Synthesis of novel [1,2,4]triazolo[1,5-b][1,2,4,5]tetrazines and investigation of their fungistatic activity

A series of novel [1,2,4]triazolo[1,5-b][1,2,4,5]tetrazines has been synthesized through oxidation reaction of the corresponding 3,6-disubstituted 1,2,4,5-tetrazines bearing amidine fragments. It is shown that the heterocyclic systems obtained can be modified easily at C(3) position in the reactions with aliphatic alcohols and amines. Also, the reactivity of [1,2,4]triazolo[1,5-b][1,2,4,5]tetrazines towards CH-active compounds has been studied. The obtained triazolo[1,5-b]annulated 1,2,4,5-tetrazines proved to be active in micromolar concentrations in vitro against filamentous anthropophilic and zooanthropophilic dermatophyte fungi (Trichophyton, Microsporum and Epidermofiton), which cause skin and its appendages (hair, nails) diseases.


Introduction
Azolo-annulated azines can be regarded as purine isosteres and are of great interest for modern medicinal chemistry as potential biologically active compounds. In particular, imidazo [1,2-a]pyridines and imidazo [1,2-a]pyrimidines exhibit a wide spectrum of biological activity, including antiviral and antibacterial ones [1,2].
A promising approach for the development of new drugs is the synthesis and bioscreening of high nitrogen-containing azoloazines, including azolo-annulated tetrazines. These compounds, bearing a large number of heteroatoms in their structures, have additional opportunities for non-covalent bonding with a variety of biological targets. In addition, a high electrophilic character of the tetrazine ring can provide chemical binding to pathogenic objects, thus leading to a disturbance of their functions.
It has been shown that the oxidative cyclization of 1,2,4,5tetrazines 2a-i into triazolo [1,5-b] [1,2,4,5]tetrazines 3a-i takes place by action of (diacetoxyiodo)benzene on heating in trifluoroethanol. The target products were obtained in 10-86% yield. According to TLC data, the synthesis of derivatives containing 4-methylphenyl (3e), phenyl (3b) and 4-chlorophenyl (3d) substituents proved to be accompanied by the formation of a large number of byproducts of unknown structure, thus decreasing the yield to 15-30%. The yield of triazolotetrazine 3i unsubstituted at C(7) was only 10%. It can be explained by a low stability of the starting tetrazine 2i containing the formamidine moiety, since the latter on heating is easily transformed into the amino group, thus leading to 3-amino-6-(3,5dimethylpyrazol-1-yl)-1,2,4,5-tetrazine. It has been found that N-bromosuccinimide can also be used as an oxidant for the oxidation of benzamidine derivatives 2b,c in acetonitrile under microwave irradiation. Along with the cyclization reaction, bromination of the pyrazole ring proved to occur at position C(4) to give the products 3j,k, as evidenced by disappearance of the characteristic singlet of H(4) of the pyrazolyl substituent in the region of 6.38-6.41 ppm in the 1 H NMR spectrum. A small amount of N-(6-(4-bromo-3,5-dimethylpyrazol-1-yl)-1,2,4,5tetrazin-3-yl)benzamide, as a byproduct, was isolated from the reaction mixture, the structure of the latter was confirmed by the XRD data ( Figure 1).  In order to select optimal conditions for the synthesis of compounds 3, the oxidative systems have been varied for the reactions of tetrazines 2a,b,g bearing methyl, phenyl, and pyrazolyl substituents. Attempts to use Pb(OAc) 4 as an oxidizing agent in CHCl 3 resulted in the formation of difficult-to-separate reaction mixtures with a low content of the target products 3a,b,g, which could not be isolated in a pure form. Heating of tetrazines 2 with N-bromosuccinimide in refluxing acetonitrile without microwave irradiation made it possible to obtain the product 3j in a low yield (10%), but did not afford the products 3a,g. The reactions of 2 with (diacetoxyiodo)benzene in acetonitrile gave the target products 3a,b,g, however, both yields and purities of these compounds proved to be higher when trifluoroethanol was used as a solvent.
It has been found that triazolo[1,5-b][1,2,4,5]tetrazines 3 do not enter the characteristic for isomeric [4,3-b]-annulated derivative reactions with malonic ester, leading to expansion of the tetrazine ring and the formation of triazolotetrazepines [36]. At the same time, a more active malononitrile reacts easily with compounds 3a,j to afford triazolopyrimidines 6a,b (Scheme 4). In the reaction of ethyl cyanoacetate with triazolotetrazine 3j, a mixture of products is formed, in which, along with the diamino compound 7 and triazolotetrazepine 8 (which is characteristic for the behavior of [4,3-b]-annulated systems) triazolopyrimidine 9 has been obtained (Scheme 4).
Product 9, like products 6a,b, appears to be derived from double addition of the reagent, accompanied by opening of the tetrazine ring and recyclization (Scheme 4). In the reaction of 7-methyl-substituted triazolotetrazine 3a with ethyl cyanoacetate, a rather complicated mixture of several products has been obtained, none of the latter failed to be isolated in a pure form. Thus, it has been shown that new triazolo [1,5b] [1,2,4,5]tetrazines retain electrophilic centers inherent in the known triazolo [4,3-b] [1,2,4,5]tetrazine system. However, their reactions have a low regioselectivity, thus leading to a large number of byproducts, thus making it difficult to separate the reaction mixture.
For a comparative analysis of antifungal activity, the previously described [37] 3-methyl-and 3-phenyltriazolo [4,3b] [1,2,4,5]tetrazines 10a,b were used as structural analogues of compounds 3a,b. Furthermore, in the reactions of compound 10a with methanol, heptylamine and morpholine, new derivatives 11a-c have been obtained, which can be regarded as isomers of compounds 4a-c (Scheme 5). Analysis of the data on fungistatic activity has revealed that in the series of compounds 2, 10 and 11 no derivatives exhibit a pronounced activity (Table 1).