BG65190B1 - Flavivirus infections chemotherapy and chemoprophylaxis means - Google Patents

Abstract

The invention relates to the application of compound 1-(4-morpholinomethyl)-tetrahydro-2-[1H]-pyrimidinon with the formula for making preparations of chemotherapeutic and chemoprophylactic effect against infections caused by flaviviruses of the Flavivirus species, such as the virus of the tick encephalitis, of the diseases of the Kiasanurski forest, of the Omsk haemorrhagic fever, the Langat virus, the Western Nile virus, the Japanese encephalitis, the encephalitis of the Valley of Murray, the St Luis encephalitis, the Denga virus of the mucous diseases - viral bovine diarrhoea, the border sheep disease, and of the species of the hepatitis C virus.

Description

The invention relates to an antiviral agent against flaviviruses, which will find application for the treatment and prevention of flavivirus infections.

BACKGROUND OF THE INVENTION

Flaviviruses are a family of viruses that cause infectious diseases in humans and pets. Of particular importance are the hepatitis C viruses, tick-borne encephalitis, Japanese encephalitis, yellow fever, dengue, and some other arbovirus infections, as well as the causes of bovine viral diarrhea, swine fever, sheep disease, etc. Some of these diseases are particularly widespread and cause significant health and economic damage, such as hepatitis C in the temperate zone, arbovirus infections mainly in the subtropical and tropical areas.

Immunoprophylaxis (administration of vaccines) has found a place in yellow fever, dengue, swine fever, and to a very limited extent in some other infections (tick-borne encephalitis, mucosal disease - viral diarrhea, etc.).

At present, the use of chemotherapeutic agents (viral replication inhibitors) is considered essential in the fight against these diseases. They are mainly used for the treatment of diseases with viral etiology in the event of a clinical picture or the like. emergency prophylaxis during the incubation period of the disease (chemoprophylaxis).

The chemotherapy of flavivirus infections is in the initial experimental stages. The number of substances having an effect under experimental conditions (in vitro experiments) is too limited. Units are the substances that have shown the effect of in vivo models. Several cell-free screening systems have been developed for testing substances that suppress the activity of virus-specific protease and helicase enzymes. A serious obstacle to the search for substances effective against the hepatitis C virus is the lack of a cell culture system for reproduction of this virus.

Recently, in the clinical use for the treatment of the disease caused by the human hepatitis C flavivirus, and to a very limited extent, the preparation is ribavirin. It is most commonly used in combination with interferon. Ribavirin is known to be a non-virus-specific target and its selectivity has very moderate values, which is why it is practically used in vital indications.

In view of all of the above, the creation of highly effective and selective antiviral chemotherapeutic agents has been the subject of intense research in recent years.

SUMMARY OF THE INVENTION

The chemotherapeutic activity of compound 1- (4-morpholinomethyl) -tetrahydro-2- [1H] pyrimidinone of the formula was unexpectedly found to be clearly expressed

against flaviviruses. This enables the compound to be used in the manufacture of chemotherapy and chemoprevention preparations for flavivirus infections.

This compound is known from copyright certificate 25874, which describes a method for preparing a group of compounds is a general formula

and testing for influenza viruses. The mechanism of their influenza action against influenza virus A and B - the attack on the virus-specific protein N1 - has been established. A synergistic effect on the influenza viruses of combination with rimantadine has been demonstrated [ed. witness. No. 45 569].

According to Sidzhakova D. et al ,, Arch. Pharm. 315, 509 (1982), Galabov A. S. et al., Artzneim.-Forsch./Other Res. 34 (I), 9 (1984), Karparov A. et. al., Artzneim.-Forsch./Other Res. 35 (II), 8.1269 (1985), Karparov A. et al., C. r. Acad, bulg. Sci. 39, 8,149 (1986), Galabov A. S. et al., Zschr. Naturforsch. 55c, 292 (2000) compounds in this group also show an effect against togaviruses against representatives of the genus alfaviruses (Semliki, Sindbis, Western equine encephalomyelitis) and the genus rubiviral (rubella virus). Data have been obtained showing that the mechanism of antitagavirus activity is different from that of their anti-influenza activity.

As a rule, antiviral-active chemotherapeutic agents are strictly specific. Very often, the activity of a chemotherapeutic agent varies with different viruses in the same family. Less common are cases of activity of one substance against viruses from different families. They are (a) substances of the biological response modifier type (immunomodulators, interferons and interferon inducers, antioxidants, etc.), (b) substances whose effect is mediated by the host cell (ribavirin), or (in ) substances that suppress viral replication through different mechanisms of action (with different targets) in viruses belonging to individual families.

The current knowledge of the above mentioned three families of viruses - Orthomixoviridae, Togaviridae and Flaviviridae show huge differences, as in the properties of viral particles (morphology, physicochemical and physical properties, type of nucleic acid, proteins, lipids and carbohydrates) in genomic organization and viral replication in the host cell, which is reflected in their very different biological, including pathogenic properties [Murphy, F. A. et al. - Taxonomy, Gasification and Nomenclature virus. Springer-Verlag, Wien / New York (1995); Fields, B. N., Knipe, D. M. et al. - Fields Virology, Vol. 3, Third Edition. Raven Press, Ltd., New York (1997)].

The activity of the known compound 1- (4-morpholinomethyl) -tetrahydro 2- [1H] -pyrimidinone against flaviral infections is completely unexpected.

This substance will serve for the chemotherapy and chemoprophylaxis of flavivirus infectious diseases caused by viruses from the three genera of the Flaviviridae family: (a) the genus Flavivirus - tick-borne encephalitis, Kyasanur forest disease, Omsk treghalagen, Nyamsk tregatal, , Japanese encephalitis, Murray Valley encephalitis, St. Louis encephalitis, dengue viruses, etc .; (b) the genus Pestivirus - a virus of mucosal disease - viral diarrhea in cattle, on board disease in sheep, on swine fever; (c) hepatitis C.

The agent can be administered included in various pharmaceutical compositions for general administration (oral, intravenous, intramuscular, subcutaneous) and topical administration, together with suitable carriers, solvents and other pharmaceutical excipients. Because the substance is well water soluble, it is easy to prepare solutions for various dosage forms.

Because the compound is too low toxicity, it can be administered in a wide range of effective doses depending on the mammalian type, route of administration and the severity of the disease.

Examples of carrying out the invention

Example 1. The compound of formula (I), C9H17N3O2, obtained by the method described in author's certificate No. 25 874, is a white fine crystalline powder, well soluble in water, with a slightly bitter taste, m.p. 143-1440C, IR (Nujol) VCO = 1670 cm-1, VNH = 3230,3305 cm-1. The activity of compound (I) was tested on representatives of two genera of the flavivirus family - the genus flavivirus and the genus pestivirus. The tick-borne encephalitis virus, a representative of the first genus, was operated on in vivo model - an experimental infection of white mice. Experimental models of infection with bovine viral diarrhea virus (BVD V) in vitro in cell cultures and in vivo - experimental infestation of calves, as well as in field trials in natural mucosal disease - viral diarrhea, were used in the representative of the pestivirus genus. .

In vitro experiments compared the activity of (I) and ribavirin.

Toxicological and general pharmacological studies of the compound - in vitro, in vivo - in mice, rats, Beagle dogs and calves were conducted.

Example 1.1. In an in vivo effect test on experimental infection with tick-borne encephalitis virus, white ICR mice (10-12 g, male) were infected subcutaneously with the Sophin strain (10 LD 50 / 0.2 ml).

The compound was administered subcutaneously at a single dose of 150 mg / kg in three different regimens: A once 4 h before viral inoculation. Four times, on the 4th, 24th, 48th and 72nd hour after 5 viral inoculation, C - from the first to the 5th day after viral inoculation in two doses per day.

From the results presented in Table

I, it is seen that compound (I) exhibits a distinctive protective effect applied under Schemes B and C.

Table 1.

Effect of (I) on infection is tick-borne encephalitis virus in mice

Treatment course Survival rate,% *

A (single, -4 hours **) 10.0

B (four times, +4, +24, +48, + 72h.) 30.0

With (+ 1st - 5th day, two daily intakes) 40.0

Placebo0 * 20 animals per experimental group ** Time interval before or after viral inoculation

Example 1.2. In testing the effect of (I) on bovine pestivirus, BVDV (bovin viral diarrhaea virus), the following viral strains were used: TVM (cytopathic strain); C (Veterinary Institute, Reims Island, Germany, cytopathic strain); Kableshkovo (isolated in Bulgaria cytopathic 3 θ strain); Oregon C24V (attenuated vaccine strain). Each of these strains showed approximately the same sensitivity to compound (I) when propagated in 4 different cell cultures: primary and secondary cell cultures of 3 5 kidney, primary tracheal cultures, and vein tracheal cell (CTC) cell line expressed in severe suppression. of viral replication in single-stranded viral reproduction and multi-cycle propagation production [delTaloglO CCID50 = 3.0 - 3.8 at 120. at a concentration of (I) 30 µg / ml)].

Comparison of the effects of compound (I) and ribavirin on the replication of BVDV, strain C, in CTC cells, by the method of plaque inhibition (96 well microplates), equaled IC50 values of 0.32 µg / ml. However, the selectivity of compound (I) is much higher, due to its significantly lower toxicity (Table 2).

Table 2.

Effect of (I) and ribavirin on BVDV replication in CTC cells (CPE inhibition assay)

Compound IC 50 µg / ml CGIC50 µg / ml SI (I) 0.32 51.5 156.1 Ribavirin 0.32 20,0 60.1

IC5 O, an inhibitory concentration of 50% in the test for inhibiting the cytopathic effect of the virus; CGIC50, a concentration reducing 50% of the cell count in the cytotoxicity assay by monitoring the cell culture growth curve; SI = CGIC50 / IC50, selectivity index (chemotherapeutic index).

Example 1.3. Compound (I) was tested in calves (100 kg) seronegative for BVDV Animals were infected with BVDV, Kableshkovo strain, 7.3 log10 CCID50. Compound (I) was administered orally with milk in a course starting on the day of viral inoculation and continuing for 6 days (12 h dose interval. The results are presented in Table 3, which shows the clinical records reported by the calves.

Table 3.

Clinical symptoms in calves is an experimental infection with BVDV orally treated with (I)

Exp. Tele Daily Elevated Cognac-Anorek-Decreased

Group No. dose of (I) Tivation leukocyte temperature (mg / kg) (days) count

I 1 1 ---- 2 3 II 4 3 5 6 III 7 10 8 9 IV 10 Placebo 4 + + * 11 1 + - 12 2 + +

* Leukocyte count reduced by 3000 in 1 ml

Data from clinical observations of infected groups of calves treated with different doses of compound (I) indicate no deviation from the normal physiological state. In the control group (placebo), there were moderate clinical symptoms (fever, conjunctivitis, anorexia, and in one animal a decrease in leukocytes).

From the data in Table 4 of the virus neutralization serologic study, the same antibody response was seen in calves from the different study groups (increase of antibody titer on day 15 by 2-4 log10). Obviously, the course of treatment with compound (I) does not impede the development of an immune response to BVDV infection.

Table 4.

Virus neutralizing antibody titer in serum of experimentally infected BVDV calves orally treated with (I)

Exp. group Tele No. Daily dose of (I) mg / kg Viral Neutralizing Antibodies * in Serum Samples day 0 15th day

I 1 1 <4 4/8 * 2 <4 4 3 <4 8 II 4 3 <4 8/16 5 <4 8/16 6 <4 4/8 III 7 10 <4 8 8 <4 4/8 9 <4 8 IV 10 Placebo <4 8/16 11 <4 4/8 12 <4 4

^ Reciprocal value of the final dilution of the serum sample by neutralizing 100ID50 of BVDV (Kableshkovo strain)

Example 1.4. Two field trials were conducted on the curative effect of compound (I) on calves, a natural disease of mucosal disease - viral diarrhea. In the first trial, 3 experimental groups of 6 healthy calves (3-4 months, 80-100 kg weight) were formed: group I - treated with 1 mg / kg compound (I); group II - treated with 10 mg / kg, group III placebo. The groups of animals were housed in separate boxes, each of which introduced 3 diseased animals with a well-defined clinical presentation of MB-VD and a serologically confirmed etiology. Compound (I) was administered orally with milk at a dose of 10 (or 1) mg / kg per day (at a dose interval of 12 h) for 9 days.

In the second field test, two groups of 10 calves (15-20 days weighing 50-60 kg) were formed; group I treated with compound (I) at a dose of 10 mg / kg and group II placebo, each in a separate box. The infection and treatment course were conducted as in the first field trial.

The results of the first test are presented in Table 5.

Table 5.

Clinical indicators in 4-month-old calves is a natural infection mucosal disease - viral diarrhea treated orally with (I)

Exp. The group Tele No. Daily dose of (I; (mg / kg) Increased temperature (days) Conjunctivitis Anorek- tion Decreased leukocyte count I 1 1 3 * + + • 2 4 + + + 3 6 4 + - 4 3 4- + + 5 4. ... + - 6 7 4 4 4 II 1 10 1 2 - 3 - 4 - 5 1 6 1 III 1 Placebo 4 ²⁵ + + + 2 5 4 4 4 3 6 + + + 4 4 4 ' 4 - 5 4 30 + 4 - 6 5 + + +

* Number of days with temperature above 40 ° C

In the second test, similar results were obtained. None of the treated animals became ill. There was no case of fever. All animals in the placebo group showed severe clinical symptomatology of MB-VD and fever was a characteristic two-wave curve.

Toxicological tests of compound (I) for calves followed the scheme for the determination of acute toxicity (single dose) and subacute toxicity (administration of the compound daily for 30 days). They showed very good tolerance of the compound from the body of calves and complete harmlessness at the administered therapeutic doses.

Claims (4)

Claims What is claimed is: 1. A composition comprising 1- (4-morpholinomethyl) -tetrahydro-2- [1H] -pyrimidinone of formula (I) (I) for the preparation of chemotherapeutic and chemopreventive agents against flavivirus infections. 2, The agent of claim 1 for the chemotherapy and chemoprophylaxis of infectious diseases caused by Flavivirus-type viruses, such as tick-borne encephalitis virus, Kiasanur forest disease, Omsk hemorrhagic fever, Virus Langat, encephalitis virus, Japan Murray Valley encephalitis, St. Louis encephalitis, dengue viruses, and more. 3. The agent of claim 1 for the chemotherapy and chemoprophylaxis of infectious diseases caused by viruses of the genus Pestivirus, such as the mucosal virus. 5 Disease - viral diarrhea in cattle, Borderline disease in sheep, swine fever. The agent of claim 1 for chemotherapy and chemoprophylaxis of infectious diseases caused by hepatitis C virus viruses.