Synthesis, Tautomeric Structure and Antimicrobial Activity of 3-Arylhydrazono-4-phenyl-[1,2,4]-triazepino[2,3-a]quinazoline-2,7(1H)-diones

A simple strategy for the synthesis of the hitherto unreported 3-arylazo-4-phenyl- [1,2,4]triazepino[2,3-a]quinazoline-2,7(1H)-diones is described. Spectral data indicated that the studied compounds exist predominantly in the hydrazone tautomeric form. The antimicrobial activity of the newly synthesized compounds was also evaluated. The results indicated that some of these compounds have moderate activity towards bacteria.

It has been reported recently that the azo-hydrazone tautomerism of diazonium coupling products of acyclic and heterocyclic active methylene compounds in both ground and excited states is useful in the field of material sciences [18][19][20][21][22][23][24].
The structure 4 can exist in one of the possible tautomeric forms 4A-D as shown in Figure 1. The isomeric form 4A is the predominant one based on the spectral data (MS, 1 H-NMR and IR). For example, the 1 H-NMR spectrum revealed three characteristic signals near  4.52, 7.38-8.22 and 13.15 which are assignable to CH 2 , C 6 H 5 and the ring NH protons, respectively. Also, the mass spectrum of compound 4 showed the molecular ion peak at m/z 304, which is consistent with its structural formula.
Reaction of 4 with diazotized anilines in basic medium afforded the respective arylazo derivatives 6 (Scheme 2). The mass spectra of the latter products revealed the molecular ion peaks at the expected m/z values and their elemental analyses data were consistent with their assigned structures. The infrared spectral data of compounds 6 (see Experimental) seem to be consistent more with the hydrazone tautomeric form 6A rather than the other tautomeric forms 6B-E (Scheme 2). For example, all compounds exhibit two carbonyl bands in the regions 1715-1681 and 1698-1643 cm −1 corresponding to the stretching vibrations of the pyrimidinone and the 1,2,4-triazepinone carbonyl groups, respectively. The observed wave number of the latter CO stretching band in compounds 6 seems to result from the possible strong hydrogen bond with the hydrazone NH and conjugation with the C=N double bond as required by the hydrazone form 6A (Scheme 2) [29].
To elucidate the actual tautomeric form of the studied compounds 6, we investigated their electronic absorption spectra. The data are summarized in Table 1. As shown, each of compounds 6 in dioxane exhibits three characteristic absorption bands in the regions 385-346, 328-248 and 257-231 nm. Such an absorption pattern is similar to that of typical hydrazone chromophore [23,24]. Furthermore, the spectrum of 6d, taken as a typical example of the series studied was recorded in variety solvents with different polarities. The spectra obtained showed little, if any, shift ( Table 1).
The small shifts in λ max of 6d in different solvents are due to solute-solvent interaction. In agreement with this conclusion, is the observation that the spectra of arylhydrazones derived from the reaction of quinones with N-alkyl-N-phenylhydrazine, unlike those of o-and p-hydroxyazo compounds, are largely independent of the solvent polarity [30]. This finding, while it excludes the azo tautomeric forms 6B, 6D and 6E, indicates that each of compounds 6 exist in one tautomeric form, namely 6A (Scheme 2).

Anti-Microbial Activity
In vitro anti-microbial screening of compounds 4 and 6a-j prepared in this study was carried out using two bacteria species, including a Gram negative bacterium, Escherichia coli (RCMB 000103) (EC) and a Gram positive bacterium, Staphylococcus aureus (RCMB 000106) (SA) and two fungal strains, namely Aspergillus fumigatus (RCMB 002003) (AF) and Candida albicans (RCMB 005002) (CA) using the agar diffusion well method (see Experimental). The microorganisms were tested against the activity of solutions of concentrations of 1.0 mg/mL of each compound in dimethylsulfoxide (DMSO) and using an inhibition zone diameter in cm (IZD) as criterion for the antimicrobial activity. The fungicide amphotericin B and the bactericide tetracycline were used as references to evaluate the potency of the tested compounds under the same conditions. The results, depicted in Table 2, showed that compounds 6a, e, h-j displayed moderate activities against E. coli and S. aureus, while, none of the tested compounds have any activity against A. fumigatus and C. albicans. These biological activities of compounds 6a-j are far less than the studied reference drugs (tetracycline and amphotericin B). Table 2. Antimicrobial Activity of the newly synthesized compounds.

Agar Diffusion Well Method to Determine the Antimicrobial Activity
The microorganism inoculums were uniformly spread using sterile cotton swab on a sterile Petri dish containing Malt extract agar (for fungi) and nutrient agar (for bacteria). Each sample (100 μL) was added to each well (6 mm diameter holes cut in the agar gel, 20 mm apart from one another). The systems were incubated for 24-48 h at 37 °C (for bacteria) and at 28 °C (for fungi). After incubation, microorganism growth was observed. Inhibition of the bacterial and fungal growth were measured in mm. Tests were performed in triplicate p [31].