Synthesis, molecular characterization, and biological activity of novel synthetic derivatives of chromen-4-one in human cancer cells

Article abstract of DOI:10.1021/jm051068y

The synthesis and characterization of Schiff base derivatives of 3-formylchromone 3-6 (FPA-120 to FPA-123), the minimal biologically active structural motif of soy isoflavone, genistein, and their copper(II) complexes 7-10 (FPA-124 to FPA-127) are reporte

Full text of DOI:10.1021/jm051068y

3800
J. Med. Chem. 2006, 49, 3800-3808
Synthesis, Molecular Characterization, and Biological Activity of Novel Synthetic Derivatives of
Chromen-4-one in Human Cancer Cells
Vivek Barve,†,‡ Fakhara Ahmed,‡,§ Shreelekha Adsule,§ Sanjeev Banerjee,§ Sudhir Kulkarni,| Prashant Katiyar,|
Christopher E. Anson, Annie K. Powell, Subhash Padhye,†,§ and Fazlul H. Sarkar*,§
Department of Chemistry, UniVersity of Pune, Pune 411007, India, Department of Pathology, Barbara Ann Karmanos Cancer Institute,
Wayne State UniVersity School of Medicine, 9374 Scott Hall, 540 East Canfield AVenue, Detroit, Michigan 48201, Vlife Science Technologies
PVt Ltd., 1 Akshay Residency, Plot No. 50 Anand Park, Aundh Pune 411007, India, and Institute for Inorganic Chemistry, UniVersity of
Karlsruhe, D-76128 Karlsruhe, Germany
ReceiVed October 21, 2005
The synthesis and characterization of Schiff base derivatives of 3-formylchromone 3-6 (FPA-120 to FPA-
123), the minimal biologically active structural motif of soy isoflavone, genistein, and their copper(II)
complexes 7-10 (FPA-124 to FPA-127) are reported here. These copper complexes possess distorted square-
planar geometries capable of stabilizing Cu2+/Cu+ redox forms. The molecular modeling study revealed
that the key interaction of the metal complexes was with amino acids in the pleckstrin homology (PH) and
the kinase domain of the PKB (Akt) protein. Copper complex 7 significantly forms stronger charge interactions
in the kinase domain than genistein, leading to better stabilization in the active pocket. In vitro evaluation
of copper complexes against hormone-independent and metastatic breast (BT20), prostate (PC-3), and K-ras
mutant (COLO 357) and K-ras wild-type (BxPC-3) pancreatic cancer cells revealed that 7 was the most
potent compound which exhibited PKB (Akt protein) inhibitory activities and caused NF-κB inactivation in
a well-established orthotopic pancreatic tumor model using COLO 357 cells. An inverse relationship was
observed between IC50 values of the anti-proliferative activities and the Cu2+/Cu+ redox couple for these
compounds, which may provide a rapid screen for evaluating the efficacy of active metallodrugs affecting
redox-senstitive transcription factors such as NF-κB and its upstream target, the PKB (Akt) pathway, in
multiple cancers.
Introduction
by condensing it with various amines in alcoholic medium,
yielding corresponding Schiff bases. Such compounds are
capable of forming metal complexes with several transition
metal ions among which copper is particularly effective in
yielding moieties with potent radical scavenging properties.19
Among the flavonoids and isoflavonoids found in plants with
potential beneficial effects to human health, genistein (1), a
major metabolite of soy, is believed to be one of the most potent
anticancer agents.1,2 Studies from our laboratory and others have
found that genistein can inhibit the growth of various cancer
cell lines, including leukemia, lymphoma, prostate, breast, lung,
and head and neck, both in vitro and in vivo.3-10 The compound
has been shown to inhibit growth of cancer cells through
modulation of genes that are related to the homeostatic control
of the cell cycle and apoptosis.11-13 It was also found that
genistein could inhibit the activation of nuclear transcription
factor, NF-κB,14 and Akt signaling pathways,15 both of which
are known to maintain a balance between cell survival and
programmed cell death (apoptosis). The structure-activity
correlations for the isoflavonoid compounds have indicated
certain features desirable for the antitumor properties of these
compounds which include a chromone motif, with a double bond
between C2-C3 positions and a side chain containing a phenyl
ring having metal chelating ability.16-18 These structural re-
quirements can be easily built into the compound 3-formyl-
chromone (2), which is a versatile synthon in heterocyclic
chemistry, having antiinflammatory and anticancer activities,
The antiproliferative properties of genistein have been thought
to be partly due to its strong antioxidant property for which
they may enlist the help of relevant metal ions present in the
biological system.20-25 Chelation of metal ions has been shown
to enhance antitumor effects via several different intracellular
mechanisms.26,27 For example, iron chelators have been shown
to induce apoptosis through a p53-independent pathway28 and
by the inhibition of N-myc expression.29 Similarly copper
chelators induce apoptosis of tumor cells through inhibition of
the NF-κB signaling cascade.30 However, despite many of the
active antitumor flavonoid or isoflavonoid compounds being
effective metal ion chelators,23,24 there have not been many
studies on the metal complexes of biologically active isofla-
vonoids and their correlation with antioxidant potentials or their
effects on the NF-κB signaling pathway.
In the present paper, we describe synthesis and characteriza-
tion of four Schiff base derivatives of 2. Conjugation with copper
ions by these derivatives yields compounds with enhanced
antiproliferative activities against hormone-independent BT20
and PC-3 cells as well as COLO 357 (K-ras mutant) and BxPC-3
(K-ras wild type) pancreatic cancer cell lines with IC50 values
in therapeutically achievable concentration ranges. The fluo-
rescence polarization method for the detection of serine/
threonine kinase activity showed that 7 had the lowest IC50 in
comparison with the other copper complexes in this series.
Moreover, we report in vivo results on the most active
compounds of this series, viz., 7, in an established orthotopic
* To whom correspondence should be addressed at the Department of
Pathology, Karmanos Cancer Institute, Wayne State University School of
Medicine, 715 Hudson Webber Cancer Research Center, 110 E. Warren,
Detroit, MI 48201. Phone: 313-576-8327. Fax: 313-576-8389. E-mail:
fsarkar@med.wayne.edu.
† University of Pune.
‡ V.A. and F.A. have contributed equally to this work.
§ Wayne State University.
| Vlife Science Technologies Pvt Ltd.
University of Karlsruhe.
10.1021/jm051068y CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/03/2006

IsoflaVone DeriVatiVe Anticancer ActiVity
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3801
Scheme 1. Schematic Structures of Genistein (1) and 3-Formylchromone (2), Its Schiff Bases 3-6, and Its Metal Complexes 7-10
animal model of pancreatic cancer showing that this compound
down-regulates the nuclear transcription factor NF-κB, which
is known to be regulated by the PKB (Akt) protein and is
responsible for rendering resistance against many cancer chemo-
therapeutic agents. Our present results suggest that carefully
designed metal complexes of genistein analogues may provide
useful chemotherapeutic agents for the treatment of, at least,
three of the most common human cancers.
corresponding to the -NH and -OH protons confirm the
existence of the keto form and the absence of deprotonation in
these compounds. The aromatic protons appear in the range of
6-8.2 ppm for all compounds.36 Compound 3 contains an
isothiocyanate function (although not terminal as in sul-
foraphanes), which has been shown to be capable of activating
MAPKs, NRF, ARE-mediated luciferase reporter genes and
phase II enzyme gene induction.37,38
The spectroscopic data indicate that, during metal conjugation,
ligands 3-6 behave as bidentate thionic moieties coordinating
through azomethine nitrogen and thiocarbonyl sulfur/enolic
hydroxyl (in the case of hydrazonates), respectively. The
electronic spectra confirm the square-planar geometries for the
Results and Discussion
The Schiff base ligands 3-6 (Scheme 1) were synthesized
by condensing equimolar amounts of 2 with various amines in
methanolic solvent and recrystallizing the resulting compounds
from dimethylformamide (DMF)-methanol (1:1) solvent. The
ligands were interacted further with copper chloride in the
stoichiometric ratio to precipitate out the corresponding metal
complexes 7-10, which were purified by the chromatographic
workups. The selected amines are effective pharmacophores
found in many therapeutic compounds currently used in the
clinical practice and serve as spacers in the present design,
keeping the cytotoxic metal complexes away from the isofla-
vonoid moiety. Such a strategy was found to be useful for
retaining pharmacological properties of both the carrier and
cytotoxic moieties.31
The 1H NMR spectra of ligands 3-6 were recorded in
dimethyl sulfoxide-d6 (DMSO-d6) on a Varian-Mercury 300
MHz spectrometer using Si(CH3)4 as an internal standard. The
1H NMR spectra of ligands 3-6 in DMSO-d6 exhibits signal
in the region of 11-12 ppm, which can be attributed to the
amide proton and confirms the E-isomeric form.32,33 The
presence of a downfield NH proton for them may be due to the
involvement of this group in the hydrogen bonding with DMSO-
d6, which is well-known for the amide proton.34 The downfield
shift of the -OH proton in 6 that resonates at 11.80 ppm
indicates that the -OH proton in this ligand is probably involved
in the formation of strong intramolecular hydrogen bonding.35
Thus, 1H NMR spectra of ligands 3-6 displaying signals
2
2
2
copper complexes with the presence of B1g f A1g and B1g
2
f Eg transitions, respectively.39,40 The absorption at 25 000
cm-l observed for compound 7 is ascribed to the S f Cu(II)
charge-transfer band,41 while the absorption in the region of
22 000-24 000 cm-l is due to an oxygen to copper charge-
transfer transition.42 The magnetic moments of the copper
compounds (1.74-1.94 µB) are typical of monomeric com-
pounds having distorted square-planar geometries.43 The electron
paramagnetic resonance (EPR) parameters further confirm such
geometries with a parametric relation g| > g > 2.0 and A|
values around 175-140 G.44 The distortion factor f (given by
g|/A|) calculated for the present compounds are comparable with
analogous copper complexes reported in the literature.45 This
parameter has been suggested to be important for designing
copper compounds having radical scavenging properties.46
The electrochemical profile of 3 (Supporting Information)
indicates an irreversible reduction peak centered at -1.30 V
which is due to the reduction of the azomethine (CdN)
function.47 A similar peak is also observed in the case of
compounds 3-6. An additional quasireversible peak at -0.65
V is assigned to the reduction of the aroylhydrazone moiety in
the case of the hydrazonate ligands. We calculated the redox
reversibility for all copper complexes, and these are shown in
Table 1 on page 9 of the Supporting Information. The ipa/ipc

3802 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
BarVe et al.
ratio for all copper complexes falls in the range of 0.75-0.95,
indicating that the couples are 75-95% reversible. To confirm
this, we also carried out the scan rate dependence of all the
copper complexes using cyclic voltammetric methods, and this
shows that the redox peaks observed for 7 (FPA-124) are indeed
reversible as compared to the parent ligand 3 (FPA-120), which
does not show any redox peak (Figure 1c in the Supporting
Information on page 6).
of 0.1 kcal/(mol‚Å) using MMFF94 force-field in VLifeMDS
2.0. The binding energy was then calculated as
Ebinding ) Ecomplex - (Eapo + Eligand
)
The kinase domain cavity is comprised of a buried hydro-
phobic interior and a relatively less buried hydrophilic surface.
While the polar exposed portion of the cavity is characterized
by the presence of charged residues such as glutamates (Glu274,
Glu310), aspartates (Asp270, Asp288), and Thr156, the buried
hydrophobic interior of the cavity is characterized by the
presence of hydrophobic residues such as Phe289, Leu291,
Leu152, Val160, and Ala173. Unlike the kinase domain, the
PH domain ligand binding cavity is primarily shallow and
hydrophilic in nature. The presence of an ample number of
lysine and arginine residues such as Arg25, Arg23, Lys14,
Arg86, Arg15, and Lys39 renders a positively charged nature
to the cavity surface. However, one negatively charged residue,
Glu17, is also present on the cavity surface, thereby conferring
an acidic nature to a relatively smaller region of the cavity
surface. It is worth noticing that the PH domain cavity lacks a
hydrophobic buried interior as observed in the kinase domain.
Hence, we believe that these fundamental differences, viz.,
electrostatics and shape-size characteristics of the kinase and
PH domain, form the underlying basis for variable binding
affinity as well as specificity of different ligands docked into
these two domains.
It was observed that the aromatic chromone ring of genistein
interacts with Arg25 and the phenolic -OH of the B-ring of
genistein interacts with the carboxyl group of Glu17, leading
to stabilization in the PH domain. Hydrophobic interactions were
not found to contribute to genistein binding in any manner
because of a lack of any significant hydrophobic residues in
the PH domain cavity. Unlike genistein, metal complex 7
binding in the PH domain is stabilized by an additional hydrogen
bond with key amino acids Arg25, Glu17, and Ile19 that are
involved in the stabilization of the receptor-ligand complex
(see the Supporting Information for docking figures). Since
different PH domains normally share less than 20% sequence
identity, this may facilitate the development of drugs that bind
specifically to the PH domain of the PKB protein. It was
observed that some charged residues such as Arg86, Asn53,
and Lys14 are yet unexploited and provide an ideal opportunity
for further modifications to the benzopyran motif, which may
yield highly potent molecules with enhanced PKB inhibitory
activity.
The crystal structure of the kinase domain of the PKB protein
bound to AMP-PNP, which is an inhibitor analogous to natural
substrate ATP, is reported in the literature (PDB ID: 1O6K).
The AMP-PNP ligand does not directly bind to THR308 yet
prevents its phosphorylation (see Supporting Information). It
was seen that the genistein molecule interacts with the hydro-
phobic cavity of the the kinase domain of the PKB protein,
where its single phenolic ring structure orients itself to hydrogen
bond with the CdO of the Met223 and the -OH group of B
ring binds to the carboxyl group of Glu274 (Figure 1a, where
hydrogen bonds are shown in pink).
The docking studies of 7 have shown that the metal complex
has three kinds of interactions with the receptor protein. The
hydrophobic chromone end of 7 interacts with the hydrophobic
interior of the cavity, namely, the hydrophobic side chains of
the residues Phe289, Leu(291,152), and Val160. Second, there
exists a hydrogen bond between the backbone N-H of Gly290
and the ethereal oxygen atom of 7. As the copper atom carries
a partial positive charge, the metal complex interacts with the
All copper complexes show a reversible Cu(II)/Cu(I) redox
couple in the range of +0.28 to +0.35 V, indicating a facile
reduction of the cupric center. One of the properties of the metal
complexes is increased liposolubility. This increases the ability
of the drug molecules in crossing the cell membrane, thus
increasing the biological availability and hence its activity. Our
result suggests that the structure and conformation of the ligand
has influence on the redox potential of the central atom of the
coordination compound. This is observed with the present
isoflavone derivatives 3-6, where changes in the coordination
sphere are connected with the change of biofunction of
compounds, as shown in Figure 4.
Previously, 3′,4′-O-substituted derivatives of isoflavones have
been developed as therapeutic agents against protein tyrosine
kinase (PTK) and molecular modeling studies have confirmed
that isoflavonoids have the structural features important for
binding to SH2 domain of p56lck protein kinase.48 The PDB
structure of the PH domain of the human protein kinase B-R
isoform was available in protein databank (PDB code: 1UNP).49
However, the X-ray crystal structure for the kinase domain of
the PKBR isoform was not available in the protein databank.
To investigate the interactions of the parent genistein and the
metal complex 7 with the active domains of the PKB (Akt)
protein, namely, PH and the catalytic kinase domain, we carried
out homology modeling studies for 7 using the program
VLifeMDS 2.0 (VLifeMDS 2.0, Molecular Design Suite
developed by VLife Sciences technologies Pvt Ltd.)50 in order
to obtain a model for the kinase domain of PKBR by using
X-ray crystal structure of PKBâ (PDB code: 1GZK, sequence
identity 86% and similarity 94% with the R-isoform) as
template.51,52 The conformation analysis of genistein (1) and
its complexes 7-10 was done by using the Monte Carlo
conformation method, and conformers satisfying steric require-
ment of the each cavity were considered for docking.53 The
MMFF94 force field was utilized for energy minimization to a
gradient root mean square (RMS) of 0.1 kcal/(mol‚Å) in order
to remove local structural defects such as steric hindrance and
unusual bond lengths, bond angles, and torsions.54,55 All the
minimizations were carried out in the absence of water,
assuming a dielectric constant (ꢀ) equal to 1.The charge
equilibrium Qeq method provided the functions to calculate
forces on atoms and energy of atoms and metals such as Cu2+
explicitly bonded to the other four atoms. We used certain
parameters for selecting the conformers. After the ligands were
placed in the active site, the residues were flexed within 10 Å,
and residues beyond 10 Å were frozen. We chose this method
because residues beyond 10 Å are unlikely to interact with the
ligand. A second criterion used was based on the similarity
between the force field and root-mean-square distance (RMSD)
calculated over heavy atoms and polar atoms of the ligand
conformers. Conformers with a difference in energy (based on
the nature of potential energy) by at least 1 kcal/mol were
chosen. The conformers were selected and placed in the active
site, and the atoms of the ligand that form a ring with Cu2+
along with two chlorine atoms were aggregated and the resultant
complex structures were energy-minimized to a gradient-RMS

IsoflaVone DeriVatiVe Anticancer ActiVity
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3803
Figure 1. (a) Docking of genistein (1) into the kinase domain of the PKB (Akt) protein, showing the interactions within the hydrophobic cavity
where the phenolic ring structure of genistein hydrogen bonds with CdO of the Met223 and the -OH group of B ring binds to the carboxyl group
of Glu274 in the kinase domain of the PKB protein. (b) Key interactions of 7 with the active site of the kinase domain of the PKB protein, showing
stronger charge interactions as well as hydrogen bonding with the N-H of Gly290, electrostatic interactions with carboxyl groups of Glu274,
Glu310, Asp270, and Asp288 as well as hydrophobic interactions with residues Phe289, Leu(291,152), and Val160.
carboxyl groups of Glu274, Glu310, Asp270, and Asp288
through electrostatic interactions (Figure 1b). From these
modeling experiments, we found that genistein had one extra
hydrogen bond interaction over 7 with the PKB protein, whereas
the 7 showed charge interactions within the kinase domain.
Hence, we conclude that the metal complex 7 could lead to
better stabilization because charge interactions are stronger than
hydrogen bonding; it leads to better stabilization of the parent
genistein, yielding higher potent kinase inhibitory activity as
supported by our fluorescence polarization IC50 data (Table 1).
When we examined the biological effects of these compounds
against the hormone-independent breast (BT20) and prostate
(PC-3) cancers as well as K-ras mutant COLO 357) and K-ras
Table 1. IC50 Values of 7, 8, and 10 by MTT and Fluorescence
Polarization Assay in BT20, PC-3, BxPC-3, and COLO 357
Cancer Cell Lines
fluorescence
polarization
Akt kinase
MTT cell proliferation assay IC50 (µM)
Compounds COLO357 BxPC3
BT20
PC3 assay IC50 (µM)
genistein
7
8
10
50
34
30
16
30
55
20
22
46-70
50
10
15
14
>70
0.1
15
8
7
12
12
wild-type (BxPC-3) pancreatic cancer cell lines, the metal
complexes exhibited dose-dependent growth inhibitory effects

3804 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
BarVe et al.
Figure 2. Inhibition of cell growth after treatment for 72 h with copper complexes 7, 8, and 10 on cancer cell lines: (a) K-ras mutant pancreatic
COLO 357 cancer cells; (b) K-ras wild-type pancreatic BxPC-3 cancer cells; (c) estrogen-independent BT20 breast cancer; (d) androgen-independent
PC-3 prostate cancer cells. (IC50 is defined as the concentration of the compound required to inhibit proliferation of cells by 50%.)
in all of the cell lines. Most importantly, 7(FPA-124) showed
50% cell kill at concentrations of 7 µM in BT20 and 10 µM in
PC-3 cells (Figure 2). The metal complexes 7, 8, and 10
inhibited growth of BxPC-3 and COLO 357 cells at concentra-
tion > 20 µM, whereas the metal complex 9 showed a cell kill
in all of the four cell lines at very high doses (>50 µM). We
observed a synergistic enhancement in the antiproliferative
activities upon metal conjugation compared to their correspond-
ing parent ligands 3-6 (Figure 5 on Supporting Information
page 8) as well as to the parent genistein (positive control),
which has IC50 values > 40 µM in these multiple cancer cell
lines.
In our earlier work, it was observed that metal conjugation
renders a pleiotrophic characteristic to the organic ligands which
are essential for treating a heterogeneous disease like cancer.56
Compounds with well-defined but singular targets have been
found to have limited therapeutic value due to associated but
unanticipated side effects and rapidly built resistance. On the
other hand, metal complexes are able to assert effects on
multitarget sites (whether in intact or dissociated configurations)
and hence exhibit superior activity and comparatively less
frequency of resistance. A comparison of the IC50 values of
present metal complexes with those of genistein revealed
substantial decrease indicating therapeutically achievable ef-
ficacy (Table 1). Since all copper compounds were redox active
metal complexes, we believe that redox triggered oxidative stress
may be one of the underlying mechanisms for the observed
apoptotic cell death in the present case.
with the favorable distortion factor calculated for the present
copper compounds using EPR spectra as suggested by Cao et
al.,46 which helps stabilize cuprous and cupric species without
dissociation of the complex.
Interestingly, we observed an inverse relationship between
the metal redox couple and the IC50 values for the present metal
complexes against the hormone-independent breast and prostate
cancer cell lines (Figure 4a,b). Further to show the correlation
between Eo and liposolubilities, we calculated the log P values
of the metal complexes 7-10 (FPA-124 to FPA-127). Our
results show that although the copper complex FPA-124 has a
lower log P than other complexes, a high positive redox value
E1/2 of the metal complex FPA-124 correlates with higher
liposolubility and thus higher biological activity (inhibition of
cell growth in vitro) as shown in Figure 4c.
We have calculated the stability constants of these copper
complexes via Job’s ratio method57 (mole ratio method) as log
K ) 4.10, which confirms that these complexes are mildly stable
at neutral pH. Various copper complexes have similar log K
values which sustain their structures under physiological condi-
tions as described previously.58 These results are supported by
our additional findings as shown in the scan-dependent experi-
ments by cyclic voltammetry (Figure 1b,c on Supporting Infor-
mation page 6). The intactness of the copper complexes during
redox cycling of the copper center through the Cu2+/Cu+ couple
is also supported by the distortion factors (F) calculated for these
compounds from their EPR spectra (average value ) 150 G)
(Supporting Information Table 2b on page 10). From our results
it follows that the structure and conformation of the ligand has
influence on the redox potential of the central atom in the
coordination compound, indicating that this parameter can
indeed be used as a guideline for developing effective antitumor
metal complexes against these cancers.
The quantitative evaluation of the apoptosis by ELISA for
the metal complexes 7, 8, and 10 has been carried out in four
cancer cell lines COLO 357, BxPC-3, BT20, and PC-3,
respectively (Figure 3). It was observed that metal complexes
7, 8, and 10 induced apoptosis in the entire cancer cell lines
tested and 7 showed the highest index of apoptotic cells in PC-3
cells. The bar graph in Figure 3 is the mean of three separate
experiments carried out. This observation is also in accordance
In further experiments using fluorescence polarization-based
assays,59,60 we investigated whether the effects of the present
metal complexes 7, 8, and 10 could be mediated by inactivation

IsoflaVone DeriVatiVe Anticancer ActiVity
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3805
Figure 3. Induction of apoptosis in cancer cells by ELISA, showing data from (a) K-ras mutant pancreatic COLO 357 cancer cells, (b) K-ras
wild-type pancreatic BxPC-3 cancer cells, (c) estrogen-independent BT20 breast cancer, and (d) androgen-independent PC-3 prostate cancer cells
(control, 20 µM (7, 8, and 10) treated cells).
of serine/threonine kinase activity, since PKB (Akt) signaling
is important in cancer development as it promotes cell survival
by inhibiting apoptosis through inactivation of proapoptotic
factors. The PKB (Akt) protein has been shown to influence
many transcription factors that are involved in controlling the
cell growth and survival such as E2F, NF-κB,61 and CREB and
are also known to cross-talk with the RAF/Erk signaling
pathways.62 The PKB (Akt) protein is activated by phospholipid
binding and phosphorylation at Thr308 by PDK1 or Ser473 by
PDK2, respectively.
In conclusion, the knowledge of the 3D structure of the bio-
logical target as well as the specialized computational software
constitutes a powerful approach to design libraries of inhibitors
with specific activity for PKB (Akt) protein. Our structure-based
focused approach has led to the identification of bioactive
thiosemicarbazonate or hydrazonate pharmacophores appended
to the chromone motif capable of copper conjugation as novel
antitumor molecules compared to genistein, which are capable
of inhibiting NF-κB activation through the PKB (Akt) pathway
in the animal model. Further work is ongoing in our laboratory
to unravel the underlying mechanism of their superior cell killing
action and the role played by cupric ions in these novel metal-
based antitumor agents.
The IC50 values of our compounds are presented in Table 1.
Compound 7 exhibited the lowest IC50 value compared to other
copper complexes in inhibiting Akt kinase activity. It is
interesting to note that the Akt kinase activity could be inhibited
with 100 nM (IC50) of 7 but the IC50 for inhibiting cell growth
is 70-100-fold greater than the inhibition of kinase activity.
Further studies are underway to understand these results.
Activation of NF-κB in cancer cells has been shown to
attenuate apoptosis induced by chemotherapeutic agents resulting
in lower cell killing and drug resistance. Since the NF-κB
pathway is regulated by Akt protein, we decided to investigate
the effects of the lead copper complex of the present series, 7,
on the NF-κB activity through an in vivo experiment in a well-
established orthotropic pancreatic tumor model using COLO
357 cells.63 The parent genistein compound has been shown to
inhibit the activity of NF-κB and the growth of hormone-
dependent (LNCaP) and hormone-independent (PC-3) human
prostate cancer cell lines in vivo without causing systemic
toxicity.64 Recently, we have also observed potentiating effects
of genistein leading to inhibition of tumor growth by radiation
in a prostate cancer orthotopic model65 and the effect of
chemotherapy in orthotopic pancreatic cancer model.66 In this
study, our results showed that 7 had no apparent animal toxicity,
as indicated by no change in the body weight of the treated
animals (Figure 5a) but caused a decrease in the tumor load
(Figure 5b). Most importantly, the NF-κB activity was sig-
nificantly decreased in 7-treated animal tumor tissues, sup-
porting the in vivo effect of 7 through the Akt/NF-κB pathway
(Figure 5c).
Experimental Section
Chemical Methods. Unless otherwise noted all solvents, chemi-
cals, and reagents were obtained commercially and used without
1
purification. The H NMR spectra were carried out on a Varian-
Mercury 300 Hz spectrophotometer using Si(CH3)4 as an internal
standard.
Synthesis of 3-Formylchromone Schiff Bases (3-6). The Schiff
base ligands 3-6 (Scheme 1) were synthesized by mixing equimolar
amounts of 2 with thiosemicarbazide hydrochloride (3), benzoyl
hydrazide (4), isonicotinoyl hydrazide (5), and salicylic hydrazide
(6), respectively, in methanolic solvent and maintaining the reaction
mixture at reflux temperature for 1 h. The products obtained were
filtered off, recrystallized from (1:1) DMF-methanol, and finally
dried in a vacuum desiccator over anhydrous CaCl2. 1H NMR data
for 3-6 are listed in the Supporting Information.
3-Formylchromone Thiosemicarbazone (3). (C17H10N2O3): C,
53.88%; H, 3.32%; N 16.50%; S, 12.76% (calcd: C, 53.44%; H,
3.64%; N, 17.00%; S, 12.95%).
3-Formylchromone Benzoyl Hydrazone (4). (C16H9N3O3): C,
69.86%; H, 4.10%; N 9.58% (calcd: C, 63.43%; H, 4.38%; N,
9.01%).
3-Formylchromone
Isonicotinyl
Hydrazone
(5).
(C16H9N3O3): C, 65.00%; H, 3.48%; N, 14.06% (calcd: C, 65.00%;
H, 3.75%; N, 14.33%).
3-Formylchromone Salicylic Hydrazone (6). (C17H11N2O4): C,
65.68%; H, 3.89%; N 9.03% (calcd: C, 66.23%; H, 3.87%; N,
9.09%).

3806 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
BarVe et al.
Figure 5. Data representing an average of two independent experiments
with a total of nine animals in each group. In vivo data showing (a)
the relative body weight of control vs treated animals (treatment with
7 (25 mg/kg) body weight per mouse) (%), (b) the relative pancreas
weight (%) of primary pancreatic tumors harvested from control versus
treated mice with 7, and (c) the gel shift assay showing down-regulation
of NF-κB DNA binding activity in primary pancreatic tumors from
two representative mice derived from control and treated groups.
phosphopeptide in the reaction results in a lesser amount of
fluorescent phosphopeptide available for binding to antibody,
thereby causing less polarization, which reflects higher Akt kinase
activity. On the other hand with fewer unlabeled phosphopeptides
due to Akt kinase inhibition, more fluorescent phosphopeptide will
bind to the antibody, resulting in a higher polarization value, which
reflects the inhibition of the Akt kinase activity.
For specific assays, the Akt kinase assay reaction was set up in
a total volume of 40 µL in the presence of different concentrations
of inhibitor. Stock solutions of test compounds were made in
DMSO. From DMSO stocks, 4× solutions of compounds 7, 8, and
10 were prepared in 1× assay dilution buffer; 10 µL of the
respective drug solution was used in the assay. Each reaction well
had 25 ng of Akt1/PKBR, active enzyme (Upstate Biotechnology
Catalog No. 14-276), 0.4 mM GSK-3 peptide (custom synthesized),
ATP (100 µM) from Sigma Catalog No. A5394, and the test
compound to be tested. A plate was incubated at 30 °C for 30 min.
The enzyme reaction was quenched with 10 µL of 50 mM
ethylenediaminetetraacetic acid (EDTA) for 5 min. Antiphospho-
serine antibody was added at 25 µL/well followed by 10 µL of
fluorescent tracer.
The total volume was made up to 100 µL by adding 15 µL of
fluorescence polarization assay dilution buffer. Appropriate controls
and blanks were prepared. The plate was incubated for 1 h at room
temperature and was analyzed for polarization at 435 nm excitation
and 535 nm emission wavelength on the Tecan Ultra microplate
reader. Polarization values were normalized to the control and were
plotted against the log concentration of the test compounds in Graph
Pad Prism software. Sigmoidal dose response curve analysis was
used to calculate the IC50 of the test compounds.
In Vivo Studies. Experimental Animals. Female nude mice
(ICR-SCID) were purchased from Taconic Farms (Germantown,
NY). The mice were housed and maintained under sterile conditions
in facilities accredited by the American Association for the
Accreditation of Laboratory Animal Care and in accordance with
current regulations and standards of the United States Department
of Agriculture, United States Department of Health and Human
Services, and the National Institutes of Health (NIH). The mice
were used in accordance with Animal Care and User Guidelines
of Wayne State University under a protocol approved by the
Institutional Animal Care and Use Committee. The mice received
Lab Diet 5021 (Purina Mills, Inc., Richmond, IN).
Figure 4. Plot showing correlation between IC50 (µM) values and the
metal redox couple (E1/2) for copper complexes 7-10.
Synthesis of Metal Complexes (7-10). The copper(II) com-
plexes of 7-10 were synthesized by mixing equimolar amounts of
the ligands and CuCl2‚2H2O in methanol with a trace amount of
dimethylformamide. The resulting mixture was refluxed at room
temperature for 1 h. The precipitates formed were removed by
filtration, washed with the methanol solvent, and dried in a vacuum
over anhydrous CaCl2. The analytical elemental data for the copper
complexes (7-10) of 3-formylchromome are given in the Sup-
porting Information.
Biology. Akt Kinase Assay by Fluorescence Polarization.
Serine/threonine Akt kinase reaction was carried out in 96-well
black plates in a total volume of 100 µL using a Ser/Thr kinase
polarization based assay kit from Invitrogen Part No. P310353. The
Akt kinase assay was done in vitro using fluoroscence polarization
based on the following principle. Briefly, enzyme is incubated with
the substrate in the presence of ATP and ideal buffer conditions.
In the absence of enzyme inhibitor, complete enzymatic reaction
takes place, resulting in the production of a maximum amount of
phosphopeptide, while in the presence of enzyme inhibitor it blocks
the reaction, resulting in the reduction of phosphopeptide product.
This phosphopeptide competes with fluorescently tagged phospho-
peptide for its binding to the anti-phospho antibody in the assay
plate. Therefore, the availability of a greater amount of unlabeled
Orthotopic Implantation of Tumor cells. COLO 357 cells were
harvested from subconfluent cultures after a brief exposure to 0.25%
trypsin and 0.2% EDTA. Trypsinization was stopped with medium
containing 10% fetal bovine serum (FBS). The cells were washed
once in serum-free medium and resuspended in phosphate buffer
saline (PBS). Only suspensions consisting of single cells with >90%
viability were used for the injections. The pancreas of anaesthetized

IsoflaVone DeriVatiVe Anticancer ActiVity
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3807
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mice was exposed through a midline laparotomy incision and by
retraction of the spleen. 2 × 105 cells in 20 µL PBS were injected
into the parenchyma of the pancreas with a 27 gauge hypodermic
needle and a Hamilton syringe as previously described by our
laboratory.65 The abdominal wound was sutured using a 5.0 chromic
gut suture in a running fashion. On the basis of our previous
experience with this model, we found a tumor take rate approaching
>90%.
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Mice were randomized into two treatment groups (n ) 9, in each
group): (a) untreated control; (b) 7-treated (25 mg/kg, body weight)
once every alternate day by intravenous (iv) injection (total of six
injections). All mice were sacrificed on day 10 following the last
dose of treatment, and their body weight was determined. The
pancreas from all animals were excised and weighed. For routine
H&E staining, one part of the tissue was fixed in formalin and
embedded in paraffin and another part was rapidly frozen in liquid
nitrogen and stored at -70 °C. H&E staining confirmed the
presence of tumor(s) in each pancreas (data not shown).
Tumor Tissue Nuclear Protein Extraction and Electro-
phoretic Mobility Shift Assay (EMSA). Nuclear proteins were
extracted from the tumor tissue as described previously.66 The
supernatant (nuclear proteins) was collected and kept at -70 °C
until use. Protein concentration was determined using the bicin-
chonic acid assay kit with BSA as the standard (Pierce Chemical
Co., Rockford, IL). The electrophoretic mobility shift assay (EMSA)
was performed by incubating 8 µg of nuclear proteins with IR
DyeTM-700 labeled NF-κB oligonucleotide. The incubation mixture
included 2 µg of poly(dI-dC) in a binding buffer. The DNA-
protein complex formed was separated from free oligonucleotide
on 8.0% native polyacralyamide gel using buffer containing 50 mM
Tris, 200 mM glycine, pH 8.5, and 1 mM EDTA and then visualized
by Odyssey infrared imaging system using Odyssey software release
1.1.
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Acknowledgment. S.A. gratefully thanks Dr. Jayendra
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Supporting Information Available: Full experimental infor-
mation for Schiff base isoflavone derivatives 3-6 and the metal
complexes 7-10, analytical characterization, and biological ex-
perimental assays. This material is available free of charge via the
Internet at http://pubs.acs.org.
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