Antiproliferative activity of cycloalkanecarboxamide derivatives possessing sulfonate or sulfamate moiety

Article abstract of DOI:10.1016/j.bioorg.2020.103677

A series of cycloalkanecarboxamide-containing sulfonate and sulfamate derivatives were prepared, and their antiproliferative activity was tested against NCI-60 cancer cell lines panel. Compound 1f possessing cyclohexyl and p-(tert-butyl)benzenesulfonate moieties was the most active among all the target compounds. It exerted broad-spectrum anticancer activity against all the nine cancer types involved in the NCI-60 panel. Additionally, compound 1g containing cyclohexyl and p-fluorobenzenesulfonate moieties was the most potent against HT29 colon cancer cell line (IC₅₀ = 4.73 μM) with selectivity index more than 4.23 towards HT29 than normal fibroblasts. It exerts its antiproliferative activity against HT29 through the induction of apoptosis (increasing caspase 3/7 activity) but not necrosis. Structure-activity relationship studies are presented in detail.

Full text of DOI:10.1016/j.bioorg.2020.103677

Journal Pre-proofs
Antiproliferative activity of cycloalkanecarboxamide derivatives possessing
sulfonate or sulfamate moiety
Mohammed I. El-Gamal, Hany A. Omar, Mohammad H. Semreen, Israa A.
Younes, Youmna Y. Zaghloul, Ayat E. Abbas, Iman G. Moussa, Fatima
Hersi, Chang-Hyun Oh
PII:
S0045-2068(20)30125-5
DOI:
https://doi.org/10.1016/j.bioorg.2020.103677
YBIOO 103677
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
16 January 2020
13 February 2020
18 February 2020
Please cite this article as: M.I. El-Gamal, H.A. Omar, M.H. Semreen, I.A. Younes, Y.Y. Zaghloul, A.E. Abbas,
I.G. Moussa, F. Hersi, C-H. Oh, Antiproliferative activity of cycloalkanecarboxamide derivatives possessing
sulfonate or sulfamate moiety, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103677
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Antiproliferative activity of cycloalkanecarboxamide derivatives possessing
sulfonate or sulfamate moiety
Mohammed I. El-Gamal 1,2,3,*,$, Hany A. Omar1,2,4,*,$, Mohammad H. Semreen 1,2, Israa A.
1
1
1
1
2
Younes , Youmna Y. Zaghloul , Ayat E. Abbas , Iman G. Moussa , Fatima Hersi , and
Chang-Hyun Oh 5,6
1 College of Pharmacy, University of Sharjah, Sharjah 27272, United Arab Emirates.
2
Sharjah Institute for Medical Research, University of Sharjah, Sharjah 27272, United Arab
Emirates.
3 Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura
35516, Egypt.
4 Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt.
5
Center for Biomaterials, Korea Institute of Science and Technology, PO Box 131,
Cheongryang, Seoul 130-650, Republic of Korea.
6
Department of Biomolecular Science, Korea University of Science and Technology, 113
Gwahangno, Yuseong-gu, Daejeon 305-333, Republic of Korea.
*Corresponding authors: e-mail addresses: drmelgamal2002@gmail.com [M.I. El-Gamal] &
hanyomar@sharjah.ac.ae [H.A. Omar]; address: College of Pharmacy, University of Sharjah,
Sharjah 27272, United Arab Emirates.
$ Co-first authors.
Abstract
A series of cycloalkanecarboxamide-containing sulfonate and sulfamate derivatives were
prepared, and their antiproliferative activity was tested against NCI-60 cancer cell lines panel.
Compound 1f possessing cyclohexyl and p-(tert-butyl)benzenesulfonate moieties was the most
active among all the target compounds. It exerted broad-spectrum anticancer activity against all
the nine cancer types involved in the NCI-60 panel. Additionally, compound 1g containing
cyclohexyl and p-fluorobenzenesulfonate moieties was the most potent against HT29 colon
cancer cell line (IC50 = 4.73 µM) with selectivity index more than 4.23 towards HT29 than
normal fibroblasts. It exerts its antiproliferative activity against HT29 through the induction of
Page 1 of 21

apoptosis (increasing caspase 3/7 activity) but not necrosis. Structure-activity relationship studies
are presented in detail.
Keywords: Anticancer; Antiproliferative; Apoptosis; Sulfamate; Sulfonate.
1. Introduction
Cancer usually originates from cells that transform and lose control of cell growth and
division into unrestrained aggressive behavior with the ability to invade surrounding tissues.
This transformation is linked to changes and mutations in the genetic material caused by
different intrinsic or extrinsic mutagens [1]. Cancer is classified as the second cause of death
after heart disease internationally. The World Health Organization (WHO) reported that cancer
caused 9.6 million deaths in 2018. One in each six death cases worldwide occur due to cancer
[2]. Consequently, it is one of the most serious diseases that required urgent development of
more efficient and selective anticancer agents.
Several sulfonate and sulfamate derivatives have been reported as antiproliferative agents
[3-12]. Examples of them are illustrated in Figure 1. We have previously reported a series of
cycloalkanecarboxamide-possessing sulfonate and sulfamate derivatives as inhibitors of steroid
sulfatase or nucleotide pyrophosphatase enzymes (Figure 1) [13, 14]. In this article, we report the
cell-based antiproliferative activity of the target compounds 1a-z against NCI-60 tumor cell lines
of 9 types of cancer [15]. The most active compounds were tested for potency against cancer and
normal cell lines to investigate their selectivity indexes.
Page 2 of 21

O
S
O
O
S
O
S
O
O
O
O
O
S
O
O
O
O
N
Coumarin sulfonate derivative
Raloxifene bis-sulfonate derivative
O
O
S
O
NH2
H
O
O
O
O
O
H
H
S
S
H2N
O
O
O
H2N
O
STX 140
Irosustat (STX64)
H
N
n
O
O
S
O
R
O
The target compounds 1a-z
R = alkyl, aryl, NH2, NHMe, NMe2
n = 1, 2, 3, 4
Figure 1. Structures of reported antiproliferative sulfonate or sulfamate derivatives and the target
compounds 1a-z.
2. Results and discussion
2.1.
Chemical synthesis
The final compounds 1a-z were prepared via the 2-step synthetic pathway shown in
Scheme 1. The reaction of 4-aminophenol (2) with the appropriate cycloalkanecarbonyl chloride
3a-d produced the relevant phenolic intermediates 4a-d. In order to avoid diacylation and retain
the OH group free, the acid chloride was diluted in acetone and added slowly to a mixture of 4-
aminophenol and potassium carbonate in acetone while swirling. Sulfonate derivatives 1a-h and
1l-z were synthesized through the reaction of the phenolic compounds 4a-d with appropriate
Page 3 of 21

sulfonyl chloride reagents in the presence of triethylamine. Furthermore, sulfamate derivatives
1i-k were obtained through the interaction of 4b with the appropriate (substituted) sulfamoyl
chloride using NaH. All the final compounds were purified by flash column chromatography and
were characterized by spectral analysis. Table 1 illustrates the target compounds’ structures.
H
N
H
R1
N
R1
NH2
ii
i
Cl
R1
O
R2
O
+
O
S
O
HO
HO
O
O
3a-d
2
4a-d
1a-h, 1l-z
R2 = alkyl, aryl
4a: R1 = cyclopentyl
4b: R1 = cyclohexyl
4c: R1 = cycloheptyl
4d: R1 = cyclooctyl
iii
H
N
R1
O
O
R2
S
O
N
O
R3
1i-k
1i: R2, R3 = H
1j: R2 = H, R3 = Me
1k: R2, R3 = Me
Scheme 1. (i) anhydrous K2CO3, acetone, 0 ºC, rt, 4 h, 65-80%; (ii) appropriate sulfonyl chloride
derivative, Et3N, anhydrous THF, 0 ºC, rt, 2 h, 43-91%; (iii) (substituted) sulfamoyl chloride,
NaH, anhydrous, DMF, 0 ºC, rt, overnight, 83-90%.
Page 4 of 21

Table 1. Structures of the target compound 1a-z.
H
N
n
O
O
S
O
R
O
Compound No.
n
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
3
3
3
3
3
4
4
4
R
Me
1a
1b
1c
1d
1e
1f
Et
n-Pr
Ph
4-Me(C6H4)
4-tert-butyl(C6H4)
4-F(C6H4)
4-CF3(C6H4)
NH2
1g
1h
1i
1j
NHMe
1k
1l
NMe2
Ph
1m
1n
1o
1p
1q
1r
1s
4-Me(C6H4)
4-tert-butyl(C6H4)
4-F(C6H4)
4-CF3(C6H4)
Ph
4-Me(C6H4)
4-tert-butyl(C6H4)
4-F(C6H4)
4-CF3(C6H4)
Ph
1t
1u
1v
1w
1x
4-Me(C6H4)
4-tert-butyl(C6H4)
Page 5 of 21

1y
1z
4
4
4-F(C6H4)
4-CF3(C6H4)
2.2.Structure-activity relationship
In the beginning, we synthesized the cyclohexyl analogues 1a-k and tested the compounds
selected by the National Cancer Institute (NCI, Bethesda, Maryland, USA) against the NCI-60
cancer cell line panel of nine different cancer types [15]. The mean inhibition percentages of
each tested compound against the whole panel are shown in Figure 2. It is obvious that the aryl
sulfonate derivatives 1d-h are more active than the alkyl sulfonates 1b,c and the sulfamate
derivatives 1i-k. As the aryl derivatives are more hydrophobic than alkyl or (substituted)amino,
it could be hypothesized that the aryl sulfonates could be more able to cross the cell membrane
and produce stronger antiproliferative effects due to higher cellular exposure to the compound.
Furthermore, the aryl rings might form stronger hydrophobic interaction with the receptor site(s)
such as nucleotide pyrophosphatases (NPP1 and NPP3) [14]. Any or both of these two
explanations could rationalize the stronger activity of aryl sulfonates than alkyl sulfonates or
sulfamate analogues.
Figure 2. Mean % inhibition values of compounds 1b-k over the NCI-60 cell line panel at a
single-dose concentration of 10 µM. They were calculated by dividing the summation of %
inhibition values over the number of tested cell lines.
Page 6 of 21

The inhibitory effects of compounds 1d-h against each single cell line of the NCI-60 panel
are illustrated in Figure 3. Compounds 1d, 1e, and 1g demonstrated the highest activity against
HT29 colon cancer cell line. Among them, compound 1e possessing p-toluenesulfonate moiety
was the most active (% inhibition = 87.0%). Compound 1f containing p-(tert-
butyl)benzenesulfonate exerted broad-spectrum anticancer activity against all the nine subpanels.
Its highest inhibitory effect was against T-47D breast cancer cell line and RPMI-8226 leukemia
cell line (% inhibition values are 99.1% and 86.38%, respectively). The tert-butyl group is
bulkier and more hydrophobic than the other substituents in the other arylsulfonate derivatives,
so it might enhance the activity by its hydrophobicity and/or steric effect(s). Compound 1h
possessing p-(trifluoromethyl)benzenesulfonate moiety exerted selectivity towards the CNS
cancer subpanel compared to its results against the cell lines of the other eight cancer types.
Page 7 of 21

Figure 3a
Page 8 of 21

Figure 3b
Page 9 of 21

Figure 3c
Page 10 of 21

Figure 3d
Page 11 of 21

Figure 3e
Figure 3. % inhibition values of compounds 1d (Fig. 3a), 1e (Fig. 3b), 1f (Fig. 3c), 1g (Fig. 3d), and 1h (Fig. 3e) against the NCI-60
cancer cell lines.
Page 12 of 21

After that, we decided to focus on the aryl sulfonate derivatives only as they were more
promising than the corresponding alkyl sulfonates and sulfamates. The cyclohexyl ring of
compounds 1d-h was replaced with smaller (cyclopentyl ring, compounds 1l-p) or expanded
(cycloheptyl derivatives 1q-u and cyclooctyl 1v-z) rings. These derivatives that were accepted
by the NCI were tested over the NCI-60 panel also, and the effect of ring size on activity was
investigated. The mean % inhibition values of the target compounds with different ring sizes are
depicted in Figure 4. The effect of cycloalkane ring size on activity was found to be parabolic.
Increasing the ring size from cyclopentyl to cyclohexyl increased the activity, but further ring
expansion to cycloheptyl or cyclooctyl was detrimental to the activity.
It was also noticeable that the tert-butylbenzenesulfonate analogues 1s and 1x were more
active than the corresponding analogues possessing the same cycloalkyl ring size and different
terminal aryl moieties. This finding is similar to the results of compound 1f upon comparison
with the other cyclohexyl analogues.
Figure 4. Comparison of mean % inhibition values of all the tested compounds. Data of
compounds 1b-k are taken from Figure 2.
The most active compounds 1d-h were further tested in 5-dose testing mode by MTT
assay against A-549 lung cancer, HT29 colorectal cancer, and MCF7 breast cancer cells in order
to investigate their potency. We have followed the protocol reported in the literature [5]. They
Page 13 of 21

were also tested against fibroblasts to study the selectivity indexes of these compounds against
cancer cells compared to normal cells. The results are summarized in Table 2. Among the five
tested compounds, compound 1f was the most potent against MCF7 breast cancer cell line. Its
selectivity towards cancer cells than fibroblasts is higher than that of doxorubicin. In addition,
compound 1g possessing p-fluorobenzenesulfonate was the most potent against HT29 colon
cancer cell line. It was more selective against it than fibroblasts and even than the other two
tested cancer cell lines. Its selectivity index is more than 2.6 times the selectivity index of
doxorubicin.
Table 2. Potency and selectivity indexes of compounds 1d-h against three cancer cell lines
compared to doxorubicin. a
Compound
No.
IC50 (µM)
Selectivity Selectivity Selectivity
Index
Index
Index
HT29 A549 MCF7 Fibroblasts
(HT29)
(A549)
(MCF7)
1d
8.11
5.45
9.71
4.73
>20
>20
9.32
>20
>20
>20
7.25
>20
>20
0.94
>20
9.21
16.42
>20
>2.47
1.69
-
-
1e
-
-
2.26
-
1f
1.69
1.76
-
1g
1h
>4.22
1.09
13.64 15.91
1.20 1.32
14.83
1.94
0.93
1.47
-
Doxorubicin
1.62
1.72
a
The selectivity indexes were calculated by dividing the IC50 value against fibroblasts by the
IC50 value against the cancer cell line (n=6).
Furthermore, compound 1g was further tested for the induction of apoptosis and necrosis
over HT29 colon cancer cells. It was tested at 5 µM and 10 µM concentrations in caspase-3/7
and lactate dehydrogenase (LDH) release assays in order to investigate its ability to induce
Page 14 of 21

apoptosis and necrosis, respectively. In caspase-3/7 apoptosis assay, compound 1g increased
caspases activity by 68% and 123% at 5 µM and 10 µM concentrations, respectively, compared
to untreated cells (Figure 5). On the other hand, the compound was unable to cause LDH release
at both of the tested concentrations. So it can be concluded that compound 1g could exert its
antiproliferative activity against HT29 cells through induction of apoptosis but not necrosis.
Fold of Caspase-3/7 activity
2.5
2
1.5
1
0.5
0
0
5 µM
10 µM
Figure 5. Caspase-3/7 activity after treatment of HT29 cells with compound 1g (5 µM or 10
µM).
It is noteworthy that compounds 1f-h exerted potential inhibitory activity against NPPs
[14] but showed weak inhibitory effect against steroid sulfatase (STS) [13]. Compound 1f
showed selectivity towards NPP3 (IC50 = 0.807 µM) than NPP1 and NPP2. Compound 1g is a
dual inhibitor of NPP1 and NPP3 (IC50 values are 0.564 µM and 0.254 µM, respectively). In
addition, compound 1h is more selective against NPP1 (IC50 = 0.387 µM) than NPP2 and NPP3
[14]. These inhibitory effects against NPPs could be, at least partially, a potential molecular
mechanism of anticancer activity of compounds 1f-h.
Page 15 of 21

3. Conclusion
In conclusion, structure-activity relationship studies of this series of compounds showed
that cyclohexanecarboxamide and aryl sulfonate moieties are optimal for anticancer activity.
Compound 1f showed a broad-spectrum activity over the NCI-60 panel, and this could be
affected, at least in part, by its inhibition of NPP3 enzyme. Moreover, compound 1g
demonstrated high potency and selectivity towards HT29 colon cancer cell line than normal
fibroblasts. Its antiproliferative activity could be induced by its ability to enhance caspase-3/7
activity and inhibition of NPP1/3 enzymes. Its selectivity index was more than 2.6 times higher
than that of doxorubicin. So this work could be a step towards the development of safer
anticancer agents.
4. Experimental
4.1.
Synthesis of compounds 4a-d [13, 14]
4-Aminophenol (100 mg, 0.916 mmol) was dissolved in acetone (15 mL), and anhydrous
K2CO3 (152 mg, 1.1 mmol) was added to the solution. The mixture was stirred at ambient
temperature for 15 min, and then cooled to 0 °C. Relevant cycloalkanecarbonyl chloride (0.833
mmol) was dissolved in acetone (10 mL), and the solution added dropwise to the reaction
mixture at 0 °C with continuous stirring. The reaction temperature was then raised to room
temperature, and stirring was continued for 4 h. The mixture was filtered, and the filtered solid
was washed with acetone (2 x 10 mL). The combined filtrate and wash were completely
evaporated. The remained residue was dissolved in ethyl acetate (10 mL) and extracted with
dilute aqueous hydrochloric acid. The organic layer was then washed with saturated saline
solution (2 x 10 mL), and dried using anhydrous Na2SO4. The organic solvent was evaporated
under reduced pressure to get the title compounds. They were used in the next steps as such
without further purification.
4.2.
General method for synthesis of the target sulfonate derivatives 1a-h and 1l-z [13,
Page 16 of 21
14]

Compound 4a-d (0.456 mmol) was dissolved in dry THF (10 mL) was cooled to 0 °C, and
triethylamine (0.25 mL, 2.47 mmol) was added thereto. The appropriate sulfonyl chloride (0.90
mmol) was dissolved in dry THF (3 mL), and the solution was added dropwise to the reaction
mixture at 0 °C. The reaction mixture was stirred at ambient temperature for 2 h. The mixture
was quenched with ethyl acetate (10 mL) and water (10 mL). The organic layer was separated,
and the aqueous layer was extracted with ethyl acetate (3 x 5 mL). The combined organic
extracts were washed with saturated saline (3 x 10 mL), and dried over anhydrous sodium
sulfate. The organic solvent was evaporated under reduced pressure, and the crude residue was
purified by normal phase column chromatography (silica gel, an appropriate ratio of hexane :
ethyl acetate) to isolate pure product. The yield percentages and the spectral data are provided in
the supplementary file.
4.3.
General method for synthesis of the target sulfamate derivatives 1i-k [13]
Compound 4b (100 mg, 0.456 mmol) was dissolved in dry DMF (10 mL) and cooled to 0
°C. Sodium hydride (60% dispersion in mineral oil, 18.2 mg, 0.456 mmol) was added to the
reaction mixture under inert atmosphere. The appropriate sulfamoyl chloride (2.0 mmol) was
dissolved in dry DMF (3 mL), and the resultant solution was added dropwise to the reaction
mixture at 0 °C. The reaction mixture was stirred at ambient temperature overnight. The mixture
was quenched with ethyl acetate (10 mL) and water (10 mL). The organic layer was separated,
and the aqueous layer was extracted with ethyl acetate (3 x 5 mL). The combined organic extract
was washed with saline (3 x 10 mL), and dried over anhydrous sodium sulfate. Ethyl acetate was
evaporated under reduced pressure, and the remained crude residue was purified by column
chromatography (silica gel, an appropriate ratio of hexane: ethyl acetate) to isolate pure product.
The yield percentages and the spectral data are provided in the supplementary file.
4.4.
NCI-60 screening protocol
It was carried out as per the standard protocol of the NCI [15].
Page 17 of 21

4.5.
MTT assay protocol [5]
MTT assay was carried out to assess the effect of the synthesized compounds on cell
viability and to determine the half-maximal inhibitory concentration (IC50) of each compound as
mentioned before with few modifications. A549, HT-29, MCF7, or fibroblast cells were seeded
in 96-well tissue culture plates with a density of 4 × 104/well and incubated overnight. After that,
the cells were treated with the newly synthesized compounds or doxorubicin as a positive control
for 48 h. DMSO was used as a negative control. After treatment, the media was removed and the
o
cells were incubated for 2 h at 37 C with 200 µL media containing 0.5 mg/mL of MTT
tetrazolium dye (Sigma-Aldrich). At last, the media were removed and 200 µL of DMSO was
added to solubilize the formed violet crystals. Absorbance was measured at 570 nm using a
microplate reader (Thermo Scientific, Massachusetts, USA).
4.6.
Caspase-3/7 apoptosis assay protocol
Compound 1g was dissolved in DMSO at 10 mM stock. Caspase-Glo 3/7 assay reagent was
purchased from Promega (Madison, WI). HT29 cells were grown in RPMI-1640 medium
supplemented with 10% FBS. 100 µg/mL penicillin and 100 µg/mL streptomycin were added to
the culture medium. Cultures were maintained at 37 C in a humidified atmosphere of 5% CO2
and 95% air. 5 µM and 10 µM concentrations of compound 1g were prepared. 250 nL of
compound 1g was delivered from the source plate to each well of a cell culture 384-well plate by
Echo 550. 25 µL of culture medium containing 5000 of HT29 cells was added to the wells of the
cell culture plate. The cells were incubated with the compound at 37 C in presence of 5% CO2
for 24 hours. 25 µL of Caspase-Glo 3/7 assay reagent was added to each well and incubated at 37
C, 5% CO2 for 1 hour. Luminescence of each sample well on the plate was measured by
Envision 2104 Multilabel Reader (PerkinElmer, Santa Clara, CA).
4.7.
LDH release assay
Compound 1g was dissolved in DMSO at 10 mM stock. Pierce LDH cytotoxicity assay kit
was purchased from Thermo Scientific. HT29 cells were grown in RPMI-1640 medium
Page 18 of 21

supplemented with 10% FBS. 100 µg/mL penicillin and 100 µg/mL streptomycin were added to
the culture medium. Cultures were maintained at 37 C in a humidified atmosphere of 5% CO2
and 95% air. 20000 of HT29 cells in 90 µL of 1% FBS culture media in duplicates were seeded
in the wells of a 96-well plate in an incubator at 37 C, 5% CO2 for overnight incubation. 5 µM
and 10 µM concentrations of compound 1g were added to the wells containing cells and
incubated at 37 C, 5% CO2 for 3 hours. 10 µL of 10x lysis buffer was added to duplicate wells
containing cells and incubated at 37 C, 5% CO2 for 45 minutes as a positive control. 50 µL of
each sample medium was transferred to a 96-well flat-bottom plate in duplicate wells. 50 µL of
the reaction mixture was transferred to each sample well and mixed by gentle shaking. The plate
was incubated at room temperature for 30 minutes protected from light. The absorbance at 492
nm was measured immediately using an Envision 2104 Multilabel Reader (PerkinElmer, Santa
Clara, CA).
The LDH release (%) was calculated using the formula below:
LDH release (%) = (Compound treated sample LDH activity-DMSO treated sample LDH
activity) x 100/(Lysis buffer treated sample LDH activity- DMSO treated sample LDH activity)
Acknowledgment
The authors are thankful to Boehringer-Ingelheim company and the University of Sharjah,
United Arab Emirates, for financial support.
Supplementary file
The experimental procedures, charts of the NCI results, and representative spectral charts are
available at
Declaration of interest: None.
Page 19 of 21

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Page 20 of 21

H
H
N
N
O
O
O
O
S
O
S
O
O
O
F
1g
1f
Broad-spectrum antiproliferative activity
High selectivity toward HT29 than fibroblasts
► Antiproliferative activity of a series of cycloalkanecarboxamides is reported.
► Compound 1f is the most active with broad-spectrum antiproliferative activity.
► Compound 1g is the most potent against HT29 colon cancer cell line (IC50 = 4.73 µM).
► Compound 1g is 4.23 times more selective toward HT29 than normal fibroblasts.
► Compound 1g induces apoptosis in HT29 colon cancer cell line.
Declaration of interest: None.
Page 21 of 21