Synthesis, Characterization, Antibacterial, α -Glucosidase Inhibition and Hemolytic Studies on Some New N-(2,3-Dimethylphenyl)benzenesulfonamide Derivatives

Purpose: To synthesize a series of new N-(2,3-dimethylphenyl)benzenesulfonamide derivatives with pharmacological analysis. Methods: N-(2,3-Dimethylphenyl)benzenesulfonamide (3) was synthesized by the reaction between 2,3-dimethylaniline (1) and benzenesulfonyl chloride (2) in aqueous basic medium. Compound 3 was further treated with various alkyl/aralakyl halides (4a-m) to yield new compounds, 5a-m, in a weak basic aprotic polar organic medium. The proposed structures of synthesized compounds were confirmed using proton-nuclear magnetic resonance (1H-NMR), infra-red spectroscopy (IR) and electron impact mass spectrometry (EIMS). The synthesized compounds were screened for in vitro antibacterial, anti-enzymatic and hemolytic activities using standard procedures. Results: All the synthesized compounds showed moderate to high activity against Gram-positive and Gram- negative bacterial strains. The molecules 5g and 5j exhibited good inhibition of α -glucosidase enzyme with half-maximal inhibitory concentration (IC 50 ) of 59.53 ± 0.01 and 55.31 ± 0.01 μmoles/L, respectively, relative to acarbose with IC 50 of 38.25 ± 0.12 μmoles/L. All the compounds exhibited cytotoxicity levels ranging from 27.20 ± 0.24 to 5.20 ± 0.41 %, relative to Triton X-100. Conclusion: Compound 5f is the most potent antibact erial while 5j is the best α -glucosidase inhibitor; 5e showed the least cytotoxicity.


EXPERIMENTAL General
The chemicals solvents were of commercial grade and were used without purification.Reaction progress and product purity was monitored by pre-coated TLC silica gel G-25-UV254 plates with different solvent systems using ethyl acetate and n-hexane.Melting points were noted on Gallenkamp apparatus by open capillary tube and were uncorrected.FTIR spectra were recorded on MIDAC M 2000 spectrometer.1H-NMR spectra were recorded in CDCl 3 on Bruker (400 MHz).Mass spectra (EI-MS) were measured on Finnigan MAT-312 instruments.

Procedure for the synthesis of N-(2,3dimethylphenyl)benzenesulfonamide (3)
An equimolar mixture of 2,3-dimethylaniline (1, 0.02 mol) was dispersed in 25 mL waterfollowed by the addition ofbenzenesulfonyl chloride (2, 0.02 mol).The pH of the suspension was maintained at 9-10 during the whole reaction by adding Na 2 CO 3 at RT.The reaction solution was stirred for 3 h.After complete reaction, the concentrated HCl (2-3 mL) was added slowly to adjust the pH to 2. Product formation was confirmed by TLC.The reaction mixture was kept still for 3-5 min.The off-white precipitates were collected by filtration, washed with distilled water and dried to acquire the compound 3.

General procedure for the synthesis of Nalkyl/aralakyl substituted sulfonamides (5am)
The calculated amount of 3 (0.7 mmol; 0.2 g) was taken in a round bottom flask (50 mL), then N,N-dimethyl formamide (DMF) was added to dissolve it followed by the addition of lithium hydride LiH (0.004 g).The mixture was stirred for 45 min at RT and then alkyl/aralakyl halides (4am; 0.7 mmol) were added to the mixture.The solution was further stirred for 3-4 h.The progress of reaction was monitored by TLC.The product was precipitated by adding cold water, filtered, washed with distilled water and dried.

Evaluation of antibacterial activity
The antibacterial activity was performed in sterile 96-wells microplates under aseptic environments by the methods of Kaspady et al [17] and Yang et al [18].

α-Glucosidase assay
The α-glucosidase inhibition activity was performed according to the method of Chapdilaine et al [10].

Determination of hemolytic activity
Hemolytic activity was studied by the reported method of Powell et al [11,19].3.0 mL fresh heparin added human blood obtained from volunteers after guidance from the Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad, Pakistan.The study protocol was approved by the institutional ethical committee (approval no.DGS/8786-89, dated 09-03-2015), University of Agriculture, Faisalabad, Pakistan and was conducted in accordance with 1964 Declaration of Helsinki and its subsequent amendments [20].

Data analysis
All the measurements were performed in triplicate and statistical analysis was carried out by Microsoft Excel 2010.The results are presented as mean ± SEM with CL 80 % (for antibacterial), 85 % (for α-glucosidase) and 95 % (for hemolytic analysis).MIC (minimum inhibitory concentration and IC 50 (50 % inhibitory concentration) were calculated using EZ-Fitz software (Perrella Scientific Inc. Amherst, USA).

Antibacterial activity
The synthesized compounds were evaluated against two Gram-positive and four Gramnegative bacterial strains using ampicillin and ciprofloxacin as standards.(Table 1).The compounds showed moderate to significant activity against bacterial strains except compounds, 5a, 5d and 5e.The compound 3 was active against one strain of gram-positive bacteria only.The preliminary screening showed that compound, 5c, 5f, 5g, 5i, 5l and 5m were the most active ones against gram-positive and compounds, 5b, 5f, 5j and 5k were more active against gram-negative bacterial strains.

Anti-enzymatic activity
The screening against α-glucosidase enzyme demonstrated that the most of compounds were active with good inhibitory potential except 5a, 5b, 5c and 3 (Table -2).The molecules 5g and 5j were found to be excellent inhibitors with their IC 50 values of 59.53 ± 0.008 and 55.31 ± 0.01 μmoles/L respectively, relative to acarbose a reference standard with IC 50 value 38.25 ± 0.12μmoles/L.

DISCUSSION
Sulfonamide 3 was obtained as off-white powder.Although its structure has been established by the x-ray crystallography [21] but more structural details are presented here for the first time.Its molecular formula, C 14 H 15 NO 2 S, was determined through CHNS analysis with percentage of elements as C: 64.03 %; H: 6.40 %; N: 5.52 % and S: 11.73 %.The antibacterial activity results declared the compound 5f bearing aliphatic straight chain with moderate length at nitrogen atom was the most active molecule against all the bacterial strains used in this study.The compounds with sulfamoyl moiety have been introduced as valuable biologically active compounds and in accordance with our previous findings [13][14][15][16], the overview of synthesized compounds hereby showed that N-substitution of sulfamoyl group with aliphatic chains and halogenated aromatic compounds presented best activity because of best fit to active site of enzyme and better π-π interactions.The two molecules, 5g and 5j, showed good activity against α-glucosidase probably because of branched long alkyl chain in 5g and p-substituted halogenated aralkyl group in 5j, presented better binding interactions with the active site of enzyme.The compound 5e was the least cytotoxic in this series but it was inferred that all the compounds can be used as possible therapeutic agents.

CONCLUSION
All the compounds have been structurally identified by spectral data and screened for antibacterial, α-glucosidase and hemolytic analysis.The synthesized compounds exhibit cytotoxicity and antibacterial potentials.