Synthesis and Antimicrobial activity of Bulky molecules comprising Benzothiazole and Sulphonamide moieties

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Synthesis of N-(substituted 1, 3-benzothiazol-2-yl)-4-(substituted aryl diazenyl) benzenesulphonamide
0.01 mol of 4-amino-N-(substituted1, 3-benzothiazol-2-yl) benzenesulphonamide was added in 20 ml Hydrochloric acid (50%), stirred and cooled to 0 °C to 5 °C. 8 mL of aqueous sodium nitrite (10.88 mmole, 0.7507 g) was added in drops maintaining the temperature between 0-5 °C. The diazonium chloride formed was consecutively coupled with 0.01 mol of 1-Naphthol or 2-Naphthol or Phenol that was dissolved in 15 mL of 10% sodium hydroxide solution. The reaction mixture was stirred at 0 °C to 5 °C for 1 hour. The product that precipitated was recrystallized from ethanol (65%).6

N-(benzothiazol-2-yl)-4-((4-hydroxynaphthalen-1-yl) diazenyl) benzenesulphonamide (SK5a)
Yield (%): 64.8; M.P. (°C): 262-264; Rf: 0.72 (Hexane : Ethyl acetate, 8:2); IR (KBr, cm-1): 3600 (OH), 3250 (NH), 1630 (C=N), 1430 (N=N), 1370, 1140 (SO2), 1300 (C-N), 650 (C-S); 1H- NMR (DMSO, ppm): 12.60 (s, 1H, NHSO2), 6.59- 8.21 (m, 14H, Ar-H), 5.60 (s, 1H, Ar-OH); Mass spectra (m/z): 462 (M+1)+.

N-(6-chlorobenzothiazol-2-yl)-4-((2-hydroxynaphthalen-1-yl) diazenyl) benzenesulphonamide (SK5b)
Yield (%): 63.8; M.P. (°C): 270-274; Rf: 0.75 (Hexane : Ethyl acetate, 8:2); IR (KBr, cm-1): 3300 (OH),  3200 (NH), 1652 (C=N), 1400 (N=N), 1375, 1090 (SO2), 1292(C-N), 707 (C-Cl), 608 (C-S); 1H- NMR (DMSO, ppm): 11.90 (s, 1H, NHSO2), 9.97 (s, 1H, Ar-OH), 7.22-8.30 (m, 13H, Ar-H); Mass spectra (m/z): 497.2 (M+2)+.

4-((2-hydroxynaphthalen-1-yl) diazenyl)-N-(6-methylbenzthiazol-2-yl) benzenesulphonamide (SK5c)

Yield (%): 59.7; M.P. (°C): 254-258; Rf: 0.68 (Hexane : Ethyl acetate, 8:2); IR (KBr, cm-1):
3675 (OH), 3200 (NH), 1635 (C=N), 1410 (N=N), 1360, 1100 (SO2), 1275(C-N), 711 (C-S); 1H- NMR (DMSO, ppm): 12.18 (s, 1H, NHSO2), 8.81 (s, 1H, Ar-OH), 7.40-8.28 (m, 13H, Ar-H), 2.20 (s, 3H, CH3); Mass spectra (m/z): 475.4 (M)+.

N-(7-chloro-6-fluorobenzohiazol-2-yl)-4-((2-hydroxynaphthalen-1-yl) diazenyl) benzenesulphonamide (SK5d)
Yield (%): 73.1; M.P. (°C): 274-280; Rf: 0.76 (Hexane : Ethyl acetate, 8:2); IR (KBr, cm-1):
3310 (OH), 3150 (NH), 1652 (C=N), 1447 (N=N), 1368, 1197 (SO2), 1268(C-N), 1033 (C-F), 715 (C-Cl),  667 (C-S); 1H- NMR (DMSO, ppm): 12.30 (s, 1H, NHSO2), 9.81 (s, 1H, Ar-OH), 6.97- 8.30 (m, 12H, Ar-H); Mass spectra (m/z): 512.9 (M)+.

4-((4-hydroxyphenyl) diazenyl)-N-(6- nitrobenzothiazol-2-yl) benzenesulphonamide (SK5e)
Yield (%): 66.3; M.P. (°C): 284-288; Rf: 0.78 (Hexane : Ethyl acetate, 8:2); IR (KBr, cm-1): 3270 (OH), 3150 (NH), 1652 (C=N), 1480, 1337 (NO2), 1430 (N=N), 1375, 1140 (SO2), 690 (C-S); 1H- NMR (DMSO, ppm): 12.68 (s, 1H, NHSO2), 6.77-8.94 (m, 11H, Ar-H), 5.57 (s, 1H, Ar-OH); Mass spectra (m/z): 456.1 (M+1)+.

4-((2-hydroxynaphthalen-1-yl) diazenyl)-N-(6-methoxybenzothiazol-2-yl) benzenesulphonamide (SK5f)
Yield (%): 65.8; M.P. (°C): 270-276; Rf: 0.74 (Hexane : Ethyl acetate, 8:2); IR (KBr, cm-1): 3518(OH), 3334 (NH), 1653 (C=N), 1456 (N=N), 1339, 1144 (SO2), 1224 (C-O), 668 (C-S); 1H- NMR (DMSO, ppm): 12.10 (s, 1H, NHSO2), 9.75 (s, 1H, Ar-OH), 6.61-8.29 (m, 13H, Ar-H), 2.06 (s, 3H, OCH3); Mass spectra (m/z): 491.7 (M)+.

BIOLOGICAL EVALUATION
The microbiological assay is based upon a comparison of inhibition of growth of microorganisms by measured concentrations of test compounds with that produced by known concentration of a standard antibiotic. Two methods generally employed are turbidometric (tube-dilution) method and cylinder plate (cup-plate) method. In the turbidometric method inhibition of growth of microbial culture in a uniform ablution of antibiotic in a fluid medium is measured. It is compared with the synthesized compounds. Here the presence or absence of growth is measured. The cylinder plate method depends upon diffusion of antibiotic from a vertical cylinder through a solidified agar layer in a Petridis or plate to an extent such that growth of added micro-organisms is prevented entirely in a zone around the cylinder containing solution of the antibiotics. The cup-plate method is simple and measurement of inhibition of microorganisms is also easy. Name of organisms for antimicrobial activity Gram Positive microorganisms [Staphylococcus aureus (MTCC No. 88), Bacillus subtilis (MTCC No. 10518)]; Gram Negative microorganisms [Escherichia coli (MTCC No. 44), Pseudomonas aeruginosa(MTCC No. 3541)]; Name of organisms for antifungal activity [Candida albicans (MTCC No. 10231)]

Preparation of test solution:
10 mg of the test compound was dissolved in 10 mL of DMSO. From this 2 mL of solution was taken and diluted to 10 mL with DMSO. Now the concentration of the test compound is 200 µg/mL. From this 5 mL of solution was taken and diluted to 10 mL with DMSO. Now the concentration of the test compound is 100 µg/mL. These sample solution were made in suitably labeled sterilized test tubes.

Preparation of standard solution:
The standard drug used for the testing is Ciprofloxacin. It is water soluble; the concentration of this drug is adjusted so as to contain 100 µg/mL and 200 µg/mL.The standard drug Griseofulvin was dissolve in appropriate quantity of Methanol to obtain the concentration of 100 μg/mL and 200 μg/mL.

Minimum inhibitory concentration test
This is the lowest concentration of the drug that will inhibit the growth and the replicating of the invading pathogen. This test is performed as a Tube Dilution Test. A standard amount of bacteria is placed within a series of tubes that have been diluted with a specific antibiotic or antimicrobial. These tubes are incubated overnight at 37 °C. No cloudiness means; no bacterial growth, whereas if the broths are cloudy, there is the presence of bacterial growth. Moving from right to left, you check the smaller dilutions of the broth for cloudiness. The first tube after the last cloudy tube is the MIC value.7

RESULTS AND DISCUSSION
All synthesized compounds were evaluated for antibacterial activity against E. coli, P. aeruginosa, B. subtilis, S. aureus andantifungal activity against C. albicans.

Table 1.  Zone of Inhibition of Synthesized compounds for antibacterial and antifungal activity

Comp.

Code


Diameter of zone of inhibition (mm)

S.

aureus

B.

subtitis

E.

coli

P.

aeruginosa

C.

albicans

Conc.

(µg/mL)

100

200

100

200

100

200

100

200

100

200

SK3g

8

14

-

9

7

11

-

9

-

-

SK3h

9

13

-

10

7

14

-

9

-

8

SK3i

-

8

-

9

-

8

-

10

-

-

SK3j

9

12

-

10

9

13

-

8

-

12

SK5a

10

15

9

14

11

15

10

14

-

11

SK5b

15

19

13

17

15

19

14

20

10

15

SK5c

11

15

10

16

9

13

12

16

9

13

SK5d

16

21

14

19

17

21

15

20

14

21

SK5e

13

19

14

17

12

16

13

18

12

16

SK5f

11

17

10

15

13

19

11

15

14

17

Ciprofloxacin

23

28

21

29

25

33

24

30

-

-

Griseofulvin

-

-

-

-

-

-

-

-

22

29


(-) indicates no inhibition zone (no activity)

Table 2. Minimum inhibitory concentration of synthesized compounds

Comp.

Code

Minimum inhibitory concentration (μg/mL)

S.

aureus

B.

subtitis

E.

coli

P.

aeruginosa

C.

albicans

SK3g

62.5

125

62.5

125

250

SK3h

62.5

125

62.5

125

125

SK3i

125

125

125

125

250

SK3j

62.5

125

62.5

125

125

SK5a

62.5

62.5

62.5

62.5

125

SK5b

31.25

31.25

31.25

31.25

62.5

SK5c

62.5

62.5

62.5

62.5

62.5

SK5d

31.25

31.25

15.5

31.25

31.25

SK5e

31.25

62.5

31.25

31.25

62.5

SK5f

62.5

62.5

31.25

62.5

62.5

Ciprofloxacin

7

<5

<5

7

-

Griseofulvin

-

-

-

-

7

Figure 2: Biological activity (Zone of inhibition) of synthesized compounds

The objective of the present work was to Synthesis and biological evaluation of Bulky molecules comprising Benzothiazole and Sulphonamide moieties as antimicrobial agents. The yield of different synthesized compounds was found to be in the range of 59-72%. The physical characterization was done by melting point and TLC and spectral characterization was done by FTIR, 1H NMR, and Mass spectral studies.

From the in vitro antibacterial activity studies against the Gram-positive bacteria Staphylococcus aureus, Bacillus subtilus, the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa and antifungal activity studies against Candida albicans,it was found that the synthesized compounds showed significant activity. Hence these synthesized compounds appear to be promising antibacterial and antifungal agents. The standard drug used was Ciprofloxacin for antibacterial and Griseofulvin as standard drug for antifungal activity.

Among the all synthesized compounds, compound 5dwith MIC value of (31.25 μg/mL) for S. aureus, (31.25 μg/mL) for B. subtilis, (15.5 μg/mL) for E. coli and (31.25 μg/mL) forP. aeruginosa gives better antibacterial activity than other synthesized compounds. And other compounds SK5b and SK5e with MIC value of (31.25 μg/mL) for S. aureus, (31.25 μg/mL, 62.5 μg/mL) for B. subtilis, (31.25 μg/mL) for E. coli and (31.25 μg/mL) forP. aeruginosa give good antibacerial activity. The compoundSK5d with MIC value of (31.25 μg/mL) against fungi (C. albicans) gives a better antifungal activitythan other synthesized compounds.

The in vitro antibacterial and antifungal data in the(Table 1, 2) indicate that the all the synthesized compounds were found to posses significant antibacterial and antifungal activity. Perhaps the presence of strong electron withdrawing groups such as -F, -Cl, and -NO2 at 6th position on 2-amino benzothiazole (SK5b, SK5d, and SK5e)showed highly significant antibacterial and antifungal activity. The presence of electron donating group such as -CH3 at6th position on 2-amino benzothiazole (SK5c) shown moderate activity. Based on in vitro antibacterial activity and antifungal activity, synthesized compounds SK5a to SK5f(Azo derivatives) showed better antibacterial and antifungal activity than SK3g to SK3j (Acetamide derivatives).Overall none of the compounds showed higher activity than the standards.

CONCLUSION
Result of present study demonstrate that, a new class of different Bulky molecules comprising Substituted Benzothiazole and Sulphonamide were synthesized and evaluated for antimicrobial activity by agar disk diffusion method. All the newly synthesized compounds showed moderate to good potency against different bacterial strains and fungal strain. Among them SK5b, SK5d and SK5e showed better antimicrobial activity. It can be concluded that this class of compounds certainly holds great promise towards good active leads in medicinal chemistry. A further study to acquire information concerning biological activity is in progress.

REFERENCES
1.    Bhusari K, Amnerkar N, Khedekar P, Kale M “Synthesis and in vitro Antimicrobial Activity of Some New 4-Amino-N-(1, 3-Benzothiazol-2-yl) benzenesulphonamide Derivatives.” Asian J. Res. Chem. 2008, 1(2), 53-59.
2.    Bhusari S, Pawar R and Vibute Y, Synthesis and Anti-microbial activity of 2-(substituted phenylsulfonamido)-6-substituted benzothiazoles.” Indian J. Hetero. Chem., 2001, 11, 79-88.
3.    Chaudhary M, Pareek D, Ojha K and Pareek A, “Synthesis and Antibacterial Activity of 1-(2-Diazo-6-ethoxybenzothiazolyl) Substituted Benzene Derivatives.” Int. J. Curr. Chem., 2010, 1(3), 175-179.
4.    Mkpenie V, Ebong G, Obot I and Abasiekong B, “Evaluation of the Effect of Azo Group on the Biological Activity of 1-(4-Methylphenylazo)-2-naphthol.” E-Journal of Chemistry, 2008, 5(3), 431-434.
5.    Soni B, Bhandari A, Ranawat M, Sharma P, Sharma S and Prajapat R, “Synthesis and antimicrobial activity of some 2-substituted benzothiazoles containing azomethine linkage.” Pharmacophore, 2011, 2 (1), 36-45.   
6.    Eaton DC., Laboratory Investigation in Organic Chemistry, McGraw-Hill Inc, USA, 1989, pp 429-435.
7.    Pelczar MJ., Chan ECS., and Krieg NR. In Microbiology; 5th Edn; Tata McGraw Hill Publishing Company Limited, New Delhi, 1993, pp 73-98.

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