Chemotherapy of leishmaniasis. Part XII: Design, synthesis and bioevaluation of novel triazole integrated phenyl heteroterpenoids as antileishmanial agents

Article abstract of DOI:10.1016/j.bmcl.2013.03.055

A novel series of triazole integrated phenyl heteroterpenoids have been synthesized and screened for their in vitro activity against intracellular amastigote form of Leishmania donovani. Among all tested compounds, compound 3a was found to be the most active with IC₅₀ 6.4 μM and better selectivity index (SI) 18 as compared to reference drugs, miltefosine and miconazole. When evaluated in vivo in L. donovani/hamster model, 3a has exhibited 79 ± 11% inhibition of parasite multiplication at 50 mg kg ^-1 × 5 days on day 7 post treatment.

Full text of DOI:10.1016/j.bmcl.2013.03.055

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2925–2928
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chemotherapy of leishmaniasis. Part XII: Design, synthesis and
bioevaluation of novel triazole integrated phenyl heteroterpenoids
as antileishmanial agents q
a
a
b
S. N. Suryawanshi a, , Avinash Tiwari , Santosh Kumar , Rahul Shivahare
⇑
Monika Mittal b, Padam Kant c, 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
c Department of Chemistry, University of Lucknow, 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:
Available online 26 March 2013
A novel series of triazole integrated phenyl heteroterpenoids have been synthesized and screened for
their in vitro activity against intracellular amastigote form of Leishmania donovani. Among all tested com-
pounds, compound 3a was found to be the most active with IC50 6.4 lM and better selectivity index (SI)
Keywords:
Triazole
Pyrazoline
Leishmania donovani
In vivo trial
Hamster
18 as compared to reference drugs, miltefosine and miconazole. When evaluated in vivo in L. donovani/
hamster model, 3a has exhibited 79 11% inhibition of parasite multiplication at 50 mg kgÀ1 Â 5 days on
day 7 post treatment.
Ó 2013 Elsevier Ltd. All rights reserved.
Leishmaniasis is a family of parasitic diseases with extensive
morbidity and mortality in 88 tropical and subtropical countries.1
These parasitic infections are caused by an obligate intracellular
protozoan parasite belonging to the genus Leishmania. Leishmani-
asis manifests mainly in three clinical forms: cutaneous leishman-
iasis (CL), mucocutaneous leishmaniasis (MCL) and visceral
leishmaniasis (VL). VL is generally lethal if left untreated. The situ-
ation has become complicated with the co-infection of AIDS with
leishmaniasis. According to the World Health Organisation, leish-
maniasis currently affects 12 million people worldwide and there
around 2 million new cases per year with growing tendency. More-
over, it is estimated that approximately 350 million people live at
risk of Leishmania infection.1 With no immediate prospect for vac-
cines, chemotherapy is the only way to cure the patients suffering
with this parasitic infection.
At present, only drugs in practice include pentavalent antimoni-
als, paromomycin, amphotericin-B and miltefosine. High toxicity
and increasing resistance to the current chemotherapeutic regi-
mens have further complicated the situation in VL endemic regions
of the world.2 Since the chemotherapy against leishmaniasis is still
inefficient; there is an urgent need for development of new thera-
peutic agents from natural sources. Chalcones represent an impor-
tant class of naturally occurring small molecules, biologically
active against leishmaniasis.3 Their recognized synthetic utility in
the preparation of pharmacologically-interesting heterocyclic sys-
tems like pyrazoline is of great importance as these pyrazolines
have been recognized owing to their pharmacological activities,
which includes antitumor,4 antiinflammatory,5 antiinfective,6 anti-
microbial7 as well as antileishmanial activity.8
Sterol biosynthesis is a very important biosynthetic pathway in
protozoan parasites. Ergosterol is one of the most important sterols
and cell membrane components of protozoan parasites and fungi.
Based on this sterol biosynthetic pathway, most of the azoles
(miconazole, ketoconazole, fluconazole and itraconazole) (Fig. 1)
were designed as antifungal drugs and their antileishmanial poten-
tial9 was reported in 1981 and later years.10,11 Antifungal agents
containing triazole ring inhibit the Leishmania amastigotes growth
by preventing the 14a-demethylation of lanosterol and effectively
block synthesis of ergosterol.12 Taking the above reports in consid-
eration and in continuation of our efforts to synthesize novel
antileishmanial agents,13a,b we designed some novel triazole inte-
grated phenyl heteroterpenoids and evaluated for their in vitro
and in vivo antileishmanial activity.
The synthetic routes followed for the preparation of the target
compounds have been outlined in Scheme 1. In the first step, tria-
zole integrated chalcones 2 and 5 were synthesized by stirring
commercially available b and
a ionone respectively with 4-(1H-
1,2,4-triazol-1-yl)benzaldehyde at room temperature via Claisen
Schmidt condensation reactions catalysed by phase transfer
catalyst, cetyltrimethyl ammonium bromide (CTABr), in aqueous
q
CSIR–CDRI Communication no.: 8443.
Corresponding author. Tel.: +91 2212411 18; fax: +91 0522 2223405/938/504.
E-mail address: shivajins@yahoo.co.in (S.N. Suryawanshi).
⇑
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.055

2926
S. N. Suryawanshi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2925–2928
R
7'
2'
NH2
N
O
5
1
2''
H
N
N
1''
1'
6'
3''
3'
3
4
2
2'''
N
1'''
4'
N
9'
6''
N
N
4'' N
3'''
5'
5''
R= H, F
(ii)
8'
N
5''
4'''
2
R
O
(a) R = H
(b) R = F
3
O
(i)
1
N
N
N
4-(1H-1,2,4-triazol-1-yl)
benzaldehyde
O
(i)
4
R
NH2
O
N
H
N
N
N
N
N
N
N
R= H, F
(ii)
N
5
R
(a) R = H
(b) R = F
6
Scheme 1. Synthesis of triazole integrated phenyl heteroterpenoids. Reagents and conditions: (i) cetyltrimethyl ammonium bromide (CTABr), NaOH, H2O, rt, 24 h; (ii) EtOH,
reflux, 8 h.
O
H
N
N
N
N
O
N
N
N
N
O
O
Cl
Itraconazole
Cl
N
O
N
N
N
Cl
Cl
N
O
Cl
OH
N
N
O
N
O
N
O
N
H
N
Cl
F
F
N
Cl
Fluconazole
Ketoconazole
Cl
Miconazole
Figure 1. Chemical structures of azole containing drugs.

S. N. Suryawanshi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2925–2928
2927
Figure 2. In vivo efficacy of compound 3a and 6a against L. donovani/hamster model.
solution of NaOH. In the second step, chalcones (2, 5) were refluxed
with phenyl hydrazine and 4-fluorophenyl hydrazine hydrochlo-
ride in ethanol to furnish 1,3,5-trisubstituted pyrazolines (3a, 3b,
6a and 6b) in quantitative yield. The structures of the synthesized
compounds were determined by means of IR, NMR and mass spec-
trometery. For example IR spectrum of triazole integrated chalcone
2 (intermediate of the most active compound, 3a) revealed a strong
band at 1643 cmÀ1, along with resonance in its 13C NMR at d 188.6
indicating the presence of a keto carbonyl function. Its 1H NMR re-
vealed, besides aromatic proton resonances in the region d 7.67–
6a,
a
ionone based triazole integrated phenyl pyrazoline, were
M and SI value
found to show significant activity with IC50 6.4, 9.2
l
18, 32, respectively. When compared with standard antileishma-
nials, miltefosine and miconazole, the compound 3a showed com-
parable in vitro activity to miltefosine and miconazole. It is
interesting to note that compound 3a and 6a, synthesized using
phenyl hydrazine have shown increased activity as compared to
the compound 3b and 6b, synthesized using 4-flurophenyl hydra-
zine hydrochloride. This decrease in activity of
a, b ionone based
triazole integrated phenyl pyrazoline (3a, 6a) on para fluoro substi-
tution indicates that replacement of para-hydrogen of phenyl ring
with more electronegative fluorine atom diminishes the antileish-
manial activity.
8.56, two b-protons of
up as doublets at 7.61 and 7.48 with trans olefinic-H coupling con-
stant J = 16 Hz and two -protons appeared as doublets at d 6.96
a,b-unsaturated carbonyl moiety showed
a
and 6.42 (J = 16 Hz). Chemical characterization of compounds 3a
and 6a is given in Ref. 14. Spectral data for all the synthesized com-
pounds are given in the Supplementary data.
The leishmanicidal activity of triazole integrated phenyl hete-
roterpenoids 2, 3a–b, 5, 6a and 6b was evaluated against L. dono-
vani intracellular amastigotes and results have been presented in
Table 1. All the synthesized compounds have shown more than
Based on the in vitro screening profile, compound 3a and 6a
were selected for in vivo trial in L. donovani/golden hamster mod-
el.17 These compounds were evaluated at the dose of 50 mg/
kg  5 days by intraperitoneal (ip) route. Compound 6a has shown
58 08% inhibition of parasite multiplication while compound 3a
was found to show significant parasitic inhibition at same dose
regimen. The compound 3a has shown 79 11% inhibition of par-
asitic growth which is more or less similar to first line drug, So-
dium stibogluconate (SSG) and inferior to miltefosine. In
summary, we have found compound 3a as a promising hit mole-
cule for antileishmanial chemotherapy and it was not toxic to
mammalian cells at parasite inhibitory concentration.
90% inhibition at 40
mania parasite in vitro.15,16Compound 2, b ionone based triazole
integrated chalcone, and compound 6b, ionone based triazole
lM concentration against intracellular leish-
a
integrated 4-fluorophenyl pyrazoline, showed marginal in vitro
activity. Compound 3b, b ionone based triazole integrated 4-fluo-
rophenyl pyrazoline, and compound 5,
integrated chalcone, exhibited good antileishmanial activity with
IC50 12.4 and 11.6 M respectively. However, compound 3a, b io-
a
ionone based triazole
All the synthesized compounds were also checked for compli-
ance to the Lipinski rule of five, and the results are summarized
in Table 2. According to this rule a molecule likely to be developed
as an orally active drug candidate should show no more than one
violation of the following four criteria: logP (octanolÀwater parti-
tion coefficient) 65, molecular weight 6500, number of hydrogen
bond acceptors 610 and number of hydrogen bond donors 65.
Molecular properties of synthesized compounds were calculated
l
none based triazole integrated phenyl pyrazoline, and compound
Table 1
In vitro antileishmanial activity of triazole integrated phenyl heteroterpenoids
against intracellular amastigotes and their cytotoxicity
Compound no. In vitro antiamastigote activity Cytotoxicity Selectivity
CC50
(
lM)
index (SI)
% Inhibition at
40
IC50 (lM)
Table 2
l
M
Molinspiration calculation of molecular properties for the Lipinski rule
2
3a
3b
5
6a
6b
100
100
98.65
98.18
98.03
90.54
15.3 2.2
6.4 1.2
12.4 2.0
11.6 1.6
9.2 1.7
16.9 3.1
8.6 0.4
5.4 1.5
72.4 5.8
112.4 10.9 18
58.6 4.9
38.5 5.1
296 15.9 32
72.2 8.4
54.7 6.8
37.4 4.1
5
Compound
nViol MW
miLogP nON nOHNH natoms nrotb
5
3
Acceptable
range
2
3a
3b
5b
6a
6b
61
6500
65
610 65
—
—
0
1
1
0
1
1
347.462
4.22
4
5
5
4
5
5
0
0
0
0
0
0
26.0
33.0
34.0
26.0
33.0
34.0
5
5
5
5
5
5
4
6
7
437.591
455.581
347.462
437.591
455.581
6.051
6.215
3.9
5.731
5.895
Miltefosine
Miconazole
100
100
IC50 and CC50 values are the average (mean SD) of two independent experiments.
The selectivity index (SI) is defined as the ratio of CC50 on Vero cells to IC50 on L.
donovani intramacrophagic amastigotes.
IC50, half maximum inhibitory concentration; CC50, half maximum cytotoxic con-
centration; SD, standard deviation.
nViol, no. of violations; MW, molecular weight; miLogP, molinspiration predicted
LogP; nON, no. of hydrogen bond acceptors; nOHNH, no. of hydrogen bond donors;
natoms, no. of atoms; nrotb, no. of rotatable bond.

2928
S. N. Suryawanshi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2925–2928
14. Chemical characterization of compounds 3a and 6a: Chemical characterization of
all the compounds including compounds 3a and 6a was done through spectral
data (IR, 1H NMR, 13C NMR, mass) and elemental analysis. IR spectrum of the
by www.molinspiration.com software, and it was found that al-
most all the synthesized compounds followed the above criteria
(Table 2). Therefore, these triazole integrated phenyl heteroterpe-
noids have a good potential for eventual development as oral
agents and can be potentially active drug candidate.
This finding indicates that triazole moiety with chalcone and
pyrazoline scaffold should be investigated for the development of
highly selective leishmanicidal agent.
best compound of the series (3a) showed a C@N stretching band at 1597 cmÀ1
.
In its 1H NMR spectra, three protons of the pyrazoline ring were seen as
doublet of doublets at 2.86 ppm (J = 16, 7 Hz), 3.63 ppm (J = 16, 12 Hz) and 5.16
(J = 12, 7 Hz). Two protons of the triazole ring were appeared downfield at d
8.02 and d 8.45. This downfild shift is due to the presence of three electron
withdrawing nitrogen atoms in the triazole ring. Simillarly compound 6a
showed a C@N stretching band at 1601 cmÀ1. In its 1H NMR spectra, three
protons of the pyrazoline ring were seen at 2.88 ppm (J = 17, 8 Hz), 3.64 ppm
(J = 17, 12 Hz) and 5.20 (J = 12, 8 Hz) and triazole ring’s protons were appeared
at d 8.08 and 8.51. Finally, mass spectra of compounds 3a and 6a showed well
defined [M+1] ions.
Acknowledgments
15. In vitro antileishmanial assay: In order to assess the activity of compounds
against amastigote stage of the parasite, the murine macrophage cell line (J-
774A.1) infected with luciferase-transfected promastigotes was used. Briefly,
The authors are thankful to the division of Sophisticated Analyt-
ical Instrument Facility (SAIF), CSIR–CDRI for providing spectral
and elemental analysis data. Financial support from U.G.C. is grate-
fully acknowledged. Technical assistance by Mrs. Manju is grate-
fully acknowledged. The transgenic L. donovani promastigotes
were originally procured from Dr. Neena Goyal, Division of Bio-
chemistry, CSIR–CDRI, Lucknow, India.
cells (4 Â 103/100
lL/well) were seeded in 96-well plates. After 24 h
incubation in CO2 incubator, cells were infected with promastigotes (4 Â 104/
100 lL/well). Promastigotes phagocytised by the macrophage and transformed
into amastigotes. The test compounds in appropriate concentration (0.62–
40 M) were added and plates were incubated in CO2 incubator for 72 h. At the
end of the incubation, the supernatants were removed and 50 L PBS was
l
l
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 a luminometer
(Pandey et al., 2007). 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. (Pandey, S.;
Suryawanshi, S. N.; Nishi; Goyal, N.; Gupta, S. Eur. J. Med. Chem. 2007, 42, 669).
16. Cytotoxicity assay: The cytotoxicity of compounds was determined by following
the method of Mossman (1983) with some modifications. The 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 described by Huber
and Koella (1993). (Mosmann, T. J. Immunol. Methods 1983, 65, 55). (Huber, W.;
Koella, J. C. Acta Trop. 1993, 55, 257).
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.
03.055.
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17. 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. Pre-
treatment spleen biopsies were performed after 15–20 days to assess the
degree of infection. The animals with +1 infection (5–15 amastigotes/100 liver
cell nuclei) will be included in the in vivo trials. After establishment of
infection, drug treatment by either intraperitoneal (ip) or per oral (p.o.) route
was initiated for
5 consecutive days. Sodium stibogluconate (SSG) and
miltefosine are used as reference 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 per cent inhibition (PI) (Kumar, S.; Tiwari,
A.; Suryawanshi, S. N.; Mittal, M.; Vishwakarma, P.; Gupta, S. Bioorg. Med.
Chem. Lett. 2012, 22, 6728).
In vivo result is presented as means standard deviations (SD) for two
experiments (Fig. 2), and analysis of data was carried out by one-way ANOVA
using Graphpad Prism (version 5.0).