4-aryl-4h-chromene-3-carbonitrile derivatives: Evaluation of Src kinase inhibitory and anticancer activities

Article abstract of DOI:10.2174/157340611796799258

Src kinase mutations and/or overexpression have been implicated in the development of a number of human cancers including colon, breast, and lung cancers. Thus, designing potent and selective Src kinase inhibitors as anticancer agents is a subject of majo

Full text of DOI:10.2174/157340611796799258

466
Medicinal Chemistry, 2011, 7, 466-472
4-Aryl-4H-Chromene-3-Carbonitrile Derivatives: Evaluation of Src Kinase
Inhibitory and Anticancer Activities
Asal Fallah-Taftia, Rakesh Tiwarib, Amir Nasrolahi Shirazib, Tahmineh Akbarzadeha, Deendayal Mandalb,
Abbas Shafieea, Keykavous Parangb,* and Alireza Foroumadia,*
aDepartment of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran Uni-
b
versity of Medical Sciences, Tehran 14147, IRAN; Department of Biomedical and Pharmaceutical Sciences, College of
Pharmacy, The University of Rhode Island, Kingston, Rhode Island 02881, USA
Abstract: Src kinase mutations and/or overexpression have been implicated in the development of a number of human
cancers including colon, breast, and lung cancers. Thus, designing potent and selective Src kinase inhibitors as anticancer
agents is a subject of major interest. A series of 4-aryl substituted derivatives of 2-amino-7-dimethylamino-4H-chromene-
3-carbonitrile were synthesized using one-pot reaction of appropriate substituted aromatic aldehydes, malononitrile, and
3-(dimethylamino)phenol in the presence of piperidine. All 23 compounds were evaluated for inhibition of Src kinase and
cell proliferation in human colon adenocarcinoma (HT-29) and leukemia (CCRF-CEM) cell lines. Among the tested
compounds, 2-chlorophenyl- (4c), 3-nitrophenyl- (4h), 4-trifluoromethyphenyl- (4i), and 2,3-dichlorophenyl- (4k) substi-
tuted chromenes showed Src kinase inhibitory effect with IC50 values of 11.1-18.3 μM. Compound 4c was relatively se-
lective against Src (IC50 = 11.1 μM), when compared with selected kinases, epidermal growth factor receptor (EGFR, IC50
> 300 μM), C-terminal Src kinase (Csk, IC50 = 101.7 μM), and lymphocyte-specific protein tyrosine kinase (Lck, IC50
=
46.8 μM). 3-Chlorophenyl substituted thiazole (4v) and 2-chlorophenylsubstituted thiazole (4u) chromene derivatives in-
hibited the cell proliferation of HT-29 and CCRF-CEM by 80% and 50%, respectively, at a concentration of 50 ꢀM. The
data indicate that 4H-chromene-3-carbonitrile scaffold has the potential to be optimized further for designing more potent
Src kinase inhibitors and/or anticancer lead compounds.
Keywords: Anticancer activity, benzopyranones, carbonitrile, chromenes, inhibitor, protein kinase, Src kinase.
INTRODUCTION
Over the years, a diverse number of compounds have
been identified as Src kinase inhibitors [8]; among them
there are several different structural classes, including
staurosporine as a standard nonselective protein kinase in-
hibitor, pyrazolopyrimidine (e.g., PP1, PP2) [9,10], anilino-
quinazoline [11,12], and quinolinecarbonitrile [13-15] de-
rivatives. The Saracatinib (AZD0530) [12,16] and Bosutinib
(SKI-606) [17,18] with anilinoquinazoline and 3-
quinolinecarbonitrile cores, respectively (Fig. 1), are among
potent Src kinase inhibitors, which are currently in clinical
development for the treatment of a wide range of tumor
types. Designing selective Src kinase inhibitors still remains
a challenge. Because of the highly conserved nature of the
ATP-binding site within the Src family kinase, nonselective
inhibitors may provoke toxicity in vivo. Thus, additional
research is needed to identify new chemical scaffolds that
can inhibit selectively Src kinase domain.
Protein tyrosine kinases (PTKs) catalyze the transfer of
ꢁ-phosphate group from ATP to specific tyrosine residue of
many protein substrates. Src family tyrosine kinases (SFKs)
are subject of major interest because of their critical roles in
signal transduction pathways and cellular functions, such as
division, differentiation, survival, adhesion, migration, and
invasion. SFKs are a family of eleven different non-receptor
PTKs, including Blk, Brk, c-Fgr, Frk, Fyn, Hck, Lck, Lyn, c-
Src (Src), Srm, c-Yes [1] of which Src is the prototype.
There is significant evidence demonstrating that overexpres-
sion or mutation of Src kinase correlate with tumor growth
and metastasis in several human malignancies, including
colon, breast and pancreatic cancers [2-6]. Src has also been
implicated to have key roles in other pathologic disorders,
such as osteoporosis, cardiovascular diseases, and neurode-
generation [7]. Thus, Src kinase has become an attractive
target for designing new lead compounds against cancer and
other diseases.
There is evidence that the presence of a nitrile group in
different series of compounds like quinolinecarbonitrile
(e.g., Bosutinib), and benzylidene-malononitriles is crucial
for protein kinase inhibitory activity [19]. Within this cate-
gory, compound A (Fig. 2) [20] has been reported as an in-
hibitor of Lck (IC50 = 7-10 ꢀM). Dicycanosubstituted com-
pound B (Fig. 2) [21] has been shown to reduce the steady-
state levels of Src in NIH 3T3 cells.
*Address correspondence to these authors at the Faculty of Pharmacy and
Pharmaceutical Sciences Research Center, Tehran University of Medical
Sciences, Tehran, Iran Tel: +98 21 66954708; Fax: +98 21 66461178;
E-mail: aforoumadi@yahoo.com and 41 Lower College Road, Department
of Biomedical and Pharmaceutical Sciences, College of Pharmacy, Univer-
sity of Rhode Island, Kingston, Rhode Island, 02881, USA; Tel: +1-401-
874-4471; Fax: +1-401-874-5787; E-mail: kparang@uri.edu
Furthermore, several natural benzopyranone-containing
compounds (flavonoids), such as quercetin and genistein
1573-4064/11 $58.00+.00
© 2011 Bentham Science Publishers Ltd.

4-Aryl-4H-Chromene-3-Carbonitrile Derivatives
Medicinal Chemistry, 2011, Vol. 7, No. 5 467
HN
O
N
N
NH2
N
N
Me
OMe
N
N
O
Cl
NHMe
PP2
Staurosporine
O
N
N
CN
Cl
O
O
N
O
N
N
H
O
Cl
O
N
NH
N
O
N
O
Cl
Bosutinib
Saracatinib
Fig. (1). Chemical structures of some of the Src kinase inhibitors.
NH2
O
several chromenes such as LY294002 (Fig. 3) [22] were syn-
thesized and evaluated for their ability to inhibit tyrosine
kinases. LY294002 was found to be a potent and selective
inhibitor of PtdIns 3-kinase.
CN
CN
OH
CN
Considering the fact that a series of variously substituted
4-aryl-4H-chromens-3-carbonitrile have anticancer and
apoptosis-inducing effect [23,24] and in continuation of our
efforts to design or identify new scaffolds as kinase inhibi-
tors [25-32], herein we report the synthesis and evaluation of
selected 2-amino-4-aryl-7-dimethylamino-4H-chromene-3-
carbonitrile derivatives 4a-w (Fig. 3) that contain both nitrile
and chromene core for their protein kinase inhibitory activity.
HO
HO
OH
OH
A
B
Fig. (2). Benzylidene-malononitrile derivatives as PTK inhibitors.
OH
O
OH
O
OH
OH
MATERIALS AND METHODS
1. General
O
HO
All starting materials, reagents, and solvents were pur-
chased from Merck AG (Germany). The purity of the syn-
thesized compounds was confirmed by thin layer chromatog-
raphy (TLC) using various solvents of different polarities.
Merck silica gel 60 F254 plates were applied for analytical
TLC. Column chromatography was performed on Merck
silica gel (70–230 mesh) for purification of the intermediate
and final compounds. Melting points were determined on a
Kofler hot stage apparatus (Vienna, Austria) and are uncor-
HO
OH
O
OH
Quercetin
Genistein
O
Ar
CN
O
N
1
rected. H NMR spectra were recorded using a Bruker 500
O
O
Me2N
NH2
MHz spectrometer (Bruker, Rheinstatten, Germany), and
chemical shifts are expressed as d (ppm) with tetramethylsi-
lane (TMS) as an internal standard. The IR spectra were ob-
tained on a Shimadzu 470 (Shimadzu, Tokyo, Japan) spec-
trophotometer (potassium bromide disks). The mass spectra
were run on a Finnigan TSQ-70 spectrometer (Thermo Fin-
nigan, West Chester, OH, USA) at 70 eV. Elemental analy-
ses were carried out on a CHN-O-rapid elemental analyzer
(Heraeus GmbH, Hanau, Germany) for C, H, and N, and the
results were within ± 0.4% of the theoretical values.
4a-w
LY294002
Fig. (3). Chemical structures of natural protein kinase inhibitors
containing benzopyranone core (quercetin and genistein), chormene
core (LY294002), and synthesized 4-aryl-4H-chromene-3-
carbonitrile derivatives (4a-w).
(Fig. 3) [19] have been shown to serve as general inhibitors
for protein kinases. Using quercetin as a model compound,

468 Medicinal Chemistry, 2011, Vol. 7, No. 5
Fallah-Tafti et al.
2. General Procedure for the Preparation of 2-amino-4-
aryl-7-dimethylamino-4H-chromene-3-carbonitrile
derivatives 4a-w
protocol from Millipore described in www.millipore.com/
drugdiscovery/KinaseProfiler. In brief, Csk (h) was incubated
with 50 mM Tris (pH 7.5), 0.1 mM EDTA, 0.1 mM Na3VO4,
and 0.1% ꢁ-mercaptoethanol. EGFR (h) and activated Lck (h)
were incubated with 8 mM MOPS (pH 7.0) and 0.2 mM
EDTA. To the mixture was added substrate (0.1 mg/mL
poly(Glu, Tyr) 4:1 for Csk and EGFR, 500 μM KVEKI-
In general, piperidine (10 mmol) was added to a mixture
of the 3-dimethylaminophenol (5 mmol), substituted aryl-
benzaldehyde (5 mmol), and malonitrile (5 mmol) in ethanol
(20 mL). The reaction mixture was stirred at 35 ºC for 12 h.
After cooling, the precipitated solid was filtered, washed
with cold ethanol, and crystallized from the same solvent.
33
GEGTYGVVYK for Lck), 10 mM MgAcetate, and [ꢂ- P-ATP]
(specific activity approx. 500 cpm/pmol, 10 μM). The reactions
were initiated by the addition of the MgATP mix. After incuba-
tion for 40 min at room temperature, the reaction is stopped by
the addition of 3% phosphoric acid solution. 10 μL of the reac-
tion is then spotted onto a P30 filtermat and washed three times
for 5 minutes in 75 mM phosphoric acid and once in methanol
prior to drying and scintillation counting.
1
The synthesized compounds were characterized by IR, H
NMR, mass spectral data and elemental analysis. Physico-
chemical and spectral properties of compounds 4a-w were
consistent with the previously reported data [23,24,33].
3. c-Src Kinase Activity Assay
5. CELL CULTURE AND CELL PROLIFERATION
ASSAY
The effect of synthesized compounds on the activity of c-
Src kinase was determined by HTScan Src Kinase Assay Kit,
catalogue number 7776 from Cell Signaling Technology
(Danvers, MA, USA); according to manufacturer’s protocol.
Streptavidin-coated plates were purchased from Pierce
(Rockford, IL, USA). In brief, the kinase reaction was
started with the incubation of the 12.5 ꢀL of the reaction
cocktail (0.5 ng/ꢀL of GST-Src kinase in 1.25 mM DTT)
with 12.5 ꢀL of prediluted compounds (dissolved in 1%
DMSO) for 5 min at room temperature. ATP/substrate (25
ꢀL, 20 ꢀM/1.5uM) cocktail was added to the mixture. The
biotinylated substrate (catalogue number 1366) contains the
residues surrounding tyrosine 160 (Tyr160) of signal trans-
duction protein and has a sequence of EGIYDVP. The reac-
tion mixture was incubated for 30 min at room temperature.
The kinase reaction was stopped with the addition of 50 ꢀL
of 50 mM EDTA (pH 8.0). The reaction solution (25 ꢀL)
was transferred into 96-well streptavidin plates (Pierce, part
number 15125), diluted with 75 ꢀL double distilled water,
and incubated at room temperature for 60 min. At the end of
the incubation, the wells were washed three times with 200
ꢀL of 0.05% Tween-20 in PBS buffer (PBS/T). After that to
the each well was added 100 ꢀL of phosphotyrosine anti-
body (P-Tyr-100) (1:1000 dilution in PBS/T with 1% BSA)
and the wells were incubated for another 60 min. After
washing three times with 0.05% Tween-20 in PBS/T, the
wells were incubated with 100 ꢀL secondary anti-mouse IgG
antibody, which was HRP-conjugated (1:500 dilution in
PBS/T with 1% BSA) for next 30 min at room temperature.
The wells were washed five times with 0.05% Tween-20 in
PBS and then were incubated with 100 ꢀL of 3,3',5,5'-
tetramethylbenzidine dihydrochloride (TMB) substrate for 5
min. The reaction was stopped by adding 100 ꢀL/well of stop
solution to each well and mixed well and read the absor-
bance at 450 nm using a microplate reader (Molecular de-
vices, spectra Max M2). IC50 values of the compounds were
calculated using ORIGIN 6.0 (origin lab) software. IC50 is
the concentration of the compound that inhibited enzyme
activity by 50%. All the experiments were carried out in trip-
licate.
5.1. Cell Culture
Human colon adenocarcinoma HT-29 (ATCC no. HTB-
38) and leukemia CCRF-CEM (ATCC no. CCL-119) cell
lines were obtained from American Type Culture Collection.
Cells were grown on 75 cm2 cell culture flasks with RPMI-
16 medium for leukemia and EMEM medium for colon ade-
nocarcinoma and supplemented with 10% fetal bovine serum
(FBS), and 1% penicillin-streptomycin solution (10,000 units
of penicillin and 10 mg of streptomycin in 0.9 % NaCl) in a
humidified atmosphere of 5% CO2, 95% air at 37 ºC.
5.2 Cell Proliferation Assay
Cell proliferation assay of compounds was evaluated in
HT-29 and CCRF-CEM cells, and was compared with that of
doxorubicin (DOX). Cell proliferation assay was carried out
using CellTiter 96 aqueous one solution cell proliferation
assay kit (Promega, USA). Briefly, upon reaching about 75-
80% confluency, 5000 cells/well were plated in 96-well mi-
croplate in 100 ꢀL media. After seeding for 72 h, the cells
were treated with 50 ꢀM compound in triplicate. DOX (10
ꢀM) was used as the positive control. Incubation was carried
out at 37 ºC in an incubator supplied with 5% CO2 for 72 h.
At the end of the sample exposure period (72 h), 20 ꢀL
CellTiter 96 aqueous solution was added. The plate was re-
turned to the incubator for 1 h in a humidified atmosphere at
37 °C. The absorbance of the formazan product was meas-
ured at 490 nm using microplate reader. The blank control
was recorded by measuring the absorbance at 490 nm with
wells containing medium mixed with CellTiter 96 aqueous
solution but no cells. Results were expressed as the percent-
age of the control (DMSO without compound set at 100%).
6. Molecular Modeling
Simulations were performed with the Accelrys Discovery
Studio 2.5 modeling package, with the CHARMm-based
force field [34]. Model of PP1 bound to Src was constructed
based on the X-ray crystal structures of PP1 bound to Hck
(1QCF) and AMP-PNP bound to c-Src (2SRC) templates
from RCSB Protein Data Bank. The coordinates and positi-
ons of the backbone atoms of PP1 was superimposed on the
corresponding atoms in AMP-PNP after which Hck was de-
leted. For refinement, the PP1-Src complex underwent
4. Selectivity Assay of 4c against Csk, EGFR, and Lck
The inhibitory activity of compound 4c was determined
against Csk, EGFR, and Lck according to the KinaseProfiler

4-Aryl-4H-Chromene-3-Carbonitrile Derivatives
Medicinal Chemistry, 2011, Vol. 7, No. 5 469
Aryl
CN
CN
Piperidine
EtOH
Aryl CHO
+
+
CN
O
Me2N
OH
Me2N
NH2
2
3
4a-w
1
Fig. (4). One-pot synthesis of 4-aryl-4H-chromenes 4a-w.
CHARMm minimization. All default parameters were used
in the minimization process.The complex first underwent 50
steps of steepest descent and 150 steps of conjugate gradient
minimization. Next, the complex was equilibrated briefly
with 200 steps molecular dynamics each at 300 K. The final
structure was then minimized with 300 steps with the conju-
gate gradient algorithm. For the molecular modeling of 4c,
after initial minimization, the coordinates and positions of
the backbone atoms of 4-(2-chlorophenyl) group was supe-
rimposed on the corresponding atoms in PP1 in complex
with c-Src after which PP1 was deleted. The minimization
was carried out as described above.
Table 1. Inhibition of Src Kinase Activity by 2-amino-7-
dimethylamino-4H-chromene-3-carbonitrile Deriva-
tives
Ar
CN
O
Me2N
NH2
Compounds
Ar
IC50 (ꢀM)a
4a
4b
4c
4d
4e
4f
3-F-Ph
4-F-Ph
21.1
37.1
11.1
32.3
>150
37.0
31.1
18.3
13.0
26.1
18.2
23.9
44.6
45.7
>150
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
3-Br-Ph
2-NO2-Ph
3-NO2-Ph
3-CF3-Ph
4-CF3-Ph
RESULTS AND DISCUSSION
Chemistry
An array of 23 diversely 4-aryl substituted derivatives of
2-amino-7-dimethylamino-4H-chromene-3-carbonitrile were
synthesized and evaluated against Src kinase. The com-
pounds were synthesized according to the previously re-
ported general procedure by a one-pot reaction [23,24,33]
through the condensation of a substituted aromatic aldehyde
(1), malonitrile (2), and 3-(dimethylamino) phenol (3) in
ethanol in the presence of piperidine (Fig. 4).
4g
4h
4i
4j
4k
4l
2,3-di(Cl)-Ph
2,6-di(Cl)-Ph,
2-MeO-Ph
4m
4n
4o
2,3-di(MeO)-Ph
2,5-di(MeO)-Ph
Src Kinase Inhibitory Activity
The results of Src kinase inhibitory activity of com-
pounds 4a-w are shown in Table 1. Compounds 4c, 4h, 4i,
and 4k exhibited higher Src inhibitory activity (IC50 =11.1–
18.3 ꢀM) when compared with other compounds. 2-
Chlorophenyl substituted analog 4c showed an IC50 value of
11.1 ꢀM and was the most potent compound in this series. In
comparison, 3-chlorophenyl substituted analog 4d demon-
strated 3-fold decrease in inhibitory potency when compared
to 4c. It is noteworthy that movement of chlorine atom to
position 4 of phenyl ring in compound 4e led to loss of activ-
ity (IC50 >150 ꢀM). Similarly 4-phenylsubstituted analogs 4b
(Ar = 4-F- Ph), 4j (Ar = 4-CF3-Ph), and 4p (Ar = 2,3,4-
tri(MeO)-Ph) showed less inhibitory potency when com-
pared with the corresponding 2- or 3-phenylsubstituted ana-
logs 4a (Ar = 3-F-Ph), 4i (Ar = 3-CF3-Ph), and 4n (Ar = 2,3-
di(MeO)-Ph), respectively. Other analogs with chlorophenyl-
thiazolyl bulky groups (4u-w) did not show any significant
inhibitory activity (IC50 > 300 ꢀM) against Src. These data
suggest that incorporation of bulky phenylsubstituted thia-
zole groups or 4-aryl groups with substitution at position 4 is
less tolerated in the ATP-binding site cavity of the Src kinase
domain.
4p
4q
4r
2,3,4-tri(MeO)-Ph
2,4-diMe-Ph
>300
>150
28.8
4-bi-Ph
N
4s
32.7
OMe
O
4t
30.4
O
S
4u
>300
N
Cl
S
4v
>300
>300
N
Cl
Cl
S
4w
N
Staurosporine
PP2
0.3
2.8
-
-
2-Chloro- (4c), 2,3-dichloro- (4k), and 2,6-dichloro- (4l)
substituted analogs with IC50 values of 11.1-23.9 ꢀM con-
taining an electron withdrawing group showed significantly
aThe concentration that inhibited enzyme activity by 50%.

470 Medicinal Chemistry, 2011, Vol. 7, No. 5
Fallah-Tafti et al.
(4n, IC50 = 45.7 μM), and 2,5-dimethoxy (4o, IC50 > 150
μM) containing an electron donating methoxy group. Other
compounds containing fluorine (4a, 4b), nitro (4g, 4h) and
trifluoromethyl (4i) were moderate inhibitors, suggesting
that the presence of electron withdrawing groups is preferred
for optimal Src kinase inhibitory activity in these analogs.
The 2-biphenyl (4r), isoquinoline analog (4s), and benzodi-
oxolyl analog (4t) exhibited moderate to week inhibitory
activity (IC50 =28.8–30.4 ꢀM).
Compound 4c (IC50 = 11.1 ꢀM) was selected for inhibi-
tory selectivity assays against EGFR, a receptor tyrosine
kinase, Csk, a tyrosine kinase that phosphorylates Src, and
Lck, a member of Src family kinase. IC50 values were >300
ꢀM, 101.7 μM, and 46.8 μM for EGFR, Csk, and Lck, re-
spectively. These data suggest that compound 4c was rela-
tively selective against Src when compared with the selected
kinases (EGFR > Csk > Lck).
Fig. (5). Structural complex of Src kinase with compound 4c based
on molecular modeling. Compound 4c and side chains of amino
acids are rendered in stick styles. Compound 4c is in the lowest
energy conformer predicted. The carbon skeleton of compound 4c
and side chains are gray, oxygen atoms are red, nitrogens are blue,
and the chlorine atom is green. The Figure is drawn using the Ac-
celrys visualization system.
Molecular modeling and minimization was used to ex-
plore how 4c would fit within the ATP-binding site of the
enzyme (Fig. 5). The modeling studies indicated that 2-
chlorophenyl group in 4c occupies the hydrophobic binding
pocket similar to tolyl group of PP1 with slightly different
orientations. The 2-cholorophenyl group contributed to Src
kinase inhibitory activity possibly through hydrophobic in-
higher inhibitory potency when compared with the 2-
methoxysubstituted (4m, IC50 = 44.6 μM), 2,3-dimethoxy
(a)
140
120
100
80
60
40
20
0
(b)
140
120
100
80
60
40
20
0
Fig. (6). Inhibition of HT-29 (a) and CCRF (b) cell proliferation by compounds 4a-w (50 ꢀM) after 72 h incubation. The results are shown
as the percentage of the control DMSO that has no compound (set at 100%). All the experiments were performed in triplicate.

4-Aryl-4H-Chromene-3-Carbonitrile Derivatives
Medicinal Chemistry, 2011, Vol. 7, No. 5 471
teractions with a large hydrophobic pocket in the ATP bind-
ing site. The chromene core mimics the adenine base of ATP
and is oriented towards the cavity formed from side chains of
helix ꢀC and helix ꢀD. This cavity is normally occupied by
carbohydrate moiety followed by triphosphate group of ATP.
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CONCLUSION
A series of 4-aryl substituted derivatives of 2-amino-7-
dimethylamino-4H-chromene-3-carbonitrile were evaluated
for inhibition of Src kinase and cell proliferation in human
colon adenocarcinoma (HT-29) and leukemia (CCRF-CEM)
cell lines. In summary, structure-activity relationship studies
revealed that the incorporation of less bulkier electron with-
drawing groups such as chloro, nitro, and trifluoromethyl at
positions 2 or 3 of 4-aryl is preferred and well tolerated
compared to other groups in generating Src inhibitory activ-
ity. The data provide insights for key structural requirements
for further optimization of chromene-carbonitrile derivatives
as a scaffold and generating more potent and selective Src
kinase inhibitors and/or anticancer agents.
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ACKNOLEDGEMENTS
This work was supported by grants from the American
Cancer Society Grant # RSG-07-290-01-CDD, National Sci-
ence Foundation, Grant Number CHE 0748555, and Re-
search Council of Tehran University of Medical Sciences
and Iran National Science Foundation (INSF).
[16]
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We also acknowledge National Center for Research Re-
sources, NIH, and Grant Number 1 P20 RR16457 for spon-
soring the core facility.

472 Medicinal Chemistry, 2011, Vol. 7, No. 5
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Received: December 16, 2010
Revised: January 21, 2011
Accepted: January 26, 2011