Design, synthesis, and biological evaluation of prenylated chalcones as 5-LOX inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.06.107

Ten novel mono- and di-O-prenylated chalcone derivatives were designed on the basis of a homology derived molecular model of 5-lipoxygenase (5-LOX). The compounds were docked into 5-LOX active site and the binding characteristics were quantified using LUD

Full text of DOI:10.1016/j.bmc.2010.06.107

Bioorganic & Medicinal Chemistry 18 (2010) 5807–5815
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of prenylated chalcones as
5-LOX inhibitors
Nimmanapalli P. Reddy a, Polamarasetty Aparoy a, T. Chandra Mohan Reddy a, Chandrani Achari a,
P. Ramu Sridhar b, Pallu Reddanna a,
*
a School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India
b School of Chemistry, University of Hyderabad, Hyderabad 500 046, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Ten novel mono- and di-O-prenylated chalcone derivatives were designed on the basis of a homology
derived molecular model of 5-lipoxygenase (5-LOX). The compounds were docked into 5-LOX active site
and the binding characteristics were quantified using LUDI. To verify our theoretical assumption, the
molecules were synthesized and tested for their 5-LOX inhibitory activities. The synthesis was carried
out by Claisen–Schmidt condensation reaction of mono- and di-O-prenylated acetophenones with appro-
priate aldehydes. 5-LOX in vitro inhibition assay showed higher potency of di-O-prenylated chalcones
than their mono-O-prenylated chalcone analogs. Compound 5e exhibited good inhibition with an IC50
Received 28 April 2010
Revised 29 June 2010
Accepted 30 June 2010
Available online 6 July 2010
Keywords:
Prenylated chalcone
5-LOX
Docking
at 4
agreement with the experimental data. Further, the compound 5e showed potent anti-proliferative
effects (GI50 at 9 M) on breast cancer cell line, MCF-7.
lM. The overall trend for the binding energies calculated and LUDI score was in good qualitative
l
LUDI
Ó 2010 Elsevier Ltd. All rights reserved.
Claisen–Schmidt condensation reaction
Anti-proliferative
1. Introduction
tions, the anti-inflammatory activity of O-prenylated chalcones
may be mediated by the inhibition of the arachidonic acid (AA)-
metabolizing enzyme, LOX.
Chalcones or 1,3-diaryl-2-propene-1-ones, belong to the flavo-
noid family. Chemically they consist of open chain flavonoids in
LOXs (linoleate: oxygen oxido reductase, EC 1.13.11.12) are a
group of closely related non-heme iron containing dioxygenases.
These enzymes catalyze the addition of molecular oxygen into
poly-unsaturated fatty acids (PUFAs) containing cis, cis-1,4 penta-
diene structures to give their hydroperoxy derivatives. LOXs are
further classified into 5-, 8-, 9-, 11-, 12-, and 15-LOXs according
to the positional specificity of arachidonate oxygenation.16 One of
the LOX pathways of AA metabolism, the 5-LOX pathway is the
source of potent pro-inflammatory mediators.17 The 5-LOX acts
preferentially upon unesterified AA, inserting molecular oxygen
at the fifth carbon and forming the hydroperoxy intermediate, 5-
hydroperoxyeicosatetraenoic acid (5-HPETE).18 The same enzyme
then catalyzes a dehydration reaction, forming the unstable epox-
ide intermediate, leukotriene A4 (LTA4). LOX metabolites are potent
physiological effectors in a variety of cellular responses, associated
with normal host defense and inflammation. Products of the 5-LOX
pathway are thus important mediators of inflammation. Inhibitors
of the 5-LOX pathway, therefore, have a therapeutic potential in a
variety of inflammatory and allergic diseases. These efforts have
resulted in the release of Zileuton (5-LOX inhibitor) and Monteluk-
ast [LT receptor antagonist] into the market for the treatment of
asthma. 5-LOX is also associated with Gastro Esophageal Reflux
Disease (GERD). Elevated levels of LTB4 have been found in blood
and joint fluid from patients with rheumatoid arthritis19 and in
which the two aromatic rings are joined by a three-carbon
a,b-
unsaturated carbonyl system. There is currently a great deal of
interest in the health benefits of phytochemicals, in particular
prenylated flavonoids, because of their interesting biological activ-
ities. As revealed by structure–activity relationship (SAR) studies,
the presence of isoprenoid chains of varying length and type is a
major determinant of the bioactivity of prenylated flavonoids.
The remarkable properties of these compounds are thought to re-
side in their enhanced interaction with biological membranes
and increased affinity for target proteins compared with their
non-prenylated analogs. Several prenylated flavonoids became
the focus of attention as a result of growing interest in isoprenoid
chemistry, of these, prenylated chalcones are an abundant sub-
class of flavonoids that are widely distributed in nature.1–4 They
are associated with a wide range of biological activities, such as
anti-bacterial,5 anti-malarial,6 anti-fungal,7 anti-diabetic,8 anti-tu-
mor,9–11 anti-oxidative,12 anti-inflammatory,13 and NF-
jB inhibi-
tory activity.14 Prenylated chalcones and their cyclic analogs
were found to possess moderate to good antimicrobial, antiviral,
anti-invasive, and anti-insecticidal activities.15 Among these ac-
* Corresponding author. Tel.: +91 40 23134542; fax: +91 40 23010745.
E-mail addresses: prsl@uohyd.ernet.in, preddanna@yahoo.com (P. Reddanna).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.107

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N. P. Reddy et al. / Bioorg. Med. Chem. 18 (2010) 5807–5815
colonic mucosa from patients with ulcerative colitis or Crohn’s dis-
ease. LOX and their products are shown to play an important role
in tumor formation and cancer metastasis.20 High expression of
5-LOX was found in prostate, lung, colon, breast, and other cancer
cell lines.21–23 Recently it has been shown that 5-LOX (ALOX5) is
critical regulator for leukemia cancer stem cells (LSCS) in chronic
myeloid leukemia (CML). Treatment of CML mice with a 5-LOX
inhibitor also impaired the function of LSCS.24 Inhibition of the
5-LOX pathway may also be useful for cancer therapy. 5-LOX inhib-
itor has been shown to prevent lung tumorigenesis in carcinogen-
treated mice.25 As 5-LOX has been shown to have a major role in
the pathogenesis of various inflammatory disorders, including can-
cer, inhibitors of 5-LOX will have wide ranging therapeutic
applications.23
Prenylated flavonoids have become the focus of attention as a
result of growing interest in isoprenoid chemistry. Thus, in this
study, prenylated chalcones were designed, synthesized, and
tested for their 5-LOX inhibition activity. After evaluating the com-
pounds for inhibition of 5-LOX in vitro, the compounds were fur-
ther tested for their anti-proliferative effects on breast (MCF-7)
and colon (COLO-205) cancer cell lines. These cell lines were se-
lected as 5-LOX inhibitors are known to exhibit anti-proliferative
effects in these cell lines by inducing apoptosis.26,27 Further, to
check the toxicity of the best compound, it was tested on human
keratinocyte cell line, HaCaT.
were around 16 Hz (trans-configuration). There is no need for pro-
tection of hydroxyl groups during this procedure and it also re-
sulted in good yield. The structures of all the 10 prenylated
chalcones were established on the basis of IR, 1H NMR, 13C NMR,
and LCMS spectral data.
2.2. Docking and scoring
The structures of the ligands were energy minimized to a RMSD
of 0.001 kcal/mol using MMFF94X forcefield. The ligands (preny-
lated chalcones and NDGA) were docked into 5-LOX active site
and the best and the most energetically favorable conformation
of each ligand were selected. A four-stage protocol was set up for
energy minimizations of the protein–inhibitor complex using AM-
BER. Minimization methods were used for predicting relative bind-
ing affinities of the inhibitors using energies obtained both in
solvent as well as complex phases of each inhibitor. The binding
affinities of the prenylated chalcones in the optimized complexes
was further estimated using LUDI. The di-O-prenylated chalcones
were predicted to be more effective than their mono-O-prenylated
chalcones analogs (Table 1). The high affinity of compound 5e
(Fig. 1), as calculated in the Ludi module and by minimization
methods indicates that it can form a potential 5-LOX inhibitor
candidate.
A comparison of SAR data show that the LUDI score and binding
energies increase as the number of hydrogen bonds increase. In the
case of the most highly scored compound 5e most of the functional
groups interacted favorably with the 5-LOX active site. Strong
hydrogen bond interactions were observed between 3-methoxy
of compound 5e and Thr784 (Oꢀ ꢀ ꢀHO, 3.7 Å), 4-methoxy and
His271 (Oꢀ ꢀ ꢀHN, 2.4 Å), and 5-methoxy and Asp560 (Oꢀ ꢀ ꢀHO,
2.3 Å), respectively. The 20-hydroxyl is replaced with 20-O-prenyl
group in compound 5e in comparison to compound 4e. In mono-
O-prenyl compounds, it has been observed that the 20-hydroxyl
group formed hydrogen bond interactions with Ser567 (Oꢀ ꢀ ꢀHO,
2.5 Å). The 40-O-prenyl group in compound 4a–e formed strong
hydrophobic interactions with Trp526 (3.4 Å) and Leu572 (4 Å).
In compound 5a–e, it has been observed that the prenyl group of
2. Results and discussion
2.1. Synthesis
Prenylated chalcones (4a–e and 5a–e) were synthesized by a
base catalyzed Claisen–Schmidt condensation of appropriately
substituted prenylated acetophenone with substituted benzalde-
hyde in EtOH (Scheme 1). The prenylated acetophenones 228 and
329 were prepared from 2,4-dihydroxy acetophenone with prenyl
bromide and anhydrous K2CO3 in acetone. This method for the
preparation of prenylated chalcones is attractive, as observed from
1H NMR spectra, the coupling constants of the two olefinic protons
R4
R3
R2
R4
H
R3
R2
Br
PreO
1 eq.
R1
O
PreO
5 h
50%KOH
Reflux
R1
OH
O
O
4a-4e
HO
K2CO3
HO
OH
O
2
Acetone
R4
R3
R2
Br
PreO
R4
R3
R2
H
2.5 eq.
1
R1
O
PreO
24 h
50%KOH
Reflux
PreO
O
R1
PreO
O
3
5a-5e
4a, 5a R1 = R2 = R4 = H, R3 = OMe
4b, 5b R1 = R2 = OMe, R3 = R4 = H
4c, 5c R1 = R2 = R3 = OMe, R4 = H
4d, 5d R1 = R4 = H, R2 = R3 = OMe
4e, 5e R1 = H, R2 = R3 = R4 = OMe
Scheme 1. Route for the synthesis of prenylated chalcones (pre denotes prenyl group).

N. P. Reddy et al. / Bioorg. Med. Chem. 18 (2010) 5807–5815
5809
Table 1
gen-bonding interactions with the main chain of Asp276 (OHꢀ ꢀ ꢀO,
0
Binding energies, LUDI scores, and in vitro 5-LOX enzyme inhibition assay data for the
prenylated chalcones
2.4 ÅA), the third hydroxyl group formed hydrogen bonding with the
0
side chain atom of Ile564 (OHꢀ ꢀ ꢀO, 2.86 ÅA) and two bonds with
0
Compound
IC50
(
lM)
LUDI score
DEbind (total) (kcal/mol)
Asn565 (Oꢀ ꢀ ꢀN, 3.16 ÅA). The fourth hydroxyl group is in close prox-
0
imity to the His530 (Oꢀ ꢀ ꢀN, 3.9 ÅA). Hence it exhibits very low IC50
and possesses high affinity. The binding energies calculated and
the LUDI scores estimated for the compound 5e indicate that it
may be as potential as NDGA. To verify our theoretical findings
the molecules were further synthesized and tested for their 5-
LOX inhibitory activities.
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
>100
>100
>100
>100
>100
>100
22
49
9
4
1.5
752
613
690
726
768
710
766
779
785
875
882
ꢁ14.4
ꢁ14.5
ꢁ15.1
ꢁ16.6
ꢁ17.5
ꢁ21.2
ꢁ21.6
ꢁ22.9
ꢁ24.4
ꢁ25.1
ꢁ25.5
2.3. In vitro 5-LOX enzyme assay
NDGA
The potential 5-LOX inhibitory molecules synthesized were
tested in vitro for their inhibitory properties against 5-LOX enzyme
using the assay described by Reddanna et al.30 Out of the 10 mol-
ecules tested compounds 5d and 5e showed inhibition at a concen-
tration <10
mono-O-prenyl chalcones, on the other hand, showed IC50
>100 M. The most active molecule, compound 5e, showed potent
inhibition of 5-LOX with an IC50 value of 4 M (Table 1). The bind-
lM. Compounds 5b and 5c showed IC50 <50 lM. All the
l
l
ing energies estimated were in good qualitative agreement with
the experimental data than the LUDI scores calculated. As esti-
mated the di-O-prenylated chalcones were more potent than the
mono-O-prenyl chalcones. The number of hydrogen bonds in the
molecules showed inverse correlation with the experimental IC50
.
Further, the inhibitory activity of the best inhibitor, compound
5e, was analyzed and confirmed by analysis of products on straight
phase-HPLC (SP-HPLC). It has been observed in HPLC studies that
the compound 5e at 4 lM effectively inhibited the production of
5-HPETE from 5-LOX as shown in Figure 3.
2.4. In vitro anti-proliferative effects on cancer cell lines
Figure 1. Structure of compound 5e.
Prenylated chalcones were further tested for their anti-prolifer-
ative effects on MCF-7 and COLO-205 cell lines. Cells were incu-
bated with different concentrations of compounds for 24 h and
the cell viability was measured by MTT assay. The GI50 values for
different compounds obtained are shown in Table 2 and Figure 4.
The compounds showed anti-proliferative effects in a concentra-
tion dependent manner. The di-O-prenyl compounds (5a–e)
showed better anti-proliferative effects than their mono-O-prenyl
derivatives (4a–e). The toxicity of the potential compound 5e when
tested on normal cell line (HaCaT), showed no appreciable effects
the 20-O-prenyl group formed strong hydrophobic interactions
with Leu255 (3.1 Å) and Lys283 (3.4 Å) along with strong hydrogen
bond interactions between oxygen atom of 20-O-prenyl and Ser567
(Fig. 2). The interaction analysis of the compounds suggests that
compound 5e is the most potential molecule.
NDGA is firmly bound in the open cavity, that is, in the sixth
coordination of the iron atom as reported in our earlier studies.31
NDGA has four hydroxyl groups, the first hydroxyl group formed
strong hydrogen bonding interactions with the side chain of
0
His271 (Oꢀ ꢀ ꢀN
e, 2.75 ÅA), the second hydroxyl group formed hydro-
up to 100 lM.
Figure 2. Hydrogen bonding of the compounds with 5-LOX active site residues: (a) compound 4e and (b) compound 5e (hydrogen bonding interactions are shown as dotted
line, hydrogen bond forming amino acids in active site are labeled in white and hydrophobic interactions forming amino acids are labeled in pink).

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N. P. Reddy et al. / Bioorg. Med. Chem. 18 (2010) 5807–5815
Figure 3. HPLC product profile of 5-HPETE generated from the reaction of potato 5-LOX with AA as the substrate. (a) Standard 5-HPETE. (b) 5-LOX products in the control. (c)
5-LOX products in the presence of compound 5e (4
l
M). HPLC conditions were as described under experimental procedures. UV absorbance of the eluate was monitored at
235 nm.
trend for the binding energies estimated is in good qualitative
agreement with the experimental data than the LUDI scores. In
3. Conclusions
addition, all of the lead molecules were designed on the basis of
a homology derived molecular model of 5-LOX. Compound 5e
inhibited 5-LOX with an IC50 of 4 lM. Further it showed potent
A new class of prenylated chalcones has been designed and syn-
thesized by Claisen–Schmidt condensation reaction and evaluated
for their 5-LOX inhibitory and anti-proliferative effects. The overall

N. P. Reddy et al. / Bioorg. Med. Chem. 18 (2010) 5807–5815
5811
Table 2
(3 g) and prenyl bromide (4.07 mL, 1 equiv, 35 mmol). The reaction
mixture was refluxed for 5 h. On completion of the reaction (TLC),
solvent was evaporated under reduced pressure and ice cold water
(75 mL) was added to it, and extracted with ethyl acetate
(3 ꢂ 100 mL). The combined organic layer was separated, dried
over Na2SO4, and concentrated under reduced pressure. The resi-
due was purified by silica gel column chromatography eluting with
hexane/EtOAc (8:2) to afford the title compound 2 as white crystal-
line solid (5.42 g) in 70.3% yield.
Cytotoxicity of prenylated chalcone derivatives against
human cancer cell lines, MCF-7 and COLO cells
Compound
GI50 (lM)
MCF-7
COLO
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
97.5
>100
>100
>100
81
67.5
73
92
>100
>100
>100
>100
96
97
99
>100
90
78
1H NMR (400 MHz, CDCl3): d 12.76 (1H, s, OH-2), 7.62 (1H, d,
J = 7.6 Hz, H-6), 6.48–6.43 (2H, m, H-5, H-3), 5.51–5.46 (1H, m,
CH-20), 4.56 (2H, d, J = 6.8 Hz, CH2-10), 2.57 (3H, s, COCH3), 1.82
(3H, s, prenyl-CH3), 1.79 (3H, s, prenyl-CH3). LCMS (positive mode)
m/z 221 [M+H]+.
66.5
9
4.1.3. Synthesis of 2,4-di-O-prenylacetophenone (3)
To a solution of 2,4-dihydroxyacetophenone (1) (5.32 g, 1 equiv,
35 mmol) in acetone (100 mL) was added freshly ignited K2CO3
(10 g) and prenyl bromide (10.18 mL, 2.5 equiv, 87.5 mmol). The
reaction mixture was refluxed for 24 h. On completion of the reac-
tion (TLC), cooling to room temperature, filtration, and the residue
was purified by silica gel column chromatography eluting with
hexane/EtOAc (7:3) to afford the title compound 3 as pale yellow
solid (8.12 g) in 80.5% yield.
1H NMR (400 MHz, CDCl3): d 7.82 (1H, d, J = 8.4 Hz, H-6), 6.50
(1H, dd, J = 8.4, 2.0 Hz, H-5), 6.47 (1H, d, J = 2.0 Hz, H-3), 5.50
(2H, br s, CH-20, CH-200), 4.58–4.54 (4H, m, CH2-10, CH2-100), 2.58
(3H, s, COCH3), 1.80 (3H, s, prenyl-CH3), 1.75 (3H, s, prenyl-CH3),
1.74 (3H, s, prenyl-CH3), 1.70 (3H, s, prenyl-CH3). LCMS (positive
mode) m/z 289 [M+H]+.
4.1.4. General procedure for the synthesis of prenylated
chalcones
Figure 4. MCF-7, COLO-205, and HaCaT cells were cultured for 24 h with or without
5e (1–100 M) and cell viability was analyzed by MTT assay. Each data point
represents the mean of four independent experiments with error bars indicating
SE.
Prenylated chalcones (4a–e and 5a–e) were synthesized by a
base catalyzed Claisen–Schmidt condensation reaction of mono-
and di-O-prenylated acetophenone (1.0 equiv, 1 mmol) and a
substituted benzaldehyde (1.0 equiv, 1.0 mmol). The starting
materials dissolved in a minimum volume of ethanol (10–15 mL)
and added aqueous solution of 50% KOH (5 equiv). The reaction
mixture was refluxed for 3–4 h. Until the completion of reaction
by TLC, the reaction mixture was concentrated to one-fourth of
its original volume and pH was adjusted to 3–4 with 2 N HCl solu-
tion and then extracted with ethyl acetate (3ꢂ100 mL). The com-
bined organic layer was separated, dried over anhydrous Na2SO4,
and concentrated under reduced pressure. The residue was puri-
fied by column chromatography over silica gel, eluting with step
gradient of hexane/EtOAc to afford pure prenylated chalcone deriv-
atives 4a–e and 5a–e.
l
anti-proliferative effects on breast cancer cell line, MCF-7. These
novel inhibitors developed could form leads for further inhibitor
development against 5-LOX. This study assumes importance in
the light of key role played by 5-LOX in various pathological
manifestations.
4. Experimental section
4.1. Chemistry
4.1.1. General information
4.1.4.1. 20–Hydroxy-4-methoxy-40-O-prenylchalcone (4a). Or-
Melting points were determined on a Kofler hot-stage appara-
tus and are uncorrected. The reactions were monitored by thin
layer chromatography (TLC) on precoated Merck Silica gel 60F254
aluminum plates. IR spectra were recorded in KBr disks on a JASCO
FT/IR-5300. 1H NMR spectra were recorded on Bruker Avance 400
spectrometer using CDCl3 with TMS as internal standard. Coupling
constant (J) values are calculated in hertz (Hz) and spin multiples
are given as s (singlet), d (double), t (triplet), q (quartet), m (mul-
tiplet), and br (broad). Mass spectra were recorded on either
VG7070H mass spectrometer using EI technique or Shimadzu-
LCMS-2010A. Column chromatography was performed on silica
gel (Merk) 100–200 mesh.
ange color semi-solid (0.27 g); yield 79.8%; IR (KBr)
m
;
max: 3350,
2930, 2858, 1633, 1568, 1367, 1253, 1032 cmꢁ1
1H NMR
(400 MHz, CDCl3): d 13.41 (1H, s, OH-20), 7.86 (1H, d, J = 16.0 Hz,
H-b), 7.62 (1H, d, J = 8.7 Hz, H-60), 7.48 (1H, d, J = 16.0 Hz, H-
a),
7.31 (2H, d, J = 8.0, H-2, H-6), 6.96 (2H, d, J = 8.0 Hz, H-3, H-5),
6.50–6.46 (2H, m, H-50, H-30), 5.49 (1H, t, J = 6.6 Hz, H-200), 4.56
(2H, d, J = 6.6 Hz, CH2-100), 3.87 (3H, s, OMe), 1.81 (3H, s, prenyl-
CH3), 1.76 (3H, s, prenyl-CH3); 13C NMR (100 MHz, CDCl3): d
191.85 (C@O), 166.58 (C-20), 165.22 (C-40), 161.80 (C-4), 144.20
(C-b), 139.15 (C-300), 132.26 (C-60), 130.36 (C-2 and C-6), 128.66
(C-1), 118.74 (C-200), 117.90 (C-
a), 114.48 (C-3 and C-5), 113.98
(C-10), 108.20 (C-50), 101.72 (C-30), 65.18 (C-100), 55.44 (OMe),
25.82 (Me-400), 18.25 (Me-500). Anal. Calcd for C21H22O4: C, 74.54;
H, 6.55. Found: C, 74.61; H, 6.51. LCMS (positive mode) m/z 339
[M+H]+.
4.1.2. Synthesis of 2-hydroxy–4-O-prenylacetophenone (2)
To a solution of 2,4-dihydroxyacetophenone (1) (5.32 g, 1 equiv,
35 mmol) in acetone (100 mL) was added freshly ignited K2CO3

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N. P. Reddy et al. / Bioorg. Med. Chem. 18 (2010) 5807–5815
4.1.4.2. 20–Hydroxy-2,3-dimethoxy-40-O-prenylchalcone (4b). Yel-
500). Anal. Calcd for C23H26O6: C, 69.33; H, 6.58. Found: C, 69.25;
low solid (0.305 g); yield 82.8%; mp 95–96 °C; IR (KBr)
m
;
max: 3439,
H, 6.52. LCMS (positive mode) m/z 399 [M+H]+.
2930, 2843, 1641, 1575, 1359, 1269, 1072 cmꢁ1
1H NMR
(400 MHz, CDCl3): d 13.49 (1H, s, OH-20), 8.18 (1H, d, J = 15.6 Hz,
4.1.4.6. 4-Methoxy-20,40-di-O-prenylchalcone (5a). Yellowish oil
H-b), 7.84 (1H, d, J = 9.0 Hz, H-60), 7.68 (1H, d, J = 15.6 Hz, H-
a
),
(0.305 g); yield 75.1%; IR (KBr)
1255, 1020 cmꢁ1 1H NMR (400 MHz, CDCl3): d 7.85 (1H, d,
J = 15.6 Hz, H-b), 7.62 (1H, d, J = 8.5 Hz, H-60), 7.52 (1H, d,
J = 15.8 Hz, H- ), 7.42 (2H, d, J = 8.2 Hz, H-2, H-6), 6.95 (2H, d,
mmax: 2930, 2849, 1687, 1575, 1425,
7.30 (1H, dd, J = 9.0, 2.0 Hz, H-6), 7.16 (1H, dd, J = 9.0, 9.0 Hz, H-
5), 7.00 (1H, dd, J = 9.0, 2.0 Hz, H-4), 6.52–6.49 (2H, m, H-50, H-
30), 5.51 (1H, t, J = 6.7 Hz, H-200), 4.58 (2H, d, J = 6.7 Hz, CH2-100),
3.91 (3H, s, OMe), 3.89 (3H, s, OMe), 1.82 (3H, s, prenyl-CH3),
1.77 (3H, s, prenyl-CH3); 13C NMR (100 MHz, CDCl3): d 192.10
(C@O), 166.58 (C-20), 165.48 (C-40), 153.22 (C-3), 148.98 (C-2),
;
a
J = 8.4 Hz, H-3, H-5), 6.51–6.48 (2H, m, H-50, H-30), 5.49 (2H, t,
J = 6.7 Hz, H-200, H-2000), 4.57 (4H, d, J = 6.7 Hz, CH2-100, CH2-1000),
3.89 (3H, s, OMe), 1.81 (3H, s, prenyl-CH3), 1.79 (3H, s, prenyl-
CH3), 1.77 (3H, s, prenyl-CH3), 1.69 (3H, s, prenyl-CH3); 13C NMR
(100 MHz, CDCl3): d 190.05 (C@O), 163.44 (C-20), 161.07 (C-40),
159.96 (C-4), 141.37 (C-b), 138.87 and 138.70 (C-300 and C-3000),
132.00 (C-60), 130.36 (C-2 and C-6), 128.51 (C-1), 119.21 and
139.18 (C-b and C-300), 131.21 (C-60), 128.98 (C-
a), 124.17 (C-5),
121.86 (C-1), 119.74 (C-200), 118.65 (C-6), 114.27 (C-4), 114.06
(C-10), 108.20 (C-50), 101.68 (C-30), 65.15 (C-100), 61.31 (OMe),
55.88 (OMe), 25.78 (Me-400), 18.21 (Me-500). Anal. Calcd for
C
22H24O5: C, 71.72; H, 6.57. Found: C, 71.65; H, 6.52. LCMS (posi-
119.09 (C-200 and C-2000), 118.58 (C-
a), 114.32 (C-3 and C-5),
tive mode) m/z 369 [M+H]+.
113.63 (C-10), 106.07 (C-50), 100.38 (C-30), 65.54 and 65.04 (C-100
and 1000), 55.36 (OMe), 25.84 and 25.76 (Me-400 and Me-4000), 18.27
and 18.24 (Me-500 and Me-5000). Anal. Calcd for C26H30O4: C, 76.82;
H, 7.44. Found: C, 76.71; H,7.52. LCMS (negative mode) m/z 405
[MꢁH]ꢁ.
4.1.4.3. 20–Hydroxy-2,3,4-trimethoxy-40-O-prenylchalcone (4c).
Light yellowish oil (0.315 g); yield 79.1%; IR (KBr)
m
;
max: 3445,
2932, 2849, 1631, 1494, 1359, 1257, 1097 cmꢁ1
1H NMR
(400 MHz, CDCl3): d 13.60 (1H, s, OH-20), 8.05 (1H, d, J = 15.6 Hz,
H-b), 7.81 (1H, d, J = 8.8 Hz, H-60), 7.63 (1H, d, J = 15.6 Hz, H-
a),
4.1.4.7. 2,3-Dimethoxy-20,40-di-O-prenylchalcone (5b). Yellow
7.38 (1H, d, J = 8.8 Hz, H-6), 6.72 (1H, d, J = 8.8 Hz, H-5), 6.49–
6.47 (2H, m, H-50, H-30), 5.48 (1H, br s, H-200), 4.56 (2H, d, J =
6.4 Hz, CH2-100), 3.97 (3H, s, OMe), 3.91 (3H, s, OMe), 3.89 (3H, s,
OMe), 1.81 (3H, s, prenyl-CH3), 1.75 (3H, s, prenyl-CH3); 13C NMR
(100 MHz, CDCl3): d 192.19 (C@O), 166.57 (C-20), 165.33 (C-40),
155.95 (C-4), 153.90 (C-2), 142.49 (C-3), 139.73 and 139.02 (C-b
gummy oil (0.341 g); yield 78.2%; IR (KBr)
1645, 1601, 1469, 1267, 1012 cmꢁ1 1H NMR (400 MHz, CDCl3): d
8.10 (1H, d, J = 15.5 Hz, H-b), 7.80 (1H, d, J = 9.0 Hz, H-60), 7.62
(1H, d, J = 15.6 Hz, H- ), 7.25 (1H, dd, J = 8.8, 2.2 Hz, H-6), 7.06
mmax: 2974, 2930,
;
a
(1H, dd, J = 8.8, 8.8 Hz, H-5), 6.91 (1H, dd, J = 8.8, 2.2 Hz, H-4),
6.58–6.45 (2H, m, H-50, H-30), 5.52 (2H, t, J = 6.7 Hz, H-200, H-2000),
4.58 (4H, d, J = 6.7 Hz, CH2-100, CH2-1000), 3.88 (3H, s, OMe), 3.86
(3H, s, OMe), 1.81 (3H, s, prenyl-CH3), 1.77 (3H, s, prenyl-CH3),
1.72 (3H, s, prenyl-CH3), 1.62 (3H, s, prenyl-CH3); 13C NMR
(100 MHz, CDCl3): d 190.22 (C@O), 163.58 (C-20), 160.07 (C-40),
153.19 (C-3), 148.81 (C-2), 139.10 (C-b), 138.87 and 138.64 (C-300
and C-300), 131.12 (C-60), 128.83 (C-
a), 124.20 (C-6), 119.45
(C-200), 118.78 (C-10), 114.15 (C-1), 108.08 (C-50), 107.80 (C-5),
101.71 (C-30), 65.15 (C-100), 61.38 (OMe), 60.90 (OMe), 56.17
(OMe), 25.80 (Me-400), 18.22 (Me-500). Anal. Calcd for C23H26O6: C,
69.33; H, 6.58. Found: C, 69.21; H, 6.62. LCMS (positive mode) m/
z 399 [M+H]+.
and 3000), 130.95 (C-60), 128.88 (C-
a), 124.23 (C-5), 121.89 (C-1),
119.79 and 119.20 (C-200 and C-2000), 118.72 (C-6), 114.28 (C-4),
113.48 (C-10), 106.12 (C-50), 100.31 (C-30), 65.55 and 65.05 (C-100
and C-1000), 61.42 (OMe), 55.87 (OMe), 25.83 and 25.73 (Me-400
and Me-4000), 18.24 (Me-500 and Me-5000). Anal. Calcd for C27H32O5:
C, 74.29; H, 7.39. Found: C, 74.21; H, 7.33. LCMS (positive mode)
m/z 437 [M+H]+.
4.1.4.4. 20–Hydroxy-3,4-dimethoxy-40-O-prenylchalcone (4d). Or-
ange color semi-solid (0.29 g); yield 78.8%; IR (KBr)
m
;
max: 3410,
2930, 2847, 1633, 1514, 1367, 1257, 1024 cmꢁ1
1H NMR
(400 MHz, CDCl3): d 13.40 (1H, s, OH-20), 7.87–7.83 (2H, m, H-b,
H-60), 7.44 (1H, d, J = 15.6 Hz, H-
a), 7.25 (1H, d, J = 2.1 Hz, H-2),
7.17 (1H, dd, J = 8.4, 2.1 Hz, H-6), 6.90 (1H, d, J = 8.4, Hz, H-5),
6.51–6.49 (2H, m, H-50, H-30), 5.48 (1H, br s, H-200), 4.56 (2H, d,
J = 6.5 Hz, CH2-100), 3.97 (3H, s, OMe), 3.94 (3H, s, OMe), 1.81 (3H,
s, prenyl-CH3), 1.76 (3H, s, prenyl-CH3); 13C NMR (100 MHz,
CDCl3): d 191.71 (C@O), 166.59 (C-20), 165.42 (C-40), 151.57 (C-4),
149.28 (C-3), 144.76 (C-b), 139.13 (C-300), 131.09 (C-60), 128.84
4.1.4.8. 2,3,4-Trimethoxy-20,40-di-O-prenylchalcone (5c). Light
yellowish oil (0.361 g); yield 77.4%; IR (KBr)
1630, 1494, 1365, 1284, 1097 cmꢁ1 1H NMR (400 MHz, CDCl3): d
8.04 (1H, d, J = 15.6 Hz, H-b), 7.82 (1H, d, J = 9.2 Hz, H-60), 7.63
(1H, d, J = 15.6 Hz, H- ), 7.39 (1H, d, J = 8.8 Hz, H-6), 6.74 (1H, d,
mmax: 2930, 2854,
;
a
(C-1), 123.34 (C-6), 118.71 (C-200) 118.04 (C-
a
), 114.02 (C-10),
J = 8.8 Hz, H-5), 6.50–6.43 (2H, m, H-50, H-30), 5.49 (2H, br s, H-
200, H-2000), 4.57 (4H, d, J = 6.0 Hz, CH2-100, CH2-1000), 3.97 (3H, s,
OMe), 3.94 (3H, s, OMe), 3.90 (3H, s, OMe), 1.81 (3H, s, prenyl-
CH3), 1.76 (3H, s, prenyl-CH3), 1.72 (3H, s, prenyl-CH3), 1.68 (3H,
s, prenyl-CH3); 13C NMR (100 MHz, CDCl3): d 192.18 (C@O),
166.52 (C-20), 165.29 (C-40), 155.90 (C-4), 153.80 (C-2), 142.55
(C-3), 139.72 (C-300 and C-3000), 139.04 (C-b), 131.08 (C-60), 128.80
111.16 (C-5), 110.22 (C-2), 108.20 (C-50), 101.69 (C-30), 65.19 (C-
100), 55.99 (OMe), 55.88 (OMe), 25.81 (Me-400), 18.25 (Me-500). Anal.
Calcd for C22H24O5: C, 71.72; H, 6.57. Found: C, 71.58; H, 6.45.
LCMS (positive mode) m/z 369 [M+H]+.
4.1.4.5. 20–Hydroxy-3,4,5-trimethoxy-40-O-prenylchalcone (4e).
Yellow amorphous powder (0.31 g); yield 77.8%; mp 113–
(C-a
), 124.20 (C-6), 119.47 (C-200 and C-2000), 118.71 (C-10), 114.11
115 °C; IR (KBr)
1124 cmꢁ1 1H NMR (400 MHz, CDCl3): d 13.51 (1H, s, OH-20),
7.84–7.79 (2H, m, H-b, H-60), 7.46 (1H, d, J = 15.6 Hz, H-
), 6.88
m
max: 3440, 2939, 2841, 1639, 1572, 1371, 1271,
(C-1), 108.08 (C-50), 107.58 (C-5), 101.66 (C-30), 65.12 (C-100 and
C-1000), 61.35 (OMe), 60.88 (OMe), 56.05 (OMe), 25.77 (Me-400 and
Me-4000), 18.19 (Me-500 and 5000). Anal. Calcd for C28H34O6: C, 72.08;
H, 7.35. Found: C, 72.21; H, 7.38. LCMS (positive mode) m/z 467
[M+H]+.
;
a
(2H, s, H-6, H-2), 6.49 (2H, m, H-50, H-30), 5.50 (1H, br s, H-200),
4.58 (2H, d, J = 6.8 Hz, CH2-100), 3.94 (6H, s, 2 ꢂ OMe), 3.91 (3H, s,
OMe), 1.82 (3H, s, prenyl-CH3), 1.77 (3H, s, prenyl-CH3); 13C NMR
(100 MHz, CDCl3): d 191.58 (C@O), 166.68 (C-20), 165.57 (C-40),
153.51 (C-3 and C-5), 144.48 (C-b), 140.59 (C-4), 139.27 (C-300),
4.1.4.9. 3,4-Dimethoxy-20,40-di-O-prenylchalcone (5d). Yellow-
ish oil (0.338 g); yield 77.5%; IR (KBr)
1514, 1375, 1261, 1020 cmꢁ1 1H NMR (400 MHz, CDCl3): d 7.81
(1H, d, J = 15.5 Hz, H-b), 7.78 (1H, d, J = 8.4 Hz, H-60), 7.72 (1H, d,
J = 15.5 Hz, H- ), 7.52 (1H, d, J = 2.2 Hz, H-2), 7.18 (1H, dd, J = 9.0,
mmax: 2928, 2866, 1645,
131.14 (C-60), 130.31 (C-1), 119.53 (C-200), 118.64 (C-
a), 113.07
;
(C-10), 108.35 (C-50), 105.80 (C-2 and C-6), 101.69 (C-30), 65.23
(C-100), 61.03 (OMe), 56.27 (2 ꢂ OMe), 25.84 (Me-400), 18.26 (Me-
a

N. P. Reddy et al. / Bioorg. Med. Chem. 18 (2010) 5807–5815
5813
2.2 Hz, H-6), 6.87 (1H, d, J = 9.0 Hz, H-5), 6.58–6.52 (2H, m, H-50, H-
30), 5.50 (2H, br s, H-200, H-2000), 4.57 (4H, d, J = 6.7 Hz, CH2-100, CH2-
1000), 3.97 (3H, s, OMe), 3.91 (3H, s, OMe), 1.81 (3H, s, prenyl-CH3),
1.76 (3H, s, prenyl-CH3), 1.74 (3H, s, prenyl-CH3), 1.72 (3H, s, pre-
nyl-CH3); 13C NMR (100 MHz, CDCl3): d 190.71 (C@O), 167.63 (C-
20), 163.35 (C-40), 150.81 (C-4), 149.10 (C-3), 144.48 (C-b), 138.88
and 138.29 (C-300 and C-3000), 132.22 (C-60), 128.83 (C-1), 122.45
and final stage, all the atoms of residues within 15 Å from the
center of the inhibitor (solvent molecules, protein atoms, and
the ligand) were allowed to relax.
A four-stage protocol was also established for energy minimiza-
tion of the solvated inhibitor. These minimizations were carried out
using periodic boundary conditions in all directions and each stage
involved 100 steps of steepest descent and 1500 steps of conjugate
gradient optimization. In the first stage of minimization, only the
waters were minimized keeping the inhibitor (i.e., the solute) fixed.
In the second stage, only hydrogens in the system were allowed to
relax. In the third stage, atoms common to the ligand in the crystal
structure complex and the modified ligand were also fixed, while
allowing the solvent and the modified group in the ligand to move
during optimization. In the fourth stage of the solvent calculation,
all water molecules and the solute (ligand) were allowed to relax.
The minimized structures for all the inhibitors in the complexed
and solvated states were used for calculating the following energy
variables:
(C-6), 119.20 and 119.05 (C-200 and C-2000) 118.68 (C-
a), 114.20
(C-10), 111.09 (C-5), 110.36 (C-2), 106.05 (C-50), 100.51 (C-30),
65.74 and 65.02 (C-100 and C-1000), 55.95 (OMe), 55.86 (OMe),
25.81 and 25.74 (Me-400 and Me-4000), 18.28 and 18.22 (Me-500 and
Me-5000). Anal. Calcd for C27H32O5: C, 74.29; H, 7.39. Found: C,
74.36; H, 7.31. LCMS (positive mode) m/z 437 [M+H]+.
4.1.4.10. 3,4,5-Trimethoxy-20,40-di-O-prenylchalcone (5e). Yel-
lowish gummy oil (0.368 g); yield 78.9%; IR (KBr)
2934, 1633, 1583, 1379, 1273, 1008 cmꢁ1 1H NMR (400 MHz,
CDCl3): d 7.80 (1H, d, J = 8.4 Hz, H-60), 7.61 (1H, d, J = 16.0 Hz, H-
b), 7.44 (1H, d, J = 16.0 Hz, H- ), 6.83 (2H, s, H-6, H-2), 6.59–6.44
mmax: 2970,
;
a
D
EbindðIntraÞ ¼ EcomðIntraÞ ꢁ EsolðIntraÞ
ð1Þ
(2H, m, H-50, H-30), 5.49 (2H, br s, H-200, H-2000), 4.58 (4H, d,
J = 6.6 Hz, CH2-100, CH2-1000), 3.94 (3H, s, OMe), 3.85 (3H, s, OMe),
3.83 (3H, s, OMe), 1.82 (3H, s, prenyl-CH3), 1.77 (3H, s, prenyl-
CH3), 1.77 (6H, s, prenyl-2ꢂCH3) 13C NMR (100 MHz, CDCl3): d
191.60 (C@O), 166.50 (C-20), 163.55 (C-40), 153.53 and 153.39 (C-
3 and C-5), 144.49 (C-b), 141.70 (C-4), 138.94 and 138.20 (C-300
and C-3000), 132.98 (C-60), 131.20 (C-1), 119.57 and 119.16 (C-200
D
EbindðInterÞ ¼ EcomðInterÞ ꢁ EsolðInterÞ
ð2Þ
where
DEbind(intra) and DEbind(inter) are relative intra and intermo-
lecular binding interaction energies of a ligand, respectively, and
Ecom(Intra), Ecom(Inter), Esol(intra), and Esol(Inter) are intra and inter-
molecular interaction energies of a ligand in the complexed and sol-
and C-2000), 118.67 (C- ), 113.10 (C-10), 106.80 (C-50), 105.87 (C-2
a
vated states, respectively. The total binding energy,
an inhibitor is given by
D
Ebind (Total), of
and C-6), 101.73 (C-30), 65.89 and 65.08 (C-100 and C-1000), 60.98
(OMe), 56.28 (2 ꢂ OMe), 25.81 (Me-40 and Me-4000), 18.24 (Me-500
and Me-5000). Anal. Calcd for C28H34O6: C, 72.08; H, 7.35. Found: C,
72.18; H, 7.29. LCMS (negative mode) m/z 465 [MꢁH]ꢁ.
D
EbindðTotalÞ ¼
D
EbindðIntraÞ þ EbindðInterÞ
D
ð3Þ
In Table 1, the experimentally measured IC50’s are compared
with the binding energies calculated using minimization methods
and with scores obtained using LUDI. After four-stage protocol en-
ergy minimizations, the optimized structures of protein and ligand
were visualized and studied using InsightII.
4.2. Computational studies
As the crystal structure of 5-LOX is not yet available, the homol-
ogy model of 5-LOX reported by us earlier was used in the study.31
Ten novel mono- and di-O-prenylated chalcone derivatives were
designed. The structure of the molecules was sketched and mini-
mized using cerius2 and the final structure was obtained in confor-
mational analysis. GOLD (Genetic Optimization of Ligand Docking),
a docking program based on genetic algorithm32 was used to dock
the inhibitors. During docking, the default algorithm speed was se-
lected. The number of poses for each inhibitor was set to 100, and
early termination was allowed if the top three bound conforma-
tions of a ligand were within 1.5 Å RMSD.
After docking, the individual binding poses of each ligand were
observed and their interactions with the protein were studied.
The best and the most energetically favorable conformation of
each ligand were selected and complexed with 5-LOX model. A
four-stage protocol33,34 was set up for energy minimizations of
the protein–inhibitor complex using AMBER.35 Minimization at
each stage was performed using 200 steps of steepest descent
and 1500 steps of conjugate gradient algorithms for minimiza-
tion. In the first stage, only the solvent molecules were mini-
mized, keeping the inhibitor and the protein (in the complex
calculation) fixed. The purpose of this step is to relieve any bad
contacts involving solvent molecules in the initially solvated sys-
tem. In the second stage, only hydrogens in the system were al-
lowed to relax. This step relaxes the hydrogen atoms prior to
relaxing heavy atoms. It was performed because the adjustments
in hydrogen atom locations are necessary to improve hydrogen
bond geometries. In the third stage, all atoms of the protein were
fixed, while allowing all the atoms of the inhibitor and the sol-
vent to move during optimization. This stage allows for the relax-
ation of the inhibitor with respect to the protein and establishes
the preferred interactions (e.g., hydrogen bonds). In the fourth
The LUDI method for de novo design of ligands for proteins (i.e.,
enzyme inhibitors) is a method for screening a large number of
compounds by analyzing the geometrical fit of given chemicals in
the designated protein binding site. Other determinants of good
binding are also calculated and include hydrogen bond formation,
lipophilic interactions, ionic interactions, and acyclic interactions.
However, LUDI can also score protein ligand interactions by statis-
tically evaluating the fit of potential ligands. The LUDI scoring
method36 of interactions between a protein and its ligand was used
in this study to quantify the binding characteristics of the com-
pounds to 5-LOX. In general a higher LUDI score represents a high-
er affinity and stronger binding of a ligand to the receptor:
LUDI score ¼ ꢁ73:33mol=kcal
DG
where
D
G ¼
D
Go þ
GrotNR
Go represents the contribution to the binding energy that does not
directly depend on any specific interactions with the receptor, Ghb
and Gion represent the contribution from an ideal hydrogen bond
and unperturbed ionic interactions, respectively, Glipo represents
the contribution from lipophilic interactions which is proportional
to the lipophilic surface Alipo GrotNR represents the contribution
due to freezing of internal degrees of freedom in the fragment, NR
the number of acylic bonds, R the deviation of the hydrogen bond
length from the ideal value of 1.9 Å, and a is the deviation of the
D
Ghbfð
D
RÞfð
D
aÞ þ
D
Gionfð
D
RÞfð
D
aÞ þ
DGlipoAlipo
þ
D
D
G;
D
D
D
D
,
D
D
D
hydrogen bond angle from the ideal value of 180°. In general, a
higher LUDI score (0–1100 in range) represents higher affinity
and stronger binding of a ligand to the receptor.

5814
N. P. Reddy et al. / Bioorg. Med. Chem. 18 (2010) 5807–5815
4.3. In vitro 5-LOX inhibitory assay
Acknowledgments
5-LOX was purified and assayed by the method described by
Reddanna et al.30 Enzyme activity was measured using polari-
graphic method with a Clark’s oxygen electrode on Strathkelvin
Instruments, model 782, RC-300. Typical reaction mixture con-
We thank Centre for Modelling, Simulation, and Design (CMSD),
University of Hyderabad for permitting us to use the SGI worksta-
tion and Catalyst facilities. We duly acknowledge the funding
bodies UGC (Dr. D.S. Kothari PDF), CSIR, DBT-RA for providing
financial support to N.P.R., P.A., C.A., and T.C.M.R., respectively.
tained 50-100 lL of enzyme and 10 lL of substrate (133 lM of
AA) in a final volume of 3 mL with 100 mM phosphate buffer pH
6.3. Rate of decrease in oxygen concentration was taken as a mea-
sure of enzyme activity. Stock solutions of test compounds, pre-
pared immediately before use, were dissolved in DMSO. Various
concentrations of test drug solutions were added and the LOX reac-
tion was initiated by the addition of substrate. The reaction was al-
lowed to proceed at 25 °C and the maximum slope generated was
taken for calculating activity. Percent inhibition was calculated by
comparison of LOX activity in the presence and absence of inhibi-
tor. The concentration of the test compound causing 50% inhibition
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.107. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
References and notes
(IC50, lM) was calculated from the concentration–inhibition re-
sponse curve. Each assay was repeated thrice.
1. Shimiz, K.; Kondo, R.; Sakai, K.; Buabarn, S.; Dilokkunanant, U. Phytochemistry
2000, 54, 737.
2. Jayasinghe, L.; Rupasinghe, G. K.; Hara, N.; Fujimoto, Y. Phytochemistry 2006, 67,
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4.4. HPLC analysis
HPLC (Shimadzu SPD-6AV) was performed to analyze the inhib-
itory activity of compound 5e according to the method described
earlier with some modification (Reddanna et al. 1988).37 In brief,
5. Yin, S.; Fan, C. Q.; Wang, Y.; Dong, L.; Yue, J. M. Bioorg. Med. Chem. 2004, 12,
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Potato 5-LOX was incubated with AA (133
l
M) in 0.1 M potassium
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Med. Chem. Lett. 2005, 15, 2453.
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Kobayashi, E.; Sagawa, H.; Kato, I. J. Agric. Food Chem. 2007, 55, 6013.
9. Akihisa, T.; Tokuda, H.; Hasegawa, D.; Ukiya, M.; Kimura, Y.; Enjo, F.; Suzuki, T.;
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10. Akihisa, T.; Tokuda, H.; Ukiya, M.; Iizuka, M.; Schneider, S.; Ogasawara, K.;
Mukainaka, T.; Iwatsuki, K.; Suzuki, T.; Nishino, H. Cancer Lett. 2003, 201,
133.
11. Tabata, K.; Motani, K.; Takayanagi, N.; Nishimura, R.; Asami, S.; Kimura, Y.;
Ukiya, M.; Hasegawa, D.; Akihisa, T.; Suzuki, T. Biol. Pharm. Bull. 2005, 28, 1404.
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39.
phosphate buffer (pH 6.3) for 2 min and the reaction was termi-
nated by acidifying the reaction mixture to pH 3.0 with 6 N HCl.
The products formed were extracted with equal volumes of
hexane/ether (1:1) twice and the organic solvent was evaporated
under inert conditions. The dried products were then dissolved in
straight phase HPLC mobile phase consisting of hexane/propane-
2-ol/acetic acid in 1000:15:1 ratio and separated on the straight
phase analytical column (Type: LUNA
5
l
SILICA (2), 250 ꢂ
4.60 mm, Phenomenex) at a flow rate of 1 mL/min. Standard
5-HPETE was synthesized as per Reddy et al. (1992).38 Enzyme
without any inhibitor was taken as control. Four micrometer of
compound 5e was taken as test concentration for the study.
14. Sugii, M.; Ohkita, M.; Taniguchi, M.; Baba, K.; Kawai, Y.; Tahara, C.; Takaoka, M.;
Matsumura, Y. Biol. Pharm. Bull. 2005, 28, 607.
4.5. MTT assay
15. Parmar, V. S.; Jain, R.; Sharma, S. K.; Vardhan, A.; Jha, A.; Taneja, P.; Singh, S.;
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Inc. micro titre plate reader. Each concentration was tested in three
different experiments run in three replicates.

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