RU2256451C1 - Medicinal agent for treatment of atypical pneumonia - Google Patents

Abstract

FIELD: medicine, virology, pharmacy.

SUBSTANCE: invention proposes an agent for treatment and prophylaxis of infection caused by coronaviruses, in particular, for treatment of atypical pneumonia (SARS), and pharmaceutical composition of indicated designation based on thereof. Agent represents 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-oxybromoindole or 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-oxybromoindole monohydrate hydrochloride (arbidol) known early as an immunomodulator and preparation used against influenza viruses. Invention provides reducing accumulation of coronaviruses (on example with TOPS virus) in lung.

EFFECT: valuable medicinal properties of agent.

2 cl, 7 tbl, 9 ex

Description

The invention relates to the field of pharmacy, specifically to the use of 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole and its salts as a medicine for the treatment or prevention of SARS.

Atypical pneumonia is a relatively recent respiratory disease in Asia, North America and Europe. The disease is caused by viruses and is characterized by transient development and high mortality. The first cases were described in 2003. At the very beginning of the new virus epidemic, doctors believed that the mortality rate from it was 4%, but subsequent data showed that the mortality rate in Hong Kong, Canada and Singapore reached 7.6%, 10.7% and 9.9% respectively. Further study of SARS and a synthesis of data for all countries where SARS was registered showed that older people are much more likely to develop SARS than young people. In addition, mortality is much higher among them: up to 40% of patients die, while among patients under 60 years old - about 13%. This high mortality rate makes the search for SARS treatment an extremely urgent task.

Known drug for the treatment of coronavirus infection, used for the treatment of SARS, is ribavirin (“Medicus Amicus”, 2003, No. 3, p.1, 13). However, the use of this drug for the treatment of SARS has insufficient effect, which shows a high mortality rate. In addition, the use of ribavirin often manifests side effects and there are a number of contraindications.

1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole hydrochloride monohydrate was previously known as a drug for the treatment of influenza A and B viruses (RF Patent No. 2008004), which also has immunomodulatory effects (Patent RF №2033157).

The objective of the invention is to expand the arsenal of funds in the treatment of coronavirus infection, reduce toxicity and increase the effectiveness of the treatment of SARS.

To solve this problem, we propose to use, in addition to 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole, a number of its salts, namely: hydrochloride, citrate, maleate, succinate and fumarate. All salts are obtained by adding the corresponding acids to the base - (1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole), which, in turn, is obtained from the known substance - arbidol ( 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole hydrochloride).

The invention is illustrated by the following examples.

EXAMPLE 1. Preparation of the base: 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole

To a mixture of 20 ml of 2M Na ₂ CO ₃ , 40 ml of 1M NaHCO ₃ and 200 ml of CHCl ₃ , 10.64 g of (1-methyl-2-phenylthiomethyl-3-carbetoxy-4-dimethylaminomethyl-5-hydroxy) are added in small portions. -6-bromoindole hydrochloride). After dissolving all the hydrochloride CHCl _{3 is} separated, the aqueous layer is extracted with 50 ml CHCl ₃ . The combined CHCl ₃ extracts were washed with saturated NaCl, dried over MgSO ₄ and evaporated to dryness in vacuo. The residue is recrystallized from absolute alcohol, dried in vacuo over P ₂ O ₅ / KOH. 8.46 g (yield: 88.6%) of 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole (hereinafter referred to as the base) are obtained with a melting point of 125-126 ° C. .

EXAMPLE 2. Obtaining 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole succinate

To a solution of 509 mg of the base (obtained in Example 1) in 2 ml of warm tetrahydrofuran (THF) was added 0.5 ml of a solution of 91.7 mg of succinic acid in 1 ml of hot THF. The reaction mixture is cooled to room temperature, succinate seeded and left for 3 hours at + 4 ° C. The precipitated crystals are filtered off, washed with a small amount of THF and dried in vacuo over P ₂ O ₅ / KOH. 450 mg (75% yield) of product are obtained. Mp = 138-139 ° C. The structure of the obtained compound was confirmed by the NMR spectrum (see Table No. 2).

EXAMPLE 3. Obtaining 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole citrate

To a suspension of 978 mg of the base (obtained in Example 1) in 14 ml of absolute ethyl alcohol, a solution of 493 mg of citric acid monohydrate in 6 ml of absolute ethyl alcohol was added with stirring. The precipitate was dissolved at room temperature, the reaction mixture was evaporated to dryness. The residue was dissolved in 4 ml of absolute ethyl alcohol and the product was precipitated as a thick oil by the addition of 50 ml of ethyl ether. The oil is triturated twice with fresh portions of ether, the precipitate is filtered off, washed with ethyl ether and dried in vacuo over P ₂ O ₅ / KOH. 1.13 g (85% yield) of product are obtained. When heated to 78-82 ° C, the substance melts with decomposition. The salt structure was confirmed by NMR spectrum (see Table No. 3).

Table number 3.
NMR data for anhydrous 1-methyl-2-phenylthiomethyl-3-
carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole citrate Chemical shift Peak type Number of protons Group 1.26 triplet 3

2,50 singlet 6 N (CH ₃ ) ₂ 2.54-2.65 multiplet 4 C-CH ₂ (citrate) 3.66 singlet 3 N-CH ₃ 4.38 singlet 2 CH ₂ -N 4.15 quadruplet 2 O-CH ₂ 4.67 singlet 2 CH ₂ -S 7.27-7.35 multiplet 5 aromatic protons 8.00 singlet 1 H ₍₇₎

EXAMPLE 4. Obtaining 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole maleate

To a solution of 477.4 mg of the base obtained in Example 1 in 4 ml of absolute ethanol was added a warm solution of 105.8 mg of maleic acid in 2.5 ml of ethyl acetate. The mixture was cooled to + 4 ° C, the precipitate was filtered off, washed with cold ethyl acetate and dried in vacuo over P ₂ O ₅ / KOH. 336 mg (yield 60%) of product are obtained with a mp = 133-135 ° C. The structure of the obtained compound was confirmed by NMR spectrum (see Table No. 4).

Table No. 4.
NMR data for 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-
dimethylaminomethyl-5-hydroxy-6-bromoindole maleate Chemical shift Peak type Number of protons Group 1.26 triplet 3

2.73 singlet 6 N (CH ₃ ) ₂ 3.69 singlet 3 N-CH ₃ 4.72 singlet 2 CH ₂ -N 4.20 quadruplet 2 O-CH ₂ 4.77 singlet 2 CH ₂ -S 6.03 singlet 2 CH = CH (maleate) 7.28-7.38 multiplet 5 aromatic protons 8.13 singlet 1 H ₍₇₎

EXAMPLE 5. Obtaining 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole fumarate

To a suspension of 241.8 mg of the base obtained in Example 1 in 2 ml of absolute ethanol is added a solution of 47.7 mg of fumaric acid in 1.5 ml of warm absolute ethanol. Heated almost to a boil and cooled to room temperature. Leave overnight at a temperature of + 4 ° C, the precipitate is filtered off, washed with cold ethyl alcohol and dried in vacuum over P ₂ O ₅ / KOH. 157 mg (55% yield) of product is obtained with mp = 154-156 ° C. The structure of the obtained compound was confirmed by NMR spectrum (see Table No. 5).

Table No. 5.
NMR data for 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole fumarate Chemical shift Peak type Number of protons Group 1.25 triplet 3

2,31 singlet 6 N (CH ₃ ) ₂ 3.66 singlet 3 N-CH ₃ 4.11 singlet 2 CH ₂ -N 4.16 quadruplet 2 O-CH ₂ 4.63 singlet 2 CH ₂ -S 6.60 singlet 2 CH = CH 7.24-7.34 multiplet 5 aromatic protons 7.80 singlet 1 H ₍₇₎

EXAMPLE 6. The antiviral effect of arbidol against the virus of severe acute respiratory syndrome (SARS), strain SOD, in cell culture (VERO-E6).

Test the prophylactic effect of the drug.

The growth medium was removed from the tubes with the cell monolayer and 1.0 ml of the support medium containing various concentrations of the studied preparations were introduced into the test tubes. After 4 hours of incubation at a temperature of 37.0 ° C, the nutrient medium was poured out and 0.1 ml of a suspension of TARS virus was added to the tubes, at a concentration of 0.001-0.004 PFU per cell. The contact of the virus with cells is 60 minutes at 37.0 ° C. After that, the virus was removed from the tubes, the infected cells were washed with PS medium, then 1.0 ml of PS maintenance medium containing 2.5% cattle blood serum (Bovine) was added to the experimental and control tubes. Used 4 tubes for each concentration of the drug.

Additional controls:

a) control of cell cultures — in 1.0 ml 1.0 ml of the supporting medium were introduced into 4 tubes

b) virus dose control - 0.1 ml of a suspension of SARS virus was added to 4 tubes and after 60 minutes of incubation after removal of the virus-containing suspension, 1.0 ml of support medium was added.

Testing the therapeutic effect of the drug.

The growth medium was removed from the tubes with a monolayer of cells and 0.1 ml of a suspension of SARS virus was added at a concentration of 0.001-0.004 PFU (plaque forming unit) per cell. Contact of the virus with cells lasting 60 minutes. After the virus was removed, the cells were washed with PS medium with 2.5% cattle serum and a support medium containing various doses of the drug was added. Cell incubation was carried out at a temperature of 37.0 ° C. 72 hours after infection, infected cells were mechanically destroyed, pooled, and monolayer cell cultures in vials were then diluted tenfold, followed by agar coating to assess the level of virus accumulation by the method of negative colonies. Accounting for the formation of negative colonies was carried out 72 hours after infection. The results were evaluated by suppressing virus production in cell culture in the presence of various doses of the evaluated drugs.

Table number 6
Antiviral efficacy of arbidol against the SARS virus in VERO-E6 cell culture A drug Dose, μg / ml Scheme of drug administration Pathogen accumulation, lg PFU / ml Suppression of virus reproduction, Δ, lg Inhibition rate, percent Arbidol
50,0
In 4 hours
before infection 5.9 0.7 79.5 After initiation 6.0 0.6 77.7 Ribavirin
50,0
4 hours before infection 6.0 0.6 77.7 After infection 5,0 1,6 97.5 Virus dose control 6.6 0

Arbidol antiviral activity

The prophylactic and therapeutic efficacy of the drugs, determined by the difference in the accumulation of the SARS pathogen in the experimental and control cultures, is presented in Table 6. Arbidol had almost the same efficacy against the SARS virus in comparison with ribavirin in the same doses.

EXAMPLE 7. The antiviral effect of arbidol against the virus of severe acute respiratory syndrome (SARS), strain SOD, in an experimental model (Syrian hamsters)

Animals: Syrian hamsters weighing from 60 to 80 g, delivered from the nursery of the Virology Center NIIM MO RF (Sergiev Posad)

The virus of the causative agent of severe acute respiratory syndrome - CoD strain from the Museum of the Virology Center NIIM MO RF; inoculum is a suspension of VERO-E6 cell culture previously infected with the SARS pathogen.

The drugs were administered orally. The preparations were diluted in physiological saline with the addition of DMSO (0.01-0.005%).

Evaluation of the effectiveness of drugs: to reduce the level of accumulation of the pathogen in the lungs, normalize the level of aminotransferases and leukocytes in the blood of experimental animals compared to control groups.

The results of the study. The results are presented in table 7, from which it follows that the introduction of the drug according to the treatment and prophylactic scheme helps to reduce the level of virus accumulation in the lungs by 2.1 lg PFU, helps to reduce the activity of aminotransferases in the blood and normalizes the level of leukocytes. Arbidol has antiviral efficacy against the causative agent of SARS. In contrast to the ribavirin used to treat this infection, arbidol is low toxic.

Table number 7
Evaluation of the effectiveness of drugs on a non-lethal model - Syrian hamsters by changing the level of SARS virus accumulation in the lungs by biochemical and hematological blood parameters A drug Dose mg / kg Introduction scheme Decreased lung virus accumulation, Δlg The activity of aminotransferases in serum, mmol / tsp. The level of leukocytes in the blood, μl * 10 ³ AlAT AsAT Arbidol 60.0 -24, -1, +24, +48 2.1 0.44 0.41 4.8 Ribavirin 80.0 +24 and then 6 days 1.4 0.40 0.25 6.9 Dose control 2.17 1,58 12.8 Herd control 0.41 0.30 6.2 Note: infectious dose of 5.0 lg PFU when taken orally.

Antiviral salts of 1-methyl-2-phenylthiomethyl-3-carbetoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole can be used in various dosage forms, including using pharmaceutical carriers accepted in pharmaceutical chemistry for the preparation of means of delivery of the active component to the patient.

EXAMPLE 8. Obtaining a pharmaceutical composition for the preparation of tablet mass

A mixture is prepared consisting of 10 g of 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole maleate; 4.5 g of potato starch; 0.25 g of methyl cellulose; 0.15 g of calcium stearate; 0.10 g of stearic acid. The mass obtained after mixing is used for tabletting.

EXAMPLE 9. Obtaining a pharmaceutical composition for the preparation of capsules

To obtain the contents of the capsule, a mixture is prepared consisting of 10 g of 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromindole fumarate; 2.8 g of potato starch; 6.0 g microcrystalline cellulose; 0.9 g of collidone, 0.2 g of aerosil, 0.2 g of calcium stearate. The powder obtained after mixing is used for encapsulation.

Claims (2)

1. The use of 1-methyl-2-phenylthiomethyl-3-carbethoxy-4-dimethylaminomethyl-5-hydroxy-6-bromoindole or a salt thereof selected from the group of besylate, fumarate, maleate, citrate or hydrochloride monohydrate as a medicine for treating or prevention of coronavirus infection, in particular SARS. 2. A pharmaceutical composition for treating or preventing coronavirus infection, in particular SARS, comprising the active ingredient of claim 1 in an effective amount and a pharmaceutically acceptable carrier.