Synthesis and biological evaluation of a novel series of aryl S,N-ketene acetals as antileishmanial agents

Article abstract of DOI:10.1016/j.bmcl.2013.04.025

A series of aryl S,N-ketene acetals 7(a-f) was synthesized and evaluated for their in vitro and in vivo antileishmanial activity against Leishmania donovani. All the 6 compounds exhibited significant in vitro activity against intracellular amastigotes of L. donovani with IC₅₀ values ranging from 1.2 to 3.5 μM and were found promising as compared with reference drugs, sodium stibogluconate (SSG) and paromomycin. On the basis of good selectivity indices (SI), they were further tested for their in vivo potential against L. donovani/hamster model. Two compounds 7a and 7b showed significant inhibition of parasite multiplication, 72% and 83%, respectively. These compounds were comparable with SSG and superior to paromomycin. Preliminary in vitro metabolic investigations were also performed to assess the metabolic stability and in vitro hepatic intrinsic clearance (Cl_int) of compound 7b in hamster liver microsomes.

Full text of DOI:10.1016/j.bmcl.2013.04.025

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3979–3982
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of a novel series of aryl
S,N-ketene acetals as antileishmanial agents q
a
a
b
c
S. N. Suryawanshi a, , Santosh Kumar , Avinash Tiwari , Rahul Shivahare , Yashpal Singh Chhonker ,
⇑
Susmita Pandey a, Nishi Shakya b, Rabi Sankar Bhatta c, Suman Gupta b
a Division of Medicinal Chemistry, CSIR-Central Drug Research Institute, Chattar Manzil Palace, P.O. Box 173, Mahatma Gandhi Road, Lucknow 226001, India
b Division of Parasitology, CSIR-Central Drug Research Institute, Chattar Manzil Palace, P.O. Box 173, Mahatma Gandhi Road, Lucknow 226001, India
c Division of Pharmacokinetics and Metabolism, CSIR-Central Drug Research Institute, Chattar Manzil Palace, P.O. Box 173, Mahatma Gandhi Road, Lucknow 226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 September 2012
Revised 5 April 2013
Accepted 9 April 2013
Available online 26 April 2013
A series of aryl S,N-ketene acetals 7(a–f) was synthesized and evaluated for their in vitro and in vivo
antileishmanial activity against Leishmania donovani. All the 6 compounds exhibited significant in vitro
activity against intracellular amastigotes of L. donovani with IC50 values ranging from 1.2 to 3.5 lM
and were found promising as compared with reference drugs, sodium stibogluconate (SSG) and paromo-
mycin. On the basis of good selectivity indices (SI), they were further tested for their in vivo potential
against L. donovani/hamster model. Two compounds 7a and 7b showed significant inhibition of parasite
multiplication, 72% and 83%, respectively. These compounds were comparable with SSG and superior to
paromomycin. Preliminary in vitro metabolic investigations were also performed to assess the metabolic
stability and in vitro hepatic intrinsic clearance (Clint) of compound 7b in hamster liver microsomes.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Aryl S,N-ketene acetal
Leishmania donovani
Antileishmanial activity
Hamster
Pharmacokinetic studies
Leishmaniasis is a group of parasitic diseases that affect about
twelve million people in tropical and subtropical areas provoking
three clinical expressions: visceral leishmaniasis (VL) that is fatal
in the absence of treatment,1 muco-cutaneous leishmaniasis and
cutaneous leishmaniasis, the last one often self-curing.2 Pentava-
lent antimonials remain the first-line treatment in most parts of
the world, but in India, its use is restricted due to the high level
of drug resistance. There has been a significant improvement in
the number of treatments available for VL during the past decade,
with both new drugs and new formulations of old drugs either re-
cently approved or in clinical trial. These new treatments include:
AmBisome™, a liposomal amphotericin-B formulation, registered
for VL in the USA and Europe in the 1990s with remarkable activity,
even at a single dose in India; oral miltefosine, registered in India
in 2002 (later in Bangladesh); and a low cost intramuscular formu-
lation of paromomycin (aminosidine), registered in India in 2006
and in phase III trial in East Africa by DNDi. Unfortunately, all of
these drugs have significant drawbacks that limit their utilization
in disease endemic areas. These include route of administration,
length of treatment (21–28 days), toxicity and cost.3 Thus, the
development of new, efficient, and safe drugs for the treatment
of this disease is imperative. In this endeavour, diarylheptanoids,4
oxygenated abietanes,5 diterpenequinones6,7 and chalcones8
showed promising results as antileishmanial agents. Curcumin 1
isolated from Curcuma longa Linn., well known as an anticancer
agent,4a also showed antileishmanial activity in in vitro studies.4b,9
Exhaustive analoging of curcumin has generated some interesting
results.10 Licochalcone 2 isolated from Glycerrhiza sp., first reported
for its antibacterial activity11 has also showed promising antileish-
manial activity.12 Chemical library generated on the basis of licoch-
alcone as a lead molecule was found active in in vitro studies.8a
Phenolic diketone 3 isolated from Zingiber offıcinale,13 a structural
mimic of 1 and 2 shows radical scavenging activities, quite compa-
rable to curcumin 1. In continuation of our efforts to generate nat-
ural product based novel antileishmanial agents14 coupled with
encouraging results of 1–3 (Fig. 1), we have synthesized some no-
vel aryl S,N-ketene acetals for their in vitro and in vivo antileish-
manial activity profile and preliminary in vitro metabolic studies.
The findings are reported in this communication .
a
-Oxoketene dithioacetals of aromatic substrates are very use-
ful synthons in the synthesis of variety of heterocyclic and carbo-
cyclic compounds.15 Various aryl S,N-ketene acetals have been
synthesized from
a
-oxoketene dithioacetals.16a–c However; they
have not been fully exploited for their biological activity profile.
3,4,5-trimethoxy aryl S,N-ketene acetals 7(a–f) were prepared as
shown in Scheme 1. The reaction of 3,4,5-trimethoxybenzaldehyde
(tri-o-methylgallaldehyde) 4 with acetone based ketene dithioace-
tal 5 furnished 6 which on further reaction with primary amines
q
CDRI Communication No. 272/2011/SNS.
⇑
Corresponding author. Tel.: +91 522 221 2411 18; fax: +91 522 222 3405.
E-mail address: shivajins1952@gmail.com (S.N. Suryawanshi).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.025

3980
S. N. Suryawanshi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3979–3982
O
OH
OCH3
O
OH
HO
(CH2)nCH3
HO
OH
OCH3
HO
OCH3
O
1. Curcumin
2. Licochalcone
3. Phenolic diketone
Figure 1. A natural product based approach to synthesized novel aryl S,N-ketene acetals.
O
SMe
SMe
MeO
MeO
CHO
MeO
MeO
O
SMe
SMe
i
OMe
OMe
ii
6
5
4
O
NHR
SMe
MeO
MeO
OMe
R
R
7
(7a)
F
Cl
(7d)
(7b)
(7c)
OMe
(7e)
(7f)
Scheme 1. Reagents and conditions: (i) aq.KOH, MeOH, stirring, 12 h, rt. (ii) Amines, EtOH, heat in steel bomb, 120 °C, 24 h.
gave 7(a–f) as a yellow crystalline solids. The structures of 7(a–f)
were assigned with the help of IR, 1H NMR, 13C NMR and mass
spectrometry.
ingly, all the six compounds exhibited high inhibition (96–99%)
against promastigotes at 25 lM concentration. These compounds
were further screened 19,20 in vitro against intracellular amastigote
model and their IC50, CC50 and selectivity index (SI) were calculated
and presented in Table 1. All the compounds were found highly ac-
A series of trimethoxy aryl S,N-ketene acetals has been synthe-
sized and the compounds were evaluated against extracellular
promastigotes and intracellular amastigotes of Leishmania dono-
vani. In our earlier studies17 we have demonstrated compound 6
as antileishmanial agent which showed 66% inhibition of Leish-
mania parasite in hamster model. Therefore, our main objective
of present work was to study the effect of substitution on the bio-
logical activity profile of compound 6. Table 1 displays the percent
inhibition of these compounds against promastigotes. Interest-
tive with IC50 ranging from 1.2 to 3.5
compounds was far better than the reference drugs sodium stibo-
gluconate (IC50 = 57.3 g/mL) and paromomycin (IC50 = 24.8 g/
mL). Compounds showing SI above 10 (7a–e) were tested further
lM. The activity of these
l
l
21
for in vivo antileishmanial activity
and the results are depicted
in Table 1. Amongst them, only two compounds 7a and 7b showed
significant inhibition of parasite multiplication, 72% and 83%,
Table 1
In vitro and in vivo antileishmanial activity data of synthesized aryl S,N-ketene acetals
Compound code no.
In vitro assessment
Anti-promastigote activity Anti-amastigote
percent inhibition (PI) activity IC50 M)
at 25
Cytotoxicity
CC50 M)
Selectivity index
(SI) CC50/IC50
In vivo activity
(
l
(dose-50 mg/kg  5 days, ipa)
percent inhibition SD
(l
l
M
7a
7b
97.3–98.5 (97.9)
95.2–97.2 (96.2)
1.1–1.3 (1.2)
1.8–2.4 (2.1)
39.7–47.2 (43.4)
58.8–70.4 (64.6)
36
31
72 22
83 12
64 10 (pob)
NIc
21 16
32 25
NDd
7c
7d
7e
97.5–99.7 (98.6)
97.6–99.8 (98.7)
98.7–99.5 (99.1)
98.3–99.5 (98.9)
NI
1.5–2.5 (2.0)
1.4–1.8 (1.6)
2.1–3.2 (2.6)
2.7–4.3 (3.5)
52.6–62.1 (57.3)
22.6–27.1 (24.8)
64.1–79.0 (71.5)
53.6–62.2 (57.9)
174.8–192.9 (183.8)
18.7–26.8 (22.7)
397.5–399.2 (398.3)
49.6–57.1 (53.3)
36
36
69
7
7
2
7f
SSG*
89
46
8
9
Paromomycin
NI
PI, IC50 and CC50 values are the average of two independent experiments.
a
ip = intraperitoneal.
po = per oral.
NI = no inhibition.
ND = not done.
b
c
d
*
SSG (sodium stibogluconate) and paromomycin-concentration in lg/mL.

S. N. Suryawanshi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3979–3982
3981
respectively against L. donovani/hamster model at 50 mg/
kg  5 days dose when administered by intraperitoneal (ip) route.
The efficacy of most active compound 7b was also evaluated by
oral route and showed 64% inhibition in parasite growth at same
dose regimen. There were no toxic symptoms observed during
treatment. The efficacies of these compounds were more or less
similar to SSG and superior to paromomycin. Compound with ani-
lino substitution, 7b showed 83% inhibition, while in case of benzyl
amino substitution, the compound 7c was found to be ineffective
indicating that introduction of methylene group made the com-
pound inactive. Similar inhibition was reported in the case of
cyclohexylamino substitution 7f. Among the halogen substituted
anilines compound, 7d having chlorine at para position of phenyl
ring showed only 21% inhibition of L. donovani whereas compound,
7a with fluorine at para position showed 72% inhibition of L. dono-
vani. Hence, introduction of more electronegative halogen at para
position increased the antileishmanial potential of compound 7.
Compound, 7e having p-anisidino group showed 32% inhibition
of L. donovani. Metabolism is a major contributor of drug clearance
and it directly influences the systemic drug exposure. If metabo-
lites are observed in to systemic circulation, they may have phar-
macological and/or toxicological effects. Moreover, metabolism
also plays a role in drug efficacy by forming metabolites with spe-
cific to drug target receptors leading to synergic effects. The meta-
The determined intrinsic clearance of these probe substance was
comparable to previously reported values.18 As shown in data pre-
sented in Figure 2, compound 7b was degraded in HLM, with only
56.5% of the original amount left after 45 min incubation. The cal-
culated in vitro half-life for compound 7b was 60.5 2.7 min and
derived intrinsic clearance (CLi) was 14.3
somal protein in pooled hamster liver microsomes.
l
L/min  mg of micro-
After incubation of compound 7b with NADPH, three prominent
peaks (M1, M2 and M3) were detectable at 4.8, 6.5 and 7.0 min.
The M1, M2 and M3 were observed at 389 nm (kmax of compound
7b). These peaks were not observed in the control samples (Fig. 3).
M1, M2 and M3 were completely undetectable on incubation with-
out NADPH and with 1-ABZT (CYP suicidal inhibitor). Hence, it was
observed that compound 7b exhibited moderate metabolic stabil-
ity with three metabolites (M1, M2 and M3). These metabolites
were found to be formed by CYP enzyme. Detection of M1, M2
and M3 will be a gateway for prospective metabolic studies involv-
ing structural identification of metabolites (in vitro/in vivo), its
efficacy and pharmacokinetic evaluation.
These results clearly indicate that synthesized aryl S,N-ketene
acetals reported herein, are promising compounds and provide
useful model for further structural and biological optimization.
The study opens up the possibility of advancing this new class of
compounds as novel antileishmanial agents.
bolic stability of most active compound 7b at 25 lM was evaluated
in pooled hamster liver microsomes (HLM). Testosterone was em-
ployed as control compound to evaluate the metabolic stability.
Acknowledgments
The authors are thankful to the Division of Sophisticated and
Analytical Instrument Facility (SAIF), CSIR-CDRI for providing spec-
tral and elemental analysis data. S.K. is also grateful to CSIR, New
Delhi for awarding Senior Research Fellowship. The transgenic L.
donovani promastigotes were originally procured from Dr. Neena
Goyal, Division of Biochemistry, CSIR-CDRI, Lucknow, India.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.04.025.
References and notes
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Figure 3. Representative HPLC-PDA chromatograms of (a) compound 7b incubated
without NADPH at 389 nm. (b) Incubated compound 7b with NADPH at 10 min,
metabolites (M1, M2 and M3), and (c) compound 7b incubated with at 60 min,
metabolites (M1, M2 and M3).

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S. N. Suryawanshi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3979–3982
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amastigotes. The test compounds in appropriate concentration (0.62–40
were added and plates were incubated in CO2 incubator for 72 h. After
incubation, the compound-containing medium was aspirated and 50 L PBS
was added in each well and mixed with an equal volume of steady GloÒ
reagent. After gentle shaking for 1–2 min, the reading was taken in
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lM)
l
a
20. Cytotoxicity assay: the cytotoxicity of compounds was determined by
following the method of Mosmann (1983). The exponentially growing
monkey kidney fibroblast cells (vero cell line) were incubated with test
compounds for 72 h and MTT was used as reagent for detection of viable cells.
Fifty percent cytotoxic concentration (CC50) values were estimated as
19. In vitro antileishmanial assays: The in vitro antileishmanial activity of these
compounds against the luciferase-transfected promastigotes of the L. donovani
was assessed as described previously by Pandey et al. (2007). Briefly,
described by Huber and Koella (1993). Mosmann, T. J. Immunol. Methods
1983, 65, 55; Huber, W.; Koella, J. C. Acta Trop. 1993, 55, 257.
21. In vivo antileishmanial assay: the in vivo antileishmanial activity was carried
out in golden hamsters infected with L. donovani as described by Kumar et al.
(2012). Briefly, golden hamsters (inbred strain) of either sex weighing 40–
45 g were infected intracardiacally with 1 Â 107 amastigotes per animal.
After establishment of infection, drug treatment (50 mg/kg) by either
(intraperitoneal) ip or per oral (po) route was initiated for five consecutive
days. Sodium stibogluconate (SSG) and paromomycin are used as reference
promastigotes (5 Â 105/100
l
L) were seeded in 96-well plates and incubated
L of
with compounds at 40 M concentration for 72 h. After incubation, 50
l
l
promastigote suspension from each well was mixed with an equal volume of
steady GloÒ reagent (Promega) and luminescence was measured by
luminometer. The values are expressed as relative luminescence units (RLU).
Data were transformed into graphical program (Excel) and the inhibition of
parasitic growth is determined by comparison of the luciferase activity of drug
treated parasites with that of untreated controls. In order to assess the activity
of compounds against amastigote stage of the parasite, the mouse macrophage
cell line (J-774A.1) infected with luciferase-transfected promastigotes was
drugs. Post-treatment biopsies were done on day
7 after the last drug
administration and amastigote counts are assessed by Giemsa staining.
Intensity of infection in both, treated and untreated animals, and also the
initial count in treated animals was compared and the efficacy was
expressed in terms of percent inhibition (PI). Kumar, S.; Tiwari, A.;
Suryawanshi, S. N.; Mittal, M.; Vishwakarma, P.; Gupta, S. Bioorg. Med.
Chem. Lett. 2012, 22, 6728.
used. Briefly, cells (4 Â 103/100
incubation in CO2 incubator, cells were infected with promastigotes (4 Â 104/
100 L). Promastigotes phagocytised by the macrophage and transformed in to
lL) were seeded in 96-well plates. After 24 h
l