RU2413513C2 - Anthelminthic composition of quaternary phosphonium salt and substituted dinitrobenzofuroxane - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to veterinary science and represents an anthelminhic composition containing n-hexadecyltriphenylphosphonium bromide and 5,7-bis-(m-nitroanilino)-4,6-nitrobenzofuroxane as an active ingredient in the weight ratio 1:10 and glucose as a pharmaceutical aid.

EFFECT: invention provides development of a new nontoxic composition exhibiting high anthelminthic activity at low therapeutic doses.

4 ex, 2 tbl

Description

The invention relates primarily to veterinary medicine, namely to anthelmintic compositional agents, can be used as an anthelmintic drug in medicine.

Helminthiasis (ascariasis, esophagostomiasis, hookworm, toxocariasis, trichocephalosis, enterobiosis, etc.) are one of the main parasitic diseases of farm and domestic animals, as well as birds, causing significant damage to the economy and pose a danger to humans.

Clinical manifestations with all helminthiases vary widely depending on the massiveness of the invasion and the presence of polyinvasion. Symptoms of animal disease with various helminthiases are mostly similar. There are pains in the epigastrium, less often in the iliac region, accompanied by a deterioration in appetite, nausea, less often vomiting, salivation, intestinal dysfunction, allergies, general weakness and weight loss of the animal. Severe forms are mainly recorded against the background of immunodeficiency states and polyinvasions. Often nematodes are accompanied by the addition of a bacterial and fungal infection. During the migration of larvae (toxocariasis, ascariasis, hookworm infection, strongyloidosis, etc.), a pulmonary form of the disease develops with coughing, shortness of breath, sometimes with eosinophilic infiltrates. Intestinal form can be combined with pulmonary. In severe cases, for example, with strongyloidosis, intestinal perforation with the development of peritonitis is possible.

The development of medicines for the prevention and treatment of helminthiases that affect various parts of the pathological process is an urgent task for pharmacy.

Known anthelmintic synthetic preparations by chemical structure are divided into derivatives of ethanolamine, benzimidazole, salicylamide, piperazine salts [Mashkovsky MD Medicines - M .: New wave. T 2. - 2004; Chervyakov D.K. et al. Medicines in veterinary medicine. - M. Kolos, 1977; Pat. RF 2242976, publ. 12/27/2004; Pat. RF 2211835, publ. 09/10/2003]. Carbonyloxycyanomethyl derivatives are also proposed [US Pat. RF 2296747, publ. 10.27.2005], derivatives of milbemycin [Pat. RF 2226531, publ. 04/10/2004; RF Application 2004115744, publ. 04/10/2005], derivatives of tetrahydrofuroisoxazole [US Pat. RF 1586154, publ. December 20, 2006] as antiparasitic agents. Known composite anthelmintic agents, including fenbendazole, sulfadimezin, sulfur and zeolite [US Pat. RF 2302864, publ. July 20, 2007], closantel, novocaine, polyvinylpyrrolidone [Pat. RF 2278661, publ. 02.16.2004], albendazole, copper sulfate and cellulose acetate [Pat. RF 2195280, publ. 12/27/2002]. The disadvantages of the known drugs are:

- high therapeutic doses;

- side neurotoxic effect;

- low effectiveness of the intended use;

The objective of the claimed technical solution is to create an effective non-toxic anthelmintic drug of a new generation, mainly for veterinary medicine, which has a characteristic set of properties:

- low therapeutic doses;

- high LD _{50 50} 3500 mg / kg;

- high efficiency against helminths, providing an expansion of the arsenal of known means of this purpose;

The technical result of the claimed technical solution is the creation of a new non-toxic (LD ₅₀ 3500 mg / kg) composition with high anthelmintic activity at low (0.047-0.044 g / kg) therapeutic doses.

The claimed technical result is achieved by using a composition containing n-hexadecyltriphenylphosphonium bromide and 5,7-bis- (m-nitroanilino) -4,6-nitrobenzofuroxan as active components and glucose as an auxiliary substance with a weight ratio of active components of 1:10.

As we have shown, benzofuroxane derivatives have a wide spectrum of biological activity, for example, acaricidal and bactericidal [Pat. RF 2255935, publ. 07/10/2005], as well as fungicidal [US Pat. RF 2032678, publ. 04/10/1995]. For example, their use is proposed for the treatment of angina pectoris [RF Application 2000132204, publ. 11/10/2002; RF application 2000132206, publ. 10/20/2002]. From the studied prior art there is no information about the presence of anthelmintic activity both in benzofuroxanes and in Quaternary phosphonium salts, both individually and in combination, which is evidence of the conformity of the claimed technical solution to the “novelty” criterion for inventions. Moreover, the antibacterial, antifungal and antimicrobial properties of the above drugs are widely known [Galkina I.V., Tudriy E.V., Bakhtiyarova Yu.V., Egorova S.N., Galkin V.I. Synthesis, structure and study of the biological activity of phosphonium salts. / In: Pharmacology and toxicology of organophosphorus compounds and other biologically active substances. - Kazan, 2008. - P.23; Pat.JP 2002308713, publ. 10/23/2002; Pat.JP 2005060332, publ. 03/10/2005; Pat.JP 2000316701, publ. 11/21/2003]. The active components of the claimed composition based on the salt of the Quaternary phosphonium and substituted dinitrobenzofuroxan are used in the claimed technical solution for a new purpose - as anthelmintic drugs, which is not obvious to a person skilled in the analyzed field of technology, thus, the claimed technical solution meets the criterion of "inventive level "presented to inventions.

The claimed technical solution meets the criterion of "industrial applicability" to the invention, because The claimed composition can be obtained through the use of known components using standard equipment and well-known techniques.

The essence of the claimed technical solution is to create an anthelmintic composition containing n-hexadecyltriphenylphosphonium bromide and 5,7-bis- (m-nitroanilino) -4,6-nitrobenzofuroxan as active components with a weight ratio of 1: 10 and glucose as an auxiliary substance .

The claimed technical solution is as follows:

1. Get the first component of the claimed composition is n-hexadecyltriphenyl phosphonium bromide of the formula I

synthesized by the method (hereinafter, the signs of the method?) described in [Stark C.M. Interphase catalysis. Chemistry, Catalysts, and Application / Ed. C.M. Starks. M .: Chemistry, 1991. - 157 p.]:

2. Get the second component - 5,7-bis- (m-nitroanilino) -4,6-dinitrobenzofuroxane of formula II was synthesized according to the method [L.M. Yusupova, S.Yu. Garmonov, I.M. Zakharov and others. Means multipurpose biological protection based on chlorine derivatives of nitrobenzo-furoxan. / Bulletin of Kazan Technological University, 2005, No. 1, p.103-111]:

Crystalline medical glucose was used according to GOST 975-88 as a nutrient for helminths and filler (manufacturer Dnepropetrovsk starch and syrup and plant).

The claimed technical solution is illustrated by the following examples. Example 1. The synthesis of n-hexadecyltriphenylphosphonium bromide.

To an excess of 6.1 g (0.02 mol) of a solution of hexadecyl bromide, 2.62 g (0.01 mol) of triphenylphosphine are added. The reaction mixture is heated for 15 hours at a temperature of 120 ° C. The precipitate formed is filtered off and washed repeatedly with ethyl ether from the starting compounds. Yield 87%. Mp 103.4 ° C, T. decomp. 320.9 ° C, δ _p ³¹ = 25.8 ppm

Elemental analysis of C ₃₄ H ₄₈ Br, P. Found,%: C 71.96; H 8.47; P 5.83. Calculated,%: C 71.54; H 8.53; R 5.47.

Example 2. A synthesis of 5,7-bis- (m-nitroanilino) -4,6-dinitrobenzofuroxane is carried out.

To 2.95 g (0.01 mol) of dichlorodinitrobenzofuroxane in 10 ml of ethyl alcohol was added dropwise 2.76 g (0.02 mol) of m-nitroaniline in 15 ml of ethyl alcohol. 30 minutes after cooling the reaction mixture, a bright red crystalline mass was separated and recrystallized from a mixture of chloroform-hexane. Yield 78%. T pl. 226 ° C.

Elemental analysis of C ₁₈ H ₁₀ N ₈ O ₁₀ . Found,%: C 43.71; H 1.97; N, 22.34. Calculated,%: C 43.37; H 2.01; N, 22.51.

Example 3. Obtaining an anthelmintic composition and a drug based on it.

2.25 g of glucose, 0.25 g of n-hexadecyltriphenylphosphonium bromide (hereinafter phosphonium salt or SF) and 2.50 g of 5,7-bis- (m-nitroanilino) -4,6-dinitrobenzofuroxane are mixed and ground in a porcelain mortar then dinitrobenzofuroxan or DNBF) until a homogeneous red powder is obtained with which 10 wafers are filled (metered closable starch containers). The 0.5 g oral dosage form contains 0.025 g of phosphonium salt, 0.250 g of dinitrobenzofuroxan and 0.225 g of glucose.

Example 4. The study of angiogenic helminth activity against mixed pig helminthiasis caused by: Ascaris suum, Esophagostoma Oe-sophagostomum dentatum and Whipworm Trichocephalus suis.

The anthelmintic activity of the claimed composition was studied on pigs (pigs) in Kolos LLC of the Tetyush region of Tatarstan, as well as in the conditions of the vivarium of the Department of Parasitology and Radiobiology of Kazan State Academy of Veterinary Medicine. Feces were individually taken from each animal spontaneously invaded by nematodes, which was examined for the presence of helminth eggs by the Fulleborn method. Based on the results of helminthovoscopy, 6 pigs of 3-4 months of age (average weight from 23 to 25 kg) were selected and divided into 3 groups of 2 pigs in each: in the 1st group, the composition was used, in the 2nd group - phosphonium salt in the 3rd group - dinitrobenzofuroxan.

In the study of the anthelmintic activity of the composition and its components, the drug was administered to animals orally in cachets with feed 2 times a day - in the morning and in the evening. The drug for the 1st group of piglets is described in example 3. For the 2nd group, the dosage form weighing 0.5 g contained 0.025 g of phosphonium salt and 0.475 g of glucose. For the 3rd group, the dosage form weighing 0.5 g contained 0.250 g of dinitrobenzofuroxan and 0.250 g of glucose.

The characteristic data of the test piglets before treatment and the dose of drugs for the deworming course are presented in table 1. As follows from the data in table 1, in all groups the blood picture was characterized by anemia, leukocytopenia, erythrocytopenia.

The feces of piglets were examined 1, 2, 3 and 4 weeks after the use of the test drugs. The results of coprological studies and assessment of the condition of animals are shown in table 2.

As follows from the data presented in table 2, complete deworming of piglets occurred only in the first group taking the composition.

In the 1st group of piglets treated with the composition, the deworming process took place within 1-2 days with a further two-week gradual cleansing of the body of the remnants of helminths and dead eggs. Blood counts returned to normal after 2 weeks. A cough that appeared temporarily during the first week indicated the death of nematode larvae in the lungs, and allergic reactions were associated with the simultaneous death of a large number of nematodes. Cough and allergies were mild and resolved spontaneously. Feces of clarified color of plasticine consistency appeared once on - the tenth day at the 1st pig (microscopy revealed the remains of the Ascaris cuticle) and twice at the 2nd pig on the 7th and 10th day in the form of residues of the cuticle with esophagostom and Ascaris, respectively. Compared to pigs of groups 2 and 3, in group 1, animals after deworming were most active, diarrhea and any other side effects of drugs were not observed. It should be noted the improvement in the general condition of the animals and good appetite after taking the pharmaceutical composition. Conclusion of the pathologist: no helminths were found in the intestine.

In group 2, which received the phosphonium salt, the deworming pattern was more blurred and the purification of the host organism was smoother. Blood counts returned to normal within 3 weeks. A prolonged cleansing process took place in the 4th mumps, which casts doubt on the complete deworming. Thus, despite the destruction of the nematode cuticle under the action of the phosphonium salt, salt intake without dinitrobenzofuroxan turned out to be less effective than their combination in the composition (group 1).

In the 3rd group of piglets treated with dinitrobenzofuroxan, along with deworming (yield of dead immature roundworms, from 8 to 15 cm) after 4 weeks the number of parasite eggs did not change, and subsequently, the sixth mumps showed a tendency to their growth against the background of an attached intestinal infection, which manifested by diarrhea, emaciation of the animal and was accompanied by leukocytosis of the blood. Thus, despite the presence of anthelmintic activity in dinitrobenzofuroxan, its use is less effective compared to a composition containing dinitrobenzofuroxan and a phosphonium salt, since firstly, the cuticle of the nematodes is not destroyed and, secondly, an intestinal infection is attached.

The appearance of a large number of eggs of different maturity in group 1 and 2 of piglets is due to a violation of the integrity of the cuticles of helminths, as well as the uterus and oviducts, which makes the helminth body accessible to the digestive juices of the intestines of the host. The results of growing larvae from nematodes obtained in this way unambiguously showed a complete absence of development of the latter.

As a result of the study, it was found that the claimed composition has a high anti-helminthic effect at low therapeutic doses of 0.047-0.044 g / kg, while piperazine preparations are used in doses of 0.33 g / kg (8 times the dose of the proposed drug). No stool disorders or other signs of intestinal infection are observed.

The acute toxicity level of the claimed anthelmintic composition LD ₅₀ 3500 mg / kg in mice under the conditions of intraperitoneal and oral routes of administration corresponds to the category “relatively harmless” - VI class of toxicity according to Izmerov [Izmerov N.F., Sanotsky IV, Sidorov K.K . Toxicometry parameters of industrial poisons with a single injection (Reference). - M .: Medicine, 1977. - S. 196-197].

Thus, the composition, which includes three components - n-hexadecyltriphenylphosphonium bromide, 5,7-bis- (m-nitroanilino) -4,6-dinitrobenzofuroxan in a certain weight ratio and glucose, is highly effective in deworming infected animals at low therapeutic doses. Each component of this composition contributes to the anthelmintic effect of the drug:

- phosphonium salt - a component penetrating the cuticle membranes leading to the death of helminths and pathogenic microflora due to the availability of the host’s digestive juices inside the parasite (as evidenced by the destruction of the nematode cuticle, the plastic-like state of feces and the absence of nematodes in the feces of animals of groups 1 and 2);

- substituted nitrobenzofuroxan - a component that generates NO in vivo and causes spastic seizures of nematodes, which ultimately paralyzes the muscle tissue of nematodes (which is confirmed by the release of dead roundworms in group 3);

- glucose is the main nutrient of helminths, in this case it is a necessary "bait" for a parasite in the intestines of the host.

Claims (1)

An anthelmintic composition containing n-hexadecyltriphenylphosphonium bromide and 5,7-bis- (m-nitroanilino) -4,6-nitrobenzofuroxan as active components in a weight ratio of 1:10 and glucose as an auxiliary substance.