A triazolopiperazine compound, a preparation method and application thereof

Abstract

The application provides a triazole piperazine 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 prodrug of the application in the treatment of a disease or disorder associated with SARS-CoV-2.

Description

Technical Field

[0001] This invention relates to a derivative and its preparation method, specifically to triazole piperazine compounds, compositions containing said compounds, preparation methods, and their applications in the field of anti-coronavirus drugs, belonging to the field of organic synthesis and pharmaceutical application technology. Background Technology

[0002] The novel coronavirus continues to mutate, and new mutant strains possess significant immune evasion capabilities, able to breach the immune barrier formed by vaccines or infection, thus leading to multiple infections. It is highly likely that the novel coronavirus will coexist with humans for a long time. Therefore, continued research and development of highly effective, low-toxicity, broad-spectrum anti-drug drugs against COVID-19 is still necessary.

[0003] Main protease (M) pro It plays a crucial role in the SARS-CoV-2 replication cycle, specifically recognizing and cleaving polyproteins (pp1a and pp1ab). The main protease is a homodimeric cysteine ​​protease composed of two mutually perpendicular monomers, with its catalytic center consisting of Cys145 and His41. 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 Inhibitors are mainly peptide-like covalent inhibitors. For example, the three M inhibitors currently marketed in China... pro Inhibitor: Paxlovid TM Nematride and ritonavir, as well as senoxetine (senoxetine / ritonavir) and leretvir. However, nematride and senoxetine require combination therapy with the P450 enzyme inhibitor ritonavir, limiting their use in patients with underlying medical conditions. Furthermore, the high structural similarity of peptide-mimicking inhibitors and the emergence of clinically resistant nematride strains (E166V / T21I and E166V / L50F) have prompted researchers to continuously explore new structural types of non-covalent main protease inhibitors. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a triazole piperazine compound, 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 material. pro Inhibitor activity screening results and their applications.

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

[0007] 1. Triazole piperazine compounds

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

[0009]

[0010] in,

[0011] R can be any of the following: methyl, methoxy, halogen, or hydroxymethyl groups; the R group can simultaneously substitute for multiple positions on the benzene ring.

[0012] 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.

[0013] According to the present invention, the triazole piperazine compound is one of the following compounds:

[0014]

[0015] 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 the compound of formula I. 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.

[0016] 2. Preparation methods of triazole piperazine compounds

[0017] The preparation method of triazole piperazine compounds is as follows: 3,4-dichlorophenylboronic acid 1 and (S)-3-hydroxymethylpiperazine-1-carboxylic acid tert-butyl ester 2 are used as raw materials to obtain intermediate 3 via a coupling reaction, which is then oxidized by a Des Martin oxidant to obtain intermediate 4. Intermediate 4 undergoes an alkynylation reaction with (1-diazo-2-oxopropyl)phosphonate dimethyl ester to obtain intermediate 5. Intermediate 5 is then subjected to deprotection and acylation reactions to obtain alkynyl fragment intermediate 7. Intermediate 7 and the azide derivative are dissolved in a mixed solvent of tetrahydrofuran / water = 1:1 (v / v), with CuSO4·5H2O and sodium ascorbate as catalysts, and reacted at 55℃ to obtain the target compound.

[0018] The synthesis route is as follows:

[0019]

[0020] Reagents and conditions: (i) Acetate, pyridine, dichloromethane, oxygen, room temperature; (ii) Dys-Martin oxidant, dichloromethane, 0℃-room temperature; (iii) (1-diazo-2-oxopropyl)phosphonate dimethyl ester, potassium carbonate, methanol, room temperature; (iv) 2M hydrogen chloride-dioxane solution, dichloromethane, room temperature; (v) Nicotinic acid, HATU, N,N,-diisopropylethylamine, dichloromethane, room temperature; (vi) R-N3, CuSO4·5H2O, sodium ascorbate, 55℃, tetrahydrofuran / water = 1:1 v / v.

[0021] Wherein, R has the structure described in general formula I above; the room temperature described in this invention is 20-30℃.

[0022] According to a preferred method for preparing triazole piperazine compounds of the present invention, the specific steps are as follows:

[0023] (1) 3,4-Dichlorophenylboronic acid 1 and (S)-3-hydroxymethylpiperazine-1-carboxylic acid tert-butyl ester 2 were added to dichloromethane and dissolved until nearly clear during stirring; anhydrous copper acetate and 2 equivalents of pyridine were added to the solution at one time; after uniform suspension, the reaction was carried out for 24 h under an oxygen atmosphere, and the reaction was detected by TLC with ethyl acetate / petroleum ether = 1:2 v / v; after the reaction was complete, water was added to the system to quench the reaction; the liquid was separated and repeatedly washed with distilled water until the organic phase was free of blue color; after the organic phase was washed with saturated sodium chloride solution and separated, it was dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain intermediate 3, which was a colorless oily liquid;

[0024] (2) Dissolve intermediate 3 in dichloromethane under ice-water bath, add Dys-Martin oxidant, and then react at room temperature; after about 2 hours, wash the reaction solution with saturated sodium thiosulfate and saturated sodium bicarbonate aqueous solution in turn to separate the organic phase, dry it with anhydrous sodium sulfate, filter it, concentrate it under reduced pressure, and the crude intermediate 4 obtained is directly used for subsequent reactions.

[0025] (3) Intermediate 4 was dissolved in methanol under an ice-water bath, potassium carbonate was added, followed by a mixed solution of (1-diazo-2-oxopropyl)phosphonate dimethyl ester and methanol; the reaction was then allowed to proceed to room temperature. After about 6 hours, the reaction solution was washed sequentially with water and saturated sodium chloride solution. The reaction solution was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain intermediate 5.

[0026] (4) Dissolve intermediate 5 in dichloromethane in an ice-water bath, and add a 2M HCl-dioxane and dichloromethane mixture 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 an oily substance. After adding ethyl acetate, a large amount of white solid precipitates. The solid is filtered, washed with ethyl acetate / petroleum ether, and dried to obtain the hydrochloride salt of intermediate 6.

[0027] (5) Nicotinic acid and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate were added to dichloromethane under ice bath conditions. After activation for 30 min, the hydrochloride salt of intermediate 6 and N,N'-diisopropylethylamine were added. The mixture was then reacted at room temperature for 8 hours. After the reaction was completed by TLC monitoring, the reaction was quenched with water. The organic phase of dichloromethane was then dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and rotary evaporated to obtain intermediate 7.

[0028] (6) Intermediate 7 and the azide derivative were added to a flask and dissolved in tetrahydrofuran / water at a ratio of 1:1, v / v. CuSO4·5H2O and sodium ascorbate were then added, and the mixture was reacted at 55°C for 24 h. The reaction was monitored by TLC at a ratio of methanol / dichloromethane of 1:10, v / v. The reaction solution was concentrated under reduced pressure and extracted with ethyl acetate (3×10 mL). The organic phase was 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 the target compound. 3. Bioactivity and Application of Triazole Piperazine Compounds

[0029] This invention discloses the activity screening results of triazole piperazine compounds and their first application as main protease inhibitors of SARS-CoV-2. Experiments demonstrate that the triazole piperazine 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.

[0030] Experiments on the inhibition of SARS-CoV-2 main protease activity by the target compound

[0031] The SARS-CoV-2 main protease inhibitory activity of a class of triazole piperazine compounds synthesized according to the above method was tested, and their activity data are listed in Table 1. The piperazine non-covalent main protease inhibitor GC-14 (JMedChem.2022,65:13343.) and the marketed drug nematvir were used as positive controls.

[0032] Most of the newly synthesized triazole piperazine compounds exhibit significant main protease inhibitory activity, such as compounds N31, N43, N46, and N47. The newly synthesized triazole piperazine compounds exhibit excellent enzyme-inhibiting activity, with compound N43 showing particularly outstanding activity (IC50). 50=1.85 μM); Antiviral activity evaluation in Vero E6 cells showed that N31 had the best activity (EC). 50 =6.32μM).

[0033] The triazole piperazine 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.

[0034] This invention provides novel triazole piperazine 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 triazole piperazine 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 good 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

[0035] Figure 1 This represents the inhibition rate of the compound against the main protease at a concentration of 10 μM. Detailed Implementation

[0036] The following examples help to understand the present invention, but do not limit the scope of the invention. All percentages are mass percentages.

[0037] Example 1: Preparation of N31, N43, N46 and N47

[0038]

[0039] (1) 3,4-Dichlorophenylboronic acid (1, 7.8 g, 41 mmol, 2.0 eq.) and (S)-3-hydroxymethylpiperazine-1-carboxylic acid tert-butyl ester (2, 4.4 g, 20.5 mmol, 1.0 eq.) were added to 100 mL of dichloromethane and dissolved nearly clearly with stirring; anhydrous copper acetate (3.7 g, 20.5 mmol, 1.0 eq.) and pyridine (3.2 g, 41 mmol, 2.0 eq.) were added; after thorough reconstitution, [the solution was then added to the solution]. The reaction was carried out under an oxygen atmosphere for 24 hours. TLC analysis showed that the ethyl acetate / petroleum ether ratio was 1:2 (v / v). After the reaction was complete, 100 mL of water was added to quench the reaction. The mixture was separated, and the organic phase was repeatedly washed with distilled water until no blue color remained. The organic phase was washed with 100 mL of saturated sodium chloride solution, separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by Flash column chromatography to obtain intermediate 3, which was 4.8 g of a colorless oily liquid; yield 65.1%. ESI-MS: m / z 361.2 [M+H]+ .C 16 H 22 Cl2N2O3 (360.1).

[0040] (2) Intermediate 3 (4.8 g, 13.3 mmol, 1.0 eq.) was dissolved in 100 mL of dichloromethane under ice-water bath conditions. Dys-Martin oxidant (11.3 g, 26.6 mmol, 2.0 eq.) was added, and the reaction was allowed to proceed to room temperature. After approximately 2 hours, the reaction was quenched with a saturated sodium thiosulfate aqueous solution. The mixture was separated, and the dichloromethane phase was washed with a saturated sodium bicarbonate aqueous solution. The organic phase was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 4.28 g of crude intermediate 4, which was used directly in subsequent reactions. Yield: 90%. ESI-MS: m / z 359.1 [M+H] + .C 16 H 20 Cl2N2O3(358.1).

[0041] (3) Under ice-water bath conditions, intermediate 4 (4.0 g, 11.2 mmol, 1.0 eq.) was dissolved in 80 mL of methanol, and potassium carbonate (3.1 g, 22.4 mmol, 2.0 eq.) was added with stirring. Then, a mixture of (1-diazo-2-oxopropyl)phosphonate dimethyl ester (3.2 g, 16.8 mmol, 1.5 eq.) and 20 mL of methanol was added. The reaction mixture was then allowed to react at room temperature. After approximately 6 hours, the reaction mixture was evaporated to dryness under reduced pressure. 100 mL of dichloromethane and 100 mL of saturated sodium chloride solution were added to the residue. The dichloromethane phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by Flash column chromatography to obtain 2.89 g of pure intermediate 5; yield 73%. ESI-MS: m / z 355.1 [M+H] + .C 17 H 20 Cl2N2O2(354.1).

[0042] (4) Intermediate 5 (2.8 g, 7.9 mmol, 1.0 eq.) was dissolved in 30 mL of dichloromethane, and a mixture of 10 mL of 4 M HCl-dioxane and 10 mL of dichloromethane 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 an oily substance. After the addition of ethyl acetate, a large amount of white solid precipitated. The solid was filtered, washed with a small amount of ethyl acetate / petroleum ether, and dried to obtain 2.46 g of the hydrochloride salt of intermediate 6; yield 96%. ESI-MS: m / z 255.1 [M+H] + .C 17 H 20 Cl2N2O2(254.1).

[0043] (5) Nicotinic acid (0.85 g, 6.7 mmol, 1.1 eq.) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 3.50 g, 9.2 mmol, 1.5 eq.) were added to dichloromethane under ice bath conditions. After activation for 30 min, the hydrochloride salt of intermediate 6 (2.0 g, 6.1 mmol, 1.0 eq.) and N,N'-diisopropylethylamine (DIPEA, 3.15 g, 24.4 mmol, 4.0 eq.) were added. The mixture was then reacted at room temperature for 8 hours. The reaction was quenched with water after TLC monitoring showed completion. The organic phase of dichloromethane was then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude intermediate 7 was obtained by concentrated under reduced pressure and purified by Flash column chromatography to 1.71 g; yield 78%. ESI-MS: m / z 360.3 [M+H] + .C 18 H 15 Cl2N3O(359.1).

[0044] (6) Intermediate 7 (0.1 g, 0.28 mmol, 1.0 eq.) and 4-azido-1,2-methylenedioxybenzene (0.28 mmol, 1.0 eq.) were added to a flask and dissolved in 10 mL of tetrahydrofuran / water (1:1, v / v). CuSO4·5H2O (0.028 mmol, 0.1 eq.) and sodium ascorbate (0.14 mmol, 0.5 eq.) were then added, and the mixture was reacted at 55 °C for 24 h. The reaction was monitored by TLC (methanol / dichloromethane = 1:20, v / v). The reaction solution was concentrated under reduced pressure and extracted with ethyl acetate (3 × 10 mL). The organic phase was 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 Flash column chromatography to obtain 0.1 g of the target compound N31.

[0045] The product is a white powdery solid with a yield of 72.3% and a melting point of 90-91℃.

[0046] 1HNMR(600MHz,DMSO-d6)δ8.75–8.44(m,2H),8.34(d,J=59.3Hz,1H),7.69(d,J=199.3Hz ,1H),7.46(s,1H),7.38(d,J=9.0Hz,1H),7.31(d,J=7.3Hz,1H),7.18(s,1H),7.09(d,J =8.4Hz,1H),6.95(d,J=19.7Hz,1H),6.14(s,2H),5.38(d,J=133.1Hz,1H),4.54(d,J=1 36.8Hz,1H),3.91(d,J=10.0Hz,1H),3.74(s,1H),3.54(d,J=32.3Hz,2H),3.26(s,1H). 13 C NMR(150MHz,DMSO-d6)δ167.98,151.04,149.55,148.64(C×2),147.89,135.29,131.98,131.34(C×2),130.97, 123.82,120.16,115.56,114.30,109.09(C×2),102.62(C×2),102.40,51.14,46.01,41.82,40.52.ESI-MS:m / z 523.33[M+H] + .C 25 H 20 Cl2N6O3(522.1).HPLC purity:99.41%.

[0047]

[0048] The procedure is the same as above, except that 2,4,6-trimethylphenyl azide is used.

[0049] The product is a grayish-white solid with a yield of 75% and a melting point of 88-89℃.

[0050] 1H NMR(600MHz,DMSO-d6)δ8.68(s,2H),8.49(d,J=111.9Hz,1H),8.09(d,J=66.5Hz,1H),7.84 (d,J=38.8Hz,1H),7.55–7.49(m,1H),7.38(d,J=8.1Hz,1H),7.21–7.08(m,1H),7.05(s,2H) ,7.01(d,J=8.0Hz,1H),5.37(d,J=136.4Hz,1H),4.65–4.36(m,1H),3.96(d,J=12.2Hz,1H), 3.67–3.58(m,2H),3.50(d,J=9.6Hz,1H),3.01(d,J=11.7Hz,1H),2.30(s,3H),1.74(s,6H). 13 C NMR (150MHz, DMSO-d6) δ167.99,151.09,149.76,148.05,139.93,135.24,134.84,133.70,132.01,131.84,131.08,130. 82(C×2),129.26(C×2),124.06,122.06,120.46,118.40,117.17,79.60,51.98,48.62,40.52,21.08,16.99.ESI-MS:m / z 521.7[M+H] + .C 27 H 26 Cl2N6O(520.1).HPLC purity:99.80%.

[0051]

[0052] The procedure is the same as above, except that 4-hydroxymethylphenyl azide is used.

[0053] The product is a pale yellow foamy solid with a yield of 76% and a melting point of 163-164℃.

[0054] 1H NMR(600MHz,DMSO-d6)δ8.65(s,1H),8.59(s,1H),8.39(d,J=107.6Hz,1H), 7.86(s,1H),7.84–7.75(m,2H),7.45(dd,J=78.4,8.9Hz,4H),7.19(s,1H), 6.96(d,J=20.4Hz,1H),5.56–5.23(m,2H),4.57(d,J=5.4Hz,2H),4.45(s,1 H),3.93(d,J=8.0Hz,1H),3.75(s,1H),3.55(d,J=43.2Hz,2H),3.33(s,1H). 13 C NMR(150MHz,DMSO-d6)δ167.95,150.89,149.54,147.90,143.75,135.52,134.86,133.76,131.99,131.11,130.9 8,130.62,128.03(C×3),123.82,122.15,120.30,118.52,116.51,62.65,51.74,51.18,42.54,40.52.ESI-MS:m / z 509.41[M+H] + .C 25 H 22 Cl2N6O2(508.1).HPLC purity:99.88%.

[0055]

[0056] The procedure is the same as above, except that 4-methoxyphenyl azide is used.

[0057] The product is a white, foamy solid with a yield of 73% and a melting point of 80-81℃.

[0058] 1H NMR (600MHz, DMSO-d6) δ8.64(d,J=55.6Hz,2H),8.34(d,J=54.9Hz,1H),7.81(d,J= 77.6Hz,2H),7.53(d,J=5.7Hz,1H),7.43(dd,J=63.9,9.0Hz,2H),7.21(d,J=36.2H z,1H),7.15–7.09(m,2H),7.02–6.92(m,1H),5.39(d,J=133.6Hz,1H),4.82–4.38( m,2H),3.91(d,J=10.5Hz,1H),3.82(s,3H),3.79–3.69(m,1H),3.64–3.53(m,2H). 13 C NMR (150MHz, DMSO) δ168.15,159.74,151.07,149.77,147.85,143.75,135.03,133.82,132.01,131.08,130.97,130 .34,128.12,124.05,122.24,118.39,117.18,116.51,115.32(C×2),79.60,56.03,51.21,48.63,40.52.ESI-MS:m / z 509.52[M+H] + .C 25 H 22 Cl2N6O2(508.1).HPLCpurity:99.9%.

[0059] Example 2: The target compound's effect on the SARS-CoV-2 main protease (M pro Inhibition experiment

[0060] Experimental methods:

[0061] The inhibitory activity of the target compound against the main protease was tested using fluorescence resonance energy transfer (FRET). MCA-AVLQSGFR-Lys(Dnp)-Lys-NH2 was used as the reaction substrate. Under light-protected conditions, 1.5 μM of SARS-CoV-2 M... pro500 μM of substrate and 10 μM of compound were added to 96-well plates for initial screening. The plates were incubated at 37°C for 10 minutes. Fluorescence intensity of each group was detected using a multi-mode microplate reader with excitation wavelength of 320 nm and emission wavelength of 405 nm. Measurements were taken every 10 seconds for 10 minutes to obtain fluorescence intensity. First, the fluorescence intensity values ​​were converted into the increase in fluorescence intensity per unit time based on the standard curve. The data from the first minute was used to obtain the rate. The change in the initial reaction rate represents the degree of inhibition of enzyme activity by the inhibitor. This method is used to study enzyme inhibition. The initial reaction rate of the blank control was V0, and the rate after adding the inhibitor was V... i The degree of inhibition of enzyme activity can be expressed by the following equation: i% = (1 - V) i The experiment was divided into a blank control group, a positive control group, and an experimental group. Compounds GC-14 and nematriberi were used as positive control groups. Compounds with inhibition rates exceeding those of the positive control at a concentration of 10 μM were screened again.

[0062] Secondary screening: 1.5 μM SARS-CoV-2M was selected. pro The IC50 of the compound was measured using a substrate of 500 μM and four concentration gradients (0.1 μM, 0.5 μM, 1 μM, 5 μM). 50 Each group was configured with 3 replicates and incubated at 37°C for 10 minutes. The fluorescence intensity of each group was detected using a multi-mode microplate reader with an excitation wavelength of 320 nm and an emission wavelength of 405 nm. Measurements were taken every 10 seconds for 10 minutes. Finally, IC50 analysis was performed using a GraphPad Prism 8 microplate reader based on the inhibition rate at different concentrations. 50 The calculations were performed. The experimental results are shown in Table 1.

[0063] Table 1. Results of secondary screening for inhibition of SARS-CoV-2 main protease by triazole piperazine compounds

[0064]

[0065]

[0066] 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: Positive control; c nd: Not determined.

[0067] Analysis of experimental conclusions:

[0068] The newly synthesized triazole piperazine compounds of this invention all exhibit significant main protease inhibitory activity. Among them, compound N43 (IC 50=1.89±0.014μM), N46(IC) 50 =1.87±0.07μM) and N47 (IC 50 The activity of the triazole piperazine compound (2.09 ± 0.027 μM) is particularly outstanding, indicating that the triazole piperazine compound is worthy of further research.

[0069] Example 3: Evaluation of the Cellular-Level Anti-SARS-CoV-2 Activity of Preferred Compounds - ELISPOT Experimental Materials

[0070] SARS-CoV-2 virus strain; Vero E6 cells; test compound; PBS buffer; Dulbecco's MEM 5% fetal bovine serum; guinea pig anti-SARS-CoV-2 antibody; methylcellulose; goat anti-guinea pig IgG; positive control: nematvir.

[0071] Vero E6 cells were seeded in 96-well plates approximately 24 hours before infection at a density of 1.7 × 10⁶ cells / well. 4 Cells / wells. Dilute the test compound to a 10 mM stock solution with DMSO, and then dilute to the desired concentration with 5% fetal bovine serum in Dulbecco's MEM. After incubating the plate for 25–26 hours, remove the methylcellulose cover, wash the plate twice with PBS, add 5% paraformaldehyde in PBS to each well, and then immerse the entire plate in 5% formalin for 15 minutes. Then immerse the plate in PBS for 15 minutes, and then add fresh PBS to each well.

[0072] Plates with immobilized Vero E6 cells were washed with focus formation assay buffer and incubated with guinea pig anti-SARS-CoV-2 antibody; goat anti-guinea pig IgG (horseradish peroxidase conjugate) was used as a secondary antibody. The matrix was TrueBlue, and infected cells / lesions appeared as single blue cell clusters. After staining, the plates were read using an EliSpot plate reader, and the relative stained areas were used to calculate the inhibitory activity of the drug at specific concentration gradients. Curves were generated using Graphpad Prism 8.0, and EC50 was calculated. 50 value.

[0073] Activity Results and Discussion

[0074] Table 2. Cell-level anti-SARS-CoV-2 activity and toxicity results of triazole piperazine compounds

[0075]

[0076] a EC 50 (μM): Half-maximal effective concentration; b CC50 (μM): Half-maximal toxic concentration; c GC-14: Positive control

[0077] Analysis of experimental results:

[0078] Representative compounds with good enzyme-inhibiting activity from this invention were selected for anti-SARS-CoV-2 activity testing in Vero E6 cells. Compounds N31, N46, and N47 showed the best activity, slightly weaker than the positive control drugs GC-14 and nematvir, but provided highly valuable data for rapid screening of the S4 site.

Claims

1. A triazole piperazine compound, or a pharmaceutically acceptable salt thereof, characterized in that, It is one of the following compounds:

2. The method for preparing the triazole piperazine compound as described in claim 1 comprises the following steps: 3,4-dichlorophenylboronic acid 1 and (S)-3-hydroxymethylpiperazine-1-carboxylic acid tert-butyl ester 2 are used as raw materials to obtain intermediate 3 via a coupling reaction, which is then oxidized by a Des Martin oxidant to obtain intermediate 4. Intermediate 4 undergoes an alkynylation reaction with (1-diazo-2-oxopropyl)phosphonate dimethyl ester to obtain intermediate 5. Intermediate 5 is subsequently subjected to deprotection and acylation reactions to obtain alkynyl fragment intermediate 7. Intermediate 7 and a phenyl azide derivative are dissolved 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 the target compound. The synthesis route is as follows: Reagents and conditions: (i) Acetate, pyridine, dichloromethane, oxygen, room temperature; (ii) Dys-Martin oxidant, dichloromethane, 0℃-room temperature; (iii) (1-diazo-2-oxopropyl)phosphonate dimethyl ester, potassium carbonate, methanol, room temperature; (iv) 2M hydrogen chloride-dioxane solution, dichloromethane, room temperature; (v) Nicotinic acid, HATU, N,N,-diisopropylethylamine, dichloromethane, room temperature; (vi) Phenylated derivative, CuSO4·5H2O, sodium ascorbate, 55℃, tetrahydrofuran / water = 1:1, v / v; The phenyl azide derivatives are selected from 5-azidobenzo[d][1,3]dioxolane, 2-azido-1,3,5-trimethylbenzene, (4-azidophenyl)methanol, and 1-azido-4-methoxybenzene; in, The room temperature is 20-30℃.

3. The use of the triazole piperazine compound according to claim 1 in the preparation of a drug against SARS-CoV-2Mpro.

4. A pharmaceutical composition for treating novel coronavirus, comprising the triazole piperazine compound of claim 1 and one or more pharmaceutically acceptable carriers.

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