CN118852137A - A triazole piperazine compound and its preparation method and application - Google Patents
A triazole piperazine compound and its preparation method and application Download PDFInfo
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Abstract
本发明提供了一种三氮唑哌嗪类化合物及其制备方法和应用。所述化合物具有式I所示的结构。本发明还涉及含有式I结构化合物的制备方法，药物组合物以及提供上述化合物在制备抗SARS‑CoV‑2Mpro药物中的应用。 The present invention provides a triazole piperazine compound and a preparation method and application thereof. The compound has a structure shown in Formula I. The present invention also relates to a preparation method of a compound having a structure of Formula I, a pharmaceutical composition, and an application of the compound in the preparation of an anti-SARS-CoV-2M pro drug.
Description
Technical Field
The invention relates to a derivative and a preparation method thereof, in particular to a triazole piperazine compound, a composition containing the compound, a preparation method and application thereof in the field of anti-coronavirus medicines, and belongs to the technical fields of organic synthesis and medical application.
Background
At present, new coronaviruses are continuously mutated, and the new mutant strain has remarkable immune escape capacity, can break through the immune barrier formed by vaccine or infection of a human body, and further causes multiple infections. The high probability that a new coronavirus will coexist with humans for a long period of time. Therefore, there is still a need to continuously develop new crown resistant medicines with high efficiency, low toxicity and broad-spectrum drug resistance.
The main protease (M pro) plays an important role in the SARS-CoV-2 replication cycle and is capable of specifically recognizing and cleaving the multimeric proteins (pp 1a and pp1 ab). The main protease is a homodimeric cysteine protease consisting of two monomers perpendicular to each other, the catalytic center of which consists of Cys145 and His 41. M pro is highly conserved among coronavirus species and has low homology with human protease, so that the inhibitor aiming at the target has the advantages of good selectivity and small toxic and side effects, and becomes an important target for developing anti-coronavirus medicaments.
The currently reported SARS-CoV-2M pro inhibitor is mainly a peptidomimetic covalent inhibitor. Three inhibitors of M pro as currently marketed in China: paxlovid TM (nemaltevir and ritonavir) and minoxidil (minoxidil/ritonavir) and letrovir. However, the need for combination of Nemacleavir and minoxidil with the P450 enzyme inhibitor ritonavir limits the use of drugs in people with underlying disease. In addition, the high similarity of the structure of the peptidomimetic inhibitors and the clinically occurring Nemactetavir resistant strains (E166V/T21I and E166V/L50F) promote scientific researchers to continuously explore the noncovalent main protease inhibitors with new structure types.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a triazole piperazine compound, a composition containing the compound and a preparation method thereof, and also provides an activity screening result of the compound serving as a SARS-CoV-2M pro inhibitor and application thereof.
The technical scheme of the invention is as follows:
1. Triazole piperazine compound
The triazole piperazine compound or pharmaceutically acceptable salt thereof has a structure shown in the following general formula I:
Wherein,
R is: methyl, methoxy, halogen, hydroxymethyl, each independently; the R group may simultaneously replace multiple positions of the benzene ring.
Pharmaceutically acceptable salt forms include commonly used inorganic acid salts such as hydrochloride, sulfate or phosphate, and organic acid salts such as methanesulfonate, p-toluenesulfonate, fumarate, citrate or acetate.
According to the invention, the triazole piperazine compound is one of the following compounds:
By "pharmaceutically acceptable salts" as used herein is meant salts of the compounds which are suitable for contact with the tissues of humans or lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit to risk ratio, generally water or oil soluble or dispersible, and effective for their intended use, within the scope of sound medical evaluation. Including pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts, are contemplated herein and are compatible with the chemical nature of the compounds of formula I. A list of suitable salts is found in S.M. Birge et al, J.Pharm.Sci., pages 1977,66,1-19. Further preferred salt forms are inorganic acid salts such as hydrochloride, sulfate or phosphate, and organic acid salts such as methanesulfonate, p-toluenesulfonate, fumarate, citrate or acetate.
2. Preparation method of triazole piperazine compound
The preparation method of the triazole piperazine compound comprises the following steps: 3, 4-dichlorobenzoboric acid 1 and (S) -3-hydroxymethyl piperazine-1-carboxylic acid tert-butyl ester 2 are used as raw materials, intermediate 3 is prepared through coupling reaction, then, dess-Martin oxidant is used for oxidation to obtain intermediate 4,4 and (1-diazo-2-oxo propyl) phosphonic acid dimethyl ester are subjected to alkynyl reaction to obtain intermediate 5, and intermediate 5 is subjected to deprotection and acylation reaction in sequence to obtain alkynyl fragment intermediate 7; intermediate 7 and azide derivative are dissolved in a mixed solvent of tetrahydrofuran/water=1:1 (v/v), cuSO 4·5H2 O and sodium ascorbate are used as catalysts, and the target compound is obtained through reaction at 55 ℃.
The synthetic route is as follows:
reagents and conditions: (i) Ketone acetate, pyridine, dichloromethane, oxygen, room temperature; (ii) Dess-martin oxidant, dichloromethane, 0-room temperature; (iii) Dimethyl (1-diazo-2-oxopropyl) phosphonate, potassium carbonate, methanol, room temperature; (iv) 2M hydrogen chloride-dioxane solution, dichloromethane, room temperature; (v) Nicotinic acid, HATU, N, -diisopropylethylamine, dichloromethane, room temperature; (vi) R-N 3,CuSO4·5H2 O, sodium ascorbate, 55 ℃, tetrahydrofuran/water=1:1 v/v.
Wherein R is as described in formula I above; the room temperature of the invention is 20-30 ℃.
According to the preferred preparation method of the triazole piperazine compound, the preparation method comprises the following specific steps:
(1) 3, 4-dichlorobenzoboric acid 1 and (S) -3-hydroxymethyl piperazine-1-carboxylic acid tert-butyl ester 2 are added into methylene dichloride, and dissolved and clarified in stirring; adding anhydrous copper acetate and 2 equivalents of pyridine into the solution at one time; after being uniformly suspended, the mixture is reacted for 24 hours in an oxygen atmosphere, and TLC detection is carried out on ethyl acetate/petroleum ether=1:2v/v; after the reaction is completed, adding water into the system to quench the reaction; separating, repeatedly washing with distilled water until the organic phase has no blue color; washing the organic phase with saturated sodium chloride solution, separating, sequentially drying with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, and separating and purifying the obtained crude product by silica gel column chromatography to obtain intermediate 3 as colorless oily liquid;
(2) Dissolving the intermediate 3 in dichloromethane under ice water bath, adding a dess-martin oxidant, and then transferring to room temperature for reaction; after about 2 hours, washing the reaction solution with saturated sodium thiosulfate and saturated sodium bicarbonate water solution sequentially to separate out an organic phase, and drying, filtering and concentrating under reduced pressure sequentially through anhydrous sodium sulfate to obtain a crude product intermediate 4 which is directly used for subsequent reactions;
(3) Dissolving the intermediate 4 in methanol under ice water bath, adding potassium carbonate, and then adding a mixed solution of dimethyl (1-diazo-2-oxo-propyl) phosphonate and methanol; the reaction was carried out at room temperature. After about 6 hours, washing the reaction solution sequentially by using water and saturated sodium chloride solution, drying the reaction solution sequentially by using anhydrous sodium sulfate, filtering, concentrating under reduced pressure, and separating and purifying the obtained crude product by using silica gel column chromatography to obtain an intermediate 5;
(4) Dissolving the intermediate 5 in dichloromethane under ice water bath, and dropwise adding a mixed solution of 2M HCl-dioxane and dichloromethane; after the dripping is finished, the reaction is carried out at room temperature; after about 6 hours, the reaction solution is concentrated under reduced pressure to obtain oily matter; adding ethyl acetate, and precipitating a large amount of white solid; filtering, washing the solid with ethyl acetate/petroleum ether, and drying to obtain hydrochloride of the intermediate 6;
(5) Nicotinic acid and 2- (7-aza-benzotriazol) -N, N, N ', N ' -tetramethyl urea hexafluorophosphate are added into dichloromethane under ice bath, hydrochloride of an intermediate 6 and N, N ' -diisopropylethylamine are added after activation for 30min, the reaction is carried out for 8 hours at room temperature, and water quenching is carried out after TLC monitoring is carried out. The dichloromethane organic phase is dried by anhydrous sodium sulfate, filtered, concentrated under reduced pressure and rotary distilled to obtain an intermediate 7;
(6) Respectively adding the intermediate 7 and the azide derivative into a flask, dissolving with tetrahydrofuran/water=1:1, v/v, adding CuSO 4·5H2 O and sodium ascorbate, and reacting at 55 ℃ for 24 hours; TLC monitored reaction, methanol/dichloromethane=1:10, v/v; the reaction solution was concentrated under reduced pressure, extracted with ethyl acetate (3X 10 mL), the organic phase was washed with saturated sodium chloride solution (3X 10 mL), and then dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography to give the objective compound. 3. Biological activity and application of triazole piperazine compound
The invention discloses an activity screening result of piperazine compounds containing triazole and the first application of the piperazine compounds as SARS-CoV-2 main protease inhibitor. Experiments prove that the triazole piperazine compound can be used as a main protease inhibitor for preparing anti-coronavirus medicines. The invention also provides application of the compound in preparing anti-coronavirus medicines.
Experiment for inhibiting SARS-CoV-2 main protease activity by target compound
A class of triazolylpiperazine compounds synthesized according to the above method was tested for SARS-CoV-2 main protease inhibitory activity, and their activity data are shown in Table 1. Piperazine non-covalent main protease inhibitor GC-14 (JMed chem.2022, 65:13343.) and the marketed drug Nemactetvir were used as positive controls.
Most of the novel synthesized triazole piperazine compounds of the present invention exhibit remarkable main protease inhibitory activity, such as compounds N31, N43, N46 and N47. The novel synthesized triazole piperazine compound has excellent enzyme inhibition activity, wherein the activity of the compound N43 is particularly outstanding (IC 50 =1.85 mu M); the evaluation of antiviral activity in Vero E6 cells showed that the N31 activity was optimal (EC 50 = 6.32 μm).
The triazole piperazine compound can be used as SARS-CoV-2 main proteinase inhibitor, in particular as SARS-CoV-2 inhibitor for preparing anti-new coronavirus medicine.
The invention provides a triazole piperazine compound with a brand new structure and a preparation method thereof, and also provides a screening result of the activity of the compound against SARS-CoV-2 main protease and the first application thereof in the antiviral field. The triazole piperazine compound can be used as SARS-CoV-2 main protease inhibitor and has higher application value. Specifically, the invention discovers a main protease inhibitor with better activity and novel structure through structural optimization, and can be used as a SARS-CoV-2 main protease inhibitor for preparing anti-new coronavirus drugs.
Drawings
FIG. 1 is the inhibition of the primary protease by the compounds at a concentration of 10. Mu.M.
Detailed Description
The invention will be further understood by the following examples, which are not intended to limit the scope of the invention.
Example 1: preparation of N31, N43, N46 and N47
(1) 3, 4-Dichlorobenzoboric acid (1, 7.8g,41mmol,2.0 eq.) and (S) -3-hydroxymethylpiperazine-1-carboxylic acid tert-butyl ester (2, 4.4g,20.5mmol,1.0 eq.) were added to 100mL of dichloromethane and dissolved with stirring until clear; anhydrous copper acetate (3.7 g,20.5mmol,1.0 eq.) and pyridine (3.2 g,41mmol,2.0 eq.) were added; after being uniformly suspended, the mixture is reacted for 24 hours under an oxygen atmosphere, and TLC detection is carried out on ethyl acetate/petroleum ether=1:2 (v/v); after the reaction is completed, adding 100mL of water into the system to quench the reaction; separating, repeatedly washing with distilled water until the organic phase has no blue color; the organic phase is washed by 100mL of saturated sodium chloride solution and separated, then is dried by anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the obtained crude product is separated and purified by Flash column chromatography to obtain an intermediate 3 which is 4.8g of colorless oily liquid; the yield thereof was found to be 65.1%. ESI-MS: m/z 361.2[ M+H ] +.C16H22Cl2N2O3 (360.1).
(2) Intermediate 3 (4.8 g,13.3mmol,1.0 eq.) was dissolved in 100mL dichloromethane under an ice water bath, dess-martin oxidant (11.3 g,26.6mmol,2.0 eq.) was added and then allowed to react to room temperature; after about 2h, quenching with saturated sodium thiosulfate aqueous solution, separating liquid, washing dichloromethane phase with saturated sodium bicarbonate aqueous solution, separating organic phase, drying by anhydrous sodium sulfate, filtering, concentrating under reduced pressure to obtain 4.28g of crude intermediate 4, which is directly used for subsequent reaction. The yield thereof was found to be 90%. ESI-MS: m/z 359.1[ M+H ] +.C16H20Cl2N2O3 (358.1).
(3) Intermediate 4 (4.0 g,11.2mmol,1.0 eq.) was dissolved in 80mL methanol under ice water bath, potassium carbonate (3.1 g,22.4mmol,2.0 eq.) was added and stirred, followed by a mixed solution of dimethyl (1-diazo-2-oxopropyl) phosphonate (3.2 g,16.8mmol,1.5 eq.) and 20mL methanol; the reaction was carried out at room temperature. After about 6 hours, the reaction mixture was evaporated to dryness under reduced pressure, and 100mL of methylene chloride and 100mL of saturated sodium chloride solution were added to the residue. The methylene dichloride phase is dried by anhydrous sodium sulfate, filtered and concentrated under reduced pressure in sequence, and the obtained crude product is purified by a Flash column to obtain 2.89g of intermediate 5 pure product; the yield thereof was found to be 73%. ESI-MS: m/z 355.1[ M+H ] +.C17H20Cl2N2O2 (354.1).
(4) Intermediate 5 (2.8 g,7.9mmol,1.0 eq.) was dissolved in 30mL dichloromethane and a mixed solution of 4M HCl-dioxane mL and 10mL dichloromethane was added dropwise; after the dripping is finished, the reaction is carried out at room temperature; after about 6 hours, the reaction solution is concentrated under reduced pressure to obtain oily matter; adding ethyl acetate, and precipitating a large amount of white solid; filtering, washing the obtained solid with a small amount of ethyl acetate/petroleum ether, and drying to obtain 2.46g of hydrochloride of the intermediate 6; the yield thereof was found to be 96%. ESI-MS: m/z 255.1[ M+H ] +.C17H20Cl2N2O2 (254.1).
(5) Nicotinic acid (0.85 g,6.7mmol,1.1 eq.) was reacted with 2- (7-azabenzotriazol) -N, N' -tetramethyluronium hexafluorophosphate (HATU, 3.50g,9.2mmol,1.5 eq.) in dichloromethane under ice bath, after 30min activation, the hydrochloride salt of intermediate 6 (2.0 g,6.1mmol,1.0 eq.) and N, -diisopropylethylamine (DIPEA, 3.15g,24.4mmol,4.0 eq.) were added and the reaction was quenched with water after TLC monitoring the reaction for 8 hours at room temperature. The dichloromethane organic phase is dried by anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and concentrated under reduced pressure to obtain a crude product of the intermediate 7, and purified by a Flash column to obtain 1.71g; the yield thereof was found to be 78%. ESI-MS: m/z 360.3[ M+H ] +.C18H15Cl2N3 O (359.1).
(6) Intermediate 7 (0.1 g,0.28mmol,1.0 eq.) was added separately to 4-azido-1, 2-methylenedioxybenzene (0.28 mmol,1.0 eq.) in a flask, dissolved with 10mL (tetrahydrofuran/water=1:1, v/v), and then added with CuSO 4·5H2 O (0.028 mmol,0.1 eq.) and sodium ascorbate (0.14 mmol,0.5 eq.) and reacted at 55 ℃ for 24h; TLC monitored reaction (methanol/dichloromethane=1:20, v/v); the reaction solution was concentrated under reduced pressure, extracted with ethyl acetate (3X 10 mL), and the organic phase was washed with saturated sodium chloride solution (3X 10 mL). The organic phase is dried by anhydrous sodium sulfate, filtered and concentrated under reduced pressure, and the obtained crude product is separated and purified by a Flash column to obtain 0.1g of target compound N31.
The product was a white powdery solid in 72.3% yield, melting point: 90-91 ℃.
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＝136.8Hz,1H),3.91(d,J＝10.0Hz,1H),3.74(s,1H),3.54(d,J＝32.3Hz,2H),3.26(s,1H).13C 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]+.C25H20Cl2N6O3(522.1).HPLC purity:99.41％.
The procedure was as above, except that 2,4, 6-trimethylphenyl azide was used.
The product was an off-white solid, yield: 75%, melting point: 88-89 ℃.
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).13C 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]+.C27H26Cl2N6O(520.1).HPLC purity:99.80％.
The procedure was as above, except that 4-hydroxymethylphenyl azide was used.
The product was a pale yellow foamy solid, yield: 76%, melting point: 163-164 ℃.
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,1H),3.93(d,J＝8.0Hz,1H),3.75(s,1H),3.55(d,J＝43.2Hz,2H),3.33(s,1H).13C NMR(150MHz,DMSO-d6)δ167.95,150.89,149.54,147.90,143.75,135.52,134.86,133.76,131.99,131.11,130.98,130.62,128.03(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]+.C25H22Cl2N6O2(508.1).HPLC purity:99.88％.
The procedure was as above, except that 4-methoxyphenyl azide was used.
The product was a white foamy solid, yield: 73%, melting point: 80-81 ℃.
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＝36.2Hz,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).13C 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]+.C25H22Cl2N6O2(508.1).HPLC purity:99.9％.
Example 2: inhibition test of SARS-CoV-2 Main protease (M pro) by target Compound
The experimental method comprises the following steps:
The inhibition activity of the target compound on the main protease was tested using a fluorescence resonance energy transfer method. MCA-AVLQSGFR-Lys (Dnp) -Lys-NH 2 was used as reaction substrate. Under the condition of avoiding light, adding 1.5 mu M of SARS-CoV-2M pro, 500 mu M of substrate and 10 mu M of compound into a 96-well plate for primary screening, incubating for 10 minutes at 37 ℃, detecting the fluorescence intensity of each group by using a multifunctional enzyme-labeled instrument, wherein the excitation wavelength is 320nm, the emission wavelength is 405nm, and measuring once every 10 seconds for 10 minutes to obtain the fluorescence intensity. Firstly, converting the fluorescence intensity value into the increase of the fluorescence intensity in unit time according to the standard curve. Taking the data of the first minute to obtain the speed, wherein the change of the initial reaction speed is used for representing the inhibition degree of the inhibitor on the enzyme activity, the inhibition degree of the enzyme activity is studied, the initial reaction speed of a blank control is V 0, the inhibition degree of the enzyme activity is V i after the inhibitor is added, and the inhibition degree of the enzyme activity can be represented by the following equation: i% = (1-V i)/V0 x 100%. Experiments were divided into blank control group, positive control group and experimental group. Compound GC-14 and nemaltar were used as experimental positive control group, and compound with inhibition ratio exceeding positive control at 10 μm concentration was rescreened.
And (3) re-screening: 1.5. Mu.M of SARS-CoV-2M pro, 500. Mu.M substrate and four concentration gradients (0.1. Mu.M, 0.5. Mu.M, 1. Mu.M, 5. Mu.M) of IC 50 of the test compound were selected. 3 compound holes are arranged in each group, the incubation is carried out for 10 minutes at 37 ℃, a multifunctional enzyme-labeled instrument is used for detecting the fluorescence intensity of each group, the excitation wavelength is 320nm, the emission wavelength is 405nm, the measurement is carried out every 10 seconds, the measurement is carried out for 10 minutes, and the fluorescence intensity is obtained. And finally, calculating the IC 50 by using GraphPadprism 8 according to the inhibition rates at different concentrations. The experimental results are shown in table 1.
TABLE 1 re-screening results of triazolylpiperazine Compounds for inhibiting SARS-CoV-2 Main protease
aIC50 (Mu M) at which 50% inhibition of the enzyme was achieved, the desired compound concentration, i.e., half inhibition concentration, was the result of three tests; b GC-14: a positive control; c n.d.: not measured.
Conclusion of experiment analysis:
The novel synthesized triazole piperazine compounds all show remarkable main protease inhibition activity. The activity of the compound N43 (IC 50＝1.89±0.014μM),N46(IC50 =1.87+/-0.07 mu M) and N47 (IC 50 =2.09+/-0.027 mu M) is particularly outstanding, which indicates that the triazole piperazine compound has the value of further research.
Example 3: evaluation of anti-SARS-CoV-2 Activity at cellular level of preferred Compounds-ELISA (ELISPOT) Experimental Material
SARS-CoV-2 virus strain; vero E6 cells; a test compound; PBS buffer; dulbecco's MEM 5% fetal bovine serum; guinea pig anti-SARS-CoV-2 antibodies; methyl cellulose; goat anti-guinea pig lgG; positive control: nemactetvir experimental method
Vero E6 cells were seeded in 96-well plates at a density of 1.7x10 4 cells/well approximately 24 hours prior to infection. The test compounds were diluted with DMSO solution to 10mM stock and diluted to the desired concentration with Dulbecco's MEM of 5% fetal bovine serum. After incubating the plates for 25-26 hours, the methylcellulose coating was removed, the plates were washed twice with PBS, 5% paraformaldehyde in PBS was added to each well, and then the whole plate was immersed in 5% formalin for 15 minutes. The plate was then immersed in PBS for 15 minutes, and fresh PBS was then added to each well.
Washing the plate attached with the immobilized Vero E6 cells by using a focus forming assay buffer solution, and incubating with guinea pig anti-SARS-CoV-2 antibody; goat anti-guinea pig IgG (horseradish peroxidase conjugate) was used as the secondary antibody. The matrix was TrueBlue and the infected cells/lesions represented a single blue cluster of cells. After staining, the plates were read on EliSpot plate readers and the relative areas of staining were used to calculate the inhibitory activity of the drug at a specific concentration gradient. A curve was generated by GRAPHPAD PRISM 8.0.0 fit and EC 50 values were calculated.
Active results and discussion
TABLE 2 cellular level of triazolylpiperazine compounds anti-SARS-CoV-2 Activity and toxicity results
aEC50 (Mu M) half the effective concentration; bCC50 (μm): a cell halftoning concentration; c GC-14: positive control
Analysis of experimental results:
Representative compounds with better enzyme inhibition activity in the invention are selected to perform anti-SARS-CoV-2 activity test in Vero E6 cells. The activity of the compounds N31, N46 and N47 is optimal and slightly weaker than that of the positive medicaments GC-14 and the Namactetvir, but the rapid screening of the S4 locus provides data with great reference value.
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| CN114409643A (en) * | 2022-01-24 | 2022-04-29 | 山东大学 | Dichlorobenzene polysubstituted piperazine compound and preparation method and application thereof |
| CN117143096A (en) * | 2023-08-09 | 2023-12-01 | 山东大学 | A substituted nicotinyl piperazine compound and its preparation method and application |
| CN118047783A (en) * | 2024-01-08 | 2024-05-17 | 山东大学 | Substituted triazole compound and preparation method and application thereof |
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| CN114409643A (en) * | 2022-01-24 | 2022-04-29 | 山东大学 | Dichlorobenzene polysubstituted piperazine compound and preparation method and application thereof |
| CN117143096A (en) * | 2023-08-09 | 2023-12-01 | 山东大学 | A substituted nicotinyl piperazine compound and its preparation method and application |
| CN118047783A (en) * | 2024-01-08 | 2024-05-17 | 山东大学 | Substituted triazole compound and preparation method and application thereof |
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| CN119707919A (en) * | 2024-11-11 | 2025-03-28 | 山东大学 | Deuterated piperazine compound and preparation method and application thereof |
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