A 5-phenoxytetrazolyl compound, its reagent, preparation method and application in the preparation of antitumor drugs

Abstract

The present invention belongs to the field of biopharmaceuticals and specifically relates to a 5-phenoxytetrazolyl compound, a reagent, a preparation method, and its use in the preparation of anti-tumor drugs. The present invention first provides a novel small molecule compound with anti-tumor efficacy, the novel small molecule compound being N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride. The present invention further provides a reagent, preparation method, and use of this novel small molecule compound.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a 5-phenoxytetrazolyl compound and a reagent and a preparation method thereof, and an application thereof in the preparation of an anti-tumor drug. 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 compound that can effectively inhibit ARHGAP9, a reagent containing the small molecule compound, and a preparation method and use thereof, so as to partially solve or alleviate the above-mentioned deficiencies in the prior art. The present invention specifically adopts the following technical solutions.

[0006] In one aspect, the present invention provides a novel small molecule compound and a reagent comprising the novel small molecule compound.

[0007] A novel small molecule compound with anti-tumor efficacy, wherein the novel small molecule compound is N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride; the chemical formula is C 18 H 22 N6O˙2HCl; the structural formula is shown in formula (I):

[0008] Formula (I).

[0009] The compound of formula (I) has a 5-phenoxytetrazolyl mother ring structure, connected to a phenyl group and a [{3-(methylamino)propyl}amino]methyl group;

[0010] The structure of 5-phenoxytetrazolyl is shown below:

[0011] .

[0012] Furthermore, the novel small molecule compound can target ARHGAP9-overexpressing tumors and competitively bind to ARHGAP9 with the ARHGAP9 functional ligand RAC2, thereby exerting an efficacy against ARHGAP9-overexpressing tumors.

[0013] An anti-tumor agent comprising immune cells and a small molecule compound; the small molecule compound is N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride; the structural formula is shown in compound formula (I).

[0014] As a preference, the anti-tumor agent is an anti-tumor experimental reagent used for laboratory scientific research or preclinical research.

[0015] Preferably, the immune cells include T cells and / or NK cells.

[0016] Furthermore, the small molecule compound is configured as an in vitro immune activator of the immune cells.

[0017] Another aspect of the present invention provides a method for synthesizing the novel compound.

[0018] The synthesis method of N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride comprises the following steps:

[0019] S01: Under the action of cesium fluoride, 5-chloro-1-phenyl-1H-tetrazolyl and m-hydroxybenzaldehyde react to generate 3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzaldehyde;

[0020] S02: The 3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzaldehyde and tert-butyl (3-aminopropyl)(methyl)carbamate are condensed and reduced with sodium cyanoborohydride to obtain tert-butyl methyl [3-({3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}amino)propyl]carbamate;

[0021] S03: The tert-butyl methyl [3-({3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}amino)propyl]carbamate removes the Boc protecting group under the action of hydrochloric acid to generate the N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride.

[0022] Furthermore, 1.0 equivalent of m-hydroxybenzaldehyde and 1.0 equivalent of cesium fluoride were reacted with N,N-dimethylformamide (DMF) at room temperature under a nitrogen atmosphere to obtain a first reaction solution, and then 1.2 equivalents of 5-chloro-1-phenyl-1H-tetrazole dissolved in N,N-dimethylformamide were slowly added dropwise to the first reaction solution to continue the reaction; after completion of the reaction, the reaction was quenched with water, and then extracted, washed with an organic phase, concentrated with an organic phase, and purified with an organic phase to obtain 3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzaldehyde.

[0023] Further, 1.0 equivalents of the 3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzaldehyde and 1.2 equivalents of tert-butyl 3-(aminopropyl)(methyl)carbamate were added to methanol under a nitrogen atmosphere to react to obtain a second reaction solution; the second reaction solution was heated under reflux and then cooled to room temperature, and then 3.0 equivalents of sodium cyanoborohydride and 2.0 equivalents of acetic acid were dissolved in methanol and then slowly added to the second reaction solution to react at room temperature; after completion of the reaction, the reaction was quenched with water, and then extracted, washed with an organic phase, concentrated with an organic phase, and purified with an organic phase to obtain tert-butyl methyl [3-({3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}amino)propyl]carbamate.

[0024] Furthermore, 1.0 equivalent of the tert-butyl methyl [3-({3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}amino)propyl]carbamate was dissolved in a dichloromethane (DCM) solution to obtain a third reaction solution; HCl dissolved in 1,4-dioxane was slowly added dropwise to the third reaction solution at room temperature to react at room temperature; after the reaction was completed, the N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride was obtained by extraction, concentration, and purification.

[0025] Another aspect of the present invention provides applications of the novel small molecule compound.

[0026] A small molecule compound is used in the preparation of an anti-tumor drug targeting ARHGAP9, wherein the small molecule compound is N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride; the structural formula is shown in formula (I).

[0027] Furthermore, the types of tumors include solid tumors and hematological tumors.

[0028] Furthermore, the solid tumor or blood tumor is a solid tumor or blood tumor mediated by high expression of ARHGAP9, including:

[0029] (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%.

[0030] (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%.

[0031] Furthermore, the anti-tumor drug also includes other pharmaceutically acceptable carriers and / or adjuvants.

[0032] The core component of the above-mentioned small molecule compound is N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine, which can be combined with various typical inorganic acids and / or organic acids to form other pharmaceutically acceptable salt forms.

[0033] Furthermore, the inorganic acid includes typical inorganic acids such as sulfuric acid, bisulfate, phosphoric acid, dihydrogen phosphate, monohydrogen phosphate, hydrobromic acid, nitric acid, hydroiodic acid, carbonic acid, bicarbonate, boric acid, and sulfamic acid.

[0034] Furthermore, the organic acid includes typical organic acids such as methanesulfonic acid, citric acid, tartaric acid, maleic acid, fumaric acid, acetic 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, salicylic acid, nicotinic acid, fumaric acid, edetic acid, gluconic acid, gentisic acid, oleic acid, pamoic acid, sorbic acid, and lauric acid.

[0035] 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.

[0036] Furthermore, the dosage form of the anti-tumor drug includes a dosage form for gastrointestinal administration and / or an injection dosage form.

[0037] The "gastrointestinal dosage forms" described in the present invention include common tablets, capsules, solutions, suspensions, etc.

[0038] The "injection dosage form" mentioned in the present invention includes common intravenous injections, intramuscular injections or subcutaneous injections, etc.

[0039] The last aspect of the present invention can also provide various combinations of the above-mentioned novel small molecule compounds.

[0040] The novel small molecule compound N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride provided by the present invention can be used in combination with one or more tumor treating drugs, wherein the tumor treating drugs include but are not limited to the following drugs.

[0041] (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.

[0042] (2) Small molecule targeted drugs: EGFR inhibitors: gefitinib, erlotinib, icotinib, osimertinib, ametinib, vometinib, 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: cedabenb, etc.; BCR-ABL inhibitors: imatinib, dasatinib, nilotinib, ponatinib, etc.; MET inhibitors: savotinib, capmatinib, etc.; RET inhibitor: pralsetinib; BRAF inhibitors: dabrafenib, vemurafenib, encorafenib, etc.; C DK4 / 6 inhibitors: guabotinib, 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.

[0043] (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.

[0044] (4) Immune checkpoint inhibitors (Anti-PD1): nivolumab, pembrolizumab, toripalimab, 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, XmAb 20717, 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.

[0045] (5) Immunomodulators: such as thalidomide, lenalidomide, thymosin, retinoic acid, etc.

[0046] (6) Antibody-drug conjugates: brentuximab, U3-1402, trastuzumab emtansine, etc.

[0047] (7) Hormones: tamoxifen, toremifene, letrozole, anastrozole, enzalutamide, bicalutamide, abiraterone, etc.

[0048] (8) Cell therapy: CAR-T, CAR-NK, etc.

[0049] Beneficial technical effects:

[0050] (1) The present invention provides a novel small molecule compound, N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride, which has a novel 5-phenoxytetrazolyl mother ring structure and has been shown to have significant anti-tumor immune effects in animal experiments and also has good biosafety.

[0051] (2) The novel small molecule compound N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride provided by the present invention targets ARHGAP9-overexpressing tumors and competitively binds to ARHGAP9 with the functional ligand RAC2. In an experiment using surface plasmon resonance technology to detect the affinity with ARH, it was found that it exhibited a high binding strength with ARHGAP9 (KD value of 2.03e -7M). This novel small molecule compound was demonstrated to be a potential specific binding inhibitor of ARHGAP9.

[0052] (3) The novel small molecule compound N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride provided by the present invention has also been shown to effectively activate immune cells in in vitro experiments.

[0053] (4) The present invention also provides a method for synthesizing this novel small molecule compound. The method has a clear synthesis path, a relatively streamlined process, and the final product has stable properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] 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.

[0055] Figure 1 Schematic diagram of the synthesis process of N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride in one embodiment of the present invention;

[0056] Figure 2 This is a hydrogen spectrum of N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride synthesized in one example of the present invention;

[0057] Figure 3 This is a carbon spectrum of N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride synthesized in one of the examples of the present invention;

[0058] Figure 4 This is a mass spectrum of N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride synthesized in one example of the present invention;

[0059] Figure 5 This is a verification of the specific binding of N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride to ARHGAP9 in one of the embodiments of the present invention;

[0060] Figure 6 This is the result of qPCR detection of changes in cytokine release by T cells and NK cells enhanced by N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride in one of the embodiments of the present invention;

[0061] Figure 7 This is the result of N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride enhancing anti-tumor immunity in mice in one of the examples of the present invention;

[0062] Figure 8 This is an in vivo safety evaluation of N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride in one of the examples of the present invention. DETAILED DESCRIPTION

[0063] 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.

[0064] As used herein, "and / or" includes any and all combinations of one or more of the associated listed items.

[0065] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0066] 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.

[0067] Example 1

[0068] This embodiment provides an example of a method for synthesizing N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride (hereinafter referred to as J067-0657). The synthesis flow chart is shown in FIG. Figure 1 .

[0069] S01: Under the action of cesium fluoride, compound (1) 5-chloro-1-phenyl-1H-tetrazole and compound (2) m-hydroxybenzaldehyde react to generate compound (3) 3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzaldehyde.

[0070] Specifically, 1.0 equivalent (equiv) of compound (2) m-hydroxybenzaldehyde and 1.0 equiv of cesium fluoride were weighed into a dry round-bottom flask, and dry N,N-dimethylformamide (DMF) was added under a nitrogen atmosphere and reacted at room temperature for 30 minutes. Then, 1.2 equiv of compound (1) 5-chloro-1-phenyl-1H-tetrazole dissolved in N,N-dimethylformamide was slowly added dropwise to the reaction solution containing compound (2) and the reaction was continued for 2 hours. 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 (3) 3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzaldehyde.

[0071] S02: Compound (3) and compound (4) tert-butyl (3-aminopropyl)(methyl)carbamate are condensed and reduced with sodium cyanoborohydride to obtain compound (5) tert-butylmethyl [3-({3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}amino)propyl]carbamate.

[0072] Specifically, 1.0 equiv of compound (3) and 1.2 equiv of compound (4) tert-butyl (3-aminopropyl)(methyl)carbamate were weighed into a dry round-bottom flask, and a methanol solution was added under a nitrogen atmosphere. After heating and reflux for 24 hours, the reaction mixture was cooled to room temperature. 3.0 equiv of sodium cyanoborohydride and 2.0 equiv of acetic acid were dissolved in a methanol solution and slowly added to the reaction solution containing compound (3) and compound (4), and the reaction was carried out at room temperature for 2 hours. 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 (5) tert-butylmethyl [3-({3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}amino)propyl]carbamate.

[0073] S03: Compound (5) is treated with hydrochloric acid to remove the Boc protecting group and simultaneously form a salt to obtain the final product J067-0657.

[0074] Specifically, 1.0 equiv of compound (5) tert-butylmethyl [3-({3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}amino)propyl]carbamate was dissolved in a dichloromethane (DCM) solution, and 4N HCl dissolved in 1,4-dioxane was slowly added dropwise to the reaction solution containing compound (5) at room temperature. The reaction was allowed to proceed at room temperature for 2 hours. After the reaction was completed, the product was extracted three times with dichloromethane, the organic phase was concentrated, and finally purified by silica gel column chromatography to obtain the final product J067-0657.

[0075] The characterization data of J067-0657 are as follows, and the structural characterization diagram is shown in Figure 2-Figure 4 :

[0076] 1 H NMR (600 MHz, D2O) δ = 7.83 – 7.74 (m, 2H), 7.66 – 7.56 (m, 4H), 7.53– 7.43 (m, 3H), 4.30 (s, 2H), 3.19 (dd, J = 9.2, 6.8 Hz, 2H), 3.12 (dd, J = 9.0,6.8 Hz, 2H), 2.71 (s, 3H), 2.20 – 2.03 (m, 2H).

[0077] 13 C{ 1 H} NMR (151 MHz, D2O) δ = 159.9, 153.2, 132.8, 132.0, 131.3, 130.3,129.8, 128.6, 122.8, 121.3, 121.2, 50.5, 45.7, 44.0, 32.7, 22.4.

[0078] HRMS (ESI+) m / z: ([M]+H + ) Calculated value: C 18 H 23 N6O + = 339.1928, measured value: 339.1930.

[0079] Example 2

[0080] Validation of binding of J067-0657 to ARHGAP9.

[0081] The binding mode of compound J067-0657 to ARHGAP9 was predicted by molecular docking, and the docking score of compound J067-0657 at the RAC2 and ARHGAP9 binding sites was calculated, with a binding energy of -8.5 kcal / mol. J067-0657 forms hydrogen bond interactions with the side chain of the aspartic acid (D594) residue of ARHGAP9, and also forms hydrophobic interactions with three amino acid residues: leucine (L636), leucine (L168), and lysine (K116). Figure 5 A). Surface plasmon resonance (SPR) technology was used to detect the affinity of J067-0657 to ARH. The results showed that J067-0657 exhibited a concentration-dependent binding reaction with ARHGAP9 protein in vitro, and J067-0657 exhibited a high binding strength with ARHGAP9 (KD value was 2.03e -7 M)( Figure 5 B). The above results prove that J067-0657 can specifically bind to ARHGAP9 and is a potential specific inhibitor of ARHGAP9. Figure 5 The concentrations of analyte J067-0657 in B, from low to high, were: 0.04882813 μM, 0.09765625 μM, 0.1953125 μM, 0.390625 μM, 0.78125 μM and 1.5625 μM.

[0082] Clinically, the KD of methotrexate is 28-500 nM (equivalent to KD = 2.8 e -6 to 5e -7 M), Description 10 -6 or 10 -7 Drugs with KD values ​​of this level have clinical application value (doi: 10.1016 / j.bios.2014.09.082).

[0083] In addition, a large number of studies have reported KD values ​​of 100 nM-50 µM (1.0e -7 M to 5.0e -5 M) all bind with high affinity; related studies are as follows.

[0084] Ding et al. Anal Chem. 2013;85:5727-5733 (doi: 10.1021 / ac400273g);

[0085] Day et al. Proc Natl Acad Sci US A. 2015;112:E7266-7275 (doi:10.1073 / pnas.1421082112);

[0086] Fathi et al. Food Chem. 2018;246:228-232 (doi:10.1016 / j.foodchem.2017.11.023);

[0087] Fang et al. Talanta. 2024;1;274:125987 (doi: 10.1016 / j.talanta.2024.125987);

[0088] Gao et al. Talanta. 2023;1;255:124225 (doi: 10.1016 / j.talanta.2022.124225).

[0089] Example 3

[0090] Validation of J067-0657 in vitro activation of immune cells.

[0091] Jurkat cells (Wuhan Punosai Life Science Co., Ltd.) and NK92MI cells (Saibo Biotechnology Co., Ltd., Shanghai) were used to investigate the in vitro activation effect of J067-0657 on T cells and NK cells. Different drug concentration groups (J067-0657 concentrations were set at 0 μM, 5 μM, 10 μM, and 20 μM) were co-cultured with Jurkat cells and NK92MI cells, respectively. After 24 hours of culture, qPCR was used to detect the expression of activation markers (IFNγ and TNFα) in Jurkat cells and NK92MI cells after J067-0657 administration. The results are shown in the figure. Figure 6 .

[0092] Figure 6 A is the expression of T cell activation markers (IFNγ, TNFα) after the administration of the test compound J067-0657: The results showed that J067-0657 can increase the expression of T cell activation markers (IFNγ, TNFα), showing a good dose-effect relationship; proving that J067-0657 can effectively activate T cells. Figure 6B is the detection of the expression of NK cell activation markers (IFNγ, TNFα) after the administration of J067-0657. The results showed that J067-0657 can increase the expression of NK cell activation markers (IFNγ, TNFα), showing a good dose-effect relationship, which proves that J067-0657 can also effectively activate NK cells.

[0093] Based on this, this embodiment also provides an anti-tumor experimental reagent comprising immune cells (T cells and / or NK cells) and J067-0657; wherein the immune cells and J067-0657 are prepared separately. When used in anti-tumor laboratory research or preclinical research, J067-0657 is added to the immune cell culture medium to culture the immune cells and effectively activate the immune cells.

[0094] Example 4

[0095] Antitumor immune effect of J067-0657 in a mouse ectopic tumor model.

[0096] To further confirm the immune activation effect of J067-0657, C57BL / 6 mice (Chengdu Yaokang Biotechnology Co., Ltd.) were subcutaneously inoculated with lymphoma E.G7 cells (1×10 5 The lymphoma xenograft model was constructed by injecting J067-0657 in the experimental group and the control group was injected with an equal volume of normal saline. The results showed that daily injection of J067-0657 (10 mg / kg) significantly inhibited the growth of xenograft tumors and significantly reduced the volume and weight of tumors compared with the control group; however, it had no significant effect on the body weight of mice ( Figure 7 A- Figure 7 B). Subsequently, the infiltrating lymphocytes in the transplanted tumor tissue were separated by Percoll, and the CD8 + Changes in the proportion of T and NK cells in tumor infiltration; and flow cytometry detection of tumor infiltrating CD8 + T and NK cells release cytokines. The results show that J067-0657 can effectively increase the number of CD8 + The ratio of T cells and NK cells ( Figure 7 C), 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 D).

[0097] The above results indicate that J067-0657 can effectively increase the expression of CD8+ The number of T cells and NK cells and the tumor-infiltrating CD8 + T and NK cells release cytokines TNFα and IFNγ, which further proves that J067-0657 can significantly enhance the anti-tumor immunity of mice.

[0098] Example 5

[0099] In vivo safety evaluation of J067-0657.

[0100] No abnormalities in behavior, diet, excretion, etc. were found in mice treated with J067-0657 during the administration of lymph node transplanted tumors in mice, and H&E staining of the liver and kidneys of mice showed 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), no significant liver, kidney, or heart toxicity was found with J067-0657, nor was there a significant effect on routine blood tests, indicating that J067-0657 has a good safety profile in vivo.

[0101] 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. 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-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride; the immune cells include T cells and / or NK cells; the small molecule compound is configured as an in vitro immune activator for the immune cells; the structural formula is shown in the following formula (I): Formula (I).

2. Use of a small molecule compound in the preparation of an anti-tumor drug targeting ARHGAP9, characterized in that: The small molecule compound is N-methyl-N'-{3-[(1-phenyl-1H-tetrazol-5-yl)oxy]benzyl}propane-1,3-diamine hydrochloride; the structural formula is shown in the following formula (I): Formula (I).

3. The use according to claim 2, characterized in that The types of tumors include solid tumors and hematological tumors.

4. The use according to claim 2, characterized in that The anti-tumor drug also includes other pharmaceutically acceptable carriers and / or adjuvants.

5. The use according to claim 2, characterized in that The dosage form of the anti-tumor drug includes a dosage form for gastrointestinal administration or an injection dosage form.

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