CN113773275A

CN113773275A - Antiviral compound and preparation method thereof

Info

Publication number: CN113773275A
Application number: CN202110895286.4A
Authority: CN
Inventors: 王伟; 陆永章; 谭进辉
Original assignee: Guangdong Zhongke Drug R&d Ltd
Current assignee: Guangdong Zhongke Drug R&d Ltd
Priority date: 2020-12-29
Filing date: 2021-08-05
Publication date: 2021-12-10
Anticipated expiration: 2041-08-05
Also published as: WO2022142727A1; CN113416172A; CN113773275B; WO2022142731A1; CN113416172B; CN112707874A

Abstract

The invention discloses an antiviral compound and a preparation method thereof. The structural formula of the compound is shown as formula I, wherein in the formula I, R¹Selected from: mono-or poly-substituted H, F, methyl, trifluoromethyl, preferably H; r²Selected from: H. straight or substituted alkanes (C1-C6), preferably methyl, isopropyl; r³Selected from: mono-or polysubstituted H, Cl, Br, F, preferably Cl; r⁴Selected from: straight-chain or substituted alkanes (C1-C6), preferably methyl, ethyl or propyl. Experiments prove that the compound has a good inhibitory effect on H1N1 influenza A virus and coronavirus, does not have toxicity to normal human cells, and can inhibit the virus and the inflammatory reaction.

Description

Antiviral compound and preparation method thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to an antiviral compound and a preparation method thereof.

Background

Influenza virus is called influenza virus for short. It is classified into types A (A), B (B) and C (C), and influenza viruses discovered in recent years are classified into types D (D). The influenza virus can cause infection and morbidity of various animals such as human, poultry, pigs, horses, bats and the like, and is a pathogen of epidemic diseases of human and animals such as human influenza, avian influenza, swine influenza, horse influenza and the like.

The typical clinical symptoms of these diseases are acute hyperpyrexia, general pain, marked debilitation and respiratory symptoms. Influenza viruses are spread primarily by airborne droplets, contact between a susceptible and infected person, or contact with contaminated items. The autumn and winter season is the high-incidence period. Human influenza is mainly caused by influenza a and influenza b viruses. Influenza a viruses often have antigenic variation and can be further classified into subtypes H1N1, H3N2, H5N1, H7N9, etc. (wherein H and N represent two surface glycoproteins of influenza viruses, respectively). Influenza viruses are not very resistant to the environment. Animal influenza viruses do not normally infect humans, human influenza viruses do not normally infect animals, but swine is the exception. Pigs can be infected with both human and avian influenza viruses, but they are also predominantly infected with swine influenza virus. After a few animal influenza viruses are adapted to humans, a human influenza pandemic can be caused.

Human coronavirus causes the common cold, Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) in humans, and has certain differences in epidemiological characteristics.

Influenza a H1N1 is a highly contagious acute respiratory disease in pigs caused by one or more swine influenza a viruses. The morbidity is often high, but the mortality is low (1-4%). The virus is spread in the herd by aerosols, direct and indirect contact and asymptomatic pigs carrying the virus. Herd epidemic may occur throughout the year. In the fall and winter in temperate zones, the incidence of disease rises. Human influenza A H1N1 usually comes from infected pigs, but some cases do not have a history of exposure to pigs or the environment in which they are located. Interpersonal transmission occurs in some cases, but is limited to close contacts and people in an enclosed environment.

Coronavirus (HCoV-229E) is a species of coronavirus. Coronaviruses belong to the order of the nested viruses, the family of coronaviruses, the genus of coronaviruses, a family of large viruses, and are widely found in nature. Coronaviruses only infect vertebrates, are associated with a variety of diseases in humans and animals, and can cause diseases in the respiratory, digestive and nervous systems of humans and animals.

Therefore, the research of an effective antiviral drug has important practical significance.

Disclosure of Invention

The invention aims to provide a compound shown as a formula I and pharmaceutically acceptable salts and solvates thereof.

The structural general formula of the compound provided by the invention is shown as formula I:

in the formula (I), the compound has the following structure,

R¹selected from: mono-or poly-substituted H, F, methyl, trifluoromethyl, preferably H;

R²selected from: H. straight or substituted alkanes (C1-C6), preferably methyl, isopropyl;

R³selected from: mono-or polysubstituted H, Cl, Br, F, preferably Cl;

R⁴selected from: straight-chain or substituted alkanes (C1-C6), preferably methyl, ethyl or propyl.

In some embodiments of the present invention, the compounds of formula I according to the present invention may be exemplified by, but are not limited to, the structures shown below:

the invention also provides a preparation method of the compound shown in the formula I.

According to the document Med. chem. Commun.,2016,7, 1441-1448, the reaction of alkylhydrazines of compound 1 (formula a) with sodium thiocyanate gives thiourea compound 2 (formula b), which is then condensed with R1-substituted o-formic benzaldehyde to give compound 3 (formula c), which is finally condensed with R1-substituted o-formic benzaldehyde³Substituted bromoacetophenone is subjected to ring closure, and a thiazole compound 4 (formula d) is easily obtained; under the action of a condensing agent, the benzamide derivative shown in the formula I is obtained. The method comprises the following specific steps:

1) reacting the compound shown in the formula a with sodium thiocyanate to obtain a compound shown in a formula b;

wherein R in the formula a²Is as defined in formula I; in the formula b, R²Is as defined in formula a;

2) reacting a compound represented by the formula b with R represented by the formula e¹Carrying out condensation reaction on substituted o-benzoic acid benzaldehyde to obtain a compound shown as a formula c;

wherein R in the formula e¹Is as defined in formula I; in the formula c, R¹Is defined by the formula e, R²Is as defined in formula b;

3) reacting a compound represented by the formula c with R represented by the formula f³Substituting bromoacetophenone for carrying out a ring closure reaction to obtain a compound shown in a formula d;

wherein R in the formula f³Is as defined in formula I, formula d wherein R¹、R²Is defined by the formula c, R³Is as defined in formula f;

4) carrying out condensation reaction on the compound shown in the formula d and the compound shown in the formula g under the action of a condensing agent to obtain a compound shown in the formula I;

wherein, X, R in the formula g⁴Is as defined in formula I.

In the step 1), the reaction conditions of the reaction are as follows: the reaction temperature is 50-100 ℃, and the reaction time is 24-72 hours; the reaction is carried out in a solvent which may be methanol, ethanol, tetrahydrofuran, acetonitrile, etc., preferably ethanol.

In the step 2), the reaction conditions of the condensation reaction are as follows: the reaction temperature is 50-100 ℃, and the reaction time is 1-3 hours; the reaction is carried out in a solvent which may be methanol, ethanol, tetrahydrofuran, acetonitrile, etc., preferably ethanol.

In the step 3), the reaction conditions of the ring closure reaction are as follows: the reaction temperature is 50-100 ℃, and the reaction time is 3-6 hours; the reaction is carried out in a solvent which may be methanol, ethanol, tetrahydrofuran, acetonitrile, etc., preferably ethanol.

In the step 4), the reaction conditions of the condensation reaction are as follows: the reaction temperature is 0-25 ℃, and the reaction time is 2-8 hours; the reaction is carried out in a solvent which may be dichloromethane, tetrahydrofuran, acetonitrile, etc., preferably dichloromethane.

Other compounds claimed in the claims of the present invention can be obtained by reference to the preparation process of the examples of the present invention.

Another object of the present invention is to provide the use of the compounds of formula I as described above.

The application provided by the invention is that the compound shown in the formula I or the pharmaceutically acceptable salt, ester and solvate thereof is applied to the following (a) and/or (b) and/or (c):

(a) the application of the compound shown in the formula I or the pharmaceutically acceptable salt, ester and solvate thereof in preparing products for treating diseases caused by viruses or viral infection;

(b) the application of the compound shown in the formula I or the pharmaceutically acceptable salt, ester and solvate thereof in preparing products for preventing diseases caused by viruses or viral infection;

(c) the application of the compound shown in the formula I or pharmaceutically acceptable salt, ester and solvate thereof in preparing a virus inhibitor;

(d) the application of the compound shown in the formula I or pharmaceutically acceptable salts, esters and solvates thereof in preparing analgesic drugs.

The product may be a medicament or a pharmaceutical formulation.

The viral inhibitor is capable of inhibiting viral replication.

The virus includes influenza virus and coronavirus.

The influenza virus may be specifically influenza a virus (H1N 1);

the coronavirus may be an alpha coronavirus and/or a beta coronavirus, and is specifically selected from HCoV-229E.

In the present invention, the disease caused by the virus may be a respiratory infectious disease.

The respiratory system infection is respiratory tract infection and/or lung infection; the respiratory tract infection can be nasopharyngitis, rhinitis, pharyngolaryngitis, tracheitis and/or bronchitis; the pulmonary infection may be pneumonia.

In the present invention, the diseases caused by influenza virus generally include acute respiratory infectious diseases caused by influenza virus.

In the present invention, the diseases caused by coronavirus generally include viral pneumonia, severe acute respiratory syndrome, and the like.

In the present invention, the coronavirus infection usually causes viral pneumonia, severe acute respiratory syndrome and other diseases.

The compound of the invention has the inhibition effect on coronavirus and H1N1 influenza A virus, has no toxicity on normal cells of human, can inhibit the generation degree of inflammatory reaction, reduces the damage of pneumonia to organisms and promotes the recovery of the organisms.

The antiviral drug or analgesic drug prepared by using the compound shown in the formula I as an active ingredient also belongs to the protection scope of the invention.

The antiviral drug can be introduced into body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spray, nasal drop, eye drop, penetration, absorption, physical or chemical mediated method; or mixed or coated with other materials and introduced into body.

If necessary, one or more pharmaceutically acceptable carriers can be added into the medicine. The carrier includes diluent, excipient, filler, binder, wetting agent, disintegrating agent, absorption enhancer, surfactant, adsorption carrier, lubricant, etc. which are conventional in the pharmaceutical field.

The above medicine can be made into tablet, powder, granule, capsule, oral liquid, unguent, cream, injection, etc.; the medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.

The invention also provides a medicine or a medicine composition, and the active ingredient of the medicine or the medicine composition is a compound shown in the formula I or pharmaceutically acceptable salt, ester and solvate thereof.

The medicament or the medicament composition has at least one of the following effects:

1) treating a disease or viral infection caused by a virus;

2) preventing a disease or viral infection caused by a virus;

3) inhibiting viruses;

4) relieving pain.

The above-mentioned drugs or pharmaceutical compositions can be prepared into dosage forms such as solutions, tablets, capsules or injections according to conventional methods known to those skilled in the art.

When the compound shown in the formula I or the pharmaceutically acceptable salt thereof provided by the invention is used for preventing and/or treating infection caused by virus, an effective amount of the compound shown in the formula I or the pharmaceutically acceptable salt thereof is administered to a subject organism.

Experiments prove that the compound has good inhibitory action on H1N1 influenza A virus and coronavirus, does not have toxicity on normal human cells, and can inhibit the degree of inflammatory reaction while resisting viruses; in addition, the compound also has a remarkable analgesic effect.

Drawings

FIG. 1 is a scheme showing the synthesis of the compound of formula I according to the present invention.

Detailed Description

The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.

Examples 1 to 13

1. Synthesis of (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-methylhydrazono) methyl) benzamide (ZONK2003-1)

1) 2-Methylaminothiourea (1-2)

Methylhydrazine (23.0g, 0.5mol), ammonium thiocyanate (38.0g, 0.5mol) and ethanol (200mL) were added to a single-neck round-bottom flask, and the mixture was heated under reflux for 72 hours. The reaction solution was cooled and concentrated, and purified by column chromatography to give 2-methylaminothiourea (44.1g, 84.0%) as an off-white solid.¹H NMR(DMSO-d6 400MHz)δ7.24(s,2 H),6.85(s,2H),3.14(s,3H).ESI-MS m/z:106.1[M+H]+.

2) (E) -2- ((2-aminomethylthiono-2-methylhydrazono) methyl) benzoic acid (1-3)

2-Methylaminothiourea (40.0g, 0.38mol), 2-carbobenzoic acid (57.0g, 0.38mol) and ethanol (300mL) were added to a single-neck round-bottom flask, and the mixture was refluxed for 2 hours. The reaction solution was cooled and concentrated, and purified by column chromatography to give (E) -2- ((2-aminomethylthiaacyl-2-methylhydrazono) methyl) benzoic acid (85.6 g, 95.0%) as a pale yellow solid.¹H NMR(DMSO-d6 400MHz)δ13.0(s,1H),8.12-7.23(m,7H),2.47(s,3 H).ESI-MS m/z:238.1[M+H]+.

3) (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-methylhydrazono) methyl) benzoic acid (1-4) (ZONK 2003-0 for short)

(E) -2- ((2-aminomethylthiaacyl-2-methylhydrazono) methyl) benzoic acid (80.0g, 0.34mol), 2-bromo-1- (2-chlorophenyl) ethanone (79.2g, 0.34mol), and ethanol (400mL) were added to a single-neck round-bottom flask, followed by reflux reaction with heating for 3 hours. The reaction solution was cooled, concentrated and purified by column chromatography to give (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-methylhydrazono) methyl) benzoic acid (124g, 98.5%) as a pale yellow solid.¹H NMR(DMSO-d6 400MHz)δ13.23 (s,1H),8.60(s,1H),8.01-7.34(m,9H),3.66(s,3H).ESI-MS m/z:372.1[M+H]+.

4) Synthesis of (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-methylhydrazono) methyl) -N- (methylsulfonyl) benzamide (ZONK2003-2)

(E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-methylhydrazono) methyl) benzoic acid (3.71g, 10mmol), 4-dimethylaminopyridine (1.22g, 10mmol), aminomethanesulfonic acid (1.11g, 10mmol) were dissolved in dichloromethane (30mL), followed by addition of dicyclohexylcarbodiimide (2.27g, 11mmol) and stirring at room temperature for 5 h. The reaction was concentrated and purified by column chromatography to give (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-methylhydrazino) methyl) -N- (methylsulfonyl) benzamide (3.41g, 76.2%) as a pale yellow solid.¹H NMR(DMSO-d6 400MHz)δ8.10 (s,1H),8.00-7.43(m,9H),3.67(s,3H),3.42(s,3H).ESI-MS m/z:449.1[M+H]+.

2. Synthesis of (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-methylhydrazono) methyl) -N- (ethylsulfonyl) benzylamine (ZONK2003-4)

(E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-methylhydrazono) methyl) benzoic acid (5.0g, 13.4mmol), ethyl sulfonamide (1.46g, 13.4mol), 4-dimethylaminopyridine (1.63g, 13.4mol) were dissolved in dichloromethane (100mL), dicyclohexylcarbodiimide (2.90g, 14.1mol) was added in an ice-water bath, and the reaction was carried out at room temperature for 5 h. The reaction was concentrated and purified by column chromatography to give (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-methylhydrazono) methyl) -N- (ethanesulfonyl) benzamide (5.27g, 85.1%) as a white solid.¹H NMR(DMSO-d₆ 400MHz)δ8.60(s,1 H),8.01-7.34(m,9H),3.66(s,3H),3.43(m,2H),1.23(m,3H).ESI-MS m/z:463.5[M+H]+.

3. Synthesis of (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-methylhydrazono) methyl) -N- (propanesulfonyl) benzamide (ZONK 2003-14 for short)

(E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-methylhydrazono) methyl) benzoic acid (5.0g, 13.4mmol), propylsulfonamide (1.65g, 13.4mol), 4-dimethylaminopyridine (1.63g, 13.4mol) were dissolved in dichloromethane (100mL), and 1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride (2.70g, 14.1mol) was added in an ice-water bath and reacted at room temperature for 5 h. The reaction was concentrated and purified by column chromatography to give (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-methylhydrazono) methyl) -N- (propanesulfonyl) benzamide (5.50g, 86.2%) as a white solid.¹H NMR(DMSO-d₆400MHz)δ8.60(s,1H),8.01-7.34(m,9H),3.66(s,3H),3.43(m,2H),1.69(m,2H),1.23(m,3 H).ESI-MS m/z:477.5[M+H]⁺.

4. Synthesis of (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-isopropylhydrazono) methyl) -N- (methylsulfonyl) benzamide (abbreviated as ZONK2003-15)

By combining the procedures of examples 1-3, (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-isopropylhydrazono) methyl) -N- (methylsulfonyl) benzamide was synthesized.¹H NMR(DMSO-d₆ 400MHz)δ8.60(s,1H),8.01-7.34 (m,9H),3.34(s,3H),3.13(m,1H),1.09(d,6H).ESI-MS m/z:477.1[M+H]⁺.

5. Synthesis of (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-isopropylhydrazono) methyl) -4-fluoro-N- (methylsulfonyl) benzamide (abbreviated as ZONK2003-16)

By combining the procedures of examples 1-3, (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-isopropylhydrazono) methyl) -4-fluoro-N- (methylsulfonyl) benzamide was synthesized.¹H NMR(DMSO-d₆ 400MHz)δ8.60(s,1 H),8.01-7.34(m,8H),3.34(s,3H),3.13(m,1H),1.09(d,6H).ESI-MS m/z:495.5[M+H]⁺.

6. Synthesis of (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-isopropylhydrazono) methyl) -N- (ethylsulfonyl) -5-fluorobenzamide (abbreviated as ZONK2003-17)

By combining the procedures of examples 1-3, (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-isopropylhydrazono) methyl) -N- (ethylsulfonyl) -5-fluorobenzamide was synthesized.¹H NMR(DMSO-d₆ 400MHz)δ8.60(s,1 H),8.01-7.34(m,8H),346(m,2H),3.25(m,1H),1.,23(m,3H)，1.09(d,6H).ESI-MS m/z:509.5[M+H]⁺.

7. Synthesis of (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-methylhydrazono) methyl) -4-fluoro-N- (methylsulfonyl) benzamide (abbreviated as ZONK2003-18)

By combining the procedures of examples 1-3, (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-methylhydrazono) methyl) -4-fluoro-N- (methylsulfonyl) benzamide was synthesized.¹H NMR(DMSO-d₆ 400MHz)δ8.60(s,1 H),8.01-7.34(m,8H),3.66(s,3H),3.42(s,3H).ESI-MS m/z:467.1[M+H]⁺.

8. Synthesis of (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-methylhydrazono) methyl) -N- (ethylsulfonyl) -4-fluorobenzamide (abbreviated as ZONK2003-19)

By combining the procedures of examples 1-3, (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) -2-methylhydrazono) methyl) -N- (ethylsulfonyl) -4-fluorobenzamide was synthesized.¹H NMR(DMSO-d₆ 400MHz)δ8.60(s,1 H),8.01-7.34(m,8H),3.66(s,3H),3.12(m,2H),1.29(m,3H).ESI-MS m/z:481.5[M+H]⁺.

9. Synthesis of (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) hydrazono) methyl) -5-methyl-N- (methylsulfonyl) benzamide (abbreviated as ZONK2003-20)

By combining the procedures of examples 1-3, (E) -2- ((2- (4- (2-chlorophenyl) thiazol-2-yl) hydrazono) methyl) -5-methyl-N- (methylsulfonyl) benzamide was synthesized.¹H NMR(DMSO-d₆ 400MHz)δ8.60(s,1H),8.01-7.34 (m,8H),3.42(s,3H),2.54(s,3H).ESI-MS m/z:449.5[M+H]⁺.

10. Synthesis of (E) -2- ((2- (4- (3, 5-dichlorophenyl) thiazol-2-yl) hydrazono) methyl) -5-methyl-N- (methylsulfonyl) benzamide (abbreviated as ZONK2003-21)

By combining the procedures of examples 1-3, (E) -2- ((2- (4- (3, 5-dichlorophenyl) thiazol-2-yl) hydrazono) methyl) -5-methyl-N- (methylsulfonyl) benzamide was synthesized.¹H NMR(DMSO-d₆ 400MHz)δ8.60(s,1H),8.01-7.34 (m,7H),3.42(s,3H),2.54(s,3H).ESI-MS m/z:483.5[M+H]⁺.

EXAMPLE 11 in vitro antiviral assay of the series of Compounds

The toxicity of ZONK2003 series compounds (compounds shown in formula I) on virus-cultured MRC-5 and MDCK cells is determined by MTT method, and antiviral test is carried out by designing proper concentration. In vitro antiviral pharmacodynamic test was performed by cytopathic effect (CPE) method to evaluate the effect of ZONK2003 series compounds on viruses causing respiratory tract diseases.

And (3) test results: the ZONK2003 series compounds have no obvious toxicity to MRC-5 and MDCK cells; the two viruses have antiviral effects of different degrees, and the antiviral effect is evaluated by a half effective rate (EC 50). EC50 for coronavirus (HCoV-229E) was 12.83. mu. mol/L, 15.00. mu. mol/L; the EC50 for influenza A virus (H1N1) was 11.75. mu. mol/L, 18.19. mu. mol/L.

And (4) test conclusion: within the concentration range of 10-20 mu mol/L, the ZONK2003 series compounds have different degrees of inhibition effects on two strains of viruses, and can promote the recovery of cells after the cells are infected by the viruses.

1. Test materials

1.1 test substances

The test substance: ZONK2003-4, batch number: 20200521, purity 98.70%; ZONK2003-2, batch number: 20200513, purity 99.01%, supplied by Guangdong Zhongke pharmaceutical research Co., Ltd. Prepared into 50.0, 25.0, 12.5, 6.25, 3.13 and 1.56 mu mol/L by DMEM medium, and stored for later use at the temperature of 2-8 ℃.

1.2 Positive control

Oseltamivir phosphate granules with the specification of 15mg multiplied by 10 are bagged, and the product of Yangtong sunshine Yangtze river pharmaceutical industry Limited company with the batch number: 0371912115, expiration date to 2021.12.11, for a positive control against influenza a virus; recombinant human interferon alpha 2b gel with specification of 1.0 × 10⁵IU/g, 10 g/count, Megaku pharmaceutical industry (fertilizer combination) Co., Ltd., for use as a positive control against coronavirus.

1.3 Virus strains

Coronavirus (HCoV-229E), accession number: ATCC-VR-740, available from ATCC in the United states; influenza A virus (H1N1), accession number ATCC-VR-95, was purchased from ATCC in the United states. All cultured and stored in a class II biosafety laboratory.

1.4 cell lines

Human embryonic lung cells (MRC-5), canine kidney cells (MDCK), were all from the limited science and technology company of Wuhan Pronace.

1.5 Primary reagents

1.6 Main instruments

BSCIIB2-1101 type biological safety cabinet, manufactured by Shanghai Ruiyang purification equipment Limited, center number: 019, YXQ-50A model vertical pressure steam sterilizer, manufactured by Shanghai Bo news medical biological instruments GmbH, center code number: 584; 3111 type CO₂Incubator, manufactured by thermo fisher corporation, usa, center number: 147.

1.7 test facility

Changsha pathogenic microorganism laboratory, laboratory grade: biosafety class II (BSL-II), docket number: changwaijie Ji Zi (2019) No. B001. The test was conducted in the central biosafety class II laboratory (BSL-II-1), performed strictly in accordance with regulations and the central SOPs.

2. Test method

2.1 cell culture

Human embryonic lung cells (MRC-5) are anchorage-dependent growing cells. The culture medium is DMEM containing 10% FBS, and when the growth state is good, passage can be performed every 2-3 d. Removing the culture medium in a purification workbench, washing with 1 × PBS for 2-3 times, then adding an appropriate amount of 0.25% Trypsin-EDTA for digestion, after about 1-3 min, adding an appropriate amount of DMEM culture medium containing 10% FBS to stop the digestion of pancreatin, blowing to form a single cell suspension, transferring into an EP tube, and centrifuging at 1000rpm for 5 min. Discarding the culture medium, adding fresh culture medium for re-suspension, and proportionally (the cell density is about 10)⁵/mL) was inoculated into a new flask and placed at 37 ℃ with 5% CO₂Culturing in an incubator.

Canine kidney cells (MDCK) are anchorage-dependent growing cells. The culture medium is DMEM containing 10% FBS, and when the growth state is good, passage can be performed every 2-3 d. Removing the culture medium in a purification workbench, washing with 1 × PBS for 2-3 times, then adding an appropriate amount of 0.25% Trypsin-EDTA for digestion, after about 2-5 min, adding an appropriate amount of DMEM culture medium containing 10% FBS to stop the digestion of pancreatin, blowing and beating into single cell suspension, transferring into an EP tube, and centrifuging at 1000rpm for 5 min. Discarding the culture medium, adding fresh culture medium for resuspension, and proportioning (cell density about 10)⁵/mL) was inoculated into a new flask and placed at 37 ℃ at 5%CO₂Culturing in an incubator.

2.2 amplification of viruses

2.2.1 coronavirus (HCoV-229E): MRC-5 cell expansion

MRC-5 cells were seeded at 75cm²In a culture bottle, when the cell density reaches 80-90%, removing part of the culture medium, covering the rest cells, adding a proper amount of HCoV-229E virus, after the virus is adsorbed on the cell surface (about 3h, gently shaking the culture plate every 30min to make the virus be adsorbed uniformly), replacing the fresh culture medium without FBS, placing at 35 ℃ and 5% CO₂Culturing in a humidified constant-temperature incubator. And (3) observing that the cells start to generate lesions until the cells do not generate lesions (generally 5-7 days), performing repeated freeze thawing method, separating at 3000rpm for 10min to remove cell residues, collecting supernate, subpackaging in a freezing storage tube, labeling, and then performing medium-short term storage at-80 ℃ or medium-long term storage in liquid nitrogen for later use.

2.2.2 influenza A Virus (H1N 1): MDCK cell expansion

MDCK cells were seeded at 75cm²In a culture bottle, when the cell density reaches 70-80%, removing part of the culture medium, covering the rest cells, adding a proper amount of H1N1 virus, after the virus is adsorbed on the cell surface (about 3H, gently shaking the culture plate every 30min to make the virus adsorbed uniformly), replacing the fresh culture medium without FBS, placing at 33 ℃ and 5% CO₂Culturing in a humidified constant-temperature incubator. Observing that the cells start to generate lesions until the cells do not generate lesions (generally 2-3 days), adopting a repeated freeze thawing method, separating at 3000rpm for 10min to remove cell residues, collecting supernate, subpackaging in a freezing storage tube, labeling, and then storing at-80 ℃ for a medium-short period or in liquid nitrogen for a long period for later use.

2.3 Virus half Tissue culture infection concentration (TCID)₅₀) Measurement of (2)

TCID was performed on the virus solutions collected at 2.2.1 and 2.2.2₅₀The determination of (1): inoculating 100 μ L of corresponding cell suspension with appropriate density into 96-well cell culture plate, culturing for 24 hr, sucking out culture solution, adding 100 μ L of virus solution diluted with cell maintenance solution (virus solution)Line 10^-1、10^-2、10^-3、10^-4、10^-5、10^-6Gradient dilution) were performed in duplicate, 10 wells for each dilution, 33 ℃, 5% CO₂Adsorbing and culturing for 3h in an incubator, sucking unadsorbed virus liquid, supplementing 100 mu L of cell vitamin growth liquid to each hole, and continuing culturing. Observing cytopathic effect (CPE) day by day under an inverted microscope, wherein the CPE is characterized in that cells are rounded, have strong refractivity, are fused with cell processes, are partially separated from walls, have filamentous processes or pseudo-podoids in cytoplasm, have irregular maps in the whole shape and form large and round fused multinuclear giant cells, and the CPE is caused by viruses in cultured cells. And the number of wells with CPE was recorded, ending with the highest dilution at which no more lesions appeared, and the extent of cytopathic effect was indicated by "- + + + + + + +": no cytopathy "-", < 25% cytopathy "+", 25% -50% cytopathy "+", 50% -75% cytopathy "+ + +",>75% of the cells were diseased "+ + + + +". Calculating TCID according to Reed-Muench formula₅₀：

TCID₅₀Log (dilution of virus with CPE less than 50% + distance ratio x dilution spacing

Wherein the distance ratio is (percentage higher than 50% -50) ÷ (percentage higher than 50% -percentage lower than 50%)

2.4 concentration profiling of test substances

2.4.1 cytotoxicity assays

ZONK2003 series compound (concentration gradient of 50.0, 25.0, 12.5, 6.25, 3.13, 1.56. mu. mol/L) and oseltamivir phosphate (concentration gradient of 20.0, 10.0, 5.0, 2.5, 1.25, 0.625. mu.g/mL) and interferon alpha 2b (concentration gradient of 1 × 10) were prepared in DMEM medium in series³、5×10²、2.5×10²、 1.25×10²、6.25×10¹、3.1×10¹IU/mL), added to the cultured MRC-5 and MDCK cells, respectively, at 37 deg.C and 5% CO₂Culturing in humidified incubator for 72h, adding MTT, culturing for 4h, measuring OD value of each well at 492nm wavelength, and calculating EC of ZONK2003 series compounds on each cell₅₀。

2.4.2 treatment of cells with CompoundsWhen toxic, 1/2IC is adopted₅₀Setting 3 test concentrations for the highest concentration by diluting down at 2-fold intervals; when the compounds are not toxic to cells, the highest concentration of 20 μmol/L is used, and 3 concentrations tested are set by dilution down at 2-fold intervals.

2.5 detection of antiviral Effect

2.5.1 cell seeding: 24h before virus infection, corresponding cell strains which grow well are used for controlling the appropriate density (about 10)⁵One/well) were inoculated in a 96-well plate at 100. mu.L/well in 5% CO at 37 ℃₂Culturing in an incubator;

2.5.2 after the cells reach about 70% -80%, sucking out part of the culture medium, covering the cells (making the viruses and the cells better adsorbed), and inoculating 100TCID₅₀After culturing the virus solution (50. mu.L/well) in an incubator for 3 hours, the medium in a 96-well plate was aspirated, and a sample solution (set according to cytotoxicity) of 200. mu.L/well prepared with a serum-free cell maintenance solution and having different concentrations was added thereto at 33 ℃ and 5% CO₂Culturing in an incubator.

2.5.3 grouping

Normal control group: group not infected with virus;

model control group: a virus-infected group;

positive control group: a commercially available control drug;

test group: infection group + different concentrations of test substance.

2.5.4 evaluation of antiviral Activity (CPE method)

Cytopathic effect was observed day by day, and the number of cytopathic and non-cytopathic wells was recorded for each concentration, with continuous observation that the cytopathic effect did not increase.

2.6 evaluation of results

The rate of cell lesion inhibition (%) was 100% based on the normal control cell lesion-free rate of 100% (1-each group of cells lesion-free wells/8) × 100%.

The test data significant figure rounding was done by rounding and the software used was counted as SPSS 16.0. The measured data is averaged + -SD

Showing that the method of Leven's test is used for checking the normality and the homogeneity of variance. If it has no statistical significance (P)>0.05), statistical analysis was performed using one-way analysis of variance (ANOVA). If ANOVA has statistical significance (P is less than or equal to 0.05), the LSD method is used for comparative analysis. If the variance is not uniform (P.ltoreq.0.05), the test is carried out by Kruskal-Wallis. If the Kruskal-Wallis Test is statistically significant (P.ltoreq.0.05), a comparative analysis is performed using Dunnett's Test (nonparametric method). The statistical result takes alpha-0.05 as the test limit, wherein P <0.05 represents the statistical significance, and P <0.01 represents the significant significance of the tested difference.

3. Test results

3.1 Effect of series of Compounds on proliferation of individual cells

As shown in tables 1-1 and 1-2, both compounds have no significant cytotoxicity to MRC-5 and MDCK within the tested concentration range (1.56-50.0 mu mol/L), so the highest concentration is set to be 20.0 mu mol/L in antiviral research. As shown in Table 2, the positive drug had interferon alpha 2b (6.25X 10) in the range of concentration tested¹～1×10³IU/mL) has no obvious cytotoxicity to MRC-5 cells and oseltamivir phosphate (0.625-20.0 mu mol/L) to MDCK cells, so the highest concentration is respectively set as 1 multiplied by 10 in antiviral research³IU/mL、20.0μmol/L。

TABLE 1-1 Effect of two Compounds on MRC-5 cell proliferation

Table 1-2 Effect of two compounds on MDCK cell proliferation

TABLE 2 Effect of Positive drugs on proliferation of individual cells

3.2 Virus titer results detection

As shown in tables 3 and 4, the amounts of TCID infected with coronavirus (HCoV-229E) and influenza A virus (H1N1) were half as large as the number of viruses₅₀Are respectively 10^-3.5/0.1mL、10^-3.850.1mL, 3.16X 10 virus doses respectively⁴Multiple, 7.08 × 10⁴When diluted twice, 0.1mL of inoculated cells can cause lesion of 50% of cells. Get 100 TCIDs₅₀The virus amount was 316-fold and 708-fold diluted for in vitro antiviral assay.

TABLE 3 amount of HCoV-229E infected with half of the viruses of MRC-5 cells

TABLE 4 amount of infection of MDCK cells with half of the virus by H1N1

3.3 Effect of ZONK2003 series Compounds on cell viability following Virus infection

Compound ZONK2003-4, ZONK2003-2 anti-coronavirus (HCoV-229E) EC₅₀Respectively 12.83 mu mol/L and 15.00 mu mol/L; compound ZONK2003-4, ZONK2003-2 anti-influenza A Virus (H1N1) EC₅₀11.75. mu. mol/L and 18.19. mu. mol/L, respectively.

EC of interferon alpha 2b against coronavirus (HCoV-229E)₅₀Is 2.42 multiplied by 10²IU/mL; EC of Oseltamivir phosphate against influenza A virus (H1N1)₅₀It was 5.06. mu. mol/L.

TABLE 5 Effect of test/control on HCoV-229E infection of MRC-5 cells

TABLE 6 Effect of two compounds on H1N1 infection of MDCK cells

4. Conclusion and evaluation

Under the present test conditions:

the compounds ZONK2003-4 and ZONK2003-2 have no obvious cytotoxicity to MRC-5 and MDCK cells within the tested concentration range (1.56-50.0 mu mol/L). The positive control drug has no obvious toxicity to MRC-5 and MDCK cells within the test concentration range (0.625-20 mmol/L).

The compounds ZONK2003-4 and ZONK2003-2 have different degrees of inhibitory effects on two strains of viruses in a test, and have EC on coronavirus (HCoV-229E)₅₀Respectively 12.83 mu mol/L and 15.00 mu mol/L; EC for influenza A virus (H1N1)₅₀11.75. mu. mol/L and 18.19. mu. mol/L, respectively.

The positive control drug has different degrees of inhibition on two strains of virus in the experiment, and has EC on coronavirus (HCoV-229E)₅₀Is 2.42 multiplied by 10²IU/mL; EC of Oseltamivir phosphate against influenza A virus (H1N1)₅₀It was 5.06. mu. mol/L.

EXAMPLE 15 protective Effect of Compounds on influenza A Virus A/FM/1/47(H1N1) infected mice

The test substance: ZONK2003-0, ZONK2003-2, ZONK 2003-4; supplied by Guangdong Chinese medicine research Co.

Oseltamivir phosphate granules with the specification of 15mg multiplied by 10 are bagged, and the product of Yangtong sunshine Yangtze river pharmaceutical industry Limited company with the batch number: 0371912115, expiration date to 2021.12.11, for a positive control against influenza a virus;

experimental materials: influenza A virus mouse lung adapted strain A/FM/1/47(H1N1), inoculated with chick embryo, collected allantoic fluid and stored. ICR mice, weight 18 ~ 22 g. During administration, the patients can take food and drink water freely, and the patients are lighted for 12 hours every day and dark for 12 hours at the temperature of 22 +/-2 ℃ and the humidity of 55-70%. The experimental method comprises the following steps: after 3 days of acclimatization, the experiment was started. Except for uninfected control group, mice in each group were lightly anesthetized with ether, and inoculated intranasally with a solution of 8 × LD diluted with physiological saline₅₀50 mu L/mouse of allantoic fluid of chicken embryo of influenza A virus/FM/1/47 (H1N1), mice of the positive control oseltamivir group and the example compound group were first gavaged 2H after infection, and each compound was orally administered at a dose of 10 mu mol/kg, 20 mu mol/kg, and 30 mu mol/kg, twice daily for 5 days. Survival of mice was observed over 14 days and the mortality protection rate of drug to mice was calculated (mortality protection rate-model group mortality-experimental group mortality).

TABLE 7 protective Effect of Compounds on influenza A Virus (H1N1 influenza A Virus) infected mice

Example 16: relieving effect of drug on mouse lung inflammation caused by influenza virus H1N1 infection

The experimental method comprises the following steps: after 3 days of acclimatization, the experiment was started. Except for uninfected control group, mice in each group were lightly anesthetized with ether, and inoculated intranasally with a solution of 8 × LD diluted with physiological saline₅₀50 μ L/mouse of allantoic fluid of chick embryo of influenza A/FM/1/47(H1N1), positive control oseltamivir group mouse and test administration group after 24H of virus infectionThe administration is carried out by first intragastric administration at 80mg/kg, and then 1 time per day, and the normal saline is orally administered to the virus control group and the uninfected control group according to the same method, 1 time per day, and the administration volume is 0.1mL/10g body weight. For a total of 5 days. And 3 mice are taken out and weighed in each group on the 6 th day, the eyeballs are removed, bloodletting is performed to kill the mice, the whole lung is taken out and weighed, and the lung index and the inhibition rate of the lung index are calculated.

Percent weight loss-weight before dosing-weight after dosing/weight before dosing x 100%

Lung index ═ lung weight of mouse/body weight of mouse × 100

Pulmonary index inhibition (%) is mean pulmonary index of virus control group-mean pulmonary index of administration group/mean pulmonary index of virus control group × 100%

TABLE 8

The experimental results are as follows: therefore, the compound of the embodiment has obvious protection and inhibition effects on lung inflammation caused by influenza virus, and the effect is better than that of an oseltamivir control group and a ZONK2003-0 group.

Example 17: ZONK2003 drug rat pharmacokinetic experiment

The male rats were divided into 4 groups of 3 in ZONK2003-0 injection and oral administration, and 3 in ZONK2003-2 injection and oral administration, respectively, and 6 in ZONK2003-2 oral administration, respectively, 10 discrete time points were collected for each rat.

An LC-MS/MS analysis method for determining the concentrations of ZONK2003-0 and ZONK2003-2 in ICR mouse whole blood is established. The obtained blood concentration data adopts pharmacokinetic processing software Pharsight Phoenix WinNonlin 8.0 non-atrial chamber model to calculate related pharmacokinetic parameters.

The rats are gavaged with ZONK2003-2, the oral dosage is 25mg/kg, and the injection ZONK2003-2 is 1.21 mg/kg;

rats gavage ZONK2003-0 with an oral dose of 2.51mg/kg and an injection ZONK2003-0 of 1.00mg/kg,

detecting blood concentration, and calculating pharmacokinetic parameters:

solvent: 20% Solutol HS-15/normal saline

The detailed results are shown in the following table.

TABLE 9 measurement of plasma concentration of prototype drug (ng/mL) in rats after 1.00mg/kg of ZONK2003-0 was administered to the tail vein of rats

TABLE 10 Primary pharmacokinetic parameters of proto-drugs after 1.00mg/kg of ZONK2003-0 administered to the tail vein of rats

TABLE 11 measurement of prototype drug plasma concentration in rats (ng/mL) after intragastric administration of 2.51mg/kg of ZONK2003-0 to rats

TABLE 12 Primary pharmacokinetic parameters of proto-drug after gastric gavage of 2.51mg/kg ZONK2003-0 in rats

TABLE 13 Primary pharmacokinetic parameters of proto-drug after 1.21mg/kg of ZONK2003-2 administered to rat tail vein

TABLE 14 ZONK2003-2 gavage 25mg/kg group rats prototype drug plasma concentration test results (ng/mL)

ND: not detected, i.e. the measured value is lower than the lower limit point of the quantification after the blood concentration reaches the peak

TABLE 15 Primary pharmacokinetic parameters of proto-drugs after gastric gavage of 25mg/kg ZONK2003-2 in rats

As can be seen from the above table, the absolute bioavailability F of ZONK2003-0 was 62.5%, and the absolute bioavailability F of ZONK2003-2 group was 106.0%.

Example 18: experiment on analgesic effect of ZONK2003 series compounds

Effects of the Compounds of the examples on writhing in NIH mice caused by glacial acetic acid

NIH mice: SPF grade, half male and female, 15-17g, 110, provided by Guangdong province medical laboratory animal center, ZONK2003-0, ZONK2003-2, ZONK2003-4, ZONK2003-14, ZONK 2003-18; supplied by Guangdong Chinese medicine research Co.

2. Experimental methods and results

NIH mice 110, male, weight 15-17 g. And (3) quarantine for 3 days, and randomly dividing the plants into 10 groups according to the weight after the quarantine is finished, namely: model group, experimental drug group. In the experiment, the tail vein of each mouse is given with corresponding drugs, and the administration volume is as follows: 0.1ml/10g body weight, the model group was given an equal volume of physiological saline. 0.7% (0.7g/100mL) HAc was intraperitoneally injected 0.5 hour after administration at 10mL/kg body weight for pain, and then writhing reaction was immediately observed in each mouse for 15min, and the number of writhing was recorded and the inhibition rate was calculated.

As shown in Table 17, the number of writhing of the NIH mice caused by glacial acetic acid (p <0.05 or p <0.01) can be remarkably inhibited by each dose group and the model group, and the action intensity is better than that of the ZONK2003-0 group.

TABLE 17 Effect of Compounds on glacial acetic acid-induced writhing in NIH mice

Example 19DHODH enzyme inhibitory Activity assay

The results of the DHODH enzyme activity tests for ZONK2003-0 and ZONK2003-2, Brequinar are as follows:

watch 18

ZONK2003-2 differs from ZONK2003-0 in that ZONK2003-2 does not exert an antiviral effect through inhibition of DHODH enzyme.

Claims

1. A compound with a structural general formula shown in formula I or pharmaceutically acceptable salt and solvate thereof:

in the formula (I), the compound has the following structure,

R¹selected from: mono-or poly-substituted H, F, methyl, trifluoromethyl;

R²selected from: H. linear or substituted C1-C6 alkanes;

R³selected from: mono-or polysubstitutedH, Cl, Br, F;

R⁴selected from: straight chain or substituted C1-C6 alkanes.

2. The compound according to claim 1, or a pharmaceutically acceptable salt, solvate thereof, wherein:

the R is¹Is selected from H;

or, said R²Selected from methyl or isopropyl;

or, said R³Selected from Cl;

or, said R⁴Selected from methyl, ethyl or propyl.

3. A process for the preparation of a compound of formula I as claimed in claim 1 or 2, comprising the steps of:

3) reacting a compound represented by the formula c with R represented by the formula f³Substituting bromoacetophenone for carrying out a ring closure reaction to obtain a compound shown as a formula d;

wherein, X, R in the formula g⁴Is as defined in formula I.

4. The method of claim 3, wherein:

in the step 1), the reaction conditions of the reaction are as follows: the reaction temperature is 50-100 ℃, and the reaction time is 24-72 hours; the reaction is carried out in a solvent selected from any one of the following: methanol, ethanol, tetrahydrofuran, acetonitrile, etc., preferably ethanol;

in the step 2), the reaction conditions of the condensation reaction are as follows: the reaction temperature is 50-100 ℃, and the reaction time is 1-3 hours; the reaction is carried out in a solvent selected from any one of the following: methanol, ethanol, tetrahydrofuran, acetonitrile, preferably ethanol;

in the step 3), the reaction conditions of the ring closing reaction are as follows: the reaction temperature is 50-100 ℃, and the reaction time is 3-6 hours; the reaction is carried out in a solvent selected from any one of the following: methanol, ethanol, tetrahydrofuran, acetonitrile, preferably ethanol;

in the step 4), the reaction conditions of the condensation reaction are as follows: the reaction temperature is 0-25 ℃, and the reaction time is 2-8 hours; the reaction is carried out in a solvent selected from any one of the following: dichloromethane, tetrahydrofuran, acetonitrile, preferably dichloromethane.

5. The compound of formula I or its pharmaceutically acceptable salt, solvate, according to claim 1, wherein the compound is selected from the following (a) and/or (b) and/or (c):

6. Use according to claim 5, characterized in that:

the product is a medicament or pharmaceutical formulation; the viral inhibitor is capable of inhibiting viral replication;

the virus includes influenza virus and coronavirus.

7. Use according to claim 6, characterized in that: the influenza virus is influenza A virus; the coronavirus is HCoV-229E.

8. A medicine or pharmaceutical composition comprises compound shown in formula I or its pharmaceutically acceptable salt and solvate as active ingredient;

1) treating a disease or viral infection caused by a virus;

2) preventing a disease or viral infection caused by a virus;

3) inhibiting viruses;

4) relieving pain.

9. The medicament or pharmaceutical composition of claim 8, wherein:

the virus includes influenza virus and coronavirus.

10. The medicament or pharmaceutical composition of claim 9, wherein: the influenza virus is influenza A virus; the coronavirus is HCoV-229E.