Use of an aminonitrothiazole derivative in preparing a drug for preventing and treating viral infections and related diseases caused by them

Abstract

The present invention provides an aminonitrothiazole derivative for use in the preparation of a drug for preventing and treating viral infections and related diseases caused thereto, and belongs to the field of pharmaceuticals. The structure of the aminonitrothiazole derivative is shown in Formula I. Experimental results show that the compound can effectively inhibit the activity of respiratory syncytial virus, enterovirus 71, and the new coronavirus, and the inhibitory effect is better than that of the positive controls nitazoxanide and lauric acid. The compound provided by the present invention can be used to prepare a drug for preventing and / or treating viral infections, and can be used to prepare a drug for preventing and / or treating related diseases caused by viruses, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceuticals, and particularly relates to use of an aminonitrothiazole derivative in preparing a drug for preventing and / or treating viral infections and related diseases caused by the drug. Background Art

[0002] Viral infection refers to the process by which viruses invade the body through various pathways and multiply in susceptible host cells. Viral infection is a serious health concern. There are numerous types of viruses, approximately 4,000, of which approximately 400 are associated with human diseases. Statistics show that over 80% of human infectious diseases are caused by viruses, and one-sixth of human tumors are linked to viruses.

[0003] Enterovirus 71 (EV71) is a member of the genus Enterovirus in the family Picornaviridae. Its primary cause of hand-foot-and-mouth disease (HFMD) is hand-foot-and-mouth disease (HFMD), which primarily affects children under five years old. It is primarily transmitted through close contact, the respiratory tract, and the digestive tract. It can cause fever, rashes, and ulcers on the hands, feet, and mouth. In some cases, it can lead to complications such as myocarditis, pulmonary edema, and aseptic meningitis, even leading to death. Currently, there are no effective antiviral drugs for HFMD on the domestic market. The development of drugs to treat EV71 infection is crucial for the treatment of HFMD.

[0004] Respiratory syncytial virus (RSV), a member of the genus Pneumovirus in the family Paramyxoviridae, can cause upper and lower respiratory tract illnesses and is a common cause of acute lower respiratory tract infections in infants and young children. Almost all children under the age of two have experienced RSV infection. In recent years, RSV infection has also been found to be a significant viral disease in the elderly and immunocompromised adults.

[0005]

[0006] Nitazoxanide is a derivative of nitazoxanide. While its exact mechanism of action is unclear, it is believed to involve inhibition of the pyruvate-ferredoxin oxidoreductase-dependent electron transfer reaction, which is crucial for anaerobic energy metabolism. Studies have reported that nitazoxanide can inhibit the replication of respiratory viruses such as parainfluenza virus, coronavirus, and respiratory syncytial virus. However, the inhibitory activity of nitazoxanide against respiratory syncytial virus needs to be further improved, and the cytotoxic side effects of nitazoxanide need to be further reduced.

[0007] Therefore, there is an urgent need to develop an antiviral infection drug that can not only improve the inhibitory activity against viruses but also reduce the toxic side effects on cells. Summary of the Invention

[0008] The object of the present invention is to provide an aminonitrothiazole derivative for use in preparing a drug for preventing and / or treating viral infection, as well as a drug for related diseases caused by viruses.

[0009] The present invention provides the use of a compound represented by formula (I), or a crystal form thereof, or a salt thereof, in the preparation of a medicament for preventing and / or treating viral infections, and / or in the preparation of a medicament for preventing and / or treating diseases caused by viruses:

[0010]

[0011] Wherein, R is selected from C3, C4 straight chain alkanes, C5-C 15 Straight-chain or branched alkanes.

[0012] Furthermore, the compound is one of the following compounds:

[0013]

[0014] Furthermore, the virus is rotavirus, norovirus, adenovirus, coronavirus, enterovirus or respiratory syncytial virus.

[0015] Furthermore, the enterovirus is enterovirus 71; the respiratory virus is respiratory syncytial virus; and the coronavirus is a new coronavirus.

[0016] Furthermore, the disease caused by the virus is gastroenteritis or infectious diarrhea caused by rotavirus, norovirus or adenovirus.

[0017] Furthermore, the disease caused by the virus is hand, foot and mouth disease caused by enterovirus 71.

[0018] Furthermore, the disease caused by the virus is inflammation caused by adenovirus, respiratory syncytial virus or coronavirus.

[0019] Furthermore, the inflammation is rhinitis, pharyngitis, tracheitis, bronchitis, bronchiolitis, or pneumonia.

[0020] Furthermore, the pneumonia is pneumonia caused by infection with the new coronavirus.

[0021] Furthermore, the drug is a preparation prepared by using the compound, or its crystal form, or its salt as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0022] Furthermore, the excipients or auxiliary ingredients are selected from one or more of diluents, fillers, colorants, glidants, lubricants, adhesives, stabilizers, suspending agents and buffers.

[0023] In this invention, the novel coronavirus refers to SARS-CoV-2, also known as 2019-nCoV. Pneumonia caused by the novel coronavirus infection is referred to as COVID-19.

[0024] Experimental result shows, the compound of the present invention can effectively suppress respiratory syncytial virus activity, particularly compound I-3 is far more effective than positive control nitazoxanide and lauric acid in inhibiting activity of respiratory syncytial virus, and the half toxic concentration of the cell is far more than positive control nitazoxanide, illustrating that the compound significantly improves virus inhibition activity, and significantly decreases the toxicity of the cell, and the selection index SI significantly improves. The compound of the present invention can effectively suppress enterovirus 71 type activity, particularly compound I-3 is slightly more effective than positive control nitazoxanide in inhibiting activity of enterovirus 71 type, and toxicity is far less than nitazoxanide, and selection index SI is far greater than nitazoxanide. The compound of the present invention I-3 can significantly suppress novel coronavirus activity, is better than positive control nitazoxanide in inhibiting activity of novel coronavirus, and toxicity is far less than nitazoxanide, and selection index SI is far greater than nitazoxanide. The compound of the present invention I-3 can effectively treat mice infected by novel coronavirus, reduce mouse lung virus load, reduce mouse lung inflammatory reaction, and at equimolar doses, its therapeutic effect is significantly better than control compound nitazoxanide and lauric acid.

[0025] The compounds provided by the present invention can be used to prepare drugs for preventing and / or treating viral infections, and can be used to prepare drugs for preventing and / or treating related diseases caused by viruses, and have broad application prospects.

[0026] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0027] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0028] Figures in the specification

[0029] Figure 1 These are the lung tissue pathology scoring results of each group of mice in Example 4.

[0030] Figure 2 These are the HE staining results of some mouse lung tissues in Example 4.

[0031] Figure 3 These are the viral load results of lung tissues of each group of mice in Example 4. DETAILED DESCRIPTION

[0032] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0033] Preparation of test compounds I-1 to I-4:

[0034] The test compounds I-1 to I-4 were prepared according to the preparation method disclosed in CN114044761A. The specific operation is as follows:

[0035]

[0036] 2.0 g of tizoxanide (7.5 mmol) was placed in a 50 ml single-necked flask, 20 ml of ethyl acetate and 0.9 g of triethylamine (8.8 mmol) were added, and 0.9 g of n-butyryl chloride (8.4 mmol) was added dropwise with stirring. After the addition was complete, the mixture was heated to reflux in an oil bath and allowed to react for 2 hours. TLC confirmed the complete reaction. The mixture was cooled to room temperature and filtered to remove the solid. The filtrate was diluted with 20 ml of ethyl acetate and washed twice with 10 ml of 0.1 M dilute hydrochloric acid, once with 10 ml of 10% sodium bicarbonate solution, and finally with 10 ml of saturated brine until neutral. The ethyl acetate layer was dried over 2.5 g of anhydrous sodium sulfate for 30 minutes; the sodium sulfate was removed by filtration, and the filtrate was concentrated to dryness under reduced pressure. The resulting crude product was purified by silica gel column chromatography to obtain compound I-1.

[0037] Referring to the above method, n-butyryl chloride was replaced by octanoyl chloride, lauroyl chloride, and tetradecanoyl chloride, respectively, to prepare compounds I-2, I-3, and I-4, respectively:

[0038]

[0039] Example 1. In vitro inhibitory activity of the test compounds against respiratory syncytial virus

[0040] (1) Experimental methods

[0041] Hep2 (laryngeal squamous cell carcinoma) cells were used as the viral host to determine the inhibitory effects of test compounds I-1 to I-4 on Hep2 cytopathic effects induced by respiratory syncytial virus (RSV). Test compounds I-1 to I-4 and the positive controls, nitazoxanide and lauric acid, were prepared using diluents immediately prior to use. The stock solutions were diluted in culture medium to a specific concentration and then further diluted three-fold, starting at 200 μM. A total of eight dilutions were used.

[0042] Hep2 cells were seeded in 96-well culture plates and 24 hours later, the cells were plated at 100 TCID 50Infected with respiratory syncytial virus, and added with maintenance solution containing different dilutions of compounds I-1 to I-4 and positive control drugs and positive control drugs, set up cell control wells and virus control wells, and cultured at 37 ° C with 5% CO2. When the cytopathic effect (CPE) of the virus control group reached 4+, the cytopathic effect (CPE) of each group was observed, and the half toxic concentration (TC) of the sample to the cells was calculated by the Reed-Muench method. 50 ) and the half-maximal inhibitory concentration (IC 50 ), and calculate the selection index SI, SI = TC 50 / IC 50 .

[0043] The results are shown in Table 1 below.

[0044] (2) Experimental results

[0045] Table 1. The results of the inhibition of respiratory syncytial virus by each compound

[0046] Compound <![CDATA[IC 50 (μM)]]> <![CDATA[TC 50 (μM)]]> SI I-1 5.96 55.71 9.34 I-2 4.47 57.34 12.83 I-3 0.82 115.47 140.82 I-4 8.47 69.18 8.17 Nitazoxanide 7.41 12.83 1.73 Lauric acid >200 >200 -

[0047] The experimental results show that the test compounds of the present invention can effectively inhibit the activity of respiratory syncytial virus, and the inhibitory activity of compounds I-1 to I-4 is much stronger than that of the control compound lauric acid, and the inhibitory activity of compounds I-1 to I-3 is much stronger than that of the positive control nitazoxanide.

[0048] Further, according to the selectivity index (SI), it can be seen that the selectivity index of the test compounds of the present invention is greater than that of the positive control, nitazoxanide. In particular, compound I-3 has a much stronger inhibitory activity against respiratory syncytial virus than the positive controls, nitazoxanide and lauric acid, and its median toxic concentration for cells is much higher than that of the positive control, nitazoxanide, indicating that the compound has significantly improved antiviral activity while significantly reducing cytotoxicity, and the selectivity index (SI) is significantly improved.

[0049] The above experimental results indicate that the test compound of the present invention can be used to prevent and / or treat respiratory syncytial virus infection and related diseases caused by it.

[0050] Example 2: In vitro inhibitory activity of the test compounds against enterovirus 71

[0051] (1) Experimental methods

[0052] Vero (African green monkey kidney) cells were used as the viral host to determine the extent to which test compounds I-1 to I-4 inhibited enterovirus 71-induced Vero cytopathic effects. Test compounds I-1 to I-4 and positive controls nitazoxanide and lauric acid were prepared using diluents immediately prior to use. Stock solutions were diluted with culture medium to a specific concentration and then further diluted three-fold, starting at 200 μM. A total of eight dilutions were used.

[0053] Vero cells were cultured in 96-well plates and 100 TCID 50 Infected with enterovirus 71, adsorbed for 2 hours, discarded the virus solution, added with maintenance solution containing different dilutions of compounds I-1 to I-4 and positive control drugs, and set up cell control wells and virus control wells at the same time. Incubated at 37°C with 5% CO2. When the cytopathic effect (CPE) of the virus control group reached 4+, the cytopathic effect (CPE) of each group was observed, and the half toxic concentration (TC) of the sample to the cells was calculated using the Reed-Muench method. 50 ) and the half-maximal inhibitory concentration (IC 50 ), and calculate the selection index SI, SI = TC 50 / IC 50 .

[0054] The results are shown in Table 2 below.

[0055] (2) Experimental results

[0056] Table 2. Results of the inhibitory activity of each compound on enterovirus 71

[0057]

[0058]

[0059] The experimental results show that the test compounds of the present invention can effectively inhibit the activity of enterovirus 71, and the inhibitory activity of compounds I-1 to I-4 is much stronger than that of the control compound lauric acid, and the inhibitory activity of compounds I-1 and I-3 is stronger than that of the positive control nitazoxanide.

[0060] Further, it can be seen from the selectivity index SI that the selectivity index of the test compounds of the present invention is greater than that of the positive control nitazoxanide. In particular, compound I-3 has a slightly stronger inhibitory activity against enterovirus 71 than the positive control nitazoxanide, and is much less toxic than nitazoxanide, with a selectivity index SI much greater than that of nitazoxanide.

[0061] The above experimental results indicate that the test compound of the present invention can be used to prevent and / or treat enterovirus 71 infection and related diseases caused by it.

[0062] Example 3: In vitro inhibitory activity of the test compounds against the novel coronavirus

[0063] (1) Experimental methods

[0064] Cell culture: Vero E6 cells were cultured in complete medium at a rate of approximately 1 × 10 4Inoculate 100 μl of cells / well into a 96-well plate, culture overnight in a 37°C, 5% CO2 cell culture incubator to form a cell monolayer. Observe under an inverted microscope. When the cells are approximately 80%-90% confluent, discard the supernatant, wash with PBS, and set aside for the next experiment.

[0065] Drug addition: Test compound I-3 and control compounds nitazoxanide and lauric acid were tested in eight concentration gradients, starting at 200 μM and diluted three-fold, with four replicates per well. The test drugs were added to the cells and incubated in a 37°C, 5% CO2 incubator for 24 hours.

[0066] Viral infection: In the microbiology room of the BSL-III laboratory, the new coronavirus strain (No.: IVCAS6.7512lot20211202) was diluted with serum-free DMEM medium at a virus infection multiplicity of infection (MOI) of 0.01. The drug-containing medium in the 96-well plate was discarded, and 50 μl of the virus infection solution was added to the cell culture wells and incubated for 1 hour.

[0067] RNA extraction: Aspirate the viral fluid, wash the wells with PBS to remove unadsorbed virus, and then replace with 200 μl / well of culture medium containing the same drug concentration as in the original well. After 48 hours, remove 100 μl of cell supernatant and add it to 400 μl of Trizol LS. Total RNA from each well was extracted using an automated nucleic acid extraction kit (Vazyme Cat. RM201-02 96rxn).

[0068] Reverse transcription: The above RNA was reverse transcribed into cDNA using a reverse transcription kit (Reverse Transcriptase Kit (M-MLV) ZR102).

[0069] The cDNA generated in the above steps was used as a sample, and the sample Ct value was detected by fluorescence quantitative PCR. The infected cell group without drug treatment was used as a control to calculate the inhibition rate of the drug on the intracellular virus. The IC value of the inhibitory activity of the test compound on the new coronavirus was calculated by GraphPad. 50 The CCK8 method was used to test the half toxic concentration (TC) of the test compound to cells. 50 ), and calculate the selection index SI, SI = TC 50 / IC 50 .

[0070] (2) Experimental results

[0071] The results are recorded in Table 3 below:

[0072] Table 3. IC inhibitory activity of the test compounds of the present invention against the novel coronavirus 50

[0073] Test compound <![CDATA[IC 50 (μM)]]> <![CDATA[TC 50 (μM)]]> SI I-3 1.73 94.77 54.78 Nitazoxanide 2.33 33.62 14.43 Lauric acid >200 >200 -

[0074] The experimental results show that the test compound I-3 of the present invention can significantly inhibit the activity of the new coronavirus, and its inhibitory activity is much higher than that of the control compound lauric acid, and stronger than the positive control nitazoxanide.

[0075] Further, according to the selectivity index SI, it can be seen that the inhibitory activity of the test compound I-3 of the present invention against the new coronavirus is slightly stronger than that of the positive control nitazoxanide, and its toxicity is much less than that of nitazoxanide, and the selectivity index SI is much greater than that of nitazoxanide.

[0076] The above experimental results show that the test compounds of the present invention can be used to prevent and / or treat novel coronavirus infection and related diseases caused by it.

[0077] Example 4: Therapeutic Effects of the Test Compounds on the Novel Coronavirus Infection Model in Mice

[0078] (1) Experimental methods

[0079] B6 / JGpt-H11 em1Cin(K18-hACE2) / Gpt , K18-hACE2 KI mice, SPF grade (Jiangsu Jicui Yaokang Biotechnology Co., Ltd., strain number: T037657), 9-10 weeks old, female, a total of 30 mice, were randomly divided into 5 groups, namely blank group, model group, I-3 group, nitazoxanide group, and lauric acid group, with 6 mice in each group. After one week of adaptive feeding, the drug was given the day before the challenge. The I-3 group was given 300 mg / kg compound I-3; the nitazoxanide group was given 204 mg / kg nitazoxanide; the lauric acid group was given 45 mg / kg lauric acid, and the model group was given an equal volume of normal saline, which was administered twice a day for a total of 4 days. On the day of the challenge, the mice in each group except the blank group were challenged with the SARS-CoV-2Omicron BA.1 strain (the strain was a clinically isolated strain). The animals were sacrificed after 3 days of normal feeding after the challenge, and the samples were taken for analysis.

[0080] The left lungs of mice in each group were fixed with 10% formaldehyde and then rendered harmless. HE staining was performed to observe the inflammatory response of the mouse lungs after virus attack. The scores were scored and the results were recorded. The scoring criteria are as follows:

[0081] 0 points: No damage

[0082] 1 point: <25% of the damaged area [mild damage]

[0083] 2 points: 25% to 50% of the damaged area [moderate damage]

[0084] 3 points: 50% to 75% of the damaged area

[0085] 4 points: >75% of the injured area

[0086] Right lung tissue from each group of mice was homogenized and centrifuged, and 100 μl of the supernatant was added directly to 4 times the sample volume of Trizol LS. The sample was thoroughly mixed, and RNA was extracted according to the Trizol LS instructions. After RNA extraction, the viral N gene was directly quantitatively detected using a one-step qPCR method (Shanghai Bojie Medical Technology Co., Ltd., Novel Coronavirus 2019-nCoV Nucleic Acid Detection Kit (Fluorescence PCR Method), National Medical Device Registration No. 20203400065). The viral load in the sample was calculated using the standard curve external standard method, and the results were recorded.

[0087] (2) Experimental results

[0088] The lung tissue pathology scores of mice in each group were recorded as follows: Figure 1 As shown in Table 4, * represents P < 0.5 compared with the model group; HE staining results of some mouse lung tissues are shown in Figure 2 The results of viral load in lung tissue of mice in each group are shown as follows: Figure 3 As shown in the table, ** represents P < 0.05 compared with the model group, and ## represents P < 0.05 compared with the nitazoxanide group.

[0089] Table 4. Lung histopathological scores of mice in each group

[0090] Group score Blank group 0.2 Model Group 2.7 Group I-3 <![CDATA[1.2 * ]]> Nitazoxanide group 1.8 Lauric acid group 2.3

[0091] The experimental results showed that in the results of mouse lung tissue pathology scoring, the I-3 group significantly reduced the inflammatory response of mouse lung tissue after novel coronavirus infection, which was statistically different from the model group; the nitazoxanide group and the lauric acid group slightly reduced the inflammatory response of mouse lung tissue, but there was no statistical difference compared with the model group. In the results of mouse lung tissue viral load, the I-3 group and the nitazoxanide group significantly reduced the viral load compared with the model group, and the I-3 group viral load was significantly different from the nitazoxanide group. The above results show that the test compound of the present invention can effectively treat mice infected with novel coronavirus, reduce mouse lung viral load, reduce mouse lung inflammatory response, and at equimolar doses, its therapeutic effect is significantly better than that of the control compounds nitazoxanide and lauric acid.

[0092] The above experimental results show that the test compounds of the present invention can be used to prevent and / or treat novel coronavirus infection and related diseases caused by it.

[0093] In summary, the present invention provides an aminonitrothiazole derivative shown in Formula I for use in the preparation of drugs for preventing and / or treating viral infections and related diseases caused thereto. Experimental results show that the compound can effectively inhibit the activity of respiratory syncytial virus, enterovirus 71 and new coronavirus, and the inhibitory effect is better than that of the positive controls nitazoxanide and lauric acid. The compound provided by the present invention can be used to prepare drugs for preventing and / or treating viral infections, and can be used to prepare drugs for preventing and / or treating related diseases caused by viruses, and has broad application prospects.

Claims

1. Use of a compound or a salt thereof in the preparation of a drug for antiviral infection; the virus is enterovirus 71 or respiratory syncytial virus, and the compound is one of the following compounds:

2. The use according to claim 1, characterized in that: The medicine is a medicine for preventing and / or treating diseases caused by viruses.

3. The use according to claim 2, characterized in that: The disease caused by the virus is hand, foot and mouth disease caused by enterovirus 71.

4. The use according to claim 2, characterized in that: The disease caused by the virus is inflammation caused by respiratory syncytial virus.

5. The use according to claim 4, characterized in that: The inflammation is rhinitis, pharyngitis, tracheitis, bronchitis, bronchiolitis.

6. The use according to any one of claims 1 to 5, characterized in that: The medicine is a preparation prepared by taking the compound or its salt as the active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

7. The use according to claim 6, characterized in that: The auxiliary materials or auxiliary components are selected from one or more of diluents, fillers, colorants, glidants, lubricants, adhesives, stabilizers, suspending agents and buffers.

8. Use of a compound or a salt thereof in the preparation of a drug for antiviral infection; the virus is a novel coronavirus, and the compound is:

9. The use according to claim 8, characterized in that: The medicine is a medicine for preventing and / or treating diseases caused by viruses.

10. The use according to claim 9, characterized in that: The disease caused by the virus is inflammation caused by the new coronavirus.

11. The use according to claim 9, characterized in that: The inflammation is pneumonia.

12. The use according to any one of claims 8 to 11, characterized in that: The medicine is a preparation prepared by taking the compound or its salt as the active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

13. The use according to claim 12, characterized in that: The auxiliary materials or auxiliary components are selected from one or more of diluents, fillers, colorants, glidants, lubricants, adhesives, stabilizers, suspending agents and buffers.

Images