Nitrogen-containing heterocyclic 1,2,4-oxadiazole compounds and their applications

Abstract

The present invention relates to the field of medicinal chemistry, and more specifically to a class of nitrogen-containing heterocyclic 1,2,4-oxadiazole compounds and their applications. Pharmacological tests have shown that the compounds of the present invention have strong inhibitory effects on both SphK1 and SphK2, and can act on both SphK1 and SphK2 simultaneously. Activity results show that in terms of inhibitory activity against A549 and MDA-MB-231 cells, all compounds have superior activity to cisplatin; in terms of inhibitory activity against HCT116 cells, T16, T18, T30, T32, T33, T42, T47(R), T48(R), T55(S), and T57(S) all have superior activity to cisplatin.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular to a class of nitrogen-containing heterocyclic 1,2,4-oxadiazole compounds, pharmaceutical compositions thereof and applications thereof. Background Art

[0002] Cancer is a complex disease caused by abnormal cell proliferation and is a major global public health challenge. Normally, human cells maintain a balanced tissue function through orderly division and apoptosis. However, when cancer develops, genetic mutations cause uncontrolled cell division, forming abnormal tissue (tumors). These tumors are classified as benign (non-metastatic) or malignant (invading surrounding tissues and metastasizing through the blood or lymphatic system). According to the World Health Organization, lung cancer, breast cancer, and colorectal cancer are currently the most common types of cancer. Cancer is one of the most difficult diseases to cure worldwide. Due to the increasing number of deaths from cancer, research into the mechanisms of cancer development, progression, and metastasis, as well as the development of cancer therapeutics, has been a hot topic both domestically and internationally. The five-year survival rate for cancer patients in China has significantly improved, thanks to the widespread promotion of cancer screening and multidisciplinary treatment, the implementation of precision oncology, and continuous innovation in new drug research and development.

[0003] Sphingolipids are ubiquitous components of eukaryotic cell membranes. Sphingolipids and their metabolites participate in numerous important signal transduction pathways. Sphingosine kinases (SphKs) catalyze the conversion of sphingosine to sphingosine 1-phosphate (S1P). Sphingosine 1-phosphate (S1P) regulates diverse biological processes, including cell proliferation and differentiation, as well as immune cell trafficking. SphKs have two isoforms, SphK1 and SphK2, which share high molecular homology but differ in tissue distribution and function. While inhibiting one isoform can be therapeutic for related diseases, inhibiting both isoforms simultaneously could potentially achieve a broader therapeutic range and improved efficacy. While the mechanisms of SphK1 are more intensively studied, the physiological functions of SphK2 are more complex, and its role in pathological processes has only been gradually uncovered in recent years. The expression and activity of both SphK1 and SphK2 are significantly elevated in various tumor cells compared to normal tissues or cells. SphK1 and SphK2 can inhibit apoptosis and promote cell proliferation and angiogenesis by increasing S1P synthesis. The SphKs / S1P signaling axis plays a crucial role in regulating tumor cell proliferation and apoptosis. Inhibiting SphKs expression and activity can modulate tumor biological behavior and drug response, improving patient prognosis. Currently, several SphKs inhibitors, such as the SphK2 inhibitor ABC294640, exhibit promising oral bioavailability and pharmacokinetic properties and are currently in Phase II clinical trials, demonstrating that the application of SphKs inhibitors in cancer treatment is gradually moving from the laboratory to the clinic.

[0004] Patent CN119119016A discloses a series of substituted oxadiazole compounds, their preparation methods, and uses. Pharmacological tests have shown that these compounds exhibit strong selectivity and inhibitory effects against SphK2, as well as antiproliferative effects against cervical, prostate, and colon cancer cells. The compounds described in this patent are selective inhibitors of SphK2. If compounds with inhibitory effects against both SphK1 and SphK2 could be obtained, their application could be broadened and their potential for drug development could be enhanced. Summary of the Invention

[0005] The technical problem solved by the present invention is to provide a class of nitrogen-containing heterocyclic 1,2,4-oxadiazole compounds, their preparation methods, pharmaceutical compositions and their use in the preparation of drugs for preventing and / or treating diseases mediated by SphKs.

[0006] In order to solve the technical problems of the present invention, the present invention provides the following technical solutions:

[0007] The first aspect of the technical solution of the present invention is to provide a compound represented by general formula (I) and a solvate thereof:

[0008]

[0009] Formula (I)

[0010] Where n is 1 or 2, R 1 Cl, F, Br, CH3, CF3,

[0011] R 2 for:

[0012] .

[0013] Preferably, the nitrogen-containing heterocyclic 1,2,4-oxadiazole compound or its solvate represented by general formula (I) is:

[0014]

[0015] Formula (I)

[0016] Where n = 1, 2, R 1 = F, Br, CH3, CF3, R 2 for

[0017] .

[0018] Preferably, the above-mentioned nitrogen-containing heterocyclic 1,2,4-oxadiazole compound or its solvate has the structural formula:

[0019] .

[0020] More preferably, the structural formula of the nitrogen-containing heterocyclic 1,2,4-oxadiazole compound or its solvate is:

[0021] .

[0022] The term "solvate" refers to a complex in which the compound of the present invention is coordinated with solvent molecules to form a specific ratio.

[0023] The second aspect of the technical solution of the present invention is to provide a method for preparing the compound described in the first aspect. The compound of general formula (I) of the present invention can be prepared by the following method:

[0024] .

[0025] Substituted p-hydroxybenzonitrile reacts with hydroxylamine hydrochloride to obtain an intermediate amino oxime (2), which then undergoes a cyclization reaction with 1-Boc-azetidine-3-acetic acid / N-Boc-3-pyrrolidineacetic acid to obtain an oxadiazole intermediate (3), which is then reacted with a halogenated hydrocarbon via the Williamson synthesis to obtain an ether intermediate (4). The resulting product is treated with trifluoroacetic acid to obtain the target compound.

[0026] The third aspect of the technical solution of the present invention is to provide a pharmaceutical composition comprising the nitrogen-containing heterocyclic 1,2,4-oxadiazole compound or a solvate thereof as described in the first aspect and a pharmaceutically acceptable carrier, in any pharmaceutically acceptable dosage form, preferably an oral preparation or an injection. The composition contains 0.01g to 10g of the compound represented by general formula (I) in a physiologically effective amount, which may be 0.01g, 0.015g, 0.02g, 0.025g, 0.03g, 0.04g, 0.05g, 0.1g, 0.125g, 0.2g, 0.25g, 0.3g, 0.4g, 0.5g, 0.6g, 0.75g, 1g, 1.25g, 1.5g, 1.75g, 2g, 2.5g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10g, etc.

[0027] Any compound of the present invention can be administered orally or parenterally to a patient in need of such treatment.

[0028] The compounds of the present invention can be added with pharmaceutically acceptable carriers to prepare common pharmaceutical preparations, such as tablets, capsules, powders, syrups, liquids, suspensions, and injections, and can be added with common pharmaceutical excipients such as flavorings, sweeteners, liquid or solid fillers or diluents.

[0029] The clinical dosage of the compound of the present invention is 0.01-1000 mg / day, and may deviate from this range depending on the severity of the disease or the dosage form.

[0030] The fourth aspect of the technical solution of the present invention is to provide the use of the nitrogen-containing heterocyclic 1,2,4-oxadiazole compound or its solvate described in the first aspect and the pharmaceutical composition described in the third aspect in the preparation of drugs for preventing and / or treating diseases mediated by SphK1 and / or SphK2.

[0031] The SphK1-mediated diseases are cancers and inflammatory diseases. The cancers include colon cancer, lung cancer, breast cancer, liver cancer, gastric cancer, lung adenocarcinoma, melanoma, and esophageal cancer; and the inflammatory diseases include inflammatory bowel disease, hepatitis, asthma, chronic obstructive pulmonary disease, rheumatoid arthritis, or multiple sclerosis.

[0032] The diseases mediated by SphK2 are cancer, inflammatory diseases, ischemia-reperfusion injury, renal fibrosis, Alzheimer's disease, diabetic retinopathy, and diabetic nephropathy. The cancers are rectal cancer, gastric cancer, kidney cancer, colon cancer, uterine cancer, ovarian cancer, breast cancer, and lung cancer.

[0033] Preferably, the cancer is lung cancer, colon cancer, or breast cancer.

[0034] Beneficial technical effects:

[0035] (1) The compounds of the present invention have strong inhibitory effects on both SphK1 and SphK2, and can act on SphK1 and SphK2 simultaneously. Compared with SphK1 or SphK2 selective inhibitors, the compounds of the present invention have a wider range of possible indications and higher drug development value.

[0036] (2) At 10 μM, most of the 17 compounds tested showed inhibition rates of more than 40% or even above 96% on SphK1 and SphK2. Among them, T16, T18, T23, T29, T30, T31, T32, T33, T35, T42, T44, T47(R), T48(R), T55(S), and T57(S) showed strong inhibitory effects on both SphK1 and SphK2.

[0037] (3) The activity results showed that the compounds all showed better inhibitory effects on A549 cells than cisplatin, especially T16, T33, T42, T47(R) and T55(S), which had the best activity. 50 The inhibitory activities of T16, T18, T30, T32, T33, T42, T47(R), T48(R), T55(S), and T57(S) against HCT116 cells were better than those of cisplatin, with IC 50The inhibitory activity against MDA-MB-231 cells was better than that of cisplatin, and the activities of T16 and T47(R) were particularly significant, with IC 50 4.47±0.14 μM and 4.00±0.07 μM respectively. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention, but the present invention is not limited thereto.

[0039]

[0040] Dissolve substituted p-hydroxybenzonitrile (11 mmol, 1.0 eq), hydroxylamine hydrochloride (16 mmol, 1.5 eq), and sodium bicarbonate (16 mmol, 1.5 eq) in methanol (20 mL). Reflux for 22 hours. Cool the reaction mixture to room temperature and filter. Evaporate the solvent to obtain a crude amino oxime. Dissolve the crude product, 1-Boc-azetidine-3-acetic acid (18 mmol, 1.2 eq), and DIPEA (27 mmol, 1.8 eq) in DMF (20 mL) and add HATU (16 mmol, 1.1 eq). o C for 12 hours. The reaction solution was extracted with ethyl acetate. The organic phase was washed with water and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The crude product was separated and purified by silica gel column chromatography (mobile phase: n-hexane-ethyl acetate = 4:1) to obtain intermediate (3). Intermediate (3) (0.52 mmol, 1.0 eq), cesium carbonate (0.58 mmol, 1.1 eq), and halogenated hydrocarbon (0.58 mmol, 1.1 eq) were dissolved in DMF (20 mL). 75 oC for 8 hours. The reaction solution was extracted with ethyl acetate, and the organic phase was washed with water and saturated brine in sequence, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The crude product was separated and purified by silica gel column chromatography (mobile phase: n-hexane-ethyl acetate = 10:1) to obtain intermediate (4). The intermediate compound (4) (3 mmol, 1.0 eq) was dissolved in CH2Cl2 (6 mL), TFA (3 mL) was added dropwise with stirring, and the reaction was carried out at room temperature for 4 hours. The reaction solution was evaporated under reduced pressure to remove the solvent, and the crude product was separated and purified by silica gel column chromatography (dichloromethane-methanol = 15:1) to obtain white target compounds T1-T64 with a yield of 85%-90%.

[0041] Example 1: 5-(azetidin-3-ylmethyl)-3-(3-fluoro-4-(nonyloxy)phenyl)-1,2,4-oxadiazole trifluoroacetate (T16):

[0042]

[0043] The substituted p-hydroxybenzonitrile 3-fluoro-4-hydroxybenzonitrile, hydroxylamine hydrochloride, and sodium bicarbonate were dissolved in methanol and refluxed for 22 hours. The reaction solution was cooled to room temperature and filtered, and the solvent was evaporated under reduced pressure to obtain a crude amino oxime. The crude product, 1-Boc-azetidine-3-acetic acid, and DIPEA were dissolved in DMF, and HATU, 100 o C for 12 hours. The reaction solution was extracted with ethyl acetate and the crude product was separated and purified by silica gel column chromatography. Then the intermediate, cesium carbonate and halogenated hydrocarbon 1-bromononane were dissolved in DMF. o C for 8 hours. The reaction solution was extracted with ethyl acetate. The organic phase was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The crude product was separated and purified by silica gel column chromatography to obtain the key intermediate. The key intermediate was dissolved in CH2Cl2, and TFA was added dropwise with stirring. The reaction was allowed to react at room temperature for 4 hours. After completion of the reaction, the solvent was evaporated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography (dichloromethane:methanol = 15:1) to obtain the white target compound T16 in an 89% yield. 1 HNMR (800 MHz, DMSO- d6) δ 7.79 (d, J = 8.1 Hz, 1H), 7.75 (d, J = 11.6 Hz, 1H), 7.34 (t, J = 8.7 Hz, 1H), 4.13 (d, J = 6.9 Hz, 4H), 3.93 (s, 2H), 3.39 (d, J= 10.9 Hz, 3H), 1.76 (t, J = 7.2 Hz, 2H), 1.42 (s, 2H), 1.33 (s, 2H), 1.30 –1.24 (m, 8H), 0.85 (dd, J = 9.4, 4.6 Hz, 3H). 13 C NMR (200 MHz, DMSO- d 6) δ178.32, 166.97, 158.90 (d, J = 34.3 Hz, 1C), 151.99 (d, J = 245.7 Hz, 1C), 149.70 (d, J = 10.3 Hz, 1C), 124.59, 118.95 (d, J = 7.3 Hz, 1C), 117.65 (d, J= 299.3 Hz, 1C), 115.64, 114.81 (d, J = 19.8 Hz, 1C), 69.30(2C), 50.53, 31.73(2C), 29.39(2C), 29.16, 29.10, 28.88, 25.81, 22.56, 14.38. IR (KBr, cm -1 ):723.01, 791.47, 831.86, 1141.37, 1202.46, 1286.09, 1369.44, 1506.40, 1593.74,1698.83, 2687.61, 2857.02, 2931.20, 3433.71. HRMS (ESI): m / z calcd forC 21 H 30 FN3O2[M+H] + : 376.2400 found: 376.2360.

[0044] The preparation methods of the compounds in Examples 2-10 are the same as those in Example 1, except that different substituted p-hydroxybenzonitriles and halogenated hydrocarbons are used. The specific substituted p-hydroxybenzonitriles and halogenated hydrocarbons used in each example are indicated in the corresponding example:

[0045] Example 2: 5-(azetidin-3-ylmethyl)-3-(4-(dodec-9-en-1-yloxy)-3-fluorophenyl)-1,2,4-oxadiazole trifluoroacetate (T18):

[0046]

[0047] The substituted p-hydroxybenzonitrile used was 3-fluoro-4-hydroxybenzonitrile, and the halogenated hydrocarbon used was 1-bromodecane. T18 was obtained as a white solid powder with a yield of 87%. 1 H NMR (800 MHz, DMSO- d 6) δ 7.78 (d, J = 8.2 Hz, 1H), 7.76– 7.72 (m, 1H), 7.34 (q, J = 8.7, 7.3 Hz, 1H), 5.79 (qt, J = 10.5, 5.8 Hz,1H), 4.99 (dd, J = 17.3, 2.1 Hz, 1H), 4.93 (d, J = 9.0 Hz, 1H), 4.12 (d, J =6.2 Hz, 4H), 3.92 (t, J = 7.8 Hz, 2H), 3.38 (d, J = 5.1 Hz, 3H), 2.01 (t, J =6.5 Hz, 2H), 1.75 (s, 2H), 1.42 (s, 2H), 1.37 – 1.26 (m, 8H). 13 C NMR (200 MHz, DMSO- d 6) δ 178.32, 166.97, 152.60, 151.38, 149.70 (d, J = 10.2 Hz, 1C), 139.29, 124.59, 118.95 (d, J = 7.3 Hz, 1C), 117.69 (d, J = 294.8 Hz, 1C),115.68, 115.11, 114.82 (d, J = 20.3 Hz, 1C), 69.30, 50.54(2C), 33.64(2C),29.38, 29.24, 29.11, 28.90, 28.88, 28.70, 25.78. IR (KBr, cm -1): 720.57,793.70, 832.19, 880.94, 911.93, 993.75, 1019.03, 1138.65, 1165.42, 1203.94,1282.30, 1371.58, 1423.24, 1476.23, 1505.86, 1548.15, 1578.43, 1702.72,2518.65, 2686.60, 2852.21, 2925.86. HRMS (ESI): m / z calcd for C 22 H 30 FN3O2[M+H] + :388.2400 found: 388.2349.

[0048] Example 3: 5-(azetidin-3-ylmethyl)-3-(3-bromo-4-(non-8-en-1-yloxy)benzene)-1,2,4-oxadiazole trifluoroacetate (T23):

[0049]

[0050] The substituted p-hydroxybenzonitrile used was 3-bromo-4-hydroxybenzonitrile, and the halogenated hydrocarbon used was 1-bromoheptene. T23 was obtained as a white solid powder with a yield of 86%. 1 H NMR (800 MHz, Methanol- d 4) δ 8.18 (s, 1H), 7.98 (s, 1H),7.14 (t, J = 9.1 Hz, 1H), 5.81 (td, J = 17.0, 7.5 Hz, 1H), 4.98 (d, J = 15.0 Hz,1H), 4.91 (d, J = 10.2 Hz, 1H), 4.29 (t, J = 10.2 Hz, 2H), 4.14 – 4.06 (m, 4H), 3.54 (q, J = 8.8 Hz, 1H), 3.37 (d, J = 7.7 Hz, 2H), 2.05 (qt, J = 6.9, 2.7 Hz, 2H),1.84 (ddd, J = 12.9, 10.5, 5.3, 3.3 Hz, 2H), 1.55 (tddd, J= 9.9, 7.7, 4.6, 2.1Hz, 2H), 1.45 – 1.34 (m, 6H). 13 C NMR (200 MHz, Methanol- d 4) δ 177.25, 166.86,161.75 (d, J = 36.9 Hz, 1C), 157.91, 138.71, 131.66, 127.69, 119.81, 116.68 (d, J IR (KBr, cm -1 ): 724.26, 800.20,840.45, 905.66, 992.97, 1048.13, 1138.12, 1209.25, 1273.11, 1295.33, 1328.94,1390.97, 1429.47, 1464.71, 1559.14, 1595.36, 1686.17, 1716.86, 2852.80,2929.13. HRMS (ESI): m / z calcd for C 21 H 28 BrN3O2[M+H] + :434.1443 found: 434.1403.

[0051] Example 4: 5-(azetidin-3-ylmethyl)-3-(4-(dodecyloxy)-3-methylphenyl)-1,2,4-oxadiazole trifluoroacetate (T29):

[0052]

[0053] The substituted p-hydroxybenzonitrile used was 3-methyl-4-hydroxybenzonitrile, and the halogenated hydrocarbon used was 1-bromododecane. T29 was obtained as a white solid powder with a yield of 86%. 1 H NMR (800 MHz, Methanol- d4) δ 7.83 (d, J = 8.5 Hz, 1H),7.79 (s, 1H), 6.98 (d, J = 8.6 Hz, 1H), 4.31 – 4.26 (m, 2H), 4.10 – 4.03 (m,4H), 3.52 (p, J = 8.1 Hz, 1H), 3.35 (d, J = 7.6 Hz, 2H), 2.25 (s, 3H), 1.82(p, J = 6.7 Hz, 2H), 1.52 (p, J = 7.5 Hz, 2H), 1.40 (p, J = 7.6, 7.1 Hz, 2H),1.37 – 1.25 (m, 14H), 0.89 (t, J = 7.1 Hz, 3H). 13 C NMR (200 MHz, Methanol- d 4)δ 176.76, 168.01, 161.71 (q, J = 34.8, 34.2 Hz, 1C), 159.85, 128.94, 127.03,126.37, 117.97, 116.95 (d, J = 283.8 Hz, 1C), 110.59, 67.82, 50.73(2C),31.67, 29.41, 29.36(2C), 29.29, 29.28, 29.08, 29.03, 28.91, 28.54, 25.82,22.34, 15.00, 13.04. IR (KBr, cm -1 ): 645.94, 720.69, 753.66, 792.02, 824.38,864.73, 910.94, 1017.14, 1134.57, 1171.74, 1199.80, 1262.34, 1306.04,1340.68, 1371.21, 1394.74, 1428.70, 1468.83, 1494.37, 1574.39, 1605.81,1695.77, 2678.22, 2854.54, 2921.39. HRMS (ESI): m / z calcd for C 25 H 39 N3O2[M+H] + :414.3121 found: 414.3085.

[0054] Example 5: 5-(azetidin-3-ylmethyl)-3-(3-methyl-4-(non-8-en-1-yloxy)phenyl)-1,2,4-oxadiazole trifluoroacetate (T30):

[0055]

[0056] The substituted p-hydroxybenzonitrile used was 3-methyl-4-hydroxybenzonitrile, and the halogenated hydrocarbon used was 1-bromononene. T30 was a white solid powder with a yield of 88%. 1 H NMR (800 MHz, Methanol- d 4) δ 7.83 (d, J = 8.5 Hz, 1H),7.79 (s, 1H), 6.98 (d, J = 8.6 Hz, 1H), 5.81 (ddt, J = 17.1, 10.1, 6.8 Hz,1H), 4.98 (dd, J = 17.1, 1.9 Hz, 1H), 4.91 (d, J = 9.1 Hz, 1H), 4.30 – 4.26 (m, 2H), 4.10 – 4.03 (m, 4H), 3.53 (dt, J = 15.1, 7.6 Hz, 1H), 3.35 (d, J =7.6 Hz, 2H), 2.24 (s, 3H), 2.06 (q, J = 6.3 Hz, 2H), 1.83 (p, J = 6.7 Hz, 2H), 1.52 (p, J = 7.3 Hz, 2H), 1.44 – 1.34 (m, 6H). 13 C NMR (200 MHz, Methanol- d 4) δ 176.76, 168.01, 161.68 (q, J = 34.0 Hz, 1C), 159.85, 138.69, 128.94,127.04, 126.37, 117.97, 116.16, 113.35, 110.60, 67.81, 50.73(2C), 33.46,29.41, 28.89, 28.88, 28.74, 28.62, 28.53, 25.79, 14.98. IR (KBr, cm -1):721.18, 824.49, 909.17, 1017.36, 1135.61, 1171.31, 1199.45, 1261.30, 1306.89,1340.78, 1370.62, 1394.80, 1428.14, 1496.10, 1573.98, 1608.67, 1694.17,2698.46, 2858.94, 2925.50, 3525.53. HRMS (ESI): m / z calcd for C 22 H 31 N3O2[M+H] + :370.2495 found: 370.2459.

[0057] Example 6: 5-(azetidin-3-ylmethyl)-3-(3-methyl-4-(pent-4-en-1-yloxy)phenyl)-1,2,4-oxadiazole trifluoroacetate (T31):

[0058]

[0059] The substituted p-hydroxybenzonitrile used was 3-methyl-4-hydroxybenzonitrile, and the halogenated hydrocarbon used was 1-bromopentene. White solid, yield 87% 1 H NMR (800 MHz, Methanol- d 4) δ 7.84 (d, J = 2.3 Hz, 1H), 7.80 (s,1H), 6.98 (d, J = 8.5 Hz, 1H), 5.89 (ddt, J = 17.0, 10.2, 6.7 Hz, 1H), 5.06(dq, J = 17.1, 1.7 Hz, 1H), 4.99 (d, J = 10.2 Hz, 1H), 4.29 (dd, J = 11.7,8.8 Hz, 2H), 4.11 – 4.05 (m, 4H), 3.55 – 3.50 (m, 1H), 3.35 (d, J = 7.6 Hz, 2H), 2.31 – 2.27 (m, 2H), 2.26 (s, 3H), 1.92 (dt, J = 13.4, 6.3 Hz, 2H). 13 CNMR (200 MHz, Methanol- d4) δ 176.78, 168.00, 161.71 (d, J = 34.1 Hz, 1C), 159.76, 137.67, 128.95, 127.06, 126.37, 118.05, 116.86 (d, J = 284.2 Hz, 1C),114.22, 110.61, 67.02, 50.73(2C), 29.93, 29.41, 28.53, 28.23, 14.97. IR (KBr,cm -1 ): 433.02, 647.44, 718.57, 825.53, 912.84, 1030.83, 1135.09, 1172.70,1199.69, 1257.80, 1307.27, 1340.23, 1394.48, 1429.58, 1493.05, 1573.55,1607.85, 1695.49, 2693.62, 2954.93. HRMS (ESI): m / z calcd for C 18 H 23 N3O2[M+H] + :314.1869 found: 314.1834.

[0060] Example 7: 5-(azetidin-3-ylmethyl)-3-(3-methyl-4-(nonyloxy)phenyl)-1,2,4-oxadiazole trifluoroacetate (T32):

[0061]

[0062] The substituted p-hydroxybenzonitrile used was 3-methyl-4-hydroxybenzonitrile, and the halogenated hydrocarbon used was 1-bromononane. White solid, yield 88%, 1 H NMR (800 MHz, Methanol- d 4) δ 7.83 (dd, J = 8.5, 2.3 Hz, 1H), 7.79(s, 1H), 6.98 (d, J = 8.5 Hz, 1H), 4.30 – 4.26 (m, 2H), 4.09 – 4.04 (m, 4H), 3.55 – 3.50 (m, 1H), 3.35 (d, J = 7.7 Hz, 2H), 2.25 (s, 3H), 1.85 – 1.80 (m,2H), 1.54 – 1.50 (m, 2H), 1.40 (p,J = 7.0 Hz, 2H), 1.37 – 1.27 (m, 8H), 0.90(t, J = 7.1 Hz, 3H). 13 C NMR (200 MHz, Methanol- d 4) δ 176.76, 168.01, 161.54 (d, J = 34.4 Hz, 1C), 159.86, 128.93, 127.04, 126.37, 117.96, 116.85 (d, J = 293.0Hz, 1C), 110.60, 67.83, 50.73(2C), 31.64, 29.41, 29.27, 29.07, 28.98, 28.92,28.53, 25.84, 22.33, 14.98, 13.03. IR (KBr, cm -1 ): 720.66, 824.66, 908.59,1015.73, 1135.33, 1172.05, 1199.51, 1261.08, 1308.67, 1340.73, 1370.07,1394.78, 1428.07, 1497.91, 1572.94, 1607.96, 1693.13, 2857.14, 2923.59,2957.30. HRMS (ESI): m / z calcd for C 22 H 33 N3O2[M+H] + : 372.2651 found: 372.2614.

[0063] Example 8: 5-(azetidin-3-ylmethyl)-3-(4-(decyloxy)-3-methylphenyl)-1,2,4-oxadiazole trifluoroacetate (T33):

[0064]

[0065] The substituted p-hydroxybenzonitrile used was 3-methyl-4-hydroxybenzonitrile, and the halogenated hydrocarbon used was 1-bromododecane. White solid, yield 89%, 1 H NMR (800 MHz, Methanol- d 4) δ 7.83 (dd, J = 8.4, 2.2 Hz, 1H), 7.79(d, J= 2.2 Hz, 1H), 6.98 (d, J = 8.5 Hz, 1H), 5.80 (ddt, J = 17.0, 10.2, 6.7 Hz,1H), 4.97 (dq, J = 17.1, 1.8 Hz, 1H), 4.91 (dd, J = 10.3, 1.9 Hz, 1H), 4.87 (s,2H), 4.32 – 4.26 (m, 2H), 4.10 – 4.06 (m, 2H), 4.05 (t, J = 6.3 Hz, 2H), 3.53(hept, J = 8.0 Hz, 1H), 3.35 (d, J = 7.6 Hz, 2H), 2.24 (s, 3H), 2.04 (q, J = 7.1Hz, 2H), 1.82 (dt, J = 14.9, 6.5 Hz, 2H), 1.52 (p, J = 7.4 Hz, 2H), 1.44 – 1.30(m, 8H). 13 C NMR (200 MHz, Methanol- d 4) δ 176.77, 168.01, 161.73 (q, J = 34.5 Hz,1C), 159.85, 138.72, 128.94, 127.03, 126.37, 117.97, 113.32, 110.60, 67.82,50.72, 33.49, 29.41, 29.12, 29.01, 28.90, 28.71 (d, J = 7.4 Hz, 4C), 28.54,25.81, 14.99. IR (KBr, cm -1): 654.80, 721.51, 824.87, 909.23, 1017.59,1135.82, 1171.54, 1199.34, 1261.65, 1306.49, 1340.65, 1369.40, 1394.94,1428.47, 1468.86, 1495.17, 1574.45, 1608.01, 1693.92, 2697.84, 2857.48,2925.20. HRMS (ESI): m / z calcd for C 23 H 35 N3O2[M+H] + : 386.2808 found: 386.2808.

[0066] Example 9: 5-(azetidin-3-ylmethyl)-3-(4-(heptyloxy)-3-methylphenyl)-1,2,4-oxadiazole trifluoroacetate (T35):

[0067]

[0068] The substituted p-hydroxybenzonitrile used was 3-methyl-4-hydroxybenzonitrile, and the halogenated hydrocarbon used was 1-chloroheptane. The product was a white solid with a yield of 86%. 1 H NMR (800 MHz, Methanol- d 4) δ 7.83 (dd, J = 8.5, 2.2 Hz, 1H), 7.79(s, 1H), 6.98 (d, J = 8.5 Hz, 1H), 4.29 (dd, J = 11.7, 8.8 Hz, 2H), 4.10 – 4.06(m, 2H), 4.05 (t, J = 6.3 Hz, 2H), 3.55 – 3.50 (m, 1H), 3.35 (d, J = 7.6 Hz, 2H),2.24 (s, 3H), 1.85 – 1.80 (m, 2H), 1.52 (p, J = 7.5 Hz, 2H), 1.40 (p, J = 6.9 Hz,2H), 1.37 – 1.32 (m, 4H), 0.92 (t, J = 6.9 Hz, 3H). 13C NMR (200 MHz, Methanol- d 4) δ 176.77, 168.01, 161.73 (q, J = 34.4 Hz, 1C), 159.85, 128.93, 127.03,126.37, 117.97, 116.85 (d, J IR (KBr, cm) -1 ):647.98, 721.08, 751.65, 823.08, 909.00, 1037.68, 1131.82, 1197.52, 1255.34,1308.29, 1393.18, 1429.32, 1467.51, 1574.70, 1607.54, 1707.26, 2673.33,2860.83, 2926.91. HRMS (ESI): m / z calcd for C 20 H 29 N3O2[M+H] + : 344.2338 found:344.2301.

[0069] Example 10: 5-(azetidin-3-ylmethyl)-3-(4-(nonyloxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazole trifluoroacetate (T40):

[0070]

[0071] The substituted p-hydroxybenzonitrile used was 3-trifluoromethyl-4-hydroxybenzonitrile, and the halogenated hydrocarbon used was 1-bromononane. The product was a white solid with a yield of 89%. 1 H NMR (800 MHz, Chloroform- d ) δ 8.22 (d, J = 2.2 Hz, 1H), 8.13(dd, J = 8.7, 2.2 Hz, 1H), 7.05 (d, J = 8.8 Hz, 1H), 4.33 (s, 2H), 4.10 (t, J= 6.4Hz, 2H), 4.08 – 4.04 (m, 2H), 3.52 (h, J = 7.9 Hz, 1H), 3.35 (d, J = 7.8 Hz, 2H),1.83 (dq, J = 14.3, 7.8, 7.2 Hz, 2H), 1.48 (p, J = 7.4 Hz, 2H), 1.35 (q, J = 7.9Hz, 2H), 1.33 – 1.24 (m, 8H), 0.88 (t, J = 7.1 Hz, 3H). 13 C NMR (200 MHz,Chloroform- d ) δ 176.08, 167.35, 162.56 (d, J = 39.4 Hz, 1C), 159.40, 132.41,126.58 (q, J = 5.2 Hz, 1C), 123.18 (d, J = 272.6 Hz, 1C), 119.45 (q, J = 31.5 Hz,1C), 117.96,116.50 (d, J = 291.6 Hz, 1C), 112.94, 69.09, 50.50(2C), 31.86,29.46, 29.38, 29.34, 29.22, 29.21, 28.88, 25.74, 22.67, 14.10. IR (KBr, cm -1 ):417.28, 676.10, 721.26, 832.76, 916.04, 1003.03, 1056.38, 1136.45, 1202.72,1286.58, 1319.47, 1351.22, 1427.84, 1474.56, 1624.53, 1696.14, 2857.81,2926.00, 2968.68, 3441.82. HRMS (ESI): m / z calcd for C 22 H 30 F3N3O2[M+H] + : 426.2368found: 426.2349.

[0072] Example 11: 3-(3-bromo-4-(nonyloxy)phenyl)-5-(pyrrolidin-3-ylmethyl)-1,2,4-oxadiazole trifluoroacetate (T42):

[0073]

[0074] Dissolve substituted p-hydroxybenzonitrile 3-bromo-4-hydroxybenzonitrile, hydroxylamine hydrochloride, and sodium bicarbonate in methanol and reflux for 22 hours. The reaction solution is cooled to room temperature and filtered, and the solvent is evaporated under reduced pressure to obtain a crude amino oxime. N -Boc-3-pyrrolidineacetic acid, DIPEA was dissolved in DMF, HATU was added, 100 o C for 12 hours. The reaction solution was extracted with ethyl acetate and the crude product was separated and purified by silica gel column chromatography. Then the intermediate, cesium carbonate and halogenated hydrocarbon 1-bromononane were dissolved in DMF. o C for 8 hours. The reaction solution was extracted with ethyl acetate, and the organic phase was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The crude product was separated and purified by silica gel column chromatography to obtain the key intermediate. The key intermediate was dissolved in CH2Cl2, and TFA was added dropwise with stirring. The reaction was allowed to react at room temperature for 4 hours. After completion of the reaction, the solvent was evaporated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography (dichloromethane:methanol = 15:1) to obtain the white target compound T42 in an 85% yield.

[0075] T42 is a colorless oily liquid with a yield of 85%. 1 H NMR (400 MHz, DMSO- d 6) δ 8.12 (s, 1H),7.97 (d, J = 8.6 Hz, 1H), 7.29 (d, J = 8.7 Hz, 1H), 4.14 (t, J = 6.4 Hz, 2H), 3.18(t, J = 8.1 Hz, 3H), 2.96 – 2.90 (m, 1H), 2.75 (p, J = 7.7 Hz, 1H), 2.17 (dt, J =13.0, 6.4 Hz, 1H), 1.77 (p, J = 7.0 Hz, 2H), 1.71 – 1.62 (m, 1H), 1.46 (t, J =7.6 Hz, 2H), 1.31 (d, J= 35.9 Hz, 12H), 0.88 – 0.83 (m, 3H). 13 C NMR (100 MHz, DMSO- d 6) δ 179.24, 166.70, 162.39, 157.73, 131.71, 128.46, 120.07, 116.13,114.50, 112.02, 69.46, 49.75, 45.05, 35.54, 31.72, 30.19, 29.39, 29.08,29.06, 28.97, 28.83, 25.83, 22.56, 14.40. IR (KBr, cm -1 ): 480.07, 504.16,583.98, 638.65, 711.93, 800.04, 826.48, 901.20, 1009.88, 1046.34, 1129.73,1197.63, 1272.62, 1349.90, 1392.96, 1465.28, 1522.68, 1594.30, 1673.31,2856.89, 2924.81. HRMS (ESI): m / z calcd for C 22 H 32 BrN3O2[M+H] + :450.1756 found:450.1717

[0076] The preparation methods of the compounds in Examples 12-17 are the same as those in Example 11, except that different substituted p-hydroxybenzonitriles and halogenated hydrocarbons are used. The specific substituted p-hydroxybenzonitriles and halogenated hydrocarbons used in each example are indicated in the corresponding example:

[0077] Example 12: 3-(4-((3,5-bis(trifluoromethyl)benzyl)oxy)-3-bromophenyl)-5-(pyrrolidin-3-ylmethyl)-1,2,4-oxadiazole trifluoroacetate (T44):

[0078]

[0079] The substituted p-hydroxybenzonitrile was 3-bromo-4-hydroxybenzonitrile, and the halogenated hydrocarbon used was 1-(bromomethyl)-3,5-bis(trifluoromethyl)benzene. T44 was a white solid with a yield of 87%. 1 H NMR (400 MHz, DMSO- d 6) δ 8.23 ​​(s, 2H), 8.19 (d, J= 2.1 Hz, 1H), 8.13 (s, 1H), 8.04 (dd, J = 8.5, 2.1 Hz, 1H), 7.43 (d, J = 8.7Hz, 1H), 5.53 (s, 2H), 3.32 (s, 2H), 3.20 (t, J = 9.1 Hz, 3H), 2.99 (t, J = 9.3Hz, 1H), 2.79 (p, J = 7.8 Hz, 1H), 2.20 (dq, J = 12.8, 6.9 Hz, 1H), 1.71 (dq, J =17.1, 8.7 Hz, 1H). 13 C NMR (100 MHz, DMSO- d 6) δ 179.25, 166.60, 160.51, 156.85,140.30, 131.90, 130.89 (q, J = 32.8 Hz, 2C), 128.52 (d, J IR (KBr, cm -1 ): 417.24, 441.05, 679.01, 717.79, 802.02, 838.70, 890.99,1132.66, 1175.15, 1203.78, 1279.05, 1365.47, 1397.93, 1426.83, 1452.32,1480.94, 1593.30, 1682.47, 2990.87, 3441.86. HRMS (ESI): m / z calcd for C 22 H 18 BrF6N3O2[M+H] + :550.0565 found: 550.0538.

[0080] Example 13: (R)-3-(3-bromo-4-(nonyloxy)phenyl)-5-(pyrrolidin-3-ylmethyl)-1,2,4-oxadiazole trifluoroacetate (T47(R)):

[0081]

[0082] The substituted p-hydroxybenzonitrile is 3-bromo-4-hydroxybenzonitrile, using the halogenated hydrocarbon 1-bromononane. T47(R) is a colorless oily liquid with an 86% yield. +37.22 ( c = 0.04, MeOH). 1 H NMR (800 MHz, Methanol- d 4) δ 8.20– 8.16 (m, 1H), 8.00 – 7.95 (m, 1H), 7.14 (q, J = 9.1, 8.0 Hz, 1H), 4.12 (p, J =6.4 Hz, 2H), 3.65 (dd, J = 11.8, 7.7 Hz, 1H), 3.46 (ddd, J = 12.1, 8.3, 4.1 Hz,1H), 3.34 – 3.32 (m, 1H), 3.24 – 3.16 (m, 2H), 3.11 (dd, J = 12.0, 8.9 Hz, 1H),2.94 (hept, J = 7.4 Hz, 1H), 2.37 (dtd, J = 14.4, 7.4, 4.2 Hz, 1H), 1.85 (dq, J =13.0, 8.9 Hz, 3H), 1.54 (d, J = 7.5 Hz, 2H), 1.40 (s, 2H), 1.31 (d, J = 31.9 Hz,8H), 0.89 (ddd, J = 7.2, 4.3, 2.6 Hz, 3H). 13 C NMR (200 MHz, Methanol- d 4) δ178.13, 166.88, 161.71 (d, J = 36.1 Hz, 1C), 157.91, 131.66, 127.65, 119.87,116.70 (d, J= 290.2 Hz, 1C), 112.89, 111.86, 69.05(2C), 49.32, 44.91, 35.37,31.64, 29.65, 29.26, 28.97, 28.70, 28.24, 25.70, 22.35, 13.06. IR (KBr, cm -1 ):440.29, 472.02, 492.85, 520.71, 599.60, 639.42, 719.76, 799.92, 839.43,900.38, 1049.46, 1132.04, 1193.46, 1271.07, 1336.38, 1391.47, 1462.50,1592.68, 1673.63, 2855.52, 2924.67. HRMS (ESI): m / z calcd for C 22 H 32 BrN3O2[M+H] + :450.1756 found: 450.1708.

[0083] Example 14: (R)-3-(3-bromo-4-(heptyloxy)phenyl)-5-(pyrrolidin-3-ylmethyl)-1,2,4-oxadiazole trifluoroacetate (T48(R)):

[0084]

[0085] The substituted p-hydroxybenzonitrile was 3-bromo-4-hydroxybenzonitrile, and the halogenated hydrocarbon used was 1-bromoheptane. T48 was a colorless oily liquid with an 86% yield. +22.54 ( c = 0.04, MeOH). 1 H NMR (800 MHz, Methanol- d 4) δ 8.20– 8.17 (m, 1H), 7.98 (dd, J = 8.6, 2.1 Hz, 1H), 7.13 (d, J = 8.6 Hz, 1H), 4.12(t, J = 6.3 Hz, 2H), 3.66 (dd, J = 11.8, 7.7 Hz, 1H), 3.46 (ddd, J = 12.3, 8.5, 4.3Hz, 1H), 3.20 (qd, J= 16.2, 7.3 Hz, 2H), 3.12 (dd, J = 11.8, 9.0 Hz, 1H), 2.94(hept, J = 7.4 Hz, 1H), 2.40 – 2.35 (m, 1H), 1.86 (dq, J = 13.3, 8.9 Hz, 3H),1.57 – 1.51 (m, 2H), 1.41 (s, 2H), 1.34 (d, J = 3.6 Hz, 5H), 0.91 (dt, J = 15.2,7.0 Hz, 3H). 13 C NMR (200 MHz, Methanol- d 4) δ 178.13, 166.88, 116.73 (d, J =290.6 Hz, 1C), 157.90, 131.66, 127.66, 119.87, 116.01, 112.88, 111.85, 69.06,49.32, 44.92, 35.37, 31.57, 29.65, 28.72, 28.70, 28.24, 25.70, 22.26, 13.03.IR (KBr, cm -1 ): 424.71, 439.25, 470.86, 493.38, 520.93, 599.61, 639.21,719.86, 799.89, 839.68, 900.11, 1013.31, 1049.40, 1131.83, 1193.00, 1271.15,1336.50, 1391.64, 1462.27, 1592.84, 1673.53, 2849.11, 2926.29. HRMS (ESI): m / z calcd for C 20 H 28 BrN3O2[M+H] + :422.1443 found: 422.1400.

[0086] Example 15: (S)-3-(3-bromo-4-(heptyloxy)phenyl)-5-(pyrrolidin-3-ylmethyl)-1,2,4-oxadiazole trifluoroacetate (T54):

[0087]

[0088] The substituted p-hydroxybenzonitrile was 3-bromo-4-hydroxybenzonitrile, and the halogenated hydrocarbon used was 1-chloroheptane. T54 was a colorless oily liquid with an 89% yield. -23.81 ( c = 0.04, MeOH). 1 H NMR (800 MHz, Methanol- d 4) δ 8.22(d, J = 2.0 Hz, 1H), 8.01 (dd, J = 8.6, 2.1 Hz, 1H), 7.17 (d, J = 8.6 Hz, 1H), 4.15(t, J = 6.3 Hz, 2H), 3.66 (dd, J = 7.9, 3.9 Hz, 1H), 3.47 (ddd, J = 12.4, 8.5, 4.3Hz, 1H), 3.22 (qd, J = 16.3, 7.3 Hz, 2H), 3.12 (dd, J = 11.8, 8.9 Hz, 1H), 2.96(p, J = 8.0 Hz, 1H), 2.41 – 2.37 (m, 1H), 1.87 (dd, J = 14.5, 6.8 Hz, 2H), 1.57(p, J = 7.6 Hz, 2H), 1.46 – 1.29 (m, 8H), 0.94 (t, J = 6.8 Hz, 3H). 13 C NMR (200MHz, Methanol- d 4) δ 178.14, 166.90, 161.67 (d, J = 47.2 Hz, 1C), 157.94,131.68, 127.63, 119.87, 116.85 (d, J IR (KBr, cm -1): 722.82, 835.78, 902.85, 1014.34, 1050.45, 1133.31, 1206.20,1273.49, 1338.26, 1421.88, 1465.65, 1559.02, 1597.01, 1681.88, 2858.95,2928.07. HRMS (ESI): m / z calcd for C 20 H 28 BrN3O2[M+H] + :422.1443 found: 422.1396.

[0089] Example 16: (S)-3-(3-bromo-4-(nonyloxy)phenyl)-5-(pyrrolidin-3-ylmethyl)-1,2,4-oxadiazole trifluoroacetate (T55(S)):

[0090]

[0091] The substituted p-hydroxybenzonitrile was 3-bromo-4-hydroxybenzonitrile, and the halogenated hydrocarbon used was 1-bromononane. T55 was a white solid with a yield of 90%. -32.61 ( c = 0.04, MeOH), mp: 63-65 °C. 1 H NMR (800 MHz, Methanol- d 4) δ 8.21 (d, J = 2.1 Hz, 1H), 8.01 (dd, J = 8.6, 2.1 Hz, 1H), 7.17 (d, J = 8.6Hz, 1H), 4.15 (t, J = 6.2 Hz, 2H), 3.67 (dd, J = 11.8, 7.7 Hz, 1H), 3.48 (ddd, J =12.4, 8.5, 4.3 Hz, 1H), 3.22 (qd, J = 16.2, 7.3 Hz, 3H), 3.13 (dd, J = 11.8, 8.9Hz, 1H), 2.96 (p, J = 7.4 Hz, 1H), 2.42 – 2.37 (m, 1H), 1.57 (p, J= 7.5 Hz, 2H),1.45 – 1.29 (m, 13H), 0.92 (t, J = 7.0 Hz, 3H). 13 C NMR (200 MHz, Methanol- d 4) δ178.13, 166.89, 161.60 (d, J = 34.1 Hz, 1C), 157.92, 131.67, 127.64, 119.87,116.82 (d, J = 292.6 Hz, 1C), 112.90, 111.86, 69.06, 49.34, 44.94, 35.37,31.64, 29.63, 29.25, 28.98, 28.96, 28.69, 28.22, 25.70, 22.34, 13.04. IR(KBr, cm -1 ): 718.58, 747.77, 795.65, 828.52, 901.78, 1008.65, 1048.11,1129.77, 1167.35, 1210.21, 1271.91, 1357.32, 1395.78, 1467.60, 1592.76,1674.91, 2778.79, 2854.75, 2926.88. HRMS (ESI): m / z calcd for C 22 H 32 BrN3O2[M+H] + :450.1756 found: 450.1704.

[0092] Example 17: (S)-3-(3-bromo-4-((3-(trifluoromethyl)benzyl)oxy)phenyl)-5-(pyrrolidin-3-ylmethyl)-1,2,4-oxadiazole trifluoroacetate (T57(S)):

[0093]

[0094] The substituted p-hydroxybenzonitrile was 3-bromo-4-hydroxybenzonitrile, using the halogenated hydrocarbon 1-(chloromethyl)-3-(trifluoromethyl)benzene. T57(S) was obtained as a white solid in 87% yield. -32 ( c = 0.04, MeOH), mp: 105-107 ℃. 1 H NMR (800 MHz, Methanol-d 4) δ 8.14 (d, J = 2.0 Hz, 1H), 7.91 (dd, J = 8.6, 2.1 Hz,1H), 7.75 (s, 1H), 7.68 (d, J = 7.6 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.51 (t, J =7.7 Hz, 1H), 7.17 (d, J = 8.6 Hz, 1H), 5.25 (s, 2H), 3.56 (dd, J = 11.8, 7.8 Hz,1H), 3.36 (ddd, J = 12.4, 8.5, 4.3 Hz, 1H), 3.25 – 3.22 (m, 1H), 3.10 (qd, J =16.2, 7.3 Hz, 2H), 3.01 (dd, J = 11.8, 8.9 Hz, 1H), 2.87 – 2.82 (m, 1H), 2.30 –2.25 (m, 1H), 1.76 (dq, J = 13.2, 8.9 Hz, 1H). 13 C NMR (200 MHz, Methanol- d 4) δ 1178.23, 166.78, 161.65 (q, J = 34.9 Hz, 1C), 157.11, 137.78, 131.83, 130.54 (q, J = 32.2 Hz, 1C), 129.07, 127.67, 124.41 (q, J = 3.9 Hz, 2C), 123.43 (q, J = 3.9Hz, 2C), 120.74, 116.86 (d, J = 293.4 Hz, 1C), 113.64, 112.11, 69.62, 49.32,44.93, 35.36, 29.63, 28.23. IR (KBr, cm -1): 715.35, 747.44, 802.82, 902.36,1059.96, 1127.44, 1170.89, 1205.67, 1275.40, 1331.67, 1397.48, 1448.67,1484.21, 1532.72, 1591.08, 1676.25, 2800.71, 3003.02. HRMS (ESI): m / z calcd for C 21 H 19 BrF3N3O2[M+H] + :482.0691 found: 482.0672.

[0095] Some of the pharmacological activities of the compounds of the present invention are as follows:

[0096] Experimental Example 1: Inhibition rate and IC of the compounds of the present invention on SphK1 and SphK2 50 active

[0097] 1. Experimental Methods

[0098] The compound was dissolved in DMSO to prepare a 10 mmol stock solution and serially diluted in kinase buffer (pH = 7.4, composition: 40 mmol / L Tris, 10 mmol / L MgCl2, 0.1 g / L BSA, 1 mmol / L DTT, 10 µM / L ATP). The initial screening concentration was set at 10 µM and the IC 50 When the concentration of α-aminobutyric acid is less than 0.01 μM, 0.1 μM, 1 μM, 10 μM and 100 μM are set, with a total of 5 concentration gradients.

[0099] To a 96-well microtiter plate, first add the drug, then add kinase buffer and mix thoroughly. Next, add sphingosine substrate (0.2 mM). Finally, add SphK1 or SphK2 and mix thoroughly. Incubate at 30°C for 40 minutes. Then, add the ATP test solution, and incubate the mixture at room temperature for 5 minutes. Luminescence (Lu) is immediately measured using a microplate spectrophotometer. Substituting this value into the following formula to calculate inhibition: % activity = [(Lu drug - Lu background) / (Lu enzyme - Lu background)] × 100%.

[0100] The IC values ​​of the compounds were calculated using GraphPad Prism 10 software. 50 value.

[0101] 2. The experimental results are shown in Table 1 below.

[0102] Table 1 Inhibition rate of target compounds (10 μM) on SphK1 / 2 (%)

[0103]

[0104] Note: a IC 50 value, b ND means not tested, PF-543 is the positive control drug for SphK1, and K145 is the positive control drug for SphK2.

[0105] At 10 μM, most of the 17 compounds tested exhibited inhibition rates exceeding 40% and even exceeding 96% against SphK1 and SphK2. Among them, T16, T18, T23, T29, T30, T31, T32, T33, T35, T42, T44, T47(R), T48(R), T55(S), and T57(S) exhibited strong inhibitory effects on both SphK1 and SphK2.

[0106] Based on the inhibitory effect of the target compounds on phK1 and SphK2 activities at a concentration of 10 μM, we selected some compounds with relatively ideal activity results and tested their IC values ​​for phK1 and SphK2. 50 , the test results are shown in Table 2:

[0107] Table 2 IC values ​​of compounds against SphKs 50

[0108]

[0109] Note: a PF-543 is the positive control drug for SphK1, and K145 is the positive control drug for SphK2.

[0110] The activity results showed that the compounds of the present invention had an ideal inhibitory effect on SPhK1 or SPhK2, so the anti-proliferative effect of some compounds on tumor cells was further determined.

[0111] Experimental Example 2: Evaluation of the anti-proliferative effect of compounds on tumor cells (A549 cells, HCT116 cells, MDA-MB-231 cells)

[0112] 1. Experimental Methods

[0113] Tumor cells (A549, HCT116, and MDA-MB-231) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, with passages performed every 2 to 3 days. The cells were maintained in a cell culture incubator at 37°C and 5% CO₂. Cells in the logarithmic growth phase were used for experiments.

[0114] Tumor cell suspensions (6×10³ / well / 100 μL) were added to a 96-well plate and cultured for 24 hours. Test compounds (0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, and 100 μM) and cisplatin were then added and cultured for a further 48 hours. After the incubation period, the supernatant was removed, and 100 μL of culture medium containing 0.5 mg / mL CCK-8 was added to each well. The cells were incubated for a further 2.5 hours at 37°C and 5% CO₂. The absorbance (OD) of each well was measured at 450 nm using a multi-functional microplate reader. Cell viability was calculated using the formula "Cell viability (%) = (OD value of treatment group / OD value of control group) × 100%." ​​Data were analyzed using GraphPad Prism 10 software.

[0115] 2. Experimental Results

[0116] Table 3 Antiproliferative effects of compounds on tumor cells

[0117]

[0118] Note: Cisplatin is a positive control drug.

[0119] The activity results (Table 3) showed that the compounds all showed better inhibitory effects than cisplatin in the inhibitory activity against A549 cells, especially T16, T33, T42, T47(R) and T55(S), which had the best activity, with IC50 of 4.12±1.41 μM, 4.78±0.18 μM, 4.60±0.61 μM, 3.27±0.25 μM and 4.56±0.45 μM, respectively. In terms of inhibitory activity against HCT116 cells, T16, T18, T30, T32, T33, T42, T47(R), T48(R), T55(S), and T57(S) were all superior to cisplatin, with IC50 of 5.37±0.62 μM, 5.27±0.22 μM, 4.32±0.11 μM, 4.29±0.11 μM, 4.13±0.21 μM, 4.14±0.26 μM, 3.87±0.06 μM, 5.43±0.23 μM, 4.11±0.21 μM, and 3.74±0.14 μM, respectively. In terms of inhibitory activity against MDA-MB-231 cells, all compounds were more active than cisplatin, among which T16 and T47(R) were particularly active, with IC50 of 4.47±0.14 μM and 4.00±0.07 μM, respectively.

Claims

1. A nitrogen-containing heterocyclic 1,2,4-oxadiazole compound, characterized in that: The structural formula of the compound is: 。 2. The nitrogen-containing heterocyclic 1,2,4-oxadiazole compound according to claim 1, characterized in that The structural formula of the compound is: 。 3. A pharmaceutical composition comprising the nitrogen-containing heterocyclic 1,2,4-oxadiazole compound according to claim 1 or 2 and a pharmaceutically acceptable carrier.

4. Use of the nitrogen-containing heterocyclic 1,2,4-oxadiazole compound according to claim 1 or 2 or the pharmaceutical composition according to claim 3 in the preparation of a medicament for preventing and / or treating diseases mediated by SphK1 and / or SphK2.

5. The use according to claim 4, characterized in that The SphK1-mediated disease is cancer.

6. Use according to claim 5, characterized in that The cancer is colon cancer, lung cancer, or breast cancer.

7. The use according to claim 4, characterized in that The SphK2-mediated disease is cancer.

8. The use according to claim 7, characterized in that The cancer is lung cancer, colon cancer, or breast cancer.

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