In vitro and in vivo antifilarial activity evaluation of 3,6-epoxy [1,5]dioxocines: A new class of antifilarial agents

Article abstract of DOI:10.1016/j.bmcl.2012.01.009

A series of 3,6-epoxy [1,5]dioxocines were synthesized and evaluated for their antifilarial activity against adult parasites of human lymphatic filarial parasite Brugia malayi (sub-periodic strain) in vitro. Out of these, six compounds (4a-f) possessed im

Full text of DOI:10.1016/j.bmcl.2012.01.009

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1527–1532
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In vitro and in vivo antifilarial activity evaluation of 3,6-epoxy [1,5]dioxocines:
A new class of antifilarial agents q
Koneni V. Sashidhara a, , Abdhesh Kumar a, K. Bhaskara Rao a, Vikas Kushwaha b, Kirti Saxena b,
⇑
P.K. Murthy b
a Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226 001, India
b Division of Parasitology, CSIR-Central Drug Research Institute, (CSIR-CDRI), Lucknow 226 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3,6-epoxy [1,5]dioxocines were synthesized and evaluated for their antifilarial activity against
adult parasites of human lymphatic filarial parasite Brugia malayi (sub-periodic strain) in vitro. Out of
these, six compounds (4a–f) possessed improved in vitro anti-filarial activity and examples 4d and 4f
were also found to be active in the in vivo experiments. These results demonstrate that 3,6-epoxy
[1,5]dioxocines exhibits potent antifilarial activity and might be developed into a new class of antifilarial
drug.
Received 11 November 2011
Revised 28 December 2011
Accepted 4 January 2012
Available online 10 January 2012
Keywords:
Synthesis
Ó 2012 Elsevier Ltd. All rights reserved.
Regioselectivity
Dioxocines
Antifilarial activity
Lymphatic filariasis (LF) is caused by a group of parasitic worms
that are transmitted through the bites of infected mosquitoes. It is
commonly known as elephantiasis and one of the most prevalent,
neglected tropical diseases of the world. According to WHO report
more than 1.3 billion people in 81 countries worldwide are threa-
tened by lymphatic filariasis and over 120 million people are cur-
rently infected, with about 40 million disfigured and incapacitated
by the disease.1 In India, approximately 25 million people are
known to harbor circulating microfilariae (mf) and another 19 mil-
lion people suffer from filarial manifestations caused by lymph
dwelling filariids, Wuchereria bancrofti and Brugia malayi.2
There are only three drugs currently in use for the treatment of
LF: albendazole, ivermectin, and diethylcarbamzine (DEC) the
chemical structures of which are shown in Figure 1. Albendazole
is administered in combination with either ivermectin or DEC
through MDA (Mass drug administration) programs. Both DEC
and ivermectin kill mf but has no effect on most of the adult filarial
species and causes side effects.3 No drug yet has been found to be
effective against adult worms. Also drug resistance to ivermectin
appears to be another issue of concern, especially in areas where
DEC cannot be administrated. The search for new molecular struc-
tures associated with macro filaricidal activity as lead molecules is,
therefore, needed.4,5
In continuation of our ongoing drug discovery programme, on
oxygenated heterocycles,6 herein we have synthesized 3,6-epoxy
[1,5]dioxocines and since a number of molecules from heterocycles
are known as good antifilarials, we evaluated our oxygenated het-
erocycles for their antifilarial activity both in vitro and in vivo. The
method of synthesis is simple and efficient and does not involve
any special apparatus or reagents.The route followed for the prep-
aration of 3,6-epoxy [1,5]dioxocines and 3,6-epoxy [1,5]dioxo-
cines-chalcone hybrids is illustrated in Scheme 1. Commercially
available ortho substituted phenols (1a–c) underwent the Duff
formylation reaction in presence of hexamethylene tetraamine
(HMTA) and TFA to furnish dicarbaldehydes (2a–c) which on reac-
tion with different acetophenones in refluxing dioxane in the pres-
ence of a catalytic amount of conc.HCl give regioselective para-
condensed chalcones (3a–l)6a,g In all the chalcones synthesized
the trans double bond (on the basis of coupling constant) was ob-
tained exclusively. These chalcones (3a–l) on reaction with epi-
chlorohydrin in the presence of
a
catalytic amount of
triethylamine furnished 3,6-epoxy [1,5]dioxocine (4a–l).7 The iso-
lated product was found to be a mixture of two enantiomers, in
which the chiral centers have R,S and S,R configuration.8 All com-
pounds were characterized using 1H NMR, 13C NMR, 2D NMR, mass
spectrometry and IR spectroscopy. The purity of these compounds
was ascertained by elemental analysis and spectral analysis.
(Please refer to Supplementary data).9
q
Part XIV in the series, ‘‘Advances in drug design and discovery’’.
For the purpose of biological evaluation of synthesized (race-
mic 3,6-epoxy [1,5]dioxocine) compounds, initially efficacy of
the compounds was assessed in vitro on adult worms and micro-
⇑
Corresponding author. Tel.: +91 9919317940; fax: +91 522 2623405.
E-mail
addresses:
sashidhar123@gmail.com,
kv_sashidhara@cdri.res.in
(K.V. Sashidhara).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.009

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K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1527–1532
Figure 1. Chemical structure of clinically used antifilarial drugs.
Scheme 1. Synthesis of 3,6-epoxy [1,5]dioxocines derivatives (4a–l). Reagents and conditions: (a) (1) HMTA/TFA, 120 °C, 3 h; (2) 10% H2SO4, 90–100 °C, 2 h; (b) Conc.HCl,
p-R1C6H4COCH3, dioxane, 80–90 °C, 3.5–4.5 h; (c) epichlorohydrin, Et3N, reflux, 45–90 min.
filariae (mf) of B. malayi using motility assay according to the
method of Murthy and Chatterjee.10 Test compounds and refer-
ence drug DEC were dissolved in DMSO. The final concentration
of DMSO in the incubation medium was kept below 0.1%. DMSO
was used in place of test compounds for controls. The results of
the in vitro biological screening have been summarized in Table 1.
Among 12 compounds tested, majority of the synthesized com-
pounds exhibited considerable antifilarial activity. As evident
from Table 1 compounds 4a–c, 4d, 4e, 4f, and 4g caused complete
IC50 (SI = CC50/IC50).11 Generally a compound showing SI value of
P10 is considered safe for in vivo evaluation. The selectivity in-
dexes (SI) of these compounds were reasonably acceptable (Ta-
ble 1). Therefore, we have selected six compounds (4a–f)
showing better in vitro antifilarial activity profile in comparison
to standard drug DEC (Table 1), and these were subjected for fur-
ther studies in vivo.
Initially, the most potent compounds (4a–f) were evaluated for
their in vivo studies against B. malayi in Meriones unguiculatus (jird)
(primary model). In this model, male jirds of 8–10 weeks old were
transplanted intraperitoneally with adult worms isolated from
peritoneal cavity of infected jirds.12 Each animal received 10 fe-
male and 5 male adult worms. On day 2 or 3 post-adult worm
transplantation (p.a.t.), the peritoneal fluid was aspirated and
checked for the presence of mf. The treatment was started on
day 7 or 8-p.a.t. The animals were sacrificed on day 42 post initia-
tion of treatment (p.i.t.) and live parasites were collected and
counted.13
Effect of the compounds on the parasitological parameters is
depicted in Table 2. Majority of the synthesized compounds exhib-
ited considerable antifilarial activity. As evident from the table,
compounds 4d & 4f showed significantly (P <0.001) lesser worm
recovery at 200 mg/kg over control. In terms of embryostatic activ-
ity, the compounds 4d & 4f exerted about 24 & 68% sterility in fe-
male worms, respectively, whereas from standard drug DEC-
Citrate treated animals 11% sterile female worms were recovered.
inhibition in motility of adult worms at 1.25, 2.5, 5.0 and 10.0
mL (LC100: lethal concentration), respectively, while remaining
compounds (4h–l) required >10 g/mL concentration for com-
plete inhibition in motility of the worms. The compounds (4a–g)
have shown IC50 values in the range of 0.4–1.6 g/mL. The com-
pounds which had LC100 >10 g/mL were considered inactive
lg/
l
l
l
and IC50 value was not calculated. The compounds 4a–d showed
15–39% inhibition in MTT reduction assays while compound 4e
did not show any inhibition in MTT reduction assay. Further, the
effect of these compounds was also assessed on mf and results
are shown in Table 1. The compounds 4a–b, 4c, 4e, 4f, 4d and
4g–l showed LC100 2.5, 5.0, 5.0, 10.0 and more than 10.0
The compounds (4a–f) have shown IC50/EC50 values in the range
of 0.9–7.1 g/mL. The CC50 values of compounds (4a–f) were
found in the range of 10–40 g/mL. After confirming the in vitro
lg/mL.
l
l
antifilarial activities of the compounds, the selectivity index (SI)
values of compounds was determined as the ratio of CC50 and

K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1527–1532
1529
Table 1
In vitro activity of 3,6-epoxy [1,5]dioxocines (4a–l) on adult worms and microfilariae of Brugia malayi
c
Compounds
Effect on female adult worm
Effect on microfilariae (Mf)
g/ml) IC50 /EC50 g/ml)
in motility assayb
CC50
g/
ml)
SI w.r.t.
adults
motility
SI w.r.t.
Mf
motility
(
l
LC100
(l
g/ml) in IC50
(l
g/ml) in Mean % inhibition LC100
(
l
(l
a
motility assay
motility assayb in MTT reduction in motility
assay
O
4a
O
O
1.25
1.0
15
2.5
0.9
10
10
11
O
4b
O
O
O
1.25
1.25
10
0.6
0.4
3.5
39
22
22
2.5
1.1
1.8
3.9
10
40
33
16.7
100
9.4
9.1
O
Cl
4c
O
O
O
5
22.2
O
4d
4e
O
O
O
>10
8.5
O
OCH3
O
O
O
2.5
0.9
NI
5
0.9
31
34.4
34.4
O
O
4f
O
O
5
1.6
NI
10
3.5
37
23.1
10.6
O
Cl
(continued on next page)

1530
K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1527–1532
Table 1 (continued)
c
Compounds
Effect on female adult worm
Effect on microfilariae (Mf)
g/ml) IC50 /EC50 g/ml)
in motility assayb
CC50
g/
ml)
SI w.r.t.
adults
motility
SI w.r.t.
Mf
motility
(
l
LC100
(l
g/ml) in IC50
(l
g/ml) in Mean % inhibition LC100
(
l
(l
a
motility assay
motility assayb in MTT reduction in motility
assay
4g
4h
O
O
O
10
0.5
ND
ND
ND
ND
NI
NI
34
41
33
>10
>10
>10
>10
>10
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
O
O
O
O
>10
>10
>10
>10
ND
ND
ND
ND
ND
ND
ND
ND
O
O
O
O
O
4i
O
4j
O
O
O
O
Cl
4k
O
O
O
O
O
O
O
4l
>10
800
ND
35
64
>10
500
ND
ND
ND
ND
O
O
DEC
289
354
9103
31.5
25.7
a
b
c
100% reduction in motility indicates death of parasite.
IC50 = 50% concentration of the agent at which 50% inhibition in motility is achieved.
CC50 = concentration at which 50% of cells are killed; ND = Not Done, SI = Selectivity Index (CC50/IC50).
Nevertheless except compound 4e the other compounds 4a–c also
showed considerable antifilarial activity.
Compounds (4d and 4f) that were found most potent in the M.
unguiculatus model, were further evaluated against B. malayi in
Mastomys coucha (secondary model) for confirmation of their antif-
ilarial activity. M. coucha harboring 5–7 months old B. malayi infec-
tion and showing progressive rise in microfilaria were selected for
the study. Peripheral blood (10-lL tail blood) taken between 12:00

K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1527–1532
1531
Table 2
Antifilarial efficacy of 3,6-epoxy [1,5]dioxocines (4a–f) against Brugia malayi in jirds (Meriones unguiculatus)
Compound No.
Dose mg/kg s.c. Â 5 days (n)
Status of microfilaraemia in peritoneal cavity
No. of worms recovered (Mean SD)
% Sterilized female worms
4a
4b
4c
4d
4e
4f
200 (4)
200 (4)
200 (4)
200 (4)
200 (4)
200 (4)
25 (6)
Vehicle treated (4)
Active
Active
Active
Active
Active
Active
Active
Active
6.0 0.0**
7.5 0.7*
7.5 0.7*
5.50 0.7***
10.0 2.0
5.3 0.6***
10.3 2.9
11 1.53
29
29
45
24
27
63
11
0
DEC-Citrate
Control
n = number of animals.
*
P <0.05 (vs control).
P <0.01 (vs control).
P <0.001 (vs control).
**
***
Table 3
Antifilarial activity of compounds 4d and 4f and DEC against Brugia malayi in Mastomys coucha (values are Mean SD).
Compound No.
Dosing (mg/kg)a
No. Live worms recovered
Female
Sterilized female worms
Male
Total
Count (%)
Control
4d
4f
Vehicle treated
11.40 3.58
8.75 2.63
11.25 2.63
6.00 1.79
26.60 8.02
16.75 2.63
12.25 6.85
24.33 4.03
38.00 9.06
4.00 2.65 (19.62)
4.00 2.94 (24.60)
2.75 3.10 (17.78)
3.67 6.09 (20.59)
200
200
50
25.50 3.32 (32.89)b,*
23.50 9.47 (38.16)b,*
30.33 3.50 (20.18)b
DEC-Citrate
a
Each group consisted of 5 animals which received the agent, s.c. Â 5 days.
b
% reduction in worm burden over control; DEC: Diethylcarbamazine.
*
P <0.05 (vs Control).
noon and 1:00 PM14 was made into thick smears just before initi-
ation of treatment (day 0), on days 7/8 and 14 and thereafter at
fortnightly intervals till day 90 p.i.t. The animals were sacrificed
on day 91 p.i.t. Both the compounds, exhibited significant (P
<0.05) adulticidal activity (4d: 33%; 4f: 38%) compared to control.
However, treatment with DEC-Citrate could reduce the adult worm
burden by only 20% (Table 3).
kraborty (Director, CSIR-CDRI) for his constant support and
encouragement. This is CDRI communication number 8178.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.01.009.
A closure look into the structure–activity relationship indicates
that compounds containing naphthalene nucleus were found inac-
tive (4i–l) while compounds containing alkyl group at 10-postion
were more promising. Surprisingly, the substitution on the phenyl
ring of chalcone seems to have no effect, as the activity was con-
served in both.
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In conclusion, we report here a series of 3,6-epoxy [1,5]dioxo-
cines (4a–l) which was first time evaluated for their in vitro as well
as in vivo antifilarial activity. One of the compound 4f displayed
potent activity in vitro as well as in vivo and it exerted antifilarial
activity better than the standard drug. Thus compound 4f, found
active with no apparent signs of any toxicity in gross health of trea-
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optimization and development of new antifilarial agents. Though
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and provide an approach to develop new prototypes for further
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hydroxy-3-methyl-5-(3-oxo-3-phenylprop-1-enyl)benzaldehyde
A
solution of (E)-2-
3a
(3.0 mmol) in epichlorohydrin containing Et3N (2.3 mmol) was heated for
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pressure. The solid residue was poured in water and extracted threefold with
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(CDCl3, 300 MHz): d 8.03 (s, 1H), 8.0 (bs, 1H), 7.73 (d, J = 15.7 Hz, 1H), 7.60–7.41
Acknowledgments
Instrumentation facilities from SAIF, CDRI are gratefully
acknowledged. A.K. and K.B.R. are thankful to CSIR, New Delhi, In-
dia for financial support. We are also thankful to Dr Tushar K. Cha-

1532
K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1527–1532
(m, 5H), 7.34 (bs, 1H), 6.06 (s, 1H), 4.68 (d, J = 6.1 Hz, 1H), 4.38 (dd, J = 2.5 and
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