Anxiolytic and method for preparing it. RU patent 2519755.

FIELD: medicine.

SUBSTANCE: invention concerns an anxiolytic representing the amino acid glycine immobilised on the detonation-synthesised nanodiamond particles of 2-10 nm in size, and a method for preparing it.

EFFECT: improving properties.

4 cl, 7 dwg, 6 tbl, 3 ex

The invention relates to medicine, in particular to pharmacology and concerns of anxiolytic, representing glycine, immobilized on particles of detonation nanodiamond.

Currently, the most common psihonevroticheskih disorders are conditions "unreasonably" anxiety and sleep disorders. For their elimination, prevention and treatment of psychosomatic diseases, somatogenne disorders, the withdrawal of state, insomnia, complex syndromes (anxiety-depression and others), specific conditions, such as panic, obsessive-compulsive-impulsive, social phobia, widely used anxiolytic drug belonging to different classes of chemical compounds exhibiting a wide range of actions [1, 2].

Despite t that modern psychopharmacotherapy has in the Arsenal of more than 100 anxiolytics, continuing their active search and improvement of [2]. This is because modern anxiolytic drug does not satisfy the requirements of the clinic and have many side effects. In particular, benzodiazepine anxiolytics have miorelaksantnoe, sedative and amnestic effect, as antidepressants, including atypical, often cause increased anxiety, the occurrence of cardiotoxic and anticholinergic effects [3, 4]. It is known that certain forms of a mental pathology are rarely isolated and most mental disorders neurotic level is represented as a composition of various psychopathological syndromes, in particular comorbid depressive, anxiety, affective and cognitive impairment [4]. The treatment of such disorders is a serious problem due to the lack of use of specific drugs from different groups and the risk of increased side effects of the drugs caused by the mechanisms of their interaction with combination therapy [5]. Inability to separate anxiolytic activity from the other effects is the main restriction of the use of benzodiazepine drugs. Their use is contraindicated in patients not to stop work, especially if the latter incorporates elements of operator's work [6]. Therefore, the detection of funds with anxiolytic effect, does not have side effects and important for development anxiolytics new generation, which are promising as a means of therapy neurotic and depressive disorders.

It is known that nonessential amino acid glycine (NH 2 CH 2 COOH), being the Central neurotransmitter brake type of action has sedative activity and improves metabolic processes in the brain tissues [1, s]. In modern medical practice glycine is used as a means weakening attraction to alcohol, reduces phenomenon abstinence, depressive disorders, irritability, normalizing sleep, as well as in treatment of disorders of cerebral circulation [1, s]. In the basis of pharmacological action of glycine is the effect of amplification of metabolic and neurotransmitter processes that occur by strengthening its endogenous synthesis. To increase the intracellular synthesis glycine is possible only by way of signal transmission, due to the interaction with the receptor systems. His interaction with glycine receptors leads to the opening of the chloride channel, hyperpolarization membrane and distribution of braking. Along with this glycine is able to act as an allosteric of lagonita glutamate receptors. Linking to a specific site, it enhances the ability of glutamate and N-methyl-D-aspirate (NMDA) open cation channel [7, 8].

Used pharmacopoeial glycine is administered in tablet form (0.1 g) under the tongue 3-4 times a day.

Known glycine, immobilized on particles of detonation nanodiamond size 2-10 nm used as a binder in polymer composites [9, 10]. Its production method is the following [10]. A portion of nd placed in a reactor at a constant current of helium and annealed at temperatures 150-470 OC for 3-4 hours Then hold fluoridation samples nd at a temperature of 50-500 OC for 1-24 h contacts with a mixture of gaseous fluorine and hydrogen. To obtain glycine, immobilized on nanodiamond particles, fluorinated nanodiamond treated with ultrasound in-dichlorobenzene for 20-30 minutes, add ethyl ester hydrochloride glycine (NH 2 CH 2 COOCH 2 CH 3 ·HCl) and a few drops of pyridine. The resulting mixture was stirred at a temperature of 130-140 OC for 8-12 hours the Resulting product is filtered, washed with ethanol and dried under vacuum at 70 degrees C.

Additional feature of this matter is the size of its particles in suspension, equal, according to the dynamic light scattering (DSU), 310 nm [10].

The peculiarity of this matter is the presence on the surface of particles nd apart molecules glycine also fluorine atoms. Although their number by the authors declare less than 1% at., in fact it is experimentally established that the concentration of fluoride on the surface of the nd can reach 14% at. and more. This is because the relationship of C-F (E St. =115 kcal/g atom) is durable and ftorplasty carbon inert in relation to many substances. Therefore, when chemical immobilization of glycine on the surface of nd, containing fluorine atoms, molecules of glycine are replaced by fluorine atoms only partially. It is known that the presence of organic matter fluorine and its derivatives increases its toxicity and may change indicators microsomal system biotransformation of xenobiotics in the liver [11]. So, the presence of fluorine atoms in the near nanostructured similar nd - fullerene (60 ) increases its overall toxicity 2.4-5 times [12]. In addition, fluorine and its compounds can accumulate in different environmental objects and to present them in different quantities [13]. Therefore, glycine, immobilized on nanodiamond particles that contain fluorine atoms, it is undesirable to use in medical practice as a drug.

So getting anxiolytic, representing glycine, immobilized on particles detonation nano-diamonds that does not contain fluorine atoms, with increased dispersion in suspension used as a medicine, as well as reducing environmental and endoecological risk, simplify and reduce the cost of getting anxiolytic, are timely and important task.

The use of glycine, immobilized on particles of detonation nanodiamond as the functional in the scientific and patent literature is not described.

The problem is solved by application described in accordance with the invention of anxiolytic, representing glycine, immobilized on particles of detonation nanodiamond size 2-10 nm, not containing fluorine atoms and with a covering thickness of 1 nm, with content of glycine to 21 or minus 3% of the mass., and method of its production.

Described anksiolitik in the form of glycine, immobilized on particles detonation nano-diamonds that do not contain on its surface atoms of fluorine, is an ultra-fine powder (Figure 1) dark gray or dark gray color with greenish or dark blue shades with particle size from 2 to 10 nm, with a covering thickness of 1 nm (Figure 2), the size of the units in aqueous suspensions of up to 100 nm (Fig 3) and the content of glycine to 21 or minus 3% of the mass., part of the surface of the shell.

Figure 1 clearly shows the presence described anxiolytic ultrafine structure of the particles with a size smaller resolution used device (20 nm).

Micrograph of particles described by the functional received on field emission scanning electron microscope Zeiss ultra-high resolution Ultra Plus (Carl Zeiss, Germany).

Figure 2 shows that the size of particles described anxiolytic, coated of a thickness up to 1 nm equal 2-10 nm.

Micrograph of particles described by the functional received a transmission electron microscope Jeol 1011 (JEOL, Japan).

Figure 3. see the curve of the size distribution of particles in suspension described anxiolytic, from which it follows that the size of the particles in suspension does not exceed 100 nm.

Measurement of the size distribution of particles described by the functional suspension was performed by the method of DSU on the device ZetaSizer (Malvern Instruments, USA). On the x-axis is logarithmic scale the size of particles in nm. The ordinate axis is the percentage of particles of certain sizes.

The elemental composition of the surface of particles described by the functional according to rentgenofotoelektronnoj spectroscopy (XPS) are given in table 1.

Elemental composition and the binding energy of the surface atoms described anxiolytic

Name of characteristics

Chemical elements

Atomic %

77,5-94,5

The binding energy, eV

RUB 285.2±0,5

530,7±0,5

399,8±0,5

Surface investigation described anxiolytic carried out on the instrument LAS-3000 (Riber, France), equipped with a hemispherical analyser ORH-150. For excitation of photoelectrons used rimonabantincanadaje x-ray radiation aluminium anode (AlK? =1486,6 eV) at voltage on the tube 12 kV and current emissions 20 mA. Calibration photoelectron peak held on line C 1s with the binding energy 285 eV. The vacuum in the chamber was 6.7·10 -8 PA. To obtain high vacuum was used ion pump.

The described method of obtaining the functional consists in the following. Detonation nanodiamond annealed in a stream of hydrogen gas at a temperature of 500-1200 OC for 1-8 h, then subjected liquid-phase chlorination of molecular chlorine in photochemical exposure to visible light at temperature 50 to 70 OC for 36-60 hours with the subsequent washing carbon tetrachloride, by centrifugation and dried under vacuum. Modified chlorine nanodiamond dissolved in polar solvents with the formation of a suspension. Add tertiary amine and glycine and process the received mix ultrasound for 5-60 minutes with subsequent keeping at 50-80 OC for 12-48 hours, centrifugation, washing the solvent and drying. Processing by ultrasound are within 5-60 minutes, as tertiary amine use triethylamine and as a polar solvent used pyridine, lower aliphatic alcohol, water-alcohol mixture or water.

Described anksiolitik does not contain fluorine atoms (table 1), and other halogen atoms, in the amount exceeding the error of the instrument (0,1% at.), as in the process of obtaining anxiolytic all atoms replaced by chlorine molecules glycine and leave the surface of the nd in the form of molecules of HCl.

In the process of pharmacological research was conducted to study specific anxiolytic actions described drug and its safety in comparison with pharmacopoeial glycine.

Assessment anxiolytic activity carried out according to the Methodological guidance on the study of the activity of the substances possessing drugs is got with the activity, set out in [14]. The methodology uplifted cruciform labyrinth by Fellow, which is the baseline test and is widely used for search and study of substances with anxiolytic activity both in Russia and abroad [3, 14, 15]. The method is based on the natural fear of finding the rodents of open areas and falling from a height and skill preferences animals dark holes. Anxiolytic effect substances was evaluated by increasing the number of visits in the bright open sleeves and time spent in them and by the number of faecal boles (bowel movements).

The experiments were carried out on the white Mature mice male Balb/c mice weighing 25 to 28 grams of 10 mice each group. The total number of 60 heads.

In the group of animals treated described anxiolytic dose of 0.1 mg/kg, a statistically significantly increased the time spent in the open (dangerous) the sleeves, increased number of visits to the open arm of the maze and decreased the time spent in the closed sleeves (table 3). At doses of 0.5 and 1 mg/kg described anksiolitik also significantly changed the behavior in terms of methodology uplifted cruciform labyrinth, which was reflected in the statistically significant increase of 3.2 (a dose of 0.5 mg/kg) and 2.3 times (dose of 1 mg/kg) time spent animals in open arms.

Thus, the described anksiolitik in the dose range of 0.1-1 mg/kg has a strong anxiolytic effect in terms of methodology uplifted cruciform labyrinth, resulting in a statistically significant increase as time spent animals in open (dangerous) the sleeves, and the number of visits in the open arm of the maze in comparison with control, and reduction in the number of boles defecation. Maximum anxiolytic effect described anksiolitik exhibits in the dose of 0.5 mg/kg and exceeds anxiolytic activity pharmacopoeial glycine.

The study of the acute toxicity described anxiolytic was conducted in accordance with the Methodological guidance on the study of the acute toxicity set out in [16].

When the experiment was registered following indicators: the nature of the coat, changing the state of the mucous membranes, upper eyelid ptosis, increased urinate, increased defecation, increased salivation, piloerection, vocalization, lateral position, rhythm and depth of breathing movements, aggressiveness, fearfulness, tremor, seizures, changes in the thresholds of painful reaction, change posture, catalepsy, impaired coordination of movements in test rotating rod, hold for 5 seconds on the inverted net platform, perelozhenie with inverted mesh platform up, the presence of the pineal, corneal reflexes, sedation, stereotipi and grooming, the death of the animal.

Statistical processing of the results was carried out using statistical packages "patch BIOSTAT" for Windows. Expected average for the group and the standard errors of the parameters.

The results obtained clearly show that the described anksiolitik intraperitoneal administration to mice at doses of 75, 150 and 225 mg/kg, as well as pharmacopoeial glycine, does not cause signs of intoxication and the death of animals within 14 days of observation.

Short description of graphic materials.

Fig 1. Electron micrograph described anxiolytic, obtained by scanning electron microscope.

2. Electron micrograph described anxiolytic received a transmission electron microscope.

3. The size distribution of particles described by the functional in water suspension according to the method of the DSU.

The invention is illustrated by the following examples.

Example 1.

300 mg original detonation nanodiamond annealed in a stream of hydrogen gas with a speed of 3.0 l/h at a temperature of 1000? C for 6 hours Then annealed nanodiamond expose a liquid-phase chlorination of molecular chlorine, dissolved in 40 ml of CCl 4 to 6% of the mass. Cl 2 . The reaction of chlorination is carried out at the photochemical effect visible for 60 hours at a temperature of 60 degrees C. The sample is then washed CCl 4 centrifugation suspension at 6000 rpm and dried under pressure of 0.1 mm Hg to constant weight. Then of chlorinated nd receive suspension, with 40 ml of water-alcohol mixture (water : methanol = 1:1), which contribute to 300 mg glycine in the form of free amino acids (NH 2 CH 2 COOH) with addition of 1 ml of triethylamine. The mixture is treated with ultrasound (50 W) for 60 min and maintained at constant stirring at the temperature at 65 degrees C for 30 PM The received product is washed with a large number of ethanol, centrifuged and dried under vacuum at 70 C during the night. Residual moisture in the product is 2.2%. The output of the target product is 279 mg (93%). The product is a dark gray with bluish tint ultrafine powder (Figure 1) with the size of the primary particles 2-10 nm (Figure 2), with the shell surface layer up to 1 nm. Suspension size of the powder particles does not exceed 100 nm (Fig 3). The elemental composition of the surface of the particles of the resulting product are given in table 2.

Data XPS product obtained

Name of characteristics

Chemical elements

Atomic %,

80,1±0,1

11,5±0,1

8,4±0,1

The binding energy, eV

RUB 285.2±0,5

530,7±0,5

399,6±0,5

For determination of mass fraction of glycine in the received product is prepared 3 mixture of nd with glycine, with content of 1:1,75:3,5 respectively. For each mixture take the sample mass 0,0035 g and carefully fray in a mortar with 0,090 g KBr. 0,070 g of the mixture is pressed in a tablet and removed IR-spectrum (Figure 4). Characteristic bands choose when 1407, 1332 and 504 cm-1, respectively, and build for them calibration graphs (Figure 5). The intensity of the corresponding characteristic bands in the IR spectrum of the obtained sample mass 0,0035 g amounted to 0.23, 0.22 and 0,10 per unit, respectively. From the calibration curves a, b, C figure 5 determine the magnitude of the content of glycine in the obtained sample, which is 0,00057±8·10 -5, Therefore, the mass fraction of glycine in the ground sample is 21 or minus 3% of the mass.

Example 2.

The study of specific pharmacological action described anxiolytic.

Animals received from the Central kennel of laboratory animals "Pillar", Moscow region. The content of animals in conformity with the rules of laboratory practice in preclinical studies in Russia (GOST 351000.3-96 and 51000.4-96), to the normative document "regulations on the device, equipment and maintenance of vivarium", approved by Chief State sanitary doctor 06.04.1973, №1045-73, and the Order of MOH №267 dated 19.06.2003, "On approval of the rules of laboratory practice" (GLP) in compliance with the International recommendations of the European Convention for the protection of vertebrate animals used for experimental studies (1997). Animals were kept in vivarium at temperature 20-22 C, light cycle - 12 hours of light and 12 hours of dark periods in plastic cages T/4A size 580 x 375 x 200 mm with the upper stainless steel lid and bedding dedusted of wood shavings. The animals were kept under constant access to forage and water, using the full ration extruded briquettes feed (GOST fed R 50258-92) and drinking water. In experiments were taken into account requirements of the Commission on the ethical treatment of animals of the Russian national Committee on bioethics at the Russian Academy of Sciences and ethical guidelines in the International recommendations for the conduct of biomedical research with animals" (1985). Experiments were performed in the first half of the day.

The experiments were carried out on the white Mature mice male Balb/c mice weighing 25-28 g 10 animals in the group.

Described anksiolitik investigated in comparison with pharmacopoeial glycine. The substance was injected intraperitoneally once in a volume of 0.1 ml 10 g weight mouse for 30 minutes before the experiment. Animals of the control group was injected distilled water. Statistical processing of the results was carried out using statistical packages "patch BIOSTAT" for Windows. Expected average for the group and the standard errors of the parameters.

It is established that the animals of the control group was scared of open space and height, reflected in the increased number of bowel movements and rapid care of animals in the private arm of the maze, where they spent more time (table 3).

In the group of animals treated described anxiolytic dose of 0.1 mg/kg, a statistically significantly increased the time spent in the open (dangerous) the sleeves, increased number of visits to the open arm of the maze and decreased the time spent in the closed sleeves (table 3). Described anksiolitik at doses of 0.5 and 1 mg/kg significantly changed the behavior in terms of methodology uplifted cruciform labyrinth, which was reflected in the statistically significant increase (3.2 when using a dose of 0.5 mg/kg and in 2,3 times when using doses of 1 mg/kg) time spent animals in open sleeves, and also in increase (2.8 times when using a dose of 0.5 mg/kg and 1.7 times when using doses of 1 mg/kg) number of visits animals in the open arm of the maze in comparison with indexes in the control group (table 3).

In the group of animals treated described anksiolitik at doses of 0.5 and 1 mg/kg, decreased the number of visits animals in closed sleeves and time spent in them. The number of boles defecation animals treated described anxiolytic dose of 0.5 mg/kg, was almost 4 times less (p<0.05). In the group of animals treated described anxiolytic dose of 1 mg/kg, the number of boles defecation was 2.7 times less in comparison with this indicator in the control (table 3).

The data obtained indicate expressed anxiolytic action described anxiolytic at doses of 0.1; 0.5 and 1 mg/kg, with a maximum effect at a dose of 0.5 mg/kg

Pharmacopoeial glycine in the dose of 0.1 mg/kg only at the level trends have increased the time spent in the open arms of the labyrinth, and reduced the time spent in the closed sleeves maze, in comparison with the control (table 3). Dose of 1 mg/kg pharmacopoeial glycine statistically significantly increased compared to control the time spent in the open arms (3.5 times), the number of visits in the open arm of the maze (2.3 times), and also reduced the number of visits animals in closed sleeves and time spent in them (table 3).

Thus, the described anksiolitik in the dose range of 0.1-1 mg/kg has a strong anxiolytic effect in terms of methodology uplifted cruciform labyrinth, resulting in a statistically significant increase as time spent animals in open (dangerous) the sleeves, and the number of visits in the open arm of the maze in comparison with control, and reduction in the number of boles defecation. Maximum anxiolytic effect described anxiolytic appears in the dose of 0.5 mg/kg and exceeds activity anxiolytic effect pharmacopoeial glycine.

Example 3.

The study of side effects and toxic effects described anxiolytic.

The investigation was conducted by outbred Mature mice-males weighing 20-24 g aged 2-3 months. Just used 42 animals - 7 teams of 6 mice.

Check the possible side effects of intoxication signs and mortality conducted in 1 hour - 14 days after intraperitoneal administration described anxiolytic in comparison with pharmacopoeial glycine in the same doses.

The study found that the described anksiolitik intraperitoneal administration to mice at doses of 75, 150 and 225 mg/kg did not cause signs of intoxication and the death of animals within 14 days (table 4-6). Thus described anksiolitik did not in mice changes of the coat, the state of mucous membranes. There was also no ptosis of the upper eyelid, increased urinary, defecation, salivation, piloerection, vocalization, lateral position. Within limits were pace and depth of breathing movements, no aggressiveness, fearfulness, tremor, convulsions, catalepsy, stereotypes and grooming. Was not observed change poses. The animals were saved pineal, corneal and pain reflexes. During all 14 days of observation animals were kept inverted mesh platform within 5 (table 4-6).

The study of possible side effects and death of mice after 1 hour after administration of pharmacopoeial glycine and described anxiolytic, a measure of changes in the group in %)

Indicators

Anksiolitik/dose, mg/kg

Pharmacopoeial glycine per dose, mg/kg

The changing nature of the coat

Changing the state of the mucous membranes

Ptosis of the upper eyelid

Increased urinary

Increased defecation

Increased salivation

The presence of piloerection

The presence of vocalizations

There is lateral position

A rhythm and depth of breathing movements

The presence of aggressiveness

The change in reaction by tapping on the cell

Increased fearfulness

The presence of tremor

The presence of convulsions

The changes in the thresholds of painful reaction

Change poses, catalepsy

Poor coordination movements in the test rotating rod

Holding 5 on the inverted mesh platform

Perelozhenie with inverted mesh platform up

The presence of the pineal reflex

The presence of corneal reflex

The presence of sedation

The presence of stereotipi

The presence of grooming

The death of an animal

* - P<0.05 - relatively control by the criterion C2

The study of possible side effects and death of mice after 24 hours after the introduction of the Pharmacopoeia of glycine and described anxiolytic, a measure of changes in the group in %)

Indicators

Anksiolitik/dose, mg/kg

Pharmacopoeial glycine per dose, mg/kg

The changing nature of the coat

Changing the state of the mucous membranes

Ptosis of the upper eyelid

Increased urinary

Increased defecation

Increased salivation

The presence of piloerection

The presence of vocalizations

There is lateral position

A rhythm and depth of breathing movements

The presence of aggressiveness

The change in reaction by tapping on the cell

Increased fearfulness

The presence of tremor

The presence of convulsions

The changes in the thresholds of painful reaction

Change poses, catalepsy

Infringement of coordination of movements in the test rotating rod

Holding 5 on the inverted mesh platform

Perelozhenie with inverted mesh platform up

The presence of the pineal reflex

The presence of corneal reflex

The presence of sedation

The presence of stereotipi

The presence of grooming

The death of an animal

* - P<0.05, with respect to control on the criterion C2

Study possible side effects and death of mice after 14 days after the introduction of the Pharmacopoeia of glycine and described anxiolytic, a measure of changes in the group in %)

Indicators

Anksiolitik/dose, mg/kg

Pharmacopoeial glycine per dose, mg/kg

The changing nature of the coat

Changing the state of the mucous membranes

Ptosis of the upper eyelid

Increased urinary

Increased defecation

Increased salivation

The presence piloerection

The presence of vocalizations

There is lateral position

A rhythm and depth of breathing movements

The presence of aggressiveness

The change in reaction by tapping on the cell

Increased fearfulness

The presence of tremor

The presence of convulsions

The changes in the thresholds of painful reaction

Change poses, catalepsy

Infringement of coordination of movements in the test rotating rod

Holding 5 on the inverted mesh platform

Perelozhenie with inverted mesh platform up

The presence of the pineal reflex

The presence of corneal reflex

The presence of sedation

The presence of stereotipi

The presence of grooming

The death of an animal

* - P<0.05, with respect to control on the criterion C2

List of literature

1. PPM Mashkovsky. Medicines. - 16-e Izd., Rev., Corr. and supplementary): New wave: the Publisher Umerenkov, 2012. P.75-85.

10. Y. Liu, Zh. Gu, J.L. Margrave, V.N. Khabashesku. Functionalization of Nanoscale Diamond Powder: Fluoro-, Alkyi-, Amino-, and Amino Acid Nanodiamond Derivatives // Chem. Mater. 2004. V.16. P.3924-3930.

11. Russian encyclopedia for labour protection. 3 t,2nd ed., Rev. and supplementary V.3. - M: Izd. SC ANAS. 2007. S.

12. N.N. Karkishchenko. Biomedicine, 2009. No. 2. Pp.5-26.

13. TI Shalin, PS Vasiliev. General questions toxic effects of fluoride // Siberian journal of medicine. 2009. №5. Pp.5-9.

14. Guide to experimental (pre-clinical) studies of new pharmacological substances, Ed. RU Khabriev - M: Publishing house of Medicine, 2005. S-263.

15. S. Pellow, P. Chopin, S.E. File, M. Driley. Validation of open-close arm entries in an elevated plus maze as a measure of anxiety in the rat. J. Neurosci. Meth. 1985. V.14. P.149-167.

16. The guidelines for conducting pre-clinical research of medicines 4.1. "NCASP". - M, Publishing house of the Neck and, 2012. 244 S.

1. Anksiolitik, representing glycine, immobilized on particles of detonation nanodiamond size 2-10 nm, with a covering thickness of 1 nm, with content of glycine to 21 or minus 3% of the mass.

2. The method of receiving anxiolytic according to claim 1, characterized in that detonation nanodiamond annealed in a stream of hydrogen gas at a temperature of 500-1200 OC for 1-8 hours, subjected to liquid-phase chlorination of molecular chlorine in photochemical exposure to visible light at temperature 50 to 70 OC for 36-60 hours with the subsequent washing carbon tetrachloride, by centrifugation and dried received modified chlorine nanodiamond dissolved in polar solvent to form a suspension add tertiary amine and glycine, the mixture is treated with ultrasound with subsequent keeping at 50-80 C, centrifugation, washing the solvent and drying.

3. The method of claim 2, where the processing by ultrasound are within 5-60 minutes and conditioning at 50-80°carried out within 12 to 48 hours

4. The method of claim 2, or section 3, where the tertiary amine use triethylamine and as a polar solvent used pyridine, lower aliphatic alcohol, water-alcohol mixture or water.