INTRODUCTION:

Benzimidazole and piperazine derivatives are regarded as promising class of bioactive heterocyclic compounds exhibiting wide range of biological activities.^[1] Literature survey reveals that the benzimidazole nucleus is present in numerous antiviral, ^[1] antifungal, ^[2], antibacterial, ^[3]agents. In view of the literature led to the concept that a novel series of N¹-methyl–2-aminophenyl (4-N4-alkyl/aryl piperazinyl) benzimidazole derivatives were synthesized.^[4-7] The structural elucidation of synthesized compounds is done by IR, 1H-NMR, Mass spectral analysis. The compounds were screened for their antifungal activity. ^[8-9]

MATERIALS AND METHODS:

The melting points are taken in an open capillary tube and are uncorrected. The purity of compound was checked by TLC on pre-coated silica gel 60 F₂₅₄ aluminium plates (E Merck) and visualized in Ultraviolet Chamber.IR spectra of the compounds recorded on Shimadzu IR 480 spectrometer. The 1H-NMR spectra recorded on Brucker 300 MHz (TMS) in DMSO. Mass Spectra recorded on Perkin Elmer Autolysis Excel GC.

EXPERIMENTAL:

Ten1-methyl-2-aminophenyl-4-(N⁴-alkyl/aryl piperazinyl) benzimidazole derivatives were synthesized and characterized. The details are as follows.

1) N-methyl -2-nitroaniline (1b): 78.5 g (0.5 mol) of 2-chloronitrobenzene and 193 ml (2.5 mol, 40% solution) of methylamine were mixed thoroughly.1 l Water was added to make the stirrable volume. The mixture was heated at 80⁰C at 5 kg pressure for 5 h. The product was obtained as low melting solid. Reaction mixture was extracted with 3 x 250 ml benzene. Benzene layer was then washed with 5% hydrochloric acid. Benzene was distilled off and the residue was collected. Yield: 65 g (85%), m.p.:126-128 ⁰C (by freezing), R_f: 0.82[benzene: pet ether (9:1)]

2) N¹-methyl-o-Phenylenediamine (1c): 25 g (0.16 mol) of N¹-methyl-2-nitro aniline was dissolved in 200 ml methanol. To the clear solution, 15 g of Raney Nickel W-2 catalyst (wet cake) was suspended. The reaction mixture was hydrogenated in 2 l Stainless steel bottle at pressure of 3 kg till the hydrogen uptake is complete (4-5h.). The completion of reaction was also checked by TLC. The catalyst was filtered on a hyflow bed and washed with 50 ml methanol. The solvent was distilled under diminished pressure. The residue was treated with methanolic hydrogen chloride (10 %) for 1 h, cooled and filtered the reaction mixture to give N¹-methyl-o-phenylenediamine dihydrochloride. Yield: 22 g (85%), m.p. 31-33 ⁰C (by freezing), R_f: 0.56 [benzene: pet ether (9:1)]

3) N¹-methyl-2-benzimidazolone (1d): A mixture of 25 g (0.20 mol) of N¹-methyl-o-phenylenediamine and 36 g (0.61 mol) of urea was heated on oil bath at 150 ºC for 1 h. After completion of the reaction, reaction mixture was poured in ice-cold water. The solid precipitated was filtered and washed with 2N hydrochloric acid and then with water. The desired product N¹-methyl-2-benzimidazolone obtained was then recrystallised with isopropyl alcohol to give pale yellow crystals. Yield: 27.3 g(90%), m.p. 176-178 ºC, R_f: 0.74[benzene: acetone (8:2)]

4) N¹-methyl-2-chloro benzimidazole (1e): 25 g (0.16 mol) of N¹-methyl-2-benzimidazolone and 200g (120 ml, 0.84 mol) of Phosphorous oxychloride was taken in RBF and refluxed for 12 h. The completion of reaction was monitored by TLC. After completion of the reaction, distilled off the excess Phosphorous oxychloride and then reaction mixture was poured in crushed ice with stirring. The reaction mixture was made alkaline using chilled aqueous 4 N sodium hydroxide solution and extracted with ethyl acetate. Extract was washed with cold water, dried over anhydrous sodium sulphate and solvent was distilled off under diminished pressure. The product was then recrystallised with n-hexane to give white crystals. Yield: 13.5 g (46%), m.p.107-109 ºC, Rf: 0.58[benzene: acetone (9:1)]

5) 1-phenyl-4- (4'-nitrophenyl) piperazine (2b): In a RBF, 2.17 g (0.015 mol) p-fluoro nitrobenzene, 2.5 g (0.015 mol) N-phenyl piperazine and freshly fused 2.0 g (0.015 mol) potassium carbonate were dissolved in 30 ml dioxane. The reaction mixture was then refluxed for about 4-5 h. Meanwhile the reaction was monitored by TLC. After completion of the reaction, reaction mixture was poured in water and the solid was filtered on suction. The desired product 1-phenyl-4- (4’-nitrophenyl) piperazine was obtained as yellow crystalline solid. Yield: 3.7 g (87%), m.p. 194-196 ⁰C, R_f: 0.56 [Chloroform: acetone (8:2)]

6) 1-phenyl-4- (4’-aminophenyl) piperazine (2c): 3.0 g (0.01 mol) of 1-phenyl-4- (4’nitrophenylpiperazine) was dissolved in 100 ml methanol. 1 g Raney Nickel (wet cake) was suspended in this clear solution. This was then shaken with hydrogen at 3 kg pressure till absorption of hydrogen was complete (4-5 h.). The completion of reaction was checked by TLC. After completion of reaction the catalyst was filtered on hyflow bed. The methanol was distilled off under diminished pressure. The residue was checked for purity on GC. The residue was then treated with methanolic hydrogen chloride (30 ml, 10%). The methanol was distilled off and the residue was triturated with methylene chloride. The desired product 1-phenyl-4- (4’-aminophenyl) piperazine was then filtered on suction and dried. The purity was checked by non-aqueous titration. Yield: 1.9 g(73%), m.p. 158-160 ⁰C, R_f: 0.52[chloroform: acetone (8:2)]

7)N¹-methyl-2-aminophenyl[4-N⁴-alkyl/aryl piperazinyl] benzimidazole (3a): In a RBF, 1 g (0.006 mol) of N¹-methyl-2-chloro benzimidazole and 1.8 g (0.007 mol) of 1-phenyl-4- (4’-aminophenyl) piperazine was mixed and heated on oil bath at 180 ºC for 24 h. Meanwhile, the completion of reaction was checked by TLC. The reaction mass was dumped in aqueous 2.5 N sodium hydroxide solution (50 ml), and the resulting precipitate of desired product N¹-methyl-2-aminophenyl [4-N⁴-alkyl/aryl piperazinyl] benzimidazole was filtered on suction and recrystallised from methanol to give white crystals. The purity was checked by non-aqueous titration. Yield: 1 g (67%), m.p.296-298ºC, Rf: 0.38[benzene: acetone (8:2)]

Adopting these procedures by scheme in Figure 1 we have synthesized 10 different compounds, their physical data is presented in table 1.

Figure 1: Scheme for the synthesis of 1-methyl-2-aminophenyl-4-(N⁴-alkyl/arylpiperazinyl) Benzimidazole derivatives

Antifungal Activity

All the synthesized compounds are screened for antifungal activity by tube dilution method (Turbidimetric method) against Trichophyton rubrum ATCC9029 and Candida albicans ATCC10231 using ketoconazole as reference standard. The stock solution of (1 mmol/ml) of compounds was prepared in DMSO and water. To each tube containing sterilized Sabouraud’s liquid medium (1 ml), 1 ml of drug solution was added. Each tube was inoculated with microorganism and was kept at 30⁰C for 48 h. The serial dilutions were made to obtain concentrations (in m mol/ml) such as 0.125, 0.0625 and 0.0314. Positive control and negative control also prepared. The readings were taken as inhibition of growth is seen and not seen.MIC was considered to be the lowest concentration of the test substance, exhibiting no visible growth of fungi in tubes. The observed MIC is presented in Table 2.

RESULT AND DISCUSSION:

All the observed facts in IR, ¹H-NMR, Mass spectral analysis are consistent with the structures.

N¹-methyl-2-chloro benzimidazole

Yield: 46 %; m.p.: 107-109⁰C; IR (KBr, cm^-1): 1610(C=N), 1232(C-N), 824(C-Cl), 2928(CH in CH₃), 3051(Ar-CH). ¹H-NMR (CDCl₃) δ: 7.06-7.69(m, 4H, C₄, C₅, C₆, C_7, Ar-H), 3.76(s, 3H, -CH₃).MS m/z (M+): 166(calculated for C₈H₄ClN₂; 166.23).

1-(3’, 4’-dichlorophenyl)-4-(4’’-aminophenyl) piperazine

Yield: 71 %; m.p.: 130-132⁰C; IR (KBr, cm^-1): 1246(C-N), 3060(Ar-CH), 549 (C-Cl). ¹H-NMR (CDCl₃) δ: 6.89-7.24(m, 7H, C₂^’, C’₅, C’₆, C”_2, C”₃, C”₅, C”₆, Ar-H), 3.23-3.46 (m, 8H, C₂,C₃,C₅,C₆,-N-CH₂-C), 2.16(s, 2H, -NH₂).MS m/z (M+): 166(calculated for C₈H₄ClN₂; 166.53).

1-methyl-2-aminophenyl-4-(N⁴-phenylpiperazinyl) benzimidazole

Yield: 67 %; m.p.: 296-298⁰C; IR (KBr, cm^-1): 2951(Ar-CH), 1224(C-N), 1440(C=N). 3399(N-H).¹H-NMR (CDCl₃) δ: 6.95-7.65(m, 13H, C₄,C₅,C₆,C_7,C₂^’, C₃^’_, C’₅, C’₆, C”_2, C”₃, C”₄,C”₅, C”₆, Ar-H), 3.44(s, 3H, -CH₃),3.29-3.36 (m, 8H, C”₂,C”₃,C”₅,C”₆,-N-CH₂-C), 3.28(s, 2H, -NH₂).MS m/z (M+): 383(calculated for C₂₄H₂₅N₅; 383.18).

1-methyl-2-aminophenyl-4-(N⁴-3’, 4’-dichlorophenyl piperazinyl) benzimidazole

Yield: 64 %; m.p.: 190-192⁰C; IR (KBr, cm^-1): 3308(Ar-CH), 2928(CH in CH₃), 1232(C-N), 1687(C=N), 567(C-Cl). 3317(N-H).¹H-NMR (CDCl₃) δ: 6.97-7.40(m, 11H, C₄,C₅,C₆,C_7,C₂^’, C₃^’_, C’₅, C’₆, C”_2, C”₅, C”₆, Ar-H), 3.61(s, 3H, -CH₃),3.28-3.49 (m, 8H, C’”₂,C’”₃,C’”₅,C’”₆,-N-CH₂-C), 3.24(s, 2H, -NH).MS m/z (M+): 451(calculated for C₂₄H₂₃Cl₂N₅; 451.61).

1-methyl-2-aminophenyl-4-(N⁴-2’-methoxyphenyl piperazinyl) benzimidazole

Yield: 66 %; m.p.: 186-188⁰C; IR (KBr, cm^-1): 3059(Ar-CH), 2928(CH in CH₃), 1410(C-O), 1240(C-N), 1509(C=N), 567(C-Cl). 3321(N-H).¹H-NMR (CDCl₃) δ: 6.96-7.33 m, 12H, C₄,C₅,C₆,C_7,C₂^’, C₃^’_, C’₅, C’₆, C”_3,C”_4, C”₅, C”₆, Ar-H), 3.88(s, 3H, -CH₃),3.49-3.78 (m, 8H, C’”₂,C’”₃,C’”₅,C’”₆,-N-CH₂-C), 3.31(s, 2H, -NH), 3.23 (s, 3H, -OCH₃).MS m/z (M+): 413(calculated for C₂₅H₂₇N₅O; 413.32).

CONCLUSION:

Present study highlights the importance of condensation products of the benzimidazole and piperazine heterocycles separated by amino phenyl bridge. This skeleton of structure is responsible for the antifungal property, and therefore, may serve as a lead molecule to obtain clinically useful antifungal agent in new era.

Table 1: Characterization data for 1-methyl-2-aminophenyl-4-(N⁴-alkyl/arylpiperazinyl) Benzimidazole derivatives

Sr.No.	R	m.p. (ºC)	Yield (%)	R_f^#	Assay*	Code
1.	N-phenylpiperazine	296-298	67	0.38	90	I.
2.	2-methylphenylpiperazine	246-248	74	0.36	88	II.
3.	4-methylphenylpiperazine	264-266	71	0.58	93	III.
4.	2-methoxyphenylpiperazine	186-188	66	0.52	89	IV.
5.	3-chlorophenylpiperazine	210-212	71	0.62	97	V.
6.	N-methylpiperazine	272-274	67	0.38	94	VI.
7.	Morpholine	212-214	65	0.46	95	VII.
8.	2,3-dichlorophenylpiperazine	168-170	75	0.66	92	VIII.
9.	3,4-dichlorophenylpiperazine	190-192	64	0.60	87	IX.
10.	N-2-hydroxyethylpiperazine	208-210	72	0.54	91	X.

_* Non-aqueous assay using standard 0.1N perchloric acid in anhydrous acetic acid.

^# TLC mobile phase: - benzene: acetone (8:2)

Most of the synthesized compounds exhibited antifungal activity against tested microorganisms. Compounds IV and X displayed moderate antifungal activity, whereas, the remaining compounds showed lesser activity. MIC range of the compound is 0.031 to 0.125 μg/ml. The antifungal activity exhibited by the compounds is due to the incorporation of the hydroxyl and methoxy group substitution on piperazine moiety.

REFERENCES:

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Received on 18.01.2012 Modified on 25.02.2012

Asian J. Research Chem. 5(3): March 2012; Page 325-328

In vitro activity – MIC in mg/ml
Compound	I.	II	III	IV	V.	VI	VII.	VI	IX.	X.	Standard
C. albicans	0.062	0.125	0.125	0.031	0.125	0.125	0.062	0.062	0.125	0.031	0.015
T.rubrum	0.125	0.062	0.125	0.062	0.125	0.125	0.125	0.062	0.125	0.062	0.015