DOI: 10.1002/slct.

201701162 Full Papers

1
2 z Biological Chemistry & Chemical Biology
3
4
5
In Vitro Antimalarial Evaluation of Piperidine- and Piperazine-Based
6 Chalcones: Inhibition of Falcipain-2 and Plasmepsin II
7
8
Hemoglobinases Activities from Plasmodium falciparum
9 Hemandra Kumar Tiwari,[a] Prashant Kumar,[a] Nidhi Jatana,[b] Krishan Kumar,[a] Sandeep Garg,[c]
10
Latha Narayanan,[b] Puran Singh Sijwali,[d] Kailash Chand Pandey,[e] Nickolay Yu Gorobets,[f]
11
12
Ben M. Dunn,[g] Virinder Singh Parmar,[a, h, i] and Brajendra Kumar Singh*[a]
13
14
15 Never-ending effort to develop new treatments for malaria, strain and inhibition of plasmepsin II and falcipain-2. Degrada-
16 targeting the hemoglobin-degradation in the food vacuole of tion of hemoglobin by falcipain-2 in the presence or absence of
17 the parasite is of particular interest because it appears to be inhibitors was also demonstrated by sodium dodecyl sulphate-
18 critical for the erythrocytic stage parasite development. The polyacrylamide gel electrophoresis. The IC50 values of chalcones
19 Plasmodium aspartic proteinases plasmepsins and cysteine for cell growth inhibition were in the range of 1.60-153.51 mM.
20 proteases falcipains have been shown to be the major IC50 values for cytotoxicity of selected chalcones against
21 hemoglobin-degrading proteases and are proposed as the high Michigan Cancer Foundation (MCF) 7 human breast adenocarci-
22 priority drug targets by the World Health Organization for noma cells were in the range of 8.46-15.64 mM. Molecular
23 developing novel small molecules as inhibitors of hemoglobin docking studies revealed the binding orientation of these
24 degradation. In the present study, several piperidine and chalcones in the active sites of falcipain-2. Our results clearly
25 piperazine-based chalcones were assessed for anti-malarial depict the advantage of these chalcones as they kill P. falciparum
26 activity against the chloroquine-susceptible P. falciparum 3D7 malaria parasite in culture, most likely via inhibition of falcipain.
27
28 Introduction
29 [a] Dr. H. K. Tiwari, P. Kumar, K. Kumar, Prof. V. S. Parmar, Dr. B. K. Singh
Malaria is a mosquito-borne parasitic disease in the tropical and
30 Bio-organic Research Laboratory, Department of Chemistry
University of Delhi, Delhi-110007, India sub-tropical regions of the World. It has been challenging to
31
Fax: 91–11-2766 7206 control the disease. Although new drugs are consistently
32 E-mail: [email protected] introduced into wide spread clinical and field use resistance
33 [b] Dr. N. Jatana, Dr. L. Narayanan
appears often within a few years (we have not introduced a
34 Bioinformatics Infrastructure Facility, Sri Venkateswara College
University of Delhi single drug in last 16 years!!). The World Health Organization
35
South Campus, New Delhi-110021, India estimated 236,000-635,000 deaths in 2015, mostly in young
36 [c] Dr. S. Garg children in sub-Saharan Africa.[1] About 205,000 malaria-associ-
37 Department of Zoology, Smt. Chandibai Himathmal Mansukhani College
ated deaths were registered per year in India below the age of
38 University of Mumbai
Ulhasnagar, Thane, Maharashtra-421003, India 70 (55,000 in early childhood, 30,000 at ages 5–14 years,
39
[d] Dr. P. S. Sijwali 120,000 at ages 15–69 years); 1·8 % cumulative probability of
40 CSIR-Centre for Cellular and Molecular Biology death from malaria below the age of 70.[2] Among the five
41 Habsiguda, Hyderabad-500007, Andhra Pradesh, India
species of Plasmodium parasite responsible for the disease in
42 [e] Dr. K. C. Pandey
National Institute of Malaria Research human beings: Plasmodium falciparum, Plasmodium vivax,
43
Host-Parasite Interaction Biology Group Plasmodium ovale, Plasmodium malariae and Plasmodium
44 Lab No-219, Sector-8, Dwarka, New Delhi-110077, India knowlesi, P. falciparum is the most wide spread and deadly, as it
45 [f] Dr. N. Y. Gorobets
can lead to the cerebral and pregnancy-associated malarias,
46 SSI ‘Institute for Single Crystals’ of National Academy of Science of Ukraine
Lenina Ave 60, Kharkiv, Ukraine which often result in mortality.[3,4] The current limitations of
47
[g] Prof. B. M. Dunn vaccine and vector control as well as the increasing resistance
48 Department of Biochemistry and Molecular Biology of malaria parasite to existing drugs highlight the urgent need
49 University of Florida College of Medicine, University of Florida
for the discovery and development of new anti-malarial agents
50 P. O. Box-100245, Gainesville, Florida-32610-0245, USA
[h] Prof. V. S. Parmar with novel mode of action. Therefore, there is an urgent need
51
School of Chemical Science, Central University of Haryana to work towards the development of new potent anti-malarial
52 Mahendragarh, Haryana-123031, India molecules.
53 [i] Prof. V. S. Parmar
The Plasmodium plasmepsins and falcipains belong to
54 Institute of Advanced Sciences
North Dartmouth, 86-410 Founce Corner Mall Road, MA 02747, USA aspartic proteases and cysteine proteases family, respectively,
55
Supporting information for this article is available on the WWW under and have been shown to play critical role in hemoglobin
56
https://doi.org/10.1002/slct.201701162 degradation that takes place in acidic food vacuole during the
57

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intra-erythrocytic stage development of malaria parasite[5,6]
1 Results and Discussion
Plasmepsins and falcipains have been recently recognized as
2
suitable targets for designing novel chemotherapeutic com- Bioassays
3
pounds in the treatment of malaria.[7,8] Three different classes of
4 Plasmodium falciparum culture and inhibition assays
enzymes have been reported to digest the hemoglobin in the
5
food vacuole of the parasite; cysteine proteases (falcipains-1, 2 Anti-malarial activities of the chalcones was assessed against
6
and 3),[9] aspartic proteases (plasmepsin I (PMI), plasmepsin II the 3D7 strain of P. falciparum. Several compounds inhibited
7
(PMII), plasmepsin IV (PMIV), and histo-aspartic protease parasite growth with low micromolar IC50 values. The results are
8
(HAP))[10] and metalloprotease (falcilysin)[11] (a cathepsin C-like depicted in Table 1. Introduction of different substituents on
9
enzyme (DPAPI) and M1-family alanyl aminopeptidase (PfA M1) the aromatic rings enabled investigation of the relationship
10
are also present in the food vacuole. During asexual develop- between substitution pattern and anti-malarial activity. Out of
11
ment of malaria parasite in human erythrocytes, P. falciparum the 44 compounds tested, 18 compounds [33(a, b, g, i, j, k, l,
12
consumes up to 80 % of the hemoglobin present;[12] it is taken m, s, t and v), 31(a, e, h, i and m), 32 and 33] inhibited parasite
13
up by a cytostome like organelle and transported to the food development at concentrations lower than 10 mM. Several
14
vacuole wherein it is cleaved by multiple proteases. The amino compounds inhibite parasite development at higher IC50.
15
acids derived from hemoglobin serve as a source of amino The effect of substituent pattern in the aromatic ring on P.
16
acids for protein synthesis, and the byproduct heme is falciparum growth was studied. In piperidine series of chal-
17
polymerized into non-toxic hemozoin.[13] Malaria parasites both cones 30 a-v, electron-withdrawing groups and electron-donat-
18
in culture and in animal models are killed by the inhibitors of ing groups in the aromatic ring increased IC50 values in the
19
plasmepsins, providing the proof of the concept that these order of 2-F ~ 2-Br < 2-Cl < H < 3,4-di-F < 4-F < 3-F < 4-Cl <
20
proteases are viable drug targets.[7] Moreover, it has been also 4-CF3 < 3,4-di-Cl < 3-Cl < 4-Br < 2,6-di-Cl and 3-CH3 < 2,5-di-
21
observed that cysteine protease inhibitors arrest the erythro- OCH3 < H- < 3,4,5-tri-OCH3 < 3,4-di-OCH3 < 4-N(C2H5)2 < 2,4,6-
22
cytic life cycle of P. falciparum by blocking the hydrolysis of the tri-OCH3, respectively. In piperazine series of chalcones 31 a-t,
23
host hemoglobin and causing abnormally swollen food electron-withdrawing groups and electron-donating groups in
24
vacuoles, indicating that these proteases could be targetes for the aromatic ring increased IC50 values in the order of 2,6-di-Cl
25
controlling malaria.[14] < 3-F < 2-F < H < 2,6-di-F < 4-F < 3,4-di-F < 2-Cl < 3-Cl and
26
Chalcones or 1,3-diaryl-2-propen-1-ones (Figure 1) belong- 3,4,5-tri-OCH3 < 3-CH3 < H < 4-OCH3 < 3,4-di-OCH3 < 2,5-di-
27
ing to the flavonoid family are natural products with wide OCH3 < 4-CH3 < 4-N(CH3)2 < 2,4,6-tri-OCH3, respectively.
28
Chalcones 32 (IC50 ; 5.66 mM) and 33 (IC50; 7.96 mM) showed
29
almost the same IC50 values. Chalcones 30 p, 30 r, 31 d, 31 f and
30
31 g did not affect parasite development below 62.5 mM
31
concentration.
32
Potent anti-malarial activity of the chalcone 31 t could be
33
accounted for in terms of protonation of nitrogen of piperazinyl
34
ring of the chalcone under weakly acidic conditions, which may
35
enhance their interaction with the protease at histidine 67 in its
36
active sites or else protonated histidine 67 may form a
37 Figure 1. General structure of chalcone. hydrogen bond with chalcone, resulting in the greater
38
inhibition of parasite development. However, the exact mecha-
39
nism of action of the chalcones is not known hitherto.
40
spread distribution in fruits, vegetables, spices and tea.
41
Recently, the compounds with chalcone (1,3-diaryl-2-propen-1-
42 b–Hematin inhibition activity
one) backbone have been reported to exhibit diverse biological
43
properties, including antimalarial,[15] antiviral,[16] antibacterial,[17] To evaluate whether anti-malarial activity of chalcones was due
44
antituberculosis,[18] antifungal,[19] anticancer,[20] antileishmani- to inhibition of hemozoin formation, only nineteen chalcones
45
nal,[21] antigiardial[22] and antiinflammatory.[23] A number of [30(a, b, e, g, i, j, k, l, m, s and v), 31(a, e, h, i, m and t), 32 and
46
chalcone derivatives have also been found to inhibit several 33] were assessed for their ability to inhibit heme crystalliza-
47
important enzymes in cellular systems, including protein tion. It has been proposed that heme crystallizes spontaneously
48
tyrosine kinase,[24] malarial aspartic protease (PM II)[25] and under acidic and low oxygen conditions found in the food
49
malarial cysteine protease (FP-1).[26] Interest in development of vacuole of the parasite.[28] Compounds [30(a, e, i, j, k and v),
50
chalcones as anti-malarial drugs began after the isolation of 31(a, h and t), 32 and 33] inhibited significant heme
51
licochalcone A from the roots of the Chinese licorice (Gan Cao) crystallization (Table 1). Chloroquine[29] showed strong inhibi-
52
which inhibited the growth of both chloroquine-sensitive (3D7) tion of b-hematin formation (Figure 2), whereas Licochalcone A
53
and resistant (Dd2) strains of P. falciparum in vitro.[27] Herein, we shows weak inhibition against b-hematin formation compared
54
report anti-malarial assessment, inhibitory activities against to chloroquine. Mishra et al. have also reported that hemozoin
55
recombinant plasmepsin II and falcipain-2, mode of action and formation remains unaffected in licochalcone A treated malaria
56
molecular docking studies of known promising chalcones. parasites.[30] We speculate that these chalcones co-ordinate
57

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1 Table 1. Plasmodium falciparum cell growth inhibition and b-hematin inhibition of chalcones, 30 (a-v), 31 (a-t), 32 and 33.
2 Compound R1 R2 R3 R4 R5 P. falciparum % inhibition of b-hematin @ 25 mM
3 cell growth inhibition
IC50 (mM)
4
5 30a H H H H H 7.2 63
6 30b Cl H H H H 5.51 50
30c H Cl H H H 21.0
7
30d H H Cl H H 16.32
8 30e Cl H H H Cl 38.0 65
9 30f H Cl Cl H H 18.74
10 30 g Br H H H H 4.36 50
30 h H H Br H H 34.84
11
30i F H H H H 4.47 63
12 30j H F H H H 9.36 73
13 30k H H F H H 6.65 65
14 30 l H F F H H 7.04 50
30 m H CH3 H H H 5.32 48
15
30n H H CH3 H H 24.74
16 30o H H CF3 H H 16.63
17 30p H H N(CH3)2 H H no inhibition
18 at/below 62.5 mM
30q H H N(C2H5)2 H H 14.22
19
30r H H OCH3 H H no inhibition
20 at/below 62.5 mM
21 30 s OCH3 H H OCH3 H 6.11 58
22 30 t H OCH3 OCH3 H H 8.84
30u OCH3 H OCH3 H OCH3 18.28
23
30v H OCH3 OCH3 OCH3 H 8.16 60
24 31a H H H H H 7.40 60
25 31b Cl H H H H 15.45
26 31c H Cl H H H 18.38
31d H H Cl H H no inhibition
27
at/below 62.5 mM
28 31e Cl H H H Cl 3.27 55
29 31f H Cl Cl H H no inhibition
30 at/below 62.5 mM
31 g H H Br H H no inhibition
31
at/below 62.5 mM
32 31 h F H H H H 7.11 60
33 31i H F H H H 6.71 50
34 31j H H F H H 11.32
31k F H H H F 10.82
35
31 l H F F H H 15.11
36 31 m H CH3 H H H 5.99 50
37 31n H H CH3 H H 26.76
38 31o H H N(CH3)2 H H 35.86
31p H H OCH3 H H 15.95
39
31q OCH3 H H OCH3 H 20.03
40 31r H OCH3 OCH3 H H 17.10
41 31 s OCH3 H OCH3 H OCH3 153.51
42 31 t H OCH3 OCH3 OCH3 H 1.6 73
32 H H H – H 5.66 60
43
33 H H H – H 7.96 70
44 Chloroquine 0.026 (0.004) 95
45 Diphosphate
46 Licochalcone - 70
A
47
48
49
50
with Fe + 2 ions of the porphyrin ring of heme via piperidinyl Plasmepsin II inhibition activity
51
and piperazinyl nitrogen, resulting in inhibition of b-hematin
52
formation. The compounds were tested as inhibitors of plasmepsin II at
53
25 mM (Table 2). In general, weak inhibition was observed with
54
maximal inhibition for the compounds: 31 j, 47 %; 30 g, 40 %;
55
30 n, 32 %; and 30 s, 30 %. Tight binding inhibitors would be
56
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1 Table 2. Plasmepsin II inhibition, cyotoxicity and hemolysis assay of

chalcones, 30 (a-v), 31 (a-t), 32 and 33.
2
3 Compound % inhibi- MCF-7 cells Hemolysis Selectivity DG
tion of Cytotoxicity IC50 (mM) Indexa Kcal/
4
PMII @ IC50 (mM) mol
5 25 mM
6
30a 9 11.79 13.29 1.63
7
30b 0 11.49 17.42 2.08 -
8 47.06
9 30c 19
10 30d 21
30e 7 11.57 11.16 0.30
11
30f 9
12 30 g 40 15.64 18.24 3.58 -
13 41.44
14 30 h 23
30i 10 12.50 15.62 2.79 -
15
34.71
16 Figure 2. Inhibition of b-hematin formation by chalcones.
30j 14 12.75 14.53 1.36
17 30k 0 12.25 15.62 1.84
18 30 l 14 11.39 14.80 1.61
30 m 9 10.52 13.24 1.97 -
19
expected to show 100 % inhibition at a concentration as high 50.26
20 30n 32
as the 25 mM used in these assays.
21 30o 15
22 30p 9
30q 9
23 Falcipain-2 inhibition activity
30r 28
24 30 s 30 13.99 13.24 2.28 -
In order to validate in silico results and inhibitory activity in P.
25 39.28
falciparum culture, the compounds 30 b, 30 g, 30 i, 30 m, 30 s, 30 t 0
26
31 e, 31 i, 31 m, 32 and 33 were assessed for inhibition of 30u 23
27
hemoglobin hydrolysis by falcipain-2.[26] The sodium dodecyl 30v 19 14.52 15.32 1.77
28 31a 0 9.56 10.28 1.29
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analy-
29 31b 14
ses (Figure 3) clearly indicated that all the compounds, except 31c 10
30
31d 18
31
31e 4 11.16 14.88 3.41 -
32 46.53
33 31f 13
34 31 g 10
31 h 5 12.52 17.42 1.76
35
31i 21 11.94 13.56 1.77 -
36 46.53
37 31j 47
38 31k 21
31 l 19
39
Figure 3. Hemoglobin (Hb) hydrolysis by falcipain-2 (FP2) in the presence 31 m 23 8.46 14.26 1.41 -
40 41.7
and absence of chalcones. 200 nM FP2 was incubated with different
41 chalcones at a concentration of 15 mM before addition of 8 mg Hb and 31n 8
42 hydrolysis pattern was visualized by SDS–PAGE analysis. Lane 1: Hb alone, 31o 24
Lane 2: Marker (M: Kd), Lane 3: Hb + FP2 + 31e, Lane 4: Hb + FP2 + 30 g, Lane 31p 0
43
5: Hb + FP2 + 30 m, Lane 6: Hb + FP2 + 30i, Lane 7: Hb + FP2 + 32, Lane 8: 31q 12
44 31r 23
Hb + FP2 + 30s, Lane 9: Hb + FP2 + 30b, Lane 10: Hb + FP2 (positive control),
45 Lane 11: Marker (M: Kd), Lane 12: Hb + FP2 + 31 m, Lane 13: Hb + FP2 + 33 31 s 12
46 and Lane 14: Hb + FP2 + 31i. 31 t 12 11.57 12.25 7.23
32 11 11.79 15.24 2.08 -
47
51.1
48 33 8 12.25 13.02 1.53 -
49 50.46
30 b, significantly inhibited hemoglobin hydrolysis by falcipain- Chloroquine
50
2, with 15 mM concentration of inhibitors completely inhibiting Diphosphate
51
falcipain-2. Licochalcone 12.25 12.01
52 A
Thus, it could be inferred that the chalcones that possess
53
anti-malarial activity interact with the falcipain-2. Inhibition of a
Selectivity index is the ratio of IC50 in MCF-7 cells to IC50 in P. falciparum.
54
this enzyme has been shown to hamper digestion of
55
hemoglobin within the food vacuole, blocking the parasite
56
development.[31] Experiential inferences are supported by
57

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computational structural analyses that predict malarial cysteine cells at micromolar concentrations, at which they kill the
1
protease as the most likely target enzyme of chalcones.[32] On parasite. Further, the ratio of the cytotoxicity (IC50 in mM) and
2
the basis of vinyl sulphones as falcipain-2 inhibitors,[33] we also in vitro anti-malarial activity (IC50 in mM for 3D7 strain) enabled
3
speculate that chalcones inactivate the enzyme by nucleophilic the determination of a selectivity index (SI) for these com-
4
irreversible addition of the thiol group of the active site pounds. The chalcones, [30(a, b, e, g, i, j, k, l, m, s and v), 31(a,
5
cysteine-25 to the a,b-unsaturated ketone moiety of chalcones, e, h, i, m and t), 32 and 33] displayed a selectivity index (SI)
6
which acts as a Michael acceptor (Figure 4). It is proposed that ranging from 0.30 to 3.58, 1.29-7.23, 2.08 and 1.53, respectively
7
(Table 2).
8
Furthermore, to rule out any anti-parasitic effect due to
9
effect on erythrocytes, we performed hemolytic assay for the
10
above mentioned chalcones. The hemolytic activities (IC50) are
11
depicted in Table 2 (Figure 6), clearly indicating that all these
12
13
14
15
16
17
18 Figure 4. Proposed mechanism of inhibition of falcipain-2 by chalcones.
19
20
21
a hydrogen bond from the protonated active site histidine-159
22
to oxygen of a,b-unsaturated ketone moiety polarizes this
23
moiety for further activation of b-carbon towards nucleophilic
24
attack. The negative charge at the a-carbon will immediately
25
develop via protonation by the histidinium residue.
26
27
28 Cytotoxicity and hemolytic activity
29
The cytotoxicity of nineteen chalcones [30(a, b, e, g, i, j, k, l, m,
30 Figure 6. Hemolytic activity of different chalcones with human fresh
s and v), 31(a, e, h, i, m and t), 32 and 33] was determined
31 erythrocytes. Values given are IC50 (mM).
against human carcinoma MCF-7 cell lines by MTT colorimetric
32
assay.
33
The IC50 values are summarized in Table 2 (Figure 5). These
34
studies revealed that most of the compounds are non-toxic to chalcones, except 30 e, did not have any adverse effect on
35
erythrocytes at the concentrations at which they kill the
36
parasite.
37
38
39 Molecular docking study
40
A crystal structure of falcipain-2 with a chalcone is not known
41
so far. To find out the binding orientations of chalcones at the
42
active sites of amino acid residues of falcipain-2 enzyme, we
43
carried out docking studies of ten chalcones, [30 (b, g, i, m and
44
s), 31 (e, i and m), 32 and 33] against chain A of falcipain-2
45
(2GHU). The crystal structure of falcipain-2 (2GHU) consists of
46
four almost identical falcipain-2 monomers of which we
47
retained chain A for docking studies. The overall fold of
48
falcipain-2 resembles that of papain-like proteases showing a
49
core structure with a distorted ellipsoidal shape. The structure
50
contains two distinct domains separated by a long central
51
substrate binding cleft containing the binding site. The left
52
Figure 5. Cytotoxicity of different chalcones with human carcinoma, MCF-7
domain (L domain) is predominantly a-helical, whereas the
53
cell lines. Values given are IC50 (mM). right domain (R domain) contains a large antiparallel b-sheet
54
surrounded by peripheral helices.
55
Binding site was characterized using CASTp server and
56
validated with the experimental studies. Ligands were docked
57

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into the binding site of falcipain-2 using PATCHDOCK serv- synthesized chalcones (1H NMR, 13C NMR and HRMS) are
1
er.[34,35] The top ten solutions of every docked complex were reported in supporting information.
2
refined with FIREDOCK. The docked compounds were ranked
3
according to the global energy and are listed in Table 2.
4 Acknowledgements
The energies in the Table 2 correspond to the first complex
5
generated by PATCHDOCK. The docked complex and protein- This work was supported by Science and Engineering research
6
ligand interaction of the top hit chalcone 32 with falcipain-2 Board (SERB), India (SR/SO/BB-092/2013) and supported by
7
are shown in Figure 7. It was observed that chalcone 32 binds funding from Department of Biotechnology, Ministry of Science
8
and Technology, Govt. of India at Bioinformatics Center, Sri
9
Venkateswara College, Delhi, India.
10
11
12 Conflict of Interest
13
The authors declare no conflict of interest.
14
15
16 Keywords: Anti-malarial Activity · Chalcone · Molecular
17 Docking · Plasmepsins Falcipains · Plasmodium Falciparum
18 Figure 7. (A) The docked complex of the top hit chalcone 32 with falcipain-2
19 and (B) protein-ligand interaction map showing key amino acid residues
20 involved in binding to the ligand (chalcone 32).
[1] WHO. World Malaria Report 2015; World Health Organization, 2015.
21 http://www.who.int/malaria/publications/world- malaria-report-2015/re-
22 port/en/.
23 [2] N. Dhingra, P. Jha, V. P. Sharma, A. A. Cohen, R. M. Jotkar, P. S. Rodriguez,
with falcipain-2 with high affinity, as observed from the global D. G. Bassani, W. Suraweera, R. Laxminarayan, R. Peto, Lancet 2010, 376,
24
energy and display key interactions with the conserved amino 1768–1774.
25 [3] N. J. White, Lancet 1993, 341,793.
acid residues within the binding site. The binding modes of
26 [4] C. L. Mackintosh, J. G. Beeson, K. Marsh, Trends. Parasitol. 2004, 20, 597–
chalcone 32 (Figure 7A & B) can be generalized as follows:
27 603.
carbonyl oxygen of a,b-unsaturated ketone moiety shows [5] P. A. Moura, J. B. Dame, D. A. Fidock, Antimicrob. Agents. Chemother.
28
hydrogen bonding with Asn-173 of falcipain-2 and 4-benzyl 2009, 53, 4968–4978.
29 [6] M. Chugh, V. Sundararaman, S. Kumar, V. S. Reddy, W. A. Siddiqui, K. D.
piperidine moiety interacts with amino acid residues Gly-40,
30 Stuart, P. Malhotra, Proc. Natl. Acad. Sci. 2013, 110, 5392–5397.
Cys-42, Trp-43, Gly-82, Gly-83, Leu-84, Ile-85, Asn-86, Ser-149,
31 [7] C. Berry, Biochem. Educ. 1997, 25, 191–194.
Leu-172, Ala-175, Asp-234 and Phe-236 hydrophobically. [8] P. J. Rosenthal, Int. J. Parasitol. 2004, 34, 1489–1499.
32
[9] P. J. Rosenthal, P. S. Sijwali, A. Singh, B. R. Shenai, Curr. Pharm. Des. 2002,
33 8, 1659–1672.
34 Conclusion [10] R. Banerjee, J. Liu, W. Beatty, L. Pelosof, M. Klemba, D. E. Goldberg, Proc.
35 Natl. Acad. Sci. 2002, 99, 990–995.
In this study, several piperidine and piperazine-based chalcones [11] K. K. Eggleson, K. L. Duffin, D. E. Goldberg, J. Biol. Chem. 1999, 274,
36
were synthesized and evaluated for anti-malarial activities 32411–32417.
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