CN114681454B - Application of thiazole compound in preparation of novel coronavirus medicament for resisting SARS-COV-2 - Google Patents

Application of thiazole compound in preparation of novel coronavirus medicament for resisting SARS-COV-2 Download PDF

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CN114681454B
CN114681454B CN202210265352.4A CN202210265352A CN114681454B CN 114681454 B CN114681454 B CN 114681454B CN 202210265352 A CN202210265352 A CN 202210265352A CN 114681454 B CN114681454 B CN 114681454B
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王文龙
吴婧
李佳
臧奕
冯勃
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Shanghai Institute of Materia Medica of CAS
Jiangnan University
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Jiangnan University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/428—Thiazoles condensed with carbocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
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    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D277/46—Acylated amino or imino radicals by carboxylic acids, or sulfur or nitrogen analogues thereof
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    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/84—Naphthothiazoles

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Abstract

The invention discloses application of thiazole compounds in preparing novel coronavirus medicines for resisting SARS-COV-2, and belongs to the technical field of medicines. The thiazole compound with the structure shown in the general formula (I) is a brand new inhibitor of SARS-CoV-23CLpro protein, is a brand new molecular skeleton of a novel coronavirus drug lead of SARS-COV-2, can realize the purpose of blocking nucleic acid and protein synthesis, provides a new thought and new application for preventing and/or treating novel coronaviruses of SARS-COV-2, and has important medical research value.

Description

Application of thiazole compound in preparation of novel coronavirus medicament for resisting SARS-COV-2
Technical Field
The invention belongs to the technical field of medicines, and in particular relates to application of thiazole compounds in preparation of novel coronavirus medicines for resisting SARS-COV-2.
Background
The novel coronavirus pneumonia (COVID-19) is an infectious disease caused by the novel coronavirus (SARS-CoV-2) which is a severe threat to human health. SARS-CoV-2 is an enveloped RNA virus that consists of an outer envelope and an inner nucleocapsid. The envelope, like the cell membrane, is a phospholipid bilayer structure, containing structural proteins encoded by the viral genome, including spike glycoprotein (spike glycoprotein, S protein), envelope protein (E protein) and membrane protein (membraneprotein, M protein). The nucleocapsid is assembled from nucleocapsid proteins (nucleocapsidprotein, N proteins) and viral RNA. During the nucleic acid and protein synthesis phase, the 2 functional open reading frames (open READING FRAME, ORF1 a/b) of coronaviruses translate into polyprotein 1a (polyprotein 1a, pp1 a) and polyprotein 1ab (polyprotein 1a, pp1 ab), which are cleaved by papain-like proteases (3C-like protease, PLpro) and 3-chymotrypsin (3C-like protease,3 CLpro) to produce a number of nonstructural proteins, including RNA-dependent RNA polymerase (RNA-DEPENDENT RNA polymerase, rdRp) involved in viral transcription and replication.
The SARS-CoV-2 3CLpro and PLpro proteins are the key proteins for the SARS-CoV-2 key protein RdRp to be in active form. SARS-CoV-2 3CLpro protein is assembled into active RdRp by hydrolyzing the viral pp1a and pp1ab proteins to form the mature product nsp 1-16. PLpro can specifically recognize and cleave the bisglycine polypeptide, the LXGG sequence between nsp1-2, nsp2-3 and nsp3-4 at the N-terminus of polyprotein ppla (pp 1 ab), participate in the cleavage process of the N-terminus of 1a (1 ab) replicase protein and release the mature products nsp1, nsp2 and nsp3, thus assembling into an active RdRp. SARS-CoV-2PLpro and 3CLpro play a critical role in the viral replication cycle and have been considered as key drug targets for the treatment of coronaviruses. At present, there is a lack of effective novel anti-SARS-COV-2 coronavirus drugs.
Disclosure of Invention
The invention provides thiazole compounds with a structure shown in a general formula (I), which are brand new inhibitors of SARS-CoV-23CLpro protein, are brand new molecular frameworks of novel anti-SARS-COV-2 coronavirus drug leads, can realize the purpose of blocking nucleic acid and protein synthesis, provide new ideas and new uses for preventing and/or treating novel SARS-COV-2 coronavirus, and have important medical research values.
One aspect of the invention provides application of thiazole compounds shown in the following general formula I or pharmaceutically acceptable salts thereof in preparing novel coronavirus drugs for resisting SARS-COV-2,
Wherein,
R 1 is mono-to trisubstituted selected from: h, -OH, halogen (F, cl, br, I), C1-C6 alkyl, C1-C6 alkoxy, substituted or unsubstituted amino-NR 3R4, substituted or unsubstituted amido-NH-C (O) -R 5, substituted or unsubstituted aminoacyl-C (O) -N-R 6R7;
R 2 is selected from substituted or unsubstituted amino-NR 8R9, substituted or unsubstituted 5-7 membered heterocyclylamido-NH-C (O) -R 10, substituted or unsubstituted aminoacyl-C (O) -N-R 11R12;
R 3、R4、R8、R9 is independently selected from H, substituted or unsubstituted C1-C6 alkyl, and the substituent of the alkyl is selected from: substituted or unsubstituted aryl, heteroaryl, C3-C6 cycloalkyl; the substituent of aryl and heteroaryl is mono-substituted to tri-substituted and is selected from-OH, halogen (F, cl, br, I), C1-C6 alkyl, C1-C6 alkoxy and C1-C6 alkoxycarbonyl;
R 5、R10 is independently selected from substituted or unsubstituted 5-7 membered heterocyclic groups, aryl groups, heteroaryl groups; the substituents on the heterocyclic group, aryl group and heteroaryl group are mono-substituted to tri-substituted and are selected from the group consisting of: -OH, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkoxycarbonyl, halogen (F, cl, br, I);
R 6、R7、R11、R12 is independently selected from substituted or unsubstituted aryl and benzothiadiazolyl; the substituents of aryl and benzothiadiazolyl are mono-substituted to tri-substituted and are selected from the group consisting of: -OH, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkoxycarbonyl, halogen (F, cl, br, I);
the heterocyclyl or heteroaryl group comprises one to three heteroatoms selected from N, O, S;
is a carbon-carbon bond or a carbon-hydrogen bond;
When n is 2, A is a six-membered ring; or when n is1, A is a five-membered ring; or when n is 0, A is not cyclic.
In one embodiment of the invention, the aryl group is a benzene ring or a naphthalene ring.
The invention also provides an application of thiazole compounds shown in the general formula I or pharmaceutically acceptable salts thereof in preparing SARS-CoV-2 3CLpro protein inhibitors.
The invention also provides a medicine for preventing or treating SARS-COV-2 novel coronavirus pneumonia infection, which contains thiazole compounds shown in the general formula I or pharmaceutically acceptable salts and pharmaceutical excipients thereof.
The pharmaceutical excipients comprise any one or more of the following: solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-binding agents, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, flocculant and deflocculant, filter aids, and release retarders.
In one embodiment of the invention, the medicament further comprises a drug carrier. The drug carrier comprises a microcapsule, a microsphere, a nanoparticle and a liposome.
In one embodiment of the invention, the dosage form of the medicament comprises any one of the following: injection, freeze-dried powder for injection, suspension, implant, suppository, capsule, tablet, pill and oral liquid.
The invention also provides a synthesis method of the thiazole compound shown in the general formula I:
A. When n=1 or 2,
Reagents and conditions: a) Liquid bromine, thiourea, absolute ethyl alcohol at 100 ℃ for 5 hours; b) HATU (2- (7-azobenzotriazole) -N, N' -tetramethyluronium hexafluorophosphate), DIPEA (N, N-diisopropylethylamine), DMF (N, N-dimethylformamide), room temperature, 4h;
Weighing A, liquid bromine and thiourea, dissolving in absolute ethyl alcohol, reacting for 5 hours at the temperature of 100 ℃ in an oil bath, clarifying and then clouding the solution in the reaction process to separate out white solid, decompressing and distilling to remove 1/4 volume of solvent after TLC monitoring reaction is completed, regulating alkali by using 5% NaOH solution, and carrying out suction filtration, wherein the solid is a compound B; dissolving a compound B, a compound C and HATU (2- (7-azobenzotriazole) -N, N, N ', N' -tetramethyl urea hexafluorophosphate) in DMF (N, N-dimethylformamide), dropwise adding DIPEA (N, N-diisopropylethylamine), stirring at room temperature for 4 hours, monitoring that the reaction is complete, dripping the reaction liquid into water, precipitating a solid, and performing suction filtration and drying to obtain a compound I;
B. When n=0,
Reagents and conditions: a) Iodine simple substance, thiourea, 110 ℃ and 10 hours; b) HATU (2- (7-azobenzotriazole) -N, N' -tetramethyluronium hexafluorophosphate), DIPEA (N, N-diisopropylethylamine), DMF (N, N-dimethylformamide), room temperature, 4h;
Weighing A ', I 2, thiourea, reacting for 10 hours in a molten state at 110 ℃ through an oil bath, adding a solid obtained after pulping by methyl tertiary butyl ether into water after TLC monitoring the reaction, adjusting the pH to 9-10 through 25% ammonia water, carrying out suction filtration, dissolving the solid into ethyl acetate, washing 2-3 times through saturated NaHCO 3 solution, drying an organic phase through anhydrous sodium sulfate, carrying out reduced pressure distillation to obtain a compound B ', dissolving a compound C ', HATU (2- (7-azobenzotriazole) -N, N, N ', N ' -tetramethylurea hexafluorophosphate) into DMF (N, N-dimethylformamide), dropwise adding DIPEA (N, N-diisopropylethylamine), stirring for 4 hours at room temperature, dripping the reaction liquid into the water after monitoring the reaction completion, precipitating a solid, carrying out suction filtration, and drying to obtain the compound I.
The invention has the beneficial effects that:
The thiazole compound with the structure shown in the general formula (I) is a brand new inhibitor of SARS-CoV-2 3CLpro protein, is a brand new molecular skeleton of a novel anti-SARS-COV-2 coronavirus drug lead, can realize the purpose of blocking nucleic acid and protein synthesis, provides a new thought and a new application for preventing and/or treating the novel SARS-COV-2 coronavirus, and has important medical research value.
Detailed Description
EXAMPLE 1 preparation of thiazoles
Synthetic method 1, n=1 or 2
Reagents and conditions: a) Liquid bromine, thiourea, absolute ethyl alcohol at 100 ℃ for 5 hours; b) HATU (2- (7-azobenzotriazole) -N, N' -tetramethyluronium hexafluorophosphate), DIPEA (N, N-diisopropylethylamine), DMF (N, N-dimethylformamide), room temperature, 4h;
Weighing A, liquid bromine and thiourea, dissolving in absolute ethyl alcohol, reacting for 5 hours at the temperature of 100 ℃ in an oil bath, clarifying and then clouding the solution in the reaction process to separate out white solid, decompressing and distilling to remove 1/4 volume of solvent after TLC monitoring reaction is completed, regulating alkali by using 5% NaOH solution, and carrying out suction filtration, wherein the solid is a compound B; dissolving a compound B, a compound C and HATU (2- (7-azobenzotriazole) -N, N, N ', N' -tetramethyl urea hexafluorophosphate) in DMF (N, N-dimethylformamide), dropwise adding DIPEA (N, N-diisopropylethylamine), stirring at room temperature for 4 hours, monitoring that the reaction is complete, dripping the reaction liquid into water, precipitating a solid, and performing suction filtration and drying to obtain the compound I.
The specific synthesis process comprises the following steps:
Step 1
5-Methoxy-1-indenone A (1.62 g,10.0mmol, liquid bromine (1.76 g,11.0 mmol), thiourea (2.28 g,30.0 mmol) are weighed and dissolved in 20mL absolute ethyl alcohol, the reaction is carried out for 5 hours at the temperature of an oil bath 100 ℃, the solution is clarified firstly and then becomes turbid to separate out white solid in the reaction process, after TLC monitoring reaction is completed, 1/4 volume of solvent is removed by reduced pressure distillation, 5% NaOH solution is used for regulating alkali, suction filtration is carried out, and the solid is a compound WJ-218 (1.8 g, yield 50%).
Step 2
The compound WJ-218 (0.24 g,1.1 mmol), 3-methoxybenzoic acid (0.15 g,1.0 mmol), HATU (2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate) (0.38 g,1.0 mmol) was dissolved in 5mL DMF (N, N-dimethylformamide), DIPEA (N, N-diisopropylethylamine) (0.39 g,3.0 mmol) was added dropwise, stirred at room temperature for 4h, after completion of the reaction, the reaction solution was dropped into water to precipitate a solid, which was suction-filtered and dried to give the compound WJ-354 (0.18 g, yield 50%).
The following compounds were prepared in the same manner, substituting the corresponding substrates.
Synthesis scheme 2, n=0
Reagents and conditions: a) Iodine simple substance, thiourea, 110 ℃ and 10 hours; b) HATU (2- (7-azobenzotriazole) -N, N' -tetramethyluronium hexafluorophosphate), DIPEA (N, N-diisopropylethylamine), DMF (N, N-dimethylformamide), room temperature, 4h;
Weighing A ', I 2, thiourea, reacting for 10 hours in a molten state at 110 ℃ through an oil bath, adding a solid obtained after pulping by methyl tertiary butyl ether into water after TLC monitoring the reaction, adjusting the pH to 9-10 through 25% ammonia water, carrying out suction filtration, dissolving the solid into ethyl acetate, washing 2-3 times through saturated NaHCO 3 solution, drying an organic phase through anhydrous sodium sulfate, carrying out reduced pressure distillation to obtain a compound B ', dissolving a compound C ', HATU (2- (7-azobenzotriazole) -N, N, N ', N ' -tetramethylurea hexafluorophosphate) into DMF (N, N-dimethylformamide), dropwise adding DIPEA (N, N-diisopropylethylamine), stirring for 4 hours at room temperature, dripping the reaction liquid into the water after monitoring the reaction completion, precipitating a solid, carrying out suction filtration, and drying to obtain the compound I.
The specific synthesis process comprises the following steps:
Step 1
A' (1.64 g,10 mmol), I 2 (2.54 g,10 mmol), thiourea (0.76 g,10 mmol) were weighed out and reacted for 10h in a molten state at 110℃in an oil bath, after TLC monitoring the reaction was completed, the solid slurried with methyl tert-butyl ether was added to water, pH was adjusted to 9-10 with 25% aqueous ammonia, suction filtration was carried out, the solid was dissolved with EtOAc (50 mL), washed with saturated NaHCO 3 solution (20 mL. Times.3), the organic phase was dried over anhydrous sodium sulfate, and distilled under reduced pressure to give compound WJ220 (1.10 g, yield 50%).
Step 2
The compound WJ-220 (0.24 g,1.1 mmol), 3-methoxybenzoic acid (0.15 g,1.0 mmol), HATU (2- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate) (0.38 g,1.0 mmol) was dissolved in 5mL DMF (N, N-dimethylformamide), DIPEA (N, N-diisopropylethylamine) (0.39 g,3.0 mmol) was added dropwise, stirred at room temperature for 4h, after completion of the reaction, the reaction liquid was dropped into water to precipitate a solid, which was suction-filtered and dried to give the compound WJ-354-1 (0.16 g, yield 45%).
The following compounds were prepared in the same manner, substituting the corresponding substrates.
Example 2 inhibition of SARS-CoV-2 3CLpro protease activity by 8H-indeno [1,2-d ] thiazoles:
Based on the SARS-CoV-2 3CLpro protein as one basic characteristic of proteinase, a fluorescent screening system for detecting SARS-CoV-2 3CLpro protein activity is established. SARS-CoV-2 3CLpro protein can specifically cut the substrate with Gln (Q) at P1 position, and its activity can be detected by using fluorescent polypeptide as substrate and detecting the generation of fluorescent signal to reflect its proteolytic enzyme activity. The SARS-CoV-2-3 CLpro protease activity assay uses a fluorescent-labeled polypeptide substrate (sequence: MCa-AVLQSGFRK (DNP) K), SARS-CoV-2-3 CLpro enzyme solution (diluted to 0.5. Mu.M in the reaction solution) and the compound are incubated in the reaction solution (20mM Tris,pH7.3,150mM NaCl,1mM EDTA,1%Glycerol,0.01%Tween-20) for 10 minutes at room temperature, the substrate (40. Mu.M, total volume of the reaction solution 50. Mu.L) is added, and after the reaction is started, the fluorescence intensity (excitation light 320nm, emission light 405 nm) of the reaction solution is detected by EnVision Multilabel Reader (Perkinelmer), and three wells are provided for each dose. The fluorescence value of the control wells (DMSO) was set to 100%, and the compound-treated wells were expressed as a percentage relative to the control wells.
The 3CLpro protease activity inhibition was tested for 8H-indeno [1,2-d ] thiazoles at various concentrations (ranging from 0.08. Mu.M to 20. Mu.M), the activity dose dependence, IC 50/EC50 values, were tested, the sample concentration was non-linearly fitted by sample activity, the software used was calculated to be GRAPHPAD PRISM, the model used for fitting was a four-parameter dose effect integral model (four-PARAMETER CONCENTRATION-response model) (varible slope), and the bottom and top of the fitted curve were set to 0 and 100 for most inhibitor screening models. Typically, each sample was provided with a multiple well (n.gtoreq.3) in the test, and the results were expressed in terms of Standard deviation (Standard Deviation, SD) or Standard Error (SE). Calculate IC 50.
Example 3 inhibition of SARS-CoV-2PLpro protease activity by 8H-indeno [1,2-d ] thiazoles assay:
Based on SARS-CoV-2PLpro protein as one basic characteristic of proteinase, a fluorescent screening system for detecting SARS-CoV-2PLpro protein activity is established. SARS-CoV-2PLpro protein can specifically recognize and cut double glycine polypeptide, its activity detection can use fluorescent polypeptide as substrate, and can utilize detection of fluorescent signal generation to make reaction of its proteolytic enzyme activity. The protease activity of SARS-CoV-2PLpro was detected by using a coumarin-labeled polypeptide substrate (sequence: Z-RLRGG-AMC), a SARS-CoV-2PLpro enzyme solution (diluted to 40nM in the reaction solution) was incubated with the compound in the reaction solution (20mM Tris pH8.0,0.01%Tween20,0.5mM DTT) at room temperature for 10 minutes, the substrate (50. Mu.M, total volume of the reaction solution 50. Mu.L) was added, and after starting the reaction, the fluorescence intensity (355 nM excitation light, 460nM emission light) of the reaction solution was detected by EnVision Multilabel Reader (Perkinelmer), and three wells were provided for each dose. The fluorescence value of the control wells (DMSO) was set to 100%, and the compound-treated wells were expressed as a percentage relative to the control wells.
The inhibition of protease activity by 8H-indeno [1,2-d ] thiazole was tested at different concentrations (concentration from 0.08. Mu.M to 20. Mu.M), the activity dose dependence, IC 50/EC50 value, was tested, the sample concentration was non-linearly fitted by sample activity, the software used for calculation was GRAPHPAD PRISM, the model used for fitting was four-parameter dose-response model (varible slope), and the bottom and top of the fitted curve were set to 0 and 100 for most inhibitor screening models. Typically, each sample was provided with a multiple well (n.gtoreq.3) in the test, and the results were expressed in terms of Standard deviation (Standard Deviation, SD) or Standard Error (SE). Calculate IC 50.
5. Test method
(1) Cell culture and treatment:
Vero E6 cells were cultured in T75 cell flasks at 37 ℃, in a 5% co2 incubator, and passaged every 48 hours at a 1:3 ratio, medium formulation: 90% DMEM (Gibco Invitrogen), 10% fetal bovine serum (Gibco Invitrogen).
The day before the test, the cell culture broth was aspirated, rinsed once with extracellular fluid, and 2mL of the Trypsin solution was added and digested at room temperature for 1-2 minutes. Medium was added to neutralize pancreatin, cells were counted and transferred to 48-well plates, 50000 cells per well.
(2) Compound preparation:
Test compounds were dissolved in DMSO to 40mM stock solutions, respectively, and on the day of the test, the compound stock solutions were serially diluted 10 times with DMEM, i.e., 1. Mu.L of the compound stock solution was added to 9. Mu.L of DMSO, and after 2 dilutions 10 times, 0.4mM dilutions were obtained, and then diluted 40 times to 10. Mu.M.
Preparation of the positive control compound chloroquine phosphate, compound was dissolved in PBS to 2mM stock solution.
The final test concentration of the compound contained no more than 0.2% DMSO, which had no effect on SARS-CoV-2 replication.
(3) Detecting the effect of a compound on viral replication
The supernatant was removed from the cells of the 48-well plate, diluted compounds were added to each well, incubated for 1 hour, SARS-CoV-2 was added in a biosafety level 3 (BSL-3) laboratory, the multiplicity of infection (MOI) was 0.01, after incubation for 1 hour, the supernatant was removed, washed with PBS, diluted compounds were added, the supernatant was removed 24 hours after infection, and the well plate cells were fixed with paraformaldehyde. After the cells are fixed, incubation and staining are carried out by using SARS-CoV-2NP protein rabbit polyclonal antibody, 488 fluorescent antibody and DAPI dye, a 48-pore plate is placed under a fluorescent microscope imaging system for automatic scanning and photographing, and according to the virus load (green fluorescence) and the cell number (blue fluorescence) of the DMSO hole as a control, the fluorescent signals are counted by using Image J software, and the compound inhibition rate and the drug toxicity are respectively calculated.
(4) Data processing and statistics
The data analysis process uses Excel software, and the following formula is used for calculation:
Inhibition％＝[1–(I/Io)]×100％
wherein, inhibition% represents the percent Inhibition of SARS-CoV-2 replication by the compound, and I and Io represent the fluorescent signal areas of SARS-CoV-2 viral RNA in the cells in the compound and control wells (DMSO set), respectively.
Cytotoxicity are the same as above.
(5) Experimental results
The test results obtained during the above-described experiment are shown in Table 1.
TABLE 1

Claims (9)

1. Application of thiazole compounds shown in the general formula I or pharmaceutically acceptable salts thereof in preparing novel coronavirus medicaments for resisting SARS-COV-2,
The thiazole compound shown in the general formula I is specifically selected from:
、、、。
2. the use according to claim 1, wherein the novel anti-SARS-COV-2 coronavirus drug comprises the thiazole compound or the pharmaceutically acceptable salt thereof according to claim 1 and a pharmaceutical adjuvant.
3. The use according to claim 2, wherein the pharmaceutical excipients comprise any one or more of the following: propellants, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, tonicity modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-binders, integration agents, permeation promoters, pH adjusting agents, buffers, plasticizers, foaming agents, defoamers, thickeners, inclusion agents, humectants, flocculants and deflocculants, filter aids, and release retarders.
4. The use according to claim 2, wherein the pharmaceutical excipients comprise any one or more of the following: solvent and surfactant.
5. The use according to claim 2, wherein the medicament further comprises a medicament carrier.
6. The use according to claim 5, wherein the pharmaceutical carrier comprises microspheres, liposomes.
7. The use according to claim 2, wherein the medicament is in the form of injection, freeze-dried powder for injection or suspension.
8. The use according to claim 2, wherein the medicament is in the form of an implant, an embolic agent.
9. The use according to claim 2, wherein the pharmaceutical dosage forms are capsules, tablets, pills and oral liquids.
CN202210265352.4A 2022-03-17 2022-03-17 Application of thiazole compound in preparation of novel coronavirus medicament for resisting SARS-COV-2 Active CN114681454B (en)

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