Antimicrobial activity of a new series of benzimidazole derivatives

Article abstract of DOI:10.1007/s12272-011-0903-8

Due to antimicrobial importance of benzimidazoles and hydrazones, some benzimidazolehydrazone compounds were synthesized to screen their antimicrobial activity. Structures of the synthesized compounds were elucidated by ¹H-NMR, IR and ES-MS spe

Full text of DOI:10.1007/s12272-011-0903-8

Arch Pharm Res Vol 34, No 9, 1427-1435, 2011
DOI 10.1007/s12272-011-0903-8
Antimicrobial Activity of a New Series of Benzimidazole Derivatives
·
Yusuf Özkay1, Ya˘gmur Tunal 2
l
, Hülya Karaca2, and Ilhan Is
l
kdag˘1
¸
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470 Eskisehir, Turkey and
¸
2Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Anadolu University, 26470 Eskisehir, Turkey
¸
(Received February 24, 2011/Revised May 2, 2011/Accepted May 2, 2011)
Due to antimicrobial importance of benzimidazoles and hydrazones, some benzimidazole-
hydrazone compounds were synthesized to screen their antimicrobial activity. Structures of
the synthesized compounds were elucidated by 1H-NMR, IR and ES-MS spectral data and ele-
mental analysis. The synthesized benzimidazole-hydrazones exhibited very weak antibacterial
activity. However, antifungal activity of some of the synthesized compounds was very notable
against Candida species. The compounds displaying important antifungal activity were
screened for their toxicity. Artemia salina 96-well assay was used to determine cytotoxicity of
the compounds. Tested compounds exhibited toxicity to different extents (LD50 = 126.33-
368.72 µg/mL). Nevertheless, determination of 3-14 folds higher LD50 than minimum inhibi-
tory concentration is a significant finding, which demonstrates that the compounds display
antifungal activity at non-toxic concentration.
Key words: Benzimidazole, Hydrazone, Antibacterial, Antifungal, Artemia salina
thousands of benzimidazole analogs have been syn-
thesized and screened for their antimicrobial activity
INTRODUCTION
In the past 25 years, the incidence of microbial infec- up-to-date (Podunavac-Kuzmanovic et al., 2008). The
tions has increased on alarming levels over the world cause of a special notice on benzimidazole compounds
as a result of antimicrobial resistance (Bayrak et al., has been cyanocobalamine (vitamin B12) which bears
2009) and possible microbial implications for morbidity, 5,6-dimethylbenzimidazole fragment and is capable of
mortality and health care costs have become a serious inducing the growth of bacteria. Nevertheless, the
fear (Cosgrove, 2006). In order to overcome these benzimidazole shows structural similarity to purine
challenges, new agents should preferably consist of and hence; antibacterial capability of benzimidazoles
chemical characteristics that clearly differ from those is explained by their competition with purines result-
of existing agents. Thus, there is a pressing need for ing in inhibition of the synthesis of bacterial nucleic
development of novel antimicrobial agents. In drug acids and proteins (Spasov et al., 1999; Arjmand et al.,
design and development field, a critical part of the 2005). In addition to their antibacterial activity, benzi-
search for novel drugs is the synthesis of compounds midazoles can be classified as one of the most impor-
that are new yet resemble known biologically active tant groups of fungicides with systemic activity and
molecules by virtue of the presence of essential struc- are well-known for their pronounced ability to control
tural features (Silverman, 1992; Cunha, 1998; Levy, a large number of fungal diseases. Thiabendazole, ben-
1998; Lipsitch, 2001).
omyl, carbendazim, chlorfenazole, cypendazole, debacarb,
Benzimidazoles fit above requirement well since they fuberidazole, mecarbinzid and rabenzazole are some
have shown a chemotherapeutic importance. Several main examples of this fungicide class (Kaplancikli et
al., 2004; Ozdemir et al., 2010). In this group, the well-
known fungicide thiabendazole inhibits fungal micro-
tubular function, resulting in non-disjunction of chro-
mosomes at cell division (Allen and Gootlied, 1970;
Watanabe-Akanuma et al., 2005).
Correspondence to: Yusuf Özkay, Department of Pharmaceuti-
cal Chemistry, Faculty of Pharmacy, Anadolu University, 26470
Eskisehir, Turkey
¸
Tel: 90-222-335-0580, 3779, Fax: 90-222-335-0750
E-mail: yozkay@anadolu.edu.tr
As well as benzimidazoles, hydrazide-hydrazones
1427

1428
Y. Özkay et al.
are the other pharmacophores which have been claim-
ed to exhibit appreciable antibacterial and antifungal
Synthesis of the starting materials (1 and 2)
Methyl 4-(1H-benzimidazol-2-yl-iminomethyl) ben-
activity. Furacilin, furazolidone, ftivazide and nifuro- zoate (1) and its reduction product methyl 4-(1H-ben-
xazide are the well-known antimicrobial agents, which zimidazol-2-yl-aminomethyl) benzoate (2) were synthe-
contain hydrazide-hydrazones moieties (Chornous et al., sized in accordance with the previous method (Nawrocka
2001). Due to their chemotherapeutic potency, attention et al., 2004), in which reaction of 2-aminobenzimidazole
through the hydrazide-hydrazones has increased and with various benzaldehyde derivatives and reduction
lots of studies related with their antimicrobial activity of the obtained products by NaBH4 were described.
have been reported in recent years (Papakonstantinou-
Garoufalias et al., 2002; Rollas et al., 2002; Vicini et
al., 2002; Loncle et al., 2004; Masunari and Tavares,
2007; Salgin-Goksen et al., 2007; Joshi et al., 2008;
4-(1H-Benzimidazol-2-yl-aminomethyl) benzo-
hydrazide (3)
Methyl 4-(1H-benzimidazol-2-yl-aminomethyl) ben-
Mostafa et al., 2008; Kumar et al., 2009; Ozdemir et al., zoate (
2) (10 g, 35.58 mmol) was dissolved in 50 mL of
2009).
EtOH and 15 mL of 80% hydrazine hydrate was added.
In the light of above observations, we have recently Reaction mixture was refluxed for 12 h. Solvent and
performed a study (Ozkay et al., 2010) including the excess of hydrazine hydrate was evaporated and the
synthesis of some benzimidazole-hydrazone deriva- residue was washed with water, filtered, dried, and
tives and evaluation of a relationship between their then crystallized from EtOH. Yield 73%; M. p. 220-222
structures and antimicrobial activity. Results of such oC; IR [
ν
, cm−1, KBr]: 3416-3279 (N-H), 1678 (C=O),
investigation have encouraged us to make some struc- 1603-1457 (C=C and C=N).
tural modifications to improve biological activity. Hence,
we have designed some synthesis strategies. One of
these strategies includes the synthesis of a compact
system bearing 2-aminobenzimidazole and hydrazone
moieties. The reason for this choice can be explained
General synthesis procedure for 4-(1H-benzim-
idazol-2-yl-aminomethyl)-N'-(4-substituted benzy-
lidene) benzohydrazides (4a-4n)
Equimolar quantities of 4-(1H-benzimidazol-2-yl-
by the pharmacological importance of 2-aminobenzi- aminomethyl) benzohydrazide (
3) (0.562 g, 2 mmol)
midazole, which has approximately 30 derivatives re- and appropriate 4-substituted benzaldehyde (2 mmol)
gistered in the world as drugs exhibiting diverse phar- in 25 mL of butanol were refluxed for 3 h with the
macological activities such as antiparasitic, antifun- presence of catalytic amount of glacial AcOH. Reac-
gal, and antiviral (Nawrocka et al., 2004).
tion mixture was allowed to cool down and then result-
As a result, in connection with our recent study, we ing solid was filtered and recrystallized from EtOH to
synthesized a new class of compounds bearing 2- give the title products (4a
-4n).
aminobenzimidazole and hydrazone moieties in order
to develop new antimicrobial agents.
4-(1H-Benzimidazol-2-yl-aminomethyl)-N'-(benzy-
lidene) benzohydrazide (4a)
Yield 67%; M. p. 157-158oC; IR [ , cm−1, KBr]: 3394-3285
ν
MATERIALS AND METHODS
Chemistry
1
(N-H), 1684 (C=O), 1610-1453 (C=N and C=C); H-
NMR (500 MHz, DMSO-d6 ppm): 4.39 (2H, d, J =
,
δ
All reagents were used as purchased from commer- 4.52 Hz, NH-CH2), 7.06-7.52 (10H, m, NH-CH2 and
cial suppliers (Merck, Acros or Sigma-Aldrich) without Ar-H), 7.82-7.94 (4H, m, Ar-H), 8.39 (1H, s, N=CH), 11.73
further purification. Melting points (M.p.) were deter- (1H, s, CO-NH), 12.33 (1H, br, N-H, benzimidazole);
mined by using an Electrothermal 9100 digital melt- ES-MS (m/z): 370.5 [M+1, 100%]. Anal. calcd. for
ing point apparatus and were uncorrected. During the C22H19N5O: C, 71.53; H, 5.18; N, 18.96. Found: C, 71.69;
synthesis, the compounds were routinely checked for H, 5.15; N, 18.88.
purity by TLC on silica gel 60. IR spectra were record-
ed on a Shimadzu, 8400 FTIR spectrometer as KBr 4-(1H-Benzimidazol-2-yl-aminomethyl)-N'-[4-(di-
1
pellets. H-NMR spectra were recorded on a Bruker methylamino)benzylidene] benzohydrazide (4b)
UltraShield 500 MHz spectrometer in DMSO-d6. MS Yield 73%; M. p. 190oC (dec.); IR [ , cm−1, KBr]: 3383-
ν
data were obtained on an Agilent 1100 Series LC/ 3281 (N-H), 1677 (C=O), 1607-1451 (C=N and C=C);
MSD Trap VL&SL spectrometer. Elemental analyses 1H-NMR (500 MHz, DMSO-d6
ppm): 13.02 (6H, s,
(C, H and N) were determined on a Perkin Elmer N(CH3)2), 4.37 (2H, d, = 4.59 Hz, NH-CH2), 6.84-7.51
,
δ
J
analyser.
(9H, m, NH-CH2 and Ar-H), 7.79-7.91 (4H, m, Ar-H),
8.34 (1H, s, N=CH), 11.71 (1H, s, CO-NH), 12.39 (1H,

Novel Antimicrobial Benzimidazoles
1429
br, N-H, benzimidazole); ES-MS (m/z): 413.7 [M+1, 4-(1H-Benzimidazol-2-yl-aminomethyl)-N'-[4-
100%]. Anal. calcd. for C24H24N6O: C, 69.88; H, 5.86; (ethoxy)benzylidene] benzohydrazide (4g)
N, 20.37. Found: C, 69.78; H, 5.88; N, 20.31.
Yield 63%; M. p. 231oC; IR [ , cm−1, KBr]: 3401-3296
ν
(N-H), 1683 (C=O), 1607-1453 (C=N and C=C); 1H-NMR
4-(1H-Benzimidazol-2-yl-aminomethyl)-N'-[4-(dieth- (500 MHz, DMSO-d6 ppm): 1.17 (3H, t, = 7.22 Hz,
ylamino)benzylidene] benzohydrazide (4c) OCH2CH3), 4.02 (2H, q, = 7.26 Hz, OCH2CH3), 4.37
Yield 64%; M. p. 262-263oC; IR [ , cm−1, KBr]: 3391- (2H, d,
= 4.38 Hz, NH-CH2), 7.01-7.52 (9H, m, NH-
3287 (N-H), 1682 (C=O), 1608-1456 (C=N and C=C); CH2 and Ar-H), 7.82-7.92 (4H, m, Ar-H), 8.35 (1H, s,
1H-NMR (500 MHz, DMSO-d6
ppm): 1.12 (6H, t, N=CH), 11.73 (1H, s, CO-NH), 12.34 (1H, br, N-H, ben-
7.04 Hz, N(CH2-CH3)2), 3.43 (4H, q, = 7.11 Hz, N(CH2- zimidazole); ES-MS (m/z): 414.5 [M+1, 100%]. Anal.
CH3)2), 4.39 (2H, d, = 4.46 Hz, NH-CH2), 6.77-7.51 calcd. for C24H23N5O2: C, 69.72; H, 5.61; N, 16.94.
,
δ
J
J
ν
J
,
δ
J =
J
J
(9H, m, NH-CH2 and Ar-H), 7.81-7.93 (4H, m, Ar-H), Found: C, 69.46; H, 5.63; N, 16.96.
8.38 (1H, s, N=CH), 11.74 (1H, s, CO-NH), 12.36 (1H,
br, N-H, benzimidazole); ES-MS (m/z): 441.6 [M+1, 4-(1H-Benzimidazol-2-yl-aminomethyl)-N'-[4-
100%]. Anal. calcd. for C26H28N6O: C, 70.89; H, 6.41; (chloro)benzylidene] benzohydrazide (4h)
N, 19.08. Found: C, 70.37; H, 6.44; N, 19.06.
Yield 69%; M. p. 214-215oC; IR [ , cm−1, KBr]: 3397-3278
ν
1
(N-H), 1687 (C=O), 1608-1453 (C=N and C=C); H-
NMR (500 MHz, DMSO-d6 ppm): 4.37 (2H, d,
4.64 Hz, NH-CH2), 7.04-7.51 (9H, m, NH-CH2 and Ar-
4-(1H-Benzimidazol-2-yl-aminomethyl)-N'-[4-(hy-
droxy)benzylidene] benzohydrazide (4d)
,
δ
J =
Yield 76%; M. p. 243oC (dec.); IR [ , cm−1, KBr]: 3528 H), 7.79-7.91 (4H, m, Ar-H), 8.33 (1H, s, N=CH), 11.75
ν
(O-H), 3402-3278 (N-H), 1686 (C=O), 1605-1457 (C=N (1H, s, CO-NH), 12.32 (1H, br, N-H, benzimidazole);
and C=C); 1H-NMR (500 MHz, DMSO-d6
ppm): 4.40 ES-MS (m/z): 404.9 [M+1, 100%], 405.9 [M+2, 31%],
(2H, d, = 4.57 Hz, NH-CH2), 6.94-7.54 (9H, m, NH- 406.9 [M+3, 14%]. Anal. calcd. for C22H18ClN5O: C,
,
δ
J
CH2 and Ar-H), 7.83-7.93 (4H, m, Ar-H), 8.39 (1H, s, 65.43; H, 4.49; N, 17.34. Found: C, 65.22; H, 4.50; N,
N=CH), 9.74 (1H, s, O-H), 11.73 (1H, s, CO-NH), 12.38 17.33.
(1H, br, N-H, benzimidazole); ES-MS (m/z): 386.6
[M+1, 100%]. Anal. calcd. for C22H19N5O2: C, 68.56; H, 4-(1H-Benzimidazol-2-yl-aminomethyl)-N'-[4-
4.97; N, 18.17. Found: C, 68.86; H, 4.97; N, 18.21.
(bromo)benzylidene] benzohydrazide (4i)
Yield 71%; M. p. 202oC; IR [ , cm−1, KBr]: 3409-3286
(N-H), 1684 (C=O), 1605-1452 (C=N and C=C); 1H-NMR
ν
4-(1H-Benzimidazol-2-yl-aminomethyl)-N'-[4-
(methyl)benzylidene] benzohydrazide (4e)
(500 MHz, DMSO-d6, δ ppm): 4.35 (2H, d, J = 4.41 Hz,
Yield 74%; M. p. 226oC; IR [
(N-H), 1679 (C=O), 1601-1460 (C=N and C=C); H- 7.93 (4H, m, Ar-H), 8.34 (1H, s, N=CH), 11.76 (1H, s,
ν
, cm−1, KBr]: 3384-3279 NH-CH2), 7.03-7.50 (9H, m, NH-CH2 and Ar-H), 7.81-
1
NMR (500 MHz, DMSO-d6
,
δ
ppm): 2.29 (3H, s, CH3), CO-NH), 12.38 (1H, br, N-H, benzimidazole); ES-MS
m/z): 448.5 [M+1, 100%], 449.5 [M+2, 28%], 450.6
4.37 (2H, d, = 4.63 Hz, NH-CH2), 7.04-7.52 (9H, m,
J
(
NH-CH2 and Ar-H), 7.83-7.91 (4H, m, Ar-H), 8.38 (1H, [M+3, 12%]. Anal. calcd. for C22H18BrN5O: C, 58.94; H,
s, N=CH), 11.71 (1H, s, CO-NH), 12.36 (1H, br, N-H, 4.05; N, 15.62. Found: C, 59.11; H, 4.04; N, 15.67.
benzimidazole); ES-MS (m/z): 384.5 [M+1, 100%].
Anal. calcd. for C23H21N5O: C, 69.16; H, 5.30; N, 17.53. 4-(1H-Benzimidazol-2-yl-aminomethyl)-N'-[4-
Found: C, 69.40; H, 5.29; N, 17.45.
(fluoro)benzylidene] benzohydrazide (4j)
Yield 63%; M. p. 221-223oC; IR [ , cm−1, KBr]: 3394-
4-(1H-Benzimidazol-2-yl-aminomethyl)-N'-[4- 3281 (N-H), 1676 (C=O), 1609-1455 (C=N and C=C);
(methoxy)benzylidene] benzohydrazide (4f) ppm): 4.37 (2H, d,
1H-NMR (500 MHz, DMSO-d6
Yield 59%; M. p. 310-311oC; IR [ , cm−1, KBr]: 3389- 4.57 Hz, NH-CH2), 7.05-7.51 (9H, m, NH-CH2 and Ar-
3282 (N-H), 1681 (C=O), 1612-1455 (C=N and C=C); H), 7.84-7.93 (4H, m, Ar-H), 8.36 (1H, s, N=CH), 11.72
1H-NMR (500 MHz, DMSO-d6
ppm): 3.85 (3H, s, (1H, s, CO-NH), 12.40 (1H, br, N-H, benzimidazole);
OCH3), 4.36 (2H, d, = 4.55 Hz, NH-CH2), 6.99-7.51 ES-MS (m/z): 388.5 [M+1, 100%]. Anal. calcd. for
ν
,
δ
J =
ν
,
δ
J
(9H, m, NH-CH2 and Ar-H), 7.84-7.93 (4H, m, Ar-H), C22H18FN5O: C, 68.21; H, 4.68; N, 18.08. Found: C,
8.34 (1H, s, N=CH), 11.73 (1H, s, CO-NH), 12.42 (1H, 68.04; H, 4.69; N, 18.13.
br, N-H, benzimidazole); ES-MS (m/z): 400.4 [M+1,
100%]. Anal. calcd. for C23H21N5O2: C, 72.04; H, 5.52; 4-(1H-Benzimidazol-2-yl-aminomethyl)-N'-[4-(ni-
N, 18.26. Found: C, 72.16; H, 5.53; N, 18.29.
tro)benzylidene] benzohydrazide (4k)
Yield 82%; M. p. 254-256oC; IR [ , cm−1, KBr]: 3396-
ν

1430
Y. Özkay et al.
3282 (N-H), 1683 (C=O), 1613-1458 (C=N and C=C); 1H- Turkey), as gram-negative bacteria; Klebsiella pneu-
NMR (500 MHz, DMSO-d6 ppm): 4.36 (2H, d, moniae ATCC 13883, Escherichia coli ATCC 35218, E.
,
δ
J =
4.38 Hz, NH-CH2), 7.09-7.51 (7H, m, NH-CH2 and Ar- coli ATCC 25922, Salmonella thyphimurium NRRL
H), 7.91-8.06 (4H, m, Ar-H), 8.33-8.37 (3H, m, N=CH B-4420 and Proteus vulgaris NRLL B-123 and yeast
and Ar-H), 11.74 (1H, s, CO-NH), 12.37 (1H, br, N-H, as Candida albicans, Candida tropicalis and Candida
benzimidazole); ES-MS (m/z): 415.6 [M+1, 100%]. Anal. globrata ATCC 36583 (obtained from Faculty of Medi-
calcd. for C22H18N6O3: C, 63.76; H, 4.38; N, 20.28. cine Osmangazi University, Eskisehir, Turkey). Chlor-
Found: C, 63.85; H, 4.36; N, 20.23.
amphenicol and ketoconazole were used as control
drugs.
4-(1H-Benzimidazol-2-yl-aminomethyl)-N'-[4-(tri-
fluoro)benzylidene] benzohydrazide (4l)
Some of the synthesized compounds were tested for
their toxicity by applying Brine-Shrimp lethality
Yield 78%; M. p. 304oC; IR [
ν
, cm−1, KBr]: 3398-3285 assay. Fresh eggs of Brine-Shrimp
(
Artemia salina),
(N-H), 1691 (C=O), 1602-1451 (C=N and C=C); 1H-NMR sold as a fish food, were purchased from the local pet
(500 MHz, DMSO-d6 ppm): 4.38 (2H, d, = 4.43 Hz, shop, Eskisehir/Turkey.
NH-CH2), 7.08-7.53 (7H, m, NH-CH2 and Ar-H), 7.91-
8.02 (4H, m, Ar-H), 8.23 (2H, d, = 8.23 Hz, Ar-H),
8.34 (1H, s, -N=CH-), 11.73 (1H, s, CO-NH), 12.43 (1H,
,
δ
J
J
Antimicrobial activity assay
Antimicrobial activity test was performed according
br, N-H, benzimidazole); ES-MS (m/z): 438.6 [M+1, to CLSI reference M7-A7 and M100-S16 broth micro-
100%]. Anal. calcd. for C23H18F3N5O: C, 63.15; H, 4.15; dilution methods as described in our previous study
N, 16.01. Found: C, 62.96; H, 4.17; N, 15.96.
(Ozkay et al., 2010). Twice minimum inhibitory con-
centration (MIC) readings were carried out for each
chemical agent. The compounds were dissolved in
DMSO for antibacterial and antimycotic assays. Fur-
4-(1H-Benzimidazol-2-yl-aminomethyl)-N'-[4-
(cyano)benzylidene] benzohydrazide (4m)
Yield 81%; M. p. 198oC; IR [ , cm−1, KBr]: 3404-3283 ther dilutions of the compounds and standard drugs in
ν
(N-H), 1677 (C=O), 1606-1454 (C=N and C=C); 1H-NMR test medium were prepared at the required quantities
(500 MHz, DMSO-d6 ppm): 4.37 (2H, d, = 4.42 Hz, of 800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625
NH-CH2), 7.08-7.51 (7H, m, NH-CH2 and Ar-H), 7.92- g/mL concentrations with Mueller-Hinton broth and
8.05 (4H, m, Ar-H), 8.28 (2H, d, = 8.56 Hz, Ar-H), Sabouroud dextrose broth. In order to ensure that the
,
δ
J
µ
J
8.34 (1H, s, -N=CH-), 11.73 (1H, s, CO-NH), 12.39 (1H, solvent per se had no effect on bacteria or yeast growth,
br, N-H, benzimidazole); ES-MS (m/z): 395.5 [M+1, a control test was also performed containing inoculat-
100%]. Anal. calcd. for C23H18N6O: C, 70.04; H, 4.60; ed broth supplemented with only DMSO at the same
N, 21.31. Found: C, 69.83; H, 4.59; N, 21.33.
dilutions used in our experiments and found inactive
in culture medium.
4-(1H-Benzimidazol-2-yl-aminomethyl)-N'-[4-
(carboxy)benzylidene] benzohydrazide (4n)
Brine-Shrimp lethality assay
Artemia salina 96-well assay has been used to deter-
Yield 77%; M. p. 311oC; IR [
ν
, cm−1, KBr]: 3468 (O-H),
3399-3287 (N-H), 1681 (C=O), 1609-1451 (C=N and mine the cytotoxicity levels of the compounds. Tested
C=C); 1H-NMR (500 MHz, DMSO-d6
ppm): 4.36 (2H, compounds were prepared within the range of 1.95-
d, = 4.54 Hz, NH-CH2), 7.08-7.52 (7H, m, NH-CH2 1000 g/mL, by dissolving in DMSO. The LD50 values
and Ar-H), 7.91-8.04 (4H, m, Ar-H), 8.19 (2H, d, and 95% confidence intervals of the compounds were
,
δ
J
µ
J
=
8.53 Hz, Ar-H), 8.35 (1H, s, -N=CH-), 11.71 (1H, s, CO- calculated with the LC50 computer program (Trimmed
NH), 12.40 (1H, br, N-H, benzimidazole); ES-MS (m/ Spearman-Karber Method, Version 1.5) (Calleja and
z): 414.5 [M+1, 100%]. Anal. calcd. for C23H19N5O3 Persoone, 1992; Choudhary and Thomsen, 2001). The
(413.44): C, 66.82; H, 4.63; N, 16.94. Found: C, 66.87; experimental procedure details were explained in our
H, 4.64; N, 16.88.
recent study (Ozkay et al., 2010).
Microbiology
RESULTS AND DISCUSSION
Final products were tested for their in vitro growth
inhibitory activity against human pathogenic as
gram-positive bacteria; Staphylococcus aureus ATCC
Chemistry
In the present work, 14 novel benzimidazole-hydra-
25923, Enterococcus faecalis ATCC 29212, Bacillus zone compounds were synthesized. In the first step,
subtilis and Listeria monocytogenes (obtained from methyl 4-(1H-benzimidazol-2-yl-iminomethyl) benzoate
Faculty of Pharmacy Anadolu University, Eskisehir,
(1) was prepared by reacting 2-aminobenzimidazole

Novel Antimicrobial Benzimidazoles
1431
with methyl 4-formylbenzoate in the mixture of abso- observed at 6.84-7.52 ppm as multiplet. The aromatic
lute ethanol/benzene (5:1), a few drops of glacial acetic protons belonging to 2nd and 6th positions of both benzoic
acid used as a catalyst. Secondly, reduction of 1 with a acid hydrazide and benzylidene fragments gave multi-
chemoselective reducing agent NaBH4 in isopropanol plet at 7.79-8.06 ppm in all of the compounds. In the
gave the methyl 4-(1H-benzimidazol-2-yl-aminomethyl) compounds 4k
benzoate ( ). In the third reaction step, compound benzylidene fragment appeared at 8.19-8.33 ppm as a
-
4n, protons of 3th and 5th positions of
2
2
was treated with excess of hydrazine hydrate (80%) in doublet. CH=N, CO-NH and N-H protons of benzimi-
absolute ethanol to obtain 4-(1H-benzimidazol-2-yl- dazole appeared at 8.33-8.39 ppm, 11.71-11.76 ppm
aminomethyl) benzohydrazide (3). Finally, reaction of and 12.32-12.43 ppm, respectively. All the other aro-
3
and corresponding benzaldehyde derivatives in matic and aliphatic protons were observed at the ex-
butanol with the presence of catalytic amounts of pected chemical shifts. All compounds gave satisfactory
glacial acetic acid gave the 4-(1H-benzimidazol-2-yl- elemental analysis results. Mass spectra (ES-MS) of
aminomethyl)-N'-(4-substituted benzylidene) benzohy- the compounds showed M+1 peaks, in agreement with
drazides (4a
is given in Scheme 1.
Formulas of the compounds (4a
by elemental analyses and their structures were
elucidated by IR, H-NMR and ES-MS spectral data. of a compound that inhibits the growth of tested micro-
In the IR spectra N-H, C=O, C=N and C=C functions organisms. As shown in Table I, all of the compounds
absorbed strongly in the expected regions: N-H at were inactive against bacterial strains. However, most
3404-3278 cm−1, C=O at 1691-1676 cm−1, C=N and C=C of the synthesized compounds indicated moderate
at 1613-1451 cm−1, respectively. The 1H-NMR spectra antibacterial activity against E. coli when compared
showed methylene protons of methylamine (NH-CH2) with the positive control chloramphenicol.
-4n). Synthetic pathway of the compounds their molecular formula.
-4n) were confirmed
Microbiology
MICs were recorded as the minimum concentration
1
at 4.35-4.40 ppm, as doublet, in all of the compounds.
In the compounds 4a 4j, the amine protons of methyl- the synthesized compounds showed significant anti-
amine and the aromatic protons belonging to benzimi- fungal activity against Candida yeasts (Table II). In
dazole, 3th and 5th positions of benzoic acid hydrazide the series, the compounds 4a
4k 4l 4m, and 4n
and 3th and 5th positions of benzylidene fragments were seemed to be more potent to inhibit each of the C.
Contrary to their very poor antibacterial activity,
-
,
,
,
Scheme 1. Synthetic pathway for the 4-(1H-benzimidazol-2-yl-aminomethyl)-N'-(4-substituted benzylidene) benzohydrazides

1432
Y. Özkay et al.
Table I. MIC values (
µ
g/mL) of the compounds 4a-4n against bacterial strains
Bacterial strains
Compounds
A
B
C
D
E
F
G
H
I
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
25
25
25
25
25
25
25
25
25
50
25
25
25
25
12.5
400
400
400
400
400
400
400
400
400
400
400
400
400
400
12.5
400
400
400
400
400
400
100
200
100
200
400
200
200
100
50
400
400
400
400
400
400
400
400
400
400
400
400
400
400
12.5
200
400
400
200
200
200
100
400
200
400
100
400
400
200
12.5
400
400
400
400
400
400
400
400
400
400
400
400
400
400
50
100
50
200
200
400
200
200
100
200
100
400
400
400
200
100
400
12.5
400
400
400
400
400
400
400
400
400
400
400
400
400
400
12.5
100
100
100
100
100
50
100
100
50
100
100
100
12.5
Chloramphenicol
A: E. coli 35218, B: E. coli 25922, C: Proteus vulgaris, D: Salmonella thyphimurium, E: Klebsiella pneumoniae, F:
Listeria monocytogenes, G: Staphylococcus aureus H: Enterococcus faecalis, I: Bacillus subtilis
,
albicans, C. globrata and C. tropicalis. Nitro substi- C. albicans and equal antifungal activity to reference
tuted 4k and cyano substituted 4 m showed two-fold against C. globrata. Antifungal activity of 4a was at
better potency (MIC = 25
ketoconazole (MIC = 50
fungal strains. Besides, antifungal activity of the 4a
µ
g/mL) than reference drug same degree with the reference against C. albicans
g/mL) against all of these and C. globrata. The other compounds (4b 4j) displayed
moderate to equal antifungal activity to ketoconazole.
Achieving an important pharmacological activity is
µ
-
,
4l and 4n was greater than that of the reference
against C. tropicalis. The compounds 4l and 4n also not adequate alone to develop new agents. New drug
showed two-fold better potency than reference against candidates also should not produce toxic effect. Thus,
toxicity of the compounds 4a, 4k-4n, which have sig-
Table II. MIC values (
against fungal strains
µ
g/mL) of the compounds 4a-4n
nificant antifungal activity, was needed to be investi-
gated. For this purpose, Brine-Shrimp lethality assay,
which is considered as a useful tool for preliminary
assessment of toxicity and gives reliable results in
correlation with rodent and human acute oral toxicity
data, was used for determination of toxicity levels of
the compounds (Calleja and Persoone, 1992; Choudhary
and Thomsen, 2001; Carballo et al., 2002). The LD50
values and 95% confidence intervals of the compounds
were calculated with the LC50 computer program (Trim-
med Spearman-Karber Method, Version 1.5) (Brayn et
al., 1993). As seen in Table III, tested compounds ex-
hibited toxicity to different extents (LD50 = 126.33-
Fungal strains
Compounds
A
B
C
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
50*
50*
100
50*
100
50*
50*
100
100
50*
25**
25**
25**
25**
50
50*
100
50*
100
50*
100
100
50*
100
50*
25**
50*
25**
50*
50
25**
50*
50*
50*
50*
50*
50*
50*
50*
100
25**
25**
25**
25**
50
368.72 µg/mL). However, LD50 of the compounds was
3-14 folds higher than their MIC. This is a significant
finding which demonstrates that the compounds dis-
play antifungal activity at non-toxic concentration.
Furthermore, observation of no correlation between
antifungal activity and toxic effect implies that these
compounds may have selectively interacted with cell
of fungi instead of host cells.
4n
Ketoconazole
A: C. albicans, B: C. globrata, C: C. tropicalis
*Equal MIC value to reference; **Lower MIC value than
reference

Novel Antimicrobial Benzimidazoles
1433
Table III. Brine-shrimp toxicity results of the compounds
4a, k-n
oromethyl, cyano and carboxy substituted compounds
4k-4n indicated the best antifungal activity, whereas
the other compounds substituted with electron donat-
ing groups could not exhibit the same potency (Table
II). The reason for above observation can be explained
by electron density, which is an important factor to
reach optimum antimicrobial activity. The compounds
LD50
g/mL)
Upper 95%
limit ( g/mL)
Lower 95% limit
g/mL)
Comp.
(µ
µ
(µ
4a
4k
4l
4m
4n
368.72
126.33
144.26
211.07
193.70
688.62
171.07
183.50
302.17
351.87
222.68
109.75
123.46
162.02
134.86
4b-4j possess higher electron density than the com-
pounds 4a
,
4k 4n since they bear electron donating
-
groups, which enhance the electron density. It is
known that high electron density causes more difficult
diffusion through the cell membrane of bacteria or
fungi and substantial activity loss may occur (Hania,
Evaluation of structure-activity relationships
(SARs)
In connection with our recent study (Ozkay et al., 2009). Thus, it can be concluded that, significant anti-
2010), the synthesized compounds were designed to fungal activity of the compounds 4a 4k 4n may be
,
-
bear substituents that supply different electronic en- due to their reduced electron density, which makes
vironment to the molecules. Electron donating groups the intracellular diffusion easier.
to aromatic ring and electron withdrawing groups
Another interesting finding, which should be discussed
from aromatic ring were chosen for this purpose. In in this paper, is the antimicrobial selectivity differ-
the recent study, we used some electronic parameters ence between the compounds existing in the present
to clarify a SAR between synthesized compounds and and our recently reported (Ozkay et al., 2010) studies.
their antibacterial activity. On the other hand, in the In our recent study, antibacterial activity of the com-
present study SAR was performed easier because it pounds was noteworthy, but they showed insignificant
was very clear that potential of antifungal activity of antifungal activity. On the other hand, in the present
the compounds is related with the presence of electron study, although the synthesized compounds exhibited
donating or withdrawing substituents on benzene low antibacterial activity, their antifungal activity was
ring. Electron withdrawing and donating groups were very important. The reason for such difference can be
classified according to resonance effect constant (
R), explained by following two factors: The first one is the
which is used for the substituents belonging to an distance between the cell structures of bacteria and
aromatic ring (Hansch et al., 1973). Non-substituted yeast. For instance, while the cell wall of fungi contain
compound 4a and electron withdrawing nitro, triflu- chitin, the cell wall of bacteria contain murein (Eweis
Scheme 2. Conjugation levels of the benzimidazole-hydrazones

1434
Y. Özkay et al.
et al., 2006). Thus, lipophilic characters of cell mem- mination of 3-14 folds higher LD50 than MIC was a
branes of the bacteria and fungi differ each other. Due significant result, which demonstrates that the com-
to their cell nature, bacteria possess more lipophilic pounds display antifungal activity at non-toxic concen-
cell membrane than fungi (Patil et al., 2010). As a result tration. Determination of antimicrobial selectivity dif-
of such distance, bacteria and fungi can show selec- ference between the compounds existing in the present
tivity to substances during intracellular transport. and our recently reported studies was an important
The second factor is the structural distance between finding that indicates the effect of structural features
the benzimidazole-hydrazone series existing in the on antimicrobial activity. Consequently, results of the
present and our recently performed studies. As seen present study have highlighted antimicrobial poten-
in Scheme 2, main difference between benzimidazole- tials of both benzimidazole and hydrazone moieties
hydrazone series is the methylamine group, which once more and has directed us to develop new synthesis
exists on the chemical structure of the compounds syn- strategies. In further studies, we believe that depend-
thesized in this study. This group possesses electron ing on the findings of this study it will be probable to
donating ability, so it hinders the delocalisation of
π-
synthesize new and more active benzimidazole-hydra-
electrons between benzimidazole ring and hydrazide zones, which display selective antifungal or antibac-
moiety and causes a decrease on conjugation level terial activity.
(Scheme 2). It is known that electron delocalisation
and conjugation correlate lipophilicity, which is a key
property that influences the ability of a drug to reach
the target by transmembrane diffusion and to have a
major effect on the biological activity (Testa et al.,
2000; Patil et al., 2010). As a result of their enhancing
REFERENCES
Allen, P. M. and Gootlieb, D., Mechanism of action of the
fungicide thiabendazole, 2-(4'-thiazolyl) benzimidazole.
Appl. Microbiol., 20, 919-926 (1970).
Arjmand, F., Mohani, B., and Ahmad, S., Synthesis, antibac-
terial, antifungal activity and interaction of CT-DNA with
a new benzimidazole derived Cu(II) complex. Eur. J. Med.
Chem., 40, 1103-1110 (2005).
Bayrak, H., Demirbas, A., Karaoglu, S. A., and Demirbas,
N., Synthesis of some new 1,2,4-triazoles, their Mannich
and Schiff bases and evaluation of their antimicrobial
activities. Eur. J. Med. Chem., 44, 1057-1066 (2009).
Block, S. S., Disinfection, Sterilization, and Preservation,
5th Ed., Lippincott Williams and Wilkins, Philadelphia,
pp. 300-309, (2001).
Brayn, B., Timothy, M., and Tore, S., General and Applied
Toxicology, 2nd Ed., Vol. I, Macmillan, London, p. 52,
(1993).
Calleja, M. C. and Persoone, G., Cyst-based toxicity tests
IV,the potential of ecotoxicological tests for the prediction
of acute toxicity in man as evaluated on the first ten
chemicals of the MEIC programme. Atla, 20, 396-405
(1992).
Carballo, J. L., Hernandez-Inda, Z. L., Perez, P., and Garcia-
Gravalos, M. D., A comparison between two brine shrimp
assays to detect in vitro cytotoxicity in marine natural
products. BMC Biotechnol., 2, 17 (2002).
Chornous, V. A., Bratenko, M. K., Vovk, M. V., and Sidorchuk,
I. I., Synthesis and antimicrobial activity of pyrazole-4-
carboxylic acid hydrazides and N-(4-pyrazoyl)hydrazones
of aromatic and heteroaromatic aldehydes. Pharm. Chem.
J., 35, 203-205 (2001).
Choudhary, M. I. and Thomsen, W. J., Bioassay Techniques
For Drug Development, Harwood Academic Publishers,
pp. 9-10, (2001).
π-electron delocalisation and conjugation level, our
previously synthesized benzimidazole-hydrazone deri-
vatives would possess more lipophilicity. Depending
on described two factors, it may be suggested that pre-
viously synthesized compounds possess selective anti-
bacterial activity because of their higher lipophilicity,
which may cause an easier interaction with more lipo-
philic cell membrane of bacteria. Otherwise, benzimi-
dazole-hydrazone derivatives reported in this study
have selective antifungal activity due to their lower
lipophilic character that makes intracellular transport
easier through the cell membrane of fungi.
The resistance of fungi and bacteria to current anti-
biotic therapies is rapidly becoming a major public
health threat throughout the world in 21th century.
Hence, our research group focused on the development
of novel antimicrobial agents. A careful literature sur-
vey revealed that it would be worthwhile to synthesize
some combination products of benzimidazole and
hydrazone moieties due to their chemotherapeutic
value. Depending on this purpose, we designed some
compact systems, which bear these vital moieties. Our
first investigation on benzimidazole-hydrazone deriva-
tives (Ozkay et al., 2010) indicated that some of the
synthesized compounds possess significant antibacter-
ial activity. This result encouraged us and we performed
the present study so as to screen antimicrobial acti-
vity of a new series of benzimidazole-hydrazones.
In the present study, some of the synthesized benzi-
midazole-hydrazone derivatives exhibited notable anti-
fungal activity. Furthermore, in toxicity assay, deter-
Cosgrove, S. E., The relationship between antimicrobial
resistance and patient outcomes: mortality, length of hospi-

Novel Antimicrobial Benzimidazoles
1435
tal stay, and health care costs. Clin. Infect. Dis., 42, S82-
S89 (2006).
derivatives. J. Enzyme Inhib. Med. Chem., 24, 825-831
(2009).
Cunha, B. A., Antibiotic resistance. Drugs Today (Barc), 34,
691-698 (1998).
Eweis, M., Elkholy, S. S., and Elsabee, M. Z., Antifungal
efficacy of chitosan and its thiourea derivatives upon the
growth of some sugar-beet pathogens. Int. J. Biol. Macromol.,
38, 1-8 (2006).
Hania, M. M., Synthesis of some imines and investigation of
their biological activity. E.-J. Chem., 6, S508-S514 (2009).
Hansch, C., Leo, A., Unger, S. H., Kim, K. H., Nikaitani, D.,
and Lien, E. J., “Aromatic” substituent constants for struc-
ture-activity correlations. J. Med. Chem., 16, 1207-1216
(1973).
Joshi, S. D., Vagdevi, H. M., Vaidya, V. P., and Gadaginamath,
G. S., Synthesis of new 4-pyrrol-1-yl benzoic acid hydra-
zide analogs and some derived oxadiazole, triazole and
pyrrole ring systems: a novel class of potential antibac-
terial and antitubercular agents. Eur. J. Med. Chem., 43,
1989-1996 (2008).
Kaplancikli, Z. A., Turan-Zitouni, G., Revial, G., and Guven,
K., Synthesis and study of antibacterial and antifungal
activities of novel 2-[[(benzoxazole/benzimidazole-2-yl)sul-
fanyl] acetylamino]thiazoles. Arch. Pharm. Res., 27, 1081-
1085 (2004).
Kumar, P., Narasimhan, B., Sharma, D., Judge, V., and
Narang, R., Hansch analysis of substituted benzoic acid
benzylidene/furan-2-yl-methylene hydrazides as antimicro-
bial agents. Eur. J. Med. Chem., 44, 1853-1863 (2009).
Levy, S. B., The challenge of antibiotic resistance. Sci. Am.,
278, 46-53 (1998).
Lipsitch, M., The rise and fall of antimicrobial resistance.
Trends Microbiol., 9, 438-444 (2001).
Loncle, C., Brunel, J. M., Vidal, N., Dherbomez, M., and
Letourneux, Y., Synthesis and antifungal activity of
cholesterol-hydrazone derivatives. Eur. J. Med. Chem.,
39, 1067-1071 (2004).
Ozdemir, A., Turan-Zitouni, G., Kaplancikli, Z. A., Revial, G.,
Demirci, F., and Iscan, G., Preparation of some pyrazoline
derivatives and evaluation of their antifungal activities.
J. Enzyme Inhib. Med. Chem., 25, 565-571 (2010).
Ozkay, Y., Tunali, Y., Karaca, H., and Isikdag, I., Antimicro-
bial activity and a SAR study of some novel benzimidazole
derivatives bearing hydrazone moiety. Eur. J. Med.
Chem., 45, 3293-3298 (2010).
Papakonstantinou-Garoufalias, S., Pouli, N., Marakos, P.,
and Chytyroglou-Ladas, A., Synthesis antimicrobial and
antifungal activity of some new 3 substituted derivatives
of 4-(2,4-dichlorophenyl)-5-adamantyl-1H-1,2,4-triazole.
Farmaco, 57, 973-977 (2002).
Patil, M., Hunoor, R., and Gudasi, K., Transition metal com-
plexes of a new hexadentate macroacyclic N2O4-donor
Schiff base: inhibitory activity against bacteria and fungi.
Eur. J. Med. Chem., 45, 2981-2986 (2010).
Podunavac-Kuzmanovic, S. O., Barna, D. J., and Cvetkovic,
D. D., Quantitative structure-activity relationships to pre-
dict antibacterial effect of some benzimidazole derivatives.
Acta Period. Technol., 39, 181-191 (2008).
Rollas, S., Gulerman, N., and Erdeniz, H., Synthesis and
antimicrobial activity of some new hydrazones of 4-fluoro-
benzoic acid hydrazide and 3-acetyl-2,5-disubstituted-1,3,
4-oxadiazolines. Farmaco, 57, 171-174 (2002).
Salgin-Goksen, U., Gokhan-Kelekci, N., Goktas, O., Koysal,
Y., Kilic, E., Isik, S., Aktay, G., and Ozalp, M., 1-Acylthio-
semicarbazides, 1,2,4-triazole-5(4H)-thiones, 1,3,4-thiadia-
zoles and hydrazones containing 5-methyl-2-benzoxazo-
linones: synthesis, analgesic-anti-inflammatory and anti-
microbial activities. Bioorg. Med. Chem., 15, 5738-5751
(2007).
Silverman, R. B., Organic chemistry of Drug Desing and
Drug Action, Academic press, San Diego, pp. 7-105, (1992).
Spasov, A. A., Yozhitsa, I. N., Bugaeva, L. I., and Anisimova,
V. A., Benzimidazole derivatives: Spectrum of pharma-
cological activity and toxicological properties. Pharm. Chem.
J., 32, 232-243 (1999).
Masunari, A. and Tavares, L. C., A new class of nifuroxazide
analogues: synthesis of 5-nitrothiophene derivatives with
antimicrobial activity against multidrug-resistant Sta-
phylococcus aureus
(2007).
.
Bioorg. Med. Chem., 15, 4229-4236
Testa, B., Crivori, P., Reist, M., and Carrupt, P. A., The
influence of lipophilicity on the pharmacokinetic behavior
of drugs: Concepts and examples. Perspect Drug Discov.
Des., 19, 179-211 (2000).
Vicini, P., Zani, F., Cozzini, P., and Doytchinova, I., Hydra-
zones of 1,2-benzisothiazole hydrazides: synthesis, anti-
microbial activity and QSAR investigations. Eur. J. Med.
Chem., 37, 553-564 (2002).
Watanabe-Akanuma, M., Ohta, T., and Sasaki, Y. F., A novel
genotoxic aspect of thiabendazole as a photomutagen in
bacteria and cultured human cells. Toxicol. Lett., 158,
213-219 (2005).
Mostafa, Y. A., Hussein, M. A., Radwan, A. A., and Kfafy Ael,
H., Synthesis and antimicrobial activity of certain new
1,2,4-triazolo[1,5-a] pyrimidine derivatives. Arch. Pharm.
Res., 31, 279-293 (2008).
Nawrocka, W., Sztuba, B., Kowalska, M. W., Liszkiewicz, H.,
Wietrzyk, J., Nasulewicz, A., Pelczynska, M., and Opolski,
A., Synthesis and antiproliferative activity in vitro of 2-
aminobenzimidazole derivatives. Farmaco, 59, 83-91 (2004).
Ozdemir, A., Turan-Zitouni, G., Kaplancikli, Z. A., and Tunali,
Y., Synthesis and biological activities of new hydrazide