Synthesis and potent antimicrobial activity of some novel methyl or ethyl 1H-benzimidazole-5-carboxylates derivatives carrying amide or amidine groups

Article abstract of DOI:10.1016/j.bmc.2004.12.025

A series of benzimidazole-5-carboxylic acid alkyl ester derivatives carrying amide or amidine substituted methyl or phenyl groups at the position C-2 were synthesised and evaluated for antibacterial and antifungal activities against S. aureus, methicillin

Full text of DOI:10.1016/j.bmc.2004.12.025

Bioorganic & Medicinal Chemistry 13(2005) 1587–1597
Synthesis and potent antimicrobial activity of some novel methyl
or ethyl 1H-benzimidazole-5-carboxylates derivatives
carrying amide or amidine groups
a
Sec¸kin Ozden, Dilek Atabey, Sulhiye Yıldız and Hakan Goker
a
b
a,
*
¨
¨
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, 06100 Tandogan, Ankara, Turkey
bDepartment of Microbiology, Faculty of Pharmacy, Ankara University, 06100 Tandogan, Ankara, Turkey
Received 13April 2004; accepted 9 December 2004
Abstract—A series of benzimidazole-5-carboxylic acid alkyl ester derivatives carrying amide or amidine substituted methyl or phenyl
groups at the position C-2 were synthesised and evaluated for antibacterial and antifungal activities against S. aureus, methicillin
resistant S. aureus (MRSA), S. faecalis, methicillin resistant S. epidermidis (MRSE), E. coli and C. albicans. The results showed that
while all simple acetamides are essentially inactive, aromatic amides and amidines have potent antibacterial activities. Aromatic ami-
dine derivatives 13f–h exhibited the best inhibitory activity with 1.56–0.39 lg/mL MIC values against MRSA and MRSE.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
2. Result and discussion
Numerous benzimidazole derivatives containing ester
groups on the benzene ring have been synthesised for
their antifungal, insecticidal, herbicidal,1,2 anti-inflam-
matory3 and potential anthelmintic4 activities. It is also
well known that amides,5–7 amidines8–10 and combina-
tions of both11,12 are present in a variety of antimicro-
bial, antiparasitic, anthelmintic, antiviral and
antitumoural agents. Furthermore, our previous work
and that of others1,12–14 showed that benzimidazolecarb-
oxamides display good antibacterial and antimycotic
activity. Taking into consideration these structural fea-
tures and the expectation of low toxicity with ester func-
tion present, we planned to prepare a series of
benzimidazoles carrying the ester group at the benzene
ring of the benzimidazole core and with additional sub-
stitution at the position C-2 by alkyl or aryl groups pos-
sessing amides or amidines. Their antimicrobial activity
against bacteria and fungi will be described.
2.1. Chemistry
The synthetic pathway for preparation of the targeted
benzimidazoles 6a–h (Table 1)and 8a–f (Table 2) is
shown in Scheme 1. 4-Chloro-3-nitrobenzoic acid 1
was converted to its methyl or ethyl ester 2a and b, then
the chlorine atom of 2 was replaced with amines by aro-
matic nucleophilic substitution. Reduction of the nitro
group of 3a–h (Table 5) afforded 4b–f, 4h–j (Table 6).
Compounds 4a and g were prepared according to our
previous methods.15a,b The reaction of 4a–f with ethyl
b-amino-b-ethoxy acrylate HCl16 gave ethyl 5-methoxy-
carbonyl-1-alkylsubstituted-1H-benzimidazole-2-acetate
derivatives 5a–f (Table 7). The acetamides 6a–h were
prepared by the aminolysis of 5a–f in moderate yield
without reaction with the aromatic ester (Table 1). Com-
pounds 7a–d (Table 8) and 12a–d (Scheme 2, Table 9)
were obtained in good yield by condensation of 4g–j
with the Na2S2O5 adduct of 4-carboxybenzaldehyde or
4-cyanobenzaldehyde, respectively, in DMF.17 The car-
boxyl groups of 7a–d were converted to the amides
8a–f, first by acid chloride formation using SOCl2, then
by reaction with the several amines. Compound 9 was
prepared by heating 4a with ethyl cyanoacetate
according to a previously published method.18a,b,19
However, treatment of the other N-substituted
Keywords: Methicillin-resistant S. aureus; Methicillin-resistant S. epi-
N,N0-Dialk-
dermidis;
ylamidine.
*
Ethyl
1H-benzimidazole-5-carboxylate;
Corresponding author. Tel.: +90 312 222 0471; fax: +90 312 213
1081; e-mail: goker@pharmacy.ankara.edu.tr
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.12.025

Table 1. Yields, physicochemical and spectral properties of 6a–h
R 1
N
NHR2
MeO
N
O
O
Compd R1
R2
Yield Mp
Formulas
NMR (CDCl3) (d ppm)
MS ESI (m/z)
(M+H) (rel int 100%)
Eluant for c.c.
6a
H
Isopropyl
54
168–173C 14H17N3O3
1.09 (d, 6H, J = 6.6), 3.85 (s, 3H),
3.94 (s, 2H), 4.01 (m, 1H, J = 6.7), 7.3
(d, 1H, J = 6), 7.5 (d, 1H, Jo = 8.5),
7.9 (d, 1H, Jo = 8.5, Jm = 1.3), 8.25
(d, 1H, Jm = 1.3)
276
EtOAc–EtOH (90:10)
6b
Ethyl
n-Butyl
66
142
C17H23N3O3
0.82 (t, 3H, J = 7.3), 1.24 (m, 2H,
J = 7.2), 1.34 (t, 3H, J = 7.3), 1.42 (m,
2H), 3.19 (q, 2H, J = 6), 3.86 (s, 2H),
3.87 (s, 3H), 4.21 (q, 2H, J = 7.3),
7.33 (d, 1H, J = 8.6), 7.8 (t, 1H), 7.96
(d, 1H, Jo = 8.5, Jm = 1.2), 8.36 (d,
1H, Jm = 1.1)
318
EtOAc
a
6c
6d
Methyl
3-Diethyl
aminopropyl
21
25
C19H28N4O3Æ2HClÆ3H2O
0.95 (t, 6H, J = 7.1), 1.64–1.68 (m,
2H), 2.40–2.5 (m, 6H), 3.38 (q, 2H),
3.85 (s, 3H), 3.9 (s, 2H), 3.97 (s, 3H),
7.36 (d, 1H, Jo = 8.4), 8.05 (dd, 1H,
Jo = 8.5, Jm = 1.5), 8.2 (br s, 1H), 8.44
(d, 1H, Jm = 1)
361
365
Chloroform–isopropanol (10:2)
Chloroform–isopropanol (10:2)
Cyclopropyl
3-Pyridilmethyl
183–185 C20H20N4O3
1.01 (2H), 1.27 (2H), 3.20 (m, 1H),
3.88 (s, 3H), 4.01 (s, 2H), 4.5 (d, 2H,
J = 6), 7.2 (d, 1H), 7.5 (d, 1H,
Jo = 8.6), 7.6 (d, 1H), 7.95 (dd, 1H,
Jo = 8.5, Jm = 1.6), 8.3(d, 1H,
Jm = 1.3), 8.45 (dd, 1H, Jo = 8.5,
Jm = 1.6), 8.51 (d, 1H, J = 1.9), 8.77
(br s, 1H)
6e
Benzyl
3-Dimethyl
aminopropyl
19
139–142
C
23H28N4O3Æ2HClÆ2H2O
1.63(m, 2H), 2.02 (s, 6H), 2.30 (t, 2H,
J = 6.4), 3.33 (q, 2H, J = 6.2), 3.86 (s,
2H), 3.98 (s, 3H), 5.46 (s, 2H), 7.09
(dd, 2H, Jo = 7.7, Jm = 2.4), 7.3–7.4
(m, 4H), 8.0 (dd, 1H, Jo = 8.5,
Jm = 1.5), 8.2 (br s, 1H), 8.49 (d, 1H,
Jm = 1.2)
409
Chloroform–isopropanol (10:2)

¨
S. Ozden et al. / Bioorg. Med. Chem. 13 (2005) 1587–1597
1589
o-phenylendiamines 4h–j with ethyl cyanoacetate led to
unidentified mixtures. When we have attempted to con-
vert the cyano group of 9, to the N-butylacetamidine by
the well-known Pinner reaction,20 10 and amide com-
pound 11 were obtained instead of the expected ami-
dine. It was reported that ester has been obtained in
similar this kind of reactions.18a,b To our knowledge,
there is only one example of a benzimidazole-2-acetam-
idine in the literature and it was not prepared by Pinner
reaction (obtained by using AlCl3/NH3).21 Meanwhile,
conversion of the aromatic methyl ester to ethyl ester
was performed in a straightforward manner. By treating
the aromatic nitriles 12a–d, with dry HCl gas in ethanol
which formed the imidate esters and was followed by the
reaction with various amines in ethanol gave the ami-
dines 13a–i (Table 3) in moderate yields.
Unexpectedly, the N,N0-disubstituted amidine 13d was
obtained from the reaction at room temperature, nor-
mally higher temperatures (50–55 or 80 °C) and 4-fold
excess of amine are required to lead to the formation
of N,N0-disubstituted amidines.11,22,23 In our case we
had to use 3- to 4-fold excess of amine, in order to ob-
serve significant reaction. In contrast, 13f and g were
formed with only small amounts of N,N0-dialkylated
amidines (5% and 6%) according to the LC/MS chro-
matogram. These mixtures exhibited very close reten-
tion times of 5.29 min (m/e 647, 100%): 5.86 min (m/e
523, 100%) and 6.07 min (m/e 717, 100%): 6.85 min
(m/e 591, 100%), respectively. After column chromatog-
raphy, the mono-alkylated amidines 13f and g were ob-
tained by crystallisation from isopropanol, however, the
pure diamidines could not be isolated. The formation
ratio for the di- and monoalkylated amidines of 13h is
21% (4.05 min, m/e 783, 100%) and 79% (6.99 min, m/e
625, 100%) respectively, according to the LC/MS chro-
matogram. In contrast to the previous example the
retention times are sufficiently different that the mixture
was readily separated by column chromatography. The
dialkylated amidine derivative has been identified as 13i.
The other derivatives of series 13 do not form detectable
amounts of dialklylated amidines. To fully understand
the factors controlling the monoalkyl/dialkylamidine
ratio further studies are necessary.
2.2. Microbiology
All of the benzimidazoles series 6, 8, 11 and 13 were
tested for in vitro antibacterial activity against Gram-
negative Escherichia coli, Gram-positive Staphylococcus
aureus, methicillin-resistant Staphylococcus aureus
(MRSA, clinical isolate), Streptococcus faecalis, methi-
cillin-resistant Staphylococcus epidermidis (MRSE, clin-
ical isolate) bacteria and antifungal activity against
Candida albicans by the agar diffusion method,24 which
has been extensively used by us.24 The compounds
showing antimicrobial activity by this method were fur-
ther tested by the macro-broth dilution assay25a,b to
determine the MICs which are listed in Table 4. The
synthesised compounds and the reference drugs were
dissolved in DMSO–H2O (50%), at a concentration of
400 lg/mL. The concentration was adjusted to 100 lg/
mL by 4-fold dilution with media culture and bacteria

Table 2. Yields, physicochemical and spectral properties of 8a–f
R 1
N
O
EtO
N
NHR2
O
Compd
R1
H
R2
Formulas
Mp (°C)
Yield (%)
NMR (CDCl3) (d ppm)
MS (ESI+) m/z (rel intensity: 100%)
8a
Isopropyl
C
20H21N3O3
250–252
71
1.11 (d, 6H, J = 6.6), 1.26 (t, 3H, J = 7), 4.08
(m, 1H, J = 6.7), 4.2 (q, 2H, J = 7), 7.5 (d, 1H,
Jo = 8.5), 7.79 (dd, 1H), 7.89 (m, 3H), 8.14 (m,
3H)
352
8b
Propyl
Isopropyl
C
23H27N3O3Æ0.5H2O
145–147
45
0.78 (t, 3H, J = 7.4), 1.24 (d, 6H, J = 6.6), 1.35
(t, 3H, J = 7.2), 1.74 (m, 2H, J = 7.5), 4.15 (t,
2H, J = 7.6), 4.25 (m, 1H, J = 6.6), 4.35 (q,
2H, J = 7.2), 6.1 (d, 1H, J = 7.5), 7.38 (d, 1H,
J = 8.6), 7.72 (d, 2H), 7.86 (d, 2H), 7.95 (dd,
1H, Jo = 8.5, Jm = 1.3), 8.49 (d, 1H, J = 1.1)
394
8c
8d
8e
8f
Benzyl
Isopropyl
C27H27N3O3Æ0.75H2O
C31H26ClN3O3Æ0.1H2O
C28H30N4O3Æ2.5H2O
C28H28Cl2N4O3ÆH2O
15344
175
1.18 (d, 6H,
J = 6.6), 1.34 (t, 3H, J = 7.2), 4.25
(m, 1H, J = 6.6), 4.34 (q, 2H, J = 7.2), 5.39 (s,
2H), 6.03(d, 1H, J = 7.6), 6.96 (m, 2H), 7.3
(m, 4H), 7.68 (d, 2H), 7.76 (d, 2H), 7.92 (dd,
1H, Jo = 8.5, Jm = 1.3), 8.53 (d, 1H, J = 1.1)
1.34 (t, 3H, J = 7.1), 4.33 (q, 2H, J = 7.1), 4.54
(d, 2H, J = 5.7), 5.4 (s, 2H), 6.9 (1H), 6.95 (m,
2H), 7.22 (overlapped, 8H), 7.66 (d, 2H), 7.79
(d, 2H), 7.96 (dd, 1H, Jo = 8.5, Jm = 1.2), 8.52
(d, 1H, J = 1)
442
524
471
539
Benzyl
4-Chlorobenzyl
Ethyl aminoethyl
Ethyl aminoethyl
54
Benzyl
188–192(6H, o4v3e1r.3lapped), 2.94 (q, 2H), 2.97 (2H),
3.73 (q, 2H), 5.34 (s, 2H), 6.93 (2H), 7.11 (d,
1H), 7.14 (m, 3H), 7.63 (d, 2H), 7.87 (dd, 1H,
Jo = 8.5, Jm = 1.2), 8.0 (d, 2H), 8.43(d, 1H),
8.52 (t, 1H)
2,4-Dichlorobenzyl
100a
35
1.08 (t, 3H), 1.12 (t, 3H), 2.7 (q, 2H), 2.85
(2H), 3.52 (q, 2H), 4.33 (q, 2H), 5.38 (s, 2H),
6.54 (d, 1H), 7.04 (m, 2H), 7.3(t, 1H), 7.4 (d,
1H), 7.6 (d, 2H), 7.84 (d, 2H), 7.92 (dd, 1H,
Jo = 8.4, Jm = 1.1), 8.49 (d, 1H, Jm = 1)
a Bubbling, no sharp mp.

¨
S. Ozden et al. / Bioorg. Med. Chem. 13 (2005) 1587–1597
1591
Cl
NO2
NHR1
NO2
Cl
NO2
NH2
NH2
c
b
a
RO
RO
HO
HO
O
O
O
O
3a - 3h
1
2a R = CH3
2b R = C2H5
a
R 1
N
R 1
N
e
NHR1
d
MeO
N
NHR2
MeO
RO
N
O
O
NH2
O
EtO
O
6a - 6h
O
5a - 5f
4a - 4j
R 1
N
R 1
N
O
O
f
g
EtO
N
NHR2
EtO
N
OH
O
O
8a - 8f
7a - 7d
Scheme 1. Reagents: (a) methanolic or ethanolic HCl; (b) R1NH2; (c) H2ÆPd/C, for 4f and j NiCl2/Zn; (d) ethyl b-amino-b-ethoxyacrylateÆHCl; (e)
corresponding amines; (f) Na2S2O5 adduct of 4-carboxybenzaldehyde; (g) SOCl2/various amines.
R 1
N
NH2
NH2
NHR1
NH2
a
MeO
EtO
EtO
EtO
CN
N
O
a
O
4a
O
O
R1 = n-propyl
R1 = n-benzyl
4h
4i
H
N
H
H
N
N
N
N
O
b,c
O
CN
MeO
N
+
EtO
NHC4H9
OEt
O
11
9
O
10
R 1
N
HCl
NH
R 1
N
NHR1
NH2
d
b
N
EtO
EtO
EtO
N
N
OEt
O
O
O
12a - 12d
4g - 4j
R 1
N-R3
NHR2
e
N
EtO
N
O
13a - 13i
Scheme 2. Reagents: (a) NCCH2CO2C2H5; (b) EtOH/dry HCl gas; (c) C4H9NH2; (d) Na2S2O5 adduct of 4-cyano-benzaldehyde; (e) corresponding
amines.
solution. Data were not taken for the initial solution be-
cause of the high DMSO concentration (12.5%). Since
the compound 13i is insoluble in DMSO–H2O (50%),
its antimicrobial activity has only been determined by
the diffusion method and it has some growth inhibition
zone, however, not as well as the others. As shown in
Table 4, the acetamide derivatives 6g and h exhibit only
a modest inhibitory effect with a MIC values of 50 lg/mL
against S. aureus. All of the acetamide–benzimidazoles 6
series, are essentially inactive. Among the benzamide
series, compound 8e and f show good activity, in partic-
ular against S. aureus, MRSA and MRSE. All the aro-
matic amidine analogues 13 exhibited strong ability to
inhibit S. aureus with most of the MICs in the low
micromolar range. The most active compounds are
13f–h and exhibit MIC values of 0.78–1.56 lg/mL
against S. aureus. While ampicillin and sultamicillin is
nearly inactive against MRSA (50 and 25 lg/mL and
no inhibition zone with the diffusion method), com-
pounds 13f–h exhibited the greatest activity with MICs
between 0.78–0.39 lg/mL. Activity against drug-resis-
tant microorganisms, which are clinically difficult to
treat, is very important for the next generation of anti-
bacterial agents. Consequently the results for 13f–h are
quite encouraging. Since compounds 13f–h had superior
activity against MRSA and MRSE, when compared to

Table 3. Yields, physicochemical and spectral properties of 13a–i
R 1
N
N
R 3
EtO
N
NHR2
O
Compd
R1
H
R2
R3
H
Formulas
Mp (°C)
Yield (%) NMR (DMSO-d6) (d ppm)
LC/MS ESI(+), m/z M+H,
100% (Retention time)
13a
Isopropyl
C20H22N4O2Æ0.5C3H8OÆ0.75HOH
95a 224–225
35
1.1 (d, 6H, J = 6.3), 1.26 (t, 3H, J = 7.1), 351 (5.81)
3.77 (m, 1H, J = 6.3), 4.23 (q, 2H,
J = 7.1), 7.53(d, 1H, J = 8.4), 7.67 (dd,
1H, Jo = 8.4, Jm = 1.4), 7.78 (d, 2H,
J = 8.4), 8.09 (d, 1H, J = 1.1), 8.2 (d, 2H,
J = 8.4)
13b
H
Phenylethyl
Isopropyl
H
H
C
25H24N4O2Æ0.5C3H8OÆ0.5HOH
95a 205
33
41
1.55 (t, 3H, J = 7), 3.13 (t, 2H, J = 6.5),
3.67 (t, 2H, J = 6.1), 4.52 (q, 2H, J = 6.9),
7.4 (m, 7H), 7.77 (d, 1H, J = 8.3), 7.96 (d,
2H), 8.1 (d, 1H, J = 8.2), 8.49 (s, 1H)
413(6.35)
13cb
Propyl
C23H28N4O2Æ2HClÆ0.5EtOHÆHOH
100–135a
253–255
0.65 (t, 3H, J = 7.3), 1.21 (d, 6H, J = 5.5), 393 (5.8)
1.29 (t, 3H, J = 7.1), 1.62 (m, 2H, J = 7.4),
4.07 (m, 1H, J = 7.6), 4.29 (m, 4H,
J = 6.9), 7.9 (d, 3H), 7.93 (dd, 1H,
Jo = 8.6, Jm = 1.3), 8.01 (d, 2H, J = 8.5),
8.27 (d, 1H, Jo = 1), 9.25 (s, 1H), 9.57 (s,
1H), 9.74 (d, 1H)
13db
Propyl
Propyl
Propyl
C26H34N4O2Æ2HClÆ0.1HOH
Very
hygroscopic
40
0.67 (tt, 6H, partly overlapped), 0.9 (t,
3H, J = 7.2), 1.28 (t, 3H, J = 7.2), 1.45 (m,
2H, J = 7.3), 1.62 (m, 4H), 3.03 (q, 2H,
J = 6.3), 3.37 (q, 2H, J = 6.3), 4.29 (m,
4H), 7.76 (d, 2H, J = 8.2), 7.86 (d, 1H,
J = 8.3), 7.91 (dd, 1H, Jo = 8.2, Jm = 1.4),
8.00 (d, 2H, J = 8.2), 8.26 (s, 1H), 9.53(t,
1H), 9.76 (t, 1H)
435 (6.2)
13e
13f
Benzyl
Benzyl
Isopropyl
H
H
C27H28N4O2Æ0.75HOH
100–110a
35
1.49 (d, 6H, J = 6.4), 1.61 (t, 3H, J = 7.1), 441 (5.89)
4.22 (t, 3H, J = 7.1), 4.22 (m, 1H, J = 6.2),
4.61 (q, 2H, J = 7.12), 5.67 (s, 2H), 7.26
(m, 2H), 7.51 (m, 4H), 7.89 (d, 2H,
J = 8.2), 7.95 (d, 2H, J = 8.3), 8.2 (dd, 1H,
Jo = 8.5, Jm = 1.4), 8.76 (d, 1H, Jm = 1.1)
(With 1 drop D2O): 1.31 (t, 3H, J = 7.1), 523(5.86)
4.31 (4H, overlapped), 5.63 (s, 2H), 6.96
(d, 2H, J = 6.8), 7.25 (m, 3H), 7.36 (d, 2H,
J = 8.4), 7.45 (d, 2H, J = 8.7), 7.62 (d, 1H,
J = 8.8), 7.76 (d, 2H, J = 8.4), 7.88 (dd,
1H, Jo = 8.2, Jm = 1.1), 7.97 (d, 2H,
4-Chlorobenzyl
C31H27ClN4O2Æ0.25C3H8OÆ0.5HOH 93–95a 138–140 30
J = 8.3), 8.32 (d, 1H, J = 1.2)

¨
S. Ozden et al. / Bioorg. Med. Chem. 13 (2005) 1587–1597
1593
the clinically important agents oxacillin (diffusion meth-
od), ampicillin and sultamicillin, further studies are
needed to clarify their mechanism of action.
It was reported that natural product distamycin A, a
DNA minor-groove binding ligand, has weak antibacte-
rial activity, with moderate or high DNA binding affin-
ity.26 Nevertheless, distamycin has served as a prototype
for the development of minor-groove binding ligands
with subnanomolar binding affinity. Dimeric analogous
of distamycin A have recently been reported with strong
DNA binding properties which is thought to be neces-
sary for good antibacterial activity. Furthermore, some
researchers27 are focused on the optimisation of dista-
mycin A analogues for therapeutic application in the
treatment of severe infections caused by drug-resistant
Gram-positive bacteria. Some of the analogues exhib-
ited very potent antibacterial activity and were shown
to bind A/T rich target sequences that are commonly
found in bacterial promoters and replication origins.
These results that they inhibit DNA replication and
RNA transcription and consequently kill bacteria. Re-
cently, internal and C-terminal benzimidazole28,29 mod-
ifications of distamycin A were published by the same
group. In addition, it has been reported that benzimid-
azole ring is an important structural motif of various
types of DNA minor groove binding ligands.27,28,30–32
Hence, a similar mechanism of action may exist for
our benzimidazoles, since they have also very potent
inhibitory activity against drug resistance Gram-posi-
tive bacteria. At the same time, low cytotoxicity should
be expected, since the unmodified distamycin A has
only limited cytotoxicity33 and it has also monocationic
amidine moiety like the compounds 13a–i. It appears
that benzyl or halogenated benzyl substitution at the
position N1 enhance the antibacterial activities against
S. aureus, for both series 8 and 13. The best result
was obtained against S. faecalis with compound 13g.
All the synthesised compounds were inactive (MICs
P 50 lg/mL) against Gram-negative bacteria E. Coli
possibly due to poor permeability of the additional out-
er membrane. To study the selectivity for inhibition of
bacterial growth, all these compounds were also tested
against the fungi C. albicans. Generally all of the com-
pounds were less effective against fungi compared to
bacteria.
3. Conclusion
Introduction of aromatic amidine groups into the benz-
imidazole system gives a good profile of Gram-positive
antibacterial activity. In particular, compounds 13f–h
having chlorinated benzyl groups at the position N1
and 2-(4-N-benzylcarboxamidinophenyl) benzimidaz-
oles exhibited the greatest activity with MIC values
ranging from 1.56 to 0.39 lg/mL against S. aureus,
methicillin-resistant S. aureus (MRSA) and methicillin-
resistant S. epidermidis (MRSE), respectively. Detailed
mechanistic studies are required to understand the po-
tent activity of these compounds. In vivo studies of com-
pounds such as 13f–h are necessary to fully evaluate the
potential of these compounds.

¨
S. Ozden et al. / Bioorg. Med. Chem. 13 (2005) 1587–1597
1594
Table 4. Antibacterial and antifungal activity of compounds 6g–13h (MIC minimum inhibitory concentration lg/mL)
a
Compound
S. aureus ATCC 25923MRSA
S. faecalis ATCC 29212 MRSEb E. coli ATCC 25922 C. albicans ATCC 10231
6g
6h
50
50
>50
>50
50
>50
>50
50
50
25
> 50
>50
>50
50
>50
>50
50
8e
12.5
12.5
8f
13a
6.25
12.5
3.12
6.25
>50
6.25
12.5
>50
12.5
6.25
>50
3.12
50
25
50
>50
>50
>50
>50
>50
>50
>50
>50
13b
13c
50
50
6.25
3.12
3.12
0.78
1.56
1.56
0.39
0.78
>50
>50
50
>50
>50
>50
13d
13e
25
12.5
50
50
13f
13g
0.78
6.25
1.56
12.5
12.5
6.25
0.78
0.39
3.12
3.12
0.78
1.56
0.78
1.56
13h
Ampicillin
Sultamicillin
Gentamisin
Fluconazole
50
25
3.12
0.78
1.56
a MRSA: methicillin-resistant S. aureus (clinical isolate).
b MRSE: methicillin-resistant S. epidermidis (clinical isolate).
4. Experimental
4.1. General procedure for synthesis of 3a–h
Melting points were measured with a capillary melt-
ing point apparatus (Buchi SMP 20 and Electrotermal
9100) and are uncorrected. The IR spectra were re-
corded on a Jasco FT/IR-420 spectrometer as KBr
discs. Compounds 3a–g: 1709–1730 cmꢀ1 (C@O ester);
4a–h: 1692–1730 cmꢀ1 (C@O ester). The 1H NMR
spectra were recorded with Bruker DPX-400 and
VARIAN Mercury 400 FT-NMR spectrophotometers,
d scale (ppm) from TMS. Mass spectra were taken on
Waters Micromass ZQ by using ESI(+) method. LC/
MS analyses for 13 series were performed with
Waters Alliance and Micromass ZQ by using MeOH
and C-18 column (15 cm) with a flow rate of 0.5 mL/
min. LC equipped with a diode array UV detection
monitoring at 254 nm. The mixtures ratio were calcu-
lated from the peak area. Elemental analyses were ta-
ken on a Leco 932 CHNS-O analyzer (TUBlTAK
Instrumental Analyze Lab., Ankara). For the HCl
salts of the synthesised compounds, the free bases
were dissolved in isopropanol and dry HCl gas was
passed through the solution, some salts were highly
hygroscopic and no sharp melting points could be
observed.
To a solution of 2a or b (4.6 mmol) in DMF (1.5 mL),
the corresponding amines (9.3mmol) were added and
the mixture was heated for 3–8 h at 80 °C until starting
material was consumed. The mixture was allowed to
cool, H2O and EtOH were added for 3a–c and for 3d–
h, respectively. The resultant yellow precipitate was fil-
tered and washed with H2O, crystallised from EtOH
or EtOH–H2O (Table 5).
4.2. General procedure for synthesis of 4b–e and 4h–j
Compounds 3a–d, f–h (3.4 mmol) in EtOH (75 mL) was
reduced by hydrogenation using 40 psi of H2 and 10%
Pd–C (40 mg) until cessation of H2 uptake. The catalyst
was filtered on a bed of Celite, washed with EtOH and
the filtrate was concentrated in vacuo. The crude amines
were used for the further steps without crystallisation,
grey-purple in colour (Table 6).
4.3. General procedure for synthesis of 5a–f
To a solution of 4a–f (2 mmol) in DMF (1 mL), ethyl-b-
amino-b-ethoxyacrylate HCl (4.7 mmol, 0.74 g) was
Table 5. Yields, formulas and physical properties of 3a–h
NHR1
NO2
RO
O
Compd
R
R1
Formulas
Mp (°C)
Yield (%)
34
142–143lit. 145
95 lit.35
3a
3b
3c
3d
3e
3f
Methyl
Methyl
Methyl
Methyl
Methyl
Ethyl
Methyl
Ethyl
C9H10N2O4
C10H12N2O4
79
67.5
78
Cyclopropyl
Benzyl
C
C
C
11H12N2O4
15H14N2O4
15H13ClN2O4
80–82
101 lit.13 98
141–142 lit.1 141
78
p-Chlorobenzyl
n-Propyl
Benzyl
C12H16N2O4
C16H16N2O4
84
70
3g
3h
Ethyl
Ethyl
9371
126
2,4-Dichlorobenzyl
C
16H14Cl2N2O4

¨
S. Ozden et al. / Bioorg. Med. Chem. 13 (2005) 1587–1597
1595
Table 6. Yields, physicochemical and spectral properties of 4a–j
NHR1
NH2
RO
O
Compd
R
R1
Formulas
Mp (°C)
Yield (%)
4a
4b
4c
4d
4e
4f
Methyl
Methyl
Methyl
Methyl
Methyl
Methyl
Ethyl
H
C8H10N2O2
C9H12N2O2
C10H14N2O2
Lit.15a
Methyl
Ethyl
99–100 lit.36 102
136–137
108–110
86
94
93
Cyclopropyl
Benzyl
C
11H14N2O2
C15H16N2O2
15H15ClN2O2
126–127 lit.13 125
1
221–223lit. 221
p-Chlorobenzyl
H
C
4g
4h
4i
C9H12N2O2
C12H18N2O2
C16H18N2O2
Lit.15b
Ethyl
Ethyl
Ethyl
n-Propyl
Benzyl
2,4-Dichlorobenzyl
Oily-semisolid
103–104
119
94
93
55
4j
C16H16Cl2N2O2
added and the mixture was stirred for 8 h at 40 °C. The
mixture was cooled, water was added and then extracted
with chloroform. The extract was washed H2O and
evaporated. The residue was purified by column chro-
matography (EtOAc) to give 5a–f. All of them were ob-
tained as white solids (Table 7).
Na2CO3 solution (5%) and H2O, dried (anhydrous
Na2SO4) and evaporated. The residue was purified by
column chromatography to give 6a–h as white solids
(Table 1).
4.5. General procedure for synthesis of 7a–d and 12a–d
4.4. General procedure for synthesis of 6a–h
4-Carboxy or 4-cyanobenzaldehyde (15 mmol) were dis-
solved in 50 mL EtOH and sodium metabisulfite (1.6 g)
in 10 mL H2O was added in portions. The reaction
mixture was stirred vigorously and more EtOH was
added. The mixture was kept in a refrigerator for a
several hours. The precipitate was filtered and dried
A mixture of 5a–f (3mmol), and corresponding amines
(11 mmol) was heated for 3–8 h under reflux (for 5a; 4
days). After cooling, water was and the mixture was ex-
tracted with chloroform. The extract was washed with
Table 7. Yields, physicochemical and spectral properties of 5a–f
R
N
OEt
O
MeO
O
N
Compd
R
H
Mp (°C) Formulas
Yield (%) NMR (CDCl3) (d ppm)
IR m cmꢀ1 (C@O)
5a
113–114
C
13H14N2O4
27
1.23(t, 3H, J = 7.1), 3.86 (s, 3H), 4.06 (s, 2H),
4.19 (q, 2H, J = 7.1), 7.53(d, 1H, Jo = 8.5), 7.91
(d, 1H, Jo = 8.5), 8.25 (s, 1H)
1717 and 1721
5b
5c
5d
5e
5f
Methyl
Ethyl
120–122
C
14H16N2O4
51.5
1.14 (t, 3H, J = 7.13), 3.66 (s, 3H), 3.79 (s, 3H), 1715
3.90 (s, 2H), 4.08 (q, 2H, J = 7.2), 7.21 (d, 1H,
Jo = 8.5), 7.87 (dd, 1H, Jo = 8.5, Jm = 1.5), 8.28
(d, 1H, Jm = 1)
107–109
105–108
113–115
C15H18N2O4
65
29
64
1.17 (t, 3H, J = 7.1), 1.35 (t, 3H), 3.84 (s, 3H),
3.92 (s, 2H), 4.09–4.17 (4H overlapped), 7.26 (d,
1H, Jo = 8.5), 7.91 (dd, 1H, Jo = 8.5, Jm = 1.5),
8.34 (d, 1H, Jm = 1.3)
1715
Cyclopropyl
Benzyl
C
C
16H18N2O4
1.12–1.15 (2H), 1.22–1.33 (5H), 3.3 (m, 1H),
3.96 (s, 3H), 4.16 (s, 2H), 4.26 (q, 2H, J = 7.2),
7.58 (d, 1H, Jo = 8.5), 8.03(dd, 1H, Jo = 8.5,
Jm = 1.5), 8.44 (d, 1H, Jm = 1.4)
1713and 1723
20H20N2O4
1.11 (t, 3H, J = 7.13), 3.83 (s, 3H), 3.86 (s, 2H), 1719
4.02 (q, 2H), 5.33 (s, 2H), 6.95 ve 7.2 (m, 6H),
7.87 (dd, 1H, Jo = 8.5, Jm = 1.4), 8.38 (d, 1H,
Jm = 1.3)
p-Chlorobenzyl 128–129
C20H19ClN2O4 59
1.25 (t, 3H, J = 7.14), 3.97 (s, 3H), 3.99 (s, 2H), 1717
4.16 (q, 2H), 5.43(s, 2H), 7.02 (d, 2H, Jo = 8.4),
7.23(d, 1H, Jo = 8.6), 7.32 (d, 2H, J = 8.4), 8.0
(dd, 1H, Jo = 8.5, Jm = 1.5), 8.51 (d, 1H)

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S. Ozden et al. / Bioorg. Med. Chem. 13 (2005) 1587–1597
1596
(yield over 93%). The mixture of these salts (2 mmol)
and 4h–j in DMF (5 mL) were heated at 130 °C for
4 h. The reaction mixture was cooled, poured into
H2O, and the solid was filtered, crystallisation of crude
product was from EtOH to give 7a–d and 12a–d as white
solids (Tables 8 and 9).
4.8. General procedure for synthesis of 10, 11 and 13a–i
Compounds 9, 12a–d (2.5 mmol) were suspended in
absolute EtOH, cooled in a ice–salt bath and dry HCl
gas was then passed through the solution for 40 min.
The stoppered flask was stirred at room temperature
for 3days. The solution was diluted with dry ether.
The imidate ester hydrochloride separated as an oil. Ex-
cess solvent was decanted, and dried under vacuum at
room temperature. The imidate esters of 13e–h were pre-
cipitated as white solids, washed with ether, then dried
under vacuum at room temperature. All imidate esters
were used directly without characterisation. A suspen-
sion of imidate ester HCl in absolute EtOH was stirred
with corresponding amines (4-fold excess) for overnight
at 25–30 °C. The reaction mixture was evaporated,
stirred with dilute Na2CO3 solution then extracted
with dichloromethane. The solvent was evaporated
and the residue purified by column chromatography
4.6. General procedure for synthesis of 8a–f
Compounds 7a–d (1 mmol) were heated at reflux in benz-
ene (4 mL) with SOCl2 (3mL) for 2 h at 80 °C. Then
solvent and excess of SOCl2 were evaporated and the
residue was dissolved in chloroform (10 mL). Excess of
the corresponding amine derivatives (1 mL) was added
and the mixture was stirred and heated for 30 min at
50 °C. Chloroform was added, washed with Na2CO3
(5%), H2O and evaporated (Table 2). Compounds 8a
and d recrystallised from EtOH, 8b–c and 8e–f were
purified by column chromatography using EtOAc and
chloroform–isopropanol–isopropylamine (100:50:1) as
eluant, respectively.
using
dichloromethane–isopropanol–isopropylamine
(100:50:1.5) mixture as eluant for 13a–h. In the 13h case,
13i was obtained from column chromatography by elut-
ing with dichloromethane–isopropanol–isopropylamine
(100:50:0.5). The solid of 9 treated with butylamine as
mentioned above, gave compound 10 by column chro-
matography using EtOAc–n-hexane (1:1), yield 42%,
4.7. Methyl 2-cyanomethyl-5-benzimidazolecarboxylate 9
The mixture of 4a (0.7 g, 4.2 mmol) and 0.8 g of ethyl
cyanoacetate were heated for 2 h at 200 °C. After cool-
ing, the residue was extracted with chloroform and puri-
fied by column chromatography using EtOAc–n-hexane
(50%) as eluant and decolourised with activated carbon.
1
mp 110 °C, H NMR (CDCl3) d 1.23(t, 3H, J = 7.1),
1.33 (t, 3H, J = 7), 3.86 (s, 3H), 4.04 (s, 2H), 4.30 (q,
2H, J = 7.1), 4.33 (q, 2H, J = 7.1), 7.53(d, 1H,
Jo = 8.5), 7.91 (dd, 1H, Jo = 8.5, Jm = 1.2), 8.25 (d, 1H,
Jm = 1.1), MS (ESI) m/z (rel intensity): 277 (M+H,
100%), C14H16N2O4 Anal. (C, H, N). Then the column
was eluted a second time with EtOAc–EtOH (95:5), 11
1
Yield 0.31 g, 34%, mp 218–220 °C, H NMR (DMSO-
d6) d 3.78 (s, 3H), 4.38 (s, 2H), 7.53 (1H), 7.45 (d,
1H), 8.1 (s, 1H), 12.8 (s, 1H). MS ESI m/z (rel intensity):
216 (M+H, 100%), C11H9N3O2, Anal. (C, H, N).
Table 8. Yields, formulas and mp of 7a–d
R 1
N
O
EtO
O
N
OH
Compd
R1
Formulas
Mp (°C)
Yield (%)
IR m cmꢀ1 COOEt COOH overlapped
7a
7b
7c
7d
H
Propyl
C
17H14N2O4
>300
268–270
265
69
71
79
65
1683
1703
1702
1708
C20H20N2O4
C24H20N2O4
Benzyl
2,4-Dichlorobenzyl
C24H18Cl2N2O4
>300
Table 9. Yields, formulas and mp of 12a–d
R 1
N
N
EtO
N
O
Compd
R1
Formulas
Mp (°C)
Yield (%)
IR m cmꢀ1
COOEt
CN
12a
12b
12c
12d
H
Propyl
C
17H13N3O2
C20H19N3O2
24H19N3O2
C24H17Cl2N3O2
>300
73
68
1685
1704
2225
1711
2226
2230
130–131
161
213–215
Benzyl
2,4-Dichlorobenzyl
C
631717
61
2229

¨
S. Ozden et al. / Bioorg. Med. Chem. 13 (2005) 1587–1597
1597
was obtained, mp 194–195 °C, yield 11%. 1H NMR
(DMSO-d6) d 0.85 (t, 3H, J = 7.3), 1.32 (m, 7H,
J = 7.1, overlapped), 3.07 (q, 2H, J = 6.8), 3.76 (s,
2H), 4.3(q, 2H, J = 7.1), 7.56 (d, 1H, J = 8.3), 7.78 (d,
1H, J = 8.1), 8.09 (s, 1H), 8.27 (t, 1H), 12.5 (s, 1H),
MS (ESI) m/z (rel intensity): 304 (M+H, 100%),
C16H21N3O3, Anal. (C, H, N).
¨
15. (a) Go¨ker, H.; Olgen, S.; Ertan, R.; Akgun, H.; Ozbey, S.;
¨
¨
Kendi, E.; Topc¸u, G. J. Hetereocycl. Chem. 1995, 32,
1767; (b) Go¨ker, H.; Ertan, R.; Akgun, H.; Yulug, N.
¨
Arch. Pharm. (Weinheim) 1991, 324, 283.
16. Glickman, S. A.; Cope, A. C. J. Am. Chem. Soc. 1945, 67,
1017.
17. Ridley, H. F.; Spickett, R. G. W.; Timmis, G. M. J.
Heterocycl. Chem. 1965, 2, 453.
18. (a) Copeland, A. B. R.; Day, A. R. J. Am. Chem. Soc.
1943, 1072; (b) Zakhs, E. R.; Ponyaev, A. I.; Subbotina,
M. A.; Eltꢀsov, A. V. Russ. J. Gen. Chem. 2001, 71, 1076.
19. Gerd, R.; Wolfgang, S. DE Patent 4,107,439, 1992.
20. Roger, R.; Neilson, D. Chem. Rev. 1961, 61, 179.
21. Bukowski, L. Acta Pol. Pharm. 1978, 35, 295.
22. McCarthy, J. R.; Wright, D. L.; Schuster, A. J.; Abdallah,
A. H.; Shea, P. J.; Eyster, R. J. Med. Chem. 1985, 28,
1721.
Acknowledgements
This work was supported by Ankara University Re-
search Fund (Project No.: 0000018-2003) and TUBI-
TAK (SBAG-AYD 293) Central Lab. of Pharmacy
Faculty of Ankara University provided support for
acquisition of the NMR and Mass spectrometer used
in this work. The authors wish to thank Prof. Dr. David
Boykin (Department of Chemistry, Georgia State Uni-
versity, Atlanta, GA 30303-3083) for his helpful sugges-
tions in the preparation of this manuscript.
23. Xu, F.; Sun, J.; Shen, Q. Tetrahedron Lett. 2002, 43, 1867.
24. Go¨ker, H.; Kus, C.; Boykin, D. W.; Yildiz, S.; Altanlar,
N. Bioorg. Med. Chem. 2002, 10, 2589.
25. (a) National Committee for Clinical Laboratory Stan-
dards. Methods for Dilution Antimicrobial Susceptibility
Tests for Bacteria that Grow Aerobically, 5th ed.;
Approved Standard (M7A5); National Committee for
Clinical Laboratory Standards: Wayne, PA, 2000; (b)
Shadomy, S.; Pfaller, M. A. Laboratory Studies with
Antifungal Agents: Susceptibility Tests and Quantitation
in Body Fluids. In Manual of Clinical Microbiology, 5th
ed.; Balows, A., Hausler, W. J., Hermann, K. L., Isenberg,
H. D., Shadomy, H. J., Eds.; Washington, DC, 1991;
Chapter 117, p 1173.
26. Burli, R. W.; Ge, Y.; White, S.; Baird, E. E.; Touami, M.
¨
S.; Taylor, M.; Kaizerman, J. A.; Moser, E. H. Bioorg.
Med. Chem. Lett. 2002, 12, 2591.
27. Kaizerman, J. A.; Gross, M. I.; Ge, Y.; White, S.; Hu, W.;
Duan, J.; Baird, E. E.; Johnson, K. W.; Tanaka, R. D.;
Moser, H. E.; Bu¨rli, R. W. J. Med. Chem. 2003, 46, 3914.
References and notes
1. Go¨ker, H.; Tuncbilek, M.; Ayhan, G.; Altanlar, N.
Farmaco 1998, 53, 415.
2. Hisano, T.; Ichikawa, M.; Tsumoto, K.; Tasaki, M. Chem.
Pharm. Bull. 1982, 30, 2996.
3. Lazer, E. S.; Matteo, M. R.; Possanza, G. Z. J. Med.
Chem. 1987, 30, 726.
4. Naim, S. S.; Singh, S. K.; Satyavan, S.; Suman, G.; Fatma,
N.; Chatterjee, R. K.; Katiyar, C. J. Indian J. Chem. B
1988, 27B, 1106.
5. Ram, S.; Wise, D. S.; Wotring, L. L.; McCall, J. W.;
Townsend, L. B. J. Med. Chem. 1992, 35, 539.
6. Dubey, R.; Abuzar, S.; Sharma, S.; Chatterjee, R. K.;
Katiyar, J. C. J. Med. Chem. 1985, 28, 1748.
7. Garuti, L.;Roberti, M.;Gentilomi, G. Farmaco2000, 55, 3 5.
8. Danan, A.; Charon, D.; Kirkiacharian, S.; Bories, C.;
Loiseau, M. P. Farmaco 1997, 52, 227.
9. Stephens, C. E.; Brun, R.; Salem, M. M.; Werbovetz, K.
A.; Tanious, F.; Wilson, D. W.; Boykin, D. W. Bioorg.
Med. Chem. Lett. 2003, 13, 2065.
10. Weidner-Wells, M. A.; Ohemeng, K. A.; Nguyen, V. N.;
Fraga-Spano, S.; Macielag, M. J.; Werblood, H. M.;
Foleno, B. D.; Webb, G. C.; Barrett, J. F.; Hlasta, D. J.
Bioorg. Med. Chem. Lett. 2001, 11, 1545.
11. Dyatkina, N. B.; Roberts, D. C.; Keicher, J. D.; Dai, Y.;
Nadherny, J. P.; Zhang, W.; Schmitz, U.; Kongpachith,
A.; Fung, K.; Novikov, A. A.; Lou, L.; Velligan, M.;
Khorlin, A. A.; Chen, M. S. J. Med. Chem. 2002, 45, 805.
12. Matsunaga, A.; Nakajima, Y.; Kohno, H.; Komatsu, H.;
Edatsugi, H.; Iwata, D.; Takezawa, K. U.S. Patent
5,852,011, 1995.
28. Burli, R. W.; McMinn, D.; Kaizerman, J. A.; Hu, W.; Ge,
¨
Y.; Pack, Q.; Jiang, V.; Gross, M.; Garcia, M.; Tanaka,
R.; Moser, H. E. Bioorg. Med. Chem. Lett. 2004, 14,
1253.
29. Burli, R. W.; Jones, P.; McMinn, D.; Le, Q.; Duan, J.;
¨
Kaizerman, J. A.; Difuntorum, S.; Moser, H. E. Bioorg.
Med. Chem. Lett. 2004, 14, 1259.
30. Renneberg, D.; Dervan, P. B. J. Am. Chem. Soc. 2003,
125, 5707.
31. Minehan, T. G.; Gottwald, K.; Dervan, P. B. Helv. Chim.
Acta 2000, 83, 2197.
32. Joubert, A.; Sun, X. W.; Johansson, E.; Bailly, C.; Mann,
J.; Neidle, S. Biochemistry 2003, 42, 5984.
33. Baraldi, P. G.; Romagnoli, R.; Guadix, A. E.;
Infantas, J. P.; Gallo, M. A.; Espinosa, A.; Martinez,
A.; Bingham, J. P.; Hartley, J. A. J. Med. Chem. 2002, 45,
3630.
34. Reverdin, L. Bull. Soc. Chim. Fr 1908, 4(3), 133.
35. Setsuya, S.; Kazunori, K.; Yumiko, K.; Hiromi, N. JP
Patent 2,002,047,287, 2002.
13. Go¨ker, H.; Tebrizli, E.; Abbasoglu, U. Farmaco 1996, 51,
53.
14. Go¨ker, H.; Tuncbilek, M.; Suzen, S.; Kus, C.; Altanlar, N.
¨
36. Kurt, W.; Rudolf, H. M. DE Patent 2,733,156,
1978; Kurt, W.; Rudolf, H. M. Chem. Abstr. 1978, 89,
61044.
Arch. Pharm. Pharm. Med. Chem. 2001, 334, 148.