Synthesis, Characterization and Antimicrobial Evaluation of New 3-(Alkyl/Arylamino)benzo[d]isothiazole 1,1-Derivatives

The saccharine nucleus has long been recognized as a significant component in medicine. A series of pseudo-saccharine amines derivatives (7a-j) were synthesized and examined for their antibacterial activity. After testing all compounds, 7b, 7f, 7g, 7i and 7j were found most effective against Escherichia coli, Streptococcus aureus and Bacillus subtilis strains. The MIC of the compound was found from 4.6 to 16.1 μM. Further, compound 7f and 7i exhibited excellent activity against E.coli and Bacillus subtilis with MIC value 4.6 and 4.7 μM respectively. The compound 7b and 7i was found active against all the three bacteria. The zone inhibition was observed at 10 μM against Escherichia coli, Staphylococcus aureus and Bacillus subtilis at 0.9, 1.8, 3.9 respectively for 7b and 1.0, 1.8 and 2.0 cm respectively for 7i.


MATERIAL AND METHODS
From the commercial sources the reagents and solvent were purchased and it has been used without further purification. The melting points were taken in open capillary tubes and are uncorrected. During the reaction, the synthesis of compounds was examined using TLC on 0.5 mm thick silica-gel plates, and the location of spots was checked using iodine and UV-light. All of the chemicals were purified utilizing suitable organic solvents and a recrystallization/silica gel (100-200 mesh) gravity column. The compound's mass spectra were determined using the Shimadzu GC-MS-QP-2010 model and the direct inlet probe technique. 1 H NMR, 13 C NMR was recorded in CDCl 3 and DMSO-d6 solution on a Bruker Ac 200 0r 400 MHz spectrometer.

ExPERIMENTAL
The Psuedo-saccharinchloride (6) were synthesized by taking 18 g of saccharin with 56.25 mL of 1,4 dioxane, 56.25 mL of thionyl chloride and 4 mL of DMF in round bottomed flask and refluxing it for 24 h at 100°C. After completion of 24 h reflux add 18 ml of SOCl 2 and 4 mL of DMF and continue to reflux for another 12 hours. The formed crude product was recrystallized from toluene to afford yellow-colored fine crystals of Psuedo-saccharin chloride.

Procedure for the synthesis of Pseudo-saccharine amines analogues (7a-j)
Take 500 mg (1eq) of saccharine chloride and alkyl or aryl amine (1eq) was dissolved in 1,4-dioxan and refluxed the content for 2h at 100 o C. After completion of the reaction add water to quench the reaction to get a solid product, which then recrystallized by using toluene. The formation of the product was validated by 1 H NMR, 13 C NMR and ES-MS.

Biological evaluation
All the Pseudo-saccharine amine derivatives were check for in vitro antimicrobial activity against Escherichia coli, Bacillus subtilis and Streptococcus aureus using Disc diffusion method and time dose dependent growth inhibition assay. The results of antimicrobial activity of tested compounds (7a-j), using tetracycline as reference standard, are shown in Table1, 2 and Figure 1.

General protocol for antibacterial activity Growth of pathogenic microorganisms
Clinical isolates were growth in Luria bertini medium (pH 6.8) for 24 h for activation of cultures. The colony forming units (CFUs) were calculated from the broth. The 100 uL (100x10 2 CFUs/mL) of the medium were inoculated into fresh Luria bertini broth (5 mL) and kept for 16 h to 18 h for log phase culture. The log phase culture was used for the antimicrobial assay.

Preparation of compounds
The compounds stock solution was prepared in DMSO and diluted further for antimicrobial action.

Antimicrobial effect of compounds on pathogenic microorganism by using Disc diffusion method
For the determination of antimicrobial activities of every compound, the Disc diffusion method was used. We use a multidrug resistant strain of Escherichia coli, Staphylococcus aureus, and Bacillus subtilis culture for this. Several bacterial species were cultured on nutrient agar media. Microorganisms in broth media were used to make inoculum suspensions (100 CFU per uL), nutrient broth inoculated with bacteria species was incubated for 24 h at 37 o C. The sterile filter paper disc of 4mm in diameter were impregnated with 10 μL, 50 μL and 100 μL (stock concentration 10 ug/mL) of each compound. For drying purpose the desk were kept for 1 h at room temperature in a sterile airflow laminar chamber. After that it placed in the center of fresh nutrient agar plates which earlier seeded with 100 μL of inoculum suspension of each bacteria. The culture was kept incubation purpose either at 37 o C for 24-48 hours. The every experiment was repeated three times. The antibiotics were used as positive control and use tetracycline as reference antibacterial standard. The antimicrobial activities were checked by measuring the zone inhibition diameters (Millimetres) surrounding in each disk.

Time and dose dependent effect of compounds on the growth of the pathogenic microorganisms
The culture of MDR strain of Escherichia coli, Streptococcus aureus and Bacillus subtilis were inoculated separately into LB medium and incubated at 37 o C for 16-18 hours. After 16 to 18 h the cultured tubes were exposed to the compounds at concentration of 10, 25 and 50 μg/mL. The optical density was recorded at 660 nm after fixed interval of time. The time-dependent development of microorganisms was investigated using Graph pad prism 7.
All the newly synthesized compounds were screened for In vitro antimicrobial activity, against Escherichia coli, Streptococcus aureus and Bacillus subtilis using Disc diffusion method. Time and dose dependent growth inhibition assay with the result of antimicrobial activity of tested compounds (7a-j), using tetracycline as reference standard, are shown in Table 1   Compound 7a was found active against the pathogenic E. coli with promising zone of inhibition 1.5 and 2.5 cm at 50 μL and 100 μL respectively and not found active against Bacillus subtilis and S. aureus at any concentration. The compound 7b was found very active against all the three bacteria. The zone of inhibition was observed at 10 μL against E. coli, S. aureus and Bacillus subtilis 0.9, 1.8 and 3.4 cm, respectively. The compound 7c was found active against the pathogenic E.coli at concentration 50 μL and 100 μL with the zone of inhibition was found 1.3 and 2.5 cm respectively. The Compound 3 was also found active against S. aureus at 100 μL with zone of inhibition 1.7 cm. The compound 7d was found active against E. coli at concentration 50 μL and 100 μL with zone of inhibition 2 and 2.5 cm respectively. The zone of inhibition was recorded at 50 μL and 100 μL concentrations against Bacillus subtilis were 1.5 and 2 cm respectively. The compound 7e was found not active against any E.coli, Bacillus subtilis and S. aureus at concentration 10 μL, 50 μL and 100 μL. Compound 7f was found active against the E. coli at concentration 50 and 100 μL with zone of inhibition 1.0 and 2.2 cm respectively. It was found active with Bacillus subtilis at concentration of 10 μL with zone inhibition at 2.3 cm and shows no growth with S. aureus. Compound 7g was found active against the pathogenic E. coli at concentraion 10 subtilis at zone inhibition at 2.5 cm and it also found active for S. aureus with promising zone of inhibition 2.9 cm at 10 μL and no growth was observed at higher concentration. The compound 7h was found active against S. aureus and Bacillus subtilis with zone of inhibition 1.5 and 1.8 cm at 50 μL concentrations respectively. The compound 7i was found active against all the three bacteria. The zone of inhibition was observed at 10 μL against E. coli, S. aureus and Bacillus subtilis at 1.0, 1.8 and 2.0 cm respectively. The compound 7j was found active against Bacillus subtilis 10 μL, 50 μL and 100 μL concentrations with zone of inhibition, 1, 2 and 3.3 cm respectively. All three bacteria were shown to be susceptible to the compounds 7b and 7i. At 10μM, zone inhibition against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis was seen at 0.9, 1.8, and 3.9 cm for 7b and 1.0, 1.8, and 2.0 cm for 7i, respectively.

CONCLUSION
In summary, the eco-friendly and catalyst free protocol was developed and applied for the synthesis of saccharin derivatives. Saccharin derivatives showed significant inhibition of microbial growth. These analogues are chemically tractable and hence provide ample opportunities for further modification to obtain potent antimicrobial agents. The isolated yield of the saccharin derivatives is excellent, so gram scale synthesis is possible.

ACKNOWLEDGMENT
The authors are thankful to S.B.E.S. College of science, Aurangabad and Vasantrao Naik Mahavidyalaya, Aurangabad and IICT, Hyderabad for providing the laboratory facility and spectral data respectively.

Conflict of interest
Authors declare that they have no conflict of interest.