Molecular modeling, synthesis, antibacterial and cytotoxicity evaluation of sulfonamide derivatives of benzimidazole, indazole, benzothiazole and thiazole

Article abstract of DOI:10.1016/j.bmc.2018.05.015

A new series of heterocyclic molecules bearing sulfonamide linkage has been synthesized and screened for antibacterial activity. During antibacterial screening using broath dilution method, molecules were found to be highly active (MIC value 50–3.1 μg/mL)

Full text of DOI:10.1016/j.bmc.2018.05.015

Accepted Manuscript
Molecular modeling, synthesis, antibacterial and cytotoxicity evaluation of sul-
fonamide derivatives of benzimidazole, indazole, benzothiazole and thiazole
Farha Naaz, Ritika Srivastava, Anuradha Singh, Nidhi Singh, Rajesh Verma,
Vishal K. Singh, Ramendra K. Singh
PII:
S0968-0896(18)30618-7
https://doi.org/10.1016/j.bmc.2018.05.015
BMC 14357
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 March 2018
1 May 2018
10 May 2018
Please cite this article as: Naaz, F., Srivastava, R., Singh, A., Singh, N., Verma, R., Singh, V.K., Singh, R.K.,
Molecular modeling, synthesis, antibacterial and cytotoxicity evaluation of sulfonamide derivatives of
benzimidazole, indazole, benzothiazole and thiazole, Bioorganic & Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.bmc.2018.05.015
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Molecular modeling, synthesis, antibacterial and cytotoxicity evaluation of
sulfonamide derivatives of benzimidazole, indazole, benzothiazole and thiazole
Farha Naaz, Ritika Srivastava, Anuradha Singh, Nidhi Singh, Rajesh Verma, Vishal K. Singh
and Ramendra K. Singh*
Bioorganic Research Laboratory, Department of Chemistry, University of Allahabad, Allahabad-211002, India
*Corresponding author: Ramendra K. Singh, rksinghsrk@gmail.com
Abstract
A new series of heterocyclic molecules bearing sulfonamide linkage has been synthesized and
screened for antibacterial activity. During antibacterial screening using broath dilution method,
molecules were found to be highly active (MIC value 50 - 3.1 µg/mL) against different human
pathogens, namely B. cerus, S. aureus, E. coli and P. aeruginosa, and most effective against E.
coli. A great synergistic effect was observed during determination of FIC where molecules were
used in combination with reference drugs chloramphenicol and sulfamethoxazole. The MIC
value of the combination – varying concentration of test compounds and ½ MIC of reference
drugs or varying concentration of reference drugs and ½ MIC of test compounds, was reduced up
1
1
to /4 or /32 of the original value, indicating thereby the combination was 4 - 32 times more
potent than the test molecule. The molecules also showed low degree of cytotoxicity against
PBM, CEM and VERO cell lines. The results positively indicated towards the development of
lead antibacterials using the combination approach.
Keywords: Sulfonamides, minimum inhibitory concentration (MIC), fractional inhibitory
concentration (FIC), cytotoxicity, molecular docking
1. Introduction
The incidence of bacterial infection has been one of the most multifaceted global health issues in
past and is escalating at an alarming rate and thereby presenting an extraordinary challenge to
health care professionals.1-2 Furthermore, many drug-resistant pathogenic microbes have
emerged in recent years because of increasing use or abuse of antibacterial agents as well as
inaccurate diagnosis.3 Antibiotic resistance, a concomitant problem, has become the limiting
factor to the effectiveness of current drugs; therefore, there is an imperious need for the
development of new treatment approaches with minimal adverse effects.4-5 To combat drug-
resistant microorganism, best scientific strategies include discovery and development of new,
1

cost-effective and more potent pioneering antibacterial agents with minimum adverse effects.6-7
Sulfonamide derivatives have been the focus of attention for the chemists and biologists for a
long time due to their wide array of biological activities.8-21 Some derivatives of this class are
efficient for healing of urinary infections, arthritis and Alzheimer’s disease.22 In search of some
new antibiotics, we have focused on sulfonamide nucleus, which has significance in the area of
medicinal chemistry and drug development and used as a core substituent of antibacterial agents.
In order to overcome the resistance and to reduce the adverse effects, continuous efforts are
made to synthesize novel antibacterial compounds. In this regard, combinations of certain
sulfonamides and other drug molecules are being used to develop novel formulations. The
current work is an effort to develop certain drug regimens as effective antibacterial agents
against drug resistant/mutant bacterial strains. The, sulfonamides still represent the drugs of first
choice for the treatment of some conditions and diseases.
Sulfonamides suppress the bacterial growth by targeting the folate pathway enzyme
dihydropteroate synthase (DHPS), which catalyzes the condensation of p-aminobenzoic acid
(PABA) and 6-hydroxymethyl-7, 8-dihydropterin-pyrophosphate (DHPPP) into dihydropteroate
(DHPt). Antimicrobial sulfonamide exert their effect by acting as competitive inhibitors of
PABA and thus inhibit biosynthesis of dihydrofolic acid, which prevents the growth and
reproduction of microorganisms.23 Sulfamethoxazole is an available sulfa drug acting as PABA
competitive inhibitor. Keeping in view the wide range of pharmaceutical activities of sulfa drugs
and to explore the development of new generation sulfa drugs with improved therapeutic effect
and longevity, we report herein the design consideration of compounds introducing certain
modifications in the A and B unit of sulfur antibiotic sulfamethoxazole. ‘A’ unit is replaced by
methyl (a); chloro (b) and nitro (c) group whereas ‘B’ unit is replaced by different heterocyclic
rings as shown in Fig. 1. Thus, a series of N-heterocyclic sulfonamides 1-7 and derivatives
bearing different heterocyclic rings of versatile biological importance was designed, synthesized
and evaluated for antibacterial activity. The combined virtual screening, molecular docking and
ADMET analysis on modified derivatives of sulfamethoxazole (1-7a/b/c) were used to elucidate
the mechanism of mode of action.
2

2. Results and discussion
2.1. Physicochemical descriptors of sulfonamide derivatives
A good number of N-heterocyclic sulfonamide derivatives have been designed as probable
antibacterial agents using in silico structure based approach. The physicochemical data of all
compounds and standard antibacterial agents were calculated using online Molinspiration and
ChemDraw software using Discovery Studio 2.5 software. To find out the drug-like
characteristics, molecules were assessed using Lipinski’s rule of five, which specifies that a
probable drug molecule should have log P ≤ 5, molecular weight ≤ 500, hydrogen bond
acceptors ≤ 10 and donor ≤ 5 and polar surface area ≤ 140 Å2 24 As rule of five compliance
.
ensures the bioavailability, the molecules in the designed library were assumed to have better
intestinal permeability. The presence of C-N, N-C, S=O and S-N dipoles allowed these
molecules to function as H-bond acceptors as well as H-bond donors. The results summarized in
table 1 revealed that compounds possessed the drug-like characteristics, as standard antibiotics.
The lipophilicity of compounds was reported in the form of log P and indicated that compounds
3

should have no problem to passage through cell membrane. The intermediate TPSA values of all
compounds predicted
their good cell internalization, similar to standard drugs. Therefore, results revealed that none of
the designed ligands violated the rule of five and may be developed as potent drug-like
antibacterial agents.
Table 1
In silico Predicted Physicochemical Properties of all compounds (1-7 and derivatives)
Compound
MW
H-A
H-D
Log P
TPSA
Rotatable
ROF
bonds
Rule
1a
1b
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
<500
≤10
4
4
4
4
7
4
4
7
7
7
10
5
5
8
4
4
4
≤5
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
0
0
3
3
2
≤5
≤140
51.97
51.97
51.97
51.97
97.79
51.97
51.97
97.79
97.79
97.79
143.62
68.30
68.30
114.12
59.06
59.06
51.97
97.79
98.22
115.38
83.47
≤10
2
2
2
2
3
2
2
3
3
3
4
5
5
6
3
3
2
3
3
7
3
≤1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
272.33
292.75
306.77
327.19
337.74
272.33
292.75
303.30
317.33
337.74
348.30
348.45
368.87
379.42
254.34
274.75
464.57
495.45
253.28
323.12
281.35
2.58
2.92
3.23
3.58
2.83
2.58
2.92
2.18
2.48
2.83
2.08
3.88
4.14
3.40
2.21
2.44
4.76
4.35
1.36
0.73
0.76
6b
7a
7c
7
6
7
5
Sulfamethoxazole
Chloramphenicol
Cycloheximide
MW: Molecular weight; H-A: number of H bond acceptors; H-D: number of H bond donors; Log P:
predicted octanol-water partition coefficient; TPSA: total polar surface area; ROF: Rule of five
2.2. Chemistry
The synthetic strategy adopted for the preparation of compounds 1-7 and derivatives (a/b/c) is
outlined in Scheme 1 and that of compound 7 in Scheme 2. Commercially available different N-
heterocyclic compounds - 1 (1-H-Benzimidazole), 2 ( 2-Cl-1-H- Benzimidazole), 3 (1-H-
Indazole), 4 (5-NO2-Indazole), 5 (2-amino-6-ethoxy-benzothiazole), 6 (2-amino-thiazole),
4

indigenously synthesized 7 (5,6-dibromo-2-Cl-benzimidazole) and aromatic sulfonyl chlorides,
like p-toluene sulfonyl chloride (a), 4-chloro-benzene-sulfonyl chloride (b), 4-nitro-benzene-
sulfonyl chloride (c) have been used as starting materials. The reaction condition was optimized
using various bases and the best result - short reaction time and good yield, was obtained using
DMAP as base in place of pyridine or TEA (Scheme 1).
Scheme 1. Synthesis of compounds 1-7 and derivatives
Bromination of compound 2 in MeOH was performed using a solution of Br2/MeOH as a
brominating agent followed by addition of H2O at room temperature to synthesize compound 7
(5,6-Dibromo-2-chloro-1H-benzimidazole) (Scheme 2).25 The compounds 1-7 and derivatives
were synthesized by reacting the respective heterocyclic molecule and sulfonyl chloride. The
1
products were purified by column chromatography and crystallization and characterized by H
NMR, 13C NMR, mass spectra and elemental analysis techniques.
5

Scheme 2. Synthesis of compound 7
2.3. Antibacterial assay
2.3.1. Determination of MIC of compounds 1-7 and derivatives
The micro dilution susceptibility test in Muller-Hinton Broth was used to assess the antibacterial
activity of compounds. All compounds were screened against four common bacterial strains, viz.
B. cerus, S. aureus, E. coli and P. aeruginosa for determination of their antibacterial efficacy.
The well known commercial antibiotics chloramphenicol and sulfamethoxazole were used as
standard drugs and the results presented in Table 2. Interestingly, some of the N-heterocyclic
sulfonamide derivatives displayed better antibacterial activity compared to the reference
standards.
It is worth mentioning that all molecules exhibited excellent inhibition activity against bacterial
strain E. coli and significant activity against P. aeruginosa and good to moderate activity against
S. aureus and B. cerus strains.
Aryl sulfonamide derivatives bearing benzimidazole heterocycle, 1a-b, showed potent activity
against E. coli. Compound 1a (MIC, 6.2 µg/mL against E. coli), being equipotent to reference
antibiotic chloramphenicol was found to be two-fold more active than reference antibiotic
sulfamethoxazole whereas 1b was found to be two-fold more active than chloramphenicol (MIC,
3.1 µg/mL versus 6.2 µg/mL). Compounds 1a-b showed moderate activity against S. aureus, P.
aeruginosa and B. cerus except that 1a showed low activity against B. cerus (MIC, 50 µg/mL).
Among compounds 2a-c, 2c showed significant activity against all bacterial strains and was two-
fold more potent against E. coli than reference chloramphenicol (MIC, 3.1 µg/mL versus 6.2
µg/mL) and equipotent in activity as compared to sulfamethoxazole.
6

Table 2
In vitro antibacterial activity of compounds 1-7 and derivatives against gram +ve and gram –ve bacterial strains and cytotoxicity results
MIC (Wild type)
Gram +ve strain
MIC (Mutant
type)
Cytotoxicity: IC50 µg/mL (µM)
Compound
Gram –ve strain
E. coli P. aeruginosa
E. coli
PBM
CEM
VERO
B. cerus
S. aureus
25
50
25
25
6.2
3.1
3.1
6.2
3.1
12.5
6.2
3.1
3.1
3.1
3.1
12.5
3.1
3.1
3.1
3.1
3.1
3.1
6.2
3.1
12.5
25
12.5
12.5
6.2
6.2
25
12.5
6.2
12.5
6.2
3.1
25
12.5
6.2
3.1
6.2
6.2
3.1
6.2
3.1
12.5
50
50
50
25
12.5
50
50
25
50
--
12.5
50
12.5
12.5
50
25
25
12.5
25
50
>27.23 (>100)
>29.27 (>100
>30.67 (>100)
>32.72 (>100)
>33.73 (>100)
>27.23 (>100)
>29.27 (>100)
>30.33 (>100)
>31.73 (>100)
>33.77 (>100)
--
≥27.23 (≥100)
18.06 (61.7)
>30.67 ( >100)
>32.72 ( >100)
>33.73 ( >100)
25.87 (94.8)
>29.27 (>100)
>30.33 (>100)
>31.73 ( >100)
>33.77 ( >100)
--
>27.23 (>100)
>29.27 (>100)
>30.67 ( >100)
>32.72 ( >100)
>33.73 ( >100)
24.78 (91.3)
>29.27 ( ≥100)
>30.33 ( >100)
>31.73 (>100)
≥33.77 (≥100)
--
1a
1b
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
7a
12.5
50
25
6.2
50
12.5
6.2
25
25
25
25
6.2
25
50
25
6.2
50
12.5
6.2
25
>34.84 (>100)
>36.88 (>100)
>37.94 (>100)
>25.43 (>100)
>27.47 (>100)
>46.45 (>100)
≥49.54 (≥100)
--
>34.84 ( >100)
>36.88 ( >100)
>37.94 (>100)
>25.43 ( >100)
>27.47 ( >100)
>46.45 ( >100)
20.41 (41.6)
--
≥34.84 ( ≥100)
>36.88 ( >100)
>37.94 (>100)
>25.43 ( >100)
1.65 (6.2)
>46.45 (>100)
> 49.54 (>100)
--
12.5
12.5
6.2
3.1
6.2
3.1
6.2
6.2
12.5
50
6.2
12.5
6.2
6.2
6.2
6.2
6.2
12.5
7c
Chloramphenicol
Sulfamethoxazole
Cycloheximide
--
--
--
50
0.25 (0.9)
0.056 (0.2)
0.056 (0.2)
MIC-Minimum Inhibitory Concentration in µg/mL; IC50 = 50% Index of cytotoxicity
7

Compounds 2a and 2b showed good activity against E. coli (MIC, 3.1 µg/mL and 6.2 µg/mL,
respectively) and P. aeruginosa (MIC, 12.5 µg/mL and 6.2 µg/mL, respectively) whereas
moderate activity against B. cerus and S. aureus. Derivatives of compound 2 showed better
results than 1, probably due to presence of –Cl group. Another series of compounds, 3a-c,
bearing indazole moeity also showed significant activity against gram -ve strains as compared to
gram +ve strains. Compound 3c was two times more potent against E. coli and equipotent
against P. aeruginosa compared to chloramphenicol and equipotent to sulfamethoxazole against
E. coli. (MIC, 3.1 µg/mL versus 3.1 µg/mL), 3a showed considerable to less activity against B.
cerus, S. aureus, P. aeruginosa and displayed half the potency of chloramphenicol against E.
coli. Similarly, compounds 4a-c, bearing 5 - NO2 - indazole moieties showed good antibacterial
activity and 4c was the most active among the series. Compound 4c was equipotent to
chloramphenicol and sulfamethoxazole against B. cerus and S. aureus (MIC, 6.2 µg/mL),
equipotent to sulfamethoxazole and two-fold more active as compared to chloramphenicol
against E. coli and P. aeruginosa (MIC, 3.1 µg/mL). Compounds 4a-b also displayed significant
activity against gram –ve strains whereas less activity was observed against gram +ve strains.
Another series of compounds, 5a-c, bearing benzothiazole heterocycle, showed similar inhibition
pattern as indazoles: 5a showed considerable activity against all tested strains, 5b displayed
excellent activity against E. coli (MIC, 3.1 µg/mL), moderate activity against B. cerus and P.
aeruginosa (MIC, 12.5 µg/mL) and low activity against S. aureus (MIC, 50 µg/mL), 5c showed
high activity against S. aureus and P. aeruginosa (MIC, 6.2 µg/mL), and two-fold more potency
against E. coli compared to chloramphenicol. Interestingly, compounds 6a-b derived from 2-
amino-thiazole moiety, revealed a prominent inhibition pattern ranging from potent to
considerable activity against the entire set of tested microorganisms: 6a was equipotent to
sulfamethoxazole and two times more potent compared to chloramphenicol against E. coli and P.
aeruginosa, whereas 6b showed the same inhibition pattern against E. coli (MIC, 3.1 µg/mL).
The last series of compounds, 7a and 7c, bearing bromo group at 5th and 6th position of
benzimidazole moiety, showed the highest antibacterial activity and excellent inhibition pattern
against all tested strains. Compound 7c was found to be the most active and showed very good
antibacterial activity against B. cerus, E. coli and P. aeruginosa and 7a against E. coli (MIC, 3.1
µg/mL).
8

Analysis of antibacterial results revealed that among all compounds, 2c, 3c, 4c, 5c, 6a-b, 7a and
7c showed much better antibacterial activity compared to other compounds. SAR study clearly
showed that compounds bearing –NO2 group, i.e., 2c, 3c, 4c and 5c displayed higher antibacterial
activity, probably due to the presence of this polar substituent as it offers chances for H -
bonding. Further, Dibromo groups are more hydrophobic than nitro groups and hence
compounds 7a and 7c have more chances for hydrophobic interactions as well as H-bonding due
the presence of polar groups at N-1 position of heterocycle 7(a,c). Comparison of antibacterial
activity of compounds 1-7 and derivatives with reference antibiotic chloramphenicol and
sulfamethoxazole has been shown in Fig. 2.
60
B. cerus
50
S. aureus
E. coli
P. aeruginosa
40
30
20
10
0
Compounds
Fig. 2. Comparison of antibacterial activity (MIC in µg/mL) of compounds 1-7 and derivatives
with reference antibiotic (Chloramphenicol and Sulfamethoxazole)
2.3.2. Antibacterial activity against E. coli mutant strains
Compounds were also tested for their antibacterial activity against E. coli mutant strain and the
results were quite encouraging. Compounds 1a, 1b, 2a, 3a, 3b, 4a, 5a, 6a were equipotent in
activity as compared to reference antibiotics sulfamethoxazole and cycloheximide (MIC, 50
µg/mL). Compounds 2b, 3c, 6b and 7a were two-fold more potent (MIC, 25 µg/mL) than
reference drugs sulfamethoxazole and cycloheximide (MIC, 50 µg/mL) and equipotent in
9

activity as compared to reference drug chloramphenicol (MIC, 25 µg/mL) whereas 2c, 4c, 5b,
5c, 7c were four-fold more potent (MIC, 12.5 µg/mL) than sulfamethoxazole and cycloheximide
antibiotics (MIC, 50 µg/mL) and two-fold more active against antibiotic chloramphenicol (MIC,
25 µg/mL)
It is noteworthy to mention here that some of the compounds were even much more potent than
the reference antibiotics used.
2.4. Determination of fractional inhibitory concentration (FIC) of selected N-heterocyclic
sulfonamide derivatives
2.4.1. Method 1: Evaluation of synergistic effect using ½ MIC of reference antibiotics and
varying concentrations of test compounds
Once the antibacterial activity (MIC) of all compounds was determined, a new study was
performed for evaluating the synergistic effect of some selected compounds 1a - b, 2a - b, 3a - c,
4a – b, 5a – c, 6a and the reference antibiotics in the form of fractional inhibitory concentration
(FIC). For this purpose, a combination approach was followed where ½ MIC of reference drugs
(3.1 µg/mL in case of chloramphenicol for both gram +ve and gram –ve bacterial strains, 3.1
µg/mL for sulfamethoxazole antibiotic for gram +ve strain and 1.5 µg/mL for gram –ve bacterial
strains) and varying concentrations of compounds were used. Only the compounds having MIC
value greater than the MIC value of reference drug were undertaken for this study in order to
improve their efficacy.26 All selected compounds with ½ MIC of reference drug in combination
were tested by two-dimensional broth micro dilution method to evaluate the extent of synergism.
This combination study was performed with both the reference antibiotics (chloramphenicol and
sulfamethoxazole) against all the four bacterial strains. The FIC value of most of the selected
compounds varied between 0.12 - 0.25 against the bacterial strains tested. Results of effect of
combination approach on MIC of test compounds and FIC of all studied compounds with
antibiotic chloramphenicol and sulfamethoxazole are summarized in Table 3 and Table 4.
Table 3
FIC determination: Combination study showing synergistic effect on MIC of selected compounds with
varying concentrations of test compounds and ½ MIC of chloramphenicol
10

Compound
Gram +ve strain
B.cerus S.aureus
Gram –ve strain
E.coli P.aeruginosa B.cerus S.aureus
Gram +ve strain
Gram –ve strain
E.coli P.aeruginosa
FIC
MIC (Combination)
12.5
6.2
6.2
12.5
3.1
12.5
3.1
6.2
ND
6.2
12.5
6.2
6.2
12.5
ND
3.1
ND
ND
ND
ND
3.1
ND
ND
ND
ND
3.1
6.2
3.1
3.1
ND
12.5
3.1
ND
3.1
ND
6.2
3.1
ND
ND
0.25
0.24
0.24
0.12
0.25
0.5
0.24
0.24
0.24
0.25
0.24
0.24
--
0.50
0.24
0.25
0.24
0.24
0.24
0.24
0.25
0.49
0.24
0.25
--
--
--
--
--
0.24
--
--
--
--
0.24
--
0.24
0.24
0.24
--
0.50
0.24
--
0.24
--
0.24
0.24
--
1a + ref
1b + ref
2a + ref
2b + ref
3a + ref
3b + ref
3c + ref
4a + ref
4b + ref
5a + ref
5b + ref
5c + ref
6a + ref
3.1
12.5
12.5
6.2
6.2
6.2
12.5
3.1
3.1
ND
ND
ND
--
--
ND
0.24
--
MIC (in µg/mL) = Minimum Inhibitory Concentration, ND = Not done,
FIC (Fractional inhibitory concentration) = MIC of test compound in combination with ref/MIC of test compound
alone, ref=½ MIC of chloramphenicol (Antibiotic)
Table 4
FIC determination: Combination study showing synergistic effect on MIC of selected compounds with
varying concentrations of test compounds and ½ MIC of sulfamethoxazole
Compound
Gram +ve strain
B. cerus S. aureus E. coli P. aeruginosa B. cerus S. aureus E. coli
MIC FIC
0.50
Gram –ve strain
Gram +ve strain
Gram –ve strain
P. aeruginosa
6.2
6.2
6.2
3.1
6.2
3.1
ND
6.2
ND
6.2
12.5
3.1
6.2
6.2
ND
3.1
ND
ND
ND
3.1
ND
ND
1.5
6.2
3.1
3.1
3.1
ND
6.2
3.1
1.5
3.1
1.5
6.2
ND
1.5
ND
1.5
3.1
1.5
0.24
0.24
0.24
0.12
--
0.49
0.12
0.12
0.24
--
0.24
0.24
0.24
0.50
--
0.24
0.24
0.24
0.24
0.24
0.24
--
0.24
--
0.24
0.50
0.48
1a + ref
1b + ref
2a + ref
2b + ref
2c + ref
3a + ref
3b + ref
3c + ref
4a + ref
4b + ref
5a + ref
5b + ref
5c + ref
6a + ref
6b + ref
7a + ref
7c + ref
--
--
3.1
ND
12.5
6.2
6.2
6.2
6.2
12.5
6.2
0.24
--
0.24
0.24
--
--
--
0.24
--
--
ND
3.1
1.5
0.25
0.24
0.24
0.24
0.24
0.25
0.49
0.24
--
0.24
0.24
0.24
0.24
0.25
0.24
0.12
--
ND
ND
ND
3.1
ND
ND
ND
ND
ND
ND
3.1
ND
ND
ND
ND
ND
0.24
--
--
--
--
--
--
--
--
--
--
MIC (in µg/mL) = Minimum Inhibitory Concentration, ND = Not done,
FIC (Fractional inhibitory concentration) = MIC of test compound in combination with ref/MIC of test compound
alone, ref=½ MIC of sulfamethoxazole (Antibiotic)
11

Due to the synergistic effect, the combined MIC values were found to be reduced up to
significant level resulting in marked increment in antibacterial activity (Fig. 3). MIC of
1
compound 2b was found approximately /8 (3.1 µg/mL) of its original MIC value against B.
cerus bacterial strain. In case of compounds 1a-b, 2a, 3a, 3c, 4a-b, 5a-c, the value of combined
1
MIC was reduced up to /4 (12.5, 6.2, 6.2, 12.5, 6.2, 6.2, 6.2, 12.5, 3.1, 3.1 µg/mL, respectively)
as compared to original MIC against B. cerus. Similarly, compounds 1b, 2a-b, 3a-b, 4a, 5a-b, 6a
1
displayed reduction up to /4 in MIC (3.1, 12.5, 3.1, 6.2, 6.2, 12.5, 6.2, 12.5, 3.1 µg/mL,
respectively) against gram +ve strain S. aureus. Only two compounds, 3a and 5a, were
subjected to combination approach against gram –ve strain, E. coli and both showed diminution
1
in MIC up to /4 (3.1, 3.1 µg/mL ) of its original MIC. Similar diminution pattern was observed
in MIC by 1/4 (6.2, 3.1, 3.1, 3.1, 3.1, 6.2, 3.1 µg/mL, respectively) with compounds 1a-b, 2a, 3b,
4a, 5a-b against bacterial strain P. aeruginosa. However, the combination MIC value was
reduced to half in the case of compounds 3b, 1a / 4b and 3a against the bacterial strains B. cerus,
S. aureus and P. aeruginosa, respectively. Thus, the FIC values clearly indicated the synergistic
effect generated due to a combination of the test compounds and reference drugs.
0.6
B. cerus
S. aureus
E. coli
0.5
0.4
0.3
0.2
0.1
0
P. aeruginosa
Compounds
Fig. 3. Graphical presentation of fractional inhibitory concentration (FIC) of selected compounds in
combination with reference antibiotic (Chloramphenicol) against gram +ve and gram -ve bacterial strains
Similar experiments were performed with selected compounds (1a-b, 2a-c, 3a-c, 4a-b, 5a-c, 6a-
b, 7a, and c) and antibiotic sulfamethoxazole as reference. All compounds exhibited the same
12

diminution pattern of MIC values, however, some compounds showed better synergistic effect as
1
revealed by their FIC values (Fig. 4). MIC of compound 3b was found approximately /4 (6.2
µg/mL) of its original MIC value against B. cerus bacterial strain. Compounds 1a and 4b showed
1
the same pattern whereas compounds 2a and 5b displayed a marked declination in MIC up to /8
(6.2 and 6.2 µg/mL) as compared to its original MIC. In combination with reference drug,
1
compounds 2b and 3b showed diminution in MIC (1.5, 1.5 µg/mL, respectively) up to /4 of its
original MIC against gram –ve strain E. coli. Similar inhibition pattern was observed in MIC
(6.2, 1.5, 1.5, 1.5, 1.5 µg/mL) of compounds 3a, 3c, 4b, 5c, 6b, respectively, whereas 7a and 7c
in combination with reference drugs were found to be two fold more potent, i.e., MIC reduced to
½ (3.1, 1.5 µg/mL) compared to their original MIC, against bacterial strain P. aeruginosa. All
compounds subjected to combination study exhibited synergistic effect to a greater extent except
compounds 5b (against B. cerus), 1a (against S. aureus and E. coli), 7a and 7c (against P.
aeruginosa) (Table 4). Thus, we see that the synergistic effect of the compounds and reference
drugs has lowered the combined MIC to a much greater extent in all the cases.
0.6
B. cerus
S. aureus
0.5
E. coli
0.4
P. aeruginosa
0.3
0.2
0.1
0
Compounds
Fig. 4. Graphical presentation of fractional inhibitory concentration (FIC) of selected compounds in
combination with reference antibiotic (Sulfamethoxazole) against gram +ve and gram -ve bacterial strains
13

2.4.2. Method 2: Evaluation of synergistic effect of selected compounds using ½ MIC of test
compounds and varying concentrations of reference antibiotic
In this type of combination approach, varying concentrations of antibiotics (12.5 µg/mL to
0.0976 µg/mL) and ½ MIC of test compounds were used for determination of FIC using two-
dimensional broth micro dilution method. This type of combination study was subjected only to
those compounds whose MIC value was lower than the MIC of the reference drugs and
performed against gram –ve strains - E. coli (Wild type) and P. aeruginosa. The FIC value of all
selected compounds, ranged between 0.03 - 0.25 against the tested bacterial strains and the
results are summarized in Table 5.
Table 5
Combination study showing synergistic effect on gram –ve strains (E. coli and P. aeruginosa) on
compounds 1-7 and derivatives with varying concentrations of reference antibiotic and ½ MIC of test
compound
Compound
MIC (combination)
FIC
MIC (combination)
FIC
ref=½ MIC of tested compounds with varying
ref=½ MIC of tested compounds with varying
concentration of chloramphenicol (Antibiotic)
concentration of sulfamethoxazole (Antibiotic)
E. coli
0.7812
0.3906
0.7812
0.3906
0.3906
ND
0.3906
0.1953
0.7812
0.3906
0.1953
ND
0.7812
0.3906
0.3906
0.1953
0.3906
0.3906
P.aeruginosa E. coli P.aeruginosa
E. coli
ND
0.0976
0.7812
ND
0.1953
ND
ND
0.1953
0.3906
0.1953
0.0976
ND
0.1953
0.0976
0.1953
0.1953
0.1953
0.0976
P.aeruginosa E. coli P.aeruginosa
ND
ND
ND
0.7812
0.3906
ND
0.12
0.12
0.25
0.06
0.12
---
---
---
0.12
0.06
---
ND
ND
ND
---
---
---
---
ref (1a)
ref (1b)
ref (2a)
ref (2b)
ref (2c)
ref (3a)
ref (3b)
ref (3c)
ref (4a)
ref (4b)
ref (4c)
ref (5a)
ref (5b)
ref (5c)
ref (6a)
ref (6b)
ref (7a)
ref (7c)
0.03
0.25
---
ND
---
ND
ND
0.06
---
---
---
ND
0.3906
ND
0.3906
0.1953
ND
0.06
0.06
0.25
0.12
0.06
---
0.06
---
0.06
0.06
---
ND
---
---
ND
ND
ND
0.1953
ND
0.06
0.12
0.06
0.03
---
---
---
---
0.06
---
ND
ND
0.3906
0.1953
0.7812
0.3906
0.25
0.12
0.12
0.06
0.12
0.12
---
---
0.12
0.03
0.12
0.12
ND
ND
0.3906
ND
ND
0.06
0.03
0.06
0.06
0.06
0.03
---
---
0.121
---
---
0.0976
0.03
MIC (in µg/mL) = Minimum Inhibitory Concentration, ND = Not done,
FIC (Fractional inhibitory concentration) = MIC of test compound in combination with ref/MIC of test compound alone
14

Analysis of the results in the present case was quite rewarding, since marked reduction in MIC
values against gram –ve bacterial strains was observed. Taking chloramphenicol as reference
antibiotic (Fig. 5), it was observed that compounds 2a, 4a, and 5b displayed reduction in
combined MIC value up to1/4 (0.7812 µg/mL) against E. coli. Due to combination approach, the
1
combined MIC values were reduced up to /8 in compounds 1a (0.7812 µg/mL), 1b, 2c, 3b, 4b,
5c, 6a, 7a and 7c (0.3906 µg/mL) against E. coli and in compounds 2b, 7a (0.7812 µg/mL), and
6a, 7c (0.3906 µg/mL) against P. aeruginosa. In the case of compounds 2b, 3c, 4c and 6b,
marked diminution in MIC (0.3906, 0.1953, 0.1953 and 0.1953 µg/mL, respectively) was
1
observed up to /16 of its original MIC against E. coli, whereas similar inhibition pattern was
observed in MIC of compounds 2c, 3c, 4b (0.3906 µg/mL) and 4c (0.1953 µg/mL) against P.
aeruginosa. More interestingly, it was observed that combined MIC value of compound 6b was
thirty two-fold lower against P. aeruginosa, i.e., MIC (0.1953 µg/mL) compared to original
MIC. Thus, a significant shift in the antibacterial profile was observed using combination
approach against the entire range of bacterial strains.
0.3
E. coli
0.25
P. aeruginosa
0.2
0.15
0.1
0.05
0
Compounds
Fig. 5. Graphical presentation of FIC of selected compounds in combination with varying concentrations
of chloramphenicol and ½ MIC of test compounds against gram –ve bacterial strains
15

The same experiment was performed again with varying concentrations of antibiotic
sulfamethoxazole and ½ MIC of test compounds using serial dilution method. From Table 5, it
was observed that the combined MIC of compound 2a was reduced to 1/4 (0.7812 µg/mL) against
E. coli bacterial strain and that of compound 4a against E. coli and 6a against P. aeruginosa to
1/8 (0.3906 µg/mL) compared to their original MIC values, whereas compounds 2c, 3c, 4b, 5b,
1
6a, 6b, and 7a displayed a marked declination in MIC up to /16 (0.1953 µg/mL) of its original
MIC against E. coli and compound 4c also exhibited the same inhibition pattern against P.
aeruginosa. In the case of compounds 1b, 4c, 5c, and 7c, excellent inhibition pattern was
1
observed in MIC, i.e., a noticeable diminution up to /32 (0.0976 µg/mL) of original MIC against
E. coli and compound 7c showed the same reduction pattern (MIC 0.0976 µg/mL) against P.
aeruginosa (Fig. 6).
0.3
0.25
0.2
0.15
0.1
0.05
0
E. coli
P. aeruginosa
Compounds
Fig. 6. Graphical presentation of FIC of selected compounds in combination with varying concentrations
of sulfamethoxazole and ½ MIC of test compounds against gram –ve bacterial strains
Thus, the synergistic effect of antibiotics and compounds discovered through this type of
combinational approach, showed a marked declination in combined MIC in all cases. Significant
augmentation in antibacterial activity was observed when experiment was performed with
antibiotic sulfamethoxazole, as compared to chloramphenicol. Highly favourable results clearly
16

indicated that broad spectrum potential antibiotics can be developed using a combination
approach.
2.5. Cytotoxicity evaluation of N-heterocyclic sulfonamide analogues
The cytotoxicity of compounds was assayed by MTT assay using three different cell lines, i.e.,
PBM cell lines of human, CEM and Vero cell lines derived from kidney of African green
monkey. The results showing the viability potency of compounds 1-7 and derivatives in terms of
IC50 are summarized in Table 2. All test compounds except compounds 1a - b, 3a - b, 4b, 6b and
7c, exhibited considerably less cytotoxicity against all the three cell lines. In the case of
compounds 1a (IC50 = 27.23 µg/mL) and 1b (IC50 = 29.27 µg/mL on PBM, Vero cell line and
IC50 = 18.06 µg/mL on CEM cell lines) higher cytotoxicity values were observed against PBM
and Vero cell in comparison to CEM cell line. In the case of compounds 3a - b, 3a showed lower
toxicity on PBM cell line (IC50 = 27.23 µg/mL) followed by mild toxicity against CEM cell (IC50
= 25.87 µg/mL) and Vero cell (IC50 = 24.78 µg/mL) whereas 3b (IC50 = 29.27 µg/mL) was less
toxic against PBM and CEM cell lines compared to Vero cell. Compounds 4b, 5a and 6b (IC50 =
33.77, IC50 = 34.84 and IC50 = 27.47 µg/mL, respectively) were also found to be less toxic
against PBM and CEM cell lines in comparison to Vero cell line (6a; IC50 = 1.65 µg/mL) .
Compound 7c was found to be least toxic against PBM and Vero cell lines (IC50 = 49.54 µg/mL)
and mildly toxic against CEM cell line (IC50 = 20.41 µg/mL). Overall, the cytotoxicity assay
clearly suggested that compounds having polar groups like (-NO2, Br, Cl), showed less toxicity
in comparison to non polar –CH3 group against PBM, Vero and CEM cell lines.27 It can be
inferred from the results that cytotoxicity increased when moving from polar to non-polar
substituents, like nitro to chloro to methyl. The results also disclosed that all compounds were
less cytotoxic than the reference standard drug cycloheximide against all the three cell lines.
2.6. Molecular modeling
To study the binding of the designed ligands to the receptor binding site of DHPS, a significant
computational method, viz. molecular docking, was performed using Discovery Studio 2.5. All
molecules 1-7 and derivatives were docked within the active site of receptor to evaluate the
scoring functions (Interaction energy, Dock score, Binding energy, Predicted EC50) and assess
their mode of interaction (H-bond and π – π, π – + interactions). During analysis of the results,
compound 5 showed the highest interaction energy, i.e., I.E. of 5c was (-) 72.46 kcal/mol and
17

that of 5a and 5b was (-) 69.42 and (-) 61.59 kcal/mol, respectively, whereas compound 7
showed the lowest interaction energy, I.E. of 7a and 7c was (-) 40.17 and (-) 37.71 kcal/mol,
respectively. Rest compounds showed moderate I.E. as compared to the reference drug.
Dock Score (D.S.), another descriptor during in silico studies, again supported a better
interaction of designed ligands with the protein DHPS. The designed sulfonamides showed
significant to excellent value of dock score ranging from 30.76 to 58.51, whereas
sulfamethoxazole and chloramphenicol exhibited dock score 57.66 and 39.87, respectively.
All designed molecules showed higher binding free energy (ΔG) in comparison to reference
antibiotic sulfamethoxazole (-6.68 kcal ⁄ mol) and chloramphenicol (-6.41 kcal ⁄ mol). Ludi_2
and Ludi_3 scoring functions were used to calculate ΔG and Predicted EC50, respectively.28 It
can be concluded from the present study that molecules with higher value of ΔG were associated
with higher affinity for formation of stable protein – ligand complexes which corresponded to
lower EC50 value. Molecular docking results were explained on the basis of hydrogen bonding
and non-covalent π - π, π - + interactions, which stabilized the ligand - protein complexes.29-30
Docking results of all compounds with bacterial protein are shown in Table 6.
Docking results obtained from DS 2.5 showed that compounds were accommodated well in the
binding pocket of BaDHPS. The top-ranked docking poses reproduced the key interactions
observed in N-heterocyclic sulfonamide-BaDHPS complexes. The reference antibiotic
sulfamethoxazole formed six H - bonds with amino acids Arg68, Arg254 and Lys220 and
chloramphenicol formed four H - bonds with Lys220, Ser221 and Arg254. A close inspection of
docking results revealed that -SO2- moiety of all designed ligands formed 2 - 4 H - bonds with
amino acids Arg68 and Arg254 at a distance ranging between 2.5 - 3.1 Å and 2.5 - 3.0 Å,
respectively. In addition to H - bonds formed with Arg68 and Arg254, compound 2c formed H -
bonds with amino acid Thr67 and Lys220, compound 3c with Ser221, compound 4c with Ser221
and Asn120, compound 5a with Asn120 and Lys220, 5b with Ser221 and 5c with Asn120.
Hydrogen bond was formed between amino acid and -Cl group in 2c, -NO2 group in 3c and 4c, -
OEt group in 5a, 5b and 5c with a distance ranging between 2.5 - 3.2 Å. Most notably, docking
results revealed that all compounds interacted with active site of DHPS enzyme and formed
two-to-five π - + interactions with amino acid residues Lys220 and Arg254 at a distance ranging
between 4.4 - 6.7 Å and 3.7 - 4.7 Å, respectively, as found in reference drugs.
18

Table 6
Docking results: scoring, stability of ligands, molecular docking interaction of ligands 1-7 and derivatives with DHPS receptor in ligand-receptor docked complexes
Compound
IE
DS
∆G
Predicted EC50
No. of H-B/ Amino acid in H-B (distance)
No. of π-B/ Amino acid in π-B (distance)
-47.43
-44.10
46.60
44.70
-6.58
-6.53
5.75X10-6
6.16X10-6
2/ Arg68 (2.5), Arg254 (2.6)
2/ Arg68 (2.5), Arg254 (2.6)
3/ Lys220 (6.0), Lys220 (4.9), Arg254 (4.2)
4/ Lys220 (5.1), Arg68 (6.4), Lys220 (5.1),
Arg254 (4.3)
1a
1b
-47.78
-45.19
-52.53
40.26
36.67
30.76
-7.27
-7.27
-6.7
7.58X10-7
7.76X10-7
1.07X10-6
3/ Arg68 (2.5), Arg68 (2.9), Arg254 (2.6)
3/ Arg68 (3.1), Arg68 (2.5), Arg254 (2.6)
6/ Thr67 (3.1), Arg68 (3.0), Lys220 (2.9),
Arg254 (2.7), Arg254 (2.5), Arg254 (2.6)
3/ Lys220 (5.0), Lys220 (5.6), Arg254 (4.3)
3/ Lys220 (5.2), Lys220 (5.9), Arg254 (4.2)
3/ Arg254 (3.7), Arg254 (3.9), Lys220 (4.1)
2a
2b
2c
-56.39
-57.44
-59.87
51.26
52.89
50.90
-7.0
-7.10
-7.04
5.88X10-7
7.24X10-7
1.94X10-6
3/ Arg68 (3.0), Arg68 (2.5), Arg254 (2.6)
3/ Arg68 (3.0), Arg68 (2.6), Arg254 (2.6)
4/ Arg68 (3.0), Arg68 (2.5), Ser221 (3.2),
Arg254 (2.6)
3/ Arg254 (3.8), Arg254 (4.6), Lys220 (4.4)
3/ Arg254 (3.8), Arg254 (4.7), Lys220 (4.5)
3/ Arg254 (3.9), Arg254 (4.6), Lys220 (4.6)
3a
3b
3c
-55.99
-52.19
-58.17
51.76
49.67
45.22
-6.70
-6.59
-6.69
3.63X10-6
4.89X10-6
3.38X10-6
2/ Arg68 (2.5), Ser221 (3.2)
2/ Arg68 (2.6), Arg254 (2.6)
5/ Arg68 (2.6), Asn120 (3.2), Ser221 (3.0),
Ser221 (3.1), Arg254 (2.6)
3/ Lys220 (5.9), Lys220 (5.3), Arg254 (4.6)
3/ Lys220 (6.3), Lys220 (5.1), Arg254 (4.1)
5/ Arg254 (3.8), Arg254 (4.6), Lys220 (4.4),
Phe189 (5.8), Phe189 (6.4)
4a
4b
4c
-69.42
-61.59
52.87
58.36
-9.17
-6.97
3.54X10-8
2.39X10-7
5/ Arg68 (2.6), Asn120 (3.0), Lys220 (2.8),
Arg254 (3.1), Arg254 (2.6)
2/ Arg254 (4.5), Arg254 (4.8)
5a
5b
5/ Arg68 (2.5), Ser221 (2.9), Ser221 (2.8),
Arg254 (2.7), Arg254 (2.6)
3/ Lys220 (5.4), Lys220 (4.6), Arg254 (3.9)
-72.46
-50.35
-52.66
-40.17
57.79
53.62
56.22
42.91
-8.60
-6.99
-6.76
-7.29
6.91X10-8
2.39X10-6
3.23X10-6
3.54X10-6
3/ Arg68 (2.6), Asn120 (2.9), Arg254 (2.8)
3/ Arg68 (2.6), Arg254 (2.6), Arg254 (3.0)
3/ Arg68 (2.6), Arg254 (2.7), Arg254 (3.1)
4/ Arg68 (3.0), Arg68 (2.5), Arg254 (2.6),
Arg254 (2.9)
1/ Arg254 (4.2)
1/ Arg254 (4.1)
2/ Arg254 (4.3), Lys220 (6.7)
2/ Lys220 (5.2), Arg254 (4.2)
5c
6a
6b
7a
-37.31
-60.32
40.14
57.66
-7.26
-6.68
3.54X10-6
3.01x10-6
3/ Arg68 (2.5), Arg68 (3.1), Arg254 (2.6)
6/ Arg68 (2.6), Lys220 (2.9), Lys220 (3.0),
Arg254 (2.5), Arg254 (2.9), Arg254 (3.0)
3/ Lys220 (5.2), Lys220 (5.9), Arg254 (4.3)
2/ Lys220 (4.4), Arg254 (3.9)
7c
Sulfamethoxazole
Chlormphenicol
-77.42
39.87
-6.41
1.73x10-5
1/ Lys220 (4.8)
4/ Lys220 (2.5), Ser221 (2.9), Ser221 (2.7),
Arg254 (2.6)
IE -Total interaction energy (kcal/mol), DS - Dock Score, ΔG - predicted binding free energy (kcal⁄ mol), Predicted EC50- predicted 50% effective concentration required to inhibit
bacterial replication (μM)
19

Sulfamethoxazole formed two π - + interactions with amino acid Lys220 (4.4 Å) and Arg254
(3.9 Å) whereas chloramphenicol interacted with Lys220 (4.8 Å) to form a stable π - +
interaction .Compounds 1b and 4c, additionally, showed one π - + interaction with Arg68 (6.4 Å)
and two π - + interactions with Phe189 (5.8, 6.4 Å), which was not observed in reference drugs.
Docking interactions of some selected sulfonamide derivatives and the reference drugs
(chloramphenicol and sulfamethoxazole) with active site of DHPS binding pocket are shown in
Fig. 7. The characteristic orientation of all compounds indicated that they can occupy the PABA
pocket preventing the key substrate from binding, which is the basis of the inhibitory action of
all sulfa drugs against DHPS.31-32
Compound 1b
Compound 2c
Compound 4c
Compound 7a
20

Sulfamethoxazole
Chloramphenicol
Fig. 7. Molecular docking interactions of selected compounds 1b, 2c, 4c, 7a and reference drugs
(Sulfamethoxazole and Chloramphenicol) into the active site of DHPS binding pocket
2.7. In silico ADMET prediction of sulfonamide analogs
In silico pharmacokinetic, metabolism and toxicity parameters of all sulfonamide derivatives are
predicted using DS 2.5 software and summarized in table 7. Intestinal absorption has been
predicted to be more than 90% for all compounds except 5c (87.45%), which is greater than
reference drugs sulfamethoxazole (76.46%) and chloramphenicol (69.94%). Metabolism plays a
vital role in the bioavailability of drugs and cytochrome CYP450 enzymes are most important
class to study this effect. Compounds were studied either to act as substrate or inhibitors of
CYPs. Most of the designed compounds were found to be the substrate of CYP3A4 and
inhibitors of CYP1A2, CYP2C19, and CYP2C9. All ADMET parameters including absorption,
distribution, metabolism, excretion, and toxicity were found to be favorable in an acceptable
range for all sulfonamide derivatives and are comparable to that of the standard drugs having
lead like properties and in some cases, even better results than reference drugs were observed.
21

Table 7
In silico ADMET predictions and synthetic accessibility values of sulfonamide derivatives
Absorption
Distribution
Metabolism
Excretion
Toxicity
Maximum
Compd
Synthetic
Water
Intestinal
Skin
Blood brain
barrier
permeability
CYP
Total Renal OCT2 Oral rat acute
Acessibility
solubility absorption permeability
(human)
clearance
substrate
toxicity(LD50)
tolerated
dose
2
D
6
3
A
4
1
A
2
2C
1
9
2
C
9
2
D
6
3
A
4
(human)
Subst
rate
Inhibitor
(log S
mol/L)
Numeric
(%
Numeric
(log Kp)
Numeric
(log
Categorical
(Y/ N)
Numeric
(mol/kg)
Numeric
(log
Numeric
(log BB)
Absorbed)
ml/min/kg)
mg/kg/day)
Categorical (Y/ N)
1a
1b
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
7a
-3.05
-3.08
-3.03
-3.06
-3.0
93.43
91.97
92.16
90.70
100.00
95.50
94.04
95.40
95.90
96.61
92.31
91.95
90.49
87.45
92.99
91.53
90.84
100.00
76.46
69.94
-2.74
-2.74
-2.74
-2.74
-2.73
-2.57
-2.58
-2.70
-2.71
-2.71
-2.73
-2.79
-2.79
-2.74
-2.78
-2.79
-2.74
-2.73
-3.06
-2.75
0.05
0.04
0.02
0.01
-1.27
0.32
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
Y
N
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
N
Y
Y
N
N
N
N
N
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
N
Y
Y
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
0.43
0.42
0.27
0.25
0.03
0.14
0.19
0.15
0.15
0.21
0.62
0.13
0.06
0.20
0.06
-0.01
0.37
0.14
0.37
0.27
Y
Y
N
Y
N
N
N
N
N
N
N
Y
Y
N
N
N
N
N
N
N
2.47
2.52
2.82
2.87
2.56
2.12
2.22
2.46
2.50
2.59
2.54
2.15
2.21
3.16
2.17
2.27
3.13
2.69
2.42
2.47
0.65
0.66
0.37
0.38
0.23
0.37
0.38
0.08
-0.08
-0.08
-0.33
0.17
0.18
-0.08
0.43
0.45
0.42
0.28
0.91
-0.07
2.74
2.90
2.94
2.91
3.04
2.82
2.80
2.86
2.88
2.85
2.98
3.10
3.06
3.20
2.76
2.73
2.34
3.05
2.73
2.78
N
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
N
Y
Y
N
Y
-3.87
-3.08
-3.09
-4.29
-4.06
-4.09
-4.14
-4.08
-4.06
-3.38
-3.83
-3.16
-3.21
-2.96
-3.14
0.31
-0.09
-0.98
-1.15
-1.09
-0.69
-0.87
-1.0 2
0.24
0.23
-0.05
-0.64
-0.72
-1.12
7c
Ref 1
Ref 2
Ref 1 = Sulfamethoxazole; Ref 2 = Chloramphenicol; Y = Yes; N = No; Water solubility = < -4 soluble; Intestinal absorption = below 30% indicates poor absorbance; Skin
permeability = > -2.5 considered to be permeable; Blood brain barrier permeability = < -1 considered poorly distributed to the brain; Total clearance (logCLtot) = Lower value
indicates high drug half lifetime; LD50 (Lethal Dose) = Lower value predicts minimum toxicity; Maximum tolerated dose = ≥ 0.47 predicts lower toxicity; Synthetic Acessibility
= 1(very easy) to 10(very difficult)
22

2.8. Structure activity relationship (SAR)
It was observed that most of the N-heterocyclic sulfonamide derivatives were equipotent in
antibacterial activity with reference antibiotics and exhibited even superior activity when
subjected to combination study. Analyzing the result, it was found that antibacterial activity
depends on the type of substituents (electron withdrawing group or electron releasing group),
position of substituents and even number of substituents present in the compound. We have
investigated that compounds 6a, 6b, 7a and 7c showed the most potent inhibitory activity against
all bacterial strains. Focusing on SAR, we found that introduction of -Cl group at second position
of nucleus 2 (2-Cl-benzimidazole) in comparison to nucleus 1 (benzimidazole), enhanced the
antibacterial activity considerably. Similarly in the case of nucleus 3 (indazole) and 4 (5-NO2-
indazole), a significant augmentation is noticed in the MIC value of compound 4 against the
gram +ve and gram –ve strains, which may be attributed due to introduction of NO2 group at
fifth position. On the other hand, substitution at 5 and 6 position (nucleus 7) or 5/6 position
(nucleus 4 and 5) of heterocyclic benzene ring affected significantly the antibacterial activity.
Table 2 illustrates that all derivatives with electron withdrawing substituents such as Cl, Br, and
NO2 group, had significantly higher antibacterial activity due to high electronic effect of these
groups than electron donating CH3 group, showing moderate activity. It was also observed that
among the halo-substituted derivatives, electronegativity and atomic size of the substituents,
played an important role, i.e., the activity enhanced with decreasing electro negativity and
increasing atomic size of the substituent. Finally, experimental results revealed the order of
antibacterial activity of all compounds against different bacterial strains as: E. coli > P.
aeruginosa > S.aureus ≈ B. cerus.
3. Conclusion
As a concluding remark, a new series of aryl N-heterocyclic sulfonamide derivatives bearing
thiomide linkage was designed, synthesized on the basis of physicochemical parameters and
evaluated for in vitro antibacterial activity. The molecules showed promising in silico results,
which were in conjugation with significant in vitro antibacterial activity. Excellent antibacterial
efficacy was observed against E. coli bacterial strain in the case of test compounds. A great
synergistic effect, on the basis of determination of FIC values, on the MIC of test compounds
was observed using the combination approach, which reduced the MIC of test compounds
23

1
1
ranging from /4 to /32. This indicated four to thirty two times higher antibacterial potential of
test compounds with low cytotoxicity. Furthermore, molecular docking results represented that
binding mode of the test compounds to BaDHPS was similar to the reference drugs and the
molecules were well accommodated in the PABA pocket of DHPS indicating thereby a similar
mechanism of action as that of sulfa drugs. A deep investigation of in vitro activity supported by
docking results and in silico ADMET results clearly suggested that compounds 2c, 4c, 5c, 6a-b,
7a and 7c could be developed as lead inhibitors of folic acid synthesis. The study further
indicated towards the development of highly potent drug regimens using combination approach.
4. Experimental
4.1. Design, Physicochemical descriptors and ADME properties of sulfonamides
Using Molinspiration and ChemDraw softwares, some physically significant descriptors and
pharmaceutically relevant properties of compounds, like molecular weight, H-bond donors, H-
bond acceptors, log P, rotatable bonds and TPSA have been analyzed on the basis of Lipinski’s
rule of five. ADME properties, like aqueous solubility (Log S), skin permeability (Log Kp),
synthetic accessibility scores and Absorption (% ABS) were calculated using DS 2.5 software.
4.2. Chemistry
All chemicals and reagents were purchased from Sigma Aldrich Chemical Company, USA and
E. Merck India Ltd, India. All reactions were carried out in oven dried apparatus using dried and
distilled solvents. Column chromatography was carried out on silica gel (100-200 mess).
Compounds were confirmed by TLC, using silica gel 60F254 aluminium sheets and visualized
by ultraviolet light at 254 nm. Melting points recorded on electro thermal apparatus using open
capillary tubes are uncorrected. NMR spectra were recorded on BRUKER-AV400 spectrometer
(Bruker Co., Faellanden, Switzerland) in DMSO-d6 (1H at 400 MHz and 13C at 100 MHz).
Chemical shifts (δ) are expressed in parts per million (ppm) and J (coupling constant) values are
expressed in Hz. Multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), and
m (multiplet). Mass spectra were recorded on Micromass Q-Tof (ESI-HRMS). The elemental
analyses were performed on a Perkin-Elmer 240-C analyses equipment.
4.2.1. Synthesis of 5, 6-dibromo-2-chloro-1H-benzimidazole (7)
To a solution of compound 2 (2-chloro-1H-benzimidazole; 1g) in MeOH (30 mL), a solution of
Br2/MeOH (1.5 mL/15 mL) was added drop wise. The resulting suspension was stirred at room
24

temperature for 6 h, added H2O (30 mL) and stirring continued at room temperature for 18-20 h.
The reaction mixture was filtered and the precipitate was washed thoroughly with cold H2O until
the washing was neutral. The white coloured solid was air dried and crystallized from MeOH.
White solid, yield (72%); mp 227-228 oC; Rf - 0.52 (EtOAc:Hexane :: 3:7); 1H NMR (400 MHz,
13
DMSO-d6) δppm: 7.95 (s, 2H), 13.79 (br, s, 1H, -NH); C NMR (100 MHz, DMSO-d6) δppm
:
120.9, 121.5, 138.7, 140.2; HRMS m/z Calcd. for C7H3Br2ClN2 (M+H)+: 308.8430, Found:
308.8434; Anal. Calcd. For C7H3Br2ClN2: C, 27.05; H, 0.95; Br, 51.45; Cl, 11.42; N, 9.01%
Found: C, 27.07; H, 0.96; Br, 51.48; Cl, 11.44; N, 9.05%.
4.2.2. General synthesis of N-heterocyclic sulfonamide derivatives 1 – 7 and derivatives
A solution of compound 1 (or 2/ 3/ 4/ 5/ 6/ 7; 1 mmol) and dimethyl amino pyridine (1.5 mmol)
in acetonitrile (5 mL) was stirred at room temperature for 30 min. After 30 min. sulfonyl chloride
a-c (2 mmol) was added to the stirred solution. The reaction mixture was heated at 40-50 oC for
5-6 h. TLC analysis indicated the progress of the reaction and consumption of reactants into
product (compounds 1-7). The reaction mixture was dried under vacuum and the dry mass was
partitioned between EtOAc and aqueous NaHCO3 solution. The organic layer was dried over
anhydrous Na2SO4 and concentrated under vacuum to give the crude product. Purification of the
crude product was done by column chromatography and recrystallisation from ethanol.
4.2.2.1. 1-Tosyl-1H-benzimidazole (1a)
o
White crystalline powder, yield (65%), mp 113-114 C; Rf - 0.58 (DCM:Hexane :: 5:5); 1H
NMR (400 MHz, DMSO-d6) δppm= 2.39 (s, 3H,), 6.92-6.94 (m, 1H ), 6.98-7.01 (m, 2H, ), 7.27
13
(d, J = 8.2 Hz, 2H), 7.58 (d, J = 8.2 Hz, 2H), 8.03 (s, 1H), 9.09 (s, 1H) ; C NMR (100 MHz,
DMSO-d6) δppm= 21.2, 116.4, 117.5, 128.1, 128.2, 129.8, 130.9, 137.9, 138.5, 141.7, 145.1,
146.5; MS m/z Calcd for C14H12N2O2S (M+): 272.03; Anal. Calcd. For C14H12N2O2S: C, 61.75;
H, 4.44; N, 10.29; S, 11.77% Found: C, 61.72; H, 4.41; N, 10.27; S, 11.76%.
4.2.2.2. 1-(4-Chlorophenylsulfonyl)-1H-benzimidazole (1b)
o
1
White crystalline powder, yield (67%); mp 188-189 C; Rf - 0.56 (DCM:Hexane :: 5:5) ; H
NMR (400 MHz, DMSO-d6) δppm= 6.90-6.93 (m, 1H ), 6.99-7.01 (m, 2H), 7.28 (d, J = 8.2
13
Hz,2H), 7.59 (d, J = 8.2 Hz, 2H), 8.04 (s, 1H), 8.96 (s, 1H); C NMR (100 MHz, DMSO-d6)
δppm= 112.5, 121.1, 122.8, 126.8, 129.5, 129.8, 131.2, 135.1, 139.8, 140.6, 143.8; MS m/z Calcd
25

for C13H9ClN2O2S (M+): 292.01; Anal. Calcd. For C13H9ClN2O2S: C, 53.34; H, 3.10; N, 9.57;
Cl, 12.11; S, 10.95% Found: C, 53.32; H, 3.9; N, 9.55; Cl, 12.10, S, 10.94%.
4.2.2.3. 2-Chloro-1-tosyl-1H–benzimidazole (2a)
o
1
Cream coloured powder, yield (65%); mp 99-100 C; Rf - 0.55 (DCM:Hexane :: 5:5); H NMR
(400 MHz, DMSO-d6) δppm= 2.39 (s, 3H), 6.92-6.94 (m, 1H), 6.98-7.00 (m, 1H), 7.27 (d, J = 8.2
13
Hz, 2H), 7.58 (d, J = 8.2 Hz, 2H), 8.03 (s, 1H), 9.0 (s, 1H); C NMR (100 MHz, DMSO-d6)
δppm= 21.5, 112.9, 115.7, 123.4, 126.6, 128.5, 130.4, 130.9, 134.9, 138.9, 139.7, 141.8; MS m/z
Calcd for C14H11ClN2O2S (M+): 306.02; Anal. Calcd. For C14H11ClN2O2S: C, 54.81; H, 3.61; N,
9.13; Cl, 11.56; S, 10.45% Found: C, 54.78; H, 3.64; N, 9.11; Cl, 11.53; S, 10.55%.
4.2.2.4.2-Chloro-1-(4-chlorophenylsulfonyl)-1H-benzimidazole (2b)
o
Dull white powder, yield (68%); mp 148-149 C; Rf - 0.58 (DCM:Hexane :: 5:5); 1H NMR (400
MHz, DMSO-d6) δppm= 7.22 (d, J = 3.0 Hz, 2H), 7.60 (d, J = 2.0 Hz, 2H), 7.96 (d, J = 5.8 Hz,
2H), 8.16 (d, J = 4.8 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δppm= 112.9, 115.3, 123.4, 126.5,
129.8, 129.9, 130.9, 135.8, 138.9, 139.6, 141.8; MS m/z Calcd for C13H8Cl2N2O2S (M+): 325.97;
Anal. Calcd. For C13H8Cl2N2O2S: C, 47.72; H, 2.46; N, 8.56; Cl, 21.67; S, 9.80% Found: C,
47.70; H, 2.47; N, 8.55; Cl. 21.64; S, 9.79%.
4.2.2.5.2-Chloro-1-(4-nitrophenylsulfonyl)-1H-benzimidazole (2c)
o
1
Light yellow powder, yield (58%); mp 160-161 C; Rf - 0.54 (DCM:Hexane :: 5:5); H NMR
(400 MHz, DMSO-d6) δppm= 7.40 (d, J = 3.2 Hz, 2H), 7.79 (d, J = 2.2 Hz, 2H), 7.97 (d, J = 5.9
Hz), 8.15 (d, J = 4.8 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δppm= 112.8, 116.4, 123.8, 124.9,
126.7, 129.4, 131.2, 138.6, 140.8, 144.2, 152.7; MS m/z Calcd for C13H8ClN3O4S (M+): 336.99;
Anal. Calcd. For C13H8ClN3O4S: C, 46.23; H, 2.39; N, 12.44; Cl, 10.50, S, 9.49% Found: C,
46.21; H, 2.37; N, 12.46; Cl, 10.49; S, 9.50%.
4.2.2.6. 1-Tosyl-1H-indazole (3a)
o
1
Off white powder, yield (69%); mp 128-129 C; Rf - 0.55 (DCM:Hexane :: 5:5); H NMR (400
MHz, DMSO-d6) δppm= 2.52 (s, 3H), 7.46 (t, J = 7.0 Hz, 1H), 7.70-7.75 (m, 1H), 7.92 (d, J = 8
13
Hz, 1H), 8.17-8.22 (m, 3H), 8.38 (d, J = 7.2 Hz, 2H), 8.86 (s, 1H) ; C NMR (100 MHz,
DMSO-d6) δppm= 21.3, 112.5, 122.4, 125.0, 125.8, 128.7, 130.1, 139.6, 141.0, 143.7, 150.8; MS
m/z Calcd for C14H12N2O2S (M+): 272.06; Anal. Calcd. For C14H12N2O2S: C, 61.75; H, 4.44; N,
10.29; S, 11.77% Found: C, 61.72; H, 4.43; N, 10.27; S, 11.76%.
26

4.2.2.7. 1-(4-Chlorophenylsulfonyl)-1H-indazole (3b)
Light cream coloured powder, yield (60%); mp 135-136 oC; Rf - 0.58 (DCM:Hexane :: 5:5); 1H
NMR (400 MHz, DMSO-d6) δppm= 7.47 (t, J = 7.0 Hz, 1H), 7.70-7.75 (m, 1H), 7.88 (d, J = 8.4
13
Hz, 1H), 8.17-8.26 (m, 3H), 8.36-8.40 (m, 2H), 8.85 (s, 1H); C NMR (100 MHz, DMSO-d6)
δppm= 112.5, 122.4, 125.0, 125.8, 128.7, 130.1, 139.6, 141.0, 143.7, 150.8; MS m/z Calcd for
C13H9ClN2O2S (M+): 292.01; Anal. Calcd. For C13H9ClN2O2S: C, 53.34; H, 3.10; N, 9.57; Cl,
12.11; S, 10.9% Found: C, 53.31; H, 3.12; N, 9.55; Cl, 12.10; S, 10.94%.
4.2.2.8. 1-(4-Nitrophenylsulfonyl)-1H-indazole (3c)
Light cream coloured powder, yield (62%); mp 140-142 oC; Rf - 0.54 (DCM:Hexane :: 5:5); 1H
NMR (400 MHz, DMSO-d6) δppm= 7.47 (t, J = 7.0 Hz, 1H), 7.70-7.75 (m ,1H), 7.91 (d, J = 8Hz,
1H), 8.17-8.22 (m, 3H), 8.36-8.40 (m, 2H), 8.66 (s, 1H); 13C NMR (100 MHz, DMSO-d6) δppm
=
112.5, 122.4, 125.0, 125.8, 128.7, 130.1, 139.6, 141.0, 143.7, 150.8; MS m/z Calcd for
C13H9N3O4S (M+): 303.03; Anal. Calcd. For C13H9N3O4S: C, 51.48; H, 2.99; N, 13.85; S,
10.57% Found: C, 51.47; H, 2.97; N, 13.86; S, 10.55%.
4.2.2.9. 5-Nitro-1-tosyl-1H-indazole (4a)
Light cream coloured powder, yield (65%); mp 190-191 oC; Rf - 0.58 (DCM:Hexane :: 7:3); 1H
NMR (400 MHz, DMSO-d6) δppm= 2.34 (s, 3H), 7.43 (d, J = 8.1 Hz, 2H), 7.88 (d, J = 8.4 Hz,
2H), 8.33 (d, J = 9.2 Hz, 1H), 8.49 (d. J = 2.2 Hz, 1H), 8.75 (s, 1H), 8.85 (s, 1H) ; 13C NMR
(100 MHz, DMSO-d6) δppm= 21.09, 113.4, 119.4, 124.4, 125.5, 127.3, 130.5, 133.0, 141.7, 143.3,
144.3, 146.6; MS m/z Calcd for C14H11N3O4S (M+): 317.05; Anal Calcd For C14H11N3O4S: C,
52.99; H, 3.49; N, 13.24; S, 10.10% Found: C, 52.98; H, 3.47; N, 13.24; S, 10.09%.
4.2.2.10. 1-(4-Chlorophenylsulfonyl)-5-nitro-1H-indazole (4b)
o
1
Cream coloured powder, yield (69%); mp 198-199 C; Rf - 0.60 (DCM:Hexane:: 7:3); H NMR
(400 MHz, DMSO-d6) δppm= 7.44 (d, J = 8.0 Hz, 2H), 7.88 (d, J = 8.4 Hz, 2H), 8.33 (d, J = 9.2
13
Hz, 1H), 8.47 (d, J = 2.4 Hz, 1H), 8.85 (s, 1H), 8.86 (s, 1H); C NMR (100 MHz, DMSO-d6)
δppm= 113.4, 119.6, 124.8, 125.5, 127.6, 130.8, 133.4, 141.9, 143.5, 144.3, 146.7; MS m/z Calcd
for C13H8ClN3O4S (M+): 336.99; Anal. Calcd. For C13H8ClN3O4S: C, 46.23; H, 2.39; N, 12.44;
Cl, 10.50; S, 9.49% Found: C, 46.22; H, 2.38; N, 12.42; Cl, 10.47; S, 9.47%.
4.2.2.11. 5-Nitro-1-(4-nitrophenylsulfonyl)-1H-indazole (4c)
27

o
1
Cream coloured powder, yield (69%); mp 201-202 C; Rf - 0.60 (DCM:Hexane:: 7:3); H NMR
(400 MHz, DMSO-d6) δppm= 7.43 (d, J = 8.0 Hz, 2H), 7.89 (d, J = 8.4 Hz, 2H), 8.33 (d, J = 9.2
13
Hz, 1H), 8.50 (d, J = 2.2 Hz, 1H), 8.75 (s, 1H), 8.86 (s, 1H); C NMR (100 MHz, DMSO-d6)
δppm= 113.5, 119.4, 124.4, 125.4, 127.3, 130.5, 133.1, 141.7, 143.3, 144.3, 146.6; MS m/z Calcd
for C13H8N4O6S (M+): 348.02; Anal. Calcd. For C13H8N4O6S C, 44.84; H, 2.33; N, 16.05; S,
9.20% Found: C, 44.83; H, 2.32; N, 16.09; S, 9.21%.
4.2.2.12. N-(6-Ethoxybenzothiazol-2-yl)-4-methylbenzenesulfonamide (5a)
Creamish yellow powder, yield (66%); mp 223-224 oC; Rf - 0.54 (DCM:Hexane :: 5:5); 1H NMR
(400 MHz, DMSO-d6) δppm: 1.41 (t, J = 6.8 Hz, 3H), 2.36 (s, 3H,), 3.92-3.98 (q, J = 6.8 Hz, 2H),
6.72-6.74 (d, J = 8.4 Hz, 1H), 7.18 (d, J = 2.4 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 8.8
Hz, 2H), 8.24 (d, J = 8.8Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δppm= 21.7, 14.8, 63.4, 105.5,
113.1, 118.2, 123.8, 127.3, 142.9, 148.2, 151.4, 153.6, 155.8, 164.7; MS m/z Calcd for
C16H16N2O3S2 (M+): 348.06; Anal. Calcd. For C16H16N2O3S2: C, 55.15; H, 4.63; N, 8.04; S,
18.40%. Found: C, 55.12; H, 4.61; N, 8.02; S, 18.38%.
4.2.2.13. 4-Chloro-N-(6-ethoxybenzothiazol-2-yl)benzenesulfonamide (5b)
Creamish yellow powder, yield (62%); mp 234-235 oC; Rf - 0.56 (DCM:Hexane :: 5:5); 1H NMR
(400 MHz, DMSO-d6) δppm= 1.40 (t, J = 6.8 Hz, 3H), 4.01 (q, J = 6.8 Hz, 2H), 6.74 (d, J = 8.4
Hz,1H), 7.17 (d, J = 2.4 Hz, 1H), 7.20 (d, J = 8.4 Hz, 2H), 8.02 (d, J = 8.8 Hz, 2H), 8.25 (d, J =
13
8.8 Hz, 1H); C NMR (100 MHz, DMSO-d6) δppm= 14.7, 63.4, 105.8, 113.2, 118.2, 123.4,
127.6, 142.9, 148.2, 151.4, 152.7, 155.8, 165.7; MS m/z Calcd for C15H13ClN2O3S2 (M+):
368.01; Anal. Calcd. For C15H13ClN2O3S2: C, 48.84; H, 3.55; N, 7.59; Cl, 9.61; S, 17.39%.
Found: C, 48.82; H, 3.53; N, 7.58; Cl, 9.61; S, 17.37%.
4.2.2.14. N-(6-Ethoxybenzothiazol-2-yl)-4-nitrobenzenesulfonamide (5c)
o
Mustard yellow powder, yield (60%); mp 240-241 C; Rf - 0.50 (DCM:Hexane :: 5:5); 1H NMR
(400 MHz, DMSO-d6) δppm= 1.31 (t, J = 6.8 Hz, 3H), 3.98-4.03 (q, J = 6.8 Hz, 2H), 6.72-6.75
(d, J = 8.4 Hz, 1H), 7.17 (d, J = 2.4 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 8.8 Hz, 2H),
8.25 (d, J = 8.8 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δppm= 14.8, 63.5, 105.6, 113.2, 118.2,
123.7, 127.4, 142.9, 148.2, 151.4, 153.7, 155.8, 164.7; MS m/z Calcd for C15H13N3O5S2 (M+):
379.03; Anal. Calcd. For C15H13N3O5S2: C, 47.48; H, 3.45; N, 11.08; S, 16.90% Found: C,
47.47; H, 3.44; N, 11.06; S, 16.89%.
28

4.2.2.15. 4-Methyl-N-(thiazol-2-yl)benzenesulfonamide (6a)
Brown viscous solid, yield (60%); Rf - 0.56 (EtOAc:Hexane :: 3:7); 1H NMR (400 MHz, DMSO-
d6) δppm= 2.89 (s, 3H), 4.02 (s, 1H), 6.85 (d, J = 4.5 Hz, 1H), 7.27 (d, J = 4.6 Hz, 1H), 7.62 (d, J
13
= 8.6 Hz; 2H), 7.80 (d, J = 4.7 Hz; 2H); C NMR (100 MHz, DMSO-d6) δppm= 21.6, 113.8,
128.4, 130.2, 136.5, 137.1, 137.9, 172.3; MS m/z Calcd for C10H10N2O2S2 (M+): 254.02; Anal.
Calcd. For C10H10N2O2S2: C, 47.23; H, 3.96; N, 11.01; S, 25.22% Found: C, 47.24; H, 3.97; N,
11.01; S, 25.23%.
4.2.2.16. 4-Chloro-N-(thiazol-2-yl)benzenesulfonamide (6b)
Brown viscous solid, yield (65%); Rf - 0.54 (EtOAc:Hexane :: 3:7); 1H NMR (400 MHz, DMSO-
d6) δppm= 4.04 (s, 1H), 6.86 (d, J = 4.5 Hz, 1H), 7.28 (d, J = 4.6 Hz, 1H), 7.65 (d, J = 8.6 Hz,
2H), 7.82 (d, J = 8.6 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δppm= 112.4, 128.9, 129.4, 137.2,
137.7, 138.0, 171.6; MS m/z Calcd for C9H7ClN2O2S2 (M+): 273.96; Anal. Calcd. For
C9H7ClN2O2S2: C, 39.34; H, 2.57; N, 10.20; Cl, 12.90; S, 23.34% Found: C, 39.35; H, 2.55; N,
10.22; Cl, 12.91; S, 23.36%.
4.2.2.17. 5, 6-Dibromo-2-chloro-1-tosyl-1H-benzimidazole (7a)
White viscous oil, yield (58%); Rf - 0.58 (DCM:Hexane :: 8:2); 1H NMR (400 MHz, DMSO-d6)
δppm= 2.37 (s, 3H), 7.22 (d, J = 6 Hz, 2H), 7.49 (d, J = 3.2 Hz, 1H), 7.83 (s, 1H), 8.19 (s, 1H);
13C NMR (100 MHz, DMSO-d6) δppm= 21.5, 120.7, 121.5, 128.5, 130.4, 132.6, 134.5, 139.7,
140.4, 141.7; MS m/z Calcd for C14H9Br2ClN2O2S (M+): 463.84; Anal. Calcd. For
C14H9Br2ClN2O2S: C, 36.20; H, 1.95; N, 6.03; Cl, 7.63; Br, 34.40; S, 6.90% Found: C, 36.18; H,
1.96; N, 6.01; Cl, 7.64; Br, 34.39; S, 6.91%.
4.2.2.18. 5, 6-Dibromo-2-chloro-1-(4-nitrophenylsulfonyl)-1H-benzimidazole (7c)
1
Dirty white viscous oil, yield (55%); Rf - 0.52 (DCM:Hexane :: 8:2); H NMR (400 MHz,
DMSO-d6) δppm= 7.21 (d, J = 6 Hz, 2H), 7.50 (d, J = 3.2 Hz, 2H), 7.84 (s, 1H), 8.20 (s, 1H); 13C
NMR (100 MHz, DMSO-d6) δppm= 120.6, 120.9, 129.5, 129.8, 132.2, 135.8, 139.5, 140.2, 141.3;
MS m/z Calcd for C13H6Br2ClN3O4S (M+): 494.81; Anal. Calcd. For C13H6Br2ClN3O4S: C,
31.51; H, 1.22; N, 8.48; Cl, 7.15; Br, 32.25; S, 6.47% Found: C, 31.22; H, 1.20; N, 8.45; Cl,
7.10; Br, 32.30; S, 6.54%.
4.3 Antibacterial evaluation
4.3.1. Determination of Minimum Inhibitory Concentration (MIC)
29