New Coumarin Derivatives as Anti-Breast and Anti-Cervical Cancer Agents Targeting VEGFR-2 and p38α MAPK

Article abstract of DOI:10.1002/ardp.201700064

Breast and cervical cancers are the most common gender-specific cancers affecting women worldwide. In this investigation, we highlighted the synthesis, VEGFR-2 and p38α MAPK inhibitory activity of new series of fluorinated coumarin-based derivatives featuring a variety of bioactive chemical moieties attached or fused to the coumarin nucleus at the 3 and/or 4 position. The bioactive inhibitors were further assessed for their anti-proliferative effect against human MCF-7 breast cancer and HeLa cervical cancer cell lines, respectively. Most of the tested compounds showed potent preferential inhibition effects against human VEGFR-2 and remarkable anticancer activities in the human breast cancer cell line MCF-7. Compounds 29, 24, and 2 displayed the highest inhibitory activity against VEGFR-2 (94% inhibition) and they were the most potent anticancer agents toward MCF-7 cancer cells with IC₅₀ values of 7.90, 8.28, and 8.30 μg/mL, respectively. Compound 13 inhibited p38α MAPK phosphorylation with a significant reduction in % cell viability against HeLa cancer cells at 10 and 30 μM. Docking experiments carried out on VEGFR-2 and p38 MAPK crystallographic structures revealed that the active compounds bind to the active sites through H-bonds, arene–cation, and hydrophobic π–π interactions. QSAR analysis demonstrated considerable correlation coefficient (R² = 0.76969) and root mean square error (RMSE = 0.10446) values. Also, the residual values between the experimental pIC₅₀ and predicted pIC₅₀ are very close, indicating the reliability of the established QSAR model.

Full text of DOI:10.1002/ardp.201700064

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Full Paper
New Coumarin Derivatives as Anti-Breast and Anti-Cervical Cancer
Agents Targeting VEGFR-2 and p38a MAPK
1
2
3
4
5
Rasha Z. Batran
, Dina H. Dawood , Samia A. El-Seginy , Mamdouh M. Ali , Timothy J. Maher ,
5
5
Kuljeet S. Gugnani , and Alejandro N. Rondon-Ortiz
1
Department of Chemistry of Natural Compounds, Division of Pharmaceutical Industries Research,
National Research Center, Dokki, Giza, Egypt
Department of Chemistry of Natural and Microbial Products, Division of Pharmaceutical Industries
2
Research, National Research Center, Dokki, Giza, Egypt
Department of Green Chemistry, Division of Chemical Industries Research, National Research Center,
3
Dokki, Giza, Egypt
4
Department of Biochemistry, Division of Genetic Engineering and Biotechnology Research, National
Research Center, Dokki, Giza, Egypt
Department of Pharmaceutical Sciences, Massachusetts College of Pharmacy and Health Sciences,
5
Boston, MA, USA
Breast and cervical cancers are the most common gender-specific cancers affecting women worldwide. In this investigation,
we highlighted the synthesis, VEGFR-2 and p38a MAPK inhibitory activity of new series of fluorinated coumarin-based
derivatives featuring a variety of bioactive chemical moieties attached or fused to the coumarin nucleus at the 3 and/or 4
position. The bioactive inhibitors were further assessed for their anti-proliferative effect against human MCF-7 breast cancer
and HeLa cervical cancer cell lines, respectively. Most of the tested compounds showed potent preferential inhibition effects
against human VEGFR-2 and remarkable anticancer activities in the human breast cancer cell line MCF-7. Compounds 29, 24,
and 2 displayed the highest inhibitory activity against VEGFR-2 (94% inhibition) and they were the most potent anticancer
agents toward MCF-7 cancer cells with IC50 values of 7.90, 8.28, and 8.30 mg/mL, respectively. Compound 13 inhibited p38a
MAPK phosphorylation with a significant reduction in % cell viability against HeLa cancer cells at 10 and 30 mM. Docking
experiments carried out on VEGFR-2 and p38 MAPK crystallographic structures revealed that the active compounds bind to
the active sites through H-bonds, arene–cation, and hydrophobic p–p interactions. QSAR analysis demonstrated considerable
2
correlation coefficient (R ¼ 0.76969) and root mean square error (RMSE ¼ 0.10446) values. Also, the residual values between
the experimental pIC50 and predicted pIC50 are very close, indicating the reliability of the established QSAR model.
Keywords: Breast cancer / Cervical cancer / Fluorinated coumarins / Molecular modeling / p38a MAPK /
QSAR / VEGFR-2
Received: February 19, 2017; Revised: July 2, 2017; Accepted: July 4, 2017
DOI 10.1002/ardp.201700064
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Correspondence: Dr. Rasha Z. Batran, Department of Chemistry of Natural Compounds, Division of Pharmaceutical Industries Research,
National Research Center, 33 El Bohouth St. (Former El Tahrir St.), Dokki, Giza, P.O. Box 12622, Egypt.
E-mail: rasha_batran@yahoo.com
Fax: þ20 23 337-0931
Abbreviations: NF-kB, nuclear factor-immunoglobulin kappa light chain of activated B cells; uPA, urokinase plasminogen activator; MTS,
3
-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MOE, Molecular Operating Environment;
RCSB, The Research Collaboratory for Structural Bioinformatics; H-bond, hydrogen bonding; RMSD, root mean squared deviation;
DTT, dithiothreitol; BSA, bovin serum albumin; EDTA, ethylenediaminetetraacetic acid; HEPES, 4-(2-Hydroxyethyl)-1-piperazineetha-
0
0
nesulfonic acid; EMEM, Eagle’s minimum essential medium; SRB, sulforhodamine B; HRP, horseradish peroxidase; TMB, 3,3 ,5,5 ,-
Tetramethylbenzidine; MMFF, Merck molecular force field.
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Introduction
Blockade of MAPK signaling pathway was confirmed as a
promising strategy for the treatment of cervical cancer [10]. It
has been recently reported that inhibiting phosphorylation of
p38 MAPK disrupts the nuclear translocation of NF-kB and
reduces uPA expression, leading to suppression of migration
and invasion of human cervical cancer cells [11].
The identification and development of new anticancer
therapies with considerable activity and minimum toxicity is
still an area of significance in the anticancer research.
Recently, natural product-derived agents gained considerable
attention due to their broad spectrum of antitumor activ-
ity [12, 13]. Coumarin is a versatile nucleus of a wide variety of
naturally occurring compounds that displays various bio-
chemical and pharmacological activities [14–16]. It has been
considered as an interesting scaffold for anticancer drug
discovery [17–19]. Moreover, coumarins expressed significant
inhibitory effects against VEGFR-2 [20, 21] and proved to be
potential inhibitors of the phosphorylation of p38a MAPK [22,
23] (Fig. 1).
Cancer is a major leading cause of mortality in developed and
developing countries [1]. Breast and cervical cancers are
considered the most common gender-specific cancer repre-
senting about 33.1% of all cancers that affect women [2].
Breast and cervical cancer early detection programs provided
a service for the treatment of hundreds of thousands of
women diagnosed with malignant or pre-malignant breast
and/or cervical cancer [3]. However, the incidence and
mortality rates are still high [4, 5].
It was investigated that invasive breast cancer expresses
several angiogenic factors throughout all tumor stages [6],
among them VEGF signaling pathway was demonstrated to
play a potential role in the regulation of breast tumor
angiogenesis through vascular endothelial growth factor
receptor-2 (VEGFR-2), which was found to be extensively
expressed in invasive primary and metastatic breast carcino-
mas [7]. In addition to inhibition of angiogenesis, VEGFR-2
inhibitors retarded the resistance of selective estrogen
receptor modulators (SERMs) in breast carcinoma through
inhibition of Ras/MAPK pathway [8].
On the other hand, a great deal of literature data on the
importance of fluoro compounds as a potential chemother-
apy consider them crucial for modulating anticancer ef-
fect [24]. So far, the aforementioned information encouraged
us to assay a large array of fluorinated coumarin-based
derivatives incorporated with different bioactive nitrogen
containing heterocyclic moieties known to have anticancer
On the other hand, protein expression of p38 mitogen-
activated protein kinase (MAPK) was recognized in cervical
cancer cell lines as HeLa, HT-3, and CaSki, which suggests that
p38 MAPK may play a key role in cervical carcinogenesis [9].
Figure 1. Examples of coumarin compounds as VEGFR-2 and p38a MAPK inhibitors.
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effects as Schiff’s bases [25], pyrazoles [26], thiazolidi-
nones [27], triazoles [28], pyridines [29], and pyrimidines [30],
hoping to add some synergistic pharmacological significance
to the target molecules in order to evaluate their anti-breast
cancer effect targeting VEGFR-2 and anti-cervical cancer
effect targeting p38a MAPK. Molecular docking studies on
VEGFR-2 and p38a MAPK crystallographic structures were
conducted in order to analyze the structural requirements for
the biological activity and explain the interactions between
the synthesized compounds and the target proteins. The
structural analyses of these compounds were done using
QSAR techniques to elucidate the essential features of the
target molecules responsible for the biological activity.
compound 9 [33], which was further condensed with phenyl
isothiocyanate in refluxing pyridine to afford N-phenyl pyrazo-
line carbothioamide derivative 10, while the synthesis of
pyrazolo-indoline compound 11 was achieved through reflux-
ing the pyrazoline derivative 9 with isatin in absolute ethanol
(Scheme 2).
The 2-aminopyridin-3-carbonitrile compound 12 was pre-
pared by the condensation of chalcone compound 3 with
malononitrile in the presence of ammonium acetate in
refluxing ethanol (Scheme 3). The pyrimidin-2-thione 13
and 2-aminopyrimidine derivative 14 were obtained by
cyclocondensation of the chalcone with thiourea and guani-
dine hydrochloride, respectively, in the presence of potassium
hydroxide solution (Scheme 3).
A convenient approach for the preparation of the target 4-
hydroxycoumarinyl-3-thiazolidinone compound 18 is de-
scribed in Scheme 4. The key reactions involved the reduction
of 3-nitro-4-hydroxycoumarin 15 [34] into 3-amino derivative
16 [35] according to the reported procedures, which was
further reacted with 4-fluorobenzaldehyde to afford the
Schiff’s base 17. The thiazolidinone derivative 18 was
obtained through the cyclocondensation of the Schiff’s base
17 with mercaptoacetic acid (Scheme 4).
A facile synthesis of the key intermediate hydrazide 20 [36]
involves the hydrazinolysis reaction of ester 19 [36] and
hydrazine hydrate in absolute ethanol at room temperature.
Furthermore, the Schiff’s base 21 [37] was synthesized by the
reaction of hydrazide intermediate 20 with 4-fluorobenzal-
dehyde. Heterocyclization of the Schiff’s base 21 with
mercaptoacetic acid afforded the corresponding thiazolidi-
none derivative 22 (Scheme 5).
Results and discussion
Chemistry
Condensation of 3-acetyl-4-hydroxycoumarin 1 [31] with 4-
fluoroaniline in absolute ethyl alcohol using a catalytic amount
of glacial acetic acid afforded the corresponding Schiff’s base 2
(
Scheme 1). The chalcone derivative 3 [32] was synthesized
through a Claisen–Schmidt condensation reaction of the
precursor 3-acetyl-4-hydroxycoumarin 1 and 4-fluorobenzal-
dehyde according to the reported procedures (Scheme 1).
Cyclo-addition reaction of hydrazine derivatives with the
intermediate chalcone 3 furnished the corresponding pyrazo-
lines 4–9. Heterocyclization of chalcone 3 with methyl hydra-
zine and phenylhydrazine in ethyl alcohol gave the
corresponding N-methylpyrazoline 4 and N-phenylpyrazoline
6
[33] derivatives, respectively. Refluxing ter. butylhydrazine
hydrochloride with chalcone in ethyl alcohol in the presence of
KOH afforded the corresponding N-tert. butylpyrazoline 5.
N-Acetylpyrazoline 7 was prepared by condensing the key
chalcone compound with hydrazine hydrate in glacial acetic
acid. The target pyrazoline carbothioamide 8 was obtained by
refluxing chalcone and thiosemicarbazide in pyridine. Reaction
of chalcone 3 with hydrazine hydrate afforded the pyrazoline
Three-component one-pot reaction of 4-hydroxycoumarin,
4-fluorobenzaldehyde, and malononitrile in refluxing abso-
lute ethanol in the presence of piperidine afforded the
2-amino-3-cyanopyranocoumarin 23 [38] that was reacted
with trimethylorthoformate or triethylorthoformate to yield
the corresponding tricyclic methoxymethylene amino or
ethoxymethylene amino derivatives 24 and 25, respectively.
Scheme 1. Synthesis of compounds 2 and 3. Reagents and conditions: (i) POCl
iii) 4-F-PhCHO/piperidine/EtOH/reflux.
3
/AcOH/reflux; (ii) 4-F-PhNH
2
/EtOH/AcOH/reflux;
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Scheme 2. Synthesis of compounds 10 and 11. Reagents and conditions: (i) CH
3
NHNH
CSNHNH
2
/EtOH/reflux; (ii) C(CH
3
)
3
NHNH
NH
2
ꢀHCl/EtOH/
KOH/reflux; (iii) PhNHNH
2
/EtOH/reflux; (iv) NH
2
NH
2
ꢀH
2
O/AcOH/reflux; (v) NH
2
2
/pyridine/reflux; (vi) NH
2
2
ꢀH O/EtOH/
2
reflux; (vii) phenylisothiocyanate/pyridine/reflux; (viii) isatin/EtOH/reflux.
Cyclization of compound 25 with hydrazine hydrate gave the
corresponding tetracyclic pyranopyrimidine derivative 26.
Compound 26 was used as a key intermediate for the
preparation of the target pentacyclic triazolopyrimidine
derivatives 27–29 as described in Scheme 6. Reaction of 26
with triethylorthoformate yielded the corresponding
triazolo[1,5-c]pyrimidine 27. The 2-methyl triazolopyrimidine
derivative 28 was afforded by refluxing 26 in acetic anhydride.
The reaction of compound 26 with ethyl chloroformate gave
the corresponding triazolone derivative 29 (Scheme 6).The
proposed structures of all new prepared compounds were
1
13
confirmed by elemental analysis, IR, H NMR, C NMR, and
mass spectra.
Biology
Inhibition of VEGFR-2 kinase
The inhibitory effect of the tested compounds on VEGFR-2 as a
marker for angiogenesis was determined as triplicate
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2 2 3 4 2 2
Scheme 3. Synthesis of compounds 12–14. Reagents and conditions: (i) CH (CN) /CH COONH /EtOH/reflux; (ii) NH CSNH /KOH/EtOH/
reflux; (iii) NH
2
C(NH)NH
2
ꢀHCl/KOH/EtOH/reflux.
determinations and the data are presented in Table 1. The
results showed that most of the tested compounds displayed
strong-to-moderate inhibition against human VEGFR-2 in
human breast cancer cell line MCF-7 as compared to the
untreated cancer cells. The Schiff’s base 2, tricyclic pyrano-
coumarinyl-formimidic acid methyl ester 24, and pentacyclic
triazolopyrimidinylpyranocoumarin 29 were found to be the
most potent derivatives. They were as potent as the positive
drug, tamoxifen (95%) against human VEGFR-2 with percent-
age of inhibition values of 94%. On the other hand,
oxothiazolidinyl acetamide 22, pyrazolyl indolin-2-one 11,
N-phenylpyrazole-1-carbothioamide 10, methyltriazolpyrimi-
dine 28, and 2-amino-coumarinyl pyridine-3-carbonitrile 12
revealed moderate effects against VEGFR-2 with percentage of
inhibition values of 58, 50, 47, 40, and 39%, respectively. The
rest of compounds showed weak activity against VEGFR-2.
the phosphorylation site (Thr180/Tyr182) to the non-
phosphorylated p38 must be evaluated. For a compound to
show MAPK inhibition, it should have a lower anti-phospho
p38 MAPK value than the anti-p38 MAPK value; where anti-
phospho p38 MAPK alpha (Thr180/Tyr182) represents the
extent of MAPK phosphorylation; (Thr180/Tyr182) being the
site where phosphorylation takes place, and anti-p38 MAPK
represents the non-phosphorylated protein, which is identi-
fied by antibodies directed against nonphosphorylated p38
MAPK (Table 2).
2-Thioxopyrimidine compound 13 at a concentration of
10 mM was statistically responsible of preventing p38 MAPK
phosphorylation in the presence of a known MAPK stimulator
(30 mM anisomycin) when compared with the stimulator alone
(p38 MAPK activation fold change ¼ 0.64 ꢁ 0.2). Figure 2
illustrates the effect of compound 13 on p38 MAPK activation
at different concentrations where a significant reduction in
p38a MAPK phosphorylation, as compared to the stimulation
caused by 30 mM anisomycin, was observed at 10 mM and
30 mM.
Inhibition of p38a MAPK phosphorylation
In this study, a screening assay was designed in order to assess
the inhibitory activity of test compounds on MAPK activation.
HeLa cells were subjected to MAPK stimulation using 30 mM
anisomycin. Upon activation, p38 MAPK is phosphorylated on
Thr180 and Thr182. After cell membrane lysis, phospho p38
MAPK could be detected using kit reagents. The assay involves
two monoclonal antibodies, the anti-phospho p38 MAPK and
anti-p38 MAPK antibodies. In order to evaluate the MAPK
inhibitory activity, the ratio of phosphorylated p38 protein at
Anti-breast cancer effect of the target compounds
The anticancer effect of the tested target compounds against
breast MCF-7 cancer cells was evaluated and expressed by
median growth inhibitory concentration (IC50). As shown in
Table 1, the pentacyclic triazolopyrimidinylpyranocoumarin
29, tricyclic pyranocoumarinyl-formimidic acid methyl ester
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3 2 4 4
Scheme 4. Synthesis of compound 18. Reagents and conditions: (i) HNO /H SO /heat; (ii) Pd/HCOONH /MeOH/RT; (iii) 4-F-PhCHO/
EtOH/AcOH/reflux; (iv) HSCH COOH/benzene/reflux.
2
2
4, and the Schiff’s base 2 displayed equipotent anticancer
27 (IC50 ¼ 19.80 ꢁ 2.20 mg/mL) were found to be slightly
potent anticancer agents. N-Methylpyrazoline 4, N-tert-butylpyr-
azoline 5, 2-thioxopyrimidine 13, 2-amino-pyrimidine 14, and
ethoxymethylene amino derivative 25 had no effect on the
cancer cells. These results were consistent with VEGFR-2
activity.
effects against breast MCF-7 cells as the reference drug
tamoxifen with IC50 values of 7.90 ꢁ 0.88, 8.28 ꢁ 0.91, and
8
.30 ꢁ 0.90 mg/mL, respectively, versus 8.38 ꢁ 0.93 mg/mL for
tamoxifen. The oxothiazolidinyl acetamide 22, pyrazolyl
indolin-2-one 11, methyltriazolpyrimidine 28, N-phenylpyr-
azole-1-carbothioamide 10, and 2-aminocoumarinylpyridine-
3
-carbonitrile 12 showed moderate potency with IC50 values
of 9.90 ꢁ 1.20, 10.00 ꢁ 1.45, 11.00 ꢁ 1.37, 11.85 ꢁ 2.10, and
1.90 ꢁ 2.10 mg/mL, respectively. The pyrazolines 6, 7, and 8
Anti-cervical cancer effect of the target compounds
Effect of different concentrations of the p38a MAPK inhibitor
13 on HeLa cell viability was evaluated using MTS assay. The
thioxopyrimidine compound 13 displayed significant reduc-
tion in cell viability against HeLa cells at 10 mM (80%) and
30 mM (72%) (Fig. 3).
1
(
IC50 ¼ 31.20 ꢁ 3.40, 28.70 ꢁ 3.37, and 18.90 ꢁ 2.15 mg/mL,
respectively), the Schiff’s base 17 and its cyclized thiazolidi-
none 18 (IC50 ¼ 21.10 ꢁ 2.50 and 17.60 ꢁ 2.00 mg/mL), the pyrano-
pyrimidine 26 (IC50 ¼ 27.30 ꢁ 3.20 mg/mL), and triazolopyrimidine
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Scheme 5. Synthesis of compound 22. Reagents and conditions: (i) BrCH
RT, (iii) 4-F-PhCHO/EtOH/AcOH/reflux; (iv) HSCH COOH/benzene/ reflux.
2
2
COOCH
2
CH
3
/K
2
CO
3
/acetone/reflux; (ii) NH
2
NH
2
2
ꢀH O, EtOH/
Structure–activity relationship
activity (VEGFR-2inh% ¼ 9% and 15%, respectively). The activity
was completely lost for the N-methyl and N-tert-butylpyrazo-
line compounds 4 and 5. The N-indolylpyrazole compound 11
showedpotentanti-breastcancereffect(IC50 ¼ 10.00 ꢁ 1.45 mg/
mL) and 50% VEGFR-2 inhibition activity.
Anti-breast cancer effect targeting VEGFR-2
In the series of open chain Schiff’s base derivatives, the Schiff’s
base 3-(4-fluorophenylimino)ethyl)-4-hydroxycoumarin 2 was
as potent anti-breast cancer agent as the standard tamoxifen
with IC50 ¼ 8.30 ꢁ 0.90 mg/mL and VEGFR-2 inhibition effect
The importance of the thiazolidinone nucleus in improv-
ing the anticancer effect was clearly observed for the
thiazolidinone derivatives 22 and 18. The 4-oxycoumarinyl-
thiazolidinone acetamide 22 displayed potent anti-breast
cancer effect comparable to the reference standard, with
IC50 ¼ 9.90 ꢁ 1.20 mg/mL, and significant VEGFR-2 inhibitory
activity (VEGFR-2inh% ¼ 58%). Compound 18 showed remark-
able anti-breast cancer potency with moderate VEGFR-2
inhibition activity (IC50 ¼ 17.60 ꢁ 2.00 mg/mL and VEGFR-2inh
¼
94%. While the activity significantly decreased for the
Schiff’s base 3-(4-fluorobenzylidene-amino)-4-hydroxycou-
marin 17 (IC50 ¼ 21.10 ꢁ 2.50 mg/mL, VEGFR-2inh% ¼ 24%).
The N-phenylpyrazolinyl-carbothioamide compound 10 was
the most active anti-breast cancer derivative within the
synthesized pyrazoline series with IC50 ¼ 11.85 ꢁ 2.10 mg/mL
and VEGFR-2 inhibition¼ 47%. It was more active than the
N-unsubstituted pyrazolinyl-carbothioamide compound
8
(
IC50 ¼ 18.90ꢁ 2.15mg/mL, VEGFR-2inh% ¼ 24%). Both N-acetyl
%
¼ 33%). It is worthy to note that compound 22 with a
and N-phenylpyrazolines 6 and 7 displayed equipotent moder-
ate anti-breast cancer activity (IC50 ¼ 31.20 ꢁ 3.40mg/mL and
thiazolidinone moiety attached to the 4-oxycoumarin
scaffold through an acetamide linker was about twofolds
more active than compound 18, with a free 4-OH group and
2
8.70 ꢁ 3.37 mg/mL, respectively) and weak VEGFR-2 inhibition
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Scheme 6. Synthesis of compounds 27–29. Reagents and conditions: (i) 4-F-PhCHO/CH
2
(CN)
2
/EtOH/piperidine/reflux; (ii) CH(OR)
, benzene/reflux.
3
/
(CH
3
CO)
2
O/reflux; (iii) NH
2
NH
2
ꢀH
2
O/EtOH/RT; (iv) CH(OC
H
2 5
)
3
/DMF/reflux; (v) (CH
3
CO)
2
O/reflux; (vi) ClCOOC H
2 5
a thiazolidinone moiety directly attached to C-3 of the
coumarin scaffold.
pentacyclic triazole derivatives 27–29 significantly improved
theanticancereffect. Thetriazolone derivative 29was the most
potent anti-breast cancer compound of all the synthesized
compounds being as active as the standard tamoxifen with IC50
value of7.90 ꢁ 0.88 mg/mLand 94%VEGFR-2inhibitionactivity.
It displayed higher potency than the 2-methyltriazolopyrimi-
dine 28 (IC50 ¼ 11.00 ꢁ 1.37 mg/mL, VEGFR-2inh% ¼ 40%)
and the unsubstituted triazolopyrimidine compound 27
(IC50 ¼ 19.80ꢁ 2.20mg/mL, VEGFR-2inh% ¼ 25%).
Within the coumarin-C3-linked six-membered azahetero-
cycles, compound 12 bearing 2-aminopyridyl-3-carbonitrile
was the most active anti-breast cancer compound with
IC50 ¼ 11.90 ꢁ 2.10 mg/mL and anti-VEGFR-2 ¼ 39%. The anti-
breast cancer effect and VEGFR-2 inhibitory activity were
diminished for the 2-thioxopyrimidine and 2-aminopyrimi-
dine derivatives 13 and 14.
While the tricyclic methoxymethylene amino derivative 24
wasashighlypotentanti-breastcancercompoundandVEGFR-2
inhibitor as the reference tamoxifen (IC50 ¼ 8.28ꢁ 0.91mg/mL,
VEGFR-2inh% ¼ 94%),theethoxymethyleneaminoderivative25
completely lost any activity. The tetracyclic coumarinylpyrano-
pyrimidine 26 exhibited moderate anti-breast cancer effect
Anti-cervical effect targeting p38a MAPK
Among all the synthesized compounds, the thioxopyrimidine
derivative 13 displayed anti-p38a MAPK inhibiting effect
through inhibition of p38 MAPK phosphorylation (p38 MAPK
activation ¼ 0.64 ꢁ 0.2). The activity was diminished for the 2-
aminopyrimidine isostere 14. While the N-methylpyrazolinyl
compound 4 and the N-phenylpyrazolinyl-carbothioamide
(
IC50 ¼ 27.30ꢁ 3.20mg/mL) and decreased anti-VEGFR-2 activ-
ity (26%). Cyclization of the pyranopyrimidine 26 to the
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Table 1. Anti-breast cancer effect in MCF-7 (IC50) and VEGFR-2 kinase inhibitory activity (% inhibition) of the synthesized
compounds.
ꢂ
Compound
IC50 (mg/mL)
VEGFR-2 (ng/mL)
% Inhibition of VEGFR-2
Tamoxifen
DMSO
8.38 ꢁ 0.93
ꢃ
95.27 ꢁ 6.00
95
ꢃ
1950.00 ꢁ 210.00
122.00 ꢁ 13.60
2
4
5
6
7
8
8.30 ꢁ 0.90
ꢃ
94
4
1869.00 ꢁ 190.50
1920.70 ꢁ 211.00
1770.00 ꢁ 180.75
1650.00 ꢁ 180.30
1480.00 ꢁ 160.30
1080.00 ꢁ 125.00
980.00 ꢁ 105.00
1190.50 ꢁ 86.65
1860.70 ꢁ 190.10
1900.30 ꢁ 195.00
1490.90 ꢁ 160.75
1300.80 ꢁ 146.30
820.80 ꢁ 80.30
ꢃ
1.5
9
31.20 ꢁ 3.40
28.70 ꢁ 3.37
18.90 ꢁ 2.15
11.85 ꢁ 2.10
10.00 ꢁ 1.45
11.90 ꢁ 2.10
ꢃ
15
24
47
50
39
5
2.5
24
33
58
94
3
10
11
12
13
14
17
18
22
24
25
26
27
28
29
ꢃ
21.10 ꢁ 2.50
17.60 ꢁ 2.00
9.90 ꢁ 1.20
8.28 ꢁ 0.91
ꢃ
110.00 ꢁ 12.20
1885.00 ꢁ 197.00
1450.00 ꢁ 160.80
1470.00 ꢁ 170.60
1176.00 ꢁ 133.00
117.00 ꢁ 12.80
27.30 ꢁ 3.20
19.80 ꢁ 2.20
11.00 ꢁ 1.37
7.90 ꢁ 0.88
26
25
40
94
Data are expressed as mean ꢁ standard error (S.E.) of three independent experiments.
ꢂ
%
inhibition indicates percentage changes as compared with control cancer cells.
̊
̊
̊
compound 10 showed very weak inhibitory effect (p38 MAPK
activation ¼ 0.98 ꢁ 0.2 and 0.97 ꢁ 0.2, respectively), the other
synthesized compounds were completely inactive. A signifi-
cant reduction in % cell viability, using HeLa cells, was
observed for compound 13 at 10 mM and 30 mM concen-
trations (% cell viability ¼ 80% and 72%, respectively).
and Asp1046 with bond length of 2.86 A, 3.02 A, and 2.79 A,
respectively, in addition to hydrophobic interaction with the
hydrophobic amino acids Leu840, Val844, Ile888, Leu889, and
Phe1047. Also a closure contact with Gly922 and Lys920 was
also displayed. From docking results, compound 2 showed
two H-bonds with amino acid residues. OH of the chromene-2-
one moiety accepted H from NH of Lys868 and formed H-bond
̊
Molecular docking studies
Docking study against VEGFR-2 kinase
at bond length of 2.59 A. Carbonyl oxygen acted as H-acceptor
and formed H-bond with NH of Asp1046 at bond length of
2.78 A. The fluorophenyl moiety showed hydrophobic inter-
action with Phe1047. In addition, the chromene moiety
exhibited hydrophobic interaction with Ile888 and Leu889
(Fig. 5).
Compound 24 showed two H-bonds with amino acids of the
active site. Carbonyl oxygen of the chromen-2-one moiety
̊
To further validate our rational design, all the synthesized
compounds were docked against the kinase-binding site of
VEGFR-2. Compounds 2, 24, and 29 showed a significant
binding mode within the active site of VEGFR-2. Molecular
Operating Environment (MOE) software was used to conduct
the docking study [39]. The crystal structure of VEGFR-2
complex was retrieved from the RCSB Protein Data Bank (PDB;
̊
formed H-bond with NH of Thr916 at bond length of 2.91 A.
3
U6J) and used as a target for the molecular modeling
Nitrogen atom linked to pyrano moiety accepted H from NH
of Asp1046 and formed H-bond with bond length of 2.91 A
̊
analysis. In order to validate the MOE algorithm with VEGFR-2
crystal structure, the native ligand was re-docked with MOE,
the self-docking result showed that the re-docked ligand
showed nearly the same binding mode as the co-crystallized
ligand (Fig. 4). This result indicated the reliability of MOE
algorithm with 3U6J complex.
(Fig. 6). The chromene moiety showed arene–cation interac-
tion with Lys868 and showed hydrophobic interaction with
Leu889. The fluorophenyl exhibited hydrophobic interactions
with the four hydrophobic amino acids Val848, Val899,
Leu1035, and Phe1047.
The H-bond profiles of the three active compounds toward
active site of target protein are summarized in Table 3. The
native ligand showed three H-bonds with Lys868, Cyst919,
Compound 29 showed good binding mode within the
active site, through the formation of one H-bond, arene–
cation interaction, arene–arene interaction, and hydrophobic
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Table 2. p38a MAPK inhibition activity of the synthesized
compounds.
p38a MAPK activation
fold change vs. control)
(
(
anti-phospho p38 MAPK/
anti-p38 MAPK)
Compound
Control
1
3
0 mM anisomycin
positive control)
1.42 ꢁ 0.2
(
2
4
5
6
7
8
1
1
1
1
1
1
1
2
2
2
2
2
2
2
1.24 ꢁ 0.61
0.98 ꢁ 0.2
Figure 3. Effects of different concentrations of compound 13
ns
ns
ns
on cell viability were observed using MTS assay. Data are
ꢂ
represented as mean ꢁ SEM (n ¼ 3). Significant reduction in %
cell viability as compared to control (P ꢄ 0.001).
ns
0.97 ꢁ 0.2
ns
0
1
2
3
4
7
8
2
4
5
6
7
8
9
ns
Chromene moiety showed arene–arene interaction with
Phe1047. Also, the amino acids Asp1046 and Thr916 were
in close contact of compound 29 (Fig. 7).
a),
ꢂ
0.64 ꢁ 0.2
ns
1.12 ꢁ 0.35
It could be concluded that the three active compounds
showed similar interactions to the native ligand interactions.
The interaction of compounds 2, 24, and 29 with Asp1046 and
Lys_868 is the main reason of their promising inhibitory
activity. Compounds 2 and 24 formed H-bonds with Asp1046,
while compound 29 was in close contact of Asp1046. Also,
compounds 2 and 29 formed H-bonds with Lys868, while
compound 24 showed arene–cation interaction with Lys868.
In addition, the hydrophobic interaction of the fluorophenyl
and chromene moieties with conserved hydrophobic amino
acid residues was also displayed.
ns
ns
ns
ns
ns
1.13 ꢁ 0.64
1.07 ꢁ 0.53
1.18 ꢁ 0.52
Data are expressed as mean ꢁ standard error (S.E.) of three
independent experiments.
a)
Significant inhibition in MAPK activation as compared to
ꢂ
3
0 mM anisomycin. p ꢄ 0.05.
Docking study against p38 MAPK kinase
The promising activity of compound 13 against p38 MPAK
encouraged us to perform docking study to investigate the
binding mode of compound 13 against p38 MAPK protein
(PDB: 3FMK). The co-crystallized ligand showed H-bond with
Met109 in addition to hydrophobic interaction with Val30,
Lys53, and Leu167. Compound 13 was stabilized within the
active site of the target protein by forming H-bond through
C–O moiety which accepted H atom from NH of Met109 at
interaction with amino acid residues. Carbonyl oxygen
formed H-bond with NH of Lys868 at bond length of
̊
2
.26 A. The fluorophenyl moiety showed arene–cation inter-
action with Lys868 and hydrophobic interaction with Leu889.
Figure 2. Effect of compound 13 on p38a MAP activation at
ꢂ
different concentrations: Significant increase in p38a MAPK
ꢂꢂ
phosphorylation as compared to the control. Significant
Figure 4. Docked co-crystallized ligand (light purple, stick)
showed the same binding mode of the native ligand (green,
stick). The protein represented as cartoon (PDB:3U6J).
reduction in p38a MAPK phosphorylation as compared to the
stimulation caused by 30 mM anisomycin.
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Table 3. Docking results of the active compounds against VEGFR-2.
Main residue from the
̊
Compound no.
compound
Main residue from VEGFR-2
Distance (A)
2
OH
NH Lys_868
NH Asp_1046
NH Thr_916
NH Asp_1046
NH Lys_868
NH Lys_868
NH Cys_919
NH Asp_1046
2.59
2.78
2.91
2.91
2.26
2.86
3.02
2.79
C–O
C–O
2
2
4
9
N-pyran
C–O
Native ligand
C–O
N-pyridine
C–O
̊
distance 2.77 A (Fig. 8). Chromene moiety showed hydropho-
bic interaction with Val30 and the fluorophenyl moiety
showed hydrophobic interaction with Lys53 and Leu167. The
activity of compound 13 against p38 MAPK could be
attributed to its binding mode that is similar to the native
ligand.
(LUMO and HOMO). The correlation between biological
activity and physicochemical properties of the training set
was done by applying linear regressions protocol. The cross-
validation analysis was performed using leave-one-out (LOO)
method, in which one compound is removed from the data
set and the activity is correlated using the rest of the data
2
2
set [41]. Squared correlation coefficient R , predicted R
QSAR study
(cross-validation), root mean square error (RMSE), and
residuals between the predicted and experimental activity
of the test set and training set were used to validate QSAR
models.
Development of QSAR model
Quantitative structure–activity relationship (QSAR) is
a
technique used to investigate the correlation between the
physicochemical parameters of a set of molecules and their
biological activity. In this study, QSAR analysis was per-
formed for the anti-proliferative activity against MCF-7
cancer cell line. The analysis was run with MOE package. A set
of newly synthesized active compounds was used as training
set. The 15 compounds that showed anti-proliferative
activity against MCF-7 were selected as training set, while
inactive compounds were omitted from the analysis [40]. The
pIC50 was calculated from observed IC50 as pIC50 ¼ ꢃlogIC50. A
large number of 2D and 3D molecular descriptors were
calculated as well as the energies of the lowest unoccupied
molecular orbital and the highest occupied molecular orbital
QSAR study results
Several models were built using different descriptors, it was
found that the best molecular descriptors for the fifteen
active compounds are MW, logP (O/W), BCUT_SMR_3,
FCASAþ, and vsurf_EWmin1. Equation (1) represents the best
performing QSAR model;
pIC50 ¼ 0:01062ðMWÞ ꢃ 0:21913 logPðO=WÞ
ꢃ2:49710ðBCUTꢃSMRꢃ3Þ ꢃ 0:050432ðFCASAþÞ
þ0:05728ðvsurfꢃEWmin1Þ þ 9:61943
ð1Þ
where MW: molecular weight, logP (O/W): log (octanol/water)
partition coefficient, BCUT_SMR_3: Molar reactivity BCUT
(
3/3), FCASAþ: fractional charge-weighted positive surface
area, vsurf_EWmin1: lowest hydrophobic energy.
According to aforementioned equation, QSAR model was
represented graphically by scattering plots of the experimen-
tal pIC50 versus the predicted pIC50 for the training þþset
(
Fig. 9). The method used to build the model was partial least
2
squares method (PLS). The correlation coefficient R was
0
2
.76969, predicted R was 0.235055, RMSE was 0.10446, and
cross-validation RMSE was 0.20760. It was also found that
adding more molecular descriptors showed remarkable
increase in the correlation coefficient of the regression
equation. However, to avoid overfitting the QSAR model, it
is recommended not to use more than one molecular
descriptor for three to six observations [40]. The Z-score
was calculated for all the compounds and it was found that no
compound is an outlier (Table 4).
Figure 5. Compound 2 (yellow, stick) formed H-bonds with
Lys868 (blue) and Asp1046 (blue). The H-bond is represented as
red line. For clarity, interacted residues only displayed.
In conclusion, the anti-proliferative activity of the training
set against the MCF-7 cancer cell line correlated positively
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Figure 6. (A) Represented interactions of compound 24 (yellow, stick) with Asp1046 (blue). (B) Another view of compound 24
showing H-bond with Thr916 (blue). Protein represented as cartoon.
with MW and (vsurf_EWmin1) while it correlated negatively
with logP (O/W), (BCUT_SMR_3), and (FCASAþ).
Conclusion
A new series of fluorinated coumarin-based derivatives
featuring a variety of bioactive chemical moieties was
designed, synthesized, and screened for their kinase inhibi-
tion properties against VEGFR-2 and p38a MAPK and the
bioactive inhibitors were further assessed for their anti-
proliferative effect against human MCF-7 breast cancer and
HeLa cervical cell lines, respectively. Most of the tested
compounds showed preferential inhibition effect against
human VEGFR-2 and remarkable anticancer activities in
human breast cancer cell line MCF-7. The triazolopyrimidinyl
derivative 29, pyranocoumarinyl formimidic acid methyl ester
24, and the Schiff’s base 2 were found to be the most potent
QSAR cross-validation
Robustness of the created QSAR model was validated by using
LOO method. In our study, the established model was
2
validated with R , RMSE, and the residual between experi-
2
mental and predicted pIC50 (Table 4). R was 0.76969 and
RMSE was 0.10446, which is considered low error. The
2
2
predicted R was 0.235055; the predicted R is equivalent to
q from a LOO cross-validation. Interestingly, the residual
between the experimental and predicted pIC50 was very close,
indicating the reliability and effectiveness of the established
QSAR model (Table 4).
Figure 7. (A) 2D diagram of compound 29 represents arene–arene interaction between Phe1047 and chromene ring, arene–cation
interaction between Lys868 and fluorophenyl ring, and H-bond between C–O group and Lys868. (B) 3D view of compound 29
̊
(
yellow, stick) and its interaction with Lys868 at a distance of 2.26 A.
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Figure 8. Binding mode of compound 13 (green, stick) in the ATP-binding site of p38 MAPK. Compound 13 showed H-bond with
̊
Met109 (light blue, stick) at a distance of 2.77 A. H-bond is represented as a black line. The protein is represented as cartoon (PDB:
3
FMK).
VEGFR-2 inhibitors and displayed equipotent anticancer
effect against MCF-7 cells as tamoxifen. On the other hand,
the thioxopyrimidine derivative 13 displayed anti-p38a MAPK
inhibiting effect through inhibition of p38 MAPK phosphor-
ylation at 10 mM in the presence of 30 mM anisomycin when
compared with the stimulator alone. A significant reduction
in % cell viability against HeLa cells was observed for
compound 13 at 10 mM and 30 mM concentrations (% cell
viability ¼ 80% and 72%, respectively). The combined analysis
of molecular docking and QSAR provided significant infor-
mation on the structural features of the molecules that are
required for interactions and afforded a guide for the design
of novel lead compounds. The main interactions between
chromene derivatives and the pocket site were through
H-bond donor/acceptor, arene–cation, and hydrophobic p–p
interactions. On the other hand, the activity of compound 13
Figure 9. Correlation plot of experimental versus predicted pIC50 against MCF-7.
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Table 4. Experimental and predicted inhibitory activity of the synthesized compounds against MCF-7 by QSAR model.
Compound no.
Experimental pIC50
Predicted pIC50
Error
Z-score
2
6
7
8
4.5542
4.1080
4.1060
4.3072
4.5880
4.6564
4.4966
4.1279
4.3075
4.6219
4.6575
4.1369
4.2903
4.5611
4.7070
4.2089
4.1203
4.4434
4.6588
4.4474
4.5163
4.2042
4.2524
4.6328
4.5500
4.0953
4.4320
4.6447
4.6447
4.3751
ꢃ0.1029
ꢃ0.0123
ꢃ0.1362
ꢃ0.0708
0.2090
ꢃ0.0197
ꢃ0.0763
0.0551
0.9855
0.1181
1.3038
0.6778
2.0008
0.1891
0.7304
0.5278
0.1040
1.0288
0.3978
1.3566
0.8006
0.5961
1.7147
10
11
12
17
18
22
24
26
27
28
29
ꢃ0.0109
0.1075
0.0416
ꢃ0.1417
ꢃ0.0836
0.0623
0.1791
was related to H-bond formation with amino acids of ATP site
as well as the hydrophobic interactions of the fluorophenyl
ring and chromene moiety. Several QSAR models were
created using wide range of 2D and 3D descriptors. The
models were validated with R , RMSE, and the residual
between experimental and predicted pIC50. It was shown that
Synthesis of 3-[1-(4-fluorophenylimino)-ethyl]-4-hydroxy-
chromen-2-one (2)
A mixture of 3-acetyl-4-hydroxycoumarin 1 (1.02 g, 5 mmol),
p-fluoroaniline (0.48 mL, 5 mmol), and a catalytic amount of
acetic acid (1 mL) in absolute ethanol (20 mL) was refluxed for
8 h. The reaction mixture was cooled and the formed
precipitate was filtered off and re-crystallized from acetic
acid to give compound 2. Gray crystals, m.p. ¼ 140–142°C,
2
the best five descriptors of the selected model were MW, logP
(
O/W), BCUT_SMR_3, FCASAþ, and vsurf_EWmin1.
yield (82%). Anal. calcd. for C17
H
12FNO
3
(297.28): C, 68.68; H,
ꢃ1
4
.07; N, 4.71. Found: C, 68.84; H, 4.12; N, 4.79. IR (KBr, cm ):
1
Experimental
3432 (OH), 1711 (C–O), and 1611 (C–N). H NMR (DMSO-d
ppm): 2.59 (s, 3H, CH ), 7.30–8.02 (m, 8H, Ar-Hs), 15.40 (s, 1H,
OH, exchangeable with D O). C NMR (DMSO-d , d ppm):
6
, d
3
1
3
Chemistry
General
2
6
14.33, 83.11, 116.85, 116.87, 118.13, 122.55, 124.44, 124.54,
125.56, 126.83, 127.99, 144.55, 151.22, 159.45, 160.33, 165.44,
All melting points were uncorrected and measured using
Electrothermal IA 9000 apparatus. Infrared spectra were
measured by Nexus 670 FT-IR FT-Raman spectrometer using
KBr discs at National Research Center, Egypt. The nuclear
magnetic resonance NMR spectra were determined using
Varian mercury 300 MHz spectrometer using TMS as the
internal standard. The mass spectra were recorded on GCMS-
QP 1000EX Shimadzu gas chromatography MS spectrometer.
þ
176.38. MS (m/z): M : 297 (91%) and 296 (100%).
Synthesis of 3-(5-(4-fluorophenyl)-4,5-dihydro-1-methyl-
1H-pyrazol-3-yl)-4-hydroxy-2H-chromen-2-one (4)
A mixture of chalcone 3 (1.55g, 5 mmol) in absolute ethanol
(30 mL) and methyl hydrazine (0.26 mL, 5 mmol) was refluxed
for 6 h. Upon cooling, the obtained product was filtered off
and re-crystallized from ethanol to obtain the desired
compound 4. Yellow crystals, m.p. ¼ 157–159°C, yield (82%).
1
13
H NMR, C NMR, MS spectra, and the elemental analyses
were performed at Micro-Analytical Laboratory, Central
Services Laboratory, Faculty of Science, Cairo University,
Egypt. The reactions were followed by TLC (silica gel,
aluminum sheets 60 F254, Merck) using chloroform/methanol
Anal. calcd. for C19
Found: C, 67.54; H, 4.52; N, 8.35. IR (KBr, cm ): 3441 (OH),
H
15FN
2
O
3
(338.33): C, 67.45; H, 4.47; N, 8.28.
ꢃ1
1
1696 (C–O), and 1617 (C–N). H NMR (DMSO-d
(s, 3H, CH ), 3.29–3.35 (dd, 1H, pyrazoline), 4.14–4.19 (dd, 1H,
pyrazoline), 4.22–4.25 (dd, 1H, pyrazoline), 7.21–7.96 (m, 8H,
6
, d ppm): 2.76
(
9.5:0.5 v/v) as eluent and sprayed with iodine/potassium
3
iodide reagent. The purity of the newly synthesized com-
pounds was assessed by TLC and elemental analysis and was
found to be higher than 95%. Compounds 1 [31], 3 [32], 6 [33],
Ar-Hs), 13.40 (s, 1H, OH, exchangeable with D
M : 338 (13%) and 243 (100%).
2
O). MS (m/z):
þ
9
[33], 15 [34], 16 [35], 19 [36], 20 [36], 21 [37], and 23 [38] were
previously prepared and characterized.
The InChI codes of the investigated compounds together
with some biological activity data are provided as Supporting
Information.
Synthesis of 3-(1-tert-butyl-5-(4-fluorophenyl)-4,5-
dihydro-1H-pyrazol-3-yl)-4-hydroxy-2H-chromen-2-one (5)
To a mixture of chalcone 3 (1.55g, 5 mmol) and tert-
butylphenylhydrazine hydrochloride (0.623 g, 5 mmol) in
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ethyl alcohol (20 mL), potassium hydroxide solution (0.28 g in
with dil. HCl. The formed precipitate was filtered, washed
with water, dried, and crystallized from DMF to give
compound 10. Green crystals, m.p. ¼ 235–237°C, yield
1
2
mL H O) was added and the mixture was refluxed for 12 h.
The formed precipitate was filtered, washed several times
with water, dried, and recrystallized from the ethanol to
obtain the desired compound 5. Yellow crystals, m.p. ¼ 160–
3 3
(82%). Anal. calcd. for C25H18FN O S (459.49): C, 65.35; H,
3.95; N, 9.14; S, 6.98. Found: C, 65.42; H, 4.07; N, 9.22; S, 7.02. IR
ꢃ1
1
6
62°C, yield (82%). Anal. calcd. for C22
H
21FN
2
O
3
(380.41): C,
(KBr, cm ): 3391 (OH), 3303 (NH), 1721 (C–O), and 1609 (C–
1
9.46; H, 5.56; N, 7.36. Found: C, 69.54; H, 5.62; N, 7.42. IR (KBr,
N). H NMR (DMSO-d
6
, d ppm): 3.40–3.47 (dd, 1H, CH
pyrazoline), 5.97–6.01 (dd,
1H, CH pyrazoline), 7.15–7.98 (m, 13H, Ar-Hs), 10.22 (s, 1H, NH,
exchangeable with D O), 11.03 (s, 1H, OH, exchangeable with
O). C NMR (DMSO-d , d ppm): 38.44, 66.56, 87.88, 115.36,
2
ꢃ1
1
cm ): 3427 (OH), 1705 (C–O), and 1615 (C–N). H NMR
DMSO-d , d ppm): 1.15 (s, 9H, 3CH ), 3.24–3.31 (dd, 1H, CH
pyrazoline), 4.02–4.13 (dd, 1H, CH pyrazoline), 4.87–4.93 (dd,
H, CH pyrazoline), 7.15–7.97 (m, 8H, Ar-Hs), 13.42 (s, 1H, OH,
pyrazoline), 4.00–4.10 (dd, 1H, CH
2
(
6
3
2
2
2
1
3
1
D
2
6
þ
exchangeable with D
2
O). MS (m/z): M : 380 (23%) and 229
115.39, 117.46, 121.44, 125.11, 125.67, 126.53, 126.58, 126.95,
(
100%).
128.63, 128.77, 128.85, 129.22, 129.25, 137.45, 139.22, 150.78,
þ
1
56.34, 159.58, 161.12, 166.41, 179.55. MS (m/z): M : 459
Synthesis of 3-(1-acetyl-5-(4-fluorophenyl)-4,5-dihydro-
H-pyrazol-3-yl)-4-hydroxy-2H-chromen-2-one (7)
(24%) and 81 (100%).
1
To a solution of chalcone 3 (1.55g, 5 mmol) in glacial acetic
acid (20 mL), excess hydrazine hydrate (2 mL) was added and
the mixture was refluxed for 8 h. The reaction mixture was
cooled and poured onto ice/water. The formed precipitate
was filtered, washed with water, dried, and recrystallized
from acetic acid to give compound 7. Yellow crystals,
Synthesis of 3-(5-(4-fluorophenyl)-3-(4-hydroxy-2-oxo-2H-
chromen-3-yl)-1H-pyrazol-1-yl)indolin-2-one (11)
A mixture of compound 9 (1.62g, 5 mmol) in absolute ethanol
(20 mL) and isatin (0.74, 5 mmol) was refluxed for 8 h. Upon
cooling, the obtained product was filtered off and recrystal-
lized from acetic acid to obtain the desired compound 11.
White crystals, m.p. ¼ 280–282°C, yield (82%). Anal. calcd. for
m.p. ¼ 177–179°C, yield (82%). Anal. calcd. for C20
2 4
H15FN O
(
366.34): C, 65.57; H, 4.13; N, 7.65. Found: C, 65.63; H, 4.18; N,
C
26
H
16FN
3
O
4
(453.42): C, 68.87; H, 3.56; N, 9.27. Found: C,
ꢃ1
ꢃ1
7
1
3
.72. IR (KBr, cm ): 3389 (OH), 1707 (C–O), 1666 (C–O), and
68.94; H, 3.61; N, 9.33. IR (KBr, cm ): 3432 (OH), 3260 (NH),
1
1
617 (C–N). H NMR (DMSO-d
, d ppm): 2.31 (s, 3H, CH
), 3.29–
2
pyrazo-
1735 (C–O), 1699 (C–O), and 1619 (C–N). H NMR (DMSO-d
ppm): 6.23 (s, 1H, indole), 6.95–7.89 (m, 13H, Ar-Hs, and CH
pyrazole), 10.84 (s, 1H, NH, exchangeable with D O), 13.20 (s,
O). C NMR (DMSO-d , d ppm):
64.44, 94.25, 106.34, 116.19, 116.22, 117.54, 121.44, 122.25,
25.32, 125.66, 126.82, 127.56, 127.66, 128.78, 128.88, 129.34,
6
, d
6
3
.37 (dd, 1H, CH
2
pyrazoline), 3.95–4.03 (dd, 1H, CH
line), 5.47–5.51 (dd, 1H, CH pyrazoline), 7.13–8.02 (m, 8H,
2
þ
13
Ar-Hs), 9.65 (s, 1H, OH, exchangeable with D
66 (60%) and 121 (100%).
2
O). MS (m/z): M :
1H, OH, exchangeable with D
2
6
3
1
Synthesis of 5-(4-fluorophenyl)-4,5-dihydro-3-(4-hydroxy-
-oxo-2H-chromen-3-yl)pyrazole-1-carbothioamide (8)
129.42, 130.16, 141.67, 141.42, 144.52, 150.94, 162.33, 162.79,
166.56, 168.45. MS (m/z): M : 453 (7%) and 132 (100%).
þ
2
A mixture of chalcone 3 (1.55g, 5 mmol) and thiosemicarba-
zide (0.456 g, 5 mmol) in pyridine (20 mL) was refluxed for
Synthesis of 2-amino-4-(4-fluorophenyl)-6-(4-hydroxy-2-
oxo-2H-chromen-3-yl)pyridine-3-carbonitrile (12)
1
0 h. The reaction mixture was cooled, poured into
ice-cold water, and acidified by diluted hydrochloric acid.
The formed precipitate was filtered, dried, and crystallized
from the methanol to give compound 8. Light brown
crystals, m.p. ¼ 186–188°C, yield (82%). Anal. calcd. for
A mixture of chalcone 3 (1.55g, 5 mmol), malononitrile
(0.32 mL, 5 mmol), and ammonium acetate (0.77 g, 10 mmol)
in absolute ethanol (20 mL) was heated under reflux for 8 h.
After cooling, the formed precipitate was filtered off, washed
with water, dried, and recrystallized from DMF/water to
afford compound 12. Yellow crystals, m.p. ¼ 253–255°C, yield
C
19
H
14FN
3
O
3
S (383.40): C, 59.52; H, 3.68; N, 10.96; S, 8.36.
ꢃ1
Found: C, 59.64; H, 3.70; N, 11.02; S, 8.47. IR (KBr, cm ): 3416
1
(
(
4
OH), 3277, 3220 (NH
DMSO-d , d ppm): 3.40–3.52 (dd, 1H, CH
.20 (dd, 1H, CH pyrazoline), 4.50 (s, 2H, NH
O), 5.55–5.65 (dd, 1H, CH pyrazoline), 6.78–8.09 (m,
O). MS
2
), 1707 (C–O), and 1617 (C–N). H NMR
pyrazoline), 4.12–
, exchangeable
(82%). Anal. calcd. for C21
H12FN
3
O
3
(373.34): C, 67.56; H, 3.24;
ꢃ1
6
2
N, 11.26. Found: C, 67.64; H, 3.32; N, 11.32. IR (KBr, cm ): 3389
ꢃꢃ
), 2207 (C ꢃꢃ N), 1679 (C–O), and 1608
ꢃꢃ
6
, d ppm): 7.31–8.02 (m, 8H, Ar-Hs), 8.17
–
2
2
(OH), 3287, 3194 (NH
2
(CN). H NMR (DMSO-d
(s, 1H, pyridine), 8.52 (s, 2H, NH
1
with D
8
(
2
H, Ar-Hs), 13.40 (s, 1H, OH, exchangeable with D
m/z): M : 383 (65%) and 335 (100%).
2
2
, exchangeable with D
2
O),
þ
þ
12.80 (s, 1H, OH, exchangeable with D
44%) and 90 (100%).
2
O). MS (m/z): M : 373
(
Synthesis of 5-(4-fluorophenyl)-4,5-dihydro-3-(4-hydroxy-
-oxo-2H-chromen-3-yl)-N-phenyl-pyrazole-1-
carbothioamide (10)
2
Synthesis of 3-(6-(4-fluorophenyl)-1,2,3,6-tetrahydro-2-
thioxopyrimidin-4-yl)-4-hydroxy-2H-chromen-2-one (13)
To a mixture of chalcone 3 (1.55 g, 5 mmol) and thiourea
(1.52 g, 20 mmol) in ethyl alcohol (20 mL), potassium hydrox-
To a solution of compound 9 (1.62 g, 5 mmol) in pyridine
(
20 mL), phenyl isothiocyanate (0.60 mL, 5 mmol) was added
and the reaction mixture was refluxed for 10 h. The reaction
mixture was cooled and poured onto ice/water and acidified
ide solution (1.12 g KOH in 1 mL H
reaction mixture was refluxed for 24 h. The solvent was
2
O) was added and the
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concentrated and acidified with dilute hydrochloric acid. The
formed solid was filtered off, washed several times with
water, dried, and recrystallized from the methanol to obtain
the desired compound 13. Orange crystals, m.p. ¼ 295–297°C,
(70%).Anal.calcd.forC18
4
H12FNO S(357.36):C,60.50;H,3.38;N,
3.92; S, 8.97. Found: C, 60.64; H, 3.50; N, 4.11; S, 9.13. IR (KBr,
ꢃ1
1
cm ): 3266 (OH), 1687 (C –– O), and 1629 (C –– O). H NMR (DMSO-
, d ppm): 3.42–3.64 (dd, 2H, CH , thiazolidinone), 5.62 (s, 1H,
thiazolidinone), 7.37–7.90 (m, 8H, Ar-Hs), 12.20 (s, 1H, OH,
d
6
2
2 3
yield (82%). Anal. calcd. for C19H13FN O S (368.38): C, 61.95;
1
3
H, 3.56; N, 7.60; S, 8.70. Found: C, 61.90; H, 3.61; N, 7.55; S,
2 6
exchangeable with D O). C NMR (DMSO-d , d ppm): 34.55,
ꢃ1
8
1
.72. IR (KBr, cm ): 3415 (OH), 3180 (NH), 1675 (C–O), and
54.48, 90.16, 115.34, 115.40, 118.51, 122.35, 125.15, 126.22,
127.54, 130.22, 130.28, 135.18, 149.52, 159.87, 160.93, 161.54,
610 (C–N). 1H NMR (DMSO-d
O exchangeable), 4.75 (dd, 1H, CH-pyrimidine), 6.63–8.64
m, 9H, Ar-H, and CH-pyrimidine), 15.22 (s, 1H, OH,
6
, d ppm): 2.00 (s, 2H, 2NH,
þ
D
2
166.76. MS (m/z): M : 357 (18%) and 121 (100%).
(
13
D
2
O exchangeable).
C NMR (DMSO-d
6
, d ppm): 57.58,
Synthesis of 2-(2-oxo-2H-chromen-4-yloxy)-N-(2-(4-
fluorophenyl)-4-oxothiazolidin-3-yl)acetamide (22)
A mixture of compound 21 (1.70 g, 5 mmol) and thioglycolic
acid (0.35 mL, 5 mmol) was refluxed in dry benzene (20 mL) for
9
1
1
6.23, 99.44, 115.35, 115.38, 117.58, 122.65, 125.58, 127.22,
28.76, 128.81, 128.97, 138.65, 150.15, 150.24, 158.55, 159.87,
61.45, 174.77. MS (m/z): M : 368 (5.5%) and 121 (100%).
þ
1
0 h. The solvent was evaporated and the reaction mixture
Synthesis of 3-(2-amino-6-(4-fluorophenyl)pyrimidin-4-yl)-
-hydroxy-2H-chromen-2-one (14)
To a mixture of chalcone 3 (1.55 g, 5 mmol) and guanidine
was neutralized with cold dilute sodium bicarbonate solution,
the formed product was filtered off, washed with water,
dried, and crystallized from methanol to afford the title
compound 22. White crystals, m.p. ¼ 238–240°C, yield (80%).
4
hydrochloride (0.48 g, 5 mmol), in ethyl alcohol (20 mL),
potassium hydroxide solution (0.56 g KOH in 0.5 mL H
2
O)
2 5
Anal. calcd. for C20H15FN O S (414.41): C, 57.97; H, 3.65; N,
was added and the reaction mixture was refluxed for 10 h.
After cooling, the solution was poured onto ice/water and
acidified with diluted hydrochloric acid. The formed precipi-
tate was filtered off, washed several times with water, dried,
and recrystallized from methanol to give the title compound.
Yellow crystals, m.p. ¼ 200–202°C, yield (82%). Anal. calcd. for
6.76; S, 7.74. Found: C, 58.10; H, 3.82; N, 6.90; S, 7.82. IR (KBr,
ꢃ1
cm ): 3428 (NH), 1715 (C–O), 1677 (C–O), and 1617 (C–O).
1
H
NMR (DMSO-d
6
,
d
ppm): 3.74–3.95 (dd, 2H, CH
2
,
thiazolidinone), 4.84 (s, 2H, CH
2
), 5.70 (s, 1H, thiazolidinone),
5.84 (s, 1H, CH, H-3 coumarin), 7.16–7.79 (m, 8H, Ar-Hs), 10.55
þ
(s, 1H, NH, D
196 (100%).
2
O exchangeable). MS (m/z): M : 414 (2%) and
C
19
H
12FN
3
O
3
(349.32): C, 65.33; H, 3.46; N, 12.03. Found: C,
ꢃ1
6
5.48; H, 3.50; N, 12.22. IR (KBr, cm ): 3435 (OH), 3260, 3218
), 1679 (C–O), and 1606 (C–N). 1H NMR (DMSO-d
, exchangeable with D O), 7.15–7.98 (m,
(
NH
2
6
, d
Synthesis of N-[3-cyano-4-(4-fluorophenyl)-5-oxo-4H,5H-
pyrano[3,2-c]chromen-2-yl]-formimidic acid methyl ester
(24) and N-[3-cyano-4-(4-fluorophenyl)-5-oxo-4H,5H-
pyrano[3,2-c]chromen-2-yl]-formimidic acid ethyl ester (25)
A mixture of compound 23 (1.67g, 5 mmol), trimethylortho-
formate and/or triethylorthoformate (5 mmol), and acetic
anhydride (2 mL) was heated under reflux for 8–10 h. After
cooling, the solid formed was filtered off and washed with
water to give compounds 24 and 25, respectively.
ppm): 1.73 (s, 2H, NH
2
2
9
H, Ar-Hs, and CH-pyrimidine), 10.63 (s, 1H, OH, exchangeable
þ
with D
2
O). MS (m/z): M : 349 (100%).
Synthesis of 3-(4-fluorobenzylideneamino)-4-hydroxy-2H-
chromen-2-one (17)
A mixture of 3-amino-4-hydroxycoumarin 16 (0.89 g, 10 mmol)
and p-fluorobenzaldehyde (0.54 mL, 5 mmol) in absolute
ethanol (20 mL) in the presence of catalytic amount of acetic
acid (1 mL) was refluxed for 6 h. The reaction mixture was
cooled and the formed precipitate was filtered off and
recrystallized from the ethanol to give compound 17. Yellow
crystals, m.p. ¼ 203–205°C, yield (76%). Anal. calcd. for
N-[3-Cyano-4-(4-fluorophenyl)-5-oxo-4H,5H-pyrano[3,2-c]
chromen-2-yl]-formimidic acid methyl ester (24)
Off-white crystals, m.p. ¼ 204–206°C, yield (78%). Anal. calcd.
for C21
H
13FN
2
O
4
(376.34): C, 67.02; H, 3.48; N, 7.44. Found: C,
ꢃ1
ꢃꢃ
–
C
16
H
10FNO
3
(283.25): C, 67.84; H, 3.56; N, 4.94. Found: C,
67.14; H, 3.62; N, 7.59. IR (KBr, cm ): 2210 (C ꢃꢃ N), 1720 (C–O),
ꢃꢃ
ꢃ1
1
6
7.99; H, 3.72; N, 5.11. IR (KBr, cm ): 3434 (OH), 1673 (C–O),
and 1628 (C–N). H NMR (DMSO-d
6
, d ppm): 3.93 (s, 3H, OCH
3
),
1
and 1609 (C–N). H NMR (DMSO-d
, d ppm): 7.25–7.86 (m, 8H,
4.76 (s, 1H, pyran), 7.14–8.22 (m, 8H, Ar-Hs), 8.96 (s, 1H, CH–N).
6
1
3
Ar-Hs), 8.10 (s, 1H, CH–N), 12.23 (s, 1H, OH, exchangeable with
6
C NMR (DMSO-d , d ppm): 39.11, 52.13, 76.31, 105.53,
þ
D
2
O). MS (m/z): M : 283 (5%) and 121 (100%).
115.41, 115.45, 117.54, 117.67, 122.65, 125.58, 127.22, 128.24,
1
31.17, 131.20, 137.84, 151.24, 156.63, 159.92, 161.12, 162.24,
þ
Synthesis of 2-(4-fluorophenyl)-3-(4-hydroxy-2-oxo-2H-
chromen-3-yl)-thiazolidin-4-one (18)
170.38. MS (m/z): M : 376 (37%) and 281 (100%).
A mixture of compound 17 (1.416g, 5 mmol) and thioglycolic
acid (0.35 mL, 5 mmol) was refluxed in dry benzene (20 mL) for
N-[3-Cyano-4-(4-fluorophenyl)-5-oxo-4H,5H-pyrano[3,2-c]
chromen-2-yl]-formimidic acid ethyl ester (25)
1
0 h. The solvent was evaporated and the reaction mixture was
Off-white crystals, m.p. ¼ 211–213°C, yield (75%). Anal. calcd.
neutralized with cold dilute sodium bicarbonate solution, the
formedproductwas filteredoffandcrystallized frommethanol
to give compound 18. White crystals, m.p. ¼ 192–194°C, yield
for C22
H
2
15FN O
4
(390.36): C, 67.69; H, 3.87; N, 7.18. Found: C,
ꢃ1
ꢃꢃ
–
67.80; H, 3.92; N, 7.24. IR (KBr, cm ): 2214 (C ꢃꢃ N), 1719 (C–O),
ꢃꢃ
1
and 1625 (C–N). H NMR (DMSO-d
, d ppm): 1.33–1.37 (t, 3H,
6
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CH
3
), 4.36–4.43 (q, 2H, CH
2
), 4.75 (s, 1H, pyran), 7.14–8.21 (m,
off, dried, and crystallized from methanol to give compound
þ
8
2
H, Ar-Hs), 8.92 (s, 1H, CH¼N). MS (m/z): M : 390 (58%) and
29. Yellowish brown crystals, m.p. ¼ 298–299°C, yield (82%).
39 (100%).
Anal. calcd. for C21
H
11FN
4
O
4
(402.33): C, 62.69; H, 2.76; N,
ꢃ1
1
3.93. Found: C, 62.76; H, 2.85; N, 14.00. IR (KBr, cm ): 3423
0
1
Synthesis of 9-amino-7-(4 -fluorophenyl)-8,9-dihydro-8-
imino-6H,7H[1]benzpyrano[3 ,4 :5,6]-pyrano[2,3-d]-
pyrimidine-6-one (26)
Hydrazine hydrate 99% (0.5 mL, 10 mmol) was added to a
solution of 25 (1.95 g, 5 mmol) in absolute ethanol (20 mL) and
the reaction mixture was stirred at room temperature for 4 h.
The separated solid was filtered off, dried, and recrystallized
from acetic acid to afford compound 26. White crystals,
(NH), 1720 (C–O), 1650 (C–O), and 1607 (C–N). H NMR
(DMSO-d , d ppm): 5.48 (s, 1H, pyran), 7.05–8.08 (m, 8H, Ar-
Hs), 8.63 (s, 1H, pyrimidine), 9.77 (s, 1H, NH, exchangeable
0
0
6
1
3
2 6
with D O). C NMR (DMSO-d , d ppm): 35.22, 92.98, 105.38,
115.66, 115.72, 117.51, 122.51, 125.44, 127.33, 128.56, 130.75,
130.88, 138.21, 143.25, 151.42, 158.11, 160.12, 161.24, 162.33,
þ
163.56, 165.67. MS (m/z): M : 402 (19%) and 59 (100%).
m.p. ¼ 222–224°C, yield (75%). Anal. calcd. for C20
H
13FN
4
O
3
Biology
(
376.34): C, 63.83; H, 3.48; N, 14.89. Found: C, 63.91; H, 3.62; N,
MCF-7 cell line was obtained from the ATCC (Rockville, MD,
ꢃ1
1
TM
1
(
4.95. IR (KBr, cm ): 3416, 3316, 3231 (NH and NH
2
), 1716
USA). HeLa cell line (ATCC CCL2 ) was purchased from
ATCC (Manassas, VA, USA). Human vascular endothelial
growth factor receptor-2 (VEGFR-2) ELISA kit was purchased
from Alpha Screen System (PerkinElmer, USA). p38 MAPK
alpha (Thr180 Tyr182) ELISA Kit (cat. ab126425) was supplied
by Abcam (Cambridge, UK).
1
C–O), and 1607 (C–N). H NMR (DMSO-d
pyran), 5.74 (s, 2H, NH
, d ppm): 5.00 (s, 1H,
O), 7.04–7.94
6
2
, exchangeable with D
2
(
m, 9H, Ar-Hs and NH), 8.19 (s, 1H, CH pyrimidine). MS (m/z):
þ
M : 376 (14%) and 64 (100%).
0
Synthesis of 14-(4 -fluorophenyl)-13H,14H[1]benz-
0
0
pyrano[3 ,4 :5,6]pyrano[3,2-e][1,2,4]triazolo[1,5-c]-
pyrimidine-13-one (27)
VEGFR-2 inhibition assay
MCF-7 cell line in the culture medium was treated with 20 mL
of IC50 values of the compounds or the standard reference
drug, tamoxifen, dissolved in DMSO, then incubated for 24 h
A mixture of compound 26 (1.88 g, 5 mmol) in DMF (20 mL)
and triethylorthoformate (0.83 mL, 5 mmol) was refluxed for
8
h. The solution was left to cool. The precipitate was filtered
2
at 37°C, in a humidified 5% CO atmosphere. The cells were
off, washed with water, and crystallized from DMF to afford
harvested and homogenates were prepared in saline using a
tight pestle homogenizer until complete cell disruption. The
kinase activity of VEGFR-2 was measured using an anti-
phosphotyrosine antibody according to the manufacturer’s
instructions. Enzyme reactions were performed in 50 mM Tris-
compound 27. White crystals, m.p. ¼ 254–255°C, yield (82%).
Anal. calcd. for C21
H
11FN
4
O
3
(386.34): C, 65.29; H, 2.87; N,
ꢃ1
1
4.50. Found: C, 65.41; H, 2.99; N, 14.62. IR (KBr, cm ): 1719
C–O) and 1606 (C–N). H NMR (DMSO-d
1
(
6
, d ppm): 5.49 (s, 1H,
pyran), 7.05–8.09 (m, 8H, Ar-Hs), 8.64 (s, 1H, pyrimidine), 9.78
2 2
HCl, pH 7.5, 5 mM MnCl , 5 mM MgCl , 0.01% Tween-20, and
þ
(
s, 1H, triazole). MS (m/z): M : 386 (10%) and 291 (100%).
2 mM DTT, containing 10 mM ATP, 0.1 mg/mL biotinylated
poly-GluTyr (4:1), and 0.1 nM of VEGFR-2. Prior to catalytic
initiation with ATP, compound at final concentrations
ranging from 0 to 100 mg/mL and enzyme were incubated
for 5 min at room temperature. The reactions were quenched
by the addition of 25 mL of 100 mM EDTA, 10 mg/mL
AlphaScreen Streptavidin donor beads, and 10 mg/mL accep-
tor beads in 62.5 mM HEPES, pH 7.4, 250 mM NaCl, and 0.1%
BSA. Plate was incubated in the dark overnight and then read
by ELISA reader (PerkinElmer). Wells containing the substrate
and the enzyme without compounds were used as reaction
control. Wells containing biotinylated poly-GluTyr (4:1) and
enzyme without ATP were used as basal control. The level of
human VEGFR-2 in samples was calculated (ng/mL) as
triplicate determinations from the standard curve. Percent
inhibition was calculated by the comparison of the tested
compounds treated to control cancer cells.
0
Synthesis of 14-(4 -fluorophenyl)-2-methyl-13H,14H[1]-
0
0
benzpyrano[3 ,4 :5,6]pyrano[3,2-e][1,2,4]-triazolo[1,5-c]-
pyrimidine-13-one (28)
A mixture of compound 26 (1.88gm, 5 mmol) and acetic
anhydride (15 mL) was heated under reflux for 6 h. Upon
cooling, the reaction mixture was poured onto ice/water. The
formed precipitate was filtered, washed with water, dried,
and crystallized from acetic acid to afford compound 28.
White crystals, m.p. ¼ >300°C, yield (60%). Anal. calcd. for
C
22
H
13FN
4
O
3
(400.36): C, 66.00; H, 3.27; N, 13.99. Found: C,
ꢃ1
6
6.11; H, 3.35; N, 14.08. IR (KBr, cm ): 1725 (C–O) and 1609
1
(
C–N). H NMR (DMSO-d
6
, d ppm): 2.47 (s, 3H, CH
3
), 5.43 (s, 1H,
pyran), 7.05–8.07 (m, 8H, Ar-Hs), 9.62 (s, 1H, pyrimidine). MS
þ
(
m/z): M : 400 (44%) and 305 (100%).
0
Synthesis of 14-(4 -fluorophenyl)-2-oxo-13H,14H[1]benz-
0
0
pyrano[3 ,4 :5,6]pyrano[3,2-e][1,2,4]-triazolo[1,5-c]-
pyrimidine-13-one (29)
p38a MAPK inhibition assay
Test compounds were dissolved in DMSO. A concentration of
10 mM was selected in order to perform the screening assay. A
total of 30 mM anisomycin was used as a positive control. A
seeding density of 30000 cells/well in a 96-well plates was
selected. At 24 h after cell seeding, the cells were starved in
To a solution of compound 26 (1.88 g, 5 mmol) in benzene
(
20 mL), ethyl chloroformate (0.48 mL, 5 mmol) was added and
the reaction mixture was heated under reflux for 12 h. The
solvent was evaporated and the formed product was filtered
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the presence of serum-free EMEM media for 4 h. Then, the
cells were treated with vehicle or 10 mM test compounds,
which were incubated for 1 h followed by treatment with
24 h after cell seeding, the wells are treated with vehicle or
20 mL of different concentrations of compound 11 (3–30 mM)
added to each well, such that the final concentration of DMSO
in the well is not greater than 0.5%. The cells are incubated
for 24 h. MTS reagent is then added and cells are incubated
with MTS for 3 h. Using a UV plate reader (Synergy HT Multi-
Mode Microplate Reader, Biotek, Winooski, VT), cell viability
is determined by measuring absorbance at 490 nm. Each
measurement is done in triplicate. Three replicates are
performed for every assay. One-way ANOVA followed by
Dunnet’s test was performed (p < 0.0001).
3
0 mM anisomycin for 1 h such that the final concentration of
DMSO in well was not greater than 0.5%. Mouse anti-
phospho-p38 MAPK antibody was directed in order to
evaluate the phosphorylated protein p38 at sites Thr180/
Tyr182. Mouse anti-p38 MAPK antibody was directed to
detect the presence of nonphosphorylated protein p38. A
secondary antibody conjugated with HRP was directed
against the primary antibodies. Addition of a suitable
substrate such as TMB leads to development of blue color
which is proportional to the amount of protein. The Stop
Solution (HCl) changes the color from blue to yellow. The
intensity of the color is thus measured at 450 nm.
Molecular docking
The crystal structure of VEGFR-2 protein (PDB:3U6J) was
downloaded from Protein Data Bank (http://www.rcsb.org).
Protein is prepared in MOE (version 2008). Ligand structures
werebuiltwithMOEandenergyminimizedusingtheMMFF94x
Anti-breast cancer SRB assay
Human breast MCF-7 cells were maintained in Dulbecco’s
ꢃ1
̊
ꢃ1
force field until a RMSD gradient of 0.05 kcal mol
A
was
modified Eagle’s medium (DMEM) supplemented with
reached. Compounds were docked within the VEGFR-2 protein.
Thesiteofdockingwasconsideredusingcrystallographicligand
position. The placement criterion was chosen to be Alpha
Triangle. Rescoring 1 was set to be London G and Retain 20
poses. In our study, we prefer to make refinement with Force
field and rescoring 2 was chosen to be London G. The same
protocol was applied in docking the compound 13 against p38
MPAK (PDB: 3FMK).
1
0% heat-inactivated fetal calf serum (GIBCO), penicillin
100 U/mL), and streptomycin (100 mg/mL) at 37°C in humidi-
fied atmosphere containing 5% CO . Cells at a concentration
(
2
2
6
of 0.50 ꢅ 10 were grown in a 25-cm flask in 5 mL of complete
culture medium. The anticancer activity of the tested
compounds was measured in vitro using the Sulfo-Rhoda-
mine-B stain (SRB) assay according to Skehan et al. [42] Briefly,
4
cells were inoculated in 96-well microtiter plate (10 cells/well)
for 24 h before treatment with the tested compounds to allow
attachment of cell to the wall of the plate. Test compounds
were dissolved in DMSO at 1 mg/mL immediately before use
and diluted to the appropriate volume just before addition to
the cell culture. Different concentration of tested compounds
and tamoxifen were added to the cells. Three wells were
prepared for each individual dose. Cells were incubated with
QSAR analysis
QSAR models were conducted by QSAR software of MOE 2008.
Molecule structures were built using builder tool, energy
minimization was carried using MMFF94x force field. The IC50
values were converted to pIC50 using equation: pIC50 ¼ (ꢃlog
IC50). Physicaldescriptors,constitutionalproperties,topological
descriptors, quantum chemical descriptors, surface area,
volume and shape descriptors, and molecular properties were
calculated. Predicted pIC50 for the data set were calculated.
the compounds for 48 h at 37°C and in atmosphere of 5% CO
After48 h,cellswerefixed,washed,andstainedfor30 minwith
.4% (w/v) SRB dissolved in 1% acetic acid. Unbound dye was
2
.
2
0
QSARmodelswerevalidatedbyusingtheR , RMSEandresidual
removed by four washes with 1% acetic acid, and attached
stain was recovered with Tris-EDTA buffer. Color intensity was
measured in an ELISA reader. The relation between surviving
fraction and drug concentration is plotted to get the survival
curve for each cell line after the specified time. The
concentration required for 50% inhibition of cell viability
values between expected and predicted pIC50.
The authors have declared no conflict of interest.
References
(
IC50) was calculated and the results are given in Table 1. The
results were compared to the effect of the reference drug,
tamoxifen [43].
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