RU2443704C1 - 2-ARYL-4-DIALKYLAMINOETHYL-3-PHENYLPYRROLO[1,2-a]-BENZIMIDAZOLE SULPHATES, HAVING ANTIOXIDANT AND ANTIRADICAL PROPERTIES - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to novel pyrrolo-[1,2-a]benzimidazole derivatives of formula I

, where NR₂ assumes morpholino or diethylamino values, and Ar is 4-methoxyphenyl or 4-chlorophenyl, having antioxidant and antiradical properties.

EFFECT: obtaining novel sulphates possessing useful biological properties.

2 tbl, 5 ex

Description

The invention relates to new chemical compounds of a series of pyrrolo- [1,2-a] benzimidazole, namely to sulfates of 2-aryl-4-dialkylaminoethyl-3-phenylpyrrolo [1,2-a] benzimidazoles of the general formula I:

where NR ₂ takes the values of morpholino or diethylamino, and Ar - 4-methoxyphenyl or 4-chlorophenyl, which have antioxidant and antiradical properties and can find application in medicine.

Free radical oxidation plays an important role both in the normal functioning of cells and in the development of pathological processes. Antioxidant treatment is widely used in the pathology of the cardiovascular system (A.P. Golikov, S.A. Boytsov and others. Free radical oxidation and cardiovascular pathology: correction by antioxidants. Attending physician: Journal for the Practitioner, 2003, No. 4, p.70-74; O.N. Kovaleva, A.N. Belovol, M.V. Zaika. The role of oxidative stress in cardiovascular pathology. Journal of the Academy of Medical Sciences of Ukraine, 2005, v.11, No. 4, p.660-670) , neurological diseases (Y. A. Zozulya, V. A. Baraboy, D. A. Sutkova. Free radical oxidation and antioxidant protection in patol gii of the brain. M: Knowledge-M, 2000, 344 pp .; A.N. Dzyuba, Yu.N. Sorokin. Pro- and antioxidant mechanisms in multiple sclerosis. Ukrainian Medical Chronicle., 2004, No. 5 (43) , p.30-35), diabetes mellitus (M.I. Balabolkin, E.M. Klebanova, V.M. Kreminskaya. Treatment of diabetes and its complications. M: Medicine, 2005, 512 p .; T.P Bardymova, LI Kolesnikova, MI Dolgikh Lipid peroxidation, antioxidant system in patients with diabetes mellitus and environmental factors. Bulletin of the All-Russian Scientific Research Center SB RAMS, 2006, No. 1 (47), pp. 116-119), pancreatitis (V. G. Artyukhov, Yu. N. Chernov and others. The effect of the quamate on the properties of neutrophils, lipid peroxidation and antioxidant protection in of blood of patients with duodenal ulcer and pancreatitis. Experimental and Clinical Pharmacology, 2003, v.66, No. 6, p. 59-63), irradiation (Yu.N. Shanin, V.Yu. Shanin, E.V. Zinoviev. Antioxidant therapy in clinical practice (theoretical basis and implementation strategy). St. Petersburg: ELBI-St. Petersburg, 2003, 128 pp.), Etc.

Antioxidants by their origin are divided into two main groups: natural and synthetic (N.V. Vereshchagin, M.M. Tanashyan and others. Antioxidants in angioneurology. Atmosphere A. Nervous diseases, 2004, No. 3, pp. 8-12). The most commonly used natural antioxidants are α-tocopherol (vitamin E) and ascorbic acid (vitamin C) (A.I. Fedin, Oxidative stress and the use of antioxidants in neurology. Atmosphere A. Nervous diseases, 2002, No. 1, pp. 15-18 ) α-Tocopherol in the body performs several functions that together give an antioxidant effect (V.V. Lyakhovich, V. A. Vavilin and others. Activated oxygen metabolites in monooxidase reactions. Bulletin of RAMS, 2005, No. 4 (118), p. 7-12). However, the action of the fat-soluble vitamin is somewhat delayed in time, due to the need to incorporate it into the structure of the cell membrane (N.V. Vereshchagin et al., 2004). In addition, α-tocopherol used in clinical practice can be administered only orally or intramuscularly (but not intravenously). These circumstances significantly limit the possibilities of using the drug in intensive care practice.

The water-soluble antioxidant ascorbic acid reduces the level of superoxide anion radical, singlet oxygen, hydroxyl, peroxide and some organic radicals, hypogaloids, and also restores the oxidized forms of vitamin E and glutathione, restoring their antioxidant properties (Yu.N. Shanin et al., 2003). However, in some cases (the use of high doses, the lack of α-tocopherol and glutathione), the drug is able to induce the generation of reactive oxygen species (ROS) and promotes free radical reactions (M.I. Balabolkin et al., 2005).

Currently, synthetic antioxidant drugs are widely used in clinical practice (A.I. Fedin, 2002). One of them is dibunol (ionol, 2,6-di-tert-butyl-4-methylphenol) - a fat-soluble drug belonging to the class of screened phenols. By its antioxidant properties, dibunol is similar to α-tocopherol, although the antiradical activity of the drug is lower than that of vitamin E [Yu.N. Shanin et al., 2003]. Dibunol was first proposed for topical use in the treatment of cancer and bladder papillomatosis. At present, it is also used for cystitis, burns, frostbite, long-term non-healing trophic and radiation ulcers, sluggishly granulating wounds.

The synthetic water-soluble antioxidant Mexidol (2-ethyl-6-methyl-3-hydroxypyridine succinate) is currently widely used. It is an inhibitor of free radical processes, increases the activity of antioxidant enzymes, improves energy metabolism in the cell and protects the membrane structure (V.A. Kraineva, Features and mechanism of neuroprotective action of the drug mexidol in hemorrhagic stroke in the experiment. Bull. Experiment. Biology and medicine, 2006, Appendix 1, p.40-44). Due to its mechanism of action, Mexidol is an effective means of anti-ischemic brain protection (T. Voronina. Domestic drug of the new generation Mexidol (main effects, mechanism of action, use), M., 2005, 20 s). However, as a result of experimental studies, it was found that the antioxidant activity of the drug is still low (Kosolapov, V.A. Antioxidant agents: selection strategy, application prospects: Diss ... Dr. med. Sciences, Volgograd, 2005. - 303 s).

Thus, it can be assumed that the antihypoxic and anti-ischemic actions of Mexidol are due not so much to its antioxidant properties as to its constituent succinate, which, under conditions of hypoxia and ischemia, entering the intracellular space is capable of being oxidized by the respiratory chain.

It should be noted that, despite the presence of a wide range of drugs to suppress free radical oxidation reactions, the arsenal of antioxidant agents used in modern medicine is quite limited. In addition, problems associated with their use are described, in particular, the impossibility of prescribing for emergency care (vitamin E), a narrow range of indications (dibunol), insufficient antioxidant activity (mexidol), etc.

In this regard, the search for new compounds with antioxidant properties, on the basis of which drugs can be created for the treatment of diseases accompanied by excessive activation of peroxidation processes, is very important.

Among the derivatives of tricyclic benzimidazole systems with a bridging nitrogen atom, the most π-redundancy and, consequently, pyrrolo [1,2-a] benzimidazole derivatives (V.A. Anisimova, N.I. Avdyunina, A.F., have the ability to inhibit free radical processes Pozharsky et al., Chemistry of heterocycle compounds, 1980, No. 4, pp. 528-537; A.F. Pozharsky. Theoretical foundations of the chemistry of heterocycle. M., Chemistry, 1985, p. 528-537).

Considering the above, work was carried out to identify antioxidant and antiradical properties in new compounds related to pyrrolo [1,2-a] benzimidazole derivatives.

The closest in structure to the claimed compounds are dihydrochlorides 2- (4-methoxyphenyl) -4-morpholinoethyl-3-phenyl- (IIa) and 4- (2-diethylaminoethyl) -2- (4-chlorophenyl) -3-phenylpyrrolo [1 , 2-a] benzimidazoles (IIb), exhibiting antioxidant activity (V.A. Anisimova, A.A. Spasov and others. Synthesis and pharmacological activity of salts of 4- (2-dialkylaminoethyl) pyrrolo [1,2-a] benzimidazoles. Chem.-Pharm. Journal, 1996, T. 30. No. 1, pp. 22-25). However, their activity is still not high enough.

The objective of the invention is the identification of antioxidant and antiradical properties in new derivatives of pyrrolo [1,2-a] benzimidazole.

The technical result of the invention is new compounds in the pyrrolo [1,2-a] benzimidazole series, which have high antioxidant activity exceeding the activity of drugs used in medical practice (ionol, mexidol) and known compounds of the above series IIa, b, and also exhibit antiradical properties.

The technical result is achieved by sulfates of 2-aryl-4-dialkylamino-ethyl-3-phenylpyrrolo [1,2-a] benzimidazoles of the general formula I.

The synthesis of compounds I consists in cyclization in an alkaline medium of 2-benzyl-3-aroylmethyl-1- (2-dialkylaminoethyl) benzimidazolium bromides obtained by the action of 4-substituted phenacylbromides on 2-benzyl-1-dialkylaminoethylbenzimidazole in acetone or acetonitrile, and the subsequent translation of the obtained tricyclic bases into sulfates using an acetone solution of sulfuric acid:

where NR ₂ and Ar take the above values.

The following are examples of synthesis methods and studies of the biological activity of the proposed compounds.

EXAMPLE 1 2- (4-Methoxyphenyl-4- (2-morpholinoethyl) -3-phenyl-pyrrolo [1,2-a] benzimidazole (Ia) sulfate Solution 3.21 g (10 mmol) 2-benzyl-1 - (2-morpholinoethyl) benzimidazole (V.A. Anisimova et al. Chemical-Pharmaceutical Journal, 1996, v.30, No. 1, p.22-26) and 2.3 g (10 mmol) of 4-methoxyphenacyl bromide (V.A. Anisimova et al. Chem.-Pharm. Journal, 2005, T.39, No. 9, P.26-32) in 30 ml of acetone or acetonitrile is boiled for 5-10 minutes and left for a day at room temperature. The precipitated snow-white precipitate of 2-benzyl-3- (4-methoxyphenacyl) -1- (2-morpholinoethyl) benzimidazolium bromide is filtered off, washed with hot acetone. 4.6 g (88.6%) yield. , mp 209-210 ° C (decomp., from EtOH). Found,%: C 63.2; H 5.9; Br 14.2; N 7.8. C ₂₈ H ₃₂ BrN ₃ O ₃ Calculated,%: C 63.3; H 5.9; Br 14.5; N 7.6. IR spectrum (petrolatum, oil), ν, cm ^-1 : 1695 (C = O).

2.2 g (4 mmol) of the obtained bromide and 0.7 g (8.5 mmol) of NaHCO _{3 are} boiled in inert gas and in 50 ml of water until the reaction proceeds (control - TLC). After cooling without access to air, the light yellow oil released as a result of the reaction solidifies. It is separated and recrystallized from isooctane to give a base of 2- (4-methoxyphenyl) -4- (2-morpholinoethyl) -3-phenylpyrrolo [1,2-a] benzimidazole as slightly beige, highly electrifying crystals, so pl. 63-64 ° C. Yield 1.73 g (96%). Found,%: C 77.0; H 6.6; N, 9.6. C ₂₉ H ₂₉ N ₃ O ₂ . Calculated,%: C 77.1; H 6.5; N, 9.5. IR spectrum (petrolatum, oil), ν, cm ^-1 : 1600, 1605, 1620 (C = C, C = N).

Sulfate Ia. A solution of 4 mmol of the obtained tricyclic base in 50 ml of acetone is acidified with a solution of conc. H ₂ SO ₄ in acetone to a pH of 1-2. The precipitated salt precipitate is filtered off, washed thoroughly on the filter with acetone (3 × 10 ml), recrystallized from ~ 80% aqueous ethyl alcohol, and dried in a vacuum desiccator. Slightly greenish crystals with so pl. 213-215 ° C (decomp., When a capillary is introduced into a device for determining the melting point heated to 195 ° C). Yield 2.0 g (91%). Found,%: C 63.3; H 5.7; N, 7.8; S 5.7. C ₂₉ H ₂₉ N ₃ O ₂ · H ₂ SO ₄ . Calculated,%: C 63.4; H 5.7; N, 7.6; S 5.8.

Example 2. 4- (2-Diethylaminoethyl) -3-phenyl-2- (4-chlorophenyl) pyrrolo [1,2-a] benzimidazole sulfate (IB). A mixture of 3.1 g (10 mmol) of 2-benzyl-1- (2-diethylaminoethyl) benzimidazole (V.A. Anisimova et al. Chem.-Pharm. Journal, 1996, vol. 30, No. 1, p.22- 26) and 2.34 g (10 mmol) of 4-chloroacetophenone in 20 ml of acetone are boiled for 2 hours. A heavy precipitate of 2-benzyl-1- (2-diethylaminoethyl) -3- (4-chlorophenacyl) benzimidazole bromide is filtered off after cooling, washed with acetone. Yield 4.75 g (87.8%), mp. 167-168 ° C (decomp., From EtOH with ether). Found,%: C 62.0; H 5.9; N, 8.0.

C ₂₈ H ₃₁ BrClN ₃ O. Calculated,%: C 62.2; H 5.8; N, 7.8. IR spectrum (petrolatum, oil), ν, cm ^-1 : 1700 (C = O).

A mixture of 2.72 g (5 mmol) of the resulting bromide, 0.84 g (10 mmol) of sodium bicarbonate and 50 ml of water is boiled in an inert atmosphere until the reaction is complete (control - TLC, 1-1.5 h), with a precipitate salt gradually turns into a light yellow oil. At the end of the reaction, the flask is closed to prevent air from entering it and the mixture is allowed to cool. The next day, the hardened oil is separated and, thoroughly grinding, dried in a desiccator over P ₂ O ₅ . Recrystallization from benzene gives 4- (2-diethylaminoethyl) -3-phenyl-2- (4-chlorophenyl) pyrrolo [1,2-a] benzimidazole in the form of white or slightly green crystals with mp. 82-84 ° C. Yield 2.05 g (93%). Found,%: C 76.0; H 6.2; Cl 8.2; N, 9.8. C ₂₈ H ₂₈ ClN ₃ . Calculated,%: C 76.1; H 6.4; Cl 8.0; N, 9.5. IR spectrum (petrolatum. Oil), ν, cm ^-1 : 1595, 1605, 1618 (C = C, C = N).

Sulfate Ib. Obtained analogously to sulfate Ia described in Example 1 with a yield of 89.5%. Mp 231-233 ° C (sect., When a capillary is introduced into a device for determining mp. Heated to 210 ° C). Found,%: C 62.4; H 5.7; Cl 6.5; N, 7.9; S 5.8. C ₂₈ H ₂₈ ClN ₃ · H ₂ SO ₄ . Calculated,%: C 62.3; H 5.6; Cl 6.6; N, 7.8; S 6.6.

Example 3. The study of the antioxidant activity of compounds Ia, b. The antioxidant activity of substances was studied in vitro on the model of ascorbate-dependent lipid peroxidation (LPO) of liver homogenates (V.Z. Lankin et al. Study of ascorbate-dependent lipid peroxidation of tissues using a 2-thiobarbituric acid test. Proceedings of the Moscow Society Naturalists. M., 1975, v. 52, pp. 73-78). Compounds were investigated in a concentration range of 0.1-100 μmol / L. The inhibitory concentration of IC _{50 was} calculated by the method of regression equations in the program Statistica 6.0 (StatSoft, USA).

As a substrate, 4% rat liver homogenate was used. The reaction was initiated with 50 mmol / L ascorbic acid. The rate of LPO was judged by the accumulation of malondialdehyde (a secondary LPO product) in reaction with TBA. The optical density of the colored product was measured at a wavelength of 532 nm on a PD-303 UV spectrophotometer (APEL, Japan) in a cuvette with an optical path length of 1 cm.

The activity of the compounds was expressed as a percentage and was calculated by the formula:

% inhibition = 100- (E _on / E _counter × 100),

where E _on is the optical density of the sample with the substance,

E _counter - the optical density of the sample containing no substance.

The comparison drugs were the antioxidants dibunol (Merck, Germany) and Mexidol (Research Institute of Pharmacology RAMS, Russia), as well as hydrochlorides IIa, b. All substances except dibunol were used in the form of aqueous solutions; an alcohol solution was prepared for dibunol.

As a result of the study, it was found that sulfates Ia, b at a concentration of 1 μmol / L inhibit the reaction of ascorbate-dependent LPO by 94.4 and 93.8%, respectively. Compound IIb has less pronounced antioxidant properties (60.4%), while dihydrochloride IIb turned out to be low activity (9.6%). Dibunol and Mexidol at the same concentration showed activity at the level of 38.2 and 1.5%, respectively.

In terms of IC _{50, the} studied compounds and comparison substances can be arranged in the following sequence in decreasing activity: Ia (0.32 μmol / L) = Ib (0.32 μmol / L)> IIb (1.20 μmol / L)> Dibunol ( 3.16 μmol / L)> Mexidol (480 μmol / L).

Example 4. The study of the antiradical properties of compounds Ia, b.

The antiradical properties were investigated on the model (G. Glavind Antioxidants in animal tissue // Acta Chemica Scand. - 1963. - Vol.17, No. 6. - P.1635-1640) interactions with the stable free radical 2,2-diphenyl-1- picrylhydrazyl (DPPH •). We used an alcohol solution of DPPH • at a concentration of 1 × 10 ^-5 mol / L with the addition of the studied antioxidant substances. The antiradical activity of the compounds was recorded by the decrease in optical density using a PD-303 UV spectrophotometer (APEL, Japan) at a wavelength of 517 nm in quartz cuvettes with an optical path length of 1 cm.

The activity of substances was expressed as a percentage and was calculated by the formula:

% inhibition = 100- (ΔD ₁ / ΔD ₂ × 100),

where ΔD ₁ is the change in optical density when adding substances in the studied concentration,

ΔD ₂ - change in optical density with the addition of pyrogallol in excess.

Measurements were taken 5 minutes after the start of the reaction. During the study of the antiradical properties of the compounds, it was found that at a concentration of 1 μmol / L, sulfates Ia and IB do not show activity against the stable DPPH free radical •, while dibunol (3.3%) and mexidol (4.3%) are low active. However, an increase in the concentration of the test compounds to 100 μmol / L is accompanied by complete discoloration of the DPPH • solution. Among the reference substances, under the same conditions, dibunol was the most effective (% inhibition of the reaction was 83.9).

According to the IC ₅₀ value, the studied compounds and comparison preparations can be arranged in the following sequence in decreasing activity: Ib (7.28 μmol / L)> Ia (9.83 μmol / L)> Dibunol (15.56 μmol / L)> Mexidol ( 20.94 μmol / L).

Example 5. Determination of acute toxicity and conditional therapeutic index of the studied compounds.

The acute toxicity of the compounds was determined on 70 white non-inbred male mice weighing 18-26 g contained in vivarium conditions (temperature 22-24 ° C, relative humidity 40-50%) with natural light on a standard diet (GOST R 50258-92 ) At the time of the study, the animals were healthy, without changes in behavior, appetite, sleep and wakefulness.

In the study of acute toxicity, substances were administered once intraperitoneally. Animals were observed for two weeks from the time of administration. To calculate the value of the toxicological index LD ₅₀ , the Lichfield-Wilcoxon method was used in accordance with the requirements and instructions of the Federal Service for Supervision in Healthcare and Social Development (Methodological guidelines for studying the general toxic effects of pharmacological substances. A guide for experimental (preclinical) study of new pharmacological substances, under general editorship of R.U. Khabriev, 2nd ed., M., Medicine, 2005, 832 pp.).

The conditional therapeutic index was calculated as the ratio of LD ₅₀ to IC ₅₀ .

In the study of acute toxicity, the characteristic signs of poisoning with compounds Ia, b were limited mobility, frequent shallow breathing, and a decrease in pain and tactile sensitivity. The immediate death of animals was preceded by phenomena of deep sedation, respiratory depression, lateral position with rare clonic convulsions.

Based on the obtained LD ₅₀ values, the studied sulfates Ia, b and comparison substances can be arranged in the following sequence in decreasing toxicity: Ib (261.9 mg / kg)> IIb (350 mg / kg)> IIa (438 mg / kg)> Mexidol (475 mg / kg)> Dibunol (490 mg / kg)> Ia (792 mg / kg).

Thus, compound Ia in terms of acute toxicity LD ₅₀ exceeds compound IB and all substances of comparison.

In accordance with the classification of toxicity of compounds (I.V. Berezovskaya. Classification of chemicals according to the parameters of acute toxicity for parenteral routes of administration. Chemical and Pharmaceutical Journal, 2003, v. 37, No. 3, p. 32-34) studied derivatives of pyrrolo [1,2-a] benzimidazole can be classified as moderately toxic (class 3).

The results of the calculation of the conditional therapeutic index of antioxidant activity of substances Ia and Ib are presented in Table 1.

Table 1 Indicators of antioxidant activity, acute toxicity and conditional therapeutic index of compounds Ia, Ib and comparison drugs No. Compound LD ₅₀ Ascorbate-dependent lipid peroxidation mg / kg Inhibition of the reaction at a concentration of 1 μmol / l,% IR ₅₀ , mcmol / l Conditional therapeutic index one. Ia 792 94.4 0.32 4517.5 2. IIa 438 ¹ 9.6 - - 3. Ib 262 93.8 0.32 1534.9 four. IIb 350 ¹ 60,4 1.2 ¹ 566.5 5. Dibunol 490 38,2 3.16 701.0 6. Mexidol 475 ² 7.5 480.0 3.9 Note: “-” - for low-active compounds, the IC ₅₀ value was not calculated; ¹ - in accordance with (Anisimova V.A. et al., 1996); ² - in accordance with (Voronina T.A., 2005).

From table 1 it can be seen that according to the conditional therapeutic index on the model of ascorbate-dependent lipid peroxidation, compounds Ia (4517.5) and Ib (1534.9) significantly exceed dihydrochloride IIb (566.5), as well as comparison drugs dibunol (701.0 ) and Mexidol (3.9).

The results of the calculation of the conditional therapeutic index of the antiradical activity of substances Ia, b are presented in Table 2.

table 2 Indicators of antiradical activity, acute toxicity and conditional therapeutic index of compounds Ia, Ib and comparison drugs No. Compound LD ₅₀ mg / kg Reaction with 2,2-diphenyl-1-picrylhydrazyl IR ₅₀ , mcmol / l Conditional therapeutic index one. Ia 792 9.83 146.6 2. Ib 262 7.28 66.7 3. Dibunol 490 15,56 142.5 four. Mexidol 475 ¹ 20.94 89.0 Note: ¹ - in accordance with (Voronina T.A., 2005).

From table 2 it can be seen that according to the conditional therapeutic index of antiradical activity obtained on the model of interaction with the stable DPPH free radical •, compound Ia (146.6) is superior to dibunol (142.5) and mexidol (89.0), and substance Ib (66.7) inferior to comparison drugs.

Thus, sulfates of 2-aryl-4-dialkylaminoethyl-3-phenyl-pyrrolo [1,2-a] benzimidazoles Ia, b are superior in the conventional therapeutic index of antioxidant activity of dihydrochlorides of the same bases, as well as comparison drugs dibunol and mexidol. According to the conditional therapeutic index of antiradical activity, compound Ia is superior to all comparison preparations. Given the above, the studied compounds, primarily substance Ia, can be recommended for further detailed study of the antioxidant and antiradical properties in vitro and in vivo models.

Claims (3)

1. Sulfates of 2-aryl-4-dialkylaminoethyl-3-phenylpyrrolo [1,2-a] benzimidazoles of the general formula I:

where NR ₂ is morpholino or diethylamino, and Ar is 4-methoxyphenyl or 4-chlorophenyl. 2. Sulfates of 2-aryl-4-dialkylaminoethyl-3-phenylpyrrolo [1,2-a] benzimidazoles according to claim 1, where NR ₂ = morpholino, Ar = C ₆ H ₄ OCH ₃ -4, with antioxidant and antiradical properties . 3. Sulfates of 2-aryl-4-dialkylaminoethyl-3-phenylpyrrolo [1,2-a] benzimidazoles according to claim 1, where NR ₂ = diethylamino, Ar = C ₆ H ₄ Cl-4, with antioxidant and antiradical properties.