RU2518740C1 - METHOD, INHIBITING Na+/H+-EXCHANGE, AND DIHYDROCHLORIDE OF 2-(3,4-METHYLENEDIOXYPHENYL)-9-MORPHOLINOETHYLIMIDAZO[1,2-a]BENZIMIDAZOLE - Google Patents

Abstract

FIELD: chemistry, pharmacology.

SUBSTANCE: invention relates to medications, capable of inhibiting Na+/H+-exchange (NHE-exchangers, NHE inhibitors).

EFFECT: increased efficiency of inhibitors.

3 cl, 1 tbl

Description

The invention relates to agents having the ability to inhibit Na ⁺ / H ⁺ exchange (NHE inhibitors).

Na ⁺ / H ⁺ exchangers (NHE) constitute a group of integral membrane proteins expressed in all tissues of the body that carry transmembrane ion exchange Na ⁺ to H ⁺ ions. Currently, nine different types of Na ⁺ / H ⁺ exchanger proteins have been cloned from mammalian tissues, and these isoforms are commonly referred to as NHE-1, 2, 3, 4, 5, 6, 7, 8, 9, respectively. They differ in amino acid composition, tissue specificity, and intracellular regulation mechanisms. NHE exchangers are involved in many complex physiological and pathological processes, including regulation of cell pH, cell migration, apoptosis, hypertrophy, damage associated with ischemia and reperfusion, protecting the cell from acidification of the cytoplasm, controlling cell volume [Malo ME, Fliegel L Physiological role and regulation of the Na ⁺ / H ⁺ exchanger./ Can. J. Physiol. Pharmacol - 2006. - No. 84 (11). - p.1081-1095], endothelial dysfunction, as well as diseases such as diabetes and its complications, heart failure, cerebrovascular accident, the development of malignant tumors [Fliegel L. Regulation of the Na ( ⁺ ) / H ( ⁺ ) exchanger in the healthy and diseased myocardium. / Expert. Opin. Ther. Targets. - 2009. - No. 13 (1). - p. 55-68; Karmazyn M.,

Kilic A., Javadov S. The role of NHE-1 in myocardial hypertrophy and remodeling. / J. Mol. Cell Cardiol. - 2008. - No. 44. p.647-653; Provost JJ, Wallert MA. Inside Out: Targeting NHE1 as an Intracellular and Extracellular Regulator of Cancer Progression. // Chem Biol Drug Des. - 2013. - No. 81 (1). - p.85-101].

Various groups of NHE inhibitors are known: amiloride and its derivatives (DMA, EIPA, MIBA and NMA), benzoylguanidine derivatives, such as HOE-642 (cariporide), its derivative HOE-694 and eniporide (EMD-85131), sabiporide (BIIB-722 ), bicyclic guanidine derivatives containing quinoline (MS 31038), indole (SM 20220, SM 20550, SMP-300), benzoxazinone (KB-R9032), dihydrobenzofuran (BMS 284640), tetrahydronaphthalene (T-162559), cycloheptapyridine (TY- 12533) and others [Masereel B., Pochet L., Laeckmann D. An overview of inhibitors of Na + / H + exchanger // Eur. J. Med. Chem. - 2003. - No. 38. - P.547-554].

Derivatives of benzimidazoles containing a non-cyclic guanidine group (benzimidazol-2-yl and benzimidazol-2-ylthiomethyl benzoylguanidine) capable of inhibiting Na ⁺ / H ⁺ exchange are known [Zhang R et al. Benzimidazol-2-yl or benzimidazol-2-ylthiomethyl benzoylguanidines as novel Na ⁺ / H ⁺ exchanger inhibitors, synthesis and protection against ischemic-reperfiision injury. / Bioorg. Med. Chem. Lett. - 2007. - No. 17. - p.2430-2433].

The closest to the achieved result is Zoniporide (CP-597396) - [1- (quinolin-5-yl) -5-cyclopropyl-1H-pyrazole-4-carbonyl] guanidine hydrochloride monohydrate [A. Guzman-Perez et al. Discovery of zoniporide: A potent and selective sodium-hydrogen exchanger type 1 (NHE-1) inhibitor with high aqueous solubility. / Bioorganic & Medicinal Chemistry Letters. - 2001. - No. 11 (6). - p.803-807]. In vitro studies on rabbit platelets have shown that this drug suppresses NHE-1-dependent platelet swelling (EC ₅₀ = 56 nM) [Tracey WR et al. Zoniporide: a potent and selective inhibitor of the human sodium-hydrogen exchanger isoform 1 (NHE-1) // Cardiovasc. Drug Rev. - 2003. - No. 21. - p.17-32].

However, its properties as an NHE inhibitor remain insufficiently effective.

The technical result is to increase the effectiveness of inhibitors of Na ⁺ / H ⁺ exchange (NHE inhibitor).

The technical result is achieved by using, as inhibitors of the Na ⁺ / H ⁺ exchange, tricyclic derivatives of imidazo [1,2-a] benzimidazole, of the general formula I:

where Ar = C ₆ H ₅ , C ₆ H ₄ OCH ₃ -4, C ₆ H ₄ OH-4, C ₆ H ₃ (O ₂ CH ₂ ) -3,4

NR ₂ = N (C ₂ H ₅ ) ₂ , N (CH ₂ CH ₂ ) ₂ O

including Ia Ar = C ₆ H ₅ ; NR ₂ = N (C ₂ H ₅ ) ₂

Ib Ar = C ₆ H ₅ ; NR ₂ = N (CH ₂ CH ₂ ) ₂ O

Ic Ar = C ₆ H ₄ OCH ₃ -4; NR ₂ = N (CH ₂ CH ₂ ) ₂ O

Id Ar = C ₆ H ₄ OH-4; NR ₂ = N (CH ₂ CH ₂ ) ₂ O

Ie Ar = C ₆ H ₃ (O ₂ CH ₂ ) -3.4; NR ₂ = N (CH ₂ CH ₂ ) ₂ O.

The compounds can be used, inter alia, for the manufacture of agents having the ability to inhibit Na ⁺ / H ⁺ exchange, as well as to inhibit Na ⁺ / H ⁺ exchange in living organisms.

Compound Ia is known to have antihypertensive and anti-inflammatory activities (A.M. Simonov, A.A. Belous, V.A. Anisimova, S.V. Ivanovskaya. Chemical and Pharmaceutical Journal, 1969, No. 1, p. 7- 10).

Compound Ib is known as exhibiting antidiabetic and antiplatelet action (VAAnisimova, MVLevchenko, TBKorochina, AASpasov, SGKovalev, GPDudchenko. Nouveaux derives du benzimidazole, leur precede de preparation et les pharmaceutical pharmiquesiques que les contiennent. Fr. Pat. 2691462 (1995); Bull., 95/23. EP 0571253).

Compound Ic is known to have the ability to inhibit cAMP PDE and reduce atrial excitability (V.A. Anisimova, A.A. Spasov, V.A. Kosolapov, A.F. Kucheryavenko, O.V. Ostrovsky, N.P. Larionov, R.E. Libinson, Pharmacological activity of 2-methoxyphenyl-substituted 9-dialkylaminoethylimidazo [1,2-a] benzimidazoles, Chemical Pharmaceutical Journal, 2005, vol. 39, No. 9, p. 26-32), as well as exhibiting properties of an antagonist of purine P ₂ Y ₁ receptors and antithrombotic activity [RF patent No. 2377990, IPC A61K 31/5377, 2006)

Compound Id exhibits antioxidant and antiplatelet activity, as well as a weak antiserotonin and antipurinergic effect (V.A. Anisimova, A.A. Spasov, V.A. Kosolapov, M.V. Chernikov, A.Yu. Stukovina, L.V. Eltsova, N.P. Larionov, R.E. Libinson, O.E.Vatolkina, Synthesis and biological activity of 9-dialkylaminoethyl-2-hydroxy (dioxi) phenylimidazo [1,2-a] benzimidazoles. Chemical Pharmaceutical Journal , 2006, vol. 40, No. 10, pp. 23-26).

The technical result is also new compounds in the imidazo [1,2-a] benzimidazole series, which have the property to inhibit Na ⁺ / H ⁺ exchange.

The technical result is achieved by dihydrochloride of 2- (3,4-methylenedioxyphenyl) -9-morpholinoethylimidazo [1,2-a] benzimidazole of the formula Ie:

The synthesis of 2- (3,4-methylenedioxyphenyl) -9-morpholinoethylimidazo [1,2-a] benzimidazole dihydrochloride consists in the reaction of 1-morpholinoethyl-2-aminobenzimidazole with 3,4-methylenedioxyphenacyl bromide and further cyclization of the intermediate 1 morpholinoethyl-2-amino-3- (3,4-methylenedioxyphenacyl) benzimidazolium (without isolation of the latter) to a tricyclic base, which is converted by conventional means into the dihydrochloride Ie.

Example. Obtaining dihydrochloride Ie.

2.43 g (10 mmol) of 3,4-methylenedioxyphenacyl bromide are added to a solution of 2.46 g (10 mmol) of 1-morpholinoethyl-2-aminobenzimidazole in acetone. The mixture is stirred. The solvent from the reaction mixture with the precipitated precipitate of 1-morpholinoethyl-2-amino-3- (3,4-methylenedioxyphenacyl) benzimidazolium bromide was evaporated, 120 ml of water was added to the residue, and the mixture was boiled until the reaction was complete. The solution was cooled, basified and the precipitated base - 2- (3,4-methylenedioxyphenyl) -9- (2-morpholino-ethyl) imidazo [1,2-a] benzimidazole, was extracted with chloroform. The extract is purified on a chromatographic column. The eluate was evaporated, the residue was dissolved in acetone and acidified with HCl. The precipitated salt is filtered off, washed with acetone and dried. Yield 92.5-94.3%. Mp 183-184 ° C (decomposition).

Found,%: C 57.18; H 5.29; Cl 15.5; N, 12.17.

C ₂₂ H ₂₂ N ₄ O ₃ · 2HCl.

Calculated,%: C 57.03; H 5.22; Cl 15.30; N, 12.09.

IR spectrum, λ _max , cm ^-1 : 1660 (C = N), 2360-2650 (wide band N ⁺ H).

¹ H NMR spectrum (DMSO-d ₆ ), δ, ppm: 3.37-3.56 m (4H, N (CH ₂ ) ₂ ); 3.71 t (2H, J 6.5 Hz, CH ₂ ); 3.80-4.00 m (4H, O (CH ₂ ) ₂ ); 5.02 t (2H, J 6.5 Hz, CH ₂ ); 6.10 s (2H, OCH ₂ O); 7.06 d (1H, J 9.0 Hz, H _Ar ), 7.39-7.58 m (4H, H _Ar ), 7.90-8.00 m (2H, H _Ar ), 8.48 s (1H, H ³ _Ar ), 2H ⁺ in exchange.

Below are the results of testing the properties of the compounds as inhibitors of Na ⁺ / H ⁺ exchange.

1. Materials and methods.

1.1. Study design

Stage 1 - study of the effect of compounds on the activity of rabbit platelet Na ⁺ / H ⁺ -exchange. Calculation of IC ₅₀ .

Stage 2 - the study of acute toxicity.

1.2. Materials

Compounds Ia-e, zoniporide (SIGMA, USA).

Reagents: sodium propionate (MP Biomedicals, Inc., France), HEPES (GERBU, Germany), calcium chloride 2-water (dihydrate) CaCI ₂ · 2H ₂ O (LLC AGAT-MED, Russia), magnesium chloride 6-water “h” MgCI ₂ · 6H ₂ O (ZAO “UNIHIM”, Russia), D-glucose (anhydrous) crystalline “h” C ₆ H ₁₂ O ₆ (LLC “AGAT-MED”, Russia), sodium chloride “ChCH” NaCl (ZAO NPO ECROS, Russia), potassium chloride “ChDA” KCl (LLC “REAHIM”, Russia), potassium phosphate monosubstituted KH ₂ PO ₄ “CHDA” (OJSC “Petersburg Red Chemist”, Russia), magnesium sulfite 7-aqueous MgSO ₄ · 7H ₂ O (LLC "Reahim", Russia), sodium hydrogencarbonate NaHCO ₃ (REACHIM LLC, Russia), ethylenediaminetetraacetic acid (EDTA) (MP Biomedicals, Inc., France).

1.3. Animals.

In the experiments, male rabbits were used, weighing 2.5-3.5 kg, which were kept under vivarium conditions with natural light conditions on a standard diet of laboratory animals in accordance with GOST R 50258-92 [1993]. The study was carried out in accordance with the applicable requirements of GOST ISO / IEC 17025-2009, GOST R ISO 5725-2002 and the "Laboratory Practice Rules", approved by order of the Ministry of Health and Social Development of the Russian Federation of August 23, 2010 No. 708n, in compliance with the "European Convention for the Protection of Vertebrate Animals Used for experiments or other scientific purposes ”[Directive 2010/63 / EU of the European Parliament and of the Council of September 22, 2010 on the protection of animals used for scientific purposes Official Journal L 276, 10.20.2010 p.33-79 ( revising Directive 86/609 / EEC)].

1.4. Methods

The study The study of the effect of compounds on the activity of rabbit platelet Na ⁺ / H ⁺ exchange was carried out in vitro on rabbit platelets (according to the method of Rosskopf D. et. Al., Rapid determination of the elevated Na ⁺ / H ⁺ exchanger in platelets of patients with essential hypertension using an optical swelling assay // Journal of Hypertension. - 1991. - No. 9. - p. 231-238; Kusumoto K. et al., In vitro and in vivo pharmacology of a structurally novel Na ⁺ / H ⁺ exchanger inhibitor, T-162559 // British Journal of Pharmacology. - 2002. - No. 135. - p.1995-2003).

Rabbit platelets, on the membrane of which NHE-1 are actively expressed, were selected as the study material. The method was based on approaches based on changes in light transmission with a change in platelet shape.

Blood samples were taken from the marginal vein of the rabbit ear into a tube containing 3.8% sodium citrate, in a ratio of 1:10. To obtain platelet rich plasma (PBHT), the blood was centrifuged at 1000 rpm for 12 minutes.

For calibration, PBogT with EDTA and Krebs solution were used (NaCl - 120 mM, KCl - 4.8 mM, KH ₂ PO ₄ - 1.2 mM, MgSO ₄ - 2.5 mM, NaHCO ₃ - 25 mM, CaCl ₂ - 2.6 mM, glucose 5.4 mM, pH 7.4.).

In a control series of experiments, 600 μl of sodium propionate solution (in mmol / L: Na propionate 135, HEPES 20, CaCl ₂ 1, MgCl ₂ 1, glucose 10; pH 6.7; t = 37 ° C) was added to 200 μl of PBogT to reduce intracellular pH ) In this case, activation of the Na ⁺ / H ⁺ exchanger occurred, an increase in the influx of sodium, associated with the release of cytosolic H ⁺ , leading to platelet edema (spherulation) as a result of the accumulation of water in the cytoplasm. At the same time, light transmission was facilitated. To control changes in light transmission at physiological pH, Krebs solution was used.

Platelet shape changes were recorded by changes in light transmission using a BIOLA-220 LA laser aggregometer, Russia.

When studying new compounds and a zoniporide comparison drug (10 μl), substances were added to a cuvette with PBogT (200 μl) 3-5 minutes before adding sodium propionate solution, incubated at 37 ° C and stirring constantly with a magnetic stirrer (1000 rpm minute).

Studies were conducted in the concentration range of 10 ^-10 -10 ^-5 M.

The IC ₅₀ , the concentration of the compound blocking the Na ⁺ / H ⁺ exchange of rabbit platelets by 50%, was calculated.

1.5. Acute Toxicity Study

To calculate the breadth of the therapeutic effect of the compound, acute toxicity was determined on 50 male mice with intraperitoneal administration. The calculation of the LD- ₅₀ value was carried out graphically according to Miller and Teitner [Belenky M.L. Elements of a quantitative assessment of the pharmacological effect. - L., 1963. - 152 p.].

1.6. Statistical Processing Methods

For the test drug and the comparison drug, IC ₅₀ values were experimentally determined using the method of regression analysis of the relationship between the concentration lg and the percent inhibition of the exchanger activity in Microsoft Excel (Office XP, Microsoft, USA).

2. The results of the study

When studying the effect of new compounds and zoniporide on the activity of rabbit Na ⁺ / H ⁺ exchange, a dose-dependent increase in the inhibitory effect was shown. Calculated IC ₅₀ , which are presented in table 1.

When studying acute toxicity with intraperitoneal administration to mice, an LD _{50 was} calculated. To assess the breadth of the therapeutic effect on isolated organs calculated therapeutic index (table 1).

Thus, in terms of the NHE inhibitory effect of compound I ae, they were more active than zoniporide and exceeded it as in IC _{50 by} 19.7; 12.1; 2.1; 3.34 and 2.7 times, and according to the therapeutic index - 34.7; 9.8; 1.7; 1.3 and 1.1 times, respectively.

Table 1 Calculation of the therapeutic index of the most active inhibitors of Na ⁺ / H ⁺ exchange among derivatives of IMBI No. Substance NHE Inhibition LD ₅₀ , mg / kg Therapeutic index IC ₅₀ , M ED ₅₀ mg / L (kg) one. Id 1.37 × 10 ^-9 0.001 598.0 1002748.34 2. Ic 2.23 × 10 ^-9 0.001 282.8 282,252.71 3. Ie 8.08 × 10 ^-9 0.004 182.3 48698.21 four. Ib 1.28 × 10 ^-8 0.005 197.5 36798.68 5. Ia 1.00 × 10 ^-8 0.003 95.0 31127.13 6. Zoniporide 2.7 × 10 ^-8 0.009 250,0 28903.57

Claims (3)

1. The use as inhibitors of Na ⁺ / H ⁺ exchange of tricyclic derivatives of imidazo [1,2-a] benzimidazole, of the general formula I:

where Ar = C ₆ H ₅ , C ₆ H ₄ OCH ₃ -4, C ₆ H ₄ OH-4, C ₆ H ₃ (O ₂ CH ₂ ) -3,4
NR ₂ = N (C ₂ H ₅ ) ₂ , N (CH ₂ CH ₂ ) ₂ O
including Ia Ar = C ₆ H ₅ ; NR ₂ = N (C ₂ H ₅ ) ₂
Ib Ar = C ₆ H ₅ ; NR ₂ = N (CH ₂ CH ₂ ) ₂ O
Ic Ar = C ₆ H ₄ OCH ₃ -4; NR ₂ = N (CH ₂ CH ₂ ) ₂ O
Id Ar = C ₆ H ₄ OH-4; NR ₂ = N (CH ₂ CH ₂ ) ₂ O
Ie Ar = C ₆ H ₃ (O ₂ CH ₂ ) -3.4; NR ₂ = N (CH ₂ CH ₂ ) ₂ O. 2. The use according to claim 1 for the manufacture of agents having the ability to inhibit Na ⁺ / H ⁺ exchange, as well as to inhibit Na ⁺ / H ⁺ exchange in living organisms. 3. 2- (3,4-methylenedioxyphenyl) -9-morpholinoethyl imidazo [1,2-a] benzimidazole dihydrochloride of formula Ie: