INTRODUCTION:

Among the various types of known heterocyclic ring systems, benzimidazole nucleus was found to be present in various medicinally important compounds having antifungal and antibacterial¹ activity. 2-mercapto benzimidazole has shown antibacterial and antifungal activity². Benomyl is another antifungal compound having benzimidazole moiety with a carbamido linkage³.

It is observed that incorporation of Naphthoxy⁴ and Phenoxy⁵ moiety at the fifth position of benzimidazole molecule enhanced antibacterial and antifungal activity.

Thus, taking in consideration the above mentioned facts, it was decided to synthesize 5-substituted and 5,6-disubstituted-2-(N,N-dialkyl Thiocarbamido) benzimidazole derivatives by condensation of 5-substituted and 5,6-disubstituted-2-mercapto benzimidazoles (V) with N, N-dialkyl carbamoyl chlorides (VI) and triethyl amine in dry 1,4-dioxane. The resultant compounds depicted in Table-I have been further screened for their antibacterial and antifungal activity.

The 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.

EXPERIMENTAL:

Step I: Preparation of 5-substituted and 4,5-disubstituted Ortho Phenylene Diamine (OPD) [IV]

A mixture of 2-Naphthol (0.01 mol) or Phenol (0.01 mol) and anhydrous potassium carbonate was refluxed in dimethyl formamide (DMF) at 140° – 160°C for two hours. To this, 4,5-Dichloro or 5-chloro-2-nitro aniline (0.01 mol) dissolved in DMF was added dropwise at the same temperature and was continued to reflux for 6–8 hours. After completion of reaction, the reaction mixture was allowed to cool to room temperature and was poured into crushed ice. Product separated was filtered, dried and recrystallized from ethanol. 5-substituted or 4,5-disubstituted-2-nitro anilines (III) were formed, which were further reduced at 60⁰–80⁰C 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 vaccum distillation, and the concentrated solution remaining was poured into crushed ice and the product 5-substituted or 4,5-disubstituted OPD (IV) was filtered and dried.

Step II: Preparation of 5–substituted and 5,6-disubstituted-2-mercapto benzimidazoles (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-substituted or 4,5-disubstituted OPDA (0.01 mol) was taken in 15 ml of ethanol and was refluxed. After half an hour, potassium ethyl xanthate solution prepared as above was added dropwise and was further refluxed for 8–10 hours.

The reaction mixture was concentrated under vaccum to remove most of the ethanol. The concentrated solution was poured into crushed ice and neutralized with dil. HCl. The solid product 5-substituted or 5,6-disubstituted-2-mercapto benzimidazole (V) separated was filtered, washed with water, dried and recrystallized from ethanol.

Step III: Preparation of 5-substituted and 5,6- disubstituted-2-(N,N-dialkyl Thiocarbamido) benzimidazoles [VIIa – VIIh].

A mixture of 5-substituted or 5,6-disubstituted-2-mercapto benzimidazoles (0.002 mol) and triethyl amine (0.003 mol) in 10 ml dry 1,4-dioxane, was stirred at 40°–50°C and was refluxed. After half an hour N,N-diethyl or N,N-dimethyl carbamoyl chloride (0.003 mol) (VI) taken in 10 ml dry 1,4-dioxane was added dropwise with constant stirring. Stirring and refluxing was continued at 50°–60°C for 4–6 hours. After completion of reaction, reaction mixture was poured into crushed ice. The solid product separated was filtered, dried and recrystallized from ethanol to give the final product (VIIa–VIIh).

Adopting the above methods, 8 derivatives [VIIa – VIIh] were prepared and their physical data have been mentioned in Table-I.

Table – I: Physical Data of Compounds [VIIa – VIIh]

Compound	R	R^’	R₁=R₂	Melting Point (°C)	Yield (%)
VIIa	H	Phenoxy	Methyl	225	68
VIIb	H	Phenoxy	Ethyl	232-234	64
VIIc	H	Naphthoxy	Methyl	248-250	69
VIId	H	Naphthoxy	Ethyl	255	63
VIIe	Cl	Phenoxy	Methyl	210-212	68
VIIf	Cl	Phenoxy	Ethyl	225	66
VIIg	Cl	Naphthoxy	Methyl	238	69
VIIh	Cl	Naphthoxy	Ethyl	246	66

Elemental analysis (C, H, N) of all the compounds were within the range of ±0.4%.

Spectral Data:

VIIa: Yield: 68%; M. P.: 225°C

IR (KBr): 3120 (>N-H), 2940(-CH₃)1260 (>N-C=O), 1100 (>N-C-S), 1620 (C=C), 1680 (C=O).

¹H NMR (δ, ppm): 3.14 (s, 6H, -CH₃), 7.12-7.78 [m, 9H; (8H, Ar-H; 1H, >N-H)].

VIIb: Yield: 64%; M. P.: 232-234°C

IR (KBr): 3155 (>N-H), 2958(-CH₃), 1266 (>N-C=O), 1090 (>N-C-S), 1595 (C=C), 1686 (C=O).

¹H NMR (δ, ppm): 3.20-3.33 (t, 6H, -CH₂-CH₃), (q, 4H, -CH₂-CH₃), 6.95-7.69 [m, 9H; (8H, Ar-H; 1H, >N-H)].

VIIg: Yield: 69% ; M. P.: 238°C

IR (KBr): 3090 (>N-H), 2890

(-C₂H₅), 1268 (>N-C=O), 1110 (>N-C-S), 1575(C=C), 1691 (C=O).

¹H NMR (δ, ppm): 3.19–3.25 (d, 6H, -CH₃), 6.89–7.73 [m, 10H; (8H, Ar-H; 1H, >N-H)]

VIIh: Yield: 66%; M. P.: 246°C

IR (KBr): 3000 (>N-H), 2980

(-C₂H₅), 1240 (>N-C=O), 1120 (>N-C-S), 1590(C=C), 1670 (C=O).

¹H-NMR (δ, ppm): 1.28 (t, 6H, -CH₂ CH₃), 3.25 (q, 4H,-CH₂ CH₃), 6.90-7.85 [m, 11H; (10 H, Ar-H; 1H, >N-H)].

RESULTS AND DISCUSSION:

The title compounds [VIIa–VIIh] synthesized 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.

The Minimum Inhibitory Concentration (MIC) was determined by using tube dilution method as per standard procedure⁶. DMSO was used as a solvent with appropriate control.

Synthesized compounds [VIIa – VIIh] were found to exhibit moderate to good antibacterial and antifungal activity, the detailed results of which has been mentioned in Table-II.

Antibacterial activity:

All the eight compounds synthesized were subjected to antibacterial screening. Muller-Hinton broth was used as culture medium. It was concluded that VIIh was the better of all the compounds prepared in terms of bioactivity. Ethyl linkage derivatives were more favourable in terms of antibacterial activity than methyl linkage derivatives.

Antifungal activity:

In the manner similar to the above procedure, all the synthesized eight compounds were tested for their antifungal activity against Candida Albicans. Sabouraud-Dextrose broth was used as a culture medium. It was observed that all the compounds showed decent antifungal activity. The presence of naphthyloxy at the fifth position of all the substituted benzimidazoles showed activity at 50 µg/ml. Hence presence of naphthyloxy group was more favorable for antifungal activity.

Table-II: Results of Bioactivity of Compounds prepared and tested against pathogenic strains

Compound	R	R'	R₁=R₂	*S. aureus* *ATCC3750*	*S.typhi* *NCTC786*	*C. albicans* *ATCC 10231*
VIIa	H	Phenoxy	Methyl	100	200	100
VIIb	H	Phenoxy	Ethyl	50	100	100
VIIc	H	Naphthoxy	Methyl	100	100	50
VIId	H	Naphthoxy	Ethyl	50	100	50
VIIe	Cl	Phenoxy	Methyl	100	200	100
VIIf	Cl	Phenoxy	Ethyl	50	100	50
VIIg	Cl	Naphthoxy	Methyl	50	200	50
VIIh	Cl	Naphthoxy	Ethyl	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 of the Title Compounds prepared:

Table – I: Physical Data of Compounds [VIIa – VIIh]

Spectral Data:

ACKNOWLEDGEMENT: