A triazole-cis enamide compound, a preparation method thereof and anti-inflammatory application thereof

Abstract

The application discloses a triazole-cis enamide compound, a preparation method and anti-inflammatory application thereof, and relates to the technical field of biological medicines. The triazole-cis enamide compound has excellent effects in anti-inflammatory and treatment of allergic rhinitis diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a triazole-cis-enamide compound, its preparation method, and its anti-inflammatory applications. Background Technology

[0002] Allergic rhinitis (AR), also known as hay fever, is a non-infectious inflammatory disease caused by the inhalation of environmental allergens, which activates immunoglobulin E (IgE) and leads to its development. Its main clinical symptoms include paroxysmal sneezing, runny nose, nasal congestion, and nasal itching, and may also be accompanied by eye itching and tearing. Statistics show that approximately 10%-40% of the world's population is affected by allergic rhinitis, and this number is increasing annually, making it a global health concern.

[0003] Lansiumamide B, isolated from the kernel of wampee fruit, is a cis-enamide compound with various pharmacological activities, including treating obesity, lowering blood lipids, anti-diabetic effects, anti-snake venom activity, and anti-inflammation. Its structural characteristics include a cinnamamide moiety and a cis-enamide moiety. 1,2,4-Triazole is a stable azole compound derived by substituting a nitrogen atom at the 4-position of a pyrazole, often existing in two tautomeric forms. Due to its dipole interaction, hydrogen bonding ability, rigidity, and solubility, 1,2,4-triazole can exhibit high affinity for biological receptors, becoming an important pharmacologically active group. In addition, 1,2,4-triazole can also act as a bioisostere of amides, esters, and carboxylic acids. Therefore, 1,2,4-triazole is a superior framework in the field of drug development and is commonly found in various clinical drugs, including anticancer drugs, antiviral drugs, anticonvulsants, antifungal drugs, anxiolytics, hypnotics, skeletal muscle relaxants, antimigraines, antiplatelet drugs, antidepressants, and aromatase inhibitors.

[0004] This invention aims to develop anti-inflammatory drugs for treating allergic rhinitis by combining the cis-enamide structure with the 1,2,4-triazole structure commonly found in medicinal chemistry through structural transition. Summary of the Invention

[0005] The purpose of this invention is to provide a triazole-cis-enamide compound, its preparation method, and its anti-inflammatory application, in order to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a 1,2,4-triazole-cis-enamide compound and its pharmaceutical salt or solvate, with the structural formula shown in formula (I):

[0008]

[0009] In formula (I), R is hydrogen, C6-C14 aryl, five- to fourteen-membered heteroaryl, C1-C10 cycloalkyl, five- to fourteen-membered heterocyclic, C1-C6 straight-chain alkyl or C1-C6 branched alkyl;

[0010] The C6-14 aryl, five- to fourteen-membered heteroaryl, C6-10 cycloalkyl, or five- to fourteen-membered heterocyclic group is substituted by one or more substituents selected from halogen, halogenated C1-C6 alkyl, carbonyl, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, carboxyl, cyano, hydroxyl, carbonyl, phenyl-SO2-, nitro, and C1-C6 alkyloxycarbonyl; the five- to fourteen-membered heteroaryl or five- to fourteen-membered heterocyclic group contains 1 to 3 heteroatoms selected from N, O, and S.

[0011] Furthermore, the halogen is F, Cl, Br, or I.

[0012] Furthermore, the 1,2,4-triazole-cis-enamide compounds and their pharmaceutical salts or solvates are selected from any one of the following:

[0013]

[0014] Furthermore, the pharmaceutical salt is a pharmaceutically acceptable salt, including inorganic acid salts, organic acid salts, alkyl sulfonates, or aryl sulfonates.

[0015] Specifically, the inorganic acid salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, isothiosulfate, nitrate, sulfate, hydrogen sulfate, persulfate, phosphate, and bicarbonate;

[0016] Specifically, the organic acid salts include, but are not limited to, formate, acetate, propionate, butyrate, valerate, neovalerate, camphorate, adipate, alginate, aspartate, benzoate, maleate, fumarate, lactate, nicotinate, succinate, tartrate, citrate, diglucose, glycerophosphate, cysteine, oxalate, palmitate, pectin ester, 3-phenylpropionate, thiocyanate, glutamate, and oleate;

[0017] Specifically, the alkyl sulfonates include, but are not limited to, methyl sulfonates, ethyl sulfonates, and camphor sulfonates;

[0018] Specifically, the aryl sulfonates include, but are not limited to, benzenesulfonates, p-toluenesulfonates, and 2-naphthalenesulfonates.

[0019] Further, the solvate comprises a solvate of the 1,2,4-triazole-cis-enamide compound with water, ethanol, isopropanol, diethyl ether, ethylene glycol, acetone, or glycerol.

[0020] The present invention also provides a method for preparing the above-mentioned 1,2,4-triazole-cis-enamide compounds and their pharmaceutical salts or solvates, comprising the following steps:

[0021]

[0022] a) Dissolve compounds of formula I and formula II in acetonitrile solution at a stoichiometric ratio of 1:1 and react at room temperature for 1-3 hours;

[0023] b) Dissolve the product of formula III obtained in step a) in dry dichloromethane, and add it dropwise to a dry dichloromethane solution of NBS-dimethyl sulfide (3 equivalents) cooled to -78°C. React at -78°C for 0.5-2 h, then raise the temperature to room temperature (20-25°C) and react for 2-5 h.

[0024] c) Dissolve compounds of formula V and formula VI in water at a stoichiometric ratio of 1:1.2, add sodium carbonate in small amounts several times, and react at room temperature for 2-5 hours;

[0025] d) Dissolve compound IV obtained in step b) and compound VII obtained in step c) in dry toluene at a stoichiometric ratio of 1:1.2, add triethylamine, and react at 110℃-130℃ for 1-3 h.

[0026] e) Dissolve the product VIII obtained in step d) in an ammonia-methanol solution and react at room temperature for 2-8 days;

[0027] f) React the product compound IX obtained in step e) with compound X at a stoichiometric ratio of 1.2:1 at 40℃-80℃ for 0.5-3h;

[0028] g) The product compound XI obtained in step f) is reacted with sodium hydride (1.6 equivalents) at -20℃ to 5℃ for 0.5-1h. Then, iodomethane (1.6 equivalents) is added at this temperature. Finally, the temperature is raised to 20℃ to 50℃ and the mixture is stirred for 2-6h to obtain compound XII, which is the compound with the structure shown in formula (I).

[0029] Wherein, R is hydrogen, C6-C14 aryl, five- to fourteen-membered heteroaryl, C1-C10 cycloalkyl, five- to fourteen-membered heterocyclic, C1-C6 straight-chain alkyl or C1-C6 branched alkyl;

[0030] The C6-14 aryl, five- to fourteen-membered heteroaryl, C6-10 cycloalkyl, or five- to fourteen-membered heterocyclic group is substituted by one or more substituents selected from halogen, halogenated C1-C6 alkyl, carbonyl, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, carboxyl, cyano, hydroxyl, carbonyl, phenyl-SO2-, nitro, and C1-C6 alkyloxycarbonyl; the five- to fourteen-membered heteroaryl or five- to fourteen-membered heterocyclic group contains 1 to 3 heteroatoms selected from N, O, and S.

[0031] The present invention further provides the application of the above-mentioned 1,2,4-triazole-cis-enamide compounds and their pharmaceutical salts or solvates in the preparation of anti-inflammatory drugs.

[0032] The present invention further provides the use of the above-mentioned 1,2,4-triazole-cis-enamide compounds and their pharmaceutical salts or solvates in the preparation of drugs for treating allergic rhinitis.

[0033] The present invention discloses the following technical effects:

[0034] This invention provides a triazole-cis-enamide compound, which has excellent effects in anti-inflammatory and therapeutic applications for allergic rhinitis. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 This demonstrates the inhibitory effect of 1,2,4-triazole-cis-enamide compounds on the mRNA levels of IL-6 and IL-1β inflammatory factors in Raw264.7 cells in Example 1. Figure A shows the IL-6 RNA expression level in Raw264.7 cells stimulated with LPS after treatment with different compounds; Figure B shows the IL-1β mRNA expression level in Raw264.7 cells stimulated with LPS after treatment with different compounds; Figures C and D show the IL-6, IL-1β, and TNF-α mRNA expression levels in Raw264.7 cells stimulated with LPS after treatment with different concentrations of compound 2125 (compared to the Control group). ### P < 0.001; compared with the LPS group * P < 0.05 ** P < 0.01, *** P < 0.001).

[0037] Figure 2This indicates the effect of compound 2 in alleviating rhinitis symptoms in an AR mouse model (compared to the control group). #### P < 0.0001; compared with the LPS group ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0038] Figure 3 This indicates the total number of inflammatory cells in the nasal lavage fluid of mice in Example 2 (compared to the Control group). ## P < 0.01; compared with the AR group * P < 0.05 ** P < 0.01).

[0039] Figure 4 The images show Gimsa staining of cells in mouse nasal lavage fluid (A), the number of eosinophils (B), and the number of mast cells (C) in Example 2 (compared to the Control group). #### P < 0.0001; compared with the AR group *** P < 0.001, **** P < 0.0001.

[0040] Figure 5 Example 2 shows the HIS expression level in mouse serum (compared to the Control group) to verify the effect. #### P < 0.0001; compared with the AR group * P < 0.05 *** P < 0.001, **** P < 0.0001.

[0041] Figure 6 The image shows a pathological image of mouse lung tissue stained with HE in Example 2 (magnification: 400×; scale bar = 100μm).

[0042] Figure 7 The image shows a PAS staining image of mouse lung tissue in Example 2 (magnification: 400×; scale bar = 100μm). Detailed Implementation

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0044] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0045] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0046] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0047] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0048] The room temperature mentioned in the embodiments of the present invention refers to 20-25℃.

[0049] Example 1: Synthesis of intermediate IV of the present invention:

[0050]

[0051] Step 1: Take a 100 mL egg-shaped flask, weigh phenylhydrazine I (1.08 g, 10 mmol) into the flask and add a magnetic stir bar. Add acetonitrile (15 mL) to dilute and stir. Separately weigh ethyl glyoxylate II (2.04 g, 10 mmol, 50 wt% in toluene) into a sample bottle, dilute with acetonitrile (5 mL), and slowly add it dropwise to the egg-shaped flask. Wash the sample bottle with acetonitrile (10 mL) and add it to the egg-shaped flask. Stir the reaction at room temperature for 2 hours until the reaction is complete. Remove the solvent using a rotary evaporator, and purify by silica gel chromatography using petroleum ether as the eluent to obtain yellow solid III (1.88 g, 9.8 mmol), yield: 98%.

[0052] Step 2: Dry a 100mL two-necked flask, vacuum pump, and magnetic stir bar in an oven for later use. After assembling the flask, evacuate it and allow it to cool to room temperature before introducing argon gas. Weigh N-succinimide bromide (2.67g, 15mmol) and add it to the two-necked flask, then add 20mL of dry anhydrous dichloromethane. Under 0°C ice bath conditions, slowly add 2.25mL of dimethyl sulfide (30mmol) and react for 15 minutes. Lower the temperature to -78°C. Weigh III (961mg, 5mmol) into a sample vial, dissolve it in 10mL of dry anhydrous dichloromethane, and add it dropwise to the two-necked flask. Wash the sample vial twice with 6mL of dry anhydrous dichloromethane and add the rinsings to the two-necked flask. Maintain the reaction at -78°C for 1 hour, then allow it to warm naturally and stir for 3 hours. After the reaction was completed, water (15 mL) was added dropwise to the two-necked flask to quench the reaction. The mixture was extracted with dichloromethane (3 × 15 mL), and the organic phase was washed with water (3 × 15 mL), then washed once with saturated sodium chloride solution (20 mL), then once with saturated sodium sulfite solution (20 mL), and finally washed once with water (20 mL). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The organic phase was then purified by silica gel chromatography using petroleum ether as the eluent to give a yellow solid IV (1.19 g, 4.4 mmol), yield: 88%.

[0053] Example 2: Synthesis of the final product 2121 of the present invention

[0054] (1) Synthesis of compound 2

[0055]

[0056] Weigh benzaldehyde (531 mg, 5 mmol) into a 25 mL egg-shaped flask, add a magnetic stir bar and water (5 mL), then add hydroxylamine hydrochloride (417 mg, 6 mmol). While stirring at room temperature, add sodium carbonate (1.06 g, 10 mmol) in small portions several times, stirring at room temperature for 3 hours. After the reaction is complete, extract with diethyl ether (3 × 10 mL), wash once with saturated sodium chloride solution (10 mL), collect the organic phase, dry it with anhydrous sodium sulfate, and remove the solvent from the dried organic phase using a rotary evaporator to obtain a white solid for the next step.

[0057] (2) Synthesis of compound 3

[0058]

[0059] Take 100 mL of sealing tube, weigh out 2 (363 mg, 3 mmol) and IV (678 mg, 2.5 mmol) respectively, pour them into the sealing tube and add a magnetic stir bar. Pipette 15 mL of dry anhydrous toluene into the sealing tube, purge the sealing tube with argon gas (for 1 min), add 0.69 mL of dry triethylamine (5 mmol) dropwise into the sealing tube, tighten the sealing stopper, and place the sealing tube in a 120 °C oil bath for heating and stirring for 2 h. Remove the oil bath, allow the temperature to drop to room temperature, remove the toluene by rotary evaporation, add water (20 mL), extract with dichloromethane (3 × 20 mL), wash once with saturated sodium bicarbonate solution (20 mL), wash once with saturated sodium chloride solution (20 mL), collect the organic phase, dry with anhydrous sodium sulfate, remove the solvent by rotary evaporation, and purify by silica gel column chromatography using petroleum ether as eluent to obtain 403 mg of yellow oily liquid (compound 3), yield: 55%.

[0060] (3) Synthesis of compound 4

[0061]

[0062] Take a 15 mL sealing tube, weigh out compound 3 (403 mg, 1.37 mmol), add a magnetic stir bar, and inject 2.74 mL of ammonia-methanol solution. Purge the sealing tube with argon gas (for 1 min), tighten the stopper, and stir for 3 days. After the reaction is complete, collect the organic phase, remove the solvent using a rotary evaporator, and then purify by silica gel chromatography using petroleum ether and acetone (V / V = 1:1) as eluents to obtain 330 mg of a white solid (compound 4), yield: 90%.

[0063] (4) Synthesis of compound 5

[0064]

[0065] Take 10mL After drying the Schlenk tube and magnetic induction device, assemble them and evacuate them. Once the temperature has cooled to room temperature, replace the vacuum with argon gas. Then weigh in compound 4 (291 mg, 1.1 mmol), cesium carbonate (407 mg, 1.25 mmol), and cuprous iodide (9.5 mg, 0.05 mmol). Dilute (Z)-2-iodovinylbenzene (243 mg, 1 mmol) with 5 mL of dried tetrahydrofuran and add it to the Schlenk tube. Add DMEDA (11 μL, 0.1 mmol), seal the Schlenk tube, and heat and stir in a 60°C oil bath for 3 hours. After the reaction is complete, remove the oil bath and allow it to cool naturally to room temperature. Filter the reaction solution with diatomaceous earth and wash the diatomaceous earth with ethyl acetate (3 × 20 mL). Collect the organic phase and remove the solvent using a rotary evaporator. Then purify the solution using silica gel chromatography with petroleum ether and ethyl acetate (V / V = 3:1) as eluent to obtain 304 mg of a yellow solid (compound 5), yield: 83%.

[0066] (5) Synthesis of compound 2121

[0067]

[0068] A 25 mL two-necked flask, vacuum pump, and magnetic stir bar were dried and assembled. Vacuum was then applied, and the flask was allowed to cool to room temperature. Argon gas was introduced, and compound 5 (275 mg, 0.75 mmol) was weighed in. 3 mL of dried DMF was added to the flask. Sodium hydride (45 mg, 1.2 mmol) with a purity of 60% was added to the flask under an ice bath at 0°C. The mixture was stirred at 0°C for 30 min, and then iodomethane (68 μL, 1.2 mmol) was slowly added dropwise. The mixture was slowly brought back to room temperature and stirred for 3 h. After the reaction was complete, water was added to quench the reaction under an ice bath. The mixture was extracted with diethyl ether (3 × 20 mL), washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was then purified by silica gel chromatography using petroleum ether and ethyl acetate (V / V = 5:1) as eluents to give 228 mg of a yellow solid (compound 2121), yield: 80%.

[0069] The obtained compound 2121 was identified using nuclear magnetic resonance spectroscopy and mass spectrometry. The identification results are as follows:

[0070] Major conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.53-7.31 (m, 14H), 7.26-7.21 (m, 1H), 6.94 (d, J = 8.8Hz, 1H), 6.06 (d, J = 8.8Hz, 1H), 3.16 (s, 3H). 13C10 NMR (100MHz, CDCl3) δ: 161.9, 157.2, 154.2, 137.8, 135.1, 130.4, 130.3, 129.5 (2×C), 129.3, 129.2 (2×C), 128.8 (2×C), 128.6 (2×C), 128.5 (2×C), 127.7, 127.3, 125.5 (2×C), 121.2, 35.8. Minor conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.53-7.31 (m, 14H), 7.26-7.21 (m, 1H), 7.06 (d, J = 9.2Hz, 1H), 6.22 (d, J = 9.2Hz, 1H), 3.31 (s, 3H). 13 C NMR (100MHz, CDCl3) δ: 162.5, 157.0, 154.5, 137.8, 135.5, 130.6, 130.4, 129.6 (2×C), 129.3, 129.0 (2×C), 128.7 (2×C), 128.6 (2×C), 128.4 (2×C), 127.5, 127.1, 125.5 (2×C), 121.1, 38.7. HRMS (ESI) m / z calculated value is C 24 H 21 ON4 + [M+H] + 381.1710, measured value is 381.1716.

[0071] By selecting different aldehydes, compounds 2122-2142 can be obtained:

[0072] Example 3: Synthesis of compound 2122:

[0073] The synthesis steps were the same as in Example 2, except that the aldehyde reagent used in the first step was 4-fluorobenzaldehyde, yielding compound 2122 with a yield of 80%; the major conformational isomers were: 1 H NMR(400MHz, CDCl3)δ:7.54-7.40(m,5H),7.38-7.30(m,6H),7.26-7.21(m,1H) ,7.04-7.00(m,2H),6.91(d,J=8.8Hz,1H),6.05(d,J=8.8Hz,1H),3.15(s,3H). 13CNMR (100MHz, CDCl3) δ: 163.9 (d, J = 250Hz), 161.7, 157.1, 153.4, 137.7, 135.0, 131.3 (d, J = 8.5Hz, 2×C), 130.2, 129.6 ( 2×C),129.5,128.8(2×C),128.6(2×C),127.7,125.5(2×C),123.4(d,J=3.3Hz),121.4,115.9(d,J=21.9Hz,2×C),35.8. 19 F NMR (376MHz, CDCl3) δ: -109.0. Minor conformational isomer: 1 H NMR(400MHz, CDCl3)δ:7.54-7.40(m,5H),7.38-7.30(m,6H),7.26-7.21(m,1H) ,7.04-7.00(m,2H),7.05(d,J=9.2Hz,1H),6.22(d,J=9.2Hz,1H),3.30(s,3H). 13 C NMR (100MHz, CDCl3) δ: 163.9 (d, J = 250Hz), 162.3, 157.0, 153.6, 137.7, 135.5, 131.3 (d, J = 8.5Hz, 2×C), 130.2, 129.7 ( 2×C), 129.3, 129.0 (2×C), 128.4 (2×C), 127.5, 125.5 (2×C), 123.3 (d, J=3.3Hz), 120.3, 116.0 (d, J=22.0Hz, 2×C), 38.7. 19 F NMR (376MHz, CDCl3) δ: -109.0. HRMS (ESI) m / z calculated value is C 24 H 20 ON4F + [M+H] + 399.1616, measured value is 399.1611.

[0074] Example 4: Synthesis of compound 2123:

[0075] The synthesis steps were the same as in Example 2, except that isobutyraldehyde was used as the aldehyde reagent in the first step reaction to give compound 2123, yield: 85%; major conformational isomers: 1H NMR(400MHz, CDCl3)δ:7.52-7.45(m,3H),7.44-7.35(m,2H),7.31-7.25(m,4H),7.22-7.18(m,1H),6.8 6(d,J=8.8Hz,1H),5.98(d,J=8.8Hz,1H),3.11(sept,J=6.8Hz,1H),3.10(s,3H),1.29(d,J=6.8Hz,6H). 13 C10 NMR (100MHz, CDCl3) δ: 162.1, 161.4, 156.5, 137.0, 135.1, 130.3, 129.5 (2×C), 129.4, 128.7 (2×C), 128.5 (2×C), 127.5, 125.5 (2×C), 120.7, 35.7, 25.9, 21.6 (2×C). Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.52-7.45(m,3H),7.44-7.35(m,2H),7.31-7.25(m,4H),7.22-7.18(m,1H),7.0 2(d,J=9.2Hz,1H),6.17(d,J=9.2Hz,1H),3.22(s,3H),3.11(sept,J=6.8Hz,1H),1.31(d,J=6.8Hz,6H). 13 C NMR (100MHz, CDCl3) δ: 162.7, 161.7, 156.4, 137.0, 135.5, 130.3, 129.6 (2×C), 129.4, 128.9 (2×C), 128.3 (2×C), 127.3, 125.5 (2×C), 119.8, 38.5, 25.9, 21.6 (2×C). HRMS (ESI) m / z calculated values ​​are C 21 H 23 ON4 + [M+H] + 347.1866, the measured value is 347.1868.

[0076] Example 5: Synthesis of compound 2124:

[0077] The synthesis steps were the same as in Example 2, except that the aldehyde reagent used in the first step was 2-thiophenecarboxaldehyde, yielding compound 2124 with a yield of 85%; major conformational isomers: 1H NMR(400MHz, CDCl3)δ:7.56-7.42(m,5H),7.40-7.37(m,1H),7.34-7.29(m,4H),7.25-7.20(m,1H),7.08 (d,J=3.6Hz,1H),6.94(dd,J=4.8,4.0Hz,1H),6.91(d,J=8.8Hz,1H),6.04(d,J=8.8Hz,1H),3.13(s,3H). 13 C10 NMR (100MHz, CDCl3) δ: 161.6, 157.0, 149.9, 137.3, 135.0, 130.3, 130.2, 129.8, 129.7 (2×C), 129.6, 129.5, 128.8 (2×C), 128.5 (2×C), 127.7, 127.6, 126.7 (2×C), 121.2, 35.8. Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.56-7.42(m,5H),7.40-7.37(m,1H),7.34-7.29(m,4H),7.25-7.20(m,1H),7.10 (d,J=3.2Hz,1H),7.04(d,J=9.2Hz,1H),6.94(dd,J=4.8,4.0Hz,1H),6.21(d,J=9.2Hz,1H),3.28(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.2, 156.8, 150.1, 137.3, 135.5, 130.5, 130.2, 129.8, 129.7 (2×C), 129.6, 129.3, 129.0 (2×C), 128.4 (2×C), 127.8, 127.5, 126.7 (2×C), 120.2, 38.6. HRMS (ESI) m / z calculated value is C 22 H 19 ON4S + [M+H] + 387.1274, measured value is 387.1272.

[0078] Example 6: Synthesis of compound 2125:

[0079] The synthesis steps were the same as in Example 2, except that cyclohexylformaldehyde was chosen as the aldehyde reagent in the first step reaction to give compound 2125, yield: 88%; major conformational isomers: 1H NMR(400MHz, CDCl3)δ:7.52-7.45(m,3H),7.43-7.35(m,2H),7.32-7.26(m,4H),7.24-7.19(m,1H),6.86(d, J=8.8Hz,1H),5.98(d,J=8.8Hz,1H),3.11(s,3H),2.78-2.72(m,1H),1.81-1.65(m,7H),1.33-1.16(m,3H). 13 C10 NMR (100MHz, CDCl3) δ: 162.1, 160.6, 156.6, 137.1, 135.2, 130.4, 129.5 (2×C), 129.4, 128.7 (2×C), 128.5 (2×C), 127.5, 125.5 (2×C), 120.7, 35.8, 35.4, 31.7 (2×C), 25.9 (2×C), 25.5. Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.52-7.45(m,3H),7.43-7.35(m,2H),7.32-7.26(m,4H),7.24-7.19(m,1H),7.02(d, J=9.2Hz,1H),6.18(d,J=9.2Hz,1H),3.22(s,3H),2.78-2.72(m,1H),1.81-1.65(m,7H),1.33-1.16(m,3H). 13 C NMR (100MHz, CDCl3) δ: 162.8, 160.8, 156.5, 137.1, 135.6, 130.4, 129.6 (2×C), 129.4, 129.0 (2×C), 128.3 (2×C), 127.4, 125.5 (2×C), 119.8, 38.6, 35.4, 31.7 (2×C), 25.9 (2×C), 25.5. HRMS (ESI) m / z calculated value is C 24 H 27 ON4 + [M+H] + 387.2174, measured value is 387.2182.

[0080] Example 7: Synthesis of compound 2126:

[0081] The synthesis steps were the same as in Example 2, except that the aldehyde reagent used in the first step was 4-bromobenzaldehyde, yielding compound 2126 with a yield of 85%; major conformational isomers: 1H NMR (400MHz, CDCl3) δ: 7.52-7.29 (m, 13H), 7.25-7.21 (m, 1H), 6.89 (d, J = 8.8Hz, 1H), 6.05 (d, J = 8.8Hz, 1H), 3.15 (s, 3H). 13 C10 NMR (100MHz, CDCl3) δ: 161.6, 157.2, 153.3, 137.6, 135.0, 131.9 (2×C), 130.6 (2×C), 130.2, 129.7 (2×C), 129.6, 128.7 (2×C), 128.6 (2×C), 127.7, 126.1, 125.5 (2×C), 125.1, 121.5, 35.8. Minor conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.52-7.29 (m, 13H), 7.25-7.21 (m, 1H), 7.05 (d, J = 9.2Hz, 1H), 6.22 (d, J = 9.2Hz, 1H), 3.30 (s, 3H). 13 C NMR (100MHz, CDCl3) δ: 162.3, 157.1, 153.5, 137.6, 135.5, 132.0 (2×C), 130.6 (2×C), 130.2, 129.8 (2×C), 129.3, 129.0 (2×C), 128.4 (2×C), 127.5, 126.0, 125.5 (2×C), 125.3, 120.3, 38.7. HRMS (ESI) m / z calculated value is C 24 H 20 ON4Br + [M+H] + 459.0815, the measured value is 459.0807.

[0082] Example 8: Synthesis of compound 2127:

[0083] The synthesis steps were the same as in Example 2, except that formaldehyde was chosen as the aldehyde reagent in the first step reaction to give compound 2127, yield: 80%; major conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 8.56 (s, 1H), 7.67 (d, J = 8.0Hz, 2H), 7.57-7.49 (m, 2H), 7.46-7.40 (m, 1H), 7.36-7.31(m,4H),7.28-7.22(m,1H),6.81(d,J=8.8Hz,1H),6.07(d,J=8.8Hz,1H),3.15(s,3H). 13C10 NMR (100MHz, CDCl3) δ: 161.5, 158.4, 141.0, 136.7, 135.0, 130.2, 129.9 (2×C), 128.8, 128.7 (2×C), 128.6 (2×C), 127.8, 121.6, 120.3 (2×C), 35.9. Minor conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 8.61 (s, 1H), 7.72 (d, J = 8.0Hz, 2H), 7.57-7.49 (m, 2H), 7.46-7.40 (m, 1H), 7.36-7.31(m,4H),7.28-7.22(m,1H),7.05(d,J=9.2Hz,1H),6.23(d,J=9.2Hz,1H),3.26(s,3H). 13 C NMR (100MHz, CDCl3) δ: 161.2, 158.2, 141.2, 136.7, 135.0, 130.2, 130.0 (2×C), 129.0 (2×C), 128.9, 128.5 (2×C), 127.6, 121.6, 120.3 (2×C), 38.7. HRMS (ESI) m / z calculated value is C 18 H 17 N4O + [M+H] + 305.1397, measured value is 305.1401.

[0084] Example 9: Synthesis of compound 2128:

[0085] The synthesis steps were the same as in Example 2, except that the aldehyde reagent used in the first step was n-propionaldehyde, yielding compound 2128 with a yield of 89%; major conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.52-7.39(m,5H),7.32-7.27(m,4H),7.24-7.19(m,1H),6.89(d,J=8 .8Hz,1H),6.00(d,J=8.8Hz,1H),3.11(s,3H),2.82(q,J=7.6Hz,2H),1.31(t,J=7.6Hz,3H). 13 C10 NMR (100MHz, CDCl3) δ: 162.0, 157.6, 156.6, 137.0, 135.1, 130.4, 129.5 (2×C), 129.3, 128.8 (2×C), 128.5 (2×C), 127.6, 125.0 (2×C), 120.7, 35.8, 20.2, 12.1. Minor conformational isomers: 1H NMR(400MHz, CDCl3)δ:7.52-7.39(m,5H),7.32-7.27(m,4H),7.24-7.19(m,1H),7.03(d,J=9 .2Hz,1H),6.18(d,J=9.2Hz,1H),3.24(s,3H),2.84(q,J=7.6Hz,2H),1.29(t,J=7.6Hz,3H). 13 C NMR (100MHz, CDCl3) δ: 162.6, 157.8, 156.4, 136.9, 135.5, 130.4, 129.6 (2×C), 129.4, 129.0 (2×C), 128.3 (2×C), 127.4, 125.0 (2×C), 119.9, 38.6, 20.2, 12.1. HRMS (ESI) m / z calculated values ​​are C 20 H 21 ON4 + [M+H] + 333.1710, measured value is 333.1712.

[0086] Example 10 Synthesis of compound 2129:

[0087] The synthesis steps were the same as in Example 2, except that the aldehyde reagent used in the first step was 1-naphthaldehyde, yielding compound 2129 with a yield of 83%; major conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.93 (d, J = 7.2Hz, 1H), 7.87 (d, J = 8.0Hz, 1H), 7.83 (d, J = 8.0Hz, 1H), 7. 52-7.31(m,8H),7.26-7.15(m,6H),6.95(d,J=8.8Hz,1H),6.11(d,J=8.8Hz,1H),3.21(s,3H). 13 C10 NMR (100MHz, CDCl3) δ: 161.8, 157.3, 153.1, 137.3, 134.9, 133.6, 131.3, 131.0, 130.2, 129.2, 129.1 (2×C), 128.7 (2×C), 128.6 (2×C), 128.5, 128.4, 127.7, 127.3, 126.6, 125.2, 125.1, 124.9, 124.1 (2×C), 122.0, 35.7. Minor conformational isomers: 1H NMR (400MHz, CDCl3) δ: 7.93 (d, J = 7.2Hz, 1H), 7.87 (d, J = 8.0Hz, 1H), 7.83 (d, J = 8.0Hz, 1H), 7. 52-7.31(m,8H),7.26-7.15(m,6H),7.10(d,J=9.2Hz,1H),6.24(d,J=9.2Hz,1H),3.38(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.4, 157.2, 153.4, 137.3, 135.5, 133.6, 131.3, 131.1, 130.2, 129.3, 129.0 (2×C), 128.7 (2×C), 128.6 (2×C), 128.5, 128.3, 127.5, 127.4, 126.7, 125.2, 125.1, 125.0, 124.1 (2×C), 120.2, 38.7. HRMS (ESI) m / z calculated value is C 28 H 23 ON4 + [M+H] + 431.1866, measured value is 431.1850.

[0088] Example 11 Synthesis of compound 2130:

[0089] The synthesis steps were the same as in Example 2, except that the aldehyde reagent used in the first step was 4-methylbenzaldehyde, yielding compound 2130 with a yield of 80%; major conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.47-7.30(m,11H),7.26-7.21(m,1H),7.13(d,J=8.0H z,2H),6.94(d,J=8.8Hz,1H),6.05(d,J=8.8Hz,1H),3.15(s,3H),2.35(s,3H). 13 C10 NMR (100MHz, CDCl3) δ: 162.0, 157.1, 154.4, 140.7, 138.0, 135.1, 130.4, 129.4 (2×C), 129.3 (2×C), 129.2, 129.0 (2×C), 128.8 (2×C), 128.6 (2×C), 127.6, 125.5 (2×C), 124.4, 121.1, 35.8, 21.5. Minor conformational isomers: 1H NMR(400MHz, CDCl3)δ:7.47-7.30(m,11H),7.26-7.21(m,1H),7.13(d,J=8.0H z,2H),7.06(d,J=9.2Hz,1H),6.22(d,J=8.8Hz,1H),3.31(s,3H),2.36(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.6, 156.9, 154.6, 140.9, 138.0, 135.5, 130.5, 129.6 (2×C), 129.5 (2×C), 129.1, 129.0 (2×C), 128.8 (2×C), 128.4 (2×C), 127.4, 125.8 (2×C), 124.2, 120.1, 38.7, 21.5. HRMS (ESI) m / z calculated value is C 25 H 23 ON4 + [M+H] + 395.1866, the measured value is 395.1860.

[0090] Example 12 Synthesis of compound 2131:

[0091] The synthesis steps were the same as in Example 2. The aldehyde reagent used in the first step was 3,3-dimethylbutyraldehyde, yielding compound 2131 with a yield of 87%. Major conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.53-7.27(m,9H),7.25-7.20(m,1H),6.84(d,J=8.8Hz,1H),6.01(d,J=8.8Hz,1H),3.13(s,3H),2.72(s,2H),0.87(s,9H). 13 C10 NMR (100MHz, CDCl3) δ: 162.1, 156.6, 154.9, 137.4, 135.0, 130.4, 129.4 (2×C), 128.8 (2×C), 128.6 (2×C), 127.7, 126.3, 126.2 (2×C), 121.3, 39.0, 35.7, 32.7, 29.6 (3×C). Minor conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.53-7.27(m,9H),7.25-7.20(m,1H),7.04(d,J=9.2Hz,1H),6.19(d,J=9.2Hz,1H),3.25(s,3H),2.76(s,2H),0.91(s,9H). 13C NMR (100MHz, CDCl3) δ: 162.7, 156.5, 155.2, 136.9, 135.6, 130.7, 129.6 (2×C), 129.0 (2×C), 128.4 (2×C), 127.4, 126.3, 126.2 (2×C), 119.8, 38.7, 35.7, 32.7, 29.7 (3×C). HRMS (ESI) m / z calculated values ​​are C 23 H 27 ON4 + [M+H] + 375.2179, measured value is 375.2189.

[0092] Example 13 Synthesis of compound 2132:

[0093] The synthesis steps were the same as in Example 2, except that the aldehyde reagent used in the first step was n-butyraldehyde, yielding compound 2132 with a yield of 87%; major conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.53-7.37(m,5H),7.32-7.27(m,4H),7.23-7.19(m,1H),6.87(d,J=8.8Hz,1H) ,5.99(d,J=8.8Hz,1H),3.11(s,3H),2.75(t,J=7.6Hz,2H),1.81-1.71(m,2H),0.91(t,J=7.6Hz,3H). 13 C10 NMR (100MHz, CDCl3) δ: 162.0, 156.6, 156.5, 137.0, 135.1, 130.4, 129.5 (2×C), 129.3, 128.7 (2×C), 128.5 (2×C), 127.6, 125.2 (2×C), 120.8, 35.8, 28.4, 21.2, 13.8. Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.53-7.37(m,5H),7.32-7.27(m,4H),7.23-7.19(m,1H),7.03(d,J=9.2Hz,1H) ,6.18(d,J=8.8Hz,1H),3.23(s,3H),2.75(t,J=7.6Hz,2H),1.81-1.71(m,2H),0.91(t,J=7.6Hz,3H). 13C NMR (100MHz, CDCl3) δ: 162.6, 156.7, 156.5, 137.0, 135.5, 130.4, 129.6 (2×C), 129.3, 129.0 (2×C), 128.3 (2×C), 127.4, 125.2 (2×C), 119.9, 38.6, 28.4, 21.2, 13.8. HRMS (ESI) m / z calculated value is C 21 H 23 ON4 + [M+H] + 347.1866, the measured value is 347.1868.

[0094] Example 14 Synthesis of compound 2133:

[0095] The synthesis steps were the same as in Example 2, except that the aldehyde reagent used in the first step was trimethylbenzaldehyde, yielding compound 2133 with a yield of 78%; the major conformational isomers were: 1 H NMR(400MHz, CDCl3)δ:7.38-7.30(m,5H),7.28-7.25(m,3H),7.24-7.19(m,2H),6.87(d, J=8.8Hz,1H),6.86(s,2H),6.05(d,J=8.8Hz,1H),3.18(s,3H),2.30(s,3H),1.94(s,6H). 13 C10 NMR (100MHz, CDCl3) δ: 162.0, 157.4, 153.4, 140.3, 137.6 (2×C), 137.4, 134.9, 130.4, 129.2 (2×C), 128.8 (2×C), 128.7 (2×C), 128.6 (2×C), 128.3, 127.8, 125.1, 122.6 (2×C), 122.0, 35.7, 21.4, 19.9 (2×C). Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.38-7.30(m,5H),7.28-7.25(m,3H),7.24-7.19(m,2H),7.08(d, J=9.2Hz,1H),6.86(s,2H),6.22(d,J=9.2Hz,1H),3.30(s,3H),2.30(s,3H),1.98(s,6H). 13C NMR (100MHz, CDCl3) δ: 161.7, 157.2, 153.6, 140.5, 137.6 (2×C), 137.5, 135.6, 129.4, 129.3 (2×C), 129.1 (2×C), 128.5 (2×C), 128.4 (2×C), 128.3, 127.5, 125.0, 122.6 (2×C), 119.9, 38.8, 21.4, 19.9 (2×C). HRMS (ESI) m / z calculated values ​​are C 27 H 27 ON4 + [M+H] + 423.2179, the measured value is 423.2171.

[0096] Example 15 Synthesis of compound 2134:

[0097] The synthesis steps were the same as in Example 2, except that the aldehyde reagent used in the first step was 2-methylbenzaldehyde, yielding compound 2134 with a yield of 83%; major conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.36-7.28 (m, 8H), 7.26-7.16 (m, 6H), 6.90 (d, J = 8.8Hz, 1H), 6.06 (d, J = 8.8Hz, 1H), 3.17 (s, 3H), 2.09 (s, 3H). 13 C10 NMR (100MHz, CDCl3) δ: 161.8, 157.1, 153.9, 137.6, 137.4, 135.0, 130.8, 130.5, 130.4 (2×C), 130.3, 129.3 (2×C), 128.7 (2×C), 128.6 (2×C), 128.5, 127.7, 126.1, 123.8 (2×C), 121.7, 35.8, 19.9. Minor conformational isomers: 1 HNMR(400MHz, CDCl3)δ:7.36-7.28(m,8H),7.26-7.16(m,6H),7.07(d,J=9.2Hz,1H),6.22(d,J=9.2Hz,1H),3.32(s,3H),2.12(s,3H). 13C NMR (100MHz, CDCl3) δ: 162.4, 157.0, 154.2, 137.6, 137.4, 135.5, 130.9, 130.6, 130.4 (2×C), 130.3, 129.4, 129.3 (2×C), 129.0 (2×C), 128.4 (2×C), 127.5, 126.2, 123.8 (2×C), 120.1, 38.7, 19.9. HRMS (ESI) m / z calculated values ​​are C 25 H 23 ON4 + [M+H] + 395.1866, measured value is 395.1871.

[0098] Example 16 Synthesis of compound 2135:

[0099] The synthesis steps were the same as in Example 2, except that the aldehyde reagent used in the first step was 2-furancarbaldehyde, yielding compound 2135 with a yield of 84%; major conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.53-7.41(m,6H),7.34-7.28(m,4H),7.25-7.21(m,1H),6.90(d,J=8.8 Hz,1H),6.66(d,J=3.6Hz,1H),6.43(dd,J=3.6,1.6Hz,1H),6.04(d,J=8.8Hz,1H),3.14(s,3H). 13 C10 NMR (100MHz, CDCl3) δ: 161.6, 157.3, 146.4, 144.7, 141.9, 137.6, 135.0, 130.1, 129.9, 129.3 (2×C), 128.8 (2×C), 128.6 (2×C), 127.7, 126.0 (2×C), 121.5, 113.9, 111.8, 35.8. Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.53-7.41(m,6H),7.34-7.28(m,4H),7.25-7.21(m,1H),7.03(d,J=9.2 Hz,1H),6.66(d,J=3.6Hz,1H),6.43(dd,J=3.6,1.6Hz,1H),6.22(d,J=9.2Hz,1H),3.26(s,3H). 13C NMR (100MHz, CDCl3) δ: 162.2, 157.2, 146.7, 144.9, 141.8, 137.6, 135.5, 130.0, 129.5 (2×C), 129.2, 129.0 (2×C), 128.4 (2×C), 127.5, 126.1 (2×C), 120.2, 114.0, 111.8, 38.6. HRMS (ESI) m / z calculated value is C 22 H 19 O2N4 + [M+H] + 371.1503, measured value is 371.1496.

[0100] Example 17 Synthesis of compound 2136:

[0101] The synthesis steps were the same as in Example 2, except that the aldehyde reagent used in the first step was 2-naphthaldehyde, yielding compound 2136 with a yield of 82%; major conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 8.08 (s, 1H), 7.82 (d, J = 8.0Hz, 1H), 7.77 (d, J = 8.4Hz, 1H), 7.76 (d, J = 7.6Hz, 1 H),7.55-7.31(s,12H),7.27-7.22(m,1H),6.97(d,J=8.8Hz,1H),6.08(d,J=8.8Hz,1H),3.18(s,3H). 13 C10 NMR (100MHz, CDCl3) δ: 161.9, 157.3, 154.3, 137.9, 135.1, 133.8, 132.8, 130.3, 129.7, 129.5 (2×C), 129.4, 128.8 (2×C), 128.7 (2×C), 128.6, 128.3, 127.8, 127.7, 127.6, 126.9, 125.5 (2×C), 125.4, 124.5, 121.3, 35.8. Minor conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 8.14 (s, 1H), 7.82 (d, J = 8.0Hz, 1H), 7.77 (d, J = 8.4Hz, 1H), 7.76 (d, J = 7.6Hz, 1 H),7.55-7.31(s,12H),7.27-7.22(m,1H),7.09(d,J=9.2Hz,1H),6.24(d,J=9.2Hz,1H),3.35(s,3H). 13C NMR (100MHz, CDCl3) δ: 162.5, 157.1, 154.5, 137.9, 135.5, 133.8, 132.8, 130.3, 129.6, 129.5 (2×C), 129.4, 129.0 (2×C), 128.7 (2×C), 128.6, 128.4, 127.8, 127.7, 127.5, 126.9, 125.5 (2×C), 125.4, 124.4, 120.2, 38.7. HRMS (ESI) m / z calculated value is C 28 H 23 ON4 + [M+H] + 431.1866, the measured value is 431.1875.

[0102] Example 18 Synthesis of compound 2137:

[0103] The synthesis steps were the same as in Example 2, except that acetaldehyde was used as the aldehyde reagent in the first step reaction to obtain compound 2137, with a yield of 82%; the major conformational isomers were: 1 H NMR(400MHz, CDCl3)δ:7.52-7.43(m,5H),7.33-7.28(m,4H),7.24-7.20(m, 1H), 6.90 (d, J=8.8Hz, 1H), 6.02 (d, J=8.8Hz, 1H), 3.11 (s, 3H), 2.54 (s, 3H). 13 C10 NMR (100MHz, CDCl3) δ: 161.9, 156.6, 152.8, 137.1, 135.1, 130.4, 129.5 (2×C), 129.2, 128.8 (2×C), 128.5 (2×C), 127.6, 124.7 (2×C), 120.7, 35.8, 13.3. Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.52-7.43(m,5H),7.33-7.28(m,4H),7.24-7.20(m, 1H),7.03(d,J=9.2Hz,1H),6.19(d,J=9.2Hz,1H),3.25(s,3H),2.57(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.5, 156.5, 153.0, 137.1, 135.5, 130.4, 129.6 (2×C), 129.4, 129.0 (2×C), 128.4 (2×C), 127.4, 124.7 (2×C), 120.1, 38.6, 13.3. HRMS (ESI) m / z calculated values ​​are C19 H 19 ON4 + [M+H] + 319.1553, measured value is 319.1546.

[0104] Example 19 Synthesis of compound 2138:

[0105] The synthesis steps were the same as in Example 2, except that the aldehyde reagent used in the first step was 2-chlorobenzaldehyde, yielding compound 2138 with a yield of 86%; major conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.48-7.38 (m, 3H), 7.37-7.27 (m, 9H), 7.26-7.20 (m, 2H), 6.88 (d, J = 8.8Hz, 1H), 6.07 (d, J = 8.8Hz, 1H), 3.18 (s, 3H). 13 C10 NMR (100MHz, CDCl3) δ: 161.6, 157.3, 151.7, 137.4, 135.0, 133.9, 132.1, 132.0, 130.2 (2×C), 129.2 (2×C), 128.8, 128.7 (2×C), 128.6 (2×C), 127.9, 127.7, 127.2, 123.7 (2×C), 121.9, 35.8. Minor conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.48-7.38 (m, 3H), 7.37-7.27 (m, 9H), 7.26-7.20 (m, 2H), 7.06 (d, J = 9.2Hz, 1H), 6.23 (d, J = 9.2Hz, 1H), 3.31 (s, 3H). 13 C NMR (100MHz, CDCl3) δ: 162.2, 157.2, 152.0, 137.4, 135.5, 133.9, 132.1, 132.0, 130.3 (2×C), 129.4 (2×C), 129.3 (2×C), 129.0, 128.4 (2×C), 127.9, 127.7, 127.5, 123.8 (2×C), 120.2, 38.7. HRMS (ESI) m / z calculated value is C 24 H 20 ON4Cl + [M+H] + 415.1320, measured value is 415.1327.

[0106] Example 20: Synthesis of compound 2139:

[0107] The synthesis steps were the same as in Example 2, except that the aldehyde reagent used in the first step was 3-methylbenzaldehyde, yielding compound 2139 with a yield of 85%; major conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.47-7.31 (m, 10H), 7.25-7.14 (m, 4H), 6.93 (d, J = 8.8Hz, 1H), 6.05 (d, J = 8.8Hz, 1H), 3.15 (s, 3H), 2.30 (s, 3H). 13 C10 NMR (100MHz, CDCl3) δ: 161.9, 157.1, 154.4, 138.6, 137.9, 135.1, 131.2, 130.3, 129.9, 129.4 (2×C), 129.3, 128.8 (2×C), 128.6 (2×C), 128.4, 127.7, 127.1, 126.1, 125.5 (2×C), 121.2, 35.8, 21.4. Minor conformational isomers: 1 HNMR(400MHz, CDCl3)δ:7.47-7.31(m,10H),7.25-7.14(m,4H),7.06(d,J=9.2Hz,1H),6.22(d,J=9.2Hz,1H),3.31(s,3H),2.32(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.6, 157.0, 154.6, 138.7, 137.9, 135.6, 131.3, 130.3, 129.9, 129.5 (2×C), 129.3, 129.0 (2×C), 128.6 (2×C), 128.4, 127.5, 127.1, 126.1, 125.5 (2×C), 120.1, 38.7, 21.4. HRMS (ESI) m / z calculated values ​​are C 25 H 23 ON4 + [M+H] + 395.1866, measured value is 395.1874.

[0108] Example 21 Synthesis of compound 2140:

[0109] The synthesis steps were the same as in Example 2, except that the aldehyde reagent used in the first step was 4-chlorobenzaldehyde, yielding compound 2140 with a yield of 83%; major conformational isomers: 1H NMR (400MHz, CDCl3) δ: 7.47-7.29 (m, 13H), 7.25-7.21 (m, 1H), 6.90 (d, J = 8.8Hz, 1H), 6.05 (d, J = 8.8Hz, 1H), 3.15 (s, 3H). 13 C10 NMR (100MHz, CDCl3) δ: 161.7, 157.2, 153.2, 137.6, 136.7, 135.0, 130.4 (2×C), 130.2, 129.7, 129.6 (2×C), 129.5, 129.0 (2×C), 128.7 (2×C), 128.6 (2×C), 127.7, 125.5 (2×C), 121.5, 35.8. Minor conformational isomers: 1 H NMR (400MHz, CDCl3) δ: 7.47-7.29 (m, 13H), 7.25-7.21 (m, 1H), 7.05 (d, J = 9.2Hz, 1H), 6.22 (d, J = 9.2Hz, 1H), 3.30 (s, 3H). 13 C NMR (100MHz, CDCl3) δ: 162.3, 157.1, 153.5, 137.6, 136.9, 135.5, 130.4 (2×C), 130.2, 129.7, 129.6 (2×C), 129.5, 129.3 (2×C), 128.7 (2×C), 128.4 (2×C), 127.5, 125.7 (2×C), 120.3, 38.7. HRMS (ESI) m / z calculated value is C 24 H 20 ON4Cl + [M+H] + 415.1320, the actual measured value is 415.1313.

[0110] Example 22 Synthesis of compound 2141:

[0111] The synthesis steps were the same as in Example 2, except that the aldehyde reagent used in the first step was 3-chlorobenzaldehyde, yielding compound 2141 with a yield of 85%; major conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.57(s,1H),7.48-7.41(m,3H),7.39-7.28(m,7H),7 .26-7.21(m,3H),6.89(d,J=8.8Hz,1H),6.06(d,J=8.8Hz,1H),3.16(s,3H). 13C10 NMR (100MHz, CDCl3) δ: 161.6, 157.2, 152.8, 137.5, 135.0, 134.7, 130.5, 130.1, 129.9, 129.7, 129.6 (2×C), 129.3 (2×C), 128.7 (2×C), 128.6 (2×C), 127.7, 127.1, 125.5 (2×C), 121.6, 35.8. Minor conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.62(s,1H),7.48-7.41(m,3H),7.39-7.28(m,7H),7 .26-7.21(m,3H),7.05(d,J=9.2Hz,1H),6.22(d,J=9.2Hz,1H),3.30(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.2, 157.1, 153.1, 137.5, 135.5, 134.8, 130.7, 130.1, 129.9, 129.8, 129.6 (2×C), 129.0 (2×C), 128.9 (2×C), 128.4 (2×C), 127.5, 127.1, 125.5 (2×C), 120.3, 38.7. HRMS (ESI) m / z calculated value is C 24 H 20 ON4Cl + [M+H] + 415.1320, measured value is 415.1327.

[0112] Example 23 Synthesis of compound 2142:

[0113] The synthesis steps were the same as in Example 2, except that the aldehyde reagent used in the first step was 4-trifluoromethylbenzaldehyde, yielding compound 2142 with a yield of 85%; major conformational isomers: 1 H NMR(400MHz, CDCl3)δ:7.68-7.57(m,4H),7.52-7.43(m,3H),7.39-7.29(m,6H) ,7.26-7.22(m,1H),6.89(d,J=8.8Hz,1H),6.07(d,J=8.8Hz,1H),3.17(s,3H). 13C NMR (100MHz, CDCl3) δ: 161.5, 157.4, 152.8, 137.5, 135.0, 132.2 (q, J = 32.6Hz), 130.7, 130.1, 129.8, 129.7 (2×C), 1 29.5(2×C),128.7(2×C),128.6(2×C),127.8,125.7(q,J=3.7Hz,2×C),125.6(2×C),123.7(q,J=271Hz),121.8,35.8. 19 F NMR (376MHz, CDCl3) δ: -63.0 (3×F). Minor conformational isomer: 1 H NMR(400MHz, CDCl3)δ:7.68-7.57(m,4H),7.52-7.43(m,3H),7.39-7.29(m,6H) ,7.26-7.22(m,1H),7.05(d,J=9.2Hz,1H),6.24(d,J=9.2Hz,1H),3.31(s,3H). 13 C NMR (100MHz, CDCl3) δ: 162.1, 157.3, 153.1, 137.5, 135.5, 132.2 (q, J = 32.6Hz), 130.6, 130.0, 129.9 (2×C), 129.8, 1 29.3(2×C),129.0(2×C),128.4(2×C),127.6,125.7(q,J=3.7Hz,2×C),125.6(2×C),123.7(q,J=271Hz),120.5,38.7. 19 F NMR (376MHz, CDCl3) δ: -63.0 (3×F). HRMS (ESI) m / z calculated value is C 25 H 20 ON4F + [M+H] + 449.1584, the measured value is 449.1589.

[0114] Example 1: Screening of cytotoxic and anti-inflammatory activities of 1,2,4-triazole-cis-enamide compounds.

[0115] 1. Cell culture

[0116] Mouse monocyte-macrophage Raw 264.7 cells were cultured in DMEM high-glucose medium (Gibco, catalog number: 11995065) supplemented with 10% (v / v) FBS (Gibco, catalog number: 10270-106), 100 U / mL penicillin, and 100 μg / mL streptomycin (Gibco, catalog number: 15140122). The culture conditions were 37°C and 5% CO2. Cells were passaged when they reached 80% confluence.

[0117] 2. Cytotoxicity detection

[0118] Mouse monocyte-macrophage Raw 264.7 was selected, at a rate of 2 × 10⁻⁶. 4 The cells were seeded at a density of 100 μL / well in 96-well plates. When the cell coverage was about 80%, all synthetic compounds (10 μM) were added. Three replicates were made for each group, and a blank control group was set up. After culturing for 24 h, 10 μL of CCK8 solution was added to each well, and the cells were incubated for another 2 h in a 37°C, 5% CO2 incubator. The absorbance (OD) value was measured at 450 nm using a microplate reader, and the cell inhibition rate was calculated.

[0119] 3. Detection of the expression levels of inflammatory factors by compounds

[0120] Raw 264.7 cells were grown at a rate of 2 × 10⁻⁶. 5 Cells were seeded at a density of 1 mL / well in 12-well plates. When the cell coverage was approximately 80%, the positive control drug (dexamethasone, final concentration 1 μM) and the candidate compound (DMSO solvent control, candidate compound 10 μM) were added. After 1 hour, LPS was added at a final concentration of 100 ng / mL. LPS stimulation continued for 4 hours.

[0121] (1) RNA extraction and qPCR assay

[0122] After completely discarding the culture medium from the cultured cells, add 0.5 mL of RL from the RNA extraction kit to each well for lysis; collect the cell lysate and extract Total RNA according to the kit instructions (Vazyme, catalog number: RC112), and measure its concentration using an ultra-micro UV-Vis spectrophotometer.

[0123] (2) Reverse transcription reaction

[0124] A 20 μL reverse transcription system was established, including 4 μL of 5×Buffer, X μL of Total RNA (500 ng), and μL of DEPC-H2O (16-X). After being placed in 8-tube sets, the tubes were labeled and placed in a PCR instrument for reverse transcription at 50℃ for 15 min; 85℃ for 5 s; and 16℃ for ∝.

[0125] (3) Quantitative fluorescence

[0126] Add 80 μL DEPC-H2O to the transcribed cDNA to a final volume of 100 μL, centrifuge to mix, and store at -20℃. Take 18 μL of the mixture (SYBR 10 μL + DEPC-H2O 10 μL + primer 0.5 μL) multiplied by the number of samples and transfer it to a 96-well plate. Add 2 μL of cDNA to make a 20 μL mixture, centrifuge at 1200 rpm for 1 min to mix. Place the plate in a CFX Connect Real-Time System for detection, cycling 39 times (95℃, 2 min; 95℃, 20 s; 57℃, 20 s; 72℃, 20 s) followed by a cycle of 95℃, 1 min; 55℃, 30 s; 95℃, 30 s.

[0127] (4) Analysis

[0128] Use 2 -ΔΔCt The experimental results were analyzed using the following formulas: △Ct target gene = Ct target gene - Ct internal reference gene; △△Ct target gene = △Ct experimental group target gene - △Ct control group target gene. 2 -ΔΔCt This indicates the fold increase in the expression of the target gene in the experimental group compared to the control group.

[0129] The primers are shown in Table 1 below:

[0130] Table 1 Primer sequences.

[0131]

[0132] The results are as follows Figure 1 As shown, none of the synthesized 1,2,4-triazole-cis-enamide compounds, except for 2133, exhibited significant toxicity. Inflammation screening results showed that most compounds significantly inhibited the secretion levels of inflammatory factors IL-1β and IL-6, with compound 2125 exhibiting the strongest in vitro anti-inflammatory activity and suppressing inflammatory expression in a concentration-dependent manner.

[0133] Example 2: Anti-inflammatory effect of compound 2125 on OVA-induced rhinitis in mice.

[0134] I. Experimental Objective

[0135] The therapeutic effect of compound 2125 on an OVA-induced mouse rhinitis model was investigated.

[0136] II. Experimental Materials

[0137] Experimental animals: 30 male Balb / c mice, aged 6-8 weeks and weighing approximately 18-22g, purchased from Zhuhai Baishitong Biotechnology Co., Ltd.

[0138] Experimental instruments: heating stirrer, paraffin microtome, microscope, syringe, cell counter, balance, oven, refrigerated centrifuge, etc.

[0139] Experimental reagents: HE staining reagents, PBS solution, etc.

[0140] III. Animal Experiments

[0141] 1. Establishment of an OVA-induced rhinitis mouse model: ① Modeling drug: OVA (Sigma, catalog number: S7951); ② Modeling method: Balb / c mice were used to establish the rhinitis model. 50 μg of OVA was dissolved in 100 μL of 4% Al(OH)3 in 100 μL of physiological saline. 100 μL of the sensitizing solution was injected intraperitoneally every 2 days for 14 days. From days 15 to 22, 5% OVA was administered intranasally for 7 consecutive days. The control group mice were treated with an equal volume of physiological saline.

[0142] 2. Animal grouping: Control group, AR group, positive control group (dexamethasone, Dex), and treatment group (three doses of compounds 2125-L, 2125-M, and 2125-H: 50 mg / kg, 100 mg / kg, and 150 mg / kg), with 5 animals in each group.

[0143] 3. Animal administration: The animals were administered low (50 mg / kg), medium (100 mg / kg), and high (150 mg / kg) doses of compound 2125 by gavage. The positive control group was given dexamethasone. The control group and AR group were given an equal volume of physiological saline by gavage once a day for 7 consecutive days. Blood was collected from the eyeballs 24 hours later, and the animals were euthanized by intraperitoneal injection of an excessive amount of 3% sodium pentobarbital.

[0144] 4. Animal sampling: After the mice were euthanized, nasal irrigation fluid and nasal mucosa tissue were collected.

[0145] 5. Indicator observation: total number of inflammatory cells, histopathology of mouse nasal mucosa tissue, inflammatory expression, etc.

[0146] (1) Behavioral scoring: Mice were observed 30 minutes after the last injection of OVA sensitizing solution for modeling. Allergic symptoms were recorded, such as the number of sneezes, the number of times the mice scratched their noses, and the amount of nasal discharge. Mice with a score >5 were considered successfully modeled. Specific scoring criteria are shown in Table 2. Results are as follows: Figure 2 As shown, compared with the Control group, mice in the AR group exhibited rhinitis symptoms such as nose scratching, sneezing, and runny nose. After administration, the rhinitis symptoms in the mice were relieved.

[0147] Table 2 Behavioral Scoring Criteria

[0148]

[0149] (2) The total number of cells in the nasal lavage fluid of mice was measured using a cell counter. The results are as follows: Figure 3 As shown, compared with the control group, the total number of cells in the nasal lavage fluid of mice in the AR group was significantly increased; compared with the AR group, Dex and high-dose compound 2125 reduced the total number of cells in the nasal lavage fluid.

[0150] (3) Giemsa staining

[0151] ① Add 70% ethanol and fix for 10 min.

[0152] ② Add an appropriate amount of the prepared modified Giemsa staining working solution (1X) to the smear sample and stain for 45 min.

[0153] ③Wash thoroughly from one side with distilled water, and after drying, observe and photograph under a microscope.

[0154] The results are as follows Figure 4 As shown, compared with the Control group, the AR group mice had increased cell counts in the nasal lavage fluid, with an increase in eosinophils and mast cells. All treatment groups effectively reduced inflammatory cells in the nasal cavity.

[0155] (4) Detection of serum histamine levels by enzyme-linked immunosorbent assay (ELISA).

[0156] Serum collection: Collect whole blood from mice into 1.5 mL EP tubes, centrifuge at 4000 rpm for 10 min, then centrifuge at 10000 rpm for 10 min, and use the supernatant for ELISA detection of cytokines.

[0157] The secretion of histamine HIS in mouse serum was detected using an enzyme-linked immunosorbent assay (ELISA) kit (Jianglai, catalog number: Histamine HIS: JL45802). The procedure was performed according to the kit's instructions, as follows:

[0158] a. Reagent preparation

[0159] ① Dilute the washing buffer (20×) to 1× with double-distilled water to prepare the required washing buffer. ② Add the standard diluent to one bottle of standard according to the volume indicated on the standard label and incubate at room temperature for 15 min. ③ Take five clean 1.5 mL centrifuge tubes, pre-add 500 μL of standard diluent to each tube, and perform serial dilutions of the standard to obtain seven standard concentrations: 50, 25, 12.5, 6.25, 3.12, 1.56, and 0.78 pg / mL. Finally, add the diluted standard to the wells of the pre-coated plate sequentially, adding the standard diluent directly as the 0 pg / mL concentration, for a total of seven standard concentrations. ④ Add 200 μL to each well, and repeat the washing process after approximately 15-30 seconds. Wash the plate five times in total, and then pat it dry on paper.

[0160] b. Operating steps

[0161] ① Remove the required strips from the aluminum foil bag after equilibration at room temperature for 10 minutes, and seal the remaining strips in a self-sealing bag and return them to 4℃.

[0162] ② Sample addition: Add 50 μL of sample or standard of different concentrations to each well. Add 50 μL of universal diluent to each blank well, followed by 50 μL of Biotin-antibody working solution to each well. Cover with sealing film and incubate at 37°C for 1 hour.

[0163] ③ Wash the plate 3 times: Discard the liquid, add 300μL of washing solution to each well, let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 3 times.

[0164] ④ Add enzyme conjugate working solution: Add 100 μL of enzyme conjugate working solution to each well, cover with sealing film and incubate at 37°C for 30 min.

[0165] ⑤ Wash the board 5 times, and pat it dry on thick absorbent paper on the last wash.

[0166] ⑥ Add substrate: Add 90 μL of substrate (TMB) to each well, cover with sealing film, and incubate at 37°C in the dark for 15 min.

[0167] ⑦ Add stop solution: Add 50 μL of stop solution to each well and immediately measure the OD value of each well at a wavelength of 450 nm.

[0168] The results are as follows Figure 5 As shown, compared with the AR group, compound 2125 significantly downregulated HIS levels at doses of 50 mg / kg, 100 mg / kg, and 150 mg / kg.

[0169] (5) Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of ovalbumin-specific immunoglobulin E and histamine in serum.

[0170] Serum collection: Collect whole blood from mice into 1.5 mL EP tubes, centrifuge at 4000 rpm for 10 min, then centrifuge at 10000 rpm for 10 min, and use the supernatant for ELISA detection of cytokines.

[0171] The secretion of mouse IgE in serum was detected using an enzyme-linked immunosorbent assay (ELISA) kit (Jianglai, catalog number: Mouse ovalbumin-specific IgE (OVA-sIgE): JL23405). The procedure was performed according to the kit's instructions, as follows:

[0172] a. Reagent preparation

[0173] ① Preparation of positive and negative control working solutions: Add 1 mL of universal diluent to each lyophilized control, let stand for 15 min until completely dissolved, then gently mix. ② Preparation of HRP-secondary antibody working solution: 15 min before use, centrifuge the concentrated HRP-antibody at 1000×g for 1 min, and dilute the 100× concentrated HRP-antigen to a 1× working concentration with universal diluent. ③ Preparation of 1× wash buffer: Add 10 mL of 20× wash buffer to 190 mL of distilled water. ④ Washing: Add 200 μL to each well, wash for approximately 15-30 seconds, and repeat the washing process five times in total. Pat the plate dry on paper.

[0174] b. Operating steps

[0175] ① Remove the required strips from the aluminum foil bag after equilibration at room temperature for 10 minutes, and seal the remaining strips in a self-sealing bag and return them to 4℃.

[0176] ② Sample addition: Add 100 μL of sample or positive / negative control to each well, and add 10 μL of universal diluent to the blank well. Cover with sealing film and incubate at 37°C for 1 hour.

[0177] ③ Wash the plate 3 times: Discard the liquid, add 300μL of washing solution to each well, let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 3 times.

[0178] ④ Add secondary antibody: Add 100 μL of secondary antibody working solution to each well, cover with sealing film, and incubate at 37°C for 30 min.

[0179] ⑤ Wash the board 5 times, and pat it dry on thick absorbent paper on the last wash.

[0180] ⑥ Add substrate: Add 90 μL of substrate (TMB) to each well, cover with sealing film, and incubate at 37°C in the dark for 15 min.

[0181] ⑦ Add stop solution: Add 50 μL of stop solution to each well and immediately measure the OD value of each well at a wavelength of 450 nm.

[0182] The results are shown in Table 3. Compared with the AR group, compound 2125 significantly reduced IgE positive expression at a dose of 150 mg / kg.

[0183] Table 3

[0184]

[0185] (6) Tissue fixation, dehydration and embedding

[0186] ① Fix the mouse tissues in 10% neutral formaldehyde solution for 24 hours.

[0187] ② Place the trimmed tissue block in an embedding cassette and wash with running water for 24 hours to completely remove residual formaldehyde.

[0188] ③ Perform alcohol gradient dehydration on the tissue, with the following steps: 70% alcohol for 12 hours, 80% alcohol for 1.5 hours, 95% alcohol I for 45 minutes, 95% alcohol II for 30 minutes, 100% alcohol I for 25 minutes, and 100% alcohol II for 20 minutes.

[0189] ④ After dehydration, immerse the tissue in an alcohol / xylene (1:1, v / v) solution for 20 min.

[0190] ⑤ Soak the tissue in xylene I for 20 minutes, then soak it in xylene II for 10 minutes.

[0191] ⑥ Place the tissue into paraffin I and paraffin II, which have been preheated and melted in an oven at 60-65℃, for 1 hour each.

[0192] ⑦ Pour a small amount of embedding paraffin into the preheated metal embedding frame, place the skin tissue block inside, perpendicular to the embedding frame, with the bottom flat, pour in paraffin again, embed the tissue, and let it cool.

[0193] ⑧ Paraffin Sectioning: Fix the tissue paraffin block onto a Leica microtome, sectioning to a thickness of 5 μm in a continuous manner. Use toothless forceps to immerse the sections in 40°C water for spreading, retrieve them with a glass slide, bake at 60°C for 2 hours, and then store in a slide box at room temperature for later use.

[0194] (7) HE staining

[0195] ①Dewaxing and hydration: Place paraffin sections of skin tissue into xylene I and xylene II for 15 min each, then into 100% ethanol I and ethanol II for 3 min each, 95% ethanol I and ethanol II for 3 min each, 80% ethanol for 3 min, and double-distilled water for 1 min.

[0196] ② Stain with hematoxylin for 15 minutes, then wash the slide with water to remove excess staining solution;

[0197] ③ Use 1% hydrochloric acid and ethanol (99 mL 70% ethanol + 1 mL concentrated hydrochloric acid) to separate colors for 3 seconds. Under a microscope, the cell nucleus and chromatin should be clearly visible.

[0198] ④ Rinse with running water for 15 minutes to restore blue color, then rinse with distilled water for 1 minute;

[0199] ⑤ Eosin for 2 minutes, then rinse with running water for 1 minute;

[0200] ⑥ Dehydrated with 80% and 100% ethanol for 2 seconds and 7 minutes respectively;

[0201] ⑦ Xylene I and II, 5 min each;

[0202] ⑧ Mounting: Remove the slide from xylene II, drop neutral resin onto the tissue, gently cover with a coverslip, and allow to air dry naturally;

[0203] ⑨ Observation: Observe pathological changes under a microscope, take pictures, and analyze.

[0204] like Figure 6 As shown, in the Control group mice, the nasal mucosa showed no epithelial thickening, no vascular dilation or congestion, and the nasal mucosa structure was clear, with no inflammatory cell infiltration. In the AR group mice, significant proliferation of nasal mucosal epithelial cells, obvious small vascular dilation and interstitial edema were observed, along with inflammatory cell infiltration dominated by eosinophils; inflammation of the nasal mucosa tissue was reduced to varying degrees in all drug-treated groups.

[0205] (8) PAS staining

[0206] ① Paraffin sections are routinely dewaxed and dissolved in distilled water.

[0207] ② Follow the Beyotime PAS kit instructions for periodic acid oxidation: Remove the periodic acid solution and equilibrate to room temperature. Add 100 μL of periodic acid solution to each sample and react in a humidified chamber in the dark for 10 min. Remove the periodic acid solution, soak in distilled water, and wash on a shaker for 5 min.

[0208] ③Schiff reagent staining: Add 100 μL of Schiff reagent to each sample, place in a humidified chamber, and stain in a 37℃ oven in the dark for 30 min to 1 h. Remove the staining solution, soak in distilled water, and wash on a shaker for 5 min. Note: The optimal staining time may vary for different tissues; please adjust the staining time according to the actual staining effect.

[0209] ④ Hematoxylin staining: Add 100 μL of hematoxylin staining solution to each sample and stain for 30 seconds. Remove the staining solution and rinse with distilled water at least twice, 3 seconds each time, until the excess stain is removed. For smears, after proper drying, mount with mounting solution and observe under a microscope. Cells can be washed with distilled water to remove excess stain before photographing under a microscope.

[0210] ⑤ Rapid dehydration with 95% ethanol for 2-3 seconds, followed by dehydration twice with anhydrous ethanol, each time for 5 seconds.

[0211] ⑥ Apply xylene twice, 2 minutes each time, and then seal with neutral resin.

[0212] Microscopic observation shows (see) Figure 7 In the Control group mice, the nasal mucosa showed no epithelial thickening, no vascular dilation or congestion, and the nasal mucosa structure was clear, with no goblet cells observed. In the AR group mice, significant proliferation of nasal mucosal epithelial cells and increased goblet cell expression were observed; inflammation of the nasal mucosa tissue was reduced to varying degrees in all drug-treated groups.

[0213] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A 1,2,4-triazole-cis-enamide compound and its pharmaceutically acceptable salt, characterized in that, The structural formula is shown in equation (I): （I）； The compound shown in formula (I) is selected from any one of the following compounds: 。 2. The 1,2,4-triazole-cis-enamide compound and its pharmaceutical salt according to claim 1, characterized in that, The medicinal salt is a pharmaceutically acceptable salt, including inorganic acid salts or organic acid salts.

3. The method for preparing the 1,2,4-triazole-cis-enamide compound and its pharmaceutical salt as described in any one of claims 1-2, characterized in that, Includes the following steps: a) Dissolve compounds of formula I and formula II in acetonitrile solution at a stoichiometric ratio of 1:1, and react for 1-3 h to obtain compound III: ； b) Dissolve the product of formula III obtained in step a) in dry dichloromethane, add it dropwise to a dry dichloromethane solution of NBS-dimethyl sulfide cooled to -78°C, react at -78°C for 0.5-2 h, then raise the temperature to 20-25°C and react for 2-5 h to obtain the product of formula IV. ； c) Compounds of formula V and formula VI are stoichiometrically 1: 1.2 Dissolve in water, add sodium carbonate, and react for 2-5 h to obtain compound VII; ； d) Dissolve the product compound IV obtained in step b) and the product compound VII obtained in step c) in dry toluene at a stoichiometric ratio of 1:1.2, add triethylamine, and react at 110℃-130℃ for 1-3 h to obtain compound VIII. ； e) Dissolve the product of formula VIII obtained in step d) in ammonia-methanol solution and react for 2-8 days to obtain compound of formula IX; ； f) React the product of formula IX obtained in step e) with formula X at a stoichiometric ratio of 1.2:1 at 40℃-80℃ for 0.5-3 h to obtain formula XI; ； g) The product compound XI obtained in step f) is reacted with sodium hydride at -20℃ to 5℃ for 0.5-1 h, then iodomethane is added at this temperature, and finally the temperature is raised to 20℃ to 50℃ and stirred for 2-6 h to obtain compound XII, which is the compound with structure (I). ； Wherein, R is a substituent as defined in claim 1.

4. The use of the 1,2,4-triazole-cis-enamide compounds and their pharmaceutical salts as described in any one of claims 1-2 in the preparation of anti-inflammatory drugs.

5. The use of the 1,2,4-triazole-cis-enamide compounds and their pharmaceutical salts as described in any one of claims 1-2 in the preparation of drugs for treating allergic rhinitis.

Images