A 1,2,4-heterodiazole derivative, its synthesis method and uses

Abstract

This invention provides a 1,2,4-heterodiazole derivative, its synthesis method, and its uses, belonging to the pharmaceutical field. The structural formula of the 1,2,4-heterodiazole derivative of this invention is shown in Formula I: where n is selected from integers 0 to 3; ring A is selected from substituted or unsubstituted 3- to 6-membered cycloalkanes and substituted or unsubstituted 4- to 6-membered heterocyclic alkanes, wherein the 4- to 6-membered heterocycle in ring A contains at least one N, O, or S heteroatom; and the substituents of the substituted 3- to 6-membered cycloalkanes and substituted 4- to 6-membered heterocyclic alkanes in ring A are selected from halogens, carbonyl groups, hydroxyl groups, C1- to C6 alkoxy groups, C1- to C6 alkyl groups, and halogen-substituted C1- to C6 alkyl groups. This invention also provides a synthesis process and uses for this 1,2,4-heterodiazole derivative. The novel 1,2,4-heterodiazole derivative developed by this invention, while maintaining high CB2 selectivity and high activity, reduces its CYP inhibition, improves its drug-likeness, and can be prepared for the treatment of diseases related to the CB2 target.

Description

Technical Field

[0001] This invention provides a 1,2,4-heterodiazole derivative, its synthesis method, and its uses, belonging to the pharmaceutical field. Background Technology

[0002] Cannabinoid receptors belong to the class A GPCR family and play an important role in regulating various pathological and physiological activities such as pain perception, inflammatory response, and learning and memory.

[0003] Cannabinoid receptors mainly include two subtypes, CB1 and CB2. CB1 is primarily expressed in the central nervous system, such as the cerebral cortex and hippocampus, and its agonists are often associated with addictive and other psychoactive side effects. CB2, on the other hand, is mostly distributed in peripheral tissues, such as the immune system, cardiovascular tissue, and spleen, and is also found in microglia of the central nervous system. Studies have shown that CB2 dysfunction is significantly associated with inflammatory diseases, neurodegenerative diseases, pain-related diseases, and cardiovascular diseases. Targeting CB2 agonists is considered a novel approach for treating various related diseases. However, existing small-molecule CB2 agonists face challenges related to activity, selectivity, and druggability; therefore, no small-molecule drugs in this field have yet been successfully marketed.

[0004] Cytochrome P450 (CYP450) enzymes are the main enzymes in drug metabolism. CYP 2D6, 2C9, and 3A4 are three common isoforms of CYP450 enzymes, which can catalyze reactions such as epoxidation, monohydroxylation, or polyhydroxylation of drug molecules. Some drugs exhibit strong inhibitory activity against CYP450 enzymes, affecting the binding and metabolism of CYP450 enzymes with other drugs, leading to drug accumulation and increased toxicity in the body. Therefore, the inhibitory activity of a drug against CYP450 enzymes is an important parameter for evaluating drug dependence (DDI). This study aims to screen and optimize novel CB2 small molecule agonist backbones with high activity, high selectivity, weak CYP inhibitory activity, and low toxicity.

[0005] The literature Entropy drives the ligand recognition in G-protein-coupled receptor subtypes, Proceedings of The National Academy of Sciences of The United States of America, 2024, 121(30), e2401091121, reports a 1,2,4-heterodiazole derivative YL-035 with selective CB2 agonist activity and an EC50 of 8.1 nM. This compound corresponds to patent publication number CN117756793A, invention title: 1,2,4-heterodiazole derivatives and their uses, which discloses a type of 1,2,4-heterodiazole derivative of formula I, which has the activity of activating cannabinoid receptor 2 and can be used to prepare small molecule CB2 agonists. It typically consists of three key structural units: a 1,2,4-heterodiazole ring at one end as the core skeleton, a nitrogen-containing heterocyclic structure (such as piperidine or a similar structure) at the other end, and an aromatic ring structure (Ring C) in the middle connected by a linking group (such as methylene, carbonyl, or sulfonyl). In this type of structure, the aromatic ring (corresponding to the ring C position described in this invention) is usually limited to a benzene ring or an aromatic heterocyclic ring and can be modified by substituents such as halogens and alkoxy groups.

[0006] However, although YL-0355 (corresponding to compound 34 in patent CN117756793A) has high CB2 selectivity and high activity, it also has high CYP inhibitory activity (CYP3A4 IC50 = 535.9 ± 5.8 nM). The main reason may be the indazole group in ring A, the pyridine group in ring C, and the overall hydrophobicity of the compound. Optimizing the structure to address these issues will yield a more selective small molecule CB2 agonist with better drug-like properties, which is of great significance for the development of innovative drugs targeting CB2. Summary of the Invention

[0007] The present invention aims to provide a novel class of 1,2,4-heterodiazole derivatives. By modifying key structural units in the molecule, the CYP inhibitory activity is reduced while maintaining high CB2 selectivity and high activity, thereby improving drug-likeness and enhancing the overall pharmacological performance and application value of the compound.

[0008] This invention provides a 1,2,4-heterodiazole derivative with the structural formula shown in Formula I:

[0009]

[0010] Wherein, n is selected from integers from 0 to 3; ring A is selected from substituted or unsubstituted 3- to 6-membered cycloalkanes and substituted or unsubstituted 4- to 6-membered heterocyclic alkanes, wherein the 4- to 6-membered heterocycle in ring A contains at least one N, O, or S heteroatom, and the substituents of the substituted 3- to 6-membered cycloalkanes and substituted 4- to 6-membered heterocyclic alkanes in ring A are selected from halogens, carbonyl groups, hydroxyl groups, C1- to C6 alkoxy groups, C1- to C6 alkyl groups, and halogen-substituted C1- to C6 alkyl groups.

[0011] Preferably,

[0012] The 3- to 6-membered cycloalkanes mentioned in ring A are selected from... , , , The 4-6 member heterocyclic alkanes are selected from , , , , , , , , .

[0013] Preferably, the substituted 3-6 membered cycloalkanes and substituted 4-6 membered heterocycloalkanes in ring A are selected from: , , , , , , , , , , , , , , , , , , , .

[0014] Preferably, ring A is selected from substituted or unsubstituted 4-6 membered bridged rings, and the substituents of the substituted 4-6 membered bridged rings in ring A are selected from halogens, hydroxyl groups, and C1-C6 alkoxy groups.

[0015] The unsubstituted 4-6 quinary bridge rings described in ring A are selected from , , The 4-6 quinary bridge rings substituted in ring A are selected from […]. , , , , , More preferably, the ring A is selected from: , , , , , , , .

[0016] The structural formula of the compound is: ;

[0017] The present invention also provides a synthetic process for the 1,2,4-heterodiazole derivative, the synthetic route of which is shown below:

[0018] ;

[0019] The present invention also provides the use of the 1,2,4-hexadiazole derivative in the preparation of a medicament for the production of a highly selective CB2 receptor agonist.

[0020] This invention provides a novel class of 1,2,4-heterodiazole derivatives that, while retaining the core skeleton of 1,2,4-heterodiazole and the piperidine ring structure, replace the key structural unit in the prior art—the aromatic ring (C-ring) connected to one end of the piperidine ring. Specifically, this invention replaces the aromatic ring with a non-aromatic aliphatic ring structure. The aliphatic ring can be a C3-C10 saturated or partially unsaturated carbon ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl, or it can be an aliphatic heterocycle containing one or more heteroatoms (such as nitrogen, oxygen, or sulfur), and this aliphatic ring can be further modified by substituents. In this structural system, the aliphatic ring is connected to the heterodiazole core through a linking group (such as an amide group, methylene group, carbonyl group, or sulfonyl group), while the other end remains connected to the nitrogen-containing heterocycle structure. Through the above structural modification, the overall molecule is transformed from a planar aromatic system into a structure with more three-dimensional conformational features, thereby significantly changing its interaction mode with the receptor. In the embodiments of the present invention, the representative compound 16 adopts the above-mentioned aliphatic ring substitution strategy, which is characterized by replacing the original aromatic ring with an aliphatic ring structure. Experimental results show that it has good activity on the CB2 receptor.

[0021] The beneficial effects of this invention are:

[0022] 1. This invention breaks through the limitation of "aromatic ring (ring C)" in the prior art and develops a novel 1,2,4-heterodiazole derivative, which reduces its CYP inhibition and improves drug-likeness while maintaining high CB2 selectivity and high activity.

[0023] 2. It can be formulated for the treatment of diseases related to the CB2 target, including inflammatory diseases (rheumatoid arthritis), pain-related diseases, neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Huntington's disease) and nervous system diseases (epilepsy). Attached Figure Description

[0024] Figure 1 The proton NMR spectrum of compound 16;

[0025] Figure 2 Inhibitory effect of compound 16 on cell growth at different doses;

[0026] Figure 3 Excitation curve of compound 16 on CB2. Detailed Implementation

[0027] Example 1: Synthesis process of the compound of the present invention

[0028] The process route is as follows:

[0029]

[0030] Reagents and conditions: i) Hydroxylamine hydrochloride, sodium carbonate, EtOH : H2O = 2 : 1, 75℃, 5h; ii) HATU, DIPEA, dried 1,4-dioxane, 100℃, 14h; iii) HCl-1,4-dioxane solution (4mol / L), dichloromethane, 25℃, 2h; iv) HATU, DIPEA, dichloromethane, 25℃, 3h; iv) Triphosgene, triethylamine, dichloromethane, 25℃, 6h.

[0031] Synthesis of intermediate a (4-fluoro-N'-hydroxy-1H-indazole-7-formamidinamide): 4-fluoro-1H-indazole-7-carboxynitrile (0.81 g, 5 mmol) and hydroxylamine hydrochloride (2.6 g, 40 mmol) were refluxed in ethanol (10.0 mL) for 3 hours. The reaction was monitored by TLC. The reaction mixture was concentrated under reduced pressure, and the resulting residue was partitioned between water and ethyl acetate. The organic layer was dried over sodium sulfate and concentrated under vacuum. The crude product was purified by silica gel chromatography to give 0.7 g of a yellow solid, intermediate a, in 72% yield.

[0032] Synthesis of Intermediate 2 (®-3-(4-fluoro-1H-indazol-7-yl)-5-(3-methylpiperidin-3-yl)-1,2,4-oxadiazole hydrochloride): ®-1-(tert-butoxycarbonyl)-3-methylpiperidin-3-carboxylic acid (1.22 g, 5 mmol), Intermediate 1 (971 mg, 5 mmol), HATU (2.28 g, 6 mmol), and N,N-diisopropylethylamine (1.29 g, 10 mmol) were dissolved in DMF. The mixture was stirred at 100 °C for 14 hours. After the reaction was complete, the mixture was diluted with ethyl acetate and washed successively with saturated aqueous sodium carbonate solution and saturated brine. The solution was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was then purified by silica gel chromatography to give 1.07 g of a yellow solid condensate, in 50% yield. The condensate (1 g, 2.5 mmol) was dissolved in DCM, and a 1,4-dioxane solution of HCl (1 M, 3 mL, 12 mmol) was added. The mixture was stirred at 25 °C for 2 hours. After concentration under reduced pressure, the residue was slurryed with ethyl acetate and dichloromethane to intermediate b, which was a white solid.

[0033] Synthesis of (3-(3-(4-fluoro-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-3-methylpiperidin-1-yl)((1S,4S)-4-methoxycyclohexyl) ketone (1): Intermediate b (1 eq) was dissolved in dichloromethane with commercial starting materials (1S,4S)-4-methoxycyclohexane-1-carboxylic acid (1 eq) and HATU (1.2 eq). DIPEA (4 eq) was slowly added dropwise and the reaction was carried out at room temperature for 3 h. After the reaction was complete as shown by TLC, the reaction solution was concentrated under reduced pressure and separated by preparative thin-layer chromatography to obtain a white solid with a yield of 61%. 1 H NMR (400 MHz, DMSO-d6) δ 7.92-7.84 (m, 1H), 7.15 (d, J = 11.5Hz, 1H), 7.00-6.90 (m, 1H), 4.31 (d, J = 13.8 Hz, 0.5H), 4.14-3.97 (m, 1H),3.89 (s, 3H), 3.58 (s, 0.5H), 3.46-3.39 (m, 1.5H), 3.16 (d, J = 11.6 Hz, 3H), 2.88 (d, J = 12.3 Hz, 1H), 2.62 (s, 0.5H), 2.34-2.17 (m, 1H), 1.88-1.55 (m,6H), 1.48-1.17 (m, 9H). MS (ESI) m / z: 432.2 [M + H] + .

[0034] Synthesis of 4-(3-(3-(4-fluoro-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-3-methylpiperidin-1-carbonyl)cyclohexane-1-one (2):

[0035] Compound 2 was synthesized in the same way as compound 1, using the commercially available starting material 4-oxocyclohexane-1-carboxylic acid to react with intermediate b, with a reaction yield of 58%. 1 H NMR (400 MHz, DMSO-d6) δ 7.89 (q, J = 8.7 Hz, 1H), 7.15 (dd, J = 10.3, 5.2 Hz, 1H), 6.99-6.90 (m, 1H), 4.41 (d, J = 13.9 Hz, 0.5H), 4.19-4.11 (m, 0.5H), 4.06-3.98 (m, 0.5H), 3.88 (s, 3H), 3.69 (s,0.5H), 3.46 (dd, J = 13.3, 8.7 Hz, 1.5H), 3.14 (d, J = 11.4 Hz, 0.5H), 2.97(t, J = 11.6 Hz, 0.5H), 2.62-2.52 (m, 0.5H), 2.46-2.09 (m, 5H), 2.03-1.90 (m,1H), 1.88-1.51 (m, 6H), 1.43-1.35 (m, 3H). MS (ESI) m / z: 416.2 [M + H] + .

[0036] Synthesis of 4-(3-(3-(4-fluoro-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-3-methylpiperidin-1-carbonyl)-4-methylcyclohexane-1-one (3): The synthesis of compound 3 was the same as that of 1, using commercially available raw material 1-methyl-4-oxocyclohexane-1-carboxylic acid to react with intermediate b, with a reaction yield of 65%. 1H NMR (400 MHz, Chloroform-d) δ8.02-7.93 (m, 1H), 6.79-6.69 (m, 2H), 4.48 (d, J = 13.3 Hz, 1H), 3.95 (s,3H), 3.88-3.78 (m, 1H), 3.57-3.41 (m, 2H), 2.64-2.49 (m, 2H), 2.44-2.32 (m,2H), 2.32-2.21 (m, 2H), 2.19-2.08 (m, 1H), 1.86-1.75 (m, 3H), 1.72-1.62 (m,2H), 1.47 (s, 3H), 1.35 (s, 3H). MS (ESI) m / z: 430.2 [M + H] + .

[0037] Synthesis of 5-(3-(3-(4-fluoro-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-3-methylpiperidin-1-carbonyl)piperidin-2-one (4): The synthesis of compound 4 was the same as that of 1, using commercially available raw material 6-oxoperidin-3-carboxylic acid to react with intermediate b, with a reaction yield of 54%. 1 H NMR (400 MHz, Chloroform-d) δ 8.03-7.81 (m,1H), 6.80-6.62 (m, 2H), 6.08-5.81 (m, 1H), 4.47-4.09 (m, 2H), 3.89 (s, 3H),3.60-3.11 (m, 4H), 2.98-2.64 (m, 1H), 2.58-2.22 (m, 3H), 1.93 (s, 2H), 1.71(s, 3H), 1.38 (d, J = 5.1 Hz, 3H). MS (ESI) m / z: 417.2 [M + H] + .

[0038] Synthesis of 4-(3-(3-(4-fluoro-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-3-methylpiperidin-1-carbonyl)piperidin-2-one (5): Compound 5 was synthesized in the same way as 1, using commercially available 2-oxoperidin-4-carboxylic acid reacting with intermediate b, with a yield of 57%. 1H NMR (400 MHz, DMSO-d6) δ 7.92-7.83 (m, 1H),7.50-7.34 (m, 1H), 7.15 (d, J = 11.5 Hz, 1H), 6.95 (t, J = 8.7 Hz, 1H), 4.43-4.03 (m, 1.5H), 3.89 (d, J = 1.8 Hz, 3H), 3.72-3.57 (m, 0.5H), 3.55-3.42 (m,1H), 3.26-2.97 (m, 3H), 2.92-2.80 (m, 0.5H), 2.33-2.04 (m, 3H), 1.91-1.50 (m,5H), 1.37 (dd, J = 15.6, 2.2 Hz, 3H). MS (ESI) m / z: 417.2 [M + H] + .

[0039] Synthesis of (3-(3-(4-fluoro-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-3-methylpiperidin-1-yl)(4-hydroxybicyclo[2.2.2]octane-1-yl) ketone (6): Compound 6 was synthesized in the same way as 1, using commercially available 4-hydroxybicyclo[2.2.2]octane-1-carboxylic acid reacted with intermediate b, with a yield of 43%. 1 H NMR (400 MHz, DMSO-d6) δ 7.87 (dd, J = 8.7, 6.9 Hz, 1H), 7.14 (dd, J = 11.5, 2.4 Hz, 1H), 6.96 (td, J = 8.4, 2.5 Hz, 1H), 4.31 (s, 1H), 4.04 (d, J = 13.0 Hz, 1H), 3.89 (s, 3H), 3.70 (s, 1H), 3.48 (d, J = 12.8 Hz, 2H), 2.18 (t, J = 8.0 Hz, 1H), 1.85-1.62 (m, 9H), 1.50 (t, J = 7.8 Hz, 6H), 1.33 (s, 3H). MS (ESI) m / z:444.2 [M + H] + .

[0040] Synthesis of (3-(3-(4-fluoro-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-3-methylpiperidin-1-yl)(4-hydroxypiperidin-1-yl) methyl ketone (7): Intermediate b (1 eq) and triphosgene (0.33 eq) were dissolved in dichloromethane, and DIPEA (4 eq) was added dropwise. The mixture was stirred at room temperature for 30 minutes, followed by the addition of piperin-4-ol (1 eq). The reaction system was stirred at room temperature for another 5.5 hours. After the reaction was complete as shown by TLC, the reaction solution was concentrated under reduced pressure and separated by preparative thin-layer chromatography to obtain a white solid with a yield of 44%. 1 H NMR (400 MHz, DMSO-d6) δ 7.87 (dd, J = 8.7, 6.9 Hz, 1H), 7.15 (dd, J = 11.5, 2.4 Hz, 1H), 6.95 (td, J = 8.4, 2.4 Hz, 1H), 4.64 (d, J =4.1 Hz, 1H), 3.88 (s, 4H), 3.60-3.51 (m, 1H), 3.27 (d, J = 13.0 Hz, 3H), 3.07(d, J = 13.0 Hz, 2.5H), 2.89-2.67 (m, 2.5H), 2.26 (s, 1H), 1.82-1.50 (m, 6H),1.35 (s, 3H). MS (ESI) m / z: 419.2 [M + H] + .

[0041] Synthesis of (3-(3-(4-fluoro-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-3-methylpiperidin-1-yl)(4-hydroxy-4-methylpiperidin-1-yl) methyl ketone (8): Compound 8 was synthesized in the same way as 7, using commercially available 4-methylpiperidin-4-ol and intermediate b in a yield of 36%. 1H NMR (400 MHz, Chloroform-d) δ 8.03(dd, J = 8.7, 6.6 Hz, 1H), 6.81-6.70 (m, 2H), 3.96 (s, 4H), 3.38 (dd, J =10.8, 7.3 Hz, 1H), 3.33-3.05 (m, MS (ESI) m / z: 433.2 [M + H] + .

[0042] Synthesis of 1-(3-(3-(4-fluoro-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-3-methylpiperidin-1-carbonyl)piperidin-4-one (9): Compound 9 was synthesized in the same way as 7, using commercially available raw material piperidin-4-one and intermediate b, with a reaction yield of 39%. 1 H NMR (400 MHz, DMSO-d6) δ 7.87 (dd, J = 8.7, 6.9 Hz, 1H), 7.15 (dd, J = 11.4, 2.5 Hz, 1H), 6.95 (td, J = 8.4, 2.4 Hz, 1H), 3.88(s, 4H), MS (ESI) m / z: 417.2[M + H] + .

[0043] Synthesis of (3-(3-(4-fluoro-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-3-methylpiperidin-1-yl)(morpholino) ketone (10):

[0044] Compound 10 was synthesized using the same method as 7, by reacting the commercially available starting material morpholine with intermediate b, with a yield of 40%. 1H NMR (400 MHz, DMSO-d6) δ 7.92-7.83 (m, 1H), 7.19-7.10 (m, 1H), 7.01-6.91 (m, 1H), 3.94-3.83 (m, 4H), 3.59-3.44 (m, 5H), 3.15-3.06 (m, 3H), 3.05-2.94 (m, 3H), 2.25 (d, J = 12.1 Hz, 1H), 1.80-1.62 (m, 3H), 1.35 (s, 3H). MS (ESI) m / z: 405.2 [M + H] + .

[0045] Synthesis of (1,1-thiomorpholine(3-(3-(4-fluoro-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-3-methylpiperidin-1-yl)methyl ketone (11): Compound 11 was synthesized by the same method as 7, using the commercially available raw material thiomorpholine-1,1-dioxide and intermediate b, with a reaction yield of 43%. 1 H NMR (400 MHz, DMSO-d6) δ7.86 (t, J = 7.8 Hz, 1H), 7.21-7.09 (m, 1H), 7.02-6.89 (m, 1H), 3.99-3.85 (m,4H), 3.62-3.38 (m, 5H), 3.20-2.96 (m, 6H), 2.26 (d, J = 13.0 Hz, 1H), 1.80-1.59 (m, 3H), 1.36 (s, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 181.76, 166.71,166.01, 164.20, 163.14, 159.74, 159.64, 132.70, 132.59, 111.83, 111.80,107.82, 107.61, 100.23, 99.97, 56.28, 54.88, 51.23, 47.94, 45.48, 37.90,34.92, 24.85, 21.80. MS (ESI) m / z: 453.2 [M + H] + .

[0046] Synthesis of 1-(3-(3-(4-fluoro-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-3-methylpiperidin-1-yl)-2-morpholinoacetone (12): Compound 12 was synthesized in the same way as 1, using commercially available 2-morpholinoacetic acid to react with intermediate b, with a reaction yield of 53%. 1 H NMR (400 MHz, Chloroform-d) δ 8.08-7.93 (m, 1H), 6.77 (d, J = 8.8 Hz, 2H), 4.51-4.32 (m, 1H), 4.07 (d, J = 12.7 Hz, 1H), 3.96 (s, 3H), 3.72 (s, 3H), 3.60-3.46 (m, 2.5H), 3.43-3.31 (m, 1.5H), 3.24 (d, J =13.9 Hz, 1H), 3.07 (d, J = 14.0 Hz, 1H), 2.53 (s, 2H), 2.49-2.24 (m, 3H),1.92 (s, 2H), 1.43 (s, 3H). MS (ESI) m / z: 419.2 [M + H] + .

[0047] Synthesis of (3-(3-(4-fluoro-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-3-methylpiperidin-1-yl)(3-fluorobicyclo[1.1.1]pentan-1-yl) methyl ketone (13): Compound 13 was synthesized by the same method as 1, using the commercially available starting material 3-fluorobicyclo[1.1.1]pentan-1-carboxylic acid reacted with intermediate b in a yield of 63%. 1 H NMR (400 MHz, DMSO-d6)δ 7.89 (q, J = 7.1 Hz, 1H), 7.15 (d, J = 11.4 Hz, 1H), 6.97 (t, J = 8.4 Hz,1H), 4.15-4.00 (m, 1H), 3.89 (s, 3H), MS (ESI) m / z: 404.2 [M + H] + .

[0048] Synthesis of (3-(3-(4-fluoro-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-3-methylpiperidin-1-yl)(3-(trifluoromethyl)bicyclo[1.1.1]pentan-1-yl) methyl ketone (14): Compound 14 was synthesized by the same method as 1, by reacting 3-fluorobicyclo[1.1.1]pentan-1-carboxylic acid with intermediate b, with a yield of 58%. 1 H NMR (400 MHz, DMSO-d6) δ7.95-7.81 (m, 1H), 7.15 (d, J = 11.4 Hz, 1H), 6.96 (td, J = 8.3, 2.5 Hz, 1H), 4.21 (d, J = 13.7 Hz, 0.5H), 4.00 (d, J = 13.0 Hz, 0.5H), 3.89 (s, 3H), 3.68-3.44 (m, 2.5H), 3.12-3.02 (m, 0.5H), 2.27 (s, 7.5H), 1.87-1.60 (m, 2.5H), 1.46-1.32 (m, 3H). MS (ESI) m / z: 454.2 [M + H] + .

[0049] Synthesis of (4,4-difluorocyclohexyl)(3-(3-(4-fluoro-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-3-methylpiperidin-1-yl) methyl ketone (15): Compound 15 was synthesized by the same method as 1, by reacting 4,4-difluorocyclohexane-1-carboxylic acid with intermediate b, with a yield of 59%. 1 H NMR (400 MHz, DMSO-d6) δ 7.92-7.84 (m, 1H),7.15 (d, J = 11.6 Hz, 1H), 6.96 (s, 1H), 4.34 (d, J = 13.9 Hz, 0.5H), 4.15(d, J = 13.0 Hz, 0.5H), 4.03-3.93 (m, 0.5H), 3.88 (s, 3H), 3.70-3.60 (m,0.5H), 3.45 (s, 1H), 3.10-2.77 (m, 2H), 2.33-2.18 (m, 1H), 2.08-1.90(m, 3H),1.84-1.68 (m, 4H), 1.66-1.43 (m, 4H), 1.40-1.34 (m, 3H). MS (ESI) m / z: 438.2[M + H] + .

[0050] Synthesis of (3-(3-(4-fluoro-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-3-methylpiperidin-1-yl)((1S,4S)-4-hydroxycyclohexyl) ketone (16): Compound 16 was synthesized by the same method as 1, by reacting (1S,4S)-4-hydroxycyclohexane-1-carboxylic acid with intermediate b, with a yield of 63%. 1 H NMR (400 MHz, Chloroform-d) δ8.12-7.92 (m, 1H), 6.84-6.69 (m, 2H), 4.32 (d, J = 13.5 Hz, 0.5H), 4.22-4.06(m, 1H), 4.03 (s, 0.5H), 3.96 (s, 3H), 3.81-3.68(m, 0.5H), 3.49(s, 1H), 3.30(d, J = 13.5 Hz, 0.5H), 3.11-2.82 (m, 1H), 2.50 (d, J = 14.5 Hz, 1H), 2.29(s,0.5H), 2.05-1.72 (m, 6H), 1.69-1.47 (m, 6.5H), 1.43 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 182.45, 174.36, 165.60, 159.97, 159.86, 132.86, 112.31, 107.79,107.60, 101.32, 101.01, 64.21, 64.01, 56.90, 53.33, 49.33, 45.31, 41.33,38.30, 37.42, 34.90, 34.50, 32.06, 24.56, 23.92, 23.82, 23.62, 23.46, 23.16,22.56, 22.20.MS (ESI) m / z: 418.2 [M + H] + The proton NMR spectrum is as follows: Figure 1 As shown.

[0051] Synthesis of (3-(3-(4-fluoro-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-3-methylpiperidin-1-yl)((1R,4R)-4-hydroxycyclohexyl) ketone (17): Compound 17 was synthesized by the same method as 1, by reacting (1R,4R)-4-hydroxycyclohexane-1-carboxylic acid with intermediate b, with a yield of 60%. 1H NMR (400 MHz, Chloroform-d) δ8.09-7.92 (m, 1H), 6.77 (t, J = 8.1 Hz, 2H), 4.36 (d, J = 13.5 Hz, 0.5H), 4.21-4.06 (m, 1H), 3.96 (s, 3H), 3.78-3.58 (m, 1.5H), 3.54-3.43 (m, 1.5H), 3.28 (d, J = 13.6 Hz, 0.5H), 3.06-2.78 (m, 1.5H), 2.57-2.42 (m, 1H), 2.29 (s, 0.5H), 2.19-1.92 (m, 4H), 1.89-1.61 (m, 6H), 1.57-1.50 (m, 1H), 1.37-1.25 (m,3H). MS (ESI) m / z: 418.2 [M + H] + .

[0052] Synthesis of cyclohexyl(3-(3-(4-fluoro-2-methoxyphenyl)-1,2,4-oxadiazol-5-yl)-3-methylpiperidin-1-yl) ketone (18): Compound 18 was synthesized by the same method as 1, by reacting cyclohexanecarboxylic acid with intermediate b, with a yield of 66%. 1 H NMR (400 MHz, DMSO-d6) δ 7.94-7.83(m, 1H), 7.15 (d, J = 11.4 Hz, 1H), 6.95 (t, J = 7.9 Hz, 1H), 4.30 (d, J = 13.8 Hz, 0.5H), 4.14-3.99 (m, 1H),3.89 (s, 3H), 3.56 (s, 0.5H), 3.51-3.39 (m, 1.5H), 2.94-2.74 (m, 1H), 2.57(s, 0.5H), 2.37-2.12 (m, 1H), 1.91-1.43 (m, 8H), 1.42-1.31(m, 5H), 1.25-1.11(m, 3H). MS (ESI) m / z: 402.2 [M + H] + .

[0053] The following efficacy tests demonstrate the beneficial effects of this invention.

[0054] Experimental Example 1: Evaluation and Verification of CB2 and CB1 Activity

[0055] 1. Evaluation of CB2 and CB1 activity based on Glosensor cAMP assay

[0056] HEK 293T cells were selected, and human CB2 or CB1 receptors were co-introduced into these cells along with the pGloSensor-22F cAMP plasmid. After transfection, the cells were seeded evenly into 96-well plates at a density of 15,000 cells per well. The seeded 96-well plates were then placed in a cell culture incubator at 37°C with 5% CO2. The next day, the culture medium in the 96-well plates was aspirated, and 80 μL of D-fluorescein potassium salt solution (0.5 mg / mL) prepared with GloSensor test buffer was added to each well. The cells were incubated at room temperature for 30 minutes. The test compound solution diluted with test buffer and the trichosamine solution diluted with test buffer (3 μM) were added to the cells, and the chemiluminescence value of the cells was detected using a microplate reader.

[0057] In this invention, after CB2 or CB1 is activated by the agonist, it primarily couples to G. i Proteins cause a decrease in cAMP levels; therefore, the stronger the agonist effect of a compound, the smaller the detected luminescence value. The agonist rate of a compound for CB2 or CB1 is calculated as follows: Agonist rate (%) = (1 – Sample reading / Negative reference reading) × 100%. Finally, the dose-response curves were fitted using Graphpad Prism software, and the EC50 values ​​of the test compounds for CB2 and CB1 were calculated.

[0058] 2. Activity verification experiment

[0059] This invention uses PTX (pertussis toxin) to verify the agonistic activity of compounds for CB2. PTX is a G... i / o Protein inhibitors are often used to verify whether GPCRs are G-dependent. i / o The protein pathway regulates cAMP. The specific experimental procedure is as follows: HEK 293T-CB2 cells were evenly seeded into 96-well plates and incubated overnight. Then, half a volume of the supernatant was aspirated, and PTX solution was prepared using culture medium without fetal bovine serum. The same volume of PTX solution was added to each well to achieve a final PTX concentration of 100 ng / mL. The 96-well plates were incubated in a cell culture incubator for 16 hours. Afterward, the agonistic activity of the compound on CB2 cells was measured according to the corresponding procedure in step 1.

[0060] 3. Experimental Results:

[0061] Table 1. Activities of all compounds against CB2 / CB1

[0062]

[0063] Experimental results showed that compounds 15, 16, and 18 exhibited high CB2 agonist activity, with EC50 values ​​all below 10 nM. Compound 16 showed good CB2 selectivity, but did not exhibit significant agonist activity towards CB1 at a concentration of 10 μM, with a CB2 / CB1 selectivity >1140-fold.

[0064] Table 2 Comparison data between compound 16 and the publicly disclosed compound YL-035:

[0065]

[0066] Compared with the previously reported compound YL-035 (PNAS, 2024, 121(30), e2401091121; CN117756793A), compound 16 has comparable CB2 selectivity and agonist activity.

[0067] Experimental Example 2: CYP 450 Enzyme Inhibition Experiment

[0068] Drug-drug interactions (DDIs) refer to the phenomenon where the potency, duration, or even properties of one drug are significantly affected by another drug when two or more drugs are used simultaneously or sequentially. Because DDIs are easily overlooked in the early stages of drug discovery, and because some interactions can affect drug metabolism and increase blood drug concentrations, leading to serious adverse reactions, they are a key factor influencing the progress of preclinical research and clinical trials. Cytochrome P450 (CYP450) enzymes are the main enzymes involved in drug metabolism. CYP450 enzymes are primarily involved in the metabolism of endogenous substances and exogenous substances, including drug molecules. Clinically, over 90% of drug molecules can be metabolized by CYP450, which mainly consists of three subtypes: CYP3A4, CYP2C9, and CYP2D6. Cytochrome P450 can catalyze reactions such as epoxidation, monohydroxylation, or polyhydroxylation of drug molecules. Excessive inhibitory activity of drug molecules against CYP450 enzymes can slow down the metabolism of the drug molecule itself or other drugs, leading to drug accumulation in the body and a series of toxic side effects. Some drugs have a strong inhibitory effect on CYP450 enzymes, affecting the binding and metabolism of CYP450 enzymes with other drugs, thus leading to drug accumulation and increased toxicity in the body. Therefore, the inhibitory activity of a drug against CYP450 enzymes is an important parameter for assessing drug-induced drug dependence (DDI).

[0069] CYP450 enzyme inhibitory activity was tested using the CYPP450-GloSensor assay kit (Promega, USA). The test compound was prepared as a 10 mM stock solution (2% DMSO + 98% acetonitrile). This stock solution was then diluted four-fold with the same mixture to create seven concentration gradients. The positive control (ketoconazole) was prepared as a 10 mM stock solution with DMSO, then diluted with 2% DMSO, followed by four-fold dilutions with 2% DMSO and 98% acetonitrile, also creating seven concentration gradients. In each well of a white 96-well plate, 5.52 μL of water, 6 μL of protease solution, 0.48 μL of the compound, and 12 μL of NADPH solution were added. After incubation with ketoconazole for 10 minutes, 24 μL of substrate was added, followed by incubation for another 20 minutes before bioluminescence was measured. Higher inhibitory activity against CYP450 enzymes was associated with lower fluorescence values; the inhibitory activity was calculated from the measured fluorescence values.

[0070] Table 3. Inhibitory activity of highly active compounds against CYP 3A4

[0071]

[0072] a The numerical value represents the average of three independent experiments.

[0073] Experimental results: Compound 16, which replaced the p-fluorophenyl substituted compound with a cis-4-hydroxycyclohexyl substituted compound, exhibited greater than 100 μM inhibitory activity against CYP3A4 and showed almost no inhibition of the CYP3A4 isoform.

[0074] Experimental Example 3: Cytotoxicity Evaluation

[0075] MTT assays were performed on four normal human cell lines, including HUVEC, HK-2, BEAS-2B, and LX-2, to determine the cytotoxicity of compound 16.

[0076] The experimental procedure was as follows: First, 100 μL of culture medium containing different concentrations of compound 16 was added to a 96-well plate. Then, 100 μL of cells in the logarithmic growth phase was quickly added to each well, adjusting the cell density to 3000-5000 cells / well. To reduce evaporation of the solution in the wells, 200 μL of injectable physiological saline was added to the side wells of the 96-well plate. The 96-well plate was then placed in a cell culture incubator and incubated for 72 hours. After the incubation period, 20 μL of MTT solution was added to each well, and the plate was returned to the cell culture incubator for another 2-4 hours. Finally, the absorbance of each well was measured at 570 nm using a microplate reader. The inhibition rate of cell growth by different concentrations of the compound was calculated based on the absorbance measured in each well, and the CC of the compound was calculated using GraphPad Prism software. 50 value.

[0077] The results are as follows Figure 2 The effects of different doses of compound 16 on the growth and survival of four cell lines (HUVEC, HK-2, BEAS-2B, and LX-2) were measured using the MTT assay. Compound 16 did not show significant toxicity to any of the four cell lines at a dose of 100 μM, demonstrating that compound 16 has low cytotoxicity and good in vitro safety.

[0078] Table 4: Data on the activity and selectivity of preferred compound 16, and its activity against drug-metabolizing enzymes.

[0079]

[0080] The activation curve of compound 16 to CB2 is shown in Figure 16. Figure 3 As shown.

[0081] This invention addresses the design approach of using aromatic rings as key pharmacodynamic structural units in existing technologies. By replacing the aromatic ring (ring C) with a non-aromatic aliphatic ring, the conformational characteristics of the molecule and its interaction with the receptor are altered. This reduces the inhibitory activity of drug-metabolizing enzymes (cytochrome P450 enzymes) while maintaining high CB2 selectivity and high agonistic activity, effectively improving the drug-likeness of the compound. The representative compound 16 validates the effectiveness of this structural modification strategy and is an important technical support for this invention.

Claims

1. A 1,2,4-hexadiazole derivative, characterized in that: The structural formula is shown in Formula I: ； Wherein, n is selected from integers from 0 to 3; ring A is selected from substituted or unsubstituted 3- to 6-membered cycloalkanes and substituted or unsubstituted 4- to 6-membered heterocyclic alkanes, wherein the 4- to 6-membered heterocycle in ring A contains at least one N, O, or S heteroatom, and the substituents of the substituted 3- to 6-membered cycloalkanes and substituted 4- to 6-membered heterocyclic alkanes in ring A are selected from halogens, carbonyl groups, hydroxyl groups, C1- to C6 alkoxy groups, C1- to C6 alkyl groups, and halogen-substituted C1- to C6 alkyl groups.

2. The 1,2,4-hexadiazole derivative according to claim 1, characterized in that: The 3- to 6-membered cycloalkanes mentioned in ring A are selected from... , , , The 3- to 6-membered heterocyclic alkanes are selected from , , , , , , , , .

3. The 1,2,4-hexadiazole derivative according to claim 1, characterized in that: The substituted 3-6 membered cycloalkanes and substituted 4-6 membered heterocycloalkanes mentioned in ring A are selected from: , , , , , , , , , , , , , , , , , , , .

4. The 1,2,4-hexadiazole derivative according to claim 1, characterized in that: Ring A is selected from substituted or unsubstituted 4-6 membered bridged rings, and the substituents of the substituted 4-6 membered bridged rings in ring A are selected from halogens, hydroxyl groups, and C1-C6 alkoxy groups. The unsubstituted 4-6 quinary bridge rings described in ring A are selected from , , The 4-6 quinary bridge rings substituted in ring A are selected from... , , , , , .

5. The 1,2,4-hexadiazole derivative according to any one of claims 1-4, characterized in that: The ring A mentioned above is selected from: , , , , , , , .

6. The 1,2,4-hexadiazole derivative according to claim 5, characterized in that: The structural formula of the compound is: 。 7. A synthetic process for the 1,2,4-heterodiazole derivative according to any one of claims 1-5, characterized in that: The synthesis route is shown below: ； in, .

8. Use of the 1,2,4-hexadiazole derivative according to any one of claims 1-6 in the preparation of a medicament for a highly selective CB2 receptor agonist.

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