Chemotherapy of leishmaniasis. Part IX: Synthesis and bioevaluation of aryl substituted ketene dithioacetals as antileishmanial agents

Article abstract of DOI:10.1016/j.bmcl.2012.08.096

A new series of aryl substituted ketene dithioacetals 6a-h was synthesized and evaluated for their in vitro and in vivo antileishmanial activity against Leishmania donovani. Two compounds exhibited significant in vitro activity against intracellular amastigotes of L. donovani with IC₅₀ values 3.56 and 5.12 μM and were found promising as compared with reference drug, miltefosine. On the basis of good Selectivity Indices (S.I.), they were further tested for their in vivo response against L. donovani/hamster model and showed significant inhibition of parasite multiplication 78% and 83%, respectively. These compounds were better than the existing antileishmanials in respect to IC₅₀ and SI values, but were less active than miltefosine in vivo.

Full text of DOI:10.1016/j.bmcl.2012.08.096

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6728–6730
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chemotherapy of leishmaniasis. Part IX: Synthesis and bioevaluation of
aryl substituted ketene dithioacetals as antileishmanial agents q
Santosh Kumar a, Avinash Tiwari a, S. N. Suryawanshi a, , Monika Mittal b, Preeti Vishwakarma b,
⇑
Suman Gupta b
a Division of Medicinal Chemistry, Central Drug Research Institute, CSIR, Lucknow 226001, India
b Division of Parasitology, Central Drug Research Institute, CSIR, Lucknow 226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of aryl substituted ketene dithioacetals 6a–h was synthesized and evaluated for their
in vitro and in vivo antileishmanial activity against Leishmania donovani. Two compounds exhibited sig-
nificant in vitro activity against intracellular amastigotes of L. donovani with IC50 values 3.56 and 5.12 lM
and were found promising as compared with reference drug, miltefosine. On the basis of good Selectivity
Indices (S.I.), they were further tested for their in vivo response against L. donovani/hamster model and
showed significant inhibition of parasite multiplication 78% and 83%, respectively. These compounds
were better than the existing antileishmanials in respect to IC50 and SI values, but were less active than
miltefosine in vivo.
Received 25 January 2012
Revised 21 August 2012
Accepted 24 August 2012
Available online 13 September 2012
Keywords:
Ketene dithioacetal
L. donovani
Leishmaniasis
In vitro/in vivo
Hamster
Ó 2012 Elsevier Ltd. All rights reserved.
Leishmaniasis comprises
a
spectrum of parasitic illnesses
restricted due to teratogenicity. In addition, it has a long half-life,
which might encourage the emergence of resistance once its use
becomes widespread.9 Taken together, current armamentarium
of leishmaniasis is still limited and alternative therapies are
strongly warranted. In this endeavour, diarylheptanoids,10 oxygen-
ated abietanes,11 diterpene quinones12,13 and chalcones14 are
showing promise in the area of in vitro antileishmanial studies.
Curcumin 1 isolated from Curcuma longa Linn has not only shown
promise as anticancer agent10a but it is also showing antileishma-
nial activity profile in in vitro studies.10b,15 Exhaustive analoging of
curcumin has generated some interesting results.16 Lichochalcone
2 isolated from the root of Chinese licorice plant is also showing
promising antileishmanial activity.17 Chemical Library generated
on the basis of Lichochalcone as a lead molecule is showing prom-
ise in in vitro antileishmanial studies.14a Phenolic diketone 3 iso-
lated from Zingiber offıcinale,18 a structural mimic of 1 and 2
shows radical scavenging activities, quite comparable to Curcumin
1. In continuation of our efforts19a and others19b at CDRI to gener-
ate novel antileishmanial agents coupled with encouraging results
of 1, 2, and 3, we synthesized some aryl substituted ketene dithio-
acetals and evaluated for their in vivo antleishmanial activity pro-
file and the results are reported in this Letter Figure 1.
caused by several species of the protozoan kinetoplastid parasites.
Leishmania spp., affects 12 million people around the world with an
annual death rate of approximately 80,000 people.1 In various clin-
ical manifestations, disseminated visceral infection is most fatal, if
left untreated. This form of leishmaniasis is present in 61 coun-
tries2 and having second rank after malaria in the top 10 fatal par-
asitic diseases worldwide. The first-line therapy includes sodium
stibogluconate (SbV) which has unfortunately developed resistance
in some areas of Bihar, (India) where failure rates up to 65% have
been reported and the use of antimony has been abandoned.3 Pent-
amidine also went out of favor for treatment of VL.4 The drawback
of the clinical use of pentamidine is the route of administration
(injection) and the toxicity of the compound. Administration by
injection increases the expense of the treatment and makes the
use of the drug less practical in developing nations, where cost is
a major factor. The clinical side effects of pentamidine include real
and hepatic toxicity, pancreatitis, hypotension; dysglycemia, and
cardiac abnormalities.5,6 The use of liposomal Amphotericin-B is
limited due to high market price.7 A major milestone in chemo-
therapy of VL is the discovery of miltefosine, an analogue of phos-
phatidylcholine initially developed as an anticancer agent.8 It is an
effective oral drug but its use in women of child-bearing age is
a-Oxoketene dithioacetals of aromatic substrates are very use-
ful synthons in the synthesis of variety of heterocyclic and carbo-
cyclic compounds20 However, they have been not fully exploited
q
CDRI communication no. 155/2011/SNS.
Corresponding author. Tel.: +91 522 2212411 18; fax: +91 522 2223405.
E-mail address: shivajins@yahoo.co.in (S.N. Suryawanshi).
for their biological activity profile.
a-Oxoketene dithioacetals
⇑
6(a–h) were prepared as shown in Scheme 1. The required starting
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.096

S. Kumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6728–6730
6729
OH
O
O
OH
(CH2)nCH3
HO
OH
OCH3
HO
OCH3
HO
OCH3
O
1
2
3
Figure 1. Structures of naturally occurring & synthetic antileishmanial chalcones 1, 2, and 3.
were initially dissolved in DMSO and further diluted with fresh
complete medium. After incubation, 25 L of MTT reagent (5 mg/
ml) in PBS medium, followed by syringe filtration was added to
each well and incubated at 37 °C for 2 h. At the end of the incuba-
tion period, the supernatant were removed by inverting the plate
SMe
SMe
O
O
SMe
SMe
CHO
R1
aq.KOH,
l
R3
R1
MeOH, stirr. R3
R2
4a-h
R2
6a-h
12h, r. t.
5
4a, 6a) R1 = R2 = H, R3= Cl
completely without disturbing cell layer and 150 lL of pure DMSO
4b, 6b) R2 = R3 = H, R1 = Cl
are added to each well. After 15 min of shaking the readings were
recorded as absorbance at 544 nm on a micro plate reader. The
cytotoxic effects were expressed as 50% lethal dose, that is, as
the concentration of a compound which provoked a 50% reduction
in cell viability compared to cell in culture medium alone. IC50 val-
ues were estimated as described by Huber et al.26The selectivity in-
dex (SI) for each compound was calculated as ratio between,
4c, 6c) R1 = R2 = H, R3 = NO2,
4d, 6d) R1 = R3 = H, R2 = NO2,
4e, 6e) R1 = R2 = H, R3 = F
4f, 6f) R1 = H, R2= OCH3, R3 = OBn
4g, 6g) R1 = R2 = H, R3 = C2H2N3
4h, 6h) R1 = R2 = H, R3 = C3H3N2
cytotoxicity (CC50
amastigotes.
)
and activity (IC50
)
against Leishmania
Scheme 1. General synthesis of E-5-(4-Substitutedphenyl)-1, 1-bis(methylthio)
penta-1,4-dien-3-one (6a–h).
The in vivo leishmanicidal activity was determined in golden
hamsters (Mesocricetusauratus) infected with MHOM/IN/80/Dd8
strain of Leishmania donovani obtained through the courtesy of
P.C.C. Garnham, Imperial College, London (UK). The method of Bev-
eridge et al27 as modified by Bhatnagar et al28 and Gupta et al29
was used for in vivo evaluation. Golden hamsters (Inbred strain)
of either sex weighing 40–45 g were infected intracardially with
1 ꢀ 107amastigotes per animal. The infection is well adapted to
the hamster model and establishes itself in 15–20 days. Mean-
while, hamsters gain weight (85–95 g) and can be subjected to re-
peated spleen biopsies. Pre-treatment spleen biopsy in all the
animals was carried out to assess the degree of infection. The ani-
mals with +1 infection (5–15 amastigotes/100 spleen cell nuclei)
were included in the chemotherapeutic trials. The infected animals
were randomized into several groups on the basis of their parasitic
burdens. Five to six animals were used for each test sample. Drug
treatment by intraperitoneal (ip) route was initiated after 2 days of
biopsy and continued for five consecutive days. Post-treatment
biopsies were done on day 7 of the last drug administration and
amastigote counts were assessed by Giemsa staining. Intensity of
infection in both, treated and untreated animals, as also the initial
count in treated animals was compared and the efficacy was ex-
pressed in terms of percentage inhibition (PI) using the following
formula:-
material 1,1-bis(methylthio)-1-en-3-one 5 was prepared following
a known procedure.21 The reaction of 4-chlorobenzaldehyde 4a
with acetone based ketene dithioacetal 5 furnished 6a as a yellow
crystalline solid, melting at 157–158 °C. The structure of 6a was as-
signed on the basis of IR, 1H, 13C NMR and mass spectrometry.
Similarly the reaction of substituted benzaldehydes 4(b–h) with
ketene dithioacetal 5 in the presence of methanolic potassium
hydroxide furnished ketene dithioacetals 6(b–h) in good yield as
pale yellow crystalline solids.
For assessing the activity of compounds against the amastigote
stage of the parasite, mouse macrophage cell line (J-774A.1) in-
fected with promastigotes expressing luciferase firefly reporter
gene was used. Cells were seeded in a 96-well plate (4 ꢀ 104 cell/
100lL/well) in RPMI-1640 containing 10% foetal calf serum and
the plates were incubated at 37 °C in a CO2 incubator. After 24 h,
the medium was replaced with fresh medium containing station-
ary phase promastigotes (4 ꢀ 105 cell/100
lL/well). Promastigotes
invade the macrophage and are transformed into amastigotes.
The test compounds were added at two fold dilutions up to seven
points in complete medium starting from 40 lM conc after replac-
ing the previous medium and the plates were incubated at 37 °C in
a CO2 incubator for 72 h. After incubation, the drug containing
medium was decanted and 50 lL PBS was added in each well
PI ¼ 100 ꢁ ½ANAT ꢀ 100=ðINAT ꢀ TIUCÞꢂ
and mixed with an equal volume of Steady Glo reagent. After gen-
tle shaking for 1–2 min, the reading was taken in a lumino-
meter.22–24 The values are expressed as relative luminescence
units (RLU). Data were transformed into a graphic program (Excel).
IC50 of antileishmanial activity was calculated by nonlinear regres-
sion analysis of the concentration response curve using the four
parameter Hill equations.
Where PI is percent inhibition of amastigotes multiplication,
ANAT is actual number of amastigotes in treated animals, INAT is
initial number of amastigotes in treated animals and TIUC is time
increase of parasites in untreated control animals.
The antileishmanial activity of aryl substituted dithioacetals
6(a–h) has been displayed in Table 1. Of the 6 compounds, 6d
and 6f have shown interesting antiamastigote activity with IC50’S
for L. donovani intramacrophagic amastigotes of 5.12 and 3.56
and selectivity indices of 1.92 and 57.26, respectively. The
in vitro antileishmanial response of both the compounds were bet-
The cell viability was determined using the MTT assay.25 Expo-
nentially growing cells (KB Cell line) (1 ꢀ 105 cells/100
lL/well)
were incubated with test compounds for 72 h. The test compounds
were added at three fold dilutions up to seven points in complete
medium starting from 400 lM concentration, and were incubated
ter than the reference drug, miltefosine (IC50 = 12.5 lM, S.I. = 0.2).
at 37 °C in a humidified mixture of CO2 and 95% air in an incubator.
Podophyllotoxin was used as a reference drug and control wells
containing dimethyl sulfoxide (DMSO) without compounds were
also included in the experiment. Stock solutions of compounds
The compounds 6d and 6f were also selected for in vivo efficacy
evaluation against L. donovani/hamster model at the ip dose of
50 mg kgꢁ1 ꢀ 5 days. Both the compounds exhibited significant
inhibition of parasite multiplication (83% and 78%, respectively).

6730
S. Kumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6728–6730
Table 1
Antileishmanial activity of aryl substituted ketene dithioacetals against L. donovani
Sr.
No.
Compound In vitro Antiamastigote activity IC50
M)
CC50
M)
Selectivity index (S.I.) CC50
IC50
/
In vivo activity (%inhibition) (50 mg/kg ꢀ 5 ip
Remarks
(
l
(
l
dose)
1.
2.
3.
4.
5.
6.
7.
8.
9.
6a
6b
6c
6d
6e
6f
>20
>40
>40
5.12
>40
3.56
>20
>40
ND
ND
ND
9.83
ND
203.85
ND
ND
—
—
—
1.92
—
57.26
—
—
—
—
—
Inactive
Inactive
Inactive
Active
Inactive
Active
Inactive
Inactive
Active
82.8 13.00 (n = 9)
—
77.97 12.7 (n = 9)
—
6g
6h
—
Miltefosine 12.50
3.23
0.26
95.28 2.49* (n = 8)
*
PI at 30 mg/kg ꢀ 5 po, Miltefosine: reference drug.
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the compounds showed promising activity, it could not exceed
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Acknowledgments
The authors are thankful to the Division of Sophisticated Ana-
lytical Instrument Facility (SAIF), CDRI for providing spectral and
elemental analysis data. Santosh Kumar is also grateful to CSIR,
New Delhi for awarding Junior Research Fellowship. The transgenic
L. donovani promastigotes were procured from Neena Goyal, Divi-
sion of Biochemistry, Central Drug Research Institute, Lucknow,
India.
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Supplementary data
Supplementary data associated with this article can be found, in
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08.096.
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