RU2654062C2 - Derivatives of quinazolin-4(3h)-one inhibiting sodium-hydrogen exchange - Google Patents

Abstract

FIELD: chemical-pharmaceutical industry.

SUBSTANCE: invention relates to novel quinazolin-4(3H)-one derivatives of general formula

,

where R¹=H or CH₃; R²=H, CH₃, Cl or Br; R³=H or Br; R⁴=H or CH₃, which have inhibitory activity against sodium-hydrogen exchanger of the first isoform. During in vitro experiments on rabbit platelets the compounds demonstrate inhibitory concentration of IC₅₀ in the range of 1 × 10^-8 to 1 × 10^-12 mol/l and significantly exceed the zoniporide, comparator drug.

EFFECT: new quinazoline-4(3H)-one derivatives can be used in the development of effective and safe neuro- and cardioprotective drugs that implement their therapeutic benefits by affecting sodium-hydrogen metabolism.

1 cl, 2 ex, 4 tbl

Description

The invention relates to the field of pharmacology, to new agents with the ability to inhibit sodium-hydrogen metabolism.

The pathobiochemical cascade of ischemic damage to the myocardium, nervous tissue is a series of numerous interrelated processes. First of all, these are metabolic disorders that cause an imbalance between the processes of glycolysis and glucose oxidation, leading to intracellular accumulation of lactate and acidosis. Lowering the pH promotes activation of the Na ⁺ / H ⁺ exchanger (NHE) [Orlowski J., Grinstein S. Na ⁺ / H ⁺ exchangers // Compr. Physiol. - 2011 .-- Vol. 1. - No. 4. - P. 2083-2100], and the energy deficit disrupts the most energy-dependent enzyme, sodium-potassium-ATPase. The consequence of these processes is excessive penetration of sodium ions into the cell, followed by activation of a reverse calcium current, increased extracellular Ca ²⁺ entry into the cell, and the release of intracellular Ca ²⁺ from the depot. Calcium overload of the cell activates various enzyme systems, leads to the formation of free radicals, increases NO synthesis, promotes cytotoxic edema, and the development of oxidative stress, which ultimately contributes to cell death via necrotic or apoptotic pathways. Despite the abundance of mechanisms leading to calcium overload and cell death, NHE of the first isoform (NHE-1) is one of the main factors in the regulation of homeostasis under conditions of ischemia / reperfusion [Fliegel L. Molecular biology of the myocardial Na + / H + exchanger // J. Mol. Cell. Cardiol. - 2008. - Vol. 44. - No. 2. - P. 228-237].

R.,

U.H.,

K. The Na+/H+ exchanger modulates long-term potentiation in rat hippocampal slices //Naunyn Schmiedebergs Arch. Pharmacol. - 2009. - Vol. 379. - No. 3. - P. 233-239] и эпилепсии [Verma V., Bali A., Singh N. Implications of sodium hydrogen exchangers in various brain diseases // J. Basic. Clin. Physiol. Pharmacol. - 2015. - Vol. 26. - No. 5. - P. 417-426].NHE-1 takes part in many pathological processes of the central nervous and cardiovascular systems: ischemic-reperfusion injury to the myocardium and brain [Spasov AA, Muravyova V.Yu., Gurova N.A., Cheplyaeva N.I., Reznikov E.V., Anisimova V.A. Neuroprotective properties of a new inhibitor of the Na ⁺ / H ⁺ exchanger of compound RU-1355 on a model of focal ischemia in rats // Experiment. the clinic. pharmacol. - 2016. - T. 79. - No. 4. - S. 3-7; Karmazyn M. NHE-1: still a viable therapeutic target // Rev. Bras. Cir. Cardiovasc. - 2013 .-- No. 61. - P. 7-82], rhythm disturbance [Ayoub IM, Kolarova JD Zoniporide preserves left ventricular compliance during ventricular fibrillation and minimizes postresuscitation myocardial dysfunction through benefits on energy metabolism // Crit. Care Med. - 2007. - Vol. 35. - No. 10. - P. 2329-2336] and the development of heart hypertrophy, heart failure [Wakabayashi S., Hisamitsu T., Nakamura TY Regulation of the cardiac Na + / H + exchanger in health and disease // J. Mol. Cell. Cardiol. - 2013 .-- Vol. 61. - No. 1. - P. 68-76], endothelial dysfunction, Alzheimer's disease [

R.,

Uh

K. The Na + / H + exchanger modulates long-term potentiation in rat hippocampal slices // Naunyn Schmiedebergs Arch. Pharmacol - 2009. - Vol. 379. - No. 3. - P. 233-239] and epilepsy [Verma V., Bali A., Singh N. Implications of sodium hydrogen exchangers in various brain diseases // J. Basic. Clin. Physiol. Pharmacol - 2015. - Vol. 26. - No. 5. - P. 417-426].

Currently, the current direction is the search and study of NHE-1 inhibitors as potential cytoprotectors that protect cells from ischemic and reperfusion damage. An analysis of the chemical structure of known NHE-1 inhibitors revealed that most of them contain a guanidine group. NHE-1 inhibitors are found among pyrazine derivatives (amiloride, DMA, EIPA, MIBA); benzoylguanidines (HOE-642, HOE-694, EMD-96785); monocyclic derivatives of guanidine - derivatives of methacryloylguanidine (S1611, S3226); bicyclic derivatives of guanidine: indoloylguanidine derivatives (SM-20220) and quinoline (zoniporide) [Orlowski J., Grinstein S. Na ⁺ / H ⁺ exchangers // Compr. Physiol. - 2011 .-- Vol. 1. - No. 4. - P. 2083-2100]. NHE-1 inhibitory activity was shown in a number of benzimidazole derivatives [RF Patent No. 2518740. Means inhibiting Na ⁺ / H ⁺ exchange and 2- (3,4-methylenedioxyphenyl) -9-morpholinoethylimidazo [1,2- a ] benzimidazole dihydrochloride // Spasov AA, Anisimova VA, Gurova N .A., Timofeeva A.S., Fedorchuk V.Yu., Minkin V.I. - Publ. 06/10/2014. - Bull. Fig. - 2014. - No. 16; Zhang R., Lei L., Xu Y. Benzimidazol-2-yl or benzimidazol-2-ylthiomethyl benzoylguanidines as novel Na + / H + exchanger inhibitors, synthesis and protection against ischemic-reperfusion injury // Bioorg. Med. Chem. Lett. - 2007. - Vol. 17. - No. 9. - P. 2430-2433] and aminobenzimidazoles [RF Patent No. 2518741. Means inhibiting Na ⁺ / H ⁺ exchange and 1-dialkylaminoethyl-3- [substituted (disubstituted) phenacyl] -2-aminobenzimidazolium halides // Anisimova V.A., Spasov A.A., Gurova N.A., Tolpygin I.E., Timofeeva A.S., Fedorchuk V.Yu., Petrov V.I., Minkin V.I. - Publ. 06/10/2014. - Bull. Fig. - 2014. - No. 16]. According to the database [Thomson Reuters Integrity, 2011], there are 481 compounds that exhibit NHE-inhibitory activity, which are at various stages of preclinical and clinical studies. As possible therapeutic groups, they are recommended for the treatment of angina pectoris, cardiac arrhythmias, cerebral blood flow disorders and heart failure.

Currently, the most active for selective pharmacological inhibition of NHE-1 is the benzoylguanidine derivative, zoniporide - [1- (quinolin-5-yl) -5-cyclopropyl-1H-pyrazole-4-carbonyl] guanidine hydrochloride monohydrate. In studies on rabbit platelets, this drug inhibited NHE-1-dependent platelet swelling; on an isolated heart, according to Langendorff and in vivo, reduced the size of myocardial infarction, weakened post-reperfusion contractile dysfunction, and also reduced the frequency and duration of ventricular fibrillation [Tracey W.R., Allen M.S., Frazier D.E. 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]. The neuroprotective effects of zoniporide are associated with the inhibition of the so-called glutamate-calcium cascade both in vitro and in vivo when modeling focal ischemic damage [Lee V.K., Lee DH, Park S. Effects of KR-33028, a novel Na + / H + exchanger -1 inhibitor, on glutamate-induced neuronal cell death and ischemia-induced cerebral infarct // Brain Res. - 2009. - Vol. 1248. - No. 6. - P. 22-30]. Currently, clinical trials of the drug by Pfizer are being conducted in patients with myocardial infarction, coronary artery disease, and atherosclerosis.

Search, study and creation of new cytoprotective agents is an urgent task in solving the problem of improving the prognosis of cardiovascular diseases. Cardio and neuroprotectors used in Russia have insufficiently high clinical efficacy. Search and study of compounds with NHE-1-inhibitory properties will allow the development of drugs for the pathogenetic therapy of ischemic tissue damage [Spasov A.A., Gurova N.A., Kharitonova M.V. The structure and biological role of NHE-1. Pharmacological regulation of activity // Experiment. the clinic. pharmacol. - 2013. - T. 76. - No. 1. - S. 43-48; Madonna R., De Caterina R. Sodium-hydrogen exchangers (NHE) in human cardiovascular diseases: interfering strategies and their therapeutic applications // Vascul. Pharmacol - 2013 .-- Vol. 59. - No. 5-6. - P. 127-130].

The aim of the invention is the development of new highly active and low toxic inhibitors of sodium-hydrogen metabolism to create effective and safe neuro- and cardioprotective drugs based on them.

The invention consists in the synthesis and use as inhibitors of sodium-hydrogen exchange of new derivatives of quinazolin-4 (3H) -one containing fragments of N-acetylguanidine as substituents at the nitrogen atom N ^{3 of the} quinazoline heterocyclic system. The target compounds were synthesized by amination of 2- [chanazolin-3 (4H) -yl] acetic or propionic acid esters containing various substituents at positions 2, 6 and 8 of quinazolin-4 (3H) -one with the guanidine base obtained in situ from the corresponding hydrochloride in anhydrous methyl alcohol in the presence of potassium hydroxide.

The following examples illustrate the essence of the invention.

Example 1. N- [2-Methyl-6-bromquinazolin-3 (4H) -yl] acetylguanidine (compound VI).

A. Methyl ester of 2-acetylaminobenzoic acid. To a solution of 25.0 ml (0.193 mol) of anthranilic acid methyl ester and 20.0 ml (0.247 mol) of anhydrous pyridine in 100 ml of anhydrous 1,2-dichloroethane, cooled in an ice bath, was added with vigorous stirring over 15 minutes 25, 0 ml (0.265 mol) of acetic anhydride. Stir while cooling for another 15 minutes and leave overnight at room temperature. The reaction mass is washed successively with 100 ml of water, 100 ml of 1 M hydrochloric acid, 100 ml of water, dried with magnesium sulfate, filtered and evaporated on a rotary evaporator at a temperature of 70-80 ° C in a vacuum of a water-jet pump. It is cooled, the crystallized residue is dried in air for one day at room temperature and 36.6 g (98%) of a white crystalline substance are obtained, mp. 98-100 ° C, which is used further without further purification.

B. Methyl ester of 5-bromo-2-acetylaminobenzoic acid. To a stirred solution of 15.0 g (0.078 mol) of 2-acetylaminobenzoic acid methyl ester in 100 ml of anhydrous dimethylformamide, 14.2 g (0.082 mol) of N-bromosuccinimide are added at room temperature in one step, stirred for 1 hour and left for one day in protected from the light of the place. The reaction mass is poured into 500 ml of ice water, after 15 minutes the precipitate formed is filtered off, washed on the filter 3 times with 50 ml of water, dried in air at room temperature for 24 hours, and 19.9 g (94%) of a white needle-like crystalline substance are obtained, T. pl. 132.5-134 ° C.

¹ H NMR spectrum, δ, ppm (DMSO-D ₆ ): 2.12 s (3H, CH ₃ ); 3.87 s (3H, CH ₃ ); 7.69-7.74 m (1H, H ³ ); 7.95 t (1H, 4 Hz, H ⁶ ); 8.17-8.21 m (1H, H ⁴ ); 10.38 ears s (1H, NH).

¹³ C NMR spectrum, δ, ppm (DMCO-D ₆ ): 27.86; 55.94; 117.79; 123.02; 126.53; 135.87; 139.64; 142.34; 169.58; 171.83.

B. 2-Methyl-6-bromquinazolin-4 (3H) -one. A mixture of 15.0 g (0.055 mol) of 5-bromo-2-acetylaminobenzoic acid methyl ester and 50 ml of concentrated ammonium hydroxide is heated in a steel sealed vessel in a boiling water bath for 8 hours with periodic stirring and left overnight at room temperature. The precipitate formed is filtered off, washed on the filter 2 times with 10 ml of cold water, dried in air for a day and 7.3 g (55%) of a white crystalline substance are obtained, so pl. 298-302 ° C.

¹ H NMR spectrum, δ, ppm (DMSO-D ₆ ): 2.33 s (3H, CH ₃ ); 7.47 d (1H, 4 Hz, H ⁸ ); 7.84 d (1H, 4 Hz, H ⁷ ); 8.12 s (1H, H ⁵ ); 11.85 ears s (1H, NH).

¹³ C NMR spectrum, δ, ppm (DMSO-D ₆ ): 24.84; 121.41; 125.73; 131.18; 132.27; 140.26; 151.32; 158.42; 163.94.

D. [2-Methyl-6-bromoquinazolin-3 (4H) -yl] acetic acid benzyl ester. 3.0 g (0.013 mol) of 2-methyl-6-bromoquinazolin-4 (3H) -one and 4.0 g (0.029 mol) of anhydrous potassium carbonate are stirred in 40 ml of anhydrous dimethylformamide at a temperature of 80-85 ° C. for 15 min, add a solution of 3.0 g (0.013 mol) of bromoacetic acid benzyl ester in 10 ml of dimethylformamide and stir at the same temperature for 1 h. The reaction mass is cooled, filtered, the filtrate is evaporated on a rotary evaporator at a residual pressure of 10 mm Hg. Art. at a bath temperature of 90-95 ° C, the residue is cooled, triturated with 25 ml of water, filtered, washed on the filter 2 times with 10 ml of cold water, dried in air for one day and crystallized from 100 ml of isopropyl alcohol. Obtain 3.0 g (62%) of golden yellow needle crystals, T. pl. 158.5-160.5 ° C.

¹ H NMR spectrum, δ, ppm (DMSO-D ₆ ): 2.51 s (3H, CH ₃ ); 5.00 s (2H, CH ₂ ); 5.22 s (2H, CH ₂ ); 7.30-7.42 m (5H, phenyl); 7.56 d (1H, 4 Hz, H ⁸ ); 7.95 d (1H, 4 Hz, H ⁷ ); 8.16 s (1H, H ⁵ ).

¹³ C NMR spectrum, δ, ppm (DMSO-D ₆ ): 26.21; 49.06; 70.16; 122.31; 124.38; 131.36; 131.61; 131.63; 131.85; 132.53; 138.83; 141.01; 145.39; 158.90; 163.39; 171.22.

D. N- [2-Methyl-6-bromoquinazolin-3 (4H) -yl] acetylguanidine (compound VI). To a boiling solution of 2.50 g (6.46 mmol) of [2-methyl-6-bromquinazolin-3 (4H) -yl] acetic acid benzyl ester and 0.75 g (7.85 mmol) of guanidine hydrochloride in 50 ml of anhydrous methyl alcohol is added in one go 0.55 g (8.84 mmol) of granular potassium hydroxide and boiled for 30 minutes The hot solution is filtered, separating the precipitate of potassium chloride formed, the filtrate is cooled and incubated for one day at room temperature. The precipitated precipitate is filtered off, washed 2 times with 10 ml of cold methyl alcohol, 10 ml of diethyl ether, dried in the air for 24 hours at room temperature and 1.8 g (83%) of a white crystalline substance are obtained, so pl. 281-284 ° C (decomp.).

¹ H NMR spectrum, δ, ppm (DMSO-D ₆ ): 2.46 s (3H, CH ₃ ); 4.47 s (2H, CH ₂ ); 7.51 d (1H, 8 Hz, H ⁸ ); 7.60 ears. s (4H, NH); 7.88 d (1H, 8 Hz, H ⁷ ); 8.13 s (1H, H ⁵ ).

¹³ C NMR spectrum, δ, ppm (DMSO-D ₆ ): 23.24; 47.76; 118.44; 121.86; 128.56; 129.20; 137.14; 146.53; 156.98; 159.13; 160.22; 170.91.

The remaining compounds are obtained in a similar manner. The output and physico-chemical properties of the substances are presented in table. 1, spectral characteristics are given in table. 2 and 3.

Example 2. The study of the effect of new derivatives of quinazoline on the activity of the Na ⁺ / H ⁺ exchanger (NHE-1 inhibition). The effect of the compounds on the activity of the Na ⁺ / H ⁺ exchanger was studied in vitro on rabbit platelets, on the membrane of which NHE-1 is actively expressed, according to the method of [Rosskopf D., Morgenstern E., Scholz W. Rapid determination of the elevated Na ⁺ / H ⁺ exchanger in platelets of patients with essential hypertension using an optical swelling assay // J. Hypertens. - 1991. - No. 9. - P. 231-238; Kusumoto K., Igata H., Abe A. In vitro and in vivo pharmacology of a structurally novel Na ⁺ / H ⁺ exchanger inhibitor, T-162559 // Brit. J. Pharmacol. - 2002. - No. 135. - P. 1995-2003]. 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), blood was centrifuged at 1000 rpm for 12 minutes. For calibration, PBogT with EDTA and Krebs solution (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: sodium propionate -135, HEPES - 20, CaCl ₂ - 1, MgCl ₂ - 1, glucose - 10; pH 6, was added to 200 μl of PBogT to reduce intracellular pH) 7; temperature 37 ° C). In this case, the activation of the Na ⁺ / H ⁺ exchanger and an increase in the influx of sodium, associated with the release of the cytosolic, leading to edema (spherulation), of the platelet as a result of the accumulation of water in the cytoplasm, took place. Light transmittance was observed. To control changes in light transmission under physiological pH values, Krebs solution (600 μl) was used. Changes in the shape of platelets were recorded by changes in the level of light transmission using a laser aggregometer "BIOLA-220 LA" (Russia). When studying new compounds and a zoniporide comparison drug (SIGMA, USA), 10 μl of the substance was added to a cuvette with PBogT (200 μl) 3-5 min before the addition of sodium propionate solution, incubated at 37 ° C and with constant stirring using a magnetic stirrer ( 1000 rpm). The studies were carried out in a concentration range of 1 × 10 ^-8 -1 × 10 ^-12 M. For the test substances and the reference 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 suite XP, Microsoft, USA). In the experiments, male rabbits (n = 15) weighing 2.5-3.5 kg were used (Elektrogorsky Branch of FSBI NTsBMT RAS, Veterinary Certificate 250 No. 0380791 of January 14, 2013). The research results are presented in table. four.

The studies were conducted in accordance with the applicable requirements of GOST ISO / IEC 17025-2009, GOST R ISO 5725-2002 and GOST 33044-2014 “Interstate Standard. Principles of Good Laboratory Practice ”(enforced by the Order of Rosstandart of November 20, 2014 No. 1700-st) in compliance with the“ European Convention for the Protection of Vertebrate Animals Used for Experiments or for Other Scientific Purposes ”[Directive 2010/63 / EU of the European Parliament and of the Council of 22 September 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). All experiments were approved by the local Ethics Committee (protocol No. 126-2011 of February 2, 2011).

As follows from the data table. 4, the most active compounds VI and VII in their ability to inhibit NHE-1 in vitro, respectively, are 3250 and 8500 times superior to the reference drug.

Thus, the research results indicate that, based on new quinazoline derivatives containing guanidine fragments, it is possible to create new, more effective and safe neuro- and cardioprotective drugs that realize their therapeutic effects by affecting sodium-hydrogen metabolism.

Claims (4)

Quinazolin-4 (3H) -one derivatives of the general formula

where R ¹ = H or CH ₃ ; R ² = H, CH ₃ , Cl or Br; R ³ = H or Br; R ⁴ = H or CH ₃ , inhibiting sodium-hydrogen metabolism.