Cyclocondensation Reactions of Hydrazonoyl Chlorides with Some Azines : Synthesis of New Fused Heterocycles of Expected Microbiological Activity

New functionalized fused heterocycles, such as, 1,3,6,9,11-pentasubstituted-pyrido[3,2-f:6,5-f']bis([1,2,4]triazolo[4,3-a]-pyrimidin-5(1H)-ones (6) and 1,3-disubstituted-7-[(E)-2-(thiophen-2-yl)ethenyl]-1,4,9,9a-tetrahydro-6H-[1,2,4] triazino[4,3-b][1,2,4,5]-tetrazin-6-ones (16) were synthesized via reaction of the hydrazonoyl chlorides (1) with 1,3,6-triphenyl-9-thioxo-9,10-dihydro-pyrimido [4,5-b]pyrido[4,5-d][1,2,4]triazolo[4,3-a]pyrimidin-5,7(1H,8H)-di-one (5) and 4-amino-6-[(2-thiophen-2-yl)ethenyl]-3-thioxo-3,4-dihydro-[1,2,4]triazin-5(2H)-one (11), respectively. The mechanism and the regioselectivity of the studied reactions have been discussed. The biological activity of the products has been evaluated against some fungi and bacteria species. The tested compounds exhibited moderate activity against the bacteria species.


Introduction
The interest in the chemistry of hydrazonoyl halides is a consequence of the fact that they undergo a wide variety of reactions which provide routes to a number of both heterocyclic and acyclic compounds [1] [2] [3] [4].In addition, diverse biological activities such as anthelmintic, antiviral, antimicrobial, pesticidal, etc., have been found to be associated with hydrazonoyl halides.In recent years, interest to the chemistry of this class of compounds has been renewed because of the development of novel synthetic routes and their use as versatile synthon in synthesis of other compounds that found many applications in both industrial and pharmaceutical fields [5]- [19].In continuation of our studies dealing with the utility of hydrazonoyl halides for synthesis of various heterocycles [3] [16] [20] [21] [22], we wish to report herein a new facile synthesis of 1,3,6,9,11-pentasubstituted pyrido[3,2-f:6,5-f']bis( [1,2,4]triazolo [4,3-a]-pyrimidin-5(1H)-ones (6) and 1,3-disubstituted-7-[(E)-2-(thiophen-2-yl)ethenyl]-1,4,9,9a-tetrahydro-6H- [1,2,4]triazino [4,3-b] [1,2,4,5]-tetrazin-6-ones (16) that have not been reported hitherto, via cyclocondensation reactions of hydrazonoyl chlorides with some azines.Our interest in exploring a new simple synthetic strategy for the latter ring system is due to the fact that literature search reveals that the design and synthesis of novel mono-, di-, and poly-cyclic fused nitrogen heterocyclic compounds are among the active principles in chemical materials, particularly those displaying strategic roles in the development of different industries, especially from the biological point of view.Development of heterocycles with antimicrobial and antitumor activities represents one of the most important researches in therapeutical chemistry [23].

Chemistry
All melting points were measured on an electrothermal melting point apparatus and are uncorrected.The 1 H and 13 C NMR spectra were recorded in deuterated dimethyl sulphoxide (DMSO-d 6 ) at 300 MHz on a Varian Mercury VXR-300 NMR spectrometer (Cairo University, Egypt).600 MHz Bruker NMR spectrometer (King Abdelaziz University, Saudi Arabia) and chemical shifts were related to that of the solvent.The infrared spectra were recorded in potassium bromide discs on a Pye-Unicam, SP300 and Shimadzu, FT IR 8101 PC infrared spectrophotometers.Biological activity was carried out at the Microanalytical Center of Cairo University, Cairo, Egypt.Mass spectra were recorded on a Shimadzu GC MS-QP 1000 EX mass spectrometer at 70 e.V. Elemental analyses were carried out at the Microanalytical Center of Cairo University, Cairo, Egypt.The starting compounds, N-phenyl benzenecarbohydrazonoylchloride (1a) [28] [29] [30], N-phenyl-2-phenylami-no-2oxoethanehydrazonoyl chloride (1b), N-phenyl-2-oxopropanehydrazonoyl chloride (1c), ethyl N-phenylhydrazonochloroacetate (1d) and ethyl N-p-nitro-phenylhydrazonochloroacetate (1e) [20] [31] were prepared as previously reported.
To a solution of 4 (1 g, 1.99 mmol) and dioxane in acetic acid (20 ml) and drops 10% HCl, potassium thiocyanate (0.1 g, 1.03 mmol) was added and the mixture was refluxed for 7 h.After cooling, the solvent was evaporated and 20 ml H 2 O was added.The solid formed was filtered, washed with H 2 O and crystallized from ethanol and drops of dioxane to give 5 as pale yellow crystals.

Antimicrobial Assay
Cultures of two bacterial species namely, Escherichia coli EC, and Staphylococcus aureus SA as well as well as two fungal species, namely Aspergillus flavus AF, and Candida albicans CA were used to investigate the antimicrobial activity of eight products, namely 4, 5, 6a, 6b and 16a-16d.The antimicrobial activity tests were carried out in the Microbiology Division of Microanalytical Center of Cairo university using the diffusion plate technique [34] [35].The latter technique was carried out by pouring a spore suspension of the fungal species (1 cm 3 of sterile water contains approximately 108 conidia) or spreading bacterial suspension over a solidified malt agar medium.The layer is allowed to set for 30 min.A solution of the test compounds (1.0 g = cm 3 ) in DMSO was placed onto sterile 5mm filter paper discs and allowed to dry, then the discs were placed on the centre of the malt agar plate and incubated at optimum incubation temperature 28˚C ± 2˚C.The bactericide Ampicillinand, the fungicide Amphotericin B were used as standards under the same conditions.Measurements were considered after 72 h for fungi and 24 h for bacteria.The results are summarized in table.
The chemical shift values of the annelatedpyrimidinones A and B are shown in Chart 1 [21] [22].Since the values found for the isolated products are similar to those of A and not B, the products were assigned structure 6.
On the basis of this similarity, the isolated products were assigned structure 6 and the isomeric structure 7 was excluded.As depicted in Scheme 1.The reaction proceeded through S-alkylation [37] to give S-alkylated products 8 followed by Smiles rearrangement [38], afforded intermediate 9 which consumed insitu via elimination of hydrogen sulfide gas to give the desired products 6a,b (Scheme 2).The assignment for the structure products and reaction mechanism can be manifested by alternate synthesis.Thus, refluxing of 7-amino-1,3-diphenyl [1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one (3) with benzaldehyde in acetic acid afforded directly the product 6a (Scheme 3) [22].
Also the reaction of 6-aminouracil-2-thione (2) with benzaldehyde in acetic acid afforded the compound 10 [39], which reacted with hydrazonoyl chloride (1a) to yield the product 6a [22] (Scheme 3).The product 6a obtained by the alternate synthesis found to be identical in all respects (mp, mixed mp, IR and 1 HNMR) with the product produced from the reaction of 5 with 1a in Scheme 1.
Our study was extended to introduce 1,2,4-triazine containing thiophene moiety which has wide applications in different biological and medicinal fields beside its application in organic chemistry [40]  3,4-dihydro-[1,2,4]triazin-5(2H)-one (11) [40] in chloroform and triethylamine at room temperature for 15 -24 h (according to TLC, no start was found) to give chromatographically impure product.The latter was purified by column chromatography by using eluent of a mixture of ethyl acetate/CHCl 3 (10: 90) to give one single pure product.The structure of the product obtained may be 13 or 16 (Scheme 4).
It was initially anticipated that such reactions would yield the respective 1,2,4triazino[2,1-b][1,3,4,5]thiatriazine (13) by analogy to the reactions of 1a-d with 2-aminothiophenol, which were reported to afford benzothiadiazine derivatives [22] [39].Unexpectedly, the products isolated from the reactions of 1a-d with 11 The structures of the latter products were elucidated on the basis of their spectra ( 1 H & 13 C NMR, IR, and MS) and microanalyses.For example, the 1 HNMR spectra of the product 16a showed NH proton signal at δ 8.8, and it was lacking the N-NH 2 signal at δ 6.5, which is characteristic of the starting substrate 11.The mass spectra of 16a were also consistent with its assigned structures, especially its molecular ion peak.The formation of 16a-d from 1a-d and 11 can be rationalized in terms of the pathway outlined in Scheme 4. It is suggested that the reaction begins with the formation of the respective hydrazide 14, that cyclized to 15, which in turn give 16 with concurrent elimination of hydrogen sulfide (Scheme 4) [41] [42].

Antimicrobial Activity
The test results revealed that all compounds exhibited moderate activity against the two bacterial species except, 5, 6b and all compounds showed no activity against all fungal species, except compounds 5, and 16c against Candida albicans (CA) (Table 1).Table 1.Antibacterial and antifungal activities of some of the synthesized compounds.