Synthesis of thiophenylazolyl pyrrolylsulfamoyl acetamides as potential antimicrobial agents

A variety of thiophenylazolyl pyrrolylsulfamoyl acetamides were prepared by the reaction of azolylsulfamoyl acetate with pyrrolylamine in the presence of sodium methoxide in methanol under ultrasonication. Chloro, nitro and dinitro substituted thiophenylthiazolyl pyrrolylsulfamoyl acetamides (9d, 9e, 9f) and dinitro substituted thiophenylimidazolyl pyrrolylsulfamoyl acetamide (10f) exhibited potential antibacterial activity against B. subtilis. The compound 9f also displayed prominent antibacterial activity against S. aureus. The compounds 9f, chloro and nitro substituted thiophenylimidazolyl pyrrolylsulfamoyl acetamide (10d, 10e) and 10f exhibited prominent antifungal activity against A.niger.


Biological evaluation
Antibacterial activity All the compounds are tested for antibacterial activity at four different concentrations 12.5, 25, 50, and 100 µg/well. All the compounds displayed higher activity on Gram-positive bacteria than on Gram-negative bacteria ( Table 1). Thiophenylthiazolyl pyrrolylsulfamoyl acetamides (9) showed higher activity than thiophenyloxazolyl pyrrolylsulfamoyl acetamides (8) and thiophenylimidazolyl pyrrolylsulfamoyl acetamides (10). Amongst the latter compounds 10 exhibited greater activity than 8. It was also observed that the presence of electron withdrawing substituents enhanced the activity and the activity increased with increasing electronegativity. The compounds having nitro substituents displayed higher activity than those with chloro substituents. In fact, dinitro substituted compounds 8f, 9f and 10f displayed higher activity in the respective series. It was also observed that those having chloro and nitro substituents 8d, 9d, 10d and 8e, 9e, 10e showed slightly higher activity when compared with the compounds having two chloro substituents 8c, 9c and 10c. Further it was noticed that there was not much difference in activity amongst the compounds 8d, 9d, 10d and 8e, 9e, 10e which showed that the presence of more electron withdrawing nitro substituent either at 4 position of thiophene or at 4 position of aromatic ring has similar effect. Amongst all the tested compounds 9d, 9e, 9f and 10f displayed higher activity on B. subtilis greater than the standard drug Chloramphenicol at all tested concentrations. Further the compound 9f displayed equal activity to the standard drug on S. aureus at 50 and 100 µg/well.

Antifungal activity
All the compounds effectively inhibited the spore germination of tested fungi ( Table 2). It was noticed that compounds having imidazole unit (10) displayed grater activity than those with oxazole (8) and thiazole (9) moieties. Further the activity increased with increasing electronegativity of the substituents. The compounds 9f, 10d, 10e and 10f showed higher activity on A. niger greater than the standard drug, Ketoconazole at all tested concentrations.
The compounds which showed greater antibacterial and antifungal activities are further assayed for minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) and the values are listed in Table 3. MIC is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism. (But it is not sure that the microorganisms are completely killed). The MBC/MFC is the lowest concentration of antibiotic required to kill a particular bacterium/fungi. The MBC/MFC involves an additional set of steps performed once the MIC is determined. The antimicrobials are usually regarded as bactericidal/fungicidal if MBC/MFC is not greater than four times MIC. The compounds 9d, 9e, 9f and 10f displayed low MIC against B. Subtilis. The MIC of these compounds is equal to standard drug chloramphenicol and MBC is 2 x MIC. Further 9f showed low MIC on S. aureus and MBC is found to be 2 x MIC. Moreover the compounds 9f, 10d, 10e and 10f exhibited low MIC against A. niger and MFC is 2 x MIC.

Conclusion
A variety of thiophenylazolyl pyrrolylsulfamoyl acetamides were prepared by the reaction of azolylsulfamoyl acetate with pyrrolylamine in the presence of sodium methoxide in methanol under ultrasonication. The presence of electron withdrawing substituents increased antimicrobial activity. Chloro, nitro and dinitro substituted thiophenylthiazolyl pyrrolylsulfamoyl acetamides (9d, 9e, 9f) and dinitro substituted thiophenylimidazolyl pyrrolylsulfamoyl acetamide (10f) exhibited potential antibacterial activity against B. subtilis. These compounds exhibited low MIC against B. Subtilis equal to standard drug chloramphenicol and MBC is 2 x MIC. Further 9f showed low MIC on S. aureus and MBC is 2 x MIC. The compounds 9f, 10d, 10e and 10f displayed prominent antifungal activity against A. niger and MFC is 2 x MIC.

Apparatus and analysis
Melting points were determined in open capillaries on a Mel-Temp apparatus and are uncorrected. The purity of the compounds was evaluated by TLC (silica gel H, BDH, ethyl acetate/hexane, 1:3). The IR spectra were recorded on a Thermo Nicolet IR 200 FT-IR spectrometer as KBr pellets and the wave numbers were given in cm − 1 . The 1 H and 13 C NMR spectra were recorded in DMSO-d 6 on a Bruker spectrometer operating at 400 and 100 MHz. The chemical shifts are reported in δ (ppm) using TMS as an internal standard. The high-resolution mass spectra are recorded on micromass Q-TOF micromass spectrometer using electrospray ionization. Ultrasonication was performed in a Bandelinsonorex RK 12 H ultrasonic bath operating at a frequency of 35 KHz. The microanalyses were performed on a Perkin-Elmer 240C elemental analyzer. The progress of the reaction was monitored by TLC using silica gel plates (silica gel 60 F254 0.25 mm), and components were visualized by observation under UV light (254 and 365 nm). The compounds 4-thiophenyloxazolyl-2-amine (1) and 4-thiophenylthiazolyl-2-amine (2) 4thiophenylimidazolyl-2-amine (3) were prepared as per the literature procedures [Taterao et al. 2008]. The compound 4-(4-chlorophenyl)-1H-pyrrol-2-amine (7) was purchased from Aldrich.

Preparation of dispersed sodium
Clean sodium metal (10 g) was weighed under dry ether and introduced into a 500 ml round-bottomed ask containing sodium-dried xylene (100 cm 3 ) tted with an air condenser carrying a calcium chloride guard tube and placed on a sand bath. The ask was enveloped with a dry cloth and the sand bath was heated cautiously. The ring of condensed vapour of xylene was carefully observed. When the ring of condensed vapor had risen to the neck of the ask the ame was extinguished. The condenser was replaced by the stopper and the ask was wrapped with a pre-dried cloth. The stopper was then held rmly and shaken vigorously for 2-3 min until the molten sodium was converted into a ne dispersion. Immediately the stopper was removed and the ask was placed on the cork ring. The sodium was obtained in the form of small spheres depending upon the time and rapidity of shaking. Then the contents were cooled to room temperature, xylene was decanted and the sodium was washed with sodium-dried ether. The dispersed sodium as small spheres was preserved in absolute ether.
General procedure for the synthesis of methyl 4-thiophenylazolylsulfamoyl acetate (4/5/6) A mixture of methyl 2-chlorosulfonylacetate (0.001 mol), dispersed sodium (0.0414 g, 1.8 mg atom) and tetrahydrofuran (3 ml) was sonicated for 8 min in a sonic bath at a frequency of 35 KHz at 25 0 C. To this azolyl-2amine (1/2/3) was added and continued sonication for 12-16 min. After completion of the reaction (monitored by TLC), the organic matter was ltered, washed with water, extracted with ether and dried. Removal of the solvent under reduced pressure gave a solid which was recrystallized from ethanol.

Biological Activity Assays
The in vitro antimicrobial studies were determined by agar well diffusion method against test organisms [Mounyr et al. 2016]. Nutrient broth (NB) plates were swabbed with 24 h old broth culture (100 µL) of test bacteria. Wells (6 mm) were made into each petriplate using the sterile cork borer. The compounds were dissolved in DMSO (5 mg/mL) and from this 2.5, 5, 10, and 20 µL (12.5, 25, 50, 100 µg/well) were added into the wells by using sterile pipettes. The standard antibiotics (positive control), chloramphenicol for antibacterial activity and ketoconazole for antifungal activity were tested against the pathogens simultaneously. The samples were dissolved in DMSO which showed no zone of inhibition acts as negative control. The plates were incubated at 37 o C for 24 h for bacteria and at 28 o C for 48 h for fungi. The diameter of zone of inhibition of each well was measured after appropriate incubation. Duplicates were done and the average values were calculated for eventual antibacterial activity. Broth dilution test was used to determine minimum inhibitory concentration (MIC) of the above samples [Bishnu et al. 2009]. Freshly prepared nutrient broth was used as diluents. The 24 h old culture of the test bacteria Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Klebsiella pneumoniae and the test fungi Aspergillus niger and Penicillium chrysogenum were diluted 100 folds in nutrient broth (100 µL bacterial cultures in 10 mL NB). The stock solution of the compounds was prepared in DMSO by dissolving 5 mg of the compound in 1 mL of DMSO. Increasing concentrations of the test samples (2.5, 5, 10, 20 µL of stock solution contains 12.5, 25, 50, 100 µg of the compounds) were added to the test tubes containing the bacterial and fungal cultures. All the tubes were incubated at 37 o C for 24 h for bacteria and at 28 o C for 48 h for fungi. The tubes were examined for visible turbidity and using NB as control. Simultaneously control without test samples and with solvent was assayed. The MIC was recorded as the lowest concentration that inhibited visible growth of the tested organisms.
To determine the minimum bactericidal concentration (MBC) [Pennsylvania et al. 2006] and minimum fungicidal concentration (MFC) [Pennsylvania et al. 2002] for each set of test tubes in the MIC determination, a loopful of broth was collected from those tubes which did not show any growth and inoculated on sterile nutrient broth (for bacteria) and PDA (for fungi) by streaking. These inoculated plates were incubated at 37 o C for 24 h (bacteria) and at 28 o C for 48 h (fungi). After incubation, the lowest concentration was noted as MBC or MFC at which no visible growth was observed.    The invitro antibacterial activity of compounds 8-10 Page 17/17

Figure 2
The in vitro antifungal activity of compounds 8-10

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Table2.docx Supplementaryinformation.docx