SYNTHESIS, STRUCTURE AND RESEARCH OF THE PHARMACOLOGICAL ACTIVITY OF METHYL ESTERS OF 6-NITRO-N-PHENILANTHRANILIC ACIDS

Analysis of scientific and patent literature shows the promising results of searching biologically active compounds among derivatives of aromatic aminoacids. For many years at the Medical Chemistry department of the National University of Pharmacy the research has been conducted in the field of development of synthetic methods and study of physico-chemical and pharmacological properties of aromatic acids, in particular, N-phenylanthranilic acids and products of their transformation in order to search active and harmless medicines. The synthesis of methyl esters of 6-nitro-N-phenylanthranilic acids has been carried out by Fisher esterification in the absolute methanol medium in the presence of concentrated sulfuric acid. Substituted 6-nitro-N-phenylanthranilic acids have been obtained by Ullmann reaction by the interaction of 6-nitro-2-chlorobenzoic acids with arylamines and by arylation of 6-nitro-N-phenylanthranilic acids by halogenobenzenes derivatives in the medium of n-amylalcohol, in the medium of dimethylformamide, without a solvent in the presence of copper or CuO. The structure of the compounds has been confirmed by elemental analysis, IRand NMR-spectroscopy. The purity has been controlled by the method of thin-layer chromatography in methanol-hexane (1:1.5) and ethylacetate-methanol-ammonia (8.5:1:0.5). The computer prognosis of possible types of the biological activity of 9 methyl esters of 6-nitro-N-phenylanthranilic acids synthesized for the first time has been conducted with the help of PASS programme. It has been found experimentally that the substances synthesized possess the anti-inflammatory, analgesic, diuretic, bacteriostatic, fungistatic and antidiuretic activities. According to the classification by K.K.Sydorov the compounds synthesized when introduced intragastrically belong to low toxic compounds (DL50=1200-2500 mg/kg). Some regularities of the “structure – biological activity – toxicity” relationship have been determined.

Derivatives of N-phenylanthranilic acids (N-PAA) are used in medicine as nonsteroidal anti-inflammatory drugs. Some of them have proven to be the most effective by the nonspecific action, and do not cause degradation of glycosaminoglycans and collagen in joints [7,14,15]. Long-term studies in the field of the series of N-arylanthranilic acids and their derivatives by researchers have led to the creation of effective medicines (mefenamic acid and its alkali salt, antral, diphtorant), which are widely applied in medical practice as antiinflammatory, hepatoprotective and antipsoriatic medicines [7,8]. Data of the research of domestic and foreign scientists indicate that derivatives of N-phenylanthranilic acids have a wide synthetic and pharmacological potental [2-4, 6, 8, 10-15]. These circumstances determined to synthesize methyl esters 6-nitro-N-phenylanthranilic acids and study their biological activity.
The synthesis of methyl esters of 6-nitro-N-PAA (4a-i) has been carried out by Fisher esterification in the medium of the absolute methanol in the presence of the concentrated sulfuric acid (Scheme).
In the NMR-spectra of esters the signals of aromatic protons in the range of 6.59-7.55 ppm have been identified. The proton signal of the secondary aminogroup appears as a broad singlet in the region of 7.44-7.64 ppm. The characteristic signal of methyl esters is the signal of COOCH 3 -group, which is registered in the range of 3.95-4.15 ppm (Tab. 2).
IR-spectra of methyl esters of 6-nitro-N-PAA are characterized by a number of intense bands, which correspond to the main structural fragments of molecules of the substances synthesized. In the region of 1728-1698 cm -1 an intense band corresponding to stretching vibrations of the ester group carbonyl has been interpreted (v C=O ). In the regions of 1282-1270 cm -1 and 1155-1145 cm -1 there are stretching vibration bands of C-O-C-( , ), respectively. The first band refers to the stretching vibration of C-O-C -group, to which the main contribution is made by fluctuations of the acidic fragment of the molecule, the main contribution to the second band vibrations is made by the alcoholic fragment of the molecule. Symmetric and asymmetric vibrations of the nit-Scheme Table 1 Physical and chemical properties of methyl esters of 6-nitro-N-phenylanthranilic acids  rogroup in the spectrograms appear in the regions of 1540-1528 cm -1 ( ) and 1357-1328 cm -1 ( ). Deformation vibrations of NH-group (δ NH ) are presented in the spectrograms by peak in the region of 1584-1570 cm -1 (Tab. 2). Using the PASS programme the analysis of the computer prognosis results shows that 6-nitro-N-PAA (4a-i) methyl esters synthesized for the first time are most likely to reveal the anti-inflammatory, antitumor, diuretic, bacteriostatic, fungistatic activity.
According to Sydorov K.K. classification, methyl esters belong to the class of low toxic substances, their DL 50 with the internal administration in mice is within the range of 1200-2500 mg/kg (Tab. 3). As expected, methyl esters are more toxic than initial acids [6].
Among the esters of 6-nitro-N-PAA (Tab. 3) the strongest diuretic activity is revealed by compound (4g), but it is inferior to hypothiazide. It has been found that the carboxyl group esterification in 6-nitro-N-PAA (4a-i) leads to decrease of the diuretic, analgesic, anti-inflammatory effect and increase of acute toxicity. Compounds (4b, 4e) possess the antidiuretic activity, but they are inferior to adiurecrine (65%). The pharmacological screening on the anti-inflammatory and analgesic activity (Tab. 3) in the dose of 20 mg/kg has revealed the substances (4h, 4i) with the anti-exudative effect at the level of mefenamic acid (30%). It should be noted that methyl esters of 6-nitro-N-PAA (4a-i) are less active than initial acids (3). The anti-inflammatory activity of N-PAA esters and their derivatives (4a-i) is shown to be in close connection with their structure; by the anti-exudative action they can be ranged in the following way: D-glucosylammonium salts o0f N-PAA > acids > methyl esters. The microbiological research shows that the substances synthesized inhibit the growth of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeuruginosa in the concentrations of 31.2-500 µg/ml (Tab. 3). Esters (4h, 4i), which contain covalently bound chlorine and bromine in their structure in the neoanthranilic fragment of the molecule, reveal the most expressed bacteriostatic activity. These substances inhibit Staphylococcus aureus and Escherichia coli more selectively (MIC=31.2 µg/ml). The fungistatic activity of methyl esters of 6-nitro-N-PAA (4a-i) is 31.2-500 µg/ml in relation to Candida albicans and Candida tropicalis. Compounds (4h, 4i) exceed twice the effect of nitrofural by their fungistatic activity (Tab. 3) and are less toxic.
Experimental Part IR-spectra of the substances synthesized were measured on a "Specord M-80" spectrophotometer in КВr tab- lets (with the concentration of 1%). The chromatographic analysis was performed by the method of thin-layer chromatography on "Silufol UV-254" plates produced by "Avalier" firm (Czech Republic); chromatograms were developed by iodine vapours or using UV-radiation. The NMR-spectra were registered on a "Varian M-200" spectrophotometer with the operation frequency of 200 MHz, DMSO-d 6 as a solvent and TMS as an internal standard were used. Methyl ester of 6-nitro-N-phenylanthranilic acid (4a). Heat the mixture of 2.58 g (0.01 mol) of 6-nitro-N-phenylanthranilic acid, 0.75 ml of the concentrated sulfuric acid in 30 ml of absolute methanol with a reflux condencer for 5 hours. After cooling pour the reaction mixture into water and neutralize by sodium hydrocarbonate. Filter the precipitate and dry. The product's yield was 2.20 g (81%). Compounds 4b-i were obtained similarly.
To reveal the anti-inflammatory activity of new compounds their ability to inhibit edema development in acute inflammation caused by carrageenin subplantary injection in the mouse paw was researched [5]. The compounds examined were taken orally as a suspension stabilized by emulsifier Tween-80 in the dose of 20 mg/kg of the animal's body mass. The analgesic action of substances (4a-i) was studied in white rats (weighing 160-200 g) using the acetic acid-induced writhing test in the dose of 20 mg/kg [5]. The diuretic effect of N-PAA esters (4a-i) was studied by Berkhin Ye.B. method [1]. The substances under research and the reference medicine (hydrochlorothiazide) were injected intraperitoneally 30 min before the water load in the dose of 50 mg/kg (Tab. 3). The study of the bacteriostatic and fungistatic activity of substances (4a-i) in vitro was performed by the twofold serial dilution method in the liquid nutrient medium.
Acute toxicity of the substances synthesized was studied in white mice when introduced intragastrically [5].
The PASS programme was used for computer prognosis of the biochemical activity of 6-nitro-N-PAA methyl esters [10]. CONCLUSIONS 1. To search biologically active substances the synthesis of 6-nitro-N-phenylanthranilic acids methyl esters has been done, and their structure and purity has been studied.
2. Using the PASS programme computer prognosis of possible types of the biological activity has been conducted. It has been found experimentally that the substances synthesized possess a moderate anti-inflammatory, analgesic, diuretic, antidiuretic, bacteriostatic and fungistatic antidiuretic activities. Some regularities of the "structure -biological activity -toxicity" relationship among the N-phenylanthranilic acid derivatives have been determined.