Development of novel anti-filarial agents using carbamo(dithioperoxo)thioate derivatives

Article abstract of DOI:10.1016/j.ejmech.2017.11.047

A series of novel carbamo(dithioperoxo)thioate derivatives have been prepared in excellent yield using a significantly fast, one-pot three component reaction and experimented for their potential as anti-filarial agents against model filarial nematode Setaria cervi. Among 23 compounds (4a-w) evaluated for the anti-filarial activities, five compounds (4a, 4b, 4c, 4d and 4h) have shown promising anti-proliferative effects on the juvenile stage microfilariae (mf) as well as in adults in a time and dose dependent manner. Compound 4a was found most active against oocytes, mf and adult nematods as well as non-cytotoxic to the normal cells. It has been established that the anti-filarial activity of the compounds were observed due to the involvement of reactive oxygen species (ROS) and apoptosis. Several biochemical and microscopic experiments have been carried out to establish the fact that both intrinsic and extrinsic pathways of apoptosis contribute to the compound 4a mediated death phenomenon of the filarial nematodes.

Full text of DOI:10.1016/j.ejmech.2017.11.047

Accepted Manuscript
Development of novel anti-filarial agents using carbamo(dithioperoxo)thioate
derivatives
Arin Gucchait, Nikhilesh Joardar, Pravat Kumar Parida, Priya Roy, Niladri Mukherjee,
Ananya Dutta, Ravichandran Yesuvadian, Santi P. SinhaBabu, Kuladip Jana, Anup
Kumar Misra
PII:
S0223-5234(17)30950-9
10.1016/j.ejmech.2017.11.047
EJMECH 9922
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 September 2017
Revised Date: 27 October 2017
Accepted Date: 18 November 2017
Please cite this article as: A. Gucchait, N. Joardar, P.K. Parida, P. Roy, N. Mukherjee, A. Dutta, R.
Yesuvadian, S.P. SinhaBabu, K. Jana, A.K. Misra, Development of novel anti-filarial agents using
carbamo(dithioperoxo)thioate derivatives, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.11.047.
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ACCEPTED MANUSCRIPT
Graphical abstract
Development of novel anti-filarial agents using carbamo(dithioperoxo)thioate
derivatives
Arin Gucchait, Nikhilesh Joardar, Pravat Kumar Parida, Priya Roy, Niladri Mukherjee,
Ananya Dutta, Ravichandran Yesuvadian, Santi P. SinhaBabu, Kuladip Jana,* Anup
Kumar Misra*
S
CS2
RSH
N
S SR
NCS, CH Cl
2
2
o
R = Aryl, heteroaryl
Anti-filarial agents
0
8
C, 2-5 min
2-96%
NH

ACCEPTED MANUSCRIPT
Development of novel anti-filarial agents using
carbamo(dithioperoxo)thioate derivatives
a1
b1
a
b
b
Arin Gucchait, Nikhilesh Joardar, Pravat Kumar Parida, Priya Roy, Niladri Mukherjee,
a
c
b
a
Ananya Dutta, Ravichandran Yesuvadian, Santi P. SinhaBabu, Kuladip Jana, * Anup
a
Kumar Misra *
a
Bose Institute, Division of Molecular Medicine, P-1/12, C.I.T. Scheme VII-M, Kolkata
7
00054, India; E-mail: akmisra69@gmail.com; kuladip.jana@gmail.com
b
Parasitology Laboratory, Department of Zoology (Centre for Advanced Studies), Visva-
Bharati University, Santiniketan 731235, West Bengal, India
c
Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur –
03203, Tamil Nadu, India
6
1
Contributed equally.
Abstract: A series of novel carbamo(dithioperoxo)thioate derivatives have been prepared in
excellent yield using a significantly fast, one-pot three component reaction and experimented
for their potential as anti-filarial agents against model filarial nematode Setaria cervi. Among
2
3 compounds (4a-w) evaluated for the anti-filarial activities, five compounds (4a, 4b, 4c, 4d
and 4h) have shown promising anti-proliferative effects on the juvenile stage microfilariae
mf) as well as in adults in a time and dose dependent manner. Compound 4a was found most
(
active against oocytes, mf and adult nematods as well as non-cytotoxic to the normal cells. It
has been established that the anti-filarial activity of the compounds were observed due to the
involvement of reactive oxygen species (ROS) and apoptosis. Several biochemical and
microscopic experiments have been carried out to establish the fact that both intrinsic and
extrinsic pathways of apoptosis contribute to the compound 4a mediated death phenomenon
of the filarial nematodes.
Key words: carbamo(dithioperoxo)thioate, anti-filarial, Setaria cervi, ROS, apoptosis.
Introduction
Lymphatic filariasis (LF) has remained a exigent health hazard and has affected over 110
million people in 51 countries throughout the tropics and sub-tropics of Asia, Africa, the
Western Pacific, and parts of the Caribbean and South America (WHO, 2016) [1]. The
causative agents of LF are Wuchereria bancrofti, Brugia malayi and Brugia timori of which
W. bancrofti and B. malayi are majorly responsible for lymphatic filariasis in tropical
countries and particularly in India [2]. The immunobiology of the disease and the mode of
progression have been so far well documented earlier [3-5]. Diethylcarbamazine (DEC),
Ivermectin and Albendazole have remained as the drug of choice but their effectiveness is
limited to microfilariae (mf) only [3]. However, combination of DEC and ivermectin

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treatment are assisted with systemic and inflammatory adverse reactions [6,7]. DEC and
ivermectin showed promising efficacy in controlling the mf in combination to Albendazole
[
8,9]. The emergence of anthelmintics resistant filarial nematodes is a serious concern to
eliminate lymphatic filariasis. Factors that have been considered to mitigate the development
of drug resistance in lymphatic filariasis include the combined use of drugs with different
modes of action and the long and complex life cycle of filarial nematodes [10]. Currently
available therapeutics target the microfilarial stage of the filarial worm and this enhances the
opportunity to investigate for effective macrofilarial agents. In search of this, few synthetic
compounds such as trans-stilbene derivatives [11], C-cinnamoyl glycosides [12], 4-
oxycoumarin derivatives [13], quinolone derivatives [14], alkaloid derivative [15], a number
of natural products [16-18] as well as nanoparticle formulations such as polymer inspired
silver nano materials [19], gold nanoparticles [20] have shown promise as potent antifilarial
candidates. However, most of the promising antifilarial compounds are not equally effective
against mf as well as adult worms. As a consequence, there is a strong need to develop
therapeutics, which could be effective against both mf as well as adult worms.
th
Disulfirams, members of the dithiocarbamate family were introduced in the late 18 centuries
to the rubber industries for vulcanization purpose [21]. Later on its therapeutic role as a
scabiescide has been established against the intestinal worms too [22]. Disulfirams and their
analogs have also been used for discouraging alcohol intake [23]. Apart from these,
disulfirams have the potential to control fungal infections [24] and several cancers [25,26].
Inspired by the therapeutic potential of disulfirams, a series of carbamo(dithioperoxo)thioate
derivatives have been synthesized and found to have effective therapeutic potential against
cancer cells [27]. It has been envisaged that carbamo(dithioperoxo)thioate derivatives could
possess anti-filarial activities through anti-proliferative effects on the filarial nematods as
they do in the cancer cells. With this proposition, a series of carbamo(dithioperoxo)thioate
derivatives were synthesized and evaluated against model filarial nematode Setaria cervi (S.
cervi), which are found in the bovine abdominal cavity. Although, W. bancrofti is the major
parasite causing filarial infection in humans, it is quite difficult to get the parasite in the
laboratory experimental set-up. Whereas, S. cervi resembles with W. bancrofti in its nocturnal
periodicity and antigenic profile and can easily be used as a model parasite for the screening
of the compounds for their anti-filarial potential [11,12,28]. The activities of the promising
compounds (MIC, IC and LC values) against S. cervi were validated against W. bancrofti.
5
0
50
Synthesis of a series of novel carbamo(dithioperoxo)thioate derivatives using a one-pot three

ACCEPTED MANUSCRIPT
component reaction methodology and its bioevaluation against filarial nematodes is presented
in this report. An extensive study at the molecular level on the mechanism of action of the
active molecules has also been included in the present study. To the best of our knowledge,
this is the first report on the therapeutic potential of carbamo(dithioperoxo)thioate derivatives
against lymphatic filariasis.
Results and discussion
Chemistry
Due to the usefulness of the carbamo(dithioperoxo)thioate derivatives in the drug discovery
program, a few reports have appeared in the literature [27,29] for the synthesis of these class
of compounds. Earlier, this class of compounds were synthesized by carbon tetrabromide
(
CBr ) mediated condensation of amines, carbondisulfide (CS ) and thiols using a one-pot
4
2
multi-component reaction condition considering the fact that CBr reacts with thiols to
4
generate sulfenyl bromides, which react with the dithiocarbamate salts to produce the desired
compounds. Following literature reported reaction condition [27], piperidine (1a) (1.0 equiv.)
was treated with CS (2) (1.0 equiv.) and p-thiocresol (3a) (1.0 equiv.) in the presence of
2
CBr (2.0 equiv.) and Et N (1.1 equiv.) in CH Cl at room temperature. However, the desired
4
3
2
2
product was obtained in low yield together with the formation of disulfide derivative of 3a in
significant quantity. We believed that the aromatic sulfenyl bromide generated in situ from 3a
by the treatment with CBr might be very reactive, which reacts with another molecule of
4
thiol (3a) to furnish disulfide derivative (self coupling) instead of coupling with a
dithiocarbamate salt. In order to overcome this situation, it was decided to carry out the
reaction stepwise as well as without using any extra base (Et N). At first, the neat equimolar
3
mixture of amine (1a) and CS (2a) was stirred at room temperature to form the
2
dithiocarbamate salt instantly. The reaction mixture was diluted with CH Cl and thiol (3a)
2
2
and CBr were added to it sequentially and it was stirred at room temperature for upto 2 h.
4
Unfortunately, formation of the desired product was not observed after prolonged reaction
time. It was decided to use alternative halogenating agents such as N-chlorosuccinimide
(NCS) and N-bromosuccinimide (NBS) etc. for the expected outcome. To our satisfaction,
treatment of the reaction mixture with NBS and NCS furnished the expected product in
significant short interval of time (2-5 min) at 0 °C in absence of a base (Table S1, supporting
information). Although, NCS and NBS were equally effective in terms of the yield of the
product formation, NCS has been chosen as the halogen source with the presumption that aryl

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sulfenyl chloride might be less reactive than aryl sulfenyl bromide which allows formation of
the desired product reducing the chance of disulfide formation. Optimizing the reaction
condition, it was observed that treatment of 1a (1.0 mmol) with CS (1.1 mmol) followed by
2
addition of thiol (3a) and NCS (1.1 mmol) in CH Cl furnish desired compound 4a in 92%
2
2
yield at 0 °C in 2 min (Scheme 1). Among several commonly used organic solvents, CH Cl2
2
was found superior and used for the reaction. Following the optimized reaction condition, a
series of carbamo(dithioperoxo)thioate derivatives (4a-w) were prepared in excellent yield by
a significantly fast reaction of a variety of amines (1a-e) with CS (2) and aromatic thiols (3a-
2
e) in one-pot (Table 1). All synthesized compounds were characterized using NMR and mass
spectral analysis.
S
NCS
+
CS + RSH
2
NH
N
S SR
o
CH Cl , 0 C
2
2
R = Aryl, heteroaryl
Scheme 1: Synthesis of carbamo(dithioperoxo)thioate derivatives by NCS mediated multi-
component reaction of amines, CS and thiols in one-pot.
2
Table 1: Synthesis of carbamo(dithioperoxo)thioate derivatives in one-pot.
Sl.
No.
1
Amine
Thiol
Product
Time
(min)
2
Yield
(%)
92
S
NH
SH
1
a
N
S
S
3
a
4
a
2
1a
SH
5
90
S
N
S S
3
b
4
b
3
4
5
1a
1a
1a
S
2
5
5
90
88
86
H CO
SH
3
S
S
N
3
c
OCH3
4
c
N
S
S
SH
N
N
N
S S
N
3
d
N
4
d
N
SH
S
S
3
e
N
4
e

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6
7
3c
S
2
5
95
90
O
NH
O
N
S
S
S
1
b
OCH3
4
f
1b
3b
S
S
O
N
4
g
8
9
1
1
1b
1b
1b
3d
3a
3e
3c
S
5
2
5
2
90
92
90
92
N
O
N
S
S
N
4
h
S
O
N
S
S
[30]
4
i
0
1
S
S
S
O
N
N
4
j
S
NH
S
S
N
1
c
OCH3
4
k
12
13
14
1c
1c
1c
3a
3d
3b
S
2
5
5
96
87
88
S
S
N
4
l
S
N
S
S
N
N
4
m
S
S
S
N
N
4
S
n
1
5
6
1c
3e
3c
5
2
85
94
S
S
N
4
o
1
S
NH
N
S
S
1
d
OCH3
4
p

ACCEPTED MANUSCRIPT
1
7
8
1d
1d
3a
S
2
5
86
84
S
S
N
4
S
q
1
3b
S
S
N
4
r
1
2
2
9
0
1
1d
1d
3d
3e
3b
S
5
5
5
82
86
92
N
N
S
S
S
S
N
4
s
S
N
S
N
4
t
NH
N
S
S
1
e
4
u
2
2
2
3
1e
3c
3a
S
2
2
95
96
S
S
N
OCH3
4
v
1e
S
N
S
S
4
w
Biology
The fundamental effects of the synthesized compounds (4a-w) on cellular viability and
mortality of microfilaria of S. cervi were conducted through relative movability (RM)
assessment and inhibition of proliferation (MTT) assay. The observation from the MTT assay
is in terms of MIC (Minimum Inhibitory Concentration), IC50 and LC50 are presented in
Table 3. The elementary data collected from these in vitro experiments showed that among
the compounds tested (4a-w), five compounds (4a, 4b, 4c, 4d and 4h) were found to be
promising lead compounds against different developmental stages (oocytes, mf and adults) of
S. cervi and compound 4a had shown its maximum effectiveness due to its significant low
MIC, IC and LC values 0.358 ± 0.02 µg/mL, 0.708 ± 0.021 µg/mL and 3.89 ± 0.18 µg/mL
5
0
50

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respectively in comparison to the standard drug Ivermectin (IVM) (MIC, IC50 and LC50
values 3.12 ± 0.78 µg/mL, 6.25 ± 0.14 µg/mL and 16.57 ± 0.21 µg/mL respectively) against
mf (Table 2 and Figure 1a).
Table 2: Evaluation of anti-filarial activity of the synthesized carbamo(dithioperoxo)thioate
derivatives.
Compound
MIC (µg/mL)
IC (µg/mL)
LC (µg/mL)
5
0
50
4
a
0.358 ± 0.02
0.583 ± 0.006
2.30 ± 0.075
2.39 ± 0.061
8.69 ± 0.02
6.69 ± 0.27
0.708 ± 0.021
2.15 ± 0.049
4.61 ± 0.146
4.75 ± 0.11
3.89 ± 0.18
6.49 ± 0.21
4
b
4
c
6.94 ± 0.09
4
d
5.10 ± 0.05
4
e
24.32 ± 0.035
20.08 ± 0.74
22.23 ± 0.54
5.50 ± 0.655
19.91 ± 0.043
47.66 ± 0.025
31.40 ± 0.06
32.12 ± 0.038
31.08 ± 0.054
32.71± 0.286
24.79 ± 0.53
62.11 ± 0.012
19.65 ± 0.187
60.70 ± 0.25
59.92 ± 0.056
59.13 ± 0.86
22.57 ± 0.346
24.25 ± 0.06
23.58 ± 0.765
6.25 ± 0.14
97.28 ± 0.08
80.32 ± 0.28
88.92 ± 0.38
6.17 ± 0.10
4
f
4
g
7.41 ± 0.654
0.97 ± 0.019
6.63 ± 0.21
4
h
4
i
79.64 ± 0.034
190.64 ± 0.02
125.63 ± 0.32
128.48 ± 0.45
124.32 ± 0.67
130.82 ± 0.39
99.18 ± 0.076
248.45 ±0.065
22.54 ± 0.33
242.82 ± 0.34
239.68 ± 0.7
236.53 ± 0.45
90.31 ± 0.487
97.01 ± 0.15
94.32 ± 0.067
16.57 ± 0.21
4
j
15.88 ± 0.054
10.46 ± 0.065
10.70 ± 0.48
10.36 ± 0.83
10.90 ± 0.342
8.26 ± 0.64
4
k
4
l
4
m
4
n
4
o
p
q
4
20.70 ± 0.3
4
1.22 ± 0.116
20.23 ± 0.054
19.97 ± 0.048
19.71 ± 0.054
7.52 ± 0.053
8.08 ± 0.039
7.86 ± 0.569
3.12 ± 0.78
4
r
4
s
4
t
4
u
4
v
4
w
Ivermectin (IVM)
Standard drug)
(

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From the trypan blue dye exclusion test death percentages for each of the compounds was
measured and seemed to increase dose dependently in both mf and adults. The synthetic
compounds were much effective in combating mf and adult of S. cervi in a dose and time
dependent manner as was evident from the MTT reduction assay thus confirming their role as
emerging novel anti-filarials. The percentage of both mf and adult cells of S. cervi that were
inhibited were calculated from the MTT reduction test and found to increase dose
dependently in both mf and adult S. cervi after 24 h of treatment (Figure 1b and 1c). A dose
dependent increase in the death percentage in adult nematodes was also evident (Figure 1c).
In addition, when the cytotoxic effect of the five potent compounds (4a, 4b, 4c, 4d and 4h)
were evaluated in normal human lung fibroblast cell line i.e. WI-38, it was observed that
compounds 4a and 4d had the comparatively low IC50 values 19.83±2.50 µg/mL and
1
0.61±1.19 µg/mL respectively against WI-38 cell lines (Figure 1f) in comparison to
Ivermectin (IVM) 39.91±1.48 µg/mL. It is noteworthy that compound 4d and IVM showed
almost similar cytotoxic effect (respectively 5 fold and 6 fold lower cytoxicity against the
normal cell line (WI-38) as compared to S. cervi). Whereas, compound 4a showed
significantly lower cytotoxicity (25 fold against the normal cell line (WI-38) as compared to
S. cervi), which is 4 fold better than IVM itself. Hence, 4a has been considered as the best
lead amongst the tested compounds (4a-w) and chosen for its further biological studies at the
molecular level. The dose dependent accrual in the conversion of nitroblue tetrazolium salt
(
NBT) into dark formazan crystals proved the involvement of reactive oxygen species (ROS)
31] in the treated samples of both mf and adult (Figure 1d, 1e). Therefore it can be assumed
[
that the death of the parasite may be mediated through increased reactive oxygen species
·-
(
ROS) especially the superoxide anions (0 ).
2

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Figure 1. Evaluation of antifilarial potential of the compounds 4a, 4b, 4c, 4d and 4h
along with Ivermectin (IVM). (a) Comparative IC50 value of the projected compounds
compared to the standard IVM. (b) Percentage inhibition in compound 4a, 4b, 4c, 4d, 4h
along with IVM treated microfilaria S. cervi (c) Death and inhibition percentage of adult S.
cervi treated with compound 4a and IVM. (d) Dose dependent evaluation in the absorbance
at 620 nm, indicating generation of increased superoxide anions in microfilaria S. cervi. (e)
4
a and IVM treated adult worm of S. cervi indicating generation of increased superoxide
anions. (f) Showing the effect of Compound 4a, 4d and IVM on normal human lung
fibroblast cell line WI-38. All the data represent minimum of three independent experiment
and the bar graph shows ±SEM (*p <0.05**p < 0.01, ***p < 0.001, ns = not significant).

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Apoptosis or programmed cell death is of prime importance during the development, immune
regulation and in defense against pathogens. In order to keep consistency in an organism, cell
death and cell survival and/or proliferation are linked intimately through several
interconnected molecular aspects [32]. It is a vital process in multi-cellular organisms for
getting rid of spoiled and unwanted cells to maintain tissue homeostasis [33]. Apoptosis is the
fastest occurring phenomenon among the several cell death pathways typically induced
within the same tissue, while other forms, like necrosis or autophagy are visible only when
apoptosis is inhibited [34,35]. There are several distinct characteristic features of apoptosis
which can be designated as the hallmarks such as chromatin condensation, shrinkage and loss
of cellular attachment towards the substratum, blebbing of cell membrane etc.[36,37] During
apoptosis, outward translocation of phosphatidylserine (PS) takes place, which is a
phospholipid normally embedded inside the plasma membrane. This outwardly translocated
PS exert a pull on macrophages, and are efficiently phagocytosised [36,37]. Both extrinsic
and intrinsic pathways of apoptosis are present in the filarial nematodes as evidenced by
Mukherjee et al., 2016 [11]. It was decided to further determine the specific branch on which
these novel compounds are working in causing death in the filarial parasites. Interestingly,
the Hoechst 33342 (Sigma, USA) stained mf has confirmed that chromatin condensation had
occurred in all samples after treatment with compounds 4a, 4b, 4c, 4d and 4h. However,
maximum chromatin condensation was observed in the 4a treated samples (5 µg/mL) in
comparison to the control (Figure 2a). Moreover, quite importantly the compound 4a showed
its promising effect within 3 h of treatment on mf as evident from the phase contrast
microscopy (Figure 2b). The stain specifically binds to the condensed chromatin of apoptotic
cells more intensely than that of normal cells, which is regarded as the hallmark of apoptotic
cell death further strengthens the hypothesis of cell death in the treated filarial nematodes.
Acridine orange (AO) and ethidium bromide (EtBr) double staining technique added extra
pillars to confirm the apoptotic death mechanism of the compound 4a (1 and 5 µg/mL)
treated oocytes and mf. AO is a vital dye and can move into both live and dead cells whereas
the EtBr can penetrate only where the membrane integrity has been lost. Increase in the
intensity of orange fluorescence due to EtBr in the 4a (1, 5 µg/mL) treated samples further
corroborate with the finding that membrane rupture enhances with the increase of the dose
(Figure 2c).

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Figure 2: Microscopical studies of mf and oocytes and treated with compounds. (a)
Hoechst stained images of S. cervi mf. The DNA fragmentation and chromatin condensation
were found in all the treated parasites but absent in control. (b) The compound 4a treated
nematodes appeared as blue due to the incorporation of trypan blue inside the body in
comparison to the control one in a dose dependent manner; Magnification 20X; (c) Acridine
orange (AO) and ethidium bromide (EtBr) double staining of mf (upper panel) and oocytes
(
Lower panel).

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In addition to the observed results mentioned above, apoptotic incidence was confirmed
through the flow cytometric data of the 4a (50 µg/mL) treated oocytes and mf. The entire plot
area has been divided into four quadrants UL (necrotic cells), UR (late apoptosis), LL (viable
cells) and LR (early apoptotic cells) [38]. After 6 h of treatment with compound 4a (50
µg/mL) 24.6% oocytes showed apoptotic morphology among which 13.3% (LR) in early
stage of apoptosis and 11.3% (UR) in the late apoptotic phase. However, same dose of 4a
caused 31.44% apoptotic characteristics in mf of which 15.82% late apoptotic (UR) and
1
5.62% in the early phase of the apoptotic event (LR) (Figure 3a). The flow cytometric data
were also accounted as the strongest evidence for establishing apoptotic process. Moreover
the fluorescence microscopic images of the 4a treated oocytes (1, 10 µg/mL) showed green
fluorescence of FITC labeled Annexin-V, which binds to the phosphatidylserine (PS) of the
cell membrane that normally resides in the inner leaflet of the plasma membrane, and is
translocated outward due the disintegrity of the cellular membrane. Propidium iodide usually
excluded by the intact cell, intercalates with the base pairs of the double stranded DNA when
it gets entry inside as a consequence of membrane rupture (Figure 3b).
The apoptosis mediated death of compound 4a treated S. cervi was further confirmed by the
expression of EGL-1, CED-3 and CED-9 proteins using western blot experiments after
treatment of compound 4a in a dose dependent (10 µg/mL, 50 µg/mL and 100 µg/mL)
manner for 24 h. EGL-1 and CED-3 proteins are pro-apoptotic in nature whereas; CED-9 is
anti-apoptotic, which is homologous to proto-oncogene Bcl-2 [39]. EGL-1 shows its pro-
apoptotic function in conjugation with BH3 motif that are present in death promoting Bcl-
2
/CED-9 family members [40,41]. During apoptosis events the activation of CED-3 protein
forms cysteine protease complex that leads to the death of the nematodes. Changes in the
protein expression patterns clearly showed the dose dependent increase in the pro apoptotic
proteins EGL-1 and CED-3. On the contrary, anti apoptotic protein CED-9 expression was
decreased in a dose dependent manner (Figure 3c). Collectively these data suggested that
compound 4a induced apoptosis in S. cervi involving EGL-1, CED-3 and CED-9 proteins.

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Figure 3. Evaluation of anti-apoptotic potential of compound 4a in S. cervi (a) Annexin-
V FITC and PI labeling confirmed induction of apoptosis in mf (upper panel) and oocytes
(lower panel) of S. cervi treated with compound 4a (50 µg/ml); (b) Fluorescence microscopy
showing concentration dependent increase in Annexin-V FITC and PI oocytes population in
response to compound 4a; (c) Western immune-blot demonstrating apoptogenic change in
CED-3, CED-9 and EGL-1 with respect to the loading control GAPDH along with pixel
density. All the data represent minimum of three independent experiment and the bar graph
shows ±SEM (*p <0.05**p < 0.01, ***p < 0.001, ns = not significant).

ACCEPTED MANUSCRIPT
Quantitative structure activity relationship analysis
In addition to the biochemical assays, structure activity relationship (3D-QSAR) analyses
were also performed using Accelry’s discovery studio [42] to understand the structural
features of the test compounds those elicit anti-filarial activity. In order to analyze the anti-
filarial activity, four features pharmacophore model has been developed [43] using data set
compounds (23 molecules) that were categorized into training set (16 molecules) and test set
(7 molecules) by considering their drug like characters and ADMET properties [44]. Almost
all data set compounds complied with Lipinski’s rule [45] except 7 molecules where AlogP
value exceeds 5.6 and those molecules were taken in test set. The statistical significance of
this model is given in Table 3. The training set molecules have shown higher fit value,
correlation coefficient and lower RMSD value which denote the fitting of active
pharmacophoric features in the training set molecules with high integrity. The correlation
between the experimental IC and estimated IC values of training set and test set molecules
5
0
50
are
shown
in
Figure
4A.
Overlaying
of
molecules
confirmed
that
carbamo(dithioperoxo)thioate moiety is present in the skeleton of all molecules is the
essential feature of eliciting anti-filarial activity. All compounds have heterocyclic ring in one
end of carbamoaromatic rings and heterocycle or aryl group at the other end (Figure 4B).
Mapping of the most active compounds 4a and 4b with the pharmacophore model has shown
excellent interactions to all pharmacophoric features (Figure 4C and 4D). Whereas, mapping
of the least active compounds 4j and 4s were exhibited constraints in interactions with
hydrogen bond acceptor features (Figure 4E and 4F). The activity of the compounds may be
reduced when heterocyclic group present in both end of carbamo(dithioperoxo)thioate
moiety, substituted heterocycle with aryl group and bulkier aryl group on the other end that
deter the hydrogen bond acceptor features due to reduction of electron density on the
carbamo(dithioperoxo)thioate functional groups. Whereas heterocycle fused with aryl group
exhibited moderate activity. Heterocycle fused with aryl group and bulkier aryl group on the
other end improve the hydrophobicity features.
Table 3: Statistical Result of 3D-QSAR Model
Training set
Test set
N1 = 16
N2 = 7
Corr. Coefficient = 0.9467
RMSD = 0.1455
Max. fit value = 8.5094
Weight = 1.2139
Corr. Coefficient = 0.7351
RMSD = 0.2376
Max. fit value = 7.897
Weight = 1.3361

ACCEPTED MANUSCRIPT
Figure 4: 3D QSAR analysis of carbamo(dithioperoxo)thioate derivatives with anti-filarial
activities. (A) Regression plot representing the statistical analysis with reference to the
predicted IC value and theoretical IC values; (B) Alignment of the molecules showing
5
0
50
conserved carbamo(dithioperoxo)thioate moiety and variable alkyl or aryl/heteroaryl groups;
C and D): Pharmacophoric mapping of active compounds (4a and 4b); (E and F):
(
Pharmacophoric mapping of inactive compounds (4j and 4s).
Conclusion
In summary, a series of novel carbamo(dithioperoxo)thioate derivatives have been
synthesized in excellent yield using NCS mediated one-pot multicomponent reaction of
amines, CS and thiols. The synthesized compounds were evaluated for their potential to act
2
as anti-filarial agants against bovine S. cervi microfilaria (mf) and oocytes using a number of
biological assays. Among twenty three tested compounds six compounds showed promising
anti-filarial activities to be considered as promising leads. However, based on the significant

ACCEPTED MANUSCRIPT
less cytotoxic nature on the normal cells, effect of compound 4a on the mf and adult filarial
nematods has been further studied in cellular level. It has been established that compound 4a
showed significant detrimental effect on the oocytes, mf and adult worms following apoptotic
mechanism, which were justified by the chromatin condensation and DNA fragmentation and
differential protein expressions. A 3D-QSAR study has also been carried out to establish the
structure-activity relationships of active compounds, which supported the biological and
microscopic experimental findings. Collectively these experimental findings suggest that
carbamo(dithioperoxo)thioate derivatives and particularly compound 4a has tremendous
filaricidal potential and can be considered for further development for the advancement of
anti-filarial therapy.
Experimental
General methods: All reactions were monitored by thin layer chromatography over silica
gel coated TLC plates. The spots on TLC were visualized by warming ceric sulphate (2%
Ce(SO ) in 2N H SO ) sprayed plates on a hot plate. Silica gel 230-400 mesh was used for
4
2
2
4
1
13
column chromatography. H and C NMR spectra were recorded on Bruker Avance 500
MHz spectrometer using CDCl as solvent and TMS as internal reference unless stated
3
otherwise. Chemical shift values are expressed in δ ppm. ESI-MS were recorded on a
Micromass mass spectrometer. Elementary analysis was carried out on Carlo Erba analyzer.
Biological experiments were carried out in a Shimadzu UV-2401PC spectrophotometer.
Commercially available grades of organic solvents of adequate purity are used in many
reactions. Solvents of highest purity grade were purchased from Merck India and Milli-Q
water (Milli-Q Academic with 0.22 µm Millipak R-40) was utilized for the assays.
Antibiotics, fetal bovine serum (FBS) (Gibco), Hoechst 33342, Acridine orange, Ethidium
bromide were purchased from Sigma-Aldrich, USA, and AnnexinV-FITC Apoptosis
Detection Kit were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). RPMI-1640,
MTT, NBT were obtained from Hi-Media Laboratories, Mumbai, India. Egl-1, Ced-9 and
Ced-3 primary antibodies, alkaline phosphatase conjugated secondary antibodies were
procured from Santa Cruz Biotechnology (Santa Cruz, CA). GAPDH primary antibodies
were purchased from BioBharati Life Science Pvt. Ltd., India.
Typical experimental condition for the preparation of carbamo(dithioperoxo)thioate
derivatives: Piperidine (100 µL, 1.01 mmol) was added drop wise to CS (65 µL, 1.07 mmol)
2
and the mixture was stirred for 2 min and diluted with anhydrous CH Cl (2 mL). To the
2
2

ACCEPTED MANUSCRIPT
reaction mixture was added p-thiocresol (125 mg, 1.0 mmol) and it was cooled to 0 °C. To
the cooled reaction mixture was added NBS (200 mg, 1.12 mmol) and the reaction mixture
was allowed to stir at 0 °C for 2 min. The reaction mixture was diluted with CH Cl (20 mL)
2
2
and successively washed with 5% Na S O (10 mL), H O (10 mL). The organic layer was
2
2
3
2
dried (Na SO ) and concentrated. The crude product was purified over SiO using hexane-
2
4
2
EtOAc as eluant to give pure compound 4a (260 mg, 92%). Compounds (4b-w) were
prepared following the similar reaction condition (Table 1).
Analytical data of the synthesized compounds:
p-Tolyl piperidine-1-carbo(dithioperoxo)thioate (4a): Yellow oil; IR (neat): 1486.7 (N-
-1 1
C=S), 1277.6 (C-N), 1106.6 (C=S) cm ; H NMR (500 MHz, CDCl ):
δ
7.47 (d, J = 9.0 Hz,
3
2
1
5
H, Ar-H), 7.05 (d, J = 9.0 Hz, 2 H, Ar-H), 4.30-3.85 (m, 4 H, -CH N), 2.30 (s, 3 H, CH ),
2
3
1
3
.75-1.66 (m, 6 H, -CH -); C NMR (125 MHz, CDCl ):
δ
194.8 (C=S), 138.0-129.6 (Ar-C),
2
3
+
5.3, 51.9, 26.3 (2 C), 25.6, 21.2 (CH ); ESI-MS: 306.0 [M+Na] ; Anal. Calcd. for
3
C H NS (283.47): C, 55.08; H, 6.04; N, 4.94; found: C, 54.90; H, 6.18; N, 4.70.
13
17
3
Naphthalen-1-yl piperidine-1-carbo(dithioperoxo)thioate (4b): Yellow oil; IR (neat):
-
1 1
1
487.2 (N-C=S), 1279.0 (C-N), 1103.4 (C=S) cm ; H NMR (500 MHz, CDCl ):
δ
8.04-7.40
3
1
3
(
m, 7 H, Ar-H), 4.35-3.92 (m, 4 H, NCH -), 1.75-1.70 (m, 6 H, -CH -); C NMR (125 MHz,
2
2
CDCl3):
δ 194.4 (C=S), 133.4-126.3 (Ar-C), 55.4, 52.0, 26.3 (2 C), 25.6; ESI-MS: 342.0
+
[
M+Na] ; Anal. Calcd. for C H NS (319.50): C, 60.15; H, 5.36; N, 4.38; found: C, 60.00;
16 17 3
H, 5.50; N, 4.15.
p-Methoxyphenyl piperidine-1-carbo(dithioperoxo)thioate (4c): Yellow oil; IR (neat):
-
1 1
1
488.7 (N-C=S), 1282.7 (C-N), 1108.2 (C=S) cm ; H NMR (500 MHz, CDCl ): δ 7.60 (d, J
3
=
9.0 Hz, 2 H, Ar-H), 6.80 (d, J = 9.0 Hz, 2 H, Ar-H), 4.36-3.87 (m, 4 H, NCH -), 1.75-1.65
2
13
(
m, 6 H, -CH -); C NMR (125 MHz, CDCl ):
δ
195.1 (C=S), 160.3-114.4 (Ar-C), 55.2 (2
2
3
+
C, NCH , OCH ), 51.9 (NCH ), 26.2 (2 C), 25.6; ESI-MS: 322.0 [M+Na] ; Anal. Calcd. for
2
3
2
C H NOS (299.47): C, 52.14; H, 5.72; N, 4.68; found: C, 52.00; H, 5.90; N, 4.50.
13
17
3
Pyrimidin-2-yl piperidine-1-carbo(dithioperoxo)thioate (4d): Yellow oil; IR (neat):
-
1 1
1
486.7 (N-C=S), 1280.7 (C-N), 1107.2 (C=S) cm ; H NMR (500 MHz, CDCl ):
δ
8.59-8.55
3
(
m, 2 H, Ar-H), 7.08-7.05 (m, 2 H, Ar-H), 4.35-4.10 (m, 4 H, NCH -), 1.78-1.67 (m, 6 H, -
2
1
3
CH -); C NMR (125 MHz, CDCl ):
δ 191.7 (C=S), 156.6-116.9 (Ar-C), 54.3 (NCH ), 51.1
2
2
3

ACCEPTED MANUSCRIPT
+
(
NCH ), 26.2 (2 C), 25.6; ESI-MS: 294.0 [M+Na] ; Anal. Calcd. for C H N S (271.42): C,
2
10 13
3 3
4
4.25; H, 4.83; N, 15.48; found: C, 44.10; H, 5.00; N, 15.25.
Pyridin-2-yl piperidine-1-carbo(dithioperoxo)thioate (4e): Yellow oil; IR (neat): 1488.9
-1 1
(
N-C=S), 1278.4 (C-N), 1110.2 (C=S) cm ; H NMR (500 MHz, CDCl ):
δ
8.43-7.04 (m, 4
3
1
3
H, Ar-H), 4.32-4.05 (m, 4 H, NCH -), 1.76-1.68 (m, 6 H, -CH -); C NMR (125 MHz,
2
2
CDCl3):
δ 191.4 (C=S), 157.9-118.4 (Ar-C), 54.3 (NCH ), 51.1 (NCH ), 25.2 (2 C), 24.6;
2 2
+
ESI-MS: 293.0 [M+Na] ; Anal. Calcd. for C H N S (270.43): C, 48.85; H, 5.22; N, 10.36;
1
1
14
2 3
found: C, 48.70; H, 5.30; N, 10.50.
p-Methoxyphenyl morpholine-4-carbo(dithioperoxo)thioate (4f): Yellow oil; IR (neat):
-1 1
1
489.0 (N-C=S), 1112.9 (C=S) cm ; H NMR (500 MHz, CDCl ):
δ
7.61 (d, J = 9.0 Hz, 2 H,
3
Ar-H), 6.80 (d, J = 9.0 Hz, 2 H, Ar-H), 4.32-3.90 (m, 4 H, OCH ), 3.77 (s, 3 H, OCH ), 3.76-
2
3
1
3
3
6
.73 (m, 4 H, NCH -); C NMR (125 MHz, CDCl ):
δ
196.9 (C=S), 160.5-114.5 (Ar-C),
2
3
+
6.2 (2 C, OCH ), 55.2 (OCH ), 51.5 (2 C, NCH ); ESI-MS: 324.0 [M+Na] ; Anal. Calcd.
2
3
2
for C H NO S (301.44): C, 47.81; H, 5.02; N, 4.65; found: C, 47.65; H, 5.20; N, 4.80.
1
2
15
2 3
Naphthalen-1-yl morpholine-4-carbo(dithioperoxo)thioate (4g): Yellow oil; IR (neat):
-
1 1
1
464.9 (N-C=S), 1280.5 (C-N), 1111.2 (C=S) cm ; H NMR (500 MHz, CDCl ):
δ
8.05-7.43
3
1
3
(
m, 7 H, Ar-H), 4.34-3.98 (m, 4 H, OCH -), 3.77-3.74 (m, 4 H, NCH -); C NMR (125 MHz,
2
2
+
CDCl3):
δ 196.1 (C=S), 133.3-126.6 (Ar-C), 66.3 (2 C), 53.4, 51.4; ESI-MS: 344.0 [M+Na] ;
Anal. Calcd. for C H NOS (321.48): C, 56.04; H, 4.70; N, 4.36; found: C, 55.90; H, 4.90;
1
5
15
3
N, 4.05.
Pyrimidin-2-yl morpholine-4-carbo(dithioperoxo)thioate (4h): Yellow oil; IR (neat):
-
1 1
1
472.6 (N-C=S), 1281.3 (C-N), 1113.5 (C=S) cm ; H NMR (500 MHz, CDCl ):
δ
8.67-8.64
3
(
m, 2 H, Ar-H), 7.14-7.11 (m, 1 H, Ar-H), 4.35-3.90 (m, 4 H, OCH -), 3.88-3.74 (m, 6 H,
2
13
NCH -); C NMR (125 MHz, CDCl3):
δ
186.5 (C=S), 157.6-117.9 (Ar-C), 66.1 (2 C,
2
+
OCH ), 52.9 (2 C, NCH ); ESI-MS: 296.0 [M+Na] ; Anal. Calcd. for C H N OS (273.39):
2
2
9
11
3
3
C, 39.54; H, 4.06; N, 15.37; found: C, 39.40; H, 4.22, N, 15.05.
p-Tolyl morpholine-4-carbo(dithioperoxo)thioate (4i): Yellow oil; IR (neat): 1488.9 (N-
-1 1
C=S), 1114.4 (C=S) cm ; H NMR (500 MHz, CDCl ):
δ
7.47 (d, J = 9.0 Hz, 2 H, Ar-H),
3
7
.07 (d, J = 9.0 Hz, 2 H, Ar-H), 4.30-4.05 (m, 4 H, -OCH ), 3.79-3.74 (m, 4 H, -NCH ), 2.31
2
2
1
3
(
s, 3 H, CH ); C NMR (125 MHz, CDCl ): δ 196.6 (C=S), 138.5-129.7 (Ar-C), 66.3 (2 C,
3
3

ACCEPTED MANUSCRIPT
+
OCH ), 52.9, 51.1, 21.2 (CH ); ESI-MS: 308.0 [M+Na] ; Anal. Calcd. for C H NOS
2
3
12 15
3
(285.44): C, 50.49; H, 5.30; N, 4.91; found: C, 50.30; H, 5.45; N, 4.75.
Pyridin-2-yl morpholine-4-carbo(dithioperoxo)thioate (4j): Yellow oil; IR (neat): 1489.9
-1 1
(
N-C=S), 1280.2 (C-N), 1108.2 (C=S) cm ; H NMR (500 MHz, CDCl ):
δ
8.44-7.04 (m, 4
3
1
3
H, Ar-H), 4.30-4.05 (m, 4 H, OCH -), 3.79-3.72 (m, 4 H, NCH -); C NMR (125 MHz,
2
2
CDCl3):
δ 190.4 (C=S), 157.9-118.4 (Ar-C), 66.3 (OCH ), 52.8 (ONH ), 51.1 (NCH ); ESI-
2 2 2
+
MS: 295.0 [M+Na] ; Anal. Calcd. for C H N OS (272.41): C, 44.09; H, 4.44; N, 10.28;
1
0
12
2
3
found: C, 43.92; H, 4.60; N, 10.00.
p-Methoxyphenyl
3,4-dihydroisoquinoline-2(1H)-carbo(dithioperoxo)thioate
(4k):
-1 1
Yellow oil; IR (neat): 1491.8 (N-C=S), 1108.2 ( C=S) cm ; H NMR (500 MHz, CDCl ):
δ
3
7
.64 (d, J = 9.0 Hz, 2 H, Ar-H), 7.23-7.12 (m, 4 H, Ar-H), 6.78 (d, J = 9.0 Hz, 2 H, Ar-H),
.29-5.00 (m, 2 H, NCH ), 4.41-4.10 (m, 2 H, NCH ), 3.76 (s, 3 H, OCH ), 3.00-2.96 (m, 2
5
2
2
3
1
3
H, CH ); C NMR (125 MHz, CDCl ):
δ
195.8 (C=S), 160.4-114.5 (Ar-C), 55.7 (NCH2),
2
3
+
5
5.2 (OCH ), 51.9 (NCH ), 48.2 (CH ); ESI-MS: 370.0 [M+Na] ; Anal. Calcd. for
3
2
2
C H NOS (347.52): C, 58.75; H, 4.93; N, 4.03; found: C, 58.60; H, 5.10; N, 4.20.
17
17
3
p-Tolyl 3,4-dihydroisoquinoline-2(1H)-carbo(dithioperoxo)thioate (4l): Yellow oil; IR
-1 1
(
neat): 1472.1 (N-C=S), 1280.0 (C-N), 1107.1 (C=S) cm ; H NMR (500 MHz, CDCl ): δ
3
7
.49-7.04 (m, 8 H, Ar-H), 5.29-5.10 (m, 2 H, NCH ), 4.40-4.13 (m, 2 H, NCH ), 3.00-2.96
2 2
1
3
(
m, 2 H, CH ), 2.30 (s, 3 H, CH ); C NMR (125 MHz, CDCl ):
δ
195.8 (C=S), 157.8-127.7
2
3
3
+
(
Ar-C), 55.8 (NCH ), 52.1 (NCH ), 48.3 (CH ), 21.0 (CH ); ESI-MS: 354.0 [M+Na] ; Anal.
2
2
2
3
Calcd. for C H NS (331.52): C, 61.59; H, 5.17; N, 4.23; found: C, 61.45; H, 5.30; N, 4.00.
1
7
17
3
Pyrimidin-2-yl 3,4-dihydroisoquinoline-2(1H)-carbo(dithioperoxo)thioate (4m): Yellow
-1 1
oil; IR (neat): 1490.3 (N-C=S), 1108.2 (C=S) cm ; H NMR (500 MHz, CDCl ):
δ
8.57-7.06
3
(
m, 7 H, Ar-H), 5.35-5.20 (m, 2 H, NCH ), 4.48-4.23 (m, 2 H, NCH ), 3.10-2.96 (m, 2 H,
2
2
13
CH ), 2.30 (s, 3 H, CH ); C NMR (125 MHz, CDCl ):
δ
192.3 (C=S), 168.3-113.2 (Ar-C),
2
3
3
+
5
4.8 (NCH ), 51.1 (NCH ), 47.3 (CH ); ESI-MS: 342.0 [M+Na] ; Anal. Calcd. for
2
2
2
C H N S (319.46): C, 52.63; H, 4.10; N, 13.15; found: C, 52.47; H, 4.25; N, 13.32.
14
13
3 3
Naphthalen-1-yl 3,4-dihydroisoquinoline-2(1H)-carbo(dithioperoxo)thioate (4n): Yellow
-1 1
oil; IR (neat): 1489.9 (N-C=S), 1110.2 (C=S) cm ; H NMR (500 MHz, CDCl ):
δ
8.05-7.08
3
(
m, 11 H, Ar-H), 5.29-5.12 (m, 2 H, NCH ), 4.43-4.15 (m, 2 H, NCH ), 3.05-2.96 (m, 2 H,
2
2
1
3
CH ); C NMR (125 MHz, CDCl ):
δ 195.1 (C=S), 133.3-126.4 (Ar-C), 55.8 (NCH ), 52.1
2
2
3

ACCEPTED MANUSCRIPT
+
(
NCH ), 48.3 (CH ); ESI-MS: 390.0 [M+Na] ; Anal. Calcd. for C H NS (367.55): C,
2
2
20 17
3
6
5.36; H, 4.66; N, 3.81; found: C, 65.20; H, 4.80; N, 3.55.
Pyridin-2-yl 3,4-dihydroisoquinoline-2(1H)-carbo(dithioperoxo)thioate (4o): Yellow oil;
-1 1
IR (neat): 1488.8 (N-C=S), 1280.7 (C-N), 1108.2 (C=S) cm ; H NMR (500 MHz, CDCl ):
δ
3
8
.46-7.07 (m, 8 H, Ar-H), 5.30-5.20 (m, 2 H, NCH ), 4.43-4.28 (m, 2 H, NCH ), 3.15-2.98
2 2
1
3
(
(
(
m, 2 H, CH2); C NMR (125 MHz, CDCl3):
δ
195.1 (C=S), 148.4-118.4 (Ar-C), 54.8
+
NCH ), 52.2 (NCH ), 48.4 (CH ); ESI-MS: 341.0 [M+Na] ; Anal. Calcd. for C H N S
2
2
2
15 14
2
3
318.48): C, 56.57; H, 4.43; N, 8.80; found: C, 56.40; H, 4.56; N, 8.58.
p-Methoxyphenyl 4-benzylpiperidine-1-carbo(dithioperoxo)thioate (4p): Yellow oil; IR
-1 1
(
neat): 1487.7 (N-C=S), 1280.7 (C-N), 1107.4 (C=S) cm ; H NMR (500 MHz, CDCl ): δ
3
7
.62-7.03 (m, 9 H, Ar-H), 3.76 (s, 3 H, OCH ), 3.26-3.10 (m, 2 H), 2.65-2.50 (m, 2 H,
3
1
3
NCH ), 1.98-1.60 (m, 2 H), 1.50-1.20 (m, 5 H); C NMR (125 MHz, CDCl ):
δ
195.1 (C=S),
2
3
+
1
39.4-114.5 (Ar-C), 55.6 (OCH ), 54.5, 42.7, 38.1 (2 C), 31.2; ESI-MS: 412.0 [M+Na] ;
3
Anal. Calcd. for C H NOS (389.59): C, 61.66; H, 5.95; N, 3.60; found: C, 61.50; H, 6.12;
2
0
23
3
N, 3.40.
p-Tolyl 4-benzylpiperidine-1-carbo(dithioperoxo)thioate (4q): Yellow oil; IR (neat):
-
1 1
1
489.8 (N-C=S), 1279.9 (C-N), 1107.6 (C=S) cm ; H NMR (500 MHz, CDCl ):
δ
7.48-7.03
3
(
m, 9 H, Ar-H), 2.67-2.50 (m, 4 H), 2.31 (s, 3 H, CH ), 1.96-1.60 (m, 2 H), 1.50-1.20 (m, 5
3
1
3
H); C NMR (125 MHz, CDCl ):
δ
195.1 (C=S), 139.4-114.5 (Ar-C), 54.4, 42.7, 38.1 (2 C),
3
+
3
2.2, 21.2; ESI-MS: 396.0 [M+Na] ; Anal. Calcd. for C H NS (373.59): C, 64.30; H, 6.21;
2
0
23
3
N, 3.75; found: C, 64.18; H, 6.38; N, 3.50.
Naphthalen-1-yl 4-benzylpiperidine-1-carbo(dithioperoxo)thioate (4r): Yellow oil; IR
-1 1
(
neat); 1487.2 (N-C=S), 1255.9 (C-N), 1103.7 (C=S) cm ; H NMR (500 MHz, CDCl ):
δ
3
8
1
4
6
.04-7.09 (m, 12 H, Ar-H), 3.26-3.10 (m, 2 H), 2.58-2.54 (m, 2 H), 1.96-1.80 (m, 3 H), 1.50-
1
3
.20 (m, 4 H); C NMR (125 MHz, CDCl ):
δ
194.6 (C=S), 139.4-125.8 (Ar-C), 54.6, 51.1,
3
+
2.6, 38.1, 32.2 (2 C); ESI-MS: 432.0 [M+Na] ; Anal. Calcd. for C H NS (409.63): C,
2
3
23
3
7.44; H, 5.66; N, 3.42; found: C, 67.27; H, 5.80; N, 3.20.
Pyrimidin-2-yl 4-benzylpiperidine-1-carbo(dithioperoxo)thioate (4s): Yellow oil; IR
-1 1
(
neat); 1486.7 (N-C=S), 1256.5 (C-N), 1114.4 (C=S) cm ; H NMR (500 MHz, CDCl ): δ
3
1
3
8
.58-7.05 (m, 8 H, Ar-H), 2.68-2.56 (m, 4 H), 1.96-1.78 (m, 3 H), 1.50-1.36 (m, 4 H); C
NMR (125 MHz, CDCl3):
δ
192.9 (C=S), 157.7-118.0 (Ar-C), 42.6 (2 C), 38.1 (2 C), 30.9 (2

ACCEPTED MANUSCRIPT
+
C); ESI-MS: 384.0 [M+Na] ; Anal. Calcd. for C H N S (361.54): C, 56.47; H, 5.30; N,
17
19
3 3
1
1.62; found: C, 56.30; H, 5.46; N, 11.45.
Pyridin-2-yl 4-benzylpiperidine-1-carbo(dithioperoxo)thioate (4t): Yellow oil; IR (neat):
-
1 1
1
487.4 (N-C=S), 1256.8 (C-N), 1107.4 (C=S) cm ; H NMR (500 MHz, CDCl ):
δ
8.45-7.05
3
(m, 9 H, Ar-H), 3.35-3.16 (m, 2 H), 2.60-2.51 (m, 2 H), 2.05-1.78 (m, 3 H), 1.55-1.30 (m, 4
1
3
H); C NMR (125 MHz, CDCl ):
δ
192.0 (C=S), 158.0-118.4 (Ar-C), 53.5, 50.2, 41.5, 37.1,
3
+
3
0.9 (2 C); ESI-MS: 383.0 [M+Na] ; Anal. Calcd. for C H N S (360.56): C, 59.96; H,
1
8
20
2 3
5
.59; N, 7.77; found: C, 59.80; H, 5.75; N, 7.55.
Naphthalen-1-yl pyrrolidine-1-carbo(dithioperoxo)thioate (4u): Yellow oil; IR (neat):
-
1 1
1
486.7 (N-C=S), 1257.5 (C-N), 1114.4 (C=S) cm ; H NMR (500 MHz, CDCl ):
δ
8.08-7.43
3
1
3
(
m, 7 H, Ar-H), 3.99-3.95 (m, 2 H), 3.81-3.77 (m, 2 H), 2.15-1.98 (m, 4 H); C NMR (125
MHz, CDCl3):
δ 191.3 (C=S), 133.4-126.8 (Ar-C), 56.8, 50.5, 26.6, 24.2; ESI-MS: 328.0
+
[
M+Na] ; Anal. Calcd. for C H NS (305.48): C, 58.98; H, 4.95; N, 4.59; found: C, 58.84;
1
5
15
3
H, 5.10; N, 4.40.
p-Methoxyphenyl pyrrolidine-1-carbo(dithioperoxo)thioate (4v): Yellow oil; IR (neat):
-
1 1
1
486.9 (N-C=S), 1256.2 (C-N), 1110.4 (C=S) cm ; H NMR (500 MHz, CDCl ):
δ
7.65 (d, J
3
=
9.0 Hz, 2 H, Ar-H), 6.79 (d, J = 9.0 Hz, 2 H, Ar-H), 3.96-3.92 (m, 2 H), 3.79 (s, 3 H,
1
3
OCH ), 3.72-3.68 (m, 2 H), 2.11-1.96 (m, 4 H); C NMR (125 MHz, CDCl ):
δ
191.9 (C=S),
3
3
+
1
60.4-114.4 (Ar-C), 56.9, 55.2 (OCH ), 50.4, 26.6, 24.2; ESI-MS: 308.0 [M+Na] ; Anal.
3
Calcd. for C H NOS (285.44): C, 50.49; H, 5.30; N, 4.91; found: C, 50.32; H, 5.46; N,
12
15
3
4
.72.
p-Tolyl pyrrolidine-1-carbo(dithioperoxo)thioate (4w): Yellow oil; IR (neat): 1487.7 (N-
-1 1
C=S), 1255.5 (C-N), 1111.4 (C=S) cm ; H NMR (500 MHz, CDCl ):
δ
7.52 (d, J = 9.0 Hz,
3
2
H, Ar-H), 7.09 (d, J = 9.0 Hz, 2 H, Ar-H), 3.96-3.94 (m, 2 H), 3.76-3.71 (m, 2 H), 2.31 (s, 3
1
3
H, CH3), 2.11-1.96 (m, 4 H); C NMR (125 MHz, CDCl ):
δ
191.9 (C=S), 138.3-128.4 (Ar-
3
+
C), 56.7, 50.4, 26.6, 24.2, 21.2; ESI-MS: 292.0 [M+Na] ; Anal. Calcd. for C H NS
1
2
15
3
(269.45): C, 53.49; H, 5.61; N, 5.20; found: C, 53.34; H, 5.78; N, 5.00.
Collection and in vitro treatment procedure of parasites
Motile adult Setaria cervi worms (males and females) were collected from the peritoneal
cavity of freshly slaughtered cattle (Bovis indica Linn.) and brought into the laboratory in

ACCEPTED MANUSCRIPT
normal saline (0.85%). The worms were repeatedly washed in PBS (50 mM, pH 7.0) in order
to remove the extraneous materials. The mature female worms were kept in PBS (50 mM, pH
7
.0) at 37 °C in humidified chamber till dissection. Compounds were dissolved in Dimethyl
sulfoxide (DMSO) (Merck, Germany) with gentle heating and mild sonication. Treatments of
mf and adults were given in 24 well plates (Tarson, India) and 60mm plates (Tarson, India)
respectively maintaining proper sterility of the culture condition as well as media (RPMI-
1
640) (Himedia). Culture media was supplemented with 10% Fetal Bovine Serum (FBS)
(
Gibco). In vitro treatment of mf and adults were done following the method of Kaushal et al.
46] and Mukherjee et al.[47] The doses were standardized on the basis of the MIC and LC50
[
values obtained. The entire treatment setup was placed inside CO incubator (New
2
Brunswick, Germany) with 5% CO flow for 24 hrs and repeatedly monitored for activity.
2
Assessment of in vitro parasite viability through MTT assay
Parasite viability was quantitatively assessed by performing 3-(4, 5-dimethyl-thiazol-2-yl)-2,
5
-diphenyl-tetrazolium bromide (MTT, Sigma) reduction assay [48]. After stipulated time of
incubation with the compounds and succinct microscopic observations, mf and adult worms
were carefully removed from culture RPMI-1640 and washed twice with PBS at 7500 rpm at
3
2
5°C for 5 mins. Approximately 1×10 mf and adult worms were suspended in 0.3 ml of 0.25
mg/ml of MTT (Himedia, India) followed by incubation at 37 °C for 2 hrs in dark. The dark
blue crystals of formazan thus formed by the mitochondrial dehydrogenase enzyme were then
solubilized in 100 µL of DMSO in a 96-well microtiter plate. The absorbance intensity was
measured with the help of a microtiter plate reader (Biorad, USA) at a 595-nm wavelength
keeping DMSO as a blank. The percent inhibition calculation was carried out following the
method of Nayak et al. [49]. Each experiment was repeated for at least four times and the
results were used to calculate mean ± SE [50]. The LC values of the compounds were
5
0
calculated by using Origin Pro 8.0 software.
Microfilaricidal activity in vitro and relative movability (RM) study
Based on the LC data, compounds with maximum efficacy were chosen and triggered for
5
0
determining the in vitro microfilaricidal efficiency. The compounds were evaluated through
relative movability assessment of the treated mf with respect to the control. Mf of S. cervi
5
(
~1× 10 mf/ well) were incubated with the compounds in a series of doses in a 24-well plate
Tarsons, India). The entire experiment was repeated thrice in triplicate manner. The
(
movement of the worms was examined microscopically at every 6 h interval up to 24 h. The

ACCEPTED MANUSCRIPT
movements of the worms were categorized depending on different stages and a score was
given such as 0 for the dead, 4 for highly motile mf, 3 (moderately active and motile); 2
(slightly active and motile); 1 (less active but response to stimuli) following the method of
Mukherjee et al.[47] The IC and Minimum inhibitory concentration (MIC) values were
5
0
calculated using the Origin Pro 8.0 software. The obtained results were used to determine
mean ± SE.
Trypan blue dye exclusion test
Upon treatment with the compounds, compound 4a caused rapid mobility loss within 3 h of
treatment. In order to confirm whether the parasites have become paralyzed or died, the entire
culture was vigorously mixed to homogeneity and finally 100µL of culture media was taken
out containing adequate number of parasites through pipetting and transferred into fresh
media followed by shifting the plate in the CO incubator. Viability assessment of S. cervi
2
microfilariae was checked primarily by trypan blue, because it can stain dead cells blue and
live one transparent. The microfilariae viability (%) was determined by dividing the number
of live ones by the total number of microfilariae following the method of Mukherjee et
al.[47]
Evaluation of cytotoxicity against human normal lung fibroblast cell line WI-38
4
In order to carry out this experiment 2.0 × 10 WI-38 cells were grown in 48 well plates for
2
4 h. Post incubation cells were treated with varying concentrations (0-50 µg/mL) of the
compounds for 24 h. Later, 5% FBS containing phenol red free RPMI with MTT (400 µL;
.5 mg/mL) was added to each well and incubated at 37 °C for 4 h in a humidified incubator
0
containing 5% CO . The purple colored formazan crystals formed in the wells were dissolved
2
in DMSO and their absorbance was measured at 570 nm with a microplate reader (Multiscan
GO).
Assessment of generated superoxide anion through NBT assay
Following requisite time of incubation with the selected compounds (4a, 4b, 4c, 4d and 4h)
the cultures of mf and only 4a treated adult were collected and centrifuged at 7500 rpm for 12
mins at 25 °C and the supernatants were discarded and the parasites pellets were washed
thoroughly in PBS (50 mM, pH 7.0). The parasites pellets were incubated in 2% nitroblue
tetrazolium salt (NBT) solution at 37 °C for 2 h. The tetrazolium salt reacts with the
generated superoxide anion and forms formazan crystals. After stipulated time the pellets

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were again centrifuged and washed with PBS following one methanol wash. The crystals
were dissolved in a mixture of 240 µL of 2M KOH and 480 µL of DMSO with placid shaking
for 10-15 mins at room temperature [47]. The absorbance was measured at 620 nm using a
micro-plate reader (BioRad, USA).
Assessment of chromatin condensation through Hoechst 33342 staining in mf
Chromatin condensation in the treated mf was detected microscopically by nuclear staining
with Hoechst 33342 [12]. Briefly after treatment with the compound, the mf were washed in
PBS (50 mM, pH 7.2) and were fixed in paraformaldehyde. Then the treated and untreated mf
were stained with 1 mg/mL Hoechst 33342 stain and incubated for 5 mins followed by a
gentle wash in PBS. Poly-L-Lysine coated glass slides were prepared and mf were fixed on
to it and caught in the fluorescence microscope at 490 nm excitation (Leica, Germany).
Double staining of oocyte and mf
AO/EtBr double staining was performed following the method described in Roy et al.[17]
Briefly 1:1 ratio of AO and EtBr from the stock of 100 mg/mL in 0.1 M PBS was prepared
and added on the oocyte and mf. The suspensions were taken on to clean glass slides, covered
by cover glass and photographed under fluorescence microscope (Dewinter, Italy).
Fluorescence microscopy of Annexin V-FITC and PI stained oocytes
Oocytes were collected from the gravid females of S. cervi and they were cultured in RPMI-
1
640 supplemented with FBS and treated with the test compound 4a at 1 µg/mL and 10
µg/mL doses for stipulated time period. After proper incubation period oocytes were properly
washed and suspended in PBS (50 mM, pH 7.2). The suspension was centrifuged at 7500
rpm, at 25 °C for 5 mins and incubated with Annexin V- FITC and PI (Sigma Aldrich, MO,
USA) following the manufacturer’s guideline followed by photography under inverted
fluorecence microscope (Leica, Germany) and Las-X software.
Flow cytometry analysis of oocyte and mf
In order to study quantitatively the apoptotic effects of the targeted compounds on S. cervi,
3
4
oocytes (~1×10 ) and mf (1×10 ) were treated with two different concentrations (50 and 100
µg/mL) of compound 4a and cultured for 8 h and 16 h at 37 °C for oocyte and mf
respectively. The treated oocyte and mf were obtained following centrifugation and washing
with PBS twice accordingly and resuspended in PBS (50 mM, pH 7.0) at 4 °C overnight.

ACCEPTED MANUSCRIPT
Oocyte and mf were both stained with AnnexinV-FITC Apoptosis Detection Kit (Sigma,
USA) following Mukherjee et al.[11]. Quantitative flowcytometry analysis was further done
through BD FACSDiva version 6.1.2.
Western blotting of the apoptogenic protein in the adults of S. cervi
Profiling of nematode specific apoptogenic proteins i.e. EGL-1, CED-3 and CED-9 were
assessed using western blot experiments with GAPDH as loading control [29]. Briefly,
proteins from control and treated adult nematodes (100 µg) were estimated following Lowry
et al. [51]. Each proteins (100 µg) resolved by 12.5% SDS-PAGE, transferred on poly
vinylidene difluoride (PVDF) membranes, blocked with 10% skimmed milk and incubated
with different primary antibodies overnight at 4 °C. The membranes were thereafter washed
and incubated with alkaline phosphatse conjugated secondary antibody for 6 h. Alterations in
the expressions of the said apoptogenic proteins were examined by specific substrate
BCIP/NBT (Amresco, USA).
Quantitative structure activity relationship (QSAR) analysis
In order to elucidate a quantitative structure-activity relationship, the molecular structures
were compared with the IC values obtained from the experimental data. 3D-QSAR module
5
0
of Accelry’s discovery studio [42] was used for deriving the atomic analysis. All molecular
structures were prepared and minimized using Maestro GUI software. All 23 molecules were
categorized into test set and training set. The model divides the space of grids into uniform-
sized cubes that reflect the biological relationship using structural features, corresponding
through positive (blue) and negative (red) contours. To reflect statistically significant results
the characterization of molecules was performed using independent variables for structural
components. Models were built up using partial least-squares regression (PLS).
Acknowledgement
A.G. and P.K.P. thank Council of Scientific and Industrial Research (CSIR), India for
providing Junior and Senior Research Fellowships respectively. N.J. thanks DST, Govt. of
India for providing fellowship. This work is supported by CSIR, India [Grant No.
0
2(0237)/15/EMR-II dated 26.11.2015 (AKM)] and Department of Science and Technology
(DST), Govt. of India [(Grant No. SR/SO/AS-006/2014, (SPS)] and Bose Institute.

ACCEPTED MANUSCRIPT
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