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

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

Sulfonamide Chalcones: Synthesis and in vitro exploration for therapeutic

potential against Brugia Malayi

Sandeep P. Bahekar^a†, Sneha V. Hande^b†, Nikita R. Agrawal^a, Hemant S. Chandak^a*, Priyanka S.

Bhoj^b, Kalyan Goswami^b**, MVR Reddy^b

^aDepartment of Chemistry, G. S. Science, Arts and Commerce College, Khamgaon 444303, India

^bDepartment 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^-1and 1330 cm^-1respectively. 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 IC₅₀value. 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 IC₅₀value.

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-NO₂showed 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*

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*

82.697 ± 1.783

1.580 ± 0.090

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 IC₅₀and LD₅₀values 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. IC₅₀and LD₅₀values 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-d₆as 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 (ν_max, 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 (ν_max, 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 (ν_max,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 (ν_max, 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 (ν_max, 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 (ν_max, 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 (ν_max,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 (ν_max,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 (ν_max, 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 (ν_max, 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 (ν_max, 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⁻¹gentamycin, 100 ꢀg mL⁻¹penicillin, 100 ꢀg mL⁻¹streptomycin) 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% CO₂incubator (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 IC₅₀value 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 (1x10⁵PBMCs/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 LD₅₀concentration.

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

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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