Fluorinated 2′-hydroxychalcones as garcinol analogs with enhanced antioxidant and anticancer activities

Article abstract of DOI:10.1016/j.bmcl.2010.07.128

Chalcones are involved in the synthesis of flavonoids and are themselves known to exhibit multiple pharmacological properties. However, compared to other structurally similar phytochemicals like garcinol and curcumin, the therapeutic use of chalcones is l

Full text of DOI:10.1016/j.bmcl.2010.07.128

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5818–5821
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fluorinated 20-hydroxychalcones as garcinol analogs with enhanced antioxidant
and anticancer activities
Subhash Padhye a,b, Aamir Ahmad a, Nikhil Oswal c, Prasad Dandawate d, Rukhsana A. Rub d,
Jyoti Deshpande e, K. Venkateswara Swamy e, Fazlul H. Sarkar a,
*
a Department of Pathology, Barbara Ann Karmanos Cancer Center and Wayne State University School of Medicine, Detroit, MI 48201, USA
b Dr. D. Y. Patil University, Pune 411018, India
c Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pune 411018, India
d M.C.E.S.’s Allana College of Pharmacy, Pune 411001, India
e Dr. D. Y. Patil Institute of Biotechnology and Bioinformatics, Pune 411044, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Chalcones are involved in the synthesis of flavonoids and are themselves known to exhibit multiple phar-
macological properties. However, compared to other structurally similar phytochemicals like garcinol
and curcumin, the therapeutic use of chalcones is limited because of their lower bioavailability and rapid
metabolic clearance from biological system. In the present work, we have attempted to overcome these
limitations in case of 20-hydroxychalcones through bioisosteric substitution of fluoro groups in place of
phenolic hydroxyls. The fluorinated chalcones were found to be more potent antioxidant and anti-prolif-
erative compounds than their hydroxyl counterparts indicating the influence of metabolically stable C–F
bonds towards bioavailability. The difluoro derivatives were found to be most effective against human
pancreatic BxPC-3 cancer cells which possess up-regulated COX-2 expression and also showed activity
against human breast cancer BT-20 cells with triple negative phenotype, suggesting that these com-
pounds will have broader application in the future.
Received 21 June 2010
Revised 27 July 2010
Accepted 28 July 2010
Available online 3 August 2010
Keywords:
Hydroxychalcones
Fluorochalcones
Antioxidant
Antiproliferative
Pancreatic cancer
Breast cancer
Ó 2010 Elsevier Ltd. All rights reserved.
Chalcones are the natural precursors of flavonoid compounds
that are produced in plants by the action of enzyme chalcone syn-
thase. Compounds belonging to both these classes have been shown
to possess several pharmacological activities including antioxidant,
anti-inflammatory, and anticancer, respectively.1–4 Chemically,
chalcones are 1,3-diphenylpropenones and benzylideneacetophe-
nones which are made up of two aromatic rings in trans configura-
hance its lipophilicity and help promote cell internalization. Both
the compounds are known to exhibit antioxidant and anti-inflam-
matory activities. Since preparation of tri-isoprenylated chalcone
like garcinol is difficult to achieve through synthetic route with
good yields, we have been interested in mimicking its biological
tion separated by three carbon
a,b-unsaturated carbonyl system.
The compounds can be broadly classified based on substitution pat-
tern of the parent chalcone as: hydroxylated chalcones, methoxylat-
ed chalcones, aminochalcones, N-containing chalcones, etc.5 Our
interest in following structural and biological chemistry of 20-
hydroxychalcones stems from the remarkable anticancer property
exhibitedby tri-isoprenylated hydroxychalcone, viz. garcinol, which
is the active principle compound from Garcinia indica.6 The com-
pound structurally resembles with well-known antioxidant and
anticancer compound of plant origin, viz. curcumin (Fig. 1), whose
chemical and biological properties have been reviewed recently by
us.7 The presence of the isoprenyl groups in garcinol is known to en-
* Corresponding author. Address: Department of Pathology, Karmanos Cancer
Institute, 740 HWCRC Bldg, 4100 John R. Street, Detroit, MI 48201, USA. Tel.: +1 313
576 8327; fax: +1 313 576 8389.
E-mail address: fsarkar@med.wayne.edu (F.H. Sarkar).
Figure 1. Structures of chalcone, garcinol, and curcumin.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.128

S. Padhye et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5818–5821
5819
properties through corresponding hydroxychalcone (without the
isoprenyl groups) by addition of hydroxyl/fluoro groups in the B ring
since appending these groups has led to highly potent anticancer
compounds in our lab in case of curcumin.8
Our results suggest that chalcones fluorinated in the B ring exhibit
enhanced anti-proliferative activities than their hydroxylated coun-
terparts against these cell lines and bear a good correlation with
their antioxidant activities.
The bioisosteric substitution of fluorine in place of hydrogen in
many biologically active molecules has led to potent compounds
without extensive stereochemical changes due to its small size.9
However, such substitution is also known to modulate overall
reactivity and stability of the compounds due to resistance of the
carbon–fluorine bond toward metabolic transformations and
changes in acidity due to electronegativity differences between
the two atoms.10 There have been a few reports on the improved
biological activities of fluorinated methoxy chalcones11 and chro-
mones12 than their non-fluorinated analogs in the past. Following
our work on fluorocurcumins, Dou and co-workers have recently
described enhanced antitumor and proteasome-inhibitory activi-
ties for epigallocatechins in breast cancer xenograft model.13
We were thus motivated to examine whether such effects can
also be brought about in structurally similar chalcone group of com-
pounds. For this purpose we selected 20-hydroxychalcone as the par-
ent compound since it is one of the biologically active chalcone with
moderate anticancer activity. Several reports suggested that
2-hydroxyl substitution on A ring is crucial for its antioxidant,14
anti-inflammatory,15 and anticanceractivities.16 There is also an evi-
dence suggesting that these chalcones can modulate antioxidant
systems leading to mitochondrial oxidative stress which can result
in apoptosis.17 Hence in our syntheticstrategy we have kept this part
of the molecule untouched and have used only the B ring for substi-
tution of hydroxy/fluoro groups. Our literature search revealed that
B ring of the chalcone has been substituted with chloro, bromo,
methoxy as well as hydroxyl groups17,18 but not so frequently with
the fluoro groups. In the present work, we describe preparation,
characterization, and molecular docking studies on 20-hydroxychal-
cones appended with hydroxy/fluoro groups in the B ring along with
evaluation of their radical scavenging potential and anti-prolifera-
tive activities against human pancreatic and breast cancer cells.
The chalcones were synthesized by procedures reported ear-
lier.19,20 All synthesized hydroxychalcones (Fig. 2) 1, 3, 5 were red-
dish brown while the corresponding fluoro derivatives, 2, 4, 6,
were yellow in color. The IR spectra of all synthesized chalcones
showed a medium intensity band in the region 3300–3200 cmꢀ1
due to stretching frequency of intra-molecularly H-bonded hydro-
xyl group at C-2 in ring A. The broad band attributable to the car-
bonyl stretch of
a
,b-unsaturated ketone was observed in all
compounds at 1700–1650 cmꢀ1 21
.
The aromatic (C@C) stretches
m
could be located at 1620–1580 cmꢀ1. The hydroxy- and fluorochal-
cones can be differentiated by the presence of strong C–F stretch-
ing absorption at 1350–1150 cmꢀ1 and weaker peak in the region
800–400 cmꢀ1 (Table 1).22
The electronic spectra of all synthesized chalcones in methanol
show intra-ligand transitions for the hydroxyl compounds in
the range 400–220 nm which undergoes shift towards lower
wavelength in their fluoro counterparts. NMR spectra for the com-
pounds 3 and 4 reveal the aromatic protons in ring B at d
6.90–7.64 ppm while the ethylene proton is observed as a doublet
at d 7.39 and d 8.17 ppm, respectively. The three phenolic hydroxyl
groups in compound 3 appear as a singlet at d 5.0 ppm while only
one appears in case of 4.23 The compositional and spectral data on
all synthesized compounds is summarized in Table 1.
Since anti-inflammatory property is one of the important char-
acteristics of chalcone compounds, we evaluated the COX-2 selec-
tivity of the new chalcone derivatives by performing molecular
docking studies on them in the COX-2 protein cavity.24 All hydro-
xyl and fluorochalcone compounds were found to dock into the ac-
tive site of COX-2 ( Fig. 3), confirming that hydroxy and fluoro
substitution does not introduce any major steric changes in the
parent 2-hydroxychalcone moiety 7 except for allowing additional
hydrogen bonding interactions (Table 2). The higher log P values
Figure 2. Schematic representation of synthesis of fluoro- and hydroxyl-substituted chalcone.
Table 1
Compositional and spectral data on fluorinated 20-hyrdroxycalcones
Compounds
Molecular formula
Molecular weight
Electronic absorptions
IR frequencies (KBr,
m
, cmꢀ1
)
1H NMR (300.40 MHz, CDCl3, d, ppm)
(k, nm)
*
*
p
?
p
n ?
p
O–H
C@O
C@C
C–F
1
2
3
C
C
C
15H12O4
15H10F2O2
15H12O4
256.25
260.24
256.25
282
254
289
323
321
324
3228
3236
3258
1675
1689
1682
1596
1612
1619
—
1280
—
—
—
5.0 (s, –3OH), 6.90–7.64 (benzylic),
7.39 (d, @CH), 8.17 (d, @CH)
4
C
15H10F2O2
260.24
254
322
3222
1693
1612
1277
5.0 (s, –OH), 6.90–7.64 (benzylic),
7.39 (d, @CH), 8.17 (d, @CH)
5
6
C
C
15H12O5
15H9F3O2
272.25
278.23
295
254
325
321
3232
3251
1680
1693
1612
1600
—
1274
—
—

5820
S. Padhye et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5818–5821
Figure 3. Binding of chalcone analogs into the active site of COX-2 as assessed by computer modeling studies.
Table 2
observed for the fluorochalcone compounds 2, 4, and 6 clearly re-
flect their higher lipophilicities. Subsequently their binding ener-
gies were found to be in the range of ꢀ8.48 to ꢀ9.10 kcal/mol
compared to ꢀ8.10 kcal/mol observed for the parent compound 7
indicating their tight binding in the active site of COX-2 than par-
ent compound. Similarly, the parent compound exhibited only one
hydrogen bonding interaction involving ARG 513 residue, while
compounds 1 and 5 exhibited four and two hydrogen bonding
interactions, respectively. The hydroxyl groups on rings A and B
of compound 1 favored Van der Waals interactions with PHE 518
(1.913 Å), GLN 192 (1.780 Å). GLN 192 (2.105 Å), MET 522
(1.753 Å) residues, while for the compounds 2, 4, 5, 6, and 7 the hy-
droxyl group on ring A undergoes similar interactions with ARG
513 (2.212 Å), GLN 192 (2.042 Å), GLN 192 (2.022 Å), VAL 523
(2.163 Å), and ARG 513 (2.036 Å), respectively. Thus, it can be con-
cluded that the hydroxyl groups on either ring A or B favor hydro-
gen bonding interactions with amino acid residues in COX-2
protein which contribute in stabilizing the ligand–enzyme com-
plexes (Fig. 3).
Docking results and consensus scores of synthesized chalcone analogs
Lig. Binding
no. energy
(kcal/
Docking
energy
(kcal/
log P No of
Hydrogen
Distance
(Å)
hydrogen bonding
bonds
4
residues
mol)
mol)
1
ꢀ9.05
ꢀ10.12
2.42
PHE 518, GLN 1.913,
192, MET 522, 1.780,
GLN 192
1.753,
2.105
2.212
2.175
2.042
2.022,
2.220
2.163
2.036
2
3
4
5
ꢀ9.10
ꢀ8.57
ꢀ8.48
ꢀ8.88
ꢀ9.96
ꢀ9.60
ꢀ9.56
ꢀ9.93
3.51
2.42
3.51
2.03
1
1
1
2
ARG 513
ARG 513
GLN 192
GLN 192, SER
530
6
7
ꢀ8.63
ꢀ8.10
ꢀ9.62
ꢀ8.73
3.67
3.20
1
1
VAL 523
ARG 513
The IC50 values calculated for the SOD scavenging activity of
present compounds by the NBT method25,26 are in the range 5.27–
13.68 lM (Fig. 4) where fluoro derivatives are found to be more
potent reflecting higher metabolic stability of the C–F bond.
Compounds 2 and 4 were found to be the most potent compounds
whose activity is comparable with that of ascorbic acid used as a ref-
erence antioxidant compound27 in the studies (Fig. 4). Since the syn-
thesized chalcones exhibited significant antioxidant activity, we
checked for their efficacy in inhibiting the growth of COX-2 positive
human pancreatic cancer cell line BxPC-3 as well as against the
estrogen receptor-negative BT-20 breast cancer cell line (Fig. 5).28
We found that in case of pancreatic BxPC-3 cells the IC50 values29
for the fluorochalcones were three to seven times lower than their
Figure 4. IC50 values of superoxide dismutase activity of the synthesized chalcone
(1–6) and ascorbic acid (AA) using NBT spectrometric assay.
Figure 5. IC50 values of the hydroxy and fluoro chalcones (1–6) against human pancreatic cancer cell line, BxPC-3, and breast cancer cell line, BT-20.

S. Padhye et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5818–5821
5821
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Lee, S. H.; Ryu, J. C.; Kim, Y.; Jung, S. H. Bioorg. Med. Chem. 2007, 15, 104.
19. Synthesis of chalcones: the chalcones were synthesized by condensing
equimolar quantities of o-hydroxyacetophenone with corresponding
hydroxy/fluoro benzaldehyde in methanol with a drop of aqueous potassium
hydroxide solution (50%) ensuring neutral pH. Reaction mixture was stirred
overnight after which it was poured on the crushed ice and acidified with
hydrochloric acid. The resulting precipitates were filtered, washed with cold
methanol and re-crystallized from acetone. The final products were
characterized by detailed spectroscopic and micro analytical data.
20. Li, R.; Kenyon, G. L.; Cohen, F. E.; Chen, X.; Gong, B.; Dominguez, J. N.; Davidson,
E.; Kurzban, G.; Miller, R. E.; Nuzum, E. O. J. Med. Chem. 1995, 38, 5031.
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hydroxycounterparts(Fig. 5). The mostsubstantial lowering was ob-
served for the trifluoro compound than that of trihydroxychalcone.
The lowest IC50 value was observed for the compound 4. A similar
trend was observed in breast cancer BT-20 cells as well ( Fig. 5),
although differences between hydroxyl and fluoro-substituted ana-
logs were not as dramatic as in pancreatic cells. Additionally,
potency of anticancer activity of these compounds correlates well
with their antioxidant property.
Our research group has been exploring different natural products
containing novel classes of active phytochemicals which can effec-
tively modulate inflammatory pathways.6,30,31 As pointed out by
Hong et al.32 most of natural antioxidants exhibit biphasic action
in modulating cell growth in vitro. At low concentrations (<1 lM),
they stimulate cell proliferation possibly by activating ERK1/2 and
AKT through generation of hydrogen peroxide, but at higher concen-
trations they inhibit cell growth possibly by inducing apoptosis. The
net effects of these compounds, therefore, depend upon the tissue
levels of these compounds and their metabolites. Interestingly three
structurally resembling phytochemicals, viz. garcinol, chalcones,
and curcumin (Fig. 1) have very low bioavailability and rapid meta-
bolic clearance from biological system. In order to make them useful
as therapeutic agents, appropriate structural modifications are nec-
essary which can address these two problems. Recently, we have
shown that bioisosteric substitution of fluoro groups in place of phe-
nolic hydroxyls in such phytochemicals can not only enhance their
bioavailability but improve metabolic rates as well.8 In the present
work we have successfully extended such strategy to chalcones
which are garcinol mimics for all practical purposes; however, their
bioavailability must be tested in future studies.
The modified chalcone derivatives exhibited potent antioxidant
activities and are capable of blocking the inflammatory pathway
as revealed from the molecular modeling studies although further
biochemical studies would be required to prove the point. These
compounds also exhibited potent inhibitory activities against hu-
man pancreatic BxPC-3 cancer cells which possess up-regulated
COX-2 expression as well as against human breast cancer BT-20 cells
with triple negative phenotype. Both these cancers are known for
their insensitivities to conventional chemotherapeutics, resulting
in poor prognosis. The results of the current study indicates that
fluorochalcones might beable to provide a startingpoint for building
effective strategy to overcome many limitations especially the bio-
availability and improve cell growth inhibitory activity against
aggressive cancers and, thus, further studies are warranted.
22. Liu, H. Q.; Jiang, F. C. Guang Pu Xue Yu Guang Pu Fen Xi 2007, 27, 2243.
23. Silva, A. M. S.; Cavaleiro, J. A. S.; Tarrago, G.; Marzin, C. New J. Chem. 1999, 23,
329.
24. Docking studies: AutoDock 4.0 software was used to analyze ligand
interactions with the crystal structure binding site of COX-2 obtained from
PDB ID (6COX). Autodock calculates a rapid energy evaluation through pre-
calculated grids of affinity potentials with a variety of search algorithms to find
appropriate binding positions. The 3-D grid box has been generated a grid
center co-ordinates 21.804 Å, 21.72 Å, 49.4 Å (X, Y, and Z axis) with grid
spacing 0.37 Å considering active site residues included within it. Stable
docking conformation of compounds achieved by implementing energy
minimization parameter AMBER force field until the gradient convergence
value of 0.05 kcal/mol was reached with distance-dependent dielectric
function (e = 4r). New designed compounds were placed in grid box of COX-2
for docking process. Customized docking parameters were set in Autodock for
best results for understanding interaction studies with new designed
compounds. Parameter settings were set to 1500 iterations, 50 population
sizes, 100.0 kcal/mol of energy threshold for pose generation, 300 simplex
evolution steps, and 1.0 neighbor distance factor. For preparing the AutoDock
docking parameter file we used default settings (genetic algorithm parameters:
population size = 150, number of energy evaluations = 2,500,000, rate of gene
mutation = 0.02,
rate
of
crossover = 0.8,
maximum
number
of
generations = 27,000, number of GA runs = 10, initial dihedrals were
randomly specified, elitism value was set to 1). Prior to docking, total
Kollman and Gasteiger charges were added to the protein and the ligand.
25. Nitroblue tetrazolium (NBT) assay: the hydroxy/fluoro derivatives of 20-
hydroxychalcone were evaluated for the SOD like activities by the NBT
method in triplicate following the lab protocol established in our lab. Briefly,
the test solution (400
ll) consisted of 2.1 ml of 0.2 M Tris buffer and 1 ml of
56 M NBT. The tubes were kept in ice for 15 min followed by addition of
l
1.5 ml of KO2 with stirring and measuring the absorbance of solution at
560 nm with an interval of 30 s for 5 min. Ascorbic acid was used as a standard.
26. Bhirud, R. G.; Srivastava, T. S. Inorg. Chim. Acta 1990, 173, 121.
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28. Cell culture: pancreatic cancer cell line BxPC-3 was maintained in RPMI culture
medium (Invitrogen) while the breast cancer cell line BT-20 was maintained in
DMEM medium (Invitrogen). Both the culture media contained penicillin
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