Synthesis and biological evaluation of chalcones as potential antileishmanial agents

Article abstract of DOI:10.1016/j.ejmech.2014.05.034

Antileishmanial activities of thirty-five synthetic chalcones have been examined. Among them, ten compounds (4, 6, 16, 22, 23, 24, 25, 29, 35 and 37) exhibited potent in vitro activity (IC₅₀ range from 1.70 to 8 μM) against extracellular promastigotes and intracellular amastigotes form of Leishmania donovani. Two promising compounds 22 and 37 were tested in vivo in L. donovani/hamster model. Chalcone 37 showed 83.32% parasite inhibition at a dose of 50 mg/kg for 10 days whereas, 75.89% parasite inhibition at 100 mg/kg dose for 5 days by intraperitoneal route at day 7 post-treatment.

Full text of DOI:10.1016/j.ejmech.2014.05.034

Accepted Manuscript
Synthesis and Biological Evaluation of Chalcones as Potential Antileishmanial Agents
Shweta Gupta , Rahul Shivahare , Venkateswarlu Korthikunta , Rohit Singh , Suman
Gupta , Narender Tadigoppula
PII:
S0223-5234(14)00448-6
10.1016/j.ejmech.2014.05.034
EJMECH 6990
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 December 2013
Revised Date: 9 May 2014
Accepted Date: 11 May 2014
Please cite this article as: S. Gupta, R. Shivahare, V. Korthikunta, R. Singh, S. Gupta, N. Tadigoppula,
Synthesis and Biological Evaluation of Chalcones as Potential Antileishmanial Agents, European Journal
of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.05.034.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis and Biological Evaluation of Chalcones as Potential
Antileishmanial Agents
1

ACCEPTED MANUSCRIPT
Synthesis and Biological Evaluation of Chalcones as Potential
Antileishmanial Agents
a
b
a
a
Shweta Gupta , Rahul Shivahare , Venkateswarlu Korthikunta , Rohit Singh ,
b
a,
Suman Gupta and Narender Tadigoppula *
a
b
Division of Medicinal and Process Chemistry, Division of Parasitology,
CSIR-Central Drug Research Institute, Lucknow-226 031, India
*
Corresponding author:
Tel.: +91 522 2612411; fax: +91 522 2623405.
E-mail: t_narendra@cdri.res.in or tnarender@rediffmail.com (T. Narender)
Abstract
Antileishmanial activities of thirty five synthetic chalcones have been examined.
Among them, ten compounds (4, 6, 16, 22, 23, 24, 25, 29, 35 and 37) exhibited potent in vitro
activity (IC range from 1.70 to 8 µM) against extracellular promastigotes and intracellular
5
0
amastigotes form of Leishmania donovani. Two promising compounds 22 and 37 were tested
in vivo in L. donovani / hamster model. Chalcone 37 showed 83.32% parasite inhibition at a
dose of 50 mg/kg for 10 days whereas, 75.89% parasite inhibition at 100 mg/kg dose for 5
days by intraperitoneal route at day 7 post-treatment.
Keywords: Chromenochalcones; Chromenodihydrochalcones; Antileishmanial; Leishmania
donovani / Hamster model
1
. Introduction
Leishmaniasis is a group of disease transmitted by the bite of Leishmania-infected
female sand flies. It is classified as cutaneous, muco-cutaneous and visceral leishmaniasis
VL) depending on the parasite species and cellular immune system of the patient [1,2]. This
(
disease has been recognized as an increasing health problem worldwide by the World Health
Organization (WHO) [3]. Many parts of Asia and Africa are vulnerable to leishmaniasis [4].
The first line treatment options for the visceral form of leishmaniasis are limited and involve

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the administration of pentavalent antimonials (sodium stibogluconate (SSG) and meglumine
antimoniate) and amphotericin B [5]. Second line drugs include, pentamidine, paromomycin
and miltefosine, but these drugs have not experienced widespread use due to the severe
toxicities, parenteral administration and resistance issues [5]. Presently, more than 60% VL
patients in Bihar (India) are unresponsive to the antimonials [6]. Amphotericin B and its
formulations are quite effective for VL; however, these are very expensive, highly toxic and
have a longer half-life [7]. Pentamidine presents several side effects, including renal and
hepatic toxicities, pancreatitis, hypotension and cardiac abnormalities [8]. Paromomycin has
limited use for the treatment of VL [7]. Miltefosine, an orally effective drug also suffers from
nephrotoxicity, hepatotoxicity and teratogenicity [7]. So far, no vaccine has been clinically
approved for human use [9]. Therefore, there is an urgent need for the development of new,
low-cost, effective and safe drugs for the treatment of VL. The discovery of new lead
compounds for this disease is a pressing concern for global health programs. In this respect,
several synthetic or natural plant / marine product inspired compounds have shown promising
efficacy for the treatment of visceral leishmaniasis [10].
Chalcones, or 1,3-diaryl-2-propen-1-ones (Figure 1), are prominent secondary
metabolite precursors of flavonoids and isoflavonoids in plants. Natural and synthetic
chalcones are described in the literature with different pharmacological profiles, such as
antiinflammatory [11], antibacterial [12], antiviral [13], antimalarial [14], anticancer [15],
antileishmanial [16], antituberculosis [17], anti-HIV [18] and antifungal activities [19]. A
thorough assessment of structural requirements for antileishmanial activities of chalcones is
vital to develop and designing of novel drug like candidate [20]. Licochalcone A (II), is an
oxygenated chalcone (Figure 1), isolated from Chinese licorice, efficiently inhibits
proliferation of Leishmania donovani and L. major promastigotes and amastigotes in vitro by
interfering with the function of the parasite mitochondria. As a part of our drug discovery
program on antileishmanial agents from Indian medicinal plants, we have reported the
isolation of three chromenodihydrochalcones, crotaramosmin (III), crotaramin (IV) and
crotin (V) (Figure 1) from Crotalaria ramosissima [21], their synthesis and in vitro
antileishmanial activity of III–V and analogues thereof [22,23]. Our previous work
demonstrated that synthetic analogues, chromenochalcones enhanced the in vitro and in vivo
antileishmanial activity [23] over the natural products, in which the benzopyran core and
substitutions on ring A played an important role. Therefore, we enthusiastically moved to
further work on chromenochalcones and analogues thereof with the goal of enhanced

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antileishmanial activity and improved cytotoxic profiles. A series of chalcones with various
structural features were synthesized and evaluated for their in vitro antileishmanial activity.
Several of these compounds were found active in in vitro screening; few of them were further
evaluated for in vivo antileishmanial efficacy in L. donovani / hamster model. Herein, we
report a comprehensive assessment of antileishmanial activity and structure-activity
relationship (SAR) analyses of promising chalcones.
Figure 1
2
2
2
. Results and Discussion
.1. Chemistry
.1.1. Synthesis of Chromenochalcones
A series of chromenochalcones were prepared to evaluate their antileishmanial
activity. The acetyl chromene 2 was synthesized using pyridine-catalyzed condensation
between 2,4-dihydroxyacetophenone (1) and 3-methyl-but-2-enal [24]. The resultant acetyl
chromene 2 and substituted aromatic aldehydes or p-hydroxybenzene-1,3-dicarbaldehyde
[
25] were subjected to Claisen-Schmidt condensation using either aqueous KOH in ethanol
or NaH in dry THF at room temperature to furnish the corresponding chromenochalcones 3–
8 in good yields (Scheme 1, and Table 1) [23]. To improve the bioavailability of
1
compounds, the alkylated amine groups were introduced in chromenochalcone core. The
synthesis of alkylated amines containing-chromenochalcones (19–24) were accomplished by
the replacement of hydrogen from phenolic hydroxyl group of the chromenochalcone 12
(Table 1) with the alkyl part of the corresponding amines using K CO in dry acetone
2 3
(Scheme 1) [26].
Scheme 1
.1.2. Synthesis of Chromenochalcones with Hetero Atoms in Ring-A.
Chromenochalcones 25–29 which have hetero atoms in ring-A (Scheme 2) were
2
synthesized using Claisen-Schmidt condensation from acetyl chromen [24] 2 and various
heteroaryl aldehydes to afford the compounds 25–29 in good yields (Scheme 2).
Scheme 2

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2
.1.3. Synthesis of O-Prenyl/Geranyl/Farnesyl/Alkylamino Chalcones
To study the role of the alkenyl (prenyl, geranyl, farnesyl) and alkylated amino groups
on hydroxyl group of normal chalcones, O-prenyl, -geranyl, -farnesyl and alkylated amines
containing chalcones 35–42 were prepared as shown in scheme 3. The hydroxychalcones 31–
3
4 were synthesized through Claisen-Schmidt condensation of 4-hydroxy acetophenone (30)
and corresponding aromatic aldehydes. The resulting chalcones 31–34 were then smoothly
alkylated with various alkenyl/aminoalkyl halides using K CO in dry acetone under reflux to
2
3
furnish the desired O-alkenyl/alkylated amines containing chalcones 35–42 in good yields
26].
[
Scheme 3
2
.2. Biological Activity
In our efforts to develop new antiparasitic agents, we had earlier reported the in vitro
and in vivo antileishmanial activity of natural and synthetic chromenochalcones [22,23]. Our
previous work had shown that nature of substitutions on ring A and B are very crucial for the
potent antileishmanial activity. On the basis of these optimistic results, we wanted to find out
the influence of various substituents on ring A and B with the goal of synthesizing the various
synthetic chalcones with enhanced antileishmanial activity and improved cytotoxic profiles.
A series of chalcones were prepared and evaluated for antileishmanial activity against WHO
reference strain (MHOM/IN/80/Dd8) of promastigotes (expressing luciferase firefly reporter
gene) at 25 µM. The compounds which exhibited more than 70 % inhibition were screened
for intracellular amastigote form (expressing luciferase firefly reporter gene) of L. donovani
and their IC were determined. In parallel, the cytotoxicity of the compounds, which have
5
0
IC < 20 µM against amastigotes, was also tested using mammalian kidney fibroblast cells
5
0
(Vero cell line). Among these, two promising compounds were further evaluated against the
MHOM/IN/80/Dd8 strain of L. donovani in hamster model. Standard antileishmanials,
miltefosine and sodium stibogluconate (SSG) were included in this study as reference drugs.
2
.2.1. In Vitro Antileishmanial Activity of Chromenochalcones
In continuation of our work [22,23], chromanochalcones 3–18 were synthesized and
evaluated for their in vitro activity. Chalcones 3−16, which have the electron-donating groups
EDGs), electron-withdrawing groups (EWGs) and halogen groups on ring-A, appear to be
more potent against both promastigotes (70–100 % inhibition at 25 ꢀM) and amastigotes
(

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(
IC = 5.1–20 µM) with good selectivity, with the exception of 3 and 10 (IC > 20 µM).
5
0
50
While the introduction of aldehyde group as in 17 and fused hetero cycles as in 18 were also
showed poor activity against amastigotes (IC > 20 µM). It is noteworthy to mention that the
5
0
analogues, 4−16 have shown improved antileishmanial activity and selectivity (Table 1).
Table 1
2
.2.2. In Vitro Antileishmanial Activity of Alkylated amines containing-
Chromenochalcones
To improve the bioavailability of chromenochalcones, alkylated amines containing-
chromenochalcones 19–24, which have an alkylated amine substituents either on ring A and
B or on both rings were synthesized (Scheme 1) and evaluated for antileishmanial activity as
shown in Table 2. All these mono- and di-alkylated amino substituted analogues showed
9
9.9–100% inhibition of promastigotes at 25 µM concentration. Conversely, the di-alkylated
amino substituted analogues 22–36 have outstanding activity (IC50 = 2.5–4 µM) and
therapeutic profiles (SI = 31–105) (Table 2). The introduction of alkylated amino substituents
on ring A and B (22–24: IC = 2.5–4.0 µM versus 3; IC > 20 µM against amastigotes)
5
0
50
significantly increased the activity profile due to better bioavailability than their parent
compounds. Remarkably, the di-alkylated amino substituted analogues 22–24 were more
effective than standard drugs, miltefosine (IC = 8.40 µM), and SSG (IC = 49.7 µM) or
5
0
50
other tested compounds (Table 2).
Table 2
.2.3. In Vitro Antileishmanial Activity of Chromanochalcones with Hetero Atoms on Ring
2
A.
After studying the effect of various substitutions nature and their positions on ring A and
benzopyran moiety (Ring B) of chalcones (Table 1–2), we further moved to work on
chromenochalcones with hetero atoms on ring A. We synthesized some chromenochalcones
2
5–29 with hetero atom in ring A (Scheme 2) and evaluated for in vitro antileishmanial
activity (Table 3). Among these, most of the analogues exhibited potent activity against

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promastigotes (99.8–100% inhibition at 25 µM), with the exception of chromenochalcone 27
(62.2% inhibition at 25 µM) (Table 3). On the other hand, all analogues 25–29, with
exception of 26 (IC = 22.2 µM) and 28 (IC = 12.1 µM), were found extremely potent
5
0
50
(
IC < 5.4 µM) against amastigotes and also have good selectivity index.
50
Table 3
2
.2.4. In Vitro Antileishmanial Activity of O-Alkylated Chalcones
Finally, we wanted to study the role of the O-alkenyl (prenyl, geranyl and farnesyl)
and alkylated amine substituent on ring A and B of normal chalcones, therefore various
lipophilic monoterpenes (prenyl, geranyl, farnesyl), and hydrophilic alkyl amino substituents
containing chalcones 35–42 were generated (Scheme 3). In this series, all compounds (35–
4
2) showed good activity against promastigotes (Table 4). The analogues 35−39, in which the
ring B was substituted with O-alkenyl groups showed excellent activity (IC = 4.1–9.3 ꢀM),
50
with the exception of 38 (IC > 20 ꢀM) and 39 (toxic to cells) against amastigotes (Table 4).
5
0
While the introduction of alkylated amine substituents either on ring A and B significantly
decreased the activity, with the exception of 41 (IC > 9.3 ꢀM) (Table 4).
50
Table 4
2
.2.5. In Vivo Efficacy of Chalcones in L. donovani / Hamster Model
Large number of compounds (4, 6, 16, 22, 23, 24, 25, 29, 35 and 37) exhibited potent
in vitro activity against extracellular promastigote and intracellular amastigote (IC 1.70 to 8
5
0
µM) form of L. donovani (Table 1–4). Among these, compounds 22 (IC = 2 µM and SI =
5
0
1
62.5; table 2) and 37 (IC50 = 3.1 µM and SI = 47.2; table 4), which have potent
antiamastigote activity with high selectivity index were chosen for further in vivo studies and
tested against the MHOM/IN/80/Dd8 strain of L. donovani in hamster model [27]. The
aqueous suspension of tested compounds was administered for 5 to 10 consecutive days at 50
and/or 100 mg/kg/day dose by intraperitoneal (IP) route. The post treatment splenic biopsies
were done on day 7 after the last dose administration and amastigote counts were assessed by
Giemsa staining. Compound 22 showed 48.54 ± 10.55 % inhibition on day 7 post treatment
(p.t.) at 50 mg/kg/day by IP route (Table 5), where as the chalcone 37 exhibited 83.32 ±

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1
2.37 % suppression of parasites at 50 mg/kg/day for 10 days treatment and 75.89 ± 10.78%
at 100 mg/kg/day for 5 days treatment by IP route (Table 5).
Table 5
3
. Conclusion
In conclusion, a series of chalcone analogues with various structural features such as
chromenochalcones and O-prenyl/geranyl/farnesyl/aminoalkyl groups containing chalcones
were prepared based on natural product lead and tested for their antileishmanial activity.
Among all, ten compounds (4, 6, 16, 22, 23, 24, 25, 29, 35 and 37) found to be significantly
more active than the standard antileishmanial drugs, miltefosine and sodium stibogluconate
(
SSG) in in vitro evaluation against Leishmania amastigotes. The in vivo studies of promising
compounds 22 and 37 were performed in L. donovani / hamster model, in which compound
7 showed good antileishmanial activity (83.32 % parasite inhibition at the dose of 50 mg/kg
x 10 days and 75.89% parasite inhibition at 100 mg/kg x 5 days by IP route).
3
4
. Experimental Section
4
.1. General Methods.
Melting points were recorded on Buchi-530 capillary melting point apparatus and are
1
uncorrected. IR spectra were recorded on Perkin-Elmer AC-1 spectrometer. H NMR spectra
were recorded on Bruker Avance DPX 200 FT, Bruker Robotics and Bruker DRX 300,
1
13
spectrometers at 200, 300 MHz ( H) and 50, 75 MHz ( C). Experiments were recorded in
CDCl , CD OD, and DMSO-D at 25ºC. Chemical shifts were given in parts per million
3
3
6
(
ppm) downfield from internal standard Me Si (TMS). ESI mass spectra were recorded on
4
JEOL SX 102/DA-6000. Chromatography was executed with silica gel (60–120 or 230–400
mesh) using mixtures of ethyl acetate and hexane as eluants. Reactions, which required the
use of anhydrous, inert atmosphere techniques, were carried out under an atmosphere of
nitrogen. 1, 4-Dioxane was distilled over sodium. Commercially available reagents, solvents
and starting materials were used without further purification.
4
.1.1. Representative Procedure for the Synthesis of 1-(5-Hydroxy-2, 2-dimethyl–2H-
chromene-6-yl)-ethanone (2). To a magnetically stirred solution of 1 (12.0 g, 79 mmol) in dry
pyridine (8.04 mL) was added gradually 3-methyl-but-2-enal (6.8 g, 79 mmol) at rt. The

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o
reaction mixture was refluxed for 4 h at 150 C, additional equivalent of 3-methyl-but-2-enal
(6.8 g, 79 mmol) was added and refluxed for further 6 h. Excess pyridine, in the reaction
mixture was evaporated by rotary evaporator under reduced pressure. The crude product was
subjected to silica gel column chromatography using hexane/ethyl acetate as mobile phase to
afford the desired compound 2.
°
1
Yield: 65%; MP: 102-104 C; IR (KBr cm-1): 3435, 2862, 1633, 1486, 1333, 1109,751; H
NMR (CDCl , 300 MHz) δ: 12.90 (s, 1H), 7.52 (s, d, J = 8.8 Hz, 1H), 6.72 (d, J = 10.1Hz,
3
1
H), 6.35 (d, J= 8.8 Hz, 1H), 5.57 (d, J= 10.0 Hz, 1H), 2.55 (s, 3H), 1.47(s, 6H); MS(ESI):
m/z: 219 (M+H)+.
4
.1.2. Representative Procedure for the Synthesis of (E)-1-(5-hydroxy-2,2-dimethyl-2H-
chromen-6-yl)-3-(4-hydroxyphenyl)prop-2-en-1-one (3). To a stirred solution of 2 (500 mg,
.2 mmol) in aqueous KOH solution in ethanol (5 mL) was added 4-hydroxybenzaldehyde
279 mg, 2.2 mmol). The reaction mixture was stirred for 48 h at rt, and quenched in ice-cold
2
(
water, acidified with 1 N HCl, extracted with ethyl acetate (3 × 30 mL). The combined
organic layers were washed with water, brine solution, dried over anhydrous Na SO and the
2
4
solvent was evaporated under reduced pressure. The crude product was subjected to silica gel
column chromatography using hexane/ethyl acetate as mobile phase to afford the
chromenochalcone 3.
o
-1
1
Yield 35%; mp 201–205 C; FT-IR (KBr, cm ) 3436, 1635; H NMR (CDCl , 200 MHz) δ
3
1
3.76 (s, 1H), 7.83 (d, J = 15.8 Hz, 1H), 7.66 (d, J = 9.0 Hz, 1H), 7.54 (d, J = 8.6 Hz, 2H),
7
.42 (d, J = 15.8 Hz, 1H), 6.75 (d, J = 10.0 Hz, 1H), 6.39 (d, J = 9.0 Hz, 1H), 6.38 (d, J = 8.6
+
Hz, 2H), 5.58 (d, J = 10.0 Hz, 1H), 1.46 (s, 6H); MS (FAB) m/z: 323 (M + H) .
4
.1.3. Representative Procedure for the Synthesis of 1-(5-Hydroxy-2, 2-dimethyl-2H-
chromen-6-yl)-3-(4-nitro-phenyl)-propenone (16). To a stirred solution of 2 (575 mg, 2.6
mmol) in anhydrous THF (5 mL) was added portion wise NaH (121 mg, 5.2 mmol) and
stirred for 20 min at rt under nitrogen. Then, p-nitro-benzaldehyde (398 mg, 2.2 mmol) in 2
mL of THF was added to the reaction mixture and stirred for 8 h at rt. The mixture was
poured into ice-cold water, acidified with 1N HCl and extracted with ethyl acetate (3 × 50
mL). The combined organic layers were washed with water, brine solution, dried over
anhydrous Na SO , and evaporated under reduced pressure. The crude product was subjected
2
4

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to silica gel column chromatography using hexane/ethyl acetate as mobile phase to afford the
chromenochalcone 16.
-1
1
Yield 57%; FT-IR (KBr, cm ) 3425, 1625; H NMR (CDCl , 200 MHz) δ 13.29 (s, 1H), 8.28
3
(d, J = 8.7 Hz, 2H), 7.79 (d, J = 15.4 Hz, 1H), 7.78 (d, J = 8.7 Hz, 2H), 7.70 (d, J = 8.8 Hz,
1
H), 7.65 (d, J = 15.4 Hz, 1H), 6.74 (d, J = 10.0 Hz, 1H), 6.40 (d, J = 8.8 Hz, 1H), 5.61 (d, J
+
=
10.0 Hz, 1H), 1.47 (s, 6H); MS (FAB) m/z: 352 (M + H) .
4
.1.4. Representative Procedure for the Synthesis of 1-[2,2-Dimethyl-5-(2-piperidin-1-yl-
ethoxy)-2H-chromen-6-yl]-3-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-propenone (24). To a
stirred solution of chalcone, 3 (250 mg, 1.0 mmol) in dry acetone (20 mL) were added
anhydrous K CO (2.85 g, 20.8 mmol), 1-(2-Chloro-ethyl)-piperidine hydrochloride (953 mg,
2
3
5
.2 mmol), and the reaction mixture was refluxed for 5 h. The mixture was filtered off under
suction and solvent was evaporated under reduced pressure. The crude product was subjected
to silica gel column chromatography using hexane/ethyl acetate as mobile phase to afford the
compound 24.
−
1
1
Yield: 47%; FT-IR (neat, cm ) 1652; H NMR (CDCl , 200 MHz) δ 7.67 (d, J = 14.7 Hz,
3
1
8
4
2
1
1
H), 7.55 (d, J = 8.7 Hz, 1H), 7.47 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 14.7 Hz, 1H), 6.91 (d, J =
.6 Hz, 2H), 6.78 (d, J = 10.0 Hz, 1H), 6.63 (d, J = 8.7 Hz, 1H), 5.67 (d, J = 10.0 Hz, 1H),
.13 (t, J = 5.9 Hz, 2H), 3.93 (t, J = 5.7 Hz, 2H), 2.78 (t, J = 5.9 Hz, 2H), 2.62 (t, J = 5.7 Hz,
13
H), 2.53–2.34 (m, 8H), 1.61–1.25 (m, 18H); C NMR (CDCl , 50 MHz) δ 191.3, 161.1,
3
57.6, 155.6, 143.2, 131.5, 130.7, 130.5, 128.2 (2C), 126.6, 124.5, 117.3, 115.4, 115.3 (2C),
13.0, 77.1, 73.9, 66.4, 58.9, 58.1, 55.4 (2C), 55.2 (2C), 28.4 (2C), 26.2 (4C), 24.6, 24.5; MS
+
(
FAB) m/z: 545 (M + H) .
4
4
.2. Experimental Section for Biology
.2.1. Promastigote Growth Inhibition Assay
The antileishmanial activity of tested compounds against the promastigote form of L.
donovani (strain: MHOM/IN/80/Dd8) was assessed as described earlier [23]. The late log /
stationary phase of promastigotes (expressing firefly luciferase gene) were seeded with
5
complete M-199 medium at 5 x 10 /mL/100ꢀL/well in 96-well plates and incubated with
tested compounds in a 24ºC incubator for 96 h. Miltefosine and SSG were used as standard
drugs. After 96 h of incubation, 50 ꢀL of promastigote suspension was pipette out from each

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®
well in to another 96-well plate and mixed with an equal volume of Steady Glo reagent
Promega) and luminescence was measured by using a luminometer. The values were
(
expressed as relative luminescence unit (RLU). The inhibition of parasitic multiplication is
determined by comparison of the luciferase activity of compound treated parasites with that
of untreated control.
4
.2.2. Antiamastigote Assay
Mouse macrophage cell line (J-774A.1) infected with promastigotes (expressing
luciferase firefly reporter gene) was used for the assessment of the activity of compounds
against the amastigote form of Leishmania parasite. J-774A.1 cells were seeded in a 96-well
4
plate (4 x 10 /mL/100µL/well) in complete RPMI-1640 medium (containing 10% heat
inactivated foetal calf serum) and the plates were incubated at 37ºC in a 5% CO incubator.
2
After 24 h, the medium was replaced with fresh medium containing stationary phase
5
promastigotes (4 x 10 /mL/100µL/well). Promastigotes were phagocytized by the
macrophages and inside the phagolysosomes; they were transformed into amastigotes form.
Each well of the plate was washed with plain RPMI medium after 24 h of incubation to
remove the un-internalized promastigotes. The test compounds were added at dilutions up to
7
points starting from 40 µM concentration in complete RPMI medium and the plates were
incubated at 37ºC in a CO incubator for 72 h. After incubation, the drug containing medium
2
was aspirated and 50 µL phosphate buffer saline (PBS) was added in each well and mixed
®
with an equal volume of Steady Glo reagent. After gentle shaking for 2 min, the reading was
taken in a luminometer [23]. The values are expressed as RLU and data were transformed
into a graphic program (Excel). IC value of each compound was calculated by nonlinear
5
0
regression analysis of the concentration response curve using the four parameter Hill
equations.
4
.2.3. Cytotoxicity Assessment Assay
The cytotoxicity of the compounds was determined by following the method of
Mosmann [28] with some modifications. As described previously [23], mammalian kidney
5
fibroblast cells (Vero cell line) (1x 10 /mL/100µL/well) were incubated with test compounds
(
in 7 concentrations starting from 400 µM) at 37ºC in a CO incubator. After 72 h of
2
incubation, 25 ꢀL of MTT (3-(4,5-Dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide)

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reagent (5mg/mL) in PBS medium was added to each well and incubated at 37°C for 2 h. At
the end of the incubation period, the supernatant were removed and 150 µL of pure DMSO
was added to each well for solubilizing the formazan crystal. After 15 min of shaking, the
readings were recorded as absorbance at 540 nm on a micro plate reader. Fifty percent
cytotoxic concentration (CC ) values were estimated as described by Huber and Koella [29].
50
The selectivity index (SI) for each compound was calculated as ratio between cytotoxicity
(
CC ) in Vero cells and activity (IC ) against Leishmania amastigotes.
50 50
4
.2.4. In Vivo Evaluation in L. donovani / Hamster Model
The in vivo antileishmanial activity was determined in golden hamsters (Mesocricetus
auratus) infected with MHOM/IN/80/Dd8 strain of L. donovani as described by Gupta et al
7
[
27]. Golden hamsters of both sexes were infected intracardiacally with 1 x 10 amastigotes
per animal. After establishment of infection in 15 days, pre-treatment splenic biopsies were
performed to assess the degree of infection in all the animals. The animals with +1 grade
infection (5-10 amastigotes/ 100 spleen cell nuclei) were included in the chemotherapeutic
studies. Five infected animals were used for each test compound. Drug treatment in different
dose regimen by intraperitoneal (IP) or per oral (PO) route was initiated after 2 days of pre-
treatment biopsies and continued for 5-10 consecutive days. Miltefosine and SSG were used
as reference drugs. Post-treatment biopsies were done on day 7 after the last dose
administration and amastigote counts are assessed by Giemsa staining [23]. Intensity of
infection in both, treated and untreated animals, and also the initial parasite count in treated
animals was compared and the efficacy was expressed in terms of percentage inhibition (PI)
using the following formula:-
PI = 100- [(ANAT x 100) / (INAT x TIUC)]
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.
Acknowledgements
We are grateful to the Director, CSIR-CDRI, Lucknow, for the constant
encouragement for the program on antiparasitic drug discovery, SAIF Division for spectral
data and Department of Science and Technology (DST), New Delhi for financial support in

ACCEPTED MANUSCRIPT
the form of ad-hoc project (SR/S1/OC-58/2008). The transgenic L. donovani promastigotes
were originally procured from Dr. Neena Goyal, Division of Biochemistry, CSIR-CDRI,
Lucknow, India.
Appendix. Supplementary information
Supplementary data associated with this article can be found in the online version at doi:
References
[
1] F. Chappuis, S. Sundar, A. Hailu, H. Ghalib, J. Alvar M. Boelaer, Visceral leishmaniasis:
what are the needs for diagnosis, treatment and control? Nat. Rev. Microbiol. 5 (2007)
8
73-885.
2] H.W. Murray, J.D. Berman, C.R. Davies, Advances in leishmaniasis, Lancet 366 (2005)
561-1577.
[
[
1
3] WHO, Report of a meeting of the WHO expert committee on the control of the
leishmaniases, Geneva, 2010.
[
[
4] B.L. Herwaldt, Leishmaniasis, Lancet 354 (1999) 1191-1199
5] T.K. Jha, Drug unresponsiveness & combination therapy for kala-azar, Indian J. Med.
Res. 123 (2006) 389-398.
[
[
[
6] S. Sundar, D.K. More, M.K. Singh, V.P. Singh, S. Sharma, A. Makharia, Failure of
pentavalent antimony in visceral leishmaniasis in India: report from the center of the
Indian epidemic, Clin. Infect. Dis. 31 (2000) 1104-1107.
7] L.H. Freitas, E. Chatelin, H.A. Kim, Visceral leishmaniasis treatment: What do we have,
what do we need and how to deliver it? Int. J. Parasitol: Drugs Drug Resist. 2 (2012) 11-
1
9.
8] J. Rybniker, V. Goede, J. Mertens, M. Ortmann, W. Kulas, M. Kochanek, T. Benzing,
J.R. Arribas, G. Fätkenheuer, Treatment of visceral leishmaniasis with intravenous
pentamidine and oral fluconazole in an HIV-positive patient with chronic renal failure-a
case report and brief review of the literature, Int. J. Infect. Dis.14 (2010) 522-525.
9] J. M. Mutiso, J. C. Macharia, M. M. Gicheru, A review of adjuvants for Leishmania
vaccine candidates, J. Biomed. Res. 24 (2010) 16-25.
[
[
10] (a) M. Sharma, K. Chauhan, R. Shivahare, P. Vishwakarma, M.K. Suthar, A. Sharma,
S. Gupta, J.K. Saxena, J. Lal, P. Chandra, B. Kumar, P.M.S. Chauhan, Discovery of a
New Class of Natural Product-Inspired Quinazolinone Hybrid as Potent Antileishmanial

ACCEPTED MANUSCRIPT
agents, J. Med. Chem. 56 (2013) 4374-4392. (b) C. Marín, M.P. Clares, I. Ramírez-
Macías, S. Blasco, F. Olmo, C. Soriano, B. Verdejo, M.J. Rosales, D. Gomez-Herrera, E.
García-España, M. Sánchez-Moreno, In vitro activity of scorpiand-like azamacrocycle
derivatives in promastigotes and intracellular amastigotes of Leishmania infantum and
Leishmania braziliensis, Eur. J. Med. Chem. 62 (2013) 466-477. (c) Y. Fromentin, N.
Gaboriaud-Kolar, B.N. Lenta, J.D. Wansi, D. Buisson, E. Mouray, P. Grellier, P.M.
Loiseau, M.C. Lallemand, S. Michel, Synthesis of novel guttiferone A derivatives: In
vitro evaluation toward Plasmodium falciparum, Trypanosoma brucei and Leishmania
donovani, Eur. J. Med. Chem. 65 (2013) 284-294.
[
11] (a) H.K. Hsieh, T.H. Lee, J.P. Wang, J.J. Wang, C.N. Lin, Synthesis and anti-
inflammatory effect of chalcones and related compounds, Pharm. Res. 15 (1998) 39-46.
(b) C.Q. Meng, X.S. Zheng, L. Ni, Z. Ye, J.E. Simpson, K.J. Worsencroft, M.R. Hotema,
M.D. Weingarten, J.W. Skudlarek, J.M. Gilmore, L.K. Hoong, R.R. Hill, E.M. Marino,
K.L. Suen, C. Kunsch, M.A. Wasserman, J.A. Sikorski, Discovery of novel heteroaryl-
substituted chalcones as inhibitors of TNF-α-induced VCAM-1 expression. Bioorg. Med.
Chem. Lett. 14 (2004) 1513-1517.
[
[
12] S.F. Nielsen, T. Boesen, M. Larsen, K. Schønning, H. Kromann, Antibacterial
chalcones bioisosteric replacement of the 4′-hydroxy group, Bioorg. Med. Chem. 12
(2004) 3047-3054.
13] (a) F. Uchiumi, T. Hatano, H. Ito, T. Yoshida, S.I. Tanuma, Transcriptional
suppression of the HIV promoter by natural compounds. Antiviral Res. 58 (2003), 89-98.
(
b) J. H. Wu, X. H. Wang, Y. H. Yi, K. H Lee, Anti-AIDS agents 54. A potent anti-HIV
chalcone and flavonoids from genus Desmos, Bioorg. Med. Chem. Lett. 13 (2003) 1813-
815.
1
[
[
14] M.L. Go, M. Liu, P. Wilairat, P.J. Rosenthal, K.J. Saliba, K. Kirk, Antiplasmodial
chalcones inhibit sorbitol-induced hemolysis of Plasmodium falciparum-infected
erythrocytes, Antimicrob. Agents Chemother. 48 (2004) 3241-3245.
15] (a) Y. Xia, Z.Y. Yang, P. Xia, K.F. Bastow, Y. Nakanishi, K.H. Lee, Antitumor
agents. Part 202: Novel 2’-amino chalcones: design, synthesis and biological evaluation,
Bioorg. Med. Chem. Lett. 10 (2000) 699-701. (b) F. Bois, C. Beney, A. Boumendjel,
A.M. Mariotte, G. Conseil, A. Di Pietro, Halogenated chalcones with high-affinity
binding to P-glycoprotein: potential modulators of multidrug resistance, J. Med. Chem.
4
1 (1998) 4161-4164.

ACCEPTED MANUSCRIPT
[
16] (a) C.R. Andrighetti-Fröhner, K.N. de Oliveira, D. Gaspar-Silva, L.K. Pacheco, A.C.
Joussef, M. Steindel, C.M.O. Simões, A.M.T. de Souza, U.O. Magalhaes, I.F. Afonso,
C.R. Rodrigues, R.J. Nunes, H.C. Castro, Synthesis, biological evaluation and SAR of
sulfonamide 4-methoxychalcone derivatives with potential antileishmanial activity, Eur.
J. Med. Chem. 44 (2009) 755-763. (b) P. Boeck, C.A. Bandeira Falcão, P.C. Leal, R.A.
Yunes, V.C. Filho, E.C. Torres-Santos, B. Rossi-Bergmann, Synthesis of chalcone
analogues with increased antileishmanial activity, Bioorg. Med. Chem. 14 (2006) 1538-
1
545.
[
[
17] Y.M. Lin, Y.S. Zhou, M.T. Flavin, L.M. Zhou, W.G. Nie, F.C. Chen, Chalcones and
flavonoids as anti-tuberculosis agents, Bioorg. Med. Chem. 10 (2002) 2795-2802.
18] S. Cheenpracha, C. Karalai, C. Ponglimanont, S. Subhadhirasakul, S. Tewtrakul,
Anti-HIV-1 protease activity of compounds from Boesenbergia pandurata, Bioorg. Med.
Chem. 14 (2006) 1710-1714.
[
[
19] L. Svetaz, A. Tapia, S.N. Lopez, R.L.E. Furlan, E. Petenatti, R. Pioli, G. Schmeda-
Hirschmann, S.A. Zacchino, Antifungal chalcones and new caffeic acid esters from
Zuccagnia punctata acting against soybean infecting fungi, J. Agric. Food Chem. 52
(2004) 3297-3300.
20] (a) M.L. Bolognesi, F. Lizzi, R. Perozzo, R. Brun, A. Cavalli, Synthesis of a small
library of 2-phenoxy-1, 4-naphthoquinone and 2-phenoxy-1, 4-anthraquinone derivatives
bearing anti-trypanosomal and anti-leishmanial activity, Bioorg. Med. Chem. Lett. 18
(2008) 2272-2276. (b) A. Cavalli, F. Lizzi, S. Bongarzone, R. Brun, R. Luise Krauth-
Siegel, M.L. Bolognesi, Privileged structure-guided synthesis of quinazoline derivatives
as inhibitors of trypanothione reductase, Bioorg. Med. Chem. Lett. 19 (2009) 3031-3035.
21] J.K. Kumar, T. Narender, M.S. Rao, P.S. Rao, G. Toth, B. Balazs, H.J. Duddeck,
Further Dihydrochalcones from Crotolaria ramosissima, J. Braz. Chem. Soc. 10 (1999)
[
[
[
2
78-280.
22] T. Narender, Shweta, S. Gupta, A convenient and biogenetic type synthesis of few
naturally occurring chromeno dihydrochalcones and their in vitro antileishmanial activity,
Bioorg. Med. Chem. Lett. 14 (2004) 3913-3916.
23] (a) T. Narender, T. Khaliq, Shweta, Nishi, N. Goyal, S. Gupta, Synthesis of
chromenochalcones and evaluation of their in vitro antileishmanial activity, Bioorg. Med.
Chem. 13 (2005) 6543-6550. (b) R. Shivahare, K. Venkateshwarlu, H. Chandasana, M.K.
Suthar, P. Agnihotri, P. Vishwakarma, T.K. Chaitanya, P. Kancharla, T. Khaliq, S. Gupta,

ACCEPTED MANUSCRIPT
R.S. Bhatta, J.V. Pratap, J.K. Saxena, S. Gupta, T. Narender, Synthesis, structure-activity
relationships and biological studies of chromenochalcones as potential antileishmanial
agents, J. Med. Chem. 56 (2013) 4374-4392.
[
24] W.M. Bandaranayake, L. Crombie, D.A Whiting, Pyridine-catalysed chromenylation
of mono-chelated meta-dihydric phenols with mono-, sesqui- and di-terpene aldehydes:
synthesis of rubranine and flemingins A-, B- and C-methyl ethers, J. Chem. Soc. C (1971)
8
04-810.
[
[
25] J.R. Stille, J.A. Ward, C. Leffelman, K.A. Sullivan, 5-Formyl salicylaldehyde as a
linker for the synthesis of benzofuran containing insulin sensitivity enhancer compounds,
Tetrahedron Lett. 37 (1996) 9267-9270.
26] A. Kumar, V.D. Tripathi, P. Kumar, L.P. Gupta, Akanksha, R. Trivedi, H. Bid, V.L.
Nayak, J.A. Siddiqui, B. Chakravarti, R. Saxena, A. Dwivedi, M.I. Siddiquee, U.
Siddiqui, R. Konwar, N. Chattopadhyay, Design and synthesis of 1,3 biarylsulfanyl
derivatives as new anti-breast cancer agents, Bioorg. Med. Chem. 19 (2011) 5409-5419.
27] S. Gupta, Ramesh, S.C. Sharma, V.M.L. Srivastava, Efficacy of picroliv combination
with miltefosine, an orally effective antileishmanial drug against experimental visceral
leishmaniasis, Acta Trop. 94 (2005) 41-47.
[
[
[
28] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: Application
to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55-63.
29] W. Huber, J. C. Koella, A comparison of three methods of estimating EC50 in studies
of drug resistance of malaria parasites, Acta Trop. 55 (1993) 257-261.

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FIGURE CAPTIONS
Table 1. In vitro antileishmanial activity of chromenochalcones against promastigotes and
amastigotes of L. donovani and their cytotoxicity
Table 2. In vitro antileishmanial activity of alkylated amines containing-chromenochalcones
against promastigotes and amastigotes of L. donovani and their cytotoxicity
Table 3. In vitro antileishmanial activity of chromenochalcones with hetero atoms on ring A
against promastigotes and amastigotes of L. donovani and their cytotoxicity
Table 4. In vitro antileishmanial activity of O-alkylated chalcones against promastigotes and
amastigotes of L. donovani and their cytotoxicity
Table 5. In vivo efficacy of chalcones against L. donovani / hamster model
Figure 1. General skeleton and naturally occurring antileishmanial chalcones
Scheme 1. Synthesis of Chromenochalcones (3–18) and Alkylated amine containing-
chromenochalcones (19–24)
Scheme 2. Synthesis of chromenochalcones containing the hetero atoms in ring A (25–29)
Scheme 3. Synthesis of O-prenyl/geranyl/farnesyl/alkylatedamines containing chalcones (35–
4
2)

ACCEPTED MANUSCRIPT
Table 1. In vitro antileishmanial activity of chromenochalcones against promastigotes and
amastigotes of L. donovani and their cytotoxicity
In vitro activity
Cyto-
Antipromastigote
(
Index
(SI)
Compd
R1
R2
R3
R4
Antiamastigote
IC50 (µM)
toxicity
d
% inhibition at
b
c
a
CC (µM)
50
2
5 µM)
e
3
4
5
6
7
8
9
H
H
H
H
OH
OEt
H
H
70.2
88.9
97.1
98.7
98.2
99.0
99.9
100
> 20
5.1
ND
-
42.1
> 20.2
45.6
22.4
9.5
215
> 400
278.5
266.6
158.4
92.2
H
H
NMe
H
19.8
6.1
2
H
H
NHAc
OMe
F
H
H
OMe
H
H
11.9
16.7
17.9
> 20
17.0
13.9
9.7
H
H
H
H
Cl
H
5.1
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
H
H
Br
H
ND
-
H
F
F
H
99.9
99.9
99.9
99.9
99.6
97.4
94.5
145.7
141.7
130.9
184.9
80.2
8.6
H
Cl
H
Cl
H
10.2
13.5
15.3
9.8
Cl
H
Cl
H
OEt
H
F
H
12.1
8.2
H
CN
H
H
H
NO
H
H
5.2
106.2
ND
20.4
-
2
OH
H
CHO
> 20
H
H
91.2
> 20
49.7
ND
-
SSG
-
-
-
-
-
-
-
-
No inhibition
100
> 400
> 8.0
6.2
Miltefosine
8.4
52.5
a
b
c
Values are represented as average of at least duplicate measurements (SD ± 2%). IC and CC values are
50
50
d
e
represented as average of at least duplicate measurements (SD ± 10%), SI (selectivity index) = CC / IC . ND:
50
50
not determined.

ACCEPTED MANUSCRIPT
Table 2. In vitro antileishmanial activity of alkylated amines containing-chromenochalcones
1
9–24 against promastigotes and amastigotes of L. donovani and their cytotoxicity
In vitro activity
Antipromastigote
% inhibition at
Cytotoxicity
d
Compd
R1
R2
Antiamastigote
IC50 (µM)
c
Index (SI)
(
b
CC (µM)
50
a
2
5 µM)
1
2
9
0
H
H
100
100
14.9
> 20
341.2
22.9
ND
e
ND
2
2
2
2
1
2
3
4
H
100
100
99.9
100
17.7
2.0
> 400
325.4
258.1
86.5
> 22.6
162.5
103.2
30.9
2.5
2.8
SSG
Miltefosine
-
-
-
-
No inhibition
00
49.7
8.4
> 400
> 8.0
6.2
1
52.5
a
b
c
Values are represented as average of at least duplicate measurements (SD ± 2%). IC and CC values are
50
50
d
e
represented as average of at least duplicate measurements (SD ± 10%), SI (selectivity index) = CC / IC ND:
50
50,
not determined.

ACCEPTED MANUSCRIPT
Table 3. In vitro antileishmanial activity of chromenochalcones with hetero atoms on ring A
against promastigotes and amastigotes of L. donovani and their cytotoxicity
In vitro activity
Antipromastigote Antiamastigote CC (µM)
Cytotoxicity Index
c
d
Compd
R1
R2
X
Y
(SI)
50
b
(
% inhibition at
IC50 (µM)
a
2
5 µM)
2
2
2
2
2
5
6
7
8
9
-
-
-
N
CH
99.9
2.0
20.5
8.6
10.2
0.4
-
-
CH
N
-
100
62.2
100
22.2
e
H
Cl
Cl
H
H
Me
-
-
-
ND
ND
-
12.1
5.3
256.8
399.4
21.2
75.3
-
99.8
SSG
-
-
-
-
-
-
-
-
No inhibition
100
49.7
8.4
> 400
52.5
> 8.0
6.2
Miltefosine
a
b
c
Values are represented as average of at least duplicate measurements (SD ± 2%). IC and CC values
50
50
d
are represented as average of at least duplicate measurements (SD ± 10%), SI (selectivity index) = CC /
IC . ND. not determined.
50
e
5
0

ACCEPTED MANUSCRIPT
Table 4. In vitro antileishmanial activity of O-alkylated chalcones against promastigotes and
amastigotes of L. donovani and their cytotoxicity
R2
1
R O
O
35-42
In vitro activity
Antipromastigote
% inhibition at
Cytotoxicity
Index
(SI)
Compd
R1
R2
Antiamastigote
IC50 (µM)
c
d
(
b
CC (µM)
50
a
2
5 µM)
3
5
NMe
OH
2
84.2
4.1
> 400
> 97.6
3
3
3
3
6
7
8
9
95.2
93.0
76.1
100
9.3
3.1
12.9
1.4
OMe
146.5
47.2
e
NMe
>20
ND
-
-
2
Toxic for cells
ND
ND
4
4
4
0
1
2
OEt
99.9
99.9
99.9
>20
9.3
-
NMe
251.3
ND
27.0
-
2
>20
SSG
-
-
-
-
No inhibition
100
49.7
8.4
> 400
52.5
> 8.0
Miltefosine
6.2
a
b
Inhibition at 25 µM and values are represented as average of at least duplicate measurements (SD ± 2%). IC
5
0
c
and CC50 values are represented as average of at least duplicate measurements (SD ± 10%),ND = not
determined, SI (selectivity index) = CC / IC . ND. not determined.
d
e
5
0
50

ACCEPTED MANUSCRIPT
Table 5. In vivo efficacy of chalcones against L. donovani / hamster model
Dose
Treated
days
Route
% Inhibition ± SD
Compd
(
mg/kg)
at day 7 post treatment
2
3
3
2
7
7
50
50
5
10
5
IP
IP
48.54 ± 10.55
83.32 ± 12.37
75.89 ± 10.78
89.47 ± 4.72
98.50 ± 1.10
100
40
IP
SSG
5
IP
Miltefosine
30
5
PO
SD, standard deviation; IP, intraperitoneal; PO, per oral, SSG, sodium stibo-gluconate
Figure 1. General skeleton and naturally occurring antileishmanial chalcones
Scheme 1. Synthesis of chromenochalcones (3–18) and alkylated amine containing-
chromenochalcones (19–24)

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Scheme 2. Synthesis of chromenochalcones containing the hetero atoms in ring A (25–29)
Scheme 3. Synthesis of O-prenyl/geranyl/farnesyl/alkylated amines containing chalcones
35–42)
(

ACCEPTED MANUSCRIPT
Research Highlights
•
•
•
•
Chalcone derivatives were prepared based on the natural products lead molecules.
Compounds were assessed for in vitro and in vivo antileishmanial potential.
Ten compounds exhibited promising antileishmanial activity.
The potent compound showed >83% inhibition at dose 50 mg/kg x 10d in hamster
model.

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Supporting Information
Synthesis and Biological Evaluation of Chalcones as Potential
Antileishmanial Agents
a
Shweta Gupta, Rahul Shivahare, Venkateswarlu Korthikunta, Rohit Singh,
b
a
a
b
Suman Gupta and Narender Tadigoppula*
a
a
Division of Medicinal and Process Chemistry,
b
Division of Parasitology,
CSIR-Central Drug Research Institute, Lucknow-226 031, India
*
Corresponding author:
Tel.: +91 522 2612411; fax: +91 522 2623405.
E-mail: t_narendra@cdri.res.in or tnarender@rediffmail.com (T. Narender)
Table of Contents:
1
2
. Compounds characterization data
S-2
. NMR spectra of the selected chalcones
S-18
S-1

ACCEPTED MANUSCRIPT
Compounds characterization data
(E)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-(4-hydroxyphenyl)prop-2-en-1-one (3):
o
Yield 35%; mp 201–205 C; FT-IR (KBr, cm ) 3436, 1635; H NMR (CDCl
-1
1
3
, 200 MHz) δ
1
3.76 (s, 1H), 7.83 (d, J = 15.8 Hz, 1H), 7.66 (d, J = 9.0 Hz, 1H), 7.54 (d, J = 8.6 Hz, 2H), 7.42
(d, J = 15.8 Hz, 1H), 6.75 (d, J = 10.0 Hz, 1H), 6.39 (d, J = 9.0 Hz, 1H), 6.38 (d, J = 8.6 Hz, 2H),
+
.58 (d, J = 10.0 Hz, 1H), 1.46 (s, 6H); MS (FAB) m/z: 323 (M + H) .
5
3
-(4-Ethoxy-phenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-propenone (4):
-
1
1
Yield 56%; FT-IR (KBr, cm ) 3395, 1631; H NMR (CDCl
3
, 200 MHz) δ 13.56 (s, 1H), 7.86 (d,
J = 15.8 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.61 (d, J = 8.6 Hz, 2H), 7.42 (d, J = 15.8 Hz, 1H),
6
1
1
1
.92 (d, J = 8.6 Hz, 2H), 6.75 (d, J = 10.0 Hz, 1H), 6.37 (d, J = 8.8 Hz, 1H), 5.68 (d, J = 10.0 Hz,
1
3
H), 4.08 (q, J = 6.0 Hz, 2H), 1.60–1.46 (m, 9H); C NMR (CDCl , 50 MHz) δ 192.4, 161.6,
3
61.3, 160.0, 144.6, 131.0 (2C), 130.8, 128.5, 127.8, 118.0, 116.4, 115.3 (2C), 114.5, 109.8,
+
08.6, 78.2, 64.1, 28.8 (2C), 15.1; MS (FAB) m/z: 351 (M + H) .
(E)-3-(4-(dimethylamino)phenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)prop-2-en-1-
one(5):
S-2

ACCEPTED MANUSCRIPT
-
1
1
Yield 55%; FT-IR (KBr, cm ) 3432, 1630; H NMR (CDCl
3
, 200 MHz) δ 13.80 (s, 1H), 7.64 (d,
J = 14.5 Hz, 1H), 7.53 (m, 2H), 7.51 (d, J = 14.5 Hz, 1H), 7.49 (d, J = 8.9 Hz, 1H), 6.75 (d, J =
1
6
0.0 Hz, 1H), 6.82-6.66 (m, 2H), 6.48 (d, J = 8.9 Hz, 1H), 5.89 (d, J = 10.0 Hz, 1H), 3.01 (s,
+
H), 1.46 (s, 6H); MS (FAB) m/z: 350 (M + H) .
(E)-N-(4-(3-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-3-oxoprop-1-
enyl)phenyl)acetamide (6):
-
1
1
Yield 75%; FT-IR (KBr, cm ) 3436, 1635; H NMR (CDCl , 200 MHz) δ 14.00 (s, 1H), 10.22
3
(s, 1H), 8.20(d, J = 8.9 Hz, 2H), 7.87(d, J = 14.0 Hz, 1H), 7.86-7.83 (m, 2H), 7.72-7.67 (m, 2H),
6
.65 (d, J = 10.0 Hz, 1H), 6.46 (d, J = 8.8 Hz, 1H), 5.78 (d, J = 10.0 Hz, 1H), 2.10 (s, 3H), 1.46
1
3
s, 6H); C NMR (CDCl
(
3
, 50 MHz) δ 19.4, 169.2, 160.3, 159.5, 144.5, 142.1, 132.3, 130.4(2C),
1
29.5, 129.2, 119.2(3C), 115.2, 114.0, 108.8, 108.4, 78.1, 28.2(2C), 24.4; MS (FAB) m/z: 364
+
M + H) .
(
3
-(3,4-Dimethoxy-phenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-propenone (7):
o
-1
1
Yield 45%; mp 150–155 C; FT-IR (KBr, cm ) 3437, 1648; H NMR (CDCl , 200 MHz) δ
3
1
3.62 (s, 1H), 7.84 (d, J = 15.3 Hz, 1H), 7.73 (d, J = 8.9 Hz, 1H), 7.42 (d, J = 15.3 Hz, 1H), 7.24
(d, J = 8.3 Hz, 1H), 7.15 (d, J = 1.6 Hz, 1H), 6.90 (d, J = 8.3 Hz, 1H), 6.76 (d, J = 10.0 Hz, 1H),
1
.38 (d, J = 8.9 Hz, 1H), 5.59 (d, J = 10.0 Hz, 1H), 3.97 (s, 3H), 3.94 (s, 3H), 1.47 (s, 6H); C
3
6
NMR (CDCl
3
, 50 MHz) δ 192.3, 161.3, 160.1, 151.9, 149.7, 144.8, 130.9, 128.4, 128.2, 123.7,
S-3

ACCEPTED MANUSCRIPT
1
18.4, 116.3, 114.5, 111.6, 110.6, 109.8, 108.5, 78.0, 56.4 (2C), 28.7 (2C); MS (FAB) m/z: 367
+
M + H) .
(
3
-(4-Fluoro-phenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-propenone (8):
-
1
1
Yield 59%; FT-IR (KBr, cm ) 3429, 1627; H NMR (CDCl , 200 MHz) δ 13.50 (s, 1H), 7.83 (d,
3
J = 15.5 Hz, 1H), 7.72–7.60 (m, 3H), 7.47 (d, J = 15.5 Hz, 1H), 7.11 (m, 2H), 6.74 (d, J = 10.0
1
Hz, 1H), 6.38 (d, J = 8.9 Hz, 1H), 5.60 (d, J = 10.0 Hz, 1H), 1.47 (s, 6H); C NMR (CDCl , 50
3
3
MHz) δ 192.1, 167.0, 162.0, 161.4, 160.3, 143.3, 132.0, 131.5, 131.0, 130.9, 130.7, 128.6, 120.4,
1
16.8, 116.3, 114.4, 109.8, 108.8, 78.3, 28.8 (2C), Note– More number of carbon signals appear
due to long range coupling with Fluorine (F) substitution on phenyl ring; MS (FAB) m/z: 325
+
M + H) .
(
3
-(4-Chloro-phenyl)-1-(5-hydroxy-2, 2-dimethyl-2H-chromen-6-yl)-propenone (9):
-
1
1
Yield 52%; FT-IR (KBr, cm ) 3436 1628; H NMR (CDCl , 200 MHz) δ 13.61 (s, 1H), 7.73 (d,
3
J = 15.5 Hz, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.8 Hz, 1H), 7.43 (d, J = 15.5 Hz, 1H),
7
1
.31 (d, J = 8.4 Hz, 2H), 6.67 (d, J = 10.0 Hz, 1H), 6.30 (d, J = 8.8 Hz, 1H), 5.52 (d, J = 10.0 Hz,
+
H), 1.39 (s, 6H); MS (FAB) m/z: 341 (M + H) .
3
-(4-Bromo-phenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-propenone (0):
S-4

ACCEPTED MANUSCRIPT
o
-1
1
Yield 46%; mp 152–153 C; FT-IR (KBr, cm ) 3440, 1634; H NMR (CDCl , 200 MHz) δ
3
1
3.63 (s, 1H), 7.79 (d, J = 15.4 Hz, 1H), 7.70 (d, J = 8.9 Hz, 1H), 7.55 (d, J = 7.8 Hz, 2H), 7.53
(d, J = 15.4 Hz, 1H), 7.52 (d, J = 7.8 Hz, 2H), 6.74 (d, J = 10.0 Hz, 1H), 6.38 (d, J = 8.9 Hz, 1H),
1
3
.60 (d, J = 10.0 Hz, 1H), 1.47 (s, 6H); C NMR (CDCl
5
1
3
, 50 MHz) δ 192.0, 161.4, 160.4, 143.2,
34.1, 132.6 (2C), 131.1, 130.2 (2C), 128.6, 125.3, 121.3, 116.2, 114.4, 109.8, 108.8, 78.4, 28.8
+
2C); MS (FAB) m/z: 385 (M + H) , 387 (M + 3H) .
+
(
3
-(3,4-Difluoro-phenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)-propenone (11):
o
-1
1
Yield 38%; mp 138–139 C; FT-IR (KBr, cm ) 3430, 1639; H NMR (CDCl , 200 MHz) δ
3
1
3.54 (s, 1H), 7.89 (d, J = 15.6 Hz, 1H), 7.68 (d, J = 9.1 Hz, 1H), 7.61 (d, J = 15.6 Hz, 1H), 7.62
(d, J = 8.5 Hz, 1H), 6.97 (dd, J = 8.1, 7.6 Hz, 1H), 6.87 (dd, J = 9.2, 8.7 Hz, 1H), 6.75 (d, J =
1
3
0.0 Hz, 1H), 6.38 (d, J = 8.8 Hz, 1H), 5.60 (d, J = 10.0 Hz, 1H), 1.40 (s, 6H); C NMR (CDCl ,
1
5
1
3
0 MHz) δ 191.7, 161.4, 160.5, 142.1, 132.5, 131.0, 128.6, 125.8, 121.7, 118.5, 118.1, 117.1,
+
16.7, 116.1, 114.3, 109.8, 108.9, 78.4, 28.8 (2C); MS (FAB) m/z: 343 (M + H) .
(
E)-3-(3,4-dichlorophenyl)-1-(5-hydroxy-2,2-dimethyl-2H-chromen-6-yl)prop-2-en-1-one
12):
(
S-5