RU2502739C1 - THROMBOCYTE AGGREGATION-INHIBITING N-CARB(GLUTAMINYL)OXYMETHYLIMIDAZO[4,5-e]BENZO[1,2-c;3,4-c']DIFUROXANE - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to N-carb(glutaminyl)oxymethylimidazo[4,5-e]benzo[1,2-c; 3,4-c']difuroxane of formula

.

EFFECT: obtaining a novel compound which can be used as a medicinal preparation which inhibits thrombocyte aggregation.

1 dwg, 1 tbl

Description

The invention relates to pharmacy, in particular to the synthesis of pharmacologically active compounds.

Currently, cardiovascular diseases are steadily taking the first place among the causes of death and disability. The main importance in this group of diseases is arterial insufficiency of the heart and brain, the cause of which is vascular thrombosis.

In some cases, vascular thrombosis leads to excessive activation of the platelet aggregation process. As a result, blood flow to tissues decreases, tissue ischemia develops. The consequence of ischemia is cell death - necrosis. Thrombosis can be the cause of such serious complications as myocardial infarction (coronary artery thrombosis), ischemic stroke (cerebral vascular thrombosis). Venous thrombosis can cause pulmonary embolism.

Given the prevalence and progressive increase in circulatory system pathologies in the world, as well as the relatively high mortality rate of patients, the development of new classes of drugs with antiplatelet activity is relevant.

Antiplatelet agents include drugs of various mechanisms of action, belonging to different groups of chemical compounds.

It is known that as a result of the interaction of nitric oxide (NO) with platelets and leukocytes, their aggregation and adhesion on the walls of blood vessels decrease, which leads to inhibition of thrombogenesis. Disorders associated with the normal course of the above reactions underlie the pathophysiological processes characteristic of the development of various diseases of the cardiovascular system. In such diseases, multiple disturbances in the synthesis of endogenous NO, its reception by the soluble form of guanylate cyclase (rHC), and the regulation of the level of cyclic nucleotides and calcium ions are observed [Dessy C., Ferron O. Pathophysiological Roles of Nitric Oxide: In the Heart and the Coronary Vasculature. Current Medical Chemistry - Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry. 2004. Vol. 3. P.207-216].

To date, the formation of nitric oxide as a result of biotransformation of nitroglycerin and other nitrates, which are used to treat cardiovascular diseases as anti-ischemic and antianginal drugs, has been proven. However, their significant drawback is the emergence of tolerance and other side effects with prolonged use [Granik VG, Grigoryev NB Nitric oxide (NO). A new path to the search for medicines: a monograph. - M.: University Book, 2004, 360 pp.].

In this regard, a search is being made for new compounds capable of forming NO in a living organism by a non-enzymatic pathway. This approach is considered as a relevant and promising direction of creating new, more effective in comparison with previously known antihypertensive and antiaggregant pharmaceuticals with antianginal and anti-ischemic activity.

One of the classes of chemical compounds, derivatives of which are donors of nitric oxide - furoxanes. Furoxans are considered as prodrugs that realize their biological activity through the rHC-cGMP pathway [Granik VG, Grigoryev NB Nitric oxide (NO). A new path to the search for medicines: a monograph. - M.: University Book, 2004, 360 s; Granik V.G., Kaminka M.E., Grigoriev M.B., Severina I.S., Kalinkina M.A., Makarov V.A., Levina V.I. Furoxanopyrimidines as exogenous nitric oxide donors. // Chem. Farm. Journal. 2002, Volume 36, No. 10, pp. 7-11].

In US patent No. 2012/021007 2012, furoxans were screened, however, their anti-aggregation activity was not investigated.

Patents RU 2123046 1998 and RU 2139932 1998 describe furoxans as nitric oxide donors and activators of RHC, but data on their anti-aggregation activity are not presented.

In US patent No. 7838023 2010, it is mentioned that biologically active derivatives of furoxan have anti-aggregation activity, however, no research data are provided.

An object of the present invention is to provide new effective platelet aggregation inhibitors.

The problem is solved H-carb (glutaminyl) oxymethylimidazo [4,5-e] benzo [1,2-s; 3,4-c '] difuroxan inhibiting platelet aggregation.

Synthesis of the target compound

The synthesis of N-carb (glutaminyl) oxymethylimidazo [4,5-e] benzo [1,2-s; 3,4-s'] difuroxan was performed on the solid phase under the conditions of an automatic peptide synthesizer ABI 433A Peptide Synthesizer (AppliedBiosystems, USA), using FastMoc 0.25 strategy. The FastMoc 0.25 strategy involves the sequential addition of amino acid residues to an insoluble polymer substrate. The basic labile group Fmoc - 9-fluorenylmethoxycarbonyl is used to protect the N-groups of each amino acid residue. Residues that have potentially reactive side chains are protected by acid-resistant groups.

After removal of the Fmoc group with piperidine, the following protected amino acid is added using either a coupling reagent or a preactivated amino acid derivative.

Dicyclohexylcarbodiimide (DCC) acts as an activator of the first amino acid in the Fmoc strategy in the reaction of its addition to the resin. The reaction proceeds in the presence of 4-dimethylaminopyridine (DCC / DMAP), which plays the role of a catalyst in the process.

The activator of the second and subsequent amino acids in the Fmoc strategy is 1-hydroxybenzotriazole in dicyclohexylcarbodiimide

(HOBt / DCC). The reaction proceeds with the formation of an activated amino acid and N, N-dicyclohexylurea (DCU).

All of the above operations proceed in a peptide synthesizer. To remove the peptide from the resin in a fume hood in a polypropylene tube, a mixture was prepared with the following ratio of components: TAN - 2.5%, TIPS - 2.5%, EDT - 5%, TFA - 90%. The prepared mixture was placed on ice until cooling for 20-30 minutes. Then, a peptide resin was placed in a cold peptide remover mixture and mixed. The peptide-resin tube was placed in a polypropylene tube, closed and kept on a rocking chair for 4 hours.

Peptide extraction from the resin was carried out on a glass funnel filter in a fume hood. The funnel was pre-rinsed twice with MTBE (methyl tertiary butyl ether). At the end of removal, the mixture with resin and peptide was poured onto a Schott filter. The tube in which the TFA reaction was washed was washed, the wash was poured onto the filter. It was filtered under low vacuum until the resin was dried. Cold MTBE was added, the resin was thoroughly washed and filtered until dry. The tube containing the peptide resin was washed three times with cold MTBE and a wash was added to the main drain. The suspension was thoroughly mixed, left for at least 1 hour at -20 ° C. The precipitate was filtered off and dried.

The peptide was purified using a PuriFlash 450 high performance liquid chromatograph (InterCMm).

HPLC conditions: Interchim PF-C18 cartridge (20g), 15 μm. Other inverted C18 or C8 cartridges may be used.

a. Detector Cell: Preparative

b. Flow rate - 20.0 ml / min

c. Wavelength: range 200-400 nm

d. Detection Range: 2 AUFS

e. Loop size: 2 ml (injection volume 1.5-1.8 ml of sample)

f. Eluent A: 5.0% acetonitrile, 0.1% trifluoroacetic acid in water

g. Eluent B: 0.1% trifluoroacetic acid in acetonitrile

h. Gradient: 10-90% B for 12 min.

The structure of the synthesized compound was confirmed by ¹ H NMR and chromatography-mass spectroscopy. ¹ H NMR spectra were recorded on a Bruker Avance 600 mhz instrument; chemical shifts were measured relative to the solvent signal (DMSO-d ₆ , δ _n 2.5 ppm).

Chromatography-mass spectrometric analysis was performed on a Waters MSD SQD-ESI instrument with UV and mass spectrometric detectors: wavelength 220 nm, sampler temperature 15 ° C, column thermostat temperature 40 ° C. MSD - parameters: source temperature 130 ° С, gas temperature 400 ° С, capillary voltage 3kV; column Waters Acquity 1.7 dt 2.1-50 mm. A gradient of 5 to 100% B in 4 minutes (A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile).

¹ H NMR spectrum (δ, ppm) 1.98 (m, 2H, C (21) H ₂ ); 2.26 (m, 2H, C (22) H ₂ ); 3.86 (m, 1H, C (20) H); 4.22 (m, 1H, C (16) H); 6.94 (m, 2H, NH ₂ ); 7.44 (s, 1H, NH); 8.84 (m, 1H, C (13) H); 9.03 (s, 1H, COOH).

Mass spectrum [MS (ES)] m / z 421.19 [M + H] ⁺

pharmachologic effect

Assessment of the specific activity of anti-aggregation action of K-carb (glutaminyl) oxymethylimidazo [4,5-e] benzo [1,2-s; 3,4-c ′] difuroxan was performed in vitro using the blood of healthy donors. Blood sampling was carried out immediately before the study using sodium citrate (3.8%) as an anticoagulant. The ratio of anticoagulant: blood corresponded to 1: 9.

The anti-aggregation activity of the obtained compound was studied in platelet-rich plasma using adenosine diphosphate (ADP) as an inducer of platelet aggregation.

To prepare platelet-rich plasma, blood immediately after preparation was centrifuged for 10 minutes at 1000 rpm, after which the upper plasma layer was transferred to another tube, and the residue was centrifuged for 20 minutes at 3000 rpm to obtain platelet-free plasma. All procedures were carried out in polystyrene dishes with thromboresistant properties. Throughout the study period, rich and platelet-free plasma was at room temperature, and platelet aggregation was recorded at 37 ° C.

To study the specific anti-aggregation activity of N-carb (glutaminyl) oxymethylimidazo [4,5-e] benzo [1,2-s; 3,4-c '] difuroxane was guided by the requirements for preclinical studies of pharmacological substances of this class, approved by the Pharmacological Service for Supervision of Health and Social Development.

Platelet aggregation was studied using the Born turbidimetric method (Born, 1962), based on the change in light transmission (540 nm) through the plasma under study with constant stirring (1000 rpm). Platelet-free plasma was used as a reference sample. Light transmission through platelet-free plasma was taken as 100%, and light transmission through platelet-rich plasma was taken as 0%. The platelet concentration was adjusted in a platelet-rich plasma to 2.5 · 10 ^-8 cells / ml by dilution with platelet-poor plasma.

A two-channel laser platelet aggregation analyzer / counter 230LA-2 (NPF Biola) was used for the study. The sample volume was 300 μl. The measurement time is 8 minutes. ADP at a concentration of 50 μM was used as an inductor. The resulting aggregograms are the dependence of the degree of aggregation on the time elapsed after the addition of the aggregation inducer. The studied compound (in the form of an aqueous solution, if necessary, containing DMSO up to 0.2%) at various concentrations (0.15 · 10 ^-6 , 0.3 · 10 ^-6 , 0.45 · 10 ^-6 , 0.55 · 10 ^-6 M) was added to the sample making aggregation inducer (ADP). The results of studies on the blood of healthy donors are presented in the table.

Results of a study of the specific anti-aggregation activity of N-carb (glutaminyl) oxymethylimidazo [4,5-e] benzo [1,2-s; 3,4-c '] difuroxane

The concentration of N-carb (glutaminyl) oxymethylimidazo [4,5-e] benzo [1,2-s; 3,4-s'] difuroxane, M % aggregation in the presence of
N-carb (glutaminyl) oxymethylimidazo [4,5-e] benzo [1,2-s; 3,4-c '] difuroxane 0 50.4 0.15 · 10 ^-6 44.1 0.3 · 10 ^-6 30.5 0.45 · 10 ^-6 24.3 0.55 · 10 ^-6 19.2

In the study of the inhibitory effect

N-carb (glutaminyl) oxymethylimidazo [4,5-e] benzo [1,2-s; 3,4-s'] difuroxan on platelet aggregation of healthy donors, an inhibition effect of ADP-induced platelet aggregation was found.

The dependence of platelet aggregation on the concentration of the compound is shown in Fig. 1.

The compound inhibited ADP-induced platelet aggregation with an IC _{50 of} 0.41 μM, which demonstrates a pronounced anti-aggregation effect.

Claims (1)

N-carb (glutaminyl) oxymethylimidazo [4,5-e] benzo [1,2-s; 3,4-s'] difuroxan of the formula

,
inhibiting platelet aggregation.

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