Design and synthesis of anti-MRSA benzimidazolylbenzenesulfonamides. QSAR studies for prediction of antibacterial activity

Article abstract of DOI:10.3390/molecules16010175

A series of benzimidazolylbenzenesulfonamide compounds containing electronreleasing and electron-withdrawing substituents were synthesized and tested for their in vitro antibacterial activity. Two BZS compounds showed strong antibacterial activity against methicillin-resistant Staphylococcus aureus and Bacillus subtilis. Quantitative studies of their structure-activity relationship using a simple linear regression analysis were applied to explore the correlation between the biological activity and the charges on acidic hydrogen atoms in the synthesized compounds.

Full text of DOI:10.3390/molecules16010175

Molecules 2011, 16, 175-189; doi:10.3390/molecules16010175
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Design and Synthesis of Anti-MRSA Benzimidazolylbenzene-
sulfonamides. QSAR Studies for Prediction of
Antibacterial Activity
Marco Martín González-Chávez 1,2,*, Francisco Méndez 3, Roberto Martínez 4,
Cuaúhtemoc Pérez-González 5 and Fidel Martínez-Gutiérrez 2
1
Programa de Doctorado en Ciencias Biológicas, Universidad Autónoma Metropolitana,
México D.F., Mexico
2
Facultad de Ciencias Químicas-CIEP, Universidad Autónoma de San Luis Potosí, San Luis Potosí,
SLP, Mexico
3
Departamento de Química, División de Ciencias Básicas e Ingeniería, Universidad Autónoma
Metropolitana, Unidad Iztapalapa, México D.F., Mexico
4
Instituto de Química, Universidad Nacional Autónoma de México, México D. F, Mexico
5
Departamento de Sistemas Biológicos, Universidad Autónoma Metropolitana, Unidad Xochimilco,
México D. F., Mexico
* Author to whom correspondence should be addressed; E-Mail: gcmm@uaslp.mx;
Tel.: +52 444 826 2440; Fax: +52 444 826 2372.
Received: 22 November 2010; in revised form: 24 December 2010 / Accepted: 28 December 2010 /
Published: 29 December 2010
Abstract: A series of benzimidazolylbenzenesulfonamide compounds containing electron-
releasing and electron-withdrawing substituents were synthesized and tested for their in
vitro antibacterial activity. Two BZS compounds showed strong antibacterial activity
against methicillin-resistant Staphylococcus aureus and Bacillus subtilis. Quantitative
studies of their structure-activity relationship using a simple linear regression analysis were
applied to explore the correlation between the biological activity and the charges on acidic
hydrogen atoms in the synthesized compounds.
Keywords: benzimidazole; sulphonamide; MRSA; antibacterial activity

Molecules 2011, 16
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1. Introduction
Drug resistance in Gram-positive bacteria is a worldwide public health problem with serious
morbidity and mortality consequences. For example, the incidence of penicillin-resistant Streptococcus
pneumoniae continues to increase worldwide, except in cities that apply good antibiotic use policies.
An additional problem is the emergence of multi-drug resistant strains in America, Europe and Asia
[1-3], for example, vancomycin resistance in Enterococcus. These infections have emerged as severe
complications, mainly in critically ill patients in tertiary care hospital wards [4,5]. Staphylococcus
aureus is one of the most prominent and widespread human pathogens, causing skin and tissue
infections, deep abscess formation, pneumonia, endocarditis, osteomyelitis, toxic shock syndrome and
bacteremia. Methicillin-resistant Staphylococcus aureus (MRSA) has been recognized as one of the
main pathogenic causes of nosocomial infections throughout the world [6,7]. Infections caused by
MRSA are a problem in health care institutions in the United States and worldwide, especially for
intensive care unit patients [8]. Solving the problem of drug resistance will depend partially on the
development of chemotherapeutic agents that selectively attack new bacterial targets.
Some benzimidazoles [9,11] and sulphonamides [12,13] have recently presented promising
antimicrobial activities. Benzimidazolylbenzenesulfonamides show a variety of pharmacological
activities due to their affinity toward various cellular protein targets or enzyme inhibitors, such as the
peptide-like thrombin inhibitor [14], NR2B-selective N-methyl-D-aspartate antagonists [15], inhibitors
of the enzyme 11-hydroxysteroid dehydrogenate [16], and the human immunodeficiency virus type I
integrase inhibitor [17].
Benzimidazolylbenzenesulfonamide (BZS) compounds fulfill the structural requirements for
affinity toward metalloproteinases: a ligand group that may be directed toward a metal center, a
sulfonamide group that may provide a hydrogen bonding interaction with the amino acids of the
enzyme backbone, and an aryl group that may have effective Van der Waals interactions with enzyme
subsites (Figure 1). Based on these considerations, we have focused on the synthesis of novel BZS
compounds in search of synthetic chemotherapeutic agents.
Figure 1. Design of BZS derivatives and the structure-activity relationships considered in
this study.
Sidechain SAR and
stereoelectronic effects
3
N
Metal binding group
NH
1
(R)n
2'
1'
O
HN
hydrogen-bond donor atom
S
O
hydrogen-bond acceptor atom
3'
BZS
group with affinity to enzime hydrophobic subsite

Molecules 2011, 16
177
The present work establishes the regio-chemical, stereo-chemical, and electronic requirements of
the benzimidazole ring of BZS for achieving activity toward Gram-positive bacteria, such as MRSA.
The electronic requirements are modulated by the presence (or absence) of electron-releasing or
electron-withdrawing substituents at positions 4, 5 and 7 of the benzimidazole ring of novel
BZS derivatives.
1.1. Chemistry
BZS compounds are usually obtained via benzimidazolamines. The synthesis of benzimidazol-
amines 10-13 proceeded from the corresponding R3-dinitroaniline compounds 1-4. The formation of
the imidazole ring was carried out by reductive cyclization with acetic acid and tin (II) chloride
dihydrate at reflux temperature, followed by acid hydrolysis in the presence of 10% hydrochloric acid
at reflux temperature. The primary amine of benzimidazolamine 14 was obtained by nitration of
benzimidazole 8 using a mixture of nitric and sulfuric acids, followed by reduction with tin (II)
chloride dihydrate in hydro-chloric acid at reflux temperature. Synthesis of benzimidazolamine 15
proceeded from the starting material nitroaniline 5. The second nitro group necessary for formation of
the imidazole ring was obtained by nitration of 5 with fuming nitric acid. Formation of the imidazole
ring was done by reductive cyclization, similar to the procedure for formation of the imidazole ring in
derivatives 10-13. Finally, the benzimidazolamine 15 was obtained by acid hydrolysis of the amide
group (Scheme 1).
Scheme 1. Synthesis of the benzimidazolamines 10-15.
NO2
NH2
NO2
NH2
N
R1
R2
NO2
R1
R2
NH
NO2
a,b
a,b
c
10 R1=H, R2=H
11 R1=H, R2=F
12 R1=Br, R2=H
13 R1=Cl, R2=H
14 R1=H, R2=Cl
15 R1=H, R2=OCH3
O
O
R3
NH2
HN
5
O
1 R1=H, R2=H, R3=NH2
2 R1=H, R2=F, R3=NO2
3 R1=Br, R2=H, R3=NO2
4 R1=Cl, R2=H, R3=NO2
6
e
N
Cl
.
NH2
N
NH
NH
NO2
a
d
O2N
Cl
Cl
8
9
7
Reagents and conditions: (a) SnCl2 2H2O/CH3CO2H, reflux; (b) 10% HCl, reflux; (c) fuming
.
HNO3, 0-5 °C; (d) H2SO4/HNO3, RT; (e) SnCl2 2H2O/HCl, reflux.
The BZS 16-21 were obtained by reaction of benzimidazolamines 10-15 with benzenesulfonyl
chloride in the presence of pyridine as a base and acetone as a solvent under N2 atmosphere, at room
temperature for 24 h. Benzimidazolamines are bidentate nucleophiles, and produced the BZS

Molecules 2011, 16
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derivatives 22 y 23 (Scheme 2). This type of products was not obtained after of purification when the
benzimidazole ring contained a halogen atom.
Scheme 2. Synthesis of the BZS 16-23.
O
N
N
N
R1
R2
R1
R2
N S O
NH
R1
R2
NH
a
+
O
O
HN
HN
NH2
S
S
22 R1=H, R2=H
16 R1=H, R2=H
17 R1=H, R2=F
O
O
10-15
23 R1=H, R2=OCH3
18 R1=Br, R2=H
19 R1=Cl, R2=H
20 R1=H, R2=Cl
21 R1=H, R2=OCH3
Reagents and conditions: (a) C6H5SO2Cl/pyridine, acetone, 24 h RT.
The derivatives of BZS that contained nitro groups, i.e., 24 and 26-31, were obtained from their 16-
21 precursors via nitration with fuming nitric acid. Only one mononitrated product was obtained in all
cases, with the exception of derivative 16. The BZS derivative containing an amino group were
obtained by reduction of the nitro group of 24 using tin (II) chloride dihydrate in a 1:1 ethyl
acetate/ethanol mixture (Scheme 3).
Compounds 10-26 were purified by column chromatography on silica gel using ethyl acetate/
hexane as the eluent. Compounds 27-31 did not require chromatographic purification.
Scheme 3. Synthesis of the BZSs 24-31.
24 R1=H, R2=H, R3=NO2
N
N
b
25 R1=H, R2=H, R3=NH2
26 R1=H, R2=NO2, R3=H
27 R1=H, R2=F, R3=NO2
28 R1=Br, R2=NO2, R3=H
29 R1=Cl, R2=NO2, R3=H
30 R1=H, R2=Cl, R3=NO2
31 R1=H, R2=OCH3, R3=NO2
R1
R2
NH
R1
R2
NH
R3
a
O
O
HN
HN
S
S
O
O
16-21
.
Reagents and conditions: (a) fuming HNO3, 0-5 °C, 1 h; (b) SnCl2 2H2O, AcOEt/EtOH 1:1, reflux.
1.2. Microbiology
1.2.1. Microbial strains and culture media
The Gram-positive microorganisms Bacillus subtilis (ATCC 6051), Staphylococcus aureus (ATCC
25923), methicillin-resistant S. aureus (MRSA) (ATCC 43300) and Enterococcus fecalis (ATCC
29212) were assayed. All microorganisms were grown in brain hear infusion (BHI) broth (BD, CA).
Solid media was obtained by adding 1.5% (w/v) agar to the liquid media.

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1.2.2. Antibacterial activity
The antimicrobial activity of the synthesized compounds was determined on 96-well microdilution
plates according to the protocols (published by the Clinical and Laboratory Standards Institute [18])
for determining the minimum inhibitory concentration (MIC) of each compound that results in
inhibition of bacterial growth. Disposable micro titration plates were used for the antimicrobial tests.
All compounds were dissolved in ethanol and diluted 2–512 times with 100 μL of Mueller–Hinton and
bacteria were inoculated at a concentration of 105 colony forming units (CFU mL-1). The MIC was
measured after 20 h incubation at 37 °C with each substance tested. Growth inhibition of the bacterial
strains was assessed by visual inspection. The antibacterial activities of the synthesized compounds
were compared to oxacillin and ciprofloxacin as positive control. All assays were carried out
in triplicate.
1.3. Theoretical calculations
Quantum chemical calculations of BZS 16-31 in the gas phase were performed using GAUSSIAN
03 [19] in conjunction with density functional theory (DFT). An extensive search for the lowest energy
conformer on the potential energy surfaces of the BZS compounds was carried out at the B3LYP/3-
21G(d) level, and geometry optimization of the lowest energy BZS conformer, followed by frequency
calculations, was performed at the B3LYP/6-31G+(d,p) level. The atomic charges were obtained from
a Mulliken population analysis. A simple linear regression analysis was performed using the
StatGraphics Plus statistical software package.
2. Results and Discussion
All of the BZS compounds described above have not been previously reported. The structures of all
1
synthesized compounds were confirmed by spectral data, and the IR, H-NMR, and mass spectra
agreed with the proposed structures.
The synthesized compounds 16-31 were tested in vitro against four Gram-positive bacteria using a
standard microdilution method and the control drugs (oxacillin and ciprofloxacin), as shown in Tables
1 and 2. The results reported in Table 1 indicate that compounds 16-23 inhibited growth of the
screened microorganisms, yielding MIC values in the range 125-500 μg/mL. However, the synthesized
compounds exhibited low antibacterial potencies.
Table1. MIC (μg/mL) values for BZS compounds 16-23.
S. aureus
MRSA
E. fecalis
B. subtilis
Compounds
ATCC 25923 ATCC 43300 ATCC 29212 ATCC 6633
16
17
18
19
20
21
22
23
>500
500
>500
500
500
>500
500
500
500
500
500
500
>500
500
>500
250
125
250
250
125
250
250
500
500
500
>500
>500
500
500
375
>500
>500

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Table 2 reveals that the compounds 24-31 showed antibacterial activity against S. aureus, MRSA,
E. fecalis and B. subtilis with MIC values in the range 2-500 μg/mL.
Table 2. MIC (μg/mL) values for BZS compounds 24-31.
Compounds
S. aureus
MRSA
E. fecalis
B. subtilis
ATCC 25923 ATCC 43300 ATCC 29212 ATCC 6633
24
25
26
27
28
29
30
31
187
>500
187
>500
16
31
500
>500
187
>500
125
500
10
18
500
500
125
250
250
500
125
125
500
500
8
500
8
86
2
2
86
78
Oxacillin
0.25
2
8
>125
Ciprofloxacin
1 µg/mL
≤0.25 µg/mL
1 µg/mL
≤0.25 µg/mL
The structure-activity relationship (SAR) of compounds 16-31 was determined using the data
presented in Tables 1 and 2. SAR studies revealed that the presence of an electron-withdrawing group
on the benzimidazole ring increased the antimicrobial activity, and activity decreased in the presence
of electron-releasing atoms or groups. Specifically, compounds with a nitro group in either position 5
or position 7 of the benzimidazole core significantly increased potency against S. aureus, MRSA and
B. subtilis. The dependence of compounds efficacy (biological activity) on the position of the nitro
group did not appear to be important. The transformation of the nitro group in position 7 to an amino
group led to loss of antibacterial activity. The substitution and region of a third atom or group in the
nitro-BZS was decisive for increasing activity in MRSA and B. subtilis. The highest result antibacterial
activity was exhibited by BZS containing a nitro group at position 5 and either chlorine or bromine
atoms at position 4 of the benzimidazole ring. The importance of nitro group in enhancing the
antimicrobial activity against Gram-positive bacteria is supported by similar results observed by Ören
et al. [20] and Moro et al. [21] Exchange of the groups or atoms in the benzimidazole ring did not
strongly inhibit the growth of E. fecalis.
The SAR study suggested that the acidity of hydrogens 1 and 1’ in the BZS molecule plays an
important role in activity of against Gram-positive bacteria. Romero and Mendez showed that the
charge on the hydrogen atom, calculated using DFT, is indicative of the gas phase acidity of
substituted phenols, in agreement with the local hard and soft acids-bases principle (HSAB).[22]
Based on this antecedent and with the purpose of quantifying a substituent’s effect on antibacterial
activity, a study was carried out to investigate the quantitative structure-activity relationship (QSAR)
by linear regression analysis of the –log MIC (μM) as a function of the charge on acidic hydrogen
atoms and on BZS hydrogen bond acceptors.
The charge on hydrogen atoms (qH) that were hydrogen-bonded to the nitrogen atoms of BZS, as
determined from the energetically favorable conformers of each BZS structure at the B3LYP/6-
31+G(d,p) level, showed a larger charge for compounds containing Cl or Br atoms at position 4 and a
nitro group at position 5 (Table 3) of the benzimidazole. These two compounds presented higher
activities against MRSA and B. subtilis. The main differences between the conformations of BZS and
with a NO2 group lay in the orientation of the NHSO2 hydrogen atom with respect to the NO2 group.

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Table 3. CMI and atomic charges on substituent atoms of BZS.
B. subtilis
MIC (M) MIC (M)
MRSA
BZS
q H1 q N3 q H1’ q O2’ q O3’
16
17
18
19
20
21
24
25
26
27
28
29
30
31
1740
1639
-
1740
1639
1365
1554
1554
-
562
-
376
1427
24
0.304 -0.317 0.324 -0.533 -0.528
0.305 -0.315 0.334 -0.530 -0.532
0.308 -0.267 0.326 -0.523 -0.530
0.308 -0.302 0.325 -0.522 -0.529
0.305 -0.321 0.320 -0.516 -0.526
0.301 -0.325 0.345 -0.531 -0.528
0.335 -0.321 0.382 -0.489 -0.467
0.297 -0.346 0.348 -0.509 -0.559
0.308 -0.320 0.374 -0.470 -0.538
0.333 -0.315 0.387 -0.494 -0.448
0.310 -0.232 0.365 -0.464 -0.479
0.312 -0.284 0.367 -0.479 -0.539
0.335 -0.346 0.375 -0.421 -0.455
0.332 -0.335 0.397 -0.453 -0.462
-
1554
1575
24
1655
24
245
5
5
49
1363
1380
234
215
The linear regression analysis, which correlated the antibacterial activity with the atomic charges in
the BZS derivatives containing a variety of substituents at positions 4, 5 and 7 of the benzimidazole
ring (n = 12), showed that the best correlation between charge and antibacterial activity occurred
between the qN3 and the –log MIC in B. subtilis and MRSA, with r > 0.6 (Table 4).
Table 4. Correlation coefficients (r) for the linear regression analysis of atomic charges
with antimicrobial activity of BZS derivatives. -log MIC = a + bqk.
Bacteria
N
q H1
q N3 q H1’ q O2’ q O3’
MIC
B. subtilis
12a 0.332 0.651 0.596 0.591 0.270
7b 0.916 0.747 0.888 0.956 0.337
9c 0.889 -0.049 0.808 0.607 0.814
12a -0.126 0.691 0.296 0.361 -0.039
6d 0.938 0.882 0.798 0.868 0.530
MIC
MRSA
7e 0.579 -0.048 0.487 0.232 0.446
n = number of compounds. a BZS: 16, 17, 20, 21, 24-31; BZS: 16, 17, 20, 21, 26, 28, 29; BZS:
16, 17, 20, 21, 24, 25, 27, 30, 31 ; d BZS: 16, 17, 20, 26, 28, 29; e BZS: 16, 17, 20, 24, 27, 30, 31.
b
c
In general, the parameters showed a high co-linearity with a correlation coefficient of r > 0.8 if the
linear regression was performed by splitting the compounds into two groups: the first group comprised
BZS with substituents in the 4 and 5 positions, and the second group comprised BZS with substituents
in the 5 and 7 positions. This method yielded the best r values between qH1, qH1’, qO2’, and the –log
MIC in B. subtilis, and between qH1, qN3, qO2’, and the –log MIC in MRSA, both for the BZS
substituent positions 4 and 5.
The best correlation models for the BZS derivatives that showed the greatest antibacterial activity
are described in Equations 1-8 (Table 5) along with the statistical parameters for the linear
regression analysis. The overall quality of the models was assessed by the correlation coefficient, r, the
squared correlation, r2, the standard error, S, and the Fisher ratio, F.

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Table 5. Linear regression analysis and quality of correlation for modeling the
antimicrobial activity of BZS. –log MIC = a + bqk.
Eq QSAR Model (-log MIC=)
n
r
r2
F
S
B. subtilis
1
2
3
4
-94.5331 + 301.216 qH1
6.07449 + 27.504 qN3
-19.6851 + 50.298 qH1’
17.0515 + 38.3147 O2’
MRSA
7
7
7
7
0.840 26.20 0.545
0.558 6.31 0.905
0.788 18.57 0.599
0.914 53.31 0.398
0.916
0.747
0.888
0.956
5
6
7
8
-77.1726 + 242.811 qH1
3.63887 + 20.752 qN3
-12.128 + 27.5796 qH1’
8.95649 + 23.0719 qO2’
6
6
6
6
0.880 29.40 0.321
0.778 14.05 0.436
0.938
0.882
0.798
0.868
0.637 7.02
0.559
0.754 12.27 0.460
n = number of compounds.
The results suggested that the higher charges on H1 and H1’ in conjunction the lower charges on
N3 and O2 promoted the activity of BZS derivatives against B. subtilis and MRSA.
3. Experimental
3.1. General
Melting points were determined using a Mel-Temp melting point apparatus in open capillary tubes
and are uncorrected. IR spectra were collected on a Nexus 470 FT-IR spectrophotometer. 1H-NMR
spectra were recorded on Varian Gemini-200 MHz and Eclipse 300 MHz JEOL spectrometers in
deuterated dimethyl sulfoxide (DMSO-d6) solutions using an internal TMS standard (0 ppm). The
chemical shifts are reported in parts per million (/ppm). The peak patterns are indicated as follows: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. The coupling constants (J) are reported
in Hertz (Hz). Mass spectra were detected by electronic impact (EI) and recorded using a JEOL JEM-
AX505HA spectrometer by electronic impact (EI) of lower resolution at 70 eV. Flash column
chromatography was carried out using silica gel 60 (230-400 mesh ASTM) from Merck.
3.2. General procedure for preparing benzimidazolamine (10-13)
A mixture of R3-nitroaniline 1-4 (13.06 mmol) and tin (II) chloride dihydrate (58.77 mmol for every
nitro group) in acetic acid (20 mL) was heated under reflux for 4 h. The excess acetic acid was
removed under reduced pressure, and the crude product was dissolved in 10% hydrochloric acid. The
reaction mixture was heated under reflux for 1 h, was made alkaline by NaOH addition, and was
extracted with ethyl acetate. The organic layer was dried over Na2SO4, and the solvent was evaporated
under reduced pressure to given a crude product, which was purified by flash column chromatography
on silica gel, using ethyl acetate:ethanol (90:10) as the eluent.
2-Methyl-1H-benzimidazol-6-amine (10). Yield: 84%; m.p. 86-88 °C. FT-IR (KBr, cm-1): 3399 (N-H),
1
3299 (N-H), 1639 (C=N, imidazole). H-NMR (200 MHz):  2.37 (s, 3H, CH3), 3.45 (s br, 2H, NH2

Molecules 2011, 16
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exchangeable with D2O), 6.43 (dd, 1H, Ar-H, J = 7.7 Hz), 6.59 (d, 1H, Ar-H, J = 1.8 Hz), 7.11 (d, 1H,
Ar-H, J = 8.2 Hz). MS, m/z (Rel. Int.): 147 (M+,100), 119(8), 105 (12), 79 (13), 52 (13).
5-Fluoro-2-methyl-1H-benzimidazol-6-amine (11). Yield: 72%; m.p. 157-158 °C. FT-IR (KBr, cm-1):
1
3417 (N-H), 3371 (N-H), 1645 (C=N, imidazole). H-NMR (300 MHz):  2.36 (s, 3H, CH3), 4.69 (s
br, 2H, NH2 exchangeable with D2O), 6.76 (d, 1H, Ar-H, J = 8.1 Hz), 7.08 (d, 1H, Ar-H, J = 11.4 Hz).
MS, m/z (Rel. Int.): 165 (M+, 100), 137(5), 124(6), 97(9), 83 (7).
4-Bromo-2-methyl-1H-benzimidazol-6-amine (12). Yield: 66%; oil. FT-IR (KBr, cm-1): 3379 (N-H),
1
3338 (N-H), 1633 (C=N, imidazole). H-NMR (200 MHz):  2.38(s, 3H, CH3), 4.99 (s br, 2H, NH2
exchangeable with D2O), 6.54(d, 1H, Ar-H, J = 1.6 Hz), 6.65, (d, 1H, Ar-H, J = 1.8 Hz), 11.87 (s br,
1H, N-H exchangeable with D2O). MS, m/z (Rel. Int.): 225 (M+,100), 146 (19), 119 (9), 77 (10), 52 (7).
4-Chloro-2-methyl-1H-benzimidazol-6-amine (13). Yield: 69%; oil. FT-IR (KBr, cm-1): 3338 (N-H),
1
3391 (N-H), 1637 (C=N, imidazole). H-NMR (200 MHz):  2.38 (s, 3H, CH3), 5.07 (s br, 2H, NH2
exchangeable with D2O), 6.5 (s, 2H, Ar-H), 11.84 (s br, 1H, N-H exchangeable with D2O). MS, m/z
(Rel. Int.): 181 (M+,100), 146 (13), 105(5), 78(11), 52 (5).
3.3. Procedure for preparing 5-chloro-2-methyl-1H-benzimidazol-6-amine (14)
To a suspension of 4-chloro-2-nitroaniline (4.7 mmol) in acetic acid (14.0 mL), was added tin (II)
chloride dihydrate (0.021 mmol). The mixture was heated under reflux for 4 h. To the reaction solution
was added NaHCO3 followed by extraction with ethyl acetate. The organic layers were dried over
Na2SO4 and removed by evaporation under vacuum. The pure benzimidazole 8 was obtained in
74% yield.
To the benzimidazole 8 (1.8 mmol) was added a mixture of H2SO4/HNO3 (3:1, 1.2 mL). The
reaction was stirred at room temperature for 1 h. To the mixture was added cold water. The resulting
solid was filtered, washed with cold water, and dried under vacuum to afford pure benzimidazole 9 in
98% yield.
To a suspension of benzimidazole 9 (1.8 mmol) in HCl (5 mL) was added tin (II) chloride dihydrate
(8.0 mmol). The mixture was heated under reflux for 4 h. The resulting solid was filtered, dissolved in
aqueous 10% NaOH and the product extracted with ethyl acetate. The extract was dried and under
vacuum to gave the desired benzimidazolamine 14. Yield: 78%; m.p. 108 °C. FT-IR (KBr, cm-1): 3429
(N-H), 3354 (N-H), 1639 (C=N, imidazole). 1H-NMR (200 MHz):  2.40 (s, 3H, CH3), 4.94 (s br, 2H,
NH2 exchangeable with D2O), 6.85 (s, 1H, Ar-H), 7.32 (s, 1H, Ar-H). MS, m/z (Rel. Int.): 181 (M+,
100), 146 (11), 105(4), 78(11), 52 (8).
3.4. Procedure for preparing 2-methyl-5-methoxy-1H-benzimidazol-6-amine (15)
To 2-methoxy-4-nitroaniline 5 (29.8 mmol) was added acetic anhydride (9.2 mL) and acetic acid
(11.0 mL). The reaction was stirred under reflux for 2 h. To the mixture was added cold water, and the
resulting solid was filtered, washed with cold water, and dried under vacuum. To this solid was added
fuming HNO3 (12.0 mL), the reaction was stirred at 0 to 5 °C for 1 h. To the mixture was added cold

Molecules 2011, 16
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water, and the resulting solid was filtered, washed with cold water, and dried under vacuum, to afford
the pure acetamide 6 in 77% yield. From the acetamide 6, the benzimidazolamine 15 was obtained
following the general procedure for preparing benzimidazolamines 10-13. Yield: 54%; oil. FT-IR
(KBr, cm-1): 3417 (N-H), 3342 (N-H), 1637 (C=N, imidazole), 1140 (O-CH3). 1H-NMR (200 MHz): 
2.36 (s, 3H, CH3), 3.76 (s, 3H, O-CH3), 4.4 (s br, 2H, NH2 exchangeable with D2O), 6.68 (s, 1H, Ar-
H), 6.86 (s, 1H, Ar-H). MS, m/z (Rel. Int.): 177 (M+, 100), 162 (78), 134 (37), 89 (6), 52 (4).
3.5. General procedure for preparing benzimidazolylbenzenesulfonamides 16-23
To a solution of benzimidazolamine (10-15, 6.8 mmol) and pyridine (6.8 mmol, 0.55 mL) in
anhydrous acetone was added benzenesulfonyl chloride (6.8 mmol, 0.87 mL). The reaction mixture
was stirred at room temperature under a N2 atmosphere for 24 h. Aqueous NaHCO3 was then added,
and the mixture was extracted with ethyl acetate. The organic layer was dried over Na2SO4, filtered,
concentrated, and purified by flash column chromatography on silica gel (gradient elution 50:50
hexane/ethyl acetate to 70:30 ethyl acetate/ethanol) to give 16-21 and 22-23.
N-(2-methyl-1H-benzimidazol-6-yl)benzenesulfonamide (16). Yield: 68%; m.p. 221-222 °C. FT-IR
1
(KBr, cm-1): 3302 (N-H), 1633 (C=N, imidazole), 1340 and 1155 (O2S-NH). H-NMR (300 MHz): 
2.41 (s, 3H, CH3), 6.83 (d, 1H, Ar-H, J = 7.9 Hz), 7.14 (s, 1H, Ar-H), 7.5 (dd, 1H, Ar-H, J = 6 Hz),
7.54 (m, 3H, Ar-H), 7.68 (dd, 2H, Ar-H, J = 7.8 Hz). MS, m/z (Rel. Int.): 287 (M+, 36), 146 (100),
119 (7), 105 (4), 77 (5).
N-(5-fluoro-2-methyl-1H-benzimidazol-6-yl)benzenesulfonamide (17). Yield: 45%; m.p. 152 °C. FT-IR
1
(KBr, cm-1): 3253 (N-H), 1635 (C=N, imidazole), 1323 and 1167 (O2S-NH). H-NMR (200 MHz): 
2.43 (s, 3H, CH3), 7.17 (d, 1H, Ar-H, J = 10.2 Hz), 7.17 (d, 2H, Ar-H, J = 7.0 Hz), 7.57 (m, 5H, Ar-
H), 9.91 (s br, 1H, NH exchangeable with D2O), 12.27 (s br, 1H, NH exchangeable with D2O). MS,
m/z (Rel. Int.): 305 (M+, 18), 164 (100), 137 (12), 96 (7), 77 (11).
N-(4-bromo-2-methyl-1H-benzimidazol-6-yl)benzenesulfonamide (18). Yield: 66%; m.p. 272-273 °C.
FT-IR (KBr, cm-1): 3290 (N-H), 1631 (C=N, imidazole), 1317 and 1163 (O2S-NH). 1H-NMR (200 MHz):
 2.43 (s, 3H, CH3), 7.02 (d, 1H, Ar-H, J = 1.8 Hz), 7.15 (d, 1H, Ar-H, J = 2.0 Hz), 7.53 (m, 3H, Ar-
H), 7.69 (dd, 2H, Ar-H, J = 8.2 Hz), 10.19 (s, 1H, NH exchangeable with D2O), 12.45 (s br, 1H, NH
exchangeable with D2O). MS, m/z (Rel. Int.): 365 (M+, 27), 224 (100), 145 (14), 104 (5), 77 (17).
N-(4-chloro-2-methyl-1H-benzimidazol-6-yl)benzenesulfonamide (19). Yield: 57%; m.p. 358 °C. FT-
IR (KBr, cm-1): 3284 (N-H), 1633 (C=N, imidazole), 1319 and 1162 (O2S-NH). 1H-NMR (200 MHz):
 2.43 (s, 3H, CH3), 6.89 (d, 1H, Ar-H, J = 1.6 Hz), 7.11 (d, 1H, Ar-H, J = 1.8 Hz), 7.53 (m, 3H), 7.70
(dd, 2H, Ar-H, J = 7.9 Hz), 10.2 (s, 1H, NH exchangeable with D2O), 12.41 (s br, 1H, NH
exchangeable with D2O). MS, m/z (Rel. Int.): 321 (M+, 26) 180 (100), 153 (6), 103 (2), 77 (8).
N-(5-chloro-2-methyl-1H-benzimidazol-6-yl)benzenesulfonamide (20). Yield: 53%; m.p. 224 °C. FT-
1
IR (KBr, cm-1): 3275 (N-H), 1631(C=N, imidazole), 1292 and 1151 (O2S-NH). H-NMR (200 MHz):

Molecules 2011, 16
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 2.45 (s, 3H, CH3), 7.20 (s, 1H, Ar-H), 7.46 (s, 1H, Ar-H), 7.6 (m, 5H, Ar-H), 9.82 (s br, 1H, NH
exchangeable with D2O), 12.36 (s br, 1H, NH exchangeable with D2O). MS, m/z (Rel. Int.): 321 (M+,
20), 180 (100), 153 (7), 103 (2), 77 (7).
N-(2-methyl-5-methoxi-1H-benzimidazol-6-yl)benzenesulfonamide (21). Yield: 22%; m.p. 206-207 °C.
1
FT-IR (KBr, cm-1): 3282 (N-H), 1633 (C=N, imidazole), 1325 and 1167 (O2S-NH). H-NMR
(300 MHz):  2.42 (s, 3H, CH3), 3.38 (s, 3H, O-CH3), 6.86 (s, 1H, Ar-H), 7.26 (s, 1H, Ar-H), 7.47 (m,
2H, Ar-H, J = 7.6 Hz), 7.57 (m, 1H, Ar-H, J = 7.3 Hz), 7.61(dd, 2H, Ar-H, J = 7.8 Hz), 9.30 (s, 1H,
NH exchangeable with D2O), 12.06 (s br, 1H, NH exchangeable with D2O). MS, m/z (Rel. Int.): 317
(36), 176 (100), 148 (32), 107 (6), 77 (7).
N-(2-methyl-1-phenylsulfonyl-1H-benzimidazol-6-yl)benzenesulfonamide (22). Yield: 2%; m.p. 146-148 °C.
FT-IR (KBr, cm-1): 3427 (N-H), 1622 (C=N, imidazole), 1163 (O2S-NH). 1H-NMR (300 MHz):  2.76
(s, 3H, CH3), 7.23 (dd, J = 8.8 Hz), 7.14 (dd, J = 8.8 Hz), 7.07 (dd, 1H, J = 8.7 Hz), 10.12 (s, 1H, NH
exchangeable with D2O). MS, m/z (Rel. Int.): 427 (M+, 52), 286 (100), 141 (41), 97 (6), 77 (45).
N-(2-methyl-5-methoxi-1-phenylsolfonyl-1H-benzimidazol-6-yl)benzenesulfonamide (23). Yield: 15%;
m.p. 186-187 °C. FT-IR (KBr, cm-1): 3261 (N-H), 1601 (C=N, imidazole), and 1176 (O2S-NH), 1091
(O-CH3). 1H-NMR (200 MHz):  2.73 (s, 3H, CH3), 3.48 (s, 3H), 7.09 (s, 1H), 7.49 (m, 2H, J = 7 Hz),
7.63 (m, 5H), 7.78 (m, 1H, J = 7.6 Hz), 7.81 (s, 1H), 7.91 (dd, 2H, J = 8.2 Hz), 9.73 (s. 1H, NH
exchangeable with D2O). MS, m/z (Rel. Int.): 457 (M+, 20), 316 (100), 175 (19), 141 (26), 77 (98).
3.6. General procedure for the synthesis of benzimidazolylbenzenesulfonamides 24, 26-31
To benzimidazolylbenzenesulfonamide (16-21, 3.5 mmol) was added fuming HNO3 (2.0 mL). The
reaction mixture was stirred at 0-5 °C for 1 h. Cool water was then added to provide a yellow solid.
The solid was filtered, washed twice with water and dried. The mixture of 24 and 26 was purified by
flash column chromatography on silica gel, using gradient elution, 75:25 to 55:45 hexane/ethyl acetate.
N-(2-methyl-7-nitro-1H-benzimidazol-6-yl)benzenesulfonamide (24). Yield: 76%; m.p. 182 °C. FT-IR
(KBr, cm-1): 3410 (N-H), 3222 (N-H), 1630 (C=N, imidazole), 1493 and 1356 (NO2), 1311 and 1161
1
(O2S-NH). H-NMR (300 MHz):  2.71 (s, 3H, CH3), 7.19 (d, 1H, J = 8.7 Hz), 7.54 (dd, 2H,
J = 8.1 Hz), 7.67 (m, 3H), 7.93 (d, 1H, J = 8.7 Hz), 10.47 (s br, 1H, NH exchangeable with D2O). MS,
m/z (Rel. Int.): 332 (M+, 64), 191 (100), 131 (29), 104 (12), 77 (34).
N-(2-methyl-5-nitro-1H-benzimidazol-6-yl)benzenesulfonamide (26). Yield: 22%; m.p. 184 °C. FT-IR
(KBr, cm-1): 3261 (N-H), 1643 (C=N, imidazole), 1464 and 1358 (NO2), 1307 and 1174 (O2S-NH).
1H-NMR (200 MHz):  2.49 (s, 3H, CH3), 7.13 (s, 1H), 7.58 (m, 5H), 8.05 (s, 1H), 10.04 (s br, 1H,
NH exchangeable with D2O), 12.72 (s br, 1H, NH exchangeable with D2O). MS, m/z (Rel. Int.): 332
(M+, 58), 191 (100), 131 (26), 104 (6), 77 (59).

Molecules 2011, 16
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N-(5-fluoro-2-methyl-7-nitro-1H-benzimidazol-6-yl)benzenesulfonamide (27). Yield: 75%; m.p.
242 °C. FT-IR (KBr, cm-1): 3348 (N-H), 3265, 1639 (C=N, imidazole), 1522 (NO2), 1325 and 1167
(O2S-NH). 1H-NMR (200 MHz):  2.51 (s, 3H, CH3), 7.55 (m, 6H), 10.35 (s br, 1H, NH exchangeable
with D2O), 12.95 (s br, 1H, NH exchangeable with D2O). MS, m/z (Rel. Int.): 350 (M+, 47), 209 (100),
192 (51), 122 (8), 77 (12).
N-(4-bromo-2-methyl-5-nitro-1H-benzimidazol-6-yl)benzenesulfonamide (28). Yield: 71%; m.p.
251 °C. FT-IR (KBr, cm-1): 3373 (N-H), 3215 (N-H), 1624 (C=N, imidazole), 1463 (NO2), 1309 and
1
1161 (O2S-NH). H-NMR (300 MHz):  2.53 (s, 3H, CH3), 7.13 (s, 1H), 7.54 (m, 2H), 7.64 (m, 3H),
10.33 (s br, 1H, NH exchangeable with D2O), 13.11 (s br, 1H, NH exchangeable with D2O). MS, m/z
(Rel. Int.): 410 (M+, 92), 269 (82), 239 (21), 132 (14), 77 (34).
N-(4-chloro-2-methyl-5-nitro-1H-benzimidazol-6-yl)benzenesulfonamide (29). Yield: 92%; m.p.
140-141 °C. FT-IR (KBr, cm-1): 3394 (N-H), 3255 (N-H), 1624 (C=N, imidazole), 1369 (NO2), 1338
1
and 1172 (O2S-NH). H-NMR (300 MHz):  2.55 (s, 3H, CH3), 6.99 (s, 1H), 7.54 (m, 2H), 7.65 (m,
3H), 10.39 (s br, 1H, NH exchangeable with D2O). MS, m/z (Rel. Int.): 366 (M+, 100), 224 (97), 208
(33), 132 (12), 77 (37).
N-(5-chloro-2-methyl-7-nitro-1H-benzimidazol-6-yl)benzenesulfonamide (30). Yield: 67%; m.p.
277-278 °C. FT-IR (KBr, cm-1): 3379 (N-H), 3288 (N-H), 1631 (C=N, imidazole), 1520 (NO2), 1338
and 1163 (O2S-NH). 1H-NMR (300 MHz):  2.53 (s, 3H, CH3), 7.60 (m, 5H), 7.82 (s, 1H), 10.41 (s br,
1H, NH exchangeable with D2O), 13.04 (s br, 1H, NH exchangeable with D2O). MS, m/z (Rel. Int.):
366 (M+, 42), 224 (100), 208 (74), 132 (13), 77 (16).
N-(5-methoxy-2-methyl-7-nitro-1H-benzimidazol-6-yl)benzenesulfonamide (31). Yield: 94%; m.p.
182 °C. FT-IR (KBr, cm-1): 3290 (N-H), 1622 (C=N, imidazole), 1544 (NO2), 1346 and 1140 (O2S-
1
NH). H-NMR (300 MHz):  2.65 (s, 3H, CH3), 3.26 (s, 3H), 7.35 (s, 1H), 7.53 (m, 5H), 10.16 (s br,
1H, NHexchangeable with D2O). MS, m/z (Rel. Int.): 362 (M+, 58), 221 (100), 173 (78), 133 (3), 77 (11).
3.7. Procedure for preparing N-(7-amino-2-methyl-1H-benzimidazol-6-yl)benzenesulfonamide (25)
A mixture of benzimidazolilbenzenesulfonamide (24, 2.9 mmol) and tin (II) chloride II dihydrate
(13.0 mmol) in ethyl acetate/ethanol 1:1 (100 mL) was heated under reflux for 4 h. The solvent was
removed under reduced pressure and the crude product was added to aqueous NaHCO3.The mixture
was subsequently extracted with ethyl acetate. The organic layer was dried over Na2SO4, filtered, and
concentrated to give 25. Yield: 65%; m.p. 225 °C. FT-IR (KBr, cm-1): 3471 (N-H), 3319 (N-H), 1628
(C=N, imidazole), 1311 and 1159 (O2S-NH). 1H-NMR (300 MHz):  2.39 (s, 3H, CH3), 4.89 (s br, 2H,
NH2 exchangeable with D2O), 6.31 (d, Ar-H, J = 8.4 Hz), 6.43 (d, Ar-H, J = 8.4 Hz), 7.49 (m, 2H, Ar-
H, J = 7.8 Hz), 7.59 (m, 1H, Ar-H, J = 7.5 Hz), 7.64 (dd, 2H, Ar-H, J = 7.6 Hz), 9.14 (s br, 1H, NH
exchangeable with D2O), 11.91(s br, 1H, NH exchangeable with D2O). MS, m/z (Rel. Int.): 302 (M+,
9), 161 (100), 134 (9), 107 (4), 77 (14).

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4. Conclusions
We have synthesized and evaluated the antibacterial activities of BZS analogs containing electron-
releasing and electron-withdrawing substituents on the benzimidazole ring. SAR and theoretical
studies of this series of compounds have shown that the antibacterial activity of BZS compounds may
be related to the charges on the acidic hydrogen atoms in the BZS structure. Two compounds, 28 and
29, have shown antibacterial activity against Gram-positive bacteria, which suggests that they may be
members of a promising new class of BZS anti-MRSA agents. These compounds present a good
starting point for modelling antimicrobial activity. Future work will focus on calculation of the
absolute gas phase acidity of the hydrogens H1 and H1’ of BZS and a QSAR between the acidity of
these hydrogens and antimicrobial activity.
Acknowledgements
This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACyT) FM grant
61626, DGAPA-PAPIIT RM project IN2049103, Programa de Mejoramiento del Profesorado
scholarship and a Ph. D. Scholarship (M. M. González-Chávez) from the CONACyT. We also thank
R. Patiño, A. Peña, E. Huerta, E. Hernández, L. Velasco, and J. Pérez from the Instituto de Química,
for technical support.
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© 2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).