RU2458060C2 - 5-(4'-hydroxyphenyl)-and 5-(2',5'-dihydroxyphenyl)-6 h-1,3,4-thiadiazine-2-amines, hydrohalogenides inhibiting thrombocyte aggregation in intravenous introduction - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to 5-(4'-hydroxyphenyl)- and 5-(2',5'-dihydroxyphenyl)-6H-1,3,4-thiadiazine-2-amines, hydrohalogenides of general formulas

and

inhibiting thrombocyte aggregation in the intravenous introduction NR₁R₁-morpholino-; thiomorpholino-; pyrrolidino-; piperidino-; hexamethyleneimino-, HHal - HCl; HBr.

EFFECT: given compounds may be used for preparing cardiologic drugs and enable better treatment of various cardiovascular diseases, including myocardial infarction and thrombotic apoplexy.

1 cl, 6 tbl, 2 dwg, 10 ex

Description

The invention relates to the field of biologically active compounds and relates to the development of new derivatives of the class of 1,3,4-thiadiazines with antiplatelet action and capable of effectively affect hemostasis - inhibit platelet aggregation.

Platelet aggregation plays an important role in human physiology and pathology, as it is an important etiological moment in the pathogenesis of circulatory disorders (in particular, capillary). Aggregated platelets (glued together) and platelets adhered to the vascular wall serve as the basis for the formation of a blood clot, which leads to cardiovascular diseases (impaired coronary and cerebral circulation, vascular pathology of the limbs, abdominal cavity, etc.), to the development of thrombosis after surgery, circulatory disorders in injuring humans and animals.

The development of effective antiplatelet agents inhibiting platelet aggregation is an important problem of modern medicine, the solution of which will significantly improve the treatment of myocardial infarction, thrombotic stroke (these diseases are the leading cause of death in developed countries). Therefore, modulation of platelet aggregation ability is one of the main directions in the correction of hemostatic system disorders. Currently, the world is actively developing methods for targeted chemical synthesis of new generation antiplatelet agents with high efficiency and selectivity.

In recent years, effective platelet aggregation inhibitors from the group of thienopiperidine derivatives (ticlopidine, clopidogrel) have been developed and introduced into medical practice. They are by far the best available drugs. However, their therapeutic effect develops only on the second day and reaches a maximum only on the 4-7th day of treatment. Ticlopidine often causes complications such as diarrhea, rash, and leukopenia. Both ticlopidine and clopidogrel are not intended for intravenous administration, i.e. cannot be ambulances.

The present draft patent application involves the development of a synthesis of an agent capable of effectively inhibiting platelet aggregation, including when administered intravenously.

State of the art

Various representatives of the thiadiazine class are known, differing in the position of heteroatoms in the thiadiazine ring, which have an effect on the blood coagulation system. So, in the work of [N. Campillo] et al. [1], he noted a decrease in platelet aggregation induced by adenazine diphosphate (ADP), arachidonic acid (AA) and collagen both in vitro and ex vivo when exposed to heteroarylpyrazino [2,3-c] [1,2,6] thiadiazine-2,2-dioxides. However, the antiplatelet properties of these compounds are several times lower than that of the claimed compounds as in the aggregation of platelets of AK, and ADP.

[Rehse K] showed that 2-nitrosoimino-3,6-dihydro-2H-1,3,4-thiadiazines also reduce platelet aggregation induced by collagen [2].

Other studies [Ozcelic A.B.] [3] established antifibrinolytic activity and some inhibition of platelet aggregation of 3-substituted-5- (4-carboxy-cyclohexylmethyl) -tetrahydro-2H-1,3,5-thiadiazine-2-thione.

The patent [O. Chupakhin et al.] Provides convincing evidence of the antiplatelet and anticoagulant effects of 5-aryl-1,3,4-thiadiazin-2 amines [4].

Thus, thiadiazines are a class of compounds on the basis of which it is possible to create effective antiplatelet agents that act on various ways of platelet activation and aggregation.

The aim of this work was to develop synthesis and study the anti-aggregation properties of new groups of the 1,3,4-thiadiazine class: 5- (4′-hydroxyphenyl) - and 5- (2 ′, 5′-dihydroxyphenyl) -6H-1,3,4 -thiadiazine-2-amines, which could satisfy modern medical requirements for drugs of this kind. The drugs must be stable, able to effectively inhibit platelet aggregation, including in the conditions of intravenous administration and, thus, could be ambulances.

The specified technical result is achieved by the fact that according to the invention, hydrobromides of 5- (4′-hydroxyphenyl) -6H-1,3,4-thiadiazin-2 amines of the general formula (I) and hydrochlorides of 5- (2 ′, 5′-dihydroxyphenyl ) -6H-1,3,4-thiadiazin-2 amines of the general formula (II) having antiplatelet activity under the conditions of intravenous administration.

NR ₁ R ₁ = morpholino-; thiomorpholino-; pyrrolidino; piperidino; hexamethyleneimino

HHal = HCl

These compounds can be used in cardiology and will significantly improve the treatment of various cardiovascular diseases, including myocardial infarction and thrombotic stroke.

Close in structure (prototype), in relation to the claimed "5- (4′-hydroxyphenyl) - and 5- (2 ′, 5′-dihydroxyphenyl) -6H-1,3,4-thiadiazin-2 amines, hydrohalides, inhibiting platelet aggregation by intravenous administration are 5-aryl-1,3,4-thiadiazin-2 amines [4] of the general formula III:

where R ₁ = H; Cl; Br; C ₁ -C ₄ alkoxy; C ₁ -C ₄ alkyl groups

NR ₂ R ₂ = cycloalkylamines

The claimed compounds differ from the prototype in that the phenol and hydroquinone residues are introduced in the 5 position of the 1,3,4-thiadiazine ring instead of the aryl residue. The presence of such substituents at position-5 of the thiadiazine ring led to new positive properties of these drugs: a qualitative improvement in the solubility of the compounds, their hydrophilicity and lipophilicity, as well as the presence, due to phenols, of probable antioxidant properties.

All tested compounds inhibited platelet aggregation induced by AK and ADP (Tables 1-4) in a concentration range of 0.001-0.1 mM.

Examples of synthesis and physico-chemical characteristics of the claimed compounds

All compounds were obtained with a yield of 50-80% by condensation of the corresponding α-bromo-2-acetylthiophenes and 4,4-cycloalkylaminoosemicarbazides by heating in absolute ethanol and subsequent precipitation of the reaction products from ether from cooled solutions. The structure of the compounds was proved by ¹ H NMR spectral data. The purity of the compounds is confirmed by elemental analysis, thin layer chromatography.

experimental part

Synthesis of starting compounds: α-bromo-2,5-dihydroxyacetophenone

2 ′, 5′-Dioxyacetophenone - a synton for producing α-bromo-2,5-dihydroxyacetophenone, obtained by heating a mixture of 25 g (13 mmol) of hydroquinone diacetate with 58 g (20 mmol) of anhydrous AlBr ₃ at a temperature of 110-120 ° C to stop the rapid evolution of HBr, then the reaction mass was kept at a temperature of 160-165 ° C for 3 hours. The remainder of AlBr _{3 was} decomposed by the addition of crushed ice, and then by the addition of 125 ml of conc. HCl. Dried in air, crystallized from a large volume of water. Yield 15 g (75%).

A suspension of 14.8 g (66 mmol) of anhydrous CuBr ₂ in 60 ml of dry ethyl acetate was heated until the precipitate was completely dissolved, and a solution of 5 g (33 mol) of 2,5-dioxiacetophenone in 80 ml of ethyl acetate and 80 ml of chloroform was added. Heated with stirring until the dark green color of the solution turns yellow. The hot reaction mass was filtered, the filter cake was washed with a hot mixture of ethyl acetate and chloroform. The solvent was distilled off, the residue was recrystallized from benzene. Yield 5.0 g (66%). Mp 113-115 ° C. Lit. so pl. 112-113 ° C.

α-Bromo-4-hydroxyacetophenone

To 15.6 g (70 mmol) of anhydrous CuBr ₂ in 80 ml of anhydrous ethyl acetate was added 5 g (40 mmol) of 4-hydroxyacetophenone in a mixture of anhydrous, 40 ml of ethyl acetate and 40 ml of chloroform heated to boiling. The suspension was boiled with stirring for 1 h until the color of the precipitate changed from black to colorless CuBr. The precipitate of CuBr was filtered off, the solvent was distilled off, the residue was a crystalline precipitate of a slightly yellow color, and darkened upon standing. Yield 5.2 g (68%). Mp 127-128 ° C (crystallized from dry benzene).

Example 1

2-Morpholino-5- (4′-hydroxyphenyl) -6H-1,3,4-thiadiazine, hydrobromide (1)

To a solution of 1.08 g (5 mmol) of α′-bromo-4-hydroxyacetophenone in 40 ml of absolute ethanol was added 0.81 g (5 mmol) of thiocarbazic acid morpholinide and 0.5 ml of concentrated HBr, boiled for 40 min, the precipitate was filtered off, and the precipitate was filtered off. slightly yellow. Dry diethyl ether was added to the filtrate while cooling, a slightly yellow precipitate formed, was filtered off, dried, combined with the main product and crystallized from absolute ethanol with the addition of a few drops of HBr conc. Yield 1.40 g (78%). Mp 228-229 ° C. R _f = 0.32 (eluent, butanol: acetic acid: water 4: 1: 5). Found,%: C 43.54; H 4.44; N, 11.71. C ₁₃ H ₁₆ BrN ₃ O ₂ S. Calculated,%: C 43.58; H 4.47; N, 11.73; ¹ H NMR (DMSO-d ₆ ), δ, ppm: 3.81 (m, 4H, morpholino), 3.90 (m, 4H, morpholino); 4.37 (s, 2H, CH ₂ S), 6.91 and 7.85 (dd, 4H, C ₆ H ₄ ); 10.05 (br.s, 1H, OH).

Example 2

2-Hexamethyleneimin-1-yl-5- (4′-hydroxyphenyl) -6H-1,3,4-thiadiazine, hydrobromide (2).

To a solution of 8.4 g (40 mmol) of α-bromo-4-hydroxyacetophenone in 100 ml of absolute ethanol was added a solution of 7 g (40 mmol) of 4-hexamethyleneiminothiosemicarbazide in 150 ml of absolute ethanol and 1.5 ml conc. HBr, boiled for 50 minutes. After hot filtration, 350 ml of dry diethyl ether was added to the dark brown filtrate upon cooling, and a slightly yellow precipitate formed, which was filtered off, dried, and recrystallized from absolute ethanol. Yield 6.5 g (44.8%). Mp 221-222 ° C. Found,%: C 48.57; H, 5.48: N, 11.28. C ₁₅ H ₂₀ BrN ₃ OS. Calculated,%: C 48.65; H 5.41; N, 11.35. NMR ¹ H (DMSO-d ₆ ), δ ppm:

1.76 (6H, s, hexamethyleneimin-1-yl), 2.50 (2H, s, hexamethyleneimin-1-yl), 3.88 (4H, s, hexamethyleneimin-1-yl), 4.29 (2H s, CH ₂ S), 7.35 (4H, dd, C ₆ H ₄ ), 10.02 (1H, br.s., NH), 13.32 (1H, br.s, IT).

Example 3

2-Thiomorpholino-5- (4′-hydroxyphenyl) -6H-1,3,4-thiadiazine, hydrobromide (3)

To a solution of 2.16 g (10 mmol) of α-bromo-4-hydroxyacetophenone in 60 ml of absolute ethanol was added 1.77 g (10 mmol) of thiocarbazinic acid thiomorpholide and 1 ml of conc. HBr, boiled for 40 min, the precipitate was filtered off slightly yellow. Under ice-cooling, 100 ml of dry diethyl ether was added to the filtrate, the precipitate formed was filtered off, dried, combined with the main product, and recrystallized from absolute ethanol. Yield 2.51 g (73%). Mp 217-218 ° C. Found,%: C 41.62; H, 4.22; N, 11.34. C ₁₃ H ₁₆ BrN ₃ OS ₂ . Calculated,%: C 41.71; H 4.28; N, 11.23. ¹ H NMR (DMSO-d ₆ ), δ, ppm: 2.85 (4H, m, thiomorpholino), 4.14 (4H, m, thiomorpholino), 4.31 (2H, s, CH ₂ S ), 7.41 (4H, dd, C ₆ H ₄ ), 10.23 (1H, br s, NH), 13.25 (1H, br s, OH).

Example 4

2-Pyrrolidin-1-yl-5- (4-hydroxyphenyl) -6H-1,3,4thiadiazine, hydrobromide (4)

To a solution of 2.16 g (10 mmol) of α-bromo-4-hydroxyacetophenone in 60 ml of absolute ethanol was added 1.45 g (10 mmol) of 4-tetramethyleneiminothiosemicarbazide in 50 ml of absolute ethanol and 0.5 ml of concentrated HBr, boiled for 45 min . A slightly yellow precipitate formed after hot filtration and subsequent cooling, was filtered off, dried, and crystallized from absolute ethanol. Yield 1.65 g (48.4%). Mp 223-224 ° C. R _f = 0.61 (butanol: acetic acid: water 3: 1: 3). Found,%: C 45.42; H 4.94; N, 12.31. C ₁₃ H ₁₆ BrN ₃ OS. Calculated,%: C 45.64; H 4.72; N, 12.36. ¹ H NMR (DMSO-d ₆ ), δ, ppm: 2.04 (4H, m, pyrrolidin-1-yl), 3.76 (4H, m, pyrrolidin-1-yl), 4.14 (2H, s, CH ₂ S), 7.39 (4H, dd, C ₆ H ₄ ). 10.18 (1H, br.s., NH); 13.10 (1H, br.s., NH).

Example 5

2-piperidino-5- (4-hydroxyphenyl) -6H-1,3,4-thiadiazine, hydrobromide (5)

Under mild heating, 1.59 g (10 mmol) of 4-pentamethyleneiminothiosemicarbazide in 40 ml of absolute ethanol was dissolved and 2.16 g (10 mmol) of α-bromo-4-hydroxyacetophenone and 0.5 ml of concentrated HBr were added, boiled for 40 min, filtered the precipitate was slightly yellow, dried and crystallized from absolute ethanol. Yield 1.81 g (50.6%). Mp 253-255 ° C. R _f = 0.52 (butanol: acetic acid: water 4: 1: 5). Found,%: C 47.44; H 5.04; N, 12.02. C ₁₄ H ₁₈ BrN ₃ OS. Calculated,%: C 47.24; H, 5.12; N, 11.84. ¹ H NMR (DMSO-d ₆ ), δ, ppm: 1.81 (6H, m, piperidino), 3.92 (4H, m, piperidino), 4.27 (2H, s, CH ₂ S ), 7.18 (4H, dd, C ₆ H ₄ ), 10.15 (1H, br.s., NH), 13.11 (1H, br.s, OH).

Example 6

2-Morpholino-5- (2 ′, 5′-dihydroxyphenyl) -1,3,4-thiadiazine, hydrochloride (6)

To a solution of 1.62 g (7 mmol) of α′-bromoquinacetophenone in 30 ml of ethanol was added 1.13 g (7 mmol) of thiocarbazinic acid morpholide, boiled for 1 hour. The precipitated bright yellow precipitate was 2-morpholino-5- (2 ′, 5′-dihydroxyphenyl) -1,3,4-thiadiazine, the hydrobromide was filtered off, washed with ethanol. Yield 1.3 g (47%). Mp ° C (decomp.). To obtain A-38 hydrochloride, 0.4 g of A-38 hydrobromide was dissolved in 15 ml of hot water, cooled to 25 ° C, and 7% aqueous ammonia was added to pH 5-6. The precipitated thiadiazine base was dissolved in a minimum amount of conc. HCl. After cooling with ice, 2-morpholino-5- (2 ′, 5′-dihydroxyphenyl) -6H-1,3,4-thiadiazine hydrochloride was isolated. Mp 242 ° C (from water). Found,% C 46.80; H 4.76; N, 12.75. C ₁₃ H ₁₆ ClN ₃ SO ₃ . Calculated,%: C 47.34; H 4.85; N, 12.74. ¹ H NMR (DMSO-d ₆ ), δ, ppm: 4.2 (s, 2H, SCH ₂ ), 3.9 (m, 4H, morpholino), 3.7 (m, 4H, morpholino), 5 2-6.6 (br.s, 2H, OH); 6.8-7.8 (m, 3H, C ₆ H ₃ ).

Example 7

2-Hexamethyleneimin-1-yl-5- (2 ′, 5′-dihydroxyphenyl) -6H-1,3,4-thiadiazine, hydrobromide (7)

To 100 ml of concentrated HBr was added 6.9 g (30 mmol) of α-bromo-2,5-dioxiacetophenone and 5.1 g (30 mmol) of 4-hexamethyleneiminothiosemicarbazide, heated for 1 h at 80 ° C. The precipitated crystalline product was filtered off and crystallized from water. Yield 8.0 g (80%). Mp 232 ° C. Found,%: C 45.61; H 4.90; N, 11.41. C ₁₅ H ₂₀ BrN ₃ O ₂ S. Calculated,%: C 46.63; H 5.22; N, 10.87. ¹ H NMR (DMSO-d ₆ ), δ, ppm: 1.81 (6H, s, hexamethyleneimin-1-yl), 2.5 (2H, s, hexamethyleneimin-1-yl), 3.88 (4H, s, hexamethyleneimin-1-yl), 4.24 (2H, s, CH ₂ S), 6.80-6.95 (3H, m, C ₆ H ₃ ), 9.0-10.2 (2H, broad, s., OH).

Example 8

2-Thiomorpholino-5- (2 ′, 5′-dihydroxyphenyl) -6H, -1,3,4-thiadiazine, hydrobromide (8)

To 100 ml conc. HBr was added 6.9 g (30 mmol) of α-bromo-2,5-dioxiacetophenone and 5.3 g (30 mmol) of thiocarbazinic acid thiomorpholide, heated at 80 ° C for one hour. The crystalline precipitate formed by heating was filtered off and crystallized with coal from water acidified with HBr to pH 4. Yield 7.0 g (64%). Mp 214-215 ° C. Found,%: C 40.15; H 4.40; N, 10.77. C ₁₃ H ₁₆ BrN ₃ O ₂ S ₂ . Calculated: C 40.0; H 4.13; N, 10.76. ¹ H NMR (DMSO-d ₆ ), δ, ppm: 2.85 (4H, m, thiomorpholino), 4.15 (4H, m, thiomorpholino), 4.3 (2H, s. CH ₂ S ), 6.70-7.05 (3H, m, C ₆ H ₃ ), 8.50-9.40 (2H, br.s., OH).

Example 9

2-Pyrrolidin-1-yl-5- (2 ′, 5′-dihydroxyphenyl) -6H-1,3,4-thiadazine, hydrochloride (9)

With gentle heating, 2.9 g (20 mmol) of 4-tetramethyleneiminothiosemicarbazide were dissolved in 100 ml conc. HCl and 4.6 g (20 mmol) of α-bromo-2,5-dioxiacetophenone were added to a colorless solution, heated for 30 min at 80 ° C. The yellow precipitate was cooled and filtered off and crystallized from water acidified to pH 5. Yield 5.0 g (80%). Mp 228-229 ° C. Found,%: C 49.44; H 4.98; N 13.16. C ₁₃ H ₁₆ ClN ₃ O ₂ S. Calculated,%: C 49.75; H 5.14; N, 13.49. ¹ H NMR (DMSO-d ₆ ), δ, ppm: 2.08 (4H, br.s., pyrrolidino), 3.82 (4H, s, pyrrolidino), 4.18 (2H, s, CH ₂ S), 6.80-6.81 (3H, m, C ₆ H ₃ ), 9.06 (1H, br.s., OH), 10.6 (1H, br.s, OH) .

Example 10

2-Piperidino-5- (2 ′, 5′-dihydroxyphenyl) -6H-1,3,4-thiadiazine, hydrobromide (10)

To a solution of 2.4 g (15 mmol) of 4-pentamethyleneiminothiosemicarbazide in 50 ml of isopropyl alcohol, 3.4 g (15 mmol) of α-bromo-2,5-dioxiacetophenone in 50 ml of isopropyl alcohol was added, the mixture was refluxed for 1 h. yellow color was filtered and washed with chilled isopropyl alcohol, dried and recrystallized from acidified HBr water (pH 4), dried at 100 ° C. Yield 4.5 g (80%). Found,%: C 45.23; H 5.02; N, 11.32. C ₁₄ H ₁₈ BrN ₃ O ₂ S. Calculated,%: C 45.16; H 4.87; N, 11.29. NMR ¹ H (DMSO-d ₆ ), δ, ppm; 1.76 (6H, s, piperidino), 3.87 (4H, s, piperidino), 4.24 (2H, s, CH ₂ S), 6.80-6.94 (3H, m, C ₆ N ₃ ), 8.90 (1H, br.s., OH), 11.18 (1H, br.s., OH).

2. The study of antiplatelet activity in vitro

Materials and methods:

Working solutions of the studied compounds were prepared by diluting them in distilled water at room temperature or by heating to 50-70 ° C until the required concentration was reached.

Experiments to study the anti-aggregation ability of the compounds were performed using venous blood from healthy donors (n = 50), which was obtained by puncture of the cubital vein and stabilized with a 3.8% solution of sodium citrate in a ratio of 9: 1. To prepare platelet-rich plasma, blood was centrifuged for 10 minutes at 1000 rpm, after which the upper plasma layer was transferred to another tube, and the residue was re-centrifuged for 20 minutes at 3000 rpm to obtain platelet-poor plasma. Platelet aggregation was studied on an aggregometer of the company Chrono-Log Corporation (USA) according to the method of G. Born [5]. For this purpose, 250 μl of platelet-rich plasma was placed in the cuvette of the device. The same volume of platelet-free plasma was used as optical control. The degree of aggregation was judged by the maximum value of the optical density drop after the end of the reaction (A _max ) compared with the initial value. The experiments were carried out as follows: in a cuvette containing 250 μl of platelet-rich plasma, 28 μl of the studied drug was added and the resulting mixture was incubated for 5 min at 37 ° C and platelet aggregation was induced. In control experiments, only a proaggregant was added to the platelet-rich plasma. Arachidonic acid (AK) at a final concentration of 1 × 10 ^-3 M and ADP (Boehringer Mannheim, Germany) at a final concentration of 1 × 10 ^-5 M, arachidonic acid (AK), free of oxidation products, were used as proaggregants in the work. provided by the staff of the laboratory of oxilipids of the Institute of bioorganic chemistry named after M.M.Shemyakin and Yu.A. Ovchinnikov of the Russian Academy of Sciences.

The effect of 5- (4′-hydroxyphenyl) -6H-1,3,4-thiadiazin-2 amines (1-5) on in vitro aggregation of human platelets

- The effect on platelet-mediated aggregation of platelets of 1,3,4-thiadiazines (1-5), containing hydroxyphenyl as a substituent at position-5 of the thiadiazine ring, was studied (Table 1).

As can be seen from the table, all the 1,3,4-thiadiazines studied have antiaggregatory activity, but their activity is different and depends on the substituent at position-2 of the thiadiazine ring.

So, the presence of pyrrolidine in the structure of the substance leads to a sharp decrease in anti-aggregation activity. Compound 4 inhibited AK-induced platelet aggregation only at the maximum studied concentrations (0.25-0.1 mM). Replacing an oxygen atom with a sulfur atom in the morpholine cycle also worsened antiaggregation activity: the H-75 substance significantly reduced platelet interaction at concentrations of 0.25-0.1 mM. At the same time, in a minimum effective concentration (0.25 tM), N-75 inhibited platelet aggregation by a factor of 2 compared with the control experiment.

Substances 1, 2, and 5 inhibited platelet aggregation most effectively. Compounds 1 and 2 reduced aggregation by half compared with the control in concentrations of less than 0.1 mM and less than 0.025 mM, respectively. The anti-aggregation effect of compound 5 continued when it was used in lower concentrations (up to 0.001 mM).

Thus, the most effective in the case of AK-induced aggregation was a substance containing piperidine in its structure: 2-piperidino-5- (4′-hydroxyphenyl) -6H-1,3,4-thiadiazine, hydrobromide (5)

Compounds 3 and 4 inhibited AK-induced platelet aggregation only at the maximum studied concentrations (0.25-0.1 mM).

Table 1 Effect of 5- (4′-hydroxyphenyl) -6H-1,3,4-thiadiazin-2 amines (1-5) on human platelet aggregation induced by arachidonic acid (AK 1 × 10 ^-3 M) in vitro (A _max , %) No. Test substance AK 1 × 10 ^-3 M Final concentration, mM 0.25 0.1 0.05 0,025 0.01 0.001 one

54.5 ± 1.7 4,5 ± 1,1 * 25.2 ± 2.3 * - 35.9 ± 2.7 * 45.6 ± 2.8 - 2

73.3 ± 1.8 0 * 15.0 ± 3.2 * 46.3 ± 4.0 * 57.3 ± 5.4 * 57.7 ± 4.1 * 61.7 ± 4.2 * 3

68.6 ± 4.1 1.5 ± 0.9 * 29.3 ± 5.3 * 55.3 ± 4.3 - 61.2 ± 2.3 63.3 ± 4.5 four

69.9 ± 2.6 0 * 18.3 ± 5.5 * 66.4 ± 3.7 - 59.1 ± 3.6 68.8 ± 2.7 5

65.5 ± 2.6 0 * 6.8 ± 1.8 * 7.7 ± 2.1 * - 49.5 ± 3.4 * 55.7 ± 2.3 * * - significant in relation to control (p <0.05)

- The effect on ADP-induced platelet aggregation of 1,3,4-thiadiazines (1-5) containing hydroxyphenyl as a substituent in the thiadiazine ring was studied (Table 2).

table 2 The effect of 5- (4′-hydroxyphenyl) -6H-1,3,4-thiadiazin-2 amines (1-5) on human platelet aggregation induced by ADP (ADP 1 × 10 ^-5 M) in vitro (A _max ,% ) Test substance ADP 1 × 10 ^-5 M Final concentration, mM one 0.5 0.1 0.01 0.001 one

57.7 ± 3.2 35.0 ± 1.9 * - 36.8 ± 1.9 * 45.6 ± 2.4 * - 2

73.7 ± 2.4 34.0 ± 2.7 * 44.3 ± 3.8 * 59.8 ± 2.4 * 67.1 ± 2.1 66.4 ± 3.7 3

59.7 ± 3.7 43.4 ± 3.9 * 43.3 ± 2.5 * 51.4 ± 3.4 51.8 ± 4.1 51.1 ± 3.1 four

64.9 ± 2.0 25.7 ± 2.5 * 36.9 ± 3.1 * 43.6 ± 2.6 * 44.9 ± 2.7 * 42.8 ± 2.3 * 5

64.2 ± 3.7 23.0 ± 2.2 * 32.5 ± 1.5 * 43.2 ± 2.4 * 46.7 ± 2.2 * 46.7 ± 1.9 * • - significantly with respect to control (p <0.05)

Compound 5 proved to be the most effective in the case of inducing platelet aggregation of ADP (table 2). At the maximum studied concentration (1 mM), it inhibited platelet interaction by more than two times compared with the control value. A significant decrease in ADP-induced aggregation was observed over the entire concentration range of compound 5 (0.001-1 mM), and even at minimal doses, this compound reduced platelet activity by 20-22%.

Another compound having a pronounced anti-aggregation effect on ADP-induced aggregation was compound 4, which showed activity comparable to compound 5.

Thiadiazines containing morpholine, thiomorpholine, hexamethyleneimine in their structure were less effective and significantly inhibited ADP-induced aggregation at concentrations of 0.1-1 tM and 0.5-1 mM, respectively.

Thus, among all the studied compounds containing hydroxyphenyl in their structure, the most effective, in the case of ADP-induced aggregation, are the leader compounds:

2-pyrrolidino-5- (4′-hydroxyphenyl) -6H-1,3,4-thiadiazine, hydrobromide (4) and

2-piperidino-5- (4′-hydroxyphenyl) -6H-1,3,4-thiadiazine, hydrobromide (5).

The effect of 5- (2 ′, 5′-dihydroxyphenyl) -6H-1,3,4-thiadiazin-2 amines (6-10) on in vitro aggregation of human platelets

- The effect on platelet-mediated aggregation of platelets of 1,3,4-thiadiazines (6-10) containing dihydroxyphenyl as a substituent at position-5 of the thiadiazine ring was studied (Table 3).

Table 3 The effect of 5- (2 ′, 5′-dihydroxyphenyl) -6H-1,3,4-thiadiazin-2-amines (6-10) on human platelet aggregation induced by arachidonic acid (AK 1 × 10 ^-3 M) in vitro (A _max ,%) No. Test substance AK 1 × 10 ^-3 M Final concentration, mM 0.25 0.1 0.05 0,025 0.01 0.001 6

74.2 ± 2.0 0 8.1 ± 1.8 * 37.6 ± 8.4 * 41.3 ± 5.3 * 66.1 ± 1.8 - 7

70.5 ± 2.6 0 * 0 * 0.4 ± 0.2 * 10.6 ± 4.1 * 54.0 ± 1.8 * 73.3 ± 1.8 8

77.5 ± 1.8 0 * 0 * 2.0 ± 1.1 * 11.6 ± 3.1 * 64.7 ± 3.0 * 68.9 ± 2.6 9

71.6 ± 2.0 0 * 2.4 ± 1.2 * 5.1 ± 1.3 * 45.3 ± 4.5 * 69.3 ± 2.6 71.5 ± 2.3 10

75.9 ± 2.5 0 1.2 ± 0.8 * 2.9 ± 1.1 * 19.1 ± 4.6 * 64.2 ± 3.0 * 75.6 ± 1.9 * - significant in relation to control (p <0.05)

- The effect on ADP-induced platelet aggregation of 1,3,4-thiadiazines (6-10) containing dihydroxyphenyl as a substituent at position-5 of the thiadiazine ring was studied (Table 4).

Table 4 The effect of 5- (2 ′, 5′-dihydroxyphenyl) -6H-1,3,4-thiadiazin-2 amines (6-10) on human platelet aggregation induced by (ADP 1 × 10 ^-5 M) in vitro (A _max ,%) Test substance ADP 1 × 10 ^-5 M Final concentration, mM one 0.5 0.1 0.01 0.001 6

61.8 ± 1.6 22.6 ± 2.0 29.1 ± 1.9 * 40.4 ± 2.0 * 44.3 ± 1.7 * 42.8 ± 1.2 * 7

62.4 ± 1.9 - 34.9 ± 1.4 * 41.8 ± 2.0 * 46.7 ± 1.4 * 48.6 ± 1.9 * 8

58.5 ± 2.0 36.8 ± 2.2 * 39.7 ± 0.9 * 40.3 ± 1.9 * 46.5 ± 1.7 * 51.3 ± 1.8 9

68.6 ± 2.5 37.5 ± 2.3 44.7 ± 1.9 45.4 ± 2.5 * 47.8 ± 3.1 * 46.0 ± 3.4 * 10

64.9 ± 2.0 37.7 ± 1.1 * 40.8 ± 1.1 * 41.6 ± 1.5 * 46.9 ± 1.8 * 48.1 ± 1.6 * * - significant in relation to control (p <0.05)

All investigated 1,3,4-thiadiazines (6-10), containing dihydroxyphenyl in their structure, had a close-effective anti-aggregation effect, both in the case of AK- and in the case of ADP-induced aggregation. All compounds inhibited AK-induced aggregation at concentrations of 0.01-0.25 mM and ADP-induced aggregation at concentrations of 0.001-1 mM. The maximum anti-aggregation effect (a 2-fold decrease in aggregation) was observed for leader compound 6 at a concentration of 0.5 mM and 1.7 times for all other compounds at a concentration of 1-0.5 mM.

Thus, in the case of AK and ADP-induced platelet aggregation, the presence in the chemical structure of morpholine, pyrrolidine, hexamethyleneimine, thiomorpholine or piperidine cycles does not significantly affect the antiaggregation activity of 5- (2 ′, 5′-hydroxyphenyl) -6H-1 , 3,4-thiadiazin-2 amines (6-10).

- The integral parameters of plasma hemostasis of rabbit blood were studied for two representatives (1 and 6) of different groups of the studied compounds.

Table 5 The effect of 2-morpholino-5- (4′-hydroxyphenyl) -6H-1,3,4-thiadiazine, hydrobromide (1) (20 mg / kg, w / w) on the integral parameters of rabbit blood plasma hemostasis Test indicator Time after administration, h 0 (control) 1 hour 2 hours 3 hours 4 hours 24 hours APTT, s 16.63 ± 1.51 16.30 ± 0.98 16.19 ± 1.61 17.48 ± 2.49 15.77 ± 2.17 16.13 ± 1.46 Prothrombin time, s 13.61 ± 0.40 13.64 ± 0.55 13.27 ± 0.64 12.91 ± 0.57 * 12.80 ± 0.74 * 12.30 ± 0.84 * Thrombin time, s 14.35 ± 1.99 14.43 ± 1.13 14.67 ± 1.45 13.85 ± 0.97 13.86 ± 2.31 12.25 ± 0.92 The concentration of fibrinogen, g / l 2.86 ± 0.44 3.18 ± 0.93 3.33 ± 1.30 3.15 ± 0.45 3.11 ± 0.99 3.08 ± 0.49 * - significant in relation to control (p <0.05)

A significant decrease in ADP-induced aggregation was observed 4 hours after injection of compound 1. In this case, platelet aggregation decreased by 7.5% relative to the initial level. A day after the introduction, the aggregation index was restored (table 5, Fig. 1).

With bolus intravenous administration to rabbits of compound 1 at a dose of 20 mg / kg, a tendency to shorten prothrombin time was established (table 4). A significant decrease in thrombin time was noted from 14.35 ± 1.99 s in the control to 13.86 ± 2.31 s 4 hours after injection. Further, the thrombin time continued to shorten, one day after administration, this indicator was 12.25 ± 0.92 s.

With a bolus of intravenous administration to rabbits of compound 6 at a dose of 20 mg / kg, in contrast to compound 1, a significant elongation of thrombin time by 20% was noted compared with the initial level 4 hours after injection (table 6, Fig. 2)

Table 6 Effect of 2-morpholino-5- (2 ′, 5′-dihydroxyphenyl) -6H-1,3,4-thiadiazine, hydrobromide (6) (20 mg / kg, iv) on the integral parameters of plasma hemostasis in rabbit blood Parameter Before administration (control) 20 min after administration 1 hour after administration 2 hours after administration 3 hours after administration 4 hours after administration APTT, s 16.4 ± 0.5 12.6 ± 1.8 15.5 ± 0.9 15.5 ± 1.0 15.7 ± 1.1 16.7 ± 1.6 Prothrombin time, s 11.3 ± 1.0 10.2 ± 0.9 11.5 ± 0.9 11.0 ± 1.0 10.6 ± 0.9 11.2 ± 1.0 Thrombin time, s 29.1 ± 0.8 27.6 ± 2.3 30.6 ± 1.5 29.8 ± 1.4 28.3 ± 0.9 35.1 ± 0.7 * The concentration of fibrinogen, g / l 2.7 ± 0.2 2.4 ± 0.2 2.5 ± 0.2 2.4 ± 0.2 2.4 ± 0.9 2.1 ± 0.1 * - significant in relation to control (p <0.05)

Industrial Applicability of the Invention

These compounds can be used as pharmacologically active substances in the manufacture of drugs that can effectively and fairly quickly inhibit thrombus formation, which is extremely important in the treatment of cardiovascular diseases. As medications, they can be used for intravenous administration and can be emergency medicines.

Brief Description of the Drawings

Fig. 1 - effect of 2-morpholino-5- (4′-hydroxyphenyl) -6H-1,3,4-thiadiazine, hydrobromide (1) on ADP- and AK-induced aggregation of rabbit platelets at a dose of 20 mg / kg, ex vivo (aggregates: ADP at a final concentration of 1 × 10 ^-5 M; AK at a final concentration of 1 × 10 ^-3 M).

Fig. 2 - effect of 2-morpholino-5- (2 ′, 5′-hydroxyphenyl) -6H-1,3,4-thiadiazine, hydrobromide (6) (20 mg / kg, w / w) on the thrombin time “ex vivo. "

Claims (1)

5- (4′-hydroxyphenyl) - and 5- (2 ′, 5′-dihydroxyphenyl) -6H-1,3,4-thiadiazin-2-amines, hydrohalides that inhibit platelet aggregation upon intravenous administration

NR ₁ R ₁ = morpholino-; thiomorpholino-; pyrrolidino; piperidino; hexamethyleneimino
HHal = HCl; HBr.

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