RU2552529C1 - Vasopressor agent - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention refers to N, S-substituted isothiourea derivatives of the general formula (1) in the form of salts with pharmacologically acceptable acids as a NOS-inhibitory and vasopressor agent

wherein: n=1, 3, i.e. an acyl substitute represents cyclobutanoyl or cyclohexanoyl; R represents an alkyl group: C₂H₅, i-C₃H₇; HX represents a pharmacologically acceptable inorganic or organic acid: HCl, HBr, HI, hydrogen methyl sulphate, oxalic, succinic acids, etc.

EFFECT: extending the range of products of the vasopressor action.

6 cl, 7 tbl, 10 ex

Description

The invention relates to the field of meeting the vital needs of a person, namely to medicines that can find application in the pharmaceutical industry and medicine.

The development of new effective means and methods of treating hypotension (conditions with low blood pressure) remains an urgent task. For example, in North America, only septic shock develops annually in 700 thousand people, which, despite the high level of medicine on this continent, leads to the death of 30-50% of such patients (Ward R.A., Bosmann M.A. historical perspective on sepsis // Am. J. Pathol. 2012. v.181, N.1. p. 2-7). The difficulties in the treatment of such conditions are associated not only with their severity, but also with the shortcomings of available drugs.

In the treatment of hypotension, an important role is played by drugs that affect blood pressure by increasing vascular tone - vasopressor drugs. Α ₁ -adrenomimetics (norepinephrine, dopamine, epinephrine, phenylephrine, midodrin, etc.) and peptide vasoconstrictors (vasopressin, terlipressin, angiotensin, octapressin, etc.) are currently used as such drugs (Mashkovsky M.D. 2 vol., 14th ed. / M .: New Wave, 2002; Brunton L., Chabner B., Knollman W. Goodman & Gilman’s The Pharmacological Basis of Therapeutics / 12th ed. NY: McGraw Hill Professional, 2010, 1808 pp).

The common disadvantages of these drugs are the short duration of action and the presence of many side effects in many of them, especially in adrenergic agonists. Thus, with the use of adrenomimetics, along with an increase in vascular tone, the development of arrhythmias and metabolic acidosis, central nervous system excitation, and in the case of peptide vasopressors, deepening of myocardial ischemia (Delmas A., Leone M., Rousseau S. et al. Clinical review: Vasopressin and terlipressin in septic shock patients // Crit. Care. 2005. v.9, N.2. p. 212-22; Boerma EC, Ince C. The role of vasoactive agents in the resuscitation of microvascular perfusion and tissue oxygenation in critically ill patients // Intensive Care Med. 2010. v. 36, N 12. p. 2004-18; Ferquson-Myrthil N. Vasopressor use in adult patients // Cardiol. Rev. 2012. v.20, N.3. p .153-58).

According to pharmacological properties, the prototype is the drug mesatone (phenylephrine) - 1- (m-hydroxyphenyl) -2-methylaminoethanol hydrochloride (Mashkovsky M.D. T.1. S.233-234). It causes a narrowing of arterioles and increases blood pressure, selectively acting on α ₁ -adrenoreceptors.

The disadvantage of mesatone, like other vasopressors, is the short duration (not more than 20 minutes), which reduces its therapeutic effectiveness and significantly limits the possibility of its use, for example, in the provision of emergency, emergency care at the prehospital stage, as well as in the treatment of severe shock conditions (Avakyan O.M. Pharmacological regulation of the function of adrenergic receptors. / M .: Medicine, 1988. 256 s .; Volkov V.E., Volkov S.V. Shock. Sepsis. Multiple organ dysfunction. / Cheboksary: PBUYUL L. Naumov. , 2009.348 s.).

The technical result of the invention is to create new vasopressor drugs with a significantly longer duration of antihypertensive action, compared with the existing prototype, which can significantly expand the treatment of hypotonic conditions, especially when providing emergency medical care and treatment of critical conditions.

In recent decades, it has been shown that the tone of blood vessels depends on the activity of the synthesis of nitric oxide (NO) in the vascular endothelium (Dudzinski D., Michel T. Life history of eNOS: Partners and pathway // Cardiovasc. Res. 2007. v.75, N.2. P. 247-60; Bian K., Doursout MF, Murad F. Vascular system: role of nitric oxide in cardiovascular diseases // J. Clin. Hypertens. (Greenwich). 2008. v.10, N. 4, p. 304-10). In this regard, a vasopressor effect can be expected in chemical compounds with a special biochemical property, namely, the ability to inhibit the catalytic activity of NO synthases (NOS) - enzymes that synthesize nitric oxide.

Previously, the authors of the invention showed that some linear and cyclic N, S-disubstituted isothioureas are able to inhibit NOS and exhibit a vasopressor effect in various hypotonic conditions (Proskuryakov S.Ya., Kucherenko N.G., Filimonova M.V. et al. NO -inhibiting and vasotropic activity of some compounds containing a thioamidine group. // Bull. expert biol. honey. 2002. t.134, No. 10. S.393-96; Proskuryakov S.Ya., Filimonova M.V., Verkhovsky Yu.G. et al. Effect of nitric oxide synthase inhibitor, 2-ADT on endotoxin inducer bath changes in the hemodynamics and respiration of rats // Bull.Expert Biol.med. 2004. v.138, No. 10. p.446-49; Filimonova M.V., Shevchenko L.I., Surinova V.I. and et al. Effect of N-propionyl-S-isopropyl-isothiourea on the production of biogenic nitric oxide (NO) and hemodynamic parameters of animals with endotoxic shock // Issues of Biol. Medical and Pharmaceutical Chemistry. 2011. No. 9. p.35- 9; Filimonova M.V., Trofimova T.P., Borisova G.S. et al. Antihypertensive activity of N-2-acetylamino-5,6-dihydro-4H-1,3-thiazine. // Chem.-farm. Journal. 2012. V.46, No.3. S.39-41). The synthesis experience we acquired (Filimonova M.V., Shevchenko L.I., Makarchuk V.M., Shevchuk A.S., Lushnikova G.A. Vasoconstrictor agent. Patent for invention No. 2475479. 2013) revealed a group of compounds possessing more favorable pharmacological properties than mesatone and available vasopressor drugs.

The essence of the invention lies in the synthesis of new N, S-substituted derivatives of isothioureas of the general formula (1) in the form of salts with pharmacologically acceptable acids and in their use as inhibitors of NOS and vasopressor agents.

The term "pharmacologically acceptable acids" covers all pharmacologically acceptable acids, both inorganic (for example, hydrochloric, hydrobromic, phosphoric, sulfuric, etc.), and organic (for example, oxalic, succinic, tartaric, methylsulfuric, salicylic, etc. .).

The synthesized agent exhibits a pronounced and prolonged vasopressor effect due to inhibition of the catalytic activity of NOS.

Enumeration of figures.

Figure 1. Structural Formula 1.

, в которой: n=1, 3, то есть ацильный заместитель имеет циклическую углеводородную насыщенную группу с 4 или 6 кольцевыми атомами углерода, и представляет собой циклобутаноил и циклогексаноил;

in which: n = 1, 3, that is, the acyl substituent has a cyclic hydrocarbon saturated group with 4 or 6 ring carbon atoms, and is cyclobutanoyl and cyclohexanoyl;

R represents an alkyl group: C ₂ H ₅ , iC ₃ H ₇ ;

HX is a pharmacologically acceptable acid: HCl, HBr, HI, methyl sulphate, oxalic, tartaric, succinic, etc., as described above.

Figure 2. The effect of mesatone in a dose of 0.5 mg / kg on the dynamics of systolic (BP) and diastolic (BP) blood pressure of animals in a state of severe hemorrhagic shock: 1 - indicators of blood pressure _from control animals (not receiving "treatment"), 2 - indicators of blood pressure _D control animals, 3 - indicators of blood pressure _From animals treated with mesatone, 4 - indicators of blood pressure _{D of} animals treated with mesatone.

Figure 3. Effect of compound T-1020 at 10 mg / kg on the dynamics of the animals blood pressure in a state of severe hemorrhagic shock: 1 - BP readings _to control animals, 2 - BP readings _D control animals 5 - indicators BP _C of animals receiving T-1020, 6 - indicators of blood pressure _D animals receiving T-1020.

Figure 4. The effect of T-1049 compound in a dose of 10 mg / kg on the dynamics of the animals blood pressure in a state of severe hemorrhagic shock: 1 - BP readings _to control animals, 2 - BP readings _D control animals 7 - indicators BP _C of animals receiving T-1049, 8 - indicators of blood pressure _D animals receiving T-1049.

Figure 5. The effect of T-1059 compound in a dose of 10 mg / kg on the dynamics of the animals blood pressure in a state of severe hemorrhagic shock: 1 - BP readings _to control animals, 2 - BP readings _D control animals 9 - indicators BP _C of animals receiving T-1059, 10 - indicators of blood pressure _D animals receiving T-1059.

The synthesis of salts of N, S-substituted isothioureas of the formula (1)

The compounds of formula (1) according to the invention are prepared using methods known in the art for the preparation of similar substances. The basis for obtaining compounds of formula (1) is the reaction, which consists in the fact that the thiourea of the formula (TM), in which n = 1, 3,

they are reacted with an equivalent amount or excess (up to 200%) of the corresponding alkylating agent of formula RX (in which R represents an alkyl group C ₂ H ₅ or iC ₃ H ₇ ; and X represents a leaving group such as a halide, sulfate, alkyl sulfonate, arylsulfonate , triflate, alkyl sulfate, alkyl phosphate, dialkyl phosphate, acetate) in a polar inert solvent (such as dimethylformamide, dimethyl sulfoxide, sulfolane, acetone, acetonitrile) at room temperature to the boiling point of the reaction mixture.

Intermediate thioureas of the formula (TM) (1-cyclobutanoylthiourea and 1-cyclohexanoylthiourea) are obtained by acylation of thiourea with an equimolar amount of the corresponding carbonyl chloride in toluene solution.

The structures of the compounds and their purity are confirmed by the data of chemical, elemental and spectral analysis. The reaction progress and the purity of the obtained compounds are monitored by thin-layer chromatography on Silufol UV-254 plates in an ammonia-chloroform-isopropanol system. PMR spectra were obtained on a Bruker DRX-500 spectrometer at a frequency of 500 MHz. Examples of the synthesis of compounds of formula (1)

Example 1. Synthesis of 1-cyclobutanoyl-2-isopropylisothiourea hydrobromide (hereinafter T-1049).

A mixture of 2.0 g (12.6 mmol) of 1-cyclobutanoylthiourea, 3.0 g (25.2 mmol) of isopropyl bromide and 8.0 ml of dry acetonitrile in a sealed ampoule was heated in a boiling water bath for 30 hours. The solvent was evaporated, the residue was filtered off and recrystallized from 4-methyl-2-pentanone. Yield 1.0 g (39%). T _mp 118-120 ° C.

PMR spectrum (DMSO-d ₆ , δ): 1.3 (t, 6H); 1.95 (q, 2H); 2.2 (m, 4H); 3.8 (s, 1H); 4.1 (q, 1H); 10.9 (w, 3H).

Calculated,%: C 38.43; H 6.09; N, 9.96.

Found,%: C 38.52; H 6.27; N, 10.14.

Example 2. Synthesis of 1-cyclobutanoyl-2-ethylisothiourea hydrobromide (hereinafter T-1020).

A mixture of 2.0 g (12.6 mmol) of 1-cyclobutanoylthiourea, 2.8 g (26 mmol) of ethyl bromide and 7 ml of dry acetonitrile in a sealed ampoule was heated in a boiling water bath for 15 hours. The solvent was evaporated, the residue was filtered off and recrystallized twice from 4-methyl-2-pentanone. Yield 2.5 g (73.5%). T _mp 120-123 ° C.

PMR spectrum (DMSO-d ₆ , δ): 1.3 (t, 3H); 1.95 (q, 2H); 3.28 (q, 2H); 3.38 (m, 1H); 11.0 (w, 3H).

Calculated,%: C 35.96; H 5.66; N, 10.48.

Found,%: C 36.19; H 5.78; N, 10.48.

Example 3. Synthesis of 1-cyclohexanoyl-2-ethylisothiourea hydrobromide (hereinafter T-1059).

A mixture of 3.7 g (20 mmol) of 1-cyclohexanoylthiourea, 4.4 g (40 mmol) of ethyl bromide and 10 ml of dry acetonitrile was heated in a sealed ampoule in a boiling water bath for 20 hours. The precipitate was filtered off and recrystallized twice from 4-methyl-2-pentanone. Yield 2.8 g (48.5%). T _mp 123-125 ° C.

PMR spectrum (DMSO-d ₆ , δ): 1.3 (m, 9H); 1.62-1.84 (m, 5H); 3.65 (m, 1H); 10.8 (w, 3H).

Calculated,%: C 40.68; H 6.49; N, 9.5. C ₁₀ H ₁₉ BrN ₂ OS.

Found,%: C 40.6; H 6.56; N, 9.44.

Example 4. Synthesis of 1-cyclohexanoyl-2-isopropylisothiourea hydrobromide (hereinafter T-1064).

A mixture of 3.7 g (20 mmol) of 1-cyclohexanoylthiourea, 4.8 g (40 mmol) of isopropyl bromide and 10 ml of dry acetonitrile was heated on a boiling water bath in a sealed ampoule for 70 hours. The precipitate was filtered off, washed with a large volume of ether and recrystallized from 4-methyl-2-pentanone. Yield 2.0 g (36%). T _pl, 163-165 ° C.

PMR spectrum (DMSO-d ₆ , δ): 1.3 (m, 11H); 1.63-1.84 (m, 5H); 4.1 (q, 1H); 10.9 (w, 3H).

Calculated,%: C 42.71; H 6.84; N, 9.05. C ₁₁ H ₂₁ BrN ₂ OS.

Found,%: C 42.77; H 7.0; N, 9.06.

Another aspect of the invention is the presence in the compounds of formula (1) of the ability to inhibit the catalytic activity of NO synthases (NOS), enzymes that synthesize nitric oxide in biological tissues, which is confirmed by the results of biological studies.

Example 5. The evaluation of the NOS-inhibitory activity of the compounds of formula (1) presented in examples 1, 2, 3 and 4 was carried out in vitro experiments on isolated NOS isoforms by radiometric method according to the accumulation rate of [ ³ H] -L-citrulline (van Eijk N.M., Luiking YC, Deutz NE Method using stable isotopes to measure nitric oxide synthesis in the L-arginine / NO pathway in health and disease // J. Chromatogr. In Analyt. Technol. Biomed. Life Sci. 2007. v .851, N.1-2. P.172-85). In this case, recombinant isoforms of human NOS (Alexis Biochemicals) and a set of reagents NOSdetect Assay Kit (Alexis Biochemicals) were used. The measurements were carried out on a radiometric complex Wollac-1414 (Vinspectral).

The results of the studies indicate that all of the claimed compounds exhibit the ability to inhibit NOS. Moreover, as shown in Table 1, the obtained compounds have significant selectivity (selectivity) for inducible (iNOS) and endothelial (eNOS) isoforms, suppressing them 8-20 times more actively than the neuronal isoform (nNOS).

The selectivity of the compounds of formula (1) is essential to achieve the technical result of the present invention, since it provides high vasopressor activity and minimal risks of side effects (caused by nNOS suppression), such as increased myocardial excitability, skeletal muscle hypertonia, disorders of the central nervous system (Bian K. et al. Vascular system: role of nitric oxide in cardiovascular diseases // J. Clin. Hypertens. (Greenwich). 2008. v.10, N.4. P. 304-10; Alderton WK et al. Nitric oxide synthases: structure, function and inhibition // Biochem. J. 2001. v.357, Pt.3. p.593-615).

Table 1 Concentrations of compounds of formula (1) inhibiting 50% activity of various NOS isoforms Compound nNOS iNOS eNOS micromole mg / l micromole mg / l micromole mg / l T-1020 32 8.5 2.7 0.7 3.8 1,0 T-1049 38 10.7 2,5 0.7 3,1 0.9 T-1059 37 10.9 1.8 0.5 2,3 0.7 T-1064 41 12.7 4.7 1,5 6.5 2.0

A necessary condition for achieving the technical result of this invention is the determination of the toxic properties of the compounds of formula (1).

Example 6. The toxicity of compounds (1) was studied on outbred white male mice weighing 21-23 g according to the standard acute toxicity test with a single injection. The substances were dissolved in physiological saline and administered intraperitoneally. The observation period for the experimental animals was 14 days. Assessment of toxicity was carried out according to the Litchfield-Wilcoxon method (Litchfild JT, Wilcoxon FA Asimplified method of evaluating dose effect experiments // J. Pharm. Exp. Ther. 1947. v.95. P.99-113). The research results showed that the LD _{50 of the} compounds of formula (1) is 400-700 mg / kg (Table 2).

table 2 Indicators of acute toxicity of the compounds of formula (1). Chemical compound LD ₅₀ (LD ₁₆ ÷ LD ₈₄ ), mg / kg T-1020 542 (136 ÷ 1150) T-1049 708 (528 ÷ 868) T-1059 380 (246 ÷ 475) T-1064 650 (324 ÷ 962)

In accordance with the classification of toxicity of chemicals, the compounds of general formula (1) belong to the classes of moderately toxic and low toxicity. Moreover, the toxicity of the claimed compounds is significantly lower than that of adrenergic agonists used in the treatment of hypotonic conditions (for example, with this method of administration, LD _{50 of} mesatone is 191 mg / kg). In addition, a comparison of the range of toxic and NOS-inhibiting doses (presented in Table 1) indicates that the test substances are capable of providing a high suppression of the eNOS and iNOS activity necessary for the development of a vasopressor effect, even at doses 50-100 times lower minimum lethal doses (LD ₁₆ ).

To study the vasopressor properties of the claimed compounds, we used an experimental model of hemorrhagic shock caused by acute, massive blood loss (47-50% of the circulating blood volume for 5-8 minutes), similar in its course to severe clinical forms of hypotension.

The studies were performed on male Wistar rats weighing 300-400 g. Before surgery, the animal was anesthetized with sodium thiopental (60 mg / kg, ip). Then a tracheostomy was installed, a jugular vein and both carotid arteries were catheterized, an invasive blood pressure sensor, ECG recording electrodes were connected. 100 units of heparin were administered intravenously. After stabilization condition of the animal via RM-6000 polygraph (Nihon Kohden) baseline recorded heart rate (HR), respiratory rate (RR), systolic and diastolic blood pressure (BP _C and BP _D) in the left carotid artery. To create severe hemorrhagic shock from the right carotid artery, blood was taken in a volume of 2.5 ml per 100 g of animal mass. At the end of the blood loss, the registration of external respiration and central hemodynamics was repeated. Then the animals of the experimental groups were intraperitoneally injected with one of the test compounds in the form of a solution - at the rate of 0.1 ml per 100 g of body weight. Monitoring of physiological parameters was continued for the next 120 minutes.

The acute blood loss used in this model leads to the development of a severe degree of shock (Table 3) and, in the absence of treatment, is accompanied by the death of many experimental animals within the first two hours (Sarkisov D.S., Remezov P.I. Reproduction of human diseases in the experiment / Edited by A.A. Vishnevsky, Moscow: 1960.780 s.).

As shown in Table 3, the end of blood loss in the experimental animals was observed very marked drop in blood pressure: blood pressure _With decreased by more than 3-fold (from 186.3 ± 3.5 to 56,7 ± 6,9), _D BP - almost 5 times (from 132.9 ± 2.3 to 28.1 ± 9.0). During the first 60-70 minutes, the severity of hypotension was partially reduced due to a compensatory increase in vascular tone and heart rate. However, significant compensation for blood flow deficiency did not occur, and deep hypotension subsequently led to impaired pumping function of the heart, which caused the death of many experimental animals. Of 11 control animals, 5 individuals (45%) died by 120 minutes of observation.

Table 3 Hemodynamics and respiration of anesthetized animals after severe acute blood loss (control group; n = 11) Time after blood loss _With BP, mm Hg. Art. HELL _D , mmHg Art. Heart rate, beats.min NPV, dv / min Originally 186.3 ± 3.5 132.9 ± 2.3 413.1 ± 10.4 62.4 ± 4.9 Blood loss 56.7 ± 6.9 28.1 ± 9.0 348.1 ± 10.0 74.1 ± 8.2 2 minutes 59.7 ± 6.9 27.9 ± 9.0 5 minutes 65.2 ± 6.9 27.0 ± 6.7 355.3 ± 12.2 73.8 ± 8.4 10 minutes 67.4 ± 4.9 31.1 ± 5.0 359.5 ± 12.9 73.4 ± 8.7 20 minutes 76.4 ± 8.0 42.9 ± 8.3 358.7 ± 13.3 68.6 ± 7.7 30 minutes 88.4 ± 10.0 53.5 ± 9.4 372.2 ± 17.1 68.5 ± 7.8 40 minutes 96.8 ± 10.1 57.7 ± 9.6 383.2 ± 14.3 62.4 ± 6.7 50 minutes 101.3 ± 8.4 58.5 ± 7.6 387.9 ± 11.9 67.4 ± 7.7 60 minutes 108.3 ± 6.9 62.9 ± 6.3 402.7 ± 11.5 67.7 ± 7.8 70 minutes 113.0 ± 5.5 65.5 ± 5.1 416.3 ± 8.4 69.3 ± 7.3 80 minutes 116.7 ± 5.5 67.5 ± 5.0 429.3 ± 8.9 69.4 ± 5.2 90 minutes 117.1 ± 4.7 67.3 ± 4.1 434.2 ± 9.4 70.8 ± 6.3 100 minutes 118.0 ± 5.9 67.5 ± 6.1 439.4 ± 9.7 68.3 ± 7.4 110 minutes 118.9 ± 6.5 68.5 ± 6.3 440.3 ± 8.8 68.5 ± 6.9 120 minutes 119.8 ± 7.9 68.8 ± 7.7 439.1 ± 8.5 68.9 ± 6.7

Example 7. To obtain an objective assessment of the vasopressor properties of the claimed compounds in this model of hemorrhagic shock, the therapeutic possibilities of the prototype drug, α ₁ -adrenomimetic mesatone, were investigated. Mesatone was administered intraperitoneally to experimental animals at a high dose (0.5 mg / kg) immediately after the loss of blood.

The research results showed (Table 4, Figure 2) that the introduction of mesatone in animals in a state of severe hemorrhagic shock was accompanied by a rapid increase in blood pressure. 5 minutes after injection parameters BP _C and BP _D in the experimental animals were significantly higher than in the controls. The maximum effect developed for 10 minutes, the degree of increase in blood pressure was 25-40% _C, BP _D - 65-75% relative to the corresponding indices of control animals. However, a significant effect of mesatone was observed for a short period - from 5 to 20 minutes after injection. From 30 minutes to the end of the observation period, all controlled parameters in the experimental and control animals were statistically indistinguishable. Thus, with the used method of introducing mesatone, the duration of the vasopressor action is about 20 minutes. The short and relatively weak improvement in hemodynamic parameters under the influence of the mesatone in this experiment did not essentially affect the course of the shock - mortality in this group was 50% within 120 minutes of observation.

Table 4 Hemodynamics and respiration of anesthetized animals after severe acute blood loss when exposed to mesatone at a dose of 0.5 mg / kg (n = 8) Time after blood loss / injection _With BP, mm Hg. Art. HELL _D , mmHg Art. Heart rate, bpm NPV, dv / min Originally 181.9 ± 3.8 128.2 ± 5.9 434.1 ± 11.5 66.3 ± 5.7 Blood loss / injection 55.9 ± 4.0 26.4 ± 3.7 364.4 ± 12.9 70.3 ± 8.5 2 minutes 70.5 ± 4.2 37.5 ± 5.1 5 minutes 80.5 ± 4.5 ^* 43.7 ± 5.4 ^* 362.6 ± 10.0 72.2 ± 8.1 10 minutes 96.0 ± 6.8 ^* 58.7 ± 7.9 ^* 369.5 ± 9.7 70.5 ± 8.9 20 minutes 107.9 ± 11.1 * 67.3 ± 10.6 ^* 365.8 ± 5.1 69.6 ± 7.9 30 minutes 112.0 ± 11.9 71.6 ± 13.1 372.1 ± 9.8 69.3 ± 6.8 40 minutes 114.1 ± 13.6 71.3 ± 14.3 378.7 ± 7.4 66.1 ± 6.3 50 minutes 115.3 ± 13.2 70.6 ± 14.4 387.9 ± 8.2 69.2 ± 7.6 60 minutes 115.4 ± 14.6 67.9 ± 14.6 381.1 ± 9.5 69.7 ± 7.0 70 minutes 122.2 ± 5.4 73.7 ± 6.5 391.0 ± 15.7 68.8 ± 7.9 80 minutes 130.3 ± 12.3 80.7 ± 13.8 395.9 ± 18.0 67.9 ± 7.2 90 minutes 123.2 ± 12.1 73.2 ± 11.9 380.3 ± 24.5 68.3 ± 6.7 100 minutes 122.1 ± 12.5 73.7 ± 10.5 397.3 ± 27.1 69.2 ± 7.3 110 minutes 124.7 ± 11.8 75.5 ± 11.7 399.6 ± 24.5 68.5 ± 6.9 120 minutes 125.2 ± 12.2 76.3 ± 12.3 408.2 ± 20.3 68.7 ± 6.6 * - statistically significant difference (p <0.05) with the control group (Table 3) according to the nonparametric Q-criterion of Dunn multiple comparisons.

Example 8. A different picture of the course of shock was observed with the use of compounds of formula (1). The introduction of experimental animals in a state of severe hemorrhagic shock of the compound T-1020 at a dose of 10 mg / kg (14 times less than LD ₁₆ and 50 times less than LD ₅₀ ) caused a rapid increase in blood pressure (Table 5, Figure 3).

From the table. 5 shows that already at 2 minutes post-injection parameters BP _C and BP _D in the experimental animals were significantly higher than in the controls. The completely vasopressor effect of T-1020 developed by about 20 minutes, while the degree of pressure increase was significantly higher than that under the action of the mesatone - AD _C increased by 100-120%, and AD _D - by 200-220% relative to the control animals. The duration of the antihypotonic effect of the T-1020 compound was also 5-6 times longer than the duration of the action of mesatone. The effect began to weaken from only 100 minutes, and the statistically significant effect of T-1020 on blood pressure persisted until 110 minutes of the experiment. A prolonged and powerful increase in blood pressure under the action of T-1020 stabilized hemodynamics and qualitatively changed the course of shock - during 120 minutes of observation. None of the 8 experimental animals that received T-1020, despite heavy blood loss, did not die.

Table 5. Hemodynamics and respiration of anesthetized animals after severe acute blood loss when exposed to compound T-1020 at a dose of 10 mg / kg (n = 8) Time after blood loss / injection _With BP, mm Hg. Art. HELL _D , mmHg Art. Heart rate, bpm NPV, dv / min Originally 184.5 ± 2.8 130.3 ± 1.9 429.8 ± 9.1 66.3 ± 5.7 Blood loss / injection 55.1 ± 1.3 26.1 ± 1.8 348.1 ± 9.9 70.3 ± 8.5 2 minutes 79.1 ± 5.0 ^* 53.5 ± 5.7 ^* 5 minutes 92.1 ± 8.2 ^* 68.2 ± 8.3 ^{* m} 371.9 ± 10.1 72.2 ± 8.1 10 minutes 118.9 ± 7.5 ^* 94.7 ± 6.8 ^{* m} 397.5 ± 10.7 ^* 70.5 ± 8.9 20 minutes 175.5 ± 15.3 ^{* m} 134.0 ± 7.8 ^{* m} 404.4 ± 12.8 ^* 69.6 ± 7.9 30 minutes 177.9 ± 7.3 ^{* m} 140.0 ± 4.6 ^{* m} 407.9 ± 10.7 69.3 ± 6.8 40 minutes 181.5 ± 7.6 ^{* m} 143.5 ± 3.9 ^{* m} 410.9 ± 14.4 66.1 ± 6.3 50 minutes 168.7 ± 7.6 ^{* m} 134.7 ± 4.5 ^{* m} 414.8 ± 13.9 69.2 ± 7.6 60 minutes 166.4 ± 5.9 ^{* m} 134.0 ± 4.2 ^{* m} 417.9 ± 16.2 69.7 ± 7.0 70 minutes 171.9 ± 7.5 ^{* m} 135.5 ± 4.2 ^{* m} 433.8 ± 18.0 68.8 ± 7.9 80 minutes 155.6 ± 5.5 ^{* m} 125.5 ± 4.7 ^{* m} 422.6 ± 18.1 67.9 ± 7.2 90 minutes 162.4 ± 6.6 ^* 131.4 ± 5.1 ^{* m} 448.0 ± 20.9 68.3 ± 6.7 100 minutes 157.8 ± 7.4 ^{* m} 124.5 ± 4.5 ^{* m} 419.4 ± 16.3 69.2 ± 7.3 110 minutes 146.3 ± 10.0 ^* 114.8 ± 8.4 ^{* m} 439.2 ± 19.5 68.5 ± 6.9 120 minutes 138.1 ± 10.5 106.3 ± 10.0 ^{* m} 449.9 ± 15.8 68.7 ± 6.6 * - statistically significant difference (p <0.05) with the control group (Table 3) according to the nonparametric Q-criterion of Dunn multiple comparisons; ^m is a statistically significant difference (p <0.05) with the group receiving mesatone (Table 4), according to the nonparametric Dunn Q-test of multiple comparisons.

Example 9. The introduction of experimental animals in a state of severe hemorrhagic shock compounds T-1049 at a dose of 10 mg / kg (50 times less than LD ₁₆ and 70 times less than LD ₅₀ ) also caused a rapid increase in blood pressure (Table 6, Figure 4 )

The dynamics of the development of the effect was similar to the action of compound T-1020: by 10 minutes, AD _C increased by 100%, and AD _D by 220% relative to the control indices. The duration of the vasopressor action of the T-1049 compound was more than 5 times the duration of the action of mesatone. The effect began to weaken only from 110 minutes, however, a significant effect on the blood pressure of the experimental animals remained until the end of the experiment. Moreover, long-term pressure stabilization under the influence of T-1049 prevented the development of compensatory tachycardia, pronounced in control animals in the late stages of observation. Like T-1020, the T-1049 compound in these experiments provided 100% anti-shock protection - during the 120 minutes of observation, no experimental animals that received T-1049 died.

Table 6 Hemodynamics and respiration of anesthetized animals after severe acute blood loss when exposed to compound T-1049 at a dose of 10 mg / kg (n = 4) Time after blood loss / injection _With BP, mm Hg. Art. HELL _D , mmHg Art. Heart rate, bpm NPV, dv / min Originally 185.5 ± 3.7 126.3 ± 4.3 414.3 ± 8.6 72.3 ± 5.8 Blood loss / injection 52.8 ± 3.5 31.3 ± 4.8 366.0 ± 32.5 60.5 ± 5.9 2 minutes 77.3 ± 7.3 53.5 ± 8.1 ^* 5 minutes 101.5 ± 14.1 ^* 78.8 ± 14.8 ^{* m} 394.0 ± 33.0 67.5 ± 6.9 10 minutes 134.0 ± 21.6 ^* 108.5 ± 20.2 ^{* m} 406.0 ± 37.6 63.1 ± 5.5 20 minutes 133.5 ± 20.6 ^* 108.5 ± 18.3 ^{* m} 389.0 ± 27.3 65.3 ± 7.1 30 minutes 150.3 ± 16.8 ^{* m} 122.5 ± 12.5 ^{* m} 401.3 ± 21.5 79.5 ± 7.0 40 minutes 163.3 ± 5.8 ^{* m} 132.5 ± 4.5 ^{* m} 391.3 ± 13.7 77.3 ± 7.9 50 minutes 160.0 ± 5.9 ^{* m} 129.0 ± 5.4 ^{* m} 381.5 ± 8.5 68.4 ± 5.9 60 minutes 163.8 ± 7.6 ^{* m} 132.5 ± 5.0 ^{* m} 403.0 ± 14.3 71.5 ± 6.1 70 minutes 157.8 ± 7.6 ^{* m} 127.0 ± 7.5 ^{* m} 401.3 ± 15.6 78.8 ± 9.2 80 minutes 158.3 ± 9.3 ^{* m} 126.8 ± 7.3 ^{* m} 391.5 ± 19.3 75.0 ± 9.3 90 minutes 146.3 ± 14.1 ^{* m} 120.8 ± 13.2 ^{* m} 395.3 ± 14.5 ^* 78.0 ± 10.4 100 minutes 147.3 ± 4.9 ^{* m} 119.3 ± 2.9 ^{* m} 386.0 ± 13.5 ^* 75.8 ± 10.5 110 minutes 151.5 ± 5.0 ^{* m} 121.8 ± 2.9 ^{* m} 382.5 ± 6.6 ^* 74.1 ± 10.5 120 minutes 140.8 ± 8.0 ^* 111.0 ± 6.9 ^{* m} 373.8 ± 2.4 ^* 74.3 ± 9.6 * - statistically significant difference (p <0.05) with the control group (Table 3) according to the nonparametric Q-criterion of Dunn multiple comparisons; ^m is a statistically significant difference (p <0.05) with the group receiving mesatone (Table 4), according to the nonparametric Dunn Q-test of multiple comparisons.

Example 10. Among the compounds of formula (1), the most potent vasopressor effect was demonstrated by the derivative T-1059. The introduction of experimental animals in a state of severe hemorrhagic shock of compound T-1059 at a dose of 10 mg / kg (25 times less than LD ₁₆ and 38 times less than LD ₅₀ ) also caused a sharp increase in blood pressure (Table 7, Figure 5).

Starting Dynamics vasopressor action of T-1059 was similar to the action of T-1049, but the degree of pressure rise was significantly higher - to _C 20 minute AD increased by 140%, and BP _D - 250% of control parameters. The duration of action of the T-1059 compound was also more than 5 times longer than the duration of the action of mesatone — a significant vasopressor effect was observed from 2 minutes from the injection to the end of the observation period. As shown in FIG. 4, as a result of the powerful pressor action of T-1059, blood pressure indicators of animals that lost 50% of the circulating blood rose to the blood pressure level of healthy animals and were maintained at this level until the end of the experiment. Long-term stabilization of hemodynamic parameters under the influence of T-1059 also provided 100% anti-shock protection - not a single experimental animal died within 120 minutes of observation.

Table 7 Hemodynamics and respiration of anesthetized animals after severe acute blood loss when exposed to compound T-1059 at a dose of 10 mg / kg (n = 6) Time after blood loss / injection HELL _C , mmrt. Art. HELL _D , mmHg Art. Heart rate, bpm NPV, dv / min Originally 179.2 ± 3.6 124.8 ± 3.1 409.0 ± 8.2 63.3 ± 4.5 Blood loss / injection 44.5 ± 3.5 18.3 ± 2.8 348.7 ± 11.2 63.3 ± 5.5 2 minutes 63.5 ± 5.0 37.0 ± 4.6 ^* 5 minutes 86.7 ± 7.2 ^* 59.0 ± 8.7 ^* 367.3 ± 17.0 66.3 ± 5.6 10 minutes 141.2 ± 12.8 ^{* m} 107.8 ± 9.4 ^{* m} 389.3 ± 16.0 58.0 ± 4.1 20 minutes 181.5 ± 9.3 ^{* m} 136.7 ± 5.9 ^{* m} 416.6 ± 16.3 ^* 63.0 ± 8.9 30 minutes 166.8 ± 11.8 ^{* m} 127.5 ± 7.5 ^{* m} 404.3 ± 16.7 59.7 ± 5.9 40 minutes 157.5 ± 20.9 ^{* m} 113.5 ± 15.9 ^{* m} 384.5 ± 15.0 58.7 ± 5.3 50 minutes 160.2 ± 19.6 ^{* m} 117.3 ± 12.4 ^{* m} 377.8 ± 17.3 58.0 ± 5.2 60 minutes 174.5 ± 17.4 ^{* m} 128.8 ± 10.0 ^{* m} 389.0 ± 19.2 62.7 ± 6.2 70 minutes 187.5 ± 6.4 ^{* m} 141.8 ± 4.8 ^{* m} 409.8 ± 16.5 58.7 ± 5.9 80 minutes 189.7 ± 12.2 ^{* m} 137.5 ± 7.6 ^{* m} 396.7 ± 14.3 ^* 59.0 ± 5.8 90 minutes 188.3 ± 11.9 ^{* m} 138.8 ± 5.3 ^{* m} 399.5 ± 15.1 ^* 63.7 ± 5.5 100 minutes 169.8 ± 18.9 ^{* m} 124.8 ± 16.0 ^{* m} 396.7 ± 16.6 ^* 65.7 ± 6.5 110 minutes 180.2 ± 10.1 ^{* m} 132.2 ± 8.9 ^{* m} 399.2 ± 16.1 ^* 62.7 ± 5.1 120 minutes 173.7 ± 6.2 ^{* m} 130.3 ± 6.1 ^{* m} 397.0 ± 17.8 ^* 63.0 ± 6.4 * - statistically significant difference (p <0.05) with the control group (Table 3) according to the nonparametric Q-criterion of Dunn multiple comparisons; ^m is a statistically significant difference (p <0.05) with the group receiving mesatone (Table 4), according to the nonparametric Dunn Q-test of multiple comparisons.

Examples 7-10 demonstrate not just a significant advantage, but the qualitative superiority of the claimed compounds over the adrenomimetic drug according to the prototype, since they prove the ability of the compounds of formula (1) to provide effective assistance in shock conditions (traumatic, hemorrhagic) at the prehospital stage, which cannot be achieved by mesatone and others vasopressor drugs currently used.

Proof of achievement of the claimed result

As a result of studies of new derivatives of isothiourea, compounds of the general formula (1) were first synthesized and characterized, which are capable of selectively inhibiting the catalytic activity of eNOS and iNOS, those NOS isoforms whose activity leads to the development of hypotension. Due to this ability, the claimed compounds possess pronounced vasopressor properties with minimal risks of undesirable effects. The compounds of formula (1) are moderately toxic, and their toxicity is 2 times lower than that of the widely used adrenomimetic vasopressor drug mesaton.

The study of the vasopressor properties of the compounds of formula (1) was carried out on an experimental model of shock reproducing severe clinical form of hypotension in acute, massive blood loss. The results of the studies indicate that a single administration to animals in a state of severe shock (blood pressure at 55/25) of compounds of formula (1) in a known non-toxic dose of 10 mg / kg (1 / 50-1 / 14 LD ₁₆ ) is accompanied by a powerful and prolonged vasopressor effect . The effect develops from the first minutes after the injection, without visible changes in the condition of the animals. After 10-20 minutes BP values _C are increased by 100-140% and BP _D - by 200-250% compared to control animals not receiving "treatment", and correspond to 90-100% of the figures to bleeding. The stabilization of blood pressure under the influence of compounds of formula (1) is very long - the vasopressor effect of compounds T-1020 and T-1049 begins to weaken only 100-110 minutes after injection, and the compounds T-1059 does not weaken after 120 minutes. A persistent improvement in hemodynamics qualitatively changes the course of shock - during 120 minutes of observation, none of the animals that received the claimed compounds, despite heavy blood loss (1/2 of the volume of circulating blood), did not die, while in the control mortality by this time reaches 45 %

In these studies, the compounds of formula (1) show significant superiority over mesatone. Although this drug is one of the most long-acting α ₁ -adrenomimetics, a single injection of mesatone in a high dose (0.5 mg / kg) causes a vasopressor effect in animals for no more than 20 minutes, and the degree of blood pressure rise is 1.5 -2 times weaker than with the claimed compounds. A short and relatively weak improvement in hemodynamics under the action of mesatone does not essentially affect the course of shock - by 120 minutes of observation, mortality in this group of animals reached 50%.

It is known that the most effective way to treat hemorrhagic shock is an emergency compensation of the lost volume by donor blood or blood substitutes. However, these activities are far from always available, especially outside the clinical setting. Therefore, it should be emphasized that the long-term and powerful "anti-shock" protection provided in these studies by single injections of the claimed compounds clearly demonstrates their qualitative advantage - the ability to provide effective assistance in case of shock conditions (at least in traumatic and hemorrhagic) at the prehospital stage, which cannot be achieved with vasopressor drugs currently used. This creates new opportunities in the treatment of acute and possibly chronic arterial hypotension, as well as in cases of emergency and first aid, and in the treatment of critical conditions.

In this regard, the authors have reason to propose, as another aspect of the present invention, a new vasopressor agent containing, as an active substance, a compound of formula (1). It is advisable to have a new vasopressor agent in several dosage forms that are convenient for use in various cases: in the form of a solid dosage form with a dose of the active substance 5-30 mg, in the form of an injection solution containing 1-10% of the active substance, in the form of an infusion solution containing 0.01-0.1% of the active substance. The new tool can be used to treat hypotonic conditions on their own at a dosage that provides a dose of the active substance of 0.1-20 mg / kg or as part of a pharmaceutical composition that provides a dose of the active substance of 0.1-20 mg / kg. The new tool can be used to increase blood pressure in acute arterial hypotension caused by injuries, injuries, burns, poisoning, bleeding, and surgical interventions. Moreover, the ability of the compounds of formula (1) to realize a vasopressor effect, bypassing α ₁ -addernoreceptors, allows one to expect therapeutic efficacy of the proposed agent for hypotension developed against the background of epidural anesthesia, with an overdose of ganglion blockers, α-blockers, antipsychotics, anesthetics, and refractory stages of shock and other conditions when adrenergic agonists are ineffective or contraindicated.

The inventive tool can be used in the form of a solid or liquid dosage form containing, as an active substance, a compound of formula (1), used alone or as part of a pharmaceutical composition for the treatment of hypotonic conditions. Such a vasopressor agent creates new opportunities in the treatment of acute and possibly chronic arterial hypotension, as well as in cases of emergency and emergency care, and in the treatment of critical conditions.

The new vasopressor agent can be used to increase blood pressure in acute arterial hypotension caused by injuries, injuries, burns, poisoning, bleeding, and surgical interventions. The full range of pharmacological possibilities of the proposed vasopressor agent, of course, can only be evaluated with further detailed studies. Nevertheless, the ability of the compounds of formula (1) to realize a vasopressor effect, bypassing α ₁ -addernoreceptors, allows one to expect therapeutic efficacy in the proposed agent for hypotension that develops against the background of epidural anesthesia, with an overdose of ganglion blockers, α-blockers, antipsychotics, anesthetics, and refractory stages of shock and other conditions when adrenergic agonists are ineffective or contraindicated.

Claims (6)

1. N, S-substituted isothiourea derivatives of the general formula (1) in the form of salts with pharmacologically acceptable acids as an NOS-inhibiting and vasopressor agent:

wherein:
n = 1, 3, that is, the acyl substituent is cyclobutanoyl or cyclohexanoyl;
R - represents an alkyl group: C ₂ H ₅ , i-C ₃ H ₇ ;
HX - is a pharmacologically acceptable inorganic or organic acid: HCl, HBr, HI, methylsulfuric, oxalic, succinic, etc. 2. The compound of formula (1) according to claim 1, characterized in that it has the ability to inhibit the catalytic activity of NO synthases. 3. The compound of formula (1) according to claim 1, characterized in that it has a vasopressor effect, manifested by an increase in vascular tone and an increase in blood pressure in hypotonic conditions. 4. The compound of formula (1) according to claims. 1 and 3 to obtain a pharmaceutical composition in the form of solid dosage forms at a dose of the active substance of 5-30 mg for the treatment of hypotonic conditions. 5. The compound of formula (1) according to claims. 1 and 3 to obtain a pharmaceutical composition in the form of an injection solution containing 1-10% of the active substance for the treatment of emergency conditions. 6. The compound of formula (1) according to claims. 1 and 3 to obtain a pharmaceutical composition in the form of an infusion solution containing 0.01-0.1% of the active substance for the treatment of emergency conditions.

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