Synthesis and insight into the structure-activity relationships of chalcones as antimalarial agents

Article abstract of DOI:10.1021/jm300588j

Licochalcone A (I), isolated from the roots of Chinese licorice, is the most promising antimalarial compound reported so far. In continuation of our drug discovery program, we isolated two similar chalcones, medicagenin (II) and munchiwarin (III), from Crotalaria medicagenia, which exhibited antimalarial activity against Plasmodium falciparum. A library of 88 chalcones were synthesized and evaluated for their in vitro antimalarial activity. Among these, 67, 68, 74, 77, and 78 exhibited good in vitro antimalarial activity against P. falciparum strains 3D7 and K1 with low cytotoxicity. These chalcones also showed reduction in parasitemia and increased survival time of Swiss mice infected with Plasmodium yoelii (strain N-67). Pharmacokinetic studies indicated that low oral bioavailability due to poor ADME properties. Molecular docking studies revealed the binding orientation of these inhibitors in active sites of falcipain-2 (FP-2) enzyme. Compounds 67, 68, and 78 showed modest inhibitory activity against the major hemoglobin degrading cysteine protease FP-2.

Full text of DOI:10.1021/jm300588j

Article
pubs.acs.org/jmc
Synthesis and Insight into the Structure−Activity Relationships of
Chalcones as Antimalarial Agents
Narender Tadigoppula,*,† Venkateswarlu Korthikunta,† Shweta Gupta,† Papireddy Kancharla,†
Tanvir Khaliq,† Awakash Soni,‡ Rajeev Kumar Srivastava,‡ Kumkum Srivastava,‡ Sunil Kumar Puri,‡
Kanumuri Siva Rama Raju,§ Wahajuddin,§ Puran Singh Sijwali,∥ Vikash Kumar,⊥
and Imran Siddiqi Mohammad⊥
†Medicinal and Process Chemistry Division, CSIRCentral Drug Research Institute, Lucknow-226 001, UP, India
‡Parasitology Division, CSIRCentral Drug Research Institute, Lucknow-226 001, UP, India
§Pharmacokinetics and Metabolism Division, CSIRCentral Drug Research Institute, Lucknow-226 001, UP, India
∥CSIRCentre for Cellular and Molecular Biology, Habsiguda, Hyderabad-500007, AP, India
⊥Molecular and Structural Biology Division, CSIRCentral Drug Research Institute, Lucknow-226 001, UP, India
S
* Supporting Information
ABSTRACT: Licochalcone A (I), isolated from the roots of
Chinese licorice, is the most promising antimalarial compound
reported so far. In continuation of our drug discovery program,
we isolated two similar chalcones, medicagenin (II) and
munchiwarin (III), from Crotalaria medicagenia, which
exhibited antimalarial activity against Plasmodium falciparum.
A library of 88 chalcones were synthesized and evaluated for
their in vitro antimalarial activity. Among these, 67, 68, 74, 77,
and 78 exhibited good in vitro antimalarial activity against P.
falciparum strains 3D7 and K1 with low cytotoxicity. These
chalcones also showed reduction in parasitemia and increased survival time of Swiss mice infected with Plasmodium yoelii (strain
N-67). Pharmacokinetic studies indicated that low oral bioavailability due to poor ADME properties. Molecular docking studies
revealed the binding orientation of these inhibitors in active sites of falcipain-2 (FP-2) enzyme. Compounds 67, 68, and 78
showed modest inhibitory activity against the major hemoglobin degrading cysteine protease FP-2.
of great interest for their pharmacological activities.3 Some of
INTRODUCTION
■
the chalcones have been reported to possess many biological
About 3.3 billion people, almost half of the world’s population,
are at risk of malaria. Every year, this leads to about 250 million
cases and nearly one million deaths. People living in poor
countries are the most vulnerable, particularly children below
five years of age and pregnant women. Malaria is especially a
serious problem in Africa, where it is responsible for one in
properties including anticancer,4 antimalarial,5 antiinflamma-
tory,6 antileishmanial,7 antituberclulosis,8 nitric oxide inhib-
ition,6,9 antimitotic,10 analgesic,6 antioxidant,6 antifungal,11 anti-
HIV,12 and antiprotozoal activities.13 They are also reported to
be gastric protectant,14 antimutagenic, and antitumorogenic.15
every five (20%) childhood deaths.1 An African child has on
Licochalcone A (I), an oxygenated chalcone isolated from the
roots of Chinese licorice,16 is the most promising antimalarial
average 1.6−5.4 episodes of malaria fever each year and every
30 seconds a child dies from malaria. Malaria-endemic countries
in South East Asia account for 15% (34 million) of the total
estimated cases worldwide, the second highest number after
Africa. India, Indonesia, and Myanmar comprise most of the
region’s reported cases (94%) in South East Asia. According to
compound reported so far. Natural chalcone I have been shown
to inhibit the development of both chloroquine-susceptible
(CQS) 3D7 and chloroquine-resistant (CQR) Dd2 Plasmodium
falciparum strains. The growth of the parasites at all stages was
inhibited by compound I. The chalcone I administered
intraperitoneally (IP) or orally for 3−5 days also protected
mice from lethal Plasmodium yoelii infection. Hence, the
antimalarial activity of chalcone I has stimulated interest in
antimalarial potential of other chalcones from natural and
synthetic sources.5,17 Chalcones are thought to act against
WHO, India contributes about 70% of the total malaria cases in
the South East Asian region.2 One of the major hurdles to
control malaria is resistance of malaria parasites to most of the
commonly used antimalarials. Hence, new antimalarials are
urgently needed to combat the disease.
Chalcones are one of the major classes of natural products
with widespread distribution in fruits, vegetables, spices, tea,
and soy-based food products. They have been recently subjects
Received: April 27, 2012
© XXXX American Chemical Society
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malarial papain-like cysteine proteases as well,17 which degrade
hemoglobin to generate amino acids for erythrocytic stage
development of the parasite. Chalcones can be readily
synthesized, and contrasting substitution pattern in the two
rings (Figure 1) could be envisaged to produce a large number
evaluated for their in vitro antimalarial activity. Several of these
compounds were found active in the in vitro screening, which
were further evaluated for in vivo antimalarial activity in a Swiss
mice model. Herein, we report a comprehensive assessment of
antimalarial activity, structure−activity relationship (SAR)
analyses, pharmacokinetics (PK), mode of action, and
molecular docking studies of promising chalcones.
RESULTS AND DISCUSSION
■
Chemistry. Synthesis of Prenylated Chalcones. Prenylated
chalcones 5−15 and II were prepared from 2,4-dihydrox-
yacetophenone (1). Prenylation of 1 with 2-methyl-but-3-en-2-
ol in the presence of BF3·OEt2 in dry dioxane resulted in the
mixture of three compounds: 2,4-dihydroxy-5-C-prenylaceto-
phenone (2), 2,4-dihydroxy-3-C-prenylacetophenone (3), and
2,4-dihydroxy-3,5-C-diprenylacetophenone (4).19,20 The pre-
nylacetophenones 2−4 and various substituted benzaldehydes
were subjected to Claisen−Schmidt condensation21 using
aqueous KOH in ethanol to afford the corresponding
prenylated chalcones 5−15 and II (Scheme 1).
Synthesis of Chromanochalcones. We wanted to study the
cyclization effect of prenyl or geranyl group of chalcones;
therefore, few chromanochalcones in which the C-prenyl or C-
geranyl groups are involved in cyclization with phenolic
hydroxyl group were synthesized. Initially, chromanochalcones
18−22 were prepared through a short and convenient route
developed by us, which involves the one-pot synthesis of acetyl
chromans 16 and 17 by carrying out a reaction between 1 and
isoprene using BF3·Et2O (Scheme 2)22 and Claisen−Schmidt
condensation of resultant chromans 16 and 17 with various
substituted aromatic aldehydes. The chromanochalcones 25
and 26 were synthesized by pyridine-catalyzed condensation
between 1 and citraldimethylacetal to provide the acetylchro-
mene 23.23 Then, the intermediate 23 was smoothly reduced to
acetyl chroman 24 with Pd/C24 and subsequently subjected to
Claisen−Schmidt condensation with aromatic aldehydes to
form the desired chromanochalcones 25 and 26 (Scheme 2).
Synthesis of Chromenochalcones. A large number of
chromenochalcones were prepared to find out the role of the
Figure 1. General skeleton of chalcone and naturally occurring
antimalarial chalcones I−III.
of potential analogues. A thorough assessment of structural
requirements for antimalarial activities of chalcones is vital to
develop and optimize drug design efforts aimed at chalcones.
In continuation of our drug discovery program on
antimalarial agents from Indian medicinal plants, we isolated
a diprenylated chalcone, known as medicagenin (II),18 and a
triprenylated chalcone, known as munchiwarin (III)19 (Figure
1), from the roots of Crotalaria medicagenia. These two
chalcones exhibited in vitro antimalarial activity (MIC = 5.10
and 4.34 μM, respectively) against P. falciparum.18,19 This
motivated us to synthesize various analogues of these lead
molecules to obtain more active compounds. A library of 88
chalcones with various structural features were synthesized and
Scheme 1. Synthesis of Chalcones Containing C-Prenyl Substituents on Ring-B (5−15 and II)
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Scheme 2. Synthesis of Chromanochalcones (18−22, 25, and 26)
Scheme 3. Synthesis of Chromenochalcones (30−72) and Prenylated Chromenochalcone (74)
olefinic bond in benzopyran moiety. The acetyl/carboxalde-
hyde chromenes 23, 28, and 29 were synthesized using
pyridine-catalyzed condensation between 1 or 27 and 3-methyl-
but-2-enal/citraldimethylacetal.23 The resultant acetyl chro-
menes 23 and 28 and substituted aromatic aldehydes or p-
hydroxy-benzene-1,3-dicarbaldehyde25 and chromene carbox-
aldehyde 29 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 30−47 and 49−71 in good
yields (Scheme 3).24 To improve the bioavailability of 33 and
52, the aminoalkyl groups were introduced as in 48 and 72 (see
Tables 3 and 4). The synthesis of chalcones 48 and 72 was
accomplished by the replacement of the hydrogen of the
phenolic hydroxyl group of the chromenochalcones 33 and 52
with the alkyl part of the corresponding amines using K2CO3 in
dry acetone (Scheme 3).26 We also synthesized a prenylated-
chromenochalcone 74, which has an extra prenyl group on
benzopyran moiety. The intermediate prenylatedacetylchro-
mene 73 was synthesized using pyridine-catalyzed condensa-
tion between 2 and citraldimethylacetal. The intermediate 73
and p-hydroxy-benzene-1, 3-dicarbaldehyde were subjected to
Claisen−Schmidt condensation using NaH in dry THF at room
temperature to give the desired chalcone 74 (Scheme 3).
Chromenochalcones 77 and 78, which are isomeric to 67
and 68 (Table 4), and a dichromenochalcone 81 were also
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Scheme 4. Synthesis of Chromenochalcone (77, 78, and 81)
Scheme 5. Synthesis of Chromanochalocones 83−88 and Chromenochalcones Containing Hetero Atoms in Ring-A (90−100)
synthesized as outlined in Scheme 4 from 2,6-dihydroxyaceto-
phenone (75), 2,4,6-trihydroxyacetophenone (79), and cit-
raldimethylacetal via intermediates 76 and 80, respectively.
Synthesis of Chromeno and Chromanochalcones with
Hetero Atoms in Ring-A. Chromanochalcones 83−88 and
chromenochalcones 90−100 which have hetero atoms in ring-
A (Figure 1) were synthesized using Claisen−Schmidt
condensation from acetyl chromans22 16, 17, 24, and 82 and
acetyl chromens23 23, 28, and 89, respectively, and various
heteroarylaldehydes as shown in Scheme 5.
Synthesis of Chromenodihydrochalcones. The synthesis of
chromenodihydrochalcones 109−114 were carried out to
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Scheme 6. Synthesis of Dihydrochromenochalcones (109−114)
determine the role of the α−β olefinic bond, which connects
the ring-A and carbonyl carbon (Figure 1). Initially, the
chalcones 101−104 were prepared by Claisen−Schmidt
condensation between 1 and substituted benzaldehydes. The
resultant chalcones were hydrogenated using Pd/C to give
dihydrochalcones 105−108 and subsequent chromenylation
using 3-methyl-but-2-enal in presence of pyridine provided the
chromenodihydrochalcones 109−112 (Scheme 6).23 Com-
pounds 113 and 114 were synthesized by regioselective
hydrogenation of 67 and 78 by using NaBH4/NiCl2·6H2O
(Scheme 6).
Biological Activity. As a part of our ongoing interest in
developing new antiparasitic agents, we have recently reported
the in vitro antimalarial activity of prenylated chalcones II and
III (Figure 1) from the roots of C. medicagenia.18,19 On the
basis of these optimistic results, a library of 88 chalcones were
prepared and their antimalarial activity was evaluated27−29 in
the following protocols:
activity; MIC = between 6 and 50 μM, moderate activity;
MIC = between 0.25 and 6 μM, good activity).
2. The compounds, which have MIC = 0.25−1.2 μM, were
further studied to determine their IC50 values against
CQS strain (3D7) as well as CQR strain (K1)27 with
chloroquine (CQ) and arteether as a reference drug.
3. The compounds with IC50 values between 70 and 350
nM were assessed for cytotoxicity,30 in vivo efficacy,31
pharmacokinetic studies, inhibition of the P. falciparum
cysteine proteases falcipain-2 and falcipain-3,32,33 and
molecular docking studies.34,35
Determination of MIC of 88 Compounds Library. In
Vitro Antimalarial Activity of Prenylated Chalcones. Initially,
a series of simplified analogues 5−15 of II were synthesized
(Scheme 1) and evaluated for their in vitro antimalarial activity
and the results are shown in Table 1. Chalcones 5−15, which
contain either the electron-donating groups (EDGs) or halogen
substituents on ring-A and a prenyl group on ring-B, showed
moderate activity (MIC = 25.12−29.58 μM) with the exception
of 8 and 14 (MIC = 142.45 and 146.19 μM, respectively; Table
1), which demonstrated that the monoprenylated compounds
are less potent than diprenylated medicagenin II (MIC = 5.10
μM).18,19
In Vitro Antimalarial Activity of Chromanochalcones. To
find out the role of the effect of cyclization of a prenyl/geranyl
group in the form of a chroman ring (dihydrobenzopyran
moiety), chromanochalcones 18−22, 25, and 26 were prepared
(Scheme 2) and screened for their in vitro antimalarial activity
(Table 2). Chalcones 18−22, which have the electron-donating
1. All of these chalcones were preliminarily screened for
their in vitro antimalarial activity against P. falciparum
chloroquine sensitive (CQS) strain 3D7 at three
concentrations (2, 10, and 50 μg/mL) to determine
the lowest concentration of the compound that inhibits
100% parasitemia (minimum inhibitory concentration,
MIC) and the respective activity results presented in μM.
The compounds activity profile of this library was
defined on the basis of their MIC values (MIC > 155
μM, inactive; MIC = between 50 and 155 μM, poor
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(Table 1: 5−15) indicated that cyclization of a prenyl/geranyl
group has a negative impact on antimalarial activity.
Table 1. In Vitro Antimalarial Activity of Chalcones
Containing C-Prenyl Substituents on Ring-B (5−15 and II)
In Vitro Antimalarial Activity of Chromenochalcones. A
large number of chromenochalcones (30−48; Table 3) were
Table 3. In Vitro Antimalarial Activity of Chromeno-
chalcones (30−48)
Table 2. In Vitro Antimalarial Activity of Chromano-
chalcones (18−22, 25, and 26)
a
Not inhibited 100% parasitemia even at 50 μg/mL concentration.
synthesized to find out the activity profile of chromene
(benzopyran) moiety instead of chroman. Chromenochalcones
30−36 contain the EDG’s on ring-A. Out of these, 30−33 and
35 exhibited poor activity (MIC = 126−155 μM range),
whereas 34 (MIC = 27.47 μM) and 36 (MIC = 25.25 μM)
have moderate antimalarial activity. Except chromenochalcone
42 (MIC = 5.34 μM), all other chalcones (37−41), which
contain either mono- or 3,4-dihalogen substituents on ring-A,
have poor activity (MIC = 105−155 μM). Chromenochalcone
43 (MIC = 135.86 μM) and its regioisomer 44 (>135.86 μM),
which have the combination of EDGs and halogen substituents
on ring-A, exhibited poor activity. Compounds 45 and 46,
which contain the EWGs, were also showed poor activity, while
47, in which the ring-A is substituted with combination of
EDGs and EWGs, showed moderate activity (MIC = 28.57
μM). The presence of an aminoalkyl substituent as in 48 (MIC
= 3.67 μM) on the hydroxyl group of 33 (MIC = 155.27 μM)
significantly increased the activity profile (Table 3).
a
Not inhibited 100% parasitemia at 10 μg/mL concentration and they
were not tested at 50 μg/mL concentration.
Another series of chromenochalcones 49−72 were also
synthesized in which benzopyran core contains an additional
alkenyl substituent (prenyl/C-5 unit: Table 4). Of these, 49−
57 contain EDGs on ring-A. Compounds 49−51, 53, and 56
exhibited poor activity, and 52, 54, 55 and 57 showed moderate
activity (MIC = 21.55−25.77 μM: Table 4). It is noteworthy to
mention here that the activity was retained even after shifting
the pyran moiety to ring-A as in 54 (MIC = 25.77 μM) and 55
(MIC = 24.87 μM). Chalcones 58−63, which contain either
groups (EDGs), electron-withdrawing groups (EWGs), and
halogen groups on ring-A, appear to be less potent (MIC >25
μM: these compounds were not tested at a higher
concentration). Chalcones 25 (MIC = 126.90 μM) and 26
(MIC = 122.54 μM), which contain an extra alkyl chain on
benzopyran core also exhibited poor activity (Table 2).
Diminished antimalarial activity of chromanochalcones (Table
2: 18−22, 25, and 26) compared to C-prenylated chalcones
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chalcones 70 and 71, in which the hydroxyl at 4-position and
aldehyde at 3-position of ring-A were masked in the form of
furan (fused to aromatic ring), showed moderate to poor
activity (MIC = 19.30 and 91.24 μM, respectively). These
results indicated that aldehyde and hydroxyl groups in ring-A
have a pivotal role in antiparasitic activity. The presence of an
aminoalkyl substituents on the hydroxyl group as in 72 led to
improvement in activity (see 72, MIC = 3.26 μM, versus 52,
MIC = 25.64 μM; Table 4). A careful review of the in vitro
antiparasitic activities presented in Tables 3 and 4 and their
structural features suggested that both hydroxyl and aldehyde
substituents on ring-A an extra alkenyl (prenyl or C-5 unit)
substituent on benzopyran core and an aminoalkyl substituent
on hydroxyl groups are playing an important role for good
antimalarial activity. Because the chromenochalcones 67 and 68
exhibited good activity (MIC = 1.19 and 0.59 μM,
respectively), we prepared chalcone 74 with an additional
prenyl substituent on ring-B of 67, as outlined in Scheme 3, and
screened for in vitro antimalarial activity, which led to improved
activity (MIC = 0.25 μM: Table 4).
Table 4. In Vitro Antimalarial Activity of
Chromenochalcones (49−72, 74, 77−78, and 81)
Two chromenochalcones (77 and 78) that are isomeric to 67
and 68 (Table 4) and a dichromenochalcone (81) were
synthesized as outlined in Scheme 4. Compounds 77 and 78
also showed good antimalarial activity (MIC = 0.39 and 0.31
μM, respectively) similar to those of 67 and 68, which again
supports the role of hydroxyl, aldehyde substituents on ring-A,
and an extra alkenyl (prenyl or C-5 unit) substituent on a
benzopyran core in antiparasitic activity. However, the
dichromene 81 showed moderate activity (MIC = 15.19 μM).
In Vitro Antimalarial Activity of Chromano and
Chromenochalcones with Hetero Atoms in Ring-A. Chroma-
nochalcones 83−88 and chromenochalcones 90−100, which
have the heteroatoms in ring-A, were prepared (Scheme 5) and
screened for their in vitro antimalarial activity (Table 5). The
chalcones with heteroatom at 3-position in ring-A with alkenyl
(prenyl or C-5 unit) side chain on chroman 86 (MIC = 5.27
μM) or chromene 93 (MIC = 5.33 μM) and 94 (MIC = 4.11
μM)) showed good antiparasitic activity and thus indicated that
3-pyridyl ring and extra alkenyl substituents (prenyl or C-5
unit) are required for antimalarial activity (Table 5). The
chromenochalcones 95−100 with substituted 3-quinolyl ring
(ring-A) exhibited poor activity (105.70−140.05 μM), with the
exception of 98 (MIC = 23.52 μM). In vitro activities of 95−
100 indicated that methyl or chloro substituents have a
negative effect, whereas an extra alkenyl (prenyl or C-5 unit)
side chain on the benzopyran moiety (98 versus 95) has a
positive effect on activity (Table 5).
In Vitro Antimalarial Activity of Dihydrochromenochal-
cones. To determine the role of the α−β olefinic bond,
chromenodihydrochalcones 109−114 were synthesized
(Scheme 6). The chalcones 109−112, in which the α−β
unsaturated bond is in reduced form with a monohydroxyl
(109: MIC = 30.86 μM)) or dihydroxyl (111: MIC = 29.41
μM)) system on ring-A exhibited moderate activity, whereas
their O-methyl derivatives (110 and 112) were less active (MIC
>147.92 and >135.86 μM; Table 6). It is noteworthy to
mention here that the compound 113 and 114, which are
dihydroderivatives of 67 and 78, showed moderate activity with
MIC of 10.3 and 9.12 μM, respectvely. These results supported
the importance of Michael acceptor (α,β-unsaturated ketone)
moiety in the inhibition of parasitemia.
a
Not inhibited 100% parasitemia even at 50 μg/mL concentration.
mono- or dihalogen sustituents on ring-A, were poorly active or
inactive, with the exception of 61 (MIC =22.62 μM). The
chalcones 64 (MIC = 5.01 μM) and 66 (MIC = 4.77 μM),
which contained the EWDs (4-cyano and 3-nitro, respectively)
on ring-A, exhibited good activity except 65 (4-nitro, MIC =
119.33 μM). Chalcone 64 (Table 4; MIC = 5.01 μM) was more
active than its lower homologue 45 (Table 3; >151.05 μM).
Chalcones 67 and 68, which have the combination of hydroxyl
and aldehyde substituents on ring-A, showed good in vitro
activity (MIC = 1.19 and 0.59 μM, respectively: Table 4).
Masking the hydroxyl group of 68 with methyl-oxy-methyl
(MOM) substituent as in 69 dramatically decreased the activity
(69, MIC = 21.64 μM, versus 68, MIC = 0.59 μM). The
Determination of IC50 for Antimalarial Chalcones with
MIC = 0.251−1.196 μM. The most active compounds, 67, 68,
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and 78 have an IC50 of 347.36, 125.35, 73.25, 94.73, and 87.55
nM, respectively, against the CQS strain (3D7) and an IC50 of
935, 1093.37, 366.25, 84.21, and >1196.17 nM, respectively,
against CQR strain (K1). The marketed drug chloroquine has
an IC50 of 17 nM against CQS and 443.57 nM against CQR
strains, where as arteether has an IC50 of 1.37 nM against CQS
and 1.08 nM against CQR strain. It is noteworthy to mention
here that chalcone 77 was almost equally effective against both
(CQS and CQR) strains (IC50 = 94.73 nM against 3D7 strain
and 84.21 nM against K1 strain) and exhibited better in vitro
activity profile than chloroquine against CQR strain.
Table 5. In Vitro Antimalarial Activity of Chromano and/or
Chromenochalcones with Hetero Atoms on Ring-A (83−88,
90−100)
Toxicity Studies of Potent Antimalarial Chalcones of
Library. Toxicity is an important consideration in any drug
development program, therefore we studied cytotoxicity of
these compounds against (Table 7) Vero cell lines (C1008;
monkey kidney fibroblast).30 Given the reasonably good
potencies of the compounds against malaria, the low
cytotoxicities are encouraging, especially for 74 and 77. In
fact, 74 and 77 have a very good “selectivity index”
(cytotoxicity/efficacy) of 1210, 334 for 3D7 strain and 242,
376 for K1 strain, respectively. Chalcones 67, 68, and 78 also
showed good activity against 3D7 strain with good selectivity
index.
In Vivo Activity against the CQ Resistant P. yoelii (N-67
strain) in Swiss Mice. The compounds (67, 68, 74, 77, and
78), which showed good potency and low cytotoxicity (Table
7), were evaluated in Swiss mice infected with P. yoelii (N-67
strain: CQ resistant).31 The aqueous suspensions of test
compounds was administered from day 0 to day 3 at 50 mg/
kg/day by IP/oral route, and parasitemia levels were recorded
from thin blood smears on day 4 (Table 8). The results
indicated a rapid parasite clearance (71−98%), however, none
of the treated animals was completely cured. The mean survival
time of these animals was increased from 12.33 1.45 to 18.40
2.73 days (Table 8). The compound 67 exhibited 98%
parasitemia suppression on day four, while compounds 68, 74,
77, and 78 suppressed parasitemia by 89%, 71%, 88%, and 87%
(Table 8), respectively. However, a curative effect was not
obtained at the tested dose. The compound 67 when
administered by oral route showed very poor in vivo efficacy
compared to IP route. It suppressed only 23% parasitemia on
day 4, and mean survival time decreased to 9.23 0.81 due to
rapid parasite recrudescence. The marketed drugs chloroquine
(oral administration) and arteether (intramuscular adminis-
tration) suppressed 99 and 100% parasitemia at a dose of 20
and 3 mg/kg body weight, respectively, and all the animals
survived up to 28 days in both experiments.
a
Not inhibited 100% parasitemia at 2 and 10 μg/mL concentrations
b
and they were not tested at 50 μg/mL concentration. Not inhibited
100% parasitemia even at 50 μg/mL concentration.
Table 6. In Vitro Antimalarial Activity of
Dihydrochromenochalcones (109−114)
In Vivo Oral Pharmacokinetic Studies. In vivo oral
pharmacokinetic study was performed in male Sprague−
Dawley rats (n = 5, weight range 220−240 g) to find out the
reason for poor antiparasitic activity by oral route. Chalcone 67
was administered as a single oral dose of 50 mg/kg, and analysis
was done using LC-MS/MS method to get the plasma
concentration−time profile. Along with the plasma samples,
quality control samples were distributed among calibrators and
unknown samples. Results showed its slow (Tmax, 3.0 h) and
poor absorption with a peak plasma level of 78.27 5.92 ng/
mL (Cmax) (Table 9). The plasma concentration of the
compound 67 decreased drastically after 6 h of administration,
and it was detectable in plasma up to 11 h. The overall
systemically available area under curve0−t [AUC0−t] of the
a
Not inhibited 100% parasitemia even at 50 μg/mL concentration.
74, 77, and 78 of this library, were further subjected to
determine their IC50 values against P. falciparum CQS (3D7)
and CQR (K1) strains (Table 7). The chalcones 67, 68, 74, 77,
compound was 389.18
17.08 h·ng/mL (Figure 2). The
plasma elimination half-life and clearance were 2.32 0.81 h
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Table 7. IC50 of Chalcones, 67, 68, 74, 77, and 78 against P. falciparum Strains 3D7 and K1
IC50 (nM)
selectivity index
a
compd
3D7 (CQS)
K1 (CQR)
cytotoxicity CC50 (nM)
IC50 ratio (K1/3D7)
3D7
64
K1
67
68
74
77
78
347.36
125.35
73.25
94.73
87.55
17.00
1.37
935
1093.37
366.25
84.21
22200
9904
2.6
8.7
23.7
9.0
79
1210
334
88621
31674
102679
235109
136057
5.0
242
0.9
376
>1196.17
443.57
1.08
ND
25.2
0.79
1173
13393
98720
ND
chloroquine
arteether
530
124857
a
Cytotoxicity against Vero cell line (C1008; Monkey kidney fibroblast). ND: not determined.
a
Table 8. In Vivo Antimalarial Efficacy of Chromenochalcones in P. yoelii N-67 (Resistant to CQ)
dose mg/kg/day
(3 days)
route of
administration
no. of treated
mice
% parasitemia reduction vs controls;
day 4
mean survival time ( SE)
(days)
compd
controls
5
5
5
5
5
5
5
5
5
9.20 0.44
17.40 2.50
9.20 0.81
12.33 1.45
12.60 0.87
16.75 1.55
18.40 2.73
>28
67
50
50
50
50
50
50
20
3
IP
98
23
67
oral
IP
68
89
74
IP
71
77
IP
88
78
IP
87
chloroquine
arteether
oral
IM
99
100
>28
a
IP: Intraperitoneal; IM: Intramuscular.
Table 9. Pharmacokinetic Parameters of 67 after Single
Administration at 50 mg/kg (Oral) in Male Sprague−
Dawley Rats
fluid and simulated intestinal fluid and found to be stable in
both.
Cysteine Protease Inhibition Studies. Because chalcones are
known to inhibit the cysteine proteases, 67, 68, and 78 were
evaluated for their inhibitory activity against the cysteine
proteases falcipain-2 (FP-2) and falcipain-3 (FP-3), which are
validated drug targets for developing new antimalarial chemo-
therapy.32 Recombinant FP-2 (1 nM) and FP-3 (2 nM) were
used to determine cysteine protease inhibiting activity.
Compounds 67 and 78 did not inhibit FP-3 and, rather,
activated the enzyme at higher concentrations. The compound
68 showed marginal inhibition of FP-3 at lower concentrations
but, intriguely, stimulated the enzyme activity at higher
concentrations. However, compound 67, 68, and 78 inhibited
FP-2 with an IC50 of 13.5, 5.5, and 4.6 μM, respectively, which
correlates with their in vitro antimalarial activity (IC50 of 347.3,
125.3, and 87.5 nM). The FP-2 inhibiting activity of these
compounds is comparable with that of the chalcones reported
in the literature (Table 10).33
parameter
oral at 50 mg/kg
AUC0−t (h·ng/mL)
Tmax (h)
389.18 17.08
3.00
Cmax (ng/mL)
T1/2 (h)
78.27 5.92
2.32 0.81
433.45 163.81
128.53 5.49
3.89 0.72
Vd/F (L/kg)
CL/F (L/h/kg)
MRT (h)
Docking of Chromenochalcones to Falcipin-2 Protein
Crystal Structure. Chalcones have been reported to act by
competitive inhibition at the malarial cysteine proteases (FP-2).
We therefore carried out docking studies to find out the
binding orientations of these inhibitors at active sites of amino
Table 10. Chalcones 67, 68, and 78’s Falcipain-2 Inhibiting
Activity and Docking Studies against Falcipain-2 Protein
Crystal Structure
Figure 2. Mean plasma concentration−time profile of 67 in male
Sprague−Dawley rats (220−240 g) after a single oral administration of
67 (50 mg/kg). Results are presented as mean SEM.
IC50 of falcipain-2 inhibiting
docking scores (Flexi X total
a
compd
activity (μM)
score)
and 128.53 5.49 L/h/kg, respectively. Thus the pharmaco-
kinetic study indicated very low oral bioavailability of
compound 67 could be the reason for its poor in vivo
antimalarial activity to clear 100% parasitemia in experimental
animals. The compound 67 was also tested in simulated gastric
67
68
78
13.5
5.5
−13.3
−14.00
−15.55
4.6
a
Higher the score means strong binding interactions with Falcipain-2.
I
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Figure 3. Surface representation of falcipain-2 (FP-2) showing subsites occupied by docked compounds with enlarged view (right). Only polar
hydrogen atoms of compounds are shown. FP-2 has surface representation colored in gray.
acid residues of falcipain-2 enzyme (Figure 3; Table 10). The
binding pockets of cysteine protease, falcipain-2 (FP-2), is well
explored and its crystal structure (3BPF and 2GHU) has also
been reported with small molecule inhibitors.34,35 However,
crystal structure of FP-2 with chalcone-based inhibitor is not
available in structural database. In this context, in silico study
provides valuable insight into binding mode of these inhibitors.
FP-2 active site is divided into four subsites: S1, S1′, S2, and
S3.36,37 Any nonsubstrate molecule interacting with these
subsites may have potential to become FP-2 inhibitor. In
docking simulation by Flex X, we observed two populations of
docked conformations of chalcones (Figure 4), one consisting
of 67 and 78, has shown H-bond interactions with residues
Tyr206 of S1′, Gln36 (except compound 67), Cys39, Cys42,
and His174 of S1. In the S3 subsite, only 67 formed H-bond
with Gly83. The alkenyl (prenyl or C-5 unit) unit of 67 was
found to be in close proximity to hydrophobic residues Trp78
and Gly83 of the S3 subsite. However, the alkenyl (prenyl or C-
5 unit) unit of 78 optimally occupied the hydrophobic cavity
formed by Leu84 and Ile85 in S3 and S2 subsites, respectively.
Another population consisted of compounds 68 and
Licochalcone (I) occupying mainly S1′ and S1 subsites.
Compounds 68 appeared to form H-bond with Gln36,
Cys39, His174, Trp206, and Gln209. In this population, the
alkenyl (prenyl or C-5 unit) unit of 68 was away from the
hydrophobic S2 subsite and ring-A was involved in π−π
interaction with Trp206. Licochalcone A (I) showed weak H-
bonding only with Cys39, Cys42, and His174 and π−π
interactions with Trp206.
−15.5) and 68 (Flex X total score: −14.00) showed better
interaction than 67 (Flex X total score: −13.41) in terms of H-
bonding. These docking results correlate with the experimental
FP-2 inhibition results (IC50 of 78, 4.6 μM; 68, 5.5 μM; and 67,
13.5 μM) and their in vitro antimalarial activity. Further 67, 68,
and 78 have better interactions than Licochalcone A (Flex X
total score: −10) in our docking studies (Table 10).
CONCLUSIONS
■
In summary, a library of 88 chalcones with various structural
features such as prenylated chalcones, chromanochalcones,
chromenochalcones, and chromenodihydrochalcones were
synthesized based on the natural products lead molecule. Our
SAR studies indicated that both the hydroxyl and aldehyde
substituents on ring-A, an extra alkenyl (prenyl or C-5 unit)
substituent on the benzopyran core (as in 67, 68, 74, 77, 78),
aminoalkyl substituent on hydroxyl groups (as in 48 and 72),
and a heteroatom at the 3-position in ring-A (as in 86, 93, 94)
are required for good antimalarial activity of chalcones. Several
compounds of this synthetic series are equipotent to natural
products, and a few of them showed several-fold (>20) higher
order of in vitro activity. Some of these compounds also
suppressed the parasitemia and increased survival time of Swiss
mice infected with the chloroquine resistant P. yoelii N-67
strain. PK Studies revealed that very low oral bioavailability due
to poor absorption. Inhibition of recombinant FP-2 by
compounds 67, 68, and 78 suggested that hemoglobin
degradation is one of the mechanisms of antimalarial activity
of these compounds. Molecular docking studies against protein
crystal structure of FP-2 revealed, binding of these inhibitors at
active sites of enzyme and these results correlate with the
experimental FP-2 studies data and in vitro antimalarial activity.
The correlation between in vitro and in vivo was not observed
during our studies that might be due to varied pharmacokinetic
properties of tested compounds in in vivo system. Further
optimization studies are in progress in our laboratory to
improve bioactivity of these compounds to achieve better
curative effect and oral bioavailability.
Overlay of docked conformations of all compounds revealed
the interesting point that one aryl ring (ring-A of 67 and 78 and
ring-B of 68 and I) and carbonyl group (formyl group of 67
and 78 and ketone of 68 and I) of these chalcones clustered at
almost the same position (Figure 5). We hypothesized that aryl
ring clustering was observed due to the presence of
hydrophobic residue Trp206 and carbonyl group clustering
was due to formation of H-bond with main chain of Cys39.
Recently similar docking result was reported by Sahu and co-
workers,38 where they have shown the role of Cys39 and Gln36
in chalcone binding. Overall, compound 78 (Flex X total score:
J
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Figure 4. Predicted bound conformation of I (a), 67 (b), 68 (c), and 78 (d) in the active site of FP2 (mixed surface, ribbon-and-stick representation
in gray color). For clear appearance, only polar hydrogens are shown in both FP2 and docked compounds. Residues were labeled using 2D label
option in Chimera. Hydrogen bonds are shown in blue dotted line. Some H-bonds may not be visible (for detail, see Supporting Information).
(13C). Experiments were recorded in CDCl3, CD3OD, pyridine-d5,
D2O, 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. Chromatog-
raphy 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
Figure 5. Overlay of docked conformations of 67 (cornflower blue),
68 (green), 78 (magenta), and I (orange). Molecules were colored
individually for clear understanding.
analyses were performed on a Shimadzu 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.
EXPERIMENTAL SECTION
Synthesis of Prenyl Acetophenones (2−4). To a magnetically
stirred solution of 1 (1.0 g, 6.5 mmol) in anhydrous 1,4-dioxane (15
mL) maintained under nitrogen atmosphere was added gradually
BF3·Et2O (0.8 g, 6.5 mmol) at rt. When the solution acquired pink−
red, a solution of 2-methyl-but-3-en-2-ol (790 mg, 7.9 mmol) in
anhydrous 1,4-dioxane (10 mL) was added and the whole solution was
■
General Methods. Melting points were recorded on Buchi-530
capillary melting point apparatus and are uncorrected. IR spectra were
1
recorded on Perkin-Elmer AC-1 spectrometer. H NMR spectra were
recorded on Bruker Avance DPX 200 FT, Bruker Robotics and Bruker
DRX 300, spectrometers at 200, 300 MHz (1H), and 50, 75 MHz
K
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stirred for 1 h at rt. After dilution with diethyl ether (100 mL), the
solution was washed with water (3 × 50 mL) to discharge the color.
The combined ethereal solution obtained after extraction was dried
over anhydrous Na2SO4 and evaporated under reduced pressure. Then
the crude product was chromatographed on silica gel to afford 2 (307
mg, 27%), 3 (183 mg, 16%), and 4 (397 mg, 21%) as white solids.
1-[2,4-Dihydroxy-5-(3-methyl-but-2-enyl)-phenyl]-ethanone (2).
Melting point 145−146 °C. FT-IR (KBr, cm−1) 3340, 1635. 1H
NMR (CDCl3, 200 MHz) δ 7.43 (s, 1H), 6.35 (s, 1H), 5.29 (t, J = 7.1
Hz, 1H), 3.29 (d, J = 7.2 Hz, 2H), 2.54 (s, 3H), 1.78 (s, 6H). MS
(FAB) (m/z) 221 (M + H)+.
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 23 (14.45,
1
64%). Semisolid; FT-IR (neat, cm−1) 3436, 1695. H NMR (CDCl3,
200 MHz) δ 6.48 (d, J = 8.8 Hz, 1H), 6.74 (d, J = 10.1 Hz, 1H), 6.31
(d, J = 8.8 Hz, 1H), 5.51 (d, J = 10.1 Hz, 1H), 5.08 (t, J = 8.1 Hz, 1H),
2.51 (s, 3H), 2.07 (q, J = 7.4 Hz, 2H), 1.69−1.80 (m, 2H), 1.67 (s,
3H), 1.59 (s, 3H), 1.40 (s, 3H). MS (FAB) (m/z) 287 (M + H)+.
Representative Procedure for the Synthesis of 1-(5-
Hydroxy-2-methyl-2-(4-methylpentyl)-chroman-6-yl) Etha-
none (24). To a solution of 23 (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 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 24 (375 mg,
1-[2,4-Dihydroxy-3-(3-methyl-but-2-enyl)-phenyl]-ethanone (3).
Melting point 154−157 °C. FT-IR (KBr, cm−1) 3330, 1630. 1H
NMR (CDCl3, 200 MHz) δ 7.53 (d, J = 8.8 Hz, 1H), 6.37 (d, J = 8.8
Hz, 1H), 5.25 (t, J = 7.2 Hz, 1H), 3.43 (d, J = 7.2 Hz, 2H), 2.55 (s,
3H), 1.82 (s, 3H), 1.76 (s, 3H). MS (FAB) (m/z) 221 (M + H)+.
1-[2,4-Dihydroxy-3,5-bis-(3-methyl-but-2-enyl)-phenyl]-etha-
1
none (4). Melting point 110 −112 °C. FT-IR (KBr, cm−1)1633. H
1
64%); semisolid. FT-IR (neat, cm−1) 3430, 1714. H NMR (CDCl3,
NMR (200 MHz, CDCl3) δ 7.21 (s, 1H), 6.14 (s, 1H), 5.18 (m, 2H),
3.34 (d, J = 7.0 Hz, 2H), 3.20 (d, J = 7.0 Hz, 2H), 2.46 (s, 3H), 1.74
(s, 3H), 1.70 (s, 3H), 1.68 (s, 6H). 13C NMR (50 MHz, CDCl3) δ
203.1, 161.4, 160.1, 135.5, 135.1, 129.9, 122.2, 121.8, 119.3, 114.6,
113.8, 29.4, 26.6, 26.2 (2C), 22.2, 18.3 (2C). MS (FAB) (m/z) 289.0
(M +H)+.
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)+.
Representative Procedure for the Synthesis of 1-[2-Methyl-
2-(4-methyl-pent-3-enyl)-5-(2-piperidin-1-yl-ethoxy)-2H-chro-
men-6-yl]-3-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-propenone
(72). To a stirred solution of chalcone, 52 (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 the compound
72. Yield: 51%. FT-IR (neat, cm−1) 1652. 1H 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, 21H). 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)+.
Representative Procedure for the Synthesis of 1-[2,4-
Dihydroxy-5-(3-methyl-but-2-enyl)-phenyl]-3-(4-methoxy-
phenyl)-propenone (6). To a stirred solution of 2 (500 mg, 2.2
mmol) in aqueous KOH solution in ethanol (5 mL) was added 4-
methoxybenzaldehyde (615 mg, 4.5 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, brine solution,
dried over anhydrous Na2SO4, and the solvent evaporated under
reduced pressure. The crude product was subjected to silica gel
column chromatography using hexane/ethyl acetate as mobile phase to
afford the prenylated chalcone 6 (307 mg, 40%). Melting point 158−
160 °C. FT-IR (KBr, cm−1) 3315, 1637. 1H NMR (CDCl3, 200 MHz)
δ 13.61 (s, 1H), 7.84 (d, J = 15.4 Hz, 1H), 7.60 (d, J = 8.6 Hz, 2H),
7.43 (d, J = 15.4 Hz, 1H), 6.94 (d, J = 8.6 Hz, 2H), 6.41 (s, 1H), 6.12
(s, 1H), 5.32 (t, J = 6.9 Hz, 1H), 3.86 (s, 3H), 3.34 (d, J = 6.9 Hz,
2H), 1.79 (s, 6H). 13C NMR (CDCl3, 50 MHz) δ 192.3, 165.2, 162.1,
161.9, 144.5, 135.6, 131.4, 130.7 (2C), 128.0, 122.0, 119.2, 118.9,
118.4, 114.8 (2C), 104.4, 55.8, 29.5, 26.1, 18.3. MS (FAB) (m/z) 339
(M + H)+.
Representative Procedure for the Synthesis of 2-Hydroxy-5-
{3-[7-hydroxy-2-methyl-2-(4-methyl-pent-3-enyl)-2H-chro-
men-8-yl]-3-oxo-propenyl}-benzaldehyde (77). To a stirred
solution of 76 (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 1 N 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 77 (210 mg, 25%). FT-IR (neat, cm−1) 3493,
3392, 2923, 2854, 2359, 1730, 1599, 1554, 1478, 1358, 1209, 1096,
760. 1H NMR (300 MHz, CDCl3) δ 13.06 (s, 1H), 11.23 (s, 1H), 9.92
(s, 1H), 7.94 (d, J = 15.7 Hz, 1H), 7.77 (d, J = 15.7 Hz, 1H), 7.74 (m,
2H), 7.08 (s, 1H), 7.05 (d, J = 8.2 Hz, 1H), 6.47 (d, J = 8.2 Hz, 1H),
6.33 (d, J = 9.8 Hz, 1H), 5.48 (d, J = 9.8 Hz, 1H), 5.07 (t, J = 6.7 Hz,
1H), 2.15 (m, 2H), 1.85(m, 2H), 1.63 (s, 3H), 1.47 (s, 6H). 13C NMR
(75 MHz, CDCl3) δ 196.7, 194.3, 164.7, 163.5, 155.4, 141.2, 136.2,
134.7, 134.1, 132.8, 128.1, 127.1, 125.4, 123.3, 121.1, 119.1, 113.2,
110.9, 110.1, 81.2, 42.2, 30.1, 27.4, 26.0, 23.4, 18.0. MS (FAB) (m/z)
419 (M + H)+.
Representative Procedure for the Synthesis of Acetylchro-
mans 16 and 17. To a stirred solution of 1 (1.0 g, 6.6 mmol) in dry
1,4-dioxane (15 mL) was added gradually BF3·Et2O (0.8 g, 6.6 mmol)
at rt. When the solution acquired a pink−red, solution of isoprene
(450 mg, 6.6 mmol) in dry 1,4-dioxane (10 mL) was added and the
whole solution was stirred for 12 h at rt. After dilution with moist ether
(100 mL), the solution was washed with water (3 × 50 mL) to
discharge the color. The combined ethereal solution obtained after
extraction was dried over anhydrous Na2SO4 and evaporated under
reduced pressure. The crude mixture was isolated on column
chromatography to afford the desired acetyl chromans 16 (521 mg,
1
36%). Melting point 72−73 °C. FT-IR (KBr, cm−1) 3429, 1629. H
NMR (200 MHz, CDCl3) δ 13.11 (s, 1H), 7.49 (d, J = 8.9 Hz, 1H),
6.33 (d, J = 8.9 Hz, 1H), 2.68 (t, J = 6.7 Hz, 2H), 2.54 (s, 3H), 1.80 (t,
J = 6.7 Hz, 2H), 1.34 (6H, s). MS (FAB) m/z 220 (M)+, 221 (M +
H)+, and 17 (530 mg, 37%), mp 116−118 °C. FT-IR (KBr, cm−1)
1
3429, 1642. H NMR (200 MHz, CDCl3) δ 12.32 (s, 1H), 7.43 (s,
1H), 6.31 (s, 1H), 2.73 (t, J = 6.7 Hz, 2H), 2.53 (s, 3H), 1.82 (t, J =
6.7 Hz, 2H), 1.34 (s, 6H). MS (FAB) (m/z) 220 (M)+, 221 (M + H)+.
Representative Procedure for the Synthesis of 1-(5-
Hydroxy-2-methyl-2-(4-methylpent-3-enyl)-2H-chromen-6-yl)
Ethanone (23). 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
Representative Procedure for the Synthesis of 4-Hydroxy-3-
(3-(5-hydroxy-2-methyl-2-(4-methylpent-3-enyl)-2H-chromen-
6-yl)-3-oxopropyl) benzaldehyde (113). To a magnetically stirred
L
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solution of 67 (50 mg, 0.11 mmol) in methanol (5 mL) was added
gradually NiCl2·6H2O (28 mg, 0.11 mmol) at rt. The whole reaction
mixture was brought to 0 °C, and NaBH4 (2 mg, 0.05 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 113 (30 mg, yield 60%): FT-IR (neat,
concentration (CC50) was determined using nonlinear regression
analysis. Selectivity index (SI) was calculated as: SI = CC50/IC50.
In Vivo Antimalarial Efficacy Assay. The in vivo drug response
was evaluated in Swiss mice infected with P. yoelii (N-67 strain), which
is innately resistant to chloroquine (CQ). Mice (22
2 g) were
inoculated with 1 × 106 parasitized RBC on day 0, and treatment was
administered to a group of five mice from day 0−3, once daily. The
aqueous suspensions of compounds were prepared with a few drops of
Tween 80. Initially, the efficacy of test compounds was evaluated at 50
mg/kg/day and the required daily dose was administered in 0.2 mL
volume via oral and/or intraperitoneal (IP) route. Parasitemia levels
were recorded from thin blood smears between day 4. The mean value
determined for a group of five mice was used to calculate the percent
suppression of parasitemia with respect to the untreated control group.
Animals were followed till day 28 to determine curative effect of the
length of survival. Mice treated with chloroquine (CQ) and arteether
served as reference controls.31
In Vivo Oral Pharmacokinetic Studies. In vivo oral
pharmacokinetic study was performed in male Sprague−Dawley rats
(n = 5, weight range 220−240 g). Compound to be studied was
administered as a single oral dose of 50 mg/kg, and blood samples
were collected from the retro-orbital plexus of rats under mild ether
anesthesia in microfuge tubes containing heparin as an anticoagulant at
0.25, 0.50, 1, 3, 6, 9, 11, 24, 30, and 48 h postadministration. Plasma
was separated and stored frozen at −70 10 °C until analysis. Each
plasma sample (100 μL) was processed using a protein precipitation
method employing 200 μL of acetonitrile as a protein precipitant, and
analysis was done using LC-MS/MS method to get the plasma
concentration−time profile. Along with the plasma samples, quality
control samples were distributed among calibrators and unknown
samples.
Assays with Recombinant Falcipains. Recombinant falcipain-2
(FP-2) and falcipain-3 (FP-3) were produced as described earlier.38
FP-2 and FP-3 concentrations were determined by active site titration
using E64. Briefly, enzymes were incubated with DMSO (1% final) or
varying concentrations of E64 in sodium acetate buffer (100 mM
sodium acetate, 10 mM DTT, pH 5.5) for 30 min at room
temperature, Z-LR-AMC was added (25 μM final) to the reactions,
and enzyme activity was determined by monitoring the release of
AMC upon substrate hydrolysis over 30 min at 37 °C using a
microplate reader (excitation 355 nm; emission 460 nm; SpectraMax
M5 (Molecular Devices)). Enzyme activities as the relative
fluorescence units/min (RFU) for DMSO and inhibitor-containing
reactions were plotted against inhibitor concentrations and analyzed
using the ORIGIN program (OriginLab) to determine enzyme
concentrations.
To determine if antiparasitic effects of chalcones are due to
inhibition of hemoglobin degrading cysteine proteases, compounds
were assessed for inhibition of the major hemoglobin degrading
cysteine proteases FP2 and FP3. FP2 (1 nM) or FP3 (2 nM) was
incubated with DMSO (1%) or with varying concentrations of the
compounds in sodium acetate assay buffer for 30 min at room
temperature. Z-LR-AMC was added to the reactions (25 μM final),
and enzyme activity was measured by monitoring substrate hydrolysis
for 30 min at 37 °C as described above. Enzyme activities of inhibitor-
containing reactions were compared with those of DMSO-containing
reactions, expressed as percent inhibition, and plotted against inhibitor
concentrations using nonlinear regression analysis (Graph Pad Prism)
to determine IC50 values.32
1
cm−1) 3434, 2925,2885, 1632, 1489, 1447,1376, 1020, 770, 671. H
NMR (CDCl3, 300 MHz) δ 12.38 (s, 1H), 9.87(s, 1H), 7.74 (s, 1H),
7.69 (d, J = 8.2 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.04 (d, J = 8.2 Hz,
1H), 6.76 (d, J = 10.0 Hz, 1H), 6.36 (d, J = 8.8 Hz, 1H), 5.56 (d, J =
10.0 Hz, 1H), 5.10 (t,1H), 3.45 (t, J = 5.5 Hz, 2H), 3.10 (t, J = 5.5 Hz,
2H), 2.11 (m, 2H), 1.81 (m, 2H), 1.68 (s, 3H), 1.63 (s, 3H), 1.59 (s,
3H). 13C NMR (CDCl3, 50 MHz) δ 204.4, 190.9, 161.0, 160.5, 159.6,
132.4, 131.9, 131.2, 131.1, 128.2, 127.2, 123.6, 117.7, 116.0, 114.0,
112.8, 109.1, 108.8, 80.6, 41.7, 38.9, 27.2, 25.6, 23.2, 22.7, 17.6. MS
(FAB) m/z: 421 (M + H)+.
In Vitro Antimalarial Assay. The in vitro antimalarial activity of
the compounds was assessed against chloroquine sensitive (CQ) 3D7
strain of P. falciparum and compared with that of chloroquine. The
schizontocidal activities (MIC) as well as 50% inhibitory concentration
(IC50) were obtained as per the method we described earlier,27,28
respectively. In brief, the parasites were maintained in vitro in RPNI
medium29 supplemented with gentamycin at 40 μg/mL (Sigma),
Fungizone at 0.25 μg/mL (Gibco), and 10% fetal bovine serum (pH
7.2) at 37 °C in a CO2 incubator.
Compounds were dissolved in DMSO at 5 mg/mL, and required
dilutions were made in a template plate with RPMI medium. Then 20
μL from each dilution was transferred, in duplicate, in the test plate
and two wells receiving 20 μL of vehicle were kept as untreated
control. For evaluation of schizontocidal activity, parasite culture was
synchronized using 5% D-sorbitol to obtain ring stages only and 180
μL of 3% cell suspension containing 1% parasitized cells was added to
each well containing test compounds. The plates were incubated at 37
°C in CO2 incubator for more than 40 h, after which thin smears were
prepared from each well on grease-free glass slides. These were fixed in
methanol, stained with Giemsa’s stain, and examined under light
microscope, 100× oil immersion.
The minimum inhibitory concentration (MIC) of test compound
was designated as the minimum concentration required producing
100% inhibition of schizont maturation.
⎛
⎜
⎞
⎟
CS − TS
percent inhibition of maturation =
× 100
⎝
⎠
CS
CS: Number of schizonts in untreated culture.
TS: Number of schizonts in treated culture.
For evaluation of IC50 of the compounds, SYBR Green I-based
fluorescence (MSF) assay was used. For the assays, fresh dilutions of
all compounds in screening medium were prepared and 50 μL of
highest starting concentration was dispensed in duplicate wells in row
“B” of 96-well tissue culture plate. Subsequently, 2-fold serial dilutions
(seven concentrations) were prepared up to row “H” and finally 50 μL
of 2% parasitized cell suspension containing 0.8% parasitemia was
added to each well except four wells in row “A” received noninfected
cell suspension. After incubating the plates for 72 h, 100 μL of lysis
buffer containing 2× concentration of SYBR Green-I was added to
each well and incubated for 1 h at 37 °C. The plates were examined for
the relative fluorescence units (RFUs) per well using the fluorescence
plate reader (FLX800, Biotek). The 50% inhibitory concentration
(IC50) was determined using Logit regression analysis of dose−
response curves.
Docking of Chromenochalcones to Falcipain-2. Docking of
the active compounds 67, 68, and 78 to crystal structure of FP-2 (PDB
3BPF) was carried out with FlexX39 module of Sybyl7.1.40 FlexX uses
incremental approach while docking of compounds. Initially these
compounds were sketched and then minimized using 200 steps of
steepest descent and finally by conjugate gradient up to convergence.
In FlexX active site was constructed by selecting all residues lying
within 5 Å radius of reported chalcones binding residues. Total 30
docked conformations were generated for each compound. Final
visualization and image processing was done using UCSFchimera1−
641 and Sybyl7.1
Cytotoxicity Assay. Cytotoxicity assays of the compounds were
carried out using Vero cell line (C1008; monkey kidney fibroblast).30
The cells were incubated with different dilutions of test agents for 72 h
and MTT was used for detection of cytotoxicity. 50% cytotoxic
M
dx.doi.org/10.1021/jm300588j | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
596. (e) Vianaa, G. S.; Bandeiraa, M. A.; Matosa, F. J. Analgesic and
antiinflammatory effects of chalcones isolated from Myracrodruon
ASSOCIATED CONTENT
* Supporting Information
Final compounds characterization data and NMR spectra of the
selected chalcones and molecular docking results of 67, 68, 74,
77, and 78. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
urundeuva Allemao. Phytomedicine 2003, 10, 189−195. (f) Cheng, J.
̃
H.; Hung, C. F.; Yang, S. C.; Wang, J. P.; Won, S. J.; Lin, C. N.
Synthesis and cytotoxic, anti-inflammatory, and anti-oxidant activities
of 2′,5′-dialkoxylchalcones as cancer chemopreventive agents. Bioorg.
Med. Chem. 2008, 16, 7270−7276. (g) Heidari, M. R.; Foroumadi, A.;
Amirabadi, A.; Samzadeh-Kermani, A.; Azimzadeh, B. S.;
Eskandarizadeh, A. Evaluation of anti-inflammatory and analgesic
activity of a novel rigid 3,4-dihydroxy chalcone in mice. Ann. N. Y.
Acad. Sci. 2009, 1171, 399−406. (i) Kim, Y. H.; Kim, J.; Park, H.; Kim,
H. P. Anti-inflammatory activity of the synthetic chalcone derivatives:
inhibition of inducible nitric oxide synthase-catalyzed nitric oxide
production from lipopolysaccharide-treated RAW 264.7 cells. Biol.
Pharm. Bull. 2007, 30, 1450−1455.
AUTHOR INFORMATION
Corresponding Author
*Phone: +91 522 2612411, Fax: +91 522 2623405. Email: t_
narendra@cdri.res.in or tnarender@rediffmail.com.
Notes
■
The authors declare no competing financial interest.
(7) Zhai, L.; Chen, M.; Blom, J.; Theander, T. G.; Christensen, S. B.;
Kharazmi, A. The antileishmanial activity of novel oxygenated
chalcones and their mechanism of action. J. Antimicrob. Chemother.
1999, 43, 793−803.
ACKNOWLEDGMENTS
■
We are thankful to the Director, CSIRCDRI, Lucknow, for
the constant encouragement for the program on antiparasitc
drug discovery, SAIF Division for spectral data, J. P. Chaturvedi
for technical support, and DST, New Delhi, for financial
support in the form of ad-hoc project (SR/S1/OC-58/2008).
This is CDRI manuscript no. 92/2012/TN and communication
no. 8375.
(8) Lin, Y. M.; Zhou, Y. S.; Flavin, M. T.; Zhou, L. M.; Nie, W. G.;
Chen, F. C. Chalcones and flavonoids as anti-tuberculosis agents.
Bioorg. Med. Chem. 2002, 10, 2795−2802.
́ ́
(9) (a) Rojas, J.; Paya, M.; Dominguez, J. N.; Ferrandiz, M. The
synthesis and effect of fluorinated chalcone derivatives on nitric oxide
production. Bioorg. Med. Chem. Lett. 2002, 12, 1951−1954. (b) Here-
ncia, F.; Ferrandiz, M. L.; Ubeda, A.; Guillen, I.; Dominguez, J. N.;
Charris, J. E.; Lobo, G. M.; Alcaraz, M. J. 4-Dimethylamino-3′,4′-
dimethoxychalcone down regulates iNOS expression and exerts anti-
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(10) Ducki, S.; Forrest, R.; Hadfield, J. A.; Kendall, A.; Lawrence, N.
J.; McGown, A. T.; Rennison, D. Potent antimitotic and cell growth
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1998, 8, 1051−1056.
ABBREVIATIONS USED
■
SAR, structure−activity relationship; PK, pharmacokinetics; IP,
intraperitoneally; MIC, minimum inhibitory concentration;
IC50, half-maximal inhibitory concentration; CQ, chloroquine;
CQS, chloroquine-sensitive; CQR, chloroquine-resistant;
EDG’s, electron-donating groups; EWG’s, electron-withdrawing
groups; MOM, methyl-oxy-methyl; ADME, adsorption, dis-
tribution, metabolism and excretion; FP, falcipian
́ ́
(11) (a) Svetaz, L.; Tapia, A.; Lopez, S. N.; Furlan, R. L.; Petenatti,
E.; Pioli, R.; Schmeda-Hirschmann, G.; Zacchino, S. A. Antifungal
chalcones and new caffeic acid esters from Zuccagnia punctata acting
against soybean infecting fungi. J. Agric. Food. Chem. 2004, 52, 3297−
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V. M.; Sibirny, A. A. Antifungal activity of chalcones: a mechanistic
study using various yeast strains. Eur. J. Med. Chem. 2008, 43, 2220−
2228.
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