Application of thiazole compound in preparation of medicine for resisting hMPV virus infection

Abstract

The invention discloses an application of thiazole compounds in preparation of drugs for resisting hMPV virus infection, and belongs to the field of organic small molecule drugs. The thiazole compound (nitazoxanide and tizoxanide as shown in formula I) has the functions of protecting cells infected by hMPV and inhibiting hMPV replication, can be used for preparing the medicine for resisting hMPV virus infection, and has a very good treatment effect in the aspect of treating diseases caused by hMPV. In an in-vitro experiment, the thiophene compound disclosed by the invention can be used for efficiently inhibiting the replication of the hMPV virus at a low dosage, has obvious advantages in the aspect of inhibiting the hMPV compared with other known compounds with antiviral activity, such as ribavirin and EIDD2801, and has better potential in the treatment of hMPV infection. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of organic small molecule drugs, and specifically relates to the application of thiazole compounds in the preparation of drugs for resisting hMPV virus infection. Background Art

[0002] Nitazoxanide, chemically known as 2-(acetyloxy)-N-(5-nitro-2-thiazolyl)benzamide, is a thiazole drug first synthesized in the 1970s (Rossignol, JF, Cavier, R., 1975. Synthesis and antiparasitic activity of 2-benzamidonitrothiazoles. Chem. Abstr. 83, 28216n.). Nitazoxanide is a prodrug that is quickly hydrolyzed in vivo to its active metabolite tizoxanide (TIZ) after administration, thereby exerting its pharmacological effects. The structural formulas of nitazoxanide and tizoxanide are shown below:

[0003]

[0004] Nitazoxanide was initially studied as an oral antiparasitic drug, and has good antiprotozoal activity in vitro and in different animal models. Its first clinical study was to treat human intestinal tapeworms. Subsequent in vitro and in vivo studies found that it has good inhibitory effects on new protozoa and Cryptosporidium. In 1998, the U.S. FDA approved nitazoxanide as a clinical treatment drug for adults and children over one year old infected with Cryptosporidium parvum and Giardia. In 2006, clinical studies confirmed that nitazoxanide can effectively treat diarrhea in children caused by rotavirus infection. In addition, nitazoxanide has also been shown to have good clinical therapeutic effects on adult viral gastroenteritis and hepatitis C (Rossignol JF, Nitazoxanide: a first-in-class broad-spectrum antiviral agent, Antiviral Res. 2014; 110: 94-103.).

[0005] In recent years, a number of laboratory studies have confirmed that nitazoxanide has broad-spectrum antiviral activity. A series of laboratory studies have shown that nitazoxanide can block the maturation of influenza virus hemagglutinin protein at the post-translational stage and has high inhibitory activity against both influenza A and influenza B. Clinical research results show that nitazoxanide, taken twice a day, 600 mg each time, for 5 consecutive days, can effectively reduce the course of common influenza (Haffizulla J, Hartman A, Hoppers M, et al, Effect of nitazoxanide in adults and adolescents with acute uncomplicated influenza: a double-blind, randomised, placebo-controlled, phase 2b / 3 trial, Lancet Infect Dis. 2014; 14(7): 609-18.).

[0006] Human metapneumovirus (hMPV) was discovered in a laboratory in the Netherlands in 2001 and has been traced back to the 1950s (van den Hoogen et al., Analysis of the genomic sequence of a humanmetapneumovirus.). Due to its slow replication in vitro, its existence remained undetected for a long time. Today, it is considered an important cause of pneumonia in children, in addition to other important pathogens such as human respiratory syncytial virus (HRSV) and human parainfluenza virus (HPIV) (Choi et al., Outcomes of severe human metapneumovirusassociated community-acquired pneumonia in adults.). HMPV is a negative-sense virus that contains a non-segmented single-stranded linear RNA genome and belongs to the family Paramyxoviridae. This family is further divided into two genera, Orthopneumovirus and Metapneumovirus, which include HRSV and HMPV, respectively (Afonso et al., 2016). Two HMPV serotypes, A and B, have been identified based on nucleotide sequence diversity of the fusion protein (F) and attachment protein (G) (van den Hoogen et al., Antigenic and genetic variability of human metapneumoviruses.). Antigenic analysis has highlighted differences between the different serotypes, and A and B are further divided into HMPV A1, A2, A3, B1, and B2 subtypes. A meta-analysis of studies published between August 2017 and August 2019 found that the overall prevalence of HMPV in children ranged from 1% to 86%, depending on the country (Divarathna et al., A review of onepidemiology and impact of human metapneumovirus infections in children using TIAB search strategy on PubMed and PubMed Central articles.). In the United States and Canada, where the number of infections is highest, the economic burden on the healthcare system is very high, with associated costs reaching $277 million per year in the United States alone (Davis et al., Incidence, morbidity, and costs of human metapneumovirus infection in hospitalized children.).

[0007] Most complications caused by hMPV-related viral infections occur in children who are premature, under 1 year of age, or immunocompromised, and later in life (>65 years), patients with severe underlying medical conditions (such as chronic lung disease, congenital heart disease, and neuromuscular disease) are more likely to develop severe HMPV-induced pneumonia (Principi et al., Paediatric humanmetapneumovirus infection: epidemiology, prevention and therapy.). In these susceptible populations, especially immunocompromised patients, the mortality rate of hMPV-related pneumonia is similar to that of severe intravenous infection, 54.5% and 54.3%, respectively. Therefore, there is a high but unmet need for novel preventive and therapeutic approaches. To date, only one candidate live attenuated vaccine has been evaluated in a phase I clinical study (registered with ClinicalTrials.gov, number NCTO1255410). However, the candidate vaccine appeared too weak to provide protection in children infected with hMPV, so further studies were terminated (Karron et al., Evaluation of a live attenuated humanmetapneumovirus vaccine in adults and children.).

[0008] There are currently few small molecule drugs for the treatment and prevention of hMPV, among which ribavirin is the main representative. Ribavirin is a guanosine analog that inhibits viral purine metabolism and viral RNA polymerase and has been approved for the treatment of severe hRSV (human respiratory syncytial virus) infection in children (Crotty et al., Ribavirin's antiviral mechanism of action: lethal mutagenesis). Since hMPV is phylogenetically and phenotypically related to hRSV, hMPV infection was evaluated in vitro (Wyde et al., Comparison of the inhibition of human metapneumovirus and respiratory syncytial virus by ribavirin and immune serum globulin invitro.). In vivo evaluation of ribavirin showed a reduction in HMPV symptoms and viral load 4 days after treatment started 12 hours after infection. However, there has not been a randomized controlled study on the use of ribavirin for the treatment of HMPV to date, and the potential teratogenic effects and high production costs of ribavirin and IVIG demonstrate the need for safer and more effective antiviral treatments (Wen et al., New approaches for immunization and therapy against human metapneumovirus. Clin. Vaccine Immunol.).

[0009] Therefore, it is still a lot of work and difficulty to find small molecule compounds that have a good effect on hMPV, and the inhibitory or preventive effects of existing small molecules on hMPV are difficult to predict. Summary of the invention

[0010] The present invention aims at the problems existing in the prior art and provides the use of thiazole compounds in the preparation of drugs for resisting hMPV virus infection. The purpose of the present invention is to find drugs with anti-hMPV virus activity for the treatment of hMPV virus infection. The present invention finds through creative research that nitazoxanide and tizoxanide have the functions of protecting hMPV-infected cells and inhibiting hMPV replication, and can be used to prepare drugs for resisting hMPV virus infection, and have good therapeutic effects in treating diseases caused by hMPV.

[0011] To achieve the above objectives, in a first aspect, the present invention provides the use of a thiazole compound in the preparation of a drug for resisting hMPV virus infection, wherein the thiazole compound is a compound represented by Formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate or hydrate thereof;

[0012]

[0013] Wherein, R is selected from H or COCH3.

[0014] In a preferred embodiment, the drug against hMPV virus infection is a drug for preventing or treating a disease or infection caused by hMPV virus.

[0015] In a preferred embodiment, the disease or infection caused by the hMPV virus is a respiratory disease.

[0016] In a preferred embodiment, the disease or infection caused by the hMPV virus is simple infection, pneumonia, acute respiratory infection, severe acute respiratory infection, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis and / or septic shock; the simple infection includes at least one of fever, cough, and sore throat.

[0017] In a preferred embodiment, the pharmaceutically acceptable salt includes an inorganic acid salt, an organic acid salt, an inorganic base salt or an organic base salt.

[0018] In a preferred embodiment, the pharmaceutically acceptable salt includes at least one of sodium salt, potassium salt, calcium salt, lithium salt, meglumine salt, hydrochloride, hydrogen oxalate, hydrogen iodide, nitrate, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, acetate, propionate, butyrate, oxalate, pivalate, adipate, alginate, lactate, citrate, tartrate, succinate, maleate, fumarate, picrate, aspartate, gluconate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate.

[0019] According to the present invention, the thiazole compound shown in Formula I can protect cells from cytopathic effect (CPE) caused by hMPV virus infection, inhibit hMPV virus replication on cells, and reduce the viral nucleic acid load in cell culture. After creative invention and research, the inventors discovered new characteristics of the thiazole compound shown in Formula I in cells: the thiazole compound shown in Formula I can reduce the level of cells infected by hMPV virus at micromolar concentrations.

[0020] In a second aspect, the present invention provides a pharmaceutical composition, comprising a thiazole compound, wherein the thiazole compound is a compound represented by Formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate or hydrate thereof;

[0021]

[0022] Wherein, R is selected from H or COCH3.

[0023] In a preferred embodiment, the pharmaceutical composition is an oral tablet, capsule, powder, oral solution, injection or transdermal preparation.

[0024] In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0025] In a preferred embodiment, the pharmaceutically acceptable carrier or excipient includes a diluent, a filler, a disintegrant, a wetting agent, a lubricant, a colorant, a flavoring agent or other conventional additives, preferably at least one of microcrystalline cellulose, starch, cross-linked polyvinylpyrrolidone, povidone, polyvinylpyrrolidone, maltitol, citric acid, sodium lauryl sulfate or magnesium stearate.

[0026] In a third aspect, the present invention provides use of the aforementioned pharmaceutical composition in the preparation of a drug for resisting hMPV virus infection.

[0027] In a preferred embodiment, the drug against hMPV virus infection is a drug for preventing or treating a disease or infection caused by hMPV virus.

[0028] In a preferred embodiment, the disease or infection caused by the hMPV virus is a respiratory disease.

[0029] In a preferred embodiment, the disease or infection caused by the hMPV virus is simple infection, pneumonia, acute respiratory infection, severe acute respiratory infection, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis and / or septic shock; the simple infection includes at least one of fever, cough, and sore throat.

[0030] In a fourth aspect, the present invention provides the use of a thiophene compound or a pharmaceutical composition containing a thiophene compound in the preparation of a drug for inhibiting the replication or reproduction of hMPV virus in a cell; the thiazole compound is a compound represented by formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate or hydrate thereof;

[0031]

[0032] Wherein, R is selected from H or COCH3.

[0033] In a preferred embodiment, the cell is a mammalian cell.

[0034] In a preferred embodiment, the use comprises administering a therapeutically and / or prophylactically effective amount of the thiophene compound or a pharmaceutical composition containing the thiophene compound to a mammal.

[0035] In a preferred embodiment, the pharmaceutical composition is an oral tablet, capsule, powder, oral solution, injection or transdermal preparation.

[0036] In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0037] In a preferred embodiment, the pharmaceutically acceptable carrier or excipient includes a diluent, a filler, a disintegrant, a wetting agent, a lubricant, a colorant, a flavoring agent or other conventional additives, preferably at least one of microcrystalline cellulose, starch, cross-linked polyvinylpyrrolidone, povidone, polyvinylpyrrolidone, maltitol, citric acid, sodium lauryl sulfate or magnesium stearate.

[0038] The beneficial effects of the present invention are:

[0039] 1. The present invention provides the use of thiophene compounds (nitazoxanide and tizoxanide) in the preparation of drugs for resisting hMPV virus infection. The thiazole compounds can protect cells from cytopathic effect (CPE) caused by hMPV virus infection, inhibit hMPV virus replication on cells, and reduce the viral nucleic acid load in cell culture. They have the functions of protecting hMPV-infected cells and inhibiting hMPV replication. They can be used to prepare drugs for resisting hMPV virus infection and have good therapeutic effects in treating diseases caused by hMPV.

[0040] 2. The present invention has discovered a new characteristic of the action of the thiazole compounds in cells: the thiazole compounds shown in Formula I can reduce the level of hMPV virus-infected cells at micromolar concentrations.

[0041] 3. In in vitro experiments, the thiophene compounds (nitazoxanide and tizoxanide) of the present invention can effectively inhibit the replication of hMPV virus at low doses, and have significant advantages in hMPV inhibition compared with other known compounds with antiviral activity such as ribavirin and EIDD2801. DETAILED DESCRIPTION

[0042] It is worth noting that the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0043] Example 1: Nitazoxanide and tizoxanide reduce the level of hMPV virus-infected cells

[0044] 1. Cell recovery and subculture:

[0045] Prepare DMEM complete medium (Gibco) containing 10% FBS (V / V) and 100μg / mL streptomycin and 100 IU / mL penicillin and T25 cell culture plates, and sterilize them under ultraviolet light in a clean bench for 15 min; at the same time, turn on the water bath and preheat to 37°C. Take out the frozen LLC-MK2 cells from -80°C, put them in EP gloves and immediately immerse them in a water bath, thaw for 2 minutes, and shake them slightly during the period. Centrifuge at 800 g for 5 minutes at room temperature, discard the supernatant, add 1 mL10% DMEM complete medium, and use a 1mL pipette to blow 10 times. Move the cell suspension to a T25 cell culture flask, add 5mL10% DMEM complete medium, shake well, observe the cell state and density under a microscope, and move to a 5% CO2 incubator with saturated humidity and 37°C for culture. Observe the cell state and density under a microscope after 24 hours. When the cell state is full and the morphology is clear, and the cell density is 90%, it can be passaged. Discard the supernatant, add 3 mL PBS (Gibco) to wash the cells, add 1 mL 0.25% trypsin (Gibco), shake the culture flask to evenly cover the cells, and place it in the incubator for 5 minutes. Observe the changes of cells under a microscope. After the cells become round, add 4 mL DMEM complete medium to end the digestion. After blowing to a single cell suspension state, transfer the cell suspension to a T75 cell culture flask, add 10% DMEM complete medium to 10 mL, shake evenly, and place in the incubator for culture.

[0046] 2. Cell plating:

[0047] LLC-MK2 cells in the logarithmic growth phase were digested with 0.25% (W / V) trypsin and diluted to 2.5×10 5 cells / mL cell suspension, spread into a 24-well plate, 500 μL / well, shake to mix, and culture in an incubator for 16 h.

[0048] 3. Drug antiviral screening:

[0049] After discarding the supernatant, add 2% DMEM (containing 2% FBS (V / V) and 100 μg / mL streptomycin, 100 IU / mL penicillin, 2 μg / mL TPCK is added to the 2% DMEM medium of LLC-MK2 cells) containing the compound of the present invention, the final concentration of the compound of the present invention is 10 μM, and each treatment is repeated in 1 well. Set up control wells with DMSO and virus, virus, and no virus. Continue to culture for 1 hour, except for the control wells without virus, add virus hMPV virus to all other wells, add 20 μL of virus stock solution to each well, shake to mix, and culture in an incubator for 1.5 hours (shake every 30 minutes during this period), discard the supernatant, add 2% complete medium, and continue to culture in an incubator for three days.

[0050] 4. Trizol method to extract RNA:

[0051] Discard the supernatant, add 500μL Trizol lysis buffer to each well, pipette to fully lyse the cells, and combine the Trizol cell lysis buffers of two wells into a 2mL EP tube without enzyme. Add 100μL chloroform and vibrate vigorously for 15s to allow the two solutions to fully contact each other. Centrifuge at 4℃, 13000g for 15min, take 180μL of supernatant into a new 1.5mL EP tube without enzyme, add an equal volume of isopropanol and gently mix by inversion. After standing at 4℃ for 10min, centrifuge at 4℃, 13000g for 15min, gently pour off the supernatant, add 500μL pre-cooled freshly prepared 75% ethanol, and flick the bottom of the test tube to float the precipitate in the solution. After centrifugation at 4℃, 13000g for 5min, gently pour off the supernatant, open the lid and dry the liquid in the tube in the clean bench. Add 20μL RNase-free DEPC water to each tube and centrifuge briefly to dissolve the RNA precipitate.

[0052] 4. RNA Reverse Transcription

[0053] According to the instructions of the reverse transcription kit (Nanjing Novozyme Biotechnology Co., Ltd., IIIAII-in-one RT SuperMix Perfect for qPCR, Catalog No. R333), prepare the reverse transcription system in a 200μL PCR tube: add 8μL RNase-free ddH2O, 4μL 5×AII-in-one qRT SuperMix, 1μL EnzymeMix and 5μL template RNA (i.e., sample RNA) to each tube, a total of 20μL system, centrifuge briefly to mix, put it into a 96-well fast PCR instrument, and reverse transcribe according to the set program (see Table 1 for reverse transcription program settings). After the reverse transcription is completed, the sample is stored in a 4℃ refrigerator for a short period of time.

[0054] Table 1 Reverse transcription procedure

[0055] temperature time 50℃ 15min 85℃ 5sec 4℃ +∞

[0056] 6. Fluorescence quantitative PCR experimental operation

[0057] First, prepare the qPCR reaction system in a 96-well PCR plate: 10μL TB Green Premix Ex Taq Ⅱ (TliRNaseH Plus) (TaKaRa), 6μL RNase-free ddH2O, 1μL upstream primer, 1μL downstream primer and 2μL template cDNA (i.e., sample cDNA). After preparation, centrifuge the PCR plate to remove bubbles in the tube, put the plate into the PCR instrument, and amplify cDNA according to the program in Table 2-3. Perform fluorescent quantitative PCR reaction on the cDNA obtained by reverse transcription. GAPDH is used as the transcription standard indicating the relative expression level of mRNA, and the results are calculated using the 2-ΔΔCt method, and 1-infection rate = inhibition rate is used.

[0058] Table 2 qPCR reaction procedure

[0059]

[0060] Table 3 qPCR primer sequences

[0061]

[0062] The inhibition rate of the compounds provided by the present invention on hMPV infected cells is shown in Table 4:

[0063] Table 4 Inhibition rate of compounds on hMPV infected cells

[0064]

[0065] The solutions of the present invention are not limited to the technical means disclosed by the above technical means, but also include technical solutions composed of any combination of the above technical features. The above is a specific implementation of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications are also regarded as the protection scope of the present invention.

Claims

1. The use of thiazole compounds in the preparation of drugs for resisting hMPV virus infection, characterized in that: The thiazole compound is a compound represented by Formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate or hydrate thereof; Wherein, R is selected from H or COCH3.

2. The use according to claim 1, characterized in that The drug for resisting hMPV virus infection is a drug for preventing or treating diseases or infections caused by hMPV virus; preferably, the disease or infection caused by hMPV virus is a respiratory disease; more preferably, the disease or infection caused by hMPV virus is a simple infection, pneumonia, acute respiratory tract infection, severe acute respiratory tract infection, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis and / or septic shock; most preferably, the simple infection includes at least one of fever, cough, and sore throat.

3. The use according to claim 1, characterized in that The pharmaceutically acceptable salt includes an inorganic acid salt, an organic acid salt, an inorganic base salt or an organic base salt. More preferably, the pharmaceutically acceptable salt includes at least one of a sodium salt, a potassium salt, a calcium salt, a lithium salt, a meglumine salt, a hydrochloride salt, a hydrobromide salt, a hydroiodate salt, a nitrate salt, a sulfate salt, a hydrogen sulfate salt, a phosphate salt, a hydrogen phosphate salt, an acetate salt, a propionate salt, a butyrate salt, an oxalate salt, a trimethylacetate salt, an adipate salt, alginate salt, a lactate salt, a citrate salt, a tartrate salt, a succinate salt, a maleate salt, a fumarate salt, a picrate salt, an aspartate salt, a gluconate salt, a benzoate salt, a methanesulfonate salt, an ethanesulfonate salt, a benzenesulfonate salt, a p-toluenesulfonate salt and a pamoate salt.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a thiazole compound, wherein the thiazole compound is a compound represented by Formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate or hydrate thereof; Wherein, R is selected from H or COCH3.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition is an oral tablet, capsule, powder, oral liquid, injection or transdermal preparation; the pharmaceutical composition also contains a pharmaceutically acceptable carrier or excipient; preferably, the pharmaceutically acceptable carrier or excipient includes a diluent, a filler, a disintegrant, a wetting agent, a lubricant, a colorant, a flavoring agent or other conventional additives; more preferably, the pharmaceutically acceptable carrier or excipient includes at least one of microcrystalline cellulose, starch, cross-linked polyvinylpyrrolidone, povidone, polyvinylpyrrolidone, maltitol, citric acid, sodium lauryl sulfate or magnesium stearate.

6. Use of the pharmaceutical composition according to any one of claims 4 or 5 in the preparation of drugs for resisting hMPV virus infection.

7. The use according to claim 6, characterized in that The drug for resisting hMPV virus infection is a drug for preventing or treating diseases or infections caused by hMPV virus; preferably, the disease or infection caused by hMPV virus is a respiratory disease; more preferably, the disease or infection caused by hMPV virus is a simple infection, pneumonia, acute respiratory tract infection, severe acute respiratory tract infection, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis and / or septic shock; most preferably, the simple infection includes at least one of fever, cough, and sore throat.

8. Use of a thiophene compound or a pharmaceutical composition containing a thiophene compound in the preparation of a drug for inhibiting the replication or reproduction of hMPV virus in cells, characterized in that: The thiazole compound is a compound represented by Formula I, or a pharmaceutically acceptable salt, stereoisomer, tautomer, solvate or hydrate thereof; Wherein, R is selected from H or COCH3.

9. The use according to claim 8, characterized in that The cell is a mammalian cell; the use comprises administering a therapeutically and / or preventively effective amount of the thiophene compound or a pharmaceutical composition containing the thiophene compound to a mammal.

10. The use according to claim 8, characterized in that The pharmaceutical composition is an oral tablet, capsule, powder, oral liquid, injection or transdermal preparation; the pharmaceutical composition also contains a pharmaceutically acceptable carrier or excipient; preferably, the pharmaceutically acceptable carrier or excipient includes a diluent, a filler, a disintegrant, a wetting agent, a lubricant, a colorant, a flavoring agent or other conventional additives; more preferably, the pharmaceutically acceptable carrier or excipient includes at least one of microcrystalline cellulose, starch, cross-linked polyvinylpyrrolidone, povidone, polyvinylpyrrolidone, maltitol, citric acid, sodium lauryl sulfate or magnesium stearate.

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