RU2699658C2 - Acylhydrazone (2,3,4-trimethoxy-n'-(8-methyl-8-azabicyclo[3.2.1.]octane-3-ylidene) benzohydrazide hydrochloride), having antimigraine and anxiolytic activity - Google Patents

Abstract

FIELD: chemistry.SUBSTANCE: invention relates to acyl hydrazone (2,3,4-trimethoxy-N'-(8-methyl-8-azabicyclo[3.2.1.]octane-3-ylidene) benzohydrazide hydrochloride) of structural formula (I), which can be used in medicine:EFFECT: disclosed is a new compound with antimigraine and anxiolytic activity.1 cl, 3 ex, 3 tbl, 1 dwg

Description

The invention relates to the chemistry of biologically active compounds, and relates specifically to 2,3,4-trimethoxy-N '- (8-methyl-8-azabicyclo [3.2.1.] Octane-3-ylidene) benzohydrazide hydrochloride, a compound with the structural formula (I ):

When studying the pharmacological activity of compound I, it was found that it has pronounced anti-migraine and anxiolytic properties.

The search and study of the pharmacological agents for treating migraine patients is one of the priority problems of modern medicine, since migraine is one of the most common and socially significant diseases. In the developed countries of Europe and America, from 3 to 16% of the population suffer from migraine [P.M. Rist, J.E. Buring, C.S. Kaseetal., Migraine and functional out come from ischemic cerebral eventsin women. Circulation, 2010, 122, 24, 2551-2557; W. Dent, B. Stelzhammer, M. Meindl, W.B. Matujaetal, Migraine attack frequency, duration, and pain intensity: disease burden derived from a community-based survey in northern Tanzania, Headache, 2011, 51, 10, 1483-1492, doi: 10.1111 / j. 1526-4610. 2011; A. Pezzini, E. Del Zotto, A. Giossi, et al., The migraine-ischemic stroke relation in young adults, Stroke Res. Treatment, 2010, 9, 304921, doi: 10.4061 / 2011 /]. Despite differences in understanding of the pathogenesis of migraine, most researchers point to the significant role of serotonin, the neurogenic and cerebrovascular components in the pathogenesis of this disease [M.A. Moskowitz, Genes, proteases, cortical spreading depression and migraine: impact on pathophysiology and treatment. FunctionalNeurology, 2007, 22.3, 133-136].

Currently the treatment of patients with migraine widely used drugs acting on serotonin receptors - agonists of 5HT _{1B / D} ^- antagonists and 5HT _{2A / 2B / 2C} -type. The most common drugs for treating migraine patients are drugs that act on the serotonin receptors of the first (5HT _{1Aa / 1B / 1D / 1F} ) and second (5HT _{2A / 2B / 2C} ) types, which indicates the important role of serotonin in the pathogenesis of this disease. Agonists of 5-HT _{1B / 1D / 1F} receptors (sumatriptan, zolmitriptan, naratriptan, etc.) are used to stop a migraine attack [SJ Tepper, AM Rapoport, FD Sheftell, Mechanisms of action of the 5-HT _{1B / 1D} receptor agonists. Arch Neurol., 2002, 59, 1084-1088; T. Muller, L. Lohse, Efficacy of parecoxib, sumatriptan, and rizatriptan in the treatment of acute migraine attacks. Clin. Neuropharmacol., 2011, 34, 6, 206-209], and for intermittent treatment, 5HT _{2B / 2C} receptor antagonists (metisergide, pizotifen, cyproheptadine, nicergoline), which interfere with the vasoconstrictor effect of serotonin [EJ Mylecharane, J. Misbach, JW Duckworth, JW Lance, Effects of methysergide, pizotifen and ergotamine in the monkey cranial circulation. Eur. J. Pharmacol., 1978; 48, 1-9; K. Schmuck, C. Ullmer, HO Kalkman, et al., Activation of meningeal 5-HT _2B receptors: an early step in the generation of migraine headache? Eur. J. Neurosci., 1996, 8, 5, 959-967; G. Casucci, V. Villani, F. Frediani, Central mechanism of action of antimigraine prophylactic drugs. Neurol. Sci., 2008, 29, 123-126; JA Charles, Pathophysiology of Chronic Migraine and Mode of Action of Preventive Medications: A Comment. Headache: The Journal off leadand Face Pain, 2011, 51, 10, 1550-1551].

However, these drugs used to treat migraines do not always have sufficient effectiveness and have pronounced undesirable side properties. This determines the need to find new drugs for the prevention and treatment of migraine attacks.

Therefore, the search for new drugs for the prevention and treatment of migraine attacks is the most important task of modern pharmacology and neurology.

At the Federal State Budgetary Scientific Institution Scientific Research Institute of Pharmacology named after V.V. Zakusova "created an original antagonist of serotonin receptors with anti-migraine activity - tropoxin, which is currently undergoing clinical trials [TS. Ganshina et al., Tropoxin is a new treatment for migraine. Chemical pharmacist. Journal 2016, 50, 1, 19-23]. Tropoxin, blocking 5HT _{2B / 2C} receptors, prevents the development of constrictor reactions of cerebral vessels caused by serotonin [R.S. Mirzoyan et al., Tropoxin is a new serotonin antagonist and a potential anti-migraine agent. Experiment and wedge. pharmacol., 1998, 61, 2, 28-31]. At the same time, when studying the pharmacokinetics of the compound, it was found that tropoxin in the body is rapidly metabolized, which, apparently, is due to its short-term effect. Tropoxin does not have an anxiolytic effect. However, it is desirable for anti-migraine drugs to have anxiolytic activity in the drug, as migraine sufferers experience anxiety when approaching and during an attack.

In the literature there is information about several acyl derivatives of tropan-3-one hydrazones, claimed as agents with anti-tuberculosis, stimulating and antidepressant effects [U.S. Pat. 2, 838, 914], as well as with anti-migraine properties [R.S. Mirzoyan et al., Cerebrovascular antagonists and serotonin agonist among tropane derivatives. Experiment and wedge. pharmacol., 2008, 71, 1, 26-30]. However, among the acyl derivatives of hydrazones of tropan-3-ones, compounds exhibiting both anti-migraine and anxiolytic activity have not been discovered to date.

An object of the present invention is to provide a novel anti-migraine agent having antiserotonin activity with an anxiolytic action component.

The technical result. A new chemical compound with pronounced antiserotonin cerebrovascular activity is proposed: 2,3,4-trimethoxy-N '- (8-methyl-8-azabicyclo [3.2.1.] Octan-3-ylidene) benzohydrazide hydrochloride. A distinctive feature of the present invention is the presence in the spectrum of action of anxiolytic activity. The invention extends the arsenal of funds for the treatment of migraine.

The result of the use of the proposed compound is the presence in one preparation of two effects important for the treatment of migraine patients: the elimination of cerebral vasospasms characteristic of the first phase of the attack caused by the blockade of serotonin receptors in the brain vessels and the elimination of anxiety due to the anxiolytic activity of the drug.

The claimed compound has a pronounced anti-migraine and anxiolytic effect and may find application in medical practice as a drug for the treatment of migraine.

The claimed compound I was synthesized in the form of hydrochloride according to the following scheme:

The acylation of tropan-3-one hydrazone 2,3,4-trimethoxybenzoyl chloride in ether at room temperature in three stages gave the final product 2,3,4-trimethoxy-N- (8-methyl-8-azabicyclo [3.2.1] octane -3-ylidene) benzohydrazide hydrochloride (I). Compound I is a high-melting white crystals, soluble in water and insoluble in ether and aromatic solvents.

Used as intermediates: 2,3,4-trimethoxybenzoic acid chloride obtained by reaction of the corresponding acid with thionyl chloride in dry benzene, and tropan-3-one hydrazone by reaction of the latter with hydrazine hydrate in alcohol.

Experimental chemical part

The purity of the obtained product was determined using NMR spectroscopy and elemental analysis. The NMR spectrum was recorded on a Fourier-300 instrument (Bruker) with the internal TMS standard.

Obtaining 2,394-trimethoxy-N '- (8-methyl-8-azabicyclo [3.2.1.] Octane-3-ylidene) benzohydrazide hydrochloride

Preparation of 3-hydrazono-8-methyl-8-azabicyclo [3.2.1.] Octane (II).

10.0 g of tropinone (0.070 m) are dissolved in 100 ml of alcohol, and hydrazine hydrate dissolved in 100 ml of alcohol at room temperature is added dropwise slowly with stirring to 50.0 g (1,000 m). The solution was evaporated, the residue was distilled in vacuo to give 8.6 g (78.00% based on tropinone), because 115/3 mm and 2.0 g of dimer (by-product, because 171/1 mm).

Preparation of 2,3,4-trimethoxybenzoyl chloride (III).

6.0 g (0.028 m) of 2,3,4-trimethoxybenzoic acid are dissolved in 40 ml of dry benzene and 6.7 g (0.056 m) of thionyl chloride are added dropwise while boiling benzene for 3 hours, the benzene is evaporated, the residue is distilled into vacuum and get 4.8 g (73.71%), because 183/20 mm.

Preparation of 2,3,4-trimethoxy-N- (8-methyl-8-azabicyclo [3.2.1.] Octan-3-ylidene) benzohydrazide hydrochloride (I).

3.0 g (0.019 m) of hydrazonatropan-3-one is dissolved in 30 ml of absolute ether, and 4,5,4 (0,019 m) 2,3,4-trimethoxybenzoic acid chloride, dissolved in 10 ml of ether, are slowly added at room temperature. The precipitate obtained is filtered off, washed with absolute ether, dried and recrystallized from acetone and alcohol.

Obtain 3.2 g (42.60%), so pl. 210 ° C.

Found in% -C-56.39; H-7.08; N-10.94; Cl-8.88, C ₁₈ H ₂₅ N ₃ O ₄

Calculated in% - C-56.32; H-6.77; N-10.95; Cl-9.35.

, м.д., JГц): 1.81, 2.13, 2.34 (м, 4Н; Н-6 и Н-7); 2.73, 3.52 (м, 4Н, Н-2 и Н-4); 3.92 (м, 2Н; Н-1 и Н-5); 3.85, 3.889, 3.99 (с.9Н, 3 ОСН₃); 6.79, 7.94 (д. J=9, 2Н, аром. прот.); 10.84 (с, 1Н, NH группа).NMR spectrum - 'H (CDL ₃ ,

, ppm, JHz): 1.81, 2.13, 2.34 (m, 4H; H-6 and H-7); 2.73, 3.52 (m, 4H, H-2 and H-4); 3.92 (m, 2H; H-1 and H-5); 3.85, 3.889, 3.99 (s. 9H, 3 OCH ₃ ); 6.79, 7.94 (d. J = 9, 2H, arom. Prot.); 10.84 (s, 1H, NH group).

Pharmacological study of the claimed compound I.

To assess the pharmacological effects of the claimed compounds, we used basic certified methods recommended by the Pharmacological Committee of the Ministry of Health of the Russian Federation for the study of substances with anti-migraine, anxiolytic effects (Guidelines for the experimental (preclinical) study of substances with pharmacological activity. M., Grif and K, 2012, p. 255, p. 485).

Example 1. The study of antiserotonin cerebrovascular activity of compound I.

It is known that to simulate a migraine attack, constrictor reactions of cerebral vessels caused by serotonin are used. With intravenous administration of serotonin, a short-term decrease in cerebral blood flow and blood pressure is observed. This reaction persists with the repeated administration of serotonin [R.S. Mirzoyan et al., Tropoxin is a new serotonin antagonist and a potential anti-migraine agent. Experiment and wedge. pharmacol., 1998, 61, 2, 28-31; T.S. Ganshina, Experiment. and wedge. Pharmacol., 2003, 66, 3, 17-20].

It is known that in the vessels of the brain, mainly 5HT _{2B / 2C} receptors are present. Therefore, to study the constrictor reactions of cerebral vessels of a serotonergic nature, it is advisable to use, along with serotonin, a selective 5HT _{2B / 2C} receptor agonist - meta-chlorophenylpiperazine (mCPP). According to published data, meta-chlorophenylpiperazine provokes a headache attack in migraine patients, in contrast to healthy volunteers [A. Pancoesi, Headache induced by serotoninergic agonists - a key to the interpretation of migraine pathogenesis? Cefalalgia, 1997.17, 3-14]. It was also shown that with intravenous administration of mCPP at a dose of 0.1 mg / kg, experimental animals showed an increase in the sweating of plasma albumin from meningeal vessels, which is a quantitative marker of neurogenic inflammation in the dura mater. In this case, the activation of sensitive fibers of the trigeminovascular system and the release of vasoactive substances into the vessel wall, such as substance P, glutamate, etc. [A.V. Amelin et al., Migraine - pathogenesis, clinic and treatment of St. Petersburg medical publishing house, 2001; A.Yu. Sokolov et al., Role of serotonin receptors in the mechanisms of migraine formation, Neurochemistry, 2011, 2, 104-112].

We studied the effect of compound I on changes in blood flow in the cerebral cortex of rats caused by the intravenous administration of mCPP (Sigma-Aldrich) at a dose of 0.1 mg / kg [A.V. Amelin et al., Migraine - pathogenesis, clinic and treatment, St. Petersburg Medical Publishing House, 2001].

To assess the state of microcirculation, local blood flow was recorded in the parietal region of the rat cerebral cortex using an ALF-21 laser Doppler flowmeter from Transonic Systemlnc. (USA). For this purpose, a 0.8 mm diameter flowmeter needle sensor was installed on the parietal region of the cerebral cortex at a distance of 6-7 mm distal to the base of the middle cerebral artery in the direction of its central branch using a micromanipulator and rocker arm. In parallel, the level of blood pressure was recorded through a polyethylene catheter previously inserted into the femoral artery. Recording of blood flow and blood pressure was performed on a polygraph company "BIOPAK" USA, connected to a personal computer. Test compound I at a dose of 10 mg / kg and mCPP at a dose of 0.1 mg / kg were administered intravenously through a polyethylene catheter inserted previously into the femoral vein.

Statistical data processing was performed using the Stastistika 8.0 software package (Statistikalnc, USA). The normality of the distribution was determined using the Shapiro-Wilk test. For further data processing, a non-parametric two-sample sign-ranking Wilcoxon test was used. The average data in the text is presented as a median. The results were considered significant at p≤0.05.

The study was conducted on 20 anesthetized (chloral hydrate 350 mg / kg intraperitoneally) non-linear male rats weighing 250-300 g. All animals were kept in laboratory vivarium at a 12-hour light regime with free access to water and standard food. The experiments were carried out in accordance with the ethical rules for the humane treatment of animals, approved by the ethics committee of the Federal State Budget Scientific Educational Institution “V.V. Zakusov ".

The experiments showed that after the introduction of mCPP there is a short-term decrease in local blood flow in the rat cerebral cortex by an average of 18% and 11%) (Table 1, Fig. 1). After registration of the control reactions, compound 1 was administered. It was shown that mCPP, administered 20 minutes after injection of compound I, caused an average 5.5% decrease in blood flow in the rat cerebral cortex (Table 1, Fig. 1). The difference in the effects of control reactions on CPP and after compound1 is statistically significant (p≤0.05). The weakening of the constrictor reaction of cerebral vessels in response to the introduction of CPP under the influence of compound I is observed for 90-120 minutes.

In the control, when mCPP was administered in rats, a short-term decrease in blood pressure by an average of 22.5% was observed (Table 2, Fig. 1). 20 minutes after the administration of compound I, mCPP caused a decrease in blood pressure by an average of 3.5%, and by the end of the experiment, the blood pressure response to mCPP administration was completely absent (Table 3, Fig. 1).

It was previously shown that with the preliminary administration of the tropoxin comparison drug, mCPP caused a decrease in blood flow in the vessels of the brain on average 19% and 11% after 20 minutes and 40 minutes, respectively. In the control, the decrease in blood flow under the influence of mCPP was 25% (p≤0.05). However, 60 minutes after tropoxin, the response of the brain vessels to mCPP is even somewhat enhanced [A. Gorbunov, T. S. Ganshina, R.S. Mirzoyan, The effect of tropoxin on the cerebrovascular effects of metachlorophenylpiperazine and serotonin. Experiment and wedge, pharmacol., 2010, 73, 9, 13-16]. It should be noted that with the introduction of compound I, the effect of weakening the constrictor responses of the brain vessels on mCPP continues for 2 hours. Tropoxin attenuates the mCPP-induced blood pressure response, and Compound I almost completely eliminates it.

Thus, the study showed that compound I has antiserotonin cerebrovascular activity, as it causes a statistically significant attenuation of local cerebral blood flow and blood pressure reactions caused by mCPP. Compound I is not inferior to tropoxin in the intensity of antiserotonin vascular effects, but surpasses it in duration.

Example 2. The study of the anxiolytic effect of compound I

The experiments were carried out on 100 white outbred male mice weighing 20-25 g. All animals were kept in laboratory vivarium at a 12-hour light regime with free access to water and standard food. To exclude the influence of daily biorhythms on the rate of biosynthesis and metabolism of neurotransmitters, experiments were carried out between 10 and 12 hours of the day. The experiments were carried out in accordance with the ethical rules for the humane treatment of animals, approved by the ethics committee of the Federal State Budget Scientific Educational Institution “V.V. Zakusov ".

To assess the anxiolytic effect, the standard technique of the elevated cruciform labyrinth (PCL) was used, based on the occurrence of stress and fear in the animal during the implementation of orientational research behavior and a mink reflex in difficult conditions (novelty of the environment, fear of heights and light) [T.A. Voronina, S.B. Seredenin, M.A. Yarkova et al., Guidelines for the preclinical study of the tranquilizing (anxiolytic) action of drugs. "Guidelines for preclinical studies of drugs." Part one. - M .: Grif and K, 2012.S. 264-275; S.Pellow, P.Chopin, S.E. File, M. Briley et al., Validation of openxlosed arm entries in an EPM as a measure of anxiety in the rat. J. Neurosci. Meth., 1985, 14, 3, 149-167].

The cross-shaped labyrinth is located at a height of 50 cm from the floor and consists of two open (not having walls) (OR) and two closed (having walls 20 cm high) sleeves (SR) located at an angle of 90 °, as well as the central platform (CPU) 5 × 5 cm in size. The mouse was placed on the central PCL site with its head facing the open sleeve. Within 5 minutes, the time spent in the OP, ZR, and the number of approaches (crossing the arm boundary with four paws) in the OR and ZR were recorded. The entry into the PCL sleeves was counted in the case when the animal crossed the boundary of the sleeve with four paws. It is known that the more active the mice enter the labyrinthine OR, the lower the degree of their anxiety.

Compound I was studied in doses of 5, 10, 20 and 40 mg / kg, each dose of the substance in 10 animals. Statistical processing of the results and the significance of differences were determined using Student's criterion. The average values of the number of visits and the time spent in the compartments for each group and standard deviations were calculated.

It was found that compound I at a dose of 10 and 20 mg / kg (intraperitoneally) shows a clear anxiolytic activity, which is expressed in a significant, statistically significant increase in the length of stay of animals in the open arms of the PKL, the number of visits in them and the reduction in the latency of the first entry into the open sleeve (tab. 3). At doses of 5 and 40 mg / kg, compound I does not exhibit anxiolytic activity (Table 3). Tropoxin at a dose of 10 mg / kg does not have an anxiolytic effect in the PKL test. Afobazole at a dose of 5 mg / kg statistically significantly increases the length of stay of animals in the open arms of the PKL, the number of visits in them and reduces the latent period of the first visit to the open sleeve, which indicates its anxiolytic effect. No significant differences between the effects of compound I and afobazole were observed.

Thus, compound I, as well as afobazole, has a pronounced anxiolytic activity, while tropoxin has no anxiolytic activity.

Example 3. The study of toxicity and possible side effects of compound I.

The acute toxicity and possible side effects of compound I with a single intraperitoneal injection at a dose of 200 mg / kg was evaluated in experiments on outbred male mice weighing 20-25 g [E.V. Arzamastsev, I.V. Berezovskaya, T.A. Guskova et al. Guidelines for preclinical studies of drugs. Part One, Moscow, 2012, 13-25]. Assessment of the general condition, behavior and death of animals was carried out after 1 h, 24 h, 4 days, 10 days. and 14 days after administration of the compound. The degree of impaired coordination of movements was evaluated by the ability of animals to stay on a rotating rod of RotaRod (UgoBasile, Italy). Animals were placed on a horizontal rod with a diameter of 2.5 cm, rotating at a constant speed of 10 revolutions per minute. The retention time of animals on a rotating rod was recorded for 300 s.

It was found that after 1 h, 24 h, 4 days, 10 days. and 14 days after the administration of compound 1 at a dose of 200 mg / kg, the death of animals was not observed. When studying the possible side effects of compound I at a dose of 200 mg / kg (i.e., 10 times higher than the therapeutic - 20 mg / kg), it was shown that this substance does not cause the development of side effects and signs of neurological deficiency both after 1 h. and at a longer time (24 hours, 4 days, 10 days and 14 days) after administration of the compounds. Compound I at a dose of 200 mg / kg does not interfere with the coordination of animal movements (100% of the mice are kept on a rotating rod for 5 minutes).

Thus, the study made it possible to establish that compound I at a dose of 10 mg / kg has antiserotonin activity, causes statistically significant attenuation of local cerebral blood flow and blood pressure reactions caused by mCPP to the same extent as tropoxin, but exceeds its duration of effect . Unlike tropoxin, compound I has anxiolytic activity in doses of 10 and 20 mg / kg, and does not cause side effects and death of animals at a dose 10 times higher than effective.

Description of the drawing.

Figure 1. The effect of compound I on changes in blood flow in the cerebral cortex of rats in arbitrary units and blood pressure in mm RT. Art., caused by mCPP. Designations from top to bottom: the upper curve is the local cerebral blood flow, the lower curve is the blood pressure; along the ordinate axis, in the upper part of the figure - conventional units of tissue perfusion (TPU - tissue perfusion unit); on the bottom - mmHg, along the abscissa - time in minutes, the arrows indicate the introduction of substances.

Claims (3)

The compound acylhydrazone (2,3,4-trimethoxy-N '- (8-methyl-8-azabicyclo [3.2.1.] Octan-3-ylidene) benzohydrazide hydrochloride):

, possessing anti-migraine and anxiolytic activity.

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