THE SYNTHESIS OF NEW BIOLOGICALLY ACTIVE SUBSTANCES AMONG 4-AMINO-5-ALKYL-1 , 2 , 4-TRIAZOLE ( 4 Н )-3-YL-THIOACETANILIDES AND THEIR CHEMICAL MODIFICATION

The synthesis of a series of new 4-amino-5-alkyl-1,2,4-triazole(4H)-3-yl thioacetanilides and 4-pyrrolyl-5-alkyl-1,2,4-triazole(4H)-3-yl thioacetanilides has been described in the article. The key intermediates – 4-amino-5-alkyl-3-mercapto-1,2,4-triazoles(4H) 4a-c were synthesized started from the corresponding carboxylic acids (acetic, propanoic and butanoic) after their esterification followed by hydrazinolysis, CS2 reaction and cyclisation under hydrazine hydrate. The first group of substances 6a-p was obtained by alkylation of the key intermediate 4a-c with chloroacetic acid anilides in the presence of basic catalysts. The subsequent modification under conditions of Paal-Knorr reaction led to the corresponding pyrrolyl derivatives 7a-p. The structure of the compounds synthesized has been proven by the data of elemental analysis and NMR spectra. In NMR-spectra the result of alkylation has been confirmed by disappearance of the chemical shift of the mercapto group. All compounds both intermediate 4а-с and end products 6a-u and 7a-u contain signals of the alkyl protons of substituents in the triazole (methyl or methyl and methylene) ring; the 4-aminogroup protons are in the spectra of compounds 6 as singlet signals at 5.87-5.92 ppm. Modification of amino derivatives 6 into pyrrolyl substituted 7 is accompanied with the appearance of the characteristic signals of methyne protons of the pyrrole moiety instead of the signal of the amino group – triplet (positions 3,4) at 6.306.31 ppm and doublet (positions 2,5) at 7.21-7.24 ppm. Substances 7a, 7f, 7j and 7m were tested on the antitumour activity in vitro. As the result of this investigation it was noted that unfortunately all substances selected were not effective inhibitors of tumour cells in this dose.

The synthesis of substituted derivatives of 1,2,4-triazole is well studied and described in many scientific articles.There are many convenient and simple methods for introduction of different substituent into this heterocycle.For several years we engaged in the synthesis of 3-mercapto-1.2.4-triazole derivatives.It has been found that some of these substances are very promising thanks to their pharmacological activity [12][13][14].
There is a lot of information about the synthesis of new promising medicines among 4-aminosubstituted derivatives of 1,2,4-triazole-3-thiol.Different methods for their synthesis are described.Thus, the following procedure is used as general: the ester of carboxylic acid (corresponding to the substituent in position 5) is transformed into hydrazide treated with carbon disulfide (for introduction of the mercapto group) and potassium hydroxide.Sodium dithiocarbazinate obtained is cyclized into the triazole ring with the excess of hydrazine hydrate (introduction of the amino group in position 4).Due to this methods aryl [5,6], substituted benzyl [4], diphenylmethyl [3], pyrydyl [8,9], phthalazine [7] moieties were introduced in position 5 of 3-mercapto-1,2,4tiazole.All compounds synthesized were tested on the biological activity after some chemical transformations (usually the synthesis of Schiff bases or/and fused heterocycles).It has been shown that they can be used as antibacterial and antifungal agents.
We used this method before for the synthesis of similar cyclohexyl [11] and aryl [2] derivatives.The preliminary prognosis of the pharmacological activity using the PASS software [10] has shown that substances of such structure might have the anti-inflammatory, antiviral, membranoprotective action [1].For alkyl (methyl, ethyl, and propyl) derivatives their activity as monoamine oxidase inhibitors is the most likely.Thus, the next synthesis was planned to be carried out in the group of 3-mercapto-4-amino-5-alkyl derivatives and their pyrrolyl products.
At next step Paal-Knorr condensation was used for modification of the chemical structure of the amino group into the pyrrolyl moiety.As a result of this reaction amides 7a-p were obtained.Because of the absence of any activity predicted for amino compounds 6a-p [1], we used them without additional purification for the next step in the synthesis.Their structure was proven by the NMR method.The absence of the initial triazole 4 impurity was assessed chromatographically.All compounds synthesized were obtained with good yields (Table 1).
The structure of the substances 7a-l synthesized has been proven by the data of elemental analysis and NMR spectra (Table 2).
All compounds both intermediate 4 and end products 6a-u and 7a-u contain signals of the alkyl protons of substituents in the triazole ring (methyl or methyl and methylene); the 4-aminogroup protons are in the spectra of compounds 6 as singlet signals at 5.87-5.92ppm.In NMR-spectra the results of alkylation were proven by disappearing the chemical shift of the mercapto group and signals of the acetamide moiety (CONH at about 10 ppm).Signals of aromatic protons of the phenyl ring can be found due to their intensity and multipleticity in accordance with the nature, positions and the number of substituents (Table 2).
Modification of amino derivatives 6 into pyrrolyl substituted 7 is accompanied by changes in the NMR spectra: instead of the amino group the triplet signals of methyne protons of the pyrrole ring (protons in positions 3,4) at 6.30-6.31ppm appear.As to the second pair of the pyrrole ring protons (in positions 2, 5), they sometimes can not be distinguished among other signals of aromatic protons.In other cases they are doublet at 7.21-7.24ppm.
Due to prognosis and logical analysis of the data the substances synthesized will be examined as possible antitumour agents and MAO inhibitors.
From the compounds synthesized such substances as 7a, 7f, 7j, and 7m (Table 1) were selected by the National Cancer Institute (NCI) within the Developmental Therapeutic Programme (www.dtp.nci.nih.gov) for in vitro cell line screening.Anticancer assays were performed according to the US NCI protocol [15].Compounds were evaluated in one dose for the primary anticancer assay towards approximately 60 cell lines (with the concentration of 10 −5 M).The human tumour cell lines represent all forms of cancer (such as non-small cell lung cancer, colon cancer, breast cancer, ovarian cancer, leukemia, renal cancer, melanoma, prostate cancer).In the screening protocol, each cell line was inoculated and pre-incubated for 24-48 h on a microtiter plate.Test agents were then added with the single concentration, and the culture was incubated for an additional 48 h.The end point determinations were made with a protein binding dye, sulforhodamine B (SRB).The results for each test agent were reported as the percent growth of the treated cells compared to the untreated control cells.
The next step of the study according to the PASSprognosis for these compounds will be investigation of the CNS activity.

Experimental Part
Melting points were determined by an open capillary tube.NMR 1 H spectra were recorded on a Bruker WM spectrometer (300 MHz); solvents -CDCl 3 or DMSO-d 6 ; chemical shifts were in ppm, TMS was used as an internal standard.The purity of the compounds synthesized was monitored by TLC.
2. The structure of the compounds synthesized has been proven by the data of elemental analysis and NMRspectra.
3. When studying in vitro four of the compounds synthesized have no high potential in cancer cell inhibition.Due to prognosis and logical analysis of the data the substances synthesized will be examined as possible MAO inhibitors.

Table 1
Yields, melting points of the substances synthesized with general formulas: