Identification of antibacterial and antifungal pharmacophore sites for potent bacteria and fungi inhibition: Indolenyl sulfonamide derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.11.029

Synthesis of seven new indolenyl sulfonamides, have been prepared by the condensation reaction of indole-3-carboxaldehyde with different sulfonamides such as, sulphanilamide, sulfaguanidine, sulfathiazole, sulfamethoxazole, sulfisoxazole, sulfadiazine and

Full text of DOI:10.1016/j.ejmech.2009.11.029

European Journal of Medicinal Chemistry 45 (2010) 1189–1199
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Identification of antibacterial and antifungal pharmacophore sites for potent
bacteria and fungi inhibition: Indolenyl sulfonamide derivativesq
,
b
c
b,
*
Zahid H. Chohan a , Moulay H. Youssoufi , Aliasghar Jarrahpour , Taibi Ben Hadda
*
a Department of Chemistry, Bahauddin Zakariya University, Multan 60800, Pakistan
b
´
´
Laboratoire de Chimie des Materiaux, Universite Med. Premier, Av. Mohammed VI, Elqodss, Oujda 60000, Morocco
c Department of Chemistry, College of Sciences, Shiraz University, Shiraz 71454, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of seven new indolenyl sulfonamides, have been prepared by the condensation reaction of
indole-3-carboxaldehyde with different sulfonamides such as, sulphanilamide, sulfaguanidine, sulfathia-
zole, sulfamethoxazole, sulfisoxazole, sulfadiazine and sulfamethazine. These synthesized compounds
have been used as potential ligands for complexation with some selective divalent transition metal ions
(cobalt, copper, nickel & zinc). Structure of the synthesized ligands has been deduced from their physical,
analytical (elemental analyses) and spectral (IR, 1H NMR and 13C NMR & UV–vis) data. All the compounds
have also been assayed for their in vitro antibacterial and antifungal activities examining six species of
pathogenic bacteria (Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Salmonella typhi, Staphy-
lococcus aureus and Bacillus subtilis) and six of fungi (Trichophyton longifusus, Candida albicans, Aspergillus
flavus, Microsporum canis, Fusarium soloni and Candida glabrata). Antibacterial and antifungal results
showed that all the compounds showed significant antibacterial activity whereas most of the compounds
displayed good antifungal activity. Brine shrimp bioassay was also carried out for in vitro cytotoxic
properties against Artemia salina.
Received 14 April 2009
Received in revised form
4 November 2009
Accepted 12 November 2009
Available online 20 November 2009
Keywords:
Indolenyl sulfonamide
Antibacterial
Antifungal
Cytotoxicity
Virtual screening
Petra
Osiris
Molinspiration
Bioinformatics
Crown Copyright Ó 2009 Published by Elsevier Masson SAS. All rights reserved.
1. Introduction
Based on the significant biological and pharmacological prop-
erties [12] & Ref therein the indole moiety possesses, a new class of
Sulfonamides represent an important class of medicinally
important compounds which are extensively used as antibacterial
agent. It interferes with PABA (p-aminobenzoic acid) in the biosyn-
thesis of tetrahydrofolic acid, which is a basic growth factor essential
for the metabolic process of bacteria. Many activities apart from
carbonic anhydrase have been recently reviewed that include
endotelin antagonism, anti-inflammatory, tubular transport inhibi-
tion, insulin release and saluretic activity [1]. It is well documented
[2–5] that toxicological and pharmacological properties are
enhanced when sulfonamides are administered in the form of their
metal complexes [6–10]. Bulk of literature reveals that several
compounds containing indole ring form compounds of pharmaco-
logical interest [11].
such compounds is reported by combining the chemistry of
sulfonamides [13] with indole-3-carbaldehyde and to explore their
biological activities with the aim of obtaining more potent anti-
bacterial and/or antifungal compounds.
These indolenol sulfonamides reported in this paper formulate
a new class of antibacterial and antifungal agents that may become
excellent candidates for globally alarming drug resistance issues in
clinically used therapeutics.
There is considerable interest in these compounds as combined
antibacterial and antifungal agents, exhibiting potency similar
pharmacophore pockets to that observed for imipenem (IMP).
Several groups have reported on the failed attempts to get a clear
idea concerning the origin of each activity in this standard reference,
casting doubt over the structural needs assignment. We present
here the results of our virtual screening investigation into possible
alternative structures for these compounds. A comparison between
experiment and theoretical predictions of the antibacterial and
antifungal activity has enabled us to identify alternative combined
pharmacophore sites structures.
q
This work is dedicated to Prof. Dr. Johann Gasteiger for his bioinformatic
multiple services.
* Corresponding authors. Tel.: þ212 66 13 4178; fax: þ212 56 74 4749 (T.B. Hadda).
E-mail addresses: dr.zahidchohan@gmail.com (Z.H. Chohan), taibi.ben.hadda@
gmail.com, tbenhadda@yahoo.fr (T. Ben Hadda).
0223-5234/$ – see front matter Crown Copyright Ó 2009 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.11.029

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Z.H. Chohan et al. / European Journal of Medicinal Chemistry 45 (2010) 1189–1199
Scheme 1. Preparation of sulfonamides (1)–(7).
The most predicted potent of these compounds (2), (3) and (5)
varying degree of inhibitory effect on the growth of different tested
strains (Tables 1 and 2). A significant activity was observed by all
the compounds against E. coli. Compound (1) & (6) showed
moderate activity. Whereas, compounds (2), (3), (4), (5) & (7)
showed significant activity against S. flexenari. Compound (2) & (4)
showed moderate activity whereas compounds (1), (3), (5), (6) &
(7) showed significant activity against P. aeruginossa. All the
compounds showed significant activity against S. typhi except
compound (6), where moderate activity was observed. Compound
(1) against S. aureus copoumnd (5) & (7) against B. subtilis showed
moderate activity, whereas, all other compounds showed signifi-
cant activity against both the positive bacterial stains. Although
most of the compounds showed moderate to significant activity
against all Gram-negative and Gram-positive bacterial strains but
compounds (3) was the most active one (Figs. 2 and 3).
(high inhibition) were tested in bacterial cultures for their ability to
present a potential antibacterial pharmacophore site. Compounds
(3) and (5) were the most antibacterial agents. All these synthesized
indolenyl sulfonamides (1)–(7) were then tested for their antifungal
activity. They display too a potential antifungal activity against six
fungi, Trichophyton longifusus, Candida albicans, Aspergillus flavus,
Microsporum canis, Fusarium soloni and Candida glabrata; may be
because they have an antifungal pharmacophore site. Results from
all aspects of this bioinformatic approaches will be discussed, as will
our experience with screening candidates.
2. Chemistry
2.1. Synthesis of compounds (1)–(7)
3.1.2. Antifungal activity (in vitro)
The antifungal screening of all compounds was carried out
against T. longifusus, C. albicans, A. flavus, M. canis, F. soloni and
All synthesized compounds were prepared by refluxing an
equimolar ratio of indole-3-carbaldehyde and the respective
sulfonamide such as sulphanilamide, sulfaguanidine, sulfathiazole,
sulfamethoxazole, sulfisoxazole, sulfadiazine and sulfamethazine in
ethanol (Scheme 1) which successfully lead to a new series of
sulfonamides, containing indole moiety in good yield (80–86%). The
reaction was rapid, and no observation supported any kind of side
product. All the products were obtained as solids and their purities
were checked by thin layer chromatography (eluent ¼ n-hexane/
chloroform/ethylacetate, 1/2/1 v/v). All the synthesized compounds
were characterized by spectroscopic techniques (IR 1H NMR & 13C
NMR) and their elemental analyses.
3. Pharmacology
3.1. Biological activity
3.1.1. Antibacterial activity (in vitro)
All compounds were tested against four Gram-negative (E. coli,
S. flexneri, P. aeruginosa, S. typhi) and two Gram-positive (S. aureus,
B. subtilis) bacterial strains according to the literature protocol
[14,15]. The results were compared with those of the standard
drugs imipenem (Fig. 1). All the synthesized compounds exhibited
Fig. 1. Structure of standard reference (Imipenum) and indolenol SDZ analogue of
sulfonamides (1)–(7). The entity in blue color are a potential antibacterial pharma-
cophore site. That one in red color is a potential antifungal pharmacophore site.IMP:
Imipenum; SDZ: 4-amino-N-(pyrimidin-2yl)-benzenesulfonamide.

Z.H. Chohan et al. / European Journal of Medicinal Chemistry 45 (2010) 1189–1199
1191
Table 1
Antibacterial bioassay (concentration used 1 mg/mL in DMSO) and Antifungal bioassay (concentration used 200
m
g/mL).
Compd
Gram (ꢀ)a
Gram (þ)a
Fungi (%Inhibition)
E. coli
S. flexenari
P. aeruginosa
S. typhi
S. aureus
B. subtilis
T. longifucus
C. albicans
A. flavus
M. canis
F.soloni
C. glaberata
1
2
3
4
5
6
7
SD
21
23
24
22
23
19
24
30
18
22
23
20
19
18
19
27
19
18
20
17
19
20
20
26
20
23
23
19
20
17
22
27
18
22
23
19
20
19
23
30
20
23
24
23
17
19
18
28
00
20
35
00
20
00
25
A
40
35
40
25
45
35
60
B
55
30
30
45
55
25
55
C
00
00
25
10
00
20
00
D
43
35
80
45
65
70
75
E
25
55
60
40
55
20
28
F
a
[zone of inhibition (mm)]: <10: weak; >10:moderate; >16: Significant. SD ¼ Standard Drug (Imipenum); MIC
m
g/mL; A ¼ Miconazole (70
m
g/mL: 1.6822 ꢁ 10ꢀ4 M),
B ¼ Miconazole (110.8
m
g/mL; 2.6626 ꢁ 10ꢀ4 M), C ¼ amphotericin B (20
m
g/mL; 2.1642 ꢁ 10ꢀ5 M), D ¼ Miconazole (98.4
m
g/mL; 2.3647 ꢁ 10ꢀ4 M), E ¼ Miconazole (73.25
mg/
mL; 1.7603 ꢁ 10ꢀ4 M), F ¼ Miconazole (110.8
m
g/mL; 2.66266 ꢁ 10ꢀ4 M).
C. glabrata fungal strains according to the literature protocol [15]. All
synthesized compounds showed good antifungal activity against
different fungal strains. Although compound (3) showed good anti-
fungal activity against all the fungal strains but significant activity
against F. solani, compounds (5), (6), and (7) also showed significant
activity against F. solani. No activity was observed for compounds (1),
(4), and (6) against T. longifusus and (1), (2), (5) and (7) against M.
canis. All other compounds showed weak to moderate activity
against all other fungal stains. All individual synthesized compounds
were compared with each other (Fig. 2) and compound (3) was found
to be the most active one (Fig. 4). The inhibition results were
compared with those for the standard drugs miconazole and
amphotericin B (Table 1).
multiplet at 7.0–7.4 ppm and in compound (7), the pyrimidinyl, H-5
proton appeared as a singlet at 6.7 ppm. The spectra of compounds
(4), (5) and (7) also displayed the methyl proton peaks as singlet.
Furthermore, the number of the proton calculated from the inte-
gration curves [21] and those obtained from the values of the
expected CHN analysis agreed well with each others.
4.1.3. 13C NMR spectra
13C NMR spectra of compounds (1)–(7) displayed azomethine-C
(CH]N) peak at 162.0 ppm which further supported the results
obtained in IR and 1H NMR. Compounds (1)–(7) displayed C2, C3, C4,
C5, C6, C7, C8 and C9 carbons of indole moiety at 139.7, 115.4, 120.2,
121.3, 121.6, 114.4, 138.7 and 126.8 ppm respectively. Also, C1-
phenyl, C2, C6-phenyl, C3, C5-phenyl, C4-phenyl appeared at 138.2,
128.6, 122.6 and 156.4 respectively in all the compounds. Carba-
mimidoyl carbon in compound (2) appeared at 167.0 ppm and in
compound (3), C4, C5, C2 carbons of thiazole appeared at 108.0,
137.8, 117.7 respectively. In compound (4), CH3, C5, C4, C3 of iso-
xazole carbons appeared at 12.8, 159.6, 95.0 and 150.0 ppm
respectively. In compound (5), CH3-isoxazole, CH3-isoxazole
C3-isoxazole, C4-isoxazole, C5-isoxazole carbons appeared at 15.1,
9.5, 159.9, 100.5 and 158.9 respectively. Pyrimidine carbons, C4, C6,
C5, C2 appeared at 157.9, 110.2, 159.3 ppm respectively. In
compound (6) and (7), 2CH3- pyrimidine, C4, C6-pyrimidine, C5-
pyrimidine, C2-pyrimidine carbons appeared at 25.1, 165.2, 103.0,
168.5 respectively. Furthermore, the number of carbons calculated
from the integration curves [22]. And those obtained from the
values of the expected CHN analyses agree well with each other.
4. Results and discussion
4.1. Spectroscopic characterization of (1)–(7)
4.1.1. IR spectra
In the IR spectra of compounds (1)–(7), a sharp band at
1595 cmꢀ1 is assigned [16] to the stretching of
n(C]N). Presence of
this band is an evidence of the formation of condensation product.
nasymm(SO2) and nsymm(SO2) appeared at 1345 and 1110 cmꢀ1
respectively [17,18]. n(C–S), n(C–N), n(NH) of indole ring appeared at
833, 952, 3230 cmꢀ1 respectively, in all compounds. The IR spectra
of compounds (1) and (2) exhibited peaks at 3390 and 3395 cmꢀ1
respectively, due to –NH2 stretching. In addition, the spectra of (2)–
(7) showed bands resulting from the guanidine (C]N), thiazole
(C]N), isoxazolyl (C]N), isoxazolyl (C]N), pyrimidinyl (C]N), and
pyrimidinyl (C]N), stretching at 1580, 1615, 1610, 1605, 1585 and
1580 cmꢀ1 respectively. The IR spectra of (2)–(7), exhibited the
–SO2NH stretchings at 3255 cmꢀ1, the presence of all these
frequencies was supportive [19,20] of the formation of compounds
(1)–(7).
4.2. Molecular properties calculations
For SDZ and certainly for its analogues (1)–(7), depending on the
pH and position of the dissociate amidogen hydrogen atom, nine
possible SDZ conformations can be described for the neutral (N),
4.1.2. 1H NMR spectra
1H NMR spectra of the compounds (1)–(7) displayed azomethine
(CH]N) peak at d 9.4 and H-4, H-7 protons of indole ring at 8.1 and
Table 2
7.6 ppm as doublets. H-5 and H-6 protons appeared at 7.1 and
7.3 ppm as doublets of triplet and, H-2, H-1 protons at 8.3
and 12.1 ppm as singlet, respectively. A multiplet was observed at
7.7–7.8 ppm assigned for four protons of phenyl ring. The SO2NH2 or
SO2NHꢀ protons in all cases appeared as a singlet in the region at
11.2–11.30 ppm 1H NMR spectrum of compound (2) also displayed
–C]NH and HN]C–NH2 protons as a singlet at 8.1 and 7.6 ppm
respectively. Also, compound (3) displayed thiazole, H-4 and H-5
protons as doublet at 3.3 and 3.1 ppm and in the case of compound
(4), the isoxazolyl, H-4 proton appeared as a singlet at 5.8 ppm. In
compound (6), the pyrimidinyl, H-4, H-5, H-6 protons appeared as
Antibacterial minimum inhibitory concentration (MIC in M) and Brine shrimp
bioassay data for compounds (1)–(7).
Compd. Gram (ꢀ)
Gram (þ)
LD50 (M)
E. coli
S. flexenari
S. typhi
B. subtilis
1
2
3
4
5
6
7
–
–
–
–
–
>3.341
1.465 ꢁ 10ꢀ4 2.929 ꢁ 10ꢀ4 1.465 ꢁ 10ꢀ4 >2.929
2.615 ꢁ 10ꢀ4 1.307 ꢁ 10ꢀ4 2.615 ꢁ 10ꢀ4 6.537 ꢁ 10ꢀ5
0.980
–
–
–
–
–
–
–
–
–
–
2.629 ꢁ 10ꢀ4 >2.629
–
–
–
>2.535
>2.650
>2.466
1.233 ꢁ 10ꢀ4
2.466 ꢁ 10ꢀ4

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Z.H. Chohan et al. / European Journal of Medicinal Chemistry 45 (2010) 1189–1199
Fig. 2. Comparison of antibacterial activity of compounds (1)–(7).
Fig. 3. Average antibacterial activity..
Fig. 4. Comparison of antifungal activity of compounds (1)–(7).

Z.H. Chohan et al. / European Journal of Medicinal Chemistry 45 (2010) 1189–1199
1193
Fig. 5. Average antifungal activity.
deprotonated (D), and protonated (P) forms. These relevant struc-
tures are sketched in Fig. 5. In past, attention was mainly devoted to
the N-1 structure. However, from a chemical point of view, all other
structures are possible.
In brief, the objective of this study is to investigate the potential
pharmacophore sites of SDZ species using antibacterial and anti-
fungal screenings dependence on pH and comparison with the
calculated molecular properties. To verify these structures, further
Petra/Osiris/Molinspiration (POM) analyses were carried out for
example calculation of net atomic charges, bond polarity, atomic
valence, electron delocalization and lipophicity.
Finally, to investigate the combined antibacterial/antifungal
bioactivity of the SDZ species, tautomeric structure were performed.
Current thinking in the generation of specific drug leads
embodies the concept of achieving high molecular diversity within
the boundaries of reasonable drug-like properties [24]. Natural and
semi-natural products, examples penicillin and, imipenem have high
chemical diversity, biochemical specificity and other molecular
properties that make them favourable as lead and standard refer-
ences (SR) structures for drug discovery, and which serve to differ-
entiate them from libraries of synthetic and combinatorial
compounds.
For the development of binding approaches for SDZ and its
analogues (1)–(7) in the environment, the identification of the active
sulfonamide structures present is important. Neither experimental
nor theoretical data is available for the identification of water-solved
SDZ species. Theoretically, NMR spectroscopy could be useful for
identifying chemical structures. Theoretical ab initio studies could
supplement these measurements. Additionally, calculations of
energetics, atomic charges, minimum energy structures, geometry,
and natural bond orbital (NBO) could indicate the electronic density
distribution of each atom. Finally, by taking NBO results showing the
presence of S–O single bonds in consideration, realistic Lewis
structures can be determined. These systematic data, regarding the
variation of molecular properties, are important for the chemical
structure and could therefore provide first insights into the still
poorly understood chemical bonding of SDZ complexes to soil [23 &
ref therein].
Various investigators have used computational methods to
understand differences between natural products and other sources
Fig. 6. Protonated (P), neutral (N), deprotonated (D) and Lewis (L) structures of sulfadiazine.

1194
Z.H. Chohan et al. / European Journal of Medicinal Chemistry 45 (2010) 1189–1199
of drug leads [25]. Modern drug discovery is based in large part on
high throughput screening of small molecules against macromo-
lecular disease targets requiring that molecular screening libraries
contain drug-like or lead-like compounds. We have analyzed known
standard references (SR) for drug-like and lead-like properties. With
this information in hand, we have established a strategy to design
specific drug-like or lead-like indolenol sulfonamides (1)–(7).
activity, more, and this is in good agreement with the mode of
antbacterial action of the compounds bearing (Xdꢀ/Ydþ) phar-
macophore site. It was hypothesized that difference in charges
between two heteroatoms of the same dipolar pharmacophore site
(Xdꢀ/Ydþ) may facilitate the inhibition of bacteria, more than
viruses. It is further found that the activity increases with increase
in negative charge of one heteroatom of the common pharmaco-
phore fragment of the potential tautomers (III and VI). The presence
of tautomerism phenomena in sulfonamide analogues was pre-
sented previously since 1999, in few papers by some rare authors
[27] (Figs. 7 and 8).
On the basis of this analogue system described above, in
compound 3, sets of isomeric and tautomeric thiazole derivatives
I–IV could be generated in-situ in the presence of bacteria of fungi.
This synergistic and streamlined working procedure led to
highly active isomeric/tautomeric Gram(þ/ꢀ) and fungi receptor
ligands. However, a little difference in their respective binding
affinities was consistently found for all isomeric pairs I–IV. The
analysis of conformational differences due to heteroatom interac-
tions in tautomers I–IV revealed a favorable (S]O/S) interaction
in tautomers I and III, whereas thiazoles II and IV showed a repul-
sive (S]O/N) interaction.
4.2.1. Petra calculations
PETRA is a program package comprising various empirical
methods for the calculation of physicochemical properties in
organic molecules. All methods are empirical in nature and have
been developed over the last 20 years in the research group of Prof.
J. Gasteiger. The following chemical effects can be quantified: heats
of formation, bond dissociation energies, sigma charge distribution,
p-charge distribution, inductive effect, resonance effect and delo-
calization energies and polarizability effect [26].
The series (1)–(7) of indolenol sulfonamides have been subjected
to delocalised-charge calculations using Petra method of the non-
hydrogen common atoms (Fig. 6), obtained from the partial pi-
charge of the heteroatoms, have been used to model the bioactivity
against bacteriaand fungi. We give here, as example, the compound 3.
It is found that the negative charges of the oxygen of SO2 group
and sulfur contribute positively in favour of an antibacterial
So the antifungal activity is related with possible secondary
electronic interaction with the positively charged side chains of the
Fig. 7. Possible potential tautomeric forms of compound 3.

Z.H. Chohan et al. / European Journal of Medicinal Chemistry 45 (2010) 1189–1199
1195
Fig. 8. The structural parameters do not indicate a tautomeric equilibrium but a single imino form. The main differences between the two crystalline forms lie in the intramolecular
hydrogen bonding and the relative orientation of the methoxy groups. Attractive intermolecular interactions occur and are responsible for the crystalline cohesion.
virus target(s). Attempt was made to evaluate steric and indicator
parameters which emerged as important contributors from
previous pharmacologic analysis. The present results support the
previous observations that heterocyclic ring in adjacent position of
NH could generate four tautomeric forms, in less and two distinct
four-member pharmacophore sites are conducive to the activity to
both antibacterial (Oꢀ/Xþ) and antifungal activity (Oꢀ/Xꢀ).
very important class of enzymes, responsible for many ADMET
problems, is the cytochromes P450. Inhibition of these or produc-
tion of unwanted metabolites can result in many adverse drug
reactions. Of the most important program, Osiris is already avail-
able online.
With our recent publication of the drug design combination of
various pharmacophore sites by using spiro-heterocyclic structure,
it is now possible to predict activity and/or inhibition with
increasing success in two targets (bacteria and HIV). This is done
using a combined electronic/structure docking procedure and an
example will be given here (Table 4). The remarkably well behaved
4.2.2. Osiris calculations [28]
Structure based design is now fairly routine but many potential
drugs fail to reach the clinic because of ADME-Tox liabilities. One
Table 3
Selected Petra calculations of compounds.
Tautomer
Partial
N1
p
-charge of heteroatoms (in eꢀ)
N2
N3
0.225
ꢀ0.155
ꢀ0.099
ꢀ0.155
0.128
N4
S1O2
S2
O1
O2
3 (I)
3 (II)
3 (III)
3 (IV)
Imipenem
0.339
0.337
0.3431
0.3374
0.187
ꢀ0.065
ꢀ0.066
ꢀ0.0656
ꢀ0.0655
ꢀ0.145
ꢀ0.088
0.329
ꢀ0.139
0.329
–
ꢀ0.002
ꢀ0.009
ꢀ0.024
ꢀ0.009
0.087
0.049
0.106
0.047
0.106
0.082
ꢀ0.187
ꢀ0.233
ꢀ0.201
ꢀ0.233
ꢀ0.147
ꢀ0.187
ꢀ0.233
0.158
ꢀ0.233
ꢀ0.217

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Z.H. Chohan et al. / European Journal of Medicinal Chemistry 45 (2010) 1189–1199
Table 4
Osiris calculations of compounds.
Tautomer
Toxicity Risks
Mutagenic
Osiris calculations
Tumorigenic
Irritant
Reproductive effective
MW
CLP
Sa
DLa
D-Sa
0.63
3 (I)
382
2.87
ꢀ4.75
1.96
3 (II)
3 (III)
3 (IV)
382
382
382
299
2.87
2.13
ꢀ4.89
ꢀ3.97
ꢀ4.89
ꢀ1.93
0.9
ꢀ0.94
0.9
0.56
0.49
0.56
0.93
2.87
Imipenem
ꢀ1.36
5.34
a
CLP: cLogP, S: Solubility, DL: Druglikness, DS: Drug-Scor.
Table 5
Molinspiration calculations of compounds.
Tautomer
Molinspiration calculations
Drug-likeness
cLogP
TPSA
87.22
90.45
90.71
90.45
116.22
OH/NH
N viol.
Vol.
GPCRL
ICM
1.07
ꢀ0.84
ꢀ0.86
ꢀ0.84
ꢀ0.38
KI
NRL
3 (I)
3 (II)
3 (III)
3 (IV)
Imipenem
3.44
4.37
1.93
4.37
ꢀ0.86
2
2
2
2
4
0
0
0
0
0
309
309
309
309
255
ꢀ0.71
ꢀ0.79
ꢀ0.73
ꢀ0.79
ꢀ0.38
ꢀ1.05
ꢀ1.25
ꢀ0.94
ꢀ1.25
ꢀ0.57
ꢀ1.36
ꢀ1.30
ꢀ1.16
ꢀ1.30
ꢀ0.41
mutagenicity of divers synthetic molecules classified in data base of
CELERON Company of Swiss can be used to quantify the role played
by various organic groups in promoting or interfering with the way
a drug can associate with DNA.
properties. The positive results we have recorded, while
encouraging for purposes of new drug design, confirm that very
likely most of these compounds could be used as potential
antibacterial activity after minor modifications. Based on their
structural properties, these compounds may be useful as
chelating agents with potential activity. These results prompt
several pertinent observations: (i) This type of sulonamides can
furnish an interesting model for studying the interaction of
antibiotics with viral target because the possible charge modifi-
cation of substituents and O/N of pharmacophore group; (ii) The
future flexible pharmacophore site (s) geometric conformation
enables us to prepare molecules for multi-therapeutic materials
with high selectivity.
4.2.3. Molinspiration calculations [29]
CLogP (octanol/water partition coefficient) is calculated by the
methodology developed by Molinspiration as a sum of fragment-
based contributions and correction factors (Table 5).
The method is very robust and is able to process practically all
organic, and most organometallic molecules. Molecular Polar
Surface Area TPSA is calculated based on the methodology published
by Ertl et al. as a sum of fragment contributions. O– and N– centered
polar fragments are considered. PSA has been shown to be a very
good descriptor characterizing drug absorption, including intestinal
absorption, bioavailability, Caco-2 permeability and blood–brain
barrier penetration. Prediction results of compounds 1–7 molecular
properties (TPSA, GPCR ligand and ICM) are valued (Table 5).
6. Experimental protocols
6.1. Materials and methods
5. Conclusions
All reagents and solvents were used as obtained from the supplier
or recrystallized/redistilled as necessary. Thin layer chromatography
was performed using aluminium sheets (Merck) coated with silica
gel 60 F254. Elemental analysis was carried out with a LECO-CHNS-
9320 model. 1H NMR and 13C NMR spectra of compounds were
recorded with a Bruker Spectrospin Avence DPX-400 using TMS as
internal standard and DMSO as solvent. IR spectra of compounds
were recorded on a Philips Analytical PU 9800 FTIR spectropho-
tometer. The melting points of compounds were determined with
a Gallenkamp melting point apparatus. In vitro antibacterial, anti-
fungal and cytotoxic properties were studied at HEJ research
Institute of Chemistry, International Center for Chemical Sciences,
university of Karachi, Pakistan.
The results of present investigation support the suggested
structures of indolenyl sulfonamides. It has been suggested that
some functional groups such as azomethine or hetero-aromatics
present in these compounds displayed [30–34] role of biological
activity that may be responsible for the increase of hydrophobic
character and liposolubility of the molecules. This in turn, enhances
activity of the compounds and biological absorbance, so as, all the
synthesized sulfonamides have good antibacterial and antifungal
properties.
A number of important points emerge concerning the elec-
tronic and steric factors which have direct impact on bioactivity

Z.H. Chohan et al. / European Journal of Medicinal Chemistry 45 (2010) 1189–1199
1197
6.2. Minimum inhibitory concentration (MIC) and cytotoxic activity
(in vitro)
(d, H-7, indole), 8.3 (s, H-2, indole), 12.1 (s, NH, indole), 3.3 (d, H-4,
thiazol), 3.1 (d, H-4, thiazol), 7.7–7.8 (m, 4H, N-Ph), 9.4 (s, 1H,
azomethine), 11.3 (s, 1H, SO2NH2); 13C NMR (
d, ppm): 139.7
The preliminary antibacterial screening showed that compound
(2), (3), (4) and (7) were the most active one. Above 80% these
compounds were therefore selected for minimum inhibitory
concentration (MIC) studies (Table 3). The MIC of all the four active
compounds varied from 6.537 ꢁ 10ꢀ5 to 1.33 ꢁ10ꢀ4 M compound (3)
again proved to be the most active. It inhibited the growth of B.
subtilis at 6.537 ꢁ 10ꢀ5 M.
(C2-indole), 115.4 (C3-indole), 120.2 (C4-indole), 121.3 (C5-indole),
121.6 (C6-indole), 114.4 (C7-indole), 138.7 (C8-indole), 126.8
(C9-indole), 162.0 (C]N, azomethine), 108.0 (C4-thiazol), 137.8
(C5-thiazol), 171.7 (C2-thiazol), 138.2 (C1-phenyl), 128.6
(C2, C6-phenyl), 122.6 (C3, C5-phenyl), 156.4 (C4-phenyl). SM
[C18H14N4O2S2] [382.5]. Elemental analyses Found (Calcl.): C: 56.5
(56.7); H: 3.7 (3.6); N: 14.7 (14.6).
All the synthesized compounds were screened for their cyto-
toxicity (brine shrimp bioassay) using the protocol of Meyer et al.
[35] From the data recorded in Table 5, it is evident that only
compound (3) displayed potent cytotoxic activity against Artemia
salina, while the other compounds were almost inactive in this assay.
Compound (3) showed activity (LD50 ¼ 0.980 M) in the present series
of compounds.
6.3.4. N-(5-methylisoxazol-3-yl)-4-[(1H-indol-3-yl
methylene)amino]benzenesulfonamide (4)
Yellow powder. Mp ¼ 262–264 ꢂC. Yield: 1.64 g (86%). IR (KBr,
cmꢀ1): 3230 (NH), 3255 (NH), 1595 (HC]N), 1345, 1110 (S]O), 952
(S–N), 833 (C–S), 1610 (isoxazolyl, C]N); 1H NMR (DMSO-d6,
d,
ppm): 8.1 (d, H-4, indole), 7.1 (dt, H-5, indole), 7.3 (dt, H-6, indole), 7.6
(d, H-7, indole), 8.3 (s, H-2, indole), 12.1 (s, NH, indole), 2.3 (s, 3H,
CH3), 5.8 (S, 1H, isoxazol), 7.7–7.8 (m, 4H, N-Ph), 9.4 (s, 1H, azome-
6.3. General procedure for the synthesis of compounds (1)–(7)
thine), 11.3 (s, 1H, SO2NH2); 13C NMR (
d, ppm): 139.7 (C2-indole),
To an ethanol (20 mL) solution of the respective sulfanilamide
(0.005 mol), an indole-3-carbaldehyde (0.005 mol) solution in
ethanol (10 mL) was added with stirring. Later on the solution was
refluxed for 2 h. The precipitates formed during refluxing, were
cooled at room temperature and collected by suction filtration.
Washing thoroughly with ethanol (2 ꢁ 8 ml), afforded TLC pure
products in good yield. The same procedure was used for the prep-
aration of all ligands.
115.4 (C3-indole), 120.2 (C4-indole), 121.3 (C5-indole), 121.6
(C6-indole), 114.4 (C7-indole), 138.7 (C8-indole), 126.8 (C9-indole),
162.0 (C]N, azomethine), 12.8 (CH3-isoxazol), 159.6 (C5-isoxazol),
95.0 (C4-isoxazol), 150.0 (C3-isoxazol), 138.2 (C1-phenyl), 128.6
(C2, C6-phenyl), 122.6 (C3, C5-phenyl), 156.4 (C4-phenyl). SM
[C19H16N4O3S] [380.4]. Elemental analyses Found (Calcl.): C: 60.0
(59.9); H:4.2 (4.3); N: 14.7 (14.6).
6.3.5. N-(3,4-dimethylisoxazol-5-yl)-4-[(1H-indol-3-yl
methylene)amino]benzenesulfonamide (5)
6.3.1. 4-[(1H-indole-3-ylmethylene)amino] benzenesulfonamides (1)
Yellow powder. Mp ¼ 220–221 ꢂC. Yield: 1.29g (86%). IR (KBr,
cmꢀ1): 3230 (NH), 3255 (NH), 3399 (NH2), 1595 (HC]N), 1345, 1110
Yellow powder. Mp ¼ 299–304 ꢂC. Yield: 1.64 g (83%). IR (KBr,
cmꢀ1): 3230 (NH), 3255 (NH), 1595 (HC]N), 1345, 1110 (S]O), 952
(S]O), 952 (S–N), 833 (C–S); 1H NMR (DMSO-d6,
d, ppm): 8.1 (d, H-
(S–N), 833 (C–S), 1605 (isoxazolyl, C]N); 1H NMR (DMSO-d6,
d,
4, indole), 7.1 (dt, H-5, indole), 7.3 (dt, H-6, indole), 7.62 (d, H-7,
indole), 8.3 (s, H-2, indole), 12.1 (s, NH, indole), 7.6–7.8 (m, 4H, N-
ppm): 8.1 (d, H-4, indole), 7.1 (dt, H-5, indole), 7.3 (dt, H-6, indole),
7.6 (d, H-7, indole), 8.3 (s, H-2, indole), 12.1 (s, NH, indole), 2.4 (m,
6H, CH3), 7.7–7.8 (m, 4H, N-Ph), 9.4 (s, 1H, azomethine), 11.3 (s, 1H,
Ph), 9.4 (s, 1H, azomethine), 11.3 (s, 2H, SO2NH2); 13C NMR (
d, ppm):
139.7 (C2-indole), 115.4 (C3-indole), 120.2 (C4-indole), 121.3
(C5-indole), 121.6 (C6-indole), 114.4 (C7-indole), 138.7 (C8-indole),
126.8 (C9-indole), 162.0 (C]N, azomethine), 138.2 (C1-phenyl),
128.6 (C2, C6-phenyl), 122.6 (C3, C5-phenyl), 156.4 (C4-phenyl). SM
[C15H13N3O2S] [299.3]. Elemental analyses Found (Calcl.): C: 60.2
(60.1); H: 4.4 (4.3); N:14.1 (14.0).
SO2NH2); 13C NMR (
d, ppm): 139.7 (C2-indole), 115.4 (C3-indole),
120.2 (C4-indole), 121.3 (C5-indole), 121.6 (C6-indole), 114.4 (C7-
indole), 138.7 (C8-indole), 126.8 (C9-indole), 162.0 (C]N, azome-
thine), 15.1 (CH3-isoxazol), 9.5 (CH3-isoxazol), 159.6 (C3-isoxazol),
100.5 (C4-isoxazol),158.9 (C5-isoxazol),138.2 (C1-phenyl), 128.6 (C2,
C6-phenyl), 122.6 (C3, C5-phenyl), 156.4 (C4-phenyl). SM
[C20H18N4O3S] [394.5]. Elemental analyses Found (Calcl.): C: 60.9
(60.8); H: 4.6 (4.6); N: 14.2 (14.2).
6.3.2. N-Carbamimidoyl-4-[(1H-indol-3-ylmethylene)-
amino]benzenesulfonamide (2)
Yellow powder. Mp ¼ 255–260 ꢂC. Yield: 1.43g (84%). IR (KBr,
cmꢀ1): 3230 (NH), 3255 (NH), 3399 (NH2), 1595 (HC]N), 1345, 1110
(S]O), 952 (S–N), 833 (C–S), 1580 (guanidine, C]N); 1H NMR
6.3.6. N-(pyrimidin-2-yl)-4-[(1H-indol-3-yl
methylene)amino]benzenesulfonamide (6)
Yellow powder. Mp ¼ 277–283 ꢂC. Yield: 1.51g (80%). IR (KBr,
(DMSO-d6, d, ppm): 8.1 (d, H-4, indole), 7.1 (dt, H-5, indole), 7.3 (dt, H-
cmꢀ1): 3230 (NH), 3255 (NH), 1595 (HC]N), 1345, 1110 (S]O), 952
6, indole), 7.6 (d, H-7, indole), 8.3 (s, H-2, indole), 12.1 (s, NH, indole),
7.6–7.8 (m, 4H, N-Ph), 7.6 (s, 2H, NH2), 8.1 (s, 1H, NH), 9.4 (s, 1H,
(S–N), 833 (C–S), 1585 (pyrimidine, C]N); 1H NMR (DMSO-d6,
d,
ppm): 8.1 (d, H-4, indole), 7.1 (dt, H-5, indole), 7.3 (dt, H-6, indole),
7.6 (d, H-7, indole), 8.3 (s, H-2, indole), 12.1 (s, NH, indole), 7.0–7.4
(m, 3H, pyrimidine), 7.7–7.8 (m, 4H, N-Ph), 9.4 (s, 1H, azomethine),
azomethine), 11.3 (s, 1H, SO2NH2); 13C NMR (
d, ppm): 139.7
(C2-indole), 115.4 (C3-indole), 120.2 (C4-indole), 121.3 (C5-indole),
121.6 (C6-indole), 114.4 (C7-indole), 138.7 (C8-indole), 126.8
(C9-indole), 162.0 (C]N, azomethine), 167.0 (C-carbamimidoyl),
138.2 (C1-phenyl), 128.6 (C2, C6-phenyl), 122.6 (C3, C5-phenyl), 156.4
(C4-phenyl). SM [C16H15N5O2S] [299.4]. Elemental analyses Found
(Calcl.): C: 56.3 (56.2); H: 4.4 (4.5); N: 20.5 (20.4).
11.3 (s, 1H, SO2NH2); 13C NMR (
d, ppm): 139.7 (C2-indole), 115.4 (C3-
indole), 120.2 (C4-indole), 121.3 (C5-indole), 121.6 (C6-indole), 114.4
(C7-indole), 138.7 (C8-indole), 126.8 (C9-indole), 162.0 (C]N, azo-
methine),157.9 (C4, C6-pyrimidine),110.2 (C5-pyrimidine),159.3 (C2-
pyrimidine), 138.2 (C1-phenyl), 128.6 (C2, C6-phenyl), 122.6 (C3, C5-
phenyl), 156.4 (C4-phenyl). SM [C19H15N5O2S] [377.4]. Elemental
analyses Found (Calcl.): C: 60.5 (60.4); H: 4.0 (3.9); N: 18.6 (18.5).
6.3.3. N-(thiazole-2-yl)-4-[(1H-indol-3-ylmethylene)-
amino]benzenesulfonamide (3)
Yellow powder. Yiels : 1.62 g (85%). Mp ¼ 268–273 ꢂC. IR (KBr,
6.3.7. N-(4,6-dimethylpyrimidin-2-yl)-4-[(1H-indol-3-yl
methylene)amino]benzene-sulfonamide (7)
cmꢀ1): 3230 (NH), 3255 (NH), 1595 (HC]N), 1345, 1110 (S]O), 952
(S–N), 833 (C–S), 1615 (thiazol, C]N); 1H NMR (DMSO-d6,
d, ppm):
Yellow powder. Mp ¼ 283–288 ꢂC. Yield: 1.70 g (84%). IR (KBr,
8.1 (d, H-4, indole), 7.1 (dt, H-5, indole), 7.3 (dt, H-6, indole), 7.6
cmꢀ1): 3230 (NH), 3255 (NH), 1595 (HC]N), 1345, 1110 (S]O), 952

1198
Z.H. Chohan et al. / European Journal of Medicinal Chemistry 45 (2010) 1189–1199
(S–N), 833 (C–S), 1580 (pyrimidine, C]N); 1H NMR (DMSO-d6,
d
,
After two days nauplii were collected by a pipette from the light side.
A sample of the test compound was prepared by dissolving 20 mg of
each compound in 2 mL of DMF. From this stock solutions 500, 50
and 5 ug/mL were transferred to 9 vials (three for each dilution were
used for each test sample and LD50 is the mean of three values) and
one vial is kept as control having 2 mL of DMF only. The solvent is
allowed to evaporate overnight. After two days, when shrimp larvae
were ready, 1 mL of sea water and ten shrimps were added to each
vial (30 shrimps/dilution) and the volume was adjusted with sea
water to 5 mL per vial. After 24 h the number of survivors was
counted. Data were analyzed by Finny computer program to deter-
mine the LD50 values [35,39].
ppm): 8.1 (d, H-4, indole), 7.1 (dt, H-5, indole), 7.3 (dt, H-6, indole),
7.6 (d, H-7, indole), 8.3 (s, H-2, indole),12.1 (s, NH, indole), 2.3 (s, 6H,
CH3), 6.7 (s, 1H, pyrimidine), 7.7–7.8 (m, 4H, N-Ph), 9.4 (s, 1H,
azomethine), 11.3 (s, 1H, SO2NH2); 13C NMR (
d, ppm): 139.7
(C2-indole), 115.4 (C3-indole), 120.2 (C4-indole), 121.3 (C5-indole),
121.6 (C6-indole), 114.4 (C7-indole), 138.7 (C8-indole), 126.8
(C9-indole), 162.0 (C]N, azomethine), 25.1(2CH3-pyrimidine),
165.2 (C4, C6-pyrimidine), 103.0 (C5-pyrimidine), 168.5 (C2-pyrim-
idine), 138.2 (C1-phenyl), 128.6 (C2, C6-phenyl), 122.6 (C3, C5-
phenyl), 156.4 (C4-phenyl). SM [C21H19N5O2S] [405.5]. Elemental
analyses Found (Calcl.): C: 62.2 (62.3); H: 4.7 (4.7); N: 17.3 (17.3).
7. Biological properties
Acknowledgements
7.1. Antibacterial activity (in vitro)
We are grateful to HEJ research Institute of Chemistry, University
of Karachi for the help in undertaking NMR spectra and biological
assay. Prof. T. Ben Hadda would like to thank the ACTELION; the
Biopharmaceutical Company of Swiss, for the online molecular
properties calculations and to Ministry of High Science and Educa-
tion for financial support.
Contract/grant sponsor: CNRST and the MoroccanMinistry of
High Science and Education; Contract/grant number: PGR-BH-2005
and Protars I.
All the synthesized compounds (1)–(7) were screened in vitro
for their antibacterial activity against four Gram-negative (E. coli,
S. flexneri, P. aeruginosa, S. typhi) and two Gram-positive (S.aureus,
B. subtilis) bacterial strains by the agar-well diffusion method
[36,37]. The wells (6 mm in diameter) were dug in the media with
the help of a strile metallic borer with center at least 24 mm apart.
Two to eight hours old bacterial inocula containing approximately
104–106 colony-forming units (CFU/mL) were spread on the surface
of the nutrient agar with the help of sterile cotton swab. The rec-
ommended concentration of the test sample (1 mg/mL in DMSO)
was introduced in the respective wells. Other wells supplemented
with DMSO and reference antibacterial drug, imipenem, served as
negative and positive controls respectively. The plates were incu-
bated immediately at 37 ꢂC for 24 h activity was determined by
measuring the diameter of zones showing complete inhibition
(mm). In order to clarify any participating role of DMSO in the
bacterial screening, separate studies were carried out with the
solutions alone of DMSO and they showed no activity against any
bacterial strains.
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