RU2540070C1 - 2-ethyl-6-methyl-3-hydroxypyridine dichloroacetate, its stable crystalline form and method of obtaining thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to novel substance - 2-ethyl-6-methyl-3-hydroxypyridine dichloroacetate of formula I

its stable crystalline form and method of obtaining thereof. Substance (I) possesses antiatherosclerotic, hypolipidemic, antihypoxic, nootropic, anxiolytic and adaptogenic action with low toxicity (LD₅₀=30000 mg/kg, rats, intragastrically) and absence of hygroscopicity. Substance is obtained with interaction of equimolar quantities of 2-ethyl-6-methyl-3-hydroxypyridine and dichloroacetic acid in presence of solvent.

EFFECT: increase of stability.

9 cl, 8 dwg, 10 tbl, 12 ex

Description

The invention relates to medicine, namely to a new biologically active substance - dichloroacetate 2-ethyl-6-methyl-3-hydroxypyridine, which has antiatherosclerotic, hypolipidemic, antihypoxic, nootropic, anxiolytic and adaptogenic effects with low toxicity (LD ₅₀ = 30,000 mg / kg, rat, intragastric), storage stability and lack of hygroscopicity.

State of the art

2-Ethyl-6-methyl-3-hydroxypyridine (hereinafter EMHP) is an analogue of vitamin Bb, the study of it and its salts was started in the 80s of the last century, in particular EMHP hydrochloride was proposed as a geroprotector [Obukhova L.K. , Emanuel N.M. The role of free radical oxidation reactions in the molecular mechanisms of aging of living organisms // Uspekhi khimii. - 1983. - T. 52. - S.353-372; Emanuel L.M., Obukhova L.K. Types of experimental delay in aging patterns // Exp. Gerontol. - 1978. - Vol.13. - P.25-29], plant growth regulator with an anti-stress effect [RU 801829, publ. 02/07/1981].

Currently, salts of EMGP with glycolic, malonic, tartaric and other organic carboxylic acids have been proposed [RU 2246486, publ. 02/20/2005; RU 2284993, publ. 10/10/2006; RU 2357955, publ. 06/10/2009; RU 2365582, publ. 08/27/2009; RU 2377237, publ. 12/27/2009; RU 2394815, publ. 07/20/2010; RU 2442774, publ. 02/20/2012; RU 2454406, publ. 06/27/2012.

The most widely used hydrochloride is EMGP (emoxipin) [radar. Encyclopedia of drugs. - 16th issue / Ch. ed. G.L. Vyshkovsky. - M .: "RLS-2008", 2007. - P.1014], amber acid salt of EMHP (mexidol, mexicor) [http://medi.ru/doc/a070196.htm, RLS. Encyclopedia of drugs. - 16th issue / Ch. ed. G.L. Vyshkovsky. - M .: "RLS-2008", 2007. - P.537] and malonate EMGP [http://www.vidal.ru/poisk_preparatov/etoxydol.htm].

EMGP salts possess an antioxidant [http://www.stm-journal.ru/_resources/item/77/fields/File/STM_2_2010_p22_25.pdf; RU 2444358, publ. 03/10/2012; RU 2398583, publ. 09/10/2010; RU 325908, publ. 06/10/2008], antihypoxic [RU 2284993, publ. 10/10/2006; RU 2442774, publ. 02/20/2012; RU 2440132, publ. 01/20/2012; RU 2398583, publ. 09/10/2010; RU 2326665, publ. 06/20/2008], hypoglycemic, hypolipidemic, neuroprotective [RU 2454406, publ. June 27, 2012], metabolic and cardioprotective [RU 2365582, publ. August 27, 2009], anxiolytic [RU 2284993, publ. 10/10/2006] activity.

The search for new salts of EMHP reflects the desire to enhance and expand the biological effects of EMHP by introducing a physiologically active anion into the composition of the used salt of EMHP.

Dichloroacetates as drugs have long been known. In the 70 years in the form of a salt of 2-isopropylaminopyrimidine, it was proposed for use in neuropathy [GB 1351535, publ. 05/01/1974]; in the form of a biguanidine salt, it was indicated for metabolic disorders [GB 1473256, publ. 05/11/1977; US 4,801,597, publ. 01/31/1989]. Sodium dichloroacetate in US 4112188, publ. 09/05/1978 proposed for use as a lipid-lowering agent.

In the 80s, diisopropylammonium dichloroacetate was studied. In the treatment of embryonic distress syndrome, it was proposed in DE 3712239, publ. 06/09/1988. Salt diisopropylammonium, as well as sodium and potassium, were presented as the active substance in the treatment of cerebral ischemia US 4631294, publ. 12/23/1986. No. 4,558,050, publ. 12/10/1985, sodium salt is proposed for use in metabolic disorders.

In the 90s, further research was conducted on sodium and diisopropylammonium salts. Sodium dichloroacetate is proposed for use in the field of cardiovascular diseases [US 5587397, publ. 12/24/1996; US 6011017, publ. 01/04/2000; WO 9917763, publ. 04/15/1999], diisopropylammonium dichloroacetate in dermatology [JP 6100430, publ. 04/12/1994; JP 10330252, publ. 12/15/1998].

In FR 2663508, publ. 12/27/1991, as an antidote, methyldichloroacetate was proposed as an additive to the herbicide. In addition, since 1997, Japan has been actively developing dichloroacetate-based cosmetics for hair and skin.

In 2000, amlodipine dichloroacetate was introduced as an agent in the treatment of anemia and hypertension [CN 1343663, publ. 04/10/2002]. The study of sodium dichloroacetate as a lipid-lowering agent, begun as early as the 70s, was continued [WO 2005063761, publ. 07/14/2005; W02005063761, publ. July 14, 2005], and later it was presented as a hypoglycemic agent [CN 102058569, publ. 05/18/2011].

Over the past decade, the study of diisopropylammonium dichloroacetate has continued. In 2007, it was proposed for use in the treatment of cancer as an anti-blast agent [JP 2007016008, publ. 01/25/2007]; later presented as an active substance in the treatment of acute and chronic hepatitis [CN 101422438, publ. 05/06/2009] and the cleaning of the liver and gall bladder [CN 101757315, publ. 06/30/2010].

Potassium and sodium salts are also investigated as anticancer agents [WO 2006108276, publ. 10/19/2006; US2008221211, publ. 09/11/2008; US 2009028791, publ. September 11, 2007], and already in 2012, imatinib dichloroacetate was proposed for use in this field [US 2012295917, publ. 11/22/2012] and erlotinib [WO 2012101846, publ. 01/19/2012]. In Russia, for the treatment of cancer, pharmaceutical compositions are proposed containing sodium dichloroacetate and theophylline [RU 2463053, publ. 10.10.2012] and sodium dichloroacetate and xanthine (theobromine, caffeine, paraxanthin) [RU 2484814, publ. 06/20/2013].

It should be noted that sodium dichloroacetate is extremely hygroscopic, which makes the manufacture of convenient oral dosage forms (tablets) very difficult. In practice, for the most part, concentrated aqueous solutions are used, but they are difficult to dose due to their significant viscosity and have a very unpleasant taste.

Disclosure of invention

The aim of the present invention was to obtain a new storage-stable and non-hygroscopic salt of EMHP and dichloroacetic acid, technologically convenient, suitable for use in experimental medicine, having a spectrum of biological activity characteristic of EMHP and dichloroacetate anion. The problem was solved by the synthesis of a new salt of dichloroacetate EMHP of the formula:

The synthesized salt has very good technological properties: lack of hygroscopicity, good flowability and compressibility during tabletting; it is chemically stable.

The synthesis of the claimed substance is carried out by the interaction of dichloroacetic acid and EMHP in the presence of a solvent. The reaction of equimolar amounts of reagents is carried out with stirring at room temperature; for completeness of crystallization incubated for 12-18 hours.

A protic or aprotic solvent is used as a solvent, water or ethanol, or 2-propanol is used as a proton solvent; as an aprotic solvent, acetone or 2-butanone, or dioxane, or acetonitrile, or diethyl ether, or tetrahydrofuran is used.

The obtained stable crystalline form of EMHP dichloroacetate is characterized by an X-ray powder diffraction spectrum (see FIG. 1), expressed in terms of interplanar distance d, Bragg's angle 2θ, intensity and relative intensity:

Angle (2θ ± 0.2) ° Interplanar distance d, A Intensity, s ^-1 Relative intensity,% 8.1200 10,87978 454 four 8.2800 10.66990 886 8 11.8938 7.43485 2572 23 15,8610 5.58302 373 3 16,3800 5,40728 3409 thirty 16.6423 5,32264 10853 96 16.8275 5,26447 1564 fourteen 17.8471 4,96595 1383 12 18,2200 4,86514 653 6 18,5400 4.78189 379 3 18,7200 4,73631 398 four 20,2000 4.39251 1289 eleven 20,4200 4,34568 2093 eighteen 20,6600 4,29574 668 6 22,5759 3,93532 925 8 23,0800 3,85050 639 6 24,0000 3.70494 1040 9 24.3624 3,65064 2032 eighteen 25.1812 3,53376 11361 one hundred 25,5600 3,48224 1045 9 26,1200 3,40884 1286 eleven 26,5800 3,35088 824 7 29,3000 3,04570 525 5 29.5600 3,01950 342 3 29,9200 2,98399 354 3 32,4000 2.76102 405 four 32.6922 1,73701 2411 21 33,1200 2.70262 814 7 33,4600 2,67594 376 3 33.6800 2.65896 568 5 33.8400 2,64675 1159 10 36,5800 2.45454 573 5 36.9200 2,43271 935 8 39.9682 2025393 368 3 42.6733 2,11710 378 3

The X-ray powder diffraction spectrum was measured under the conditions:

- Thermo ARL X′TRA diffractometer, semiconductor detector;

- radiation of CuK _a voltage of 40 kV, intensity 30 mA;

- shooting in θ-2θ mode;

- measuring range from 6.02 ° to 50.02 °;

- an increase between each measurement of 0.02 °;

- measurement speed 0.25 ° / min;

- deflecting slot of the tube 1.0 °;

- diffusing slit of the tube 1.0 °;

- a diffusing slit of the detector 0.3 mm.

The products obtained as a result of the syntheses were characterized by IR, ¹³ C NMR, ¹ H spectroscopy, elemental and thermal analyzes.

The results of the study of biological activity show that the claimed substance has a lipid-lowering, anti-atherosclerotic, antihypoxic, nootropic, adaptogenic and anxiolytic effect (see examples 8, 9). EMHP dichloroacetate protects the central nervous system from the most common traumatic effects - hypoxia and psycho-emotional overstrain and allows you to correct disorders of higher nervous activity (cognitive functions) caused by pathological factors (see example 8). It turned out that the claimed substance exhibits an order of magnitude less acute toxicity (LD ₅₀ = 30,000 mg / kg, rat, intragastrically) than other known salts of EMHP and salts of dichloroacetate (see example 10). In comparison, for the well-known Mexidol® (succinate EMHP) LD ₅₀ more than 3000 mg / kg [T. Voronina Domestic drug of the new generation Mexidol: main effects, mechanism of action, application. - M .: 2004. - 248 p.], For Ethoxidol® (malate EMHP) LD ₅₀ = 3566 mg / kg [Berezovskaya IV, Rymartsev VI, Volkova L.I. Preclinical study of the general toxic effects of ethoxidol // Toxicological Bulletin. - 2010. - No. 5, P.42-45], for Emoxipin® (EMHP hydrochloride) LD ₅₀ = 360 mg / kg [http://www.rusnauka.com/ 21_DNIS_2009 / Biologia / 49568.doc], for dichloroacetate sodium LD50 = 5281 mg / kg [http://datasheets.scbt.com/sc-203275.pdf]. It can be assumed that the ability of the dichloroacetate anion to suppress intracellular milk acidosis [http://med-books.info/vnutrennie-bolezni_718/laktat-atsidoz.html. Stacpoole PW, Lorenz AC, Thomas RG. Dichloroacetateinthetreatmen toflacticacidosis. // Ann. Intern. Med. - 1988. - Vol.108 - P.58-63] protects the tissues of internal organs in case of blood supply disturbance caused by toxic doses of EMHP.

Thus, the claimed substance has the properties of salts of EMHP (antihypoxic, nootropic, hypolipidemic, anxiolytic, adaptogenic) and is at the same time a convenient source of dichloroacetate ion used in experimental oncology for stopping lactic acidosis. Since the proposed salt is storage stable and non-hygroscopic, it can be used as a replacement for sodium dichloroacetate, which is inconvenient to use. Given the low toxicity of the claimed substance (an order of magnitude lower than the currently used antioxidant salts of EMHP), it can be successfully used in cardiology, vascular surgery, neurology, geriatrics, as well as complex therapy of diseases of various origins.

Brief Description of the Drawings

Figure 1 - Diffraction pattern of dichloroacetate EMHP

Figure 2 - Thermal analysis of dichloroacetate EMHP (example 1)

Curve 1 - Thermogravimetric analysis (TG) curve

Curve 2 - differential scanning calorimetry (DSC) curve

Curve 3 - differential thermogravimetric analysis (DTG) curve

Figure 3 - Thermal analysis of EMHP dichloroacetate (example 2)

Curve 1 - TG Curve

Curve 2 - DSC Curve

Curve 3 - DTG Curve

Figure 4 - Thermal analysis of EMHP dichloroacetate (example 3)

Curve 1 - TG Curve

Curve 2 - DSC Curve

Curve 3 - DTG Curve

Figure 5 - Thermal analysis of dichloroacetate EMHP (example 4)

Curve 1 - TG Curve

Curve 2 - DSC Curve

Curve 3 - DTG Curve

6 - Thermal analysis of dichloroacetate EMHP (example 5)

Curve 1 - TG Curve

Curve 2 - DSC Curve

Curve 3 - DTG Curve

7 - Thermal analysis of dichloroacetate EMHP (example 6)

Curve 1 - TG Curve

Curve 2 - DSC Curve

Curve 3 - DTG Curve

Fig - Thermal analysis of dichloroacetate EMHP (example 7)

Curve 1 - TG Curve

Curve 2 - DSC Curve

Curve 3 - DTG Curve

The implementation of the invention Example 1. Obtaining dichloroacetate EMHP from distilled water. In a beaker equipped with a fluoroplastic stirrer with an electromechanical drive, load 25 ml of distilled water, 5 g of EMHP (0.036 mol) and 4.58 g (0.036 mol) of dichloroacetic acid, include a stirrer. The reaction mass is stirred for half an hour at room temperature. The resulting suspension is incubated for 12-18 hours. Filtered on a Schott filter, dried. 8.8 g of product (0.033 mol) are obtained. Yield 91.9% of theory.

Elemental analysis:

Calculated,%: C 45.13; H 4.93; N, 5.26; O 18.04; Cl 26.64.

Found,%: C 45.10; H 5.01; N, 5.30; O 18.01; Cl 26.58.

NMR ¹ H (DMSO d ⁶ ), δ, ppm: 1.17 (-CH ₃ , t, 3H); 2.45 (CH ₃ -C ₆ , s, 3H); 2.80 (-CH ₂ -, k, 2H); 6.34 (-CHCl ₂ , s, 1H); 7.24 (C ₄ -H, d, 1H); 7.50 (C ₅ -H, d, 1H).

NMR ¹³ C (DMSO d ⁶ ), δ, ppm: 12.95 (-CH ₃ ); 20.87 (CH ₃ -C ₆ ); 23.68 (-CH ₂ -); 68.60 (-CHCl ₂ ); 123.74 (C ₅ ); 126.86 (C ₄ ); 144.84 (C ₆ ); 147.90 (C ₂ ); 150.87 (C ₃ ); 166.36 (-COO ^- ).

IR (cm ^-1 ): 3250-3700 (valen. -OH, assoc. Group); 3000 (valence Ar-H); 2860-2973 (valence. -CH ₂ -, -CH ₃ ); 2000-2800 (valency NH ⁺ ); 1820, 1650 (valent. COO ^- ); 1630 (valent. C = N ⁺ pyridine, ring); 1600, 1550, 1455 (-C = C- pyridine, ring); 1391 (deform. Assim. -CH ₃ ); 1430 (scissor. -CH ₂ ); 1380 (deform. Sym. -CH ₃ ); 1350, 1330 (fan, torsional. -CH ₂ -); 1308 (valent. -CO- pyridine ring); 1233 (deform. -C-OH pyridine, ring); 1000, 1150, 1177, 1050, 950 (flat, deform. CH pyridine, ring); 900, 837 (deform. COO ^- ); 710 (pendulum -CH ₂ -); 772, 800, 820 (non-flat. Deform. CH pyridine, ring); 624 (valency C-Cl); 509 (deform. C-Cl).

X-ray phase analysis:

Characteristic peaks at (2θ ± 0.2) °: 8.14; 8.28; 11.68; 11.88; 12.03; 16.28; 16.58; 16.90; 17.85; 18.18; 18.58; 19.48; 19.74; 20.39; 22.54; 23.06; 23.26; 23.98; 24.38; 25.19; 25.58; 25.76; 26.08; 26.36; 26.54; 27.03; 29.52; 32.68; 33.14; 33.75; 36.74; 37.00.

Thermal analysis (Figure 2):

At a temperature of 120.0 ° C, a pronounced endothermic effect is observed corresponding to the melting of the sample (the sample melts without decomposition). At temperatures above 183.0 ° C, the decomposition of salt begins, accompanied by an endothermic effect (maximum at 256.0 ° C). At a temperature of 289-342 ° C, further decomposition of the resulting products occurs with a pronounced endothermic effect (maximum at 327.0 ° C). With a further increase in temperature, the sample oxidizes and burns out.

Example 2. Obtaining dichloroacetate EMHP from ethanol.

The experiment is carried out as in example 1, but 25 ml of ethanol is used as a solvent. 3.3 g of product (0.012 mol) are obtained. Yield 34.4% of theory. Elemental analysis:

Calculated,%: C 45.13; H 4.93; N, 5.26; O 18.04; Cl 26.64.

Found,%: C 45.07; H 4.95; N, 5.30; O 17.98; Cl 26.70.

IR (cm ^-1 ): 3250-3700 (valen. -OH, assoc. Group); 3000 (valence Ar-H); 2870-2976 (valent. -CH ₂ -, -CH ₃ ); 2000-2800 (valency NH ⁺ ); 1819, 1650 (valent. COO ^- ); 1630 (valent. C = N ⁺ pyridine, ring); 1600, 1551, 1460 (-C = C- pyridine, ring); 1400 (deform. Assim. -CH ₃ ); 1430 (scissor. -CH ₂ ); 1381 (deform. Sym. -CH ₃ ); 1350, 1330 (fan, torsional. -CH ₂ -); 1300 (valent. -CO- pyridine ring); 1237 (deform. -C-OH pyridine, ring); 1170, 1150, 1047, 1000, 950 (flat, deform. CH pyridine, ring); 900, 830 (deform. COO ^- ); 713 (pend. -CH ₂ -); 775, 805, 820 (non-flat. Deform. CH pyridine, ring); 630 (valency C-Cl); 509 (deform. C-Cl).

X-ray phase analysis:

Characteristic peaks at (2θ ± 0.2) °: 8.10; 8.28; 11.88; 16.10; 16.32; 16.64; 17.06; 17.85; 18.22; 18.58; 20.22; 20.42; 20.68; 22.64; 22.92; 23.06; 24.02; 24.35; 24.98; 25.18; 25.58; 25.80; 26.06; 26.42; 26.58; 29.38; 31.54; 32.69; 33.10; 33.81; 36.56; 36.89; 39.97; 42.68.

Thermal analysis (Figure 3):

At a temperature of 121.0 ° C, a pronounced endothermic effect is observed corresponding to the melting of the sample (the sample melts without decomposition). At temperatures above 188.0 ° C, the decomposition of salt begins, accompanied by an endothermic effect (maximum at 258.0 ° C). At a temperature of 290-346 ° C, further decomposition of the resulting products occurs with a pronounced endothermic effect (maximum at 328.0 ° C). With a further increase in temperature, the sample oxidizes and burns out.

Example 3. Obtaining dichloroacetate EMHP from 2-propanol.

The experiment is carried out as in example 1, but 25 ml of 2-propanol are used as a solvent. 7.7 g of product (0.029 mol) are obtained. Yield 80.4% of theory.

Elemental analysis:

Calculated,%: C 45.13; H 4.93; N, 5.26; O 18.04; Cl 26.64.

Found,%: C 45.12; H 4.96; N, 5.25; O 17.99; Cl 26.68.

IR (cm ^-1 ): 3250-3700 (valen. -OH, assoc. Group); 3000 (valence Ar-H); 2860-2976 (valence. -CH ₂ -, -CH ₃ ) 2000-2800 (valence. NH ⁺ ); 1826, 1650 (valent. COO ^- ); 1630 (valent. C = N ⁺ pyridine, ring); 1598, 1550, 1460 (-C = C- pyridine, ring); 1400 (deform. Assim. -CH ₃ ); 1430 (scissor. -CH ₂ ); 1382 (deform. Sym. -CH ₃ ); 1350, 1330 (fan, torsional. -CH ₂ -); 1304 (valent. -CO- pyridine ring); 1250 (deform. -C-OH pyridine, ring); 1178, 1150, 1047, 1000, 950 (flat, deform. CH pyridine, ring); 900, 830 (deform. COO ^- ); 710 (pendulum -CH ₂ -); 775, 800, 820 (non-flat. Deform. CH pyridine, ring); 630 (valency C-Cl); 509 (deform. C-Cl).

X-ray phase analysis:

Characteristic peaks at (2θ ± 0.2) °: 8.12; 8.28; 11.89; 15.86; 16.38; 16.64; 16.83; 17.85; 18.22; 18.54; 18.72; 20.20; 20.42; 20.66; 22.58; 23.08; 24.00; 24.36; 25.18; 25.56; 26.12; 26.58; 29.30; 29.56; 29.92; 32.40; 32.69; 33.12; 33.46; 33.68; 33.84; 36.58; 36.92; 39.97; 42.67.

Thermal analysis (Figure 4): At a temperature of 115.0 ° C there is a pronounced endothermic effect corresponding to the melting of the sample (the sample melts without decomposition). At temperatures above 191.0 ° C, the decomposition of salt begins, accompanied by an endothermic effect (maximum at 256.0 ° C). At a temperature of 281-304 ° C, further decomposition of the resulting products occurs with a pronounced endothermic effect (maximum at 289.0 ° C). With a further increase in temperature, the sample oxidizes and burns out.

Example 4. Obtaining dichloroacetate EMHP from acetone.

The experiment is carried out as in example 1, but 25 ml of acetone is used as a solvent. 8.9 g of product (0.033 mol) are obtained. Yield 92.9% of theory.

Elemental analysis:

Calculated,%: C 45.13; H 4.93; N, 5.26; O 18.04; Cl 26.64.

Found,%: C 45.20; H 4.99; N, 5.26; O 18.02; Cl 26.53.

IR spectrum (cm ^-1 ): 3200-3700 (valent-OH, assoc. Group); 3000 (valence Ar-H); 2870-2976 (valence. -CH ₂ -, -CH ₃ ) 2000-2800 (valence. NH ⁺ ); 1823, 1650 (valent. COO ^- ); 1630 (valent. C = N ⁺ pyridine, ring); 1600, 1553, 1460 (-C = C- pyridine ring); 1396 (deform. Assim. -CH ₃ ); 1441 (scissor. -CH ₂ ); 1380 (deform. Sym. -CH ₃ ); 1350, 1330 (fan, torsional. -CH ₂ -); 1308 (valent. -CO- pyridine ring); 1230 (deform. -C-OH pyridine ring); 1180, 1150, 1044, 1000, 956 (flat, deform. CH pyridine, ring); 900, 830 (deform. COO ^- ); 710 (pendulum -CH ₂ -); 775, 800, 820 (non-flat. Deform. CH pyridine, ring); 630 (valency C-Cl); 509 (deform. C-Cl).

X-ray phase analysis:

Characteristic peaks at (2θ ± 0.2) °: 8.14; 8.28; 11.90; 15.43; 15.84; 16.39; 16.67; 16.86; 17.86; 18.18; 18.73; 20.42; 20.72; 22.50; 22.77; 22.99; 23.08; 24.01; 24.24; 24.39; 25.18; 25.58; 25.80; 26.07; 26.43; 26.59; 28.18; 28.92; 29.40; 29.89; 32.68; 33.13; 33.82; 34.12; 36.92; 37.16; 40.07; 41.28; 42.68; 49.99.

Thermal analysis (Figure 5):

At a temperature of 120.0 ° C, a pronounced endothermic effect is observed corresponding to the melting of the sample (the sample melts without decomposition). At temperatures above 185.0 ° C, the decomposition of salt begins, accompanied by an endothermic effect (maximum at 257.0 ° C). At a temperature of 302-348 ° C, further decomposition of the resulting products occurs with a pronounced endothermic effect (maximum at 329.0 ° C). With a further increase in temperature, the sample oxidizes and burns out.

Example 5. Obtaining dichloroacetate EMHP from 2-butanone.

The experiment is carried out as in example 1, but 25 ml of 2-butanone are taken as a solvent. 8.8 g of product (0.033 mol) are obtained. Yield 91.9% of theory.

Elemental analysis:

Calculated,%: C 45.13; H 4.93; N, 5.26; O 18.04; Cl 26.64.

Found,%: C 45.20; H 4.90; N, 5.28; O 18.02; Cl 26.60.

IR (cm ^-1 ): 3250-3700 (valen. -OH, assoc. Group); 3000 (valence Ar-H); 2879-2976 (valence. -CH ₂ -, -CH ₃ ) 2000-2800 (valence. NH ⁺ ); 1826, 1650 (valent. COO ^- ); 1630 (valent. C = N ⁺ pyridine, ring); 1600, 1550, 1460 (-C = C- pyridine, ring); 1393 (deform. Assim. -CH ₃ ); 1438 (scissor. -CH ₂ ); 1380 (deform. Sym. -CH ₃ ); 1350, 1330 (fan, torsional. -CH ₂ -); 1308 (valent. -CO- pyridine ring); 1234 (deform. -C-OH pyridine ring); 1177, 1150, 1050, 1000, 952 (plane, deform. CH pyridine, ring); 900, 840 (deform. COO ^- ); 710 (pendulum -CH ₂ -); 774, 800, 820 (non-flat. Deform. CH pyridine, ring); 627 (valency C-Cl); 509 (deform. C-Cl).

X-ray phase analysis:

Characteristic peaks at (2θ ± 0.2) °: 8.16; 8.30; 11.89; 15.84; 16.30; 16.64; 16.88; 17.85; 18.22; 18.64; 20.20; 20.40; 20.64; 22.58; 22.90; 23.10; 24.04; 24.40; 25.19; 25.58; 25.76; 26.02; 26.16; 26.46; 26.60; 28.16; 28.90; 29.26; 29.54; 32.68; 33.00; 33.16; 33.82; 36.60; 36.78; 36.94; 40.25; 42.69.

Thermal analysis (Fig.6): At a temperature of 121.0 ° C there is a pronounced endothermic effect corresponding to the melting of the sample (the sample melts without decomposition). At temperatures above 198.0 ° C, the salt decomposition process begins, accompanied by an endothermic effect (maximum at 252.0 ° C). At a temperature of 290-332 ° C, further decomposition of the resulting products occurs with a pronounced endothermic effect (maximum at 313.0 ° C). With a further increase in temperature, the sample oxidizes and burns out.

Example 6. Obtaining dichloroacetate EMHP from dioxane.

The experiment is carried out as in example 1, but 25 ml of dioxane are used as a solvent. 9.3 g of product (0.035 mol) are obtained. Yield 97.1% of theory.

Elemental analysis:

Calculated,%: C 45.13; H 4.93; N, 5.26; O 18.04; Cl 26.64.

Found,%: C 45.17; H 5.00; N, 5.32; O 17.96; Cl 26.55.

IR (cm ^-1 ): 3250-3700 (valen. -OH, assoc. Group); 3000 (valence Ar-H); 2870-2979 (valence. -CH ₂ -, -CH ₃ ); 2000-2800 (valency NH ⁺ ); 1816, 1650 (valen. COO ^- ); 1630 (valent. C = N ⁺ pyridine, ring); 1604, 1551, 1460 (-C = C- pyridine, ring); 1400 (deform. Assim. -CH ₃ ); 1430 (scissor. -CH ₂ ); 1388 (deform. Sym. -CH ₃ ); 1350, 1330 (fan, torsional. -CH ₂ -); 1308 (valent. -CO- pyridine ring); 1230 (deform. -C-OH pyridine ring); 1174, 1150, 1047, 1000, 950 (flat, deform. CH pyridine, ring); 900, 843 (deform. COO ^- ); 710 (pendulum -CH ₂ -); 772, 800, 820 (non-flat. Deform. CH pyridine, ring); 633 (valency C-Cl); 512 (deform. C-Cl).

X-ray phase analysis:

Characteristic peaks at (2θ ± 0.2) °: 8.04; 8.18; 11.89; 15.83; 16.40; 16.63; 17.85; 18.64; 19.92; 20.36; 22.56; 23.00; 24.07; 24.36; 25.18; 25.76; 26.09; 26.50; 28.21; 28.90; 29.47; 32.74; 33.08; 33.79; 34.26; 35.96; 36.91; 40.08; 40.64; 41.28; 42.67; 43.80; 43.99; 49.99.

Thermal analysis (Fig. 7): At a temperature of 121.0 ° C, a pronounced endothermic effect is observed corresponding to the melting of the sample (the sample melts without decomposition). At temperatures above 182.0 ° C, the decomposition of salt begins, accompanied by an endothermic effect (maximum at 254.0 ° C). At a temperature of 227-297 ° C, further decomposition of the resulting products occurs with a pronounced endothermic effect (maximum at 283.0 ° C). With a further increase in temperature, the sample oxidizes and burns out.

Example 7. Obtaining dichloroacetate EMHP from acetonitrile.

The experiment is carried out as in example 1, but 25 ml of acetonitrile is used as a solvent. 8.9 g of product (0.033 mol) are obtained. Yield 92.9% of theory.

Elemental analysis:

Calculated,%: C 45.13; H 4.93; N, 5.26; O 18.04; Cl 26.64.

Found,%: C 45.23; H 4.98; N, 5.25; O 17.95; Cl 26.59.

IR (cm ^-1 ): 3250-3700 (valen. -OH, assoc. Group); 3000 (valence Ar-H); 2870-2979 (valence. -CH ₂ -, -CH ₃ ); 2000-2800 (valency NH ⁺ ); 1823, 1650 (valent. COO ^- ); 1630 (valent. C = N ⁺ pyridine, ring); 1600, 1550, 1460 (-C = C- pyridine, ring); 1390 (deform. Assim. -CH ₃ ); 1430 (scissor. -CH ₂ ); 1388 (deform. Sym. -CH ₃ ); 1350, 1330 (fan, torsional. -CH ₂ -); 1311 (valent. -CO- pyridine ring); 1233 (deform. -C-OH pyridine, ring); 1177, 1150, 1050, 1000, 949 (flat, deform. CH pyridine, ring); 900, 840 (deform. COO ^- ); 710 (pendulum -CH ₂ -); 775, 800, 820 (non-flat. Deform. CH pyridine, ring); 627 (valency C-Cl); 509 (deform. C-Cl).

X-ray phase analysis:

Characteristic peaks at (2θ ± 0.2) °: 8.06; 8.28; 11.89; 15.86; 16.30; 16.62; 16.84; 17.84; 18.26; 18.58; 18.70; 19.86; 20.20; 20.40; 22.55; 23.04; 24.00; 24.36; 25.18; 25.44; 25.58; 25.72; 25.98; 26.08; 26.36; 26.52; 28.24; 28.90; 29.26; 29.40; 29.90; 32.70; 33.12; 33.80; 34.06; 36.42; 36.58; 36.94; 37.14; 40.13; 49.96.

Thermal analysis (Fig. 8): At a temperature of 121.0 ° C, a pronounced endothermic effect is observed corresponding to the melting of the sample (the sample melts without decomposition). At temperatures above 181.0 ° C, the decomposition of salt begins, accompanied by an endothermic effect (maximum at 256.0 ° C). At a temperature of 289-337 ° C, further decomposition of the resulting products occurs with a pronounced endothermic effect (maximum at 325.0 ° C). With a further increase in temperature, the sample oxidizes and burns out.

Example 8. Antihypoxic activity of EMHP dichloroacetate in a hermetic volume hypoxia model.

Since the known salts of 2-ethyl-6-methyl-3-hydroxypyridine have an antihypoxic effect, it is likely to suggest the same effect with the new salt.

The antihypoxic activity of EMHP dichloroacetate was studied using a hermetic hypoxia model [Bobkov Yu.G., Losev A.S. and other guidelines for screening and preclinical testing of antihypoxic drugs. - M., 1989. - 20 p.], Against the background of hypoxic injury, the effect of the drug on the functional state of animals was evaluated, namely:

- indicative research behavior under the conditions of the "open field" methodology [Y. Bureshova, O. Bureshova, J.P. Houston. Methods and basic experiments on the study of the brain and behavior. / Ed. Batueva A.S. - M.: Higher School, 1991.- 398 p., Walsh R.N., Cummins R.A. The open-field test: A critical review. // Psychol. Bull. - 1976. - V.83. - P.482-504];

- emotional response according to the method of Brady & Nauta [Brady J.V., Nauta WJ.H. Subcortical mechanisms in emotional behavioral affective changes following septal forebrain lesions in albino rat. // J. Comparative And Phisiol. Psychol. - 1953. - V.46. - N 3. - P.339-341] in the modification for mice [Suslov N.I. Pathogenetic substantiation of the psychopharmacological effects of drugs of natural origin (experimental study): Diss. Dr. med. sciences. / Institute of Pharmacology. - Tomsk, 1995. - 406 p.].

- the ability to develop and reproduce the conditioned reflex of passive avoidance (passive avoidance reaction) [Buresh Y., Bureshova O., Houston J.P. Methods and basic experiments on the study of the brain and behavior. / Ed. Batueva A.S .. - M.: Higher School, 1991. - 398 p.].

The experimental procedure:

Hermetic volume hypoxia model:

Animals were placed in a heat chamber, which is a glass vessel with a calibrated volume of 500 ml (± <1.0%), with a hermetically sealed lid. When the first atonal sigh or a near-death convulsive seizure occurs, the animals are removed and allowed to "breathe". Functional disorders from the side of the central nervous system caused by hypoxic exposure were assessed by the change in the orientation and research behavior in the "open field" and the safety of passive avoidance reaction.

1. The study of indicative research behavior

The experimental setup "open field" was a camera measuring 40 × 40 × 20 cm. The walls of the camera are white; the chamber floor is divided into 16 squares, each of which has a circular hole with a diameter of 3 cm. From above, the chamber was illuminated by an electric incandescent lamp with a power of 100 watts, located at a height of 1 m from the floor. The animal was placed in one of the corners of the experimental setup. We recorded the number of movements from square to square (horizontal activity), the number of upsets on the hind legs (vertical activity), the number of examinations of holes (mink reflex), the number of washings (grooming) and the number of bowel movements (according to the number of fecal balls) in the first minute and in two subsequent ones. The results of the first and two subsequent minutes of the experiment were evaluated separately and in total. Activity in the first minute characterizes instinctive behavior, in the next two minutes

- the degree of adaptation of the animal to changing conditions.

Based on the results of the study, the coefficients of the ratio of the activity of animals in the first and two subsequent minutes of the experiment were calculated: the coefficient of total motor activity (K _SDA ), the coefficient of horizontal activity (K _GOR ), the coefficient of the mink reflex (K _NOR ), which are the ratio of the indicator of animal activity in the first minute of the experiment to its activity in the next two minutes according to relevant indicators. The coefficients of asymmetry of behavior were calculated for the first (K _{ACC (1)} ) and second (K _{ACC (2)} ) observation periods in the form of the ratio of the number of horizontal movements to the total motor activity, expressed as a percentage.

The number of crossed squares characterizes the spontaneous locomotor activity of animals, the sum of the racks and peeping in the holes - the total tentative research activity. The behavior of the animal during grooming reflects the degree of its emotional stress: frequent short-term (up to 5 s)

- An anxiety state, rare and long-lasting - a state of comfort. Vegetative indicators of stress (the number of boluses of bowel movements and urination) also characterize the emotional stress of animals.

2. Emotional reaction according to the method of Brady & Nauta

An emotional reaction was recorded every day twice - before the study of orientational-research behavior and after.

The following indicators were evaluated:

- resistance to capture in the cage where the animal constantly lives;

- reaction to hand grip (when performing this test, the animal was placed on a flat surface and captured in the palm of the hand);

- muscle tone (evaluated when taking the animal in the palm of the hand);

- reaction to the approach of the tweezers (when performing this test, the animal was placed on a flat surface, after which tweezers were brought to his nose to a distance of 0.5 cm, so that the animal could see its approach);

- reaction to the push with tweezers (when performing this test, the animal was placed on a flat surface, after which it was pushed with tweezers; the tweezers were brought up so that the animal did not see its approach);

- defecation and urination (recorded throughout the experiment);

- vocalization (recorded throughout the experiment);

- characteristic tail movement.

The entire procedure was performed for each animal separately. Each indicator was evaluated according to a four-point system (0 points - absence of squeaking and defecation with urination). The indicator was the total score for all test effects without differentiation for individual tests, which gave a general assessment of the emotional reaction.

3. Development and reproduction of passive avoidance reaction

This technique is based on the suppression of the innate reflex of preference for the dark space present in rodents.

The experimental setup was a camera consisting of two compartments: large - lighted and small - dark. The animal was placed in the bright compartment and soon (after 10-20 s), due to the innate reflex of preference for dark space, it passed into the dark compartment. After that, the door connecting both compartments was closed, and an electric current was supplied to the floor of the dark compartment, consisting of parallel alternating electrodes, with pulses of 50 ms duration, 5 Hz frequency, and 50 mA amplitude. After 10 seconds, the door was opened, and the animal could jump out into the bright compartment with the usual floor. As a result of the described procedure, a conditioned reflex of avoiding dark space was developed in animals. When checking the reproducibility of the reflex, the animals were placed in the light compartment at an angle opposite from the entrance to the dark compartment and observed for three minutes. The reflex was considered developed if, within three minutes of observation, the animal had never visited the dark compartment or the time of the first call (latent time of call) exceeded 150 s. The quality of the reflex was judged by the proportion of animals with the presence of a reflex. Animals that, after being placed in the light compartment, remained motionless and did not approach the entrance to the dark compartment, were not taken into account when calculating the results.

Reflex testing was carried out 48 hours after production. On the day of checking the safety of the reflex, the drug was administered to animals to exclude the influence of the phenomenon of dissociation. When checking the reproducibility of the reflex, the animals were placed in the light compartment at an angle opposite from the entrance to the dark compartment and observed for three minutes. The latent time of entry and the total time spent in the dark compartment (time of the dark compartment) were recorded. In addition, the number of animals that retained the developed reflex (did not enter the dark compartment for 150 s) was recorded (Table 1).

The experiment compared the indicators that determine the degree of training of animals and the state of memory - the more the latent period of entry of the animal into the dark compartment increases, the greater the training and preservation of the memorial trail. The total time spent in the dark compartment, recorded during the reproductions of the skill, also reflects the safety of the animal’s memorial trail, the less time the animal spends in the dark compartment, the better it remembers the electric pain irritation caused to it.

EMGP succinate, known as Mexidol, was used as the reference drug.

Studies were conducted on 100 white outbred mice of both sexes. Experimental animals were kept in cells of 6-8 pcs. on a bed of sawdust of coniferous trees under natural light conditions in conditions of free access to water and food. Animals received standard vivar feed. 2 hours before the administration of the test compounds, the food was removed from the cells. Two active doses of 50 mg / kg and 100 mg / kg were investigated. Suspensions obtained from two powder formulations by dissolving in 1% starch mucus were administered intragastrically using an atraumatic probe daily for 4 days. The last administration was carried out on day 5 an hour before hypoxia.

The significance of the differences was judged by testing the probability of the null hypothesis by the Student-Fisher method and by the Wilkinson method.

Quantitative values of the coefficients and indicators characterizing the functional state of animals after hypoxic injury are presented in tables 1-4.

Table 1

; n=10)The influence of the studied substances on the approximate reaction and the preservation of the conditioned reflex of passive avoidance of outbred male mice after suffering a hypoxic injury ( $$\overline{X}\pm m$$

; n = 10) Observation groups, dose Verification (after 48 hours) Latent time of call, s Dark compartment time, s The presence of a reflex Intact control 149.0 ± 19.6 ^# 8.9 ± 5.4 0.8 ± 0.13 ^# Hypoxic control 76.6 ± 12.2 * 10.5 ± 4.1 0.1 ± 0.1 * EMGP succinate, 50 mg / kg 126.4 ± 19.2 15.2 ± 8.3 0.5 ± 0.17 ^# EMGP succinate, 100 mg / kg 105.5 ± 14.5 29.5 ± 12.8 0, b ± 0.16 ^# The inventive substance, 50 mg / kg 126.0 ± 12.1 20.2 ± 12.6 0.5 ± 0.17 * The inventive substance, 100 mg / kg 158.1 ± 13.2 ^# 6.44 ± 3.03 0.9 ± 0.1 * * - the differences are significant with respect to intact control at P≤0.05 ^# - differences are significant in relation to hypoxic control at P≤0.05

- в первую минуту наблюдения

- in the first minute of observation

- во вторую и третью минуты наблюдения

- in the second and third minutes of observation

Table 3 The values of the coefficients of the mink reflex, horizontal activity, total motor activity and asymmetry of behavior Observation groups K _{ACC (1)} ,% K _{ACC (2)} ,% TO _MOUNTAIN TO _NOR TO _SDA Intact control 62,4 54.8 0.52 0.32 0.45 Hypoxic control 73.8 58.7 1,11 0.75 0.89 EMGP succinate, 50 mg / kg 82.6 57.6 0.73 0.30 0.50 EMGP succinate, 100 mg / kg 73,2 55.8 0.82 0.29 0.62 The inventive substance, 50 mg / kg 66.0 57.5 0.64 0.46 0.56 The inventive substance, 100 mg / kg 68.5 57.2 0.57 0.33 0.47

Table 4

; n=12)Indicators of the emotional response of outbred mice according to the Brady & Nauta method after suffering hypoxic injury in arbitrary units (average of four days of observation; $$\overline{X}\pm m$$

; n = 12) Observation groups Total reaction rate Cell capture response Reaction to capture on a flat flat surface Muscle tone Reaction to
approaching tweezers Tweezer response Squeak Defecation Tail Intact control 5.7 ± 0.9 0.40 ± 0.16 1.1 ± 0.31 1.80 ± 0.29 1.40 ± 0.31 0.70 ± 0.21 0.30 ± 0.15 0 0 Hypoxic control 3.11 ± 1.10 * 1.00 ± 0.44 1.56 ± 0.41 0.56 ± 0.24 0.78 ± 0.22 0.56 ± 0.24 0 0 0 EMGP succinate, 100 mg / kg 9.6 ± 1.4 ^# 1.56 ± 0.41 3.00 ± 0.17 2.67 ± 0.33 1.67 ± 0.24 1.22 ± 0.28 0.22 ± 0.22 0 0.33 ± 0.17 The inventive substance, 100 mg / kg 9.0 ± 1.3 ^# 1.44 ± 0.38 2.33 ± 0.29 2.11 ± 0.35 2.11 ± 0.35 1.89 ± 0.2 0 0 0.11 ± 0.11 Notes: * - differences are significant with respect to intact control at P <_0.05 # - differences are significant with respect to hypoxic control at P <_0.05

Results:

In the group of test animals that were injected with EMHP dichloroacetate, at the stage of reproducing the passive avoidance reaction skill, there was an increase in the latent time of animals entering the dark compartment and a decrease in the time spent in it relative to the control (Table 1), the proportion of animals with a reflex 48 hours later was higher , even than in the intact control. Such a positive effect of EMHP dichloroacetate on the learning and memory of animals, the slowing down of the extinction of the commemorative trace in dynamics indicate the presence of a nootropic effect in the claimed substance. The nootropic effect of EMHP dichloroacetate is comparable to the effect of EMHP succinate.

In the “open field” method in animals that were injected with EMHP dichloroacetate, in the second and third minutes of observation, the total motor activity (an indicator characterizing the rate of orientational reactions in animals), the mink reflex, and vertical activity (research activity) increased, while characterizing the emotional stress of animals (grooming, sniffing), restored to intact control (Table 2). These data suggest a positive effect of EMHP dichloracetate on the adaptation of animals after stress and its antihypoxic effect.

In the first minute of observation in animals of the hypoxic control group, the value of the coefficient of behavior asymmetry is significantly higher than the value of a similar coefficient for the group of intact control (Table 3). This circumstance speaks of the observed increase in the level of anxiety and the deterioration of cognitive functions in animals after hypoxic trauma. A group of animals treated with EMHP dichloroacetate showed a decrease in the coefficient of behavior asymmetry to almost the level of intact control (Table 3). That is, the introduction of the claimed substance leads to a decrease in the level of anxiety of animals, which allows us to talk about the anxiolytic effect of dichloroacetate EMHP. This is also confirmed by the analysis of the emotional response of animals to external stimuli after a hypoxic injury (Table 4). The studied substances (EMHP dichloroacetate and EMHP succinate) equally enhanced the emotional response of animals to external stimuli with a clear decrease in the level of emotional response in animals of hypoxic control.

Moreover, the anxiolytic effect is not accompanied by impaired memory (see reproduction of passive avoidance reaction), as, for example, in benzodiazepine tranquilizers. In addition, EMHP dichloroacetate, even at a dose of 100 mg / kg, does not have a sedative effect (it does not inhibit orientative-research behavior in the “open field” method and retains a response to external stimuli). For comparison, in traditional tranquilizers, the anxiolytic effect is accompanied by a sedative effect. So, diazepam at a dose of 2 mg / kg, causing an anxiolytic effect, reduces locomotor activity in an open field by 5 times, and alprazolam (0.5 mg / kg) by 2.8 times [T. Voronina. Antioxidant Mexidol. The main neuropsychotropic effects and mechanism of action. // Psychopharmacology. Biology. Narcology. - 2001. - T.1. - No. 1. - S.2-12].

The values of the coefficients K _GOR , K _NOR and K _SDA for the group receiving EMHP dichloroacetate are comparable with the values of these coefficients of the group of intact animals (Table 3). That is, the introduction of the inventive substance leads to the restoration of the indicative and research functions of animals after a hypoxic injury, which suggests the adaptogenic effect of dichloroacetate EMHP.

The research results indicate the ability of EMHP dichloroacetate to protect the central nervous system from the most common traumatic effects - hypoxia and psychoemotional overstrain and to correct disorders of higher nervous activity (cognitive functions) caused by pathological factors. Thus, the proposed tool exhibits antihypoxic, nootropic (antiamnestic), adaptogenic and anxiolytic effects.

A study of the antihypoxic activity of EMHP dichloroacetate in the presented model showed that the claimed substance has a more pronounced antihypoxic and nootropic effect than the comparison drug EMHP succinate.

Example 9. Antiatherosclerotic and hypolipidemic effect of dichloroacetate EMHP.

The biological effect of EMHP dichloroacetate was studied in three models:

1. experimental hyperlipidemia caused by the introduction of a detergent;

2. experimental atherosclerosis caused by hyperlipidemic diet;

3. experimental lipolysis caused by the introduction of adrenaline.

The studies were carried out in accordance with:

- An experimental study of lipid-lowering and anti-atherosclerotic drugs: guidelines. USSR Ministry of Health, Pharmacological Committee. - M., 1988. - 16 p .;

- Guidelines for the study of hypolipidemic and antiatherosclerotic effects of drugs [Guidelines for preclinical studies of drugs. Part One / Ed. A.N. Mironova. - M .: Grif and K, 2012. - p.445-453];

- Guidelines for the preclinical study of drugs for the correction of diabetes mellitus, obesity and metabolic syndrome [Ibid., S.685-700].

EMGP succinate was used as a comparison drug.

The experimental procedure:

1. Model experimental hyperlipidemia caused by the introduction of detergent

The study was carried out on white male rats weighing 220-240 g.

Hyperlipidemia was caused by the administration of WR-1339 triton at a dose of 250 mg / kg ip once.

Suspensions obtained from two powder formulations (EMHP dichloracetate and EMHP succinate) by dissolving in 1% starch mucus were administered intragastrically using an atraumatic probe at a dose of 20 mg / kg for 10 days before the detergent was introduced. The last introduction of the formulations is simultaneously with the introduction of the detergent.

Blood sampling was carried out after decapitation of the animals 12 hours after the introduction of the detergent. During this period, animals were deprived of food.

The content of total lipids (OL) in the blood serum was determined using the unified method of Zolner, Kirsch, and Knaight; total cholesterol (OX) - Ilk's enzymatic method; triglycerides (TG) - the method of Handel and Zilversmith [Laboratory research methods in the clinic. Handbook / Ed. V.V. Menshikov. - M .: Medicine, 1987. - 368 p.].

Statistical processing of the experimental results was performed according to Student-Fisher.

The results of the study are presented in table 5.

Table 5 Indicators of lipid metabolism in male rats in experimental hyperlipidemia caused by the introduction of detergent Observation groups Serum content, M ± m OL, g / l OH, mmol / l TG, mmol / l Intact control 3.5 ± 0.3 1.5 ± 0.1 2.5 ± 0.1 Hyperlipidemic control 12.5 ± 1.3 5.7 ± 0.4 19.7 ± 2.4 EMGP succinate 4.2 ± 0.4 * 2.1 ± 0.4 * 5.5 ± 0.3 * The inventive substance 4.3 ± 0.5 * 2.4 ± 0.3 * 5.9 ± 0.4 * * - significant difference from animals of the intact control group

2. Model experimental atherosclerosis caused by hyperlipidemic diet

The study was conducted on white male rats weighing 220-240 g. As a model for inducing atherosclerosis in animals, a diet supersaturated with fats was used [Reproduction of diseases in animals for experimental, therapeutic studies / Ed. N.V. Lazarev. - L .: Med-GIZ, 1954. - p. 54-55, 221-225]. Experimental animals received a homogeneous feed of the following composition:

casein - 5%; sunflower oil mixed with pork fat - 40%; glucose - 45%; agar - 2%; salts, vitamins - four%; cholic acid - one%; thiouracil - one%; cholesterol - four%;

in addition, animals were additionally intragastrically injected with a 10% cholesterol oil solution at a dose of 100 mg / kg.

Suspensions obtained from two powder formulations (EMHP dichloracetate and EMHP succinate) by dissolving in 1% starch mucus were administered intragastrically using an atraumatic probe at a dose of 20 mg / kg one hour before cholesterol administration for 1.5 months against the background of the above diet .

Intact animals received vivar feed.

Blood sampling was carried out after decapitation of the animals 18 hours after the last administration of the formulations. Animals killed by decapitation were subjected to postmortem autopsy, a histological examination of the aorta was performed.

The serum content of B-lipoproteins (B-PL) was determined by the turbometric method; free (unesterified) fatty acids (NEFA) - titrometrically, total bilirubin (OB) - spectrophotometrically; phospholipids (PL) - according to the content of total phosphorus; Ol; OH; and TG [Laboratory research methods in the clinic. Handbook / Ed. V.V. Menshikov. - M .: Medicine, 1987. - 368 p., Kolb V.G., Kamyshnikov B.C. Clinical biochemistry. - Minsk: Belarus, 1976. - S. 150-171]. The dynamics of the weight of animals was also recorded.

Statistical processing of the experimental results was performed according to Student-Fisher.

The results of the study are presented in table 6.

Table 6 Lipid metabolism in male rats with experimental atherosclerosis caused by hyperlipidemic diet Serum content, M ± m Observation groups OL, g / l OH, mmol / l TG, mmol / l V-LP, g / l OB, mmol / l FL, mmol / l Atherogenic coefficient, OX / FL Body weight g Intact control 3.3 ± 0.3 1.6 ± 0.1 2.8 ± 0.1 1.9 ± 0.1 3.2 ± 0.4 2.9 ± 0.2 0.55 ± 0.06 220 ± 11 Atherosclerotic control 5.3 ± 0.5 * 3.1 ± 0.3 * 5.0 ± 0.4 * 4.4 ± 0.5 * 5.1 ± 0.3 * 2.0 ± 0.2 * 1.55 ± 0.11 * 308 ± 13 * EMGP succinate 3.5 ± 0.4 * 1.3 ± 0.2 * 2.1 ± 0.2 * 1.5 ± 0.1 * 3.3 ± 0.3 * 3.2 ± 0.2 * 0.41 ± 0.03 * 248
± 9 * The inventive substance 3.8 ± 0.2 * 1.6 ± 0.3 * 2.7 ± 0.5 * 1.6 ± 0.2 * 3.4 ± 0.2 * 3.3 ± 0.4 * 0.42 ± 0.04 * 250 ± 10 * * - significant difference from the animals of the intact control group ^# - significant difference from animals of the experimental control group

3. Experimental adrenaline-induced lipolysis model

The study was conducted on white male rats weighing 200 g.

To stimulate lipolysis, animals were administered adrenaline at a dose of 1.5 mg / kg ip once.

Suspensions obtained from two powder formulations (EMHP dichloroacetate and EMHP succinate) by dissolving in 1% starch mucus were prophylactically administered intragastrically using an atraumatic probe at a dose of 20 mg / kg for 10 days.

2 hours after the last administration of the formulations, adrenaline was administered to the animals. 30 minutes after the adrenaline injection, animal blood was taken after decapitation. The serum content of NEFA was determined.

Statistical processing of the experimental results was performed according to Student-Fisher.

The results of the study are presented in table 7.

Table 7 The content of non-esterified fatty acids in the blood serum of male rats during experimental lipolysis caused by the introduction of adrenaline Observation groups The content of NEFA in serum, mmol / g Intact control 0.52 ± 0.04 Lipolysis control 2.44 ± 0.31 EMGP succinate 1.17 ± 0.10 The inventive substance 1.23 ± 0.11

The introduction of detergent to animals causes them significant hyperlipidemia, as evidenced by a multiple increase in the blood levels of animals of the hyperlipidemic control group OL, OX and TG (Table 5). This is due to the ability of triton WR-1339 to bind blood plasma lipids with the formation of micelles isolated from the action of lipase. In this case, lipid metabolism between blood and tissues is disrupted, resulting from lipid deficiency by the feedback mechanism, stimulates the synthesis of lipids in the liver, which leads to a marked increase in their level in the blood. The introduced formulations (EMHP dichloroacetate and EMHP succinate) equally decreased the content of OL, OX, and TG in the blood of animals after experimental hyperlipidemia (Table 5), which indicates the presence of the lipid-lowering effect of the studied substances.

On the used model of atherosclerosis, it was shown that in the blood of animals of the atherosclerotic control group the content of OL, OX, TG, B-PL, and OB significantly increased; and the PL content, on the contrary, decreased with an increase in the atherogenic coefficient (Table 6). Autopsy of animals from the atherosclerotic control group revealed the presence of pale yellow stripes and spots on the aorta of animals, and atherosclerotic plaques with xanthoma cells were determined during histological examination. In groups of animals treated with the formulation, normalization of lipid metabolism was observed (Table 6). Animals were active, with no signs of obesity. Morphologically, the aorta of the animals treated with the formulation did not differ from the aorta of the animals of the intact control group. Thus, the studied substances have antiatherosclerotic effect

On the model of experimental lipolysis caused by the introduction of adrenaline, it was shown that inhibition of lipolysis plays a role in the mechanism of action of the studied substances, as evidenced by the data presented in table 7.

Example 10. The study of the toxicity of dichloroacetate EMHP. Studies were carried out according to:

- Guidelines for the study of the general toxic effects of pharmacological substances [Guide to the experimental (preclinical) study of new pharmacological substances / Ed. R.Yu. Khabrieva. - 2nd ed., Revised. and add. - M .: Medicine, 2005. - 832 p.];

- Determination of the safety and effectiveness of biologically active food additives [MUK 2.3.2. 721-98. - M .: Federal Center for State Sanitary and Epidemiological Supervision of the Ministry of Health of Russia, 1999. - 87 p.];

- Rules for preclinical safety assessment of pharmacological agents (GLP) [RD 64-126-91. - M .: USSR Ministry of Health, 1992. - 45 p.]; The experimental procedure:

Suspensions obtained from two powder formulations (EMHP dichloroacetate and EMHP succinate) by dissolving in 1% starch mucus were administered intragastrically using an atraumatic probe in increasing doses according to Litchfield-Wilcoxon.

White rats of both sexes of standard weight were used in the studies. Animals were randomly assigned to groups using randomization. Each dose was studied in two experimental groups consisting of 6 animals of the same sex. Equivalent volumes of distilled water were administered intragastrically in a similar number and tender composition to the control groups of animals.

Mortality, time of death of animals were recorded. We described the results of daily observation of the general condition and behavior of animals, the symptoms of poisoning. The observation period was 5 days. Animals that were killed and killed by decapitation underwent postmortem autopsy, and morphological changes in the internal organs were revealed.

Statistical processing of the experimental results was carried out according to Student-Fisher.

The dependence of lethal effects on the doses of the test substances is presented in table 8.

The results of daily observation of the general condition and behavior of animals:

The introduction of the formulation in toxic doses after 30-40 minutes was accompanied by the development of inhibition, hypodynamics, ataxia, tremor, ruffled hair, and hypersalivation. Within 2 hours, attacks of clonicotonic seizures with a violation of the act of breathing developed. The death of animals was observed within 7-9 hours with the appearance of shortness of breath and paralysis. The surviving animals were inhibited, hypodynamic, had an untidy appearance.

Results of pathological dissection of animals:

Morphologically, the internal organs of animals treated with EMHP dichloroacetate did not differ from the internal organs of animals treated with EMHP succinate.

Table 8 Lethal effects (died / total) Dose mg / kg 6000 10,000 14000 20000 30000 40,000 When given to male rats The inventive substance 0/6 0/6 0/6 1/6 3/6 6/6 Succinate 0/6 1/6 3/6 6/6 - - EMGP In the case of iv administration to female rats The inventive substance 0/6 0/6 0/6 1/6 3/6 6/6 EMGP succinate 0/6 1/6 3/6 6/6 - -

At autopsy, the day after acute administration was detected; pleura and chest organs are not changed. Light pale pink, airy, without seals to the touch. The size of the heart is within normal limits. In the cavities of the heart contains a small amount of liquid blood. The heart muscle is dense, brown in color. The stomach is filled with a small amount of solid food. The mucous membrane is shiny, folded, slightly pinkish in color. The mucosa of the small intestine is shiny, smooth, pinkish in color. The size and shape of the liver do not differ from the control. The surface of the liver is smooth. The capsule is thin, transparent. The liver pattern in the section is not changed. Liver tissue is moderately full-blooded. The kidneys are of normal size and shape, brownish in color, dense, with a distinct cortical and medulla on the cut. The thyroid gland, adrenal glands and pancreas do not differ in appearance from the control.

The vessels of the lungs are moderately full-blooded. The epithelium of the alveoli and intrapulmonary bronchi did not represent any changes. The alveoli were filled with air. Edema or pneumonia was not observed. Myofibrils of the left ventricle of the heart and interventricular septum had a distinct transverse striation, the nuclei of cardiomyocytes were light. Myocardial vessels are moderately full-blooded. The lobular structure of the liver persisted. The boundaries of hepatocytes were clear, the cytoplasm of hepatocytes was weakly basophilic, granular; nuclei with sufficient chromatin content and a thin nuclear membrane.

Thus, the results of toxicometry, observation data for experimental animals for 5 days after acute administration and necropsy data allow us to classify the new salt of EMHP to class VI [N. Hodge et al. Clinical Toxicology of Commercial Products. Acute Poisoning Ed. IV, Baltimore, 1975, 427 p.] - relatively harmless drugs. Those. EMHP dichloroacetate has less toxicity than EMHP succinate, classified as class V — practically non-toxic drugs.

Example 11. Determination of the stability of dichloroacetate EMHP.

To determine stability, the sample obtained in example 3 is stored in a dark glass jar under ordinary conditions, in a dark place. EMGP succinate obtained in laboratory conditions by a known method [RU 2210568, publ. 08/20/2003]. The results are shown in table 9, from which it is seen that the inventive substance does not have hygroscopicity and during prolonged storage is not subjected to oxidation with the formation of colored products. A substance similar in structure and properties - EMGP succinate turned out to be significantly less stable.

Table 9 Changing the parameters of samples of EMHP dichloroacetate and EMHP succinate during prolonged storage under normal conditions Parameter Substance Analysis Date 01/02/2006 01/15/2007 02/01/2008 01/16/2009 03/02/2010 02/14/2011 02/06/2012 01/16/2013 09/16/2013 The content of the main substance,% EMHP dichloroacetate 99.8 99.9 99.8 99.8 99.9 99.9 99.8 99.8 99.9 EMGP succinate 99.7 99.6 99.3 98.7 98.1 98.0 97.5 96.9 96.3 The amount of impurities (HPLC),% EMHP dichloroacetate 0.005 0.005 0.006 0.006 0.006 0.006 0.006 0.007 0.007 EMGP succinate 0.008 0.11 0.18 0.21 0.26 0.27 0.33 0.46 0.55 The optical density of a 10% solution in water at 400 nm EMHP dichloroacetate 0.010 0.010 0.011 0.012 0.012 0.012 0.014 0.016 0.016 EMGP succinate 0.015 0.018 0.58 0,099 0.12 0.22 0.58 0.85 0.99 Moisture by Fisher,% EMHP dichloroacetate 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 EMGP succinate 0.05 0.08 0.11 0.15 0.16 0.22 0.66 1.10 1.16

Example 12. Determination of the stability of EMHP dichloroacetate.

1900 ml of water for injection is charged into a 2-liter three-necked flask equipped with a fluoroplastic paddle stirrer. When the stirrer is on, 100 g of EMHP dichloroacetate are added and mixed until completely dissolved. The resulting solution was poured into a 2 L volumetric flask, adjusted to the mark with distilled water, and stirred. Then the solution is filtered through a packet of membrane filters (0.45 and 0.22 μm), poured into 2 ml ampoules of HC-33 glass in a laminar cabinet, sealed and autoclaved at (122 ± 1) ° C for 8 minutes. After viewing the mechanical inclusion receive 995 ampoules.

In a similar manner, an EMHP succinate solution is prepared and amplified; after viewing mechanical inclusions, 982 ampoules are obtained.

Solutions from ampoules are analyzed for color (optical density at 400 nm) immediately after autoclaving and during storage. Ampoules are stored in a dark place under normal conditions.

The results are shown in table 10. From them it follows that a 5% solution of EMHP dichloroacetate is stable during storage; while a 5% solution of EMGP succinate, obtained and stored under identical conditions, is colored, while the color of individual ampoules varies greatly (which is considered marriage by the heterogeneity of the series).

Table 10 Changing the parameters of injection solutions of EMHP dichloroacetate and EMHP succinate during prolonged storage under normal conditions Parameter Test solution Analysis Date Note 01/16/2009 03/02/2010 01/17/2011 02/06/2012 01/16/2013 The content of the main substance,% 5% solution of dichloroacetate EMHP 5.05 ± 0.05 5.05 ± 0.04 5.05 ± 0.05 5.04 ± 0.06 5.02 ± 0.07 5% solution of EMGP succinate 5.03 ± 0.03 4.9 ± 0.08 4.8 ± 0.09 4.7 ± 0.08 4,5 ± 0,09 Solution color (absorbance at 400 nm) 5% solution of dichloroacetate EMHP Not determined 0.01 0.01 0.02 0.02 After 4 years of storage, the solution remained colorless (better than standard Y7, does not differ from water) 5% solution of EMGP succinate 0.02 0.045 ± 0.035 0.086 ± 0.061 0.110 ± 0.09 0.15 ± 0.083 The solution after 3 years of storage is highly colored

Claims (9)

1. Dichloroacetate of 2-ethyl-6-methyl-3-hydroxypyridine of the formula:

2. The substance according to claim 1, having antiatherosclerotic, hypolipidemic, antihypoxic, nootropic, anxiolytic and adaptogenic effects. 3. The stable crystalline form of the substance according to claim 1, which is characterized by a powder X-ray diffraction pattern, with characteristic peaks at the following values of the angle (2θ ± 0.2) °: 8.12; 8.28; 11.89; 15.86; 16.38; 16.64; 16.83; 17.85; 18.22; 18.54; 18.72; 20.20; 20.42; 20.66; 22.58; 23.08; 24.00; 24.36; 25.18; 25.56; 26.12; 26.58; 29.30; 29.56; 29.92; 32.40; 32.69; 33.12; 33.46; 33.68; 33.84; 36.58; 36.92; 39.97; 42.67. 4. The stable crystalline form according to claim 3, characterized by the following x-ray powder diffractogram recorded using a diffractometer (CuK _a anticathode), presented in units of 2θ angle with the following interplanar distances d, corresponding intensities and relative intensities:
Angle (2θ ± 0.2) ° Interplanar distance d, Å Intensity, s ^-1 Relative intensity,% 8.1200 10,87978 454 four 8.2800 10.66990 886 8 11.8938 7.43485 2572 23 15,8610 5.58302 373 3 16,3800 5,40728 3409 thirty 16.6423 5,32264 10853 96
16.8275 5,26447 1564 fourteen 17.8471 4,96595 1383 12 18,2200 4,86514 653 6 18,5400 4.78189 379 3 18,7200 4,73631 398 four 20,2000 4.39251 1289 eleven 20,4200 4,34568 2093 eighteen 20,6600 4,29574 668 6 22,5759 3,93532 925 8 23,0800 3,85050 639 6 24,0000 3.70494 1040 9 24.3624 3,65064 2032 eighteen 25.1812 3,53376 11361 one hundred 25,5600 3,48224 1045 9 26,1200 3,40884 1286 eleven 26,5800 3,35088 824 7 29,3000 3,04570 525 5 29.5600 3,01950 342 3 29,9200 2,98399 354 3 32,4000 2.76102 405 four 32.6922 1,73701 2411 21 33,1200 2.70262 814 7 33,4600 2,67594 376 3 33.6800 2.65896 568 5 33.8400 2,64675 1159 10 36,5800 2.45454 573 5 36.9200 2,43271 935 8 39.9682 2025393 368 3 42.6733 2,11710 378 3 5. A method of obtaining a stable crystalline form according to claim 3, comprising reacting equimolar amounts of 2-ethyl-6-methyl-3-hydroxypyridine and dichloroacetic acid in the presence of a solvent. 6. The method according to claim 5, characterized in that a protic solvent is used as a solvent. 7. The method according to claim 6, characterized in that water or ethanol or 2-propanol is used as a proton solvent. 8. The method according to claim 5, characterized in that the aprotic solvent is used as the solvent. 9. The method according to claim 8, characterized in that acetone or 2-butanone, or dioxane, or acetonitrile, or diethyl ether, or tetrahydrofuran are used as the aprotic solvent.

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