A class of quinolone compounds with anti-SARS-CoV-2 activity and their applications in drugs

Abstract

The present invention discloses a class of quinolone compounds with anti-SARS-CoV-2 activity and their applications in drugs. The quinolone compounds are obtained by chemical synthesis methods, and their structures are shown in formula (I). The compounds of the present invention exhibit single-digit micromolar inhibitory activity against the SARS-CoV-2 Omicron BA.2 virus, have excellent inhibitory activity against the novel coronavirus, and provide a new direction for the treatment and prevention of COVID-19. At the same time, they may have broad-spectrum antiviral activity against other coronaviruses, and new lead compounds are sought for drug development.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical uses, and particularly relates to quinolone compounds having anti-SARS-CoV-2 virus activity and their applications in drugs. Background Art

[0002] Coronaviruses (CoVs; Coronavirinae subfamily, Coronaviridae family, Coronavirales order) are a highly diverse group of enveloped positive-sense single-stranded RNA viruses and are common pathogens causing various diseases in animals and humans. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), is a non-double-stranded positive-sense RNA virus. While vaccines and monoclonal antibodies are being applied in humans, the SARS-CoV-2 virus itself is also constantly changing, giving rise to multiple mutant strains (also known as variant strains). On November 26, 2021, the World Health Organization (WHO) announced a new variant of the severe acute respiratory syndrome coronavirus (SARS-CoV-2), OMICRON (B.1.1.529). As a variant of concern (VOC), the OMICRON variant has been classified into four different sub-lineages: BA.1, BA.1.1, BA.2, and BA.3.

[0003] So far, there is no specific therapeutic drug for human coronavirus infection. The early antiviral treatment of COVID-19 mainly draws on the experience of combating SARS and MERS, and a large number of clinical trials are carried out to verify the efficacy of drugs. Interferon, lopinavir / ritonavir, ribavirin, chloroquine, and arbidol are among the drugs recommended in the current diagnosis and treatment plan. The strategy of "repurposing old drugs" has great limitations in clinical treatment, and currently, SARS-CoV-2 is still raging globally, and no drug has played a good role in clinical treatment. There is an urgent need to develop safe and effective antiviral drugs. Summary of the Invention

[0004] The object of the present invention is to disclose a class of quinolone compounds having anti-SARS-CoV-2 virus activity, and to provide an application of a quinolone compound as an inhibitor against SARS-CoV-2 virus infection. Experiments have proved that the compound can effectively inhibit the activity of Omicron, which can provide a new idea for the development of small molecule inhibitors of the virus for treating COVID-19.

[0005] A class of quinolone compounds with anti-SARS-CoV-2 virus activity, the general structural formula of which is shown in Formula I:

[0006]

[0007] Wherein R1 represents H, C1-C6 substituted alkyl; the substituents in C1-C6 substituted hydrocarbon groups are H, halogen, nitro, amino, substituted amino, hydroxyl, ether group, substituted phenyl, substituted heterocyclic group, carboxyl, ester group or amide group; the substituted amino is R3NH or R3R4N, wherein R3 or R4 is a C1-C6 hydrocarbon group, R3 and R4 can be independent of each other, R3 and R4 can also be connected into a ring or connected into a ring through 1-3 heteroatoms;

[0008] R2 represents a benzene ring, a 4-7 membered aromatic heterocyclic group, a substituted benzene ring, and the substituents in the substituted benzene ring can be H, F, Cl, Br, C1-C6 alkyl, hydroxyl, C1-C6 alkoxy, nitro or amino.

[0009] In some embodiments, R1 represents H, substituted or unsubstituted C1-C3 alkyl; the substituents are selected from H, halogen, nitro, amino, hydroxyl or carboxyl;

[0010] In some embodiments, R2 represents a substituted or unsubstituted benzene ring, and the substituents are selected from H, F, Cl, Br, C1-C3 alkyl, hydroxyl, C1-C3 alkoxy, nitro or amino.

[0011] The present invention also provides a quinolone compound 54 with anti-SARS-CoV-2 virus activity, the structure of which is shown as follows:

[0012]

[0013] The present invention also discloses a preparation method of the quinolone compound 54 with anti-SARS-CoV-2 virus activity.

[0014] Furthermore, the preparation method of the quinolone compound 54 with anti-SARS-CoV-2 virus activity according to the present invention has the following specific synthesis route:

[0015]

[0016] In some examples, compound I-1 is dissolved in anhydrous DMF and undergoes a benzylation reaction with benzyl bromide under the condition of using NaH as a base to obtain intermediate I-2.

[0017] In some examples, intermediate I-2 undergoes Dakin oxidation under the conditions of methanol, water, NaOH solution and hydrogen peroxide to obtain intermediate I-3.

[0018] In some examples, intermediate I-3 is dissolved in anhydrous DMF and undergoes a methylation reaction with dimethyl sulfate under alkaline conditions to obtain intermediate I-4.

[0019] In some examples, intermediate I-4 is reduced with iron powder in a mixed solvent of acetic acid and water to obtain intermediate I-5.

[0020] In some examples, intermediate I-5 and ethyl benzoylacetate undergo a nucleophilic substitution reaction in toluene as the solvent and p-toluenesulfonic acid as the catalyst to obtain intermediate I-6.

[0021] In some examples, intermediate I-6 undergoes a high-temperature cyclization reaction in diphenyl ether as the solvent to obtain intermediate I-7 with a protected phenolic hydroxyl group.

[0022] In some examples, intermediate I-7 undergoes selective demethylation and debenzylation in acetonitrile as the solvent and aluminum trichloride as the catalyst to obtain intermediate I-8.

[0023] In some examples, intermediate I-8 and 1,2-dibromoethane are refluxed in the presence of an acid-binding agent in acetone as the solvent to obtain intermediate I-9.

[0024] In some examples, intermediate I-9 and pyrrolidine are refluxed in acetonitrile to obtain the target compound 54.

[0025] In some examples, the high temperature for the high-temperature cyclization of intermediate I-6 to form intermediate I-7 is 210-215 °C.

[0026] The present invention also provides a pharmaceutical composition, which contains a therapeutically effective amount of a quinolone compound having anti-SARS-CoV-2 virus activity as described in the present invention, and a pharmaceutical excipient or carrier.

[0027] The present invention also discloses the application of the quinolone compound having anti-SARS-CoV-2 virus activity in the preparation of a drug with broad-spectrum anti-β-coronavirus activity.

[0028] The present invention also discloses the application of the quinolone compound having anti-SARS-CoV-2 virus activity in the preparation of a drug for inhibiting novel coronavirus.

[0029] In some examples, the novel coronavirus described in the present invention is a variant strain OMICRON (B.1.1.529), and the variant strain OMICRON (B.1.1.529) is a BA.1, BA.1.1, BA.2 or BA.3 sub-lineage. For example, in one example, it is the BA.2 sub-lineage.

[0030] The quinolone compounds with anti-SARS-CoV-2 activity disclosed in the present invention exhibit single-digit micromolar inhibitory activity against the SARS-CoV-2 Omicron BA.2 virus, showing excellent inhibitory activity against the novel coronavirus, providing a brand-new direction for the treatment and prevention of COVID-19. At the same time, they may have broad-spectrum antiviral activity against other coronaviruses, searching for new lead compounds for drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the toxicity test result of compound 54 in Vero E6 cells;

[0032] Figure 2 It is the toxicity test result of compound 54 in Calu-3 cells;

[0033] Figure 3 It is the anti-SARS-CoV-2 Omicron BA.2 activity test result of compound 54 in Calu-3 cells. DETAILED DESCRIPTION OF THE INVENTION

[0034] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The reagents and raw materials used in the present invention are all commercially available or can be prepared according to the methods in the literature. The experimental methods without specific conditions noted in the following examples are generally carried out under conventional conditions or according to the conditions recommended by the manufacturers.

[0035] Example 1 Preparation Method of Quinolone Compounds with Anti-SARS-CoV-2 Activity

[0036]

[0037] Step i, add 73.21 g (0.4 mol) of I-1 and 1460 mL of DMF into a 2000 mL reaction flask, stir to dissolve the solid, cool to 0 °C in an ice bath, slowly add 48 g (0.6 mol) of 60% NaH. After adding, stir for 30 min, and dropwise add a total of 57 mL (0.48 mol) of benzyl bromide at 0 °C. After dropping, continue to react at 0 °C. Stop the reaction after detecting the disappearance of the raw materials in the reaction solution by TLC, slowly add 100 mL of acetic acid, and stir for 10 min. Pour the reaction solution into about 7300 mL of stirred water, and a large amount of yellow solid precipitates. Stir, filter, and drain the filter cake to obtain a yellow solid. Pulp with a solvent of ethanol:ethyl acetate = 2:1 (900 mL), and obtain a crude yellow solid after filtration. Recrystallize with ethyl acetate to obtain 109.24 g of yellow solid 3-benzyloxy-4-hydroxy-5-nitrobenzaldehyde, with a yield of 94%.

[0038]

[0039] Step ii: In a 2000 ml reaction flask, 60.11 g (0.22 mol) of I-2, 480 mL of methanol, 480 mL of water, 220 mL of 1N NaOH solution and 133 mL of 30% H2O2 were added successively. Stir the mixture and heat it to 35 - 40 °C for reaction. Stop the reaction after monitoring by TLC that the raw materials have disappeared. Pour the reaction solution into about 4800 mL of ice water, adjust the pH to 3 - 4 with acetic acid, stir, filter, and obtain 47.60 g of red solid 2-benzyloxy-6-nitrobenzene-1,4-diol, with a yield of 83%.

[0040]

[0041] Step iii: Add 47.02 g of the crude product of compound I-3 and 470 mL of acetone to a 2000 mL three-necked round-bottom flask. Stir to dissolve the solid, then add 124.39 g (0.9 mol) of dry K2CO3 and 51.2 mL (0.54 mol) of dimethyl sulfate. Heat the mixture to 55 - 60 °C for reaction. After reacting for 6 hours, add an additional 17.1 mL (0.18 mol) of dimethyl sulfate and continue to react at 55 - 60 °C overnight. Stop the reaction after monitoring by TLC that the raw materials have disappeared. Filter to remove K2CO3, wash the filter cake with ethyl acetate, combine the washing solution and the filtrate, concentrate to dryness, and perform column chromatography (petroleum ether:ethyl acetate = 85:15) to obtain 31.24 g of yellow solid 1-benzyloxy-2,5-dimethoxy-3-nitrobenzene, with a yield of 60%.

[0042] of I-4 1 1H-NMR and HRMS: 1 1H-NMR (300 MHz, CDCl3): δ 7.36 (m, 5H, Ph-H), 6.82 (s, 1H, Ar), 6.71 (s, 1H, Ar-H), 3.95 (s, 3H, OCH3), 3.78 (s, 3H, OCH3). HRMS (ESI + ): m / z [M + H] +

[0043] calcd for C 15 H 16 NO5, 290.1023; found 290.1027.

[0044]

[0045] Step iv, add 20.25 g (0.07 mol) of compound I-4, 27.93 g (0.5 mol) of iron powder, 406 mL of acetic acid and 203 mL of water into a 1000 mL three-necked round-bottom flask, stir, and heat up to 55 - 60 °C for reaction. Stop the reaction after monitoring by TLC that the raw materials in the reaction solution disappear. Filter while it is hot, wash the filter cake with a small amount of dichloromethane, combine the filtrate and the washing liquid after cooling, extract with dichloromethane (200 mL × 3), wash with water (200 mL × 3), wash with saturated brine (200 mL × 3), and dry over anhydrous sodium sulfate.

[0046] Concentrate to dryness to obtain 17.45 g of 3-benzyloxy-2,5-dimethoxyaniline as a brown oily substance, with a yield of 96%.

[0047]

[0048] Step v, add 16.85 g (0.065 mol) of compound I-5 and 170 mL of dry benzene into a 500 mL three-necked round-bottom flask, stir, then add 2.24 g (0.013 mol) of p-toluenesulfonic acid and 24.99 g (0.13 mol) of ethyl benzoylacetate, and heat up to 60 °C for reaction. Stop the reaction after 12 hours. Filter to remove the insoluble substances, wash the filter cake with ethyl acetate, combine the washing liquid and the filtrate, concentrate to dryness, and subject the residue to column chromatography (petroleum ether:ethyl acetate = 95:5) to obtain 14.65 g of ethyl 3-(3-benzyloxy-2,5-dimethoxyanilino)-3-phenylacrylate as a yellow solid, with a yield of 52%.

[0049]

[0050] Step vi, add 200 mL of diphenyl ether into a 500 mL reaction flask, heat to 210 °C, add 13.45 g (0.031 mol) of compound I-6, and react at 210 - 215 °C. Stop the reaction after monitoring by TLC that the raw materials disappear. Let the reaction solution cool to room temperature. Distill off diphenyl ether under reduced pressure, and recrystallize the residue with ethyl acetate - petroleum ether to obtain 8.41 g of 7-benzyloxy-5,8-dimethoxy-2-phenylquinolin-4(1H)-one, with a yield of 70%.

[0051] For I-7 1 HNMR and HRMS: 1 H-NMR (300 MHz, CDCl3): δ 7.60 (br, 2H, Ph-H), 7.52 (m, 3H, Ph-H), 7.34 (m, 5H, Ph-H), 6.59 (s, 1H, Ar-H), 6.43 (s, 1H, Ar-H), 5.27 (s, 2H, OCH2), 4.02 (s, 3H, OCH3), 3.92 (s, 3H, OCH3). HRMS (ESI +): m / z [M+H] + calcd for C 24 H 22 NO4, 388.1543; found 388.1553.

[0052]

[0053] Step vii: Add 0.77 g (2 mmol) of Compound I-7 and 39 mL of anhydrous acetonitrile to a 100 mL reaction flask, stir, add 2.67 g (20 mmol) of AlCl3, heat under reflux. After refluxing for about 24 hours, add an additional 1.33 g (4 mmol) of AlCl3. Continue the reaction for 24 hours and then stop the reaction. Concentrate the reaction solution to remove the solvent to obtain a black residue. Add 40 mL of 50% (v / v) HOAc, stir to obtain a homogeneous black solution, extract with dichloromethane (30 mL × 3), combine the organic layers, wash with water (30 mL × 3), wash with saturated brine (30 mL × 3), and dry over anhydrous sodium sulfate. Concentrate to dryness, and subject the residue to column chromatography (petroleum ether: ethyl acetate = 88:12 → 0:100) to obtain 0.28 g of yellow solid 5,7-dihydroxy-8-methoxy-2-phenylquinolin-4(1H)-one, with a yield of 50%.

[0054] of I-8 1 1H-NMR and HRMS: 1 1H-NMR (300 MHz, DMSO-d6): δ 14.21 (s, 1H, 5-OH), 11.07 (s, 1H, NH), 10.33 (s, 1H, 7-OH), 7.73 (d, 2H, J = 5.43 Hz, Ar-H), 7.55 (d, 3H, J = 5.43 Hz, Ar-H), 6.14 (s, 1H, Ar-H), 6.18 (s, 1H, Ar-H), 3.76 (s, 3H, OCH3). HRMS (ESI + ): m / z [M+H] + calcd for C 16 H 13 NO4, 284.0917; found 284.0919.

[0055]

[0056] Step viii: Add 2.27 g (8 mmol) of compound I-8, 1.66 g (12 mmol) of anhydrous K2CO3, 1.80 g (9.6 mmol) of 1,2-dibromoethane and 227 mL of anhydrous acetone to a 100 mL reaction flask, and heat under reflux. Monitor the reaction by TLC and stop the reaction after the raw materials disappear. Filter to remove the insoluble substances, concentrate the filtrate to dryness, and perform column chromatography (petroleum ether: ethyl acetate = 3:1) to obtain 1.04 g of yellow solid 7-(2-bromoethoxy)-5-hydroxy-8-methoxy-2-phenylquinolin-4(1H)-one, with a yield of 32%.

[0057]

[0058] Step ix: Add 117.1 mg (0.3 mmol) of compound I-9, 10 mL of pyrrolidine and 10 mL of anhydrous acetonitrile to a 50 mL reaction flask, stir, and heat under reflux. Monitor the reaction by TLC and stop the reaction after the raw materials disappear. Concentrate the reaction solution to dryness, and perform column chromatography (ethyl acetate: methanol = 8:1) to obtain 58 mg of yellow solid 5-hydroxy-8-methoxy-2-phenyl-7-(2-(tetrahydropyrrol-1-yl)ethoxy)-quinolin-4(1H)-one, which is compound 54, with a yield of 51%.

[0059] of 54 1 1H-NMR and HRMS: 1 1H-NMR (300 MHz, DMSO-d6): δ 14.38 (s, 1H, 5-OH), 11.20 (s, 1H, NH), 7.73 (t, 2H, J = 3.84 Hz, Ph-H), 7.56 (d, 3H, J = 2.19 Hz, Ph-H), 6.51 (s, 1H, Ar-H), 6.20 (s, 1H, Ar-H), 4.39 (t, 2H, J = 5.22 Hz, ArOCH2), 3.80 (s, 3H, OCH3), 3.31 (br, 2H, N-CH2), 2.73 (br, 4H, 2×N-CH2), 1.89 (br, 4H, 2×NCH2CH2CH2CH2N). HRMS (ESI + ): m / z [M + H] + calcd for C 22 H 25 N2O4, 381.1809; found 381.1812.

[0060] Example 2 Toxicity test of quinolone compound 54 against Vero E6 and Calu-3 cells

[0061] 1. Cell lines: African green monkey kidney cells (ATCC, CRL-1586) and human lung adenocarcinoma cells Calu-3 (ATCC, HTB-55).

[0062] 2. Method: Taking Vero E6 as an example

[0063] 1) Add 100 μL of cells to each well of a 96-well plate the day before, so that the number of cells per well is 2×10 4 ; Initial infection titer 0.01 MOI

[0064] 2) Perform 3-fold serial dilution according to the highest concentration of Vero E6 (300 μM, 8 dilutions) and Calu-3 (300 μM, 8 dilutions), and set 3 replicates for each dilution;

[0065] 3) Use the Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega) to detect the cell viability of Vero E6 (48 h) and Calu-3 (24 h) cells respectively, and calculate the concentration at which the drug causes cytotoxicity to 50% of the cells (half-cytotoxic concentration, CC 50 )

[0066] The test results are as shown in Figure 1 and Figure 2 : Compound 54 has very low cytotoxicity to Vero E6 cells and Calu-3 cells.

[0067] Example 3 Activity test of compound 54 against SARS-CoV-2 Omicron BA.2 in Calu-3 cells

[0068] The EC of the drug was determined by the cytopathic titer method 50 . Seed Calu-3 cells at 5×10 4Inoculate 48-well plates by hole. On the second day, compound 54 was serially diluted 3-fold at the highest concentration of 20 μM, with 3 replicates for each dilution, and a total of 6 dilutions were set. Aspirate and discard the cell culture supernatant, add drugs at different concentrations (150 μL / well), pre-treat at 37 °C for 1 hour, then supplement each well with 150 μL of virus suspension containing 0.01 MOI, and set up virus control and normal cell control groups. Place in a 37 °C, 5% CO2 incubator for culture. On the second day, collect the cell culture supernatant, centrifuge at 5000 g for 10 minutes at 4 °C, take 100 μL of the supernatant, and use Reed & Muench serial dilution to measure the virus titer. Observe the cytopathic effect of the cells every day. After 3 days of infection, use a full-field cell analyzer (Celigo Image Cytometer, Celigo) to read the cytopathic rate, and then calculate the inhibition rate. Inhibition rate = (1 - proportion of cytopathic effect in the drug group / proportion of cytopathic effect in the DMSO group) × 100%. According to the inhibition rate results, perform four-parameter fitting to obtain the half-maximal effective concentration (EC 50 ).

[0069] The results are as Figure 2 and Figure 3 shown, indicating that compound 54 exhibits excellent anti-SARS-CoV-2 activity on human lung cancer cells (Calu-3) and has good safety at the same time.

Claims

1. Use of quinolone compound 54 in the preparation of a drug for inhibiting novel coronavirus, 。 2. The application according to claim 1, wherein wherein the novel coronavirus is the variant OMICRON (B.1.1.529).

3. The application according to claim 2, characterized in that, The variant OMICRON (B.1.1.529) is the BA.1, BA.1.1, BA.2 or BA.3 sub-lineage.

4. The application according to claim 3, wherein The variant OMICRON (B.1.1.529) is BA.2.

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