Synthesis and Evaluation of Antibacterial Activities of some Important Bridge-head Nitrogenous Heterocyclic Compounds

The key intermediates in the present study to synthesize the title bridge-head nitrogen heterocycle compounds were N-substituted semicarbazides 2(a-c), which prepared by hydrazinolysis of the ethyl substituted carbamates 1(a-c) with hydrazine hydrate. These carbamates were prepared by the reaction of proper amines and ethylchloroformate. The reaction of these semicarbazides with ethanolic solution of carbon disulfide under strong basic conditions at room temperature, followed by acidification resulted in the formation of the corresponding potassium (2-arylcarbamoyl)hydrazine carbodithioate 3(ac), while refluxing the ethanolic solution for three hours afforded 5-(arylamino)-1,3,4oxadiazole-2thioles 4(a-c). The potassium salts 3(a-c) were cyclized with hydrazine hydrate to 4-amino-5-arylamino-1,2,4-triazole-3-thiones(thioles) 5(a-c). Compounds 5(a-c) were excellent precursors for 3-(arylamino)-[1,2,4]triazolo[3,4b][1,3,4]thiadiazoles 6(a-c) by dehydrative ring closure of the proper triazole with formic acid in benzene in presence of phosphorous oxychloride or using microwave irradiation technique. Also, refluxing of the proper triazoles with carbon disulfide under basic conditions afforded 3-(arylamino)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole-6-thiones 7(a-c). Finally 4amino-3-(4-(5-(4-amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-ylamino)-1,3,4-thiadiazol2-yl)phenylamino)-1H-1,2,4-triazole-5(4H)-thione 5(d) were synthesized by the same procedures that were followed to synthesize its analogs starting from 5-(4-aminophenyl)1,3,4-thiadiazole-2-amine and duplicated all the scales of the reactants. The structures of these compounds were confirmed by their physical properties in addition to the IR, UV and NMR spectra. All synthesized compounds were tested for their in vitro growth inhibitory activity against a panel of standard strains of pathogenic microorganism including bacteria Staphylococcus, Streptococcus and Salmonella. All these compounds were practically inactive against the tested microorganisms.


INTRODUCTION
As part of the continuous program directed toward the synthesis of important heterocyclic compounds, oxygenous and nitrogenous five and six membered rings (Saied et al., 2010), it was became of interest to investigate the preparative routs to synthesize the title fused heterocyclic compounds.All the target analogues heterocyclic compounds have been reported to exert notably antibacterial activity (Subramanian et al., 2009).Also, new series of 1,3,4-thiadiazole-thione derivatives were synthesized and tested for their antibacterial activities.It was therefore thought worthwhile to incorporate the oxadiazole, thiadiazole and or triazole moieties into the title Bridge-head nitrogen.Semicarbazides were prepared by condensing carbamates with hydrazine hydrate (Mruthunjayaswamy et al., 2009), these carbamate esters were prepared by the reaction of appropriate amines with ethyl chloroformate.It was worthy to mention that semicarbazides converted to carbodithioate salts or cyclized to oxadiazole-2-thiones by the same reagents (carbon disulfide and potassium hydroxide solution in ethanol), but by two different procedures, while the former involved the agitation at room temperature of the reaction mixture which led to precipitate of quantitative yield of potassium salt which was used in the next step without further purification (Joshi et al., 1989).The latter was carried out by heating the reaction mixture until the evolution of hydrogen sulphide was ceased.These procedures were very popular since ease in workup and high yields were consistently observed but with long reaction time (Somani and Shirodkar, 2009).
Bridge-head nitrogen heterocycles were obtained either by cyclization of these salts with hydrazine hydrate in refluxing ethanol then mixing the triazole products with carboxylic acids at room temperature (Hirpara et al., 2003) or by refluxing ethanolic solution of amidothiosemicarbazides with acetyl acetone (Al-Abdullah, 2007).

EXPERIMENTAL
All melting points were determined on a Gallen Kamp and Electro thermal 1A9300 Digital-Series (1998) apparatus and were uncorrected.The IR spectra (ν cm -1 , KBr disc) were recorded on Perkin -Elmer 590B Spectrophotometer.UV measurements were carried out on Shimadzu UV-160 spectrophotometer using EtOH as a solvent.NMR spectra were obtained on a BRUKER AVANCE DPX 400 MHz.spectrophotometer as d 6 -DMSO or CdCl 3 solutions using TMS as an internal standard at the Department of Chemistry, Donnan and Robert Robinson Laboratories, University of Liverpool, U.K. Finally, microwave irradiation was performed using unmodified domestic Samsung oven (300 MHz).

Table 1 : Physical and spectral data of compounds 1(a-d).
N-substituted semicarbazides 2(a-d), (Mruthunjayaswamy et al., 2009) A mixture of (0.01 mole) of appropriate carbamate 1(a-c), excess of hydrazine hydrate (80%) and triethyl amine (1 ml.) in ethanol was stirred for an hour, then poured on ice-water and acidified with diluted HCl.The precipitate was filtered and washed with water and crystallized from ethanol.

Potassium (2-arylcarbamoyl) hydrazine carbodithioates 3(a-d).
Carbon disulphide (11.4 g, 0.15 mole) was added dropwise to a solution of proper semicarbazide 2(a-c) (0.1 mole) and potassium hydroxide (8.4 g, 0.15 mole) in ethanol (250 ml).The mixture was stirred at room temperature for 30 minutes.Dry ether (200 ml) was then added to the mixture and the precipitate was filtered, washed with ether and dried to yield the title salts which were used without further purification in the next step.
Irradiation method B: (Omprakash et al., 2011) A mixture of proper compound 5(a-c) (0.01mole) and formic acid (0.46ml, 0.01 mole) was placed in 25 ml open round bottom flask and irradiated in the microwave oven for 4 minutes.On cooling, an oily product was obtained which was worked up as in method (A).a-c).(Fiona et al., 2008) To a solution of the proper 4-amino-5-arylamino-1,2,4-triazole-3-thiol 5(a-c) (0.001 mole) in ethanol 20 ml, KOH (0.5 gm) and CS 2 (1ml) were added and the mixture was refluxed for 2hours.The solvent was removed under reduced pressure.Ice-water was added to the reaction mixture with stirring.The solid was separated, washed with water and crystallized from ethanol to afford the title compound.

Biological Test
All the synthesized compounds in the present investigation were screened for their antibacterial activity by subjecting the compounds to a standard procedure.Antibacterial activities were tested on nutrient medium against Staphylococcus, Streptococcus and Salmonella.The antibacterial activity of the compounds was assessed by disc diffusion method.The extent diameter of inhibition after 24 hours was measured as the zone of inhibition in millimeters.

RESULTS AND DISCUSSION
A convenient synthesis of target compounds were accomplished by the routes outlined in Schemes (1 and 2).Ethyl substituted carbamates 1(a-c) were prepared from ethylchloroformate and a suitable amines.The IR spectra of these compounds showed broad bands at around 3190 -3162 cm -1 due to NH bond stretching while a strong bands at 1722 -1728 cm -1 was attributed to C=O bond stretching carbonyl group and at 1596-1636 cm -1 related to aromatic heterocyclic C=N.(Table 1).The 1 HNMR spectra were supporting the presence of two thiazole protons around δ7.17 and 7.38 ppm, while the NH proton appeared as a singlet signal at δ11.68.The CH 2 protons appeared quartet at δ2.12ppm and the CH 3 appeared as triplet at δ2.12ppm.
The key intermediates N-substituted semicarbazides 2(a-d) were prepared by hydrazinolysis of carbamates 1(a-c) with hydrazine hydrate (Varvounisa and Giannopoulos, 1996).The IR spectra showed broad stretching vibrations at around 3240 -3182 cm -1 due to NH 2 and a strong band at 1697 -1672 cm -1 attributed to amide carbonyl and at 1639-1610 cm -1 due to aromatic heterocyclic C=N, (Table 2).The 1 HNMR spectra were introducing an evidence to the presence of the two thiazole protons appeared around δ7.90 ppm, and the NHCONH protons appeared multiplet at δ7.46 ppm, the semicarbazide terminal nitrogen protons appeared doublet at δ2.25 ppm.
These compounds were present in thione-thiol tautomeric forms, (Scheme 2). the ultraviolet spectra of methanolic solution of these triazoles showed absorption bands at 288-300 nm due to the presence of the chromophoric C=S group (Fiona et al., 2008).
The IR spectram disclosed the presence of C=S at (1210 cm -1 ) which was responsible to those thiones in addition to C=N bands at (1678-1672 cm -1 ), NH (3166-3055 cm -1 ) and of a primary amine bands at (3440-3324cm -1 ) with no absorbance at (2600-2400 cm -1 ) of thiole form, (Table 4) (Almajan et al., 2008).These results did not agree with the 1 HNMR (DMSO-d 6 , δ ppm) for compound 5(c) which was characterized by presence of the one SH proton appeared as singlet at δ12.30 ppm and four aromatic protons with NH proton appeared as multiplet around δ7.50-7.92ppm, while the NH 2 proton appeared as singlet at δ4.26 ppm, indicated that the thiole tautomer was existed predominantly in DMSO solution.This result was due to solvent effects (Looker et al.,1978).(Scheme 2).Thiol-thione tautomerism exists in compounds 5(a-c), in the 1 HNMR the signal of the SH proton was recorded, and as it has been reported that the crystal structures of these compounds correspond to the thione form, but they showed thiol-thione tautomerism in solution (Koparır et al., 2005).
As the structural skeleton of compounds 5(a-c) was established spectroscopically, the chemical behavior of these compounds was also used for assigning their structure, as expected it was found that refluxing of compound 4(c) with carbon disulfide under strong basic conditions followed by acidification with dilute hydrochloric acid resulted in the formation of the authentic sample 5(c) (Artemov and Shvaika, 2008).
The formation of the same product from two different compounds indicated the correct assignment for these compounds.
All the synthesized compounds were tested for their in vitro growth inhibitory activity against a panel of standard strains of pathogenic microorganism including bacteria Staphylococcus, Streptococcus and Salmonella.
All these compounds were practically inactive against the tested microorganisms.