RU2769523C1 - 4-(3,4-dibromothyophene carbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12 hexaazaisowurtzitane as an analgesic agent and method for its preparation - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to a new chemical substance: 4-(3,4-dibromothyophene carbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12 hexaazaisowurtzitane (I), as well as to a method for its preparation, which consists in the acylation of 4-(3,4-dibromothyophencarbonyl)-2,6,8,12-tetraacetyl-2,4,6,8,10,12- hexaazaisowurtzitane with acetyl chloride.

EFFECT: a new compound 4-(3,4-dibromothyophene carbonyl)-2,6,8,10,12-pentaaacetyl-2,4,6,8,10,12 hexaazaisowurtzitane with analgesic activity comparable to and/or superior to the effectiveness of Tramadol was obtained.

2 cl, 4 tbl, 2 ex

Description

The invention relates to pharmaceutical chemistry and pharmacology, and in particular to a new chemical substance - 4-(3,4-dibromothiophenecarbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12-hexaazaisowurtzitane with analgesic activity:

By its biological origin, pain is one of the most important signals that informs the body about tissue damage and triggers a whole range of protective reactions aimed at minimizing and eliminating damage. The formation of a complex, multicomponent pain sensation, including not only sensory and motivational-affective components, but also vegetative, motor reactions, is mediated by a nociceptive system consisting of sensitive neurons that have a complex hierarchical organization and provide perception, encoding, conduction and analysis of damaging stimuli. Depending on the leading pathogenetic development factor, all pain syndromes were divided into three main groups: nociceptive, neurogenic and psychogenic. Pain syndromes occupy a leading place among the most common problems faced by almost every person. Many acute and chronic diseases, injuries and medical interventions are associated with pain that requires the use of analgesics. The choice of painkillers, the corresponding analgesic potential, is determined by a number of factors, which include the etiology, intensity, type of pain, individual characteristics of the patient. According to various studies, the consumption of these medicines is steadily growing both in Russia and abroad [Tabeeva G.R. Difficult simple analgesics, or what to remember when choosing an analgesic // Russian Medical Journal. 2013, 21 (10): 470-475]. Currently existing analgesics are divided into two main groups - narcotic (opioid) and non-narcotic, various combinations of them are used in multimodal therapy schemes, analgesics from the cannabinoid class are used [Mashkovsky M.D. Medicines: 15th ed. M: 00 New Wave Publishing House. 2008, 1206; Mikhailova A.S. Analgesic arsenal of the clinician. Pharmateka. 2018, (3): 50-56; Sosnov A.V., Sadovnikov S.V., Semchenko F.M., Rufanov K.A., Tokhmakhchi V.N., Sosnova A.A., Tyurin I.A. Potent non-narcotic analgesics as a direction in the development of pharmaceuticals. Development and registration of medicines. 2016, 14(1): 196-206; Chernyakov A.V. The problem of pain and anesthesia in outpatient surgery // Russian Medical Journal. 2016, 2 (14); 927-931; Cawich S.O., Deonarine U., Harding H.E., Dan D., Naraynsingh V., Cannabis and Postoperative Analgesia. Handbook of Cannabis and Related Pathologies. Biology, Pharmacology, Diagnosis, and Treatment. 2017, 450-458]. The former have a powerful effect against pain of medium and high intensity, however, the turnover of such drugs is limited due to the development of physiological and mental dependence against the background of their use [Palekhov A.V., Vvedenskaya E.S. The tactics of choosing and using opioid analgesics for the relief of chronic pain // Palliative Medicine and Rehabilitation. 2018, (3): 18-24]. Non-opioid analgesics, which are non-narcotic drugs and available without a prescription, are used to treat acute pain and chronic pain syndromes of low intensity [Mikhailova A.S. Analgesic arsenal of the clinician. Pharmateka. 2018, (3): 50-56]. Non-opioid analgesics include drugs of five pharmacological groups: NSAIDs, selective cyclooxygenase-2 (COX-2) inhibitors, pyrazolone derivatives, paraacetaminophenol derivatives and flupirtine. According to the scientific literature for more than 130 years, hundreds of substances of non-narcotic analgesics and non-steroidal anti-inflammatory drugs (NSAIDs) have been synthesized in the world, some of which are due to severe side effects (gastroduodenopathy, nephrotoxicity, cardiovascular effects, hepatotoxicity, teratogenicity, hematotoxicity) and insufficient efficiency in many countries it is forbidden to produce and use [Kolokolov O.V., Sitkali I.V., Kolokolova A.M. Nociceptive pain in the practice of a neurologist: diagnostic algorithms, adequacy and safety of therapy // Russian Medical Journal. 2015, 23(12): 664-667. Mashkovsky M.D. Medicines: 15th ed. M.: NGO "New Wave Publishing House". 2008, 1206; Mikhailova A.S. Analgesic arsenal of the clinician. Pharmateka. 2018, (3): 50-56; Guidelines for conducting preclinical studies of drugs. Ed. Mironova A.N., Bunatyan N.D. and others. M., CJSC Grif and K. 2012.; Sosnov A.V., Sadovnikov SV., Semchenko F.M., Rufanov K.A., Tokhmakhchi V.N., Sosnova A.A., Tyurin I.A. Potent non-narcotic analgesics as a direction in the development of pharmaceuticals. Development and registration of medicines. 2016, 14(1): 196-206; Cawich S.O., Deonarine U., Harding H.E., Dan D., Naraynsingh V., Cannabis and Postoperative Analgesia. Handbook of Cannabis and Related Pathologies. Biology, Pharmacology, Diagnosis, and Treatment. 2017: 450-458; Shiels M.S., Chernyavskiy P., Anderson W.F. et al. Trends in premature mortality in the USA by sex, race, and ethnicity from 1999 to 2014: an analysis of death certificate data // The Lancet. 2017, 389 (10073): 1043-1054].

One of the most commonly used and the closest in technical result to the proposed tool is Tramadol - an analgesic with a mixed mechanism of action, including opioid and non-opioid components, which is selected as the comparison drug [Mashkovsky M.D. Medicines: 15th ed. M.: NGO "New Wave Publishing House". 2008, 1206; Mikhailova A.S. Analgesic arsenal of the clinician. Pharmateka. 2018, (3): 50-56].

The objective of the present invention is to expand the arsenal of analgesics by creating new substances with analgesic activity and low toxicity.

The problem is solved by creating a new compound - 4-(3,4-dibromothiophenecarbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12-hexaazaisowurtzitane (I) and using it as analgesic agent.

The proposed analgesic 4-(3,4-dibromothiophenecarbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12 hexaazaisowurtzitane (I) was first synthesized at the Institute of Problems of Chemical and Energy Technologies ( Biysk). Compound I has been fully confirmed by physical and chemical methods of analysis (IR spectroscopy, elemental analysis, 1H and 13C NMR spectroscopy), has a pronounced analgesic activity and belongs to the 3rd hazard class "low toxicity".

2,6,8,12-Tetraacetyl-2,4,6,8,10,12-hexaazaisowucitane is an industrial intermediate and is easily acylated. The introduction of the fifth acetyl group into 4-(3,4-dibromothiophenecarbonyl)-2,8,10,12-tetraacetyl-2,4,6,8,10,12-hexaazaisowurtzitane does not cause difficulties, the reaction proceeds at moderate temperatures and with high the yield of the target product I is more than 90% (in terms of the original 4-(3,4-dibromothiophenecarbonyl)-2,6,8,12-tetraacetyl-2,4,6,8,10,12-hexaazaisowucitane). In addition, the introduction of the fifth acetyl group into the 4-(3,4-dibromothiophenecarbonyl)-2,6,8,12-tetraacetyl-2,4,6,8,10,12-hexaazaisowucitane molecule increases the solubility of the target compound I (in compared with the original product), which makes it even more attractive in terms of practical use.

At the Research Institute of Pharmacology and Regenerative Medicine named after E.D. Goldberg of the Tomsk Research Center (Tomsk), as a result of the experimental studies, the analgesic activity and low toxicity of the first obtained compound I were identified and confirmed.

The technical result of the invention is a new substance I, a method for its preparation, the identification of the analgesic properties of substance I and confirmation of its analgesic effect. The identified property of substance I, proposed as an analgesic agent, does not explicitly follow from the prior art in this field and is not obvious to a specialist. An identical set of features was not found in the study of the state of the art in the patent and medical literature [Tolstikova T.G., Morozova E.A., Sysolyatin S.V., Kalashnikov A.I., Zhukova Yu.I., Surmachev V. N. Synthesis and biological activity of 2,4,6,8,10,12-hexaazatetracyclo[5,5,0,03,11,05,9] dodecane derivatives. Chemistry for sustainable development. 2010, 18: 511-516; Sergey V. Sysolyatin, Alexander I. Kalashnikov, Valeriy V. Malykhin, Irina A. Surmacheva, Gennady V. Sakovich. Reductive Debenzelation of 2,4,6,8,10,12 -Hexaazaisowurtzitane/ International Journal of Energetic Materials and Chemical Propulsion. 2010, 9 (4): 365; Krylova S.G., Lopatina K.A., Nesterova Yu.V., Povet'eva T.N., Kulpin P.V., Afanas'eva O.G., Kulagina D.A., Safonova E.A., Zueva E. .P., Suslov N.I., Sysolyatin S.V. Some aspects of the study of the central mechanism of the antinociceptive action of a new analgesic from the class of hexaazaisowurtzitane // Bulletin of experimental biology and medicine. 2020(12): 739-746; Kulagina, D.A., Sysolyatin, S.V., Kalashnikov, A.I. et al. Synthesis and Analgesic Activity of 4,10-Bis((±)-5-Benzoyl-2,3-Dihydro-1hpyrrolo[1,2-a]Pyrrole-1-Carbonyl)-2,6,8,12-Tetraacetyl- 2,4,6,8,10,12-Hexaazatetracyclo [5,5,03,11,05,9]Dodecane. Pharm. Chem. J. 54, 1140-1144 (2021); Tomilova, E., Kurgachev, D., Kulagina, D.. Sergey Sysolyatin, S., Krylova, S., Novikov, D. Development of HPLC-Method for Simultaneous Determination of API and Related Components in Thiowurtzine: A New Non- Narcotic Analgesic. Chromatographia 84, 147-154 (2021)].

The possibility of implementing the claimed invention in terms of the substance and the method of its production is confirmed by an example of a specific implementation of the method for producing compound I, which has a pronounced analgesic activity.

Example 1

Preparation of 4-(3,4-dibromothiophenecarbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12-hexaazaisowurtzitane.

To 18.12 g (0.03 mol) 4-(3,4-dibromothiophenecarbonyl)-2,6,8,12-tetraacetyl-2,4,6,8,10,12-hexaazaisowurtzitane in 220 ml of dry acetonitrile at 4.75 g (0.06 mol, 4.4 ml) of acetyl chloride was added while stirring. The reaction mass was boiled with stirring for 24 hours, then cooled to room temperature and filtered. The filtrate was evaporated to 3/4 parts and diluted with 120 ml of isopropyl alcohol. The precipitated product was filtered off, washed with 2×20 ml of isopropyl alcohol. The precipitate was dried in air.

18.8 g (97.0%) of 4-(3,4-dibromothiophenecarbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12-hexaazaisowurtzitane was obtained as a colorless crystalline product with a basic substance content of 99.6% (HPLC method) and a melting point of 255-256°C.

C ₂₁ H ₂₂ Br ₂ N ₆ O ₆ S.

Found (%): C 39.18; H 3.39; N 12.97; S 4.96.

Calculated (%): From 39.03; H 3.43; Br 24.73; No. 13.00; About 14.85; S 4.96.

IR spectrum, v/cm-1: 3072, 3030, 1666, 1530, 1403, 1360, 1306, 1287, 1256, 1163, 1050, 970, 949, 884, 785, 751, 727, 707, 687, 633.

1H NMR spectrum (DMSO-d6, δ, ppm): 1.98-2.20 (m, 15H, _CH3CO ), 6.47-7.07 (m, 6H, CH), 8.15(d, 1H, CH).

SPEKTRO NMR 13C (DMSO-D6, δ, M.D.): 21.27, 21.34, 21.6, 22.64, 65.6, 67.43, 69.6, 67.43, 69.6, 67.43, 12 168.10, 168.27, 168.58, 168.76, 170.21.

The possibility of implementing the claimed invention in terms of analgesic activity is confirmed by an example of a specific study of the analgesic activity of 4-(3,4-dibromothiophenecarbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12-hexaazaisowurtzitane (I) in comparison with the reference drug Tramadol, proving that the identified signs solve the problem.

Example 2

Experiments were performed on 198 outbred CD1 stock mice of both sexes (7-8 weeks, weight 22-27 g) of the first category of conventional, obtained from the Department of Experimental Biomodeling of the Research Institute of Medicine and Medicine. E.D. Goldberg of the Tomsk NIMC (certificate of animal health, veterinary certificate 270 No. 0008633). The maintenance of the animals and the design of the experiments were approved by the Bioethical Committee of the NIIFiRM. E.D. Goldberg and complied with Directive 2010/63/EU of the European Parliament and of the Council of the European Union on the protection of animals used for scientific purposes [Directive 2010/63/EU of the European Parliament and of the Council of the European Union on the protection of animals used for scientific purposes (translated from English. ). SP. 2012, 48]; Order of the Ministry of Health of the Russian Federation of August 1, 2016 N 199n.

Each animal within the group was assigned an individual number from 1 to 11, labeled with fucorcin. Mice were divided into groups randomly, using body weight as a criterion, so that the individual weight value did not deviate from the mean value within one sex by more than ±10%. The data (weight and number) of the selected animals were ranked in descending order of weight using an Excel computer program with the SORT command.

Statistical processing of the obtained results was carried out by the methods of variation statistics using the Statistica 6.0 software. For all data, the mean value (X) and standard error of the mean (m) were calculated, which, together with the value of n (the number of options in the group), are presented in the final tables. The difference between the compared values was considered significant if the probability of their identity was less than 5% (P<0.05). Using the sample coefficients of asymmetry and kurtosis, the degree of approximation of the distribution law of the studied trait to the normal one was estimated. In cases of normal distribution of signs, a parametric Student's t-test was used for statistical evaluation. When the distributions of the trait deviated from the normal form for independent samples, the nonparametric Wilcoxon-Mann-Whitney U-test was used. To identify the reliability of differences in qualitative indicators used the criterion of angular transformation of Fisher [Guidelines for conducting preclinical studies of drugs. Ed. Mironova A.N., Bunatyan N.D. and others. M., CJSC Grif and K. 2012].

Determination of acute toxicity of 4-(3,4-dibromothiophenecarbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12-hexaazaisowurtzitane (I).

When determining acute toxicity, 4-(3,4-dibromothiophenecarbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12-hexaazaisowurtzitane (I) was administered to 7 outbred female mice and 7 to outbred male mice, CD1 stock once intragastrically at a maximum dose of 2000 mg/kg (due to poor water solubility of 6.25 g/l) in a volume of 0.5 ml of a water-tween solution. Within 2 weeks of observation, the death of animals was not observed, the mice had no deviations in the state of health and behavior, and pathological reflexes were not recorded. Due to the impossibility of determining the LD50, the test substance (I) is assigned to the 3rd hazard class - “low-toxic agents” in accordance with GOST 12.1007-76 “Harmful substances. Classification and general safety requirements” and “Guidelines for conducting preclinical trials of medicinal products, 2012” [Guidelines for conducting preclinical trials of medicinal products. Ed. Mironova A.N., Bunatyan N.D. and others. M., CJSC Grif and K. 2012].

To assess antinociceptive activity in the conditions of the "Vinegar writhing" model, the "Mechanical compression of the paw" test (Rendal-Selitto), in the "Hot plate" thermal stimulation test of central genesis [Guidelines for conducting preclinical studies of drugs. Ed. Mironova A.N., Bunatyan N.D. and others. M., CJSC Grif and K. 2012; Barrot M. Tests and models of nociception and pain in rodents. neuro-science. 2012, 211: 39-50] the test substance (I) was intragastrically administered to mice through a tube in the dose range of 25, 50, 100 and 200 mg/kg once daily and for 3 days. The used daily dose of the reference drug Tramadol for mice (10 mg/kg, solvent - purified water) corresponded to the average daily dose for humans (according to the instructions for the use of Tramadol). Animals of the negative control group received the solvent (purified water) in a similar mode and route of administration. The body weight values of the animals were used to calculate the doses of the test substances. The selection of a certain amount of the test agent was carried out in a working room with a bactericidal recirculator, which ensures the safety of procedures and the cleanliness of the preparation of test substances for administration. The required amount of substances for injections was prepared by the laboratory employee responsible for pharmaceutical work. The preparation of doses was based on the values of the doses and the indicated volume of administration. Euthanasia of the animals at the end of the experiments was carried out by cranio-cervical dislocation.

Study of the analgesic activity of 4-(3,4-dibromothiophenecarbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12-hexaazaisowurtzitane (I) in comparison with the reference drug Tramadol in the test "Vinegar cramps" (Abdominal constriction test).

The test "Vinegar writhing" (Abdominal constriction test) is aimed at the study of acute visceral and somatic deep pain [Guidelines for conducting preclinical studies of drugs. Ed. Mironova A.N., Bunatyan N.D. and others. M., CJSC Grif and K. 2012; Barrot M. Tests and models of nociception and pain in rodents. neuroscience. 2012, 211: 39-50]. A specific pain reaction - "writhing" (characteristic movements of animals, including contractions of the abdominal muscles, alternating with their relaxation, stretching of the hind limbs and arching of the back) - was induced by intraperitoneal injection of 0.75% acetic acid solution (GOST 61-75) to mice in dose of 0.1 ml/10 g of body weight. The analgesic effect was assessed by the ability of a potential drug (within 15 minutes after injection) to reduce (in%) the number of "writhing" in comparison with the control group of animals (the criterion of effectiveness is a decrease in pain response by at least 50%) and the latent time of onset of pain reactions. In this series of experiments, the following groups of outbred male stock CD1 mice were used:

1. Negative control - mice were injected with purified water in a volume of 0.2 ml / 20 g; .

2. Positive control - mice were injected with Tramadol at a dose of 10 mg / kg in a volume of purified water 0.2 ml / 20 g mouse intragastrically through a tube 1 hour before intraperitoneal injection of 0.75% acetic acid in a volume of 0.1 ml / 10 g (n=9).

3. Analgesic - mice were injected with (I) at a dose of 25 mg/kg in a solvent volume of 0.2 ml/20 g mouse intragastrically through a tube 1 hour before intraperitoneal injection of 0.75% acetic acid solution in a volume of 0.1 ml/ 10 g (n=10).

4. Analgesic - mice were injected (I) at a dose of 50 mg/kg in a solvent volume of 0.2 ml/20 g mouse once intragastrically through a tube 1 hour before intraperitoneal injection of 0.75% acetic acid solution in a volume of OD ml/10 g (n=10).

5. Analgesic - mice were injected with (I) at a dose of 100 mg/kg in a solvent volume of 0.2 ml/20 g mouse once intragastrically through a probe 1 hour before intraperitoneal injection of 0.75% acetic acid solution in a volume of 0.1 ml/ 10 g (n=10).

6. Analgesic - mice were injected with (I) at a dose of 200 mg/kg in a volume of solvent 0.2 ml/20 g mouse once intragastrically through a tube 1 hour before injection of a 0.75% solution of acetic acid in a volume of 0.1 ml/10 g (n=10).

The average number of acetic "writhings" 22.8±2.0 over a 15-minute observation period, the latent time of their onset 191.7±23.2 s indicates the development of a pronounced visceral pain reaction in animals of the negative control group after the introduction of a chemical stimulus (table one).

A single administration of Tramadol to mice at a therapeutic dose of 10 mg/kg stopped the pain response to 57.5%, since the number of "writhing" decreased by 2.4 (P<0.01) times compared with the corresponding indicator of the negative control group (table 1) . The latent time for the development of a pain reaction increased statistically significantly by 1.7 times relative to that of the negative control group. The obtained results of the reference control correspond to the literature data.

The dose of 25 mg/kg was ineffective (3.5%) with a single use (I) 1 hour after administration in this model (table 1).

In the case of the introduction of (I) at a dose of 50 mg/kg, its analgesic effect was noted (30.5%), which was expressed in a 1.4-fold decrease in the number of "writhings", an increase in the latent time of pain response by 2.0 times (P <0.05) compared to the corresponding negative control value (Table 1). It should be noted the predominant value of this indicator relative to the similar value of the Tramadol group (P<0.05).

A further increase in the dose of a potential analgesic to 100 mg/kg led to an increase in the antinociceptive effect. But, if as a result of the introduction of (I) at a dose of 100 mg/kg, the analgesic activity was 58.8% (P<0.01), then an increase in the dose to 200 mg/kg did not contribute to an increase in efficiency, since the analgesic activity turned out to be 50.9 % (P<0.01).

Thus, the test substance 4-(3,4-dibromothiophenecarbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12-hexaazaisowurtzitane (I) in the "Acetic writhing" test ( Abdominal constriction test) shows a pronounced analgesic activity at doses of 100 and 200 mg / kg, comparable to Tramadol. The decrease in pain response by more than 50% under the conditions of the "Vinegar writhing" model, which correlates with the clinical situation of acute peritonitis, indicates a high potential of the new molecule as an analgesic agent.

Study of the analgesic activity of 4-(3,4-dibromothiophenecarbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12-hexaazaisowurtzitane (I) in comparison with the reference drug Tramadol in the test "Mechanical compression of the paw" (Randall-Selitto test).

To study the analgesic activity of the 4-(3,4-dibromothiophenecarbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12 hexaazaisowurtzitane (I) molecule in the “Mechanical paw compression” test, we used analgesimeter (Ugo Basile, Italy), which is designed to perform accurate testing of analgesics on normal and inflamed paws of rats and mice according to the Randall-Selitto method [Guidelines for conducting preclinical studies of drugs. Ed. Mironova A.N., Bunatyan N.D. and others. M., CJSC Grif and K. 2012; Barrot M. Tests and models of nociception and pain in rodents. neuroscience. 2012, 211: 39-50]. With increasing pressure on the limb, we successively observed: the spinal limb withdrawal reflex, a complex of supraspinal motor reactions of the animal aimed at releasing the limb, and vocalization. The threshold of vocalization occurrence (weight in grams), the latent time of the pain reaction, and the number of animals without pain reaction were recorded. The criterion for the analgesic effect was a significant decrease in the intensity of pain reactions, estimated in grams by the strength of the effect on a healthy paw, the latent time of the pain reaction, the number of animals without pain reaction in dynamics (in 1 after the administration of the drugs) after a single and course daily three-day administration.

In two series of experiments (series 1 - single injection; series 2 - course administration for 3 days), the following groups of outbred male stock CD1 mice were used:

1. Negative control - mice were injected with purified water in a volume of 0.2 ml / 20 g mouse intragastrically through a tube once and daily for three days, on the first and last day of the experiment, the administration was carried out 1 hour before the fixation of vocalization, the latent time of the pain reaction, number of animals without pain reaction (n=11);

2. Positive control - mice were injected with Tramadol at a dose of 10 mg / kg in a volume of purified water 0.2 ml / 20 g mouse intragastrically through a tube once and daily for three days, on the first and last day of the experiment, the administration was carried out 1 hour before fixation vocalization, latent time of pain reaction, number of animals without pain reaction (n=l 1);

3. Analgesic - mice were injected with (I) at a dose of 25 mg/kg in a solvent volume of 0.2 ml/20 g mice intragastrically through a tube once and for three days, on the first and last day of the experiment, the administration was carried out 1 hour before the fixation of vocalization , latent time of pain reaction, number of animals without pain reaction (n=11);

4. Analgesic - mice were injected with (I) at a dose of 50 mg/kg in a solvent volume of 0.2 ml/20 g mouse intragastrically through a tube once and for three days, on the first and last day of the experiment, the administration was carried out 1 hour before the fixation of vocalization , latent time of pain reaction, number of animals without pain reaction (n=10);

5. Analgesic - mice were injected with (I) at a dose of 100 mg/kg in a solvent volume of 0.2 ml/20 g mouse intragastrically through a tube once and for three days, on the first and last day of the experiment, the administration was carried out 1 hour before the fixation of vocalization , latent time of pain reaction, number of animals without pain reaction (n=10);

6. Analgesic - mice were injected with (I) at a dose of 200 mg/kg in a solvent volume of 0.2 ml/20 g mouse intragastrically through a tube once and for three days, on the first and last day of the experiment, the administration was carried out 1 hour before the fixation of vocalization , latent time of pain reaction, number of animals without pain reaction (n=10).

On the model of "mechanical hyperalgesia" according to Randall-Selitto, the pain threshold (g) was on the first (258.5±57.9) and (184.9±59.8) third days of observation in animals of the negative control group (Table 2, 3). There was a decrease in the latent time of pain response from 5.0±1.4 s (1 day) to 3.2±1.4 s (3 days) with the same number of animals with the maximum exposure (9.1%). The decrease in the studied control parameters is typical for this experimental model with repeated use of animals.

The reference drug Tramadol increased the threshold of pain sensitivity with a single application by 2.1 times (P<0.05), by 2.5 times (P<0.05) - with a three-time administration relative to the corresponding indicators of the negative control (table 2, 3 ). It should be noted an increase in the 1-day observation of the pain response time indicator by 2.1 times (P<0.05) and the number of mice without pain reaction up to 54.5% (P<0.01) relative to similar values of the negative control. A similar picture was recorded in the subsequent observation period in the Tramadol group: the pain threshold increased by 2.5 times (P<0.01), the latent time of the pain response increased by 2.9 times (P<0.01), while the number of mice with 15 exposures turned out to be the same (Table 2, 3).

The maximum analgesic activity (I) in the first observation period was observed in the 25 mg/kg dose group: an increase in pain threshold - 1.9 times (P<0.05), an increase in latent time - 2 times (P<0.05 ) relative to the corresponding values of the negative control group, while the number of mice with the maximum exposure was 54.5% (P<0.01) versus 9.1% of the negative control (table 2, 3). Three times the use of the compound at a dose of 25 mg/kg led to a statistically significant increase in its antinociceptive action, as evidenced by all the parameters studied. There was an increase in the threshold of pain sensitivity by 2.9 times (P<0.01), latent time - by 3.7 times with the same number of mice from 15 with an exposure of 54.5% (P<0.01). There was no statistically significant difference between all parameters of the 25 mg/kg group and those of the Tramadol group at both follow-up periods.

In animals treated with substance (I) at doses of 50 and 100 mg/kg with a single application, there was a tendency to increase the threshold of pain sensitivity by 1.7 times, the time of onset of pain reaction - by 1.8 times relative to the corresponding values of the negative control, respectively. However, a statistically significant increase in the number of animals without pain reaction in these groups was found, comparable with this indicator of the Tramadol group. Three times daily application of (I) at doses of 50 and 100 mg/kg resulted in a statistically significant antinociceptive effect. A statistically significant increase in the pain threshold by 2.3 and 2.6 times, latent pain response time - by 2.7 (P<0.05) and 3.1 times (P<0.05) was revealed. It should be noted a statistically significant number of mice without pain response (50%) only in the group of animals treated with 100 mg/kg. There was no statistically significant difference between all values of the 50 and 100 mg/kg groups and those of the Tramadol group after three doses.

When using a dose of 200 mg/kg (I) showed no analgesic effect.

Based on the totality of the results of the study of the pain sensitivity threshold, we can conclude that the activity of compound (I) is similar to Tramadol, which demonstrated statistically significant activity during all periods of observation, at a dose of 25 mg / kg - during all periods of observation, at doses of 50 and 100 mg / day. kg - after a daily three-fold injection.

Thus, as a result of the study of the analgesic activity of substance (I) in the test "Mechanical compression of the paw" (Randall-Selitto test) in outbred mice stock CD1 with a single and three-day application, a pronounced antinociceptive activity was demonstrated in the dose range of 25-100 mg/kg , comparable to the effectiveness of the reference drug Tramadol.

Study of the analgesic activity of 4-(3,4-dibromothiophenecarbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12-hexaazaisowurtzitane (I) in comparison with the reference drug Tramadol in the test studies of antinociceptive action on the model of pain of the central genesis "Hot plate".

The main standard operating procedure for measuring the threshold of pain sensitivity and the potential analgesic effect of the studied pharmacological drugs in response to thermal stimulation is the "Hot plate" test. The "Hot plate" test is the basis for the study of analgesic activity and is based on behavioral responses controlled by supraspinal structures in response to thermal pain exposure. In studies, the HOTPLATE ANALGESIA METER (Columbus Instruments, USA) was used to quickly and effectively test the level of analgesia in small laboratory animals. Mice were placed on a plate heated to 54.00±0.5°C. This temperature creates a feeling of discomfort without causing serious damage. The endpoints of this experiment were: licking the hind legs (head turned towards the hind leg, and the ventral surface angled up). At the same time, other behavioral reactions are ignored. If the animal does not show activity during the 30-s interval, then it is removed from the hot surface, and 30 s is taken as the bouncing latent time.

When processing the obtained results, the magnitude of the maximum possible effect (MPE) was calculated. % MVE=LP ₀ - LP _to /(30-LP _to ) × 100, where LP ₀ is the latent period of the nociceptive reaction in the experimental group; LPK - latent period of nociceptive reaction in the control group; 30 s is the maximum exposure time for mice. One of the criteria characterizing the analgesic activity of the test substance is the number of animals with the maximum manifestation of the analgesic effect - exposure for 30 s on the hot plate of the device.

Indicators of the latent period of the pain response of the negative control group and the number of animals with maximum exposure after 1 h (21.6±2.4 s; 36.4%) and 2 h (22.6±2.1 s; 36.4%) observations indicate a high threshold pain sensitivity of this batch of outbred CD1 stock male mice (Table 4).

The analysis of the values of the latent period and the number of animals with 30 exposures revealed a trend towards an increase 1 hour after the administration of Tramadol, (I) at doses of 25, 50, 200 mg/kg. During this observation period, the test compound (I) at a dose of 100 mg/kg showed a pronounced antinociceptive effect, which consisted in a statistically significant increase in the latent time for the development of a pain reaction, an increase in the number of mice from 30 with exposure (P<0.05) relative to the corresponding values of the negative control , as a result of which the most pronounced effect was 81% and statistically significantly exceeded this indicator of Tramadol (40.5%).

Thus, 4-(3,4-dibromothiophenecarbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12-hexaazaisowurtzitane at a dose of 100 mg/kg 1 hour after injection demonstrated efficiency superior to the activity of Tramadol.

2 hours after administration, in the group of animals treated with Tramadol, a statistically significant increase in latent time was revealed, an increase in the number of animals from 30 with exposure (P<0.05), resulting in the most pronounced effect of 58%o. The result obtained corresponds to the literature data (Table 4).

In the second observation period, compound (I) showed the most pronounced effect (67.6%) at the minimum used dose of 25 mg/kg. At the same time, there was a statistically significant increase in latent time, the number of animals without pain - up to 80%) (2 hours, P<0.05) versus 40% (1 hour). It should be noted the comparable results of the 25 mg/kg group and the group of animals treated with the reference drug.

Analysis of the results of a study of the analgesic activity of 4-(3,4-dibromothiophenecarbonyl)-2,6,8D0,12-pentaacetyl-2,4,6,8,10,12 hexaazaisowurtzitane (I) in comparison with the reference drug Tramadol in tests of somatogenic pain of various origins "Acetic writhing" (Abdominal constriction test), "Mechanical compression of the paw" (Randall-Selitto test), thermal pain stimulation "Hot plate" allows us to conclude that the newly obtained compound (I) has a pronounced antinociceptive effect even after a single application, depending on the experimental situation in the dose range of 25-200 mg / kg, comparable and / or superior in a number of indicators to the activity of the reference drug Tramadol.

The data obtained on the pronounced antinociceptive effect of low-toxic 4-(3,4-dibromothiophenecarbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12 hexaazaisowurtzitane (I) allow us to conclude that the development is promising analgesic agent based on an innovative compound for the relief of moderate and high intensity of pain of various origins and, to a certain extent, to predict the nature of its analgesic effect in humans.

Claims (2)

1. 4-(3,4-dibromothiophenecarbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12-hexaazaisowurtzitane (I), which has analgesic activity. 2. The method for producing 4-(3,4-dibromothiophenecarbonyl)-2,6,8,10,12-pentaacetyl-2,4,6,8,10,12-hexaazaisowurtzitane according to claim 1, which consists in acylation of 4-( 3,4-dibromothiophenecarbonyl)-2,6,8,12-tetraacetyl-2,4,6,8,10,12-hexaazaisowurtzitane with acetyl chloride.