Synthesis and Potent In vitro Activity of Novel 1H-Benzimidazoles as Anti-MRSA Agents

Article abstract of DOI:10.1111/j.1747-0285.2012.01393.x

A new class of 1H-benzimidazolecarboxamidines was synthesized and evaluated for in vitro antibacterial and antifungal activities, including drug-resistant bacterial strains. The most potent compound (32) has the same ratio of anti-MRSA activity as Vancomy

Full text of DOI:10.1111/j.1747-0285.2012.01393.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01393.x
Chem Biol Drug Des 2012; 80: 237–244
Research Article
Synthesis and Potent In vitro Activity of Novel
1H-Benzimidazoles as Anti-MRSA Agents
Hacer Karatas1
Yıldız and Hakan Goker *
¸
, Mehmet Alp1, Sulhiye
amidines has been reported (14,15) and evaluated for in vitro
2
1,
antibacterial activities, including drug-resistant bacteria strands.
Anti-MRSA and anti-VRE activities of the most potent compound IV
were found be superior to that of vancomycin (Figure 1). Some
novel amidinobenzimidazoles (V) have been identified as inhibitors
of bacterial KinA ⁄ SpoOF two-component system that includes a his-
tidine protein kinase and a response regulator (RR). Histidine pro-
tein kinase and RR enable bacteria to adapt to the environmental
changes and survival within the host (16). These amidino inhibitors
display good in vitro antibacterial activity, in particular, against
Gram-positive bacteria (i.e., S. aureus, MRSA, VRE).
¨
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Ankara University, 06100 Tandogan, Ankara, Turkey
2Department of Microbiology, Faculty of Pharmacy, Ankara
University, 06100 Tandogan, Ankara, Turkey
*Corresponding author: Hakan Gçker, goker@ankara.edu.tr
A new class of 1H-benzimidazolecarboxamidines
was synthesized and evaluated for in vitro antibac-
terial and antifungal activities, including drug-
resistant bacterial strains. The most potent
compound (32) has the same ratio of anti-MRSA
activity as Vancomycin (minimal inhibitory concen-
trations value 0.78 lg ⁄ mL). The mechanism of
action for 1H-benzimidazolecarboxamidine appears
to be different from existing antibacterial agents.
These compounds have potential for development
as a new class of potent anti-MRSA agent.
Potent antifungal activity of dicationic molecules against Candida
albicans and Cryptococcus neoformans has been reported (17). The
antifungal activities of a large number of compounds were compa-
rable with amphotericin B and fluconazole, which are the commonly
used antifungal drugs.
These results prompted us to investigate a series of new benzimi-
dazoles substituted with aliphatic amidines (11–18), benzamides
(26–30), and benzamidines (32–33), with multiple chloro substitu-
ents at different positions to evaluate their antistaphylococcal and
antifungal activities.
Key words: 1H-Benzimidazole, antifungal and antibacterial activities,
benzamide, benzamidine, methicillin-resistant Staphylococcus aureus
Received 5 January 2012, revised 29 March 2012 and accepted for pub-
lication 31 March 2012
Chemistry
The rate of isolation of multiple-resistant Gram-positive cocci
including methicillin-resistant Staphylococcus aureus (MRSA), methi-
cillin-resistant coagulase-negative staphylococci (MR-CNS), penicil-
lin-resistant Streptococcus pneumoniea, and vancomycin-resistant
enterococcus (VRE) continues to increase (1–3). In particular, there
has been intensive focus on S. aureus, as many strains of this
organism are now resistant against methicillin (4). In addition, clini-
cal failures have been reported with linezolid (5–7), an oxazolidindi-
one derivative that is one of the most effective agents against
multi-drug-resistant Gram-positive bacteria, including staphylococci,
streptococci, and enterococci (8,9). The emergence of these resis-
tant bacteria has created a major concern and an urgent need for
antibacterial agents in structural classes distinct from the known
antibacterial agents.
The synthetic pathway for preparation of 1H-benzimidazoles 11–18
is shown in Scheme 1. The reaction of 1 with sodium bisulfite
adduct of 3,4-dichlorobenzaldehyde gave methyl 1-(4-chloro-benzyl)-
2-(3,4-dichlorophenyl)-1H-benzimidazole-5-carboxylate, 4. 1H-Benz-
imidazole-carboxylic acids 5–7 were prepared from alkaline
hydrolysis of esters 2–4. These acids were converted into acyl
chlorides with SOCl2, and reaction between the corresponding acyl
chlorides and 3-amino-propionitrile fumarate salt resulted in the
required benzamides 8–10. The CN group of these benzamides
was converted into aliphatic amidine by using well-known Pinner
method, 11–18. Nucleophilic displacement of the chloro group of
2,4,5-trichloronitrobenzene with 1-naphtylmethylamine in DMF gave
19 (Scheme 2). Reduction of 19 with Zn ⁄ HCl produced 20. Cycliza-
tion of 20–22 with Na2S2O5 adduct of 3-carboxybenzaldehyde
afforded 1H-benzimidazolecarboxylic acids 23–25. These com-
pounds were converted into the required acyl chlorides and reacted
with corresponding amines yielding benzamides 26–30. Compound
31 was prepared by cyclization of 22 and sodium bisulfite adduct
of 4-cyanobenzaldehyde. Next, the cyano group in 31 was con-
verted into the imidate ester, using the Pinner method, and the
In our previous studies, we have reported the synthesis of benzimi-
dazoles I (10) and II (11) carrying amide or amidine (12) functional-
ities at different positions (Figure 1) and their promising
antimicrobial activities. Additionally, antibacterial activity of a series
of 2-aminobenzimidazole dimers III (13) with multiple chloro substit-
uents was described. Recently, a new class of bis-benzimidazole di-
237

Karatas
¸
et al.
Cl
Cl
Cl
Cl
O
N
N
N
H
N
Cl
N
N
N
H
(C2H5)2N
I
O
II
H
N
H
Cl
Cl
Cl
Cl
N
N
N
H
N
H
N
H
N
III
NH
H
NH
N
N
H
N
N
N
H
N
H
IV
HO
H
N
Figure 1: Structures of pre-
viously reported benzimidazoles
possessing antibacterial activities..
H2N
N
NH
V
Cl
Cl
Cl
NH
NaO3S
HO
N
DMF
135 0C
CH3
O
CH3
O
+
Cl
Cl
N
NH2
Cl
O
O
4
1
R
N
R
N
3-Aminopropionitrile
fumarate salt
R1
O
NaOH/H2O
Cl
Cl
HO
N
N
Cl
Cl
O
O
5 R= H
2 R1= Et R= H
6 R= n-butyl
3 R1= Me R= n-butyl
7 R= p-Cl-benzyl
4 R1= Me R= p-Cl-benzyl
R
R'
H2N
R
N
11
12
13
H
H
H
H
N
Cl
Cl
NH
N
NC
H
N
O
8 R= H
9 R= n-butyl
10 R= p-Cl-benzyl
NH
H
N
dry HCl gas/abs. EtOH
N
H
N
R
N
14 n-butyl
H
N
Cl
NH
H
C2H5O
N
N
Cl
15 n-butyl
NH
O
NH
(not isolated)
H
N
corresponding amines
16 n-butyl
N
R
N
H
N
H
N
17 p-Cl-benzyl
18 p-Cl-benzyl
Cl
N
R'
NH
Cl
Scheme 1: Synthesis of benz-
imidazoles with aliphatic carbox-
amidines (11–18).
H
N
O
11 - 18
NH
238
Chem Biol Drug Des 2012; 80: 237–244

Potent 1H-Benzimidazoles as anti-MRSA agents
1-naphtyl methyl
amine
Cl
Cl
Cl
Cl
Cl
NH
Zn/HCl
Cl
Cl
NH
NO2
NO2
NH2
20
19
R1
NH
R1
Cl
Cl
Cl
Cl
N
N
HO
SOCl2
DMF
+
135 °C
H2NCH2CH2R2
NaO3S
NH2
O
O
20 R1= 1-naphtyl
21 R1= phenyl
23 R1= 1-naphtyl
24 R1= phenyl
HO
HO
22 R1= p-Cl-phenyl
25 R1= p-Cl-phenyl
R1
26 R1= 1-naphtyl
27 R1= phenyl
R2= NHEt
R2= NHEt
Cl
Cl
N
N
28 R1= p-Cl-phenyl R2= NHEt
29 R1= p-Cl-phenyl R2= NH-isopropyl
30 R1= p-Cl-phenyl R2= NH(Me)2
R2
N
H
O
Cl
Cl
Cl
Cl
NH
Cl
Cl
HO
N
N
DMF
dry HCl gas
abs. EtOH
+
CN
CN
135 °C
NaO3S
NH2
31
22
R
Cl
Cl
R'
Cl
Cl
Cl
Cl
OC2H5
NH
NH
NH
N
N
N
N
Scheme 2: Synthesis of 1H-
benzimidazoles with carboxamides
(26–30) and carboxamidines
(32–33).
not isolated
32 R= Cl R'=H
33 R= Cl R'=Cl
imidate ester was directly used without characterization to make
Methyl 1-(4-chlorobenzyl)-2-(3,4-
the corresponding aromatic amidines 32–33.
dichlorophenyl)-1H-benzimidazole-5-carboxylate
4
3,4-Dichlorobenzaldehyde (15 mmol) was dissolved in 50 mL EtOH.
Sodium bisulfite (1.6 g) in water (10 mL) was added to a cooled
solution in portions, stirred vigorously, and EtOH was added and
cooled. The precipitate was filtered off and dried (yield 91%). The
mixture of this salt (3 mmol) and compound 1 (0.87 g, 3 mmol) in
DMF (3 mL) was stirred at 135 ꢀC for 4 h. The reaction mixture
was cooled, poured into the water, and the solid was filtered and
recrystallised from EtOH. Yield 69%, mp 170–173 ꢀC, 1H-NMR
(CDCl3) d: 3.96(s,3H), 5.4(s,2H), 6.99(d,2H,J = 8.4), 7.24(s,1H),
7.33(d,2H,J = 8.4), 7.44(dd,1H,J = 8.4,J = 1.2), 7.53(d,1H,J = 8.4),
7.8(d,1H,J = 1.6), 8.03(d,1H, J = 8.4), 8.57(s,1H), MS (ESI+) m ⁄ z (rel
intensity): 445(M + H, 100), 447(M + H + 2, 98), 449(M + H + 4,30),
C22H15Cl3N2O2.
Experimental
Uncorrected melting points were measured on a Bꢀchi B-540 capil-
lary melting point apparatus. 1H (400 MHz) and 13C (100 MHz)
NMR spectra were recorded employing a Varian Mercury 400 MHz
FT spectrometer, chemical shifts (d) are in ppm relative to tetra-
methylsilane (TMS), and coupling constants (J) are reported in
Hertz. Mass spectra were taken on a Waters Micromass ZQ con-
nected with Waters Alliance HPLC, using ESI(+) method, with C-18
column. Elemental analyses were performed by Leco CHNS-932.
The compounds are reported as salts frequently analyzed correctly
for fractional moles of water and ⁄ or ethanol of solvation; proton
NMR confirmed the presence of solvent. All the reagents and sol-
vents were purchased from Aldrich Chemical Co. or Fischer Scien-
tific. Compounds 1(11), 2 (12), 3 (18), 5 (12), 21 (19), and 22 (10)
were synthesized as described in literature. For the HCl salts, the
oily free base was dissolved in EtOH and dry HCl gas was passed
through the solution.
1-Butyl-2-(3,4-dichlorophenyl)-1H-
benzimidazole-5-carboxylic acid 6
Compound 3 (0.754 g, 2 mmol) was dissolved in 3% NaOH in 50%
EtOH–HOH (10 mL) and heated under reflux for 1 h and cooled;
Chem Biol Drug Des 2012; 80: 237–244
239

Karatas¸ et al.
water and acetic acid were added to obtain the carboxylic acid,
which was filtered, and recrystallized from EtOH. Yield 87%, mp
186–187 ꢀC, H-NMR (DMSO-d6) d: 0.69(t,3H), 1.06(q,2H), 1.56(q,2H),
= 1.6), 8.31(s,1H), 8.94(t,1H), MS (ESI+) m⁄ z (rel intensity): 483(M + H,
100), 485(M + H + 2, 78), 487(M + H + 4, 15), C24H17Cl3N4O.
1
4.25(t,2H), 7.68–7.72(m,4H), 7.99(d,1H), 8.18(d,1H), MS (ESI+) m ⁄ z
(rel intensity): 363(M + H, 100), 365(M + H + 2, 65), 367(M + H + 4,
11), C18H16Cl2N2O2.
General synthesis of 11–18, 32, 33
Compounds 8–10 and 31 (0.5 mmol) were suspended in abso-
lute EtOH and cooled in an icesalt bath, and dry HCl gas was
passed through the solution for 20 min. The solution was stirred
in a stoppered flask at room temperature for 3 h and then diluted
with dry ether. The imidate esters were precipitated, washed with
ether and dried under vacuum at room temperature. All imidate
esters were used directly without characterization. A suspension
of the imidate ester HCl in absolute EtOH was stirred with the
corresponding amine (1.5- to twofold excess) overnight at 25–
30 ꢀC. The reaction mixture was evaporated and diluted with
ether; the precipitate was filtered, washed with ether, and then
dried.
1-(4-Chlorobenzyl)-2-(3,4-dichlorophenyl)-1H-
benzimidazole-5-carboxylic acid 7
Compound 4 (0.89 g, 2 mmol) was dissolved in 3% NaOH in 50%
EtOH–HOH (10 mL) and heated under reflux for 1 h and cooled;
water and acetic acid were added to obtain the carboxylic acid, fil-
1
tered, and recrystallized from EtOH, yield 95%, mp 242–244 ꢀC, H-
NMR (DMSO-d6) d: 5.61(s,2H), 6.99(d,2H), 7.33(d,2H), 7.53(d,1H),
7.66(dd,1H), 7.77(d,1H), 7.88(d,1H), 7.95(d,1H), 8.27(s,1H), MS (ESI+)
m ⁄ z (rel intensity): 431(M + H, 100), 433(M + H + 2, 97),
435(M + H + 4, 29), C21H13Cl3N2O2.
N-(3-Amino-3-iminopropyl)-2-(3,4-
dichlorophenyl)-1H-benzimidazole-5-
General synthesis of 8–10
carboxamide 11
Compounds 5–7 (1 mmol) were refluxed in benzene (5 mL) with
SOCl2 (5 mL) for 2 h at 80 ꢀC (only for compound 8, 0.5 mL DMF
was added). Then, solvent and excess of SOCl2 were evaporated
completely, and the residue was dissolved in chloroform. 3-Amino-
propionitrile fumarate salt (0.256 g, 1 mmol) and triethylamine
(0.202 g, 2 mmol) were added, and mixture was stirred at room
temperature until the starting materials were used up, and then
solvent was evaporated.
The residue was purified by column chromatography using chloro-
form ⁄ methanol ⁄ ammonium hydroxide 25% (11:4:1) as eluant, yield
10%, mp 220–222 ꢀC (dec), 1H-NMR (CD3OD) d: 2.79(t,2H),
3.77(t,2H), 7.64(d,1H,J = 8), 7.71(d,1H,J = 8.2), 7.79(dd,1H,J = 8.8,
J = 1.2), 8.00(dd,1H, J = 8.4, J = 2.2), 8.14(s,1H,J = 1.6),
8.26(s,1H,J = 1.8), 13C-NMR (CD3OD) d: 169.8, 169.5, 151.9, 141.0,
139.3, 134.5, 133.2, 131.3, 129.7, 128.9, 128.6, 126.3, 122.5,
115.36, and 114.39 (Both of them are br.s, as the tautomeric
effect of imidazole ring, C-4a and C-7a), 37.1, 33.1, MS (ESI+)
m ⁄ z (rel intensity): 376(M + H, 90), 378(M + H + 2, 53), 229(100),
C17H15Cl2N5O.
N-(2-cyanoethyl)-2-(3,4-dichlorophenyl)-1H-
benzimidazole-5-carboxamide 8
The residue was purified by column chromatography using chloro-
form ⁄ isopropanol ⁄ isopropyl-amine (40:5:1) as eluant. yield 60%,
mp 257–260 ꢀC, H-NMR (DMSO-d6) d: 2.82(t,2H), 3.53(q,2H), 7.62–
8.25(m,5H), 8.4(s,1H), MS (ESI+) m ⁄ z (rel intensity): 359(M + H,100),
361(M + H + 2,65), 363(M + H + 4,11), C17H12Cl2N4O.
2-(3,4-Dichlorophenyl)-N-{3-imino-3-[(1-
methylethyl)amino]propyl}-1H-benzimidazole-5-
carboxamide 12
1
The residue was purified by column chromatography using dichlo-
romethane ⁄ methanol ⁄ ammonium hydroxide 25% (8:4:1) as elu-
1
ant, yield 53%, mp 213–214 ꢀC, H-NMR (DMSO-d6) d: 1.10(d,6H),
2.55(t,2H), 3.55(q,2H), 3.73(m,1H), 7.43(m,2H), 7.63(d,1H,J = 8.4),
7.99(s,1H), 8.17(dd,1H,J = 8.4, J = 1.7), 8.34(t,1H), 8.37(d,1H,J = 1.7),
MS (ESI+) m ⁄ z (rel intensity): 418(M + H, 100), 420(M + H + 2, 63),
422(M + H + 4,10), Anal for C20H21Cl2N5O, Calc. C, 56.2, H, 5.18, N,
16.4 Found C, 56.54, H, 5.08, N,15.9.
1-Butyl-N-(2-cyanoethyl)-2-(3,4-dichlorophenyl)-
1H-benzimidazole-5-carboxamide 9
The residue was washed with dilute sodium carbonate solution and
water and recrystallised from EtOH, yield 35%, mp 200–202 ꢀC,
1H-NMR (DMSO-d6) d: 0.67(t,3H), 1.06(m,2H), 1.56(m,2H), 2.72(t,2H),
3.43(q,2H), 4.26(t,2H), 7.71(m,2H), 7.79(m,2H), 7.99(d,1H,J = 2),
8.16(d,1H,J = 1.2), 8.80(t,1H), MS (ESI+) m ⁄ z (rel intensity): 415(M
+ H, 100), 417(M + H + 2, 65), 419(M + H + 4, 10), C21H20Cl2N4O.
2-(3,4-Dichlorophenyl)-N-[2-(1,4,5,6-
tetrahydropyrimidin-2-yl)ethyl]-1H-
benzimidazole-5-carbox-amide 13
The residue was purified by column chromatography using chloro-
form ⁄ methanol ⁄ ammonium hydroxide 25% (11:4:1) as eluant, yield
35%, mp 168–170 ꢀC (bubbling), 1H-NMR (CD3OD) d: 1.9(m,2H),
2.59(t,2H), 3.31(t,4H), 3.64(t,2H), 7.56(d,1H,J = 8.4), 7.64(d,1H,J = 8.4),
7.68(dd,1H, J = 8.8, J = 1.6), 7.93(dd,1H,J = 8, J = 2), 8.05(s,1H),
8.19(s,1H,J = 1.8), MS (ESI+) m ⁄ z (rel intensity): 416(M + H, 100),
418(M + H + 2, 68), 420(M + H + 4,12), C20H19Cl2N5O.
1-(4-Chlorobenzyl)-N-(2-cyanoethyl)-2-(3,4-
dichlorophenyl)-1H-benzimidazole-5-
carboxamide 10
The residue was washed with dilute sodium carbonate solution and
water and recrystallised from EtOH, yield 65%, mp 210 ꢀC, H-NMR
1
(DMSO-d6) d: 2.82(t,2H), 3.53(q,2H), 5.66(s,2H), 7.01(d,2H,J = 8.4),
7.36(d,2H,J = 8.4), 7.66(d,1H), 7.71(dd,1H), 7.83(m,2H), 7.97(d,1H,J
240
Chem Biol Drug Des 2012; 80: 237–244

Potent 1H-Benzimidazoles as anti-MRSA agents
1-Butyl-2-(3,4-dichlorophenyl)-N-{3-imino-3-[(1-
methylethyl)amino]propyl}-1H-benzimidazole-5-
carboxamide 14
131.5, 131.2, 129.2, 129.1, 128.2, 126.3, 124.5, 116.8, 112.4, 48.3,
45, 37.5, 33.9, 20.3, MS (ESI+) m ⁄ z (rel intensity): 542(M + H, 100),
544(M + H + 2, 95), 546(M + H + 4, 40), Anal for C27H26Cl3N5OÆH-
ClÆ2HOH, Calcd, C, 52.69, H, 5.08, N, 11.38, Found C, 52.8, H, 5.02,
N, 10.93.
The residue was purified by column chromatography using dichlo-
romethane ⁄ methanol ⁄ ammonium hydroxide 25% (16:4:1) as eluant,
yield 61%, mp 123–124 ꢀC (bubbling), 1H-NMR (CD3OD) d:
0.84(t,3H), 1.22(m, overlapped,8H), 1.75(m,2H), 2.74(t,2H), 3.77
(m,overlapped,3H), 4.37(t,2H), 7.71(m,2H), 7.78(d,1H,J = 8.4),
7.9(dd,1H,J = 8.8, J = 1.2), 7.95(d,1H,J = 1.8), 8.22(s,1H), 13C-NMR
(CD3OD + one drop D2O) d: 169.6, 164.6, 153.3, 141.7, 137.9, 134.8,
133.1, 131.3, 131.2, 129.9, 128.99, 128.9, 122.9, 118.56, 111.3, 45,
44.6, 37.4, 33.8, 31.5, 20.4, 19.5, 12.6, MS (ESI+) m ⁄ z (rel intensity):
474(M + H, 100), 476(M + H + 2, 64), 478(M + H + 4,12), Anal for
C24H29Cl2N5O. 3.5 HOH, Calcd. C, 53.63 H, 6.75, N, 13.03, Found C,
53.69, H, 6.74, N, 12.46.
1-(4-Chlorobenzyl)-2-(3,4-dichlorophenyl)-N-[3-
(butylamino)-3-iminopropyl]-1H-benzimidazole-
5-carboxamide HCl 18
The residue was purified by column chromatography using chloro-
form ⁄ methanol ⁄ ammonium hydroxide 25% (11:4:1) as eluant, yield
17%, mp 168–170 ꢀC (bubbling), 1H-NMR (CD3OD) d: 0.89(t,3H),
1.38(m,2H), 1.59(m,2H), 2.79(t,2H), 3.24(q,2H), 3.79(t,2H), 5.7(s,2H),
7.19(d,2H,J = 8.4), 7.37(d,2H,J = 8.4), 7.78(dd,1H,J = 8.8,J = 1.8),
7.88(m,2H), 8.06(d,1H,J = 1.8), 8.13(d,1H,J = 8.8), 8.45(s,1H),
8.58(br.s), 9.12(br.s), 9.39(t,1H), 13C-NMR (CD3OD) d: 167.5, 165.9,
151.2, 137.8, 135.4, 134.5, 133.9, 132.9, 132.9, 132.6, 131.9, 131.8,
129.4, 129.3, 128.5, 125.8, 123.3, 115, 113.5, 48.9, 42.4, 37.6, 33.5,
29.4, 19.9, 12.8. MS (ESI+) m ⁄ z (rel intensity): 556(M + H, 100),
1-Butyl-2-(3,4-dichlorophenyl)-N-{3-imino-3-[(3-
methylbutyl)amino]propyl}-1H-benzimidazole-5-
carboxamide 15
558(M + H + 2,
95),
560(M + H + 4,
43),
Anal
for
The residue was purified by column chromatography using di-
chloromethane ⁄ methanol ⁄ ammonium hydroxide 25% (16:4:1) as
eluant, yield 27%, mp 121–123 ꢀC(bubbling), 1H-NMR (CD3OD)
d: 0.832(t,3H), 0.87(d,6H), 1.21(m,2H), 1.49(q,2H), 1.61(m,1H),
1.75(m,2H), 2.77(t,2H), 3.25(t,2H), 3.77(t,2H), 4.38(t,2H), 7.71(m,2H),
7.78(d,1H,J = 8.4), 7.91(dd,1H,J = 8.4, J = 1.2), 7.96(d,1H,J = 1.8),
8.22(d,1H,J = 1.6), 13C-NMR (CD3OD) d: 169.3, 166, 153.3, 141.8,
138, 134.7, 133.1, 131.3, 131.2, 130.1, 128.9, 122.9, 118.6,
111.1, 44.6, 41, 37.4, 36.1, 33.7, 31.5, 25.6, 21.4, 19.6, 12.6,
MS (ESI+) m ⁄ z (rel intensity): 502(M + H, 100), 504(M + H + 2,
69), 506(M + H + 4,11), Anal for C26H33Cl2N5O. 3.7 HOH, Calcd.
C, 54.86 H, 7.15, N, 12.30, Found C, 54.83, H, 6.77, N, 12.21.
C28H28Cl3N5O.2HCl.2HOH, Calcd, C, 50.5, H, 5.14, N,10.52, Found C,
50.48, H, 5.39, N, 10.29.
4,5-Dichloro-N-(naphthalen-1-ylmethyl)-2-
nitroaniline 19
To a solution of 1,2,4-trichloro-5-nitrobenzene (22.2 mmol, 5 g) in
DMF (3 mL), 1-naphthylmethylamine (7 mL) was added and heated
for 1 h at 80 ꢀC. The hot reaction mixture was allowed to crystal-
lize from EtOH. Second time crystallization of crude product from n-
hexane gave pure 19, yield 77%, mp 143–144 ꢀC, yellow in color,
1H-NMR (CDCl3) d: 4.91(d,2H,J = 5.2), 7.04(s,1H), 7.45(m,2H),
7.56(m,2H), 7.86(m,1H), 7.93(m,2H), 8.29(t,1H), 8.32 (s,1H),
C17H12Cl2N2O2.
1-Butyl-2-(3,4-dichlorophenyl)-N-[2-(1,4,5,6-
tetrahydropyrimidin-2-yl)ethyl]-1H-
benzimidazole-5-carboxamide 16
The residue was purified by column chromatography using dichlo-
romethane ⁄ methanol ⁄ ammonium hydroxide 25% (16:4:1) as elu-
ant, yield 22%, mp 210–212 ꢀC, 1H-NMR (CD3OD) d: 0.84(t,3H),
1.21(m,2H), 1.75(m,2H), 1.99(m,2H), 2.69(t,2H), 3.41(t,4H),
3.74(t,2H), 4.38(t,2H), 7.72(m,2H), 7.79(d,1H,J = 8), 7.9(dd,1H,J
= 8.8,J = 1.6), 7.96(d,1H,J = 1.6), 8.21(d,1H,J = 1.6), 13C-NMR
(CD3OD) d: 169.5, 162.49, 153.3, 141.8, 137.9, 134.7, 133.1,
131.3, 131.2, 130.1, 129.05, 128.9, 122.8, 118.5, 111.1, 44.6,
38.8, 37.2, 33.8, 31.5, 19.5, 17.6, 12.6, MS (ESI+) m ⁄ z (rel inten-
sity): 472(M + H, 100), 474(M + H + 2, 69), 476(M + H + 4,11),
C24H27Cl2N5O.
4,5-Dichloro-N1-(naphthalen-1-
ylmethyl)benzene-1,2-diamine 20
To a solution of 19 (0.45 g, 5 mmol) in EtOH (30 mL), Raney Ni
(100 mg) was added, and the solution hydrogenated at room tempera-
ture at 40 psi. The reaction was stopped after cessation of H2 uptake.
The catalyst was filtered through a bed of Celite, washed with EtOH,
and concentrated to provide brown colored solid product, mp 147–
148 ꢀC, yield 71%, 1H-NMR (CDCl3) d: 3.27(br.s,2H), 3.68(br.s,1H),
4.65(s,2H), 6.77(s,1H), 6.81(s,1H), 7.4–7.55(m,4H), 7.84(d,1H),
7.9(m,1H), 8.04(m,1H), MS (ESI+) m ⁄ z (rel intensity): 317(M + H, 46),
319(M + H + 2, 31), 321(M + H + 4, 5), 141(100), C17H14Cl2N2.
1-(4-Chlorobenzyl)-2-(3,4-dichlorophenyl)-N-{3-
imino-3-[(1-methylethyl)amino]propyl}-1H-
General synthesis of 23–25
benzimidazole-5-carboxamide 17
3-Carboxybenzaldehyde (15 mmol) was dissolved in 50 mL EtOH.
Sodium bisulfite (1.6 g) in water (10 mL) was added to a cooled
solution in portions, stirred vigorously, and EtOH was added and
cooled. The precipitate was filtered off and dried, yield 90%. The
mixture of this salt (2 mmol) and appropriate 1,2-phenylenediamines
(20–22, 2 mmol) in DMF (2–3 mL) was heated at 135 ꢀC for 4 h.
The reaction mixture was cooled and poured into water, and the
solid was collected and recrystallized from EtOH.
The residue was purified by column chromatography using chloro-
form ⁄ methanol ⁄ ammonium hydroxide 25% (16:4:1) as eluant, yield
29%, mp 162–164 ꢀC (bubbling), 1H-NMR (CD3OD) d: 1.22(d,6H),
2.74(t,2H), 3.77(m,overlapped,3H), 5.7(s,2H), 7.1(d,2H,J = 8.4), 7.34(d,2H,J
= 8.4), 7.69(m,2H), 7.78(d,1H,J = 8.4), 7.96(m,2H), 8.35(s,1H),
8.58(br.s), 8.98(t,1H), 9.1(br.s), 9.3 and 9.32(s,s), 13C-NMR (CD3OD) d:
168.3, 164.7, 152.4, 137.1, 136.8, 136.4, 134.2, 133.9, 133.5, 131.5,
Chem Biol Drug Des 2012; 80: 237–244
241

Karatas¸ et al.
3-[5,6-dichloro-1-(1-naphthylmethyl)-1H-
benzimidazol-2-yl]benzoic acid 23
J = 8.4), 8.18(s,1H), 13C-NMR (CD3OD) d: 168, 155.7, 141.7, 135.9,
135.4, 135.4, 132, 129.5, 129.3, 129.2, 128.9, 128.4, 127.9, 127.3,
126.9, 126.2, 120.1, 112.8, 48.2, 43.3, 39.3, 13.5, MS (ESI+) m ⁄ z
(rel intensity): 467(M + H, 100), 469(M + H + 2, 61), 471(M + H + 4,
10), Anal for C25H24Cl2N4OÆEtOH (1H-NMR visible) Calc. C, 63.26, H,
5.90, N, 10.97, Found C, 63.04, H, 5.96, N, 11.11.
Yield 89%, mp 304–306 ꢀC, 1H-NMR (DMSO-d6) d: 6.1(s,2H),
6.54(d,1H,J = 7), 7.32(t,1H,J = 8), 7.51(t,1H,J = 7.8), 7.57(m,2H),
7.84(m,2H), 7.87(s,1H), 7.96(m,2H), 8.05(m,1H), 8.09(s,1H), 8.27(s,
1H), MS (ESI+) m ⁄ z (rel intensity): 447(M + H, 100), 449(M + H + 2,
61), 451(M + H + 4,12), C25H16Cl2N2O2.
3-[5,6-Dichloro-1-(4-chlorobenzyl)-1H-
benzimidazol-2-yl]-N-(2-
ethylaminoethyl)benzamide 28
3-(1-Benzyl-5,6-dichloro-1H-benzimidazol-2-
yl)benzoic acid 24
Yield 86%, mp 271–273 ꢀC, 1H-NMR (DMSO-d6) d: 5.62(s,2H),
6.94(d,2H), 7.24(m,3H), 7.62(t, 1H,J = 8), 7.93(s,1H), 7.95(s,1H),
8.02(s,1H), 8.07(d,1H,J = 7.6), 8.26(s,1H), MS (ESI+) m ⁄ z (rel inten-
sity): 397(M + H, 100), 399(M + H + 2, 58), 401(M + H + 4,10),
C21H14Cl2N2O2.
Yield 46%, mp 193–195 ꢀC, 1H-NMR (CD3OD) d: 1.13(t,3H),
2.69(q,2H), 2.80(t,2H), 3.52(t,2H), 5.56(s,2H), 7.00(d,2H,J = 8),
7.29(d,2H,J = 8.2), 7.64(t,1H,J = 7.6), 7.71(s,1H), 7.82(td, 1H,J = 8),
7.89(s,1H), 8.02(td,1H,J = 8), 8.15(t,1H), MS (ESI+) m ⁄ z (rel inten-
sity): 501(M + H, 60), 503(M + H + 2, 59), 125(100), Anal for
C25H23Cl3N4O, Calcd. C, 59.83, H, 4.62, N, 11.16, Found C, 59.71, H,
4.72, N, 11.1
3-[5,6-dichloro-1-(4-chlorobenzyl)-1H-
benzimidazol-2-yl]benzoic acid 25
Yield 89%, mp 320–322 ꢀC, 1H-NMR (DMSO-d6) d: 5.63(s,2H),
3-[5,6-Dichloro-1-(4-chlorobenzyl)-1H-
benzimidazol-2-yl]-N-(2-
6.98(d,2H,J = 8.4),
7.34(m,2H,
J = 8.4),
7.65(t,1H,J = 7.2),
7.94(d,1H,J = 8), 7.97(s,1H), 8.05(s,1H), 8.07(d,1H,J = 8), 8.24(s,1H),
MS (ESI+) m ⁄ z (rel intensity): 431(M + H, 100), 433(M + H + 2, 98),
435(M + H + 4,30), C21H13Cl3N2O2.
isopropylaminoethyl)benzamide 29
Yield 48%, mp 190–191 ꢀC1H-NMR (DMSO-d6) d: 0.94(d,6H),
2.65(t,2H), 2.71(m,1H), 3.31(q,2H), 5.63(s,2H), 6.96(d,2H, J = 8.4),
7.30(d,2H,J = 8.4),
7.59(t,1H,J = 7.6),
7.80(d,1H,J = 7.4Hz),
7.97(m,3H), 8.19(s,1H), 8.56(t, 1H), 13C-NMR (DMSO-d6) d: 166.1,
155.7, 142.8, 136.2, 135.98, 135.95, 132.9, 131.9, 129.92, 129.75,
129.63, 129.5, 128.8, 128.7, 126.2, 125.8, 121.3, 113.6, 48.6, 47.8,
46.8, 40.5, 23.5, MS (ESI+) m ⁄ z (rel intensity): 515(M + H, 80),
517(M + H + 2, 79), 125(100), Anal for C26H25Cl3N4OÆ0.5 HOH,
Calcd. C, 59.49, H, 4.99 N, 10.67, Found C, 59.64, H, 5.25, N,
10.54.
General synthesis of 26–30
Related benzoic acids 23–25 (1 mmol) were refluxed in benzene
(5 mL) with SOCl2 (5 mL) for 2 h at 80 ꢀC. Then, the solvent and
excess of SOCl2 were evaporated completely. The residue was dis-
solved in chloroform (15 mL) and mixed with excess of corresponding
amine (1 mL) for 30 min at 50 ꢀC. Chloroform was added to the reac-
tion mixture, washed with Na2CO3 (5%), then with water, and purified
by column chromatography. The mixture of chloroform ⁄ metha-
nol ⁄ ammonium hydroxide 25% (11:4:1) was used as eluant.
3-[5,6-Dichloro-1-(4-chlorobenzyl)-1H-
benzimidazol-2-yl]-N-(2-
dimethylaminoethyl)benzamide 30
3-(5,6-Dichloro-1-naphthalen-1-ylmethyl-1H-
benzimidazol-2-yl)-N-(2-
ethylaminoethyl)benzamide 26
Yield 39%, mp 164–165 ꢀC, 1H-NMR (CD3OD) d: 2.31(s,6H),
2.56(t,2H), 3.52(t,2H), 5.57(s,2H), 7.00(d,2H,J = 8.8), 7.29(d,
2H,J = 8.8), 7.64(t,1H,J = 8), 7.71(s,1H), 7.81(td,1H,J = 8), 7.88(s,1H),
8.02(td,1H,J = 8), 8.15(t,1H), 13C-NMR (CD3OD) d: 167.8, 155.7,
141.7, 135.4, 135.3, 134.8, 133,7, 132.1, 129.4, 129.3, 129.2, 129,
128.2, 127.9, 127.4, 127, 120.2, 112.7, 57.9, 44.3, 37.4, MS (ESI+)
m ⁄ z (rel intensity): 501(M + H, 100), 503(M + H + 2, 89), Anal for
C25H23Cl3N4O, Calcd. C, 59.83 H, 4.62 N, 11.16 Found C, 59.7 H,
4.59 N, 11.13.
Yield 55%, mp 183–185 ꢀC, 1H-NMR (DMSO-d6) d: 0.97(t,3H),
2.52(t,2H), 2.58(t,2H), 3.28(q,2H), 6.1(s,2H), 6.54(d,1H), 7.33(t,1H),
7.46(t,1H), 7.59 (m,2H), 7.71(d,1H), 7.84(d,1H), 7.89–8.09(m,5H),
8.28(s,1H), 8.49(t,1H), 13C-NMR (DMSO-d6) d: 166, 156, 142.8,
136.6, 136, 133.9, 132.5, 131.3, 130.4, 129.95, 129.7, 129.5, 129.3,
128.9, 128.7, 127.2, 127, 126.3, 126.2, 125.9, 123.7, 122.8, 121.3,
113.7, 49.1, 46.9, 43.9, 40.2, 15.9, MS (ESI+) m ⁄ z (rel intensity):
517(M + H, 50), 519(M + H + 2, 33), 521(M + H + 4, 6), 141(100),
Anal for C29H26Cl2N4OÆ0.25 HOH, Calc. C, 66.73, H, 5.12, N, 10.73,
Found C, 66.79, H, 5.37, N, 10.84.
4-[5,6-Dichloro-1-(4-chlorobenzyl)-1H-
benzimidazol-2-yl]benzonitrile 31
4-Cyanobenzaldehyde (5 mmol) was dissolved in 20 mL EtOH.
Sodium bisulfite (0.533 g) in water (3 mL) was added to a cooled
solution in portions, stirred vigorously and EtOH was added and
cooled. The precipitate was filtered off and dried, yield 97%. The
mixture of this salt (2 mmol) and 22 (2 mmol) in DMF (2–3 mL)
was heated at 135 ꢀC for 4 h. The reaction mixture was cooled
and poured into the water, and solid was collected and recrystallized
3-(1-Benzyl-5,6-dichloro-1H-benzimidazol-2-yl)-
N-(2-ethylaminoethyl)benzamide 27
Yield 49%, mp 159–160 ꢀC, 1H-NMR (CD3OD) d: 1.13(t,3H),
2.66(m,2H), 2.8(t,2H), 3.53(t,2H), 5.6(s,2H), 7.01(d,2H,J = 7.6),
7.28(m,3H), 7.64(m,2H), 7.81(d,1H,J = 8), 7.87(s,1H), 8.03(d,1H,
242
Chem Biol Drug Des 2012; 80: 237–244

Potent 1H-Benzimidazoles as anti-MRSA agents
from EtOH, yield 66%, mp 268 ꢀC, 1H-NMR (DMSO-d6) d:
5.66(s,2H), 6.99(d,2H,J = 8), 7.33(d,2H,J = 8), 7.91(d,2H,J = 8.4),
8.01(d, 2H,J = 8.4), 8.03(s,1H), 8.09(s,1H), 13C-NMR (DMSO-d6) d:
47.8, 113.5, 113.7, 118.9, 121.6, 126.1, 126.6, 128.7, 129.5, 130.6,
132.9, 133.5, 134.3, 135.8, 136.4, 142.7, 154.4, MS (ESI+) m ⁄ z (rel
intensity): 412(M + H, 100), 414(M + H + 2, 95), C21H12Cl3N3.
Table 1: The in vitro antibacterial activity of the synthesized
compounds (MIC, lg ⁄ mL)
Antibacterial and antifungal activities
Candida
Staphylococcus
MRSA
albicans
C. krusei
No
aureus ATTC25923 ATTC43300 ATTC10231 ATCC6258
11
12
25
>50
25
>50
12.5
25
12.5
12.5
6.25
12.5
6.25
6.25
6.25
0.78
1.56
0.39
0.78
0.78
–
12.5
>50
25
25
6.25
12.5
6.25
6.25
3.12
25
25
25
25
25
12.5
25
12.5
12.5
25
N-(4-Chlorobenzyl)-4-[5,6-dichloro-1-(4-
12.5
12.5
12.5
25
12.5
6.25
12.5
25
25
12.5
12.5
25
6.25
6.25
–
–
–
chlorobenzyl)-1H-benzimidazol-2-yl]benzamidine
32
13
14
1
Yield 56%, mp 234–235 ꢀC H-NMR (DMSO-d6 + one drop D2O) d:
15
16
4.30(s,2H), 5.57(s,2H), 6.94(d,2H,J = 8), 7.31(t,4H), 7.4(d,2H,J = 8.4),
7.70(d,2H,J = 8.4), 7.84(s,1H), 7.91(d,2H,J = 8), 7.97(s,1H), MS (ESI+)
m ⁄ z (rel intensity): 553(M + H, 80), 555(M + H + 2, 100), Anal for
C28H20Cl4N4Æ2C3H8O (1H-NMR visible) Calcd. C, 60.69, H, 5.39, N,
8.33 Found C, 60.44, H, 5.37, N, 8.54.
17
18
26
27
12.5
25
28
29
6.25
3.12
6.25
0.78
1.56
50
25
0.78
12.5
12.5
12.5
3.12
6.25
–
–
–
64
30
32
N-(3,4-Dichlorobenzyl)-4-[5,6-dichloro-1-(4-
chlorobenzy l)-1H-benzimidazol-2-
yl]benzamidine 33
33
Ampicillin
Sultamicillin
Vancomycin
Fluconazole
1
Yield 42%, mp 214–216 ꢀC, H-NMR (DMSO-d6 + one drop D2O) d:
4.32(s,2H), 5.67(s,2H), 6.99(d,2H,J = 8.4), 7.36(d,2H, J = 8.4),
7.42(d,1H,J = 8.4), 7.58(d,1H,J = 8.4), 7.66(s,1H), 7.77(d,2H,J = 8),
7.89(s,1H), 7.97(d,2H, J = 6.8), 8.05(s,1H), MS (ESI+) m ⁄ z (rel inten-
sity): 587(M + H, 60), 589(M + H + 2, 100), Anal for C28H19Cl5N4,
Calcd. C, 57.12, H, 3.25, N, 9.52 Found C, 56.97, H, 3.24, N, 9.52.
–
1.56
MRSA, methicillin-resistant Staphylococcus aureus.
(11–18) or amides (26–30) were less active than the benzimidaz-
oles having aromatic amidine group (32, 33). These results clearly
indicate that the aromatic amidine group of 1H-benzimidazole is
very important for antimicrobial activity.
Results and Discussion
All the benzimidazole compounds prepared herein were screened
for their potential in vitro antibacterial activities against S. aureus,
MRSA, and antifungal activities against C. albicans, C. krusei. The
in vitro minimal inhibitory concentrations (MIC100) of the com-
pounds were determined using micro-broth dilution method
reported by the National Committee for Clinical Laboratory Stan-
dard (20,21). Ampicillin, sultamicillin, vancomycin, and fluconazole
were used as references. The MIC100 results for the test com-
pounds are shown in Table 1. The synthesized compounds and ref-
In conclusion, we have identified the mono cationic aromatic ben-
zimidazolecarboxamidine compounds 32 and 33 as potent anti-
MRSA agents. It is predicted that the mechanism of action of these
compounds could be different from that of existing antibiotics,
because of the difference in chemical structures. Further investiga-
tion including in vivo studies is needed to establish the mechanism
of action.
erence drugs were dissolved in DMSO-H2O (50%) at
a
Acknowledgment
concentration of 200 lg ⁄ mL. The concentration was adjusted to
50 lg ⁄ mL by fourfold dilution with media culture and bacteria
solution (DMSO concentration was 12.5% in the first tube). Bacte-
rial and fungal tubes were incubated at 36 ꢀC for 18 and 48 h,
respectively. Among the 15 compounds tested against the Gram-
positive aerobic strains, six compounds 26, 28, 29, 30, 32, and
33 showed good antibacterial activity, including potent anti-MRSA
(MIC value £ 1 lg ⁄ mL) activity. Compounds 32 and 33 were
found to have moderate activity against Candida species. The rest
of the analogues (11–18, 27) showed poor or no antibacterial
and antifungal activity.
This work was supported by TUBITAK Project No: 106S202 (SBAG-
3450). Central Instrumental Analysis Lab. in Faculty of Pharmacy,
Ankara University provided the support for acquisition of the NMR,
Mass and elemental analysis data used in this work.
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