Substituted triazoles and methods for their preparation and use

Abstract

The application provides a substituted triazole compound and a preparation method and application thereof. The compound has a structure shown in formula I. The application also relates to a preparation method of a compound containing the structure of formula I, a pharmaceutical composition and application of the compound in preparation of an anti-SARS-CoV-2 M pro protease inhibitor. The application provides a substituted triazole compound and a preparation method and application thereof. The compound has a structure shown in formula I. The application also relates to a preparation method of a compound containing the structure of formula I, a pharmaceutical composition and application of the compound in preparation of an anti-SARS-CoV-2 M pro protease inhibitor.

Description

Technical Field

[0001] This invention relates to a derivative and its preparation method, specifically to a substituted triazole compound, a composition containing the compound, a preparation method, and its application in the field of anti-coronavirus drugs, belonging to the field of organic synthesis and pharmaceutical application technology. Background Technology

[0002] SARS-CoV-2 remains a major public health problem threatening human life and health. Considering different target populations and drug interactions, researchers still need to develop highly effective, low-toxicity, broad-spectrum, drug-resistant, cost-effective, and easy-to-use anti-COVID-19 drugs.

[0003] SARS-CoV-2 main protease (M) pro ) is a homodimeric cysteine ​​protease composed of two mutually perpendicular monomers, with its catalytic center consisting of Cys145 and His41. The main protease plays a crucial role in the SARS-CoV-2 replication cycle, specifically recognizing and cleaving 11 cleavage sites of the polyproteins (pp1a and pp1ab) Nsp4–Nsp16. pro Coronaviruses are highly conserved among species and have low homology with human proteases. Therefore, inhibitors targeting this target have the advantages of good selectivity and low toxicity, making them an important target for the development of anti-coronavirus drugs.

[0004] Currently reported SARS-CoV-2M pro Most inhibitors target active sites, and are predominantly peptide-like covalent inhibitors. For example, the three currently marketed M... pro Inhibitor: Paxlovid TM Nematevir and ritonavir, as well as Cenostatin (Cenostatin / ritonavir) and Leretevir. Paxlovid and Cenostatin are conditionally approved for marketing in China; however, they require combination therapy with the P450 enzyme inhibitor ritonavir, limiting the applicable population. Furthermore, these peptide-mimicking inhibitors have similar structures, and SARS-CoV-2 is gradually developing resistance mutations to nematevir, thus there is an urgent need to develop non-covalent main protease inhibitors with novel structural types. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a substituted triazole compound for inhibiting the main protease, a composition comprising the compound, and a method for preparing the same. This invention also provides the above compound as a SARS-CoV-2 malignant protein. pro Inhibitor activity screening results and their applications.

[0006] The technical solution of the present invention is as follows:

[0007] 1. Substituted triazole compounds

[0008] Substituted triazole compounds, or pharmaceutically acceptable salts thereof, have the structure shown in general formula I:

[0009]

[0010] in,

[0011] Y represents phenyl, sulfinylphenyl, sulfonylphenyl, thiophene, acetamide, methylacetamide, phthalimide, or methyl acetate.

[0012] R1 represents: independent halogen, nitro, and methanesulfonyl groups;

[0013] R2 represents: independent halogens;

[0014] X1 is either -CH2-CH2- or does not exist;

[0015] R3 represents either an independent H or a methyl group located at the 5-position of the pyridine carboxyl group;

[0016] n is an integer selected from 1 or 2.

[0017] Pharmaceutically acceptable salt types include inorganic acid salts such as commonly used hydrochloride, sulfate, or phosphate, as well as organic acid salts such as methanesulfonate, p-toluenesulfonate, fumarate, citrate, or acetate.

[0018] According to a preferred embodiment of the present invention, Y is any one of the following substituents I(a) or I(b) or I(c):

[0019]

[0020] R4, R5, and R6 are each independently represented by a nitro group, a halogen group, a methanesulfonyl group, and an H group, respectively, each time they appear.

[0021]

[0022] According to a further preferred embodiment of the present invention, the substituted triazole compound is one of the following compounds:

[0023]

[0024]

[0025] The term "pharmaceutically acceptable salt" as used in this invention refers to a salt of a compound that, within the scope of reliable pharmaceutical evaluation, is suitable for contact with tissues of humans or lower animals without undue toxicity, irritation, or allergic reactions, possesses a reasonably reasonable benefit-risk ratio, is typically water- or oil-soluble or dispersible, and is effectively used for its intended purpose. This includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts, which are suitable for the intended use and chemically compatible with compounds of formulas I and II. For a list of suitable salts, see SM Birge et al., J. Pharm. Sci., 1977, 66, pp. 1-19. Further preferred salt types are inorganic acid salts such as hydrochlorides, sulfates, or phosphates, and organic acid salts such as methanesulfonates, p-toluenesulfonates, fumarates, citrates, or acetates.

[0026] 2. Preparation method of substituted triazole compounds

[0027] The preparation method of substituted triazole compounds comprises the following steps: Starting with 3-fluorobenzaldehyde 1 and magnesium acetylene bromide 2, intermediate 3 is obtained through Grignard reaction; then, intermediate 4 is obtained through substitution reaction with sulfoxide dichloroethylene; intermediates 7a and 7b are obtained by amide condensation of tert-butyl 3,8-diazabicyclo[3.2.1]octane-8-carboxylate 5 and nicotinic acid 6a or 5-methylnicotinic acid 6b, and then the tert-butoxy carbonyl group is removed to obtain intermediate 8a or 8b; intermediate 4 is obtained by substitution reaction with 8a or 8b to obtain alkynyl fragment intermediate C6 or intermediate C5; intermediate C6 is dissolved with an azide derivative in a mixed solvent of tetrahydrofuran / water = 1:1, v / v, with CuSO4·5H2O and sodium ascorbate as catalysts, and reacted at 55°C to obtain target compound C6NX; intermediate C5 is subjected to a click chemistry reaction catalyzed by monovalent copper ions in the same manner to obtain target compound C5NX.

[0028] Starting with 1-Boc-piperazine 11 and nicotinic acid 6a, and using dichloromethane as the reaction solvent and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate as the condensing agent, intermediate 12 was obtained through an amide condensation reaction. Then, intermediate 13 was obtained by removing the tert-butyloxycarbonyl group. Intermediate 13 and intermediate 4 underwent a substitution reaction to obtain the alkyne fragment intermediate C4. Intermediate C4 was then reacted with the azide fragment in CuSO4·5H2O, with sodium ascorbate as the catalyst, in a mixed solvent of tetrahydrofuran / water = 1:1, v / v, at 55°C to obtain the target compound C4NX.

[0029] The synthesis route is as follows:

[0030]

[0031] Reagents and conditions: (i) ultra-dry tetrahydrofuran, ice-salt bath, N2; (ii) sulfoxide dichloromethane, ice bath; (iii) EDCI, HOBt, NMM, dichloromethane, room temperature; (iv) 4 mol / L dioxane hydrochloride solution, dichloromethane, room temperature; (v) CH3COOK, methanol, 70℃; (vi) azide compound, CuSO4·5H2O, sodium ascorbate, 55℃, tetrahydrofuran / water = 1:1, v / v; (vii) HATU, N,N,-diisopropylethylamine, dichloromethane, room temperature.

[0032] Wherein, R3 has the structure described in general formula I above; R3 is: an independent H or methyl group located at the 5-position of the pyridine carboxyl group. The structure described in R7 includes 3-nitrobenzyl, 4-nitrobenzyl, phenethyl, thiopheneethyl, phthalimide ethyl, methyl butyrate, methylbenzenesulfinyl, methylbenzenesulfonyl, methanesulfonylbenzyl, N-methylacetamide, and 3-fluorobenzyl. The room temperature described in this invention is 20-30°C.

[0033] According to a preferred method for preparing substituted triazole compounds of the present invention, the specific steps are as follows:

[0034] (1) 3-fluorobenzaldehyde 1 was added to ultra-dry tetrahydrofuran, and magnesium acetylene bromide 2 was added dropwise under N2 protection. The reaction was detected by TLC. Ethyl acetate / petroleum ether = 1:8, v / v. After the reaction was complete, saturated ammonium chloride was added to the system to quench the reaction. The organic phase was extracted with ethyl acetate (3×20mL). The obtained organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain intermediate 3, which was a yellow oily substance.

[0035] (2) Intermediate 3 was dissolved in dichloromethane, and a sulfoxide solution diluted with dichloromethane was added dropwise under ice bath conditions. The reaction was carried out at room temperature under the catalysis of a small amount of DMF, and the reaction was detected by TLC (ethyl acetate / petroleum ether = 1:5, v / v). The reaction was quenched by adding saturated sodium bicarbonate. The organic phase was separated and dried successively with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude intermediate 4, which was a yellow-brown oily substance.

[0036] (3) Under ice bath conditions, raw material 6a and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) were added to dichloromethane. After activation for 15 min, 1-hydroxybenzotriazole (HOBt), N-methylmorpholine (NMM) and raw material 3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (5) were added. The mixture was then reacted at room temperature for 20 hours until the solution turned dark yellow. The reaction was monitored by TLC (methanol / dichloromethane = 1:30, v / v). After the reaction was complete, the reaction solution was washed with water (3 × 20 mL). The organic phase was then washed with saturated sodium chloride solution (3 × 10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain intermediate 7a. Intermediate 7b was obtained by the same operation as 6b.

[0037] (4) Intermediate 7a was dissolved in dichloromethane, and 4 mol / L dioxane hydrochloride solution was added dropwise. After the addition was complete, the mixture was allowed to react at room temperature. After about 6 hours, the reaction solution was concentrated under reduced pressure to obtain a white powder. Saturated sodium bicarbonate was added to adjust the pH, and the aqueous phase was concentrated under reduced pressure to obtain a white solid. The obtained solid was added to tetrahydrofuran and stirred. After filtration, the mother liquor was separated and concentrated under reduced pressure to obtain intermediate 8a. Intermediate 8b was obtained by the same experimental procedure.

[0038] (5) Dissolve intermediate 8b in methanol, add anhydrous potassium acetate and stir for 15 min, then add intermediate 4, heat to reflux at 70 °C, and monitor the reaction by TLC (methanol / dichloromethane = 1:10, v / v); after the reaction is complete, concentrate the reaction solution under reduced pressure, add 20 mL of water, extract with dichloromethane (3 × 10 mL), wash the obtained organic phase with saturated sodium chloride solution (3 × 10 mL), dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure, and purify the crude product by silica gel column chromatography to obtain intermediate C5; 8a was subjected to the same experimental operation to obtain intermediate C6;

[0039] (6) Under ice-water bath conditions, nicotinic acid 6a and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) were dissolved in dichloromethane and activated for 15 min. Then, N,N-diisopropylethylamine and 1-Boc-piperazine (11) were added and the mixture was allowed to react at room temperature for 16 hours. The solution turned light yellow. The reaction was monitored by TLC (methanol / dichloromethane = 1:30, v / v). After the reaction was complete, the reaction solution was washed with water (3 × 20 mL). The organic phase was taken and washed with saturated sodium chloride solution (3 × 10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain intermediate 12.

[0040] (7) Dissolve intermediate 12 in dichloromethane, and add dioxane hydrochloride solution dropwise; after the addition is complete, the reaction is brought to room temperature; after about 6 hours, the reaction solution is concentrated under reduced pressure to obtain a white powder; add saturated sodium bicarbonate to adjust the pH, and concentrate the aqueous phase under reduced pressure to obtain a white solid. Add tetrahydrofuran to the obtained solid and stir, filter, separate the mother liquor and concentrate under reduced pressure to obtain intermediate 13;

[0041] (8) Dissolve intermediate 13 in methanol, add anhydrous potassium acetate and stir for 15 min, then add intermediate 4, heat to reflux at 70 °C and monitor by TLC (methanol / dichloromethane = 1:10, v / v); after the reaction is complete, concentrate the reaction solution under reduced pressure, add 20 mL of water to extract dichloromethane (3 × 10 mL), wash the obtained organic phase with saturated sodium chloride solution (3 × 10 mL), dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure, and the crude product is purified by silica gel column chromatography to obtain intermediate C4;

[0042] (9) Intermediate C6, C5, or C4 and the azide derivative were added to a flask, dissolved in 10 mL of (tetrahydrofuran / water = 1:1, v / v), and then CuSO4·5H2O and sodium ascorbate were added. The reaction was carried out at 55 °C for 24 h. The reaction was monitored by TLC (methanol / dichloromethane = 1:10, v / v). The reaction solution was concentrated under reduced pressure and extracted with ethyl acetate (3 × 10 mL). The organic phase was taken and washed with saturated sodium chloride solution (3 × 10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain the target compounds C6NX, C5NX, and C4NX series of final products. 3. Bioactivity and application of substituted triazole compounds

[0043] This invention discloses the activity screening results of substituted triazole compounds and their first application as inhibitors of the SARS-CoV-2 main protease. Experiments demonstrate that the substituted triazole compounds of this invention can be used as main protease inhibitors in the preparation of anti-coronavirus drugs. This invention also provides the application of the above compounds in the preparation of anti-coronavirus drugs.

[0044] Experiments on the inhibition of SARS-CoV-2 main protease activity by the target compound

[0045] The inhibitory activity of a class of substituted triazole compounds synthesized according to the above method on the SARS-CoV-2 main protease was tested, and their activity data are listed in Table 2. Piperazine non-covalent main protease inhibitor GC-14 (JMedChem.2022,65:13343.) and the marketed drug nematvir were used as positive controls.

[0046] The newly synthesized substituted triazole compounds of this invention mostly exhibit significant main protease inhibitory activity, such as compounds C5N17, C5N21, C5N57, and C5N58. Among them, C5N21 shows particularly outstanding activity (IC50). 50 =0.156±0.03μM), compared to GC-14 (IC). 50 =0.75±0.03μM) increased nearly 5 times, compared with the marketed drug nematvir (IC50). 50 =0.091±0.001μM). Screening results for antiviral activity in Calu-3 cells showed that most compounds exhibited significant anti-SARS-CoV-2 activity, such as C5N16 and C5N17, which showed outstanding inhibitory activity against the main protease, with C5N17 exhibiting the best activity (EC). 50 =0.16±0.02μM, compared to GC-14(EC) 50 =1.5±0.06μM) increased by 9-fold. After chiral resolution of the racemic C5N17, the enantiomer C5N17B (EC) was obtained. 50 The activity of the drug (0.08 ± 0.006 μM) at the Calu-3 cell level was increased by 100%, far exceeding that of the marketed drug nematvir (EC). 50 =1.0±0.04μM), far exceeding the levels of the marketed drug nematvir (EC). 50 =1.0±0.04μM). Therefore, substituted triazole compounds can be further developed as lead compounds against SARS-CoV-2.

[0047] One class of substituted triazole compounds of the present invention can be used as SARS-CoV-2 main protease inhibitors, specifically, as SARS-CoV-2 inhibitors for the preparation of anti-COVID-19 drugs.

[0048] An anti-COVID-19 pharmaceutical composition comprising the substituted triazole compound of the present invention and one or more pharmaceutically acceptable carriers or excipients.

[0049] This invention provides novel substituted triazole compounds and their preparation methods. It also provides screening results of the compounds' activity against the SARS-CoV-2 main protease and their first application in the antiviral field. The substituted triazole compounds of this invention can be used as inhibitors of the SARS-CoV-2 main protease and have high application value. Specifically, this invention, through structural optimization, discovered a main protease inhibitor with higher activity and a novel structure, which can be used as a SARS-CoV-2 main protease inhibitor in the preparation of anti-COVID-19 drugs. Attached Figure Description

[0050] Figure 1 It is M proThree-dimensional structure and its active site diagram;

[0051] Figure 2 It is SARS-CoV-2M pro A schematic diagram illustrating the principle of drug screening targeting specific targets;

[0052] Figure 3 This represents the inhibition rate of the compound against the main protease at a concentration of 10 μM. Detailed Implementation

[0053] The following examples help to understand the present invention, but do not limit the scope of the invention. All percentages are mass percentages.

[0054] Example 1: Preparation of C5N5~C5N64

[0055]

[0056] (1) 3-fluorobenzaldehyde (1, 5.0 g, 40 mmol, 1.0 eq.) was added to ultra-dry tetrahydrofuran, followed by vacuum protection with N2. Acetylene magnesium bromide (2, 3.5 g, 41 mmol, 1.0 eq.) was added dropwise, and the reaction was monitored by TLC (ethyl acetate / petroleum ether = 1:8, v / v). After the reaction was complete, saturated ammonium chloride was added to quench the reaction. The mixture was extracted with ethyl acetate (3 × 20 mL), and the resulting organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain intermediate 3, a pale yellow oil, 5.2 g, with a yield of 85.9%. ESI-MS: m / z 133.1 [M+H] + .C9H8O(132.1).

[0057] (2) Intermediate 3 (2.0 g, 13 mmol, 1.0 eq.) was dissolved in dichloromethane. A solution of sulfoxide diluted with dichloromethane (2.38 g, 20 mmol, 1.5 eq.) was added dropwise under ice bath conditions. The reaction was carried out at room temperature under micro-catalysis of DMF (ethyl acetate / petroleum ether = 1:5, v / v). The reaction was quenched by adding saturated sodium bicarbonate. The organic phase was separated and dried successively over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude intermediate 4, 1.7 g of a yellowish-brown oily substance, with a yield of 76%. ESI-MS: m / z 169.01 [M+H] + .C9H6ClF(168.01).

[0058] (3) Under ice bath conditions, raw material 6a (2.13 g, 15.5 mmol, 1.1 eq.) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 2.0 g, 10.4 mmol, 1.5 eq.) were added to dichloromethane. After activation for 15 min, HOBt (0.95 g, 7.0 mmol, 0.5 eq.) and NMM (4.3 g, 42.5 mmol, 3.0 eq.) and raw material 5 (3.0 g, 14 mmol, 1.0 eq.) were added. The mixture was then transferred to room temperature and reacted for 20 hours. The solution turned dark yellow. The reaction was monitored by TLC (methanol / dichloromethane = 1:15, v / v). After the reaction was complete, the reaction solution was washed with water (3 × 20 mL). The organic phase was then washed with saturated sodium chloride solution (3 × 10 mL). The organic phase was successively dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain intermediate 7a, a yellow solid (3.6 g), with a yield of 76.9%. ESI-MS: m / z 318.2 [M+H] + .C 17 H 23 Following the same experimental procedure, N3O3(317.2).6b yielded intermediate 7b, a yellow solid weighing 3.8 g, with a yield of 78.9%. ESI-MS: m / z 332.2 [M+H] + .C 18 H 25 N3O3(331.2).

[0059] (4) Intermediate 7a (2.0 g, 6 mmol, 1.0 eq.) was dissolved in dichloromethane under ice-water bath conditions. 4 mol / L dioxane hydrochloride solution (10.5 mL, 42 mmol, 6–7 eq.) was added dropwise. After the addition was complete, the reaction mixture was allowed to return to room temperature. After approximately 6 hours, the reaction solution was concentrated under reduced pressure to obtain a white powder. Saturated sodium bicarbonate was added to adjust the pH. The resulting aqueous phase was concentrated under reduced pressure to obtain a white solid. Tetrahydrofuran was added and stirred. The solid was filtered, washed, concentrated under reduced pressure, and dried to obtain intermediate 8a, which was 1.2 g of a yellow viscous solid, with a yield of 89%. ESI-MS: m / z 218.1 [M+H] + .C 12 H 15 N3O (217.1). Intermediate 8b, obtained by the same experimental procedure, was a pale yellow solid, 1.3 g in yield (92%). ESI-MS: m / z 232.1 [M+H] + .C 13 H 17 N3O(231.1).

[0060] (5) Intermediate 8b (1.5 g, 6.5 mmol, 1.1 eq.) was dissolved in methanol, and anhydrous potassium acetate (1.2 g, 12 mmol, 2.0 eq.) was added and stirred for 15 min. Then, intermediate 4 (1.0 g, 6 mmol, 1.0 eq.) was added. The reaction was monitored by TLC at 70 °C (methanol / dichloromethane = 1:10, v / v). After the reaction was complete, the reaction solution was concentrated under reduced pressure, 20 mL of water was added, and the solution was extracted with dichloromethane (3 × 10 mL). The organic phase was separated and washed with saturated sodium chloride solution (3 × 10 mL). The solution was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain intermediate C5. 0.7 g of yellow solid was obtained after purification, with a yield of 40.5%. 1 H NMR (400MHz, DMSO-d6): δ8.45(d,J=11.8Hz,1H),8.37(d,J=8.9Hz,1H),7.62 (d,J=10.3Hz,1H),7.48–7.24(m,3H),7.15(d,J=8.5Hz,1H),4.40–4.26(m,1 H),3.85–3.55(m,1H),3.44(d,J=13.6Hz,2H),3.31–3.08(m,2H),3.08–2.76 (m,2H),2.32(d,J=12.0Hz,3H),1.92(s,2H),1.70–1.29(m,2H).ESI-MS:m / z 364.2[M+H] + .C 22 H 22 FN3O (363.2). 8a was subjected to the same operation to obtain intermediate C6, a yellow solid, 0.75 g, in yield 48.8%. ESI-MS: m / z 350.2 [M+H] + .C 21 H 20 FN3O(349.2).

[0061] (6) Intermediate C5 and the screened azide derivative were added sequentially to a flask and dissolved in 15 mL of (tetrahydrofuran / water = 1:1, v / v). CuSO4·5H2O and sodium ascorbate were then added, and the reaction was carried out at 55 °C for 24 h. The methanol / dichloromethane ratio was monitored by TLC at 1:10, v / v. The reaction solution was concentrated under reduced pressure and extracted with ethyl acetate (3 × 10 mL). The organic phase was collected and washed with saturated sodium chloride solution (3 × 10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain the target compound C5N5.

[0062] The product is an orange powdery solid with a yield of 80% and a melting point of 95-96℃.

[0063] 1 H NMR (400MHz, DMSO-d6): δ8.45(s,1H),8.36(s,1H),8.26(s,1H),8.18(d,J=7.9Hz,1H),8.07(s,1H ),7.78–7.63(m,2H),7.61(s,1H),7.38(dd,J=17.6,9.8Hz,3H),7.07(s,1H),5.76(s,1H),5.72(d ,J=11.3Hz,2H),4.18(t,J=12.7Hz,1H),3.42(t,J=12.5Hz,1H),3.23–3.06(m,2H),3.06–2.91(m, 2H),2.31(s,3H),1.96(s,2H),1.57(d,J=11.0Hz,1H),1.41(d,J=15.2Hz,1H).HRMS(ESI)m / z[M+H] + calcd forC 29 H 28 FN7O3541.5874; found 542.231.HPLC purity:96.25%.

[0064]

[0065] The procedure is the same as above, except that p-nitrobenzyl azide is used.

[0066] The product is an orange solid with a yield of 82% and a melting point of 96.5-98℃.

[0067] 1 H NMR (600MHz, DMSO-d6): δ8.45(s,1H),8.37(s,1H),8.29–8.13(m,3H),7.62(s,1H),7.48( q,J=8.3Hz,2H),7.43–7.30(m,3H),7.07(s,1H),5.73(d,J=16.7Hz,2H),4.80(s,1H),4.25 –4.13(m,1H),3.43(t,J=12.1Hz,1H),3.18(d,J=5.2Hz,1H),3.15(d,J=20.2Hz,1H),2.99 (q,J=17.3Hz,2H),2.31(s,3H),2.05–1.89(m,2H),1.66–1.37(m,2H).HRMS(ESI)m / z[M+H] + calcd for C 29 H 28FN7O3541.5874; found 542.231.HPLC purity:97.35%.

[0068]

[0069] The procedure is the same as above, except that phenylethyl azide is used.

[0070] The product is a pale yellow solid with a yield of 85% and a melting point of 90-93℃. 1 H NMR (400MHz, DMSO-d6): δ8.46(d,J=13.7Hz,1H),8.36(s,1H),7.82(s,1H),7.62( d,J=8.8Hz,1H),7.41–7.26(m,3H),7.24–6.97(m,6H),5.75(s,1H),4.68(d,J=6.5 Hz,1H),4.56(d,J=6.7Hz,2H),4.15(t,J=11.6Hz,1H),3.10(s,3H),3.00–2.71(m ,3H),2.32(d,J=11.7Hz,3H),1.91(s,2H),1.57–1.32(m,2H).HRMS(ESI)m / z[M+H] + calcd for C 30 H 31 FN6O 510.6174; found 511.2616.HPLCpurity:93.70%.

[0071]

[0072] The procedure is the same as above, except that thiophene ethyl azide is used.

[0073] The product is a light orange solid with a yield of 70% and a melting point of 89.5-91℃.

[0074] 1H NMR (400MHz, DMSO-d6): δ8.66–8.25(m,2H),7.85(s,1H),7.62(d,J=5.1Hz,1H),7.49–7.23 (m,4H),7.07(s,2H),6.90(d,J=4.9Hz,1H),5.75(s,1H),4.70(d,J=8.0Hz,1H),4.55(d,J= 7.4Hz,2H),4.17(t,J=12.9Hz,1H),3.13(d,J=10.4Hz,3H),3.04–2.76(m,3H),2.32(d,J=6 .2Hz,3H),1.93(s,2H),1.55(d,J=8.8Hz,1H),1.38(d,J=10.4Hz,1H).HRMS(ESI)m / z[M+H] + calcd for C 28 H 29 FN6OS 516.6394; found 517.218.HPLCpurity:93.44%.

[0075]

[0076] The procedure is the same as above, except that N-(2-azidoethyl)phthalimide is used.

[0077] The product is a pale yellow solid with a yield of 80% and a melting point of 119-120℃.

[0078] 1 H NMR (400MHz, DMSO-d6): δ8.58–8.43(m,1H),8.37(s,1H),8.12(s,1H),7.89–7.57(m, 5H),7.33(s,1H),7.31–7.25(m,2H),7.04(d,J=8.5Hz,1H),5.76(s,1H),4.69(s,1H) ,4.59(s,2H),4.14(t,J=12.1Hz,1H),3.99(s,2H),3.18–2.77(m,4H),2.42–2.26(m, 3H),1.90(s,2H),1.55(d,J=9.3Hz,1H),1.39(d,J=10.2Hz,1H).HRMS(ESI)m / z[M+H] + calcd for C 32 H 30 FN7O3579.6364; found 580.2467.HPLC purity:96.76%.

[0079]

[0080] The procedure is the same as above, except that methyl γ-azidobutyrate is used.

[0081] The product is a light orange solid with a yield of 83% and a melting point of 75.5-78℃.

[0082] 1 H NMR (400MHz, DMSO-d6): δ8.44(s,1H),8.36(d,J=2.0Hz,1H),8.08(s,1H),7.61(s,1H),7.46–7.30(m ,3H),7.07(s,1H),4.75(s,1H),4.32(q,J=8.1Hz,2H),4.17(t,J=11.6Hz,1H),3.54(d,J=7.5Hz,3H) ,3.44(d,J=13.7Hz,1H),3.14(d,J=14.3Hz,1H),3.07–2.91(m,2H),2.88(d,J=7.2Hz,1H),2.29(d,J =13.4Hz,5H),2.13–1.84(m,4H),1.57(d,J=9.0Hz,1H),1.41(d,J=12.8Hz,1H).HRMS(ESI)m / z[M+H] + calcd forC 27 H 31 FN6O3506.5824; found,507.2514.HPLC purity:97.15%.

[0083]

[0084] The procedure is the same as above, except that phenylsulfinyl azidomethane is used.

[0085] The product is a white solid with a yield of 83% and a melting point of 118-120℃.

[0086] 1H NMR (400MHz, DMSO-d6): δ8.46(s,1H),8.38(s,1H),7.75–7.65(m,1H),7.63(d,J=7.9Hz,1H),7.51(s,1H) ,7.49–7.34(m,5H),7.32(d,J=2.9Hz,1H),7.30(d,J=3.1Hz,1H),7.09(d,J=8.8Hz,1H),5.75(s,2H),4.72 (d,J=14.0Hz,1H),4.27–4.12(m,1H),3.39(d,J=12.0Hz,1H),3.15(d,J=12.2Hz,1H),3.04–2.90(m,2H),2 .85(s,1H),2.32(s,3H),1.93(s,2H),1.57(d,J=13.0Hz,1H),1.41(d,J=15.2Hz,1H).HRMS(ESI)m / z[M+H] + calcdfor C 29 H 29 FN6O2S 544.6494; found, 545.2129. HPLC purity: 98.22%.

[0087]

[0088] The procedure is the same as above, except that benzenesulfonyl azidomethane is used.

[0089] The product is a white solid with a yield of 85% and a melting point of 111-112℃.

[0090] 1 H NMR (400MHz, DMSO-d6): δ8.46(s,1H),8.38(s,1H),7.75–7.65(m,1H),7.65–7.58(m,1H),7.51(s,1H) ),7.44(d,J=13.9Hz,5H),7.31(d,J=9.2Hz,2H),7.09(d,J=8.8Hz,1H),5.84(d,J=13.3Hz,1H),5.75 (s,2H),4.28–4.09(m,1H),3.39(d,J=11.9Hz,1H),3.15(d,J=12.4Hz,1H),3.04–2.90(m,2H),2.85( s,1H),2.32(s,3H),1.93(s,2H),1.57(d,J=12.8Hz,1H),1.41(d,J=15.7Hz,1H).HRMS(ESI)m / z[M+H] + calcd forC29 H 29 FN6O3S 560.6484; found, 561.2079. HPLC purity: 97.97%.

[0091]

[0092] The procedure is the same as above, except that 4-methanesulfonylbenzyl azide is used.

[0093] The product is a white solid with a yield of 81% and a melting point of 125-128℃.

[0094] 1 H NMR (600MHz, DMSO-d6): δ8.45(s,1H),8.37(s,1H),8.22(d,J=8.0Hz,1H),7.92(q,J=7.9Hz,2H),7. 62(s,1H),7.48(q,J=7.7Hz,2H),7.39(dd,J=23.9,13.3Hz,3H),7.07(s,1H),5.69(d,J=17.1Hz,2H ),4.79(s,1H),4.19(dd,J=22.6,11.7Hz,1H),3.43(t,J=13.1Hz,1H),3.18(s,3H),3.17–3.01(m,2 H),3.01–2.85(m,2H),2.31(s,3H),1.94(d,J=31.2Hz,2H),1.65–1.37(m,2H).HRMS(ESI)m / z[M+H] + calcd for C 30 H 31 FN6O3S 574.6754; found 575.2235.HPLC purity:95.86%.

[0095]

[0096] The procedure is the same as above, except that N-methylazidoacetamide is used.

[0097] The product is a white solid with a yield of 82% and a melting point of 106-107℃.

[0098] 1H NMR (400MHz, DMSO-d6): δ8.45(s,1H),8.39–8.34(m,1H),8.27–8.16(m,1H),8.00(s,1H),7.62(s, 1H),7.44–7.31(m,3H),7.08(s,1H),5.75(s,1H),5.00(d,J=9.3Hz,2H),4.77(d,J=5.4Hz,1H),4. 18(t,J=12.6Hz,1H),3.43(t,J=12.3Hz,1H),3.15(d,J=11.7Hz,1H),3.08–2.92(m,2H),2.61(s,3 H),2.31(s,3H),1.96(s,2H),1.57(d,J=10.6Hz,1H),1.41(d,J=14.5Hz,1H).HRMS(ESI)m / z[M+H] + calcd for C 25 H 28 FN7O2477.5444; found478.2361.HPLC purity:96.27%.

[0099]

[0100] The procedure is the same as above, except that azidoacetamide is used.

[0101] The product is a yellow solid, with a yield of 83%.

[0102] 1 H NMR(600MHz,DMSO-d6)δ8.45(s,1H),8.37(s,1H),7.99(d,J=3.8Hz,1H),7.75–7 .57(m,2H),7.45–7.26(m,4H),7.08(s,1H),5.00(d,J=16.3Hz,2H),4.78(d,J=11 .5Hz,1H),4.18(t,J=15.4Hz,1H),3.44(dd,J=19.8,12.5Hz,1H),3.21–2.85(m, 4H),2.31(s,3H),1.94(d,J=33.3Hz,2H),1.67–1.36(m,2H).HRMS(ESI)m / z[M+H] + calcd forC 24 H 26 FN7O2, 463.5174;(M+H) + ,464.2205.HPLC purity:95.51%.

[0103]

[0104] The procedure is the same as above, except that 3-fluorophenylbenzyl azide is used.

[0105] The product is an orange solid with a yield of 85% and a melting point of 115-116℃.

[0106] 1 H NMR (600MHz, DMSO-d6): δ8.45(s,1H),8.37(s,1H),8.19(d,J=7.6Hz,1H),7.61(s,1H),7 .45–7.28(m,4H),7.22–7.03(m,4H),5.57(d,J=17.5Hz,2H),4.78(d,J=7.1Hz,1H),4.18( t,J=12.9Hz,1H),3.43(t,J=13.5Hz,1H),3.18(d,J=5.2Hz,1H),3.12(d,J=26.5Hz,1H),3 .04–2.86(m,2H),2.31(s,3H),2.05–1.88(m,2H),1.64–1.38(m,2H).HRMS(ESI)m / z[M+H] + calcd for C 29 H 28 F2N6O 514.5808; found 515.2365.HPLC purity:95.04%.

[0107] Example 2: Preparation of C6N17~C6N21

[0108]

[0109] The procedure is the same as above, except that intermediate C6 and thiophene ethyl azide are used.

[0110] The product is an orange solid with a yield of 75% and a melting point of 98.5-100℃.

[0111] 1H NMR (400MHz, DMSO-d6): δ8.59(d,J=14.7Hz,2H),7.85(s,1H),7.80(s,1H),7.50–7.24( m,5H),7.08(d,J=9.1Hz,2H),6.90(s,1H),4.71(d,J=9.9Hz,1H),4.55(d,J=8.0Hz,2H), 4.16(t,J=12.9Hz,1H),3.32(s,1H),3.13(d,J=10.3Hz,3H),2.94(d,J=12.8Hz,2H),2.8 2(s,1H),1.92(s,2H),1.56(d,J=8.7Hz,1H),1.39(d,J=9.9Hz,1H).HRMS(ESI)m / z[M+H] + calcd for C 27 H 27 FN6OS 502.6124; found, 503.2024. HPLC purity: 98.82%.

[0112]

[0113] The procedure is the same as above, except that intermediate C6 and N-(2-azidoethyl)phthalimide are used.

[0114] The product is a white solid with a yield of 81% and a melting point of 115-116℃.

[0115] 1 H NMR (400MHz, DMSO-d6): δ8.72–8.59(m,1H),8.58(s,1H),8.11(s,1H),7.83(s,1H),7.80(d ,J=5.0Hz,2H),7.73(s,1H),7.67(s,1H),7.56–7.41(m,1H),7.32(s,1H),7.29(d,J=8.0Hz, 2H),7.04(d,J=8.7Hz,1H),4.68(s,1H),4.59(s,2H),4.12(d,J=11.8Hz,1H),3.98(s,2H), 3.29(s,1H),3.14–2.74(m,4H),1.90(s,2H),1.54(s,1H),1.39(s,1H).HRMS(ESI)m / z[M+H] + calcd for C 31 H 28 FN7O3565.6094; found 566.231.HPLC purity:99.34%.

[0116] Example 3: Preparation of C4N17~C4N21

[0117]

[0118] (1) Add 3-fluorobenzaldehyde (1) to ultra-dry tetrahydrofuran, and add magnesium acetylene bromide (2) dropwise under N2 protection. The reaction was detected by TLC (ethyl acetate / petroleum ether = 1:8, v / v). After the reaction was complete, saturated ammonium chloride was added to the system to quench the reaction. The system was extracted with ethyl acetate (3×20mL). The resulting organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain intermediate 3, which was a yellow oily substance.

[0119] (2) Intermediate 3 was dissolved in dichloromethane, and a sulfoxide solution diluted with dichloromethane was added dropwise under ice bath conditions. The reaction was carried out at room temperature under the catalysis of a small amount of DMF, and the reaction was detected by TLC (ethyl acetate / petroleum ether = 1:5, v / v). The reaction was quenched by adding saturated sodium bicarbonate. The organic phase was separated and dried successively with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude intermediate 4, which was a yellow-brown oily substance.

[0120] (3) Under ice bath conditions, raw material 6a (1.8 g, 14.6 mmol, 1.1 eq.) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 7.6 g, 20 mmol, 1.5 eq.) were added to dichloromethane. After activation for 15 min, N,N-diisopropylethylamine (DIPEA, 5.1 g, 39.5 mmol, 3.0 eq.) and raw material 11 (2.47 g, 13.3 mmol, 1.0 eq.) were added. The mixture was then transferred to room temperature and reacted for 16 hours. The solution turned pale yellow. The reaction was monitored by TLC (methanol / dichloromethane = 1:30, v / v). After the reaction was complete, the reaction solution was washed with water (3 × 20 mL). The organic phase was then washed with saturated sodium chloride solution (3 × 10 mL). The organic phase was successively dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain intermediate 12, a yellow solid, 3.1 g, with a yield of 80%. ESI-MS: m / z 292.2 [M+H] + .C 15 H 21 N3O3(291.2).

[0121] (4) Intermediate 12 (2.0 g, 6.9 mmol, 1.0 eq.) was dissolved in dichloromethane under ice-water bath conditions, and 4 mol / L dioxane hydrochloride solution (4.8 mL, 6.8 mmol, 6–7 eq.) was added dropwise. After the addition was complete, the reaction mixture was allowed to stand at room temperature. After about 6 hours, the reaction solution was concentrated under reduced pressure to obtain a white powder. Saturated sodium bicarbonate was added to adjust the pH, and the resulting aqueous phase was concentrated under reduced pressure to obtain a white solid. Tetrahydrofuran was added and stirred, filtered, washed, concentrated under reduced pressure, and dried to obtain intermediate 13, which was a yellow viscous solid of 1.15 g, with a yield of 87.8%. ESI-MS: m / z 192.1 [M+H] + .C 10 H 13 N3O(191.1).

[0122] (5) Intermediate 13 (1.0 g, 5.2 mmol, 1.1 eq.) was dissolved in methanol, and anhydrous potassium acetate (1.0 g, 10.2 mmol, 2.0 eq.) was added. After stirring for 15 min, intermediate 4 (0.80 g, 4.8 mmol, 1.0 eq.) was added, and the mixture was heated to reflux at 70 °C. The reaction was monitored by TLC (methanol / dichloromethane = 1:10, v / v). After the reaction was complete, the reaction solution was concentrated under reduced pressure, 20 mL of water was added, and the mixture was extracted with dichloromethane (3 × 10 mL). The organic phase was separated, washed with saturated sodium chloride solution (3 × 10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain intermediate C4, which was a yellow-brown solid of 0.56 g, with a yield of 33.1%. ESI-MS: m / z 324.1 [M+H] + .C 22 H 22 FN3O(323.1).

[0123] (6) Intermediate C4 and the screened azide derivative were added sequentially to a flask and dissolved in 15 mL of (tetrahydrofuran / water = 1:1, v / v). CuSO4·5H2O and sodium ascorbate were then added, and the reaction was carried out at 55 °C for 24 h. The methanol / dichloromethane ratio was monitored by TLC at 1:10, v / v. The reaction solution was concentrated under reduced pressure and extracted with ethyl acetate (3 × 10 mL). The organic phase was collected and washed with saturated sodium chloride solution (3 × 10 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain the target compound C4N17.

[0124] The product is an orange solid with a yield of 82% and a melting point of 163-165℃.

[0125] 1H NMR (400MHz, DMSO-d6): δ8.78–8.42(m,2H),7.87(s,1H),7.79(d,J=7.8Hz,1H),7. 49–7.43(m,1H),7.40(dd,J=4.9,3.0Hz,1H),7.38–7.31(m,1H),7.19(dd,J=12.7,8 .9Hz,2H),7.09(dd,J=8.5,2.7Hz,2H),6.92(d,J=5.0Hz,1H),4.78(s,1H),4.59(t, J=7.0Hz,2H),3.62(s,4H),3.16(t,J=7.0Hz,2H),2.30(s,4H).HRMS(ESI)m / z[M+H] + calcdfor C 25 H 25 FN6OS 476.5744; found477.1867.HPLC purity:95.51%.

[0126]

[0127] The procedure is the same as above, except that intermediate C4 and N-(2-azidoethyl)phthalimide are used.

[0128] The product is a white solid with a yield of 84% and a melting point of 113.5-115℃.

[0129] 1 H NMR (400MHz, DMSO-d6): δ8.65(d,J=4.8Hz,1H),8.59(s,1H),8.13(s,1H),7. 85–7.73(m,5H),7.47(dd,J=7.8,4.9Hz,1H),7.34(td,J=8.0,6.1Hz,1H),7.2 2–7.13(m,2H),7.11–7.03(m,1H),4.84(s,1H),4.63(t,J=5.4Hz,2H),4.06–3 .96(m,2H),3.62(s,2H),3.27(s,2H),2.38–2.20(m,4H).HRMS(ESI)m / z[M+H] + calcd for C 29 H 26 FN7O3539.5714; found 540.2154.HPLC purity:96.66%.

[0130] Example 4: Chiral resolution of target compound C5N17

[0131] Preparation method:

[0132] The racemic C5N17 was resolved by HPLC using a Shimadzu LC-20AT HPLC system with a CHIRALPAKAD-H (ADH0CE-CW056) chiral column (0.46 cm ID × 25 cm L). The injection volume was 10 μL, and the mobile phase was a gradient elution of n-hexane:ethanol = 20:80 (v / v) at a flow rate of 1.0 mL / min and a temperature of 35 °C. The fraction collected at a retention time of 6.792 min was C5N17A, and the fraction collected at a retention time of 8.041 min was C5N17B. The solvent was removed by rotary evaporation to obtain the pure optical isomer.

[0133] Example 5: The target compound's effect on the SARS-CoV-2 main protease (M pro Inhibition experiment

[0134] Experimental principle:

[0135] The fluorescence resonance energy transfer (FRET) method employed used a substrate with the structure: MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2. MCA is the fluorescent donor, and Dnp is the fluorescent acceptor, or fluorescence quencher. The complete sequence contains both the fluorescent and quencher groups. Due to the close spatial distance between the two groups, the inhibitory effect of the quencher prevents the fluorescent group from fluorescing. When SARS-CoV-2M was added... pro Subsequently, because the main protease can cleave between amino acids Q and S, the fluorescent group is moved away from the quenching group, emitting fluorescence at a wavelength of 405 nm under excitation light of 320 nm. M is detected by measuring the fluorescence. pro The activity of the compound, and thus indirectly reflects its inhibitory activity (see Appendix). Figure 2 (Dai Wenhao, et al., Science. 2020, 368: 1331-1335. Qiao Jingxin, et al., Science. 2021, 371: 1374-1378.)

[0136] Experimental methods:

[0137] Using SARS-CoV-2M pro Inhibitor Screening Kit (Beyotime, Beijing) determines the effect of target compounds on M proThe inhibitory ability of the main protease was assessed. The main protease concentrate was diluted to 93-fold with Assay buffer, and the test compounds were pre-prepared as concentration gradients in DMSO and Tris buffer. First, 93 μL of diluted M... pro The solution was prepared by adding 5 μL of the target compound solution and 2 μL of DMSO solution of the fluorescent substrate (Dabcyl-KTSAVLQSGFRKME-Edans). The mixture was incubated with shaking for 5 minutes (37°C, 90 rpm) in the dark. Immediately afterwards, the fluorescence intensity (RFU, Relative Fluorescence Unit) of each well was read at a single time point using a Spectramax iD5 microplate reader. The excitation wavelength was 340 nm, and the emission wavelength was 490 nm. A 100% enzyme activity control and a blank control were also included, as shown in Table 1 below.

[0138] Table 1. SARS-CoV-2 main protease (M) pro Relevant parameters of the inhibition experiment

[0139]

[0140] The inhibition percentage for each sample is calculated based on the data read. The calculation formula is as follows:

[0141] Inhibition rate (%) = (RFU) 100％酶活性对照 -RFU 样品 ) / (RFU 100％酶活性对照 -RFU 空白对照 )×100%

[0142] Using compounds GC-14 and nematvir as positive control groups, the compounds were initially screened at a concentration of 10 μM. The results of the initial screening are shown in the appendix. Figure 3 Compounds with inhibition rates exceeding those of the positive control were re-screened. Five concentration gradients (0.1 μM, 0.5 μM, 1 μM, 2.5 μM, 5 μM) were set up for compounds with significant activity, and the inhibition rates were measured at each gradient. The IC50 was calculated using GraphPad Prism8 software. 50 Values. Each sample was measured at least three times in parallel, and the average value and standard deviation were taken. The experimental results are shown in Table 2.

[0143] Table 2. Results of secondary screening for inhibition of SARS-CoV-2 main protease by substituted triazole compounds

[0144]

[0145]

[0146]

[0147] a IC 50 (μM): The concentration of the compound required to achieve 50% inhibition of the enzyme, i.e., the half-maximal inhibitory concentration. The value is the result of three tests. b GC-14: a positive control, is a novel piperazine non-covalent main protease inhibitor (JMedChem.2022,65:13343.); c nd: Not determined.

[0148] Analysis of experimental conclusions:

[0149] The newly synthesized substituted triazole compounds of this invention mostly exhibit significant main protease inhibitory activity. The activities of all compounds are superior to GC-14, with C5N17 (IC) showing the highest activity. 50 =0.17±0.016μM), C5N21(IC 50 =0.15±0.004μM) and C5N58 (IC 50 The activity of the enzyme (C5N17) at 0.15 ± 0.006 μM was particularly outstanding, with its inhibitory activity against the main protease being 3 to 5 times higher than that of GC-14; after chiral resolution of the racemic C5N17, the enantiomer C5N17B (IC) was obtained. 50 =0.12±0.015μM) The enzyme inhibitory activity was nearly doubled, and compared with the marketed drug nematvir (IC50). 50 =0.091±0.001μM). Therefore, substituted triazole compounds have value for further research and development.

[0150] Example 6: Evaluation of the cellular-level anti-SARS-CoV-2 activity of the preferred compounds

[0151] Experimental principle:

[0152] The main method for determining anti-SARS-CoV-2 activity at the cellular level is the Image-Based Antiviral Assay, which uses fluorescence imaging technology.

[0153] Experimental materials:

[0154] SARS-CoV-2 virus strain; Calu-3 cells; test compound; PBS buffer; EMEM 10% fetal bovine serum FBS; anti-spike protein (S) antibody; goat anti-mouse IgG; positive controls: nematvir, GC-14.

[0155] Experimental methods:

[0156] Calu-3 cells were seeded into 96-well plates at a density of 1 × 10⁶ cells approximately 24 hours before infection. 5Cell wells. Cells were treated with the test compound (50 μL / well) in increments of concentration, followed by infection with an equal volume of SARS-CoV-2 (final multiplicity of infection MOI 0.01) and cultured at 37°C for 2 days in a biosafety level 3 laboratory. Cells were fixed and infiltrated with acetone / methanol (1:3) at room temperature for 10 min. Viral S protein was detected using an anti-spike protein S antibody (GTX632604; Genetex, USA) and a goat anti-mouse IgG antibody conjugated with Alexa Fluor488 (Invitrogen, USA), while cell nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI, Invitrogen). Fluorescence images were quantitatively analyzed using the Operetta high-content screening system (Perkin Elmer, USA) and the built-in Harmony software. The 50% effective concentration (EC50) was calculated by determining the chemical concentration required to reduce the intensity of the viral S protein-derived green fluorescence by 50%. 50 EC 50 The values ​​were generated using curve fitting with Graphpad Prism 8.0. Each sample was measured at least three times in parallel, and the average and standard deviation were taken. The experimental results are shown in Table 3.

[0157] Activity Results and Discussion

[0158] Table 3. Cell-level anti-SARS-CoV-2 activity and toxicity results of substituted triazole compounds

[0159]

[0160]

[0161] a EC 50 (μM): Half-maximal effective concentration, the value is the result of three tests; b CC 50 (μM): Half-maximal toxic concentration; c GC-14: a positive control, is a novel piperazine non-covalent main protease inhibitor (J Med Chem. 2022, 65: 13343.); d nd: Not determined.

[0162] Analysis of experimental results:

[0163] Representative compounds with good enzyme-inhibiting activity from this invention were selected for anti-SARS-CoV-2 activity testing in Calu-3 cells. Compounds C5N16, C5N17, and C5N39 showed superior activity compared to GC-14 and the marketed drug nematvir. Among these, C5N17 (EC... 50The activity of the sample (0.16±0.01μM) was particularly outstanding, and its anti-SARS-CoV-2 activity was 9 times that of GC-14 (EC50). 50 =1.5±0.06μM), which is 6 times that of nematvir (EC). 50 =1.0±0.04μM). After chiral resolution of the racemic C5N17, the enantiomer C5N17B (EC) was obtained. 50 The activity of this compound (0.08 ± 0.006 μM) at the Calu-3 cell level was increased by 1-fold, and was 12.5 times that of nematvir. Notably, this series of substituted triazole compounds exhibited low cytotoxicity (CC). 50 (>100μM), which has value for further research and development.

Claims

1. A substituted triazole compound, or a pharmaceutically acceptable salt thereof, having the structure shown in general formula I. Formula I It is one of the following compounds: 。 2. The substituted triazole compound according to claim 1, characterized in that, Pharmaceutically acceptable salts include inorganic salts such as hydrochloride, sulfate, or phosphate, as well as organic salts such as methanesulfonate, p-toluenesulfonate, fumarate, citrate, or acetate.

3. The preparation method of the substituted triazole compound as described in claim 1, comprising the following steps: The preparation method of substituted triazole compounds comprises the following steps: Starting with 3-fluorobenzaldehyde 1 and magnesium acetylene bromide 2, intermediate 3 is obtained through Grignard reaction; then, intermediate 4 is obtained through substitution reaction with sulfoxide dichloroethylene; intermediate 7a or 7b is obtained by amide condensation of tert-butyl 3,8-diazabicyclo[3.2.1]octane-8-carboxylate 5 and nicotinic acid 6a or 5-methylnicotinic acid 6b, and then the tert-butoxy carbonyl group is removed to obtain intermediate 8a or 8b; intermediate 4 is obtained by substitution reaction with 8a or 8b to obtain alkynyl fragment intermediate C6 or intermediate C5; intermediate C6 is dissolved with an azide derivative in a mixed solvent of tetrahydrofuran / water = 1:1, v / v, with CuSO4·5H2O and sodium ascorbate as catalysts, and reacted at 55°C to obtain target compound C6NX; intermediate C5 is subjected to a click chemistry reaction catalyzed by monovalent copper ions in the same manner to obtain target compound C5NX. Starting with 1-Boc-piperazine 11 and nicotinic acid 6a, dichloromethane was used as the reaction solvent, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate was used as the condensing agent to obtain intermediate 12 via an amide condensation reaction. Then, intermediate 13 was obtained by removing the tert-butyloxycarbonyl group. Intermediate 13 and intermediate 4 were reacted with intermediate 4 via a substitution reaction to obtain the alkyne fragment intermediate C4. Intermediate C4 was reacted with the azide fragment in CuSO4·5H2O, with sodium ascorbate as the catalyst, in a mixed solvent of tetrahydrofuran / water = 1:1, v / v, at 55 °C to obtain the target compound C4NX. The synthesis route is as follows: Reagents and conditions: (i) ultra-dry tetrahydrofuran, ice-salt bath, N2; (ii) sulfoxide dichloromethane, ice bath; (iii) EDCI, HOBt, NMM, dichloromethane, room temperature; (iv) 4 mol / L dioxane hydrochloride solution, dichloromethane, room temperature; (v) CH3COOK, methanol, 70℃; (vi) azide compound, CuSO4·5H2O, sodium ascorbate, 55℃, tetrahydrofuran / water = 1:1, v / v; (vii) HATU, N,N,-diisopropylethylamine, dichloromethane, room temperature; in, The target compound is as described in claim 1.

4. The substituted triazole compound according to any one of claims 1-2 in the preparation of anti-SARS-CoV-2 M pro Its application in medicines.

5. An anticoronavirus pharmaceutical composition comprising a substituted triazole compound as described in any one of claims 1-2 and one or more pharmaceutically acceptable carriers.

6. The anticoronavirus pharmaceutical composition of claim 5, wherein the coronavirus is selected from the novel coronavirus.

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