Biological evaluation of synthetic analogues of curcumin: Chloro-substituted-20-hydroxychalcones as potential inhibitors of tubulin polymerization and cell proliferation

Article abstract of DOI:10.1007/s00044-010-9344-z

A series of chloro-substituted-20-hydroxychalcones were prepared and evaluated for their cytotoxic effects against K562 and SK-N-MC human cancer cell lines and as the inhibitors of tubulin polymerization. The 3,50-dichloro- analogue (compound 3) inhibited the assembly of protofilaments with 89% inhibition. Compound 3 was found to be bound to tubulin with a dissociation constant of 3.7 lM and altered far-UV circular dichroism spectrum of tubulin and altered far-UV circular dichroism spectrum of tubulin.

Full text of DOI:10.1007/s00044-010-9344-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:503–510
DOI 10.1007/s00044-010-9344-z
ORIGINAL RESEARCH
Biological evaluation of synthetic analogues of curcumin:
chloro-substituted-20-hydroxychalcones as potential inhibitors
of tubulin polymerization and cell proliferation
•
Hassan Aryapour Gholam Hossein Riazi Alireza Foroumadi
•
•
•
Shahin Ahmadian Abbas Shafiee Oveis Karima Majid Mahdavi
•
•
•
•
Saeed Emami Maedeh Sorkhi Sirus Khodadady
•
Received: 12 September 2009 / Accepted: 3 March 2010 / Published online: 23 March 2010
Ó Springer Science+Business Media, LLC 2011
Abstract A series of chloro-substituted-20-hydroxychal-
cones were prepared and evaluated for their cytotoxic
effects against K562 and SK-N-MC human cancer cell
lines and as the inhibitors of tubulin polymerization. The
3,50-dichloro- analogue (compound 3) inhibited the
assembly of protofilaments with 89% inhibition. Com-
pound 3 was found to be bound to tubulin with a dissoci-
ation constant of 3.7 lM and altered far-UV circular
dichroism spectrum of tubulin and altered far-UV circular
dichroism spectrum of tubulin.
up to 100-fold as compared with those taking part in
interphase (Jiang et al., 1998). High-speed dynamic is very
sensitive to interferences and is, therefore, a moving target
of many chemotherapeutic agents in tumor cells, such
as curcumin, vinca alkaloids, chalcones, and paclitaxel
(Rowinsky and Donehower, 1991; Tishler et al., 1995).
The primary action of these compounds is to bind the sites
on tubulin subunits or microtubules and then the alteration
of microtubule architecture, dynamics and subsequently,
mitotic blockage (Jordan and Wilson, 1998). In addition to
binding with tubulin, several microtubule-targeting com-
pounds have been found to alter dynamic stability of
microtubules by binding to the microtubule associated
proteins (e.g., estramustine), (Yoshida et al., 2000) and
motor proteins (e.g., monastrol) (Mayer et al., 1999).
Curcumin, a naturally occurring polyphenolic compound,
is extracted from the rhizomes of Curcuma longa. Curcu-
min exists in equilibrium with its enol tautomer. The bis-
keto form of curcumin predominates in acidic and neutral
aqueous solutions and in the cell membrane (Wang et al.,
1997). It has been used as an important dietary component
for a long time (Aggarwal et al., 2007). It exhibits various
biological functions including antiproliferative activity
against various cancer cells, antiangiogenic, and antioxi-
dant activity (Ruby et al., 1995; Gururaj et al., 2002),
wound healing ability (Panchatcharam et al., 2006) and
antimicrobial property (Rai et al., 2008). Recently, it has
been found that curcumin binds to tubulin and perturbs
microtubule polymerization. Curcumin inhibits the prolif-
eration of HeLa and MCF-7 cells in a concentration-
dependent manner with IC50 of 13.8 0.7 and
12 0.6 lM, respectively. At higher inhibitory concen-
trations ([10 lM), curcumin induces the significant
depolymerization of interphase microtubules and mitotic
spindle microtubules of HeLa and MCF-7 cells (Gupta
Keywords Microtubule ꢀ Chalcone ꢀ Curcumin ꢀ
K562 ꢀ SK-N-MC
Introduction
Microtubules are highly dynamic assemblies of ab-tubulin.
When cells enter mitosis, microtubule dynamics increase
H. Aryapour ꢀ G. H. Riazi (&) ꢀ S. Ahmadian ꢀ O. Karima ꢀ
M. Mahdavi ꢀ S. Khodadady
Institute of Biochemistry and Biophysics, University of Tehran,
Enghelab Ave, P.O. Box: 13145-1384, Tehran, Iran
e-mail: ghriazi@ibb.ut.ac.ir
A. Foroumadi ꢀ A. Shafiee ꢀ M. Sorkhi
Department of Medicinal Chemistry, Faculty of Pharmacy and
Pharmaceutical Sciences Research Center, Tehran University of
Medical Sciences, Tehran, Iran
S. Emami
Department of Medicinal Chemistry and Pharmaceutical
Sciences Research Center, Faculty of Pharmacy, Mazandaran
University of Medical Sciences, Sari, Iran
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Med Chem Res (2011) 20:503–510
et al., 2006). During the present study, we have designed
and synthesized several new chalcone analogues of cur-
cumin family with anti-microtubule properties and evaluate
their cytotoxic activities against K562 and SK-N-MC
human cancer cell lines.
respectively. The K562 and SK-N-MC cancer cell lines
were obtained from Pasture Research Center.
General procedure for synthesis of 20-hydroxychalcones
All of the compounds 1–5, presented in Table 1, were
prepared according to the same general synthetic procedure
with some modifications (Robinson et al., 2003). A mix-
ture of the aldehyde (10 mmol, 1 equiv.) and the appro-
priately substituted aromatic ketone (10 mmol, 1 equiv.)
was dissolved in 30 ml methanol. One milliliter of 50%
NaOH aqueous solution was added to the mixture and then
allowed to stir overnight at room temperature. The pre-
cipitated solids were collected by suction filtration proce-
dure. Recrystallization from ethanol provided the pure
product. During the event when solid was not formed, the
solution was neutralized with diluted HCl solution (1 N)
and extraction was done in chloroform (2 9 50 ml). The
combined organic layers were dried (MgSO4), filtered, and
evaporated in vacuo. The residues were purified by column
chromatography.
Materials and methods
General
Curcumin was synthesized on the basis of previous pro-
cedures (Hoehle et al., 2006). Briefly, acetylacetone was
allowed to react with the substituted benzaldehyde and
boric acid anhydride as a catalyst. According to HPLC
procedure, the final products obtained after crystallization
from methanol were [99% pure. Melting points were
obtained using a Kofler hot stage apparatus and are
uncorrected. The IR spectra were recorded on a Nicollet
FT-IR Magna 550 spectrometer. The 1H NMR spectra were
recorded on a Bruker 400 spectrometer and chemical shifts
are in ppm relative to internal tetramethylsilane (TMS).
Guanosine-50-triphosphate (GTP) and piperazine-1, 4-bis
(2-ethanesulfonic acid) (PIPES) were purchased from
Sigma-Aldrich. All other chemicals were of analytical
grade. All of the test compounds, used in this study, were
dissolved in DMSO (dimethyl sulfoxide) as 1 mM stock.
For in vitro and cell culture tests, DMSO was used at the
final concentrations of \4 and \0.1% (volume/volume),
respectively. All solutions were stored in the refrigerator
and protected from light. RPMI-1640 medium and MTT kit
were purchased from Invitrogen and Roche Corporation,
(E)-1-(2-hydroxyphenyl)-3-phenylprop-2-en-1-one (1)
1
Yield 85%; m.p. 89–91°C; H NMR (400 MHz, CDCl3)
d: 12.8 (s, 1H, OH), 7.94 (d, 1H, J = 14 Hz, H alkene),
7.9–7.94 (m, 1H, H aromatic), 7.68 (d, 1H, J = 14 Hz, H
alkene), 7.66–7.7 (m, 2H, H aromatic), 7.52 (t, 1H,
J = 8 Hz, H aromatic), 7.42–7.48 (m, 3H, H aromatic),
7.04 (d, 1H, J = 8.8 Hz, H aromatic), 6.96 (t, 1H,
J = 7.2 Hz, H aromatic). IR (KBr, cm-1) mmax: 3430 (OH),
1641 (C=O).
Table 1 Structure, inhibition of tubulin polymerization, and cytotoxicity activity of synthetic chalcones 1–5 in comparison with the natural
compound, curcumin
Compound
50
2
3
4
6
(%)
(IC50, lM)
Yield
Tublin inhibition
K562
SK-N-MC
1
2
3
4
5
6
a
H
H
H
H
H
Cl
–
H
H
Cl
H
H
–
H
Cl
H
Cl
H
–
H
Cl
H
H
H
–
85
75
75
79
77
83
49.3 2.4
[50
60.7 2.6
65.6 3.3
41.1 1.2
38.7 0.9
41.8 1.3
49.2 1.3a
61.2 2.5
69.8 3.8
47.8 3.2
45.8 1.6
51.3 2.7
57.4 2.9
Cl
Cl
Cl
Cl
–
19.6 0.9
20.3 0.5
21.4 1.0
26.6 1.0
According to (Roy et al., 2002), compound 6 gave an IC50 of 54.3 lm on K562 cells. Values are the mean SD
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Med Chem Res (2011) 20:503–510
505
(E)-1-(5-chloro-2-hydroxyphenyl)-3-(2,4-
dichlorophenyl)prop-2-en-1-one (2)
in use. The protein content was estimated by Bradford
method using BSA as a standard (Bradford, 1976). The
composition of MTP and tubulin purity, before and after
DEAE chromatography, was determined using 10% (w/v)
SDS-PAGE (data not shown).
Yield 75%; m.p. 144–148 °C; 1H NMR (400 MHz, CDCl3)
d: 12.6 (s, 1H, OH), 8.26 (d, 1H, J = 16 Hz, H alkene),
7.84 (d, 1H, J = 2.4 Hz, H aromatic), 7.74 (d, 1H,
J = 2.4 Hz, H aromatic), 7.54 (d, 1H, J = 16 Hz, H
alkene), 7.51 (d, 1H, J = 2.5 Hz, H aromatic), 7.47 (dd,
1H, J = 8.8 and 2.8 Hz), 7.35 (dd, 1H, J = 8.4 and
1.6 Hz), 7.02 (d, 1H, J = 9.2 Hz, H aromatic). IR (KBr,
cm-1) mmax: 3440 (OH), 1638 (C=O).
Microtubule assembly assay
The microtubule assembly was monitored spectroscopi-
cally by turbidity measurement at 350 nm using a spec-
trophotometer (Varian Cary 100 Bio UV-Visible
Spectrometer, USA), equipped with a thermostatically
regulated liquid circulator. The assay was carried out based
on previous procedures with some modification (Ma et al.,
2008). Briefly, tubulin was incubated with DMSO and
compounds at different concentrations for 15 min at 0°C in
ice. After adding the final 1 mM concentration of GTP, the
assembly was initiated by warming the solution from 0 to
37°C and polymerization process was monitored by
observing the variations in absorbance at 350 nm. Tur-
bidity (absorbance) readings were recorded after every 10 s
throughout the incubation time and the changes in absor-
bance were used to calculate the extent of polymerization
[% inhibition = (1–A350 sample/A350 control) 9 100].
(E)-1-(5-chloro-2-hydroxyphenyl)-3-(3-chlorophenyl)prop-
2-en-1-one (3)
Yield 75%; m.p. 146–147°C; 1H NMR (400 MHz, CDCl3)
d: 12.6 (s, 1H, OH), 7.85 (d, 1H, J = 2.4 Hz, H aromatic),
7.84 (d, 1H, J = 15.6 Hz, H alkene), 7.67 (d, 1H,
J = 9.2 Hz, H aromatic), 7.55 (d, 1H, J = 15.2 Hz, H
alkene), 7.36–7.48 (m, 4H, H aromatic), 6.98 (d, 1H,
J = 9.2 Hz, H aromatic). IR (KBr, cm-1) mmax: 3425 (OH),
1638 (C=O).
(E)-1-(5-chloro-2-hydroxyphenyl)-3-(4-chlorophenyl)prop-
2-en-1-one (4)
Electron microscopic analysis
Yield 79%; m.p. 131–133°C; 1H NMR (400 MHz, CDCl3)
d: 12.7 (s, 1H, OH), 7.9 (d, 1H, J = 15.2 Hz, H alkene),
7.82–7.86 (m, 1H, H aromatic), 7.63 (d, 1H, J = 8 Hz, H
aromatic), 7.55 (d, 1H, J = 15.2 Hz, H alkene), 7.4–7.5
(m, 4H, H aromatic), 7.01 (d, 1H, J = 8.8 Hz, H aromatic).
IR (KBr, cm-1) mmax: 3435 (OH), 1646 (C=O).
The electron microscopic pictures were taken as described
by (Rai et al., 2008). Tubulin (1 mg/ml) was polymerized
in the absence and presence of different concentrations of
compound 3 in the PEM buffer at 37°C. After 15 min of
polymerization, the microtubule polymers (20 ll) were
transferred to the formvar-carbon-coated copper grids (400
meshes) for 45 s and blotted dry. The grids were subse-
quently stained negatively with a 1% uranyl acetate solu-
tion for 35 s, air-dried and observed under transmission
electron microscope (Hitachi HU-12A) at 150009 magni-
fication.
(E)-1-(5-chloro-2-hydroxyphenyl)-3-(2-chlorophenyl)prop-
2-en-1-one (5)
Yield 77%; m.p. 186–192°C; 1H NMR (400 MHz, CDCl3)
d: 12.7 (s, 1H, OH), 8.35 (d, 1H, J = 15.2 Hz, H alkene),
7.89 (d, 1H, J = 2.4 Hz, H aromatic), 7.8 (d, 1H,
J = 7.2 Hz, H aromatic), 7.56 (d, 1H, J = 15.2 Hz, H
alkene), 7.42–7.52 (m, 2H, H aromatic), 7.32–7.43 (m, 2H,
H aromatic), 7.01 (d, 1H, J = 8.8 Hz, H aromatic). IR
(KBr, cm-1) mmax: 3470 (OH), 1643 (C=O).
Fluorescence measurements
All of the fluorescence measurements were performed in a
Varian’s Cary Eclipse fluorescence spectrophotometer. The
increased fluorescence of compound 3, at 425 nm, upon
binding to tubulin was used to determine the ligand affinity
with tubulin (Rai et al., 2008; Gupta et al., 2006). Tubulin
(2 lm) was incubated with varying concentrations of
ligand (0–10 lm) in 25 mM PIPES at 25°C for 30 min in
the dark. The excitation wavelength was 371 nm. The
binding constants and stoichiometries were determined
from Scatchard equation by plotting Lbound/Lfree against
MTP preparation and purification
Microtubule protein (MTP) was prepared and purified from
fresh sheep brain through two cycles of polymerization–
depolymerization in the PEM buffer (100 mM PIPES, pH
6.9, 1 mM MgSO4, and 1 mM EGTA) (Sengupta et al.,
2005). Then, MAP-free tubulin was purified from the
microtubule protein by DEAE chromatography, (Murphy
et al., 1977) aliquot and stored in liquid nitrogen when not
Lbound, where Lbound is the bound ligand concentration and
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Lfree is the free-ligand concentration (Scatchard, 1949). The
Lbound was calculated from the following equation:
2 mM L-glutamine, 50 UI/ml penicillin, and 50 UI/ml
streptomycin. Cells were grown at 37°C and 5% CO2 in
humidified atmosphere. Ten thousand cells/well were seeded
in 200 ll of the growth medium in 96-well microtiter plates
(SPL Life Science, Korea) and incubated for 24 h before
addition of test drugs. The cells were treated with several
concentrations of test compounds in a CO2 incubator for
72 h. At the end of this period, 10 ll of MTT (final con-
centration 0.5 mg/ml) was added to each well and the plates
were incubated for 4 h at 37°C. Finally, 100 ll of the solu-
bilization solution was added into each well and overnighted
at 37°C. Absorbance (A570) was determined, at 570 nm, for
each well using microplate reader (Model Expert 96, Asys
Hitech, Ec. Austria). The IC50 (concentration reducing by
50% the absorbance at 570 nm) was calculated by linear
regression, performed on the linear zone of the dose–
response curve. Control wells contained all of the com-
pounds presented in the treated wells except compounds.
Each experiment was performed in three replicates.
Lbound ¼ Ltotal=Q ꢁ 1ðF=F0 ꢁ 1Þ
where, Q is the enhancement factor of compound 3 fluo-
rescence for bound ligand. Q was measured by plotting
1/fluorescence intensity of ligand against 1/[tubulin]. For
this, a fixed amount of compound 3 (1 lM) was incubated
with the increasing amounts of tubulin in a concentration
range of 1–12 lM. Where, F is the fluorescence intensity
of the tubulin-ligand complex at a particular ligand con-
centration and F0 is the fluorescence intensity of ligand,
alone, in the absence of tubulin. The amount of free-ligand
is obtained from the difference of total ligand and calcu-
lated bound ligand. The corrections of inner filter effect
were performed using the following equation: Fcor-
= Fobserved 9 antilog [(ODex - ODem)/2].
rected
Analysis of secondary structural changes
Tubulin (4 lM) was incubated in the absence or presence
of different concentrations of compound 3 (25 and 50 lM)
in 15 mM phosphate buffer (pH: 6.5) for 30 min at 25°C.
The far-UV (215–250 nm) circular dichroism (CD) spectra
were monitored at 25°C using Aviv spectropolarimeter
(model 215), equipped with a Peltier temperature-control
system using a 1-mm path length quartz cuvette. Decon-
volution and data analysis of the CD spectra were per-
formed using CDNN from Jasco and GraphPad Prism,
respectively.
Results
Effects of synthetic chalcones on the in vitro
polymerization of microtubules
As microtubules, in cells, are associated with many micro-
tubule-interacting proteins, we analyzed MTP assembly
from sheep brain in the absence and presence of different
concentrations of synthetic chalcones (10–50 lM). The
assembly was monitored by spectrophotometeric method
(Fig. 1). The IC50 values of polymerization inhibition were
determined from the semi-logarithmic dose–response plots
using nonlinear regression program SigmaPlot. Compounds
3, 4, and 5 exhibited more potent activity and inhibited the
assembly of purified sheep brain MTP in a concentration-
dependent manner with the IC50 values of B21.4 lM. Also,
Cell growth inhibition assay
MTT colorimetric assay was employed according to the
methods of (Scudiero et al., 1988). The K562 and SK-N-MC
human cancer cell lines were maintained in RPMI-
1640 medium, supplemented with 10% fetal calf serum,
Fig. 1 Inhibition of tubulin polymerization by compound 3. a Kinet-
ics of the inhibition of microtubule assembly by increasing concen-
trations of compound 3. Control (filled circle), 10 (open circle), 15
(filled inverted triangle), 20 (open triangle), 25 (filled square), and
30 lM (open square), respectively. b The IC50 value was obtained
from a plot of percent tubulin polymerization against log concentra-
tion (M) of compound 3 at 37°C. Inset: column scatter graph of
absorbance against compound 3 concentration in the reaction mixture.
IC50 = 19.59 lM, log IC50 SE = -4.7 0.0007
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507
The binding of compound 3 to tubulin
the inhibitory activity of these compounds was more potent
than that of curcumin (IC50 = 26.6 1.0 lM).
As tubulin is the major component of microtubules, we
wanted to test whether the effects of compound 3 on
microtubule network and microtubule assembly are due to
its binding to tubulin. Although, target compounds show
weak fluorescence in PEM buffer (Fig. 3). Following
incubation with purified tubulin, the experimental com-
pounds showed fluorescence with maximum emission of
430 nm upon excitation at 371 nm. For example, incubat-
ing a 1:1 molar ratio of compound 3 with tubulin resulted
in several-fold increase in the fluorescence intensity of
compound 3. The emission spectrum of compound 3
showed a blue-shift of 8 nm upon binding to tubulin,
indicating that compound 3 binds to a hydrophobic region
of tubulin (Fig. 3a). Figure 3b shows the titration curve of
a constant amount of tubulin (2 lM), treated with various
concentrations of compound 3. The dissociation constant
(Kd) of compound 3 to tubulin was calculated to be
3.7 0.3 lM by the Scatchard plot using GraphPad Prism
software (Fig. 3b, Inset). Also, the tryptophan residues in
tubulin are intrinsically fluorescent and modulation of
ligand-directed fluorescence of tryptophan residues, as
determined by fluorescence spectroscopy has been exten-
sively used to investigate tubulin-ligand binding. Com-
pound 3 reduces the intrinsic fluorescence of tryptophan
residue within the tubulin in a concentration-dependent
manner, suggesting that it induces conformational change
in tubulin (Fig. 4).
Among these compounds, with the ability of tubulin
assembly inhibition, compound 3 with IC50 value of
19.6 lM was selected for further studies including binding
to tubulin and affecting its CD spectra. Figure 1 demon-
strates that there is a concentration-dependent inhibition of
microtubule assembly by 30 lM concentration of com-
pound 3 with 89% inhibition. Based on transmission
electron microscopy (TEM) micrographs, the length of
microtubule filaments reduced in the presence of 20 and
30 lM of compound 3 as compared to the control experi-
ment (Fig. 2).
Preliminary cytotoxicity evaluation
All of the synthesized compounds were evaluated for their
inhibitory activity on the growth of K562 and SK-N-MC
human cancer cell lines by measuring cell number in a
concentration-dependent manner after 72 h of incubation.
IC50 values were calculated from the semi-logarithmic
dose–response plots using nonlinear regression program
SigmaPlot (data not shown). The results have indicated that
these compounds effectively inhibited the growth of K562
and SK-N-MC cancer cell lines. Generally, the inhibitory
activities of all analogues against SK-N-MC cells were less
significant than that of K562. Compound 4, followed by
compounds 3 and 5, showed more potent cytotoxic activity
in respect with natural compound curcumin (Table 1).
Fig. 2 TEM micrographs
of microtubule polymers in the
absence (a) and presence of
20 lM (b) and 30 lM (c) of
compound 3. Scale bars, 2 lm
Fig. 3 Characterization of the binding of compound 3 with tubulin.
a Change in fluorescence spectra of compound 3 after binding to
tubulin. Compound 3 (2 lM) was incubated without (filled square), or
with (open circle) tubulin (2 lM). The kex and kem values were 371
and 423 nm, respectively. b Measurement of compound 3 binding to
tubulin by fluorescence spectroscopy. 2 lM of tubulin was incubated
with different concentrations of compound 3 (0–10 lM) and the
fluorescence intensity was measured as described in the ‘‘Fluores-
cence measurements’’ section. Inset shows the scatchard plot of
compound 3 binding to tubulin
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Fig. 4 Effects of compound 3 on the intrinsic tryptophan fluores-
cence of tubulin. Tubulin (2 lM) was incubated without (filled circle)
or with 1 (open circle), 2 (filled inverted triangle), 4 (open triangle), 6
(filled square), and 8 (open square) lM of compound 3 for 30 min at
25°C. Compound 3 reduced the intrinsic fluorescence of tubulin with
the excitation wavelength of 295 nm
Fig. 5 Effect of compound 3 on the CD spectrum of tubulin. The
graph shows the far-UV CD spectra of 4 lM tubulin in the absence
(solid line) and presence of 25 (dashed line) and 50 lM (dash-dotted
line) of compound 3 in a 10 mM phosphate buffer (pH 6.8). Each
spectrum was the average of three scans
Effect of compound 3 on the Cd spectra of tubulin
target compounds are small molecules with aromatic ring
A and B, linked by a,b-unsaturated ketone and easily
prepared by convenient synthetic method. Chlorine sub-
stitution on the A and B rings was used for structural
modification and modulation of basic pharmacophore of
20-hydroxychalcones. Halogens, like chlorine, are very
useful to modulate the electronic effects on the phenyl
rings of drugs. Moreover, chlorine may also influence the
steric characteristics and hydrophilic–hydrophobic balance
of the molecules (Legraverend et al., 2000; Zhang et al.,
2003). All chloro-substituted-20-hydroxychalcones exhibit
respectable tubulin inhibition and cytotoxic activities
against two selected cancer cell lines (K562 and SK-N-
MC). Their activities were more potent than that of
curcumin. Such kind of biological activities are exhibited
by other potent antimitotic compounds (Ducki et al.,
1998). Among these compounds, 3,50-dichloro-analogue
(compound 3) with IC50 value of \20 lM in tubulin
inhibition assay was selected for further studies. Microtu-
bule assembly was significantly inhibited by compound 3
with the reduction of nucleation, polymerization rate, and
maximum absorbance. Compound 3 inhibits tubulin poly-
merization with an IC50 of 19.6 lM, which is close to those
exhibited by the compounds 5 (2,50-dichloro- analogue)
and 4 (4,50-dichloro- analogue). By comparing the unsub-
stituted analogue (compound 1) and chloro-substituted
analogues (compounds 3, 4, and 5), it has been observed
that the addition of chloro-substituent to ring A/B causes
an increase in hydrophobic interaction and biological activ-
ity of compounds. While, comparing the biological activ-
ities of compounds 4 (4,50-dichloro- analogue) and 2
Compound 3 inhibits the polymerization of microtubule
proteins and decreases tryptophan fluorescence. The effect
of compound 3 on the secondary structure of tubulin was
further examined by circular dichroism spectroscopy.
Compound 3 alters the far-UV CD spectrum of tubulin,
indicating that ligand binding altered the helical content in
tubulin (Fig. 5).
Discussion
During the last years, antimitotic agents have been used as
chemotherapeutic agents to inhibit cell proliferation and
induce cytotoxicity. The action mechanism of these agents
often involves an interaction with microtubules, particu-
larly tubulin. Microtubules are polar structures and are long
protein fibers that exist in dynamic equilibrium with the
tubulin dimer. Microtubules are vital components of the
cell and are responsible for several important functions
including intracellular transport, formation of the mitotic
apparatus, mechanically stabilizing cellular processes, and
formation of the mitotic spindle during cell division
(Brown et al., 2006). Antimitotic agents, including a
diverse group of compounds are originally taken from
natural sources (Jordan and Wilson, 2004). During the
present study, we have investigated the anti-microtubule
and anti-proliferative properties of 20-hydroxychalcones as
the synthetic analogues of curcumin. Curcumin is a natural
product and possesses two methoxyphenolic rings, con-
nected by two conjugated a,b-unsaturated ketones. Our
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Med Chem Res (2011) 20:503–510
509
(2,4,50-trichloro- analogue), it can be concluded that the
additional chloro-substitution (2-chloro) diminishes the
biological activity of compound 2, which may be due to
the steric hindrance of the substitution. On the basis of
significant inhibition of microtubules in vitro, we also
examined the cytotoxic effects of our test compounds on
K562 and SK-N-MC cancer cell lines and found that these
compounds inhibit the cell proliferation with IC50 values
higher than those of cell-free systems. These findings
(based on the experiments of microtubule and cancer cell
line) suggest that the weaker effects of these compounds,
on the cells in comparison with microtubules, may be due
to the presence of biological barrier such as cell membrane.
Also, the inhibitory activities of all analogues against K562
are slightly more significant than those of SK-N-MC cells.
Electron microscopic analysis of the microtubule polymers
showed that compound 3 strongly reduced the length and
extent of formation in microtubules. The decrease in
polymer length and bundling of microtubules may be due
to the binding of ligand molecules with tubulin. Compound
3 shows fluorescence upon binding to tubulin, although the
drug itself does not have fluorescence in aqueous solution.
The interaction of compound 3 with tubulin and microtu-
bules indicates that our test compound binds to tubulin with
a Kd of 3.7 0.3 lM. In addition, compound 3 reduced
the helical content, while increasing the random coil con-
tent of tubulin, suggesting that the perturbation in sec-
ondary structure of tubulin might also be the cause of
destabilization of microtubules (Gupta and Panda, 2002).
In brief, the results suggest that chloro-substituted-
20-hydroxychalcones inhibit cell proliferation and tubulin
assembly by inducing conformational changes in the pro-
tein. These findings may help in designing new synthetic
analogues of curcumin with potent cytotoxic activity,
improved stability, and bioavailability.
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Acknowledgments We thank Ms. Aisha Javed for the critical
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preparing photographs and cooperating in the experiments. This
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