of lymph node transplanted tumors. H&E staining of the liver and kidneys of mice revealed no obvious liver and kidney toxicity. Figure 8 A). In addition, the blood of mice was sent for examination to measure the renal function indicators urea (UREA) and creatinine (CREA), the liver function indicators alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and the cardiac toxicity indicator lactate dehydrogenase (LDH) ( Figure 8 B), and routine blood tests ( Figure 8 C), P801-0710 showed no significant liver, kidney, or heart toxicity, nor did it significantly affect any blood routine indicators, indicating that P801-0710 has good safety in vivo.

[0082] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. A novel small molecule compound specifically targeting ARHGAP9 overexpressing tumors, characterized in that: The novel small molecule compound is N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)pyridineamide; the structural formula is shown in the following formula (I): Formula (I).

2. The novel small molecule compound according to claim 1, characterized in that: The novel small molecule compound has the function of competitively binding to ARHGAP9 with the ARHGAP9 functional ligand RAC2.

3. Use of a small molecule compound or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug specifically targeting ARHGAP9, characterized in that: The small molecule compound is N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)pyridineamide; the structural formula is shown in the following formula (I): Formula (I).

4. The use according to claim 3, characterized in that The types of tumors include solid tumors and hematological tumors.

5. The use according to claim 3, characterized in that The pharmaceutically acceptable salt forms include salts formed between the compound of formula (I) and inorganic or organic acids.

6. The use according to claim 3, characterized in that The dosage form of the anti-tumor drug includes a dosage form for gastrointestinal administration and / or an injection dosage form.

7. An anti-tumor agent, characterized in that The anti-tumor agent includes immune cells and a small molecule compound; the small molecule compound is N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)pyridineamide; the structural formula is shown in the following formula (I): Formula (I).

8. The anti-tumor agent according to claim 7, wherein The immune cells include T cells and / or NK cells.

9. The anti-tumor agent according to claim 7, characterized in that The small molecule compound is configured as an in vitro immune activator of the immune cells.

10. A method for synthesizing a novel small molecule compound N-(4-fluorobenzyl)-6-(3-phenylpiperazin-1-yl)pyridineamide, characterized in that: The following steps are involved: S01: In the presence of triethanolamine and acetonitrile, ethyl α-bromophenylacetate reacts with N-Boc-ethylenediamine to generate ethyl 2-[(2-{(tert-butoxycarbonyl)amino}ethyl)amino]-2-phenylacetate; S02: the ethyl 2-[(2-{(tert-butoxycarbonyl)amino}ethyl)amino]-2-phenylacetate is first reacted in the presence of trifluoroacetic acid and dichloromethane, and then in the presence of triethylamine and ethanol to produce 3-phenylpiperazin-2-one; S03: The 3-phenylpiperazine-2-one reacts with Boc anhydride and sodium bicarbonate in the presence of a H2O / THF mixed solvent to generate 1-tert-butyloxycarbonyl-3-oxo-2-phenylpiperazine; S04: Under the action of borane-tetrahydrofuran, the amide carbonyl group of the 1-tert-butoxycarbonyl-3-oxo-2-phenylpiperazine is reduced to generate 1-tert-butoxycarbonyl-2-phenylpiperazine; S05: The 1-tert-butoxycarbonyl-2-phenylpiperazine reacts with 6-chloro-2-pyridinecarboxylic acid methyl ester under the action of cesium carbonate to generate 6-[4-(tert-butoxycarbonyl)-3-phenylpiperazin-1-yl]picolinic acid methyl ester; S06: Under the action of lithium hydroxide, the ester group of the methyl 6-[4-(tert-butoxycarbonyl)-3-phenylpiperazin-1-yl]picolinate is reduced to a carboxylic acid to generate 6-[4-(tert-butoxycarbonyl)-3-phenylpiperazin-1-yl]picolinic acid; S07: The 6-[4-(tert-butoxycarbonyl)-3-phenylpiperazin-1-yl]picolinic acid reacts with p-fluorobenzylamine to generate an amide compound, N-(4-fluorobenzyl)-6-(3-phenylpiperazin-4-tert-butoxycarbonyl-1-yl)picolinamide; S08: The N-(4-fluorobenzyl)-6-(3-phenylpiperazine-4-tert-butoxycarbonyl-1-yl)picolinamide is subjected to the action of hydrochloric acid to remove the Boc substituent to generate N-(4-fluorobenzyl)-6-(3-phenylpiperazine-1-yl)picolinamide.

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