INTRODUCTION:

The potent pharmacological past of sulphonamides is unquestionable. Sulphonamides in the recent past have shown moderate to good antibacterial as well as antifungal^1,2 activity.

A number of benzimidazole derivatives recently have displayed significant antimicrobial properties^3,4. 7-nitro-5-benzimidazole sulphonamides and 4-nitro-6-benzimidzole sulphonamide were found to be excellent antibacterials⁵.

These literature facts convinced us to synthesize a series of sulphonamido benzimidazole derivatives with potential antimicrobial properties.

The title compounds were synthesized by condensation of 5-substituted-2-(1-H-benzimidazole)-sulfonyl chloride [VI] with 2-amino heterocycles [VII] in dry acetone in presence of triethyl amine.

The structures of synthesized compounds were confirmed on the basis of their analytical and spectral data.

MATERIALS AND METHODS:

Melting points were determined in open glass capillaries using Thermonik Precision Melting Point Cum Boiling Point Apparatus Model C-PMB-2 and were uncorrected. Purity of the compounds was verified by precoated TLC plates [E. Merck Kieselgel].

Elemental analysis was carried out on Thermo Finnegan Flash EA 1112. The IR spectra were recorded using KBr pellets on BOMEM FTIR MB–10452M 5903L spectrophotometer. The ¹H-NMR spectra were recorded on JEOL–AMX–400 (300MHz) spectrophotometer using T.M.S. as an internal standard.

Step I: Preparation of 5-napthoxy Ortho Phenylene Diamine (OPD) [IV]

A mixture of 2-Naphthol (0.01 mol) [II] and anhydrous potassium carbonate was refluxed in dimethyl formamide (DMF) at 140–160°C for two hours. To this solution, 5-chloro-2-nitro aniline (0.01 mol) [I] dissolved in DMF was added dropwise at the same temperature. Reaction was further refluxed for 6–8 hours. After completion of reaction, the reaction mixture was allowed to cool to room temperature and was poured into crushed ice. The product separated was filtered, dried and recrystallized from ethanol. 5-naphthoxy-2-nitro aniline [III] was formed, which was further reduced at 60–80°C with Raney-Nickel and Hydrazine Hydrate in ethanol for 4–6 hours. After completion of reaction, Raney-Nickel was filtered off and decomposed in 10% dil. HCl. Excess of ethanol was removed by vacuum distillation, and the concentrated solution remaining was poured into crushed ice and the product 5- naphthoxy OPD [IV] was filtered and dried.

Step II: Preparation of 5-naphthoxy-2-mercapto benzimidazole [V].

Potassium hydroxide (0.01 mol) was dissolved in (1:5) mixture of water and ethanol. The mixture was allowed to cool to room temperature and to this mixture carbon disulfide (0.017 mol) was added with constant stirring and this led to the formation of potassium ethyl xanthate solution.

5-naphthoxy OPD (0.01 mol) [IV] was taken in 15 ml of ethanol and was refluxed. After half an hour, potassium ethyl xanthate solution prepared as above was added drop-wise and was further refluxed for 8–10 hours.

The reaction mixture was concentrated under vacuum to remove most of the ethanol. The concentrated solution was poured into crushed ice and neutralized with dil. HCl. The solid product 5-naphthoxy-2-mercapto benzimidazole [V] separated was filtered, washed with water, dried and recrystallized from ethanol.

Step III: Preparation of 5-naphthoxy-2-(1-H-benzimidazole)-Sulfonyl Chloride⁶

[VI]

5-naphthoxy-2-mercapto benzimidazole 2.92 g. (0.01 mol) [V] was charged to a jacketed vessel, followed by 17.3 ml (0.302 mol) of acetic acid and 2.2 ml of water (0.12 mol) stirring was commenced (300 rpm) and the mixture was warmed to 30°C to completely dissolve the solids. A nitrogen purge was then applied and the vessel content cooled to approximately 4°C. The nitrogen flow was stopped, the vessel was sealed, and chlorine gas was bubbled into the mixture over 35 minutes (T_max 13.8°C). A green coloration developed and the chlorine supply was shut off. The green coloration discharged after several seconds indicating that the reaction was not completed and addition of chlorine was resumed for 5 minutes resulting in an exothermic reaction from 11.6°C- 13.3C. The green color persisted when the chlorine supply was shut off after 3 hours indicating that end of reaction had been reached. Total amount of chlorine used was 2.56 g. (90.036 mol). The vessel was vented to the atmosphere, purged with nitrogen and raised to 15°C. The mixture was discharged, allowed to warm to room temperature and then evaporated to low volume. Toluene 10.7 ml (0.01 mol) was charged to the mixture, which was then re-evaporated.

The toluene addition/re-evaporation procedure was repeated. The mixture was filtered on suction and dried in vacuum oven at 40°C for overnight. Solid product obtained was immediately used for next procedure because derivatives of sulfonyl chlorides are highly unstable in nature.

Step IV: Preparation of 5-naphthoxy-2-(1-H-benzimidazole) sulfonamido benzothiazole [VIII]

In a dry three necked flask equipped with a water condenser, guard tube, dropping funnel, stopper and magnetic needle, 1.398 g. (0.0039 mol) of 5-naphthoxy-2-benzimidazole sulfonyl chloride [VI] was dissolved in 10 ml of dry acetone. To this was added 0.39 g. (0.55 ml, 0.004 mol) of triethyl amine. The mixture was stirred so as to reach the temperature of 60°C. At this temperature (0.0039 mol) of 2-amino benzothiazole [VII] dissolved in 5 ml of dry acetone, was added dropwise through the dropping funnel over a period of 15 minutes. The reaction mixture was then stirred at 60°C for 4-6 hours. Reaction mixture was concentrated to get the desired product. It was recrystallized from methanol.

Adopting the above methods, eight derivatives [VIII-a – VIII-h] were prepared and their physical data have been mentioned in Table-I.

VIII-a: Yield: 60%; M.P.: 190-192°C IR (KBr): 3050 (>N-H), 1325, 1139 (-SO₂NH-), 1466 (C-C of C₆H₆), 1256 (Ar-O-Ar), 1592 (C=N-), 872, 787 (5-substituted benzimidazole).

¹H NMR (δ, ppm): 6.93-7.75 [m, 9H (8H, Ar-H), (1H, -NH)], 8.81 [bs, 2H (-SO₂NH₂)]

VIII-b: Yield: 63%; M.P.: 210-212°C

IR (KBr): 3058 (>N-H), 1323, 1130 (-SO₂NH-), 1490 (C-C of C₆H₆), 1267 (Ar-O-Ar), 1582 (C=N-), 881, 779 (5-substituted benzimidazole).

¹H NMR (δ, ppm): 6.96-7.81 [m, 11H (10H, Ar-H), (1H, -NH),], 9.12 [bs, 2H (-SO₂-NH₂)].

VIII-c: Yield: 53%; M.P.: 140–142°C

IR (KBr): 3071 (>N-H), 1318, 1129 (-SO₂NH-), 1469 (C-C of C₆H₆), 1262 (Ar-O-Ar), 1579 (C=N-), 869, 788 (5-substituted benzimidazole).

¹H NMR (δ, ppm): 7.05-7.99 [m, 13H (12H, Ar-H), (1H, -NH)], 9.49 [bs, 1H (-SO₂-NH)].

VIII-d: Yield: 56%; M.P.: 180–182°C

IR (KBr): 3069 (>N-H), 1309, 1145 (-SO₂NH-), 1474 (C-C of C₆H₆), 1272 (Ar-O-Ar), 1590 (C=N-), 886, 791 (5-substituted benzimidazole).

¹H NMR (δ, ppm): 6.78-7.82 [m, 15H (14H, Ar-H), (1H, -NH)], 10.18 [bs, 1H (-SO₂-NH)].

VIII-f: Yield: 59%; M.P.: 169°C

IR (KBr): 3065 (>N-H), 1329, 1155 (-SO₂NH-), 1478 (C-C of C₆H₆), 1281 (Ar-O-Ar), 1585 (C=N-), 866, 798 (5-substituted benzimidazole).

¹H NMR (δ, ppm): 6.80-7.93 [m, 13H (10H, Ar-H), (1H, -NH), (2H. thiazole)], 9.75 [bs, 1H (-SO₂-NH-)].

VIII-g: Yield: 54%; M.P.: 163°C

IR (KBr): 3077 (>N-H), 1312, 1147 (-SO₂NH-), 1460 (C-C of C₆H₆), 1265 (Ar-O-Ar), 1579 (C=N-), 879, 790 (5-substituted benzimidazole).

¹H NMR (δ, ppm): 6.92-7.84 [m, 13H (12H, Ar-H), (1H, -NH)], 9.27 [bs, 1H (-SO₂-NH)].

VIII-h: Yield: 54%; M.P.: 163°C

IR (KBr): 3088 (>N-H), 1304, 1155 (-SO₂NH-), 1471 (C-C of C₆H₆), 1270 (Ar-O-Ar), 1586 (C=N-), 880, 787 (5-substituted benzimidazole).

¹H NMR (δ, ppm): 6.87-7.90 [m, 15H (14H, Ar-H), (1H, -NH)], 9.51 [bs, 1H (-SO₂-NH)].

RESULTS AND DISCUSSION:

The compounds were screened in vitro for their antibacterial activity against Gram Positive bacteria Staphylococcus aureus (ATCC 3750), Gram Negative bacteria Salmonella typhi (NCTC 786) and in vitro antifungal screening was carried out against Candida albicans (ATCC 10231).

The Minimum Inhibitory Concentration (MIC) was determined by using tube dilution method as per standard procedure⁷. DMSO was used as a solvent with appropriate control.

Synthesized title compounds [VIII-a – VIII-h] were found to exhibit moderate antibacterial and antifungal activity. The bioactivity of the title compounds has been displayed in Table-II.

Antibacterial activity:

The synthesized eight sulphonamido heterocycles were subjected to antibacterial screening. The culture medium used for this was Muller-Hinton broth. It was concluded from the obtained results that presence of naphthoxy group at the 5^th position of benzimidazole was responsible for the comparatively above average activity shown against both the bacterial strains. Naphthoxy benzimidazole derivative having sulphonamido benzothiazole derivative (VIII-h) was the most potent amongst them with activity upto 50 µg/ml

Antifungal activity:

Similarly, eight derivatives of sulphonamido benzimidazole derivatives were screened for their antifungal potency. Sabouraud-Dextrose broth was used as a culture medium. The results showed that all the title compounds were moderately active against the fungal strain. Naphthoxy benzimidazolo sulphonamido benzothiazole derivative (VIII-h) was slightly better amongst them.

Table – I: Physical Data of Compounds [VIII-a – VIII-h]

Compound	R₁	R₂	Melting Point (°C)	Yield (%)
VIII-a	Phenoxy	H	190-192	60
VIII-b	Naphthoxy	H	210-212	63
VIII-c	Phenoxy	Pyridine	140-142	53
VIII-d	Naphthoxy	Pyridine	180-182	56
VIII-e	Phenoxy	Thiazole	143	51
VIII-f	Naphthoxy	Thiazole	169	59
VIII-g	Phenoxy	Benzothiazole	163	54
VIII-h	Naphthoxy	Benzothiazole	189-191	62

C, H, N analysis for each compound was within the range of ±0.4

Table – II: Bioactivity of synthesized title compounds :

Sr. No.	R₁	R₂	*S. aureus* ATCC 3750	*S. typhi* NCTC 786	*C. albicans* ATCC 10231
VIII-a	Phenoxy	H	200	200	200
VIII-b	Naphthoxy	H	100	100	200
VIII-c	Phenoxy	Pyridine	200	100	200
VIII-d	Naphthoxy	Pyridine	100	50	100
VIII-e	Phenoxy	Thiazole	200	200	200
VIII-f	Naphthoxy	Thiazole	100	50	100
VIII-g	Phenoxy	Benzothiazole	100	100	100
VIII-h	Naphthoxy	Benzothiazole	50	50	50

· Ampicillin (MIC 0.04 μg/ml) used as standard against S. aureus.

· Trimethoprim (MIC 0.01 μg/ml) used as standard against S. typhi.

· Miconazole (MIC 0.01 μg/ml) as standard against C. albicans.

Schematic Representation:

Table – I: Physical Data of Compounds [VIII-a – VIII-h]

Table – II: Bioactivity of synthesized title compounds :

ACKNOWLEDGEMENT: