Synthesis and biological evaluation of novel coumarin-pyrazoline hybrids endowed with phenylsulfonyl moiety as antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2012.12.004

Two groups of coumarin-pyrazoline hybrids were synthesized. The target compounds were obtained by cyclization of the coumarin chalcones with various substituted hydrazines to produce the corresponding pyrazolines through 1,4-addition on α,β-unsaturated ca

Full text of DOI:10.1016/j.ejmech.2012.12.004

Accepted Manuscript
Synthesis and biological evaluation of novel coumarin-pyrazoline hybrids endowed
with phenylsulfonyl moiety as antitumor agents
Kamilia M. Amin, Amal A.M. Eissa, Sahar M. Abou-Seri, Fadi M. Awadallah, Ghaneya
S. Hassan
PII:
S0223-5234(12)00726-X
10.1016/j.ejmech.2012.12.004
EJMECH 5875
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 August 2012
Revised Date: 3 December 2012
Accepted Date: 5 December 2012
Please cite this article as: K.M. Amin, A.A.M. Eissa, S.M. Abou-Seri, F.M. Awadallah, G.S. Hassan,
Synthesis and biological evaluation of novel coumarin-pyrazoline hybrids endowed with phenylsulfonyl
moiety as antitumor agents, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2012.12.004.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis and biological evaluation of novel coumarin-pyrazoline
hybrids endowed with phenylsulfonyl moiety as antitumor agents
Kamilia M. Amin, Amal A. M. Eissa, Sahar M. Abou-Seri*, Fadi M.
Awadallah, and Ghaneya S. Hassan.
Two groups of coumarin-pyrazoline hybrids bearing either (un)-substituted
phenylsulfonyl or terminal sulfamoyl moiety were synthesized as potential
antitumor agents utilizing the patent compounds IX as lead.
O
O
O
N
O
N
H3CO
R
N
H3CO
Ar
N
N
N
Ar
N
O
S O
N
O
O S
R'
R1
SO2NH2
10a-f
IX
lead compounds
7a-f, R1= H
8a-f, R1= CH3
R1= Cl
9a-f,

ACCEPTED MANUSCRIPT
Highlights
•
•
•
•
Two groups of coumarin-pyrazoline hybrids were synthesized.
Selected compounds were tested towards 60 cell lines according to US NCI protocol.
All compounds were screened against HCT-116.
The most active compounds were screened for PI3K (P110α/p85α) inhibition.

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel coumarin-pyrazoline hybrids
endowed with phenylsulfonyl moiety as antitumor agents
Kamilia M. Amin, Amal A. M. Eissa, Sahar M. Abou-Seri*, Fadi M. Awadallah, Ghaneya
S. Hassan.
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-
Aini Street, P.O. Box 11562, Cairo, Egypt.
* Corresponding author
Sahar Mahmoud Abou-Seri, Associate professor of Pharmaceutical Chemistry, Faculty of
Pharmacy, Cairo University, Kasr El-Aini Street, P.O. Box 11562, Cairo, Egypt.
E-mail: saharshaarawy_69@yahoo.com
Tel: +2 0122 7059 742, Fax: +2 02 2362 8426
1

ACCEPTED MANUSCRIPT
Abstract
Two groups of coumarin-pyrazoline hybrids were synthesized. The target compounds were
obtained by cyclization of the coumarin chalcones with various substituted hydrazines to
produce the corresponding pyrazolines through 1,4-addition on α,β-unsaturated carbonyl
system. Selected compounds were investigated for their anticancer activity towards 60
cancer cell lines according to US NCI protocol where breast cancer MCF7 and colon
cancer HCT-116 were the most susceptible to the influence of compounds 7d, 8c and 9c.
Encouraged by this, all final compounds were screened against colorectal cell line HCT-
116. The tested compounds exhibited high potency with IC50 ranging from 0.01 µM to 2.8
µM. Moreover, compound 9c which possessed the highest cytotoxicity proved to have
weak enzyme inhibitory activity against PI3K (P110α/p85α).
Keywords: Coumarins; pyrazoline; phenylsulfonyl derivatives; antitumor activity.
2

ACCEPTED MANUSCRIPT
1. Introduction
Cancer is a notably complex, widespread and lethal disease accounting for 7.6 million
deaths (around 13% of all deaths) in 2008, that are projected to continue rising, with an
estimated 13.1 million deaths in 2030 [1]. Cancer can affect almost every tissue lineage in
the human body and poses great challenges to medical science. Most cancers are
characterized by uncontrolled cell proliferation, lack of cell differentiation, and loss of
contact inhibition, which confers upon the tumor cell a capability to invade local tissues
and metastasize [2,3]. Nearly all cancers are caused by abnormalities in the genetic
material of the transformed cells and involve deregulation of essential enzymes and other
proteins controlling cell division and proliferation [4]. Several lines of evidence support the
view that chemotherapy has become one of the most significant treatment modalities in
cancer management. However, the nonselectivity and acute toxicity of many antitumor
agents beside the development of cellular drug resistance have been the major deterrent in
their usage for treating human cancer, prompting the search for new antitumor agents with
improved tumor selectivity, efficiency, and safety [5-7].
Natural and synthetic coumarin derivatives have attracted intense interest in recent
years because of their diverse biological and pharmacological properties. Among these
properties, their antitumor effects were extensively examined [8-14]. Coumarin and its
metabolite,7-hydroxycoumarin I, were reported to inhibit the proliferation of a number of
human malignant cell lines in vitro [15,16] and in xenograft models [17,18]. Moreover,
coumarin was found to produce objective tumor regression in some patients with metastatic
renal cell carcinoma, metastatic prostatic carcinoma and malignant melanoma in clinical
trials [19]. Osthole II, 7-methoxy-8-isopentenyl coumarin, also showed potent activity
against lung cancer A549 cells and breast cancer cells by arresting cell cycle in G2 phase
followed by inducing apoptosis through modulating PI3K/Akt pathway [20]. Novel
coumarin 3-(N-aryl) sulfonamides such as III, displayed considerable growth inhibition
followed by cell death in different cancer cell lines with GI50 values less than 100 µM, and
proved to be activators of JNK1 alpha protein kinase [21]. The stilbene-coumarin hybrid
IV was identified as novel anticancer endowed with excellent antiproliferative and
proapotosis activities against squamous cell carcinoma A43 and melanoma JR8 [22].
3

ACCEPTED MANUSCRIPT
Cytotoxic studies on 7-methoxy-4-methylcoumarin derivatives highlighted their potential
as selective antiproliferative and multidrug resistance reversal modulators [23,24] (Fig. 1).
On the other hand, the importance of pyrazoline ring as scaffold for new
antineoplastic agents was widely investigated [25,26]. For example, 3,5-diaryl-pyrazoline
regioisomers V, and their N1-acetylated derivatives showed potent and selective activity in
the NCI 60 human cancer cell lines panel [27, 28]. Another acylated derivative VI was
chosen among its series as a selective inhibitor of B- Raf Kinase [29, 30]. Some novel N1-
phenylsulfonyl pyrazoline derivatives VII exhibited high activity against human gastric
cancer cell SGC-7901, liver cancer Hep-G2 and human prostate PC-3 cell lines by virtue of
their potential telomerase inhibitory effect [31]. In addition, coumarin-pyrazoline
conjugates VIII exhibited potentially high activity against human gastric cancer cell SGC-
7901 [32] (Fig. 2). Moreover, imidazopyridine derivatives bearing the pyrazole ring, such
as the patent compounds IX [33] were reported as potent phosphoinositol-3 kinase PI3K
inhibitors and antitumor agents [34, 35] (Fig. 3).
Finally, several sulfonamide derivatives were reported to show substantial antitumor
activity, both in vitro and/or in vivo [36-38]. Some derivatives are currently being evaluated
in clinical trials, and there is much optimism that they might lead to novel alternative drugs,
devoid of the side effects of the presently available anticancer agents [39, 40].
In the design of new drugs, the hybridization and bioisosterism approaches might
allow obtaining molecules with improved biological activity with respect to the
corresponding lead compounds. Thus, adopting these approaches, two series of coumarin-
pyrazoline hybrids comprising two types of substitution patterns; one bearing (un)-
substituted phenylsulfonyl entities 7-9, while the other type had a terminal sulfamoyl
moiety 10 were synthesized as potential antitumor agents, utilizing the patent compounds
IX [33] as lead for the design of the present work (Fig. 3).
2. Results and Discussions
2.1. Chemistry
The synthesis of the novel coumarin-pyrazoline hybrids 7a-f, 8a-f, 9a-f and 10a-f was
accomplished through reaction of the precursor chalcones, 7-methoxy-8-(arylacryloyl)-2H-
4

ACCEPTED MANUSCRIPT
chromen-2-ones 6a-f [41] with the variously (un)-substituted phenylsulfonyl hydrazines viz
phenylsulfonyl hydrazine, 4-methylsulfonyl hydrazine, 4-chlorophenylsulfonyl hydrazine
or 4-sulfamoylphenyl hydrazine (Scheme 1). These pyrazolines were obtained via 1,4-
addition to the α,β-unsaturated carbonyl system, followed by dehydration and
rearrangement.
The structure elucidation of the newly synthesized compounds was based on the spectral
data (IR, 1H NMR, 13C NMR, and mass spectrometry). IR spectra of the prepared
pyrazolines 7-10 were characterized by the presence of υ (C=N) stretching at 1604-1558
cm-1 due to ring closure and two strong bands in the region of 1342-1290 and 1168-
1087cm-1 due to (SO2) stretching. Also, strong broad peaks derived from coumarin (C=O)
stretching were observed around 1728-1700 cm-1. In addition, derivatives 10a-f showed
two typical absorption bands at 3392-3367 and 3336-3201 cm-1 due to their NH2 stretching
vibrations. 1H NMR spectra recorded for these compounds clearly supported the proposed
structures. The pyrazoline protons in compounds 7-10 showed a prominent AMX pattern
with three protons HA, HM and HX seen as doublets of doublets. The HX proton was more
deshielded by aryl group (δ 4.80-5.80 ppm) and was coupled transoid to the HM proton and
cisoid to the HA proton. The HM proton was deshielded to some extent by aryl group (δ
3.40-4.20 ppm) and was coupled transoid to the HX proton and geminally to the HA proton.
The third, HA proton (δ 2.84-3.45 ppm) was coupled cisoid to the HX proton and geminally
to the HM proton.
There were three coupling constants: geminal (JAM = 16.4-21.9 Hz), trans (JMX = 10.5-16.8
Hz) and cis coupling (JAX = 6.9-8.7 Hz). In case of compounds 7b, 8b, 9b and 10a simple
splitting patterns were observed with pyrazoline protons which appeared as two signals,
one doublet equivalent to 2H (δ 3.21-3.92 ppm, J = 9.9-11.4 Hz) and a triplet equivalent to
1H (δ 4.30-4.95 ppm).
The protons belonging to the aromatic system and phenyl substituents were observed at the
expected chemical shifts and integral values. In addition, the derivatives 10a-f showed a
singlet exchangeable signal at δ 4.20-5.10 ppm corresponding to NH2 protons.
5

ACCEPTED MANUSCRIPT
2.2. Biological evaluation
2.2.1. Anticancer activity
Seven compounds 7a, 7b, 7d, 7f, 8c, 9c and 10a were selected by the National Cancer
Institute (NCI) Developmental Therapeutic Program (www.dtp.nci.nih.gov) to be screened
for their anticancer activity in vitro. The anticancer assays were performed in accordance
with the protocol of the Drug Evaluation Branch, NCI, Bethesda [42-44].
The compounds were first evaluated at one dose primary anticancer assay towards a panel
of approximately 60 cancer lines (concentration 10-5 M). The human tumor cell lines were
derived from nine different cancer types: leukemia, melanoma, lung, colon, CNS, ovarian,
renal, prostate and breast cancers. A 48 h drug exposure protocol was used and
sulforhodamine B (SRB) protein assay was applied to estimate the cell viability and growth
[45]. Results for each test agent were reported as the percentage growth of the treated cells
compared to the untreated control cells and also, presented as mean graph of the growth
percent. The preliminary screening results showed that compounds 7a, 7b, 7f and 10a had
no significant antitumor action. Nevertheless, compounds 7d, 8c and 9c possessed
considerable activity and were passed on to further evaluation at five-dose assay [0.01 µM-
100 µM]. The calculated response parameters for the three compounds -GI50, TGI and
LC50- against each cell line were presented in Table 1. GI50 corresponds to the molar
concentration of the compound causing 50% decrease in net cell growth and is viewed as a
growth-inhibitory level of effect; TGI (cytostatic activity) is the molar concentration of the
compound leading to total growth inhibition; LC50 is the cytotoxicity parameter and reflects
the molar concentration needed to cause 50% net cell death. Values were calculated for
each of these parameters if the level of activity was reached; however, if the effect was not
reached or was exceeded, the value was expressed as more or less than the maximum or
minimum concentration tested. Furthermore, sub-panel and full panel mean graph
midpoints (MG-MID) were calculated for each parameter, giving an average activity
parameter over individual sub-panels and full panel cell lines toward each compound
(Tables 2 and 3). In general, compounds 7d, 8c and 9c exhibited superior anticancer
activities against the entire panel of tumor cell lines with the chlorophenylsulfonyl
analogue 9c being the most potent. These agents showed effective growth inhibition full
6

ACCEPTED MANUSCRIPT
panel GI50 (MG-MID) values of 2.18, 1.89 and 1.63 µM, respectively and cytostatic
activity full panel TGI (MG-MID) values of 7.32, 7.36 and 5.82 µM, respectively.
Regarding sensitivity, the NCI panel cell lines displayed comparatively homogenous
response with only few cell lines showing greater sensitivity than the average toward the
evaluated compounds. Among the tested cancer cell lines, the colon cancer, melanoma and
breast cancer panels were the most susceptible to the influence of 7d, 8c and 9c [GI50 (MG-
MID) 2.09, 1.69 and 1.45 µM for colon cancer, GI50 (MG-MID) 2.04, 1.74 and 1.51 µM
for melanoma and GI50 (MG-MID) 2.03, 1.52 and 1.67 µM for breast cancer].
As for the sensitivity against some individual cell lines, compound 7d presented an
appreciable inhibitory activity toward all tumor subpanels, GI50 (MG-MID) range of 2.03-
2.65 µM. Also, low degree of differential sensitivity between the most and least sensitive
cell lines was observed, GI50 range 1.15-3.86 µM. Breast cancer MDA-MB-468, melanoma
UACC-62, renal cancer UO-31 as well as colon cancer HCT-116 with GI50 range of 1.15-
1.62 µM were among the most sensitive cell lines.
Compared to 7d, the analogue 8c elicited a better or comparable inhibitory efficacy through
out the whole cell line spectrum. It was found especially effective against breast cancer
MCF-7 and MDA-MB-468 with GI50 0.99 and 0.50 µM, respectively. Moreover, 8c
revealed a distinctive effectiveness on colon cancer HCT-116, CNS cancer U251, renal
cancer RXF 393 and breast cancer BT-549 at both the inhibitory and cytostatic levels. On
the other hand, compound 9c showed the highest growth inhibitory potential against most
of the tested cell lines, with an outstanding activity on six different cell lines with GI50
lying in the submicromolar range. It exhibited a distinguished sensitivity profile toward
leukemia SR, colon cancer HCT-116, CNS cancer U251 and melanoma LOX IMVI, in
addition to remarkable high activity against MCF-7 and MDA-MB-468 breast cancer, GI50
0.49 and 0.34 µM, respectively.
The cytotoxicity values associated with the three compounds, measured as LD50 were less
than 100 µM for most of the tested cell lines except for leukemia.
Concerning selectivity, the index obtained by dividing the full panel MG-MID (µM) of the
compounds by their individual sub-panel MG-MID (µM) is considered as a measure of
compound selectivity. Ratios between 3 and 6 refer to moderate selectivity, ratios greater
than six indicate high selectivity towards the corresponding cell line, while compounds not
7

ACCEPTED MANUSCRIPT
meeting either of these criteria are rated as nonselective [46]. In this context, compounds
7d, 8c and 9c proved to be nonselective with broad spectrum antitumor activity against all
tumor sub-panels tested at both GI50 and TGI levels, with selectivity ratios ranges of 0.80–
1.25 and 0.54–1.70, respectively (Tables 2 and 3).
Colorectal cancer (CRC) is considered one of the most common cancers worldwide and is
commonly associated with genes mutation that control growth factor signaling, therefore,
new therapeutic agents are being developed to target such tumor [47]. According to the
results of the cytotoxicity screening against 60 human tumor cell lines performed at the
NCI (Bestheda), it was observed that the tested compounds were potent inhibitors of tumor
cell growth and had promising activity against HCT-116 colon cell. Since only seven
compounds were selected by the NCI for this screening assay, it was of interest to extend
the cytotoxicity study by testing the cell growth inhibitory activity of all target compounds,
7a-f, 8a-f, 9a-f and 10a-f against human colon cancer cell line (HCT-116). This permitted
a good structure activity relationship study. Results were expressed as IC50 in µM as shown
in Table 4 and activity was compared with that of doxorubicin as reference drug. The data
showed that sixteen compounds were found more active than doxorubicin. The most active
compounds was 9c having an IC50 of 0.01 µM, (IC50 of doxorubicin= 0.63 µM). Other
highly potent compounds were 7d, 8c, 10e and 10f having IC50 values of 0.02, 0.02, 0.03
and 0.04 µM, respectively. Correlation of the activity results of the first set of compounds,
the 1-(un)substituted phenylsulfonyl-5-arylpyrazolines 7a-f, 8a-f and 9a-f showed that
within each individual series there was no absolute relation between the activity and the
nature of the aryl substituent (Ar) viz. 5-methyfuranyl, thienyl, phenyl, 4-tolyl, 4-
trifluoromethylphenyl and 4-methylthiophenyl. As for the 1-(un)substituted phenylsulfonyl
group, it was obvious that compounds having the chloro substituent 9a-c,e,f were generally
the most active especially that all compounds in this series were more active than the
reference drug. Also, compared to the other series (7a-f and 8a-f), some derivatives were
more potent than their respective analogues as exhibited by compounds 9b, 9c and 9f
(IC50= 0.11, 0.01 and 0.11 µM, respectively). The unsubstituted phenylsulfonyl pyrazolines
7a-f and the 4-methylphenylsulfonyl derivatives 8a-f interchanged positions regarding their
order of activity. From the second set of compounds 10a-f emerged two promising
compounds, 10e and 10f, having IC50 values of 0.03 and 0.04 µM, respectively. Within this
8

ACCEPTED MANUSCRIPT
series, it was apparent that the activity was favored by the phenyl groups having electron
withdrawing substituents such as trifluoromethyl and methylthio as in compounds 10e and
10f, respectively.
Comparing the activity of the two sets of compounds, namely the 1-(un)-substituted
phenylsulfonyl-5-arylpyrazolines (7a-f, 8a-f and 9a-f) and the sulfamoylphenyl congeners
10a-f; it was important to explore the effect of flipping the benzene sulfonyl moiety in the
first set into a phenyl group with free terminal sulfamoyl function in the second one.
Analyzing the data revealed that compounds having 5-methylfuran substitution at position
5 of the pyrazoline ring 7a, 8a and 9a were more active than their corresponding analogue
10a. The same held true for the compounds with a phenyl substitution (7c, 8c, and 9c c.f.
10c). On the other hand, the activity of trifluoromethylphenyl and methylthiophenyl
derivatives, 7e,f, 8e,f and 9e,f, was enhanced in the case of their respective analogues 10e,f.
2.2.2. PI3K (p110α/p85α) protein kinase activity
Genomic studies indicated that phosphoinositide 3-kinase (PI3K) signaling is one of
the most frequently deregulated pathways in several human cancers, including CRC. PI3K
signaling has an important role in cancer cell proliferation, survival, motility, and
metabolism [47,48]. To investigate the possible mechanism of action responsible for the
observed cytotoxic effects and due to structure similarity of the newly synthesized
compounds with the PI3K inhibitors IX, sixteen compounds were selected to be evaluated
for their ability to inhibit the activity of PI3K in vitro. The compounds were initially
screened against PI3K (p110α/p85α) protein kinase at a single dose of 50 µM by
employing the standardized ADP-GIoTM assay methodology at Kinexus Labs [49]. The
profiling data in Table 5 showed potent to weak inhibition of PI3K (p110α/p85α) activity
by the 4-chlorophenylsulfonylpyrazoline derivatives 9c, 9d and 9f only. Compound 9c
bearing unsubstituted phenyl group at position 5 of pyrazoline ring exhibited the highest
inhibition percentage (100%). Meanwhile the 4-tolyl 9d and 4-trifluromethylphenyl 9f
analogs inhibited the target enzyme by 20% and 19%, respectively, which suggested poor
bulk tolerance regarding substitution on this phenyl. Based on these preliminary results, the
IC50 value of the most active enzyme inhibitor 9c was determined in comparison with the
known isoform-nonspecific PI3K inhibitor wortmannin [50] as reference. Moreover, the
9

ACCEPTED MANUSCRIPT
previously reported data for the p110α inhibitor IX was listed in table 5 for comparison
[51]. Compound 9c showed mild inhibitory activity (IC50 = 50.78 ꢀM) compared to potent
enzyme inhibition demonstrated by wortmannin (IC50 =1.61 ꢀM) and the lead compound
IX (IC50 =0.67 ꢀM).
The inconsistency between the enzyme inhibitory activity and cellular efficiency against
HCT-116 suggested that the strutcural modifications made in the target compounds might
have switched the binding affinity to other biomolecular target involved in cellular
signaling pathways. Hence, the definite mechanism needs to be further investigated.
3. Conclusion
Two groups of coumarin-pyrazoline hybrids featuring an (un)substituted
phenylsulfonyl moieties, 7a-f, 8a-f and 9a-f, or a terminal sulfamoyl function 10a-f were
prepared. Among other compounds selected and evaluated according to the protocol of the
Drug Evaluation Branch, NCI, Bethesda, compounds 7d, 8c and 9c exhibited excellent
anticancer activities against the entire panel of tumor cell lines with the
chlorophenylsulfonyl analogue 9c being the most potent, in particular against breast cancer
MCF7 and colon cancer HCT-116. Subsequently, all target derivatives were screened
against human colon cancer cell line (HCT-116) where sixteen compounds were found
more potent than doxorubicin especially compound 9c having an IC50 of 0.01 µM (IC50 of
doxorubicin= 0.63 µM). SAR study showed that grafting an electron-withdrawing chlorine
atom to the 4th position of the phenylsulfonyl moiety was profitable to the antitumor
activity of the first group of compounds, 7a-f, 8a-f and 9a-f. Meanwhile, there was no
absolute relation between the activity and the nature of the aryl substituent at the 5th
position of pyrazoline ring in the same group of compounds. Conversely, the cytotoxicity
of the second group of compounds 10a-f was enhanced by the presence of a strong
electron-withdrawing group on the 5-phenyl substituent. Moreover, the relation between
activity and the location of the sulfonyl group, either internal as in the first group of
compounds or free terminal sulfamoyl as in the second group, was not consistent
throughout all derivatives.
Finally, the investigation was extended by screening sixteen selected compounds for their
ability to inhibit PI3K (P110α/p85α) as potential molecular target. Among them, the most
10

ACCEPTED MANUSCRIPT
potent antitumor agent 9c emerged as a promising enzyme inhibitor (IC50=50.78 ꢀM).
Despite of such mild enzyme inhibitory effect, the significance of 9c is that it might
represent a new lead for future optimization of more potent PI3K inhibitors.
4. Experimental
4.1. Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers and
used without further purification. Reactions' time and purity of the products were
monitored by TLC on FLUKA silica gel TLC aluminum cards (0.2 mm thickness) with
fluorescent indicator 254 nm using chloroform: methanol (9:1) as eluent. Melting points
were performed on Stuart SMP3 version 5 digital melting point apparatus and were
uncorrected. Elemental microanalyses were performed at the microanalytical center,
Faculty of Science, Cairo University and the Regional Center for Mycology and
Biotechnology, Al-Azhar University. 1HNMR spectra were determined using Varian
Mercury VX-300 MHz or 200 MHz NMR Spectrometer (Oxford, England). 13CNMR
spectra were run at 75.46 MHz in deuterated chloroform (CDCl3) or dimethylsulphoxide
(DMSO-d6). Chemical Shifts are given in δ as parts per million (ppm) downfield from
tetramethylsilane (TMS) as internal standard. Mass spectra were recorded using Hewlett
Packard Varian (Varian, Polo, USA), Shimadzu Gas Chromatograph Mass spectrometer-
QP 1000 EX (Shimadzu, Kyoto, Japan) and Finnegan MAT, SSQ 7000 mass
spectrophotometer at 70eV. IR spectra were recorded on Bruker FT-IR spectrophotometer
as potassium bromide discs.
4.1.1. 7-Acetoxy-2H-chromen-2-one (2).
Compound 2 was prepared according to the literature procedure [52] (m.p.147oC, as
reported).
4.1.2. 8-Acetyl-7-hydroxy-2H-chromen-2-one (4).
Compound 4 was prepared according to the literature procedure [52] (m.p.167oC, as
reported).
4.1.3. 8-Acetyl-7-methoxy-2H-chromen-2-one (5).
11

ACCEPTED MANUSCRIPT
Compound 5 was prepared according to the literature procedure [53- 55] (m.p.123oC, as
reported).
4.1.4. General procedure for synthesis of 7-Methoxy-8-[arylacryloyl]-2H-chromen-2-ones
6a-f.
Compounds of this series (6a-f) were prepared according to the literature procedure [41].
4.1.5. General procedure for the preparation of 8-(5-substituted-1-(phenylsulfonyl)-4,5-
dihydro-1H-pyrazol-3-yl)-7-methoxy-2H-chromen-2-ones 7a-f:
Phenylsulphonyl hydrazine (1.72 g, 10 mmol) was added to a solution of the appropriate
propenone derivative 6a-f (10 mmol) in absolute ethanol (50 ml) and refluxed for 12 h. The
reaction mixture was filtered while hot and then left overnight. The solid product was
filtered and crystallized from ethanol.
4.1.5.1. 7-Methoxy-8-[5-(5-methylfuran-2-yl)-1-(phenylsulfonyl)-4,5-dihydro-1H-pyrazol-
3-yl]-2H-chromen-2-one (7a):
Yield: 35%, m.p. 194-196oC. IR υmax/cm-1: 3032 (CH aromatic), 2943, 2843 (CH aliphatic),
1720 (C=O), 1620, 1597 (C=C, C=N), 1300, 1141 (SO2). 1H NMR (DMSO-d6) δ ppm: 2.18
(s, 3H, CH3), 2.85 (dd, 1H, C4-HA pyrazoline, JAX = 8.4 Hz and JAM = 18.0 Hz), 3.10 (dd,
1H, C4-HM pyrazoline, JMX = 11.0 Hz and JMA = 18.0 Hz), 3.88 (s, 3H, OCH3), 5.80 (dd,
1H, C5-HX pyrazoline, JXM = 11.0 Hz and JXA = 8.4 Hz), 6.29 (d, 1H, C3-H chromen, J =
9.6 Hz), 6.32 (d, 1H, C4-H furan, J = 1.2 Hz), 6.51-7.20 (m, 7H, C3-H furan, C6-H
chromen and ArH), 7.78 (d, 1H, C5-H chromen, J = 8.7 Hz), 8.05 (d,1H, C4-H chromen, J
= 9.6 Hz). Anal. Calcd for C24H20N2O6S (464.29): C, 62.06; H, 4.34; N, 6.03. Found: C,
61.70; H, 4.22; N, 6.33.
4.1.5.2. 7-Methoxy-8-[1-(phenylsulfonyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-3-yl]-
2H-chromen-2-one (7b):
Yield: 55%, m.p. 168-170oC. IR υmax/cm-1: 3066 (CH aromatic), 2966, 2939 (CH aliphatic),
1724 (C=O), 1605, 1597 (C=C, C=N), 1342, 1168 (SO2). 1H NMR (CDCl3) δ ppm: 3.29 (d,
2H, J = 10 Hz, CH2 pyrazoline), 3.80 (s, 3H, OCH3), 5.34 (t, 1H, C5-H pyrazoline, J = 11.0
Hz), 6.31 (d, 1H, C3-H chromen, J = 9.6 Hz), 6.94 (t, 1H, C4-H thiophene, J = 8.1 Hz),
7.14 (d, 1H, C6-H chromen, J = 9.6 Hz), 7.46 (d, 1H, C3-H thiophene, J = 8.6 Hz), 7.53 (d,
12

ACCEPTED MANUSCRIPT
1H, C5-H thiophene, J = 8.7 Hz), 7.58 (d, 1H, C5-H chromen, J = 9.6 Hz), 7.69-7.84 (m,
5H, ArH), 7.99 (d, 1H, C4-H chromen, J = 9.6 Hz). 13C NMR (DMSO d6) δ ppm: 40.1 (C-
4 pyrazoline), 40.4 (C-5 pyrazoline), 56.3 (OCH3), 106.9 (C-6 chromen), 108.6 (C-3
chromen), 112.8 (C-4a chromen), 113.3 (C-8 chromen), 126.4 -132.5 (C-5 chromen,
aromatic Cs and thiophene Cs), 139.6 (C-SO2), 143.9 (C-4 chromen), 144.9 (C-8a
chromen), 152.1 (C=N pyrazoline), 159.1 (C-7 chromen), 159.6 (C=O). MS, m/z 466.15:
[M+]. Anal. Calcd for C23H18N2O5S2 (466.53): C, 59.21; H, 3.89; N, 6.00. Found: C, 58.95;
H, 4.02; N, 5.84.
4.1.5.3. 7-Methoxy-8-[5-phenyl-1-(phenylsulfonyl)-4,5-dihydro-1H-pyrazol-3-yl]-2H-
chromen-2-one (7c):
Yield: 65%, m.p. 196-198oC. IR υmax/cm-1: 3066 (CH aromatic), 2947, 2908 (CH aliphatic),
1732 (C=O), 1604, 1592 (C=C, C=N), 1303, 1145 (SO2). 1HNMR (CDCl3) δ ppm: 3.25
(dd, 1H, C4-HA pyrazoline, JAX =6.9 Hz and JAM = 21.9 Hz), 3.90 (dd, 1H, C4-HM
pyrazoline, JMX = 15.0 Hz and JMA = 21.9 Hz), 3.83 (s, 3H, OCH3), 4.80 (dd, 1H, C5-HX
pyrazoline, JXM = 15.0 Hz and JXA = 6.9 Hz), 6.21 (d, 1H, C3-H chromen, J = 9.3 Hz), 6.84
(d, 1H, C6-H chromen, J = 8.7 Hz), 7.18-7.37 (m, 10H, ArH), 7.40 (d, 1H, C5-H chromen,
J = 8.7 Hz), 7.59 (d, 1H, C4-H chromen, J = 8.7 Hz). Anal. Calcd for C25H20N2O5S
(460.50): C, 65.20; H, 4.38; N, 6.08. Found C, 65.42; H, 4.75; N, 6.01.
4.1.5.4. 7-Methoxy-8-[1-(phenylsulfonyl)-(4-methylphenyl)-4,5-dihydro-1H-pyrazol-3-yl]-
2H-chromen-2-one (7d):
Yield: 65%, m.p. 237-239 oC. IR υmax/cm-1: 3062 (CH aromatic), 2974, 2843 (CH
aliphatic), 1728 (C=O), 1647, 1604 (C=C, C=N), 1300, 1145 (SO2). 1H NMR (CDCl3) δ
ppm: 2.28 (s, 3H, CH3), 3.15 (dd, 1H, C4-HA pyrazoline, JAX =8.0 Hz and JAM = 20.4 Hz),
3.83 (s, 3H, OCH3), 3.96 (dd, 1H, C4-HM pyrazoline, JMX = 16.0 Hz and JMA = 20.4 Hz),
4.80 (dd, 1H, C5-HX pyrazoline, JXM = 16.0 Hz and JXA = 8.0 Hz), 6.25 (d, 1H, C3-H
chromen, J = 9.6 Hz), 6.81 (d, 1H, C6-H chromen, J = 8.7 Hz), 6.92 (d, 1H, C5-H
chromen, J = 8.7 Hz), 7.10-7.57 (m, 9H, ArH), 7.67 (d, 1H, C4-H chromen, J = 8.6 Hz).
MS, m/z 474.10: [M+]. Anal. Calcd for C26H22N2O5S (474.53): C, 65.81; H, 4.67; N, 5.90.
Found C, 66.00; H, 5.10; N,5.50.
4.1.5.5. 7-Methoxy-8-[1-(phenylsulfonyl)-5-(4-(trifluoromethyl)phenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-2H-chromen-2-one (7e):
13

ACCEPTED MANUSCRIPT
Yield: 65%, m.p. 222-224oC. IR υmax/cm-1: 3066 (CH aromatic), 2962, 2850 (CH aliphatic),
1732 (C=O), 1645, 1604 (C=C, C=N), 1327, 1119 (SO2). 1H NMR (CDCl3) δ ppm:
3.20 (dd, 1H, C4-HA pyrazoline, JAX =8.4 Hz and JAM = 20.4 Hz), 3.83 (s, 3H, OCH3), 3.94
(dd, 1H,C4-HM pyrazoline, JMX = 16.0 Hz and JMA = 20.4 Hz), 4.96 (dd, 1H, C5-HX
pyrazoline, JXM = 16.0 Hz and JXA = 8.4 Hz), 6.23 (d, 1H, C3-H chromen, J = 9.6 Hz), 6.82
(d, 1H, C6-H chromen, J = 8.7 Hz), 7.11 (d, 1H, C5-H chromen, J = 8.7 Hz), 7.26-7.59 (m,
9H, ArH), 7.69 (d, 1H, C4-H chromen, J = 8.6 Hz). 13C NMR (CDCl3): δ 42.4 (C-4
pyrazoline) , 45.6 (C-5 pyrazoline), 56.5 (OCH3), 107.7 (C-6 chromen), 112.9 (C-3
chromen), 114.2 (C-4a chromen and C-8 chromen), 125.8-130.5 (aromatic Cs and CF3),
134.1 (C-5 chromen), 136.6 (C-4 of C6H4CF3 and C-4 of C6H5SO2), 142.7 (C-SO2), 142.9
(C-4 chromen), 143.8 (C-1 of C6H4CF3), 150.0 (C-8a chromen), 152.1 (C=N pyrazoline),
159.0 (C-7 chromen), 159.7 (C=O). Anal. Calcd for C26H19F3N2O5S (528.50): C, 59.09; H,
3.62; N, 5.30. Found: C, 59.10; H, 3.70; N, 5.40.
4.1.5.6. 7-Methoxy-8-[5-(4-(methylthio)phenyl)-1-(phenylsulfonyl)-4,5-dihydro-1H-
pyrazol-3-yl]-2H-chromen-2-one (7f):
Yield: 50%, m.p. 189-191 oC. IR υmax/cm-1: 3020 (CH aromatic), 2950, 2850 (CH
aliphatic), 1732 (C=O), 1651, 1604 (C=C, C=N), 1304, 1078 (SO2). 1H NMR (CDCl3) δ
ppm: 2.51 (s, 3H, CH3), 3.06 (dd, 1H, C4-HA pyrazoline, JAX =8.0 Hz and JAM = 20.4 Hz),
3.40 (dd, 1H, C4-HM pyrazoline, JMX = 15.0 Hz and JMA = 20.4 Hz), 3.86 (s, 3H, OCH3),
4.80 (dd, 1H, C5-HX pyrazoline, JXM = 15.0 Hz and JXA = 8.0 Hz), 6.26 (d, 1H, C3-H
chromen, J = 9.6 Hz), 6.84 (d, 1H, C6-H chromen, J = 8.7 Hz), 6.93-7.46 (m, 9H, ArH),
7.50 (d, 1H, C5-H chromen, J = 8.7 Hz), 7.64 (d, 1H, C4-H chromen, J = 9.6 Hz). Anal.
Calcd for C26H22N2O5S2 (506.59): C, 61.64; H, 4.38; N, 5.53. Found: C, 61.59; H, 4.99; N,
5.56.
4.1.6. General procedure for the preparation of 8-[5-substituted-1-(4-methylbenzene
sulfonyl)-4,5-dihydro-1H-pyrazol-3-yl]-7-methoxy-2H-chromen-2-ones 8a-f:
The appropriate propenone derivative 6a-f (10 mmol) was dissolved in absolute ethanol (50
ml), then 4-methylphenylsulphonyl hydrazine (1.86, 10 mmol) was added and the reaction
mixture was heated under reflux for 12 h. The mixture was filtered while hot, the filtrate
was concentrated under vacuum and the separated solid was crystallized from methanol.
14

ACCEPTED MANUSCRIPT
4.1.6.1. 8-[1-(4-Methylphenylsulfonyl)-5-(5-methylfuran-2-yl)-4,5-dihydro-1H-pyrazol-3-
yl]-7-methoxy-2H-chromen-2-one (8a):
Yield: 55%, m.p. 184-186 oC. IR υmax/cm-1: 3062 (CH aromatic), 2943, 2920 (CH
aliphatic), 1732 (C=O), 1631, 1604 (C=C, C=N), 1296, 1145 (SO2). 1H NMR (CDCl3) δ
ppm: 2.26 (s, 3H, CH3), 2.48 (s, 3H, CH3), 3.20 (dd, 1H, C4-HA pyrazoline, JAX =8.0 Hz
and JAM = 20.4 Hz), 3.91 (s, 3H, OCH3), 4.15 (dd, 1H, C4-HM pyrazoline, JMX = 16.0 Hz
and JMA = 20.4 Hz), 4.95 (dd, 1H, C5-HX pyrazoline, JXM = 16.0 Hz and JXA = 8.0 Hz),
6.15 (d, 1H, furan H, J = 1.2 Hz), 6.20 (d, 1H, C3-H chromen, J = 9.4 Hz), 6.50 (d, 1H,
furan H, J = 1.2 Hz), 6.84 (d, 1H, C6-H chromen, J = 8.7 Hz), 7.26-7.57 (m, 4H, Ar H),
7.62 (d, 1H, C5-H chromen, J = 8.7 Hz), 7.81 (d, 1H, C4-H chromen, J = 9.6 Hz). Anal.
Calcd for C25H22N2O6S (478.52): C, 62.75; H, 4.63; N, 5.85. Found: C, 62.79; H, 5.51; N,
5.85.
4.1.6.2. 8-[1-(4-Methylphenylsulfonyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-3-yl]-7-
methoxy-2H-chromen-2-one (8b):
Yield: 50%, m.p. 158-160 oC. IR υmax/cm-1: 3093 (CH aromatic), 2970, 2939 (CH
aliphatic), 1724 (C=O), 1604, 1558 (C=C, C=N), 1290, 1168 (SO2). 1H NMR (CDCl3) δ
ppm: 2.45 (s, 3H, CH3), 3.50 (d, 2H, J = 10 Hz, CH2 pyrazoline), 3.86 (s, 3H, OCH3), 4.95
(t, 1H, C5-H pyrazoline, J = 11.2 Hz), 6.20 (d, 1H, C3-H thiophene, J = 8.6 Hz), 6.30 (d,
1H, C3-H chromen, J = 9.6 Hz), 6.90 (t, 1H, C4-H thiophene, J = 8.1 Hz), 7.10 (d, 1H, C5-
H thiophene, J = 8.7 Hz), 7.30 (d, 1H, C6-H chromen, J = 9.6 Hz), 7.69 (m, 3H, C5-H
chromen and ArH), 7.81 (d, 1H, C4-H chromen, J = 9.6 Hz), 7.91 (d, 2H, ArH, J = 8.1 Hz).
13C NMR(CDCl3): 22.0 (CH3), 46.0 (C-4 pyrazoline), 47.5 (C-5 pyrazoline), 56.4 (OCH3),
107.9 (C-6 chromene), 112.9 (C-3 chromen), 113.7 (C-4a chromen), 117.3 (C-8 chromen),
126.7-132.0 (C-5 chromen and aromatic Cs), 138.7 (C-SO2), 139.5 (C-4 of C6H4CH3),
143.1 (C-4 chromen), 151.8 (C-8a chromen and C=N pyrazoline), 159.3 (C-7 chromen),
159.9 (C=O). MS, m/z 481.50: [M++1]. Anal. Calcd for C24H20N2O5S2 (480.56): C, 59.98;
H, 4.19; N, 5.83. Found: C, 59.83; H, 4.08; N, 5.60.
4.1.6.3. 8-[1-(4-Methylphenylsulfonyl)-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl]-7-methoxy-
2H-chromen-2-one (8c):
Yield: 68%, m.p. 186-188oC. IR υmax/cm-1: 3090 (CH aromatic), 2947, 2912 (CH aliphatic),
1739 (C=O), 1602, 1562 (C=C, C=N), 1292, 1111 (SO2). 1H NMR (CDCl3) δ ppm: 2.40
15

ACCEPTED MANUSCRIPT
(s, 3H, CH3), 3.10 (dd, 1H, C4-HA pyrazoline, JAX =8.7 Hz and JAM = 20.4 Hz), 3.82 (s,
3H, OCH3), 3.91 (dd, 1H, C4-HM pyrazoline, JMX = 15.6 Hz and JMA = 20.4 Hz), 4.80 (dd,
1H, C5-HX pyrazoline, JXM = 15.6 Hz and JXA = 8.7 Hz), 6.23 (d, 1H, C3-H chromen, J =
9.6 Hz), 6.80 (d, 1H, C6-H chromen, J = 8.4 Hz), 7.17-7.27 (m, 9H, ArH), 7.41 (d, 1H, C5-
H chromen, J = 8.4 Hz), 7.58 (d, 1H, C4-H chromen, J = 9.6 Hz). Anal. Calcd for
C26H22N2O5S (474.53): C, 65.81; H, 4.67; N, 5.90. Found: C, 66.00; H, 4.58; N, 5.96.
4.1.6.4. 8-[1-(4-Methylphenylsulfonyl)-5-(4-methylphenyl)-4,5-dihydro-1H-pyrazol-3-yl]-
7-methoxy-2H-chromen-2-one (8d):
Yield: 65%, m.p. 204-207oC. IR υmax/cm-1: 3093 (CH aromatic), 2947, 2912 (CH aliphatic),
1732 (C=O), 1600, 1562 (C=C, C=N), 1292, 1168 (SO2). 1H NMR (CDCl3) δ ppm: 2.41 (s,
3H, CH3), 2.91 (s, 3H, CH3), 3.40 (dd, 1H, C4-HA pyrazoline, JAX =8.0 Hz and JAM = 20.4
Hz), 3.82 (s, 3H, OCH3), 4.10 (dd, 1H, C4-HM pyrazoline, JMX = 16.0 Hz and JMA = 20.4
Hz), 4.95 (dd, 1H, C5-HX pyrazoline, JXM = 16.0 Hz and JXA = 8.0 Hz), 6.20 (d, 1H, C3-H
chromen, J = 9.3 Hz), 6.82 (d, 1H, C6-H chromen, J = 8.7 Hz), 7.27-7.60 (m, 8H, Ar H),
7.76 (d, 1H, C5-H chromen, J = 8.4 Hz), 7.80 (d, 1H, C4-H chromen, J = 9.6 Hz). 13C
NMR (CDCl3): 21.4 (CH3), 37.3 (C-4 pyrazoline), 42.5 (C-5 pyrazoline), 56.1 (OCH3),
107.6 (C-6 chromen), 113.0 (C-3 chromen), 114.0 (C-4a chromen and C-8 chromen),
124.8-130.6 (C-5 chromen and aromatic Cs), 136.0 (C-4 of C6H4CH3), 140.0 (C-SO2),
142.8 (C-4 of C6H4CH3SO2), 144.0 (C-4 chromen), 151.1 (C-8a chromen and C=N
pyrazoline), 158.7 (C-7 chromen), 159.4 (C=O). Anal. Calcd for C27H24N2O5S (488.55): C,
66.38; H, 4.95; N, 5.73. Found: C, 66.10; H, 5.43; N, 5.35.
4.1.6.5. 8-[1-(4-Methylphenylsulfonyl)-5-(4-(trifluromethyl)phenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-7-methoxy-2H-chromen-2-one (8e):
Yield: 60%, m.p. 165-168oC. IR υmax/cm-1: 3028 (CH aromatic), 2893, 2839 (CH aliphatic),
1732 (C=O), 1600, 1558 (C=C, C=N), 1296, 1165 (SO2). 1H NMR (CDCl3) δ ppm: 2.44 (s,
3H, CH3), 3.45 (dd, 1H, C4-HA pyrazoline, JAX =8.4 Hz and JAM = 20.2 Hz), 3.89 (s, 3H,
OCH3), 4.15 (dd, 1H, C4-HM pyrazoline, JMX = 16.8 Hz and JMA = 20.2 Hz), 4.80 (dd, 1H,
C5-HX pyrazoline, JXM = 16.8 Hz and JXA = 8.4 Hz), 6.10 (d, 1H, C3-H chromen, J = 9.3
Hz), 6.40 (d, 1H, C6-H chromen, J = 8.7 Hz), 6.80-7.40 (m, 8H, Ar H), 7.69 (d, 1H, C5-H
chromen, J = 8.7 Hz), 7.81 (d, 1H, C4-H chromen, J = 9.6 Hz). Anal. Calcd for
C27H21F3N2O5S (542.53): C, 59.77; H, 3.90; N, 5.16. Found: C, 60.04; H, 4.20; N, 4.83.
16

ACCEPTED MANUSCRIPT
4.1.6.6. 8-[1-(4-Methylphenylsulfonyl)-5-(4-(methylthio)phenyl)-4,5-dihydro-1H-pyrazol-3-
yl]-7-methoxy-2H-chromen-2-one (8f):
Yield: 65%, m.p. 170-172oC. IR υmax/cm-1: 3059 (CH aromatic), 2989, 2920 (CH aliphatic),
1732 (C=O), 1604, 1562 (C=C, C=N), 1265, 1087 (SO2). 1H NMR (CDCl3) δ ppm: 2.40 (s,
3H, CH3), 2.49 (s, 3H, S-CH3), 3.20 (dd, 1H, C4-HA pyrazoline, JAX =8.1 Hz and JAM
=
20.4 Hz), 3.80 (dd, 1H, C4-HM pyrazoline, JMX = 16.2 Hz and JMA = 20.4 Hz), 3.85 (s, 3H,
OCH3), 4.80 (dd, 1H, C5-HX pyrazoline, JXM = 16.2 Hz and JXA = 8.1 Hz), 6.21 (d, 1H, C3-
H chromen, J = 9.3 Hz), 6.82 (d, 1H, C6-H chromen, J = 8.4 Hz), 7.07-7.43 (m, 8H, ArH),
7.45 (d, 1H, C5-H chromen, J = 8.4 Hz), 7.66 (d, 1H, C4-H chromen, J = 9.6 Hz). 13C
NMR (CDCl3): 15.0 (SCH3), 21.6 (CH3), 41.5 (C-5 pyrazoline), 56.5 (OCH3), 65.9 (C-4
pyrazoline), 108.6 (C-6 chromen), 112.7 (C-3 chromen), 112.9 (C-4a chromen), 116.6 (C-8
chromen), 125.4-130.7 (C-5 chromen and aromatic Cs) , 139.4 (C-SO2), 142.9 (C-1 of
C6H4SCH3), 144.8 (C-4 chromen), 145.8 (C-4 of C6H4CH3SO2), 151.8 (C-8a chromen and
C=N pyrazoline), 158.7 (C-7 chromen), 159.4 (C=O). MS m/z 521.25: [M+]. Anal. Calcd
for C27H24N2O5S2 (520.62): C, 62.29; H, 4.65; N, 5.38. Found: C, 62.41; H, 3.88; N, 4.96.
4.1.7. General procedure for the preparation of 8-[5-substituted-1-(4-chlorophenyl
sulfonyl)-4,5-dihydro-1H-pyrazol-3-yl]-7-methoxy-2H-chromen-2-ones 9a-f:
4-Chlorophenylsulphonyl hydrazine (2.06 g, 10 mmol) was added to a solution of the
appropriate propenone derivative 6a-f (10 mmol) in absolute ethanol (50 ml) and heated
under reflux for 8 h. The mixture was filtered while hot, the filtrate was concentrated under
vacuum and the separated solid was crystallized from absolute ethanol.
4.1.7.1. 8-[1-(4-Chlorophenylsulfonyl)-5-(5-methylfuran-2-yl)-4,5-dihydro-1H-pyrazol-3-
yl]-7-methoxy-2H-chromen-2-one (9a):
Yield: 50%, m.p. 180-182oC. IR υmax/cm-1: 3032 (CH aromatic), 2943, 2843 (CH aliphatic),
1720 (C=O), 1620, 1597 (C=C, C=N), 1300, 1141 (SO2). 1H NMR (CDCl3) δ ppm: 2.34 (s,
3H, CH3), 3.10 (dd, 1H, C4-HA pyrazoline, JAX =8.0 Hz and JAM = 20.4 Hz), 3.70 (dd, 1H,
C4-HM pyrazoline, JMX = 16.0 Hz and JMA = 20.4 Hz), 3.88 (s, 3H, OCH3), 5.90 (dd, 1H,
C5-HX pyrazoline, JXM = 16.0 Hz and JXA = 8.0 Hz), 6.10 (d, 1H, furan H, J = 1.2 Hz), 6.30
(d, 1H, C3-H chromen, J = 9.4 Hz), 6.50 (d, 1H, furan H, J = 1.2 Hz), 6.80 (d, 1H, C6-H
chromen, J = 8.4 Hz), 7.30 (d, 1H, C5-H chromen, J = 8.4 Hz), 7.50 (d, 2H, Ar H, J = 8.1
17

ACCEPTED MANUSCRIPT
Hz), 7.66 (d, 2H, ArH, J = 9.0 Hz), 7.80 (d, 1H, C4-H chromen, J = 9.6 Hz). Anal. Calcd
for C24H19ClN2O6S (498.93): C, 57.77; H, 3.84; N, 5.61. Found: C, 58.04 H, 4.26; N, 5.24.
4.1.7.2. 8-[1-(4-Chlorophenylsulfonyl)-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-3-yl]-7-
methoxy-2H-chromen-2-one (9b):
Yield: 50%, m.p. 178-180oC. IR υmax/cm-1: 3032 (CH aromatic), 2943, 2843 (CH aliphatic),
1724 (C=O), 1600, 1581 (C=C, C=N), 1320, 1168 (SO2). 1H NMR (CDCl3) δ ppm: 3.21 (d,
2H, J = 11.4 Hz, CH2 pyrazoline), 3.87 (s, 3H, OCH3), 4.30 (t, 1H, C5-H pyrazoline, J =
11.2 Hz), 6.22 (d, 1H, C3-H chromen, J = 9.0 Hz), 6.66 (d, 1H, C3-H thiophene, J = 8.7
Hz), 6.76 (d, 1H, C6-H chromen, J = 9.6 Hz), 6.90 (t, 1H, C4-H thiophene, J = 8.6 Hz),
7.10 (d, 1H, C5-H thiophene, J = 8.7 Hz), 7.30 (d, 1H, C5-H chromen, J = 9.6 Hz), 7.69 (d,
2H, ArH, J = 8.1 Hz), 7.81 (d, 1H, C4-H chromen, J = 9.0 Hz), 7.99 (d, 2H, ArH, J = 8.1
Hz). Anal. Calcd for C23H17ClN2O5S2 (500.17): C, 55.14; H, 3.42; N, 5.59. Found: C,
55.50; H, 3.73; N, 5.89.
4.1.7.3. 8-[1-(4-Chlorophenylsulfonyl)-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl]-7-methoxy-
2H-chromen-2-one (9c):
Yield: 68%, m.p. 202-204oC. IR υmax/cm-1: 3032 (CH aromatic), 2943, 2843 (CH aliphatic),
1735 (C=O), 1604, 1560 (C=C, C=N), 1310, 1114 (SO2). 1H NMR (CDCl3) δ ppm: 3.10
(dd, 1H, C4-HA pyrazoline, JAX = 6.9 Hz and JAM = 20.4 Hz), 3.82 (s, 3H, OCH3), 3.93 (dd,
1H, C4-HM pyrazoline, JMX = 15.6 Hz and JMA = 20.4 Hz), 4.80 (dd, 1H, C5-HX
pyrazoline, JXM = 15.6 Hz and JXA = 6.9 Hz), 6.23 (d, 1H, C3-H chromen, J = 9.6 Hz), 6.80
(d, 1H, C6-H chromen, J = 8.4 Hz), 7.17-7.27 (m, 9H, ArH), 7.41 (d, 1H, C5-H chromen, J
= 8.4 Hz), 7.58 (d, 1H, C4-H chromen, J = 9.6 Hz). MS, m/z 494.95: [M+]. Anal. Calcd for
C25H19ClN2O5S (494.94): C, 60.67; H, 3.87; N, 5.66. Found: C, 60.69; H, 3.63; N, 5.60.
4.1.7.4. 8-[1-(4-Chlorophenylsulfonyl)-5-(4-methylphenyl)-4,5-dihydro-1H-pyrazol-3-yl]-
7-methoxy-2H-chromen-2-one (9d):
Yield: 65%, m.p. 177-179oC. IR υmax/cm-1: 3032 (CH aromatic), 2943, 2843 (CH aliphatic),
1732 (C=O), 1604, 1560 (C=C, C=N), 1292, 1168 (SO2). 1H NMR (CDCl3) δ ppm: 2.30 (s,
3H, CH3), 3.21 (dd, 1H, C4-HA pyrazoline, JAX = 8.4 Hz and JAM = 20.4 Hz), 3.82 (s, 3H,
OCH3), 3.90 (dd, 1H, C4-HM pyrazoline, JMX = 16.8 Hz and JMA = 20.4 Hz), 4.95 (dd, 1H,
C5-HX pyrazoline, JXM = 16.8 Hz and JXA = 8.4 Hz), 6.25 (d, 1H, C3-H chromen, J = 9.6
Hz), 6.85 (d, 1H, C6-H chromen, J = 8.7 Hz), 6.80-7.50 (m, 8H, Ar H), 7.60 (d, 1H, C5-H
18

ACCEPTED MANUSCRIPT
chromen, J = 8.7 Hz), 7.76 (d, 1H, C4-H chromen, J = 9.6 Hz). 13C NMR(CDCl3): 21.4
(CH3), 37.3 (C-4 pyrazoline), 42.5 (C-5 pyrazoline), 56.1 (OCH3), 107.6 (C-6 chromen),
113.0 (C-3 chromen), 114.0 (C-4a, C-8 chromen), 124.8-130.6 (C-5 chromen and aromatic
Cs), 138.0 (C-Cl and C-SO2), 142.6 (C-1 of C6H4CH3 and C-4 chromen, 151.1 (C-8a
chromen and C=N pyrazoline), 158.7 (C-7 chromen), 159.4 (C=O). Anal. Calcd for
C26H21ClN2O5S (508.97): C, 61.35; H, 4.16; N, 5.50. Found: C, 61.62; H, 5.89; N, 5.74.
4.1.7.5. 8-[1-(4-Chlorophenylsulfonyl)-5-(4-(trifluromethyl)phenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-7-methoxy-2H-chromen-2-one (9e):
Yield: 60%, m.p. 232-234oC. IR υmax/cm-1: 3032 (CH aromatic), 2943, 2843 (CH aliphatic),
1732 (C=O), 1604, 1562 (C=C, C=N), 1327, 1118 (SO2). 1H NMR (CDCl3) δ ppm: 3.20
(dd, 1H, C4-HA pyrazoline, JAX = 8.4 Hz and JAM = 20.4 Hz), 3.83 (s, 3H, OCH3), 3.91 (dd,
1H, C4-HM pyrazoline, JMX = 16.8 Hz and JMA = 20.4 Hz), 4.80 (dd, 1H, C5-HX
pyrazoline, JXM = 16.8 Hz and JXA = 8.4 Hz), 6.10 (d, 1H, C3-H chromen, J = 9.3 Hz), 6.40
(d, 1H, C6-H chromen, J = 8.7 Hz), 6.80-7.40 (m, 8H, ArH), 7.60 (d, 1H, C5-H chromen, J
= 8.7 Hz), 7.81 (d, 1H, C4-H chromen, J = 9.6 Hz). Anal. Calcd for C26H18ClF3N2O5S
(562.94): C, 55.47; H, 3.22; N, 4.98. Found: C, 55.81; H, 3.18; N, 4.88.
4.1.7.6. 8-[1-(4-Chlorophenylsulfonyl)-5-(4-(methylthio)phenyl)-4,5-dihydro-1H-pyrazol-
3-yl]-7-methoxy-2H-chromen-2-one (9f):
Yield: 65%, m.p. 206-208oC. IR υmax/cm-1: 3032 (CH aromatic), 2943, 2843 (CH aliphatic),
1730 (C=O), 1600, 1586 (C=C, C=N), 1303, 1145 (SO2). 1H NMR (DMSO-d6) δ ppm: 2.40
(s, 3H, S-CH3), 2.84 (dd, 1H, C4-HA pyrazoline, JAX = 8.1 and JMA = 20.4 Hz), 3.20 (dd,
1H, C4-HM pyrazoline, JMX = 16.2 Hz and JMA = 20.4 Hz), 3.85 (s, 3H, OCH3), 4.10 (dd,
1H, C5-HX pyrazoline, JXM = 16.2 Hz and JXA = 8.1 Hz), 6.33 (d, 1H, C3-H chromen, J =
9.3 Hz), 7.06 (d, 1H, C6-H chromen, J = 8.4 Hz), 7.11-7.33 (m, 8H, ArH), 7.79 (d, 1H, C5-
H chromen, J = 8.4 Hz), 8.06 (d, 1H, C4-H chromen, J = 9.6 Hz). 13C NMR (DMSO- d6):
14.0 (SCH3), 40.3 (C-4 pyrazoline), 41.5 (C-5 pyrazoline), 56.5 (OCH3), 108.6 (C-6
chromen), 112.7 (C-3 chromen), 112.9 (C-4a chromen), 116.6 (C-8 chromen), 125.4-130.3
(C-5 chromen and aromatic Cs), 137.0 (C-4 of C6H4SCH3), 142.6 (C-Cl and C-SO2), 144.2
(C-1 of C6H4SCH3), 145.8 (C-4 chromen), 151.1 (C-8a chromen and C=N), 158.8 (C-7
chromen), 159.4 (C=O). MS, m/z 540.60: [M+]. Anal. Calcd for C26H21ClN2O5S2 (541.03):
C, 57.72; H, 3.91; N, 5.18. Found: C, 57.99; H, 4.12; N, 4.80.
19

ACCEPTED MANUSCRIPT
4.1.8. General procedure for the preparation of 8-[5-substituted-1-(4-sulfamoyl phenyl)-
4,5-dihydro-1H-pyrazol-3-yl]-7-methoxy-2H-chromen-2-ones 10a-f:
4-Sulphamoylphenyl hydrazine (1.87 g, 10 mmol) was added to a solution of the
appropriate propenone derivative 6a-f (10 mmol) in absolute ethanol (50 ml), refluxed for
16 h. The reaction mixture was concentrated under vacuum and the residue was crystallized
from ethanol.
4.1.8.1. 8-[4,5-Dihydro-5-(5-methylfuran-2-yl)-1-(4-sulfamoylphenyl)-4,5-dihydro-1H-
pyrazol-3-yl]-7-methoxy-2H-chromen-2-one (10a):
Yield: 70%, m.p.244-246oC. IR υmax/cm-1: 3367, 3253 (NH2), 3062 (CH aromatic), 2960,
2903 (CH aliphatic), 1728 (C=O), 1602, 1560 (C=C, C=N), 1301, 1163 (SO2). 1H NMR
(CDCl3) δ ppm: 2.35 (s, 3H, CH3), 3.89 (s, 3H, OCH3), 3.92 (d, 2H, J = 9.9 Hz, CH2
pyrazoline), 4.80 (t, 1H, C5-H pyrazoline, J = 11.3 Hz), 5.10 (s, 2H, NH2, exch.), 6.09 (d,
1H, furan H, J = 1.2 Hz), 6.24 (d, 1H, C3-H chromen, J = 9.4 Hz), 6.56 (d, 1H, furan H, J
= 1.2 Hz), 6.80-7.08 (m, 3H, C6-H chromen, ArH), 7.45 (d, 1H, C5-H chromen, J = 8.4
Hz), 7.66 (d, 1H, C4-H chromen, J = 9.4 Hz), 8.01 (d, 2H, ArH, J = 7.8 Hz). Anal. Calcd
for C24H21N3O6S (479.51): C, 60.12; H, 4.41; N, 8.76. Found: C, 60.32; H, 4.29; N, 9.03.
4.1.8.2. 8-[4,5-Dihydro-1-(4-sulfamoylphenyl)- 5-(thiophen-2-yl)-1H-pyrazol-3-yl]-7-
methoxy-2H-chromen-2-one (10b):
Yield: 66%, m.p.214-216 oC. IR υmax/cm-1: 3390, 3253 (NH2), 3086 (CH aromatic), 2970,
2918 (CH aliphatic), 1730 (C=O), 1602, 1582 (C=C, C=N), 1300, 1087 (SO2). 1H NMR
(CDCl3) δ ppm: 3.40 (dd, 1H, C4-HA pyrazoline, JAX = 8.1 Hz and JAM = 16.5 Hz), 3.90 (s,
3H, OCH3), 4.30 (dd, 1H, C4-HM pyrazoline, JMX = 10.5 Hz and JMA = 16.5 Hz), 4.60 (s,
2H, NH2, exch.), 4.80 (dd, 1H, C5-HX pyrazoline, JXM = 10.5 Hz and JXA = 8.1 Hz), 6.27
(d, 2H, C3-H thiophene, C3-H chromen, J = 9.6 Hz), 6.80 (t, 1H, C4-H thiophene, J = 8.3
Hz), 6.95 (d, 1H, C6-H chromen, J = 8.4 Hz), 7.17-7.40 (m, 3H, C5-H thiophene, ArH),
7.73 (d, 1H, C5-H chromen, J = 8.4 Hz), 7.81 (d, 1H, C4-H chromen, J = 9.6 Hz), 7.95 (d,
2H, ArH, J = 7.8 Hz). MS, m/z 483.55: [M++2]. Anal. Calcd for C23H19N3O5S2 (481.54):
C, 57.37; H, 3.98; N, 8.73. Found: C, 57.53; H, 4.12; N, 9.18.
4.1.8.3. 8-[4,5-Dihydro-1-(4-sulfamoylphenyl)- 5-phenyl-1H-pyrazol-3-yl]-7-methoxy-2H-
chromen-2-one (10c):
20

ACCEPTED MANUSCRIPT
Yield: 70%, m.p.240-242 oC. IR υmax/cm-1: 3392, 3261 (NH2), 3032 (CH aromatic), 2932,
2889 (CH aliphatic), 1739 (C=O), 1593, 1558 (C=C, C=N), 1328, 1153 (SO2). 1H NMR
(CDCl3) δ ppm: 3.20 (dd, 1H, C4-HA pyrazoline, JAX = 8.4 Hz and JAM = 16.8 Hz), 3.93 (s,
3H, OCH3), 4.20 (dd, 1H, C4-HM pyrazoline, JMX = 10.5 Hz and JMA = 16.8 Hz), 4.56 (s,
2H, NH2, exch.), 5.60 (dd, 1H,C5-HX pyrazoline, JXM = 10.5 Hz and JXA = 8.4 Hz), 6.28 (d,
1H, C3-H chromen, J = 9.6 Hz), 6.91 (d, 1H, C6-H chromen, J = 8.7 Hz), 7.05 (d, 1H, C5-
H chromen, J = 8.6 Hz), 7.21-7.46 (m, 7H, ArH), 7.64 (d, 1H, C4-H chromen, J = 9.6 Hz),
7.80 (d, 2H, ArH, J = 7.8 Hz). Anal. Calcd for C25H21N3O5S (475.52): C, 63.15; H, 4.45;
N, 8.84. Found: C, 63.10; H, 3.90; N, 8.63.
4.1.8.4. 8-[4,5-Dihydro-5-(4-methylphenyl)-1-(4-sulfamoylphenyl)-1H-pyrazol-3-yl]-7-
methoxy-2H-chromen-2-one (10d):
Yield: 68%, m.p.262-264 oC. IR υmax/cm-1: 3383, 3253 (NH2), 3084 (CH aromatic), 2945,
2920 (CH aliphatic), 1732 (C=O), 1602, 1562 (C=C, C=N), 1328, 1089 (SO2). 1H NMR
(CDCl3) δ ppm: 2.33 (s, 3H, CH3), 3.20 (dd, 1H, C4-HA pyrazoline, JAX = 8.4 Hz and JAM
16.5 Hz), 3.89 (s, 3H, OCH3), 4.15 (dd, 1H, C4-HM pyrazoline, JMX = 10.5 Hz and JMA
=
=
16.5 Hz), 4.63 (s, 2H, NH2, exch.), 5.30 (dd, 1H, C5-HX pyrazoline, JXM = 10.5 Hz and JXA
= 8.4 Hz), 6.20 (d, 1H, C3-H chromen, J = 9.6 Hz), 6.40 (d, 1H, C6-H chromen, J = 8.7
Hz), 6.80-7.40 (m, 5H, C5-H chromen and ArH), 7.50 (d, 2H, ArH, J = 7.8 Hz), 7.64 (d,
1H, C4-H chromen, J = 9.6 Hz), 7.80 (d, 2H, ArH, J = 7.8 Hz). Anal. Calcd for
C26H23N3O5S (489.54): C, 63.79; H, 4.74; N, 8.58. Found: C, 63.96; H, 4.95; N, 8.44.
4.1.8.5. 8-[4,5-Dihydro-1-(4-sulfamoylphenyl)- 5-(4-(trifluromethyl)phenyl)-1H-pyrazol-3-
yl]-7-methoxy-2H-chromen-2-one (10e):
Yield: 70%, m.p.180-182oC. IR υmax/cm-1: 3255, 3201 (NH2), 3068 (CH aromatic), 2943,
2850 (CH aliphatic), 1732 (C=O), 1604, 1562 (C=C, C=N), 1325, 1168 (SO2). 1H NMR
(CDCl3) δ ppm: 3.40 (dd, 1H, C4-HA pyrazoline, JAX = 8.2 Hz and JAM = 16.4 Hz), 3.87 (s,
3H, OCH3), 4.10 (dd, 1H, C4-HM pyrazoline, JMX = 10.5 Hz and JMA = 16.4 Hz), 4.63 (s,
2H, NH2, exch.), 5.30 (dd, 1H, C5-HX pyrazoline, JXM = 10.5 Hz and JXA = 8.2 Hz), 6.30
(d, 1H, C3-H chromen, J = 9.6 Hz), 6.40 (d, 1H, C6-H chromen, J = 8.7 Hz), 7.10-7.73 (m,
6H, Ar H), 7.76 (d, 1H, C5-H chromen, J = 8.7 Hz), 7.86 (d, 1H, C4-H chromen, J = 9.6
Hz), 8.09 (d, 2H, ArH, J = 7.8 Hz). 13C NMR (CDCl3): 40.3 (C-4 pyrazoline), 51.0 (C-5
pyrazoline), 56.5 (OCH3), 108.7 (C-6 chromen), 112.7 (C-3 chromen), 112.9 (C-4a and C-8
21

ACCEPTED MANUSCRIPT
chromen), 116.3 (C-2 and C-6 of C6H4SONH2), 120.6-130.7 (C-5 chromen, aromatic Cs
and CF3), 135.8 (C-4 of C6H4SONH2), 138.0 (C-4 of C6H4CF3), 143.7 (C-4 chromen),
144.1 (C-1 of C6H4CF3), 148.4 (C-8a chromen and C-1 of C6H4SONH2), 151.1 (C-8a
chromen and C=N pyrazoline), 158.8 (C-7 chromen), 159.3 (C=O). MS, m/z 543.20: [M+].
Anal. Calcd for C26H20FN3O5S (543.51): C, 57.46; H, 3.71; N, 7.73. Found: C, 57.91; H,
4.15; N, 8.03.
4.1.8.6. 8-[4,5-Dihydro-5-(4-(methylthio)phenyl)-1-(4-sulfamoylphenyl)-1H-pyrazol-3-yl]-
7-methoxy-2H-chromen-2-one (10f):
Yield: 65%, m.p.239-242 oC. IR υmax/cm-1: 3392, 3210 (NH2), 3032 (CH aromatic), 2920,
2850 (CH aliphatic), 1724 (C=O), 1602, 1587 (C=C, C=N), 1303, 1145 (SO2). 1H NMR
(CDCl3) δ ppm: 2.20 (s, 3H, S-CH3), 2.80 (dd, 1H, C4-HA pyrazoline, JAX = 8.1 Hz and
JAM = 16.2 Hz), 3.20 (dd, 1H, C4-HM pyrazoline, JMX = 10.5 Hz and JMA = 16.2 Hz), 3.85
(s, 3H, OCH3), 4.20 (s, 2H, NH2, exch.), 5.60 (dd, 1H, C5-HX pyrazoline, JXM = 10.5 Hz
and JXA = 8.1 Hz), 6.31 (d, 1H, C3-H chromen, J = 9.3 Hz), 6.95 (d, 1H, C6-H chromen, J
= 8.4 Hz), 7.18-7.33 (m, 6H, ArH), 7.64 (d, 2H, ArH, J = 7.8 Hz), 7.80 (d, 1H, C5-H
chromen, J = 8.4 Hz), 8.04 (d, 1H, C4-H chromen, J = 9.3 Hz). 13C NMR: 15.8 (SCH3),
44.8 (C-4 pyrazolin), 56.5 (OCH3), 63.4 (C-5 pyrazolin), 108.6 (C-6 chromen), 111.0 (C-3
chromen), 112.7 (C-4a and C-8 chromen), 112.9 (C-2 and C-6 of C6H4SONH2), 123.4-
130.3 (C-5 chromen and aromatic Cs), 136.0 (C-4 of C6H4SCH3), 140.0 (C-1 of
C6H4SCH3), 143.5 (C-4 chromen), 145.6 (C-1 of C6H4SONH2), 151.0 (C-8a chromen and
C=N), 158.8 (C-7 chromen), 159.3 (C=O). Anal. Calcd for C26H23N3O5S2 (521.61): C,
59.87; H, 4.44; N, 8.06. Found: C, 60.33; H, 4.71; N, 8.10.
4.2. Biological evaluation
4.2.1. Anticancer activity [45]
The cytotoxicity assays were performed at National Cancer Institute (NCI), Bethesda,
USA (against 60 cell lines) and at Department of Pharmacology, Faculty of Pharmacy, Ain
Shams University (against HCT-116 cell line). The human tumor cell lines of the cancer
screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and
2 mM L-glutamine. For a typical screening experiment, cells were inoculated into 96 well
22

ACCEPTED MANUSCRIPT
microtiter plates in 100 µl at plating densities ranging from 5,000 to 40,000 cells/well
depending on the doubling time of individual cell lines. After cell inoculation, the
microtiter plates were incubated at 37 °C, 5 % CO2, 95 % air and 100 % relative humidity
for 24 h prior to addition of experimental drugs. After 24 h, two plates of each cell line
were fixed in situ with trichloroacetic acid (TCA), to represent a measurement of the cell
population for each cell line at the time of drug addition (Tz). Experimental drugs were
solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration
and stored frozen prior to use. At the time of drug addition, an aliquot of frozen
concentrate was thawed and diluted to twice the desired final maximum test concentration
with complete medium containing 50 µg/ml gentamicin. Additional four, 10-fold or ½ log
serial dilutions were made to provide a total of five drug concentrations plus control.
Aliquots of 100 µl of these different drug dilutions were added to the appropriate
microtiter wells already containing 100 µl of medium, resulting in the required final drug
concentrations. Triplicate wells were prepared for each individual dose. Following drug
addition, the plates were incubated for an additional 48 h at 37 °C, 5 % CO2, 95 % air, and
100 % relative humidity. For adherent cells, the assay was terminated by the addition of
cold TCA. Cells were fixed in situ by the gentle addition of 50 µl of cold 50 % (w/v) TCA
(final concentration, 10 % TCA) and incubated for 60 minutes at 4 °C. The supernatant
was discarded, and the plates were washed five times with tap water and air dried.
Sulforhodamine B (SRB) solution (100 µl) at 0.4 % (w/v) in 1 % acetic acid was added to
each well, and plates were incubated for 10 minutes at room temperature. After staining,
unbound dye was removed by washing five times with 1 % acetic acid and the plates were
air dried. Bound stain was subsequently solubilized with 10 mM trizma base, and the
absorbance was read on an automated plate reader at a wavelength of 515 nm. For
suspension cells, the methodology was the same except that the assay was terminated by
fixing settled cells at the bottom of the wells by gently adding 50 µl of 80 % TCA (final
concentration, 16 % TCA). Using the seven absorbance measurements [time zero (Tz),
control growth (C), and test growth in the presence of drug at the five concentration levels
(Ti)], the percentage growth was calculated at each of the drug concentration levels.
Percentage growth inhibition was calculated as:
23

ACCEPTED MANUSCRIPT
[(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti>/=Tz
[(Ti-Tz)/Tz] x 100 for concentrations for which Ti<Tz.
Three dose response parameters were calculated for each experimental compound:
Growth inhibition of 50 % (GI50) was calculated when [(Ti-Tz)/(C-Tz)] x 100 = 50. The
compound concentration resulting in total growth inhibition (TGI) was calculated when Ti
= Tz. The LC50 indicating a net loss of cells following treatment was calculated when [(Ti-
Tz)/Tz] x 100 = -50.
The results reported are means of at least three separate experiments in case of cytotoxic
activity evaluated at Ain Shams University. Statistical differences were analyzed according
to one way ANOVA test wherein the differences were considered to be significant at p
<0.05.
4.2.2. PI3K (p110 α /p85 α) protein kinase assay [49]
The PI3K (p110α/p85 α) assay was performed using the ADP-GloTM assay kit from
Promega which measured the generation of ADP by the PI3K (p110 α /p85 α). Generation
of ADP by the PI3K reaction led to increase in luminescence signal in the presence of
ADP-GloTM assay kit.
The PI3K assay was performed in duplicate in a reaction mixture of final volume of 25 µl
containing: 5 µl of diluted active PI3K kinase, 5 µl of stock solution of peptide substrate, 5
µl of kinase assay buffer, 5 µl of compound (250 µM) or 10% DMSO and 5 µl of 250 µM
ATP stock solution. The assay was started by incubating the reaction mixture in a 96-well
plate at 30 ºC for 30 minutes. The reaction was then terminated by the addition of 25 µl of
ADP-GloTM reagent (Promega). The plate was shaken and then incubated for 40 minute at
ambient temperature. 50 µl of kinase detection reagent was added; the plate was shaken
and incubated for further 30 minute at ambient temperature. The reaction plate was then
read using the ADP-GloTM Luminescence Protocol on a GloMax plate reader (Promega;
Cat# E7031)). Blank and control were set up that included all the assay components except
the addition of the substrate in blank (replaced with equal volume of kinase assay buffer)
and the addition of compound in control. The corrected activity for PI3K (p110α/p85α) was
24

ACCEPTED MANUSCRIPT
determined by removing the blank control value. For IC50 Determination for compound 9c
and wortmannin, a graph of log inhibitor concentrations versus normalized responses with
variable slope was generated using the Prism software.
Acknowledgement
This study was supported by The Research Fund of Cairo University (Project number
1999/45 a).
The authors are grateful to National Cancer Institute (NCI) Developmental Therapeutic
Program (www.dtp.nci.nih.gov) for screening the anticancer activity of the newly
synthesized compounds in vitro.
References
[11 WHO website: http://www.who.int/cancer/en (2012) and
http://progressreport.cancer.gov (Cancer Trends Progress Report – 2009/2010 Update).
[2] M. Mareel, A. Leroy, Physiol. Rev. 83 (2003) 337-376.
[3] J. Wesche1, K. Haglund, E.M. Haugsten, Biochem. J. 437 (2011) 199-213.
[4] J. Cummings, T. H. Ward, M. Ranson, C. Dive, Biochim. Biophys. Acta 1705 (2004) 53-
66.
[5] S.R.D. Johnston, H. Ford, P. Ross, The Royal Marsden Hospital Hand Book of Cancer
Chemotherapy; Brighton, D., Wood, M., Eds.; Elsevier Churchill Livingstone: London, New
York, Oxford, 2005, pp 1-17.
[6] J.J. Marin, F. Sanchez de Medina, B. Castaño, L. Bujanda, M.R. Romero, Martinez- O.
Augustin, R.D. Moral-Avila, O. Briz, Drug Metab. Rev. 44 (2012) 148-172.
[7] R.C. DeConti, Semin. Oncol. 39 (2012) 145-149.
[8] I. Kostova, Curr. Med. Chem. 5 (2005) 29-46.
[9] R.K. Singh, T.S. Lange, K.K. Kim, L. Brard, Invest. New Drugs 29 (2011) 63-72.
[10] F. Carta, A. Maresca, A. Scozzafava, C.T. Supuran, Bioorg. Med. Chem. 20 (2012)
2266-2273.
[11] S.J. Jeong, W. Koh, E.O. Lee, H.J. Lee, H.J. Lee, H. Bae, J. Lü, S.H. Kim, Phytother.
Res. 25 (2011) 1-10.
[12] H. Zhao, A.C. Donnelly, B.R. Kusuma, G.E. Brandt, D. Brown, R.A. Rajewski, G.
Vielhauer, J. Holzbeierlein, M.S. Cohen, B.S. Blagg, J. Med. Chem. 54 (2011) 3839-3853.
25

ACCEPTED MANUSCRIPT
[13] Y. Dong, Q. Shi, Y.N. Liu, X. Wang, K.F. Bastow, K.H. Lee, J. Med. Chem. 52 (2009)
3586-3590.
[14] X. Wang, K. Nakagawa-Goto, K.F. Bastow, M.J. Don, Y.L. Lin, T.S. Wu, K.H. Lee, J.
Med. Chem. 49 (2006) 5631-5634.
[15] C.M. Elinos-Báez, F. León, E. Santos, Cell Biol. Int. 29 (2005) 703-708.
[16] M.E. Marshall, J.L. Mohler, K. Edmonds, B. Williams, K. Butler, M. Ryles, L. Weiss,
D. Urban, A. Beuschen, M. Markiewicz, J. Cancer Res. Clin. Oncol. 120 (1994) 39-42.
[17] G.J. Finn, B.S. Creaven, D.A. Egan, Eur. J. Pharm. Sci. 26 (2005) 16-25.
[18] L.D. Raev, E. Voinova, I.C. Ivanov, D. Popov, Pharmazie 45 (1990) 696-702.
[19] J.L. Mohler, L.G. Gomella, E.D. Crawford, L.M. Glode, C.D. Zippe, W.R. Fair, M.E.
Marshall, Prostate 20 (1992), 123- 131.
[20] S.J. Kao, J.L. Su, C.K. Chen, M.C. Yu, K.J. Bai, J.H. Chang, M.Y. Bien, S.F. Yang,
M.H. Chien, Toxicol. Appl. Pharmacol. 261 (2012) 105-115.
[21] N.S. Reddy, M.R. Mallireddigari, S. Cosenza, K. Gumireddy, S.C. Bell, E.P. Reddy,
M.V. Reddy, Bioorg. Med. Chem. Lett. 14 (2004) 4093-4097.
[22] F. Belluti, G. Fontana, L. Dal Bo, N. Carenini, C. Giommarelli, F. Zunino, Bioorg.
Med. Chem. 18 (2010) 3543-3550.
[23] M.A. Musa, V.L. Badisa, L.M. Latinwo, J. Cooperwood, A. Sinclair, A. Abdullah,
Anticancer Res. 31 (2011) 2017-2022.
[24] S.G. Kini, S. Choudhary, M.J. Mubeen, Comput. Methods Mol. Des. 2 (2012) 51-60.
[25] D. Havrylyuk, B. Zimenkovsky, O. Vasylenko, L. Zaprutko, A. Gzella, R. Lesyk, Eur.
J. Med. Chem. 44 (2009) 1396-1404.
[26] X.H. Liu, J. Li, J.B. Shi, B.A. Song, X.B. Qi, Eur. J. Med. Chem. 51 (2012) 294- 299.
[27] C. Congiu, V. Onnis, L. Vesci, M. Castorina, C. Pisano, Bioorg. Med. Chem. 18 (2010)
6238-6248.
[28] B. Insuasty, A. Tigreros, F. Orozco, J. Quiroga, R. Abonía, M. Nogueras, A. Sanchez, J.
Cobo, Bioorg. Med. Chem. 18 (2010) 4965-4974.
[29] C.Blackburn, M.O. Duffey, A.E. Gould, B. Kulkarni, J.X. Liu, S. Menon, M.
Nagayoshi, T.J. Vos, J. Williams, Bioorg. Med. Chem. Lett. 20 (2010) 4795-4799.
[30] M.O. Duffey, R. Adams, C. Blackburn, R.W. Chau, S. Chen, K.M. Galvin, K. Garcia,
A.E. Gould, P.D. Greenspan, S. Harrison, S. Huang, M. Kim, B. Kulkarni, S. Langston, J.X.
26

ACCEPTED MANUSCRIPT
Liu, L. Ma, S. Menon, M. Nagayoshi, R.S. Rowland, T.J. Vos, T. Xu, J.J. Yang, S. Yu, Q.J.
Zhang, Bioorg. Med. Chem. Lett. 20 (2010) 4800-4804.
[31] X.H. Liu, B.F. Ruan, J.X. Liu, B.A. Song, L.H. Jing, J. Li, Y. Yang, H.L. Zhu, X.B. Qi,
Bioorg. Med. Chem. Lett. 21 (2011) 2916-2920.
[32] X.H. Liu, H.F. Liu, J. Chen, Y. Yang, B.A. Song, L.S. Bai, J.X. Liu, H.L. Zhu, X.B. Qi,
Bioorg. Med. Chem. Lett. 20 (2010) 5705-5708.
[33] M. Hayakawa, H. Kaizawa, K-I. Kawaguchi, K. Matsuda, N. Ishikawa, T. Koizumi, M.
Yamano, M. Okada, M. Ohta, Patent US 6,403,588 B1, Jun. 11, (2002).
[34] G. Stephen, S.G. Ward, P. Finan, Curr. Opin. Pharmacol. 3 (2003) 426-434.
[35] C. Billottet, V.L. Grandage, R.E. Gale, A. Quattropani, C. Rommel, B.
Vanhaesebroeck, A. Khwaja, Oncogene 25 (2006) 6648-6659.
[36] A. Casini, A. Scozzafava, A. Mastrolorenzo, L.T. Supuran, Curr. Cancer Drug Tar. 2
(2002) 55-75.
[37] S. Bano, K. Javed, S. Ahmad, I.G. Rathish, S. Singh, M.S. Alam, Eur. J. Med. Chem.
46 (2011) 5763-5768.
[38] R. Bashir, S. Ovais, S. Yaseen, H. Hamid, M.S. Alam, M. Samim, S. Singh, K. Javed,
Bioorg. Med. Chem. Lett. 21 (2011) 4301-4305.
[39] Y. Luo, Y. Li, K.M. Qiu, X. Lu, J. Fu, H.L. Zhu, Bioorg. Med. Chem. 19 (2011) 6069-
6076.
[40] Z. Liu, Z. Zhou, W. Tian, X. Fan, D. Xue, L. Yu, Q. Yu, Y.Q. Long, Chem. Med.
Chem. 7 (2012) 680-693.
[41] K. M. Amin, F.M. Awadalla, A.A. Eissa, S.M. Abou-Seri, G.S. Hassan, Bioorg. Med.
Chem. 19 (2011) 6087-6097.
[42] A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. Paull, D. Vistica, C. Hose, J.
Langley, P. Cronise, A. Vaigro-Wolfe, M. Gray-Goodrich, H. Campbell, M.R. Boyd, J. Natl.
Cancer Inst. 83 (1991) 757-766.
[43] M.R. Boyd, K.D. Paull, Drug Dev. Res. 34 (1995) 91-109.
[44] M.R. Boyd, Cancer Drug Discovery and Development: Anticancer Drug Development
Guide: Preclinical Screening, Clinical Trials and Approval; B.A. Teicher, Ed.; Humana
Press: Totowa, NJ, USA, 1997, Vol. 2, pp 23–43.
27