Synthesis and evaluation of substituted pyrazoles: Potential antimalarials targeting the enoyl-ACP reductase of Plasmodium Falciparum

Article abstract of DOI:10.1080/00397910500334561

A series of 1,5- and 1,3-diarylsubstituted pyrazoles were designed, synthesized, and evaluated for their ability to inhibit enoyl-ACP reductase of Plasmodium falciparum. The inhibitory activity of these synthesized compounds was evaluated in a continuous

Full text of DOI:10.1080/00397910500334561

This article was downloaded by: [Stanford University Libraries]
On: 12 October 2012, At: 22:34
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office:
Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Synthetic Communications: An International
Journal for Rapid Communication of
Synthetic Organic Chemistry
Publication details, including instructions for authors and subscription
information:
http://www.tandfonline.com/loi/lsyc20
Synthesis and Evaluation of Substituted
Pyrazoles: Potential Antimalarials Targeting
the Enoyl‐ACP Reductase of Plasmodium
Falciparum
Sanjay Kumar a , Gyanendra Kumar a , Mili Kapoor a , Avadhesha Surolia a
Namita Surolia b
&
a Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India
b Molecular Biology and Genetics Unit, Jawaharlal Nehru Center for
Advanced Scientific Research, Jakkur, Bangalore, India
Version of record first published: 15 Aug 2006.
To cite this article: Sanjay Kumar, Gyanendra Kumar, Mili Kapoor, Avadhesha Surolia & Namita Surolia (2006):
Synthesis and Evaluation of Substituted Pyrazoles: Potential Antimalarials Targeting the Enoyl‐ACP Reductase
of Plasmodium Falciparum , Synthetic Communications: An International Journal for Rapid Communication of
Synthetic Organic Chemistry, 36:2, 215-226
To link to this article: http://dx.doi.org/10.1080/00397910500334561
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any substantial
or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or
distribution in any form to anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the
contents will be complete or accurate or up to date. The accuracy of any instructions, formulae,
and drug doses should be independently verified with primary sources. The publisher shall not
be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or
howsoever caused arising directly or indirectly in connection with or arising out of the use of this
material.

Synthetic Communicationsw, 36: 215–226, 2006
Copyright # Taylor & Francis LLC
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910500334561
Synthesis and Evaluation of Substituted
Pyrazoles: Potential Antimalarials
Targeting the Enoyl-ACP Reductase
of Plasmodium Falciparum
Sanjay Kumar, Gyanendra Kumar, Mili Kapoor,
and Avadhesha Surolia
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India
Namita Surolia
Molecular Biology and Genetics Unit, Jawaharlal Nehru Center
for Advanced Scientific Research, Jakkur, Bangalore, India
Abstract: A series of 1,5- and 1,3-diarylsubstituted pyrazoles were designed, syn-
thesized, and evaluated for their ability to inhibit enoyl-ACP reductase of Plasmodium
falciparum. The inhibitory activity of these synthesized compounds was evaluated in a
continuous spectrophotometric assay. Of all the compounds analyzed, NAS-81 and
NAS-39 inhibited the enzyme with IC50 values of 30 mM and 50 mM, respectively.
The mode of ligand binding was investigated by docking the synthetic inhibitors at
the active site of the crystal structure of the enzyme.
Keywords: Antimalarials, fatty acid synthesis, inhibitor, substituted pyrazole
INTRODUCTION
Malaria is still a leading cause of death worldwide.[1] Fatty acid biosynthesis
(FAS) has emerged as an important targeted pathway because of its different
Received in India July 11, 2005
Address correspondence to Namita Surolia, Molecular Biology and Genetics Unit,
Jawaharlal Nehru Center for Advanced Scientific Research, Jakkur, Bangalore
560 064, India. Tel.: þ91-80-22082820; Fax: þ91-80-22082767; E-mail: surolia@
jncasr.ac.in
215

216
S. Kumar et al.
characteristics in the parasite, Plasmodium falciparum, and its human host.
Enoyl-ACP (acyl-carrier protein) reductase (ENR; also known as FabI)
catalyzes the final reduction step in the elongation phase of fatty acid
synthesis to form acyl-ACP, which in turn serves as a substrate for another
round of elongation. Because of the importance of ENR in type II dissociative
FAS, it is an attractive target for developing antimalarials.[2]
The wide range of biological activities of pyrazoleshas made them
popular synthetic inhibitors for different enzymes and pathways.[3–6]
Numerous methods for the synthesis of these heterocyclic molecules have
been described, which involve the direct C–C coupling by Grignard and
Suzuki coupling[7] or the condensation of hydrazine with 1,3-diketone.
Although the condensation of 1,3-diketone with aryl hydrazine yields both
the 1,3 and 1,5-diarylsubstituted pyrazole, the 1,5-diaryl pyrazole can be
generated exclusively by carrying out condensation in the presence of hydro-
chloride salt of the phenyl/substituted phenyl hydrazine by refluxing in
ethanol.[8] Previously, the success of this method was demonstrated in the
synthesis of a limited number of analogues only. Herein, we report the
synthesis of new 1,3- and 1,5-disubstituted aryl pyrazoles that inhibit enoyl-
ACP reductase of P. falciparum.
RESULTS AND DISCUSSION
Chemistry
The 1,3- and 1,5-diarylsubstituted pyrazole derivatives (3–7 and 12, 13) were
synthesized as shown in Schemes 1 and 2. The requisite intermediate 1,1,1-
trifluoro-4-(4-nitrophenyl)-2,4-butanedione (2) and hydrazone (10, 11) were
synthesized according to our published procedure.[9] The 1,5-diarylsubstituted
pyrazoles were synthesized by treating diketone (2) with different substituted
hydrazine hydrochlorides in refluxing ethanol for 20 h to get good yields of
3–7. The synthesis of 1,3-diarylsubstituted pyrazoles began with com-
mercially available ketones (9: R ¼ 2OCH3/CH3) and hydrazine (8) as a
starting material. The condensation between ketone and hydrazine provided
hydrazones (10, 11) in good yields. The Vilsmeier–Haack reaction using
2.5 equivalents of POCl3-DMF performed a double addition at the methyl
group of acetophenone pyrimidine hydrozone (10, 11), which after hydrolysis
gave the desired aldehydes (12, 13) in 80–90% yield.[10] Although several
1,3-diarylsubstituted pyrazoles are known, these studies report for the first
time the synthesis of trifluoromethyl pyrimidine–substituted pyrazoles.
Enzyme Inhibition
The pyrazole derivatives so synthesized were evaluated for their inhibitory
activity on enoyl-ACP reductase in an in vitro spectrophotometric assay. As

Synthesis of Novel Substituted Pyrazoles
217
Scheme 1. Preparation of 1,5-disubstituted pyrazole derivatives: (i) MTBE, 25%
NaOMe in MeOH, ethyl trifluro acetate, rt 18 h; (ii) ethanol, (4-sulphamoylpheny)
hydrazine hydrachloride/3-cholro-4-fluoro pheny hydrazine hydrochloride/phenyl-
hydrazine hydrochloride/phenylhydrazine hydodrochloride/4-chlorophenyl hydrazine
hydrochloride/trifluoromethyl hydrazino pyrimidine.
shown in Table 1, some of the compounds inhibited the activity of the enzyme.
NAS-81 and NAS-39 showed the maximum inhibition with IC50 values of 30
and 50 mM, respectively (Fig. 1).
Docking Studies
We docked the two best inhibitors with P. falciparum FabI (PfFabI) in
silico using Autodock and Molecular Operating Environment (MOE) to inves-
tigate their mode of binding, which would provide a rationale for their
Scheme 2. Preparation of 1,3-disubstituted pyrazole derivatives: (i) acetic acid and
water, rt 16 h; (ii) POCl3 and dimethlyformamide at 08C for 15 h; (iii) excess of
water at rt for an additional 15 h.

218
S. Kumar et al.
Table 1. Inhibition of P. falciparum enoyl-ACP
reductase by synthesized disubstituted pyrazoles
Entry
Compound
IC50 (mM)
1
2
3
4
5
6
7
8
NAS-21
NAS-23
NAS-25
NAS-39
NAS-41
NAS-45
NAS-77
NAS-81
NIa
200
100
50
NIa
100
200
30
aNI: No inhibition.
inhibitory activity (Fig. 2). Both the inhibitors bind right into the active site.
Among the five 1,5-disubstituted pyrazoles synthesized, NAS-39 shows the
best inhibition. NAS-39 has interactions with Tyr181 and Trp35 of the
protein in the docked complex. The NO2 group of NAS-39 makes hydrogen
Figure 1. Dose response curves of the two most potent inhibitors: (a) NAS-81 and
(b) NAS-39. The IC50 was calculated from a plot of percent activity versus log
concentration of the inhibitor by fitting it to nonlinear regression analysis using
GraphPad Prismw software.

Synthesis of Novel Substituted Pyrazoles
219
Figure 2. Ribbon diagram of Plasmodium falciparum enoyl-ACP reductase docked
with NAS-81 (sticks) and NAS-39 (balls and sticks). NAD is also shown (thin sticks).
bonds with OH of Tyr181 and ribose on the nicotinamide side of NADH. The
phenyl ring of Tyr181 also has an aromatic interaction with the phenyl ring of
NAS-39. Similarly Trp35 has an aromatic interaction with the other phenyl
ring (R-group) of NAS-39, contributing to the affinity of the molecule to the
enzyme (Fig. 3a). Of the two 1,3-disubstituted pyrazoles synthesized, NAS-
81 demonstrates better inhibitory activity and is also the best inhibitor
among all the pyrazoles synthesized. On analysis of the docked complex of
this inhibitor with the enzyme, we find that NH of the pyrazole ring forms a
hydrogen bond with main chain C55O of Ala223, the oxygen of the CHO
group is hydrogen bonded with NH of the Asn122 and sulphur of Met185,
and one of the fluorine of CF3 group is hydrogen bonded with OH attached
to one of the phosphate groups of NADH. The phenyl ring with CH3 as
R-group of NAS-81 has an aromatic interaction with the phenyl ring of
Tyr181, and the other phenyl ring makes an aromatic interaction with Tyr35
(Fig. 3b). The poor inhibitory activity of NAS-77 as compared to NAS-81 is
probably due to bigger a R-group; that is, OCH3 can destabilize the interactions
occurring in the case of NAS-81.
CONCLUSION
In summary, we have described the synthesis of novel 1,3- and 1,5-
disubstituted pyrazoles that inhibit P. falciparum enoyl-ACP reductase. One
of the 1,3-disubstituted pyrazoles (NAS-81) and one of the 1,5-disubstituted

220
S. Kumar et al.
Figure 3. Interactions of (a) NAS-81 and (b) NAS-39 with Plasmodium falciparum
enoyl-ACP reductase. Inhibitors are shown in balls and sticks. Hydrogens are not
shown for sake of clarity.
pyrazoles (NAS-39) show significant in vitro inhibition of the enzyme.
Docking simulations provide insight into the binding modes of these two
molecules. These studies will pave the way for further development of more
potent inhibitors in this series, leading to new antimalarial agents.
EXPERIMENTAL
Materials
Synthetic Chemistry
4-Nitrocetophenone, (4-sulphamoylphenyl) hydrazine-HCl, 3-chloro-4-fluoro
phenyl hydrazine-HCl, phenyl hydrazine-HCl, 4-chloro phenyl hydrazine-HCl,

Synthesis of Novel Substituted Pyrazoles
221
trifluoromethyl hydrazinopyrimidine, methyl-ter-butylether, and dimethyl-
formamide were purchased from Sigma-Aldrich (USA). All other reagents
were purchased locally and purified prior to use. The synthesized molecules
were purified using 200-mesh silica-gel column chromatography and were
1
characterized by H NMR, mass spectra, 13C NMR, and/or CHN analysis.
Biological Activity
Media components for E. coli cultures were from Hi-media (Delhi, India).
b-NADH, crotonoyl-CoA, imidazole, and SDS-PAGE reagents were
obtained from Sigma Chemical Co., St. Louis, MO, USA. His-bind resin
was obtained from Novagen (Madison, USA).
General Procedure for Synthesis
The pyrazole derivatives (3–7) and (10–13) were synthesized as shown in
Schemes 1 and 2. The requisite intermediate 2 was synthesized by
following the published procedure.[9] During the course of synthesis we
synthesized two different types of diarylsubstituted pyrazoles. The
1,5-diarylsubstituted pyrazoles (3–7) and 1,3-diarylsubstituted pyrazoles
(12, 13) were different from each other in having a 3-trifluoromethyl and a
4-formyl group on the pyrazole ring, respectively. 1,5-diarylsubstituted
pyrazoles (3–7) were synthesized by a one-step condensation of intermediate
2 and the substituted aryl hydrazine hydrochlorides. The 1,3-diaryl substi-
tuted-4-formyl pyrazoles (12 and 13) were synthesized by using
intermediates (10 and 11), respectively. The intermediates 10 and 11 were
synthesized by the reaction of 2-hydrazino-4-trifluoromethylpirimidine
and p-methoxy/methyl acetophenone using the published procedure[11] and
were characterized by 1H NMR. All molecules (3–7 and 12, 13) were
purified by silica-gel column chromatography using a hexane/ethylacetate
1
gradient and were characterized by H NMR, mass spectra, and elemental
analysis.
Synthesis of 4-[5-(4-Nitrophenyl)-3-(trifluoromethyl)-1H-pyrazole-1yl]
benzenesulphonamide (NAS-23)
(4-Sulphamoylphenyl) hydrazine hydrochloride (112 mg; 0.5 mM) was
added to a stirring solution of 4,4,4-trifluoro-1-(4-nitrophenyl)butane-1,3-
dione (104 mg; 0.4 mM) in ethanol. The reaction mixture was refluxed for
20 h and, after cooling to room temperature, the mixture was concentrated
in vacuo. The residue was taken in ethyl acetate, washed with water and
brine, dried over Na2SO4, filtered, and concentrated in vacuo. The product
was purified by column chromatography using a hexane : ethyl acetate
gradient (yield 60%, 130 mg). The pure product was characterized by

222
S. Kumar et al.
1H NMR. CDCl3 d ppm 8.26 (d, J ¼ 8.4 Hz, 2H), 7.96 (J ¼ 8.00 Hz, 2H),
7.61–7.43 (m, 4H), 5.00 (s, 2H). ESMS, m/z 412 (435 sodiated peak);
calculated for C16H11N4O4F3S, 412. C, H, N analysis found C, 46.34; N,
2.71; and N, 13.54; calculated for C16H11N4O4F3S: C, 46.60%; H, 2.67%;
N, 13.59%.
Synthesis of 5-(4-Nitrophenyl)-3-(trifluoromethyl)-1-
(3-chlor-4-fluorophenyl)pyrazole (NAS-25)
(3-Chloro-4-fluorophenyl)hydrazine hydrochloride (97 mg; 0.5 mM) was
added to a stirring solution of 4,4,4-trifluoro-1-(4-nitrophenyl)butane-1,3-
dione (131 mg; 0.5 mM) in ethanol. The reaction mixture was refluxed for
20 h. After cooling to room temperature, the reaction mixture was concen-
trated in vacuo. The residue was taken in ethyl acetate washed with water
and brine, dried over Na2SO4, filtered, and concentrated in vacuo. The
product was purified using column chromatography in a hexane:toluene
gradient (starting from hexane and then 10%, 20%, 30%, 40% toluene). The
light yellow pure product (yield 55%; 110 mg) was characterized by 1H
NMR. CDCl3 d ppm 8.26 (d, J ¼ 7.80 Hz, 2H), 7.43 (d, J ¼ 7.80 Hz, 2H),
7.17–7.11 (m, 3H), 6.89 (s, 1H). ESMS m/z 385.5 (408 sodiated peak);
calculated for C16H8N3O2F4Cl; 385.5. C, H, N analysis found C, 49.66; H,
2.11; and N, 10.55; calculated for C16H8N3O2F4Cl: C, 49.80%; H, 2.07%;
N, 10.89%.
Synthesis of 5-(4-Nitrophenyl)-3-(trifluoromethyl)-1-phenyl Pyrazole
(NAS-39)
Phenylhydrazine hydrochloride (116 mg; 0.8 mM) was added to a stirring
solution of 4,4,4-trifluoro-1-(4-nitrophenyl)butane-1,3-dione (131 mg;
0.5 mM) in ethanol. The reaction mixture was refluxed for 20 h. After
cooling to room temperature, the reaction mixture was concentrated in
vacuo. The residue was taken in ethyl acetate washed with water and brine,
dried over Na2SO4, filtered, and concentrated in vacuo. The product was
purified by column chromatography using 1% ethyl acetate in hexane. The
light yellow pure product (yield 50%; 130 mg) was characterized by 1H
NMR. CDCl3 d ppm 8.24 (d, J ¼ 8.86 Hz, 2H), 7.66–7.22 (m, 7H), 6.89
(s, 1H). ESMS m/z 333; calculated for C16H10N3O2F3, 333. C, H, N
analysis found C, 57.48; H, 3.1; and N, 12.56%; calculated for
C16H10N3O2F3: C, 57.66; H, 3.02; N, 12.61%.
Synthesis of 5-(4-Nitrophenyl)-3-(trifluoromethyl)-1-(4-chlorophenyl)
Pyrazole (NAS-41)
4-Chlorophenyl hydrazine hydrochloride (179 mg; 1 mM) was added
to a stirring solution of 4,4,4-trifluoro-1-(4-nitrophenyl)butane-1,3-dione

Synthesis of Novel Substituted Pyrazoles
223
(261 mg; 0.8 mM) in ethanol. The reaction mixture was refluxed for 20 h. After
cooling to room temperature, the reaction mixture was concentrated in vacuo.
The residue was taken in ethyl acetate washed with water and brine, dried over
Na2SO4, filtered, and concentrated in vacuo. The product was purified by
column chromatography using 1% ethyl acetate in hexane. The orange pure
1
product (yield 45%; 160 mg) was characterized by H NMR. CDCl3 d ppm
8.23 (d, J ¼ 8.40 Hz, 2H), 7.51–7.18 (m, 7H), 6.89 (s, 1H). ESMS m/z
367; calculated for C16H9N3O2F3Cl, 367.5. C, H, N analysis found C,
52.31; H, 2.51, and N, 11.39%; calculated for C16H9N3O2F3Cl: C, 52.26; H,
2.47; N, 11.43%.
Synthesis of 5-(4-Nitrophenyl)-3-(trifluoromethyl)-1-
f4-trifluoromethyl(2-pyrimidyl)g pyrazole (NAS-45)
2-Hydrazino-4-trifluoromethylpyrimidine (72 mg; 0.4 mM) was added
to a stirring solution of 4,4,4-trifluoro-1-(4-nitrophenyl)butane-1,3-dione
(105 mg; 0.4 mM) in ethanol and HCl (0.5 mL of 37% w/v). The reaction
mixture was refluxed for 20 h. After cooling to room temperature, the
reaction mixture was concentrated in vacuo. The residue was taken in ethyl
acetate washed with water and sodium bicarbonate solution, dried over
Na2SO4, filtered, and concentrated in vacuo. The product was crystallized in
methanol. The orange pure product (yield 50%; 130 mg) was characterized
1
by H NMR. CDCl3 d ppm 9.09 (d, J ¼ 7.5 Hz, 1H), 8.87 (d, J ¼ 7.8 Hz,
1H), 8.29 (d, J ¼ 12.5 Hz, 2H), 7.98 (d, J ¼ 11.7 Hz, 2H), 6.90 (s, 1H).
ESMS m/z 403; calculated for C15H7N5O2F6, 404.24. C, H, N analysis
found C, 44.61; H, 1.81; and N, 17.22%; calculated for C15H7N5O2F6: C,
44.68; H, 1.75; N, 17.37%.
Synthesis of (NAS-77)
Phosphorous oxychloride (1.5 mM; 137 mL) was added to 10 mL of
dimethylformamide at 08C, and the reaction mixture was stirred for 30 min
by maintaining 08C temperature. The hydrazone (0.5 mM; 155 mg) was
added as a solid slowly to the reaction mixture and stirred for 15 h at rt.
The crude reaction mixture was quenched with excess water and stirred for
an additional 15 h. The resulting solid was filtered and dried in vacuo and
thereafter taken in dichloromethane. The resulting product was 95% pure.
For testing biological activity it was purified further by 100–200–mesh
silica-gel column chromatography using a gradient 10% ethyl acetate in
petroleum ether (yield 60%; 100 mg). The pure product was characterized
1
by H NMR. CDCl3 d ppm 10.11 (s, 1H); 9.27 (s, 1H); 9.13 (d, J ¼ 4.8 Hz,
1H); 7.87 (d, J ¼ 9.0 Hz, 2H); 7.64 (d, J ¼ 2.1 Hz, 1H); 7.02
(d, J ¼ 8.85 Hz, 2H); 3.88 (s, 3H). 13C NMR CDCl3 d ppm ¼ 184.79,
162.23, 161.05, 157.69, 156.36, 155.69, 135.54, 130.66, 124.25, 123.37,

224
S. Kumar et al.
119.17, 118.58, 115.13, 114.15, 55.40. ESMS m/z found 348, calculated for
C16H11N4O2F3, 348.28.
Synthesis of (NAS-81)
Phosphorous oxychloride (1.5 mM, 137 mL) was added to 10 mL of dimethyl-
formamide at 08C, and the reaction mixture was stirred for 30 min and main-
tained at 08C. The hydrazone (0.5 mM; 147 mg) was added as a solid slowly to
the reaction mixture and stirred for 15 h at rt. The crude reaction mixture was
quenched with excess water and stirred for an additional 15 h. The resulting
solid was filtered and dried in vacuo and then taken in dichloromethane.
The resulting product was 95% pure except for biological activity; it was
purified by 100–200–mesh silica-gel column chromatography using a
gradient of hexane : ethyl acetate :: 90 : 10 (yield 65%; 110 mg). The pure
1
product was characterized by H NMR. CDCl3 d ppm 10.12 (s, 1H); 9.29
(s, 1H); 9.14 (d, J ¼ 4.8 Hz, 1H); 7.78 (d, J ¼ 6.3 Hz, 2H); 7.66
(d, J ¼ 1.5 Hz, 1H); 7.32 (d, J ¼ 7.2 Hz, 2H); 2.44 (s, 3H). 13C NMR
CDCl3 d ppm ¼ 184.96, 162.23, 162.00, 161.41, 140.03, 136.68, 135.06,
129.43, 128.48, 127.71, 115.18, 114.30, 108.56, 21.42. ESMS m/z found
332; calculated for C16H11N4OF3, 332.09.
Spectrophotometric Assay
PfFabI was expressed and purified as described previously.[12] All exper-
iments were carried out on a Jasco V-530 UV-Vis spectrophotometer.
Enoyl-ACP reductase was assayed at 258C by monitoring the decrease in
A340 as a result of the oxidation of NADH using crotonoyl-CoA as a
substrate.[12] The standard reaction mixture in a total volume of 100 mL
contained 20 mM of Tris-Cl buffer, pH 7.4; 500 mM of NaCl; 100 mM of
crotonoyl-CoA; and 100 mL of NADH. For determining the IC50 of each
compound, assays were performed as described, with the addition of
various concentrations of the inhibitor to the reaction mixture. The concen-
tration of inhibitor that reduced the enzyme activity by half was calculated
as the IC50 of the inhibitor.
Docking Studies
All the docking studies were performed using the docking program AutoDock
3.05.[13,14]
Preparation of the Receptor and Ligand Molecules
The crystal structure of PfFabI submitted to PDB (www.rcsb.org) by Perozzo
et al.[15] was used for docking studies. Triclosan was removed from the active

Synthesis of Novel Substituted Pyrazoles
225
.
site of the pdb file 1NHG pdb and the binary complex (PfFabI with cofactor)
was converted into mol2 format with MMFF94 charges loaded using the
MOE (Molecular Operating Environment) suite of programs.[16] The script
molto2pdbqs (provided with AutoDock) was used to prepare the receptor
file. Ligands were built using MOE and energy minimized with MMFF94
charges. The script AutoTors (provided with AutoDock) was used to define
torsion angles in the ligand prior to docking. The receptor molecule was
kept rigid while the ligands were flexible for docking.
Docking Simulations
Grid maps for docking simulations were generated with 80 grid points (with
˚
0.375-A spacing) in x, y, and z direction centered in the active site using
the AutoGrid program. Lennard–Jones parameters 12–10 and 12–6
(supplied with the program package) were used for modeling H-bonds and
van der Waals interactions, respectively. The distance-dependent dielectric
permittivity of Mehler and Solmajer was used in the calculations of the elec-
trostatic grid maps.[17] The genetic algorithm (GA) and Lamarckian genetic
algorithm with the pseudo-Solis and Wets modification (LGA/pSW)
methods were used with default parameters. For all simulations the starting
population in the genetic algorithm was 50. Each simulation comprised
2.5 ꢀ 106 energy evaluations. Each docking experiment consisted of a
series of 100 simulations. Finally, the docked inhibitors were energy
minimized, keeping the cofactor and all the amino acids touching the radius
˚
of 4.5 A around the inhibitor flexible on MOE. This accounts for the minor
structural changes that might occur in amino acid side chains in the active
site upon binding of the inhibitor.
ACKNOWLEDGMENTS
This work was supported by a grant from the Department of Biotechnology,
Government of India, to N. S. and partially supported by grants from
Shantha Biotechniques Pvt. Ltd., Hyderabad, to N. S. and A. S. S. K. is
thankful to the Department of Biotechnology PDF program for a postdoctoral
fellowship.
REFERENCES
1. WHO, The World Health Report. Geneva, Switzerland, 1999, pp. 49–63.
2. Surolia, N.; Surolia, A. Triclosan offers protection against blood stages of malaria
by inhibiting enoyl-ACP reductase of Plasmodium falciparum. Nature Med. 2001,
7 (2), 167–173.

226
S. Kumar et al.
3. Patel, M. V.; Bell, R.; Majest, S.; Henry, R.; Kolasa, T. Synthesis of 4,5-diaryl-1H-
pyrazole-3-ol derivatives as potential COX-2 inhibitors. J. Org. Chem. 2004,
69 (21), 7058–7065.
4. Ashton, W. T.; Huchins, S. M.; Greenlee, W. J.; Doss, G. A.; Chang, R. S. L.;
Lotti, V. J.; Faust, K. A.; Chen, T. B.; Zingaro, G. J.; Kivlighn, S. D.;
Siegl, P. K. S. Nonpeptide angiotensin II antagonists derived from 1H-pyrazole-
5-carboxylates and 4-aryl-1H-imidazole-5-carboxylates. J. Med. Chem. 1993,
36 (23), 3595–3605.
5. Flynn, D. L.; Bellioti, T. R.; Boctor, A. M.; Conot, D. T.; Kostlan, C. R.;
Nies, D. E.; Ortwine, D. F.; Schrier, D. J.; Sircar, J. C. Styrylpyrazoles, styrylisox-
azoles, and styrylisothiazoles. Novel 5-lipoxygenase and cyclooxygenase inhibi-
tors. J. Med. Chem. 1991, 34 (2), 518–525.
6. Huang, Y. R.; Katzenellenbogen, J. A. Regioselective synthesis of 1,3,5-triaryl-4-
alkylpyrazoles: Novel ligands for the estrogen receptor. Org. Lett. 2000, 2 (18),
2833–2836.
7. Minis, W. Organic Synthesis Coll. Wiley, 1950; Vol. II, p. 35.
8. Thomas, D. P.; John, J. T.; Stephen, R. B.; Jeffery, S. C.; Paul, W. C.; Stephen, D.;
Matthew, J. G.; Len, F. L.; James, W. M.; Julie, M. M.; Roland, S. R.; Rogier, D. J.;
Stella, S. Y.; Garry, D. A.; Earl, G. B.; Cogburn, J. N.; Susan, A. G.; Carol, M. K.;
William, E. P.; Karen, S.; Amy, W. V.; Yan, Y. Z.; Peter, C. I. Synthesis and bio-
logical evaluation of the 1,5-diarylpyrazole class of cyclooxygenase-2 inhibitors:
Identification of 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]
benzenesulfonamide. J. Med. Chem. 1997, 40 (9), 1347–1365.
9. Sharma, S. K.; Kapoor, M.; Ramya, T. N.; Kumar, S.; Kumar, G.; Modak, R.;
Sharma, S.; Surolia, N.; Surolia, A. Identification, characterization, and inhibition
of Plasmodium falciparum beta-hydroxyacyl-acyl carrier protein dehydratase
(FabZ). J. Biol. Chem. 2003, 278 (46), 45661–45671.
10. Kira, M. A.; Abdel Rahman, M. O.; Gadalla, K. Z. The Vilsmeier–Haack reaction
III cyclization of hydrazones to pyrazole. Tetrahedron Lett. 1969, 10 (2),
109–110.
11. Bebernitz, G. R.; Argentieri, G.; Battle, B.; Brennan, C.; Balkan, B.; Burkey, B. F.;
Eckhardt, M.; Gao, J.; Kapa, P.; Strohschein, R. J.; Schuster, H. F.; Wilson, M.;
Xu, D. D. The effect of 1,3-diaryl-[1H]-pyrazole-4-acetamides on glucose
utilization in ob/ob mice. J. Med. Chem. 2001, 44 (16), 2601–2611.
12. Kapoor, M.; Dar, M. J.; Surolia, A.; Surolia, N. Kinetic determinants of the inter-
action of enoyl-ACP reductase from Plasmodium falciparum with its substrates
and inhibitors. Biochem. Biophys. Res. Comm. 2001, 289 (4), 832–837.
13. Goodsell, D. S.; Morris, G. M.; Olson, A. J. Automated docking of flexible ligands:
Applications of AutoDock. J. Mol. Recognit. 1996, 9, 1–5.
14. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.;
Belew, R. K.; Olson, A. J. Automated docking using a Lamarckian genetic
algorithm and an empirical binding free energy function. J. Comput. Chem.
1998, 19, 1662.
15. Perozzo, R.; Kuo, M.; Sidhu, A. S.; Valiyaveettil, J. T.; Bittman, R.;
Jacobs, W. R., Jr.; Fidock, D. A.; Sacchettini, J. C. Structural elucidation of the
specificity of the antibacterial agent triclosan for malarial enoyl ACP reductase.
J. Biol. Chem. 2002, 277 (15), 13106–13114.
16. Molecular Operating Environment (MOE 2001.07). Chemical Computing Group:
Montreal, Quebec, Canada.
17. Gasteiger, J.; Marsili, M. Iterative partial equalization of orbital electronegativity—
A rapid access to atomic charges. Tetrahedron. 1980, 36, 3219–3228.