Synthesis, antitumour activities and molecular docking of thiocarboxylic acid ester-based NSAID scaffolds: COX-2 inhibition and mechanistic studies

Article abstract of DOI:10.1080/14756366.2018.1474878

A new series of NSAID thioesters were synthesized and evaluated for their in vitro antitumor effects against a panel of four human tumor cell lines, namely: HepG2, MCF-7, HCT-116 and Caco-2, using the MTT assay. Compared to the reference drugs 5-FU, afati

Full text of DOI:10.1080/14756366.2018.1474878

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis, antitumour activities and molecular
docking of thiocarboxylic acid ester-based NSAID
scaffolds: COX-2 inhibition and mechanistic
studies
Adel S. El-Azab, Alaa A.-M. Abdel-Aziz, Laila A. Abou-Zeid, Walaa M. El-
Husseiny, Ahmad M. El_Morsy, Manal A. El-Gendy & Magda A.-A. El-Sayed
To cite this article: Adel S. El-Azab, Alaa A.-M. Abdel-Aziz, Laila A. Abou-Zeid, Walaa M. El-
Husseiny, Ahmad M. El_Morsy, Manal A. El-Gendy & Magda A.-A. El-Sayed (2018) Synthesis,
antitumour activities and molecular docking of thiocarboxylic acid ester-based NSAID scaffolds:
COX-2 inhibition and mechanistic studies, Journal of Enzyme Inhibition and Medicinal Chemistry,
33:1, 989-998, DOI: 10.1080/14756366.2018.1474878
To link to this article: https://doi.org/10.1080/14756366.2018.1474878
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 33, NO. 1, 989–998
https://doi.org/10.1080/14756366.2018.1474878
RESEARCH PAPER
Synthesis, antitumour activities and molecular docking of thiocarboxylic acid
ester-based NSAID scaffolds: COX-2 inhibition and mechanistic studies
Adel S. El-Azaba,b , Alaa A.-M. Abdel-Aziza,c , Laila A. Abou-Zeidd, Walaa M. El-Husseinyd, Ahmad M. El_Morsyb,
Manal A. El-Gendya and Magda A.-A. El-Sayedd,e
b
aDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; Department of Organic Chemistry,
c
Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt; Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University,
d
e
Mansoura, Egypt; Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt; Department
of Pharmaceutical Chemistry, Faculty of pharmacy, Horus university, New Damietta, Egypt
ABSTRACT
ARTICLE HISTORY
Received 26 March 2018
Revised 6 May 2018
Accepted 7 May 2018
A new series of NSAID thioesters were synthesized and evaluated for their in vitro antitumor effects against
a panel of four human tumor cell lines, namely: HepG2, MCF-7, HCT-116 and Caco-2, using the MTT assay.
Compared to the reference drugs 5-FU, afatinib and celecoxib, compounds 2b, 3b, 6a, 7a, 7b and 8a
showed potent broad-spectrum antitumor activity against the selected tumour cell lines. Accordingly, these
compounds were selected for mechanistic studies about COX inhibition and kinase assays. In vitro COX-1/
COX-2 enzyme inhibition assay results indicated that compounds 2b, 3b, 6a, 7a, 7b, 8a and 8 b selectively
inhibited the COX-2 enzyme (IC50 ¼ ꢀ0.20–0.69 lM), with SI values of (>72.5–250) compared with celecoxib
(IC50 ¼ 0.16 lM, COX-2 SI:> 312.5); however, all the tested compounds did not inhibit the COX-1 enzyme
(IC50 > 50 lM). On the other hand, EGFR, HER2, HER4 and cSrc kinase inhibition assays were evaluated at a
10 lM concentration. The selected candidates displayed limited activities against the various tested kinases;
the compounds 2a, 3b, 6a, 7a, 7b and 8a showed no activity to weak activity (% inhibition ¼ ꢀ0–10%).
The molecular docking study revealed the importance of the thioester moiety for the interaction of the
drugs with the amino acids in the active sites of COX-2. The aforementioned results indicated that thio-
ester based on NSAID scaffolds derivatives may serve as new antitumor compounds.
KEYWORDS
NSAID thioesters; in vitro;
antitumour; COX-1/COX-2;
kinase inhibition assay;
molecular docking
Introduction
celecoxib in colon, stomach, lung, prostate and pancreatic cancer
cells have been observed by selective COX-2 inhibition22–25. On
Malignancy is global health problem and is the leading cause
of death in children until fifteen years of age1. Non-steroidal anti-
inflammatory drugs (NSAIDs) such as sulindac, indomethacin and
celecoxib are commonly used for treating arthritis via inhibition
of the cyclooxygenase enzyme (COX)2,3. COX-2 levels are over-
expressed in human tumours, unlike in normal cells and could
develop a tumorigenic potential4. Selective enzyme inhibition
and restoration of normal apoptotic responses is known as COX-2-
dependent anticancer mechanism4–6. On the other hand, COX-2-
independent mechanisms function via apoptosis stimulation,
angiogenesis arrest, or cancer cell growth inhibition by blocking
the other hand,
a combination of drugs (NSAIDs) such as
indomethacin, sulindac, tolmetin, acemetacin, zomepirac and
mefenamic acid at non-toxic levels, and different chemotherapeu-
tic drugs such as anthracyclines (doxorubicin, daunorubicin and
epirubicin), in addition to VP-16, vincristine and teniposide, led to
a significantly synergistic cytotoxicity of these chemotherapeutic
drugs in the human COR L23R, DLKP, A549 and COR L23P lung
cancer cell lines, and the human HL60/ADR leukaemia cell line3.
Continuing our studies as an attempt to develop effective
cytotoxic agents26–42, we synthesised some NSAIDs conjugated to
thioester moieties and evaluated their cytotoxic activities.
Additionally, in vitro COX-1/COX-2 enzyme and kinase inhibitory
assays were investigated for the most active compounds, to iden-
tify their mode of action.
signal transduction pathways for cell proliferation7–10
Drug repositioning development is a more important process
for saving money and time than the production of a new drug11
.
.
NSAIDs and coxib such as naproxen, ibuprofen, indomethacin,
sulindac, celecoxib and their analogues (Figure 1) have diverse
scaffolds; modifying their basic structures is relatively safe,
applicable for oral use, associated with multiple therapeutic
features, such as analgesic, antipyretic, anti-inflammatory and
anticancer activities12–18. For example, sulindac amides (Figure 1)
showed a good activity against a panel of lymphoblastic leukemia
cell lines in nanomolar concentrations18. Additionally, celecoxib
reduced the number and size of colorectal polyps in adenomatous
A molecular docking technique was used in order to predict
the binding geometry requirements of the target molecules, which
is important for the antitumour activity.
Experimental
Melting points were recorded on a Barnstead 9100 Electrothermal
melting apparatus. IR spectra (KBr) were recorded on an FT-IR
polyposis (Figure 1)19–21. Antiproliferative and apoptosis effects of Perkin-Elmer spectrometer (m cmꢁ1). H and 13C NMR spectra were
1
CONTACT Adel S. El-Azab
adelazab@ksu.edu.sa, adelazaba@yahoo.com
College of Pharmacy, P.O. Box 2457, King Saud University, Riyadh-11451, Saudi Arabia
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

990
A. S. EL-AZAB ET AL.
Figure 1. Reported NSAIDs and celecoxib as anticancer agents (A-E) and the designed compounds.
recorded on Bruker 500 or 700 MHz spectrometers using DMSO-d6 S-cyclohexyl-2–(4-isobutylphenyl)propanethioate (1b)
or CDCl3 as the solvent. Microanalytical data (C, H and N) were
obtained using a Perkin-Elmer 240 analyser and the proposed
structures were within 0.4% of the theoretical values. Mass
spectra were recorded on a Varian TQ 320 GC/MS/MS mass
Yield, 81%; colourless oil; 1H NMR (500 MHz, CDCl3): d 7.27 (d, 2H,
J ¼ 8.0 H), 7.15 (d, 2H, J ¼ 8.0 H), 3.88 (d, 1H, J ¼ 7.0 Hz), 3.52 (s, 1H),
2.52 (d, 2H, J ¼ 7.0 Hz), 1.98 (s, 1H), 1.90–1.94 (q, 2H, J ¼ 7.0,
6.5 Hz), 1.74 (s, 1H), 1.69 (s, 1H), 1.62 (d, 1H, J ¼ 12 Hz), 1.56 (d, 3H,
spectrometer. NSAIDs thioester was obtained according to
J ¼ 7.0 Hz), 1.35–1.45 (m, 4H), 1.29 (s, 1H), 0.96 (d, 6H, J ¼ 6.5);
13 C NMR (125 MHz, CDCl3): d 18.6, 22.4, 25.6, 26.0, 30.2, 32.9, 33.1,
42.4, 45.1, 54.0, 127.6, 129.3, 137.3, 140.6, 200.9; MS; m/z (304).
reported method43.
General method for the preparation of NSAIDs thioester
S-phenyl-2–(3-benzoylphenyl)propanethioate (2a)
Trifluoroacetic acid (0.5 mmol) was added dropwise to a mixture of
NSAIDs (0.1 mmol) and thiol (0.5 mmol) in dry acetonitrile that was
heated for 10–12 h at 60 ꢂC. The reaction mixture was cooled,
quenched using ammonium chloride solution, extracted with
ethylacetate, washed with brine and dried over anhydrous sodium
sulphate; the solvent was then evaporated, and the product
obtained was chromatographed with hexane and CHCl3.
Yield, 88%; mp: 96–98 ꢂC; IR (KBr) mmax/cmꢁ1 1668.97 (CO), 746.66,
694.49 (CS); 1H NMR (500 MHz, CDCl3): d 7.73 (d, 3H, J ¼ 8.5 Hz),
7.63 (d, 1H, J ¼ 7.5 Hz), 7.50 (t, 2H, J ¼ 6.0 Hz), 7.39 (dd, 3H, J ¼ 7.5,
11.5 HZ) 7.28 (s, 5H), 3.99 (q, 1H, J ¼ 6.5, 7.0 Hz), 1.52 (d, 3H,
J ¼ 7.0 Hz); 13 C NMR (125 MHz, CDCl3): d 16.3, 51.5, 125.3, 126.0,
126.4, 126.9, 127.1, 127.4, 127.8, 129.6, 130.2, 132.1, 135.1, 135.7,
137.5, 194.0, 196.3; MS m/z (346).
S-phenyl-2–(4-isobutylphenyl)propanethioate (1a)44
S-cyclohexyl-2–(3-benzoylphenyl)propanethioate (2b)
Yield, 89%; colourless oil; IR (KBr) mmax/cmꢁ1 1700.69 (CO), 738.10,
690.48 (CS); 1H NMR (500 MHz, CDCl3): d 7.47–7.51 (m, 5H), 7.44 Yield, 81%; mp: 69–70 ꢂC; H NMR (500 MHz, CDCl3): d 7.81 (d, 2H,
(d, 2H, J ¼ 8.0 Hz), 7.30 (d, 2H, J ¼ 7.5 Hz), 4.13 (d, 1H, J ¼ 7.0 Hz), J ¼ 7.0 Hz), 7.76 (s, 1H), 7.70 (d, 1H, J ¼ 7.5 Hz), 7.55–7.60 (m, 2H),
2.64 (d, 2H, J ¼ 7.0 Hz), 2.04 (t, 1H, J ¼ 6.5 Hz), 1.73 (d, 3H, 7.43–7.49 (m, 3H), 3.93 (q, 1H, J ¼ 7.0 & 6.5 Hz), 3.48 (s, 1H), 1.91
J ¼ 7.0 Hz), 1.08 (d, 6H, J ¼ 6.5); 13 C NMR (125 MHz, CDCl3): d 18.8, (s, 1H), 1.84 (d, 1H, J ¼ 10 Hz), 1.67 (d, 2H, J ¼ 14.0 Hz), 1.55 (d, 4H,
22.6, 30.3, 45.2, 53.9, 127.3, 127.6, 127.9, 128.2, 129.2, 129.3, 129.6, J ¼ 7.0 Hz), 1.34–1.40 (m, 4H), 1.25 (s, 1H); 13 C NMR (125 MHz,
1
134.6, 136.6, 141.1, 199.1; MS; m/z (298).
CDCl3): d 18.4, 25.5, 25.9, 32.8, 32.9, 42.6, 54.0, 128.3, 128.6,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
991
129.1, 129.7, 130.1, 131.8, 132.5, 137.4, 137.8, 140.3, 196.3, 200.4; S-phenyl-2–(2-((2,6-dichlorophenyl)amino)phenyl)
ethanethioate (6a)
MS m/z (352).
Yield, 86%; mp: 101–102 ꢂC; IR (KBr) mmax/cmꢁ1 1679.27 (CO),
741.06, 688.41 (CS); 1H NMR (500 MHz, CDCl3): d 10.01 (s, 1H),
7.40–7.42 (d, 2H, J ¼ 8.0 Hz), 7.24–7.29 (m, 4H), 7.09–7.12 (t, 3H,
J ¼ 7.5 Hz), 6.99–7.00 (d, 2H, J ¼ 7.5 Hz), 6.30–6.32 (d, 1H,
J ¼ 7.5 Hz), 3.68 (s, 2H); 13 C NMR (125 MHz, CDCl3): d 35.7, 109.1,
S-phenyl-2–(2-fluoro-[1,10-biphenyl]-4-yl)propanethioate (3a)
Yield, 90%; mp: 85–86 ꢂC; IR (KBr) mmax/cmꢁ1 1694.14 (CO), 736.75,
1
687.25 (CS); H NMR (500 MHz, CDCl3): d 1.52 (d, 3H, J ¼ 7 Hz), 3.94
(dd, 1H, J ¼ 6.5 & 7.0 Hz), 7.46 (d, 2H, J ¼ 12.5 Hz), 7.29–7.41 (m, 123.0, 124.3, 124.8, 125.2, 127.7, 127.9, 129.0, 130.4, 130.8, 131.0,
9H), 7.05–7.14 (m, 2H); 13 C NMR (125 MHz, CDCl3): d 18.6, 53.4,
134.5, 135.5, 139.6, 143.3, 198.0; MS m/z (388).
115.5, 115.7, 124.0, 127.5, 127.7, 128.5, 129.0, 129.2, 129.4, 130.9,
131.0, 134.4, 135.4, 140.7, 140.8, 158.7, 160.7, 198.5; MS; m/z (336).
S-cyclohexyl-2–(2-((2,6-dichlorophenyl)amino)phenyl)
ethanethioate (6b)
S-cyclohexyl-2–(2-fluoro-[1,10-biphenyl]-4-yl)propanethioate (3b)
Yield, 83%; mp: 88–90 ꢂC; 1H NMR (500 MHz, CDCl3): d 7.38–7.40
(d, 2H, J ¼ 8.0 Hz) 7.18–7.24 (m, 2H), 7.08–7.11 (t, 1H, J ¼ 7.5 Hz),
6.97–7.00 (t, 1H, J ¼ 7.5 Hz), 6.29–6.31 (d, 1H, J ¼ 8.0 Hz), 3.70 (s,
2H), 3.01 (s, 1H), 2.01–2.00 (d, 4H, J ¼ 9.0 Hz), 1.69–1.67 (t, 2H,
J ¼ 8.5 Hz), 1.49–1.47 (t, 2H, J ¼ 8.5 Hz), 1.37–1.36 (d, 2H, J ¼ 8.5 Hz);
13 C NMR (125 MHz, CDCl3): d 22.7, 29.7, 35.7, 39.2, 41.0, 123.1,
124.3, 124.8, 127.9, 129.0, 129.5, 130.4, 130.8, 131.0, 135.5, 143.3,
198.0; MS m/z (394).
Yield, 80%; mp: 90–92 ꢂC; IR (KBr) mmax/cmꢁ1 1672.76 (CO), 751.18,
690.19 (CS); 1H NMR (500 MHz, CDCl3): d 7.45 (d, 2H, J ¼ 7.5 Hz),
7.25–7.36 (m, 4H), 7.03–7.07 (m, 2H,), 3.79 (t, 1H, J ¼ 7.0 Hz), 3.40
(s, 1H), 1.76–1.84 (m, 2H), 1.57–1.60 (d, 2H, J ¼ 13 Hz), 1.45 (d, 4H,
J ¼ 7.0 H), 1.23–1.33 (m, 4H), 1.17 (s, 1H); 13 C NMR (125 MHz,
CDCl3): d 18.4, 25.5, 25.9, 32.8, 33.0, 42.7, 53.6, 115.4, 115.6, 123.8,
123.9, 127.6, 127.9, 128.0, 128.4, 128.9, 130.7, 130.8, 135.5, 141.3,
141.4, 158.7, 160.6, 200.5; MS m/z (342).
S-phenyl-2–(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl)ethanethioate (7a)46
2-[(Phenylthio)carbonyl]phenyl acetate (4a)45
Yield, 86%; mp: 133–135 ꢂC; IR (KBr) mmax/cmꢁ1 1671.45, 1604.72
(CO), 745.04, 693.51 (CS); 1H NMR (500 MHz, CDCl3): d 7.59 (d, 2H,
J ¼ 8.5 Hz), 7.38 (d, 2H, J ¼ 8.5 Hz), 7.29 (s, 5H), 6.92 (d, 1H,
J ¼ 2.0 Hz), 6.82 (d, 1H, J ¼ 9.0 Hz), 6.61 (dd, 1H, J ¼ 2.0, 9.0 Hz), 3.87
(s, 2H), 3.76 (s, 3H), 2.36 (s, 3H); 13 C NMR (125 MHz, CDCl3): d 13.5,
39.1, 55.7, 101.2, 111.8, 127.6, 129.1, 129.2, 129.4, 130.5, 130.9,
131.2, 133.7, 134.4, 136.8, 139.4, 156.2, 168.3, 195.1; MS m/z (449),
(M þ 2, 451).
Yield, 84%; mp: 72–73 ꢂC; 1H NMR (500 MHz, CDCl3): d 7.91 (dd,
1H, J ¼ 1.0 Hz), 7.42–7.45 (m, 6H) 6.87–6.92 (m, 2H), 2.24 (s, 3H);
13 C NMR (125 MHz, CDCl3): d 29.7, 118.36, 119.4, 126.0, 128.9,
129.4, 130.0, 135.5, 136.3, 159.7, 195.8. MS m/z (272).
2-[(Cyclohexylthio)carbonyl]phenyl acetate (4b)
1
Yield, 80%; mp: 55–56 ꢂC; H NMR (500 MHz, CDCl3): d 7.78 (d, 1H,
J ¼ 8.0 Hz), 7.35 (t, 1H, J ¼ 7.0 Hz), 6.88 (d, 1H, J ¼ 8.0 Hz), 6.79 (t,
1H, J ¼ 7.0 Hz), 3.66 (s, 1H), 2.26 (s, 2H), 1.95 (d, 2H, J ¼ 10 Hz), 1.69
(t, 2H, J ¼ 4.5 Hz), 1.56 (d, 1H, J ¼ 8.5 Hz), 1.38–1.49 (m, 4H) 1.25 (d,
2H, J ¼ 8.5 Hz); 13 C NMR (125 MHz, CDCl3): d 25.5, 25.9, 29.7, 33.01,
42.5, 118.1, 119.1, 120.2, 128.8, 135.6, 159.5, 197.4; MS m/z (278).
S-cyclohexyl-2–(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl)ethanethioate (7b)46
Yield, 83%; mp: 97–98 ꢂC; IR (KBr) mmax/cmꢁ1 1672.24, 1600.15
(CO), 830.24, 749.96 (CS); 1H NMR (700 MHz, DMSO-d6): d 7.76 (d,
2H, J ¼ 5.5 Hz), 7.64 (d, 2H, J ¼ 5.5 Hz), 7.05 (s, 1H), 6.93 (d, 1H,
J ¼ 6.5 Hz), 7.72 (d, 1H, J ¼ 6.5 Hz), 3.98 (s, 2H), 3.75 (s, 3H),
2.55–2.51 (m, 1H), 2.23 (s, 3H), 1.79 (s, 2H), 1.59 (s, 2H), 1.50 (d, 1H,
J ¼ 8.5 Hz), 1.33–1.30 (m, 4H), 1.20 (s, 1H); 13 C NMR (125 MHz,
CDCl3): d 13.8, 25.4, 25.8, 32.8, 40.2, 42.4, 55.8, 102.2, 111.9, 112.8,
115.0, 129.5, 130.6, 130.9, 131.6, 134.4, 136.5, 138.2, 156.0, 168.3,
196.8; MS m/z (456), (M þ 2, 458).
S-phenyl-(S)-2–(6-methoxynaphthalen-2-yl)propanethioate (5a)44
Yield, 88%; mp: 115–117 ꢂC; IR (KBr) mmax/cmꢁ1 1694.16 (CO),
738.16, 683.87 (CS); 1H NMR (500 MHz, CDCl3): d 7.88 (s, 4H), 7.59
(d, 3H, J ¼ 7.5 Hz), 7.49 (s, 3H), 7.31(d, 1H, J ¼ 8.5 Hz), 4.28 (d, 1H,
J ¼ 6.5 Hz), 4.05 (s, 3H), 1.80 (d, 3H, J ¼ 6.0 Hz); 13 C NMR (125 MHz,
CDCl3): d 18.7, 54.1, 55.3 105.7, 119.1, 126.4, 126.9, 127.4, 128.0,
129.0, 129.1, 129.3, 129.4, 134.0, 134.5, 134.7, 157.8, 199.2; MS
m/z (322).
S-phenyl-2–(5-fluoro-2-methyl-1–(4-(methylsulfinyl)benzylidene)-
1H-inden-3-yl)ethanethioate (8a)
Yield, 78%; mp: 66–68 ꢂC; IR (KBr) mmax/cmꢁ1 1700.49 (CO), 1021
1
(SO), 734.05, 684.77 (CS); H NMR (500 MHz, CDCl3): d 7.02–7.47 (m,
S-cyclohexyl-(S)-2–(6-methoxynaphthalen-2-yl)
propanethioate (5b)
13H), 3.93–3.94 (d, 2H, J ¼ 7.0 Hz), 2.27 (s, 3H), 1.46–1.52 (dd, 3H,
J ¼ 7.0 Hz); 13 C NMR (125 MHz, CDCl3): d 18.6, 21.3, 53.3, 115.5,
115.7, 124.0, 127.7, 128.5, 129.0, 129.3, 130.0, 130.9, 134.4, 135.4,
Yield, 84%; mp: 105–106 ꢂC; IR (KBr) mmax/cmꢁ1 1679.27 (CO),
1
741.06, 688.41 (CS); H NMR (500 MHz, CDCl3): d 7.59–7.64 (m, 3H), 139.7, 140.9, 158.7, 160.7, 198.9; MS m/z (448).
7.30 (dd, 1H, J ¼ 1.0 Hz), 7.05 (dd, 1H, J ¼ 2.0 Hz), 7.03 (s, 1H), 3.88
(d, 1H, J ¼ 7.0 Hz), 3.82 (s, 3H), 3.37 (s, 1H), 1.82 (d, 1H, J ¼ 8.5 Hz),
S-cyclohexyl-2–(5-fluoro-2-methyl-1–(4-(methylsulfinyl)
benzylidene)-1H-inden-3-yl)ethanethioate (8b)
1.72 (t, 1H, J ¼ 5.0 & 5.5 Hz), 1.59–1.46 (m, 6H), 1.30–1.13 (m, 5H);
13 C NMR (125 MHz, CDCl3): d 18.5, 25.5, 26.0, 32.9, 33.0, 42.5, 54.2,
Yield, 75%; mp: 121–122 ꢂC; IR (KBr) mmax/cmꢁ1 1692.84 (CO),
55.3, 105.6, 119.0, 126.4, 126.5, 127.1, 128.9, 129.3, 133.8, 135.3,
157.7, 201.2; MS m/z (328).
1
859.17, 808.66 (CS), (SO); H NMR (700 MHz, DMSO-d6): d 7.67–7.63

992
A. S. EL-AZAB ET AL.
(m, 5H, 7.05 (s, 1H), 6.93 (d, 1H, J ¼ 9.1 Hz), 6.72 (d, 1H, J ¼ 9.1 Hz), Luminescence signal was measured using a BioTek Synergy 2
3.98 (s, 2H), 3.75 (s, 3H), 2.23 (s, 3H), 1.799 (s, 2H), 1.59 (s, 2H), 1.50 microplate reader53.
(d, 1H, J ¼ 11.9 Hz), 1.31 (t, 4H, J ¼ 10.5 & 9.8 Hz), 1.20 (s, 1H);
13 C NMR (176 MHz, DMSO-d6): d 13.8, 25.4, 25.8, 32.8, 39.3, 42.4,
55.8, 102.0, 111.9, 112.8, 115.0, 129.5, 130.6, 130.9, 131.6, 134.4,
Docking methodology
136.5, 138.2, 156.0, 168.3, 196.8; MS m/z (454).
All modelling experiments were conducted with MOE programs
running on PC computer [MOE 2008.10 of Chemical Computing
Group. Inc]54. The docking protocol is summarized in supporting
information51,52,55–57
.
Biological testing
Antitumor evaluation
The evaluation of the antitumour activity was performed using
Results and discussion
tetrazolium salt MTT (3–(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
Chemistry
tetrazolium bromide) assay as reported47–50
.
The new thioesters were synthesized by the reaction of the
carboxylic acid group of NSAIDs with thiophenol and cycloxane-
thiol in the presence of trifluoroacetic acid (TFA)43. The newly
synthesized thioesters (Scheme 1) were confirmed by the presence
of the carbonyl group (C¼O) at 1700–1669 cmꢁ1 and stretching of
the (C–S) group at 859–683 cmꢁ1 in the IR spectra. Additionally,
the newly synthesised thioesters were confirmed by a characteris-
tic peak at 201.21–195.15 ppm attributable to the (S–C¼O) group
in addition to the characteristic peaks of the cyclohexane moiety
at 25.40–42.42 ppm or aromatic peaks of the thiophenol moiety in
the aromatic region of the 13C NMR spectra. The 1H NMR spectra
of the new thioesters showed a singlet peak because of the S–CH
In vitro cyclooxygenase (COX) inhibition assay
The colorimetric COX (ovine) inhibitor screening assay kit (kit
catalogue number 560101, Cayman Chemical, Ann Arbor, MI) was
utilized according to the manufacturer’s instructions to examine
the ability of the test compounds and the reference drugs to
inhibit the COX-1/COX-2 isozymes51,52
.
Kinase inhibition assay
The assay for Kinases was performed at BPS Bioscience Inc. 6044 moiety of S-cyclohexane at 3.66–3.37 ppm, as well as the other
Cornerstone Court West, Ste. E, San Diego, CA 92121, USA using 10 protons of the cyclohexane moiety in the aliphatic region or the
Kinase-Glo Plus luminescence kinase assay kit (Promega). aromatic peaks of the thiophenol moiety in the aromatic region.
CH3
O
O
O
R
S
O
R
S
F
S
R
1a,b
2a,b
3a,b
( ) Ibuprofen
Ketoprofen
( )
Flurbiprofen
O
( )
O
O
O
SH
SH
Naproxen
( )
Aspirin
S
R
O
S
R
5a,b
4a,b
a
b
Diclofenac
Sulindac
Indomethacin
O
F
R
Cl
S
Cl
H
N
O
S
Cl
O
N
O
R
S
6a,b
8a,b
O
S
R
O
7a,b
Scheme 1. Synthesis of the designed thiocarboxylic acid esters of NSAIDs.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
993
Biological evaluation
thioesters 5b, 6b, 8b and 9b showed a moderate antitumour
activity, with IC50 values of 26.81–43.79 lM, when compared to
the reference drugs 5-FU, afatinib and celecoxib (IC50 ¼ 6.85, 7.70
and 42.74 lM, respectively).
Antitumor evaluation using MTT assay
The synthesised thioesters were evaluated for their in vitro
antitumor effects using the standard 3–(4,5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide (MTT) method47–50 against a panel
of four human tumour cell lines: hepatocellular carcinoma cell line
(HepG2), breast cancer cell line (MCF-7), colon cancer cell line
(HCT-116) and colorectal cancer cell line (Caco-2). The antitumor
activities of the newly synthesized compounds 1a,b–8a,b
compared with those of the reference drugs, 5-FU, afatinib and
celecoxib are shown in Table 1. In the present study, the active
The thioesters 3b and 7b displayed a broad-spectrum antitu-
mor activity against the HepG2 cell line (IC50 ¼ 10.52 and 14.91 lM
respectively) and MCF-7 cell line (IC50 ¼ 13.73 and 17.10 lM
respectively), while the thioester 6a showed a broad-spectrum
antitumor activity against the MCF-7 cell line and Caco-2 cell line
(IC50 ¼ 6.11 and 10.16 lM, respectively). Additionally, the thioesters
2b, 7a and 8a showed strong antitumour activities against the
HepG2 cell line (IC50 ¼ 9.36, 7.86 and 7.35 lM, respectively), MCF-7
cell line (IC50 ¼ 11.86, 9.65 and 8.62 lM respectively), HCT-116 cell
line (IC50 ¼ 18.71, 14.58 and 9.73 lM, respectively), and Caco-2 cell
line (IC50 ¼ 21.73, 18.13 and 15.44 lM, respectively). On the other
hand, the thioesters 1a, b, 2b, 3a, 4a and 6b showed a weak
antitumor activity with IC50 values from 48.11 to >100 lM.
compounds revealed
a characteristic selectivity potential, in
addition being broad-spectrum compounds. With respect to the
selectivity against the hepatocellular carcinoma cell line (HepG2),
the thioesters 2b, 3b, 5b, 7a,b and 8a showed potent antitumour
activity with IC50 values of 7.35–19.74 lM, while thioesters 5a, 6a
and 8b showed moderate antitumour activity against HepG2 cell
line, with IC50 values 22.30–36.75 lM, compared to the reference
drugs 5-FU, afatinib and celecoxib (IC50 ¼ 7.91, 5.40 and 25.60 lM,
respectively). Additionally, the MCF-7 cell line showed a high sensi-
tivity to the thioesters 2a, 3b, 6b, 7a,b and 8a, with IC50 values of
6.11–17.10 lM, whereas the thioesters 4b, 5a,b and 8b showed a
moderate antitumour activity, with IC50 values 28.90–46.52 lM,
compared with the reference drugs 5-FU, afatinib and celecoxib
(IC50 ¼ 5.43, 7.1 and 31.28 lM, respectively). Moreover, the colon
cancer cell line (HCT-116) had a strong susceptibility to the
thioesters 2a, 7a and 8a with IC50 values of 9.73–18.71 lM, but a
moderate susceptibility to the thioesters 3b, 5b, 6a, 7b and 8b,
with IC50 values of 23.76–46.92 lM compared to the reference
drugs, 5-FU, afatinib and celecoxib (IC50 ¼ 5.32, 6.20 and 29.54 lM,
respectively). Additionally, the thioesters 2b, 6a, 7a and 8a
exhibited a strong antitumour activity against colorectal cancer
cell line (Caco-2), with IC50 values of 10.16–21.73 lM, whereas the
In vitro COX inhibition assay
Compounds that showed promising and potent antitumor activ-
ities (Table 1) were subjected to in vitro COX-1/COX-2 inhibition
assays. As indicated in Table 2, seven compounds were selected
for in vitro COX-1/COX-2 evaluation (Table 2). IC50 (represented in
lM) (The half-maximal inhibitor concentration) values were
determined56–59, and the SI values were calculated51,52,55 as IC50
(COX-1)/IC50 (COX-2). Interestingly, some of the tested compounds
selectively inhibited COX-2 (IC50 ¼ 0.20–0.69 lM); however, all the
tested compounds did not inhibit COX-1 (IC50 > 50 lM).
Compounds 2b, 3b, 7b and 8b possessed good COX-2
inhibitory activity with IC50 values of 0.66, 0.69, 0.49 and 0.60 lM,
and SI values of >75.8, 72.5, 102.0 and 83.3, respectively,
comparable to that of celecoxib (IC50 ¼ 0.16 lM, COX-2 SI: > 312.5).
Furthermore, compounds 6a, 7a and 8a showed a potent selective
Table 2. In vitro COX-1/COX-2 enzyme inhibition assay.
IC50 (lM)a
Table 1. In vitro antitumor activity of 5-fluorouracil, afatinib, celecoxib, and the
tested compounds.
Compd No.
COX-1
COX-2
SIb
IC50 (lM)a
Celecoxib
>50
>50
>50
>50
>50
>50
>50
>50
0.16 0.011
0.66 0.052
0.69 0.057
0.25 0.017
0.22 0.019
0.49 0.044
0.20 0.016
0.60 0.055
>312.5
>75.8
>72.5
>200.0
>227.3
>102.0
>250.0
>83.3
Compd No.
HepG2b
MCF-7c
HCT-116d
Caco-2e
2b
3b
6a
7a
7b
8a
8b
5-FU
Afatinib
Celecoxib
1a
7.91 0.28
5.4 0.25
25.6 2.3
85.12 4.53
>100
5.43 0.20
7.1 0.49
31.28 2.5
80.41 4.58
>100
5.32 0.17
6.2 0.67
6.85 0.34
7.7 0.57
42.74 3.1
94.83 4.92
>100
72.19 4.06
21.73 1.90
76.52 4.38
26.81 2.17
80.20 4.50
68.75 3.79
63.78 3.58
35.60 2.62
10.16 0.92
>100
29.54 2.1
89.63 4.68
97.56 5.12
61.29 3.97
18.71 1.50
78.11 4.08
23.76 1.80
85.40 4.57
76.54 4.22
51.17 3.71
39.52 2.61
46.92 3.23
91.22 4.96
14.58 1.24
34.05 2.25
9.73 0.85
46.71 2.93
1b
2a
2b
3a
3b
4a
4b
5a
5b
59.83 3.55
9.36 0.79
68.75 3.87
10.52 0.98
71.08 4.11
63.62 3.91
36.75 2.70
19.74 1.57
26.76 2.08
>100
48.11 3.15
11.86 1.13
63.61 3.62
13.73 1.19
73.65 3.92
46.52 2.84
42.61 2.67
28.90 1.58
6.11 0.31
95.26 4.96
9.65 0.96
17.10 1.40
8.62 0.72
34.09 2.07
aIC50 value is the compound concentration required
to produce a 50% inhibition of COX-1 or COX-2,
calculated as the mean of two determinations using
the ovine COX-1/COX-2 assay kit (catalog no. 560101,
Cayman Chemicals Inc., USA); the deviation from the
mean is <10% of the mean value.
bSelectivity index (COX-1 IC50/COX-2 IC50).
6a
6b
Table 3. % inhibitory effect of the compounds on
kinase activities.
7a
7b
8a
8b
7.86 0.39
14.91 1.38
7.35 0.34
22.30 1.96
18.13 1.73
29.14 2.45
15.44 1.37
43.79 2.96
% inhibition of 10 lM
Compd No.
Staurosporinea
EGFR
HER2
HER4
cSrc
aIC50, compound concentration required to inhibit tumour cell proliferation by
50% (mean SD), n ¼ 3.
94
0
0
6
0
1
6
81
1
0
1
0
0
0
100
5
3
5
4
3
10
100
4
bHuman hepato-cellular carcinoma cell line (HepG2).
cHuman breast adenocarcinoma cell line (MCF-7).
dHuman colon cancer cell line (HCT-116).
2b
3b
6a
7a
7b
8a
3
5
1
3
eHuman colorectal cancer cell line (Caco-2).
IC50, (lM): 1–10 (very strong), 11–25 (strong), 26–50 (moderate), 51–100 (weak),
above 100 (non-cytotoxic).
4
5-FU: 5-Fluorouracil.
aStaurosporine used in 1 lM concentration.

994
A. S. EL-AZAB ET AL.
Figure 2. The 2D and 3D orientations of the docked compounds 6a (upper panel), 7a (middle panel), and 8a (lower panel) in COX-2 active pocket (H bonds and
hydrophobic interactions are shown as dashed green lines or arrows).
inhibition of COX-2, with IC50 values of 0.25, 0.22 and 0.20 lM, and such as compounds 2b (IC50 ¼ 0.66, lM, COX-2 SI: >75.8),
SI values of >200, 227 and 250, respectively, compared to those of 3b (IC50 ¼ 0.69 lM, COX-2 SI: >72.5), 7b (IC50 ¼ 0.49 lM, COX-2
celecoxib (IC50 ¼ 0.16 lM, COX-2 SI: > 312.5). The structure–activity SI:>102.0) and 8b (IC50 ¼ 0.60 lM, COX-2 SI: >83.3); (ii) S-phenyl-
relationships of the COX inhibition assays revealed the following: 2–(5-fluoro-2-methyl-1–(4-(methylsulfinyl)benzylidene)-1H-inden-3-yl)
(i) substituted thiophenyl derivatives, such as compounds 6a, ethanethioate (8a) was more effective than S-phenyl-2–(1-(4-
(IC50 ¼ 0.25 lM, COX-2 SI: >200), 7a (IC50 ¼ 0.22 lM, COX-2 SI: >227) chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)ethanethioate (7a)
and 8a (IC50 ¼ 0.20 lM, COX-2 SI: >250), were more effective and S-phenyl 2–(2-((2,6-dichlorophenyl)amino)phenyl)ethanethioate
COX-2 inhibitors than substituted thiocyclohexyl derivatives, (6a), while the latter was less effective than compound 7a;

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
995
(iii) the substituted thiocyclohexyl derivative, S-cyclohexyl hydrogen bonds and hydrophobic interactions formed with the
2–(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)ethanethioate surrounding amino acids are used to predict the binding modes,
(7b) was more effective than S-cyclohexyl-2–(5-fluoro-2-methyl- the energy of interaction and orientation of the docked com-
1–(4-(methylsulfinyl)benzylidene)-1H-inden-3-yl)ethanethioate (8b), pounds at the active sites of the COX-2 enzyme (Figures 2–3).
S-cyclohexyl 2–(3-benzoylphenyl)propanethioate (2b) and S- The protein–ligand complex was constructed based on the X-ray
cyclohexyl 2–(4-isobutylphenyl)propanethioate (3b).
structure of COX-2, with its bound inhibitor SC-558, which was
available through the RCSB Protein Data Bank (PDB entry 1CX-2)60.
The active site of the enzyme was defined to include residues
within a 10.0-Å radius around any of the inhibitor atoms. This
active pocket consisted of amino acid residues such as arginine
(Arg510), histidine (His90), glutamine (Gln192) or tyrosine (Tyr355),
arginine (Arg120), valine (Val523) and methionine (Met535), which
play fundamental roles by forming H-bonds and hydrophobic
interactions (Figures 2–3). In order to verify the reproducibility of
the docking calculations, the cocrystallised ligand SC-558 was
extracted from the complex and submitted for one-ligand run
calculation. This reproduced 20 top scoring conformations falling
within a root-mean-square deviation (rmsd) value between 0.4 Å
and 2.0 Å, from the bound X-ray conformation for the COX-2
enzyme, suggesting that this method is valid enough to be used
for docking studies of other compounds.
In vitro kinase assay
Accordance to the cytotoxicity activity of the newly synthesized
compounds (Table 1), six compounds were selected for further
mechanistic investigations about the kinases, EGFR, HER2, HER4
and cSrc. The results of kinase inhibition assays indicated that
compounds 2a, 3b, 6a, 7a, 7b and 8a showed limited activities
against the kinase enzymes. As shown in Table 3, all the
compounds showed no or weak activities against HER2, HER4
and cSrc, as indicated by their % inhibition when used at a con-
centration of 10 lM (% inhibition ¼ ꢀ0–10%), comparable to the
81–100% inhibition of the reference drug staurosporine, used at a
concentration of 1 lM (Table 3).
The present work is based on a comparative study to define
the selectivity of most active COX-2 inhibitors, such as the thio-
Docking studies
To highlight the inhibition selectivity of different core analogues ester derivatives 6a, 7a, 7b, 8a and 8b of well-known and well-
towards the COX-2 enzyme, automated docking studies were car- established NSAIDs, namely diclofenac, indomethacin and sulindac,
ried out using the MOE 2008.10 program54. The scoring functions, by exploring their docking and complementarity to the COX-2
Figure 3. The 2D and 3D putative binding complexes of compound 7b (upper panel) and compound 8b (lower panel) within the binding pocket of COX-2 enzyme.

996
A. S. EL-AZAB ET AL.
binding site. Generally, the results of the docking study indicated
that the thioesters based on indomethacin, sulindac and
diclofenac scaffolds matched perfectly with the configuration
of the T-shaped merged COX-2 binding site, which easily
accommodated the wide bulk SC-558 inhibitor.
Conclusions
A new series of thioesters based on NSAID scaffolds were synthe-
sized and evaluated for their in vitro antitumor effects against a
panel of four human tumour cell lines, namely HepG2, MCF-7, HCT-
116 and Caco-2 using MTT assay. The thioesters 2b, 3b, 5b, 7a,b,
and 8a showed potent antitumor activity against HepG2 cell line,
while thioesters 2a, 3b, 6b, 7a,b and 8a showed high sensitivity
against MCF-7 cell line with IC50 values of 7.35–19.74 lM and
6.11–17.10 lM, respectively, compared with the reference drug, 5-
FU, afatinib and celecoxib (IC50 ¼ 7.91, 5.40, 25.60 lM and 5.43, 7.10,
31.28lM, respectively). Additionally, thioesters 2a, 3b, 6b, 7a,b and
8a revealed the most antitumor activity against MCF-7 cell line,
whereas HCT-116 has strong susceptibility to thioesters 2a, 7a and
8a with IC50 values of 6.11–17.10 lM and 9.73–18.71 lM, respect-
ively, compared with the reference drugs 5-FU, afatinib and cele-
coxib (IC50 ¼ 5.43, 7.10, 31.28 lM and 6.85, 7.70, 42.74 lM,
respectively). Additionally, the thioesters 2b, 7a and 8a showed
strong antitumor activities against HepG2 (IC50 ffi 7.35–9.36 lM),
MCF-7 (IC50 ffi 8.62–11.86 lM), HCT-116 (IC50 ffi 9.73–18.71 lM), and
Compounds 8a (IC50 ¼ 0.20 mM), 7a (IC50 ¼ 0.22 mM) and 6a
(IC50 ¼ 0.25 mM) were the most active analogues; they showed the
highest recognition at the COX-2 binding site, which is consistent
with the experimental results of the selectivity index obtained
from the COX-2 assay (Table 2). Compound 8a was shown to have
a unique binding configuration (Figure 2, lower panel). The phenyl
thioester of sulindac showed promising binding affinity and
proper complementarity, because the E-conformer allows the
crest-configuration to embed properly within the merged active
site of the 5-flouroindenyl group, via proper hydrophobic
interactions with the amino acids of the merged cleft active site.
The thiocarbonyl function was impressively recognized with the
polar amino acid Glu524. The two terminal phenyl groups are
surrounded by hydrophobic amino acid analogs, where Leu534
showed an aromatic-aromatic interaction with the 4-methylsulfi-
nylphenyl group. The terminal 4-methylsulfinyl fragment enhanced Caco-2 cell line (IC50 ffi 15.44–21.73 lM). Thioesters 3b and 7b have
the strategic function that showed proper complementarity with
the groove wall residues via both Val344 and Val349. According to
the selectivity index and computational binding, the hydrogen-
bonded compound 8a was considered the most promising
selective lead.
broad-spectrum antitumour activity against HepG2 and MCF-7 cell
lines (IC50 ffi 10.52–17.10 lM), while thioesters 6a showed broad-
spectrum antitumor activity against the MCF-7 and Caco-2 cell lines
(IC50 ¼ 6.11 and 10.16 lM). According to their cytotoxicity activities,
compounds 2b, 3b, 6a, 7a, 7b, 8a and 8b were carefully chosen for
mechanistic studies involving COX enzyme inhibition and kinase
assays. In vitro COX-1/COX-2 enzyme inhibition assay results indi-
cated that compounds 2b, 3b, 6a, 7a, 7b, 8a and 8b selectively
inhibited the COX-2 enzyme (IC50 ¼ ꢀ0.20–0.69 lM), and SI values of
(>72.5–250), compared to celecoxib (IC50 ¼ 0.16 lM, COX-2
SI: > 312.5). Although all the tested compounds did not selectively
inhibit the COX-1 enzyme (IC50 > 50 lM). On the other hand, EGFR,
HER2, HER4 and cSrc kinase inhibition assays were evaluated at a
concentration of 10 lM. The kinase inhibition assays indicated that
compounds 2a, 3b, 6a, 7a, 7b and 8a showed no activity to negli-
gible activity (% inhibition¼ ꢀ0–10%), comparable to an 81–100%
inhibition of the reference drug Staurosporine at a concentration
of 1 lM.
Moreover, compound 7a was held by one hydrogen bond with
Tyr355, via its carbonyl thioester, apart from the electrostatic
interaction between the chloro-function and the mercapto moiety
of the corresponding Met535 (Figure 2, middle panel). Aromatic
recognition also was observed between the aromatic phenyl
thioester and the side chain of Arg513
.
Additionally, the docking studies of compound 6a revealed
outstanding interactions with one of the essential active-site
Arg120 residues formed via proper hydrogen bonding (Figure 2,
upper panel). The two aminophenyl and dichlorophenyl groups
augment the aromatic-aromatic interaction with a series of seven
hydrophobic amino acids, Leu531, Met113, Val116, Leu352, Val349 and
Ile345, arranged in a continued chain, lining the wall of the cleft.
However, because of the NH-amino group being embedded
inwards and away from the surrounding residues, it does not
interact with the active-site amino acids, owing to the bulkiness of
the two phenyl substituents. The thiophenyl function protruded
towards Val523, showing notable improvement in the net lipophilic
stabilisation (Figure 2, upper panel).
In comparison to the aforementioned derivatives, compounds
7b and 8b showed moderately selective inhibition towards COX-2.
Compound 7b revealed distinct binding wherein the cyclohexyl
group was merged with the side-pocket, and the thioester func-
tion was exposed to the surrounding binding residues for inter-
action with the conserved amino acids Val523 via proper hydrogen
bonding. Additionally, methoxy oxygen was recognized by a single
conventional hydrogen bond with the conserved Arg120 (Figure 3,
upper panel).
Acknowledgements
The authors express their appreciation to the Deanship of
Scientific Research at King Saud University for funding the work
through the research group project No. RGP-163. The authors
thank the Deanship of Scientific Research and RSSU at King Saud
University for their technical support.
Disclosure statement
No potential conflict of interest was reported by the authors.
ORCID
Similarly, the cyclohexyl group of compound 8b was merged
with the side-pocket, and the ester function was exposed to the
surrounding binding residues, to be oriented ahead of the polar
amino acids, Ser353 and Arg120 (Figure 3, lower panel). Along the
lining wall of the pocket, all the hydrophobic amino acids are
oriented complementarily with the hydrophobic-facing groups
indene, methylene, and the terminal phenyl. From another site,
the following hydrophobic amino acids are stuffed properly
and sandwiched between the cyclohexyl ring and the terminal
phenyl group.
Adel S. El-Azab
Alaa A.-M. Abdel-Aziz
Magda A.-A. El-Sayed
http://orcid.org/0000-0001-7197-1515
http://orcid.org/0000-0002-3362-9337
http://orcid.org/0000-0001-9599-9248
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