WO2015167368A1

WO2015167368A1 - 4, 6-di (3,12-diaza-6, 9-diazoniadispiro [5.2.5.2] hexandecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate for the treatment of herpetic infection and a topical pharmaceutical composition

Info

Publication number: WO2015167368A1
Application number: PCT/RU2015/000281
Authority: WO
Inventors: Vadim Albertovich MAKAROV; Natalia Sergeevna MONAKHOVA; Olga Borisovna RYABOVA
Original assignee: Limited Liability Company "Nearmedic Plus"
Priority date: 2014-04-30
Filing date: 2015-04-29
Publication date: 2015-11-05
Also published as: RU2014117294A; RU2573977C2; RU2573977C9

Abstract

The invention relates to the use of 4, 6-di(3,12-diaza-6,9-diazoniadispiro[5.2.5.2]hexadecan-1-yl)-2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate exhibiting activity against herpes virus, for the prevention and treatment of herpetic infections in humans and animals by topical administration.

Description

4 , 6-DI (3 , 12-DIAZA-6 , 9-DIAZONIADISPIRO [5.2.5.2] HEXANDECAN-1-YL) -2-METHYL-5-NITROPYRIMIDI E TETRACHLORIDE DIHYDROCHLORIDE HEXAHYDRATE FOR THE TREATMENT OF HERPETIC INFECTION AND A TOPICAL PHARMACEUTICAL COMPOSITION DESCRIPTION

Despite numerous studies in the field of virology, the problems of herpes virus infections (HVI) , especially those which are associated with herpes simplex virus (Herpes simplex) , Type 1 and Type 2 (HSV-1 and HSV-2) , are still relevant and medically and socially significant. In the structure of human infectious viral diseases in humans, HVIs rank second, behind only the flu. HVI-causing herpes viruses can affect almost all organs and systems of a macroorganism, causing latent, acute and chronic infection.

Data on the prevalence of HVIs cited in the literature vary considerably, which is likely due to differences in demographic and clinical characteristics of the study groups of patients and diagnostic methods. On the one hand, some researchers report that HVIs are clinically manifested in 20-25% of infected people, and, on the other hand, the results of scientific studies show that clinical symptoms of HVIs are manifested in 60-70% of cases. In the Russian Federation, large-scale epidemiological studies of the prevalence of HVIs have not been conducted. However, data of sampling epidemiological study among persons in the age group of from 14 to 64 years indicate that the prevalence rate of HSV type 1 reaches 99.4% [Hryanin A. A. et al . Long- term trends in the prevalence rate of the herpes simplex virus in the Siberian population. In support of practical doctor, 2010, No. 5]. Official statistics on the prevalence of HVIs caused by HSV-2 is registered in Russia only since 1993. Based on the data of the annual state statistical observation, the incidence rate of HVIs of the urogenital tract increases against the decline in the incidence of sexually transmitted infections (STI) .

The same occurs, according to WHO, as a result of mortality from viral infections: 35.8% caused by the flu virus and 15.8% caused by HSV. According to various estimates, in the United States, every year there are registered up to 98 million cases of labial herpes and 20 million cases of recurrent genital herpes, 500 thousand cases of oftalmoherpes , more than 5 thousand cases of herpetic encephalitis, and about one thousand cases of herpes in newborns. Compared with the 80' s, the number of registered patients with herpes increased at the end of the millennium by 13-40% in the United States, by 7-16% in Europe, and by 30-40% in Africa. A comparative analysis of the NHANES I and NHA ES II data showed that the incidence rate increased in equal proportions in all age groups.

At present there are the following groups of antiherpetic drugs [De Clercq E. The discovery of antiviral agents: Ten different compounds, ten different stories. Med Res Rev 2008; 28: 929-953; De Clercq E. Antiviral drug discovery: Ten more compounds, and ten more stories (part B) . Med Res Rev 2009; 29: 571-610; De Clercq E. Another ten stories in antiviral drug discovery (part C) : "Old" and "new" antivirals, strategies and perspectives. Med Res Rev 2009; 29: 611-645; De Clercq E. Yet another ten stories on antiviral drug discovery (part D) : Paradigms, paradoxes and paraductions . Med Res Rev 2010; 30: 667-707; De Clercq E. The next ten stories on antiviral drug discovery (part E) : Advents, advances and adventures. Med Res Rev 2011; 31: 118-160] : class of abnormal nucleosides, derivatives of phosphonoacetic and phosphonoformic acids, adamantane hydrochloride derivatives, heterocyclic derivatives, herbal drugs, interferon and inducers thereof, and vaccines.

The most studied class of abnormal nucleosides is a large class of antiherpetic compounds and includes pyrimidine nucleoside analogs, such as idoxuridine (IDU) used in ophthalmology for chemotherapy of herpesvirus infections caused by HSV (in treatment and prevention of herpetic keratitis); purine nucleoside analogs, among which the most famous vidarabine was recommended in the systemic drug therapy of herpes encephalitis, but has lost its importance due to a high toxicity: in high doses the drug has cancerogenic , mutagenic and teratogenic action; derivatives of nitrogen heterocycles such as virazole (ribavirin) used in chemotherapy [Smith DB, Martin JA, Klumpp K, Baker SJ, Blomgren PA, Devos R, Granycome C, Hang J, Hobbs CJ, Jiang WR, Laxton C, Le Pogam S , Leveque V, Ma H, Maile G, Merrett JH, Pichota A, Sarma K, Smith M, Swallow S, Symons J, Vesey D, Najera I, Cammack N. Design, synthesis, and antiviral properties of 4 -substituted ribonucleosides as inhibitors of hepatitis C virus replication: The discovery of R1479. Bioorg Med Chem Lett 2007; 17: 2570-2576] and is active against DNA and RNA- containing viruses (influenza A and B, parainfluenza, measles, HSV Types I and II, varicella zoster virus, etc.). Virazole has a number of severe toxic effects, such as a negative effect on the bone marrow.

The most prominent representatives of acyclic analogues of purine nucleosides are acyclovir and its derivatives, valacyclovir , penciclovir, famciclovir, ganciclovir, and cidofovir. They belong to the class of compounds being guanosine and adenosine analogs comprising a nucleic base moiety. Antiherpetic drugs of this class (for local and systemic action) are highly specific and have low toxicity. In an infected cell, during the amplification of maternal viral DNA, acyclovir, after three-step phosphorylation (by viral thymidine kinase at the first stage (monophosphorylation) , and by the cell thymidine kinase at the second and the third stages) , is picked up by viral DNA polymerase and incorporated into the DNA chains of produced daughter virions. Since acyclovir, unlike the natural guanosine, does not comprise a 3- hydroxyl group required for attachment of the next nucleoside thereto in the chain, its early introduction into the synthesis of DNA terminates the process at the beginning, assembly of daughter viral DNAs is ceased, and new viral particles are not produced. It is very important that acyclovir, being inserted into any region of a new DNA, is never replaced with the natural guanosine. The use of nucleosides breaks the replication of herpes viruses at any stage, and new generations of viruses are not formed, which actually prevents the infection from spreading. The limited number of herpes virus antigens remaining in the body allows the immune system to determine, without hindrance, the types and repertoire thereof, providing adequate immunity in humans with normally functioning immune system. Other acyclic nucleosides [Sofia MJ, Bao D, Chang W, Du J, Nagarathnam D, Rachakonda S, Reddy PG, Ross BS, Wang P, Zhang HR, Bansal S, Espiritu C, Keilman M, Lam AM, Steuer HM, Niu C, Otto MJ, Furman PA. Discovery of ad- 2-deoxy-2-fluoro-2-C-methyluridine nucleotide prodrug (PSI- 7977) for the treatment of hepatitis C virus. J Med Chem 2010; 53: 7202-7218; Furman PA. Murakami E, Niu C, Lam AM, Espiritu C, Bansal S, Bao H, Tolstykh T, Micolochick Steuer H, Keilman M, Zennou V, Bourne N, Veselenak RL, Chang W, Ross BS, Du J, Otto MJ, Sofia MJ. Activity and themetabolic activation pathway of the potent and selective hepatitis C virus pronucleotide inhibitor PSI-353661. Antiviral Res 2011; 91: 120-132; Reddy PG, Chun BK, Zhang HR, Rachakonda S, Ross BS, Sofia MJ. Stereoselective synthesis of PSI- 352938 : A. -D-2 -deoxy-2 - fluoro-2-C-raethyl-3 , 5 -cyclic phosphate nucleotide prodrug for the treatment of HCV. J Org Chem 2011; 76: 3782-3790; Lam AM, Espiritu C, Murakami E, Zennou V, Bansal S, Micolochick Steuer HM, Niu C, Keilman M, Bao H, Bourne N, Veselenak RL, Reddy PG, Chang W, Du J, Nagarathnam D, Sofia MJ, Otto MJ, Furman PA. Inhibition of hepatitis C virus replicon RNA synthesis by PSI-352938, a cyclic phosphate prodrug of D-2-deoxy-2- fluoro-2-C-methylguanosine. Antimicrob Agents Chemother 2011; 55: 2566-2575] have the same principle of action, but are focused on other enzyme groups of herpes viruses. However, it appeared that some chemotherapeutic agents had severe side effects and poor pharmacokinetic properties, limiting the indications for and/or the possibility of the administration thereof either on the age or clinical criteria. Derivatives of pyrimidine-dispirotripiperazinium (4 , 6-di (3 , 12 -diaza-6 , 9-diazoniadispiro [5.2.5.2] hexadecan-1- yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate) found as a result of the target-directed search for new drugs are capable of specifically blocking receptors containing heparan sulfate (HS) on the cell wall, thus preventing adsorption of the virus to a host cell. The most known pathogenic viruses using this mechanism of adsorption to the cell wall are herpes viruses type 1 and 2, papilloma virus, cytomegalovirus, some species of HIV, respiratory syncytial virus, and others.

The invention relates to the use of 4 , 6-di (3 , 12-diaza-

6 , 9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5- nitropyrimidine tetrachloride dihydrochloride hexahydrate exhibiting activity against herpes virus, for the prevention and treatment of herpetic infections in humans and animals by topical administration.

The mechanism of action of diazoniadispiro [5.2.5.2] hexadecane derivatives has been studied on the example of herpes virus first type (HSV-1) and kidney cells of green monkey, and it has been shown that said mechanism of action is associated with a specific property of the compounds to bind to HS, dramatically decreasing the number of viral replications. It has also been shown that the addition of the studied compounds is antagonized by · heparin. The target of diazoniadispiro [5.2.5.2] hexadecane compounds is two sulfate groups in adjacent sugar residue. Thus, for example, GlcA2S-GlcNS6S, GlcA2S-GlcNS3S , IdoA2S-GlcNAc6S, IdoA2S- GlcNH23SS6S, IdoA2S-GlcNS6S , and IdoA2SGlcNS3S demonstrate a good electrostatic interaction between the negative charge on the sulfate group and the positively charged nitrogen atoms of 4 , 6 -di (3 , 12 -diaza- 6 , 9 - diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2 -methyl-5- nitropyrimidine tetrachloride dihydrochloride hexahydrate . Further, it has been shown that the same kind of interaction can occur with the carbonyl group of octasaccharide AUA-GlcNSIdoUA2S-GlcNAc-UA2S-GlcNS-IdoUA2S- GlcNH23S, which is a necessary region of HS for the penetration of HSV-1 into a host cell. Thus, diazoniadispiro [5.2.5.2] hexadecanes block key functional groups of HS, thereby preventing viral replication and providing high antiviral activity [Schmidtke M. , Riabova 0., Dahse H.-M., Stelzner A., Makarov V., Synthesis, Cytotoxicity and Antiviral Activity of N, N'-bis-5- nitropyrimidyl Derivatives of Dispirotripiperazine , Antiviral Research, 2002, 55, 117-127; Schmidtke M . , Karger A., eerbach A., Egerer R. , Stelzner A., Makarov V. , Binding of a N, N ' -bisheteryl derivative of dispirotripiperazine to heparan sulfate residues on the cell surface specifically prevents infection of viruses from different families, Virology, 2003, V.311, p.134-143; Schmidtke, M. , Wutzler, P., Makarov, V. Novel opportunities to study and block interactions between viruses and cell surface heparan sulfates, Lett. Drug Design Discov., 2004, 1, p. 293-300] .

Systemic, mainly oral, administration of an antiherpetic agent used for the treatment requires a long- term therapy and administration of high doses (up to 1000 mg/day) of an antiviral drug. A drawback of topical dosage forms such as gels, creams, solutions, lotions, lacquers, and the like, is in that a pharmaceutically active agent is in insufficiently close contact with an affected areas.

Proper comprehensive treatment with antiviral drugs, which differ in their mechanism and spectrum of actions, is one of the major problems associated with herpetic pathology in humans .

The objective of the present invention is to find a

4 , 6-di (3 , 12-diaza-6 , 9-diazoniadispiro [5.2.5.2] hexadecan-1- yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate-containing dosage form having a high activity against herpes infections, in particular, against strains resistant to currently existing drugs.

The problem has been solved by the development of a 4 , 6-di (3 , 12-diaza-6 , 9-diazoniadispiro [5.2.5.2] hexadecan-1- yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate-containing cream having antiherpetic activity.

In the treatment or prevention of human or animal, 4 , 6-di (3 , 12-diaza-6 , 9-diazoniadispiro [5.2.5.2] hexadecan-1- yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate comprised in a pharmaceutical composition or a medicament based thereon by conventional methods in dosage forms which are suitable for topical administration, for example, in the form of ointments, creams, pastes, packed lotions and liniments, or patches.

Pharmaceutically acceptable additives used in the pharmaceutical composition according to the invention include pharmaceutically acceptable components which are compatible with the active ingredient and do not harm patients, and which are conventionally used for preparing dosage forms. Such components include, for example, fillers, binders, plasticizing agents (such as camphor, triphenyl phosphate or fentanyl diisobutyrate , diisobutyl phthalate et al.) , pigments, solubilizing agents, stabilizers, diluents, adjuvants, preservatives, components of buffer systems, solvents, dispersants, preservatives, thickeners, colorants, emulsifiers, film- forming agents (such as hydroxyethyl or propylcellulose, and hydroxypropyl chitosan) , prolongators , substances improving drug penetration (for example, benzyl alcohol) , and others.

Pharmaceutically acceptable additives include, for example, gum arabic, polyvinylpyrrolidone, polyvinyl alcohol, alginic acid, sodium alginate, anhydrous silicic acid, magnesium stearate, talc, carboxyvinyl polymer, titanium oxide, sorbitol fatty acid ester, sodium laurylsulfate, glycerol, glycerol fatty acid ester, lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax, paraffin, propylene glycol, polyethylene glycol, water, ethanol, polyols, polyoxyethylene hydrogenated castor oil, sodium chloride, sodium hydroxide, hydrochloric acid, dibasic sodium phosphate, monobasic sodium phosphate, citric acid, glutamic acid, benzyl alcohol, methyl p- hydroxybenzoate, ethyl p-hydroxybenzoate , etc. As an additive, the topical composition may comprise a vegetable oil such as jojoba oil, olive oil, sunflower oil, soybean oil, peanut oil, rapeseed oil, almond oil, palm oil, coconut oil or palm kernel oil.

As an emulsifier, the composition can comprise glycerol, optionally in admixture with sucrose laurates, or sorbitol, o/w (oil/water) emulsifier, for example, a vegetable protein hydrolyzate.

The composition can also be used in the form of liniment (lat. Linimentum) which is a thick or jelly topic dosage form rubbed into the skin. Liniment can comprise additives, such as, for example, propylene glycols and benzalkonium chloride. Emulsion fatty liniments can has a structure of "oil-in-water" or "water-in-oil" emulsions. Emulsion liniments, as well as suspension liniments, should be stabilized with an emulsifier, for example, emulsifier T-2 (emulsifier T-2 is a polymeric glycerol fatty acid ester), Tween 80, and other surfactants. In some cases, emulsifiers can be formed in a reaction between ingredients constituting the liniment.

Pharmaceutically acceptable additives used in the topical pharmaceutical compositions include, for example, natural or hardened oils, such as cocoa butter, waxes, fats, glycerol saturated fatty acid ester, glycerogelatin, macrogol, semiliquid or liquid polyols, triglycerides, etc. The basis of the topical pharmaceutical composition can also include a surfactant or a stabilizer.

Embodiments of the invention

4, 6-Di (3, 12-diaza-6, 9- diazoniadispiro [5.2.5.2] hexandecan- 1-yl ) -2-methyl-5- nitropyrimidine tetrachloride dihydrochloride hexahydrate can be prepared according to the following synthesis protocol . Preparation of N-benzoylpiperazine hydrochloride

NnOH

C₆H^:ON NH

N benzoylpiperazine

190

Acetic acid (45 mL) is added to 10 g (0.116 mol) of anhydrous piperazine under cooling and vigorous stirring. Then, acetone (20 mL) and water (20 mL) are added. After 20 minutes of stirring, 6.7 mL (0.058 mol) of benzoyl chloride are added. The solution is stirred for additional 3 hours. Then the reaction mixture is concentrated by half. 40% NaOH is added in an amount of 450 mL. The precipitated residue is filtered off, and the aqueous stock solution is extracted with 100 mL of chloroform three times, dried, and stripped off to dryness. The obtained yellow oil in an amount of 13.4 g comprises about 60% of the main substance.

The resulting oil is dissolved in 5 mL of water, the solution is heated to 45 °C under stirring, and 0.78 mL of formalin is added by batches of 0.25 mL every 10 min. After addition of formaldehyde, the mixture is stirred for 40 min at 55°C, cooled to 7°C and kept at this temperature for 1.5 hours. The residue is filtered off and washed with water three times, dried at 80 °C for 15 hours, giving 6.7 g of methylene-bis-4 (1-benzoylpiperazine) . Before the next step, the product is milled. Melting point (MP) is 125-127°C. C₆H,CO H HC1

A 12% solution of hydrogen chloride in alcohol is added by drops to a suspension of the milled methylene-bis- 4 ( l-benzoylpiperazine) (6.6 g) in 35 mL of ethanol under vigorous stirring and cooling to 10-15°C until the pH value reaches 1-2. Then, the reaction mixture is kept for 3 hours at 10°C. The residue is filtered off and washed with 50 mL of ethanol 4 times, dried at 80°C for 10 hours. The yield of the product is 5.9 g. MP is 274 °C (decomposition) .

Preparation of N-benzoyl-N- (2-oxyethyl) iperazine hydrochloride ~ CH₂CH₂OH

N-benzoylpiperazine hydrochloride N-benzoyl-N-(2-oxyethyl)piperazine

190 234

N-benzoylpiperazine hydrochloride (0.02 mol) is added to a solution of KOH (0.024 mol) in 14 mL of ethanol under stirring and further stirred for an additional hour. Ethylene chlorohydrin (0.05 mol) is added to the resulting suspension, followed by the addition of a solution of KOH (0.052 mol) in 25 mL of ethanol, while maintaining the temperature in the reaction mass of not higher than 20°C. After addition of the KOH solution, the reaction mixture is stirred for 20 hours. KC1 is filtered off, and a 12% solution of hydrogen chloride in alcohol is added to the stock solution until the pH value reaches 2-1, then the solution is cooled at -5°C for 5 hours. The precipitated residue is filtered off, washed with alcohol, and dried at 50°C for 5 hours. The yield is 70%. MP is 207-210°C. 5000281

12

Preparation of N-benzoyl-N- (2-chloroethyl) pipe

/ \ / \

H₅C₆OC N NCH₂ CH₂OH H₅C₆OC N I. ( C H2C H2 CI

\ / \ /

N-benzoyl-N-(2-oxyethyl)piperazine N-benzoyl-N-(2-chloroethyl)piperazine

234 252

Thionyl chloride (0.028 mol) is added by drops to a suspension of N-benzoyl-N- (2 -oxyethyl) piperazine (0.013 mol) in 22 mL of chloroform under stirring and slowly heating. The mixture is kept for 30 min at 45°C, then for 30 min at 45°C, and for 3 hours at 70°C, wherein the residue is precipitated first as an oil, and then it solidifies into a crystalline mass. The reaction mixture is cooled to 6-8 °C and stirred at this temperature for 1 hour. The residue is filtered off, washed with 10 mL of chloroform 3 times, and dried at 70 °C for 10 hours. The yield is 72%. MP is 226-227°C.

Preparation of N, N-dibenzoyl - ¹ , N" - dispirotripiperazinium

NaOH/C,H«OH \+

C₅H₅CON CH₂CH₂CI - C₅H₆CON N N NCOC₅H₆

/ C₂H₅OH \ / \ / \ /

2CI ^"

N-benzoyl-N-(2-chloroethyl)piperazine

_2g2 N,N-dibenzoyl-N',N"-dispirotripiperazinium

505

N-benzoyl-N- (2-chloroethyl) piperazine (0.0156 mol) is added to a solution of NaOH (0,017 mol) in 25 mL of ethanol. The suspension is stirred for 1.5 hours. NaCl is filtered off. The stock solution in alcohol is refluxed for 1 hour and then stripped off, and the dry residue is heated on an oil bath for 10 hours. The bath temperature is 120°C. Then the stock solution is cooled to room temperature, ethyl alcohol is added, and the residue is filtered off, washed with ethanol, and dried at 100 °C for 3 hours. The yield is 60%. MP is 300°C (decomposition)

Preparation of 3, 12-diaza-6, 9- diazoniadispiro [5.2.5.2] hexadecane dichloride dihydrochloride

A mixture of N, N" -dibenzoyl-N ' , N" - dispirotripiperazinium dihydrochloride (0.002 mol) and 7 mL of 10% hydrochloric acid was refluxed for 4 hours, cooled to 15°C, then the benzoic acid is filtered off and washed with water. The stock solution is stripped off to dryness, and methanol is added. The residue is filtered off, washed with methanol, and dried at 90°C for 5 hours. The yield is 85%. MP is 300°C (decomposition) .

Preparation of 3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecane dichloride

3,12-diaza-6,9-diazoniadispiro[5.2.5.2] 3,12-diaza-6,9-diazoniadispiro[5.2.5.2] hexadecane dichloride dihydrochloride hexadecane dichloride

370 297

LiOH (0,0033 mol) is added to a suspension of 3,12- diaza-6 , 9-diazoniadispiro [5.2.5.2] hexadecane dichloride dihydrochloride (0.0017 mol) in 3 mL of water, while keeping the pH value within the range of between 8.5 and 9. After 30 min, activated charcoal is added to the reaction mixture, and the mixture is stirred for 20 minutes. Charcoal is filtered off, methanol (25 mL) is added to the filtrate, and kept for 16 hours at 5-10°C. The precipitated residue is filtered off, washed with methanol and dried at 90°C for 3 hours. The yield is 90%. MP is 320°C (decomposition) .

Preparation of 4 , 6-di (3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2-methyl-5- nitropyrimidine tetrachloride dihydrochloride

3,12-diaza-E,S-diazoniadispiro[5.2.5.2]

4,6-dichloro-2-methyl-5-nitropyrimidine hexadecane dichloride

208 297

4,6-di(3,12-diara-fi,9-diazoniadispiro[5.2.5.2]hexadecan-1-yl)

-2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate

802

A solution of 3 , 12 -diaza- 6 , 9 - diazoniadispiro [5.2.5.2] hexadecane dichloride (0.24 mol) in 220 mL of water is added to a solution of 4 , 6-dichloro-2- methyl-5-nitropyrimidine (0.12 mol) in 940 mL of ethanol under vigorous stirring. The suspension is heated for 4 hours at 70 °C, cooled to room temperature, and the residue is filtered off, washed with ethanol, and dried at 110°C for 18 hours, and then in air for 4 hours. The yield of 4 , 6-di (3 , 12-diaza-6 , 9-diazoniadispiro [5.2.5.2] hexadecan-1- yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride is 87%.

MP is 216°C (decomposition) . MS (m/z) : 694 (M+) C₂9H₅₃CI₃N₁₁O₂ (M+)

Microelement analysis, %

Calculated: C₂9H₆7Cl6N₁i0₈ : C - 38.25; H - 7.42; CI - 23.36; N - 16.92

Found: C - 38.44; H - 7.38; CI - 22.98; N - 16.78

Karl Fischer water analysis:

Calculated: 11.86%

Found: 11. 72%.

Examples of preparing pharmaceutical compositions in various dosage forms suitable to be used as a topical medicament comprising 4 , 6-di (3 , 12-diaza-6, 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2-methyl-5- nitropyrimidine tetrachloride dihydrochloride.

Example 1. Preparation of pharmaceutical composition in the form of cream, comprising:

active agent, 4 , 6-di (3 , 12-diaza-6, 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2 -methyl- 5- nitropyrimidine tetrachloride dihydrochloride - 10.0 g

vaselinum - 10 g

poloxamer (emyxol 268) - 8.0 g

emulsifying wax - 3.0 g

sorbitan monostearate (span- 60) - 0.5 g

sorbic acid - 0.125 g

purified water - up to 100 mL

The active agent, poloxamer and sorbic acid are dissolved in 50 mL of water under vigorous stirring, then emulsifying wax and sorbitan monostearate are added, and the mixture is allowed to be kept. The obtained cream can be further used as an antiviral drug.

Example 2. Preparation of pharmaceutical composition in the form of gel, comprising:

active agent, 4 , 6-di (3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2 -methyl- 5- nitropyrimidine tetrachloride dihydrochloride (10 g) , methylcellulose-35 (7.5 g) , sorbic acid (0.150 g) , water - up to 100 g.

Active agent is dissolved in 50 mL of water, methylcellulose-35 and sorbic acid are added thereto, and then the remaining water is added under vigorous stirring. The mixture is kept for swelling.

The obtained gel can be further used as an antiviral drug.

Example 3. Preparation of pharmaceutical composition in the form of ointment

Ointment can be prepared as follows: the active agent in an amount of 0.5-3 wt.% based on the total weight is melted in a small amount of an ointment, cream or suppository base. The obtained melt is carefully mixed with a required amount of the base heated to 35-80 °C. The obtained mixture is homogenized. The melted resulting mixture is poured into tubes .

Example 4. Preparation of pharmaceutical composition in the form of liniment

Liniment is prepared in the form of 5% transparent yellow liquid with a slight specific odor. The liniment consists of: the active compound of formula (I) (1-2 mg) , benzalkonium chloride (50 mg) , and 1, 2-propylene glycol.

Propylene glycol is used as a humectant.

Example 5. Preparation of pharmaceutical composition in the form of emylsifying liniment, comprising:

active agent (3 g) , colloidal silica (aerosil) (5 g) , castor oil (89 g) , and optionally emilsifier, such as emulsifier T-2 or Tween 80. Description of antiviral activity of the cream according to the invention

MATERIALS AND METHODS

Studied preparations

10% cream (Example A) and 10% gel (Example B) prepared on the basis of 4 , 6-Di (3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2-methyl-5- nitropyrimidine tetrachloride dihydrochloride (further the substance) were studied.

Aciclovir [9 - ( 2 -hydroxyethoxymethyl) guanine] was used as a reference drug in the form of topical cream Zovirax comprising 5% aciclovir (5%) (from GlaxoSmithKline Consumer Healthcare, GB) .

Placebo used in in the experiments with guinea pig was cream and gel of the same composition, but without of the substance of the active compound.

Animals

Male guinea pigs weighing 250-300 g were obtained from the nursery RAMS "Andreevka" (Moscow region) . Care of experimental animals was done in accordance with "Sanitary rules for designing, equipping and keeping up experimental biological clinics (vivariums)". Animals were fed with briquetted feed in accordance with approved standards. The groups of animals were marked with color dyes (eosin) applied to the body surface. Studies were conducted in accordance with the ethical standards in force in the territory of the Russian Federation, approved by the Institutional Ethics Committee of Mechnikov Research Institute of Vaccines and Sera, and following the recommendations of the international regulatory authorities (1-4) .

Viruses and cells

Cell Culture. The study was conducted by using a continuous culture of the green monkey kidney-derived Vero cell line obtained from ATCC (American Type Culture Collection) . The cells were cultured in a DMEM growth medium (minimum Eagle's medium "PanEco" , Russia) supplemented with 10% heat- inactivated fetal bovine serum (FBS "PanEco", Russia), 2 mM L-glutamine (Sigma, USA), and antibiotics (100 g/mL penicillin and 100 g/mL streptomycin) . A support medium contained all the above ingredients and 2% FBS. The cells were incubated in a 5% C0₂ atmosphere at 37°C.

Viruses. Experiments were conducted with antigenic type of herpes simplex virus HSV-2, strain BH, obtained from the State Collection of Viruses of the Russian Federation, D.I. Ivanovsky Research Institute of Virology. The virus was maintained in the serial passages and titrated in a continuous cell culture of the green monkey kidney-derived Vero cell line. Virus titers were assessed by standard (Reed-Mench) micromethod in 96 -well tissue- culture plates by using the cell culture, and expressed as lg TCD₅₀/mL. The minimum dilution of the virus causing 50% destruction of the cell monolayer, while the cell monolayer free of infection (control) remained undamaged (50% tissue cytopathic dose of the virus TCD₅₀) was taken as TCD₅₀ of virus. In the experiments, the virus was used in the form of a virus-containing suspension with an infectious titer of 6,0 lg TCD₅₀/mL. The resulting virus was stored in frozen aliquots.

Assessment of the drug efficacy in a guinea pig model of genital herpes

Male guinea pigs were infected with the culture virus - containing liquid (HSV-2, "BH") . The virus-containing liquid (at a dose of 100 TCD₅₀) was applied with a pipette (followed by rubbing) on the previously scarified skin of the penis. Scarification was performed by a surgical lance, after the animals were anesthetized with anesthetic lidocaine (1%) . The size of the scarification area was 4-7 mm². Each group contained 4-5 animals.

Placebo used in the experiments in the form of cream and a gel had the same composition, but was free of the substance PDSTP. Furthermore, 5% ointment Zovirax (acyclovir) was used as a reference dosage form. The topical treatment was conducted according to the following scheme: 3 or 48 hours after infection (in case of pronounced manifestations of the disease) . Preparations were applied by application of a thin layer on the lesions twice a day, every day for 5 days. The area of the drug application was 1.3 cm².

The severity of the infection was assessed every day before the treatment, and monitored throughout the whole period of the disease by the following parameters: the existence and degree of specific lesions (vesicles, pustules, ulcers, erosions, crusts) ; edema, hyperemia, and orchitis. The maximum severity of each symptom was 4 scores. The total duration of the monitoring period was 21 days .

The efficacy of the preparations was assessed in the maximum manifestation of the pathological process, by standard methods. Criteria for the assessment of the therapeutic effect of the preparations were: a reduction in the intensity of clinical symptoms, index of therapeutic action (ITA) , a reduction of the disease duration in the experimental groups, compared with the control.

ITA (%) =

The total score of the control group— the total score in the group of animals treated with drug the total score in the control group

On day 5 after infection of guinea pigs, mucosal swabs from the urogenital tract were taken in sterile tubes with 1.8 mL of the DMEM culture medium.

Determination of the virus titer derived from the urogenital swabs of guinea pigs

The virus was titrated in vitro from the contents of the mucosal swabs from the guinea pig urogenital tract in a VERO cell culture by standard virological methods.

The taken samples were stored at -70°C, before analysis .

VERO cells were grown in "Costar" 96 -well cell culture plates. The cells in the complete culture medium were added to each well of the plate in an amount of 10 mL at a concentration of 10⁴ cells/mL, and incubated in 5% C0₂ at 37°C. After the formation of a complete monolayer (24 hours) , the medium was removed, and the monolayer was washed three times with the serum- free culture medium and used in further experiments.

Before the addition of the samples, all wells were filled with 90 L of a serum- free culture medium (complete DMEM culture medium (PanEco, Russia) supplemented with antibiotics, 0.008% gentamycin sulphate solution (Verein, RF) , 300 mg of glutamine (FSUE of Chumakov Institute of Poliomyelitis and Viral Encephalitis RAMS, Russia) in 500 mL of medium) . The studied samples were added in an amount 10 L/well (dilution 1:100) . Further, the titration was performed by serial dilution within a range of 10¹-10⁵ TCD₅₀/mL. The virus was titrated concurrently within a range of 10³-10⁹ TCD₅₀/mL.

Intact cell culture was used as a control (negative control) . Then the cells were incubated in a thermostat at

37°C under 5% C0₂ for 5 days. The cell monolayer was examined under the light microscope every day to assess the degree of intactness and to detect a specific viral cytopathic effect (CPE) . The results were assessed visually, using an inverted microscope LABOVERT FS ("Leitz" company) . Cytopathic changes, which were expressed in grouped, round cells increased in size, were assessed according to the following scale:

+ - slight change in the monolayer (25%) ;

++ - small gaps in the monolayer (50%) ;

+++ - about 50% desctruction to the monolayer (75%) ; and

++++ - almost complete destruction of the monolayer

(100%) .

The cytopathic effect of the samples was quantitatively measured by a modified method of counting live and dead cells, using Neutral Red (NR) dye. After washing (3 times) , 50 L of the NR solution (concentration of 1.11 pg/mL) was added to each well of the plate, and after incubation for 2 hours at 37 °C in 5% of C0₂, the plate was washed three times, and 100 L of a solution of 50% of EtOH/50% of 0.1M NH₄H₂P0₄ was added to each well (pH 3.5). After 30 min incubation, the cells were counted in a spectrophotometer at a wavelength of 490 nm.

Each studied sample was assessed in 4 repeats (wells) , and each value represents the arithmetic mean.

Statistical data were processed by standard methods of variation statistics by using program "Ecxel-5.0", with 95% confidence interval .

RESULTS

1. Study design

Treatment with topical preparations (cream and gel) comprising the substance, 4 , 6-di (3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2 -methyl- 5- nitropyrimidin tetrachloride dihydrochloride hexahydrate, and with the reference drug (cream Zovirax) was carried out by the following two variants of the treatment scheme: a) 3 hours after infection; b) 48 hours after infection (in case of expressed manifestations of the disease) . Topical drugs were applied (by the method of application) in a thin layer to lesions twice a day, every day for 5 days.

Description of the studied groups

Given the purpose of the experiment, all animals were divided into 7 groups consisting of 4 pigs, and the eighth group contained 6 animals.

The first group comprised animals infected with HSV-2 and treated with the topical cream according to the treatment scheme of 3 hours after infection, twice a day, every day for 5 days .

The second group comprised animals infected with HSV-2 and treated with the topical cream according to the treatment scheme of 48 hours after infection twice a day, every day for 5 days .

The third group comprised infected animals treated with the topical gel according to the treatment scheme of 3 hours after infection twice a day, every day for 5 days.

The fourth group comprised infected animals treated with the topical gel according to the treatment scheme of 48 hours after infection twice a day, every day for 5 days.

The fifth group comprised infected animals treated with drug "Zovirax" in the form of a topical cream according to the treatment scheme of 3 hours after infection twice a day, every day for 5 days.

The six group comprised infected animals treated with drug "Zovirax" according to the treatment scheme of 48 hours after infection twice a day, every day for 5 days.

The seventh group (positive control) comprised only HSV-2 -infected animals which did not receive treatment.

The eighth group comprised 6 animals which were subjected to only scarification, wherein two pigs were not subjected to further treatment, and 2 pigs were treated with corresponding cream and gel placebo, according to the treatment scheme of 3 hours after infection twice a day, every day for 5 days. These animals were considered as a negative control, against which the severity of virus - caused pathological process and local irritating action of the drugs were assessed.

Characteristics of the infection process in infected animals.

In the control group of infected animals (Group 7) , which did not receive treatment, typical local symptoms of genital herpes developed 24-48 hours after infection. Initially, the appearance of individual vesicular lesions on an erythematous background was observed in the mucosa of the penis and surrounding areas of the skin, and then, confluent lesions with hemorrhagic content, bleeding erosion, ulceration, and moderate orchitis emerged as the disease advanced. In the following days, erosions and ulcers were epithelized either under a crust or without its formation. The specificity of local manifestations of genital herpes (HG) was confirmed by isolation of the virus from the contents of vesicles and swabs from the urogenital area of the infected animals.

The process was characterized by the presence of pronounced edematous inflammatory reactions, long-term maintenance of hyperemia, and "sluggish" healing of erosive and ulcerative elements. Maximum development of symptoms was observed on day 5 after infection: the total symptom severity score (TSSS) in this group was 34.74, the average duration of disease (ADD) was 15.0±2.46 days.

2. Results of the experimental treatment of genital herpes in guinea pigs with the topical cream and gel, comprising the substance, 4 , 6-di (3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2-methyl-5- nitropyrimidin tetrachloride dxhydrochloride hexahydrate .

The results of the experimental treatment of genital herpes in guinea pigs with the cream and gel, comprising the substance, 4 , 6-di (3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2 -methyl- 5- nitropyrimidin tetrachloride dihydrochloride hexahydrate, and with the reference drug Zovirax are presented in Table 1, and Figures la and lb.

As follows from the data presented in Table 1, Figures la and lb, the use of the topical cream and gel, comprising the substance, 4 , 6-di (3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2-methyl-5- nitropyrimidin tetrachloride dihydrochloride hexahydrate, had an effect of various intensity on the course of genital herpes infection in male guinea pigs.

The most significant results were obtained in the group of infected animals treated with the cream according to the treatment scheme of 3 hours after infection.

Table 1. Efficacy of the topical cream and gel, comprising the substance PDSTP, in the guinea pig model of experimental genital herpes

Note: * - difference between the corresponding scores in the experimental and control groups was statistically significant at p <0.05

It has been found that the use of the preparation in the form of cream at early stages of the infectious process (3 hours after induction of the infection) resulted in a statistically significant therapeutic effect consisting in a 1.5 time reduction in the severity of symptoms and in a significant reduction in the average duration of disease, compared with analogous symptoms in the group of infected animals which did not receive treatment (positive control) . ITA was 32.64%, and ADD was 9.0±1.0 days (see Table 1, Fig. la) . The animals treated with the cream recovered more quickly by an average of 6.0 days than those who did not receive treatment. The infectious process was characterized by moderately severe exudative inflammation, a reduced duration of exudative inflammatory reactions and resolution of elements. The study revealed no significant differences between the efficacy of the cream and the reference drug Zovirax for all studied parameters (by an effect on ITA, TSSS, ADI, the nature of the course of the infection) (p>0.05). In the treatment with the reference drug Zovirax (sixth group), ADD was 8.33+0.57 days; ITA was 35.23%.

The use of the cream in infected animals having pronounced manifestations of the pathological process (group 2) also resulted in a statistically significant therapeutic effect and a reduction in the duration of disease, compared with the infected animals which did not receive treatment. At the same time, the preparation was inferior, in terms of therapeutic action (26.59%), to the preparation used in the group of animals treated with the cream at early stages of the infection (32.64%), and in the group of animals treated with the reference drug Zovirax (30.05%), but it was comparable with the preparation used in the first group, in terms of ADD. Thus, ADD in the animals of the second group was 9.33±1.52 days, and in the animals of the first group was 9.0+1.0 days (p>0.5). The comparison of ADD values between the animals of the second group and the animals treated with the reference drug showed that the infected animals treated with the studied cream recovered 1.33 days later (p <0.5) . It should be also noted that in the group of the late treatment with the cream (after 48 hours) , exudative inflammatory reactions were more pronounced and prolonged than in the animals of the first group receiving the early treatment with the same preparation, and in the animals treated with the reference drug Zovirax.

An analysis of the use of the topical gel according to the treatment scheme in the experimental guinea pig model of genital herpes showed, similarly to the cream, that this form of the preparation exhibited more pronounced activity when used early after infection.

Thus, the use of gel 3 hours after infection (group 3) resulted in a significant ITA value equal to 25.73%, and in a significant reduction in ADD by 3.34 days, compared with the group of animals which did not receive treatment. The treatment efficacy of the gel used according to the treatment scheme of 3 hours after infection was comparable with the efficacy of the cream used in case of pronounced manifestations of the infection (group 2, the initiation of treatment 48 hours after infection) , but, in terms of the disease duration, its effect was observed later by an average of 2.4 days, as compared with both the effect of the cream used according to the scheme of 3 after infection, and the action of the reference drug (see Table 1) .

The study showed that the infected animals treated with the gel 48 hours after infection for 5 days (group 4) resulted in the therapeutic effect equal to 22.01% and in a reduction of ADD by 2.66 days, compared to the infected animals which do not received treatment. Nevertheless, the use of the gel according to the scheme of 48 hours was less effective by all evaluative parameters, compared to the use of both the cream at different periods of the infection process, and the reference drug Zovirax (group 6) .

Monitoring of the animals of group 8 showed that two animals, which were subjected to only scarification, recovered more quickly (for 1-2 days) and, then no pronounced symptoms were observed in pigs of this group. In addition, in four pigs treated with the corresponding placebo of cream and gel, according to the scheme of 3 hours after infection twice a day, every day for 5 days there was no evidence of swelling, visible local irritating action of the drugs, and general side effects.

Thus, according the degree of reduction, the studied modifications of the topical preparations comprising the substance, , 6-di (3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2-methyl-5- nitropyrimidin tetrachloride dihydrochloride hexahydrate, can be arranged, in terms of reduction in the efficiency, as follows: Group 1 (cream + 3 hours after infection) > Group 3 (gel + 3 hours after infection) > group 2 (cream + 48 hours after infection) > Group 4 (gel + 48 hours after infection) .

3. The study of the effect of topical cream and gel, comprising the substance, 4 , 6-di (3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2-methyl-5- nitropyrimidin tetrachloride dihydrochloride hexahydrate, on the frequency and level of shedding herpes simplex virus type 2 male by guinea pigs .

To determine the effect of cream and gel comprising the substance, , 6-di (3 , 12-diaza-6, 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2-methyl-5- nitropyrimidin tetrachloride dihydrochloride hexahydrate, on the frequency of shedding HSV-2 and the level of its replication in the infected animals, we analyzed the samples of swabs from lesions, collected from animals on day 5 after infection, by a standard method for isolating virus, in VERO cell culture.

Table 2. The effect of the topical cream and gel, comprising the substance, 4 , 6-di (3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2 -methyl- 5- nitropyrimidin tetrachloride dihydrochloride hexahydrate, on the frequency of shedding the virus from lesions in male guinea pigs infected with HSV-2 and on the level of its replication (according to data of a virological method) .

As can be seen from Table 2, on day 5 after infection, the frequency of shedding the virus by infected animals was the same as in the experimental and control groups, and was 100%. The obtained results, in turn, confirmed the specificity of the observed symptoms of genital herpes in the experimentally infected animals.

It was found that the use of the cream and gel comprising the substance, 4 , 6-di (3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2-methyl-5- nitropyrimidin tetrachloride dihydrochloride hexahydrate, in the infected animals (Groups 1-4) resulted in a reduction in the level of replication and infectious activity of the virus isolated from the samples of swabs from lesions, as compared with the infectivity of the virus isolated from the infected animals which did not receive treatment (see Table 2). Thus, the virus titers isolated from the animals treated with the cream were 1.9+0.52 lg TCD₅₀/mL and 2,41+0,4 lg TCD₅₀/mL, respectively; and treated with the gel were 2,25±0,5 lg TCD₅₀/mL and 3.0+0.11 lg TCD₅₀/mL, respectively.

The most pronounced reduction in the infectious activity of the virus, 100 times, was observed for the cream used at early stages of the infection process, while the use of the cream, in case of pronounced manifestations of herpes infection, resulted in a 30 time reduction of the viral infectious activity.

The application of the gel according to the treatment scheme resulted in a reduction in the viral infectious activity 50 and 10 times, respectively.

Virus titers in infected animals treated with the reference drug Zovirax were significantly decreased, more than 100 times, as compared with the virus titers in the control (p<0.05) and were 1,66-1,75 lg TCD₅₀/mL.

Thus, in the experiments on the isolation and titration of the virus from samples of biological material derived from animals infected with GH in VERO cell culture, we obtained data supporting the presence of antiviral activity in an ionic gel and the gel comprising the substance, , 6-di (3 , 12-diaza-6, 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2-methyl-5- nitropyrimidin tetrachloride dihydrochloride hexahydrate, in topical administration. Data obtained in the virological method correlated with data on the efficacy of the drugs, obtained by using the characteristics of the infection process caused by HSV-2.

Thus, the cream and gel comprising the substance, 4,6- di (3 , 12-diaza-6 , 9-diazoniadispiro [5.2.5.2] hexadecan-l-yl) - 2-methyl-5-nitropyrimidin tetrachloride dihydrochloride hexahydrate, in topical administration according to the treatment scheme (two time administration for 5 days) in the guinea pig model of genital herpes, have a significant therapeutic effect that can suppress the development of herpes virus infection caused by HSV-2. The most effective treatment was the treatment with the cream comprising the substance, 4 , 6-di (3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2-methyl-5- nitropyrimidin tetrachloride dihydrochloride hexahydrate, according to the treatment scheme at early stages (after 3 hours) after infection (group 1) , which has a pronounced therapeutic effect on the course of infection in animals, providing a significant reduction in the severity of symptoms, a reduction in the terms of resolution of elements, a reduction in the average duration of the disease, and infectious activity of HSV-2. The effect of the treatment with the cream at early stages (after 3 hours) after infection is confirmed by the results of a virological method, which showed a significant reduction in titers in the treated animals, compared with the group of viral control. The efficacy of the cream used according to the treatment scheme at early stages after infection was comparable with the reference drug Zovirax used by the same scheme, in terms of both the characteristics of the infectious process, and a reduction in virus titers of the treated animals .

Claims

1. A pharmaceutical composition in the form of a cream, comprising 4, 6-di (3, 12-diaza-6, 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2-methyl-5- nitropyrimidin tetrachloride dihydrochloride hexahydrate, as an active ingredient, in an amount of from 0.5 to 10.0% for the treatment of a herpes virus infection.

2. A pharmaceutical composition in the form of a gel, comprising 4 , 6-di (3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2-methyl-5- nitropyrimidin tetrachloride dihydrochloride hexahydrate, as an active ingredient, in an amount of from 0.5 to 10.0% for the treatment of a herpes virus infection.

3. The composition according to claim 1, wherein the cream has anti-herpetic activity against strains of HSV-1 and HSV-2, including strains resistant to currently existing drugs.

4. A method of treating or preventing a herpetic infection in a human or animal, comprising topically administering to a patient the pharmaceutical composition according to claim 1 and claim 2, in an effective amount.