Turk J Chem

30 (2006) , 223 – 228.

c TÜBİTAK

Synthesis and Antifungal Properties of Some

Benzimidazole Derivatives
Gülgün AYHAN KILCIGİL1∗, Nurten ALTANLAR2
1
Department of Pharmaceutical Chemistry, Pharmacy Faculty, Ankara University,
06100 Tandoğan, Ankara-TURKEY
e-mail: kilcigil@pharmacy.ankara.edu.tr
2
Department of Microbiology, Pharmacy Faculty, Ankara University,
06100 Tandoğan, Ankara-TURKEY

Received 13.05.2005

Some benzimidazole derivatives were synthesized and their in vitro antifungal activities were tested
against Candida albicans, Candida glabrata and Candida krusei. Compounds 6 and 9 possessed activity
comparable to fluconazole against C. albicans with a minimum inhibitory concentration of 12.5 µg/mL.

Key Words: Benzimidazole, thiourea, antifungal.

Introduction
Life-threatening infections caused by pathogenic fungi are becoming increasingly common, especially in
individuals with suppressed immune systems such as cancer chemotherapy or AIDS patients. However,
there are only a limited number of antifungal compounds available for such infections, which leads to a
strong need to develop new classes of compounds having antifungal activities. In particular, Candidiasis is
the fungal infection most frequently associated with HIV-positive patients, and Cryptococcosis, which is the
cause of morbidity and mortality in AIDS patients, is caused by Cryptococcus neoformans.
Compounds bearing benzimidazole moiety are reported to possess a number of interesting biological ac-
tivities such as antitubercular1 , anticancer2,3 , anthelmintic4 , antiallergic5,6, antioxidant7−9, antihistaminic10
and antimicrobial11−17. In addition, some thiourea derivatives have been reported as antimycobacterial18
and antimicrobial19. In our previous studies we reported the synthesis and antimicrobial20−23 activities of a
large series of benzimidazole derivatives. On the basis of these reports and as a continuation of our research
program on benzimidazole derivatives, we report here the synthesis of novel benzimidazole derivatives to
evaluate their antifungal properties.

∗ Corresponding author

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Synthesis and Antifungal Properties of..., G. AYHAN KILCIGİL, N. ALTANLAR

Experimental
Chemistry
Melting points were determined with an Electrothermal (Electrothermal Eng. Ltd., Essex, UK) melting
1
point apparatus and are uncorrected. H NMR spectra were measured with a Varian Mercury 400, 400
MHz instrument (California, USA) using TMS internal standard and DMSO-d6 . All chemical shifts were
reported as δ (ppm) values. ESMS were obtained with a Waters ZQ Micromass LC-MS spectrometer
(Milford, MA, USA) with positive electrospray ionization. Elemental analyses (C, H, N, S) were determined
on a Leco CHNS 932 instrument (St. Joseph, MI, USA), and were within ± 0.4% of the theoretical values.
All instrumental analyses were performed at the Scientific and Technical Research Council of Turkey and
Ankara University, Faculty of Pharmacy, Central Laboratory. The chemical reagents used in the synthesis
were purchased from E. Merck (Darmstadt, Germany) and Aldrich (Milwaukee, MI, USA). Compound 8
was used as a crude product for further reaction.

General procedure for the preparation of the compounds 5-7

The appropriate aldehyde derivative (1.5 mmol) was dissolved in 5 mL of EtOH. Then 0.160 g of Na2 S2 O5
in 5 mL of water was added in portions to the cooled ethanolic solution. The precipitate formed was filtered
off and dried. A total of 1.2 mmol of this precipitate and 1.2 mmol of compound 3 or 4 in 6 mL of DMF
were heated at 110 ◦ C for 5 h. At the end of this period the reaction mixture was cooled, and poured into
water. The precipitate was collected and recrystallized from ethanol-water.

5-Nitro-2-phenyl-1-propylbenzimidazol (5)

Anal. Calcd. For C16 H15 N3 O2 0.5H2 O: C, 66.19; H, 5.55; N, 14.47. Found: C, 65.97; H, 5.18; N, 14.49;
Yield % (56), ES (+) 282 (M+H); 1 H NMR (DMSO-d6 ) δ 0.73 (t, 3H, CH3 ), 1.66-1.71 (m, 2H, CH2 ), 4.37
(t, 2H, CH2 ), 7.62-7.65 (m, 3H, H-30 ,40 ,50 ), 7.81-7.98 ( m, 2H, H-20 ,60 ), 7.96 (d, 1H, Jo=8.8 Hz, H-7), 8.24
(dd, 1H, Jo=8.8 Hz, Jm= 2 Hz, H-6), 8.59 (d, 1H, Jm=2 Hz, H-4).

2-(p-Fluorophenyl)-5-nitro-1-propylbenzimidazol (6)

Anal. Calcd. For C16 H14 FN3 O2 : C, 64.20; H, 4.71; N, 14.03. Found: C, 63.93; H, 4.38; N, 14.25; Yield %
(42), ES (+) 300 (M+H); 1 H NMR (DMSO-d6 ) δ 0.73 (t, 3H, CH3 ), 1.65-1.71 (m, 2H, CH2 ), 4.35 (t, 2H,
CH2 ), 7.45-7.49 (m, 2H, H-30 ,50 ), 7.88-7.91 ( m, 2H, H-20 ,60 ), 7.96 (d, 1H, Jo=8.8 Hz, H-7), 8.22 (d, 1H,
Jo=8.8 Hz, H-6), 8.58 (s, 1H, H-4).

2-(p-Fluorophenyl 5-nitro-1-cyclopentylbenzimidazol (7)

Anal. Calcd. For C18 H16 FN3 O2 : C, 66.45; H, 4.95; N, 12.91. Found: C, 66.86; H, 5.24; N, 12.57; Yield %
(75), ES (+) 326 (M+H); 1 H NMR (DMSO-d6 ) δ 1.68-2.16 (m, 8H, CH2 ), 4.85-4.89 (m, 1H, CH), 7.45-7.49
(m, 2H, H-30 ,50 ), 7.78-.7.82 (m, 2H, H-20 ,60 ), 7.89 (d, 1H, Jo=9.2 Hz, H-7), 8.17 (dd, 1H, Jo=9.2 Hz, Jm=
2 Hz, H-6), 8.58 (d, 1H, Jm=1.6 Hz, H-4).

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 Synthesis and Antifungal Properties of..., G. AYHAN KILCIGİL, N. ALTANLAR

General procedure for the preparation of the compounds 9-10

5-Nitro benzimidazole derivatives 5-7 (1 mmol) in 10 mL of hot EtOH and 10 mL of 6 N HCl were refluxed
and SnCl2 .2H2 O was added in portions until the starting material was completely exhausted. The ethanol
was removed and the residue was made alkaline with KOH, extracted with EtOAc, and washed with water.
EtOAc was evaporated and the residue crystallized from EtOH.

5-Amino-2-(p-Fluorophenyl)-1-propylbenzimidazol (9)

Anal. Calcd. For C16 H16 FN3 : C, 71.35; H, 5.98; N, 15.60. Found: C, 70.95; H, 5.73; N, 15.36; Yield % (67),
M.p. 127-129 ◦ C; ES (+) 270 (M+H); 1 H NMR (DMSO-d6 ) δ 0.7 (t, 3H, CH3 ), 1.62-1.68 (m, 2H, CH2 ),
4.12 (t, 2H, CH2 ), 4.8 (s, 2H, NH2 ), 6.63 (d, 1H, Jo=8.4 Hz, H-6), 6.79 (s, 1H, H-4), 7.29 (d, 1H, Jo=8.4
Hz, H-7), 7.36-7.40 (m, 2H, H-20 ,60 ), 7.74-7.78 (m, 2H, H-30 ,50 ).

5-Amino-(2-(p-Fluorophenyl)-1-cyclopentylbenzimidazol (10)

Anal. Calcd. For C18 H18 FN3 .0.1 H2 O: C, 72.75; H, 6.77; N, 14.13. Found: C, 73.06; H, 6.42; N, 13.76; Yield
% (85), M.p. 196 ◦ C; ES (+) 296 (M+H); 1 H NMR (DMSO-d6 ) δ 1.63-2.15 (m, 8H, CH2 ), 4.68-4.77 (m,
1H, CH), 4.83 (s, 2H, NH2 ), 6.61 (d, 1H, Jo=8.8 Hz, H-6), 6.81 (s, 1H, H-4), 7.28 (d, 1H, Jo=8.8 Hz, H-7),
7.36-7.40 (m, 2H, H-30 ,50 ), 7.65-7.69 (m, 2H, H-20 ,60 ). 13 C NMR (DMSO-d6 ) δ 25.19, 30.45, 57.74, 103.39,
112.74, 116.22, 116.43, 125.99, 128.31, 132.16, 132.25, 144.83, 145.37, 152.46, 162.06, 164.52.

General procedure for the preparation of compounds 11-14

5-Amino benzimidazole derivatives (0.25 mmol) and appropriate phenylisothiocyanate (0.375 mmol) were
refluxed in 5 mL of absolute ethanol for 8 h. The reaction mixture was poured into water. The precipitate
was collected, washed with ether and recrystallized from ethanol.

N1 -(2-phenyl-1-propyl-benzimidazol-5-yl)-N3-phenylthiourea (11)

Anal. Calcd. For C23 H22 N4 S. 1.1 H2 O: C, 67.98; H, 6.00; N, 13.78; S, 7.89. Found: C, 67.52; H, 5.71; N,
13.41; S, 7.67; Yield % (26), M.p. 140 ◦ C; ES (+) 387 (M+H); 1 H NMR (DMSO-d6 ) δ 0.74 (t, 3H, CH3 ),
1.69-1.71 (m, 2H, CH2 ), 4.27 (t, 2H, CH2 ), 7.12-7.76 (m, 13H, Ar-H), 9.71 (s, 1H, NH), 9.85 (s, 1H, NH).

N 1 -[2-(p-Fluorophenyl)-1-propyl-benzimidazol-5-yl]-N 3 -(p-chlorophenyl)-thiourea (12)

Anal. Calcd. For C23 H20 ClFN4 S. 1.1 H2 O: C, 60.21; H, 4.87; N, 12.21; S, 6.98. Found: C, 60.01; H, 4.62;
N, 12.21; S, 6.88; Yield % (27), M.p. 136 ◦ C; ES (+) 439 (M+H); 1 H NMR (DMSO-d6 ) δ 0.72 (t, 3H,
CH3 ), 1.68-1.70 (m, 2H, CH2 ), 4.29 (t, 2H, CH2 ), 7.29-7.83 (m, 11H, Ar-H), 9.76 (s, 1H, NH), 9.94 (s,
1H, NH).13 C NMR (DMSO-d6 ) δ 11.58, 23.26, 46.41, 111.40, 115.63, 116.47, 116.69, 121.33, 126.06, 127.44,
128.80, 128.87, 132.15, 132.23, 134.03, 134.49, 139.38, 142.63, 153.30, 162.35, 164.80, 180.80.

N1 -[2-(p-Fluorophenyl)-1-cyclopentyl-benzimidazol-5-yl]-N3-phenylthiourea (13)

Anal. Calcd. For C25 H23 FN4 S: C, 69.74; H, 5.38; N, 13.01; S, 7.44. Found: C, 70.12; H, 5.13; N, 12.78; S,
7.43; Yield % (18), M.p. 138-139 ◦ C; ES (+) 431 (M+H); 1 H NMR (DMSO-d6 ) δ 1.68-2.13 (m, 8H, CH2 ),

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Synthesis and Antifungal Properties of..., G. AYHAN KILCIGİL, N. ALTANLAR

4.83 (m, 1H, CH), 7.27-7.75 (m, 12H, Ar-H), 9.75 (s, 1H, NH), 9.96 (s, 1H, NH).

N1 -[2-(p-Fluorophenyl)-1-cyclopentyl-benzimidazol-5-yl]-N3-(p-chlorophenyl)-thiourea (14)

Anal. Calcd. For C25 H22 ClFN4 S. 0.8 H2 O: C, 62.63; H, 4.96; N, 11.68; S, 6.68. Found: C, 62.58; H, 4.79;
N, 11.46; S, 6.29; Yield % (34), M.p. 138 ◦ C; ES (+) 465 (M+H); 1 H NMR (DMSO-d6 ) δ 1.68-2.19 (m, 8H,
CH2 ), 4.83 (m, 1H, CH), 7.34-7.79 (m, 11H, Ar-H), 9.78 (s, 1H, NH), 9.94 (s, 1H, NH).

Antifungal activity assay

The yeasts were maintained in Sabouraud Dextrose Broth (Difco) after incubation for 48 h at 25 ± 1 ◦ C.
Testing was performed in Sabouraud Dextrose Broth at pH 7.4 and the 2-fold dilution was applied. A set of
tubes containing only inoculated broth were kept as controls. After incubation for 48 h at 25 ± 1 ◦ C, the last
tube with no yeast growth was recorded to represent minimum inhibitory concentration (MIC), expressed
in µg/mL.

Results and Discussion

Compounds 1 and 2 were prepared from 1-chloro-2,4-dinitrobenzene by reaction with propyl/cyclopentylamine
in DMF according to the literature24 . The 2-nitro group of compounds 1 and 2 was reduced to 2-amino
(3 and 4) by using Na2 S/NaHCO3 in methanol24 . Condensation of o-phenylenediamines (3 and 4) with
the Na2 S2 O5 adduct of appropriate benzaldehydes in DMF25 gave 5-7. Reduction of compounds 5-7 with
SnCl2 .2H2 O produced 8-10. Thiourea compounds 11-14 were obtained with the reaction of compounds
8-10 with appropriate phenylisothiocyanates in absolute ethanol (Scheme).
The in vitro antifungal activity of the compounds was tested by the tube dilution technique26 .
Each of the test compounds and standards miconazole and fluconazole were dissolved in 12.5% DMSO,
at concentrations of 100 µg/mL. Further dilutions of the compounds and standards in the test medium were
prepared at the required quantities of 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78 µg/mL concentrations. The final
inoculum size was 105 CFU/mL. The MICs were defined as the lowest concentrations of the compounds that
prevented visible growth. It was determined that the solvent had no antifungal activity against any of the
test microorganisms.
All the compounds were tested for their in vitro growth inhibitory activity against C. albicans ATCC
10231, patient isolate C. glabrata and C. krusei ATCC 6258 (Table).
Compounds 6 and 9 possessed comparable activity to fluconazole against C. albicans with a MIC of
12.5 µg/mL. Compounds 6, 9 and 12 were more effective against C. krusei (6.25 µg/mL) compared with the
other derivatives. However, none of the compounds was superior to the standards used against any fungi.
Generally, the thiourea compounds (12-14) were less active than their amine counterparts (9 and 10), with
the exception of compound 12 against C. krusei.

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 Synthesis and Antifungal Properties of..., G. AYHAN KILCIGİL, N. ALTANLAR

Cl NHR1 NHR1 R2
Na2S/NaHCO3
+ R1NH2
O2N NO2 O2N NO2 O 2N NH 2
1-2 3-4
R1= -C3H7 1 3
CH OH
R1= 2 4 SO3Na

R1
R1
N
N SnCl2 R2
R2
O 2N N
H 2N N
8-10 R1 R 5-7
2
-C3H7 H
X N=C=S
-C3H7 F

F
R1
X N
S R2
N N N

H H
R1 R2 X

-C3H7 H H 11

-C3H7 F Cl 12

F H 13

F Cl 14

Scheme. Synthetic route of the compounds.

Table. The in vitro antifungal activity of the synthesized compounds (MIC, µg/mL).

Compound C. albicans C. glabrata C. krusei

5 25 25 12.5
6 12.5 6.25 6.25
9 12.5 12.5 6.25
10 25 25 12.5
11 25 25 12.5
12 25 25 6.25
13 25 25 25
14 50 25 25
Fluconazole 12.5 3.125 3.125
Miconazole 6.25 3.125 1.56

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Synthesis and Antifungal Properties of..., G. AYHAN KILCIGİL, N. ALTANLAR

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