Synthesis, in vitro antiproliferative activity, and in silico studies of fused tricyclic coumarin sulfonate derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.06.064

A series of fused tricyclic coumarin sulfonate derivatives was synthesized. Their in vitro antiproliferative activities against a panel of 57 human cancer cell lines of nine different cancer types were tested at the NCI. Compounds 1e, 1f, 1h, 1i, and 1o showed the highest mean percentage of inhibition values over the 57 cell line panel at 10 μM, and they were further tested in 5-dose testing mode to determine their IC₅₀ values. Compounds 1e, 1f, and 1o were more selective against leukemia and colon cancer subpanels, while compounds 1h and 1i showed broad-spectrum anticancer activities. Compounds 1e, 1f, 1h, 1i, and 1o demonstrated high selectivity towards cancer cell lines than RAW 264.7 macrophages. Compound 1h exerted lethal effect over NCI-H522 NSCLC, SK-MEL-5 melanoma, and A498 renal cancer cell lines with percentage of inhibition values of 114.10%, 103.23%, and 100.52% at 10 μM concentration, respectively. Moreover, the IC₅₀ value of compound 1o against HT29 colon cancer cell line was 532 nM. Compounds 1e, 1f, 1h, 1i, and 1o were tested for inhibitory effect over cyclooxygenase-2 (COX-2) enzyme as a possible mechanism of action. Furthermore, in silico studies were conducted to check the compliance of those five compounds with Lipinski's rule of five, and hence estimate their oral bioavailability.

Full text of DOI:10.1016/j.ejmech.2014.06.064

Accepted Manuscript
Synthesis, in vitro antiproliferative activity, and in silico studies of fused tricyclic
coumarin sulfonate derivatives
Mohammed I. El-Gamal, Chang-Hyun Oh, Dr.
PII:
S0223-5234(14)00593-5
10.1016/j.ejmech.2014.06.064
EJMECH 7107
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 April 2014
Revised Date: 9 June 2014
Accepted Date: 27 June 2014
Please cite this article as: M.I. El-Gamal, C.-H. Oh, Synthesis, in vitro antiproliferative activity, and in
silico studies of fused tricyclic coumarin sulfonate derivatives, European Journal of Medicinal Chemistry
(2014), doi: 10.1016/j.ejmech.2014.06.064.
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ACCEPTED MANUSCRIPT
Elsevier Editorial System(tm) for European Journal of Medicinal Chemistry
Manuscript Draft
Manuscript Number: EJMECH-D-14-00875R1
Title: Synthesis, in vitro antiproliferative activity, and in silico studies of fused tricyclic coumarin
sulfonate derivatives
Article Type: Full Length Article
Keywords: Anticancer; Antiproliferative; COX-2 inhibition; Fused tricyclic coumarin; In silico studies;
Sulfonate.
Corresponding Author: Dr. Mohammed Ibrahim El-Gamal, Ph.D.
Corresponding Author's Institution: Korea Institute of Science and Technology (KIST)
First Author: Mohammed Ibrahim El-Gamal, Ph.D.
Order of Authors: Mohammed Ibrahim El-Gamal, Ph.D.
Abstract: A series of fused tricyclic coumarin sulfonate derivatives was synthesized. Their in vitro
antiproliferative activities against a panel of 57 human cancer cell lines of nine different cancer types
were tested at the NCI. Compounds 1e, 1f, 1h, 1i, and 1o showed the highest mean %inhibition values
over the 57 cell line panel at 10 μM, and they were further tested in 5-dose testing mode to determine
their IC50 values. Compounds 1e, 1f, and 1o were more selective against leukemia and colon cancer
subpanels, while compounds 1h and 1i showed broad-spectrum anticancer activities. Compound 1h
exerted lethal effect over NCI-H522 NSCLC, SK-MEL-5 melanoma, and A498 renal cancer cell lines with
%inhibition values of 114.10%, 103.23%, and 100.52% at 10 µM, respectively. Moreover, the IC50
value of compound 1o against HT29 colon cancer cell line was 532 nM. Compounds 1e, 1f, 1h, 1i, and
1o were tested for inhibitory effect over cyclooxygenase-2 (COX-2) enzyme as a possible mechanism of
action. Furthermore, in silico studies were conducted to check the compliance of those five compounds
with Lipinski's rule of five, and hence estimate their oral bioavailability.

Cover Letter
ACCEPTED MANUSCRIPT
Dear Prof. Galons,
We have revised and submitted our manuscript entitled “Synthesis, in vitro
antiproliferative activity, and in silico studies of fused tricyclic coumarin sulfonate
derivatives” to the European Journal of Medicinal Chemistry. I look forward to
consideration of this revised manuscript for publication in EJMECH. Thank you so much
in advance.
Yours sincerely,
Mohammed I. El-Gamal, Ph.D.
Assistant Professor of Medicinal Chemistry
Tel.: +82 2 958 5160; fax: +82 2 958 5308.
E-mail address: choh@kist.re.kr
Address: Center for Biomaterials, Korea Institute of Science and Technology, P.O. Box
131, Cheongryang, Seoul 130-650, Republic of Korea.
1

*Response to Reviewers
ACCEPTED MANUSCRIPT
Thank you so much for your valuable comments which helped us greatly
improve our manuscript.
In the manuscript, the corrected sentences have been converted into blue color.
Reviewer #1: This work dealt with the anticancer activities of the synthetic
compounds with a cycloketone combining with the coumarin skeleton. The comparison
on the anticancer activities leads to the discussion on the structure-activity
relationship, in which the influences of the subsituents at R on the biological
activities were explored in detail. But some short-comings should be resolved
before this work can be accepted by the Journal. As the reference compound, the
biological activity of coumarin itself should be determined. The function of the
cycloketone on the biological activity should be clarified and whether the
cycloketone connected with counarin with a single bond (C-C not the combined
style as in the present work) should be emphasized.
The results of coumarin (CAS No.: 91-64-5) against NCI-60 cancer cell line panel
was obtained from the NCI datawarehouse index through this link
http://dtp.nci.nih.gov/dtpstandard/servlet/MeanGraph?searchtype=NSC&searchlist=8
774&outputformat=HTML&outputmedium=page&chemnameboolean=AND&debugs
witch=false&assaytype=&testshortname=NCI+Cancer+Screen+Current+Data&dataar
raylength=55&endpt=GI50&SVGonly=SVG&button=Mean+Graph&highconc=-4.0
Although many references have reported anticancer activity of coumarin, it didn't
show any potential potency against the NCI-60 cancer cell line panel. Upon
comparison of its results with those of our potent compounds 1e, 1f, 1h, 1i, and 1o, it
was found that fused cycloalkane and arylsulfonate moieties are essential for activity
of this series of compounds. The results of Coumarin were inserted in the manuscript
(Tables 3 and 4), and relevant modifications of the Discussion and Conclusion
sections were done as suggested by the reviewer.
Fusion through a single bond instead of a double bond will be investigated in the
near future as suggested by the reviewer. But currently, it's almost impossible to do it
before the due date for submission of the revised manuscript. We need to find a
suitable synthetic pathway, order reagents, synthesize, purify, and analyze the
compounds, submit to the NCI, and get results. This process needs long time. Thank
you so much in advance for understanding our current situation.
Reviewer #2: The paper of El-Gamal M. I. et al is a sound one and has been
improved.
I
suggest
its
acceptance
after
minor
revision.
General remark: Have these compounds been tested on non-cancerous cell-lines?
1

ACCEPTED MANUSCRIPT
The most potent compounds 1e, 1f, 1h, 1i, and 1o were tested for cytotoxicity
over RAW 264.7 macrophages, and their IC50 values were more than 100 μM. These
results indicated high selectivity of those compounds towards cancer cell lines than
normal cells. This is another merit of those target compounds. Thank you so much for
your valuable suggestion.
P4 „%inhibition" use: percentage of inhibition
It was corrected in all parts of the manuscript as suggested by the reviewer.
P9: not now, but in the future for synthesis of 3a and 3b use toluene instead of
benzene.
Thank you so much for your valuable recommendation. Yes, toluene is less toxic
than benzene.
P20 I would suggest omitting the column graph. Title: " Mean % inhibition"
Inhibition of what? Reader does not know 1a, 1b…etc. meaning in the abscissa.
The efficiency - if necessary - can be included in the text based on the general
formula.
That title was omitted from Figure 2 as suggested by the reviewer.
All parts of the manuscript should be numbered in particular sub-titles as the
following:
6-Oxo-7,8,9,10,11,12-hexahydro-6H-cycloocta[c]chromen-3-yl
benzenesulfonate (1q)
4-(tert-butyl)
All parts have been numbered as suggested by the reviewer.
We hope that our revised manuscript will be satisfactory for the editor and reviewers,
and it will be accepted for publication in European Journal of Medicinal Chemistry.
2

Graphical Abstract (for review)
Click here to download Graphical Abstract (for review): Graphical Abstract.doc
ACCEPTED MANUSCRIPT
Synthesis, in vitro antiproliferative activity, and in silico studies of fused
tricyclic coumarin sulfonate derivatives
Mohammed I. El-Gamala,c, and Chang-Hyun Oha,b,*
a
Center for Biomaterials, Korea Institute of Science and Technology (KIST), PO Box 131, Cheongryang,
Seoul 130-650, Republic of Korea.
b
Department of Biomolecular Science, University of Science and Technology (UST), 113 Gwahangno,
Yuseong-gu, Daejeon 305-333, Republic of Korea.
c
Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516,
Egypt.
A series of new fused tricyclic coumarin sulfonate was synthesized. Their in vitro
antiproliferative activities against NCI-60 cancer cell lines, COX-2 inhibition, and in
silico studies are reported.
n
O
O
S
R
O
O
O
n = 1, 2
R = Me, Et, n-Pr, c-Pr, Ph, p-tolyl, p-(CF3)C6H4,
p-(tert-butyl)C6H4, p-(F)C6H4
Mean % Inhibition
60
50
40
30
20
10
0
1a
1b
1c
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1r
Compound No.
20

Highlights (for review)
ACCEPTED MANUSCRIPT
Synthesis, in vitro antiproliferative activity, and in silico studies of fused
tricyclic coumarin sulfonate derivatives
Mohammed I. El-Gamal, and Chang-Hyun Oh*
► Synthesis and in vitro antiproliferative activities of new coumarin sulfonates are
reported.
► Compounds 1e, 1f, and 1o were more selective against leukemia and colon cancer
subpanels.
► Compounds 1h and 1i showed broad-spectrum anticancer activities.
► IC50 value of compound 1o against HT29 colon cancer cell line was 532 nM.
► The antiproliferative effect is almost due to COX-2 inhibition.
.

*Revised Manuscript
Click here to view linked References
ACCEPTED MANUSCRIPT
Synthesis, in vitro antiproliferative activity, and in silico studies of fused
tricyclic coumarin sulfonate derivatives
Mohammed I. El-Gamala,c, and Chang-Hyun Oha,b,*
a
Center for Biomaterials, Korea Institute of Science and Technology (KIST), PO Box 131, Cheongryang,
Seoul 130-650, Republic of Korea.
b
Department of Biomolecular Science, University of Science and Technology (UST), 113 Gwahangno,
Yuseong-gu, Daejeon 305-333, Republic of Korea.
c
Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516,
Egypt.
Corresponding author: Dr. Chang-Hyun Oh.
Mailing address: Center for Biomaterials, Korea Institute of Science and Technology, PO Box 131,
Cheongryang, Seoul 130-650, Republic of Korea.
Tel.: +82 2 958 5160; fax: +82 2 958 5189; e-mail address: choh@kist.re.kr (C.-H. Oh).
E-mail addresses of the first author: drmelgamal2002@gmail.com; drmelgamal2002@yahoo.com
Abstract- A series of fused tricyclic coumarin sulfonate derivatives was synthesized.
Their in vitro antiproliferative activities against a panel of 57 human cancer cell lines of
nine different cancer types were tested at the NCI. Compounds 1e, 1f, 1h, 1i, and 1o
showed the highest mean percentage of inhibition values over the 57 cell line panel at 10
μM, and they were further tested in 5-dose testing mode to determine their IC50 values.
Compounds 1e, 1f, and 1o were more selective against leukemia and colon cancer
subpanels, while compounds 1h and 1i showed broad-spectrum anticancer activities.
Compounds 1e, 1f, 1h, 1i, and 1o demonstrated high selectivity towards cancer cell lines
than RAW 264.7 macrophages. Compound 1h exerted lethal effect over NCI-H522
NSCLC, SK-MEL-5 melanoma, and A498 renal cancer cell lines with percentage of
inhibition values of 114.10%, 103.23%, and 100.52% at 10 µM, respectively. Moreover,
the IC50 value of compound 1o against HT29 colon cancer cell line was 532 nM.
Compounds 1e, 1f, 1h, 1i, and 1o were tested for inhibitory effect over cyclooxygenase-2
(COX-2) enzyme as a possible mechanism of action. Furthermore, in silico studies were
conducted to check the compliance of those five compounds with Lipinski's rule of five,
and hence estimate their oral bioavailability.
1

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Key words: Anticancer; Antiproliferative; COX-2 inhibition; Fused tricyclic coumarin; In
silico studies; Sulfonate.
1. Introduction
Cancer is a major leading cause of death worldwide. According to the American
Cancer Society report, 577,190 cancer patients died, and more than 1.6 million new
cancer cases were identified in 2012 only in USA [1]. More than 70% of all cancer deaths
have occurred in low- and middle-income countries. Deaths from cancer worldwide are
estimated to exceed 13 million in 2030 according to the World Health Organization
(WHO) report [2]. Despite of the extensive efforts and investment in research, the
management of human cancers still constitutes a major challenge for contemporary
medicinal chemistry. There has been an urgent need for development of more efficient
anticancer agents with minimal side effects.
Natural and synthetic coumarin derivatives have recently attracted much interest
because of their diverse biological and pharmacological properties. Among these
properties, their anticancer effects were extensively examined [3-13]. Coumarin and its
metabolite, 7-hydroxycoumarin (Umbelliferone) (Fig. 1), were reported to inhibit the
proliferation of a number of human malignant cell lines in vitro [14,15] and in xenograft
models [16,17]. 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 [18]. 6-Methoxy-7-hydroxycoumarin (Scopoletin,
Fig. 1) was reported to initiate apoptosis in HL-60 cells [19]. It showed dual effects on
both concanavalin A-stimulated murine T-cell proliferation and an anti-proliferative
activity in a lymphoma cell line [20]. Irusostat (STX-64) has shown potential
antiproliferative effect against breast cancer cell lines, and is currently under clinical
trials for treatment of breast cancer [4,21]. Other fused tricyclic coumarin compounds
have been reported for cytotoxic activity [22].
Cyclooxygenase-2 (COX-2) is an inducible enzyme involved in the conversion of
arachidonic acid to prostaglandins and other eicosanoids. COX-2 and its product,
prostaglandin E2 (PGE2) play a crucial role in tumor microenvironment [23]. Several
reports have shown that PGE2 and COX-2 have a wide range of effects including
induction of cellular proliferation, promotion of angiogenesis, promotion of cancer cell
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resistance to apoptosis, stimulation of tumor invasion, and suppression of immune
responses [24,25]. Molecular pathology studies have revealed that COX-2 is over-
expressed in cancer and stroma cells during tumor progression, and anticancer chemo-
radiotherapies induce expression of COX-2 in cancer cells [26]. COX-2 has been proven
to be over-expressed in several types of human cancer including melanomas [27-30],
colon [31], non-small cell lung (NSCLC) [32], intestinal [33], colo-rectal [34], pancreatic
[35], cervical [36], breast [37], endometrial [38], laryngeal [39], papillary thyroid [40],
and gastric [41] cancers. Much attention has been focused on COX-2 inhibitors as a
beneficial avenue for cancer chemotherapy [25,26]. Several experimental and clinical
studies have established potent anticancer activity of COX-2 inhibitors such as Celecoxib
(Fig. 1). Moreover, we have recently reported dual inhibitors of COX-2 enzyme and ERK
pathway as potential antiproliferative agents [42].
In the present study, eighteen fused tricyclic coumarin sulfonate analogs 1a-r (Fig. 1)
were synthesized. The inhibitory effects of compounds 1a-g and 1j-r over LPS-induced
nitric oxide and prostaglandin E2 (PGE2) productions in RAW 264.7 macrophages were
recently reported [43]. Herein, their in vitro antiproliferative activities were investigated
against 57 cancer cell line panel. In addition, the in vitro COX-2 inhibitory effect of the
most active agents was tested. And in silico study was carried out in order to estimate the
oral bioavailability of the most active compounds.
[Figure 1]
2. Results and Discussion
2.1. Chemistry
The target compounds were synthesized by the pathway illustrated in Scheme 1.
Reaction of cycloheptanone (2a) or cyclooctanone (2b) with diethyl carbonate in
refluxing benzene in the presence of sodium hydride to produce the corresponding
ethoxycarbonate derivatives 3a,b which exist in keto-enol tautomers [44]. Cyclization to
the phenolic tricyclic intermediates 4a,b was achieved by reaction of compounds 3a,b
with resorcinol in the presence of concentrated sulfuric acid and trifluoroacetic acid [45].
Treatment of the phenolic intermediates 4a,b with the appropriate sulfonyl chloride
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derivatives in the presence of triethylamine afforded the target sulfonates 1a-r [43]. Table
1 illustrates structures of the final compounds and their yield percentages.
[Scheme 1 & Table 1]
2.2. Antiproliferative activities against 57 cell line panel at the NCI
2.2.1. Single-dose testing
Structures of the synthesized target compounds were submitted to National Cancer
Institute (NCI), Bethesda, Maryland, USA [46], and the 16 compounds shown in Figure 2
were selected on the basis of degree of structural variation and computer modeling
techniques for evaluation of their anticancer activity. The selected compounds were
subjected to in vitro antiproliferative assay against tumor cells in a full panel of 57 cell
lines taken from nine different tissues (blood, lung, colon, CNS, skin, ovary, kidney,
prostate, and breast). The compounds were tested at a single-dose concentration of 10 μM,
and the percentages of growth inhibition over the 57 tested cell lines were determined.
The mean inhibition percentages for each of the tested compounds over the full panel of
cell lines are illustrated in Figure 2.
The results showed that aromatic sulfonate derivatives 1e-i, 1n, and 1o exhibited
stronger antiproliferative activities than aliphatic and cyclopropyl analogs. Upon
comparing the activities of aliphatic sulfonate derivatives 1a-c, it was found that mean
percentage of inhibition decreased with extension of the terminal aliphatic group
(Me>Et>n-Pr). Aromatic sulfonate derivatives 1e-g and 1i possessing cycloheptane ring
showed higher mean percentage of inhibition than the corresponding compounds 1n-p
and 1r with cyclooctanone ring. Compounds 1f and 1o with para-toluenesulfonate group
and compound 1h containing para-(tert-butyl)benzenesulfonate were more active than
compounds 1g and 1p possessing para-(trifluoromethyl)benzenesulfonate, and 1i and 1r
with para-fluorobenzenesulfonate moiety. Similarly, compounds 1e and 1n possessing
terminal benzenesulfonate moiety showed more activities than compounds 1g, 1i, 1p, and
1r. So it can be concluded that electron-withdrawing groups such para-(trifluoromethyl)
and para-fluoro on the terminal benzenesulfonate moiety is unfavorable for activity.
[Figure 2]
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Among all the tested derivatives, compounds 1e, 1f, 1h, and 1o showed the highest
mean inhibitions. The percentages of inhibition of these four compounds over each tested
cell line of the NCI-57 panel at 10 µM concentration are depicted in Figure 3.
Compounds 1e, 1f, and 1o showed more activities against leukemia and colon cancer cell
lines.
Compound
1h
possessing
fused
cycloheptane
and
para-(tert-
butyl)benzenesulfonate moieties demonstrated broad-spectrum activity with more than
60% inhibition over 21 different cell lines of the nine tested cancer types. It exerted lethal
effect with more than 100% inhibition against NCI-H522 non-small cell lung cancer
(NSCLC), SK-MEL-5 melanoma, and A498 renal cancer cell lines (114.10%, 103.23%,
and 100.52%, respectively). It also inhibited the growth of HOP-92 NSCLC and SF-295
CNS cancer cell lines by 96.06% and 97.29%, respectively, at 10 µM.
[Figure 3]
2.2.2. Five-dose testing
Compounds 1e, 1f, 1h, 1i, and 1o with promising results in single-dose testing were
further tested in a ※ve-dose testing mode, in order to determine their IC50 values over the
57 cancer cell lines. The mean IC50 values of those five compounds over the nine cancer
types are summarized in Table 2. The results showed that compound 1e with
benzenesulfonate moiety and compounds 1f and 1o possessing para-toluenesulfonate
terminal ring were more selective against leukemia and colon cancer subpanels. The
selectivity indices are illustrated in Table 2 as compared with the third most susceptible
cancer type. On the other hand, the fused cycloheptane compounds 1h and 1i with para-
(tert-butyl)benzenesulfonate and para-fluorobenzenesulfonate moieties, respectively,
demonstrated broad-spectrum antiproliferative activities.
[Table 2]
The IC50 values of compounds 1e, 1f, and 1o against all the tested leukemia and colon
cancer cell lines are summarized in Table 3. The results of Coumarin, Scopoletin, and
Umbelliferone were obtained from NCI datawarehouse index [47], and are inserted in
Table 3. Compounds 1e, 1f, and 1o exerted superior potencies against leukemia and colon
cancer cell lines to the three reference compounds, Coumarin, Scopoletin, and
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Umbelliferone. Upon comparing the results of those three compounds, it was found that
compound 1e was the most potent against leukemia cell lines but compound 1o showed
the highest potency against colon cancer cell lines. Of special interest, the IC50 value of
compound 1o against HT29 colon cancer cell line was in submicromolar scale, 532 nM.
[Table 3]
Furthermore, compounds 1h and 1i demonstrated broad-spectrum antiproliferative
activities. Their IC50 values over the most sensitive cell line of each subpanel are
illustrated in Table 4. Both compounds showed higher potencies than Scopoletin and
Umbelliferone against the nine cell lines of nine different cancer types. The superior
potencies of compounds 1e, 1f, 1h, 1i, and 1o to Coumarin indicate that the fused
cycloalkane and arylsulfonate moieties are essential for antiproliferative activity of this
series of compounds. Compound 1h with para-(tert-butyl)benzenesulfonate moiety was
generally more potent than compound 1i possessing para-fluorobenzenesulfonate
terminal ring. The IC50 value of compound 1h against HOP-92 NSCLC cell line was as
low as 1.22 µM. Similarly, compound 1i showed high potency against HT29 colon and
T-47D breast cancer cell lines with IC50 values of 1.04 µM and 1.86 µM, respectively.
The IC50 values of the most active compounds 1e, 1f, 1h, 1i, and 1o against RAW 264.7
macrophages were determined [43] in order to investigate their cytotoxicity against
normal cells and check their selectivity towards cancer cells over normal cells. All the
five compounds did not inhibit 50% of the macrophage growth up to 100 μM. This
indicates superior selectivity against cancer cell lines than normal cells.
[Table 4]
2.3. In vitro cyclooxygenase (COX) inhibitory activity
In order to determine the possible mechanism of antiproliferative activity at
molecular level, the most active compounds 1e, 1f, 1h, 1i, and 1o were tested for COX-2
inhibitory effect. The IC50 values were determined, and they are depicted in Table 5. The
COX-2 inhibitory effect of compound 1g [43] encouraged us to conduct this experiment.
The results showed good potency of the five compounds over COX-2 enzyme. The
highest potencies were expressed by compounds 1e, 1f, and 1o which are selective for
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leukemia and colon cancer subpanels. The most potent compound was 1o which showed
submicromolar IC50 value over HT29 colon cancer cell line. Its IC50 value over COX-2
enzyme was comparable to that of Celecoxib. It has been reported that COX-2 is up-
regulated in HT29 colon cancer cells [48]. So the superior potency of compound 1o
against HT29 colon cancer cell line can be attributed to its strong and selective COX-2
inhibitory effect. Upon comparing the potencies of compounds 1e, 1f, 1h, and 1i, it was
found that the order of potency in correlation with the terminal substituents decreases in
the following order (PhSO2 > para-tosyl > para-fluorobenzenesulfonyl > para-(tert-
butyl)benzenesulfonyl).
In order to examine selectivity of the target compounds towards COX-2 over COX-1,
the IC50 values over COX-1 were also detected and selectivity indices were determined
(Table 5). For instance, the selectivity indices of compounds 1e and 1o were more than
50 and 60.61, respectively. So it can be estimated that the target compounds may exert
antiproliferative effect due to COX-2 inhibition with diminished or no side effects caused
by COX-1 inhibition.
[Table 5]
2.4. Lipinski’s rule of five
The bioavailability was assessed using ADME (absorption, distribution, metabolism,
and excretion) prediction methods for the most active compounds 1e, 1f, 1h, 1i, and 1o
with the highest mean percentage of inhibition values, which were selected for 5-dose
testing for determination of their IC50 values. In particular, we calculated the compliance
of those five compounds to the Lipinski’s rule of five [49]. This approach has been
widely used as a filter for substances that would likely be further developed in drug
design programs. In addition, we calculated the total polar surface area (TPSA) since it is
another key property that has been linked to drug bioavailability. Thus, passively-
absorbed molecules with a TPSA > 140 are thought to have low oral bioavailability [50].
Molecules violating more than one of these rules may have problems with bioavailability.
Predictions of ADME properties for the studied compounds are given in Table 6.
Compound 1h violated only one parameter, but the other compounds 1e, 1f, 1i, and 1o
comply with Lipinski's rule of five. Theoretically, those compounds should present good
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passive oral absorption and differences in their bioactivity can not be attributed to this
property.
[Table 6]
3. Conclusion
In this study, a series of fused tricyclic coumarin analogues possessing sulfonate
moiety was synthesized and tested for in vitro antiproliferative activities over 57 cancer
cell line panel of nine different cancer types. Among them, compounds 1e, 1f, 1h, 1i, and
1o showed the most promising results at a single-dose concentration of 10 µM. Of special
interest, compound 1h with para-(tert-butyl)phenyl and fused cycloheptane moieties
demonstrated lethal effect against NCI-H522 NSCLC, SK-MEL-5 melanoma, and A498
renal cancer cell lines with percentage of inhibition values of 114.10%, 103.23%, and
100.52%, respectively, at 10 µM. Compounds 1e, 1f, 1h, 1i, and 1o were further tested in
5-dose testing mode in order to determine their IC50 values, and the results were
compared with those of reference coumarin anticancer agents, Scopoletin and
Umbelliferone. The five tested compounds showed higher potencies than Scopoletin and
Umbelliferone. Moreover, the superior potency of those five compounds, compared with
Coumarin, indicate that fused cycloalkane and arylsulfonate moieties play important roles
in antiproliferative activity of this series of compounds. Compounds 1e possessing
benzenesulfonate moiety, and 1f and 1o with para-toluenesulfonate moiety exhibited the
merit of selectivity towards leukemia and colon cancer subpanel more than the other
seven tested cancer types. Those compounds can be utilized as leads for future
development of potential selective anticancer agents for treatment of leukemia and colon
cancer. On the other hand, compounds 1h and 1i showed broad-spectrum antiproliferative
activities. Among all the five compounds, the highest potency was expressed by
compound 1o against HT29 colon cancer cell line with IC50 value of 532 nM. The five
compounds 1e, 1f, 1h, 1i, and 1o showed higher selectivity towards cancer cell lines than
RAW 264.7 macrophages. In order to study the possible mechanism of action of the
target compounds at molecular level, compounds 1e, 1f, 1h, 1i, and 1o were tested for
COX-2 inhibitory activity. They showed high potency and selectivity towards COX-2
than COX-1. So the target compounds might exhibit potential antiproliferative activity
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due to COX-2 inhibition. In silico ADME prediction indicated that compounds 1e, 1f, 1i,
and 1o comply with Lipinski’s rule of five, and they can be passively-absorbed orally.
The structure-activity relationship (SAR) studies showed that aromatic sulfonates were
more favorable for antiproliferative activities of this series of compounds than aliphatic
or alicyclic sulfonates.
4. Experimental
4.1. General
The target compounds were purified by column chromatography using silica gel
(0.040-0.063 mm, 230-400 mesh) and technical grade solvents. The melting points were
obtained on a Walden Precision Apparatus Electrothermal 9300 apparatus and are
uncorrected. Nuclear magnetic resonance (NMR) spectroscopy was performed using a
Bruker ARX-300, 300 MHz (Bruker Bioscience, Billerica, MA, USA) and a Bruker
ARX-400, 400 MHz (Bruker Bioscience, Billerica, MA, USA) with TMS as an internal
standard. Purities of the target compounds (>95%) were determined by HPLC analysis.
LC-MS analysis was conducted using the following system: Waters 2998 photodiode
array detector, Waters 3100 mass detector, Waters SFO system fluidics organizer, Waters
2545 binary gradient module, Waters reagent manager, Waters 2767 sample manager,
SunfireTM C18 column (4.6 x 50 mm, 5 μm particle size); Solvent gradient = 95% A at 0
min, 1% A at 5 min; solvent A: 0.035% trifluoroacetic acid (TFA) in water; solvent B:
0.035% TFA in CH3OH; flow rate = 3.0 mL/min; the area under curve (AUC) was
calculated using Waters MassLynx 4.1 software. Unless otherwise noted, all solvents and
reagents were commercially available and used without further purification.
4.2. Synthesis of ethyl 2-oxocycloheptanecarboxylate (3a) and
ethyl 2-oxocyclooctanecarboxylate (3b) [44]
A 250 mL two-neck, round-bottomed flask equipped with a magnetic stirrer was
fitted with a 50 mL pressure-equalizing constant-rate dropping funnel and a condenser.
To the flask, sodium hydride (4.5 g, 112 mmol, 60% dispersion in mineral oil) was added.
The mineral oil was removed by washing the dispersion four times with 20 mL portions
9

ACCEPTED MANUSCRIPT
of dry benzene under nitrogen atmosphere. The benzene was removed with a pipette after
the sodium hydride was allowed to settle. After removal of most of the mineral oil, 60
mL of dry benzene was added to the sodium hydride, followed by diethyl carbonate (6.5
g, 55 mmol), this mixture was heated to reflux, and a solution of cycloheptanone (3a) or
cyclooctanone (3b) (30 mmol) in 10 mL of dry benzene was added dropwise over a
period of 3-4 h. After the addition was completed, this mixture was allowed to reflux
until the evolution of hydrogen gas ceases (15-20 min). The reaction mixture was allowed
to cool to room temperature, and 10 mL of glacial acetic acid was added dropwise, a
heavy pasty solid separated. Then ice-cold water (about 100 mL) was added dropwise,
and the stirring was continued until all the solid material has dissolved. The benzene
layer was separated, and the aqueous layer was extracted with benzene (3 x 50 mL). The
combined benzene extracts were washed three times with cold water (3 x 50 mL). The
organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated
under reduced pressure. The title compounds were purified by flash column
chromatography. The products were existing in equilibrating mixtures of the keto and
enol tautomers.
4.2.1. Compound 3a: it was purified by flash column chromatography (silica gel,
1
hexanes:ethyl acetate 15:1 v/v); yield: 90%; yellow oil; H NMR (CDCl3, 400 MHz) δ
12.76 (brs, 1H), 4.21-4.15 (m, 2H), 3.55-3.52 (m, 2H), 2.63-2.58 (m, 2H), 2.48-2.36 (m,
1H), 2.08 (t, 1H, J = 2.5 Hz), 1.94-1.85 (m, 4H), 1.62-1.61 (m, 2H), 1.49-1.44 (m, 2H),
1.32-1.23 (m, 4H); 13C NMR (CDCl3, 100 MHz) δ 208.8, 179.4, 173.0, 170.4, 101.5, 77.5,
77.2, 76.9, 60.9, 58.8, 43.0, 35.3, 31.9, 29.6, 27.9, 27.5, 27.3, 24.6, 24.3, 14.0.
4.2.2. Compound 3b: it was purified by flash column chromatography (silica gel,
1
hexanes:ethyl acetate 10:1 v/v); yield: 85%; yellow oil; H NMR (CDCl3, 400 MHz) δ
12.60 (brs, 1H), 4.23-4.12 (m, 3H), 2.41-2.33 (m, 4H), 2.15-2.08 (m, 1H), 1.93-1.86 (m,
1H), 1.75-1.65 (m, 3H), 1.57-1.45 (m, 8H), 1.30 (t, 3H, J = 7.1 Hz), 1.24 (t, 3H, J = 7.1
13
Hz); C NMR (CDCl3, 100 MHz) δ 212.2, 176.0, 172.9, 170.1, 99.2, 61.1, 60.1, 57.0,
41.7, 32.3, 29.9, 28.9, 28.7, 27.0, 26.5, 26.0, 25.5, 25.2, 24.5, 23.8, 14.3, 14.0.
10

ACCEPTED MANUSCRIPT
4.3. Synthesis of the hydroxyl intermediates 4a,b [45]
Resorcinol or 4-methoxyresorcinol or 4-chlororesorcinol or hydroquinone (11.0
mmol) was dissolved in hot ethyl 2-oxocycloheptanecarboxylate (3a) or ethyl 2-
oxocyclooctanecarboxylate (3b) (11.0 mmol). To this stirred mixture at ice-water
temperature was added dropwise a mixture of trifluoroacetic acid (1.7 mL, 22.0 mmol)
and conc. sulfuric acid (2.2 mL, 22.0 mmol) at such a rate that the reaction temperature
was kept below 10ºC (about 30 min). The reaction mixture was then allowed to warm to
room temperature and then stirred for an additional 3 h before being quenched cautiously
with ice-water. The product was then extracted with ethyl acetate (3 x 20 mL), and the
combined organic layer extracts were washed with brine (3 x 25 mL) and dried over
anhydrous sodium sulfate. The organic solvent was evaporated under reduced pressure,
and the product was used in the next step without further purification.
4.4. Synthesis of the target cycloalkane-fused coumarin sulfonates 1a-r
A solution of the appropriate intermediate hydroxyl compound (0.1 mmol) and
triethylamine (0.0165 mL, 0.2 mmol) in dry dichloromethane (5 mL) was cooled in an ice
bath. A solution of the appropriate sulfonyl chloride derivative (0.11 mmol) in dry
dichloromethane (2 mL) was added dropwise at the same temperature. The reaction
mixture was allowed to warm to room temperature, and the stirred for an additional 1 h.
The reaction mixture was washed with brine (3 x 5 mL) and dried over anhydrous sodium
sulfate. The organic solvent was evaporated under reduced pressure, and the product was
purified by flash column chromatography.
4.4.1. 6-Oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen-3-yl methanesulfonate (1a)
It was purified by flash column chromatography (silica gel, hexanes:ethyl acetate 5:1
v/v); mp: 172-3 ºC; 1H NMR (CDCl3, 400 MHz) δ 7.72 (d, 1H, J = 9.4 Hz), 7.27-7.24 (m,
13
2H), 3.22 (s, 3H), 2.97-2.91 (m, 4H), 1.92 (d, 2H, J = 5.4 Hz), 1.70-1.62 (m, 4H); C
NMR (CDCl3, 100 MHz) δ 161.4, 153.1, 152.8, 150.0, 129.2, 125.5, 119.1, 118.0, 110.6,
37.9, 31.9, 28.2, 26.9, 25.5, 24.9.
4.4.2. 6-Oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen-3-yl ethanesulfonate (1b)
11

ACCEPTED MANUSCRIPT
It was purified by flash column chromatography (silica gel, hexanes:ethyl acetate 4:1
1
v/v); mp: 169-72 ºC; H NMR (CDCl3, 400 MHz) δ 7.71 (t, 1H, J = 4.6 Hz), 7.24-7.23
(m, 2H), 3.35 (q, 2H, J = 7.4 Hz), 2.96-2.90 (m, 4H), 1.91 (d, 2H, J = 5.5 Hz), 1.70-1.62
13
(m, 4H), 1.58 (t, 3H, J = 7.4 Hz); C NMR (CDCl3, 100 MHz) δ 161.4, 153.1, 152.8,
150.0, 129.0, 125.4, 118.9, 117.9, 110.7, 45.6, 1.9, 28.2, 26.9, 25.5, 24.9, 8.2.
4.4.3. 6-Oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen-3-yl propane-1-sulfonate
(1c)
It was purified by flash column chromatography (silica gel, hexanes:ethyl acetate 5:1
v/v); mp: 133-5 ºC; 1H NMR (CDCl3, 300 MHz) δ 7.72-7.68 (m, 1H), 7.22-7.21 (m, 2H),
3.32-3.25 (m, 2H), 2.95-2.89 (m, 4H), 2.06-1.98 (m, 2H), 1.91 (d, 2H, J = 6.0 Hz), 1.69-
1.61 (m, 4H), 1.17-1.12 (m, 3H); 13C NMR (CDCl3, 75 MHz) δ 161.4, 153.1, 150.1,
129.0, 125.4, 118.9, 118.1, 110.6, 52.7, 31.9, 28.3, 26.9, 25.5, 24.9, 17.4, 12.9.
4.4.4. 6-Oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen-3-yl cyclopropanesulfonate
(1d)
It was purified by flash column chromatography (silica gel, hexanes:ethyl acetate 5:1
1
v/v); mp: 128-30 ºC; H NMR (CDCl3, 400 MHz) δ 7.71 (d, 1H, J = 8.4 Hz), 7.27-7.25
(m, 2H), 3.33 (q, 1H, J = 7.1 Hz), 2.97-2.92 (m, 4H), 2.69-2.63 (m, 1H), 1.93 (brs, 2H),
1.71-1.63 (m, 4H), 1.25-1.18 (m, 4H); 13C NMR (CDCl3, 100 MHz) δ 161.5, 153.0, 152.8,
150.6, 129.0, 125.3, 118.9, 118.3, 110.8, 41.5, 31.9, 28.2, 26.9, 25.5, 24.9, 14.0, 6.4.
4.4.5. 6-Oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen-3-yl benzenesulfonate (1e)
It was purified by flash column chromatography (silica gel, hexanes:ethyl acetate 7:1 v/v
1
then switching to hexanes:ethyl acetate 5:1 v/v); mp: 125-8 ºC; H NMR (CDCl3, 400
MHz) δ 7.86 (d, 2H, J = 7.8 Hz), 7.71 (t, 2H, J = 7.4 Hz), 7.63 (d, 1H, J = 8.9 Hz), 7.56 (t,
3H, J = 7.6 Hz), 2.93-2.87 (m, 4H), 1.90 (d, 2H, J = 5.5 Hz), 1.68-1.58 (m, 4H); 13C
NMR (CDCl3, 100 MHz) δ 161.4, 152.9, 152.8, 150.5, 135.0, 134.7, 129.4, 129.0, 128.4,
125.2, 118.9, 118.4, 110.7, 31.8, 28.2, 26.8, 25.4, 24.8; LC-MS: 371.1 (M+ + 1).
12

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4.4.6.
6-Oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen-3-yl
4-
methylbenzenesulfonate (1f)
It was purified by flash column chromatography (silica gel, hexanes:ethyl acetate 10:1
1
v/v then switching to hexanes:ethyl acetate 7:1 v/v); mp: 149-52 ºC; H NMR (CDCl3,
400 MHz) δ 7.73 (d, 2H, J = 7.2 Hz), 7.64-7.62 (m, 1H), 7.35-7.32 (m, 2H), 7.06 (d, 1H,
J = 8.8 Hz), 6.82 (s, 1H), 2.94-2.88 (m, 4H), 2.47 (s, 3H), 1.91-1.89 (m, 2H), 1.69-1.60
(m, 4H); 13C NMR (CDCl3, 100 MHz) δ 161.5, 152.9, 152.8, 150.6, 146.0, 132.0, 130.0,
128.9, 128.5, 125.2, 118.8, 118.6, 110.7, 31.9, 28.2, 26.8, 25.5, 24.8, 21.8.
4.4.7. 6-Oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen-3-yl 4-(trifluoromethyl)
benzenesulfonate (1g)
It was purified by flash column chromatography (silica gel, hexanes:ethyl acetate 10:1
v/v); mp: 137-40 ºC; 1H NMR (CDCl3, 300 MHz) δ 8.03 (d, 2H, J = 9.0 Hz), 7.85 (d, 2H,
J = 9.0 Hz), 7.66 (d, 1H, J = 9.0 Hz), 7.04-6.95 (m, 2H), 2.95-2.88 (m, 4H), 1.93-1.88 (m,
2H), 1.72-1.59 (m, 4H); 13C NMR (CDCl3, 75 MHz) δ 161.3, 152.9, 152.7, 150.1, 138.7,
136.4, 136.1, 129.3, 129.0, 126.6, 125.4, 119.2, 118.1, 110.7, 31.9, 28.2, 26.9, 25.4, 24.8;
LC-MS: 439.1 (M+ + 1).
4.4.8. 6-Oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen-3-yl 4-(tert-butyl)
benzenesulfonate (1h)
It was purified by flash column chromatography (silica gel, hexanes:ethyl acetate 20:1
v/v then switching to hexanes:ethyl acetate 15:1 v/v); mp: 94-7 ºC; 1H NMR (CDCl3, 400
MHz) δ 7.79 (d, 2H, J = 8.5 Hz), 7.64 (d, 1H, J = 8.9 Hz), 7.56 (d, 2H, J = 8.5 Hz), 7.08
(dd, 1H, J = 2.1 Hz, 8.8 Hz), 6.90 (d, 1H, J = 2.2 Hz), 2.91 (dt, 4H, J = 10.6 Hz, 9.8 Hz),
1.90 (q, 2H, J = 6.0 Hz), 1.69-1.59 (m, 4H), 1.36 (s, 9H); 13C NMR (CDCl3, 100 MHz) δ
161.5, 158.8, 152.9, 152.8, 150.7, 132.2, 128.9, 128.3, 126.4, 125.1, 118.8, 118.5, 110.7,
35.4, 31.9, 31.0, 28.2, 26.8, 25.5, 24.9; LC-MS: 427.2 (M+ + 1).
4.4.9.
6-Oxo-6,7,8,9,10,11-hexahydrocyclohepta[c]chromen-3-yl
4-
fluorobenzenesulfonate (1i)
13

ACCEPTED MANUSCRIPT
It was purified by flash column chromatography (silica gel, hexanes:ethyl acetate 30:1
1
v/v then switching to hexanes:ethyl acetate 25:1 v/v); mp: 147-9 ºC; H NMR (CDCl3,
400 MHz) δ 7.91-7.88 (m, 2H), 7.64 (d, 1H, J = 8.8 Hz), 7.26-7.22 (m, 2H), 7.03 (dd, 1H,
J = 2.3 Hz, 8.8 Hz), 6.89 (d, 1H, J = 2.2 Hz), 2.94-2.88 (m, 4H), 1.90 (q, 2H, J = 6.0 Hz),
1.69-1.60 (m, 4H); 13C NMR (CDCl3, 100 MHz) δ 161.4, 152.8, 152.7, 150.4, 131.4,
131.3, 129.1, 125.3, 119.0, 118.3, 117.0, 116.8, 110.7, 31.8, 28.2, 26.8, 25.4, 24.8.
4.4.10. 6-Oxo-7,8,9,10,11,12-hexahydro-6H-cycloocta[c]chromen-3-yl methanesulfonate
(1j)
It was purified by flash column chromatography (silica gel, hexanes:ethyl acetate 7:1 v/v
1
then switching to hexanes:ethyl acetate 4:1 v/v); mp: 146-8 ºC; H NMR (CDCl3, 400
MHz) δ 7.66 (d, 1H, J = 8.6 Hz), 7.27-7.22 (m, 2H), 3.22 (s, 3H), 2.99 (t, 2H, J = 6.5 Hz),
13
2.82 (t, 2H, J = 6.1 Hz), 1.85-1.71 (m, 4H), 1.56-1.42 (m, 4H); C NMR (CDCl3, 100
MHz) δ 160.8, 153.3, 149.8, 149.5, 127.3, 125.7, 118.6, 118.1, 110.7, 37.9, 29.7, 29.1,
26.9, 26.6, 26.4, 25.9.
4.4.11. 6-Oxo-7,8,9,10,11,12-hexahydro-6H-cycloocta[c]chromen-3-yl ethanesulfonate
(1k)
It was purified by flash column chromatography (silica gel, hexanes:ethyl acetate 7:1 v/v
1
then switching to hexanes:ethyl acetate 4:1 v/v); mp: 157-60 ºC; H NMR (CDCl3, 400
MHz) δ 7.65 (d, 1H, J = 9.4 Hz), 7.26-7.22 (m, 2H), 3.35 (q, 2H, J = 7.4 Hz), 2.98 (t, 2H,
13
J = 4.6 Hz), 2.81 (t, 2H, J = 3.9 Hz), 1.81-1.73 (m, 4H), 1.59-1.44 (m, 7H); C NMR
(CDCl3, 100 MHz) δ 160.8, 153.3, 149.8, 149.5, 127.2, 125.6, 118.4, 118.1, 110.6, 45.6,
29.7, 29.1, 26.9, 26.6, 26.4, 25.9, 8.3.
4.4.12.
6-Oxo-7,8,9,10,11,12-hexahydro-6H-cycloocta[c]chromen-3-yl
propane-1-
sulfonate (1l)
It was purified by flash column chromatography (silica gel, hexanes:ethyl acetate 7:1
v/v); mp: 114-7 ºC; 1H NMR (CDCl3, 400 MHz) δ 7.65 (d, 1H, J = 5.9 Hz), 7.25-7.22 (m,
2H), 3.31-3.24 (m, 2H), 2.99 (t, 2H, J = 6.0 Hz), 2.82 (t, 2H, J = 5.8 Hz), 2.09-1.98 (m,
13
2H), 1.82-1.73 (m, 4H), 1.53-1.44 (m, 4H), 1.15 (t, 3H, J = 7.4 Hz); C NMR (CDCl3,
14

ACCEPTED MANUSCRIPT
100 MHz) δ 160.8, 153.3, 149.9, 149.6, 127.2, 125.6, 120.9, 118.1, 110.6, 52.7, 29.7,
29.1, 26.9, 26.6, 26.4, 25.9, 17.4, 12.8.
4.4.13.
6-Oxo-7,8,9,10,11,12-hexahydro-6H-cycloocta[c]chromen-3-yl
cyclopropanesulfonate (1m)
It was purified by flash column chromatography (silica gel, hexanes:ethyl acetate 7:1 v/v
1
then switching to hexanes:ethyl acetate 5:1 v/v); mp: 133-6 ºC; H NMR (CDCl3, 400
MHz) δ 7.65 (d, 1H, J = 8.5 Hz), 7.27-7.24 (m, 2H), 3.00 (t, 2H, J = 6.6 Hz), 2.82 (t, 2H,
J = 6.1 Hz), 2.71-2.65 (m, 2H), 1.83-1.74 (m, 6H), 1.54-1.46 (m, 6H), 1.34-1.19 (m, 5H);
13C NMR (CDCl3, 100 MHz) δ 160.9, 153.2, 150.4, 149.6, 127.2, 125.6, 118.4, 110.9,
29.7, 29.1, 28.2, 26.9, 26.6, 26.4, 25.9, 6.4.
4.4.14. 6-Oxo-7,8,9,10,11,12-hexahydro-6H-cycloocta[c]chromen-3-yl benzenesulfonate
(1n)
It was purified by flash column chromatography (silica gel, hexanes:ethyl acetate 10:1
1
v/v then switching to hexanes:ethyl acetate 6:1 v/v); mp: 108-11 ºC; H NMR (CDCl3,
400 MHz) δ 7.88 (d, 2H, J = 8.0 Hz), 7.72 (t, 1H, J = 7.0 Hz), 7.57 (t, 3H, J = 7.6 Hz),
7.04 (dd, 1H, J = 2.1 Hz, 8.8 Hz), 6.85 (d, 1H, J = 2.1 Hz), 2.96 (t, 2H, J = 6.8 Hz), 2.79
(t, 2H, J = 6.1 Hz), 1.81-1.72 (m, 4H), 1.52-1.44 (m, 4H); 13C NMR (CDCl3, 100 MHz) δ
160.8, 153.0, 150.3, 149.6, 135.1, 134.7, 129.4, 128.4, 127.2, 125.5, 118.5, 118.4, 110.8,
29.6, 29.1, 26.9, 26.6, 26.4, 25.9.
4.4.15.
6-Oxo-7,8,9,10,11,12-hexahydro-6H-cycloocta[c]chromen-3-yl
4-
methylbenzenesulfonate (1o)
It was purified by flash column chromatography (silica gel, hexanes:ethyl acetate 12:1
1
v/v then switching to hexanes:ethyl acetate 6:1 v/v); mp: 107-10 ºC; H NMR (CDCl3,
400 MHz) δ 7.74 (d, 2H, J = 8.2 Hz), 7.58 (d, 1H, J = 8.8 Hz), 7.36-7.29 (m, 2H), 7.05
(dd, 1H, J = 1.9 Hz, 8.8 Hz), 6.82 (d, 1H, J = 1.9 Hz), 2.95 (t, 2H, J = 5.7 Hz), 2.78 (t, 2H,
13
J = 5.0 Hz), 2.47 (s, 3H), 1.80-1.71 (m, 4H), 1.50-1.42 (m, 4H); C NMR (CDCl3, 100
MHz) δ 160.9, 152.9, 150.4, 149.7, 146.0, 132.0, 130.1, 128.4, 127.1, 125.4, 118.7, 118.3,
110.8, 29.6, 29.1, 26.9, 26.6, 26.4, 25.8, 21.8; LC-MS: 399.2 (M+ + 1).
15

ACCEPTED MANUSCRIPT
4.4.16.
6-Oxo-7,8,9,10,11,12-hexahydro-6H-cycloocta[c]chromen-3-yl
4-
(trifluoromethyl)
benzenesulfonate (1p)
It was purified by flash column chromatography (silica gel, hexanes:ethyl acetate 12:1
v/v then switching to hexanes:ethyl acetate 8:1 v/v); mp: 154-7 ºC; 1H NMR (CDCl3, 400
MHz) δ 8.04 (d, 2H, J = 8.2 Hz), 7.85 (d, 2H, J = 8.3 Hz), 7.60 (d, 1H, J = 8.8 Hz), 7.03
(dd, 1H, J = 2.3 Hz, 8.8 Hz), 6.94 (d, 1H, J = 2.2 Hz), 2.97 (t, 2H, J = 6.4 Hz), 2.81 (t, 2H,
J = 6.0 Hz), 1.81-1.73 (m, 4H), 1.53-1.44 (m, 4H); 13C NMR (CDCl3, 100 MHz) δ 160.7,
153.1, 150.0, 149.5, 138.8, 136.4, 129.0, 127.5, 126.6, 125.6, 118.7, 118.2, 110.7, 29.6,
29.1, 26.9, 26.6, 26.4, 25.9.
4.4.17. 6-Oxo-7,8,9,10,11,12-hexahydro-6H-cycloocta[c]chromen-3-yl 4-(tert-butyl)
benzenesulfonate (1q)
It was purified by flash column chromatography (silica gel, hexanes:ethyl acetate 20:1
1
v/v then switching to hexanes:ethyl acetate 15:1 v/v); mp: 166-9 ºC; H NMR (CDCl3,
400 MHz) δ 7.81 (d, 2H, J = 8.5 Hz), 7.59-7.56 (m, 3H), 7.07 (dd, 1H, J = 2.2 Hz, 8.8
Hz), 6.91 (d, 1H, J = 2.2 Hz), 2.96 (t, 2H, J = 6.4 Hz), 2.79 (t, 2H, J = 6.0 Hz), 1.83-1.69
(m, 4H), 1.52-1.43 (m, 4H), 1.36 (s, 9H); 13C NMR (CDCl3, 75 MHz) δ 160.9, 158.8,
153.0, 150.5, 149.7, 132.2, 128.3, 127.1, 126.5, 125.4, 118.6, 118.3, 110.7, 35.4, 31.0,
29.7, 29.1, 26.9, 26.6, 26.4, 25.9.
4.4.18.
6-Oxo-7,8,9,10,11,12-hexahydro-6H-cycloocta[c]chromen-3-yl
4-
fluorobenzenesulfonate (1r)
It was purified by flash column chromatography (silica gel, hexanes:ethyl acetate 30:1
1
v/v); H NMR (CDCl3, 400 MHz) δ 7.92-7.89 (m, 2H), 7.58 (d, 1H, J = 8.8 Hz), 7.24-
7.22 (m, 2H), 7.05 (dd, 1H, J = 2.2 Hz, 8.7 Hz), 6.88 (d, 1H, J = 2.2 Hz), 2.97 (t, 2H, J =
6.5 Hz), 2.81 (t, 2H, J = 6.1 Hz), 1.81-1.73 (m, 4H), 1.53-1.44 (m, 4H); 13C NMR (CDCl3,
100 MHz) δ 160.8, 153.0, 150.2, 149.5, 131.4, 131.3, 127.3, 125.5, 118.5, 118.4, 117.0,
116.8, 110.8, 29.6, 29.1, 26.9, 26.6, 26.4, 25.9.
16

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4.5. Cancer cell line screening at the NCI
Screening against the cancer cell lines was carried out at the National Cancer Institute
(NCI), Bethesda, Maryland, USA [46] applying the standard protocol of the NCI [51,52].
4.6. MTT assay for RAW 264.7 macrophage cell viability
RAW 264.7 macrophages were plated at a density of 105 cells/well in 96-well plates. To
determine the appropriate concentration not toxic to cells, cytotoxicity studies were
performed 24 h after treating cells with various concentrations of tested compounds. Cell
viabilities were determined using colorimetric MTT assays, as described previously [53].
4.7. In vitro cyclooxygenase (COX) inhibition assay
The ability of the test compounds to inhibit bovine COX-1 and COX-2 was determined
using an enzyme immunoassay (EIA) (kit catalog number 560101, Cayman Chemical,
Ann Arbor, MI, USA) according to the manufacturer’s instructions. Cyclooxygenase
catalyzes the ※rst step in the biosynthesis of arachidonic acid to PGH2. PGF2α, produced
from PGH2 by reduction with stannous chloride, was measured by enzyme immunoassay
(ACETM competitive EIA). Stock solutions of test compounds were dissolved in a
minimum volume of DMSO. Brie‼y, to a series of supplied reaction buffer solutions (960
µL, 0.1 M Tris–HCl pH 8.0 containing 5 mM EDTA and 2 mM phenol) with either
COX-1or COX-2 (10 µL) enzyme in the presence of heme (10 µL) were added 10 µL of
various concentrations of test drug solutions (0.01, 0.1, 1, 10, and 50 µM in a ※nal
volume of 1 mL). These solutions were incubated for a period of 5 min at 37 ºC after
which 10 µL of arachidonic acid (100 µM) solution were added and the COX reaction
was stopped by the addition of 50 µL of 1 M HCl after 2 min. PGF2α, produced from
PGH2 by reduction with stannous chloride was measured by enzyme immunoassay. This
assay is based on the competition between PGs and a PG-acetylcholinesterase conjugate
(PG tracer) for a limited amount of PG antiserum. The amount of PG tracer that is able to
bind to the PG antiserum is inversely proportional to the concentration of PGs in the
wells since the concentration of PG tracer is held constant while the concentration of PGs
varies. This antibody–PG complex bound to a mouse anti-rabbit monoclonal antibody
that was previously attached to the well. The plate was washed to remove any unbound
17

ACCEPTED MANUSCRIPT
reagents and then Ellman’s reagent, which contains the substrate to acetylcholine esterase,
was added to the well. The product of this enzymatic reaction produced a distinct yellow
color that absorbs at 405 nm. The intensity of this color, determined
spectrophotometrically, was proportional to the amount of PG tracer bound to the well,
which was inversely proportional to the amount of PGs present in the well during the
incubation: Absorbance α [Bound PG Tracer] α1/PGs. Percent inhibition was calculated
by the comparison of compound treated to various control incubations. The concentration
of the test compound causing 50% inhibition (IC50, µM) was calculated from the
concentration–inhibition response curve (duplicate determinations).
Acknowledgments
This work was supported by Korea Institute of Science and Technology (KIST), KIST
Project (2E23720). We would like to thank the National Cancer Institute (NCI), Bethesda,
Maryland, USA, for performing the in vitro anticancer testing over the cell lines.
References
[1]
http://www.cancer.org/acs/groups/content/@epidemiologysurveilance/documents/docum
ent/acspc-031941.pdf Retrieved on Apr. 29th, 2014.
[2] http://www.who.int/mediacentre/factsheets/fs297/en/ Retrieved on Apr. 29th, 2014.
[3] I. Kostova, Curr. Med. Chem. 5 (2005) 29-46.
[4] S.J. Stanway, A. Purohit, L. W. L. Woo, S. Sufi, D. Vigushin, R. Ward, R.H. Wilson,
F. Z. Stanczyk, N. Dobbs, E. Kulinskaya, M. Elliott, B.V.L. Potter, M. J. Reed, R.C.
Coombes, Clin. Cancer Res. 12 (2006) 1585-1592.
[5] W. Li, Y.N. Sun, X.T. Yan, S.Y. Yang, E.-J. Kim, H.K. Kang, Y.H. Kim, J. Agric.
Food Chem. 61 (2013) 10730-10740.
[6] R. M. Mohareb, S. M. Sherif, A. Abou Elkair, Curr. Org. Chem. 17 (2013) 1910-1918.
[7] T. Nasr, S. Bondock, M. Youns, Eur. J. Med. Chem. 76 (2014) 539-548.
[8] K. Paul, S. Bindal, V. Luxami, Bioorg. Med. Chem. Lett. 23 (2013) 3667-3672.
[9] K.B. Puttaraju, K. Shivashankar, C.M. Mahendra, V.P. Rasal, P.N.V. Vivek, K. Rai,
M.B. Chanu, Eur. J. Med. Chem. 69 (2013) 316-322.
18

ACCEPTED MANUSCRIPT
[10] O. Dömötör, T. Tuccinardi, D. Karcz, M. Walsh, B. S. Creaven, É. A. Enyedy,
Bioorg. Chem. 52 (2014) 16-23.
[11] M. Basanagouda, V.B. Jambagi, N.N. Barigidad, S.S. Laxmeshwar, V. Devaru,
Narayanachar, Eur. J. Med. Chem. 74 (2014) 225-233.
[12] B.R.V. Avin, P. Thirusangu, V.L. Ranganatha, A. Firdouse, B.T. Prabhakar, S.A.
Khanum, Eur. J. Med. Chem. 75 (2014) 211-221.
[13] K.M. Amin, A.A.M. Eissa, S.M. Abou-Seri, F.M. Awadallah, G.S. Hassan, Eur. J.
Med. Chem. 60 (2013) 187-198.
[14] C.M. Elinos-Báez, F. León, E. Santos, Cell. Biol. Int. 29 (2005) 703-708.
[15] 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.
[16] G.J. Finn, B.S. Creaven, D.A. Egan, Eur. J. Pharm. Sci. 26 (2005) 16-25.
[17] L.D. Raev, E. Voinova, I.C. Ivanov, D. Popov, Pharmazie 45 (1990) 696-702.
[18] 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.
[19] E.K. Kim, K.B. Kwon, B.C. Shin, E.A. Seo, Y.R. Lee, J.S. Kim, J.W. Park, B.H.
Park, D.G. Ryu, Life Sci. 77 (2005) 824-836.
[20] M.G. Manuele, G. Ferraro, A.M.L. Barreiro, P. Lopez, G. Cremaschi, C. Anesini,
Life Sci. 79 (2006) 2043-2048.
[21] http://clinicaltrials.gov/ct2/results?term=stx-64&Search=Search Retrieved on April
29th, 2014.
[22] M. Khoobi, A. Foroumadi, S. Emami, M. Safavi, G. Dehghan, B.H. Alizadeh, A.
Ramazani, S.K. Ardestani, A. Shafiee, Chem. Biol. Drug Des. 78 (2011) 580-586.
[23] S. Funakoshi, H. Takaishi, T. Hibi, Surg. Trauma Immunol. Responses 20 (2011)
166-168.
[24] D. Wang, R.N. Dubois, Gut 55 (2006) 115-122.
[25] A. Krishna, A.K.C. Divvela, S.R. Challa, I.K. Tagaram, 56 (2010) 502-516.
[26] Z. Khan, N. Khan, R.P. Tiwari, N.K. Sah, G.B.K.S. Prasad, P.S. Bisen, Curr. Drug
Targets 12 (2011) 1082-1093.
19

ACCEPTED MANUSCRIPT
[27] C. Denkert, M. Kobel, S. Berger, A. Siegert, A. Leclere, U. Trefzer, S. Hauptmann,
Cancer Res. 61 (2001) 303-308.
[28] F. Bianchini, D. Massi, C. Marconi, A. Franchi, G. Baroni, M. Santucci, A. Mannini,
G. Mugnai, L. Calorini, Prostaglandins Other Lipid Mediat. 83 (2007) 320-328.
[29] B.W. Chwirot, L. Kuzbicki, Melanoma Res. 17 (2007) 139-145.
[30] C.C. Johansson, S. Egyhazi, G. Masucci, H. Harlin, D. Mougiakakos, I. Poschke, B.
Nilsson, L. Garberg, R. Tuominen, D. Linden, M.F. Stolt, J. Hansson, R. Kiessling,
Cancer Immunol. Immunother. 58 (2009) 1085-1094.
[31] M. Tsujii, S. Kawano, R.N. Dubois, Proc. Natl. Acad. Sci. USA 94 (1997) 3336-
3340.
[32] M. Huang, M. Stolina, S. Sharma, J.T. Mao, L. Zhu, P.W. Miller, J. Wollman, H.
Herschman, S.M. Dubinett, Cancer Res. 58 (1998) 1208-1216.
[33] C.S. Williams, R.L. Shattuk-Brandts, R.N. Dubois, Expert Opin. Investig. Drugs 8
(1999) 1-12.
[34] K.M. Sheehan, K. Sheahan, D.P. O’Donoghue, F. MacSweeny., R.M. Conroy, D.J.
Fitzgerald, F.E. Murray, JAMA 282 (1999) 1254-1257.
[35] M.A. Molina, M. Sitja-Arnau, M.G. Lemoine, M.L. Frazier, F.A. Sinicrope, Cancer
Res. 59 (1999) 4356-4362.
[36] H.S. Ryu, K.H. Chang, H.W. Yang, M.S. Kim, H.C. Kown, K.S. Oh, Gynecol.
Oncol. 76 (2000) 320-325.
[37] L.R. Howe, K. Subbaramaiah, A.M. Brown, A.J. Dannenberg, Endocr. Relat. Cancer
8 (2001) 97-114.
[38] R. Fujikawi, K. Iida, H. Kanasaki, T. Ozaki, K. Hata, K. Miyazaki, (2002)
Cyclooxygenase-2 expression in endometrial cancer: correlation with microvessel count
and expression of vascular endothelial growth factor and thymidine phosphorylase. Hum.
Pathol. 33 (2002) 213-219.
[39] P. Nix, M. Lind, J. Greenman, N. Stafford, L. Cawkwell, Ann. Oncol. 15 (2004)
797-801.
[40] P. Siironen, A. Ristimaki, S. Nordling, J. Louhimo, R. Haapiainen, C. Haglund,
Histopathology 44 (2004) 490-497.
20

ACCEPTED MANUSCRIPT
[41] C. Wu, H.F. Tsai, W.C. Lin, A.H. Chou, H.T. Chen, J.C. Yang, P.I. Hsu, P.N. Hsu,
World J. Gastroenterol. 11 (2005) 3197-3203.
[42] M.I. El-Gamal, H.S. Choi, K.H. Yoo, D. Baek, C.-H. Oh, Chem. Biol. Drug. Des. 82
(2013) 336-347.
[43] H.-L. Jang, M.I. El-Gamal, H.-E. Choi, H.-Y. Choi, K.-T. Lee, C.-H. Oh, Bioorg.
Med. Chem. Lett. 24 (2014) 571-575.
[44] C.J. Li, D.L. Chen, Y.Q. Lu, J.X. Haberman, J.T. Mague, Tetrahedron 54 (1998)
2347-2364.
[45] L.W.L. Woo, A. Purohit, B. Malini, M.J. Reed, B.V.L. Potter, Chemistry & Biology
7 (2000) 773-791.
[46] NCI website: www.dtp.nci.nih.gov.
[47] DTP Data Search: ‹http://dtp.nci.nih.gov/dtpstandard/dwindex/index.jsp›.
[48] S. Battu, J.L. Beneytout, M. Pairet, M. Rigaud, FEBS Lett. 437 (1998) 49-55.
[49] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Adv. Drug Deliv. Rev. 46
(2001) 3-26.
[50] D.E. Clark, S.D. Pickett, Drug Discov. Today 5 (2000) 49-58.
[51] DTP Data Search: http://dtp.nci.nih.gov/dtpstandard/dwindex/index.jsp. Retrieved
on Apr. 29th, 2014.
[52]
DTP
Human
Tumor
Cell
Line
Screen
Process:
http://www.dtp.nci.nih.gov/branches/btb/ivclsp.html. Retrieved on Apr. 29th, 2014.
[53] J.-Y. Kim, S.J. Park, K.-J. Yun, Y.-W. Cho, H.-J. Park, K.-T. Lee, Eur. J.
Pharmacol. 584 (2008) 175-184.
21

Table(s)
ACCEPTED MANUSCRIPT
Table 1. Structures of the target compounds and their yield percentages.
n
O
O
S
R
O
O
O
Compound
R
n
Yield%
No.
1a
1b
1c
1d
1e
1f
Me
Et
1
1
1
1
1
1
1
95
92
93
85
93
95
90
n-Pr
Cyclo-Pr
Ph
p-Tolyl
p-(CF3)C6H4
p-(tert-
1g
1
95
1h
butyl)C6H4
p-(F)C6H4
Me
1
2
2
2
2
2
2
2
82
94
90
88
87
92
95
90
1i
1j
Et
1k
1l
n-Pr
Cyclo-Pr
Ph
1m
1n
1o
1p
p-Tolyl
p-(CF3)C6H4
p-(tert-
2
2
94
90
1q
1r
butyl)C6H4
p-(F)C6H4

Table(s)
ACCEPTED MANUSCRIPT
Table 2. Mean IC50 values (μM) of the tested compounds over in vitro subpanel cancer
cell linesa
No. of cell
line in
Compound No.
1h
Cancer
each
Subpanel
1e
1f
1i
1o
subpanel
3.60
(8.37)c
40.56
8.48
(3.55)c
54.68
30.14
65.87
41.68
51.31
36.20
4.54
(8.06)c
60.68
20.82
(1.76)c
79.70
76.06
68.98
89.46
52.13
36.60
6.26
(8.46)c
76.21
17.46
(3.03)c
>100
Leukemia
NSCLCb
Colon
6
8
6
67.73
15.92
46.13
75.93
78.45
27.36
CNS
Melanoma
Ovarian
Renal
6
9
6
8
2
6
21.17
18.78
19.12
16.88
20.06
12.48
86.60
88.21
86.54
>100
>100
40.23
76.46
86.44
75.96
52.99
67.85
Prostate
Breast
a Mean IC50 values were calculated by dividing the summation of IC50 values of the
compound over cell lines of the same cancer type by the number of cell lines in the
subpanel.
b Non-Small Cell Lung Cancer.
c Selectivity index.