Synthesis and antimicrobial activity of some derivatives of acylhydrazine including novel benzenediazasulfonamides

Some novel derivatives of acylhydrazine such as; 5-substituted-2-mercapto-1,3,4-oxadiazoles 2a-g their corresponding S-esters 3a-g, amides 4a-g and benzenediazasulfonamides 6a-d have been prepared. Twenty four synthesized compounds were screened in vitro for their antibacterial and some for their antifungal activities. Minimum Inhibitory Concentration (MIC) values of all the twenty four synthesized compounds were also determined. Some of the synthesized compounds exhibited significant antimicrobial activity.

The IR spectra of compounds 2a-g displayed SH weak absorption at 2610−2540 cm -1 beside the C=S absorption at 1300−1124 cm -1 .In the 1 H NMR spectra, the NH and SH protons derived from tautomeric equilibrium, resonated between 13.90−8.75ppm as a broad singlet integrating for one proton.In each case the absence of C=O absorption (1690−1670 cm -1 ) of the corresponding substituted-1,3,4-oxadiazole indicated that the dehydrative cyclization reaction had occurred.In the IR spectra of compounds 3a-g, the absence of absorption bands due to SH stretching frequencies of parent compounds 2a-g and appearance of C=O strong absorption at 1745−1703 cm -1 clearly indicated the formation of the respective esters.The 1 H NMR of compounds 3a-g showed disappearance of broad singlet of (SH + NH) and corresponding appearance of a triplet (1.35-1.17ppm) and a quartet (4.11-1.35ppm) integrating for three and two protons of the methyl and methylene groups respectively.The singlet at 4.35-4.15ppm integrating for two protons was due to SCH2 group.In the IR spectra, the characteristic absorption was found at 3393-3187 cm -1 for NH of primary amides 4a-g.A shift was noted for the C=O absorption from esters (~1750) to 1693-1672 cm -1 for amides.In 1 H NMR, two broad singlet, integrating for one proton in each case in the region 7.77-6.67ppm for NH 2 group, the characteristic singlet integrating for two protons at 4.27-4.02ppm attributed to SCH 2. The aromatic protons appeared at their usual values.These observations established the formation of synthesized primary amides 4a-g.Mass spectral data of these compounds, described in the experimental protocol, further supported the structures of all the synthesized compounds.
The structures of the resulting novel benzenediazasulfonamides (N-substitutedbenzoyl-N′methyl-N′-(4-substitutedbenzenesulfonyl)hydrazines) 6a-d (Scheme 1), were established by spectral data.The IR spectra of compounds 6a-d, displayed the NH absorption at 3317-3245 cm -1 , characteristic absorption of C=O at 1682−1672 cm -1 besides the SO 2 absorption in the usual range (1376-1141 cm -1 ).In 1 H NMR spectra, the NH protons resonated between 9.67-9.54ppm as a broad singlet integrating for one proton.Moreover, the proton signals due to NCH 3 group of these compounds were resonated in the region 3.31−3.02ppm as a singlet integrating for the three protons.The different aromatic protons were found in their usual range.In the 13 C NMR of these compounds, the characteristic signal belonging to carbonyl carbon appeared between 177−171 ppm.Complete 13 C NMR data is given in the experimental protocol.Molecular ion peaks along with the base peaks in mass spectra, and elemental analysis/HRMS values, in the acceptable range, provided an additional evidence to establish the structures of the synthesized compounds.It is worth noting that crystals of compound 6a were analyzed by X-ray single crystallography to assign the exact structural geometry, and reported earlier by our research group. 17wenty four newly synthesized compounds were screened in vitro for their antibacterial and some for their antifungal activities.

Antimicrobial activity
Twenty four newly synthesized compounds have been tested in vitro for their antibacterial activity against Staphylococcus aureus (ATCC-25923), Bacillus subtilis (recultured), Escherichia coli (ATCC-25922), Pseudomonas picketti (recultured) and Micrococcus luteus (recultured) bacteria by agar well diffusion method. 18DMSO was used as a control solvent, Roxythromycin and Cefixime as a standard drug.After 24 hrs of incubation at 37 °C, the zone of inhibition was measured in mm.The investigation results are listed in Table 1.The results showed that all compounds were active against Escherichia coli except 4f and 6d.It is worth noting here that compounds 6c and 6d showed moderate (15 mm) to significant activity (18 mm) against Staphylococcus aureus, respectively.The other compounds showed none to low activity as displayed in Table 1.
The investigation on the structure-activity relationship (SAR), shows that, in general, free SH at position 2 of the oxadiazole ring, presence of the chloro group at the phenyl substituent and 3pyridyl of the oxadiazole ring enhanced the antibacterial action of the synthesized compounds.Minimum Inhibitory Concentration (MIC) values of all the twenty four synthesized compounds were determined by agar well diffusion method 18 and the results are displayed in Table 1.Five selected representatives of newly synthesized compounds 2e, 3d, 4e, 6c and 6d were screened in vitro for their antifungal activity against six species using agar plate technique. 19The linear growth of the fungus was obtained by measuring the diameter of the fungal colony after seven days.The amount of growth inhibition in each case was calculated as percentage inhibition.The screening results are given in Table 2, indicated that compounds 3d and 6c exhibit moderate activity (60%) against Aspergillus flavus and Trichphyton longifusus respectivly, whereas, compound 6d showed significant activity (90%) against Candida albicans.It is worthwhile to note that compound 6d exhibits significant (maximum) antibacterial and antifungal activities, possibly due to the presence of chloro group on both sides of the phenyl substituent of the oxadiazole ring, and the sulfonamido moiety.Whereas, in case of compounds 3 and 4 the substitution at free SH (ester and amide derivatives) diminished the antimicrobial activity as given in Table 1 and 2

Experimental Section
General Procedures.Melting points were determined on a Gallenkamp digital melting point apparatus and are uncorrected.IR spectra were recorded in KBr disc on a FT-IR model FTS 3000 MX spectrometer.NMR spectra were recorded in Acetone-d 6, DMSO-d 6 or CDCl3 on a Bruker (300, 400 and 500 MHz; 13 C, 75 MHz) spectrophotometer.The chemical shifts of proton signals are in parts per million (ppm) downfield from tetramethylsilane (TMS) as an internal standard.EI-MS spectra were recorded on MAT 311A mass spectrometer (EI at 70 eV).Elemental analysis was performed on a Carlo Erba 1106 elemental analyzer.Thin layer chromatography (TLC) was performed on precoated silica gel 60 F 254 aluminum sheets (Merck).

General procedure for the preparation of 5-substituted-2-mercapto-1,3,4-oxadiazoles (44a-w)
These compounds were prepared according to the procedure reported in the literature 13 (Scheme 1).To a solution of appropriate carboxylic acid hydrazide 1 (50 mmol) in ethanol (150-200 mL), carbon disulfide (55 mmol) was added.This was followed by the addition of potassium hydroxide (50 mmol) dissolved in 25 mL of water.The reaction mixture was stirred and subjected to reflux.The progress of the reaction was monitored by TLC in each case using pet.ether:ethyl acetate (2:1).After reaction completion, excess ethanol was distilled off.The crude solid obtained was dissolved in excess water and acidified with 4N HCl to pH 2-3 (Congo ARKAT USA, Inc. red).The separated product was filtered, washed with water and recrystallized from aqueous ethanol (20-30%).

General procedure for the synthesis of [5-(Aryl)-1,3,4-oxadiazol-2-ylthio]esters (3a-g)
These esters were prepared according to the procedure reported in the literature 14 (Scheme 1).The reaction was comprised of two steps; the first step is the salt formation by reacting corresponding 5-substituted-2-mercapto-1,3,4-oxadiazole with aqueous sodium bicarbonate solution, and in the second step the salt was treated with ethylbromoacetate to produce the respective ester.Corresponding 2-mercapto-1,3,4-oxadiazole (20 mmol) was dissolved in saturated aqueous sodium bicarbonate solution by magnetic stirring.The required ethylbromoacetate (20 mmol) in absolute ethanol (10 mL) was added to the reaction mixture with continuous stirring.The progress of the reaction was monitored by TLC (silica, ethylacetate : pet.ether 1:2) in each case.After reaction completion, the resulting solid was filtered off, washed with water and recrystallized from aqueous ethanol (60%).ARKAT USA, Inc.

General procedure for the synthesis of benzenediazasulfonamides [N-substituted-benzoyl-N′-methyl-N′-(4-substitutedbenzenesulfonyl)-hydrazines] 6a-d
These benzenediazasulfonamides were obtained during an attempt to reduce [5-substituted-1,3,4oxadiazol-2-ylthio]acetamides 4 to their corresponding amines, for the preparation of their corresponding sulfonamides (Scheme 1).The procedure adopted was reported by Umino et al. 16 To a stirred suspension of sodium borohydride (50 mmol) and corresponding amide (10 mmol) in 1,4-dioxane (25 mL) was added acetic acid (50 mmol) in dioxane (10 mL) over a period of 10 minutes at 10 o C, and the resulting mixture was stirred under reflux for 1.5-2.5 hours.The progress of the reaction was monitored by TLC (silica; pet.ether: ethylacetate; 2:1).After completion of reaction, the solvent was distilled off and the excess reagent was decomposed with water and extracted with chloroform (3 x 50 mL).The extract was washed with water and dried over anhydrous sodium sulfate.The resulting chloroform layer was treated with dry hydrogen chloride, evaporated in vacuo, and the residue was crystallized from methanol-ether to corresponding acylhydrazine hydrochloride 5.The resulting acylhydrazine hydrochlorides were used, without further purification, in the next step, for the synthesis of corresponding benzenediazasulfonamides 6a-d.Appropriate acylhydrazine hydrochloride 5 (2 mmol), triethylamine (4 mmol) and DMAP (25 mg) in anhydrous chloroform were stirred at room temperature for fifteen minutes.An appropriate sulfonyl chloride (2.2 mmol), dissolved in anhydrous chloroform, was then added to the reaction mixture in portions, with the help of dropping funnel.The reaction mixture was stirred at room temperature for 23-25 hours.The progress of the reaction was monitored by TLC (silica; methanol: chloroform; 1:2).After reaction completion, the reaction mixture was washed with saturated solution of sodium bicarbonate, brine and water followed by an extraction with chloroform (2 x 25 mL).Combined organic layer was dried over anhydrous Na 2 SO 4 and concentrated under vacuum.The isolated product was purified by recrystallization from aqueous ethanol (30%).

Table 1 .
. Antibacterial activity and minimum inhibitory concentrations (MIC) of the synthesized compounds 2b

Table 2 .
Antifungal activity of the synthesized compounds 2e,