Synthesis, structure-activity relationships, and biological studies of chromenochalcones as potential antileishmanial agents

Article

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Synthesis, Structure−Activity Relationships, and Biological Studies of

Chromenochalcones as Potential Antileishmanial Agents

Rahul Shivahare,^†,#Venkateswarlu Korthikunta,^‡,#Hardik Chandasana,^§Manish K. Suthar,^⊥

Pragati Agnihotri,^∥Preeti Vishwakarma,^†Telaprolu K. Chaitanya,^§Papireddy Kancharla,^‡Tanvir Khaliq,^‡

Shweta Gupta,^‡Rabi Sankar Bhatta,^§J. Venkatesh Pratap,^∥Jitendra K. Saxena,^⊥Suman Gupta,*^,†

and Narender Tadigoppula*^,‡

^†Division of Parasitology, CSIR−Central Drug Research Institute, Lucknow 226 031, Uttar Pradesh, India

^‡Division of Medicinal and Process Chemistry, CSIR−Central Drug Research Institute, Lucknow 226 031, Uttar Pradesh, India

^§Division of Pharmacokinetics and Metabolism, CSIR−Central Drug Research Institute, Lucknow 226 031, Uttar Pradesh, India

^∥Division of Molecular and Structural Biology, CSIR−Central Drug Research Institute, Lucknow 226 031, Uttar Pradesh, India

^⊥Division of Biochemistry, CSIR−Central Drug Research Institute, Lucknow-226 031, Uttar Pradesh, India

S

* Supporting Information

ABSTRACT: Antileishmanial activities of a library of synthetic chalcone analogues have been examined. Among them, ﬁve

compounds (11, 14, 16, 17, 22, and 24) exhibited better activity than the marketed drug miltefosine in in vitro studies against the

intracellular amastigotes form of Leishmania donovani. Three promising compounds, 16, 17, and 22, were tested in a L. donovani/

hamster model. Oral administration of chalcone 16, at a concentration of 100 mg/kg of body weight per day for 5 consecutive

days, resulted in >84% parasite inhibition at day 7 post-treatment and it retained the activity until day 28. The molecular and

immunological studies revealed that compound 16 has a dual nature to act as a direct parasite killing agent and as a host

immunostimulant. Pharmacokinetics and serum albumin binding studies also suggest that compound 16 has the potential to be a

candidate for the treatment of the nonhealing form of leishmaniasis.

transforming growth factor (TGF)-β.⁶These suppressive

molecules distort the healing responses by repression of host-

protective microbicidal molecules including cytokines like

INTRODUCTION

■

Leishmaniasis, an infectious disease caused by protozoan

parasites belonging to the genus Leishmania, is transmitted to

interferon (IFN)-γ, IL-1, IL-12, and tumor necrosis factor-α

humans through the bite of female phlebotomine sand ﬂies

infected with the parasite.¹It is classiﬁed as cutaneous,

(TNF-α) and reactive nitrogen and oxygen intermediates (RNI

and ROI).⁵⁻⁷One of the major complications for the

mucocutaneous, and visceral (kala azar) forms depending on

chemotherapeutic treatment of visceral leishmaniasis (VL) is

the parasite species and cellular immune system of the

patient.^1,2It has been recognized by the World Health

the depressed immune function exhibited by patients. There-

Organization (WHO) as an increasing health problem.³

fore, induction of protective immune responses in the infected

Many parts of Asia and Africa are vulnerable to leishmaniasis.⁴

host by any therapeutic agent/immunomodulator is essential

for the successful treatment of VL. The ﬁrst-line treatment

options for leishmaniasis are limited and involve the

Leishmania parasite has evolved several skills to inactivate the

protective immune machinery of hosts to survive inside the

cell.⁵The outcome of Leishmania infection depends on the

proliferation of T-helper 2 (Th2) cell population which is

associated with the production of interleukin (IL)-10 and

Received: December 5, 2013

Published: March 17, 2014

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administration of pentavalent antimonials (sodium stibogluco-

nate (SSG) and meglumine antimoniate (MA)) and amphoter-

icin B.⁸Second line drugs include paromomycin and

miltefosine, but these drugs have not experienced widespread

use due to the severe toxicities, parenteral administration, and

resistance issues.⁸Pentavalent antimonials, which were

developed in 1940s, are presenting high toxicity and low

eﬃcacy. Presently, more than 60% of VL patients in Bihar

(India) are unresponsive to the antimonials.⁹Amphotericin B

and its formulations are quite eﬀective for VL, however, these

are very expensive, highly toxic, and have a longer half-life.¹⁰

Pentamidine presents several side eﬀects, including renal and

hepatic toxicity, pancreatitis, hypotension, and cardiac

abnormalities.¹¹Paromomycin has limited use in the treatment

of VL and also presents ototoxicity and hepatotoxicity.¹⁰

Miltefosine, an oral drug, also suﬀers from nephrotoxicity,

hepatotoxicity, and teratogenicity.¹⁰Up to now, no vaccine is

approved for human use.¹²Therefore, there is an increasingly

urgent need for the development of new, inexpensive, eﬀective,

and safe drugs for the treatment of leishmaniasis and then the

discovery of new lead compounds for this disease is a pressing

concern for global health programs.

antituberculosis,²⁰anti-HIV,²¹and antifungal activities.²²

A

thorough assessment of structural requirements for antileish-

manial activities of chalcones is vital to the development and

design of a novel drug-like candidate. Chalcones are natural-like

compounds, and as such they can show multitarget proﬁle

similar to other compounds described in the literature.²³

Licochalcone A (II), is an oxygenated chalcone (Figure 1),

isolated from Chinese licorice, eﬃciently inhibits proliferation

of Leishmania donovani and Leishmania major promastigotes

and amastigotes in vitro by interfering with the function of the

parasite mitochondria. In vitro tests have revealed that

licochalcone A at lower concentrations inhibits phytohemmag-

glutinin A-induced proliferation of human lymphocytes.²⁴

Preliminary studies reveal that changes of the substitution

pattern of the chalcones appear to aﬀect the activities against

Leishmania promastigotes and lymphocytes diﬀerently, indicat-

ing that it should be possible to prepare chalcones with a high

selectivity.

As a part of our drug discovery program on antileishmanial

agents from Indian medicinal plants, we reported the isolation

of three chromenodihydrochalcones, crotaramosmin (III),

crotaramin (IV), and crotin (V) (Figure 1) from Crotalaria

ramosissima,²⁵synthesis, and in vitro antileishmanial activity of

III−V and analogues thereof.^26,27In continuation of that

program, a series of new chalcones with various structural

features were synthesized and evaluated for their in vitro

antileishmanial activity. Several of these compounds were found

to be more active in the in vitro screening; few of them were

further evaluated for in vivo antileishmanial eﬃcacy in a

hamster model. Herein, we report a comprehensive assessment

of antileishmanial activity, structure−activity relationship

(SAR) analyses, pharmacokinetics (PK), and mode of action

studies of promising chalcones.

Chalcones, or 1,3-diaryl-2-propen-1-ones (Figure 1), are

prominent secondary metabolites precursors of ﬂavonoids and

Figure 1. General skeleton and naturally occurring antileishmanial

chalcones.

RESULTS AND DISCUSSION

■

Chemistry. Synthesis of Chromenochalcones. A large

number of chromenochalcones were prepared to evaluate their

antileishmanial activity. The acetyl/carboxaldehyde chromenes

3 and 4 were synthesized using pyridine-catalyzed condensation

between 2,4-dihydroxyacetophenone (1) or 2,4-dihydroxy-

benzaldehyde (2) and citraldimethylacetal.²⁸The resultant

acetyl chromene 3 and substituted aromatic aldehydes or p-

isoﬂavonoids in plants. Chemically, they consist of open-chain

ﬂavonoids in which the two aromatic rings are joined by a

three-carbon α,β-unsaturated carbonyl system.¹³Natural and

synthetic chalcones are described in the literature with diﬀerent

pharmacological proﬁles, such as antiinﬂammatory,¹⁴antibacte-

rial,¹⁵antiviral,¹⁶antimalarial,¹⁷anticancer,¹⁸antileishmanial,¹⁹

Scheme 1. Synthesis of Chromenochalcones (5−13) and Alkylated Amine Containing Chromenochalcone (14)

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Table 1. In Vitro Antileishmanial Activity of Chromenochalcones against Promastigotes and Amastigotes of L. donovani and

Their Cytotoxicity

a

b

Values are represented as average of at least duplicate measurements (SD 2%). IC₅₀values are represented as average of at least duplicate

c

d

measurements (SD 10%). CC₅₀values are represented as average of at least duplicate measurements (SD 10%). SI (selectivity index) = CC₅₀/

IC₅₀, ND = not done.

Scheme 2. Synthesis of Chromenochalcones with the Hetero Atoms in Ring A (16−18)

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Scheme 3. Synthesis of Chromanochalcones (20−22)

Scheme 4. Synthesis of Chromenodihydrochalcone and N-Acetyl Pyrazoline Derivative (23 and 24)

hydroxybenzene-1,3-dicarbaldehyde²⁹and chromene carbox-

aldehyde 4 and appropriate acetophenones 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 5−13 in good yields

(Scheme 1, and Table 1).²⁷To improve the bioavailability of

compounds, the alkylated amine groups were introduced in

chromenochalcone core. The synthesis of alkylated amines

containing chromenochalcone (14) was accomplished by the

replacement of the hydrogen of the phenolic hydroxyl groups

of the chromenochalcones 7 with the alkyl part of the

corresponding amines using K₂CO₃in dry acetone (Scheme

1).³⁰

Synthesis of Chromenochalcones with Hetero Atoms in

Ring-A. Chromenochalcones 16−18, which have hetero atoms

in ring-A (Scheme 2), were synthesized using Claisen−Schmidt

condensation from acetyl chromens²⁸3 and 15 and various

heteroaryl aldehydes to oﬀer the compounds 16−18 in good

yields.

Synthesis of Chromanochalcones. We wanted to study the

benzopyran core eﬀect in chromenochalcones; therefore, a few

chromanochalcones (20−22) were synthesized. For synthesis

of chromanochalcones 20−22, intermediate 3 was used as

starting material, which smoothly reduced to acetyl chroman 19

with H₂/Pd/C²⁷and subsequently subjected to Claisen−

Schmidt condensation with aromatic aldehydes to obtain the

desired chromanochalcones 20−22 (Scheme 3).

Synthesis of Chromenodihydrochalcones and N-Acetyl

Pyrazoline Derivative. We wanted to synthesize the

chromenodihydrochalcone 23 and N-acetyl pyrazoline 24 to

determine the role of the α−β oleﬁnic bond, which connects

the ring-A and carbonyl carbon (Figure 1). The chromenodihy-

drochalcone 23 was prepared from compound 16 by

regeoselective hydrogenation with NaBH₄/NiCl₂.6H₂O as

shown in Scheme 4. Reaction of the chromenochalcone 16

with hydrazine hydrate in acetic acid under stirring and reﬂux

conditions led to the formation of the N-acetyl pyrazoline

derivative 24 (Scheme 4) in good yield.

antileishmanial activity of a few natural and synthetic

chromenochalcones.^26,27This work had shown that the

benzopyran core and nature of substitutions on ring A are

very crucial for potent antileishmanial activity. On the basis of

these optimistic results, a library of new chalcones was prepared

and their antileishmanial activity against WHO reference strain

(MHOM/IN/80/Dd8) of extracellular (62−100% of inhibition

at 25 μM) and intracellular (IC₅₀= 1.7−20 μM) form

(expressing luciferase ﬁreﬂy reporter gene) of L. donovani were

evaluated. In parallel, the cytotoxicity of the compounds was

also tested using mammalian kidney ﬁbroblast cells (Vero cell

line) for the compounds, which have IC₅₀= <20 μM against

amastigotes. Among these, a few promising compounds were

further tested for their in vivo activity against the MHOM/IN/

80/Dd8 strain of L. donovani in a hamster model. Standard

antileishmanial drugs, miltefosine and sodium stibogluconate

(SSG), were included in this study as control drugs. The

molecular, immunological, pharmacokinetics, and serum

albumin binding studies were also conducted for most

promising compound (see below) (Tables 1−4).

In Vitro Antileishmanial Activity of Chromenochalcones.

In continuation of our work²⁷(Table S1, Supporting

Information), we introduced the EWD’s and fused hetero

cyclic moieties on ring A and maintained the same prenyl unit

on benzopyran core as ring B of chromenochalcones 5−11

(Table 1). The introduction of aldehyde group into ring-A as in

chalcone 6 eﬀected the in vitro activity against amastigotes (5,

IC₅₀= 16.5 μM, vs 6, IC₅₀> 20 μM and 7 IC₅₀= 10.7 μM).

Because hydrogen bonding aﬀects membrane transport, as well

as the distribution of compound, 6 and 7 exhibited diﬀerent

activity due to presence and/or absence of chelation (between

OH and CHO). Due to presence of aldehyde group in 7, the

activity was increased; at the same time the selectivity index

(SI) was decreased by two times in comparison to 5 (Table 1).

It is noteworthy that the insertion of fused hetero cycles to ring

A as in chalcones 8−10 and the interchange of ring A and B as

in 12 and 13 led to diminished activity against amastigotes

(IC₅₀> 20 μM). Interestingly, chalcone 11, which contains the

oxazolidine moiety on ring A, has an improved in vitro activity

(IC₅₀= 5.8 μM) and toxicological proﬁle (CC₅₀> 400 μM)

Biological Activity. In our eﬀorts to develop new

antileishmanial agents, we had earlier reported the in vitro

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Table 2. In Vitro Antileishmanial Activity of Chromanochalcones with Hetero Atoms on Ring A against Promastigotes and

Amastigotes of L. donovani and Their Cytotoxicity

a

b

Values are represented as average of at least duplicate measurements (SD 2%). IC₅₀values are represented as average of at least duplicate

c

d

measurements (SD 10%). CC₅₀values are represented as average of at least duplicate measurements (SD 10%). SI (selectivity index) = CC₅₀/

IC₅₀, ND = not determined.

Table 3. In Vitro Antileishmanial Activity of Chromanochalcones against Promastigotes and Amastigotes of L. donovani and

Their Cytotoxicity

a

b

Values are represented as average of at least duplicate measurements (SD 2%). IC₅₀values are represented as average of at least duplicate

c

d

measurements (SD 10%). CC₅₀values are represented as average of at least duplicate measurements (SD 10%). SI (selectivity index) = CC₅₀/

IC₅₀, ND = not determined.

against amastigotes and Vero cells, respectively. To improve the

bioavailability of chromenochalcone 14, which has an alkylated

amine substituents on both rings, was synthesized (Scheme 1)

and evaluated for antileishmanial activity as shown in Table 1.

This disubstituted analogue 14 showed 99.87% inhibition of

promastigotes at 25 μM concentration with IC₅₀of 6.3 μM and

good toxicological proﬁle (SI = 31.9) against amastigotes and

Vero cells, respectively (Table 1). The introduction of alkylated

amine substituents on ring A and B (14, IC₅₀= 6.30 μM, vs 5,

IC₅₀= 16.5 μM against amastigotes) signiﬁcantly increased the

activity proﬁle.

In Vitro Antileishmanial Activity of Chromenochalcones

with Hetero Atoms on Ring A. After studying the eﬀect of the

nature of various substitutions and their positions on ring A and

benzopyran moiety (Ring B) of chalcones (Table 1), we

enthusiastically moved to work on chromenochalcones with

hetero atoms on ring A. We synthesized few chromenochal-

cones 16−18 with hetero atoms in ring A (Scheme 2) and

evaluated their in vitro antileishmanial activity (Table 2). All

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the analogues exhibited potent activity against promastigotes

(99.5−100% inhibition at 25 μM) (Table 2). On the other

hand, compounds 16 and 17, with the exception of 18 (toxic to

cells), were very potent (IC₅₀< 5.4 μM) against amastigotes

and also have good SI. Alkylated amine substituents on

chromanochalcons 17 (IC₅₀= 1.7 μM) increased the activity 3-

fold against amastigotes and SI was increased 6−8 times that of

reference drugs (Table 2).

Table 4. In Vitro Antileishmanial Activity of

Chromenodihydrohalcone and N-Acetyl Pyrazoline against

Promastigotes and Amastigotes of L. donovani and Their

Cytotoxicity

In Vitro Antileishmanial Activity of Chromanochalcones.

To ﬁnd out the role of the oleﬁnic bond in benzopyran moiety,

chromanochalcones 20−22 which contained the chroman ring

(dihydrobenzopyran) were prepared (Scheme 3) and screened

for their in vitro antileishmanial activity (Table 3).

Chromanochalcones 20 and 21, which have the EDGs on

ring-A, showed moderate antileishmanial activity against

promastigotes (62.3−88.9% inhibition at 25 μM). Chroma-

nochalcones 20 and 21 (IC₅₀> 20 μM) are less potent than the

in vitro activity

antipromastigote

(% inhibition at

antiamastigote

cytotoxicity

index

d

a

b

c

compd

23

25 μM)

IC₅₀(μM)

CC₅₀(μM)

(SI)

98.5

99.5

11.0

6.4

265.5

88.5

24.1

13.8

>8.0

6.2

24

SSG

no inhibition

49.7

8.4

>400

52.5

chromanchalcone, which has heteroatom in ring A (22, IC₅₀

=

miltefosine

100

0

a

5.7 μM, vs 20 and 21, IC₅₀> 20 μM) and selectivity by 3−6

times than reference drugs (Table 3). From the above data, it is

very clear that the in vitro activity and toxicological proﬁles

were signiﬁcantly improved after the incorporation of the

heteroatom in ring A in chromenochalcones (Table 2) as well

as chromanochalcones (Table 3).

Values are represented as average of at least duplicate measurements

b

(SD

2%). IC₅₀values are represented as average of at least

c

duplicate measurements (SD 10%). CC₅₀values are represented as

average of at least duplicate measurements (SD

(selectivity index) = CC₅₀/IC₅₀.

d

10%). SI

In Vitro Antileishmanial Activity of Chromenodihydro-

chalcones and N-Acetyl Pyrazoline Derivative. To determine

the role of the α−β oleﬁnic bond, chromenodihydrochalcone

23 was synthesized from 16 by selective reduction. The

chromenodihydrochalcone 23 showed a lower order of activity

(23, IC₅₀= 11.0 μM, vs 16, IC₅₀= 5.4 μM) and better toxicity

proﬁle than lead compound 16 (23, CC₅₀= 265.5 μM, vs 16,

CC₅₀= 40.7 μM). These results supported the importance of

Michael acceptor (α,β-unsaturated ketone) moiety in the

inhibition of parasite burden. N-Acetyl pyrazoline derivative 24

was prepared to mask the Michael system by reacting 16 with

hydrazine. Interestingly, N-acetyl pyrazoline derivative 24 was

equipotent to the parent compound 16 against parasites (24,

IC₅₀= 6.4 μM, vs 16, IC₅₀= 5.4 μM) and its selectivity index

increased 2-fold over parent compound 16 (24, SI = 13.8 vs 16,

SI = 7.5), which reiterates the importance of Michael system

(Table 4).

In Vivo Eﬃcacy of Chalcones against L. donovani/

Hamster Model. Out of six compounds (11, 14, 16, 17, 22,

and 24), which exhibited potent amastigote inhibiting activity,

only three compounds (16, 17, and 22) were chosen for further

in vivo studies after assessing their structural features³¹and

tested against the MHOM/IN/80/Dd8 strain of L. donovani in

a hamster model.³²The aqueous suspensions of tested

compounds were administered for 5−10 consecutive days at

50 and/or 100 mg/kg/day either by intraperitoneal (IP) or oral

(PO) route. The post-treatment splenic biopsies were done on

day 7 and day 28 after the last dose administration, and

amastigote counts were assessed by Giemsa staining.

Compounds 22 showed percentage inhibition of 43.92

10.90 on day 7 post-treatment (pt) at 50 mg/kg/day by oral

route (Table 4), and these animals did not survive until day 28

administration, respectively, and this inhibition was retained till

day 28 pt and most of the animals survived after the treatment

(Table 5).

Apoptotic−Necrotic Proﬁling to Determines the Phospha-

tidylserine Exposure in Leishmania Promastigotes by

Compound 16. Apoptosis is a physiological phenomenon

that induces the cells toward natural death. The induction of

apoptosis in a parasite by any therapeutic agent leads to

reduced parasitic burden.³³To ascertain the eﬀect of

compound 16 to induce the early apoptosis in Leishmania

promastigotes, apoptotic−necrotic proﬁling was carried out. In

the early apoptotic stage, the membrane phospholipid,

phosphatidylserine (PS) is translocated from the inner side of

the cell membrane to the cell surface and exposed to the

external cellular environment. Annexin V-FITC which labels PS

sites and represent apoptotic cell death was used with

propidium iodide (PI). PI was used as a counterstain to

diﬀerentiate apoptotic cells from necrotic cells. The dot plots of

Annexin V- FITC and PI staining have been presented in

Figure 2. The percentages of promastigotes in early apoptotic

phase (lower right quadrant) due to eﬀect of compound 16 at

various time points were determined by the comparison of

treated promastigotes with that of untreated control. It was

observed that in untreated control, 13.70% promastigotes were

annexin V positive. The promastigotes treated with IC₅₀

concentration (0.78 μM against promastigotes) of compound

16 for 72 h showed the highest number of early apoptotic cells

(>52%) in comparison to untreated control. At 6, 12, 24, and

48 h time points, compound 16 induces 13.34, 15.23, 17.09,

and 23.92% promastigotes, respectively, for early apoptosis

when compared with the untreated counterpart. It was also

observed that this compound triggers maximum PS exposure to

annexin V- FITC at late time points (between 48 and 72 h).

Interestingly, there was no role of necrosis observed during

apoptotic−necrotic proﬁling. Our results clearly indicate that

compound 16 exerts leishmanicidal activity via apoptosis in

promastigotes.

pt. The chalcone 17 (73.19

10.90 at 50 mg/kg/day) had

shown good inhibition on day 7 by oral route, unfortunately, in

this case also, inhibition was not retained until day 28 and

dropped to half (Table 5). Remarkably, compound 16 showed

the best dose dependent in vivo eﬃcacy among all the tested

analogues with an average inhibition of 48.98 12.63% and

84.74 11.95% on day 7 at 50 and 100 mg/kg/day by oral

Compound 16 Triggers the Drop of Mitochondrial

Membrane Potential in Leishmania Promastigotes. A

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a

Table 5. In Vivo Eﬃcacy of Chalcones against L. donovani/Hamster Model

compd

16

dose (mg/kg)

treated days

route

% inhibition SD at day 7 (n = 5)

% inhibition SD at day 28

50

100

50

5

PO

IP

48.98 12.63

84.74 11.95

73.19 10.90

43.92 10.90

89.47 4.72

98.50 1.10

55.37 12.15 (n = 5)

84.49 10.02 (n = 4)

49.98 14.19 (n = 4)

NA

16

17

5

22

50

10

5

SSG

40

72.51 4.14 (n = 4)

83.20 3.44 (n = 5)

miltefosine

30

5

PO

a

SD, standard deviation; NA, not available, IP, intraperitoneal; PO, per oral, SSG, sodium stibogluconate; n = number of animals.

Figure 2. Detection of apoptosis in Leishmania promastigotes by annexin V-FITC and propidium iodide (PI) double staining. (a) Untreated

promastigotes, (b) 6 h treatment with compound 16, (c) 12 h treatment with compound 16, (d) 24 h treatment with compound 16, (e) 48 h

treatment with compound 16, and (f) 72 h treatment with compound 16. The lower left quadrant shows unstained living cells (Annexin V⁻/PI⁻),

the lower right shows early apoptotic cells (Annexin V⁺/PI⁻), the upper left shows necrotic cells (Annexin V⁻/PI⁺) and the upper right indicates late

apoptotic cells (Annexin V⁺/PI⁺). Data presented here are the representative of three independent experiments.

mitochondrial enzyme, fumarate reductase (FRD), is a known

target of chalcone derivatives.²⁴As FRD does not exist in

mammalian cells, chalcones selectively inhibited protozoan

FRD, which play key role in the respiratory chain of Leishmania

parasite. The loss in mitochondria membrane potential (MMP)

is a distinctive feature of cell death by apoptosis due to the

action of drugs. The outcome of our results proved that

compound 16 was responsible for loss in membrane potential.

The drop in MMP in the treated promastigotes showed that

they are undergoing apoptosis due to loss in MMP, blocking

the entry of JC-1 dye into the mitochondria and ﬂuorescing

green. The results are depicted in the Figure 3 in the form of

dot plots. Our results demonstrated that gradual increases in

MMP drop were observed in the Leishmania parasite when

promastigotes were incubated with compound 16 at IC₅₀

concentration (0.78 μM against promastigotes) for 6−72 h,

respectively. The highest drop (lower quadrant, green

ﬂorescence) was observed in 72 h post treatment (63.72%)

when subtracted with drop in untreated promastigotes (5.87%).

The incubation of promastigotes with compound 16 for 6, 12,

24, and 48 h have shown 4.6, 11.59, 26.01, and 55.8% drop in

membrane potential, respectively, in comparison with untreated

parasite. These data indicate that compound 16 might be target

FRD to trigger MMP drop.

Compound 16 Induces a Host-Protective Cytokines

Response by Leishmania-Infected Macrophages. Host

immune responses play important role on the eﬀectiveness of

any chemotherapeutic agent against leishmaniasis. Leishmania

infection is classically associated with depression of Th1 type

immune cells and expansion of Th2 type cells.⁶Hence,

compounds that could boost up the host cell activation by Th1

biased immune responses could be exploited as therapeutic

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Figure 3. Drop in mitochondrial membrane potential in Leishmania promastigotes treated with compound 16 at diﬀerent time points. (a) Untreated

promastigotes, (b) 6 h treatment, (c) 12 h treatment, (d) 24 h treatment, (e) 48 h treatment, and (f) 72 h treatment with compound 16. Upper

quadrant shows red ﬂuorescent cells (no drop in mitochondrial membrane potential) and lower quadrant indicates drop in mitochondrial membrane

potential (green ﬂuorescent cells). Results are the representative of three independent experiments.

Figure 4. Normal, infected-untreated, and infected-treated mouse macrophages (J-774A.1) were analyzed for Th1 cytokines (a) IL-12 and (b) TNF-

α and Th2 cytokines (c) IL-10 and (d) TGF-β by sandwich ELISA after 24 h post treatment. The levels of cytokine release in the culture supernatant

were measured in pg/mL concentration. The signiﬁcance between infected control versus compound 16 treated group was calculated by Student’s t

test using graph pad Prism (IC versus 16, p < 0.01**). Results are the mean SD of two independent experiments in duplicates. Miltefosine (MF)

was used as a reference drug.

agent for VL. In this context, we have also explored the

potential of compound 16 as a host-protective immune cells

activator. We have chosen hallmark Th1 (IL-12 and TNF-α)

and Th2 (IL-10 and TGF-β) cytokines produced by macro-

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phages which have up- and down-regulated during treatment of

Leishmania infection. In Leishmania-infected mouse macro-

phages (J-774A.1) treated with compound 16, we have found

16-fold higher production of IL-12 (Figure 4a, p < 0.01**) and

11.6-fold increase in the production of TNF-α (Figure 4b, p <

0.01**) than infected control. In contrast, among the Th2 type

cytokines (Figure 4c,d), we have observed considerably (p <

0.01**) lower production of IL-10 (7.11-fold less than infected

control) and the level of TGF-β was also down-regulated

signiﬁcantly (p < 0.01**) in compound 16 treated cells (4.08-

fold less production than infected-untreated macrophages).

These results suggested that compound 16 has immunostimu-

latory potential along with direct parasite killing action.

due to poor pH stability of the drug. Dose absorption from the

GI tract will aﬀect if drug has instability in the stomach or

intestine. The stability of a drug substance in gastric and

intestinal ﬂuids provides support whether drug degradation

occur in the GI tract before membrane permeation. Compound

16 was found to be stable in gastric ﬂuids, but it shows 30%

degradation in the intestinal ﬂuids (Figure 6).

Compound 16 Provides Stimulus to Leishmania Infected

Mouse Macrophages for Nitric Oxide Production. Nitric

oxide (NO) is a potent microbicidal molecule which plays a

critical role in the controlling of Leishmania infection.⁵Along

with IL-12 and TNF-α, NO is also produced from macrophages

to kill the Leishmania parasite by oxidative burst.⁷We have

estimated the generation of NO by mouse macrophage cells (J-

774A.1) in culture supernatant after 24 h post treatment. Our

results indicate that Leishmania-infected compound 16-treated

macrophages showed 6.3-fold induction in NO generation

when compared with corresponding infected control (Figure 5,

Figure 6. Simulated gastric ﬂuid (SGF) and simulated intestinal ﬂuid

(SIF) stability of compound 16. Results are presented as mean

SEM.

In Vitro Metabolic Stability. A major part of drug loss in the

body is due to metabolism in the liver. Drug metabolism in liver

plays a signiﬁcant role in clearance of the drug from the body.

Most of the time poor oral bioavailability is due to ﬁrst pass

metabolism in the liver. Testosterone was employed to assess

the activation of the hamster liver microsomes. The half-life of

the testosterone was found as per previously reported literature

value. Compound 16 was found to be stable in the control

reaction in the absence of cofactor and conﬁrmed the chemical

stability as well as cofactor dependent degradation. The

calculated in vitro half-life for compound 16 was 37.78

5.38 min (Figure 7).

Figure 5. Generation of nitric oxide (NO) in μM concentration from

normal, infected-untreated, and infected-treated mouse macrophages

(J-774A.1) was analyzed by Griess reagent at 24 h post treatment. The

absorbance of the reaction product was measured at 540 nm using a

spectrophotometer. The signiﬁcance between infected control versus

compound 16 treated group was calculated by Student’s t test using

Graph Pad Prism (IC versus 16, p < 0.01**). Results are the mean

SD of two independent experiments. Miltefosine (MF) and lip-

opolysaccharide (LPS) were used as a reference drug and as a

stimulant, respectively.

p < 0.01**). Interestingly, it was also observed that compound

16 induces infected macrophages to production of nitric oxide

(26.53 2.6 μM) slightly higher than that of standard drug,

Figure 7. Metabolic stability of compound 16. Results are

representative of three independent experiments. Data are presented

as mean SEM.

miltefosine treated macrophages (25.35

2.5 μM). Signiﬁ-

cantly enhanced production of NO in treated macrophages also

suggests that compound 16 activated mouse macrophages for

successful elimination of Leishmania parasite.

In Vitro Pharmacokinetic (SGF−SIF and Metabolic

Stability) Investigations of Compound 16 in Golden

Hamsters. Compound 16 showed consistent in vivo activity

at day 7 and day 28 after the last dose administration (Table 5)

in the hamster model. To evaluate stability upon oral dosing,

simulated gastric ﬂuid (SGF)/simulated intestinal ﬂuid (SIF)

and metabolic stability studies of the drug candidate were

performed in vitro.

Oral Pharmacokinetics of Compound 16 in Hamsters.

Oral pharmacokinetics studies were performed in hamsters, as

it was the experimental model for VL. Chalcone 16 was

administered as a single oral dose of 100 mg/kg, and analysis

was done using the HPLC−UV method to get the plasma

concentration−time proﬁle. The pharmacokinetics studies

showed no abnormality in the animals as hamsters well

tolerated the given dose. Compound 16 was rapidly absorbed

by oral route (Figure 8). It showed high V_d/F, indicating

peripheral distribution (Table 6). The active site is usually

spleen and peripheral tissue, thus the compound may have

In Vitro SGF−SIF Stability Studies. SGF and SIF stability

studies conducted in order to determine compound stability in

gastrointestinal (GI) ﬂuids. Poor ADME properties can occur

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good distribution at the target site. The compound 16 showed

two T_max(0.625 0.3 and 0.78 0.21 h) and two C_max(1.47

0.11 and 0.82 0.18 μg/mL) at 100 mg/kg, oral dose. These

results indicated that compound 16 has high permeability

across the gastrointestinal epithelium. The plasma concen-

tration of 16 showed double peak phenomenon, possibly due to

enterohepatic recirculation or variability in absorption site

(Figure 8). Half life (t_1/2) of 16 was observed at 7.4 h in plasma.

Thus the pharmacokinetic behavior of compound 16 indicated

very good oral bioavailability to exhibit antileishmanial activity.

Binding Characteristics of Compounds 16 with Proteins.

By mean of intrinsic ﬂuorescence quenching, we can ﬁnd out

the binding characteristics of small compounds with proteins.

Emission spectra of bovine serum albumin (BSA) (excited at

280 nm) showed a characteristic peak at 343 nm. Addition of

successive amounts of compound 16 resulted in a gradual

decrease in ﬂuorescence intensity as shown in Figure 9a. The

decrease in ﬂuorescence intensity is usually described by the

Stern−Volmer equation:³⁴

Figure 8. Mean plasma concentration−time proﬁle of compound 16

in male Syrian golden hamsters (100

15 g) after a single oral

administration of (100 mg/kg). Results are presented as mean SEM.

Table 6. Pharmacokinetic Estimation of Compound 16 in

Hamsters after Single Oral Dose (100 mg/kg)

a

Administration

F /F = 1 + K_SV[Q] = 1 + K_qτo[Q]

0

parameter

units

estimates

where F₀and F are the ﬂuorescence intensities before and after

the addition of the compound 16 or quencher, respectively, K_SV

is the dynamic quenching constant, K_qis the quenching rate

constant, [Q] is the concentration of compound added, τ₀is the

average lifetime of the molecule without quencher, and its value

is considered to be 10⁻⁸s.³⁵The Stern−Volmer plot (Figure

9b) shows a good linear relationship within the experimental

concentrations.

T_max1

C_max1

T_max2

C_max2

AUC_0−∞

Cl/F

V_d/F

t_1/2

h

0.625 0.3

1.47 0.11

0.78 0.21

0.82 0.18

3.7 0.67

μg/mL

h

μg/mL

h·μg/mL

L/h/kg

L/kg

27.84 5.24

391.73 79.32

7.4 0.99

h

We observed K_SV, 2.7 × 10⁵L mol⁻¹(R, 0.9984), and K_q, 2.7

× 10¹³L mol⁻¹s⁻¹, for the compound 16. In present study, we

observed K_qvalue higher than the maximum value of the

scattering collision quenching constant (2.0 × 10¹⁰L mol⁻¹

a

T_max, time at which maximum concentration achieved in plasma; C_max

maximum concentration achieved in plasma; AUC, area under the

curve from 0 to ∞ h; V_d, volume of distribution; Cl, clearance; t_1/2

terminal half-life. Each value represents mean SD.

,

Figure 9. (a) Addition of successive amount of compound 16 resulted in to gradual decrease in ﬂuorescence intensity (b) Stern−Volmer Plots, and

(c) modiﬁed Stern−Volmer plots, and (d) double logarithm plot.

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s⁻¹) of biomolecules,³⁶indicating that the probable quenching

mechanism of compound 16−BSA interaction was initiated by

complex formation. Also, dynamic collisions contribute to the

quenching of ﬂuorescence as shown by dynamic quenching

constant.

To conﬁrm the mode of compound 16−BSA interaction as a

static complex formation indicated by quenching, the diﬀerence

absorption spectroscopy was carried out. The absorption

spectrum of BSA and the diﬀerence absorption spectrum

between BSA−compound 16 complex and compound 16 at the

same concentration could not be superposed (Figure 10).

range of 10⁵L mol⁻¹indicated a strong binding with serum

albumin and value of the binding sites (n) was approximately 1,

which showed that the ground-state complex formation is

seemed to be due to single high aﬃnity binding site on BSA.

These studies indicated a high binding aﬃnity of compound 16

toward serum protein. Thus, the bound fraction of compound

16 might be acting as reservoir from which it is slowly released

in the form of unbound fraction. It is also evident from

compound 16’s long half-life (7.4 h) in oral pharmacokinetic

studies in hamsters (Table 6).

CONCLUSION

■

In conclusion, a series of new chalcone analogues with various

structural features such as chromenochalcones, chromanochal-

cones, chromenodihydrochalcone, and chromenochalcones,

with a heteroatom in ring-A were prepared based on natural

product leads and tested for their antileishmanial activity.

Among all, compounds 11, 14, 16, 17, 22, and 24 were 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 compounds 16, 17, and 22 were performed in L. donovani/

hamster model, in which compound 16 showed consistent

activity up to day 28 post-treatment (84% parasite inhibition at

100 mg/kg × 5 days dose by oral route). The molecular and

immunological studies showed that compound 16 has a dual

nature to act as a direct parasite killing agent and as a host

immunostimulant. Pharmacokinetics and serum albumin bind-

ing studies also suggest that compound 16 has potential to be a

candidate for the treatment of the nonhealing form of

leishmaniasis.

Figure 10. Absorption spectrum between bovine serum albumin

(BSA)−compound 16 complex.

These results probably indicated that compound 16 forms a

ground-state complex with BSA. BSA absorption spectra

showed two peaks: a change in peak around 210 nm is a

result of change in peptide backbone conformation associated

with helix coil transformation, whereas a change around 280

nm is explained by a change in the microenvironment of

aromatic amino acid residues (tryptophan and tyrosine).³⁷

These results showed that compound 16 forms a ground-state

complex with BSA associated with alteration in the

conformation of BSA.

EXPERIMENTAL SECTION

General Methods. Melting points were recorded on Buchi-530

capillary melting point apparatus and are uncorrected. IR spectra were

recorded on Perkin-Elmer AC-1 spectrometer. H NMR spectra were

■

1

recorded on Bruker Avance DPX 200 FT, Bruker Robotics, and Bruker

DRX 300 spectrometers at 200, 300 MHz (¹H), and 50, 75 MHz

(¹³C). Experiments were recorded in CDCl₃, CD₃OD, and DMSO-d₆

at 25 °C. Chemical shifts were given in parts per million (ppm)

downﬁeld from internal standard Me₄Si (TMS). ESI mass spectra were

recorded on 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. Elemental analyses were performed

on a Vario EL-III C, H, N, S analyzer (Germany), and values were

within 0.5% of the calculated values; therefore, these compounds

meet the criteria of >95% purity. Analytical HPLC analyses were

performed on a Shimazadu 10ATVP HPLC instrument, Zorbax C18

column (150 mm × 4.6 mm, 5 μm). A purity of ≥95% has been

established for compounds, which showed good in vitro and in vivo

activity.

1-(5-Hydroxy-2-methyl-2-(4-methylpent-3-enyl)-2H-chromen-6-

yl) ethanone (3). To a magnetically stirred solution of 1 (12.0 g, 79

mmol) in dry pyridine (8.04 mL) was added gradually citraldimethy-

lacetal (15.6 g, 79 mmol) at rt. The whole reaction mixture was

reﬂuxed for 4 h at 150 °C, and an additional equivalent of

citraldimethylacetal (15.6 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 3 (14.45g,

64%). Semisolid; FT-IR (neat, cm⁻¹): 3436, 1695, 1486, 1430, 1373,

Binding Constant and Site. Fluorescence data were

analyzed by Modiﬁed Stern−Volmer³⁸equation for the static

quenching:

F /ΔF = {1/(f K_a[Q])} + 1/f

0

a

ΔF is the diﬀerence of ﬂuorescence in the absence and presence

of compound 16 at concentration [Q], f_ais the fraction of

accessible ﬂuorescence, and K_ais the eﬀective quenching

constant for the accessible ﬂuorophores, which is similar to the

binding constant for the quencher−acceptor systems. The

Modiﬁed Stern−Volmer plot is shown in Figure 9c. The

dependence of F₀/ΔF on the reciprocal value of concentration

[Q]⁻¹is linear with the slope equaling to the value of (f_aK_a)⁻¹.

The equilibrium in static quenching between free and bound

molecules can be given by the equation:³⁹

Log(F − F)/F = log K_A+ nlog[Q]

0

where n is the number of binding sites.

Figure 9d shows the double logarithm plot. From above two

equations, we can calculate the binding constant (K_a) and

number of binding sites (n). We observed calculated K_a, 6.9 ×

10⁵L mol⁻¹(R, 0.9981) and n, 0.90 (R, 0.9973). Our results

showed that the association constant of compound 16 in the

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1

1270, 1086, 758. H NMR (CDCl₃, 300 MHz) δ: 12.99(s, 1H), 7.54

h. After replacement of air by nitrogen, Pd/C was ﬁltered oﬀ and

methanol 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 title compound 16 (375 mg,

(d, J = 8.8 Hz, 1H), 6.76 (d, J = 10.1 Hz, 1H), 6.32 (d, J = 8.8 Hz,

1H), 5.54 (d, J = 10.1 Hz, 1H), 5.10 (t, J = 7.1 Hz, 1H), 2.52 (s, 3H),

2.14 (m, 2H), 1.69−1.80 (m, 2H), 1.67 (s, 3H), 1.59 (s, 3H), 1.40 (s,

3H). MS (ESI): m/z: 287 (M + H)⁺.

1

64%); semisolid. FT-IR (neat, cm⁻¹) 3430, 1714. H NMR (CDCl₃,

1-[5-Hydroxy-2-methyl-2-(4-methyl-pent-3-enyl)-2H-chromen-6-

yl]-3-(4-hydroxy phenyl)-propenone (5). To a stirred solution of 3

(500 mg, 2.2 mmol) in aqueous KOH solution in ethanol (5 mL) was

added 4-hydroxybenzaldehyde (311 mg, 2.2 mmol). The whole

reaction mixture was stirred for 48 h at rt and quenched in ice-cold

water, acidiﬁed with 1 N HCl, and extracted with ethyl acetate (3 × 30

mL). The combined organic layers were washed with water and brine

solution and dried over anhydrous Na₂SO₄, and the 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 5 (346 mg, 45%);

200 MHz) δ 7.48 (d, J = 8.9 Hz, 1H), 6.33 (d, J = 8.9 Hz, 1H), 2.66 (t,

J = 6.8 Hz, 2H), 2.53 (s, 3H), 1.79 (t, J = 6.4 Hz, 2H), 1.35−1.60 (m,

5H), 1.29 (s, 3H), 1.18−1.21 (m, 2H), 0.88 (s, 3H), 0.85 (s, 3H). MS

(FAB) (m/z): 291 (M + H)⁺.

1-(5-Hydroxy-2-methyl-2-(4-methylpent-3-enyl)-2H-chromen-6-

yl)-3-(pyridin-3-yl) propan-1-one (23). To a magnetically stirred

solution of 16 (100 mg, 0.27 mmol) in methanol (5 mL) was added

gradually NiCl₂·6H₂O (64 mg, 0.11 mmol) at rt. The whole reaction

mixture was brought to 0 °C, and NaBH₄(10 mg, 0.27 mmol) was

added portionwise. After addition of NaBH₄, the whole solution was

stirred for 15 min at 0 °C. Methanol was removed by vacuum, and

then the reaction mixture was dissolved in ethyl acetate and

neutralized with 10% HCl solution and the organic layer was washed

with water, dried over anhyd Na₂SO₄, and evaporated under reduced

pressure. Then the crude product was chromatographed on silica gel

to aﬀord the desired compound 24 (60 mg, yield 60%); semisolid. FT-

1

semisolid; FT-IR (Neat, cm⁻¹) 3436, 1635. H NMR (CDCl₃, 200

MHz) δ 13.65 (s, 1H), 7.82 (d, J = 15.3 Hz, 1H), 7.70 (d, J = 8.8 Hz,

1H), 7.53 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 15.3 Hz, 1H), 6.88 (d, J =

8.4 Hz, 2H), 6.79 (d, J = 10.1 Hz, 1H), 6.37 (d, J = 8.8 Hz, 1H), 5.53

(d, J = 10.1 Hz, 1H), 5.09 (t, J = 6.6 Hz, 1H), 2.08 (q, J = 6.6 Hz, 2H),

1.73 (t, J = 4.5 Hz, 2H), 1.65 (s, 3H), 1.56 (s, 3H), 1.43 (s, 3H). MS

(FAB) m/z: 391 (M + H)⁺.

1

IR (KBr, cm⁻¹) 3430, 1635. H NMR (CDCl₃, 300 MHz) δ 12.85 (s,

1H), 8.53 (s, 1H), 8.45 (broad s, 1H), 7.58 (d, J = 7.5 Hz, 1H), 7.48

(d, J = 8.8 Hz, 1H), 7.23(m, 1H), 6.74 (d, J = 10.1 Hz, 1H), 7.30 (dd, J

= 78.8 Hz, 1H), 5.52 (d, J = 10.1 Hz, 1H), 5.01 (t, J = 6.6 Hz, 1H),

3.22 (t, J = 7.5 Hz, 2H), 3.06 (t, J = 7.5 Hz, 2H), 2.05 (m, 2H), 1.74

(m, 2H), 1.65 (s, 3H), 1.56 (s, 3H), 1.41 (s, 3H). MS (FAB) m/z: 378

(M + H)⁺. HRMS: 378.2064 (MH⁺).

2-Hydroxy-5-{3-[5-hydroxy-2-methyl-2-(4-methyl-pent-3-enyl)-

2H-chromen-6-yl]-3-oxo-propenyl}-benzaldehyde (6). To a stirred

solution of 3 (575 mg, 2 mmol) in anhydrous THF (5 mL) was added

portionwise NaH (120 mg, 5 mmol) and stirred for 20 min at rt under

nitrogen. Then p-hydroxy-benzene-1,3-dicarbaldehyde (300 mg, 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 and brine solution, dried over

anhydrous Na₂SO₄, and 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 6 (210 mg, 25%); mp: 105−108 °C. FT-IR

1-(3-(5-Hydroxy-2-methyl-2-(4-methylpent-3-enyl)-2H-chromen-

6-yl)-5-(pyridin-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)ethanone (24). A

mixture of chromenochalcone 16 (100 mg, 0.27 mmol), hydrazine

hydrate (13.5 mg, 0.27 mmol), and acetic acid (2.0 mL) was heated

under reﬂux for 10 h until complete consumption of the chalcone

(TLC control). After cooling, the resulting solution was neutralized

with concentrated ammonium hydroxide. Then the adding of crushed

ice to the solution precipitated a solid which was ﬁltered and washed

with water. The crude product was subjected to silica gel column

chromatography using hexane/ethyl acetate as mobile phase to aﬀord

the title compound 23 (86 mg, 75%); semisolid. FT-IR (neat, cm⁻¹)

3425, 1648, 1640, 1578. ¹H NMR (CDCl₃, 400 MHz) δ 10.53 (s, 1H),

8.55 (m, 2H), 7.55 (d, J = 8.2 Hz, 1H), 7.31 (broad s, 1H), 6.41 (d, J =

8.7 Hz, 1H), 6.81(d, J = 10.1 Hz, 1H), 6.41 (d, J = 8.7 Hz, 1H), 5.62

(d, J = 10.1 Hz, 1H), 5.10 (t, J = 6.5 Hz, 1H), 3.87 (m, 2H), 3.25 (m,

2H), 2.31 (s, 3H), 2.12. (m, 2H), 1.78 (m, 2H), 1.67(s, 3H), 1.57 (s,

3H), 1.43 (s, 3H). MS (FAB) m/z: 432 (M + H)⁺. HRMS: 432.2267

(MH⁺).

1

(KBr, cm⁻¹) 3430, 1635, 1629. H NMR (CDCl_3,300 MHz) δ 13.66

(s, 1H), 11.28, (s, 1H), 9.98 (s, 1H), 7.88 (d, J = 15.4 Hz, 1H), 7.84

(broad s, 2H), 7.74 (d, J = 8.8 Hz, 1H), 7.53 (d, J = 15.4 Hz, 1H), 7.07

(d, J = 8.8 Hz, 1H), 6.82 (d, J = 10.1 Hz, 1H), 6.41 (d, J = 8.7 Hz,

1H), 5.55 (d, J = 10.1 Hz, 1H), 5.11 (t, J = 7.0 Hz, 1H), 2.11 (m, 2H),

1.81 (m, 2H), 1.68 (s, 3H), 1.59 (s, 3H), 1.46 (s, 3H). ¹³C NMR (75

MHz, CDCl₃) δ 196.3, 191.4, 163.3, 160.9, 160.3, 142.0, 136.0, 134.4,

131.9, 130.6, 127.1, 127.0, 123.7, 120.6, 119.3, 118.7, 116.3, 113.8,

109.2, 108.2, 80.4, 41.7, 27.2, 25.6, 22.6, 17.6. MS (FAB) m/z: 419 (M

+ H)⁺. HRMS:419.1843 (MH+).

Experimental Section for Biology. Parasite, Cell Culture, and

Animals. The WHO reference strain of L. donovani (MHOM/IN/80/

Dd8) was maintained as promastigote in vitro in medium 199

supplemented with 10% heat inactivated fetal bovine serum (HIFBS)

1-[2-Methyl-2-(4-methyl-pent-3-enyl)-5-(2-piperidin-1-yl-ethoxy)-

2H-chromen-6-yl]-3-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-prope-

none (14). To a stirred solution of chalcone 5 (250 mg, 1.0 mmol) in

dry acetone (20 mL) were added anhydrous K₂CO₃(2.85 g, 20.8

mmol) and 1-(2-chloro-ethyl)-piperidine hydrochloride (953 mg, 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

at 24

2 °C incubator and as amastigotes in golden hamsters

(Mesocricetus auratus). Adherent mouse macrophage cell line (J774-

A.1) were maintained in RPMI-1640 medium with 10% HIFBS at 37

°C in a 5% CO₂incubator.⁴⁰Healthy, inbred hamsters (initial weight

40−45 g) of both sexes were used in in vivo studies. For oral

pharmacokinetic study, male Syrian golden hamsters (weight 100 15

g) were used. Throughout the study, the animals were housed in

1

the compound 14. Yield: 51%; FT-IR (neat, cm⁻¹) 1652. H NMR

(CDCl₃, 200 MHz) δ 7.67 (d, J = 15.6 Hz, 1H), 7.58 (d, J = 8.6 Hz,

1H), 7.53 (d, J = 8.5 Hz, 2H), 7.42 (d, J = 15.6 Hz, 1H), 6.91 (d, J =

8.5 Hz, 2H), 6.80 (d, J = 10.0 Hz, 1H), 6.61 (d, J = 8.6 Hz, 1H), 5.63

(d, J = 10.0 Hz, 1H), 5.12 (d, J = 6.0 Hz, 1H), 4.14 (t, J = 6.2 Hz, 2H),

3.93 (t, J = 6.3 Hz, 2H), 2.79 (t, J = 6.2 Hz, 2H), 2.62 (t, J = 6.3 Hz,

2H), 2.52−2.38 (m, 12H), 1.73−1.25 (m, 21); ¹³C NMR (CDCl₃, 50

MHz) δ 191.4, 161.1, 158.1, 155.6, 143.2, 132.3, 131.6, 130.5 (2C),

129.7, 128.3, 126.4, 124.6, 124.2, 117.8, 115.3 (3C), 112.8, 79.6, 73.9,

59.0, 58.2, 55.4 (2C), 55.2 (2C), 41.8, 27.1, 26.2 (6C), 24.6 (2C),

23.1, 18.0. MS (FAB) m/z: 613 (M + H)⁺.

climate-controlled (23

2 °C; relative humidity 60%) and

photoperiod-controlled (12 h light−dark cycles) animal quarters.

Animals were fed standard rodent pellet and had free access to

drinking water. All the in vivo studies were performed in compliance

with the Institutional Animal Ethics Committee (IAEC) guidelines for

use and handling of animals.

Promastigote Growth Inhibition Assay. The antileishmanial

activity of these compounds on the extracellular promastigote form

of L. donovani was assessed as described earlier.⁴⁰The late log phase of

promastigotes (expressing ﬁreﬂy luciferase gene) were seeded with

complete M-199 medium at 5 × 10⁵/mL/100 μL/well in 96-well

plates and incubated with tested compounds in a 24 °C incubator for

96 h. Miltefosine was used as a standard drug. After 96 h of incubation,

1-(5-Hydroxy-2-methyl-2-(4-methylpentyl) chroman-6-yl) etha-

none (19). To a solution of 4 (575 mg, 2 mmol) in methanol (10 mL)

was added a catalytic amount of 10% Pd/C. The reaction mixture was

shaken in hydrogenation assembly under hydrogen gas at 50 lbs for 2

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50 μL of promastigote suspension was pipeted out from each well into

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.

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.

Apoptotic−Necrotic Proﬁling with Annexin V and Propidium

Iodide (PI). To assess the apoptotic−necrotic proﬁling of untreated

and drug treated L. donovani promastigotes, an apoptosis detection kit

(Sigma Aldrich, USA) was used as per manufacturer’s instructions.

Exposure of phosphatidylserine due to apoptosis was studied using

annexin V-FITC (ﬂuoroscein isothiocynate) in untreated and treated

promastigotes and a cell impermeable dye, propidium iodide (PI) was

also used to label the cellular DNA in necrotic cells to diﬀerentiate cell

death via apoptosis or necrosis. Brieﬂy, promastigotes of log phase (1

× 10⁶/ mL/well) were seeded in 24-well plates. These promastigotes

(except control) were treated with IC₅₀concentration of test

compound and incubated at 24 2 °C for diﬀerent time points (6,

12, 24, 48, and 72 h). After incubation, treated and untreated

promastigotes were washed with PBS by centrifugation followed by

addition of 5 μL of annexin V-FITC and 10 μL of PI to each tube.

Samples were incubated for 30 min at room temperature. Fluorescence

of cells immediately determined with Cell Quest FACS Calibur

(Becton Dickinson). At least 10000 cells were analyzed for each

sample. Cells stained with annexin V-FITC, and PI were analyzed by

ﬂow cytometry using excitation wavelength, 488 and 536 nm and

emission wavelength, 530 and 617 nm on FL1 and FL2 channel,

respectively.⁴³

Measurement of Mitochondrial Membrane Potential Drop. To

assess the change in mitochondrial membrane potential (ΔΨm), a cell-

permeable, cationic, and lipophilic dye, JC-1 (5′,6,6′-tetrachloro-

1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) (Invitrogen,

USA), was used following the manufacturer’s instruction. JC-1 dye

aggregates in mitochondria and gives red ﬂorescence at higher

membrane potential and at lower membrane potential, and JC-1

cannot bind with mitochondria and remains as monomer form in

cytoplasm and gives green ﬂorescence. Brieﬂy, Leishmania promasti-

gotes in log phase were centrifuged and suspended in complete M-199

medium. A population of 1 × 10⁶promastigotes/mL/well were seeded

in 24-well plates. Test compound in IC₅₀concentration was added in

Reporter Gene-Based 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 plate (4 × 10⁴/mL/100 μL/well)

in RPMI-1640 medium containing 10% HIFBS, and the plates were

incubated at 37 °C in a CO₂incubator (5% CO₂−95% air mixture).

After 24 h, the medium was replaced with fresh medium containing

stationary phase promastigotes (4 × 10⁵/mL/100 μL/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 uninternalized 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 37 °C in a CO₂incubator for 72 h. After incubation, the

drug containing medium was aspirated and 50 μL of phosphate

buﬀered saline (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.⁴⁰The values are expressed as

RLU, and data were transformed into a graphic program (Excel). IC₅₀

value of each compound was calculated by nonlinear regression

analysis of the concentration−response curve using the four-parameter

Hill equation.

Cytotoxicity Assessment Assay. The cytotoxicity of the compounds

was determined by following the method of Mosmann⁴¹with some

modiﬁcations. As described previously,⁴⁰mammalian kidney ﬁbroblast

cells (Vero cell line) (1 × 10⁵/mL/100 μL/well) were incubated with

test compounds (in seven concentrations starting from 400 μM) at 37

°C in a CO₂incubator. After 72 h of incubation, 25 μL of MTT (3-

(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) reagent

(5 mg/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 544 nm on a microplate reader. The

50% cytotoxic concentration (CC₅₀) values were estimated as

described by Huber and Koella.⁴²The selectivity index (SI) for each

compound was calculated as the ratio between cytotoxicity (CC₅₀) in

Vero cells and activity (IC₅₀) against Leishmania amastigotes.

In Vivo Evaluation in L. donovani/Hamster Model. The in vivo

antileishmanial activity was determined in golden hamsters infected

with MHOM/IN/80/Dd8 strain of L. donovani. The method as

described by Gupta et al.³²was used for in vivo evaluation. Golden

hamsters of either sex were infected intracardiacally with 1 × 10⁷

amastigotes per animal. After establishment of infection in 15−20

days, pretreatment spleen biopsy was 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 trials. Five to six infected animals were used for

each test compound. Drug treatment in a diﬀerent dose regimen by

intraperitoneal (IP) or per oral (PO) route was initiated after 2 days of

pretreatment biopsy and continued for 5−10 consecutive days.

Miltefosine and SSG were used as a reference drugs. Post-treatment

biopsies were done on day 7 and day 28 of the last dose

administration, and amastigote counts were 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 percentage inhibition (PI) using the

following formula:

each well (except control well) and incubated at 24

2 °C for

diﬀerent time points (6, 12, 24, 48, and 72 h). After incubation, cells

were washed and resuspended in 1 mL of PBS and incubated with 10

μL of 200 μM JC-1 (2 μM ﬁnal concentration) for 15 min at 37 °C

and analyzed by ﬂow cytometry using 488 nm excitation wavelength

and 530 nm emission wavelength on FL1 channel. Data acquisition

was carried out using a FACS Calibur and analyzed using Cell Quest

Pro software.⁴⁴

Estimation of Th1/Th2 Cytokines. Culture supernatant from

treated and untreated mouse macrophages (J-774A.1 cell line) were

analyzed for production of various cytokines (IL-12, TNF-α, IL-10,

and TGF-β) by using an OptEIA set ELISA kit (BD Biosciences,

USA) according to manufacturer’s instructions. Brieﬂy, 1 × 10⁶mouse

macrophages (J-774A.1) infected with Leishmania parasite (1:10 ratio)

were incubated with tested compounds at IC₅₀concentration in 6-well

plates at 37 °C in a CO₂incubator. Culture supernatants of normal,

infected-untreated, and infected-treated cells from each well collected

after 24 h incubation for estimation of the cytokines level by ELISA as

described previously.⁴⁵At the end of the experiment, the absorbance

was measured at 450 nm in a microplate reader (BioTek instruments,

USA).

Quantiﬁcation of Nitric Oxide (NO). Accumulation of nitrite in

culture supernatant of normal, infected-untreated, and infected-treated

macrophages was detected by the Griess reaction for the quantiﬁcation

of NO generation.⁴⁶Brieﬂy, Leishmania infected macrophage cells (1

× 10⁶/mL/well) were incubated with tested compounds for 24 h

before nitrite assay. LPS (10 μg/mL) was used as a stimulant. After 24

h of incubation, cell supernatant (500 μL) were collected from each

well and mixed with an equal volume of Griess reagent (Sigma Aldrich,

USA) at room temperature. The absorbance of the test samples was

measured at 540 nm in a spectrophotometer (Molecular Devices,

PI = 100 − [(ANAT × 100)/(INAT × TIUC)]

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USA). The concentration of NO in macrophage supernatants was

quantiﬁed by a standard curve generated with sodium nitrite.

absorption spectra of compound with increasing concentration of BSA

were recorded.⁴⁰

In Vitro SGF−SIF Stability. Simulated gastric ﬂuid (SGF)/simulated

intestinal ﬂuid (SIF) mimic GI tract in terms of acidity/basicity and

molarity and are a perfect media to determine the stability of the drug

candidate in vitro. The simulated gastrointestinal ﬂuids were prepared

according to USP speciﬁcations.⁴⁷Brieﬂy, 10 μM of compound was

incubated in SGF and SIF at 0, 15, 30, and 60 min time points for the

gastric stability experiment and at 0, 30, 60, 90, and 120 min for the

intestinal stability experiment. Organic content in reaction mixture was

kept less than 1%. At each time point, 200 μL of sample was taken and

quenched with 200 μL of ice-cold methanol. Samples were processed

using liquid−liquid extraction (LLE) and quantify using validated

HPLC-UV method. Stability results were expressed in % remaining vs

time graph. Calculation was done by % loss with respect to zero

minute time point. The percentage of parent compound remaining at

each time point relative to the 0 min sample is calculated from peak

area ratios.

ASSOCIATED CONTENT

* Supporting Information

Final compounds characterization data and NMR spectra of

selected chalcones, in vitro activity of previously reported

chromenochalcones, and quantiﬁcation of compound 16 by

HPLC-UV method. This material is available free of charge via

the Internet at http://pubs.acs.org.

■

S

AUTHOR INFORMATION

Corresponding Authors

*For N.T.: phone, +91 522 2771940; fax, +91 522 2771941; E-

mail, t_narendra@cdri.res.in.

*For S.G.: phone, +91 9415755899; E-mail, gupta_suman@

yahoo.com.

■

In Vitro Metabolic Stability. In vitro metabolic stability studies

were performed in early drug discovery to choose compounds with

favorable pharmacokinetics based on half-life in liver fractions. The

most commonly used liver fraction S9 consists of both soluble and

membrane bound enzymes and contains a wide variety of both phase I

and phase II enzymes. Incubations were performed in triplicate in test

tube at 37 °C in a benchtop Lab-line shaker (Julabo sw 23) for 60 min.

The incubation was performed at ﬁnal 10 μM concentration.

Incubation reaction mixture contains 2 mg/mL protein hamster liver

S9, 50 mM tris buﬀer, 20 mM MgCl₂, and 2 mM NADPH. Brieﬂy, a

mixture of Tris buﬀer, MgCl₂, protein, and NADPH were

preincubated for 10 min and reaction was initiated with tested

compound. Control reaction without NADPH was also performed to

reveal any chemical instability or noncofactor dependent enzymatic

degradation. A ﬁnal organic content was kept 1% in all the reaction

mixtures. Testosterone was used as positive control to check the

activity of liver fraction. The reaction sample was taken at 0, 5, 10, 15,

20, 30, 45, and 60 min time points. Control reaction samples were

taken at 0, 15, 30, and 60 min. Samples (200 μL) were taken at each

time point and quenched with 200 μL of ice-cold methanol. Samples

were processed using LLE.⁴⁸The S9 fraction stability data is expressed

as % parent remaining at diﬀerent time points relative to the parent at

0 min (100% parent). The data will be ﬁtted to the one-phase

exponential decay equation (A = A₀e^−kt) using GraphPad Prism

software. In vitro half-life (t_1/2) generated by software is reported.

Oral Pharmacokinetics Studies in Golden Hamsters. Oral

pharmacokinetic study was performed in male Syrian golden hamsters.

Compound was administrated to male hamsters orally by gavage (100

mg/kg). Oral formulation was prepared in aqueous suspension. The

blood samples were collected from retro-orbital plexus of the hamsters

under light ether anesthesia into microfuge tubes containing heparin as

an anticoagulant. The samples were taken at 0.08, 0.25, 0.5, 0.62, 1,

1.5, 2, 4, 6, 8, 12, and 24 h postdose. The plasma was separated and

stored at −70 °C until analysis. The amount of compound was

determined in hamster plasma by HPLC-UV method. The plasma

concentration−time proﬁle of compound was analyzed by non-

compartmental method using WinNonlin Version 5.1 (Pharsight

Corporation, USA).

Author Contributions

^#R.S. and V.K. contributed equally to this manuscript.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We are grateful to the Director, CSIR-CDRI, Lucknow, for the

constant encouragement for the program on antiparasitic drug

discovery. We are thankful to SAIF Division for spectral data, A.

L. Vishwakarma, Flow Cytometry Lab, for FACS analysis, and

M. P. S. Negi, Biometry and Statistics Division for statistical

analysis. We also thank to Department of Science and

Technology (DST), New Delhi, for ﬁnancial support in 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. This is CSIR-CDRI communication number 8645.

ABBREVIATIONS USED

■

BSA, bovine serum albumin; FITC, ﬂuoroscein isothiocynate;

FRD, fumarate reductase; IC₅₀, half-maximal inhibitory

concentration; iNOS, inducible nitric oxide synthase; IP,

intraperitoneally; MMP, mitochondrial membrane potential;

NO, nitric oxide; PI, propidium iodide; PO, per oral; SAR,

structure−activity relationship; SGF, simulated gastric ﬂuid;

SIF, simulated intestinal ﬂuid; SSG, sodium stibogluconate; pt,

post treatment; VL, visceral leishmaniasis

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