Sulfonamide chalcones: Synthesis and in?vitro exploration for therapeutic potential against Brugia malayi

Article abstract of DOI:10.1016/j.ejmech.2016.08.042

Keeping in mind the immense biological potential of chalcones and sulfonamide scaffolds, a library of sulfonamide chalcones has been synthesized and evaluated for in?vitro antifilarial assay against human lymphatic filarial parasite Brugia malayi. Experim

Full text of DOI:10.1016/j.ejmech.2016.08.042

Accepted Manuscript
Sulfonamide chalcones: Synthesis and in vitro exploration for therapeutic potential
against Brugia malayi
Sandeep P. Bahekar, Sneha V. Hande, Nikita R. Agrawal, Hemant S. Chandak,
Priyanka S. Bhoj, Kalyan Goswami, M.V.R. Reddy
PII:
S0223-5234(16)30685-7
10.1016/j.ejmech.2016.08.042
EJMECH 8838
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 June 2016
Revised Date: 18 August 2016
Accepted Date: 19 August 2016
Please cite this article as: S.P. Bahekar, S.V. Hande, N.R. Agrawal, H.S. Chandak, P.S. Bhoj, K.
Goswami, M.V.R. Reddy, Sulfonamide chalcones: Synthesis and in vitro exploration for therapeutic
potential against Brugia malayi, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.08.042.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Sulfonamide Chalcones: Synthesis and in vitro exploration for therapeutic
potential against Brugia Malayi
Sandeep P. Bahekara†, Sneha V. Handeb†, Nikita R. Agrawala, Hemant S. Chandaka*, Priyanka S.
Bhojb, Kalyan Goswamib**, MVR Reddyb
a Department of Chemistry, G. S. Science, Arts and Commerce College, Khamgaon 444303, India
b Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences and JB Tropical Disease Research
Centre, Sevagram, Wardha 442102, India
Abstract
Keeping in mind the immense biological potential of chalcones and sulfonamide scaffolds, a
library of sulfonamide chalcones has been synthesized and evaluated for in vitro antifilarial assay
against human lymphatic filarial parasite Brugia malayi. Experimental evidence showcased for
the first time the potential of some sulfonamide chalcones as effective and safe antifilarial lead
molecules against human lymphatic filarial parasite B. malayi. Sulfonamide chalcones 4d, 4p,
4q, 4t and 4aa displayed the significantly wide therapeutic window. Particularly chalcones with
halogen substitution in aromatic ring proved to be potent antifilarial agents against Brugia
malayi. Sulphonamide chalcones with lipophilic methyl moiety (4q and 4aa) at para position of
terminal phenyl rings of compounds were found to have remarkable antifilarial activities with
therapeutic efficacy. Observed preliminary evidence of apoptosis by effective chalcone
derivatives envisaged its fair possibility to inhibit folate pathway with consequent defect in DNA
synthesis.
Keywords
Sulfonamide chalcone; antifilarial; Claisen-Schimdt condensation; Brugia malayi
†
Equal contribution from both the authors
*
Corresponding author: Dr. Hemant S. Chandak, Department of Chemistry, G. S. Science, Arts and Commerce
College, Khamgaon 444303 India,
hschandak@gsck.ac.in
Fax: (+)91-7263-253844, Email address: chemants@gmail.com/
**
Corresponding author: Dr. Kalyan Goswami, Department of Biochemistry, Mahatma Gandhi Institute of Medical
Sciences & JB Tropical Disease Research Centre, Sevagram, Wardha 442102 India, Fax: (+)91-7152-284038,
Tel.: (+)91-7152-284341(ext-262), Email address: goswamikln@gmail.com
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1. Introduction
Although human lymphatic filariasis is among the six most neglected tropical diseases, it is
paradoxically endemic in over 72 countries in Africa, Asia, South and Central America and the
Pacific islands. World Health Organization (WHO) notified that 120 million people are currently
infected, with about 40 million people worldwide suffers from untoward physical manifestations
of lymphatic filariasis and associated disability [1]. Lack of safe and effective drug with
significant therapeutic window constitutes a bottleneck in the effective combat against filariasis.
Filarial parasites exhibit a variety of protective prowess including very strong antioxidant system
which protects them from the reactive oxygen species (ROS) produced by immune cells of the
host as a natural innate response leading to its existence in mammalian hosts for many years [2].
Diethyl carbamazine citrate (DEC) and ivermectin are the drugs of choice for the treatment of
filariasis. In addition to this, a large number of phenoxycyclohexane and aplysinoposin
derivatives have also been used [3,4]. Disadvantages associated with these drugs such as
undesirable side effects, lack of patient compliance and poor macrofilaricidal effectiveness have
warranted research on new antifilarial drug development [5]. WHO laid emphasis on the
screening of new libraries of synthetic and herbal drugs under the Global Programme for
Elimination of Lymphatic Filariasis (GPELF) [6]. Moreover, the intricate dynamics of filariasis
transmission and the long life span of the adult worm hindered the success of new drugs against
filarial parasite. Thus as mandated by the WHO/TDR (The special programme for research and
training in tropical diseases), the development of an effective antifilarial drug has become a
global research thrust [7].
Chalcones are important precursors in the biosynthesis of flavones and flavanones [8].
Interestingly both synthetic as well as herbal flavonoids have been found to be effective against
human lymphatic parasite [9,10]. Contemporary studies report a generous variation of significant
pharmacological activities of chalcones including antioxidant [4], anti-inflammatory [11],
antibiotic [12], anticancer [13–15], tyrosine inhibitor [3] and antimalarial [16–18] activities.
Awasthi and co-worker reported the inhibitory effects of chalcones on motility, viability and
GST (glutathione-S-transferase) activity of the parasite which support its antifilarial efficacy
against adult Setaria cervi parasites, the causative agent of cattle filariasis [19].
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Moreover, some of the compounds containing bezenesulfonamide moiety also show broad
spectrum biological properties such as elastase inhibitors [20], carbonic anhydrase inhibitors
[21], clostridium histolyticum collogenase inhibitors [22] as well as herbicides and plant growth
regulators [23]. Sulfonamide chalcones reported as potent anticancer agent against human tumor
liver cell line (HEPG-2) [24], BACE1 inhibitors [25] and α-glucosidase inhibitors [26]. Recently,
it has been proved that sulfonamide chalcone bearing nitro substitution showed genotoxic,
cytotoxic, antigenotoxic, and anticytotoxic activities [27]. This strong perspective coupled with
need of suitable antifilarial drug prompted us to test efficacy of sulfonamide chalcones as an
antifilarial agent by conducting in vitro studies on B. malayi. With the prime aim of developing
potent antifilarial agent, we herein report the synthesis of chalcones linked to sulfonamide
scaffold 4a-4aa (Scheme 1), The substitution pattern on the pendant aryl moieties in 4a-4aa
were selected so as to ensure different electronic and lipophilic environments which could
influence the activity of the target compounds.
2. Results and Discussion
2.1. Chemistry
Synthesis of sulfonamide chalcones has been achieved by Claisen-Schimdt condensation of
sulphonamide ketones (1, 2 and 3) with substituted aromatic aldehydes in moderate to high
yields (60-92%) (Scheme 1). Sulfonamide ketones (1, 2 and 3) have been synthesized from p-
aminoacetophenone and benzenesulfonyl chloride/ methanesulfonyl chloride/ tolylsulfonyl
chloride respectively [28].
Scheme 1. Synthesis of N-(4-cinnamoylphenyl)arylsulfonamide derivatives
We found that aldehydes with electron-donating moieties lead to corresponding chalcones
with higher yields than those with electron-withdrawing moieties. Electron-wi
substituent might offer some hurdle and is responsible to lower yields.
thdrawing
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1
The synthesized chalcone derivatives are characterized by IR, H NMR, 13C NMR, mass
spectra and elemental analysis. The IR spectrum of the compounds perfectly indicates the
symmetric and asymmetric stretching frequency of S=O bond, exhibiting the absorption band at
around 1160 cm-1 and 1330 cm-1 respectively. The ESMS (mass spectra) of the compounds
1
showed molecular ion peaks at their respective m/e. In the H NMR spectrum, aromatic protons
observed as complex mulitiplets in the range of δ 6.0-7.5 ppm. α- and β-H corresponding to
carbonyl group resonated in the aromatic range as multiplets (in some cases as distinct doublets)
clearly indicates the formation of chalcones from corresponding sulfonamide ketones. In
addition, there is one exchangeable secondary amide (-SO2NH) proton in the structure and is
observed as broad singlet around δ 10.80 ppm. 13C NMR spectra showed requisite number of
resonances with distinct signals for C=O, >C-H and =C- fragments. The E geometry of the
double bond was shown by X-ray crystallography (Figure 1).
Figure 1. ORTEP diagram of (CCDC No. 1484678) twin molecules of 4q in the unit cell
2.2. Biological evaluation
All the synthesized sulfonamide chalcones were assayed in vitro for their biological activity
against filarial parasite (Table 1). It is worth to mention that parent sulfonamide ketones (1/2/3;
Scheme 1) failed to record any activity even at very high dose of 500 µM. Initially, all chalcone
derivatives were screened for antifilarial activity for a range of dosage with highest concentration
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of 500 µM against Mf. Derivatives which showed complete loss of motility (100%) within this
concentration range were further tested to deduce IC50 value. Thus total twenty seven
sulfonamide chalcones were screened for their antifilarial activity. Out of them thirteen
derivatives (4c, 4d, 4e, 4g, 4n, 4o, 4p, 4q, 4t, 4u, 4v, 4y and 4aa) were found to be effective and
were further evaluated at lower doses ranging from 1-100 µM to determine IC50 value.
From Table 1, it can be seen that sulfonamide chalcones 4c, 4d, 4e, 4g, 4t and 4u which
contains electron withdrawing groups such as -4-Cl, -4-F, -3-Br -4-Br and -4-NO2 showed good
antifilarial activity. Electron-withdrawing effect of these groups favors the Michael type addition
to chalcone by an available nucleophilic side chain of any protein enzyme, which might affect
enzyme activity leading to metabolic stress causing anti-parasitic effect. These findings are in
accordance with the earlier results shown in other related models [19,29,30]. It is also observed
that bromo group at meta position (4u) shows moderate activity while the same group at para
position shows potent activity (4t). Lipophilicity of substituents has also been identified as
critical parameter to affect the activity of chalcone scaffold [31–33]. Antifilarial activity shown
by 4n, 4o, 4p, 4q, 4v, 4y and 4aa might be due to the presence of lipophilic alkyl moiety in the
structure. Chalcones 4n, 4o, 4v and 4y shows moderate activity while 4p and 4aa show potent
antifilarial activity. This might be due to the combination of lipophilic methyl group along with
electron-withdrawing nitro group at para position of aromatic ring of other end of 4p to
sulfonamidophenyl end while both terminal ends of 4aa flanged with lipophilic methyl group
proved it to be very potent antifilarial agent showing crucial therapeutic window.
Table 1. In vitro effect of chalcone derivatives on motility of B. malayi Mf
Sr. No.
1.
Entry
4a
4b
4c
Activity ‡
4.52 ± 0.349
1.809 ± 0.235
100*
2.
3.
4.
5.
4d
4e
100*
100*
6.
7.
8.
9.
10.
11.
12.
13.
4f
3.747 ± 0.485
100*
4g
4h
4i
4j
4k
4l
8.110 ± 0.311
33.311 ± 4.07
5.197 ± 0.403
13.436 ± 2.115
6.339 ± 0.697
9.001 ± 1.553
4m
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14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
4n
4o
4p
4q
4r
100*
100*
100*
100*
82.697 ± 1.783
1.580 ± 0.090
100*
100*
100*
6.239 ± 1.070
4.103 ± 0.789
100*
3.045 ± 0.424
100*
4s
4t
4u
4v
4w
4x
4y
4z
4aa
DMSO control 2.440 ± 0.335
‡ Results were expressed as mean ± SEM of % loss of motility of Mf at 500 µM concentration after 48 h.
* Complete loss of motility of all Mf (100%),
Effective chalcone derivatives were tested for their toxicity by Trypan Blue assay and LD50
value was determined for each derivative. Therapeutic range for each derivative was decided by
using IC50 and LD50 values and the difference between the two concentrations shows the
therapeutic window for each of the agents (Table 2). It was observed that total thirteen
derivatives were found to have antifilarial activity and were found to be therapeutically safe, out
of which chalcones 4d, 4p, 4q, 4t and 4aa depicted the most significant therapeutic window
(Figure 2).
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Figure 2. Sulfonamide chalcones with significant therapeutic window
Table 2. IC50 and LD50 values for chalcones obtained by in vitro filarial motility assay and the in
vitro cytotoxicity assay (against PBMCs) respectively
Sr. No. Compound IC50 (µM) LD50 (µM)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
4c
4d
4e
4h
4n
4o
4p
4q
4t
4u
4v
4y
4aa
17
4.5
70
58
32
177
217
265
210
134
194
188
335
220
80
60
4.4
9.7
4.6
15
20
82
50
270
140
8.2
Further our results showed initial evidence of possible apoptosis of the parasite by
differential fluorescent staining. The drug treated parasites appeared reddish orange (Figure 3)
similar to staurosporin (a known apoptosis inducer, Figure 3b) treated positive control as
opposed to the typical greenish fluorescence found in untreated negative control (live parasite,
Figure 3a). Therefore such result supporting drug induced apoptosis might be surmised as the
primary reason for antiparasitic effect of the synthetic compounds.
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Chalcones are reportedly known for their mitochondrial damage potential which might be a
probable cause of induction of such apoptosis [34], since mitochondrial pathway of apoptosis is
well established [35]. Alternatively DNA synthesis defect is also put forward as a possible
rationale of chalcone mediated therapeutic impact in other parasite [30]. In this context it is
worth mentioning that folate metabolism is an important prerequisite for proper DNA synthesis
[36]. Earlier work by Sharma and co-worker with certain synthetic compounds showed
promising therapeutic effect against this parasite with evidence of apoptosis, which were also
confirmed to have anti-folate effect based on folate reversal studies [37]. Interestingly, folate
synthesis and flavonoid (polyphenolics) synthesis share a common metabolic route, namely
through shikimate pathway [38]. Given the fact that chalcones are related to flavonoids [8],
therefore a structural resemblance based competitive inhibition of folate metabolism most
plausibly through dihydrofolate reductase (DHFR) enzyme might be envisaged. Similar evidence
has been reported with flavonoids of green tea that displayed apoptosis by inhibition of DHFR
enzyme [39].
Therefore taken together it appears that there is a fair possibility of chalcones to inhibit folate
pathway with consequent defect in DNA synthesis which might culminated into observed
apoptosis of the parasite. Although such mechanistic details are still elusive and not in the scope
of this study, however the observed potential and safety of these compounds with a possible
apoptotic rationale definitely demands suitable status of these chalcone derivatives as effective
antifilarial lead.
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Figure 3. Display of evidence of apoptosis by differential AO/EB staining. Fluorescent
microscopic view of microfilaria: (a) live worm (–ve control) (b) dead worm (+ve control-
staurosporine) (c) compound 4c (d) compound 4d (e) compound 4e (f) compound 4g (g)
compound 4n (h) compound 4o (i) compound 4p (j) compound 4q (k) compound 4t (l)
compound 4u (m) compound 4aa (n) compound 4y (o) compound 4v
3. Conclusion
In conclusion, we have successfully synthesized and conducted in vitro antifilarial assay of
chalcones bearing substituted sulfonamide moiety . Experimental evidence showcased for the
first time the potential of some sulfonamide chalcones as effective and safe antifilarial lead
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molecules against human lymphatic filarial parasite B. malayi. Significance of sulfonamide
chalcones with halogen moiety on phenyl ring (R2) has been revealed. Sulfonamide chalcones
with fluoro (4d), chloro (4p) and bromo (4t) substitutions at para position of phenyl ring (R2)
were found to have remarkable antifilarial activities with therapeutic efficacy. Again
sulphonamide chalcones with lipophilic methyl moiety (4q and 4aa) at para position of terminal
phenyl rings of compounds were found to have remarkable antifilarial activities with therapeutic
efficacy. Finally evidence of apoptotic effect with such chalcone derivatives offers distinctive
therapeutic rationale against human lymphatic parasite and opened new vistas for exploring
newer drug designing principle. Further work in this direction is in the pipeline by utilizing this
insight to optimize sulphonamide chalcones into a viable therapeutic option.
4. Experimental
4.1. Chemistry
4.1.1. General
All solvents and chemicals were obtained commercially from E. Merck (Germany), Himedia
Laboratories (India) and Sigma Aldrich Chemicals and were used as received. Melting points
were determined in an open capillary and are uncorrected. Reaction progress was checked on
pre-coated TLC plates and spots were visualized using UV light. The synthesized compounds
were characterized on the basis of spectral analysis. IR spectra were recorded using a Bruker
spectrometer instrument. 1H NMR spectra were taken with a Bruker Avance II at 300 MHz
/ 400
MHz and Bruker DMX spectrometer instrument operating at 500 MHz using Chloroform-d or
DMSO-d6 as the solvent. 13C NMR spectra were recorded on Bruker DMX spectrometer
instrument operating at 100 MHz/ 125 MHz respectively. All chemical shifts are reported in ppm
1
and are referenced to tetramethylsilane using residual H signals of the deuterated solvents as
internal standards. Electron spray ionization mass spectra were recorded on Brucker microTOF-
Q spectrometer. Elemental (C, H, N) analysis was carried out on Carlo Erba 1108 analyser. X-
ray data were collected with SuperNova (dual, Cu at zero, Eos) diffractometer using graphite
monochromated Mo-Kα (k = 0.71073 Å) radiation at 273 K.
4.1.2. General procedure for the synthesis of sulfonamide chalcone derivatives (4a-4aa)
N-(4-acetylphenyl)benzensulfonamide (1)/ N-(4-acetylphenyl)methanesulfonamide (2)/ N-(4-
acetylphenyl)-4-methylbenzenesulfonamide (3) (0.01 mol) and aromatic aldehyde (0.01mol)
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were dissolved in ethanol (10 mL). To this, NaOH (2 eq) in aqueous solution (4 mL) was added
drop-wise with constant stirring. The reaction mixture was kept overnight at room temperature
and then quenched by dil. HCl. The resulting solid was filtered and recrystallized from suitable
solvents like ethanol or acetic acid to obtain 4a-4aa.
4.1.2.1
Compound 4a was prepared according to reported procedure (m.p. 180°C, as reported [28]).
4.1.2.2 N-[4-(3-(4-methoxyphenyl)-acryloyl)-phenyl]-benzenesulfonamide (4b) [40]
N-(4-cinnamoylphenyl)benzenesulfonamide (4a)
Yellow solid, yield 89%, m.p. 170-172°C, Rf 0.3 (30% EtOAc:Hexane); IR (νmax, cm-1): 3207
(N-H str), 1651 (C=O str), 1336 (asymm. S=O str), 1163 (symm. S=O str); 1H NMR (500 MHz,
Chloroform-d): δ 7.93 – 7.91 (m, 2H), 7.88 – 7.86 (m, 2H), 7.77 (d, J = 15.5 Hz, 1H), 7.59 –
7.54 (m, 3H), 7.48 – 7.45 (m, 2H), 7.35 (d, J = 15.6 Hz, 1H), 7.22 – 7.21 (m, 2H), 6.96 – 6.90
(m, 2H), 3.85 (s, 3H); 13C NMR (100 MHz, Chloroform-d): δ 188.95, 161.74, 144.79, 140.59,
138.88, 134.72, 133.38, 130.00, 129.23, 127.51, 127.17, 119.37, 119.15, 114.42, 55,40; MS
(ESI): m/z [M+H]+ calculated for C22H20NO3S m/z 394.1035, observed 394.1186 [M+H]+
4.1.2.3
Compound 4c was prepared according to reported procedure (m.p. 200-202°C, as reported [41]).
4.1.2.4 N-[4-(3-(4-fluorophenyl)-acryloyl)-phenyl]-benzenesulfonamide (4d)
Compound 4d was prepared according to reported procedure (m.p. 165-167°C, as reported [41]).
4.1.2.5 N-[4-(3-(4-bromophenyl)-acryloyl)-phenyl]-benzenesulfonamide (4e)
Compound 4e was prepared according to reported procedure (m.p. 205-207°C, as reported [41])
4.1.2.6 N-(4-(3-(4-(dimethylamino)phenyl)acryloyl)phenyl)benzenesulfonamide (4f)
N-[4-(3-(4-chlorophenyl)-acryloyl)-phenyl]-benzenesulfonamide (4c)
Yellow solid, yield 84%, m.p. 140-142°C, Rf 0.3 (30% EtOAc:Hexane); IR (νmax, cm-1): 3280
1
(N-H str), 1651 (C=O str), 1325 (asymm. S=O str), 1157 (symm. S=O str); H NMR (300MHz,
DMSO-d6): δ 10.83 (s, 1H), 7.99 (d, J = 9.16 Hz, 2H), 7.84 (d, J = 7.6 Hz, 2H), 7.66-7.55 (m,
7H), 7.23 (d, J = 8.4 Hz, 2H), 6.73 (d, J = 9.16 Hz, 2H); 2.99 (s, 6H); 13C NMR (100 MHz,
DMSO-d6): δ 196.92, 152.47, 145.13, 144.14, 143.05, 142.51, 137.14, 132.36, 131.16, 130.36,
130.29, 127.25, 122.58, 118.42, 112.27, 40.73; HRMS (ESI): m/z [M+H]+ calculated for
C23H23N2O3S m/z 407.1351, observed 407.1452 [M+H]+
4.1.2.7
N-[4-(3-(4-nitrophenyl)-acryloyl)-phenyl]-benzenesulfonamide (4g)
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Yellow solid, yield 70%, m.p. 201-203°C, Rf 0.17 (30% EtOAc:Hexane); IR (νmax,cm-1): 3205
(N-H str), 1649 (C=O str), 1338 (asymm. S=O str), 1159 (symm. S=O str); 1H NMR (400 MHz,
DMSO-d6): δ 10.97 (s, 1H), 8.27 (d, J = 8.4 Hz, 2H), 8.18-8.03 (m, 5H), 7.86 (d, J = 6.88 Hz,
2H), 7.75 (d, J = 16 Hz, 1H), 7.67-7.56 (m, 3H), 7.26 (d, J = 9.16 Hz, 2H); 13C NMR (125 MHz,
Chloroform-d): δ 188.49, 143.39, 140.84, 138.88, 134.25, 133.70, 133.45, 132.22, 130.17,
129.75, 129.27, 127.18, 124.88, 121.98, 119.32; HRMS (ESI): m/z [M+H]+ calculated for
C21H17N2O5S m/z 409.0780, observed 409.2468 [M+H]+
4.1.2.8
Compound 4h was prepared according to reported procedure (m.p. 142-144°C, as reported [41]).
4.1.2.9 N-[4-(3-(2-chlorophenyl)-acryloyl)-phenyl]-benzenesulfonamide (4i)
N-(4-(3-(furan-2-yl)acryloyl)phenyl)benzenesulfonamide (4h)
Compound 4i was prepared according to reported procedure (m.p. 169-170°C, as reported [41]).
4.1.2.10 N-[4-(3-(4-hydroxyphenyl)-acryloyl)-phenyl]-benzenesulfonamide (4j)
Compound 4j was prepared according to reported procedure (m.p. 206-208°C, as reported [42]).
4.1.2.11 N-[4-(3-(2-bromophenyl)-acryloyl)-phenyl]-benzenesulfonamide (4k)
Compound 4c was prepared according to reported procedure (m.p. 185-186°C, as reported [41]).
4.1.2.12 N-(4-(3-(p-tolyl)acryloyl)phenyl)benzenesulfonamide (4l)
Compound 4l was prepared according to reported procedure (m.p. 171-172°C, as reported [41]).
4.1.2.13 N-(4-(3-(5-bromothiophen-2-yl)acryloyl)phenyl)benzenesulfonamide (4m)
Yellow solid, yield 73%, m.p. 133-135°C, Rf 0.55 (30% EtOAc:Hexane); IR (νmax, cm-1): 3278
(N-H str), 1651 (C=O str), 1321 (asymm. S=O str), 1157 (symm. S=O str); 1H NMR (500 MHz,
Chloroform-d): δ 7.91 – 7.88 (m, 2H), 7.79 (d, J = 15.2 Hz, 1H), 7.87 – 7.84 (m, 2H), 7.59 –
7.55 (m, 1H), 7.50 – 7.46 (m, 2H), 7.21 – 7.18 (m, 2H), 7.15 (d, J = 15.2 Hz, 1H), 7.09 (d, J =
3.8 Hz, 1H), 7.05 (d, J = 3.9 Hz, 1H).; 13C NMR (125 MHz, Chloroform-d): δ 187.80, 141.85,
140.80, 136.25, 134.18, 133.45, 132.42, 131.36, 130.06, 129.26, 127.81, 127.18, 120.37, 119.33,
116.48; Anal. calculated for C19H14BrNO3S2 (445.95): C, 50.90; H, 3.15; N, 3.12 %. Found: C,
50.78; H, 3.22; N, 3.25 %
4.1.2.14 N-(4-cinnamoylphenyl)methanesulfonamide (4n)
Compound 4n was prepared according to reported procedure (m.p. 169-170°C, as reported [43]).
4.1.2.15 N-(4-(3-(4-methoxyphenyl)acryloyl)phenyl)methanesulfonamide (4o)
Compound 4o was prepared according to reported procedure (m.p. 132-133°C, as reported [43]).
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4.1.2.16 N-(4-(3-(4-nitrophenyl)acryloyl)phenyl)methanesulfonamide (4p)
Yellow solid, yield 75%, m.p. 130-131°C, Rf 0.56 (30% EtOAc:Hexane); IR (νmax, cm-1): 3184
(N-H str), 1650 (C=O str), 1328 (asymm. S=O str), 1146 (symm. S=O str); 1H NMR (500 MHz,
DMSO-d6): δ 10.40 (s, 1H), 8.33 – 8.26 (m, 2H), 8.22 – 8.14 (m, 4H), 8.11 (d, J = 15.7 Hz, 1H),
7.80 (d, J = 15.7 Hz, 1H), 7.39 – 7.32 (m, 2H), 3.14 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ
187.38, 148.01, 143.37, 141.23, 140.56, 131.81, 130.54, 129.73, 126.02, 123.88, 117.55, 39.95;
Anal. calculated for C16H14N2O5S (346.06): 55.48; H, 4.07; N, 8.09 %. Found: C, 55.34; H, 4.14;
N, 7.95 %
4.1.2.17 N-(4-cinnamoylphenyl)-4-methylbenzenesulfonamide (4q)
Compound 4q was prepared according to reported procedure (m.p. 160-161°C, as reported [44]).
4.1.2.18 N-(4-(3-(4-methoxyphenyl)acryloyl)phenyl)-4-methylbenzenfenesulfonamide (4r)
Compound 4r was prepared according to reported procedure (m.p. 142-143°C, as reported [44]).
4.1.2.19 N-(4-(3-(4-chlorophenyl)acryloyl)phenyl)-4-methylbenzenesulfonamide (4s)
Yellow solid, yield 71%, m.p. 181-183°C, Rf 0.3 (30% EtOAc:Hexane); IR (νmax, cm-1): 3113
(N-H str), 1651 (C=O str), 1327 (asymm. S=O str), 1155 (symm. S=O str); 1H NMR (400 MHz,
DMSO-d6): δ 10.82 (s, 1H), 8.01 (d, J = 8.5 Hz, 2H), 7.89 (dd, J = 8.4, 5.4 Hz, 2H), 7.81 – 7.62
(m, 4H), 7.33 (d, J = 7.8 Hz, 2H), 7.29 – 7.18 (m, 4H), 2.28 (s, 3H); 13C NMR (100 MHz,
DMSO-d6): δ 187.96, 144.27, 144.22, 142.99, 142.87, 142.76, 136.96, 133.00, 131.72, 130.74,
130.33, 127.29, 122.27, 118.43, 116.55, 21.49; Anal. calculated for C22H18ClNO3S (411.90): C,
64.15; H, 4.40; N, 3.40 %. Found: C, 63.98; H, 4.62; N, 3.63 %
4.1.2.20 N-(4-(3-(4-bromophenyl)acryloyl)phenyl)-4-methylbenzenesulfonamide (4t)
Yellow solid, yield 85%, m.p. 228-230°C, Rf 0.55 (30% EtOAc:Hexane); IR (νmax,cm-1): 3119
(N-H str), 1650 (C=O str), 1324 (asymm. S=O str), 1153 (symm. S=O str); 1H NMR (400 MHz,
DMSO-d6): δ 10.80 (s, 1H), 8.00 (d, J = 8.5 Hz, 2H), 7.84 (d, J = 15.4 Hz, 1H), 7.77 (d, J = 8.3
Hz, 2H), 7.69 (d, J = 8.2 Hz, 2H), 7.67 – 7.56 (m, 3H), 7.33 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.4
Hz, 2H), 2.29 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 187.97, 144.28, 143.06, 142.59,
136.99, 134.57, 132.92, 132.39, 131.23, 130.77, 130.42, 127.28, 124.39, 123.24, 118.46, 21.50;
Anal. calculated for C22H18BrNO3S (456.35): C, 57.90; H, 3.98; N, 3.07 %. Found: C, 57.76; H,
4.04; N, 2.85 %
4.1.2.21 N-(4-(3-(3-bromophenyl)acryloyl)phenyl)-4-methylbenzenesulfonamide (4u)
13

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Yellow solid, yield 80%, m.p. 215-217°C, Rf 0.5 (30% EtOAc:Hexane); IR (νmax,cm-1): 3200 (N-
1
H str), 1658 (C=O str), 1331 (asymm. S=O str), 1155 (symm. S=O str); H NMR (500 MHz,
Chloroform-d): δ 7.94-7.92 (m, 2H), 7.76-7.75 (m, 2H), 7.72 (d, J = 15.7 Hz, 1H), 7.61 (t, J =
1.8 Hz, 1H), 7.47 – 7.38 (m, 2H), 7.37-7.35 (m, 2H), 7.26 (d, J = 8.2 Hz, 3H), 7.22-7.19 (m, 2H),
2.39 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 187.32, 143.65, 142.65, 141.57, 137.21, 136.48,
132.87, 132.21, 130.86, 130.63, 130.26, 129.82, 129.71, 128.15, 126.69, 123.38, 122.32, 117.86,
20.91; Anal. calculated for C22H18BrNO3S (456.35): C, 57.90; H, 3.98; N, 3.07 %. Found: C,
57.86; H, 4.03; N, 2.98 %
4.1.2.22 N-(4-(3-(4-isopropylphenyl)acryloyl)phenyl)-4-methylbenzenesulfonamide (4v)
Compound 4v was prepared according to reported procedure (m.p. 184-185°C, as reported [41]).
4.1.2.23 N-(4-(3-([1,1'-biphenyl]-4-yl)acryloyl)phenyl)-4-methylbenzenesulfonamide (4w)
Yellow solid, yield 81%, m.p. 243-244°C, Rf 0.6 (30% EtOAc:Hexane); IR (νmax, cm-1): 3219
(N-H str), 1649 (C=O str), 1336 (asymm. S=O str), 1163 (symm. S=O str); 1H NMR (400 MHz,
DMSO-d6): δ 8.01 (d, J = 8.5 Hz, 2H), 7.91 – 7.84 (m, 3H), 7.72 – 7.66 (m, 7H), 7.44 (t, J = 7.6
13
Hz, 2H), 7.34 (dd, J = 17.5, 7.7 Hz, 3H), 7.19 (d, J = 8.5 Hz, 2H), 2.29 (s, 3H); C NMR (100
MHz, DMSO-d6): δ 187.89, 144.17, 143.88, 143.30, 142.48, 139.76, 137.63, 134.45, 132.52,
130.70, 130.30, 129.98, 129.55, 128.50, 127.58, 127.25, 122.42, 118.63, 21.48; Anal. calculated
for C28H23NO3S (453.13): C, 74.15; H, 5.11; N, 3.09 %. Found: C, 74.27; H, 4.93; N, 2.89 %
4.1.2.24 N-(4-(3-(2-chlorophenyl)acryloyl)phenyl)-4-methylbenzenesulfonamide (4x)
Yellow solid, yield 89%, m.p. 184-185°C, Rf 0.4 (30% EtOAc:Hexane); IR (νmax, cm-1): 3223 (N-
1
H str), 1651 (C=O str), 1328 (asymm. S=O str), 1157 (symm. S=O str); H NMR (400 MHz,
DMSO-d6): δ 7.98 – 7.88 (m, 2H), 7.82 – 7.48 (m, 6H), 7.46 – 7.33 (m, 1H), 7.31 (d, J = 7.8 Hz,
2H), 7.14 (m, 2H), 6.96 (d, J = 8.3 Hz, 1H), 2.28 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ
187.72, 143.68, 138.02, 137.86, 132.32, 131.17, 130.83, 130.25, 130.16, 128.19, 127.94, 127.21,
119.93, 118.63, 118.47, 21.48; Anal. calculated for C22H18ClNO3S (411.07): C, 64.15; H, 4.40; N,
3.40 %. Found: C, 64.31; H, 4.21; N, 3.20 %
4.1.2.25 N-(4-(3-(4-hydroxyphenyl)acryloyl)phenyl)-4-methylbenzenesulfonamide (4y)
Compound 4y was prepared according to reported procedure (m.p. 206-208°C, as reported [42]).
4.1.2.26 N-(4-(3-(2-bromophenyl)acryloyl)phenyl)-4-methylbenzenesulfonamide (4z)
14

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Yellow solid, yield 77%, m.p. 208-210°C, Rf 0.55 (30% EtOAc:Hexane); IR (νmax, cm-1): 3208
1
(N-H str), 1650 (C=O str), 1338 (asymm. S=O str), 1158 (symm. S=O str); H NMR (400 MHz,
DMSO-d6): δ 10.82 (s, 1H), 8.10 (d, J = 7.7 Hz, 1H), 8.02 (d, J = 8.5 Hz, 1H), 7.92 – 7.84 (m,
1H), 7.81 – 7.76 (m, 1H), 7.71 – 7.66 (m, 3H), 7.44 (t, J = 7.6 Hz, 1H), 7.35 – 7.31 (m, 3H), 7.22
13
(d, J = 8.5 Hz, 1H), 7.16 (d, J = 8.5 Hz, 1H), 2.29 (s, 3H); C NMR (100 MHz, DMSO-d6): δ
196.97, 144.31, 144.24, 143.23, 142.87, 141.26, 136.92, 133.82, 130.91, 130.41, 129.22, 128.75,
127.27, 125.29, 118.40, 118.33, 21.50; Anal. calculated for C22H18BrNO3S (455.01): C, 57.90; H,
3.98; N, 3.07 %. Found: C, 57.84; H, 4.11; N, 2.89 %.
4.1.2.27 4-methyl-N-(4-(3-(p-tolyl)acryloyl)phenyl)benzensulfonamide (4aa)
Compound 4aa was prepared according to reported procedure (m.p. 208-209°C, as reported [41]).
4.2. Antifilarial activity assay
4.2.1.
Establishment and maintenance of B. malayi life cycle:
The human filarial parasite B. malayi life cycle was maintained in jirds (Meriones
unguiculatus) and mastomys (Mastomys caucha) using mosquitoes (Aedes aegypti) as vectors by
standard methods as described earlier [45]. The use of animals for this study was approved by
the Institutional Animal Ethics Committee which follows the norms of the Committee for the
Purpose of Control and Supervision on Experiments on Animals (CPCSEA) in India.
Microfilariae (Mf) were freshly obtained from the peritoneal cavity of the jirds exposed to
infective third stage larvae (L3) 4–5 months back. Mf were washed with RPMI 1640 medium
(containing 20 ꢀg mL−1 gentamycin, 100 ꢀg mL−1 penicillin, 100 ꢀg mL−1 streptomycin) plated
on the sterile plastic petri dishes and incubated at 37°C for 1 h to remove the peritoneal exudate
cells of the jirds. Mf were collected from the petri dishes, washed with RPMI 1640 medium and
used for in vitro experiments [46,47].
4.2.2.
In vitro screening of compounds for anti-filarial activity:
The efficacy of compounds to affect the viability of Mf in vitro was assessed by the
extent of parasite motility. A stock solution of 2 mM concentration was made for each
sulfonamide chalcone derivative in DMSO. Further dilutions were made to obtain the desired
final concentration in the range of 0.5 µM to 500 µM. The highest concentration of DMSO used
along with compound was <1% hence comparable vehicle control was also taken with 1%
DMSO. Approximately, 300 Mf in 1000 ꢀL of sterile 0.9% saline were introduced into each vial
15

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for every test drug (over a dose range of 1 µM to 100 µM) along with above mentioned vehicle
control and incubated on shaker incubator at 37ºC for 30 minutes with 150 rpm (Scigenics
Biotech, India). After incubation, Mf were washed with RPMI 1640 media and 100 Mf were
plated in each well (each individual samples in triplicates) in sterile 24 well culture plates (Nunc,
Denmark) containing 1000 ꢀL of RPMI media. The plates were re-incubated at 37ºC for 48 h in
5% CO2 incubator (pre-optimized conditions). Mf motility was assessed by microscopy (using
Nikon Diaphot, TMD inverted microscope). Each experiment was repeated thrice to check the
reproducibility. Percent inhibition in terms of loss of motility was determined as described earlier
[48]. The IC50 value was also calculated for effective compounds [49].
4.2.3.
Evaluation of the in vitro cytotoxicity of various effective agents on the human
Peripheral Blood Mononuclear Cells (PBMCs):
Whole blood samples were obtained from healthy subjects. Venous blood from each
subject was overlaid carefully on histopaque-1077 (Sigma Chem. Co., USA) in 1:1 ratio taken in
screw cap tubes centrifuged at room temperature 400 g for 30 minutes. Upper layer of plasma
was discarded and opaque layer of about 0.5 cm containing PBMC was collected. Opaque layer
was re-suspended in RPMI 1640 medium supplemented with 2 mM L-glutamine. The cell
suspension was then washed by adding RPMI 1640 medium followed by centrifugation at 250 g
for 10 min and the cell pellet was collected. The washing procedure was repeated twice and the
cell pellet was re-suspended in RPMI medium supplemented with 10% fetal bovine serum.
Cytotoxicity was evaluated against human PBMCs (1x105 PBMCs/ml) by Trypan Blue exclusion
assay following similar procedure as adapted for parasites with each of the agents. The dose at
which 50% cytotoxicity was observed has been denoted as LD50 concentration.
4.2.4.
Acridine orange/ Ethidium bromide (AO/EB) staining for the detection of apoptosis:
The primary phenomenon behind AO/EB dual staining is that Acridine Orange (AO)
permeates all cells and makes the nuclei appear green and Ethidium Bromide (EB) is only taken
up by cells when cytoplasmic membrane integrity is lost, and stains the nucleus red. Thus live
cells have a normal green nucleus; early apoptotic cells have bright green nucleus with
condensed or fragmented chromatin; late apoptotic cells display condensed and fragmented
orange chromatin; cells that have died from direct necrosis have a structurally normal orange
nucleus. Dual staining with AO/EB was carried out as per standard protocol [50].The dye mix
16

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consisted of 100 ꢀg/mL AO and 100 ꢀg/mL EB in phosphate-buffered saline. Mf (control as well
as treated with different reagents for 48 h) were washed and resuspended in 25 ꢀL cold
phosphate-buffered saline, followed by the addition of 5 ꢀL AO/EB dye mix. Stained Mf were
viewed under an epifluorescence microscope (Nikon) with the excitation filter set at 480/30 nm
and the barrier filter at 535/40 nm. Tests were carried out in triplicate, counting a minimum of 10
Mf in each observation for detection of differential staining.
Acknowledgements
All authors are thankful to DBT, India for funds to support the project “Maintenance of
Repository for Filarial Parasites and Reagents”. H S Chandak and S P Bahekar are thankful UGC
New Delhi, India (F. No. 41-335 / 2012 (SR) dt.13.07.2012) for the financial support. S V Hande
acknowledges UGC, New Delhi, India for fellowship.
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List of captions
Scheme 1. Synthesis of N-(4-cinnamoylphenyl)arylsulfonamide derivatives
Figure 1. ORTEP diagram of (CCDC No. 1484678) twin molecules of 4q in the unit cell
Figure 2. Sulfonamide chalcones with significant therapeutic window
Figure 3. Display of evidence of apoptosis by differential AO/EB staining. Fluorescent
microscopic view of microfilaria: (a) live worm (–ve control) (b) dead worm (+ve control-
staurosporine) (c) compound 4c (d) compound 4d (e) compound 4e (f) compound 4g (g)
compound 4n (h) compound 4o (i) compound 4p (j) compound 4q (k) compound 4t (l)
compound 4u (m) compound 4aa (n) compound 4y (o) compound 4v
Table 1. In vitro effect of chalcone derivatives on motility of B. malayi Mf
Table 2. IC50 and LD50 values for chalcones obtained by in vitro filarial motility assay and the in
vitro cytotoxicity assay (against PBMCs) respectively
21

ACCEPTED MANUSCRIPT
Highlights
Sulfonamide Chalcones: Synthesis and in vitro exploration for therapeutic
potential against Brugia Malayi
Sandeep P. Bahekara†, Sneha V. Handeb†, Nikita R. Agrawala, Hemant S. Chandaka*, Priyanka S.
Bhojb, Kalyan Goswamib**, MVR Reddyb
a Department of Chemistry, G. S. Science, Arts and Commerce College, Khamgaon 444303, India
b Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences and JB Tropical Disease Research
Centre, Sevagram, Wardha 442102, India
Highlights
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Synthesis and antifilarial assay of sulfonamide chalcones is reported.
In vitro cytotoxicity assay of effective compounds carried out on PBMCs.
Four of the thirteen effective compounds showed significant therapeutic window.
Chalcones possibly inhibit folate pathway with consequent defect in DNA synthesis.
Substitution with halogen and lipophilic methyl group proved to be effective.