Antiviral application of parsnip lactone derivative

Abstract

The invention provides antiviral application of a compound shown in a formula I. The compound has excellent activity in an SARS-CoV-2 pseudovirus system infection resisting model and an SARS-CoV-2XBB1.5 pseudovirus system infection resisting model, and the activity is obviously superior to that of parsnilide and radix saposhnikoviae oxalic acid. The compound also has a certain inhibition effect on HPV16 (human papillomavirus 16). According to the determination of a CCK8 method, the cytotoxicity is at a 10 <-5 > M level, and the biological safety is relatively high. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and particularly relates to the antiviral use of parsnip lactone derivatives. Background Art

[0002] Coronavirus disease 2019 (COVID-19) is a zoonotic severe infectious respiratory syndrome caused by a new coronavirus (SARS-CoV-2). SARS-CoV-2 is a single-stranded positive-sense RNA virus that belongs to the β-coronavirus family of the Coronaviridae family. As a non-cellular microorganism with infectious activity and high parasitism, it must rely on the host for proliferation. The life cycle of SARS-CoV-2 virus generally includes four processes: viral adsorption and invasion, membrane fusion and uncoating, genome transcription and replication and viral protein synthesis, and viral particle assembly and budding. During its entire life cycle, it recruits more than 300 host factors or proteins to assist in its amplification.

[0003] At present, the main targets for drug development for the treatment of SARS-CoV-2 are: 1. The functional receptors that SARS-CoV-2 needs to recognize for invading host cells - angiotensin-converting enzyme 2 (ACE2) and the key host factor - neuropilin-1 (NPR1); 2. Cellular proteases required for membrane fusion and uncoating, such as Furin, transmembrane serine protease 2 (TMPRSS2), and cathepsin (Cathepsin L and Cathepsin B). However, since SARS-CoV-2 has multiple pathways in this process, blocking a single protease of cellular proteases may not be enough to completely inhibit the virus, and the combination of multiple protease inhibitors will lead to more severe toxicity. Therefore, the application of antiviral inhibitors targeting a single cellular protease is still challenging; 3. Other host proteins, such as farnesoid X receptor, bromodomain-containing protein 2 (BRD2), and CD147. Most of these drugs are still in the preclinical development stage, and a few have entered clinical trials. Among them, Sabizabulin, a microtubule disruptor with potential antiviral and anti-inflammatory activity, has been found to reduce the mortality rate of severe COVID-19 patients.

[0004] Anti-SARS-CoV-2 drugs mainly include three categories: 1. Antiviral drugs that target viral proteins to block the viral life cycle. This type of drug selection has higher specificity, but due to the rapid mutation rate of SARS-CoV-2 itself, it has a potential risk of drug resistance; 2. Antiviral drugs that target host proteins involved in the viral life cycle. This type of drug has the potential advantage of broad-spectrum antiviral activity and overcomes the problem of drug resistance, but its specificity and safety may be poor; 3. Antiviral drugs that target the human immune system. SARS-CoV-2 infection can induce dysfunctional immune responses and aggressive inflammatory responses, and it has been found clinically that the use of commercially available immunomodulators or anticoagulants, such as cytokine antagonists, can reduce the mortality and length of hospital stay of COVID-19, but the effectiveness of this type of drug remains to be considered, especially in the early stages of COVID-19.

[0005] So far, there are a total of 6 anti-SARS-CoV-2 drugs on the market in my country: 2 imported drugs (Paxlovid and Molnupiravir) and 4 domestic drugs: Azvudine, Senoxin, Mindevi and Leruiling.

[0006] Since SARS-CoV-2 may exist for a long time and continue to mutate, the drug resistance brought by the mutant strains may still lead to highly infectious diseases and cause major epidemics. Therefore, it is of great strategic significance to develop new anti-coronavirus drugs with differentiated targets and mechanisms, or new anti-coronavirus drugs that can overcome drug resistance. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides the use of a compound as shown in the following formula I or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating a disease caused by a virus infection:

[0008]

[0009] According to an embodiment of the present invention, the compound shown in formula I is recorded in patent document CN201811511738.9, the entire text of which is incorporated herein by reference.

[0010] According to an embodiment of the present invention, the pharmaceutically acceptable salt is selected from pharmaceutically acceptable salts formed by the compound represented by Formula I and an inorganic acid or an organic acid.

[0011] According to an embodiment of the present invention, the inorganic acid or organic acid is selected from hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid, phosphorous acid, sulfurous acid, carbonic acid, boric acid, phosphomolybdic acid, selenious acid, methanesulfonic acid, phenylsulfonic acid, fumaric acid, citric acid, maleic acid, tartaric acid, oxalic acid, D-malic acid, L-malic acid, DL-malic acid, L-lactic acid, D-lactic acid, DL-lactic acid, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, oleic acid, lauric acid, p-toluenesulfonic acid, 1-naphthalenesulfonic acid, 2-naphthalenesulfonic acid, phthalic acid, malonic acid, succinic acid, glycolic acid, mercaptoic acid, glycine, sarcosine, sulfonic acid, nicotinic acid, methylpyridine acid, isonicotinic acid, dichloroacetic acid, and benzoic acid.

[0012] According to an embodiment of the present invention, the pharmaceutically acceptable salt as described above can be prepared by referring to the method described in patent document CN201811511738.9 or a method known in the art.

[0013] In a preferred embodiment, the pharmaceutically acceptable salt of the compound of Formula I is its fumarate, that is, selected from the following compounds ACT004,

[0014]

[0015] According to an embodiment of the present invention, the virus is a coronavirus or HPV16, and the coronavirus is the new coronavirus SARS-COV-2.

[0016] According to an embodiment of the present invention, the novel coronavirus SARS-COV-2 includes but is not limited to the following strains: XBB1.5.

[0017] The present invention also provides a method for treating a disease caused by a coronavirus, comprising administering a compound as shown in the above formula I or a pharmaceutically acceptable salt thereof to an individual in need thereof.

[0018] Beneficial Effects

[0019] The present invention provides the antiviral use of the compound shown in formula I, wherein the compound has excellent activity in the anti-SARS-CoV-2 pseudovirus system infection and the anti-SARS-CoV-2XBB1.5 pseudovirus system infection model, and the activity is significantly better than that of oxalis lactone and oxalis acid. The compound also has a certain inhibitory effect on HPV16. Moreover, as determined by the CCK8 method, its cytotoxicity is 10 -5 M level, with high biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The figure shows the cytotoxicity test results of ACT004, oxalic acid and oxalool.

[0021] Figure 2 This is a graph showing the test results of the effects of ACT004, oxalic acid, oxalic acid lactone, remdesivir and Fab-281 against SARS-CoV-2 pseudovirus system infection.

[0022] Figure 3 This is a graph showing the test results of the effects of ACT004, oxalic acid, oxalic acid lactone and Fab-281 on the infection of the SARS-CoV-2XBB1.5 pseudovirus system.

[0023] Figure 4 This is a graph showing the test results of the compound ACT004 against HPV16 virus infection (changes in mRNA levels were determined by qPCR).

[0024] Figure 5 This is a graph showing the test results of the compound ACT004's anti-HPV16 virus infection effect (protein level changes were determined by western blot). DETAILED DESCRIPTION

[0025] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0026] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0027] Example 1. Cytotoxicity test of ACT004, oxalicum acid and oxalicum lactone

[0028] 1.1 Experimental methods

[0029] The CCK8 method was used to determine the half toxic concentration (CC 50 ).

[0030] (1) The day before, the previously constructed Vero cells and Huh7 cells stably expressing TMPRSS2 were cultured at 2×10 4 / well were inoculated into 96-well plates.

[0031] (2) On the second day, the test sample was diluted 3-fold to the highest concentration of 100 μM, with 3 replicate wells set for each dilution, for a total of 8 dilutions.

[0032] (3) Enter the BSL-1 laboratory, aspirate and discard the cell culture supernatant, and add the above-mentioned dilution of corresponding concentration to each well (100 μL / well).

[0033] (4) Culture in a constant temperature and humidity incubator at 37°C and 5% CO2.

[0034] (5) After 24 hours of culture, 10 μL of CCK-8 solution was added to each well.

[0035] (6) After culturing in a constant temperature and humidity incubator at 37°C and 5% CO2 for 1 hour, the absorbance at 450 nm was measured using a multifunctional microplate reader to analyze the cell survival rate and calculate CC. 50 value.

[0036] 1.2 Experimental Results

[0037] The cytotoxicity of the three test compounds was determined by CCK8 method, and CC 50 Value. Figure 1 As can be seen in the figure, the cytotoxicity of ACT004, parsnipid lactone and parsnipid acid was 10 -5 M level. From the curve goodness and CC 50 After comprehensive analysis, the ranking of cellular safety effects is ACT004>parsnipid lactone>parsnipid acid.

[0038] Example 2. Test of the effect of ACT004, oxalicylic acid and oxalicylic acid on the infection of SARS-CoV-2 pseudovirus system

[0039] 2.1 Experimental methods

[0040] Flow cytometry was used to determine the half-maximal effective concentration (EC) of the compounds. 50 ).

[0041] (1) The day before, the previously constructed Vero cells stably expressing TMPRSS2 were cultured at 2×10 4 / well were inoculated into 96-well plates.

[0042] (2) On the second day, the test sample was diluted 3-fold to the highest concentration of 25 μM, the control sample (Remdesivir) was diluted 3-fold to the highest concentration of 12.5 μM, and the control sample (Fab-281) was diluted 5-fold to the highest concentration of 50 ug / mL. Three replicate wells were set for each dilution, for a total of 8 dilutions.

[0043] (3) Enter the BSL-2 laboratory, discard the cell culture supernatant, add the above-mentioned dilution of corresponding concentration to each well (100 μL / well), and add an equal volume of virus suspension (SARS-CoV-2S-VSVΔG / EYFPWuhan) containing 0.1 multiplicity of infection (MOI) to each well.

[0044] (4) Culture in a constant temperature and humidity incubator at 37°C and 5% CO2.

[0045] (5) After 48 hours of culture, the fluorescence value was read using a flow cytometer to analyze the inhibition rate and calculate the EC 50 value.

[0046] 2.2 Experimental Results

[0047] Flow cytometry was used to determine the inhibitory effects of the five test substances on SARS-CoV-2Wuhan infection at the cellular level, and the EC 50 The results showed that the three test products and two control products had good inhibitory effects on SARS-CoV-2 strains at the cellular level. The inhibition curves and EC 50 Value See Figure 2 .from Figure 2 It can be seen that ACT004, parsnip lactone and parsnip acid have anti-SARS-CoV-2Wuhan activity at the micromolar level. Obviously, these three test products can effectively inhibit SARS-CoV-2 infection at the cellular level, as shown by the curve goodness of fit and EC 50 Comprehensive analysis showed that the inhibitory effects were ranked as ACT004>oxalic acid>oxalolide.

[0048] Example 3. Test of the effect of ACT004, oxalic acid and oxalool on the infection of SARS-CoV-2XBB1.5 pseudovirus system

[0049] 3.1 Experimental methods

[0050] The half-maximal effective concentration (EC) of the compound was determined by luciferase reporter gene assay. 50 ).

[0051] (1) The day before, Huh7 cells were cultured at 2×10 4 / well were inoculated into 96-well plates.

[0052] (2) On the second day, the test sample was diluted 3-fold to the highest concentration of 25 μM, and the control sample (Fab-281) was diluted 5-fold to the highest concentration of 50 ug / mL. Three replicate wells were set for each dilution, for a total of 8 dilutions.

[0053] (3) Enter the BSL-2 laboratory, discard the cell culture supernatant, add the above-mentioned dilution of corresponding concentration to each well (100 μL / well), and add an equal volume of virus suspension (S-VSVΔG / Luc XBB1.5) containing 0.1 multiplicity of infection (MOI) to each well.

[0054] (4) Culture in a constant temperature and humidity incubator at 37°C and 5% CO2.

[0055] (5) After 24 hours of incubation, the supernatant was discarded and 100 μL of luciferase detection reagent was added. After reacting at room temperature in the dark for 2 minutes, the cells were repeatedly blown and the luminescence value was read using a multifunctional imaging microplate reader to analyze the inhibition rate and calculate the EC 50 value.

[0056] 3.2 Experimental Results

[0057] The luciferase reporter gene assay was used to determine the inhibitory effects of the five test substances on SARS-CoV-2S-VSVΔG / Luc XBB1.5 infection at the cellular level, and the EC 50 The results showed that the three test products and one control product had a certain inhibitory effect on the SARS-CoV-2 strain at the cellular level. The inhibition curves and EC 50 Value See Figure 3 .from Figure 3 It can be seen that the anti-SARS-CoV-2XBB1.5 activities of ACT004, parsnip lactone and parsnip acid reached tens of micromolar levels. Obviously, these three test products can inhibit SARS-CoV-2XBB1.5 infection at the cellular level. From the curve goodness of fit and EC 50 Comprehensive analysis showed that the inhibitory effects were ranked as ACT004>oxalic acid>parsnilide, and the inhibitory effect of ACT004 was significantly better than that of oxalic acid and parsnilide.

[0058] Example 4 Test of the effect of the compound on HPV16 virus infection

[0059] 4.1 Experimental methods

[0060] The qPCR method and western blot method were used to jointly evaluate the inhibitory effect of the compound (the Caski cell line was HPV16 type).

[0061] (1) One day in advance, Caski cells carrying HPV genomes were plated on six-well plates. The next day, serum-free culture medium containing different concentrations of drugs was replaced (three replicate wells). Six hours later, the culture medium was removed. The day before, Hep2 cells were plated at 2×10 4 / well were inoculated into 96-well plates.

[0062] (2) On the second day, the test sample was diluted 2-fold to the highest concentration of 50 μM, and the control sample (FIT-039) was diluted 2-fold to the highest concentration of 20 μM. Three replicate wells were set for each dilution, for a total of 2 to 3 dilutions, and the medium was replaced with serum-free medium containing the diluted drug concentration.

[0063] (3) Culture in a constant temperature and humidity incubator at 37°C and 5% CO2.

[0064] (4) After 6 hours of culture, the cell culture supernatant was discarded and the cells were collected. RNA was extracted and reverse transcribed. Then, qPCR was performed to detect the content of E6, E7 and Gapdh (internal reference) in the cells. The normalized results were used to check the changes of E6 and E7 after administration.

[0065] (5) For another part of the samples that were administered, the culture medium was removed 12 hours later and the cells were collected. The cells were lysed with RIPA lysis buffer. Then, the levels of pRb, p53 and Gapdh (internal reference) in the cells were detected by western blot. The normalized results were used to calculate the changes of p53 and pRB after administration.

[0066] 4.2 Experimental Results

[0067] The qPCR method was used to measure the changes in mRNA levels and the western blot method was used to measure the changes in protein levels to evaluate the inhibitory effects of the test compounds on HPV16 infection at the cellular level. Figure 4 As can be seen in Figure 2, at the mRNA level, ACT004 can only reduce the expression of HPV16 E7 mRNA in the CaSki cell line. Figure 5 It can be seen that at the protein level, ACT004 can only reduce the expression of HPV16 E7mRNA in the CaSki cell line, thereby leading to a relative increase in the pRb protein content. In summary, ACT004 has a certain inhibitory effect on HPV16.

[0068] In summary, ACT004 of the present invention has excellent activity in the anti-SARS-CoV-2 pseudovirus system infection and anti-SARS-CoV-2XBB1.5 pseudovirus system infection models, and its activity is significantly better than that of oxalis lactone and oxalis acid. In addition, the compound also has a certain inhibitory effect on HPV16. Finally, the cytotoxicity of the compound was 10 -5 M level, and high biological safety. Therefore, compound ACT004 has certain application prospects.

[0069] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of the compound represented by formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a disease caused by a virus infection, 2. The use according to claim 1, characterized in that The pharmaceutically acceptable salt is selected from pharmaceutically acceptable salts formed by the compound represented by formula I and an inorganic acid or an organic acid.

3. The use according to claim 2, characterized in that: The inorganic acid or organic acid is selected from hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid, phosphorous acid, sulfurous acid, carbonic acid, boric acid, phosphomolybdic acid, selenious acid, methanesulfonic acid, phenylsulfonic acid, fumaric acid, citric acid, maleic acid, tartaric acid, oxalic acid, D-malic acid, L-malic acid, DL-malic acid, L-lactic acid, D-lactic acid, DL-lactic acid, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, oleic acid, lauric acid, p-toluenesulfonic acid, 1-naphthalenesulfonic acid, 2-naphthalenesulfonic acid, phthalic acid, malonic acid, succinic acid, glycolic acid, mercaptoic acid, glycine, sarcosine, sulfonic acid, nicotinic acid, methylpyridine acid, isonicotinic acid, dichloroacetic acid, and benzoic acid.

4. The use according to claim 1, characterized in that The pharmaceutically acceptable salt of the compound of formula I is its fumarate, which is selected from the following compound ACT004, 5. The use according to any one of claims 1 to 4, characterized in that: The virus is coronavirus or HPV16.

6. The use according to claim 5, characterized in that The coronavirus is the new coronavirus SARS-COV-2.

7. The use according to claim 6, characterized in that The new coronavirus SARS-COV-2 is the XBB1.5 strain.

Images