PHENYLCREATINE, ITS USE AND METHOD FOR ITS PRODUCTION

Abstract

The invention relates to the field of pharmaceutical chemistry, namely to new biologically active substances and their use and to a method of production. In particular, the invention relates to a derivative of creatinephenylcreatine, its use as a functional analogue of creatine, as well as a nootropic agent and for the prevention or treatment of arrhythmia and a method of its production.

Claims

1. Phenylcreatine of formula ##STR00003##

2. The use of phenylcreatine according to claim 1 as a functional analogue of creatine.

3. The use of phenylcreatine according to claim 1 for the prevention or treatment of arrhythmia.

4. The use of phenylcreatine according to claim 1 as a nootropic agent.

5. A method of producing phenylcreatine according to claim 1, comprising mixing cyanamide, pre-exposed to ammonia in catalytic amounts, with N-benzylglycine, and exposure for 24-96 hours at a temperature from +20 C. to +65 C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIG. 1 Graphs of dynamics of the duration of mice run to complete exhaustion in the experiment described in example 3.

EXAMPLE 1. PRODUCTION OF PHENYLCREATINE

[0067] N-benzylglycine weighing 429 mg, and 0.5 ml of distilled water were mixed in a round-bottom flask of 10 ml volume. Then 152 mg of NaCl were added to the mixture. Further, using a magnetic stirrer the mixture was stirred at room temperature for 10 minutes. In a small glass 206 mg of cyanamide and 0.2 ml of distilled water were added. Then a drop of solution of ammonia was added in catalytic quantities. The mixture was quickly mixed by gentle inverting, and then a mixture of cyanamide was added to a mixture of N-benzylglycine. The resulting mixture was stirred for one hour at room temperature. After 96 hours of incubation at room temperature and normal atmospheric pressure the product, namely phenylcreatine, N-benzyl-N-carbamimidoyl glycine, was precipitated. The crystals were transferred to a clean container with a volume of 10 ml.

[0068] Purification of the sample was performed by recrystallization with the use of 1-2 ml of boiling distilled water. Then the solution was cooled to until its temperature became a room one. Then the solution was cooled on an ice bath for five minutes and dried in vacuum.

[0069] The product was received also by incubation at higher temperatures up to 65 C., it was crystallized after from 24 hours to a week. If phenylcreatine remained in the solution, the solution was filtered until the dry crystals of the substance were discovered, vacuum filtration was used. The output of phenylcreatine ultimately amounted to 65-80%. Mass spectrum, found: m/z: MYR 207.2. Calculated: M 209.

EXAMPLE 2. THE STUDY OF THE STABILITY OF PHENYLCREATINE COMPARED TO CREATINE IN AQUEOUS SOLUTION AND IN BLOOD

[0070] The study of the stability of phenylcreatine and creatine in aqueous solution and in human blood was carried out as follows.

[0071] For the preparation of solutions of test substances on an analytical balance the exactly weighed phenylcreatine and creatine were taken. The calculated amount of double-distilled water was added to them to obtain a concentration of 1 mg/ml. A part of the solution was diluted 10 times, and the sample was immediately analyzed. Further, that solution was kept at room temperature and after 3 hours the analysis was repeated.

[0072] Further according to the method described in Dunnett, Harris & Orme (1991) Reverse phase ion-pairing high performance liquid chromatography of phosphocreatine, creatine and creatinine in equine muscle. Scand. J. Clin. Lab. Invest. 51, 137-141, page 139, aliquot (c), creatinine, creatine and phosphocreatine were removed from the samples used for obtaining of the blood serum for mixing, to obtain more accurate results.

[0073] 1 ml of water or prepared according to the method above blood serum was added to 200 l of a solution in water of initial substance with a concentration of 2-3 mg/ml, shaken and a sample of 200 l volume was immediately taken and initial concentration was analyzed. Then the solution was placed in a vibration thermostat at 37 C. and an aliquot of 200 l was taken in 0.5, 1 and 3 hours of incubation. 20 l of a 10% solution of trichloroacetic acid was added to the selected sample and kept for 15 min at a temperature of minus 24 C., centrifuged at 6000 g for 5 min to precipitate the plasma proteins, supernatant was collected and its analysis was conducted.

[0074] The study of the stability of phenylcreatine compared to creatine in an aqueous solution and blood was carried out using reversed-phase HPLC using a chromatographic system Agilent 1220 Infinity LC System (USA).

[0075] Buffer A was 30% acetonitrile with 0.1% TFA

[0076] Buffer B was 70% acetonitrile with 0.1% TFA

[0077] The temperature of 50 C., detection 220 nm

[0078] Flow 1.5 ml/min.

[0079] Column XRbridge Peptide BEH C18 (Waters) 5 m 300 150*4.6 mm

[0080] The following gradient was used (Table 1).

TABLE-US-00001 TABLE 1 Gradient used for assessing phenylcreatine and creatine stability Time, min. 0 20 21 % A 100 0 100 % B 0 100 0

[0081] To assess the stability of the analytes, the peak areas of the compounds were compared at the beginning of the experiment and at selected intervals (Table 2).

TABLE-US-00002 TABLE 2 Stability of phenylcreatine in comparison with stability of creatine in blood serum Stability in a blood serum Substance 0 h 0.5 h 1 h 3 h phenylcreatine 100% 100% 99% 96% creatine 100% 98% 67% 52%

[0082] As follows from the data given, phenylcreatine has a high stability in the blood, and the concentration remained practically unchanged for 3 hours, while the creatine concentration in the human blood decreased to 52%.

EXAMPLE 3. EVALUATION OF THE FUNCTIONAL STATE OF MICE IN A TREADMILL TEST WITH CREATINE AND PHENYLCREATINE

[0083] In order to find out whether phenylcreatine is a functional analogue of creatine, and also how much its effect is related to the strength of creatine, the functional state of the mice was assessed, namely, the body weight was measured, activity and endurance in the test on white mongrel mice were assessedmales weighing 18-22 g.

[0084] Two experimental groups of mice and one control group (10 mice in each group) were selected. Initially, the animals were of equal mass. The animals were kept in accordance with the rules adopted by the European Convention for the Protection of Vertebrates used for experimental and other purposes (European Convention for the Protection of Vertebrates used for Experiments or for Other Scientific Purposes (EST No. 123), Strasbourg, 18 March 1986, M., 1990, 12 pp.). Animals were kept in standard vivarium conditions. The animals were killed by decapitation in accordance with the Rules for carrying out work using experimental animals, approved by order of the Ministry of Health of the USSR No. 742 of 13 Nov. 1984 (Bolshakov O P, Neznanov N G, Babakhnyan R V Didactic and ethical aspects of research on biomodels and on laboratory animals//Qualitative clinical practice. 2002. No. 1. P.58-61).

[0085] Within 20 days, the animals received an aqueous solution of creatine in a dosage of 0.3 mg per gram of weight. The dosage is chosen according to the data that the daily intake of creatine in the amount of 20 g for adult men of average weight 75 kg for six days leads to an increase in the concentration of muscle creatine (Daniel Santarsieri TLS., Antidepressant efficacy and side-effect burden: a quick guide for clinicians Drugs in Context. 2015; 4: 1-12.). Upon administration, the drug was dissolved in 0.3 ml of water and injected into mice through a probe into the stomach daily in the morning, on an empty stomach. The animals of the control group received a similar volume of water. Phenylcreatine was also administered for 20 days in an amount of 50 mg per kg of body weight.

[0086] Weighing of animals was performed on the 1st, 5th, 10th, 15th and 20th days of the study, on an empty stomach, immediately before the administration of creatine, phenylcreatine or distilled water in the control.

[0087] Endurance of mice under physical exertion was assessed according to a standard procedure (Emirova L R Potention by citamins of the action of medicinal substances that increase the endurance of athletes: dis . . . medical doctor: 14.00.25. M., 2004. 125 pp.) for the duration of running in the treadmill test. The animals of each group were subjected to daily training loads in a high load power mode, which was modelled by running on a treadmill at a speed of 29-31 m/min. The duration of daily mice training was 5 minutes. Endurance of mice was tested on the 1st, 5th, 10th, 15th, 20th and 25th days of training against the background of administration of drugs (or distilled water in the control). Endurance testing was conducted under the same conditions as training. Endurance was tested 1 hour after drug administration (Petrenko E R Comparative pharmacological study of adaptogenic properties of ginseng preparations: dis . . . candidate of biological sciences: 14.00.25., St. Petersburg, 1998. 126 pp.) until fatigue, the criterion of which was the lack of reaction of mice to stimulation of the legs and tail by electric current (Stratienko E N Influence of phenylethyl substituted derivatives of 3-oxypridine on the physical working capacity of mice under conditions of hypobaric hypoxia: dis . . . . Medical Candidate of Sciences Bryansk, 1996. DSP. 201 pp.). Running time was recorded in seconds. The study was carried out at rest, an hour after the administration of creatine or phenylcreatine, and immediately after the end of the run in the treadmill.

[0088] Statistical processing of data was carried out in the program Statistica, for all data groups, using the Student's criterion.

[0089] The following results were obtained on the effect of administration of creatine and phenylcreatine on the body weight of mice.

[0090] Body weight of the animals of control (initially 192 g) and the experimental groups taking creatine (initially 182 g) and phenylcreatine (originally 18.62 g), changed insignificantly. There was a tendency to increase in mass in the experimental groups, weight gain was 9% for the group of animals that received creatine and 15.4% for the group of animals receiving phenylcreatine. The increase in the body weight of mice in the control group was 6.4%, the data are reliable at 95% significance level.

[0091] The following results were obtained concerning the effect of the intake of creatine and phenylcreatine on the endurance of mice. Dosages of 10 mg of phenylcreatine per animal and 300 mg of creatine per animal were used.

[0092] During the entire period of the study, the running time to total fatigue significantly increased on the 15th day of the study 2.8 times for animals from the experimental group receiving creatine and 6.4 times for the group receiving phenylcreatine, while in the control group the endurance increased in 1.1 times, on the 20th day of the study4.5 times for animals from the experimental group that received creatine, and 6.9 times for the group receiving phenylcreatine, while in the animals of the control group, endurance increased by 1.4 times, on the 25th day of research5.6 times for animals from the experimental group that received creatine, and 6.7 times for the group that received phenylcreatine, whereas in animals of the control group, endurance increased 1.7 times (Table 3, FIG. 1).

TABLE-US-00003 TABLE 3 Running time of mice (n = 15) at administration of creatine and phenylcreatine (M m) Groups 1 d 5 d 10 d 15 d 20 d 25 d Controle, s 556.71 21.74 599.80 43.00 669.00 52.91 648.60 46.52 789.50 56.40 934.6 76.3 The group receiving 626.00 32.25 772.60 33.07 775.80 37.01 1811.30 85.82 2794.70 103.22 3697.5 1373 creatine, s The group receiving 588.23 25 747.23 17.06 1874.12 114.43 3756.77 97.02 4076.45 276.57 3981.4 202.8 phenylcreatine, s P, groups receiving P > 0.05 P 0.05 P > 0.05 P 0.05 P 0.05 P 0.05 creatine and phenylcreatine

[0093] As a result of the course receiving, both creatine and phenylcreatine, a significant increase in endurance is observed for 20 days, starting from the 15th day of intake for creatine and from the 10th day of intake for phenylcreatine, and increases until it is completed. The maximum effect from the intake of phenylcreatine is already on the 15th day, that is, it increases 2 times faster than in the experimental group receiving creatine. The results obtained allow us to conclude that the intake of creatine contributes to an increase in endurance and ability to work. Taking of phenylcreatine further enhances this effect, and also promotes the body to the peak of physical abilities in preparation for physical exertion. This effect of phenylcreatine persisted even in the absence of sleep.

EXAMPLE 4. EFFECTIVENESS OF PHENYLCREATINE IN THE THERAPY OF EXTRASYSTOLES

[0094] One of the most important factors of arrhythmogenesis and the appearance of extrasystoles is the activation of the sympathoadrenal system. This circumstance determined the necessity of investigating phenylcreatine, proposed by the author of the present invention, on the model of adrenal arrhythmia (extrasystole) in rats (Kushakovsky M S, Heart arrhythmias: a guide for physicians, St. Petersburg, Hippocrates 1992).

[0095] In a control series of experiments, in all animals, 12 seconds after injection of epinephrine hydrochloride at a dose of 50 mg/kg, polytopic ventricular extrasystole occurred in all animals. The number of ventricular extrasystoles before transition to tachycardia averaged 326. The duration of such arrhythmias was 8021 seconds. In 50% of cases, it passed into the ventricular tachycardia. The duration of tachycardia was, on average, 8612 seconds.

[0096] With the introduction of phenylcreatine, proposed by the present inventor in an amount of 20 mg per animal, half an hour before adrenaline hydrochloride, the number of ventricular extrasystoles was 124. The duration of the arrhythmia was 6014 seconds. There was no transition to tachycardia.

[0097] Additionally, the following study was carried out. Man, 36 years old, professional sportsman (15 years of experience in power triathlon). Supraventricular extrasystoles with a frequency of 4 times in 24 hours were observed according to holter monitoring. Extrasystoles were very poorly tolerated, there were complaints of discomfort and a decrease in the quality of life (neurosis-like condition). He took phenylcreatine in an amount of 2 mg per 1 kg per day, for 14 days. A gradual decrease in the amount and strength of extrasystoles since the initiation of phenylcreatine was noted, after the course extrasystoles completely disappeared. Within 3 months after the course the holter monitoring does not fix supraventricular extrasystoles.

EXAMPLE 5. EVALUATION OF NOOTROPIC ACTION OF PHENYLCREATINE

[0098] The experiments were performed on male Wistar rats born in September 2014 (experiments were conducted in November 2016). The animals were kept in standard plastic cells at an air temperature of 21-23 C. They received a balanced granular food and drinking water without restrictions. The work was carried out in compliance with the principles of the Helsinki Declaration on Humane Treatment of Animals.

[0099] Rats were divided into 2 groups. The rats of the first test group received 10 mg of phenylcreatine per animal daily for a month with drinking water. The rats of the second test group received water. As a control in the experiment, rats born in May 2016 (the third group, young rats) were used.

[0100] In the experiment, a shuttle maze was used to evaluate neuropsychiatric processes, primarily cognitive processes (Navakatikyan M A, Platonov L L, 1988). At the end of the labyrinth there was a food reinforcement (a piece of cheese with a mass of 200 mg).

[0101] The time of the experiment to find the exit from the labyrinth was 5 minutes. During the experiment, the time of passing the labyrinth, the number of rats reaching the end of the labyrinth, the number of vertical racks were recorded.

[0102] If consider age dynamics in terms of locomotor and cognitive activity, it decreases with age 5 times (from 3 months to 24 months) (Anisimov V N, 2001).

[0103] Results:

[0104] Group 1. (Old rats 25 months plus phenylcreatine)

[0105] The number of racks per minute1.20.44

[0106] The number of rats reaching the end of the labyrinth in 5 minutes50%

[0107] The transit time of the labyrinth is 20.22 minutes

[0108] Group 2. (old rats 25 months)

[0109] Number of racks per minute20.56

[0110] The number of rats reaching the end of the labyrinth in 5 minutes10%

[0111] The transit time of the labyrinth is 50.42 minutes

[0112] Group 3. (young rats 6 months)

[0113] Number of racks per minute0.30.21

[0114] The number of rats reaching the end of the labyrinth in 5 minutes70%

[0115] Time of passage of the labyrinth is 10.12 minutes

[0116] The results obtained confirm the possibility of using phenylcreatine according to the invention as a nootropic agent.

[0117] It should also be noted that, with all the studies conducted, the negative effects of phenylcreatine according to the invention were not detected, which indicates its safety.