RU2835077C1 - Obtaining a water-soluble 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)-3-(2-diethylaminoethoxy) carbonylindole dihydrochloride compound - Google Patents

Abstract

FIELD: organic chemistry.

SUBSTANCE: method includes the following synthesis steps: obtaining 1,2-dimethyl-5-methoxy-3-ethoxycarbonylindole from 1,2-dimethyl-5hydroxy-3-ethoxycarbonylindole, then obtaining 6-bromo-1,2-dimethyl-5-methoxy-3-ethoxycarbonylindole from 1,2-dimethyl-5-methoxy-3-ethoxycarbonylindole, then obtaining 6-bromo-2-bromomethyl-1-methyl-5-methoxy-3-ethoxycarbonylindole from 6-bromo-1,2-dimethyl-5-methoxy-3-ethoxycarbonylindole, then obtaining 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)-3-ethoxycarbonylindole from 6-bromo-2-bromomethyl-1-methyl-5-methoxy-3-ethoxycarbonylindole, then obtaining 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)indole-3-carboxylic acid hydrochloride from 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)-3-ethoxycarbonylindole, and obtaining 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)-3-(2-diethylaminoethoxy)carbonylindole dihydrochloride (compound II).

EFFECT: disclosed is a method of producing 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl-3-(2-diethylaminoethoxy)carbonylindole dihydrochloride compound of general formula (II), having high antiviral anti-SARS-CoV-2.

1 cl, 1 dwg, 3 tbl, 14 ex

Description

The invention relates to organic chemistry, virology and medicine, concerns the production of a low-molecular compound belonging to the class of compounds of dialkylaminoalkyl esters of indole-3-carboxylic acid derivatives, namely: 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl-3-(2-diethylaminoethoxy)carbonylindole dihydrochloride,

possessing high antiviral anti-SARS-CoV-2 (Severe acute respiratory syndrome-related coronavirus 2, previously 2019-nCoV) activity, which can be used to create an effective drug necessary to expand the possibilities of therapy for COVID-19 and other viral diseases.

Coronavirus (CoV), discovered in 1960 by D. Tyrrell and then described by J. Almeida et al. in Nature in 1968, for many years was only the cause of mild acute respiratory infections. However, the COVID-19 coronavirus pandemic has changed the attitude towards coronaviruses and led to the search for effective antiviral agents against SARS-CoV-2. At the same time, several strategies were proposed for the development of effective and safe drugs for the treatment and prevention of COVID-19 coronavirus infection. The first strategy is to continue testing already registered antiviral drugs, the activity of which against RNA viruses has been previously shown in clinical trials of varying quality and design: interferon alpha (hepatitis C virus), ribavirin (hepatitis C virus, respiratory syncytial virus, causative agent of hemorrhagic fever), lopinavir / ritonavir (HIV), favipiravir (influenza virus).

The second strategy is to use existing molecular databases to screen molecules with different mechanisms of action that may have an effect on coronavirus: chloroquine and hydroxychloroquine, remdesivir, umifenovir, etc. The third strategy involves the targeted development of new antiviral drugs based on the study of genomic information and pathogenic properties of various coronaviruses (The Lancet Digital Health, https://www.covid-trials.org/).

At the same time, three horizons are distinguished for the development of methods for the treatment and prevention of COVID-19, which include not only the etiotropic effect on SARS-CoV-2, but also the treatment of acute respiratory distress syndrome (ARDS), cytokine release syndrome or cytokine storm (cytokine release syndrome, CRS), concomitant bacterial and fungal infections (der Graaf P, Giacomini K. COVID-19: a defining moment for clinical pharmacology? PharmacolTher 2020; 108(1):11-5).

However, despite the unprecedented increase in the number of clinical trials of drugs for the treatment of this infection, the frequency of use of off-label drugs remains high, which certainly indicates an urgent need to develop new effective approaches to the prevention and treatment of COVID-19 (https://www.euro.who.int/ru/health-topics/health-emergencies/coronavirus-covid-19/news/news/2020/3/who-announces-covid-19-outbreak-a-pandemic).

The drug hydroxychloroquine is known to have been tested for the treatment of COVID-19. According to in vitro studies, hydroxychloroquine suppressed the action of the SARS-CoV-2 virus, which causes COVID-19. According to clinical studies, on the 6th day of treatment, among patients who were given hydroxychloroquine, compared to those who were not given it, there were fewer virus-positive patients (according to PCR tests) (P. Gautret et al., "Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial," Int. J. Antimicrob. Agents, p. 105949, Mar. 2020).

The disadvantage of hydroxychloroquine is its high toxicity and a large number of adverse effects, including blurred vision, nausea, vomiting, abdominal cramps, headache, diarrhea, swelling of the legs/ankles, shortness of breath (Chatre C, Roubille F, Vernhet H, et al. Cardiac complications attributed to chloroquine and hydroxychloroquine: a systematic review of the literature. Drug Saf 2018; 41(10):919-31).

A drug known as tocilizumab is used to treat the SARS-CoV-2 viral infection (AntinoriS, BonazzettiC, GubertiniG, et al. Tocilizumab for cytokine storm syndrome in COVID-19 pneumonia: an increased risk for candidemia? Autoimmun Rev 2020; 19(7):102564).

The disadvantages of tocilizumab are the difficulty of its use - in the form of a solution for subcutaneous administration or as a concentrate for the preparation of a solution for infusions, as well as side effects (from the skin and subcutaneous tissue: often - rash, itching, urticaria; from the nervous system: often - headache, dizziness; from the cardiovascular system: often - increased blood pressure; from the hematopoietic system: often - leukopenia, neutropenia, increased risk of candidemia).

Derivatives of indole-3-carboxylic acid (RU 2782931 C1), with structural formulas (1), (2), (3), are known as water-soluble immunomodulatory antitumor agents:

However, the antiviral properties of these indole-3-carboxylic acid derivatives against SARS-CoV-2 viral infection have not been studied.

Derivatives of indole-3-carboxylic acid possessing antiviral activity are known (RU 2552422 C2). This invention relates to aminoalkyl esters of 5-methoxyindole-3-carboxylic acid and their pharmacologically acceptable salts of the general formula (I),

where R1 is cyclohexyl, C _1-3 alkyl; R2 is phenylthio, phenyloxy, in which the phenyl group may have 1-2 halogen substituents or a C _1-4 alkoxy group, or R2 is 5-6-membered heterocycloalkyl containing 1-2 heteroatoms selected from nitrogen and oxygen; n is 1, 2, 3, 4; each R is independently selected from C _1-4 alkyl; with the exception of the compound specified in the claims.

However, the antiviral properties of these indole-3-carboxylic acid derivatives against SARS-CoV-2 viral infection have not been studied.

The closest (prototype) drug is umifenovir (arbidol, international name Umifenovirum (WHO Drug Information // 2001, 25 (1), 91), included in the guidelines for the prevention and treatment of COVID-19 (Temporary guidelines. Prevention, diagnosis and treatment of a new coronavirus infection (COVID-19). Version 1. January 29, 2020, Ministry of Health of the Russian Federation). Umifenovir is a hydrochloride of 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromindole monohydrate (see, for example, RU 2033156;) - it differs in its mechanism of action from many drugs used in the treatment of viral infections, such as amantadine, rimantadine, zanamivir and oseltamivir. Umifenovir has a specific antiviral effect, inhibiting fusion of the viral envelope with cell membranes, which prevents the penetration of the virus into cells and disrupts its reproduction (Leneva LA. et al. Characteristics of arbidol-resistant mutants of influenza virus: implications for the mechanism of anti-influenza action of arbidol // Antiviral Res. 2009, 81(2), 132-140). Studies of in vitro antiviral activity against the new coronavirus SARS-CoV-2 have shown that, compared with the control group, umifenovir can inhibit replication up to 60 times at a concentration of 10-30 μM and significantly reduce the pathological effect of the virus on cells. In Vero E6 cells, umifenovir was shown to inhibit SARS-CoV-2 replication by 21.73% at 3 μM and by 98.93% at 30 μM (Ge Y, Tian T, Huang S, et al. An integrative drug repositioning framework discovered a potential therapeutic agent targeting COVID-19. Signal Transduct Target Ther. 2021 Apr 24; 6(1): 165. doi: 10.1038/s41392-021-00568-6. PMID: 33895786; PMCID: PMC8065335.

The disadvantage of the prototype is its limited therapeutic efficacy due to low bioavailability and insolubility in water (Chen C, Zhang Y, Huang J, et al., Favipiravir Versus Arbidol for Clinical Recovery Rate in Moderate and Severe Adult COVID-19 Patients: A Prospective, Multicenter, Open-Label, Randomized Controlled Clinical Trial. Front Pharmacol. 2021 Sep 2;12:683296. doi: 10.3389/fphar.2021.683296; PCT/WO2010128889A1 "Pharmaceutical composition comprising Arbidol in phospholipid particles").

To overcome this drawback, it is necessary to synthesize a water-soluble compound with antiviral efficacy and a chemical structure similar to umifenovir.

The objective of the present invention is to obtain a water-soluble compound of 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)-3-(2-diethylaminoethoxy)carbonylindole dihydrochloride, which can be used to create an effective drug necessary to expand the possibilities of therapy for COVID-19 and other coronavirus diseases.

The technical problem is solved by proposing 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)-3-(2-diethylaminoethoxy)carbonylindole dihydrochloride of the general formula (II):

including the following stages of synthesis:

- obtaining 1,2-dimethyl-5-methoxy-3-ethoxycarbonylindole, melting point (mp) 113°C, from 1,2-dimethyl-5 hydroxy-3-ethoxycarbonylindole,

- obtaining 6-bromo-1,2-dimethyl-5-methoxy-3-ethoxycarbonylindole, mp 156°C, from 1,2-dimethyl-5-methoxy-3-ethoxycarbonylindole,

- obtaining 6-bromo-2-bromomethyl-1-methyl-5-methoxy-3-ethoxycarbonylindole, mp 142°C, from 6-bromo-1,2-dimethyl-5-methoxy-3-ethoxycarbonylindole,

- obtaining 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)-3-ethoxycarbonylindole, mp 124-125°C, from 6-bromo-2-bromomethyl-1-methyl-5-methoxy-3-ethoxycarbonylindole,

- obtaining 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)indole-3-carboxylic acid hydrochloride, mp 236-238°C, from 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)-3-ethoxycarbonylindole,

- obtaining 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)-3-(2-diethylaminoethoxy)carbonylindole dihydrochloride (compound II), mp 237-240°C, with characteristic spectra:

NMR ^1H (200 MHz, DMSO) δ 10.76 (br s, 1H), 10.23 (br s, 1H), 8.03 (s, 1H), 7.65 (s, 1H), 4.87 (d, J=4.8 Hz, 2H), 4.76 (t, J=5.1 Hz, 2H), 3.95 (s, 3H), 3.93 (s, 3H), 3.61 (m, 2H), 3.50-3.06 (m, 8H), 2.15-1.33 (m, 6H), 1.26 (t, J=7.2 Hz, 6H);

IR (KBr, cm-1): 859, 1041, 1114, 1148, 1197, 1303, 1393, 1426, 1449, 1483, 1650, 1694 (C=O), 2354-2700, 2942, 3397, 3588;

MS: Characteristic signal: m/z 480.1862 (molecular ion) [M+H].

The present invention is explained by a detailed description of the multi-stage production of compound II, examples of the biological activity of compound II, confirming the suitability of the obtained compound for the intended use, as well as tables and an illustration showing:

Fig. 1 - diagram of the multi-stage preparation of compound II.

The obtained compound has a dose-dependent antiviral activity against SARS-CoV-2, the concentration dependences of which indicate the specificity of the action of this compound, and at a concentration of 30 μg / ml completely inhibits the reproduction of SARS-CoV-2 with an infectious activity of 10 ⁶ TCID ₅₀ / ml, has IFN-inducing activity and inhibits syncytium formation mediated by the pin-shaped protein (S-glycoprotein) of SARS-CoV-2.

The synthesis of an indole-3-carboxylic acid derivative (II) with antiviral activity against SARS-CoV-2 in vitro is carried out as follows.

Example 1. Stage 1 – obtaining 1,2-dimethyl-5-methoxy-3-ethoxycarbonylindole (compound IV).

To a solution of 4.66 g (0.02 mol) of 1,2-dimethyl-5-hydroxy-3-ethoxycarbonylindole, a compound designated III, in 40.0 ml of dioxane is added 40.0 ml of a 10% sodium hydroxide solution, then 4.0 ml of dimethyl sulfate are added dropwise at room temperature and the mixture is stirred for 2 hours. The reaction mixture is poured into distilled water, cooled, the precipitate that forms is filtered off and washed with distilled water. The yield of the substance is 4.65 g (94%). Melting point (mp). 113°C.

Example 2. Stage 2 – obtaining 6-bromo-1,2-dimethyl-5-methoxy-3-ethoxycarbonylindole (compound V).

A mixture of 4.65 g (0.0188 mol) of 1,2-dimethyl-5-methoxy-3-ethoxycarbonylindole (compound IV), 3.36 g (0.0188 mol) of bromosuccinimide in 75.0 ml of carbon tetrachloride is boiled for 5 hours. The succinimide precipitate is filtered off while hot. The mother liquor is slightly evaporated and cooled. The precipitate is filtered off. Yield 3.3 g (54%). Mp 156°C.

Example 3. Stage 3 – obtaining 6-bromo-2-bromomethyl-1-methyl-5-methoxy-3-ethoxycarbonylindole (compound VI).

A mixture of 3.3 g (0.0101 mol) of 6-bromo-1,2-dimethyl-5-methoxy-3-ethoxycarbonylindole (compound V), 1.81 g (0.0101 mol of bromosuccinimide), 0.1 g of benzyl peroxide in 30.0 ml of carbon tetrachloride is boiled under light for 5 hours. After separation of succinimide while hot and cooling, the precipitate is filtered off. Yield 3.16 g (78%). M.p. 142°C.

Example 4. Stage 4 – obtaining 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)-3-ethoxycarbonylindole (compound VII).

A solution of 4.0 g (0.01 mol) of 6-bromo-2-bromomethyl-1-methyl-5-methoxy-3-ethoxycarbonylindole (compound VI) and 1.7 g (0.02 mol) of piperidine in 50.0 ml of benzene is kept for 10-12 hours at room temperature. The resulting precipitate of piperidine hydrobromide is filtered off. Compound VII is isolated from the filtrate. Yield 1.7 g (82.9%). Mp 124-125°C.

Calculated: % C 55.75, H 6.16, N 6.84. _{C19H25N2O3 . ​ ​}

Found: % C 55.72, H 6.20, N 7.02. M 417.7.

Example 5. Stage 5 – obtaining 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)indole-3-carboxylic acid hydrochloride (compound VIII).

A solution of 6.0 g sodium hydroxide, 4.1 g (0.01 mol) 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)-3-ethoxycarbonylindole (compound VII), 3.0 ml water and 60.0 ml ethyl alcohol is boiled for 3 hours. After cooling, 10.0 ml distilled water is added, acidified with concentrated HCl, and the precipitate is filtered off. Yield 4.1 g (98%). Mp 236-238°C

Calculated: % C 48.88; H 5.31; N6.71 C ₁₇ H ₂₆ BrN ₂ O ₃ .HCl M 417.73 Found: % C 48.68; H 5.32; N 6.65

Example 6. Stage 6 – obtaining 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)-3-(2-diethylaminoethoxy)carbonylindole dihydrochloride (compound II).

1.67 g (0.004 mol) of 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)indole-3-carboxylic acid hydrochloride (compound VIII), 3.0 ml of thionyl chloride, 2 drops of dimethylformamide in 30.0 ml of dioxane are heated at 60°C for 3 hours. After evaporation in a vacuum, the precipitate is washed with ether. To the obtained precipitate of 6-bromo-1-methyl-5-methoxy-2-(1-piperidino)indole-3-carboxylic acid chloride (compound IX) add 25.0 ml of benzene and a mixture of 1.2 ml (0.008 mol) of diethylaminoethanol and 1.12 ml of triethylamine in 5.0 ml of benzene, heat on a water bath for 2 hours, filter off the precipitate of triethylamine hydrochloride, wash with hot benzene. After evaporation of benzene in a vacuum, filter off the precipitate of the base, wash with hexane. The dihydrochloride is obtained by adding ether saturated with HCl (hydrogen chloride) to a solution of the base in acetone. Compound II 1.9 g (85.2%) was obtained from isopropyl alcohol. Mp 237 - 240 ° C

Example 7. Obtaining an NMR spectrum.

The 1H NMR spectrum of the DMSO d6 solution was recorded on a Bruker AC-200 spectrometer at 298 K.

NMR ^1H (200 MHz, DMSO) 5 10.76 (br s, 1H), 10.23 (br s, 1H), 8.03 (s, 1H), 7.65 (s, 1H), 4.87 (d, J=4.8 Hz, 2H), 4.76 (t, J=5.1 Hz, 2H), 3.95 (s, 3H), 3.93 (s, 3H), 3.61 (m, 2H), 3.50-3.06 (m, 8H), 2.15-1.33 (m, 6H), 1.26 (t, J=7.2 Hz, 6H).

Example 8. Obtaining an IR spectrum.

IR spectra were recorded on a Bruker ALPHA T Fourier spectrometer. IR (KBr, cm ^-1 ): 859, 1041, 1114, 1148, 1197, 1303, 1393, 1426, 1449, 1483, 1650, 1694 (C=O), 2354-2700, 2942, 3397, 3588.

Example 9. Obtaining an MS spectrum.

MS was studied on a SHIMADZU LCMS-8040 mass spectrometer by direct sample injection in positive ionization scanning mode (Q3+Scan). The spectra contain characteristic signals: m/z 480 (molecular ion), m/z 241, (double-charged molecular ion), m/z 410 (M-N(C2H5)2), m/z 122.5 unidentified.

Calculated: % C 49.92; H 6.56; N 7.59, C ₂₃ H ₃₆ BrCl ₂ N ₃ O ₃ M 553.37

Found: % C 49.89; H 6.76; N 7.48

Example 10. Determination of the solubility of the proposed compound II.

The solubility of 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)-3-(2-diethylaminoethoxy)carbonylindole dihydrochloride was determined using the method described in the General Pharmacopoeial Article (OFS.1.2.1.0005.15 Solubility. Pharmacopoeia. Russian Federation. https://pharmacopoeia.ru/ofs-l-2-l-0005-15-rastvorimost/).

A measured amount of solvent (in this case distilled water) is gradually added to samples of 10.0 mg, 20.0 mg, 50.0 mg, 100.0 mg and 1.0 g of the test compound ground into a fine powder until complete dissolution and the mixture is shaken continuously for 10 min at (20±2)°C.

Samples of 10.0 mg and 20.0 mg of 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)-3-(2-diethylaminoethoxy)carbonylindole dihydrochloride were completely dissolved in 0.2 ml of water; a sample of 50.0 mg was dissolved in 0.5 ml; a sample of 100.0 mg was dissolved in 1.0 ml; 1.0 g of the compound was dissolved in 8.0 ml of water.

The studied compound is easily soluble, which is confirmed in Table 1.

Example 11. Determination of cytotoxicity of the developed compound II.

The study used the transplantable cell line of the green monkey kidney (Chlorocebus aethiops) Vero E6, provided by the All-Russian Collection of Cell Cultures of the Federal State Budgetary Institution "National Research Center for Epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya" of the Ministry of Health of the Russian Federation (FSBI "National Research Center for Epidemiology and Microbiology named after N.F. Gamaleya" of the Ministry of Health of the Russian Federation). This cell line is sensitive to infection with the SARS-CoV-2 virus and is successfully used to obtain virus-containing material (viral stock), titrate the infectious activity of the virus and study the antiviral properties of substances of various natures. Cells were cultured in DMEM (Gibco) nutrient medium supplemented with 5% fetal bovine serum (FBS) (5 vol.%), L-glutamine (2 mM) and a mixture of antibiotics (150 U/ml penicillin and 150 U/ml streptomycin).

The cytotoxicity of the test compound was assessed by reducing cell viability in the MTT test (Mossman, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays // J. Immunol. Meth. - 1983. - Vol. 65. - P. 55-6). A cell monolayer was cultured in the presence of the test substance added in various concentrations for 72 hours at 37°C. The cells were then washed twice with serum-free medium, 10.0 - 20.0 μl of MTT solution (6.0-10.0 mg/ml) were added and the cells were incubated for 4 hours at 37°C. The contents of the wells were then removed and 100.0 μl of dimethyl sulfoxide (DMSO) were added to dissolve the formazan formed inside the living cells. The essence of the MTT test is to measure the ability of cells to convert highly soluble yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into insoluble intracellular crystals of MTT formazan. The efficiency of such conversion reflects the overall level of dehydrogenase activity of cells. The amount of formazan determined photometrically (at 595/630 nm) is directly proportional to the number of living cells. Based on the optical density data, the 50% cytotoxic concentration (CC ₅₀ ) of the compound was calculated, i.e., the concentration of the compound in the presence of which 50% of the cells died compared to the control.

For the compound under study, CC50 was 144.30 μmol (83.32 μg/ml).

Example 12. Determination of antiviral activity of compound II.

The experiment used a transplantable line of African green monkey kidney cells (Chlorocebus aethiops) Vero E6, which was provided by the All-Russian Collection of Cell Cultures at the N.F. Gamaleya National Research Center for Epidemiology and Microbiology of the Russian Ministry of Health.

The studies were performed using the pandemic strain of human coronavirus SARS-CoV-2 with an infectious activity of 10 ⁶ TCID ₅₀ /ml (50% tissue cytopathic infectious dose) for Vero E6 cells (clinical isolate: hCoV-19/Russia/Moscow-PMVL-12/2020 (EPI_ISL_572398). Obtained from the State Collection of Viruses of the D.I. Ivanovsky Institute of Virology, N.F. Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation.

Vero E6 cells (2⋅10 ⁵ cells/ml) were placed in a 96-well plate (100 μl/well) and cultured in a complete growth medium (CGM) at 37°C in an atmosphere of 5% CO ₂ for 24 hours until a complete monolayer was formed. Using a maintenance medium with 2% fetal bovine serum (FBS), 3 non-toxic concentrations of the test substance were prepared with a uniform decrease in concentration by 2 (or 3) times and poured into wells with a monolayer of Vero E6 cells in a volume of 100.0 μl, occupying 3 vertical rows for each of the 3 concentrations (maintaining triplicate repetition). Then 10.0 μl of the viral stock were added to the wells of the first horizontal row "A" of the plate and, using a multichannel automatic micropipette, the virus was titrated directly in the wells by transferring 10.0 μl from horizontal row "A" to row "B", then from row "B" to row "C", etc. up to row "G", thus obtaining virus dilutions from 10 ^-1 to 10 ^-7 . The infected cells were incubated in a CO ₂ thermostat for 96 hours in an atmosphere of 5% CO ₂ . The virus in the same dilutions without the addition of the developed agent served as a control. After the incubation period, the reaction results were taken into account by examining the wells of the plate in an inverted microscope. The virus titer was determined in each vertical row. The reciprocal of the last dilution in which the cytopathogenic effect (CPE) (cell death) developed was taken as the virus titer. 50% tissue cytopathic infectious doses (TCID ₅₀ ) ₍ Median Tissue Culture Infectious Dose) were calculated using the Reed-Muench method for each dilution of the preparation and control titration of the virus.

Thus, the assessment of the antiviral activity of compound II was taken into account based on the reduction in the infectious titer of the virus in the Vero E6 cell culture by cytopathic action (Table 2).

As can be seen from Table 2, the test compound has reliable dose-dependent antiviral activity in vitro, which indicates the specific nature of the compound's action, and completely suppresses the reproduction of the SARS-CoV-2 virus at a concentration of 52.0 μmol (30 μg/ml), i.e. by 6 lg TCID ₅₀ . In virological studies, the antiviral effect of drugs is considered satisfactory if Alg TCID ₅₀ ≥2.0. (See "Guidelines for Conducting Preclinical Studies of Drugs." edited by A.N. Mironov. Part One. - M.: Grif i K, 2012. - pp. 527-551).

The IC ₅₀ value for the studied compound calculated using GraphPadPrism 5.0 software was 1.84 μM (1.06 μg/mL). Δlg _max TCID ₅₀ was 6.0 at a compound concentration of 52.0 μM (30 μg/mL). The selectivity index (SI) value calculated as the ratio of CC50 to IC ₅₀ (SI=CC50/IC ₅₀ ) was 78.6.

Example 13. Studies on the efficacy of inhibiting syncytium formation induced by the spike protein (S-glycoprotein) of the SARS-CoV-2 virus.

293T cells were co-transfected for 48 hours with a plasmid containing the full-length S-glycoprotein (pVAX-1-S-glycoprotein; Evrogen, Russia) and a plasmid encoding GFP (pUCHR-IRES-GFP) using Transporter™ 5 transfection reagent. Then, various concentrations of the test compound II were added to a monolayer of Vero E6 cells grown in 96-well plates, after which a suspension of 293T-S-GFP effector cells was added to the wells (cell ratio 3:1). Two hours later, the number of formed syncytia was assessed by fluorescence microscopy. The efficiency of suppression of cell fusion induced by SARS-CoV-2 S-glycoprotein was assessed using GraphPadPrism 5.0 software compared to the control (without compound addition) and expressed as a percentage. Suppression of syncytium formation induced by the pin-shaped protein (S-glycoprotein) of SARS-CoV-2 by 89% was detected.

Example 14. Determination of the interferon (IFN)-inducing effect of compound II.

Experiments on animals were carried out in compliance with legal and ethical standards for the treatment of animals in accordance with the rules adopted by the European Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes (ETS 123). Strasbourg, 1986.

Outbred white mice, males, weighing 12.0-14.0 g, were obtained from the Nursery of NEO Market LLC (veterinary certificate 250 No. 0679392). Before the study, the laboratory animals were kept for 5 days for adaptation in group housing in cages. During this period, the clinical condition of the animals was monitored daily by visual inspection. Animals with deviations detected during the examination were not included in the experimental groups. Before the study, animals meeting the inclusion criteria for the experiment were divided into groups. Animals were selected for the experimental groups by random sampling. The cage marking encoded the sex of the animals, breed, date of drug administration, and the name of the group.

The maintenance, feeding, care of animals and their withdrawal from the experiment were carried out in accordance with the Rules of Laboratory Practice adopted in the Russian Federation: GOST 33215-2014 of 07.01.2016 "Guidelines for the maintenance and care of laboratory animals. Rules for equipping premises and organizing procedures"; GOST 33044-2014 Principles of good laboratory practice." approved by Order of the Federal Agency for Technical Regulation and Metrology No. 1700-st of 20.11.2014), entered into force on 01.08.2015; GOST 33216-2014 "Rules for working with laboratory rodents and rabbits"; Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes (EEA compliant); The study protocol was reviewed and approved by the Ethics Committee of the Centre.

Murine encephalomyocarditis virus (MEV), strain "Columbia SK-Col-SK" with a titer of 10 ⁷ TCID50/ML was obtained from the State Collection of Viruses of the D.I. Ivanovsky Institute of Virology, N.F. Gamaleya National Research Center for Epidemiology and Microbiology of the Ministry of Health of the Russian Federation.

To determine the IFN-inducing activity of compound II, blood samples were taken from animals 2, 24, 48, 72 hours after a single intraperitoneal administration of compound II at a dose of 121.2 μmol/mouse (70 μg/mouse) or distilled water (0.2 ml) (Placebo, control without the drug) (3 mice for each period). The IFN activity in the blood serum of mice was determined on the mouse fibroblast cell line L-929 obtained from the All-Russian Collection of Cell Cultures at the N.F. Gamaleya National Research Center for Epidemiology and Microbiology of the Ministry of Health of the Russian Federation. In the work we used a 3-day monolayer of the continuous cell line L-929 grown in 199 and Eagle's MEM medium (1:1) with the addition of 7% fetal calf serum (FCS), L-glutamine and antibiotics: 150 U/ml penicillin and 150 U/ml streptomycin. Circulating IFN in the blood serum was determined by titration of samples in the culture of mouse fibroblasts L-929 using the mouse VEMC virus as an indicator, finding the final dilution of IFN that protected 50% of the cells from the cytopathogenic effect of 100 TCID50 of the virus.

The titration results are presented in Table 3. It was shown that compound II had IFN-inducing activity. IFN production in mice was detected 2, 24, 48 and 72 hours after the administration of compound II. Moreover, 2 hours after the administration of the test substance, IFN was detected in the blood serum of mice in a titer of 1:40, and in titers of 1:20 after 24, 48 and 72 hours.

The results of the study showed that compound II had IFN-inducing activity when administered intraperitoneally once at a dose of 121.2 μmol/mouse (70 μg/mouse), and 2 hours after administration the IFN titer was 40 U/ml, and within 24-72 hours the IFN activity was determined at a titer of 20 U/ml.

Conclusion.

The obtained results demonstrate the presence of an antiviral effect against SARS-CoV-2 in the synthesized compound II in vitro studies. This compound has a dose-dependent antiviral activity against SARS-CoV-2 and at a concentration of 52.0 μmol completely inhibits the reproduction of the SARS-CoV-2 virus with an infectious activity of 10 ⁶ TCID ₅ o / ml (50% tissue cytopathogenic infectious dose), exhibits IFN-inducing activity and inhibits syncytium formation mediated by the pin-shaped protein (S-glycoprotein) of SARS-CoV-2. Concentration dependences indicate the specificity of the action of the studied compound and indicate the promise of the developed compound and the possibility of its further study in vivo in experimental animals.

The proposed compound II, in view of its high activity (IC ₅₀ = 1.06 μg/ml) and high selectivity index (SI = 78.6), as well as economic and synthetic availability, can be recommended as a candidate for creating an effective etiotropic antiviral drug to expand the possibilities of therapy for coronavirus diseases in humans and animals caused by modern pandemic strains of SARS-CoV-2, including as an independent agent and as part of a composition for the treatment of COVID-19.

Claims (11)

A method for obtaining a water-soluble compound of 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)-3-(2-diethylaminoethoxy)carbonylindole dihydrochloride of the general formula (II): including the following stages of synthesis: - obtaining 1,2-dimethyl-5-methoxy-3-ethoxycarbonylindole, melting point (mp) 113°C, from 1,2-dimethyl-5 hydroxy-3-ethoxycarbonylindole, - obtaining 6-bromo-1,2-dimethyl-5-methoxy-3-ethoxycarbonylindole, mp 156°C, from 1,2-dimethyl-5-methoxy-3-ethoxycarbonylindole, - obtaining 6-bromo-2-bromomethyl-1-methyl-5-methoxy-3-ethoxycarbonylindole, mp 142°C, from 6-bromo-1,2-dimethyl-5-methoxy-3-ethoxycarbonylindole, - obtaining 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)-3-ethoxycarbonylindole, mp 124-125°C, from 6-bromo-2-bromomethyl-1-methyl-5-methoxy-3-ethoxycarbonylindole, - obtaining 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)indole-3-carboxylic acid hydrochloride, mp 236-238°C, from 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)-3-ethoxycarbonylindole, - obtaining 6-bromo-1-methyl-5-methoxy-2-(1-piperidinomethyl)-3-(2-diethylaminoethoxy)carbonylindole dihydrochloride (compound II), mp 237-240°C, with characteristic spectra: NMR 1H (200 MHz, DMSO) δ 10.76 (br s, 1H), 10.23 (br s, 1H), 8.03 (s, 1H), 7.65 (s, 1H), 4.87 (d, J=4.8 Hz, 2H), 4.76 (t, J=5.1 Hz, 2H), 3.95 (s, 3H), 3.93 (s, 3H), 3.61 (m, 2H), 3.50-3.06 (m, 8H), 2.15-1.33 (m, 6H), 1.26 (t, J=7.2 Hz, 6H); IR (KBr, cm-1): 859, 1041, 1114, 1148, 1197, 1303, 1393, 1426, 1449, 1483, 1650, 1694 (C=O), 2354-2700, 2942, 3397, 3588; MS: Characteristic signal: m/z 480, 1862 (molecular ion) [M+H].

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