WO2022142727A1 - 一种抗病毒化合物及其制备方法 - Google Patents

一种抗病毒化合物及其制备方法 Download PDF

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WO2022142727A1
WO2022142727A1 PCT/CN2021/128879 CN2021128879W WO2022142727A1 WO 2022142727 A1 WO2022142727 A1 WO 2022142727A1 CN 2021128879 W CN2021128879 W CN 2021128879W WO 2022142727 A1 WO2022142727 A1 WO 2022142727A1
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formula
virus
reaction
compound shown
influenza
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French (fr)
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王伟
陆永章
谭进辉
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广东中科药物研究有限公司
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    • 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
    • C07D277/50—Nitrogen atoms bound to hetero atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/04—Drugs for disorders of the respiratory system for throat disorders
    • 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
    • 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
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6536—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having nitrogen and sulfur atoms with or without oxygen atoms, as the only ring hetero atoms
    • C07F9/6539—Five-membered rings

Definitions

  • the invention belongs to the field of medicine, and in particular relates to an antiviral compound and a preparation method thereof.
  • Influenza virus is referred to as influenza virus. It is divided into three types: A (A), B (B), and C (C). Influenza viruses discovered in recent years will be classified as type D (D). Influenza virus can cause infection and disease in humans, birds, pigs, horses, bats and other animals.
  • Influenza viruses are mainly spread through airborne droplets, contact between susceptible and infected persons, or contact with contaminated items. Generally, autumn and winter are the high incidence period. Human influenza is mainly caused by influenza A and B viruses. Influenza A virus frequently undergoes antigenic variation and can be further divided into subtypes such as H1N1, H3N2, H5N1, and H7N9 (where H and N represent the two surface glycoproteins of influenza virus, respectively). Influenza viruses are not very resistant to the outside world. Animal influenza viruses do not usually infect humans, and human influenza viruses do not usually infect animals, with the exception of pigs. Pigs can be infected with both human and avian influenza viruses, but they are mainly infected with swine influenza viruses. A small number of animal influenza viruses can cause a human influenza pandemic after being adapted to humans.
  • SARS severe acute respiratory syndrome
  • MERS Middle East respiratory syndrome
  • Influenza A (H1N1) is a highly contagious acute respiratory disease of swine caused by one or more swine influenza A viruses. Morbidity tends to be high, but mortality is low (1 to 4%). The virus spreads in herds through aerosols, direct and indirect contact, and asymptomatic pigs that carry the virus. Swine outbreaks can occur throughout the year. Incidence increases during autumn and winter in temperate zones. Influenza A (H1N1) in humans usually comes from infected pigs, but some human cases have no history of contact with pigs or their environment. Human-to-human transmission has occurred in some cases, but limited to close contacts and people in closed settings.
  • Coronavirus (HCoV-229E) is a type of coronavirus. Coronaviruses belong to the order Nesteviridae, the family Coronaviridae, and the genus Coronaviridae. They are a large virus family that exists widely in nature. Coronaviruses only infect vertebrates and are associated with a variety of diseases in humans and animals, and can cause respiratory, gastrointestinal and nervous system diseases in humans and animals.
  • the object of the present invention is to provide a compound represented by formula I and pharmaceutically acceptable salts and solvates thereof.
  • R 1 is selected from: mono- or poly-substituted H, F, methyl, trifluoromethyl, preferably H;
  • R 2 is selected from: H, straight-chain or substituted alkane (C1-C6), preferably methyl, isopropyl;
  • R 3 is selected from: mono- or poly-substituted H, Cl, Br, F, preferably Cl;
  • R 4 is selected from: linear or substituted alkanes (C1-C6), preferably methyl, ethyl or propyl.
  • the compound shown in formula I of the present invention can be enumerated as the structure shown below, but is not limited to the following structure:
  • the present invention also provides a preparation method of the compound represented by the above formula I.
  • R 2 in formula a is the same as formula I;
  • R 2 in formula b is the same as formula a;
  • R 1 in formula e is the same as formula I;
  • R 1 in formula c is the same as formula e, and the definition of R 2 is the same as formula b;
  • R 3 in formula f is the same as formula I
  • the definitions of R 1 and R 2 in formula d are the same as formula c
  • the definition of R 3 is the same as formula f;
  • the reaction conditions of the reaction are: the reaction temperature is 50-100 ° C, and the reaction time is 24-72 hours; the reaction is carried out in a solvent, and the solvent can be methanol, ethanol, tetrahydrofuran, acetonitrile, etc., preferably Ethanol.
  • the reaction conditions of the condensation reaction are: the reaction temperature is 50-100 ° C, the reaction time is 1-3 hours; the reaction is carried out in a solvent, and the solvent can be methanol, ethanol, tetrahydrofuran, acetonitrile, etc., Ethanol is preferred.
  • the reaction conditions of the ring-closing reaction are: the reaction temperature is 50-100 ° C, and the reaction time is 3-6 hours; the reaction is carried out in a solvent, and the solvent can be methanol, ethanol, tetrahydrofuran, acetonitrile, etc. , preferably ethanol.
  • the reaction conditions of the condensation reaction are: the reaction temperature is 0-25°C, and the reaction time is 2-8 hours; the reaction is carried out in a solvent, and the solvent can be dichloromethane, tetrahydrofuran, acetonitrile, etc., Dichloromethane is preferred.
  • Another object of the present invention is to provide the application of the compound represented by the above formula I.
  • the application provided by the present invention is that the application of the compound represented by formula I or its pharmaceutically acceptable salt, ester and solvate is the following (a) and/or (b) and/or (c):
  • the product may be a drug or a pharmaceutical formulation.
  • the viral inhibitor is capable of inhibiting viral replication.
  • the virus includes influenza virus, coronavirus.
  • influenza virus can specifically be influenza A virus (H1N1);
  • the coronavirus can be alpha coronavirus and/or beta coronavirus, specifically selected from HCoV-229E.
  • the disease caused by the virus may be an infectious disease of the respiratory system.
  • the respiratory infection is a respiratory infection and/or a pulmonary infection;
  • the respiratory infection can be nasopharyngitis, rhinitis, pharyngitis, tracheitis and/or bronchitis;
  • the pulmonary infection can be pneumonia.
  • the diseases caused by influenza virus generally include acute respiratory infectious diseases caused by influenza virus and the like.
  • the diseases caused by the coronavirus usually include viral pneumonia, severe acute respiratory syndrome and the like.
  • the coronavirus infection usually causes diseases such as viral pneumonia and severe acute respiratory syndrome.
  • the compound of the present invention has inhibitory effect on coronavirus and H1N1 influenza A virus at the same time, and has no toxicity to normal human cells, can inhibit the degree of inflammatory reaction, reduce the damage of pneumonia to the body, and promote the recovery of the body.
  • the antiviral or analgesic drug prepared with the compound represented by formula I as the active ingredient also belongs to the protection scope of the present invention.
  • the antiviral drug can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spray, nasal instillation, eye instillation, penetration, absorption, physical or chemical mediation; or mixed with other substances or Introduce into the body after wrapping.
  • the carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants and the like conventional in the pharmaceutical field.
  • the above-mentioned medicines can be made into various forms such as tablets, powders, granules, capsules, oral liquids, ointments, creams, injections, etc.
  • the medicines of the above-mentioned various dosage forms can be prepared according to conventional methods in the pharmaceutical field.
  • the present invention also provides a medicine or a pharmaceutical composition, the active ingredient of which is the compound represented by formula I or a pharmaceutically acceptable salt, ester or solvate thereof.
  • 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.
  • an effective amount of the compound of formula I or a pharmaceutically acceptable salt thereof is administered to a subject organism.
  • the compounds described in the present invention have been confirmed by experiments that they not only have a good inhibitory effect on the H1N1 influenza A virus, but also have a good inhibitory effect on the coronavirus.
  • the degree of simultaneous inhibition of the inflammatory response by the virus; in addition, the compound also has a significant analgesic effect.
  • Fig. 1 is the synthetic route diagram of the compound represented by formula I of the present invention.
  • the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.
  • the methods are conventional methods unless otherwise specified.
  • the raw materials can be obtained from open commercial sources unless otherwise specified.
  • the MTT method was used to determine the toxic effects of ZONK2003 series compounds (compounds shown in formula I) on MRC-5 and MDCK cells cultured with viruses, and appropriate concentrations were designed to conduct antiviral tests.
  • the cytopathic effect (CPE method) was used to conduct in vitro antiviral pharmacodynamics tests to evaluate the effects of ZONK2003 series compounds on viruses that cause respiratory diseases.
  • ZONK2003 series compounds have no obvious toxicity to MRC-5 and MDCK cells; they have different degrees of antiviral effects on both strains of viruses, and their antiviral effects are evaluated by half effective rate (EC50).
  • the EC50 for coronavirus (HCoV-229E) was 12.83 ⁇ mol/L and 15.00 ⁇ mol/L; the EC50 for influenza A virus (H1N1) was 11.75 ⁇ mol/L and 18.19 ⁇ mol/L.
  • ZONK2003 series compounds have different degrees of inhibitory effect on the two strains of viruses, and can promote the recovery of cells after virus infection.
  • Test substance ZONK2003-4, batch number: 20200521, purity 98.70%; ZONK2003-2, batch number: 20200513, purity 99.01%, provided by Guangdong Zhongke Pharmaceutical Research Co., Ltd.
  • the DMEM medium was prepared into 50.0, 25.0, 12.5, 6.25, 3.13, 1.56 ⁇ mol/L, and stored at 2-8 °C for later use.
  • Oseltamivir Phosphate Granules 15mg ⁇ 10 bags, product of Yichang Dongguang Changjiang Pharmaceutical Co., Ltd., batch number: 0371912115, valid until 2021.12.11, positive control drug for anti-influenza A virus; recombinant human interference Prime ⁇ 2b gel, specification 1.0 ⁇ 10 5 IU/g, 10g/piece, Zhaoke Pharmaceutical (Hefei) Co., Ltd., used as a positive control drug for anti-coronavirus.
  • Coronavirus (HCoV-229E), code: ATCC-VR-740, purchased from ATCC in the United States; influenza A virus (H1N1), code number ATCC-VR-95, purchased from ATCC in the United States. All were cultured and stored in a Class II biosafety laboratory.
  • MRC-5 Human embryonic lung cells (MRC-5) and canine kidney cells (MDCK) were obtained from Wuhan Proceed Life Science and Technology Co., Ltd.
  • BSCIIB2-1101 biological safety cabinet produced by Shanghai Ruiyang Purification Equipment Co., Ltd., center number: 019, YXQ-50A vertical pressure steam sterilizer, produced by Shanghai Boxun Medical Biological Instrument Co., Ltd., center number: 584; 3111 Type CO 2 incubator, produced by ThermoFisher, USA, center number: 147.
  • Human embryonic lung cells (MRC-5) are adherent growing cells.
  • the medium is DMEM medium containing 10% FBS.
  • DMEM medium containing 10% FBS.
  • passage can be carried out every 2-3 days.
  • Discard the medium in the purification bench wash with 1 ⁇ PBS for 2-3 times, and then add an appropriate amount of 0.25% Trypsin-EDTA for digestion. After about 1-3 min, when the cells fall off, add an appropriate amount of DMEM containing 10% FBS to culture To stop the digestion of trypsin, it was pipetted into a single cell suspension, transferred to an EP tube, and centrifuged at 1000 rpm for 5 min.
  • Discard the medium add fresh medium to resuspend, inoculate it into a new culture flask at a certain ratio (the cell density is about 10 5 /mL), and place it in a 37° C., 5% CO 2 incubator for cultivation.
  • Canine kidney cells are adherent growing cells.
  • the medium is DMEM medium containing 10% FBS.
  • DMEM medium containing 10% FBS.
  • passage can be carried out every 2-3 days.
  • Discard the medium in the purification workbench wash with 1 ⁇ PBS for 2-3 times, and then add an appropriate amount of 0.25% Trypsin-EDTA for digestion.
  • trypsin it was pipetted into a single cell suspension, transferred to an EP tube, and centrifuged at 1000 rpm for 5 min.
  • Discard the medium add fresh medium to resuspend, inoculate it into a new culture flask at a certain ratio (the cell density is about 10 5 /mL), and place it in a 37° C., 5% CO 2 incubator for cultivation.
  • MRC-5 cells were inoculated into a 75cm 2 culture flask. When the cell density reached 80-90%, part of the medium was removed, and the rest just covered the cells. An appropriate amount of HCoV-229E virus was added, and the virus was adsorbed on the cell surface. (About 3h, shake the culture plate gently every 30min to make the virus adsorb evenly), replace the fresh medium without FBS, and place it in a 35°C, 5% CO2 humidified constant temperature incubator. When the cells start to develop lesions and no longer develop lesions (usually 5 to 7 days), the freeze-thaw method is used repeatedly, and the cells are separated at 3000 rpm for 10 minutes to remove cell residues. -80°C for medium and short-term storage or liquid nitrogen for medium and long-term storage, for future use.
  • the MDCK cells were inoculated into a 75cm 2 culture flask. When the cell density reached 70-80%, part of the medium was removed, and the rest just covered the cells. An appropriate amount of H1N1 virus was added, and after the virus was adsorbed on the cell surface (about 3h or so) , gently shake the culture plate every 30 min to make the virus adsorb evenly), replace the fresh medium without FBS, and place it in a 33°C, 5% CO humidified incubator. Observe that when the cells begin to develop lesions and no longer develop lesions (usually 2 to 3 days), adopt repeated freezing and thawing method, and separate the cells at 3000 rpm for 10 min to remove cell residues. Short-term storage at -80°C or long-term storage in liquid nitrogen for future use.
  • the virus liquid collected in 2.2.1 and 2.2.2 was tested for TCID 50 : inoculate 100 ⁇ L of the corresponding cell suspension of appropriate density in a 96-well cell culture plate, and after culturing for 24 hours, aspirate the culture medium in the culture plate, and add cells 100 ⁇ L of virus solution diluted in maintenance solution (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 gradient dilution of virus), 10 duplicate wells for each dilution, 33°C, Adsorbed and cultured in a 5% CO 2 incubator for 3 h, sucked off the unadsorbed virus liquid, added 100 ⁇ L of cell maintenance medium to each well, and continued to culture.
  • maintenance solution 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 gradient dilution of virus
  • 10 duplicate wells for each dilution 33°C
  • CPE cytopathic effect
  • TCID 50 Log (virus dilution with CPE below 50%) + distance ratio ⁇ dilution distance
  • the distance ratio (percentage above 50% - 50) ⁇ (percentage above 50% - percentage below 50%)
  • DMEM medium was used to prepare serial concentrations of ZONK2003 series compounds (concentration gradient of 50.0, 25.0, 12.5, 6.25, 3.13, 1.56 ⁇ mol/L) and oseltamivir phosphate (concentration gradient of 20.0, 10.0, 5.0, 2.5, 1.25, 0.625 ⁇ g/mL), interferon ⁇ 2b (concentration gradient of 1 ⁇ 10 3 , 5 ⁇ 10 2 , 2.5 ⁇ 10 2 , 1.25 ⁇ 10 2 , 6.25 ⁇ 10 1 , 3.1 ⁇ 10 1 IU/mL), respectively added to The cultured MRC-5 and MDCK cells were cultured in a constant temperature incubator at 37°C, 5% CO 2 humidification for 72 hours, MTT was added, and the culture was continued for 4 hours. EC50 of each cell.
  • Normal control group no virus infection group
  • Model control group virus infection group
  • Positive control group commercial control drug
  • Test substance group infection group + test substance of different concentrations.
  • the cytopathic changes were observed day by day until the cytopathic changes no longer increased, and the number of cytopathic wells and non-lesioned wells at each concentration were recorded.
  • the inhibition rate of the cytopathic rate (%) (1-unaffected cells in each group/8) ⁇ 100%.
  • the two compounds have no obvious cytotoxicity to MRC-5 and MDCK within the tested concentration range (1.56-50.0 ⁇ mol/L), so the highest concentration in the antiviral study was set as 20.0 ⁇ mol/L.
  • interferon ⁇ 2b (6.25 ⁇ 10 1 ⁇ 1 ⁇ 10 3 IU/mL) was effective against MRC-5 cells
  • oseltamivir phosphate (0.625 ⁇ 20.0 ⁇ mol/L) was effective against MRC-5 cells.
  • MDCK cells had no obvious cytotoxicity, so the highest concentrations were set as 1 ⁇ 10 3 IU/mL and 20.0 ⁇ mol/L in antiviral research.
  • the TCID 50 of coronavirus (HCoV-229E) and influenza A virus (H1N1) are 10-3.5 /0.1mL and 10-3.85 /0.1mL respectively.
  • H1N1 influenza A virus
  • 3.16 ⁇ 10 4 times and 7.08 ⁇ 10 4 times are diluted, take 0.1 mL of cells to inoculate cells, and 50% of the cells can cause lesions.
  • the EC 50 of compounds ZONK2003-4 and ZONK2003-2 against coronavirus (HCoV-229E) were 12.83 ⁇ mol/L and 15.00 ⁇ mol/L, respectively; the EC 50 of compounds ZONK2003-4 and ZONK2003-2 against influenza A virus (H1N1) were 11.75 ⁇ mol/L and 18.19 ⁇ mol/L, respectively.
  • the EC 50 of interferon ⁇ 2b against coronavirus was 2.42 ⁇ 10 2 IU/mL; the EC 50 of oseltamivir phosphate against influenza A virus (H1N1) was 5.06 ⁇ mol/L.
  • Compounds ZONK2003-4 and ZONK2003-2 had no obvious cytotoxicity to MRC-5 and MDCK cells in the tested concentration range (1.56-50.0 ⁇ mol/L).
  • the positive control drug had no obvious toxicity to MRC-5 and MDCK cells in the tested concentration range (0.625 ⁇ 20mmol/L).
  • the compounds ZONK2003-4 and ZONK2003-2 have different degrees of inhibitory effect on the two viruses in the test, and the EC 50 for coronavirus (HCoV-229E) is 12.83 ⁇ mol/L and 15.00 ⁇ mol/L, respectively;
  • the EC 50 of the virus (H1N1) were 11.75 ⁇ mol/L and 18.19 ⁇ mol/L, respectively.
  • the positive control drug had different degrees of inhibitory effect on the two viruses in the experiment, and the EC 50 for coronavirus (HCoV-229E) was 2.42 ⁇ 10 2 IU/mL; oseltamivir phosphate was effective against influenza A virus (H1N1 ) with an EC 50 of 5.06 ⁇ mol/L.
  • Tested substances ZONK2003-0, ZONK2003-2, ZONK2003-4; provided by Guangdong Zhongke Pharmaceutical Research Co., Ltd.
  • Oseltamivir Phosphate Granules 15mg ⁇ 10 bags, product of Yichang Dongguang Changjiang Pharmaceutical Co., Ltd., batch number: 0371912115, valid until 2021.12.11, positive control drug for anti-influenza A virus;
  • Influenza A virus mouse lung-adapted strain A/FM/1/47 (H1N1) inoculated with chicken embryos, and collected allantoic fluid for preservation. ICR mice, weighing 18-22 g. During the administration period, people were allowed to eat and drink freely, with 12 hours of light and 12 hours of darkness every day, the temperature was 22 ⁇ 2°C, and the humidity was 55-70%.
  • Experimental method After 3 days of adaptive feeding, the experiment was started. Except for the uninfected control group, mice in other groups were lightly anesthetized with ether, and intranasally inoculated with chick embryo allantoies of influenza virus A/FM/1/47 (H1N1) equivalent to 8 ⁇ LD 50 diluted with normal saline.
  • Example 16 Alleviating effects of drugs on pulmonary inflammation in mice caused by influenza virus H1N1 infection
  • mice in other groups were lightly anesthetized with ether, and intranasally inoculated with chick embryo allantoies of influenza virus A/FM/1/47 (H1N1) equivalent to 8 ⁇ LD 50 diluted with normal saline.
  • the mice in the positive control oseltamivir group and the test administration group were intragastrically administered with 80 mg/kg for the first time 24 hours after virus infection, and then once a day, the same as the virus control group and the uninfected control group.
  • Oral saline, once a day the administration volume is 0.1mL/10g body weight. 5 days in total.
  • 3 mice in each group were weighed, the eyeballs were enucleated and exsanguinated, and the whole lung was taken out, weighed, and the lung index and lung index inhibition rate were calculated.
  • Lung index mouse lung weight / mouse body weight ⁇ 100
  • Lung index inhibition rate (%) mean lung index in virus control group - mean lung index in drug administration group/mean lung index in virus control group ⁇ 100%
  • the rats were given ZONK2003-2 by gavage, the oral dose was 25mg/kg, and the injection of ZONK2003-2 was 1.21mg/kg;
  • Rats were given intragastric administration of ZONK2003-0, the oral dose was 2.51 mg/kg, and the injection of ZONK2003-0 was 1.00 mg/kg, the blood concentration was detected, and the pharmacokinetic parameters were calculated:
  • ND Not detected, that is, the measured value is lower than the lower limit of quantification after the blood concentration reaches the peak
  • the absolute bioavailability F value of ZONK2003-0 was 62.5%
  • the absolute bioavailability F value of ZONK2003-2 group was 106.0%.
  • NIH mice SPF grade, half male and female, 15-17g, 110 mice, provided by Guangdong Medical Laboratory Animal Center, ZONK2003-0, ZONK2003-2, ZONK2003-4, ZONK2003-14, ZONK2003-18; Provided by Science Drug Research Co., Ltd.
  • mice 110 NIH mice, male, weighing 15-17g. Quarantine was carried out for 3 days. After the quarantine, the animals were randomly divided into groups according to their body weight, with 10 animals in each group, namely: model group and experimental drug group. During the experiment, the corresponding drugs were given to each mouse through the tail vein, and the administration volume was 0.1 ml/10 g body weight. The model group was given an equal volume of normal saline. 0.5 hours after administration, intraperitoneal injection of 0.7% (0.7g/100ml) HAc at 10mL/kg body weight induced pain, and then immediately observed the writhing response of each mouse within 15min, recorded the number of writhing, and calculated the inhibition rate.
  • each dose group can significantly inhibit the writhing times of NIH mice induced by glacial acetic acid (p ⁇ 0.05 or p ⁇ 0.01), and the effect intensity is better than that of the ZONK2003-0 group.
  • ZONK2003 does not exert its antiviral effect through the inhibition of DHODH enzyme.

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Abstract

本发明公开了一种抗病毒的化合物及其制备方法。该化合物的结构式如式I所示，式(Ⅰ)中，R 1选自：单取代或多取代的H、F、甲基、三氟甲基，优选H；R 2选自：H、直链或取代烷烃(C1-C6)，优选甲基，异丙基；R 3选自：单取代或多取代的H、Cl、Br、F，优选Cl；R 4选自：直链或取代烷烃(C1-C6)，优选甲基，乙基或丙基。本发明中所述化合物经过实验证实，不仅对于H1N1甲型流感病毒具有较好的抑制作用，并且对于冠状病毒也具有较好的抑制作用，没有观察到对于人正常细胞的毒性，且能够在抗病毒的同时抑制炎症反应的程度。

Description

一种抗病毒化合物及其制备方法 技术领域
本发明属于医药领域，具体涉及一种抗病毒化合物及其制备方法。
背景技术
流行性感冒病毒简称流感病毒。它分为甲(A)、乙(B)、丙(C)三型，近年来才发现的流感病毒将归为丁(D)型。流感病毒可引起人、禽、猪、马、蝙蝠等多种动物感染和发病，是人流感、禽流感、猪流感、马流感等人与动物疫病的病原。
这些疫病典型的临床症状是急性高热、全身疼痛、显著乏力和呼吸道症状。流感病毒主要通过空气中的飞沫、易感者与感染者之间的接触或与被污染物品的接触而传播。一般秋冬季节是其高发期。人流感主要是甲型流感病毒和乙型流感病毒引起的。甲型流感病毒经常发生抗原变异，可以进一步分为H1N1、H3N2、H5N1、H7N9等亚型(其中的H和N分别代表流感病毒两种表面糖蛋白)。流感病毒对外界抵抗力不强。动物流感病毒通常不感染人，人流感病毒通常不感染动物，但是猪比较例外。猪既可以感染人流感病毒，也可以感染禽流感病毒，但它们主要感染的还是猪流感病毒。少数动物流感病毒适应人后，可以引起人流感大流行。
人冠状病毒可对人造成普通感冒，严重急性呼吸综合征(SARS)和中东呼吸综合征(MERS)，在流行病学特征上存在一定差异。
在全球，10％～30％的上呼吸道感染由HCoV-229E、HCoV-OC43、HCoV-NL63和HCoV-HKU1四类冠状病毒引起，在造成普通感冒的病因中占第二位，仅次于鼻病毒。感染呈现季节性流行，每年春季和冬季为疾病高发期。潜伏期2-5天，人群普遍易感。主要通过人与人接触传播。
甲型H1N1流感是猪的一种高度传染性急性呼吸道疾病，由一种或多种猪流感A型病毒引起。发病率往往较高，但死亡率较低(1～4％)。通过浮质、直接和间接接触以及携带病毒但无症状的猪，病毒在猪群中传播。全年都可发生猪群疫情。在温带的秋季和冬季，发病率上升。人患甲型H1N1流感通常来自被感染的猪，但有些人患病例没有与猪或猪所在环境接触的历史。在有些情况中发生了人际传播，但仅限于密切接触者和封闭环境中的人群。
冠状病毒(HCoV-229E)是冠状病毒的一种。冠状病毒属于套式病毒目、冠状病毒科、冠状病毒属，是一个大型病毒家族，在自然界广泛存在。冠状病毒仅感染脊椎动物，与人和动物的多种疾病有关，可引起人和动物呼吸道、消化道和神经系统疾病。
因此，研究一种有效的抗病毒药物具有重要的现实意义。
发明内容
本发明的目的是提供一种式I所示的化合物及其药学上可接受的盐、溶剂合物。
本发明所提供的化合物的结构通式如式I所示：
Figure PCTCN2021128879-appb-000001
所述式(Ⅰ)中，
R 1选自：单取代或多取代的H、F、甲基、三氟甲基，优选H；
R 2选自：H、直链或取代烷烃(C1-C6)，优选甲基，异丙基；
R 3选自：单取代或多取代的H、Cl、Br、F，优选Cl；
R 4选自：直链或取代烷烃(C1-C6)，优选甲基，乙基或丙基。
在本发明的一些实施方案中，本发明所述的式I所示化合物可以列举为如下所示结构，但不局限于以下结构：
Figure PCTCN2021128879-appb-000002
本发明还提供了上述式I所示的化合物的制备方法。
根据文献Med.Chem.Commun.,2016,7,1441–1448，化合物1(式a)烷基肼与硫氰酸钠反应得到硫脲化合物2(式b)，然后与R1取代的邻甲 酸苯甲醛缩合得到化合物3(式c)，最后与R 3取代溴苯乙酮关环，很容易得到噻唑类化合物4(式d)；在缩合剂作用下，得到式I所示的苯甲酰胺衍生物。具体步骤如下：
1)将式a所示化合物与硫氰酸钠进行反应，得到式b所示化合物；
Figure PCTCN2021128879-appb-000003
其中，式a中R 2的定义同式I；式b中R 2的定义同式a；
2)将式b所示化合物与式e所示的R 1取代的邻甲酸苯甲醛进行缩合反应，得到式c所示化合物；
Figure PCTCN2021128879-appb-000004
其中，式e中R 1的定义同式I；式c中R 1的定义同式e、R 2的定义同式b；
3)将式c所示化合物与式f所示的R 3取代溴苯乙酮进行关环反应，得到式d所示化合物；
Figure PCTCN2021128879-appb-000005
其中，式f中R 3的定义同式I，式d中R 1、R 2的定义同式c，R 3的定义同式f；
4)在缩合剂作用下，使式d所示化合物与式g所示化合物进行缩合反应，得到式I所示化合物；
Figure PCTCN2021128879-appb-000006
其中，式g中X、R 4的定义同式I。
上述步骤1)中，所述反应的反应条件为：反应温度50-100℃，反应时间为24-72小时；反应在溶剂中进行，所述溶剂可为甲醇、乙醇、四氢呋喃、乙腈等，优选乙醇。
上述步骤2)中，所述缩合反应的反应条件为：反应温度50-100℃，反应时间为1-3小时；反应在溶剂中进行，所述溶剂可为甲醇、乙醇、四氢呋喃、乙腈等，优选乙醇。
上述步骤3)中，所述关环反应的反应条件为：反应温度50-100℃，反应时间为3-6小时；反应在溶剂中进行，所述溶剂可为甲醇、乙醇、四氢呋喃、乙腈等，优选乙醇。
上述步骤4)中，所述缩合反应的反应条件为：反应温度0-25℃，反应时间为2-8小时；反应在溶剂中进行，所述溶剂可为二氯甲烷、四氢呋喃、乙腈等，优选二氯甲烷。
参照本发明实施例的制备方法可以获得本发明权利要求中所保护的其它化合物。
本发明另一个目的是提供上述式I所示化合物的应用。
本发明所提供的应用是式I所示化合物或其药学上可接受的盐、酯、溶剂合物的应用为下述(a)和/或(b)和/或(c)：
(a)式I所示化合物或其药学上可接受的盐、酯、溶剂合物在制备治疗病毒所致疾病或病毒感染的产品中的应用；
(b)式I所示化合物或其药学上可接受的盐、酯、溶剂合物在制备预防病毒所致疾病或病毒感染的产品中的应用；
(c)式I所示化合物或其药学上可接受的盐、酯、溶剂合物在制备病毒抑制剂中的应用；
(d)式I所示化合物或其药学上可接受的盐、酯、溶剂合物在制备镇痛药物中的应用。
所述产品可为药物或药物制剂。
所述病毒抑制剂能够抑制病毒的复制。
所述病毒包括流感病毒、冠状病毒。
所述流感病毒具体可为甲型流感病毒(H1N1)；
所述冠状病毒可为α属冠状病毒和/或β属冠状病毒，具体选自HCoV-229E。
本发明中，所述病毒所致疾病可为呼吸系统感染性疾病。
所述呼吸系统感染为呼吸道感染和/或肺部感染；所述呼吸道感染可为鼻咽炎、鼻炎、咽喉炎、气管炎和/或支气管炎；所述肺部感染可为肺炎。
本发明中，所述流感病毒所致疾病通常包括流感病毒引起的急性呼吸道传染疾病等。
本发明中，所述冠状病毒所致疾病通常包括病毒性肺炎、严重急性呼吸综合征等。
本发明中，所述冠状病毒感染通常引起病毒性肺炎、严重急性呼吸综合征等疾病。
本发明化合物同时具有对冠状病毒和H1N1甲型流感病毒的抑制作用，并且对人正常细胞没有毒性，能够抑制炎症反应发生的程度，减小肺炎对机体的伤害，促进机体恢复。
以式I所示的化合物为活性成分制备的抗病毒药物或镇痛药物也属于本发明的保护范围。
所述抗病毒药物可通过注射、喷射、滴鼻、滴眼、渗透、吸收、物理或化学介导的方法导入机体如肌肉、皮内、皮下、静脉、粘膜组织；或是被其他物质混合或包裹后导入机体。
需要的时候，在上述药物中还可以加入一种或多种药学上可接受的载体。所述载体包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。
上述药物可以制成片剂、粉剂、颗粒剂、胶囊、口服液、膏剂、霜剂、注射液等多种形式；上述各种剂型的药物均可以按照药学领域的常规方法制备。
本发明还提供了一种药物或药物组合物，其活性成分为式I所示化合物或其药学上可接受的盐、酯、溶剂合物。
所述药物或药物组合物具有下述至少一种功效：
1)治疗病毒所致疾病或病毒感染；
2)预防病毒所致疾病或病毒感染；
3)抑制病毒；
4)镇痛。
上述药物或药物组合物可以按照本领域技术人员已知的常规方法制成溶液剂、片剂、胶囊或注射剂等剂型。
利用本发明提供的式I所示化合物或其药学上可接受的盐预防和/或治疗病毒引起的感染时，给予受试者生物体有效量的式I化合物或其药学上可接受的盐。
本发明中所述化合物经过实验证实，不仅对于H1N1甲型流感病毒具有较好的抑制作用，并且对于冠状病毒也具有较好的抑制作用，没有观察到对于人正常细胞的毒性，且能够在抗病毒的同时抑制炎症反应的程度；此外，该化合物还具有显著的镇痛作用。
附图说明
图1为本发明式I所示化合物的合成路线图。
具体实施方式
下面结合具体实施例对本发明作进一步阐述，但本发明并不限于以下实施例。所述方法如无特别说明均为常规方法。所述原材料如无特别说明均能从公开商业途径获得。
实施例1-13
1、(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-甲基亚肼基)甲基)苯甲酰胺(ZONK2003-1)的合成
1)2-甲基氨基硫脲(1-2)
Figure PCTCN2021128879-appb-000007
将甲基肼(23.0g，0.5mol)、硫氰酸铵(38.0g，0.5mol)、乙醇(200mL)分别加入单口圆底烧瓶中，加热回流反应72h。反应液冷却浓缩，柱层析纯化得到灰白色固体2-甲基氨基硫脲(44.1g，84.0％)。 1H NMR(DMSO-d6 400MHz)δ7.24(s,2H),6.85(s,2H),3.14(s,3H).ESI-MS m/z:106.1[M+H]+.
2)(E)-2-((2-氨基甲硫杂酰-2-甲基亚肼基)甲基)苯甲酸(1-3)
Figure PCTCN2021128879-appb-000008
将2-甲基氨基硫脲(40.0g，0.38mol)、2-羰基苯甲酸(57.0g，0.38mol)、乙醇(300mL)分别加入单口圆底烧瓶中，加热回流反应2h。反应液冷却浓缩，柱层析纯化得到浅黄色固体(E)-2-((2-氨基甲硫杂酰-2-甲基亚肼基)甲基)苯甲酸(85.6g，95.0％)。 1H NMR(DMSO-d6 400MHz)δ13.0(s,1H),8.12-7.23(m,7H),2.47(s,3H).ESI-MS m/z:238.1[M+H]+.
3)(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-甲基亚肼基)甲基)苯甲酸(1-4)(简称ZONK2003-0)
Figure PCTCN2021128879-appb-000009
将(E)-2-((2-氨基甲硫杂酰-2-甲基亚肼基)甲基)苯甲酸(80.0g，0.34mol)、2-溴-1-(2-氯苯基)乙酮(79.2g，0.34mol)、乙醇(400mL)分别加入单口圆底烧瓶中，加热回流反应3h。反应液冷却浓缩，柱层析纯化得到浅黄色固体(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-甲基亚肼基)甲基)苯甲酸(124g，98.5％)。 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)(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-甲基亚肼基)甲基)-N-(甲磺酰基)苯甲酰胺(ZONK2003-2)的合成
Figure PCTCN2021128879-appb-000010
将(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-甲基亚肼基)甲基)苯甲酸(3.71g，10mmol)、4-二甲氨基吡啶(1.22g，10mmol)、氨基甲磺酸(1.11g，10mmol)分别溶解于二氯甲烷(30mL)中，然后加入二环己基碳二亚胺(2.27g，11mmol)，室温搅拌5h。反应液浓缩，柱层析纯化得到浅黄色固体(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-甲基亚肼基)甲基)-N-(甲磺酰基)苯甲酰胺(3.41g，76.2％)。 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、(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-甲基亚肼基)甲基)-N-(乙基磺酰基)苯甲胺(ZONK2003-4)的合成
Figure PCTCN2021128879-appb-000011
将(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-甲基亚肼基)甲基)苯甲酸(5.0g，13.4mmol)、乙基磺酰胺(1.46g，13.4mol)、4-二甲氨基吡啶(1.63g，13.4mol)溶解于二氯甲烷(100mL)中，冰水浴加入二环己基碳二亚胺(2.90g，14.1mol)，室温反应5h。反应液浓缩，柱层析纯化得到白色固体(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-甲基亚肼基)甲基)-N-(乙磺酰)苯甲酰胺(5.27g，85.1％)。 1H NMR(DMSO-d 6 400MHz)δ8.60(s,1H),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、(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-甲基亚肼基)甲基)-N-(丙磺酰)苯甲酰胺(简称ZONK2003-14)的合成
Figure PCTCN2021128879-appb-000012
将(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-甲基亚肼基)甲基)苯甲酸(5.0g，13.4mmol)、丙基磺酰胺(1.65g，13.4mol)、4-二甲氨基吡啶(1.63g，13.4mol)溶解于二氯甲烷(100mL)中，冰水浴加入1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(2.70g，14.1mol)，室温反应5h。反应液浓缩，柱层析纯化得到白色固体(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-甲基亚肼基)甲基)-N-(丙磺酰)苯甲酰胺(5.50g，86.2％)。 1H NMR(DMSO-d 6 400MHz)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,3H).ESI-MS m/z:477.5[M+H] +.
4、(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-异丙基亚肼基)甲基)-N-(甲磺酰)苯甲酰胺的合成(简称ZONK2003-15)
Figure PCTCN2021128879-appb-000013
综合实施例1-3的制备方法，合成得到(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-异丙基亚肼基)甲基)-N-(甲磺酰)苯甲酰胺。 1H NMR(DMSO-d 6400MHz)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、(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-异丙基亚肼基)甲基)-4-氟-N-(甲磺酰)苯甲酰胺的合成(简称ZONK2003-16)
Figure PCTCN2021128879-appb-000014
综合实施例1-3的制备方法，合成得到(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-异丙基亚肼基)甲基)-4-氟-N-(甲磺酰)苯甲酰胺。 1H NMR(DMSO-d 6 400MHz)δ8.60(s,1H),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、(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-异丙基亚肼基)甲基)-N-(乙基磺酰基)-5-氟苯甲酰胺的合成(简称ZONK2003-17)
Figure PCTCN2021128879-appb-000015
综合实施例1-3的制备方法，合成得到(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-异丙基亚肼基)甲基)-N-(乙基磺酰基)-5-氟苯甲酰胺。 1H NMR(DMSO-d 6 400MHz)δ8.60(s,1H),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、(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-甲基亚肼基)甲基)-4-氟-N-(甲磺酰)苯甲酰胺的合成(简称ZONK2003-18)
Figure PCTCN2021128879-appb-000016
综合实施例1-3的制备方法，合成得到(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-甲基亚肼基)甲基)-4-氟-N-(甲磺酰)苯甲酰胺。 1H NMR(DMSO-d 6 400MHz)δ8.60(s,1H),8.01-7.34(m,8H),3.66(s,3H),3.42(s,3H).ESI-MS m/z:467.1[M+H] +.
8、(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-甲基亚肼基)甲基)-N-(乙基 磺酰)-4-氟苯甲酰胺的合成(简称ZONK2003-19)
Figure PCTCN2021128879-appb-000017
综合实施例1-3的制备方法，合成得到(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)-2-甲基亚肼基)甲基)-N-(乙基磺酰)-4-氟苯甲酰胺。 1H NMR(DMSO-d 6 400MHz)δ8.60(s,1H),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、(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)亚肼基)甲基)-5-甲基-N-(甲磺酰)苯甲酰胺的合成(简称ZONK2003-20)
Figure PCTCN2021128879-appb-000018
综合实施例1-3的制备方法，合成得到(E)-2-((2-(4-(2-氯苯基)噻唑-2-基)亚肼基)甲基)-5-甲基-N-(甲磺酰)苯甲酰胺。 1H NMR(DMSO-d 6 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、(E)-2-((2-(4-(3,5-二氯苯基)噻唑-2-基)亚肼基)甲基)-5-甲基-N-(甲磺酰)苯甲酰胺的合成(简称ZONK2003-21)
Figure PCTCN2021128879-appb-000019
综合实施例1-3的制备方法，合成得到(E)-2-((2-(4-(3,5-二氯苯基)噻唑-2-基)亚肼基)甲基)-5-甲基-N-(甲磺酰)苯甲酰胺。 1H NMR(DMSO-d 6400MHz)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] +.
实施例11、系列化合物体外抗病毒试验
采用MTT法测定ZONK2003系列化合物(式I所示化合物)对培养病毒的MRC-5、MDCK细胞的毒性作用，设计合适浓度进行抗病毒试验。采用致细胞病变法(cytopathic effect，CPE法)进行体外抗病毒药效学试验，评价ZONK2003系列化合物的对引起呼吸道疾病的病毒的作用。
试验结果：ZONK2003系列化合物对MRC-5、MDCK细胞的均无明显毒性；对两株病毒均具有不同程度的抗病毒作用，以半数有效率(EC50)评价其抗病毒作用。对冠状病毒(HCoV-229E)的EC50为12.83μmol/L、15.00μmol/L；对甲型流感病毒(H1N1)的EC50为11.75μmol/L、18.19μmol/L。
试验结论：在10～20μmol/L浓度范围内，ZONK2003系列化合物对两株病毒均具有不同程度的抑制作用，可促进细胞在受病毒感染后的恢复。
1.试验材料
1.1受试物
受试物：ZONK2003-4，批号：20200521，纯度98.70％；、ZONK2003-2，批号：20200513，纯度99.01％，由广东中科药物研究有限公司提供。以DMEM培养基配制成50.0、25.0、12.5、6.25、3.13、1.56μmol/L，2～8℃保存备用。
1.2阳性对照品
磷酸奥司他韦颗粒，规格15mg×10袋装，宜昌东阳光长江药业股份有限公司产品，批号：0371912115，有效期至2021.12.11，用于抗甲型流感病毒的阳性对照药；重组人干扰素α2b凝胶，规格1.0×10 5IU/克，10g/支，兆科药业(合肥)有限公司，用于抗冠状病毒的阳性对照药。
1.3病毒株
冠状病毒(HCoV-229E)，编号：ATCC-VR-740，购自于美国ATCC；甲型流感病毒(H1N1)，编号ATCC-VR-95，购自于美国ATCC。均在II级生物安全实验室培养并保存。
1.4细胞株
人胚肺细胞(MRC-5)，犬肾细胞(MDCK)，均来源于武汉普诺赛生命科技有限公司。
1.5主要试剂
Figure PCTCN2021128879-appb-000020
1.6主要仪器
BSCIIB2-1101型生物安全柜，上海瑞仰净化装备有限公司生产，中心编号：019，YXQ-50A型立式压力蒸汽灭菌器，上海博讯医疗生物仪器股份有限公司生产，中心编号：584；3111型CO 2培养箱，美国ThermoFisher公司生产，中心编号：147。
1.7试验设施
长沙市病原微生物实验室，实验室等级：生物安全II级(BSL-II)，备案号：长卫计实备字(2019)第B001号。本试验在中心生物安全II级实验室(BSL-II-1)进行，严格按照法规及本中心SOPs执行。
2.试验方法
2.1细胞培养
人胚肺细胞(MRC-5)为贴壁生长细胞。培养基选用含10％FBS的DMEM培养基，生长状态良好时，每隔2～3d可进行传代。在净化工作台中弃去培养基，用1×PBS清洗2～3次，然后加入适量的0.25％Trypsin-EDTA消化，约1～3min后，待细胞脱落，加入适量的含10％FBS的DMEM培养基以终止胰酶的消化作用，吹打成单细胞悬液，转入EP管中，以1000rpm离心5min。弃去培养基，加入新鲜的培养基重悬，按一定比例(细胞密度约为10 5/mL)接种到新的培养瓶中，放置于37℃、5％CO 2培养箱中培养。
犬肾细胞(MDCK)为贴壁生长细胞。培养基选用含10％FBS的DMEM培养基，生长状态良好时，每隔2～3d可进行传代。在净化工作台中弃去培养基，用1×PBS清洗2～3次，然后加入适量的0.25％Trypsin-EDTA消化，约2～5min后，待细胞脱落，加入适量的含10％FBS的DMEM培养基以终止胰酶的消化作用，吹打成单细胞悬液，转入EP管中，以1000rpm离心5min。弃去培养基，加入新鲜的培养基重悬，按一定比例(细胞密度约为10 5/mL)接种到新的培养瓶中，放置于37℃、5％CO 2培养箱中培养。
2.2病毒的扩增
2.2.1冠状病毒(HCoV-229E)：MRC-5细胞扩增
将MRC-5细胞接种于75cm 2培养瓶中，当细胞密度达到80～90％时去掉部分的培养基，剩余的刚好覆盖好细胞，加入适量的HCoV-229E病毒，待病毒吸附于细胞表面后(约3h左右，每隔30min轻轻晃动培养板，使病毒吸附均匀)，更换不含FBS的新鲜培养基，置于35℃、5％CO 2加湿的恒温培养箱中培养。观察细胞开始产生病变至不再产生病变时(一般为5～7天)采取反复冻融法，于3000rpm离10min以除去细胞残渣，收集上清液分装于冻存管中，进行标注后于-80℃中短期保存或液氮中长期保存，备用。
2.2.2甲型流感病毒(H1N1)：MDCK细胞扩增
将MDCK细胞接种于75cm 2培养瓶中，当细胞密度达到70～80％时去掉部分的培养基，剩余的刚好覆盖好细胞，加入适量的H1N1病毒，待病毒吸附于细胞表面后(约3h左右，每隔30min轻轻晃动培养板，使病毒吸附均匀)，更换不含FBS的新鲜培养基，置于33℃、5％CO 2加湿的恒温培养箱中培养。观察细胞开始产生病变至不再产生病变时(一般为2～3天)，采取反复冻融法，于3000rpm离10min以除去细胞残渣，收集上清液分装于冻存管中，进行标注后于-80℃中短期保存或液氮中长期保存，备用。
2.3病毒半数组织培养感染浓度(Tissue culture infective dose,TCID 50)的测定
将2.2.1、2.2.2收集的病毒液进行TCID 50的测定：分别在96孔细胞培养板接种适宜密度的相应细胞悬液100μL，培养24h后吸去培养板中的培养液，加入用细胞维持液稀释的病毒液100μL(病毒进行10 -1、10 -2、10 -3、10 -4、10 -5、10 -6梯度稀释)，每个稀释度做10个复孔，33℃，5％CO 2培养箱吸附培养3h，吸去未吸附的病毒液，每孔补加细胞维生长持液100μL，继续培养。逐日在倒置显微镜下观察细胞病变效应(cytopathic effect，CPE，病毒在培养细胞中所致CPE的特征：细胞变圆，折光性较强，融合细胞突起，部分脱壁，胞浆有丝状突起或伪足状，整个形状有如不规则的地图，形成大而圆的融合性多核巨细胞)。并记录出现CPE的孔数，以最高稀释度不再出现病变时为终点，细胞病变程度用“－～++++”示：无细胞病变“－”，≤25％细胞病变“+”，25％～50％细胞病变“+”，50％～75％细胞病变“+++”，>75％细胞病变“++++”。根据Reed-Muench公式计算TCID 50：
TCID 50＝Log(CPE低于50％的病毒稀释度)+距离比例×稀释度间距
其中，距离比例＝(高于50％百分数-50)÷(高于50％的百分数-低于50％的百分数)
2.4受试物的浓度设计
2.4.1细胞毒性检测
用DMEM培养基配制系列浓度的ZONK2003系列化合物(浓度梯度为50.0、25.0、12.5、6.25、3.13、1.56μmol/L)和磷酸奥司他韦(浓度梯度为20.0、10.0、5.0、2.5、1.25、0.625μg/mL)、干扰素α2b(浓度梯度为1×10 3、5×10 2、2.5×10 2、1.25×10 2、6.25×10 1、3.1×10 1IU/mL)，分别加入至培养好的MRC-5、MDCK细胞，37℃、5％CO 2加湿的恒温培养箱中培养72h，加入MTT，继续培养4h，在492nm波长下测得各孔的OD值，计算ZONK2003系列化合物对各细胞的EC 50。
2.4.2当化合物对细胞有毒性时，采用1/2IC 50为最高浓度，以2倍间距向下稀释设置3个测试浓度；当化合物对细胞无毒性时，采用20μmol/L为最高浓度，以2倍间距向下稀释设置3个测试浓度。
2.5抗病毒作用的检测
2.5.1细胞接种：病毒感染前24h，将生长良好的相应细胞株以适宜的密度(约10 5个/孔)均匀接种于96孔板中，100μL/孔，于37℃、5％CO 2培养箱中培养；
2.5.2待细胞至70％～80％左右后，吸去部分培养基，剩余刚好覆盖细胞(使得病毒与细胞更好地吸附)，接种100TCID 50的病毒液50μL/孔，放入培养箱中培养3h后，吸去96孔板中的培养基，加入用不含血清的细胞维持液配制的不同浓度的受试物溶液(根据细胞毒性设置)，200μL/孔，于33℃、5％CO 2培养箱中培养。
2.5.3分组
正常对照组：未用病毒感染组；
模型对照组：病毒感染组；
阳性对照组：市售对照药；
受试物组：感染组+不同浓度的受试物。
2.5.4抗病毒活性评价(CPE法)
逐日观察细胞病变，连续观察至细胞病变不再增加，记录每个浓度细胞病变孔数和未病变孔数。
2.6结果评价
以正常对照组细胞未病变率为100％，细胞病变率抑制率(％)＝(1-各组细胞未病变孔/8)×100％。
本试验数据有效数字修约按照四舍五入进行，统计所用软件为SPSS16.0。计量资料以均数±标准差
Figure PCTCN2021128879-appb-000021
表示，用Leven’s test方法检验正态性和方差齐性。如果没有统计学意义(P>0.05)，用单因素方差分析(ANOVA)进行统计分析。如果ANOVA有统计学意义(P≤0.05)，用LSD法进行比较分析。如果方差不齐(P≤0.05)，则用Kruskal-Wallis检验。如果Kruskal-Wallis检验有统计学意义(P≤0.05)，则用Dunnett’s Test(非参数方法)进行比较分析。统计结果以α＝0.05为检验界限，其中P≤0.05表示有统计学意义，P≤0.01表示所检验的差别有非常显著性意义。
3.试验结果
3.1系列化合物的对各细胞增殖的影响
如表1-1、1-2所示，两种化合物在测试浓度范围内(1.56～50.0μmol/L)对MRC-5、MDCK均无明显细胞毒性，故在抗病毒研究中最高浓度设置为20.0μmol/L。如表2所示，阳性药在测试浓度范围内干扰素α2b(6.25×10 1～1×10 3IU/mL)对MRC-5细胞、磷酸奥司他韦(0.625～20.0μmol/L)对MDCK细胞均无明显细胞毒性，故在抗病毒研究中最高浓度分别设置为1×10 3IU/mL、20.0μmol/L。
表1-1 两种化合物对MRC-5细胞增殖的影响
Figure PCTCN2021128879-appb-000022
Figure PCTCN2021128879-appb-000023
表1-2 两种化合物对MDCK细胞增殖的影响
Figure PCTCN2021128879-appb-000024
表2 阳性药对各细胞增殖的影响
Figure PCTCN2021128879-appb-000025
3.2病毒滴度结果检测
如表3、表4所示，冠状病毒(HCoV-229E)、甲型流感病毒(H1N1)的半数病毒感染量TCID 50分别为10 -3.5/0.1mL、10 -3.85/0.1mL，病毒分别做 3.16×10 4倍、7.08×10 4倍稀释时取0.1mL接种细胞，可使50％细胞产生病变。取100个TCID 50病毒量，即分别稀释316倍、708倍倍进行体外抗病毒试验。
表3 HCoV-229E对MRC-5细胞的半数病毒感染量
Figure PCTCN2021128879-appb-000026
表4 H1N1对MDCK细胞的半数病毒感染量
Figure PCTCN2021128879-appb-000027
3.3系列浓度ZONK2003系列化合物对感染病毒后的细胞活力的影响
化合物ZONK2003-4、ZONK2003-2抗冠状病毒(HCoV-229E)的EC 50分别为12.83μmol/L、15.00μmol/L；化合物ZONK2003-4、ZONK2003-2抗甲型流感病毒(H1N1)的EC 50分别为11.75μmol/L、18.19μmol/L。
干扰素α2b对冠状病毒(HCoV-229E)的EC 50为2.42×10 2IU/mL；磷酸奥司他韦对甲型流感病毒(H1N1)的EC 50为5.06μmol/L。
表5 供试品/对照品对HCoV-229E感染MRC-5细胞的影响
Figure PCTCN2021128879-appb-000028
表6 两种化合物对H1N1感染MDCK细胞的影响
Figure PCTCN2021128879-appb-000029
Figure PCTCN2021128879-appb-000030
4.结论与评价
在本试验条件下：
化合物ZONK2003-4、ZONK2003-2在测试浓度范围内(1.56～50.0μmol/L)对MRC-5、MDCK细胞均无明显细胞毒性。阳性对照药在测试浓度范围内(0.625～20mmol/L)对MRC-5、MDCK细胞均无明显毒性。
化合物ZONK2003-4、ZONK2003-2对试验中的两株病毒均具有不同程度的抑制作用，对冠状病毒(HCoV-229E)的EC 50分别为12.83μmol/L、 15.00μmol/L；对甲型流感病毒(H1N1)的EC 50分别为11.75μmol/L、18.19μmol/L。
阳性对照药对实验中的两株病毒均具有不同程度的抑制作用，对冠状病毒(HCoV-229E)的EC 50为2.42×10 2IU/mL；磷酸奥司他韦对甲型流感病毒(H1N1)的EC 50为5.06μmol/L。
实施例15、化合物对甲型流感病毒A/FM/1/47(H1N1)感染小鼠的保护作用
受试物：ZONK2003-0、ZONK2003-2、ZONK2003-4；由广东中科药物研究有限公司提供。
磷酸奥司他韦颗粒，规格15mg×10袋装，宜昌东阳光长江药业股份有限公司产品，批号：0371912115，有效期至2021.12.11，用于抗甲型流感病毒的阳性对照药；
实验材料：甲型流感病毒鼠肺适应株A/FM/1/47(H1N1)，接种鸡胚，收集尿囊液保存。ICR小鼠，体重18～22g。给药期间自由进食、饮水，每天12小时光照，12小时黑暗，温度22±2℃，湿度55-70％。实验方法：适应性饲养3天后，开始进行实验。除未感染对照组以外，其它各组小鼠用乙醚轻度麻醉，鼻腔内接种用生理盐水稀释的相当于8×LD 50的流感病毒A/FM/1/47(H1N1)的鸡胚尿囊液50μL/只，阳性对照奥司他韦组和实施例化合物组小鼠于感染后2h首次灌胃给药，每种化合物以10μmol/kg、20μmol/kg、30μmol/kg的剂量口服给药，每天给药两次，连续用药5天。观察14天内小鼠的存活情况，并计算药物对于小鼠的死亡保护率(死亡保护率＝模型组死亡率一实验组死亡率)。
表7、化合物对A型流感病毒(H1N1甲型流感病毒)感染小鼠的保护作用
Figure PCTCN2021128879-appb-000031
Figure PCTCN2021128879-appb-000032
实施例16：药物对流感病毒H1N1感染导致的小鼠肺部炎症的缓解作用
实验方法：适应性饲养3天后，开始进行实验。除未感染对照组以外，其它各组小鼠用乙醚轻度麻醉，鼻腔内接种用生理盐水稀释的相当于8×LD 50的流感病毒A/FM/1/47(H1N1)的鸡胚尿囊液50μL/只，阳性对照奥司他韦组小鼠和供试给药组于病毒感染24h后以80mg/kg首次灌胃给药，以后每日1次，病毒对照组及未感染对照组同法口服生理盐水，每日1次，给药体积为0.1mL/10g体重。共5天。第6天每组取3只小鼠称重，摘除眼球放血致死，取出全肺，称重，计算肺指数及肺指数抑制率。
失重百分比％＝给药前体重-给药后体重/给药前体重×100％
肺指数＝小鼠肺重/小鼠体重×100
肺指数抑制率(％)＝病毒对照组肺指数均数-给药组肺指数均数/病毒对照组肺指数均数×100％
表8
组别 失重百分比％ 肺指数 肺指数抑制率％
正常对照组   0.57  
模型对照组   2.09  
奥司他韦组 15.12 1.66 20.57
ZONK2003-0 9.89 1.52 27.27
ZONK2003-2 11.45 1.23 41.14
ZONK2003-4 9.56 1.18 43.54
实验结果：因此实施例化合物对流感病毒引起的肺部炎症有明显的保护和抑制作用，且效果优于奥司他韦对照组和ZONK2003-0组。
实施例17：ZONK2003药物大鼠药代动力学实验
雄性大鼠分成4组，分别为ZONK2003-0注射和口服、ZONK2003-2注射和口服，其中，ZONK2003-0注射和口服、ZONK2003-2注射组每组3只，ZONK2003-2口服组每组6只，分别为每只大鼠采集10个不连续的时间点。
建立测定ICR小鼠全血中ZONK2003-0、ZONK2003-2浓度的LC-MS/MS分析方法。所得血药浓度数据同时采用药动学处理软件Pharsight Phoenix WinNonlin 8.0非房室模型计算相关药代动力学参数。
大鼠灌胃ZONK2003-2，口服剂量25mg/kg，注射ZONK2003-2为1.21mg/kg；
大鼠灌胃ZONK2003-0，口服剂量2.51mg/kg，注射ZONK2003-0为1.00mg/kg，检测血药浓度，计算药代动力学参数：
溶剂：20％Solutol HS-15/生理盐水
详细结果见下表。
表9 大鼠尾静脉给药1.00mg/kg的ZONK2003-0后，大鼠体内原型药物血药浓度检测结果(ng/mL)
Figure PCTCN2021128879-appb-000033
表10 大鼠尾静脉给药1.00mg/kg的ZONK2003-0后，原型药物主要药代动力学参数
Figure PCTCN2021128879-appb-000034
表11 大鼠灌胃给药2.51mg/kg的ZONK2003-0后，大鼠体内原型药物血药浓度检测结果(ng/mL)
Figure PCTCN2021128879-appb-000035
Figure PCTCN2021128879-appb-000036
表12 大鼠灌胃给药2.51mg/kg的ZONK2003-0后，原型药物主要药代动力学参数
Figure PCTCN2021128879-appb-000037
表13 大鼠尾静脉给药1.21mg/kg的ZONK2003-2后，原型药物主要药代动力学参数
Figure PCTCN2021128879-appb-000038
表14ZONK2003-2灌胃给药25mg/kg组大鼠原型药物血药浓度检测结果(ng/mL)
Figure PCTCN2021128879-appb-000039
ND：未检测到，即血药浓度达峰后测定值低于定量下限点
表15 大鼠灌胃给药25mg/kg的ZONK2003-2后，原型药物主要药代动力学参数
Figure PCTCN2021128879-appb-000040
由上表可知，ZONK2003-0的绝对生物利用度F值为62.5％，ZONK2003-2组的绝对生物利用度F值为106.0％。
实施例18：ZONK2003系列化合物的镇痛作用实验
实施例化合物对冰醋酸致NIH小鼠扭体的影响
NIH小鼠：SPF级，雌雄各半，15-17g，110只，由广东省医学实验动物中心提供，ZONK2003-0、ZONK2003-2、ZONK2003-4、ZONK2003-14、ZONK2003-18；由广东中科药物研究有限公司提供。
2、实验方法和结果
NIH小鼠110只，雄性，体重15-17g。检疫3天，检疫结束后按体重随机分成组，每组10只，即：模型组、实验药物组。实验时各小鼠尾静脉给予相应的药物，给药容积：0.1ml/10g体重，模型组给予等容积的生理盐水。给药后0.5小时按10mL/kg体重腹腔注射0.7％(0.7g/100ml)HAc致痛，然后立即观察15min内各鼠的扭体反应，并记录扭体次数，计算抑制率。
试验结果如表17所示，各剂量组与模型组比较均能显著抑制冰醋酸致NIH小鼠的扭体次数(p<0.05或p<0.01)，作用强度优于ZONK2003-0组。
表17 化合物对冰醋酸致NIH小鼠扭体的影响
Figure PCTCN2021128879-appb-000041
实施例19 DHODH酶抑制活性测试
ZONK2003-0和ZONK2003-2、Brequinar的DHODH酶活性测试结果如下：
表18
Figure PCTCN2021128879-appb-000042
Figure PCTCN2021128879-appb-000043
ZONK2003与ZONK2003-0不同，ZONK2003-2不是通过DHODH酶的抑制发挥抗病毒作用。

Claims (10)

  1. 结构通式如式I所示的化合物或其药学上可接受的盐、溶剂合物：
    Figure PCTCN2021128879-appb-100001
    所述式(Ⅰ)中，
    R 1选自：单取代或多取代的H、F、甲基、三氟甲基；
    R 2选自：H、直链或取代的C1-C6烷烃；
    R 3选自：单取代或多取代的H、Cl、Br、F；
    R 4选自：直链或取代的C1-C6烷烃。
  2. 根据权利要求1所述的化合物或其药学上可接受的盐、溶剂合物，其特征在于：
    所述R 1选自H；
    或，所述R 2选自甲基或异丙基；
    或，所述R 3选自Cl；
    或，所述R 4选自甲基、乙基或丙基。
  3. 权利要求1或2所述式I所示的化合物的制备方法，包括下述步骤：
    1)将式a所示化合物与硫氰酸钠进行反应，得到式b所示化合物；
    Figure PCTCN2021128879-appb-100002
    其中，式a中R 2的定义同式I；式b中R 2的定义同式a；
    2)将式b所示化合物与式e所示的R 1取代的邻甲酸苯甲醛进行缩合反应，得到式c所示化合物；
    Figure PCTCN2021128879-appb-100003
    Figure PCTCN2021128879-appb-100004
    其中，式e中R 1的定义同式I；式c中R 1的定义同式e、R 2的定义同式b；
    3)将式c所示化合物与式f所示的R 3取代溴苯乙酮进行关环反应，得到式d所示化合物；
    Figure PCTCN2021128879-appb-100005
    其中，式f中R 3的定义同式I，式d中R 1、R 2的定义同式c，R 3的定义同式f；
    4)在缩合剂作用下，使式d所示化合物与式g所示化合物进行缩合反应，得到式I所示化合物；
    Figure PCTCN2021128879-appb-100006
    其中，式g中X、R 4的定义同式I。
  4. 根据权利要求3所述的方法，其特征在于：
    所述步骤1)中，所述反应的反应条件为：反应温度为50-100℃，反应时间为24-72小时；反应在溶剂中进行，所述溶剂选自下述任意一种：甲醇、乙醇、四氢呋喃、乙腈等，优选乙醇；
    所述步骤2)中，所述缩合反应的反应条件为：反应温度50-100℃，反应时间为1-3小时；反应在溶剂中进行，所述溶剂选自下述任意一种：甲醇、乙醇、四氢呋喃、乙腈，优选乙醇；
    所述步骤3)中，所述关环反应的反应条件为：反应温度50-100℃，反应时间为3-6小时；反应在溶剂中进行，所述溶剂选自下述任意一种：甲醇、乙醇、四氢呋喃、乙腈，优选乙醇；
    所述步骤4)中，所述缩合反应的反应条件为：反应温度0-25℃，反应时间为2-8小时；反应在溶剂中进行，所述溶剂选自下述任意一种：二氯甲烷、四氢呋喃、乙腈，优选二氯甲烷。
  5. 权利要求1所述式I所示的化合物或其药学上可接受的盐、溶剂合物的应用为下述(a)和/或(b)和/或(c)：
    (a)式I所示化合物或其药学上可接受的盐、酯、溶剂合物在制备治疗病毒所致疾病或病毒感染的产品中的应用；
    (b)式I所示化合物或其药学上可接受的盐、酯、溶剂合物在制备预防病毒所致疾病或病毒感染的产品中的应用；
    (c)式I所示化合物或其药学上可接受的盐、酯、溶剂合物在制备病毒抑制剂中的应用；
    (d)式I所示化合物或其药学上可接受的盐、酯、溶剂合物在制备镇痛药物中的应用。
  6. 根据权利要求5所述的应用，其特征在于：
    所述产品为药物或药物制剂；所述病毒抑制剂能够抑制病毒的复制；
    所述病毒包括流感病毒、冠状病毒。
  7. 根据权利要求6所述的应用，其特征在于：所述流感病毒为甲型流感病毒；所述冠状病毒为HCoV-229E。
  8. 一种药物或药物组合物，其活性成分为式I所示化合物或其药学上可接受的盐、溶剂合物；
    所述药物或药物组合物具有下述至少一种功效：
    1)治疗病毒所致疾病或病毒感染；
    2)预防病毒所致疾病或病毒感染；
    3)抑制病毒；
    4)镇痛。
  9. 根据权利要求8所述的药物或药物组合物，其特征在于：
    所述产品为药物或药物制剂；所述病毒抑制剂能够抑制病毒的复制；
    所述病毒包括流感病毒、冠状病毒。
  10. 根据权利要求9所述的药物或药物组合物，其特征在于：所述流感病毒为甲型流感病毒；所述冠状病毒为HCoV-229E。
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