Chemotherapy of leishmaniasis part X: Synthesis and bioevaluation of novel terpenyl heterocycles

Article abstract of DOI:10.1016/j.bmcl.2012.10.110

Some novel α and β ionone based chalcones and their dihydropyrazolidines/pyrazolidines have been synthesized and evaluated for their in vitro and in vivo antileishmanial activities against Leishmania donovani. Amongest all, one compound (4d) exhibited sig

Full text of DOI:10.1016/j.bmcl.2012.10.110

Bioorganic & Medicinal Chemistry Letters 23 (2013) 248–251
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chemotherapy of leishmaniasis part X: Synthesis and bioevaluation of novel
terpenyl heterocycles q
Avinash Tiwari a, Santosh Kumar a, S. N. Suryawanshi a, , Monika Mittal b, Preeti Vishwakarma b,
⇑
Suman Gupta b
a Division of Medicinal Chemistry, CSIR, Central Drug Research Institute, Lucknow 226 001, India
b Division of Parasitology, CSIR, Central Drug Research Institute, 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:
Some novel
a and b ionone based chalcones and their dihydropyrazolidines/pyrazolidines have been syn-
Received 10 September 2012
Revised 22 October 2012
Accepted 26 October 2012
Available online 2 November 2012
thesized and evaluated for their in vitro and in vivo antileishmanial activities against Leishmania dono-
vani. Amongest all, one compound (4d) exhibited significant in vitro activity against intracellular
amastigotes of Leishmania donovani with IC50 values of 7.49 lM and was found promising as compared
to reference drug, miltefosine. On the basis of good Selectivity Index (S.I.), the compound was further
tested for its in vivo response against Leishmania donovani/hamster model and has shown significant
inhibition of parasite multiplication (81%). The present study has helped us in identifying a new lead that
could be exploited as a potential antileishmanial agent.
Keywords:
Chalcones
Pyrazolidines
Leishmania donovani
In vitro testing
In vivo trial
Hamster
Ó 2012 Elsevier Ltd. All rights reserved.
Leishmaniasis is a family of parasitic diseases that affect about
12 million people in tropical and subtropical areas in the form of
three clinical expressions: visceral leishmaniasis, which is fatal in
the absence of treatment; muco-cutaneous leishmaniasis and cuta-
neous leishmaniasis, which is often self curing. Classical drugs such
as antimonials (Pentostam and Glucantime) are toxic and drug
resistance is increasing dangerously in the field.1 A liposomal
amphotericin B formulation (AmBisome) less toxic than amphoter-
icin B deoxycholate is gradually becoming the first-line therapy,
especially in immunocompromised patients, but this drug must
be administered by a parenteral route.2 Miltefosine (Impavido)
was the first drug registered against visceral leishmaniasis in the
last decade; however, its toxicity and the appearance of drug resis-
tance justify the search for new chemical series in order to find an
orally safe and active drug.3 Currently, efforts are being made to
search for new molecules from the natural sources and in this en-
deavor diarylheptanoids,4 oxygenated abietanes,5 diterpene qui-
nones6,7 are showing promise as new lead molecules. Rationally
designed heterocyclic ionone like molecules8 and some novel
terpenyl 2,4-diamino pyrimidines9 are showing promising antimi-
crobial and dihydrofolate reductase inhibitory activities. 2,4-dia-
minopyrimidines10 and some de novo-designed molecules11 are
also giving further inputs in the leishmanial dihydrofolatereduc-
tase activity. In continuation of our studies on terpenylpyrimidines
as novel antileishmanial agents,12 we designed some novel dihyd-
ropyrazolidines/pyrazolidines, having added aryl substitution and
evaluated for their in vitro and in vivo antileishmanial activity
and the results are reported in this communication.
The synthesis of compounds (4a–j) and (8a–e) followed the
general pathway outlined in Schemes 1 and 2. They were prepared
in two steps. Firstly, the chalcones were obtained by direct conden-
sation between the substituted aromatic aldehydes and b-ionone,
a
-ionone respectively, using phase transfer catalyzed conditions.13
Secondly, reaction of different chalcones with phenyl hydrazine in
refluxing ethanol leads to the formation of dihydropyrazolidine
derivatives. Further, compounds (8a–e) were aromatized into
compounds (9a–e) using Ag2O in refluxing ethanol. The reaction
of b-ionone with substituted benzaldehydes was very facile and
furnished chalcones in good to excellent yield.
The reaction of phenyl hydrazine with chalcones (3a–j) and
(7a–e) was not only facile but it was also regiospecific in manner.
The reaction of chalcone 3a with phenyl hydrazine in ethanol fur-
nished dihydropyrazolidine 4a in 40% yield as a crystalline solid
melting at 138–140 °C. The structure was assigned on the basis
of 1H and 13C NMR spectra.
The 1H NMR spectrum of 4a displayed doublet of doublets at
2.87 ppm (J = 17, 7 Hz, 1H) & 3.85 ppm (J = 17, 12 Hz, 1H) for two
geminal protons of the dihydropyrazolidine ring and doublet of
q
CSIR-CDRI Communication No: 172/2011/SNS.
⇑
Corresponding author. Tel.: +91 2212411 18; fax: +91 0522 2223405x938x504.
E-mail address: shivajins@yahoo.co.in (S.N. Suryawanshi).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.110

A. Tiwari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 248–251
249
R2
3''
CHO
3'
R1
R2
7'
5
R1 2''
2
R3
4''
5''
R4
2'
1'
4'
(i)
(ii)
4
+
3
5' 6'
8'
R4
1''
6''
1
R3
2
9'
O
O
3
1
R2
R1
R3
R4
4
3
5
N
N 1
2
4
3, 4 R1
R2
H
H
NO2
H
R3
H
NO2
R4
H
H
H
a
b
c
d
e
f
NO2
H
H
H
H
Cl
H
H
H
H
H
OCH3 OCH3
OCH3 OCH3 OCH3
H
H
Cl
H
H
H
Cl
H
F
OBn
H
H
H
H
H
g
h
i
j
Scheme 1. Reagents and conditions: (i) Cetyl trimethyl ammonium bromide (CTABr), NaOH, H2O, rt, 24 h; (ii) PhNHNH2, EtOH, reflux, 8 h.
doublets at 5.69 (J = 12, 7 Hz, 1H) for H-5 proton (vicinal to the
geminal protons) and it established the assigned structure 4a.
The compound 4d was synthesized by the reaction of chalcone
3d with phenyl hydrazine in ethanol (reflux, 8 h). The compound
4d, Yellow crystalline solid which melted at 90–93 °C. The com-
pound 4d was characterized by IR, NMR and mass spectrum. The
IR spectrum showed a C@N stretching band at 1597 cmꢀ1. The
1H NMR spectrum of 4d displayed three doublet of doublets at
2.85 ppm (J = 17, 7 Hz, 1H), 3.55 ppm (J = 17, 12 Hz, 1H) and 5.01
(J = 12, 7 Hz, 1H) for two geminal protons of dihydropyrazolidine
ring and one for the proton vicinal to geminal protons respectively.
Analysis of 13C, DEPT-135 and mass spectra provided the final
structural elucidation of compound 4d. Cyclization in all the syn-
thesized heterocyclic compounds took place near the aromatic ring
rather than near the ionone ring. The presence of doublet near 2.25
(d value) in 1H NMR spectrum of compounds (9a–e) indicated that
the cyclization took place near the aromatic ring. If cyclization had
taken place near the ionone ring then a singlet would have been
obtained in place of doublet in 1H NMR spectrum of compounds
(9a–e). Compounds (4b–j) and (8a–e) were synthesized in good
yields under identical reaction conditions as used for the synthesis
of compound 4a. Few dihydropyrazolidines (8a–e) were aroma-
tized using Ag2O in refluxing ethanol and their structures were as-
signed by 1H NMR and 13C NMR spectra. We used the same
reaction conditions, as used for compounds (8a–e), for aromatiza-
tion of compounds (4a–j) but we didn’t find any aromatized
product.
100 lL/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 ꢁ 105/100
lL/well). Promastigotes in-
vade the macrophage and are transformed into amastigotes. The
test compounds were added at two fold dilutions up to 7 points
in complete medium starting from 40 lM conc. after replacing
the previous medium and the plates were incubated at 37 °C in a
CO2 incubator for 72 h. At the end of the incubation, the superna-
tants were removed and 50
lL PBS was added in each well and
mixed with an equal volume of Steady Glo reagent. After gentle
shaking for 1–2 min, the reading was taken in a luminometer.14–16
The values are expressed as relative luminescence units (RLU). IC50
of antileishmanial activity was calculated by nonlinear regression
analysis of the concentration response curve using the four param-
eter Hill equations.
The cell viability was determined using the MTT assay.17 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 7 points in complete med-
ium starting from 400 lM concentration, and were incubated 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
were initially dissolved in DMSO and further diluted with fresh
complete medium. After incubation, 25 lml of MTT reagent
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/
(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 incu-
bation period, the supernatant were removed and 150
lL of pure
DMSO was added to each well. After 15 min. of shaking the

250
A. Tiwari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 248–251
R2
Table 1
CHO
Antileishmanial activity of dihydropyrazolidines/pyrazolidines against L. donovani
R1
R3
R4
R1
R2
(i)
Compd.
No.
In vitro
antiamastigote
activity IC50
CC50
M)
Selectivity
index (S.I.)
CC50/IC50
In vivo activity (%
inhibiton) (50 mg/
Kg ꢁ 5 days, i.p.
dose)
+
(
l
R4
O
R3
6
O
(lM)
7
4a
4b
4c
4d
4e
4f
4g
4h
4i
>20
>40
16.95
7.49
>20
>20
>20
>40
>40
>40
>20
>40
>40
>20
>20
>40
>40
>40
>20
>40
—
—
—
—
—
—
5
(iii)
279.49 16.48
220.66 29.46
7.13 8.95
80.80 11.91
—
—
—
—
—
—
—
—
—
—
—
—
—
—
(ii)
R2
R2
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
3.23
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
0.26
R1
R3
R4
R1
5
R3
R4
4
4
3
3
5
1
N
N
N
N
2
2
1
4j
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
9
8
7, 8 and 9 R1
R2
H
H
H
H
R3
H
R4
H
H
H
A
B
C
D
E
NO2
H
—
—
N O2
F
Miltefosine 12.50
95.28 2.49
H
H
O C H3 OCH3
OCH3 OCH3
H
OCH3
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.
Scheme 2. Reagents and conditions: (i) Cetyl trimethyl ammonium bromide
(CTABr), NaOH, H2O, rt, 24 h; (ii) PhNHNH2, EtOH, reflux, 8 h; (iii) PhNHNH2,
Ag2O, EtOH, reflux, 18 h.
The leishmanicidal activity of aryl substituted dihydropyrazoli-
dines/pyrazolidines (4a–j), (8a–e) and (9a–e) was evaluated
against L. donovani intracellular amastigotes and results have been
presented in Table 1. The dihydropyrazolidines (4a–j) prepared
from b ionone showed marginal to good in vitro antileishmanial
activity. Among all the compounds, 4d has shown promising antia-
mastigote activity with IC50 of 7.5 lM and selectivity index of 29.5.
Rest of the compounds have shown marginal activity. The in vitro
antileishmanial response of this compound was better than the ref-
erence drug, miltefosine (IC50 = 12.5 lM, S.I. = 0.26). We found very
readings were recorded as absorbance at 544 nm on a micro plate
reader. The cytotoxic effect was 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 values were estimated as described by Huber and
Koella.18 The selectivity index (SI) for each compound was calcu-
lated as ratio between, cytotoxicity (CC50) and activity (IC50
against Leishmania amastigotes.
)
The in vivo leishmanicidal activity was determined in golden
hamsters (Mesocricetus auratus) 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 al19 as modified by Bhatnagar et al20 and Gupta et al21
was used for in vivo evaluation. Golden hamsters (Inbred strain)
of either sex weighing 40–45 g were infected intracardiacally with
1 ꢁ 107 amastigotes 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 (i.p.) route was initiated after 2 days
of biopsy and continued for 5 consecutive days. Post-treatment
biopsies were done on day 7 of the last drug administration and
amastigote counts are assessed by Giemsa staining. Intensity of
infection in both, treated and untreated animals, and also the ini-
tial count in treated animals was compared and the efficacy was
expressed in terms of percentage inhibition (PI) using the following
formula:
little correlation between type of substitution on the aromatic ring
and the in vitro biological activity. The dihydropyrazolidines (8a–
e) prepared from
a ionone and their aromatized compounds (9a–
e) showed only marginal in vitro activity.
The compound 4d was also selected for in vivo efficacy evalua-
tion against L. donovani/hamster model at the intraperitoneal (i.p.)
dose of 50 mg kgꢀ1 ꢁ 5 days. The compound exhibited significant
in vivo response (81% inhibition in parasite multiplication).
In summary, synthesis and biological evaluation of these ter-
penyl heterocycles led us to discover compound 4d as good anti-
leishmanial agent which is more active than miltefosine in vitro.
Selectivity index of compound 4d is 113.3 fold higher than miltefo-
sine. These investigations revealed that these terpenyl heterocycles
can serve as prototype for development of more efficacious antile-
ishmanial agents.
Acknowledgments
The authors are thankful to the division of Sophisticated Analyt-
ical Instrument Facility (SAIF), CDRI for providing spectral and ele-
mental analysis data. Financial support from U.G.C. is gratefully
acknowledged. Technical assistance by Mrs. Manju is gratefully
acknowledged. The transgenic L. donovani promastigotes were pro-
cured from Neena Goyal, Division of Biochemistry, CSIR-Central
Drug Research Institute, Lucknow, India.
PI ¼ 100 ꢀ ½ANAT ꢁ 100=ðINAT ꢁ TIUCÞꢂ

A. Tiwari et al. / Bioorg. Med. Chem. Lett. 23 (2013) 248–251
251
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.10.110.
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