RU2522198C2 - 2-(1s,2r,5s)-6,6-dimethylbicyclo[3.1.1]hept-2yl]methyl}thionyl)ethanoic acid, having antiaggregant action - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to the chemistry of sulphur-containing and terpene compounds and specifically to 2-({[(1S,2R,5S)-6,6-dimethylbicyclo[3.1.1]hept-2-yl]methyl}thionyl)ethanoic acid of structural formula I:

. 2-({[(1S,2R,5S)-6,6-dimethylbicyclo[3.1.1]hept-2-yl]methyl}thionyl)ethanoic acid can also be used in medical practice as an agent for preventing haemophilia.

EFFECT: obtaining a novel compound which can be used in medicine as an agent having antiaggregant action, for preserving blood preparations and preventing thrombosis.

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Description

The invention relates to the chemistry of sulfur-containing and terpene compounds, in particular to terpenesulfoxide of the formula I, which can be used in medical practice as an anti-aggregation agent for the preservation of blood products and the prevention of thrombosis.

Known drugs with anti-aggregation action (2-CYCLOalkyl-amino-5-thienyl-1,3,4-thiadiazines hydrobromides possessing anti-aggregate action / Chupakhin ON [and others]; applicant and patent holder State educational institution "Ural State Technical University - UPI"; application 2008130297/04, 07/22/2008; published on 01/20/2010), (Pat. .F. [Et al.]; Applicant and patentee Saratov State Medical Qing Institute - No. 93035343/14, filed 07/07/1993, publ. 07/20/1995), (Pat. 2186782 RF, C07J 53/00, C07J 63/00, C07D 271/08, C07D 271/12, A61K 31 / 50, A61K 31/4245, A61K 31/56. INCLUSION COMPLEXES OF 1,2,5-OXADIAZOL-2-OXIDE DERIVATIVES WITH POLYCYCLIC DERIVATIVES OF GLUCOPYRANOSIS, METHOD FOR PRODUCING THEM, PHARMACEUTICAL AND COMPOSITION [Application; and patent holder, Faculty of Biology, Moscow State University M.V. Lomonosov - No. 200111367/04, declared 01.16.2001, publ. 08/10/2002), (Pat. 2189241 of the Russian Federation, A61K 35/78. Means "PATRIMIN", POSSESSING SEDATIVE, HYPOTENSIVE, ANTIKOAGULANT, ANTI-AGGREGATION AND HYPOLIPIDEMIC ACTION [Patient and. Dr. Kol. Prostanorm - No. 20011117333/14, declared 2001.06.26, published 2002.09.20).

In the course of many years of research, it was found that the main factor initiating the blood coagulation process is a change in the structure of cell membranes in contact with blood (Interaction of human factor X with tissue thromboplastin / Kiselev S.V., Zubairov D.M., Timerbaev V.N. // Biomedical Chemistry. - 2003, - Т.49, - №5, - С.443-450; The value of the structure of cell membranes in the initiation of blood coagulation // Abstract of dissertation for the degree of Doctor of Medical Sciences. - Publishing House of KSMU , - 2005, - 41 pp.). These changes are induced by various physiological and pathological agents, leading to a violation of the asymmetry of the cell membrane, the appearance of mesomorphic structures and activation of specific receptors. Therefore, the search for substances that contribute to the impact on these processes and allow them to be corrected is the main task of creating fundamentally new drugs. Modification of natural terpenes by introducing sulfur-containing biologically active fragments followed by oxidation of the sulfide function allows to obtain a new type of antiplatelet agent.

Structural analogues of the claimed compound are known: methyl ({[1S, 2R, 5S, R _S ) -6,6-dimethylbicyclo [3.1.1] hept-2-yl] methyl} sulfinyl) acetate and 2 - ({[((1S, 2R, 5S) -6,6-dimethyl-bicyclo [3.1.1] hept-2-yl] methyl} sulfinyl) ethanol (Synthesis and antifungal activity of sulfides, sulfoxides, sulfones based on β-pinene / Nikitina L.E. [ and others] // HFZh, - T.44, - No. 3, - 2010, - S.17-20).

The purpose of the invention is a new effective compound with anti-aggregation action, and a method for its preparation.

The goal is achieved by the invention, namely 2 - ({[(1S, 2R, 5S) -6,6-dimethylbicyclo [3.1.1] hept-2-yl] methyl} sulfinyl) ethanoic acid (formula I), which has anti-aggregation action. The method for producing 2 - ({[(1S, 2R, 5S) -6,6-dimethylbicyclo [3.1.1] hept-2-yl] methyl} sulfinyl) ethanoic acid consists in the oxidation of terpenesulfide 2 - ({[((1S, 2R , 5S) -6,6-dimethylbicyclo [3.1.1] hept-2-yl] methyl} thio) ethanoic acid with hydrogen peroxide in glacial acetic acid. 2 - ({[(1S, 2R, 5S) -6,6-Dimethylbicyclo [3.1.1] hept-2-yl] methyl} thio) ethanoic acid was previously obtained by a known method in the reaction of (-) - β-pinene with mercaptoacetic acid (Synthesis and antifungal activity of sulfides, sulfoxides, sulfones based on β-pinene / Nikitina L.E. [et al.] // HFZh, - T.44, - No. 3, - 2010, - P.17-20 )

The claimed compound is 2 - ({[(1S, 2R, 5S) -6,6-dimethylbicyclo [3.1.1] hept-2-yl] methyl} sulfinyl) ethanoic acid is a new compound and its properties are not described in the literature.

An example of a specific implementation.

The method for preparing compound 2 - ({[(1S, 2R, 5S) -6,6-dimethylbicyclo [3.1.1] hept-2-yl] methyl} sulfinyl) ethanoic acid consists in the oxidation of terpenesulfide - 2 - ({[((1S , 2R, 5S) -6,6-dimethylbicyclo [3.1.1] hept-2-yl] methyl} thio) ethanoic acid with 27% hydrogen peroxide in glacial acetic acid with stirring for 38 hours, with a gradual increase in temperature from 5 ° C to room.

To a solution cooled to 5 ° C, 0.4 g (20 mmol) of 2 - ({[(1S, 2R, 5S) -6,6-dimethylbicyclo [3.1.1] hept-2-yl] methyl} thio) ethanoic acid in 15 ml of glacial acetic acid was added dropwise with stirring 0.4 ml (40 mmol) of hydrogen peroxide (27%). The reaction mixture was stirred 38 h (TLC control) at room temperature, then poured into water, extracted with ether. The ether extract was washed with a saturated solution of K ₂ CO ₃ , water until neutral, extracted from water with ether. The organic layer was dried with MgSO ₄ . After distillation of the solvent, the product was isolated by column chromatography on silica gel (eluent — acetone). Received 62% of the product - 2 - ({[(1S, 2R, 5S) -6,6-dimethylbicyclo [3.1.1] hept-2-yl] methyl] sulfinyl) ethanoic acid in the form of a white powder (t _pl. = 88 ° C).

¹ H NMR Spectrum (300 MHz, CDCl ₃ , δ, ppm): 1.10 s. (3H, H-9); 1.30 s (3H, H-8); 1.29-1.31 m. (1H, H-7); 1.62-1.69 m. (1H, H-3); 1.92-2.39 m. (5H, H-1, H-2, H-3, 2H-4); 2.41-2.48 (m, 1H, H-7); 2.62-2.72 (m, 1H, H-5); 2.80-3.10 (m, 2H, H-10); 4.20-4.30 (m, 2H, SOC H ₂ ).

Elemental analysis: Found,%: C - 60.7; H 6.3; O - 20.5; S - 13.4. C ₁₂ H ₂₂ O ₃ S. Calculated,%: C - 60.2; H 6.3; O - 20.1; S - 13.3.

We have established the anti-aggregation activity of 2 - ({[((1S, 2R, 5S) -6,6-dimethyl-bicyclo [3.1.1] hept-2-yl] methyl} sulfinyl) ethanoic acid, which allows its use for the preservation of blood products and thrombosis prophylaxis.

The possibility of using the obtained substance for the correction of hemostasis was determined in vitro on human blood plasma. Venous blood was taken into a plastic centrifuge tube with a volume of more than 10 ml with an anticoagulant solution (130 mm sodium citrate). The blood was stabilized with sodium citrate in a ratio of 1: 9 and slowly mixed. It was then centrifuged at 1400 rpm for 10 minutes to obtain platelet-rich plasma. After sampling 2.5 ml of the supernatant, the remaining blood was centrifuged for 20 min at 3000 rpm to obtain platelet-poor plasma, which was used to dilute platelet-rich plasma to a fixed volume concentration of platelets and to determine coagulation hemostasis.

To assess the hemostasis system, platelet aggregation rate determination and surface-dependent standard coagulation tests were used: activated partial thromboplastin time (APTT), prothrombin and thrombin time with calculation of the International Normalized Ratio (MHO). Platelet aggregation activity was determined using a Chrono-Log Corporation)) analyzer (USA) according to the G. Born method. To do this, 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 compared to the initial value. Coagulation activity was determined on a Minilab-7001 coagulometer (Russia).

Venous blood of patients with coronary heart disease (CHD) was used to determine spontaneous platelet aggregation and plasma coagulation activity, and induced platelet aggregation was studied on plasma obtained from healthy donors. To this end, 0.05 ml of a solution containing 17 mM of the drug in 8% ethanol was added to 0.45 ml of plasma, and the resulting mixture was incubated for 5 min at 37 ° C. In control experiments, the solvent used to prepare the preparation (8% solution of ethyl alcohol) was added to the plasma. To induce platelet aggregation, the following solutions were used: ADP (5 μM), adrenaline (10 μM), collagen (2 μg / ml), arachidonic acid (0.5 mm) and ristomycin (1 mg / ml).

The research results were processed using the statistical package Microsoft Excel. Statistical analysis of the data included an assessment of the significance of differences between the compared values by Student t-test (p <0.05).

It follows from the results that the obtained substance has a high selective anti-aggregation activity: it completely suppresses spontaneous platelet aggregation (Table 1) and arachidonic acid-induced aggregation (Table 2), and also significantly (6.5 times) reduces aggregation caused by collagen (table 2). In addition, the coagulation activity of plasma is reduced: APTT is normalized. prothrombin time and MHO increase (Table 1). However, at the same time, our compound did not affect the activity of thrombin (thrombin time does not change). This means that the obtained substance inhibits the activation of coagulation factors and does not affect their enzymatic activity.

Table 1 The effect of 2 - ({[(1S, 2R, 5S) -6,6-dimethylbicyclo [3.1.1] hept-2-yl] methyl} sulfinyl) ethanoic acid on platelet aggregation and in vitro coagulation hemostasis in patients with IHD Hemostasis indicators Plasma without drug, n = 31 Plasma with the drug, n = 31 P Spontaneous aggregation rate (rel. Units / min) 0.718 ± 0.587 0.042 ± 0.0091 <0.001 Normal rate no more than 0,05 P <0.001 > 0.05 The degree of aggregation (units) 3.32 ± 0.87 1.29 ± 0.24 <0.001 Normal rate no more than 1,35 P <0.001 > 0.05 APTT (s) 25.83 ± 2.21 30.6 ± 1.51 <0.01 Normal rate 32.7 ± 3.11 P <0.01 > 0.05 Prothroma. time (s) 19.02 ± 2.11 24.8 ± 1.81 <0.01 Normal rate 12.8 ± 2.11 <0.01 <0.01 Prothrombin index (%) 76.38 ± 8.91 56.92 ± 7.58 <0.001 Normal rate 85-100% > 0.05 <0.001 Mho 1.22 ± 0.14 1.54 ± 0.11 <0.01 Normal rate 1.1 ± 0.09 P > 0.05 <0.001 Thrombin time (s) 15.1 ± 0.98 15.3 ± 0.66 > 0.05 Normal rate 14.6 ± 0.79 P > 0.05 > 0.05

table 2 The effect of 2 - ({[(1S, 2R, 5S) -6,6-dimethylbicyclo [3.1.1] hept-2-yl] methyl} sulfinyl) ethanoic acid on platelet-induced in vitro aggregation Plasma without drug, n = 29 Plasma with the drug, n = 29 R ADP Induction (%) 54.41 ± 1.56 49.33 ± 1.31 <0.01 Normal rate 50-75% P > 0.05 > 0.05 Adrenaline Induction (%) 66.5 ± 2.50 40.33 ± 7.31 <0.001 Normal rate 60-71% P > 0.05 <0.001 Induction of arachidonic acid (%) 65.5 ± 3.50 1.2 ± 0.9 <0.001 Normal rate 62-69% P > 0.05 <0.001 Collagen Induction (%) 63.33 ± 10.21 9.8 ± 4.1 <0.001 Normal rate 50-75% P > 0.05 <0.001 Ristomycin induction (%) 66.66 ± 6.31 70.66 ± 1.32 > 0.05 Normal rate 50-75% R > 0.05 > 0.05

The proposed substance by its chemical nature has no analogues among anti-aggregation drugs. Therefore, acetylsalicylic acid and clopidogrel (plavica) were chosen as the comparison drug as the most effective antiaggregation drugs currently used in clinical practice. Table 3 shows the results of induced platelet aggregation in the presence of drugs.

Table 3 Comparative characteristics of 2 - ({[(1S, 2R, 5S) -6,6-dimethylbicyclo [3.1.11 hept-2-yl] methyl} sulfinyl) ethanoic acid, and clopidogrel A drug Acetylsalicylic acid, n = 19 P The proposed connection, n = 21 P Clopidogrel. n = 17 Application Location in vivo, blockade of prostaglandin H synthetase in platelets and megakaryocytes in vitro, platelet collagen receptor antagonist Prodrug, in vivo, platelet ADP receptor antagonist ADP Induction (%) 46.3 ± 9.1 > 0.05 49.33 ± 1.31 <0.01 35.1 ± 3.2 Normal rate 50-75% P > 0.05 > 0.05 <0.001 Adrenaline Induction (%) 36.3 ± 3.1 > 0.05 33.7 ± 2.1 <0.01 51.3 ± 0.7 Normal rate 50-68% P <0.01 <0.01 > 0.05 Induction of arachidonic acid (%) 24.6 ± 2.4 <0.01 1.1 ± 0.9 <0.01 64.6 ± 2.8 Normal rate 60-75% R <0.01 <0.01 > 0.05 Collagen Induction (%) 38.4 ± 14.6 <0.01 9.8 ± 4.1 <0.01 33.1 ± 10.7 Normal rate 50-75% P > 0.05 <0.001 <0.01 Ristomycin Induction (%) 48.2 ± 19.1 > 0.05 70.66 ± 1.32 <0.01 56.4 ± 9.8 Normal rate 50-75% P > 0.05 > 0.05 > 0.05

In the case of ADP-induced aggregation, our compound has the same anti-aggregation effect with acetylsalicylic acid, but 1.4 times less than clopidogrel. With collagen and arachidonic platelet activation, the proposed substance has a more significant effect, both in comparison with acetylsalicylic acid (3.9 times), and with clopidogrel (3.4 times). Thus, it follows from the presented study that a feature of our compound is its ability to selectively block platelet aggregation caused by collagen, which is known to be a stronger inducer than ADP.

Collagen-induced platelet activation occurs primarily through their P2X1 receptors. At the same time, membrane-bound enzymes of phospholipase A2 and C are activated, which leads to the release of arachidonic acid from phospholipids. Arachidonic acid under the action of the cyclooxygenase enzyme is converted into prostaglandins G2 and H2, which inhibit the activity of this enzyme and lead to the formation of thromboxane A2. Thromboxane A2 is an ionophore for calcium ions and causes an increase in their intracellular concentration. These processes cause a change in the structure of the cell membrane of platelets, and they acquire both the ability to adhesion and aggregation, and thrombogenic properties, which, in turn, leads to the activation of plasma hemostasis. Collagen is known to be a stronger inducer than ADP. Therefore, the resulting compound can be very promising for the development of fundamentally new drugs used to prevent thrombophilia.

Considering the ability of 2 - ({[(1S, 2R, 5S) -6,6-dimethylbicyclo [3.1.1] hept-2-yl] methyl} sulfinyl) ethanoic acid to completely suppress spontaneous platelet activation and low toxicity of sulfur-containing terpenes (Synthesis and antifungal activity of the compounds of the pinane series / Nikitina L.E. [et al.] // HFZh, - 2009, - T.43, - Issue 5, - S.20-23), the obtained substance can be used as a stabilizer of drugs blood.

Claims (1)

2 - ({[(1S, 2R, 5S) -6,6-dimethylbicyclo [3.1.1] hept-2-yl] methyl} sulfinyl) ethanoic acid of the formula I:

possessing antiaggregatory action.