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

Introduction

The saccharine (1,2-Benzisothiazole-3-one 1,1-dioxide) is the main core of several compound shows biological potency.1-3 Different kind of 5HT 1a antagonists4, human leukocyte elastase  (HLE)  inhibitors5,  analgetics6,  human  mast  cell  tryptase  inhibitors7,α1-a  adrenergic receptor  antagonists8   and  aldehyde  dehydrogenase  inhibitors  have  identified  the  saccharine nucleus as the main molecular ingredient.9 Bioactive compounds harbouring Saccharine nucleus are known as inhibitors of serine proteasees10, cathepsin G proteinase 3,11 α1a and α1c adrenergic receptor atagonists,12 human mast cell tryptase inhibitors,13 analgesics,14 5-HT1a receptors,15 anti- anxiety and antibacterial.16The analogues of benzo[d]isothiazole were demonstrates the central nervous system and mycobacterium activity. 17-18TheBenzo[d]isothiazole 1,1-dioxide derivatives are known as dual functional inhibitors of 5-lipoxygenase and microsomal prostaglandin E2 synthase-1.19

Hence, considerable observation have been made to synthesize 1, 2-Benzisothiazol-3-one 1,1-dioxide derivatives, specifically in those having N-basic side chain so as add to their pharmacological value. By considering the biological importance of compounds harbouring Saccharine moiety, we decided to synthesize 3-(alkyl/arylamino)benzo[d]isothazole 1,1-dioxide analogues. This work reports synthesis, characterization and antibacterial evaluation of 3- (alkyl/arylamino)benzo[d]isothiazole 1,1-dooxide analogues.

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. 1H NMR, 13C NMR was recorded in CDCl3 and DMSO-d6 solution on a Bruker Ac 200 0r 400 MHz spectrometer.

Experimental Procedures

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 hours at 100 °C. After completion of 24 hours reflux add 18 ml of SOCl2 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-saccharinchloride.

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 2hrs at 100oC. 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 1H NMR, 13C NMR and ES-MS.

3-((2-hydroxyethyl)amino)benzo[d]isothiazole 1,1-dioxide (7a)

It is whitish solid crystal, yield 75%, 1H NMR (200 MHz, DMSO-d6) δH: 3.6 (m, 4H), 3.1 (s, 1H),7.8  (d,2H),7.9  (d,  1H),8.1  (d,  1H),  9.5  (s,1H);13   C  NMR  (50  MHz,  DMSO0d6)  δc: 45,47,53,58,121,122,132,142,159; MS (ESI) m/e = 227 (M+1)

3-((4-chlorophenyl)amino)benzo[d]isothiazole 1,1-dioxide (7b)

It is whitish solid crystal, yield 65%, 1H NMR ( 200 MHz, DMSO-d6) δH: 7.5 (d, 2H), 7.6 (d, 2H), 8.0 (d, 1H), 8.2 (d, 2H), 8.4 (d,2H), 10.7 (s, 1H); 13C NMR (50 MHz, DMSO-d6) δc: 126,128,132,133,134,138,139,141,154,161; MS (ESI) m/e = 293 ( M+1)

3-(o-tolylamino)benzo[d]isothiazole 1,1-dioxide (7c)

Off white yellow crystal, yield 62 %, 1H NMR (200 MHz, DMSO-d6) δH: 2.2 (s,3H), 7.3 (d,1H), 7.4 (d,2H), 7.8 (d,1H), 7.9 (d,2H), 8.0 (d,1H), 8.4 (d1H) 10.8 (s,1H); 13C NMR (50 MHz, DMSO-d6) δC:22, 26, 126, 128,131, 132, 133, 136, 138, 139, 140, 147, 163; MS (ESI) m/e = 273 (M+1).

3-(pyridin-2-ylamino)benzo[d]isothiazole 1,1-dioxide (7d)

Off brown crystal, yield 60 %, 1H NMR (200 MHz, DMSO-d6) δH: 5.6 (d,1H), 7.4 (d,2H), 7.5 (d,2H), 7.8 (d,2H), 8.4 (d,2H), 9.9 (s,1H) ; 13C NMR (50 MHz, DMSO-d6) δC: 60, 121, 124, 127, 129, 133, 134, 135, 136, 142, 159, 171; MS (ESI) m/e = 259 (M+1).

2-((1,1-dioxidobenzo[d]isothiazol-3-yl)amino)-3-(1H-indol-3-yl)propanoic acid (7e)

Off yellow crystal, yield 65 %, 1H NMR (200 MHz, DMSO-d6) δH: 2.5 (s,1H), 3.6 (s,1H), 5.6 (d,2H), 7.4 (d,2H), 7.5 (s,1H), 7.7 (d,2H), 8.0 (d,2H), 8.5 (d,2H), 9.9 (s,1H), 13.5 (s,1H); 13C NMR (50 MHz, DMSO-d6) δC: 60, 79, 124, 127, 128, 129, 133, 134, 135, 142, 159, 171; MS (ESI) m/e = 367 (M+1)

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 hours for activation of cultures. The colony forming units (CFUs) were calculated from the broth. The 100 uL (100×102 CFUs/mL) of the medium were inoculated into fresh Luria bertini broth (5 ml) and kept for 16 hours to 18 hours 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 hours at 37oC. The sterile filter paper disc of 4mm in diameter were impregnated with 10 μl, 50μl and 100μl (stock concentration 10 ug/ml) of each compound. For drying purpose the desk were kept for 1 hour 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 37oC 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 37oC for 16-18 hours. After 16 to 18 hours 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.

Results and discussion

The following procedure was used for the synthesis of pseudo saccharine amine derivatives (7a-j) in the Scheme 1. The starting material 2-(methoxycarbonyl)benzenesulfinic acid (3) has been prepared by diazotization of commercially available 2-amonomethylbenzoate (1). Cholorination of acid (3) followed by ammonia reaction yields methyl 2-sulfamoylbezoate (4), which when cyclized with Conc. HCl yields benzo[d]isothiazol-3-(2H)-one 1,1-dioxide (5). The compound (5) on chlorination with thionylchloride gives 3-chlorobenzo[d]isithiazole 1,1- dioxide (6). Compound (6) was treated with different substituted or non-substituted aryl or aliphatic amines in presence of triethylamine as a base in 1,4 dioxane affords the target pseudo saccharine amines derivatives (7a-j) in very good yield. After the formation of derivatives (7a-j). was justify by 1H NMR, 13C NMR and MS analysis. The compound 7b having MIC of 6.8, 8.23 and 7.49  μM of E.Coli, S.aureus and Bacillus subtilis respectively whereas compound 7i and MIC values 5.93, 8.6 and 4.7 μM for E.Coli, S.aureus and Bacillus subtilis respectively.

Scheme 1: Synthesis of Pseudo-saccharine amine derivatives(7a-j). Click here to View figure

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, 2 and Figure 1

Table 1: Antimicrobial effect of compounds (7a-j) on pathogenic microorganism by using Disc diffusion method.

		Zone of inhibition (cm)
Compound	Organism	10 μl	50 μl	100 μl
7a	E coli	0	1.5	2.5
	S aureus	0	0	0
	Bacillus	0	0	0
7b	E coli	0.9	no growth	no growth
	S aureus	1.8	no growth	no growth
	Bacillus	3.4	no growth	no growth
7c	E coli	0	1.3	2.5
	S aureus	0	0	1.7
	Bacillus	0	0	0
7d	E coli	0	2	2.5
	S aureus	0	0	0
	Bacillus	0	1.5	2
7e	E coli	0	0	0
	S aureus	0	0	2
	Bacillus	0	0	0
7f	E coli	0	1	2.2
	S aureus	0	0	0
	Bacillus	2.3	no growth	no growth
7g	E coli	2.5	no growth	no growth
	S aureus	2.9	no growth	no growth
	Bacillus	no growth	no growth	no growth
7h	E coli	0	0	0
	S aureus	0	1.5	2.5
	Bacillus	0	1.8	2.7
7i	E coli	1	no growth	no growth
	S aureus	1.8	no growth	no growth
	Bacillus	2	no growth	no growth
7j	E coli	0	0	0
	S aureus	0	0	2
	Bacillus	1	2	3.3

 

Table 2: Time and dose dependent growth inhibition assay compounds (7a-j) on pathogenic microorganism.

Compound	MIC (µM)
	E. Coli	S. aureus	Bacillus subtilis
7a	14.5	Inactive	Inactive
7b	6.8	8.23	7.49
7c	12.5	10.84	Inactive
7d	8.3	Inactive	9.25
7e	Inactive	Inactive	Inactive
7f	4.6	9.1	Inactive
7g	7.25	6.8	Inactive
7h	Inactive	16.1	12.6
7i	5.93	8.6	4.7
7j	Inactive	Inactive	7.01

The stock concentration of the compound was 10 μg/ml.

Figure 1: Time and concentration dependent effect of compounds(7a-j) on microbes. Click here to View figure

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 Bacillussubtilis 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 10 μM, 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 anti-microbial 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.

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