CN114681454A - Application of thiazole compound in preparation of anti-SARS-COV-2 novel coronavirus medicine - Google Patents

Application of thiazole compound in preparation of anti-SARS-COV-2 novel coronavirus medicine Download PDF

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CN114681454A
CN114681454A CN202210265352.4A CN202210265352A CN114681454A CN 114681454 A CN114681454 A CN 114681454A CN 202210265352 A CN202210265352 A CN 202210265352A CN 114681454 A CN114681454 A CN 114681454A
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sars
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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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Shanghai Institute of Materia Medica of CAS
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
    • 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
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • 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
    • C07D277/38—Nitrogen atoms
    • C07D277/40—Unsubstituted amino or imino radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • 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
    • C07D277/38—Nitrogen atoms
    • C07D277/44—Acylated amino or imino radicals
    • C07D277/46—Acylated amino or imino radicals by carboxylic acids, or sulfur or nitrogen analogues thereof
    • 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
    • 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

Abstract

The invention discloses application of thiazole compounds in preparing a novel anti-SARS-COV-2 coronavirus medicament, belonging to the technical field of medicines. The thiazole compound with the structure shown in the general formula (I) is a brand new inhibitor for resisting SARS-CoV-23CLpro protein and is a complete guide of a novel anti-SARS-COV-2 coronavirus medicamentThe new molecular skeleton can block the synthesis of nucleic acid and protein, and provides new idea and use for preventing and/or treating SARS-COV-2 coronavirus, and has important medical research value.

Description

Application of thiazole compound in preparation of anti-SARS-COV-2 novel coronavirus medicine
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of thiazole compounds in preparation of a novel SARS-COV-2 coronavirus resistant medicine.
Background
The novel coronavirus pneumonia (COVID-19) is an infectious disease caused by the severe acute respiratory syndrome coronavirus novel coronavirus (SARS-CoV-2) which seriously threatens human health. SARS-CoV-2 is an enveloped RNA virus consisting 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 (S protein), envelope protein (E protein), and membrane protein (M protein). The nucleocapsid is assembled from nucleocapsid protein (N protein) and viral RNA. In the nucleic acid and protein synthesis phase, 2 functional open reading frames (ORF 1a/b) of coronavirus are translated into polyprotein 1a (polyprotein 1a, pp1a) and polyprotein 1ab (polyprotein 1a, pp1ab), which are cleaved by papain-like protease (3C-like protease, PLpro) and 3-chymotrypsin-like protease (3C-like protease,3CLpro) to produce a number of non-structural proteins, including RNA-dependent RNA polymerase (RdRp) involved in viral transcription and replication.
The SARS-CoV-23CLpro and PLpro proteins are the key proteins for SARS-CoV-2 key protein RdRp to become the active form. The SARS-CoV-23CLpro protein is assembled into active RdRp by hydrolyzing the virus pp1a and pp1ab proteins to form a mature product nsp 1-16. PLpro can specifically recognize and cleave the double glycine polypeptide, the polyprotein ppla (pp1ab) LXGG sequence between the N-terminal nsp1-2, nsp2-3 and nsp3-4, participate in the cleavage process of the N-terminal of the 1a (1ab) replicase protein and release the mature products nsp1, nsp2 and nsp3, and then assemble into active RdRp. SARS-CoV-2PLpro and 3CLpro play a key role in the viral replication cycle and have been identified as key drug targets for the treatment of coronaviruses. At present, there is a lack of effective anti-SARS-COV-2 novel coronavirus drugs.
Disclosure of Invention
The thiazole compound with the structure shown in the general formula (I) is a brand-new inhibitor for resisting SARS-CoV-23CLpro protein, is a brand-new molecular framework of a novel coronavirus medicine primer for resisting SARS-COV-2, can realize the aim of blocking the synthesis of nucleic acid and protein, provides a new thought and a new application for preventing and/or treating the novel coronavirus of SARS-COV-2, and has important medical research value.
One aspect of the invention provides the application of thiazole compounds shown in the following general formula I or pharmaceutically acceptable salts thereof in preparing novel anti-SARS-COV-2 coronavirus medicaments,
Figure BDA0003551529270000021
wherein, the first and the second end of the pipe are connected with each other,
R1is mono-to tri-substituted selected from: h, -OH, halogen (F, Cl, Br, I), C1-C6 alkyl, C1-C6 alkoxy, substituted or unsubstituted amino-NR3R4Substituted or unsubstituted amido-NH-C (O) -R5Substituted or unsubstituted aminoacyl-C (O) -N-R6R7；
R2Selected from substituted or unsubstituted amino-NR8R9Substituted or unsubstituted 5-7 membered heterocyclylamido-NH-C (O) -R10Substituted or unsubstituted aminoacyl-C (O) -N-R11R12；
R3、R4、R8、R9Each independently selected from H, substituted or unsubstituted C1-C6 alkyl, the substituents of the alkyl being selected from: substituted or unsubstituted aryl, heteroaryl, C3-C6 cycloalkyl; the substituent of the aryl and the heteroaryl is mono-substituted to tri-substituted and is selected from-OH, halogen (F, Cl, Br and I) and C1-C6 alkylC1-C6 alkoxy, C1-C6 alkoxycarbonyl;
R5、R10each independently selected from substituted or unsubstituted 5-7 membered heterocyclyl, aryl, heteroaryl; the substituents on the heterocyclic, aryl, heteroaryl groups are mono-to trisubstituted and are selected from: -OH, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkoxycarbonyl, halogen (F, Cl, Br, I);
R6、R7、R11、R12each independently selected from substituted or unsubstituted aryl, benzothiadiazolyl; the substituent of the aryl and the benzothiadiazolyl is mono-to tri-substituted, and is selected from: -OH, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkoxycarbonyl, halogen (F, Cl, Br, I);
the heterocyclyl or heteroaryl group contains one to three heteroatoms selected from N, O, S;
Figure BDA0003551529270000022
is a carbon-carbon bond or a carbon-hydrogen bond;
when n is 2, A is a six-membered ring; or when n is 1, A is a five-membered ring; or when n is 0, A does not form a ring.
In one embodiment of the invention, the aryl group is a benzene ring or a naphthalene ring.
The invention also provides application of the thiazole compound shown in the general formula I or the pharmaceutically acceptable salt thereof in preparing SARS-CoV-23CLpro protein inhibitor.
The invention also provides a medicine for preventing or treating SARS-COV-2 novel coronavirus pneumonia infection, wherein the medicine contains thiazole compounds shown as the general formula I or pharmaceutically acceptable salts and pharmaceutic adjuvants thereof.
The pharmaceutic adjuvant comprises any one or more of the following components: solvents, propellants, solubilizers, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavors, preservatives, suspending agents, coating materials, fragrances, antiadherents, integration agents, permeation enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulation agents, humectants, flocculants and deflocculants, filter aids, and release retardants.
In one embodiment of the invention, the medicament further comprises a medicament carrier. The drug carrier comprises microcapsules, microspheres, nanoparticles and liposomes.
In one embodiment of the present invention, the dosage form of the drug comprises any one of the following: injection, lyophilized 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 is 1 or 2, the compound is,
Figure BDA0003551529270000031
reagents and conditions: a) liquid bromine, thiourea and absolute ethyl alcohol, the temperature is 100 ℃, and the time is 5 hours; b) HATU (2- (7-azobenzotriazol) -N, N' -tetramethyluronium hexafluorophosphate), DIPEA (N, N-diisopropylethylamine), DMF (N, N-dimethylformamide), room temperature, 4 h;
weighing A, liquid bromine and thiourea, dissolving the A, the liquid bromine and the thiourea in absolute ethyl alcohol, carrying out oil bath at 100 ℃ for reaction for 5 hours, clarifying the solution in the reaction process, then becoming turbid, and precipitating a white solid, carrying out TLC (thin layer chromatography) monitoring on the complete reaction, carrying out reduced pressure distillation to remove 1/4-volume solvent, adjusting alkali by using 5% NaOH solution, and carrying out suction filtration to obtain a solid which is a compound B; dissolving a compound B, a compound C and HATU (2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate) in DMF (N, N-dimethylformamide), dropwise adding DIPEA (N, N-diisopropylethylamine), stirring at room temperature for 4h, monitoring the reaction completion, dropwise adding reaction liquid into water, separating out a solid, and performing suction filtration and drying to obtain a compound I;
B. when n is equal to 0, the compound is,
Figure BDA0003551529270000032
reagents and conditions: a) iodine simple substance, thiourea, at 110 ℃, for 10 hours; b) HATU (2- (7-azobenzotriazol) -N, N' -tetramethyluronium hexafluorophosphate), DIPEA (N, N-diisopropylethylamine), DMF (N, N-dimethylformamide), room temperature, 4 h;
weighing A', I2Thiourea is reacted in oil bath at 110 deg.c for 10 hr, after TLC monitoring reaction, the solid after pulping with methyl tert-butyl ether is added into water, pH regulated to 9-10 with 25% ammonia solution, suction filtered, dissolved in ethyl acetate and saturated NaHCO solution is used3Washing the solution for 2-3 times, drying an organic phase by using anhydrous sodium sulfate, carrying out reduced pressure distillation to obtain a compound B ', dissolving a compound B ', a compound C ', HATU (2- (7-azobenzotriazol) -N, N, N ', N ' -tetramethylurea hexafluorophosphate) in DMF (N, N-dimethylformamide), dropwise adding DIPEA (N, N-diisopropylethylamine), stirring at room temperature for 4h, monitoring the completion of the reaction, dropwise adding the reaction solution into water, separating out a solid, carrying out suction filtration and drying to obtain a 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-23CLpro protein, is a brand-new molecular skeleton of a novel SARS-COV-2 resistant coronavirus drug guide, can realize the aim of blocking the synthesis of nucleic acid and protein, 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 thiazole Compounds
Synthetic method 1, n-1 or 2
Figure BDA0003551529270000041
Reagents and conditions: a) liquid bromine, thiourea and absolute ethyl alcohol, the temperature is 100 ℃, and the time is 5 hours; b) HATU (2- (7-azobenzotriazol) -N, N' -tetramethyluronium hexafluorophosphate), DIPEA (N, N-diisopropylethylamine), DMF (N, N-dimethylformamide), room temperature, 4 h;
weighing A, liquid bromine and thiourea, dissolving the A, the liquid bromine and the thiourea in absolute ethyl alcohol, carrying out oil bath at 100 ℃ for reaction for 5 hours, clarifying the solution in the reaction process, then becoming turbid, and precipitating a white solid, carrying out TLC (thin layer chromatography) monitoring on the complete reaction, carrying out reduced pressure distillation to remove 1/4-volume solvent, adjusting alkali by using 5% NaOH solution, and carrying out suction filtration to obtain a solid which is a compound B; dissolving a compound B, a compound C and HATU (2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate) in DMF (N, N-dimethylformamide), dropwise adding DIPEA (N, N-diisopropylethylamine), stirring at room temperature for 4h, monitoring the reaction completion, dropwise adding reaction liquid into water, separating out a solid, and performing suction filtration and drying to obtain a compound I.
The specific synthetic process comprises the following steps:
step 1
Figure BDA0003551529270000051
Weighing 5-methoxy-1-indanone A (1.62g,10.0mmol, liquid bromine (1.76g,11.0mmol), thiourea (2.28g,30.0mmol) and dissolving in 20mL of absolute ethyl alcohol, carrying out oil bath at 100 ℃ for reaction for 5h, clarifying the solution in the reaction process, then becoming turbid, and precipitating white solid, monitoring the reaction by TLC, removing 1/4-volume solvent by reduced pressure distillation, adjusting alkali by using 5% NaOH solution, and carrying out suction filtration to obtain the solid which is the compound WJ-218(1.8g, 50% yield).
Step 2
Figure BDA0003551529270000052
Weighing the compound WJ-218(0.24g, 1.1mmol), 3-methoxybenzoic acid (0.15g, 1.0mmol), HATU (2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate) (0.38g, 1.0mmol) and dissolved in 5mL DMF (N, N-dimethylformamide), dropwise adding DIPEA (N, N-diisopropylethylamine) (0.39g, 3.0mmol), stirring at room temperature for 4h, monitoring the reaction completion, dropwise adding the reaction liquid into water, precipitating a solid, and performing suction filtration and drying to obtain the compound WJ-354(0.18g, 50% yield).
The following compounds were prepared in the same manner, replacing the corresponding substrates.
Figure BDA0003551529270000053
Figure BDA0003551529270000061
Figure BDA0003551529270000071
Figure BDA0003551529270000081
Figure BDA0003551529270000091
Synthesis scheme 2, n ═ 0
Figure BDA0003551529270000092
Reagents and conditions: a) iodine simple substance, thiourea, at 110 ℃, for 10 hours; b) HATU (2- (7-azobenzotriazol) -N, N' -tetramethyluronium hexafluorophosphate), DIPEA (N, N-diisopropylethylamine), DMF (N, N-dimethylformamide), room temperature, 4 h;
weighing A', I2Thiourea is reacted in oil bath at 110 deg.c for 10 hr, after TLC monitoring reaction, the solid after pulping with methyl tert-butyl ether is added into water, pH regulated to 9-10 with 25% ammonia solution, suction filtered, dissolved in ethyl acetate and saturated NaHCO solution is used3Washing the solution for 2-3 times, drying an organic phase by using anhydrous sodium sulfate, carrying out reduced pressure distillation to obtain a compound B ', dissolving a compound B ', a compound C ', HATU (2- (7-azobenzotriazol) -N, N, N ', N ' -tetramethylurea hexafluorophosphate) in DMF (N, N-dimethylformamide), dropwise adding DIPEA (N, N-diisopropylethylamine), stirring at room temperature for 4h, monitoring the reaction completion, dropwise adding the reaction solution into water, precipitating a solid, carrying out suction filtration and drying to obtain the compound B ', and finally obtaining the compound C ', HATU (2- (7-azobenzotriazol) -N, N, N ', N ' -tetramethylurea hexafluorophosphate)A compound I.
The specific synthetic process comprises the following steps:
step 1
Figure BDA0003551529270000093
A' (1.64g,10mmol), I was weighed2(2.54g,10mmol), thiourea (0.76g,10mmol), oil bath at 110 deg.C for 10h, TLC to monitor the reaction is complete, slurried solid with methyl tert-butyl ether, adding to water, adjusting pH to 9-10 with 25% ammonia, filtering, dissolving the solid with EtOAc (50mL), dissolving with saturated NaHCO3The solution was washed (20 mL. times.3) with water, and the organic phase was dried over anhydrous sodium sulfate and distilled under reduced pressure to give Compound WJ220(1.10g, 50% yield).
Step 2
Figure BDA0003551529270000101
Weighing the compound WJ-220(0.24g, 1.1mmol), 3-methoxybenzoic acid (0.15g, 1.0mmol), HATU (2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyluronium hexafluorophosphate) (0.38g, 1.0mmol) and dissolved in 5mL DMF (N, N-dimethylformamide), dropwise adding DIPEA (N, N-diisopropylethylamine) (0.39g, 3.0mmol), stirring at room temperature for 4h, monitoring the reaction completion, dropwise adding the reaction liquid into water, precipitating a solid, and performing suction filtration and drying to obtain the compound WJ-354-1(0.16g, yield 45%).
The following compounds were prepared in the same manner, replacing the corresponding substrates.
Figure BDA0003551529270000102
Figure BDA0003551529270000111
Example 28 inhibition of SARS-CoV-23CLpro protease by H-indeno [1,2-d ] thiazoles:
based on the basic characteristic that SARS-CoV-23CLpro protein is a proteolytic enzyme, a screening system for detecting the activity of SARS-CoV-23CLpro protein by a fluorescence method is established. SARS-CoV-23CLpro protein can specifically cut off Gln (Q) substrate at position P1, and its activity detection can use fluorescent polypeptide as substrate, and can reflect its proteolytic enzyme activity by detecting fluorescent signal generation. SARS-CoV-23CLpro protease activity assay uses fluorescently labeled polypeptide substrate (sequence: MCA-AVLQSGFRK(DNP) K), SARS-CoV-23CLpro enzyme solution (diluted to 0.5. mu.M in the reaction solution) and compound incubated in reaction solution (20mM Tris, pH7.3,150mM NaCl,1mM EDTA, 1% Glycerol, 0.01% Tween-20) at room temperature for 10 minutes, substrate (40. mu.M, total volume of reaction solution 50. mu.L) was added, and after the start of the reaction, the fluorescence intensity (excitation light 320nm, emission light 405nm) of the reaction solution was measured with an EnVision Multilabel Reader (PerkinElmer), with three replicate wells per dose. The fluorescence value of the control wells (DMSO) was set to 100%, and the activity rate of the compound-treated wells was expressed as a percentage of the control wells.
8H-indeno [1,2-d ]]Thiazole compounds are detected to inhibit 3CLpro protease activity at different concentrations (from 0.08 mu M to 20 mu M), and the activity dose dependence relationship, namely IC is tested50/EC50Values, obtained by nonlinear fitting of sample concentrations by sample activity, were calculated as Graphpad Prism 8, the model used for fitting was a four-parameter dose-response integral model (variable slope), and the bottom and top of the fitted curve were set to 0 and 100 for most inhibitor screening models. In general, each sample was tested with multiple wells (n.gtoreq.3) and the results were expressed as Standard Deviation (SD) or Standard Error (SE). Computing IC50。
Example 38 inhibition of SARS-CoV-2PLpro protease by H-indeno [1,2-d ] thiazoles:
based on the basic characteristic that SARS-CoV-2PLpro protein is a proteolytic enzyme, a screening system for detecting the activity of SARS-CoV-2PLpro protein by fluorescence method is established. SARS-CoV-2PLpro protein can specifically recognize and cut diglycine polypeptide, its activity detection can adopt fluorescent polypeptide as substrate, and utilizes the detection of fluorescent signal to react its proteolytic enzyme activity. SARS-CoV-2PLpro protease assay Using coumarin-labeled polypeptide substrate (sequence: Z-RLRGG-AMC), SARS-CoV-2PLpro enzyme solution (diluted to 40nM in the reaction) was incubated with compound in reaction solution (20mM Tris pH8.0, 0.01% Tween20,0.5mM DTT) for 10 minutes at room temperature, substrate (50. mu.M, total volume of reaction solution 50. mu.L) was added, and after the reaction was initiated, the reaction solution was examined for fluorescence intensity (excitation light 355nM, emission light 460nM) using an EnVision Multilabel Reader (Perkinelmer), three replicate wells per dose. The fluorescence value of the control wells (DMSO) was set as 100%, and the activity rate of the compound-treated wells was expressed as a percentage of the control wells.
8H-indeno [1,2-d ]]Thiazole compounds are used for detecting the inhibition effect of PLpro protease activity at different concentrations (the concentration is from 0.08 mu M to 20 mu M), and the dependence relationship of the activity dose, namely IC is tested50/EC50Values, obtained by nonlinear fitting of sample concentrations by sample activity, were calculated as Graphpad Prism 8, the model used for fitting was a four-parameter dose-response integral model (variable slope), and the bottom and top of the fitted curve were set to 0 and 100 for most inhibitor screening models. In general, each sample was tested with multiple wells (n.gtoreq.3) and the results were expressed as Standard Deviation (SD) or Standard Error (SE). Computing IC50。
Fifth, test method
(1) Cell culture and treatment:
vero E6 cells were cultured in a T75 cell culture flask, maintained in a incubator at 37 ℃ and 5% CO2, subcultured every 48 hours at a ratio of 1:3, and the culture medium formulation: 90% DMEM (Gibco Invitrogen), 10% fetal bovine serum (Gibco Invitrogen).
One day before the test, the cell culture fluid was aspirated, rinsed once with extracellular fluid, and then 2mL of Trypsin solution was added and digested at room temperature for 1-2 minutes. The cells were counted by adding medium to neutralize the pancreatin and transferred to 48-well plates at 50000 cells per well.
(2) Preparation of compound:
the test compounds were dissolved in DMSO respectively in 40mM stock solution, and on the day of the test, the compound stock solution was serially diluted 10-fold with DMEM, i.e., 1. mu.L of the compound stock solution was added to 9. mu.L of DMSO, and after 2 10-fold dilutions, 0.4mM dilutions were obtained and then diluted 40-fold to 10. mu.M.
Preparation of positive control Compound chloroquine phosphate, compound was dissolved in PBS to 2mM stock.
The DMSO content of the final assay concentration of the compound did not exceed 0.2%, and this concentration of DMSO had no effect on SARS-CoV-2 replication.
(3) Detecting the Effect of Compounds on viral replication
The supernatant was removed from the cells in 48-well plates, diluted compounds were added to each well, incubated for 1 hour, SARS-CoV-2 was added to a biosafety level 3 (BSL-3) laboratory at a multiplicity of infection (MOI) of 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, incubating and staining are carried out by using SARS-CoV-2NP protein rabbit polyclonal antibody, 488 fluorescent antibody and DAPI dye in sequence, a 48-hole plate is placed under a fluorescent microscope imaging system for automatic scanning and photographing, fluorescent signals are counted by using Image J software according to the virus load (green fluorescence) and the cell number (blue fluorescence) of a DMSO hole as controls, and the compound inhibition rate and the drug toxicity are respectively calculated.
(4) Data processing and statistics
The data analysis and processing adopts Excel software, and the following formula is calculated:
Inhibition％＝[1–(I/Io)]×100％
wherein, Inhibition% represents the Inhibition percentage of SARS-CoV-2 replication by the compound, and I and Io represent the fluorescence signal area of SARS-CoV-2 virus RNA in cells in compound and control wells (DMSO group), respectively.
The method for calculating cytotoxin is the same as above.
(5) Results of the experiment
The test results obtained during the above experiment are shown in table 1.
TABLE 1
Figure BDA0003551529270000131
Figure BDA0003551529270000141
Figure BDA0003551529270000151

Claims (10)

1. The thiazole compound shown in the general formula I or the pharmaceutically acceptable salt thereof is applied to the preparation of the novel SARS-COV-2 coronavirus resistant medicament,
Figure FDA0003551529260000011
wherein the content of the first and second substances,
R1is mono-to tri-substituted selected from: h, -OH, halogen (F, Cl, Br, I), C1-C6 alkyl, C1-C6 alkoxy, amino-NR3R4amido-NH-C (O) -R5aminoacyl-C (O) -N-R6R7；
R2Selected from amino-NR8R9amido-NH-C (O) -R10aminoacyl-C (O) -N-R11R12；
R3、R4、R8、R9Each independently selected from H, substituted or unsubstituted C1-C6 alkyl, the substituents of the alkyl being selected from: substituted or unsubstituted aryl, heteroaryl, C3-C6 cycloalkyl; the substituent of the aryl and the heteroaryl is mono-substituted to tri-substituted and is selected from-OH, halogen, C1-C6 alkyl, C1-C6 alkoxy and C1-C6 alkoxycarbonyl;
R5、R10each independently selected from substituted or unsubstituted 5-7 membered heterocyclic group, aryl group, heteroaryl groupA group; the substituents on the heterocyclic, aryl, heteroaryl groups are mono-to trisubstituted and are selected from: -OH, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkoxycarbonyl, halogen;
R6、R7、R11、R12each independently selected from substituted or unsubstituted aryl, benzothiadiazolyl; the substituent of the aryl and the benzothiadiazolyl is mono-to tri-substituted, and is selected from: -OH, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkoxycarbonyl, halogen;
one to three heteroatoms of N, O, S are contained in the heterocyclic or heteroaryl group;
Figure FDA0003551529260000012
is a carbon-carbon bond or a carbon-hydrogen bond;
n is 0 to 2; when n is 2, A is a six-membered ring; or when n is 1, A is a five-membered ring; or when n is 0, A does not form a ring.
2. Use according to claim 1, wherein aryl is a benzene or naphthalene ring.
3. Use of the thiazole compound or the pharmaceutically acceptable salt thereof according to claim 1 for the preparation of a SARS-CoV-23CLpro protein inhibitor.
4. A medicament for preventing or treating SARS-COV-2 coronavirus pneumonia infection, which comprises the thiazole compound according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.
5. The medicament as claimed in claim 4, wherein the pharmaceutical excipients comprise any one or more of: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation enhancers, pH adjusting agents, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulation agents, humectants, flocculants and deflocculants, filter aids, and release retardants.
6. The medicament of claim 4, further comprising a medicament carrier.
7. The drug of claim 6, wherein the drug carrier comprises a microsphere or a liposome.
8. The drug according to claim 4, wherein the drug is in the form of injection, freeze-dried powder for injection or suspension.
9. The medicament of claim 4, wherein the medicament is in the form of an implant or an embolic agent.
10. The medicament of claim 4, wherein the medicament is in the form of capsules, tablets, pills and oral liquid.
CN202210265352.4A 2022-03-17 2022-03-17 Application of thiazole compound in preparation of anti-SARS-COV-2 novel coronavirus medicine Pending CN114681454A (en)

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Citations (3)

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CN1102528A (en) * 1993-02-15 1995-05-10 株式会社大制药工场 Phosphonic diester derivative
WO2006013054A1 (en) * 2004-08-05 2006-02-09 F. Hoffmann-La Roche Ag Substituted n-acyl-2-aminothiazoles
WO2017066225A1 (en) * 2015-10-14 2017-04-20 Texas Tech University System Pharmacoperones of the v2 receptor

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