Synthesis, antitumor activity, and molecular docking study of 2-cyclopentyloxyanisole derivatives: mechanistic study of enzyme inhibition

Article abstract of DOI:10.1080/14756366.2020.1740695

A series of 24 compounds was synthesised based on a 2-cyclopentyloxyanisole scaffold 3–14 and their in vitro antitumor activity was evaluated. Compounds 4a, 4b, 6b, 7b, 13, and 14 had the most potent antitumor activity (IC₅₀ range: 5.13–17.95 μM), compared to those of the reference drugs celecoxib, afatinib, and doxorubicin. The most active derivatives 4a, 4b, 7b, and 13 were evaluated for their inhibitory activity against COX-2, PDE4B, and TNF-α. Compounds 4a and 13 potently inhibited TNF-α (IC₅₀ values: 2.01 and 6.72 μM, respectively) compared with celecoxib (IC₅₀=6.44 μM). Compounds 4b and 13 potently inhibited COX-2 (IC₅₀ values: 1.08 and 1.88 μM, respectively) comparable to that of celecoxib (IC₅₀=0.68 μM). Compounds 4a, 7b, and 13 inhibited PDE4B (IC₅₀ values: 5.62, 5.65, and 3.98 μM, respectively) compared with the reference drug roflumilast (IC₅₀=1.55 μM). The molecular docking of compounds 4b and 13 with the COX-2 and PDE4B binding pockets was studied.Highlights Antitumor activity of new synthesized cyclopentyloxyanisole scaffold was evaluated. The powerful antitumor 4a, 4b, 6b, 7b & 13 were assessed as COX-2, PDE4B & TNF-α inhibitors. Compounds 4a, 7b, and 13 exhibited COX-2, PDE4B, and TNF-α inhibition. Compounds 4b and 13 showed strong interactions at the COX-2 and PDE4B binding pockets.

Full text of DOI:10.1080/14756366.2020.1740695

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis, antitumor activity, and molecular
docking study of 2-cyclopentyloxyanisole
derivatives: mechanistic study of enzyme
inhibition
Walaa M. El-Husseiny, Magda A.-A. El-Sayed, Adel S. El-Azab, Nawaf A. AlSaif,
Mohammed M. Alanazi & Alaa A.-M. Abdel-Aziz
To cite this article: Walaa M. El-Husseiny, Magda A.-A. El-Sayed, Adel S. El-Azab, Nawaf A.
AlSaif, Mohammed M. Alanazi & Alaa A.-M. Abdel-Aziz (2020) Synthesis, antitumor activity,
and molecular docking study of 2-cyclopentyloxyanisole derivatives: mechanistic study of
enzyme inhibition, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 744-758, DOI:
10.1080/14756366.2020.1740695
To link to this article: https://doi.org/10.1080/14756366.2020.1740695
© 2020 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 18 Mar 2020.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 744–758
https://doi.org/10.1080/14756366.2020.1740695
RESEARCH PAPER
Synthesis, antitumor activity, and molecular docking study of
2-cyclopentyloxyanisole derivatives: mechanistic study of enzyme inhibition
Walaa M. El-Husseinya, Magda A.-A. El-Sayeda,b , Adel S. El-Azabc , Nawaf A. AlSaifc, Mohammed M. Alanazic
and Alaa A.-M. Abdel-Azizc
b
aDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt; Department of Pharmaceutical
c
Chemistry, Faculty of Pharmacy, Horus University, New Damietta, Egypt; Department of Pharmaceutical Chemistry, College of Pharmacy, King
Saud University, Riyadh, Saudi Arabia
ABSTRACT
ARTICLE HISTORY
Received 14 January 2020
Revised 24 February 2020
Accepted 2 March 2020
A series of 24 compounds was synthesised based on a 2-cyclopentyloxyanisole scaffold 3–14 and their
in vitro antitumor activity was evaluated. Compounds 4a, 4b, 6b, 7b, 13, and 14 had the most potent
antitumor activity (IC50 range: 5.13–17.95 lM), compared to those of the reference drugs celecoxib, afati-
nib, and doxorubicin. The most active derivatives 4a, 4b, 7b, and 13 were evaluated for their inhibitory
activity against COX-2, PDE4B, and TNF-a. Compounds 4a and 13 potently inhibited TNF-a (IC50 values:
2.01 and 6.72 lM, respectively) compared with celecoxib (IC50¼6.44 lM). Compounds 4b and 13 potently
inhibited COX-2 (IC50 values: 1.08 and 1.88 lM, respectively) comparable to that of celecoxib
(IC50¼0.68 lM). Compounds 4a, 7b, and 13 inhibited PDE4B (IC50 values: 5.62, 5.65, and 3.98 lM, respect-
ively) compared with the reference drug roflumilast (IC50¼1.55 lM). The molecular docking of compounds
4b and 13 with the COX-2 and PDE4B binding pockets was studied.
KEYWORDS
Synthesis; 2-
cyclopentyloxyanisole scaf-
fold; antitumor activity;
enzyme inhibition assay;
docking study
HIGHLIGHTS
ꢀ Antitumor activity of new synthesized cyclopentyloxyanisole scaffold was evaluated.
ꢀ The powerful antitumor 4a, 4b, 6b, 7b & 13 were assessed as COX-2, PDE4B & TNF-a inhibitors.
ꢀ Compounds 4a, 7b, and 13 exhibited COX-2, PDE4B, and TNF-a inhibition.
ꢀ Compounds 4b and 13 showed strong interactions at the COX-2 and PDE4B binding pockets.
GRAPHICAL ABSTRACT
Introduction
Cyclooxygenase-2 isoenzyme (COX-2) inhibitors, such as cele-
coxib (A; Figure 1), have been reported to have antitumor activ-
ities8,14,15. The COX-2 isoenzyme is overexpressed in numerous
human cancers, such as breast, lung, hepatocellular, gastric, ovar-
ian, prostate, and colon cancers8,14–16. There are two anticancer
mechanisms associated with COX-2 inhibition: the first, termed
the COX-2-dependent anticancer mechanism, is selective inhibition
with the restoration of normal apoptosis; the second is the COX-2-
independent mechanism, which occurs through the induction
Cancer, the uncontrolled growth of cells that invade adjacent
healthy tissues, is the most fatal disease in the world1. Therefore,
the design and synthesis of new molecules with promising and
potential antitumor activity is of great importance1–10. The clinical
use of drug combinations has led to various side effects, whereas
the use of single molecules that target multiple molecular mecha-
nisms is the currently preferred therapeutic strategy and is under
investigation by medicinal chemists11–13
.
CONTACT Alaa A.-M. Abdel-Aziz
almoenes@ksu.edu.sa
Department of Pharmaceutical Chemistry, College of Pharmacy, P.O. Box 2457, King Saud University,
Riyadh 11451, Saudi Arabia
ß 2020 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.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
745
O
SO2NH2
NH
F
O
N
N
F
O Cl
HN
F
O
F
N
O
F
Cl
OCH3
A
celecoxib(
)
B
Rolipam(
)
C
Roflumilast(
)
O
O
O
O
O
O
HN
NH
N
O
O
O
S
O
O
)
O
O
NH
HO
OH
O
D
Ro-20-1724( )
(E
Apremilast
Curcumin (F)
Moleculardesign
X
S
HN
NH
HN
NH
O
O
O
R
O
O
O
O
O
N
O
O
O
n
n
5a,b
6a,b
3a-c
4a,b
S
R
O
NH
N
O
O
R
O
O
N
R
O
O
N
O
9-11
7a-e
8
O
N
O
O
O
NC
NC
NC
N
NH
NH
O
O
O
O
S
N
H
S
R
O
O
12a-c
13
14
Figure 1. The structures of the reported antitumor agents (A–F) with COX-2 or PDE4 and the designed compounds 3–14.
of apoptosis or inhibition of cell proliferation17. These results indi- cancer cells, such as colon cancer, melanoma, prostate cancer,
cated that COX-2 enzyme inhibition was an interesting molecular myeloma, pancreatic cancer, B cell lymphoma, kidney cancer, and
target for the treatment of cancer8,14–17. In addition, phospho- lung cancer18–27. Recently, it was reported that PDE4 inhibitors
diesterase isoenzyme 4 (PDE4) is responsible for inactivation and possess antiproliferative effects, and inhibit the tumour cell
hydrolysis of 30,50-cyclic adenosine monophosphate (cAMP) and growth of several types of cancers; thus, PDE4 inhibitors are a
subdivided into four subtypes, PDE4A to PDE4D18–20. The second- promising novel target for cancer therapy18–27. Rolipram (B; Figure
ary messenger cAMP is important for various cellular processes 1)18,22,23, roflumilast (C; Figure 1)18,22,23, Ro-20–1724 (D; Figure
such as proliferation, growth, migration, differentiation, and apop- 1)23, and apremilast (E; Figure 1)23 are PDE4 inhibitors that
tosis18–20. These isoenzymes of cAMP-PDE expressed in several reduced the growth of colon cancer cells through regulation of

746
W. M. EL-HUSSEINY ET AL.
the level of intracellular cAMP, leading to the induction of apop- antitumor activity against different human cancers: liver cancer
tosis. Roflumilast (C; Figure 1) was approved by FDA as a PDE4
inhibitor and used for the treatment of chronic obstructive pul-
monary disease26 and was successfully tested in lung cancer and
B-cell lymphoma25. In contrast, an increase in the level of intracel-
lular cAMP by the inhibition of PDE4 isoenzymes leads to inhib-
ition of the production of tumour necrosis factor-alpha (TNF-a)28.
TNF-a is a central mediator of inflammation, and thus provides a
molecular link between chronic inflammation and the develop-
ment of malignancies29–32. In addition, TNF-a is overexpressed in
various cancer cells such as liver cancer, kidney cancer, and gall-
(HePG2 cell line), colon cancer (HCT-116 cell line), breast cancer
(MCF-7 cell line), prostate cancer (PC3 cell line), and cervical can-
cer (HeLa cell line); (vi) a study of the structure–activity relation-
ship (SAR) for the synthesised 2-cyclopentyloxyanisole structure
with diverse substituent moieties regarding antitumor activities;
(vii) an evaluation of the in vitro COX-2 and PDE4B, and TNF-a
inhibitory abilities of the most promising compounds; and (viii) a
molecular modelling study of the binding mode of the target mol-
ecules in the COX-2 and PDE 4 pockets.
bladder cancer and supports tumour growth and metastasis29–32
.
The aforementioned results indicated that the inhibition of PDE4
enzyme activity18–27 and the suppression of the production of
TNF-a28–32 are an interesting target for the treatment of cancer.
Compounds containing 2-cyclopentyloxyanisole analogues are
reported to be PDE4 inhibitors with anticancer activities, such as
rolipram (B; Figure 1), roflumilast (C; Figure 1), and apremilast (E;
Figure 1)18,22,23. Meanwhile, compounds bearing chalcone structures
constitute the main building block of several natural products with
potential antitumor activity, such as curcumin (F; Figure 1)7,9,33. It
was reported that curcumin exerts antitumor activity against colon
cancer through inhibition of the COX-2 isoenzyme34. Recently, cur-
cumin was shown to have in vitro anti-angiogenic effects and
in vivo anticancer activity through the inhibition of PDE isoen-
zymes35. Indeed, several compounds possessing heterocyclic core
structures, such as quinazoline2–4, quinoline9,10, pyrimidine36, pyri-
dine9, imidazole6, have potential antitumor activity.
Based on the aforementioned data, and to continue our efforts
to develop new molecules as effective antitumor agents, we have
reported (i) the synthesis of new derivatives incorporating chal-
cone derivatives based on the 2-cyclopentyloxyanisole core struc-
ture; (ii) the preparation of 2-cyclopentyloxyanisole bearing
heterocyclic moieties such as quinazoline, quinoline, pyridine, pyr-
imidine, and imidazole ring systems; (iii) the synthesis of 2-cyclo-
pentyloxyanisole bearing thioamide moieties; (iv) a comparison of
the effectiveness of heterocyclic derivatives versus the chalcone
Experimental methods
Chemistry
Melting points were recorded by using a Fisher-Johns melting
point apparatus and were uncorrected. 1H NMR and 13C NMR
spectra (500 MHz) were obtained in DMSO-d6 and CHCl3-d on a
JOEL Nuclear Magnetic Resonance 500 spectrometer at Mansoura
University, Faculty of Science, Egypt. Mass spectrometric analyses
were performed by using a JEOL JMS-600H spectrometer at
Mansoura University, Faculty of Science (Assiut, Egypt). The reac-
tion times were determined by using a TLC technique on silica gel
plates (60 F245, Merck, Kenilworth, NJ) and the spots were visual-
ised by UV irradiation at 366 nm or 245 nm. The synthesis of 3-
(cyclopentyloxy)-4-methoxybenzaldehyde (2) and 6-(3-(cyclopenty-
loxy)-4-methoxyphenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimi-
dine-5-carbonitrile (13) are described elsewhere18,37,38
.
Synthesis of compounds 3a–c, 4a, and 4b
To a mixture of 3-(cyclopentyloxy)-4-methoxybenzaldehyde (2)
(1.0 mmol, 0.22 g) and cyclic ketones (3.0 mmol) in ethanol (15 ml),
NaOH (2.0 mmol, 0.08 g) was added whilst stirring at 0 ꢁC. The
reaction mixture was then stirred at room temperature for 24 h,
poured on crushed ice, and the obtained solid was filtered,
and thioamide derivatives; and (v) an evaluation of the in vitro washed with water, and recrystallised from methanol (Scheme 1).
O
Cyclicalkanone
O
O
NaOH, rt
24 h
3a (n=0)
3b (n=1)
3c (n=2)
n
Br
HO
O
CHO
O
O
CHO
TBAB/ THF
+
K2CO3, reflux
2
Piperidone
O
O
R
N
NaOH, rt
24 h
4a (R=Methyl)
4b (R=Ethyl)
O
Dimedone,
urea or thiourea,
EtOH, HCl,
Cycloalkanone,
thiourea, EtOH
HCl, reflux, 4 h
reflux, 12 h
S
X
HN
NH
HN
NH
O
O
6a (X= O)
6b (X= S)
5a (n=1)
5b (n=2)
O
O
O
n
Scheme 1. Synthesis of the designed compounds 3–6.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
747
2-(3-(Cyclopentyloxy)-4-methoxybenzylidene)cyclopentanone (3a)
Yield, 65%; melting point [MP] 252–254 ꢁC. 1H NMR spectrum
(DMSO-d6), d, ppm: 1.53–1.56 (2H, m), 1.62–1.65 (4H, m), 1.70–1.74
(4H, m), 1.86–1.89 (2H, m), 2.89–2.91 (2H, m), 3.87 (3H, s),
4.74–4.77 (1H, m), 7.05–7.07 (1H, d, J ¼ 8.0 Hz), 7.07–7.08 (1H, d,
J ¼ 8.0 Hz), 7.21 (1H, s), 7.74 (1H, s). IR spectrum, ꢀ, cmꢂ1: 2957,
2872, 1703, 1620, 954, 642. C18H22O3 MS: m/z 287 (Mþþ1),
286 (Mþ).
(4H, m), 3.83 (3H, s), 4.67 (1H, s), 4.78–4.93 (1H, m), 6.76 (1H, s),
6.80 (1H, s), 6.82 (1H, s), 6.83–6.86 (1H, d, J ¼ 8.0 Hz), 7.13–7.16
(1H, d, J ¼ 8.1 Hz). IR spectrum, ꢀ, cmꢂ1: 3422, 3240, 2960, 2871,
1630, 1260. C20H26N2O2S MS: m/z 360 (Mþþ2), 359 (Mþþ1),
358 (Mþ).
4-(3-(Cyclopentyloxy)-4-methoxyphenyl)-1,3,4,5,6,7,8,9-octahy-
dro-2H-cyclohepta[d]pyrimidine-2-thione (5b)
Yield, 52%; MP 205–207 ꢁC. 1H NMR spectrum (CHCl3-d), d, ppm:
0.83–0.88 (6H, m), 1.19–1.24 (2H, m), 1.25–1.29 (2H, m), 1.61–1.66
(4H, m), 1.82–1.93 (4H, m), 3.84 (3H, s), 4.67 (1H, s), 4.78–4.92 (1H,
m), 6.76 (1H, s), 6.81 (1H, s), 6.84 (1H, s), 6.81–6.85 (1H, d,
2-(3-(Cyclopentyloxy)-4-methoxybenzylidene)cyclohexanone (3b)
Yield, 60%; MP 245–247 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
1.60–1.68 (6H, m), 1.81–1.93 (8H, m), 2.91–2.93 (2H, m), 3.85 (3H,
s), 4.75–4.79 (1H, m), 7.03–7.04 (1H, d, J ¼ 8.1 Hz), 7.06–7.07 (1H, d,
J ¼ 8.0 Hz), 7.25 (1H, s), 7.77 (1H, s). IR spectrum, ꢀ, cmꢂ1: 2953,
2870, 1705, 1621, 951, 638. C19H24O3 MS: m/z 301 (Mþþ1),
300 (Mþ).
J ¼ 7.9 Hz), 7.10–7.12 (1H, d, J ¼ 8.0 Hz). IR spectrum, ꢀ, cmꢂ1
:
3426, 3243, 2963, 2873, 1632, 1262. C21H28N2O2S MS: m/z 374
(Mþþ2), 373 (Mþþ1), 372 (Mþ).
Synthesis of compounds 6a and 6b
2-(3-(Cyclopentyloxy)-4-methoxybenzylidene)cycloheptanone (3c)
Yield, 63%; MP 250–252 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
1.50–1.60 (3H, m), 1.80–1.81 (2H, m), 1.82–1.85 (6H, m), 1.89–1.91
(5H, m), 2.68–2.71 (2H, m), 3.86 (3H, s), 4.73–4.75 (1H, m), 6.84 (1H,
To a solution of 3-(cyclopentyloxy)-4-methoxybenzaldehyde (2)
(5 mmol, 1.1 g), urea or thiourea (5 mmol), and dimedone
(7.5 mmol, 1.1 g) in ethanol (25 ml), four drops of concentrated
s), 6.86–6.88 (1H, d, J ¼ 7.9 Hz), 6.89–6.90 (1H, d, J ¼ 8.0 Hz), 7.44 hydrochloric acid were added. The reaction mixture was heated
(1H, s). IR spectrum, ꢀ, cmꢂ1: 2950, 2871, 1710, 1616, 954, 639.
under reflux for 12 h and the solvent was evaporated under vac-
uum. The obtained solid was dissolved in H2O and the solution
was neutralised by using ammonia solution. The precipitated solid
was filtered, washed with water, and re-crystallised from DMF
(Scheme 1).
C20H26O3 MS: m/z 315 (Mþþ1), 314 (Mþ).
3-(3-(Cyclopentyloxy)-4-methoxybenzylidene)-1-methylpiperidin-4-
one (4a)
Yield, 70%; MP 253–255 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
1.55–1.58 (2H, m), 1.64–1.73 (4H, m), 1.79–1.86 (4H, m), 2.15 (2H,
s), 2.42 (3H, s), 2.91–2.95 (2H, m), 3.71 (3H, s), 4.74–4.78 (1H, q,
J ¼ 5.5 Hz), 6.66–6.74 (2H, m), 6.89–6.94 (2H, m). IR spectrum, ꢀ,
cmꢂ1: 2955, 2872, 1708, 1620, 956, 640. C19H25NO3 MS: m/z 317
(Mþþ2), 316 (Mþþ1), 315 (Mþ).
4-(3-(Cyclopentyloxy)-4-methoxyphenyl)-7,7-dimethyl-4,6,7,8-tet-
rahydroquinazoline-2,5(1H,3H)-dione (6a)
Yield, 80%; MP 230–232 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
0.99 (3H, s), 1.02 (3H, s), 1.05 (1H, s), 1.54–1.58 (4H, m), 1.78–1.89
(4H, m), 2.40–2.43 (3H, t, J ¼ 6.5 Hz), 3.74 (3H, s), 4.67 (1H, s),
4.72–4.76 (1H, q, J ¼ 3.5 Hz), 6.67 (1H, s), 6.68 (1H, s), 6.70 (1H, s),
6.73–6.74 (1H, d, J ¼ 6.5 Hz), 6.75–6.76 (1H, d, J ¼ 6.5 H). IR spec-
trum, ꢀ, cmꢂ1: 3420, 3243, 2957, 2872, 1620, 1260. C22H28N2O4
MS: m/z 386 (Mþþ2), 385 (Mþþ1), 384 (Mþ).
3-(3-(Cyclopentyloxy)-4-methoxybenzylidene)-1-ethylpiperidin-4-
one (4b)
Yield, 68%; MP 249–251 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
1.29–1.32 (3H, t, J ¼ 4.5 Hz), 1.52–1.54 (2H, m), 1.62–1.68 (4H, m),
1.81–1.85 (4H, m), 2.43–2.45 (2H, m), 2.88–2.92 (2H, m), 2.93–2.95
(2H, m), 3.73 (3H, s), 4.73–4.79 (1H, m), 6.66–6.77 (2H, m),
6.86–6.95 (2H, m). IR spectrum, ꢀ, cmꢂ1: 2954, 2870, 1708, 1624,
958, 644. C20H27NO3 MS: m/z 331 (Mþþ2), 330 (Mþþ1), 329 (Mþ).
4-(3-(Cyclopentyloxy)-4-methoxyphenyl)-7,7-dimethyl-2-thioxo-
2,3,4,6,7,8-hexahydroquinazolin-5(1H)-one (6b)
Yield, 78%; MP 233–235 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
0.99 (3H, s), 1.01 (3H, s), 1.55 (2H, s), 1.77–1.82 (4H, m), 1.87–1.92
(4H, m), 2.44 (2H, s), 3.73 (3H, s), 4.68 (1H, s), 4.71–4.73 (1H, m),
6.66 (1H, s), 6.68 (1H, s), 6.69 (1H, s), 6.72–6.74 (1H, d, J ¼ 7.5 Hz),
6.75–6.77 (1H, d, J ¼ 6.5 Hz). IR spectrum, ꢀ, cmꢂ1: 3425, 3245,
2960, 2870, 1623, 1264. C22H28N2O3S MS: m/z 402 (Mþþ2), 401
(Mþþ1), 400 (Mþ).
Synthesis of compounds 5a and 5b
To a solution of 3-(cyclopentyloxy)-4-methoxybenzaldehyde (2)
(5 mmol, 1.1 g), thiourea (5 mmol, 380 mg), and cyclic ketones
(7.5 mmol) in ethanol (25 ml), four drops of concentrated hydro-
chloric acid were added. The reaction mixture was heated under
reflux for 4 h, and the solvent was evaporated under vacuum. The
obtained solid was dissolved in H2O and the solution was neutral-
ised with ammonia solution. The precipitated solid was filtered,
washed with water, and crystallised from ethanol (Scheme 1).
Synthesis of compound 7a
A
mixture of 3-(cyclopentyloxy)-4-methoxybenzaldehyde (2)
(5 mmol, 1.1 g), 9,10-phenanthraquinone (5 mmol, 1.04 g), ammo-
nium acetate (15 mmol, 1.17 g), and CAS or iodine (5 mol%) in
ethanol (25 ml) was heated under reflux for 4 h. The reaction mix-
ture was cooled to room temperature, poured on crushed ice, and
extracted with ethyl acetate. The extract was evaporated under
vacuum to yield a precipitate, which was collected and re-crystal-
4-(3-(Cyclopentyloxy)-4-methoxyphenyl)-3,4,5,6,7,8-hexahydro-
quinazoline-2(1H)-thione (5a)
Yield, 55%; MP 199–201 ꢁC. 1H NMR spectrum (CHCl3-d), d, ppm:
0.80–0.86 (4H, m), 1.20–1.25 (4H, m), 1.83–1.89 (4H, m), 1.91–1.95 lised from acetone (Scheme 2).

748
W. M. EL-HUSSEINY ET AL.
R
7a (R= H)
7b (R= Ph)
N
O
O
7c (R= 4-Me-Ph)
7d (R= 4-F-Ph)
7e (R= 4-Cl-Ph)
Phenanthraquinone
N
NH4OAc, CAS
with or without anilines
EtOH, reflux, 4 h
O
CHO
O
O
NH
2
Dimedone, NH4OAc
O
O
CAS, PG, reflux, overnight
O
8
4-F-aniline
sulfur, DMF,
90°C, 24 h
Cyclic amine,
sulfur, DMF,
90°C, 24 h
N,N-Dimethylamine
sulfur, DMF,
90°C, 24 h
F
S
S
S
O
O
O
O
O
9a (X= O)
9b (X= CH2)
9c (X= N-Boc)
N
H
N
N
X
O
10
11
Scheme 2. Synthesis of the designed compounds 7–11.
2-(3-(Cyclopentyloxy)-4-methoxyphenyl)-1H-phenanthro[9,10-
d]imidazole (7a)
2-(3-(Cyclopentyloxy)-4-methoxyphenyl)-1-(4-methylphenyl)-1H-
phenanthro[9,10-d]imidazole (7c)
Yield, 85%; MP 290–292 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm: Yield, 80%; MP 291–294 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
1.62 (2H, s), 1.79–1.82 (4H, m), 1.97 (2H, s), 3.84 (3H, s), 4.97 (1H, 1.52 (4H, s), 1.64–1.68 (4H, m), 2.07 (3H, s), 3.75 (3H, s), 4.36 (1H,
s), 7.17–7.18 (1H, d, J ¼ 8.0 Hz), 7.62 (2H, s), 7.72 (2H, s), 7.87 (2H, s), 6.89 (1H, s), 6.98–6.70 (1H, d, J ¼ 8.5 Hz), 7.12–7.14 (1H, d,
s), 8.55–8.56 (2H, d, J ¼ 6.5 Hz), 8.83–8.85 (2H, d, J ¼ 7.5 Hz). 13C J ¼ 8.0 Hz), 7.32–7.37 (2H, q, J ¼ 9.0 Hz), 7.50–7.54 (3H, q,
NMR spectrum (DMSO-d6), d, ppm: 18.56, 23.68, 32.38, 55.67, J ¼ 7.5 Hz), 7.56–7.58 (2H, d, J ¼ 8.0 Hz), 7.65–7.68 (1H, t, J ¼ 7.5 Hz),
56.03, 79.95, 112.36, 112.85, 119.25, 121.88, 123.71, 125.02, 126.95, 7.74–7.77 (1H, t, J ¼ 7.5 Hz), 8.66–8.67 (1H, d, J ¼ 7.5 Hz), 8.85–8.86
127.07, 136.83, 147.14, 149.34, 150.92. IR spectrum, ꢀ, cmꢂ1: 3422, (1H, d, J ¼ 8.5 Hz), 8.90–8.92 (1H, d, J ¼ 9.0 Hz). IR spectrum, ꢀ,
2964, 2864, 930, 615. C27H24N2O2 MS: m/z 409 (Mþþ1), 408 (Mþ).
cmꢂ1: 2968, 2877, 942, 632. C34H30N2O2 MS: m/z 499 (Mþþ1),
498 (Mþ).
2-(3-(Cyclopentyloxy)-4-methoxyphenyl)-1-(4-fluorophenyl)-1H-
phenanthro[9,10-d]imidazole (7d)
Synthesis of compounds 7b–e
Yield, 86%; MP 290–292 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
1.50–1.54 (2H, m), 1.60–1.64 (4H, m), 1.68–1.71 (2H, m), 3.86 (3H,
s), 4.94–4.99 (1H, m), 6.85 (1H, s), 6.94–6.96 (1H, d, J ¼ 7.5 Hz),
7.10–7.11 (1H, d, J ¼ 7.5 Hz), 7.29–7.31 (2H, m), 7.40–7.49 (5H, m),
7.62–7.64 (1H, t, J ¼ 8.0 Hz), 7.71–7.74 (1H, t, J ¼ 8.0 Hz), 8.65–8.66
(1H, d, J ¼ 8.5 Hz), 8.81–8.83 (1H, d, J ¼ 9.0 Hz), 8.86–8.87 (1H, d,
A
mixture of 3-(cyclopentyloxy)-4-methoxybenzaldehyde (2)
(5 mmol, 1.1 g), 9,10-phenanthraquinone (5 mmol, 1.04 g), ammo-
nium acetate (15 mmol, 1.17 g), the appropriate aniline (5 mmol),
and CAS or iodine (5 mol%) in ethanol (25 ml) was heated under
reflux for 4 h. The formed precipitate was filtered, washed with
ethanol, and crystallised from DMF (Scheme 2).
J ¼ 8.5 Hz). IR spectrum, ꢀ, cmꢂ1
:
2968, 2875, 940, 636.
C33H27FN2O2 MS: m/z 505 (Mþþ3), 503 (Mþþ1), 502 (Mþ).
2-(3-(Cyclopentyloxy)-4-methoxyphenyl)-1-phenyl-1H-phenan-
thro[9,10-d]imidazole (7b)
2-(3-(Cyclopentyloxy)-4-methoxyphenyl)-1-(4-chlorophenyl)-1H-
phenanthro[9,10-d]imidazole (7e)
Yield, 82%; MP 295–297 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
1.52 (2H, s), 1.60–1.65 (4H, m), 1.70–1.71 (2H, d, J ¼ 6.5 Hz), 3.74 Yield, 84%; MP 294–296 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
(3H, s), 4.49 (1H, s), 6.96–6.98 (2H, d, J ¼ 8.0 Hz), 7.01–7.03 (1H, d, 1.52–1.56 (2H, m), 1.60–1.66 (4H, m), 1.69–1.73 (2H, m), 3.83 (3H,
J ¼ 8.0 Hz), 7.29–7.31 (2H, d, J ¼ 7.5 Hz), 7.51–7.54 (1H, t, J ¼ 7.5 Hz), s), 4.91–4.95 (1H, m), 6.80 (1H, s), 6.94–6.96 (1H, d, J ¼ 8.0 Hz),
7.62–7.77 (7H, m), 8.67–8.68 (1H, d, J ¼ 7.5 Hz), 8.85–8.87 (1H, d, 7.12–7.14 (1H, d, J ¼ 8.0 Hz), 7.25–7.29 (2H, m), 7.40–7.47 (5H, m),
J ¼ 8.5 Hz), 8.90–8.92 (1H, d, J ¼ 8.5 Hz). IR spectrum, ꢀ, cmꢂ1
2960, 2869, 932, 618. C33H28N2O2 MS: m/z 485 (Mþþ1), 484 (Mþ).
:
7.61–7.63 (1H, t, J ¼ 7.5 Hz), 7.72–7.73 (1H, t, J ¼ 7.0 Hz), 8.65–8.67
(1H, d, J ¼ 8.50 Hz), 8.79–8.81 (1H, d, J ¼ 8.5 Hz), 8.84–8.86 (1H, d,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
749
J ¼ 9.0 Hz). IR spectrum, ꢀ, cmꢂ1
:
2965, 2873, 942, 635. 4.75–4.78 (1H, t, J ¼ 5.5 Hz), 6.83–6.85 (2H, t, J ¼ 8.0 Hz), 6.92–6.94
C33H27ClN2O2 MS: m/z 520 (Mþþ2), 519 (Mþþ1), 518 (Mþ).
(1H, d, J ¼ 8.0 Hz). IR spectrum, ꢀ, cmꢂ1: 2956, 2848, 1516, 1223,
1163, 925, 813, 631. C17H23NO3S MS: m/z 323 (Mþþ2), 322
(Mþþ1), 321 (Mþ).
Synthesis of compound 8
To a solution of 3-(cyclopentyloxy)-4-methoxybenzaldehyde (2)
(5 mmol, 1.1 g), dimedone (10 mmol, 1.47 g), and ammonium acet-
ate (5 mmol, 0.39 g) in propylene glycol (20 ml), CAS or iodine
(5 mol%) was added. The reaction mixture was heated under
reflux overnight, cooled to room temperature, and poured on
crushed ice. The obtained solid was filtered, washed with water,
and re-crystallised from ethanol (Scheme 2).
(3-(Cyclopentyloxy)-4-methoxyphenyl)(piperidin-1-yl)methane-
thione (9b)
Yield, 72%; MP 193–195 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
1.50–1.57 (4H, q, J ¼ 6.5 Hz), 1.66–1.69 (8H, t, J ¼ 6.0 Hz), 1.85–1.86
(2H, d, J ¼ 4.0 Hz), 3.52–3.53 (2H, d, J ¼ 5.0 Hz), 3.75 (3H, s),
4.22–4.23 (2H, d, J ¼ 5.5 Hz), 4.75–4.78 (1H, t, J ¼ 6.0 Hz), 6.78–6.80
(2H, d, J ¼ 7.5 Hz), 6.91–6.92 (1H, d, J ¼ 8.5 Hz). IR spectrum, ꢀ,
cmꢂ1: 2955, 2846, 1512, 1225, 1166, 920, 810, 630. C18H25NO2S
MS: m/z 321 (Mþþ2), 320 (Mþþ1), 319 (Mþ).
9-(3-(Cyclopentyloxy)-4-methoxyphenyl)-3,3,6,6-tetramethyl-
3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-dione (8)
Yield, 77%; MP 286–287 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
0.86 (6H, s), 0.99 (6H, s), 1.53–1.55 (2H, m), 1.63–1.67 (4H, m),
1.69–1.71 (2H, m), 1.98–2.00 (2H, d, J ¼ 6.5 Hz), 2.14–2.15 (2H, d,
J ¼ 5.5 Hz), 2.29–2.30 (2H, d, J ¼ 5.5 Hz), 2.41–2.43 (2H, d,
tert-Butyl 4-(3-(cyclopentyloxy)-4-methoxyphenylcarbonothioyl)-
piperazine-1-carboxylate (9c)
Yield, 68%; MP 191–193 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
J ¼ 6.5 Hz), 3.63 (3H, s), 4.55–4.58 (1H, q, J ¼ 6.0 Hz), 4.72 (1H, s), 1.39 (9H, s), 1.53–1.55 (2H, m), 1.68–1.70 (4H, t, J ¼ 4.5 Hz),
6.60–6.62 (1H, d, J ¼ 8.0 Hz), 6.69–6.71 (2H, d, J ¼ 6.0 Hz), 9.26 (1H,
s). 13C NMR spectrum (DMSO-d6), d, ppm: 23.52, 26.39, 29.16,
31.89, 32.07, 32.28, 50.27, 55.38, 79.38, 111.44, 111.59, 115.02,
119.52, 139.68, 146.14, 147.58, 148.95, 149.07, 194.39. IR spectrum,
ꢀ, cmꢂ1: 3420, 2968, 2872, 1735, 1738. C29H37NO4 MS: m/z 464
(Mþþ1), 463 (Mþ).
1.86–1.87 (2H, m, J ¼ 4.5 Hz), 3.30–3.33 (4H, m), 3.56–3.59 (4H, m),
3.75 (3H, s), 4.75–4.77 (1H, t, J ¼ 5.5 Hz), 6.84–6.86 (2H, t,
J ¼ 7.0 Hz), 6.92–6.95 (1H, t, J ¼ 8.0 Hz). IR spectrum, ꢀ, cmꢂ1: 2958,
2848, 1514, 1224, 1160, 929, 812, 633. C22H32N2O4S MS: m/z 421
(Mþþ1), 420 (Mþ).
3-(Cyclopentyloxy)-N-(4-fluorophenyl)-4-methoxybenzothioa-
mide (10)
Synthesis of compounds 9a–c, 10, and 11
Yield, 75%; MP 194–196 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
1.55–1.58 (2H, d, J ¼ 6.0 Hz) , 1.70–1.74 (4H, m), 1.91–1.94 (2H, d,
J ¼ 4.0 Hz), 3.81 (3H, s), 4.83–4.85 (1H, t, J ¼ 5.5 Hz), 7.06–7.07 (1H,
d, J ¼ 7.5 Hz), 7.19–7.23 (2H, m), 7.25–7.28 (2H, m), 7.34 (1H, s),
A
solution of 3-(cyclopentyloxy)-4-methoxybenzaldehyde (2)
(5 mmol, 1.1 g), appropriate amine derivatives (25 mmol), and pre-
cipitated sulphur (12.5 mmol, 0.40 g) in DMF (15 ml) was heated at
90 ꢁC for 24 h. The reaction was monitored by TLC and, after com-
pletion, was cooled to room temperature and poured on crushed
ice. The formed precipitate was filtered, washed with water, and
re-crystallised from methanol (Scheme 2).
7.49–7.50 (1H, d, J ¼ 7.5 Hz), 8.48 (1H, s). IR spectrum, ꢀ, cmꢂ1
:
2951, 2848, 1510, 1225, 1162, 921, 814, 633. C19H20FNO2S MS: m/z
347 (Mþþ2), 346 (Mþþ1), 345 (Mþ).
(3-(Cyclopentyloxy)-4-methoxyphenyl)(morpholino)methane-
thione (9a)
3-(Cyclopentyloxy)-4-methoxy-N,N-dimethylbenzothioamide (11)
Yield, 71%; MP 189–191 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
1.52–1.55 (2H, m), 1.68–1.70 (4H, t, J ¼ 5.0 Hz), 1.71–1.73 (2H, m),
3.16 (3H, s), 3.46 (3H, s), 3.75 (3H, s), 4.75–4.77 (1H, t, J ¼ 5.5 Hz),
Yield, 70%; MP 190–192 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
1.55–1.56 (2H, d, J ¼ 2.5 Hz), 1.67–1.70 (4H, m), 1.86–1.87 (2H, d,
J ¼ 4.0 Hz), 3.58–3.59 (4H, d, J ¼ 3.0 Hz), 3.75 (3H, s), 4.27 (4H, s), 6.84–6.87 (2H, m), 6.91–6.92 (1H, d, J ¼ 8.5 Hz). 13C NMR spectrum
O
NC
NH
Acetophenones, ethyl cyanoacetate
O
NH4OAc, EtOH, reflux, 16 h
R
O
12a (R= H)
12b (R= 4-Me)
12c (R= 3,4-diCl)
O
O
CHO
2
O
N
O
O
NC
NC
Chloroacetic acid,
NaOAc, AC2O
Thiourea, K2CO3,
ethyl cyanoacetate,
N
NH
O
O
O
O
S
N
H
S
Aceic acid, reflux, 24 h
EtOH, reflux, 10 h
13
14
Scheme 3. Synthesis of the designed compounds 12–14.

750
W. M. EL-HUSSEINY ET AL.
(DMSO-d6), d, ppm: 23.53, 32.15, 43.09, 43.98, 55.57, 79.47, 111.30, 7-(3-(Cyclopentyloxy)-4-methoxyphenyl)-3,5-dioxo-2,3-dihydro-
113.04, 118.95, 135.54, 145.96, 149.78, 198.94. IR spectrum, ꢀ,
cmꢂ1: 2956, 2851, 1514, 1229, 1159, 920, 814, 636. C15H21NO2S
MS: m/z 280 (Mþþ1), 279 (Mþ).
5H-thiazolo[3,2-a]pyrimidine-6-carbonitrile (14)
Yield, 55%; MP 265–267 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
1.50–1.53 (2H, m), 1.69–1.72 (4H, m), 1.88–1.90 (2H, m), 3.79 (3H,
s), 4.21 (2H, s), 4.76–4.79 (1H, m), 7.01 (1H, s), 7.35 (1H, s), 7.38
(1H, s). IR spectrum, ꢀ, cmꢂ1: 2962, 2229, 1655, 16,450, 1217, 986.
C19H17N3O4S MS: m/z 385 (Mþþ2), 383 (Mþ).
Synthesis of compounds 12a–c
A
mixture of 3-(cyclopentyloxy)-4-methoxybenzaldehyde (2)
(2 mmol, 0.44 g), the appropriate acetophenone derivatives
(2 mmol), ethyl cyanoacetate (2 mmol, 0.23 g), and ammonium
acetate (16 mmol, 1.24 g) in ethanol (10 ml) was heated under
reflux for 16 h. The reaction mixture was cooled to room tempera-
ture, filtered, washed with ethanol, and re-crystallised from acet-
one (Scheme 3).
Biological evaluation
In vitro antitumor activity evaluation assay
The antitumor activity was performed by using the tetrazolium
salt 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium brom-
ide (MTT) assay in accordance with an established method39.
4-(3-(Cyclopentyloxy)-4-methoxyphenyl)-2-oxo-6-phenyl-1,2-dihy-
dropyridine-3-carbonitrile (12a)
In vitro COX-2 inhibition assay
Yield, 88%; MP > 300 ꢁC; 1H NMR spectrum (DMSO-d6), d, ppm:
1.57–1.58 (2H, d, J ¼ 6.0 Hz), 1.71–1.76 (4H, m), 1.89–1.91 (2H, t,
J ¼ 11.5 Hz), 3.82 (3H, s), 4.88–4.90 (1H, m), 6.77 (1H, s), 7.10–7.12
(1H, d, J ¼ 10.0 Hz), 7.30 (2H, s), 7.33 (1H, s), 7.51–7.56 (3H, m),
7.87–7.88 (2H, d, J ¼ 5.0 Hz). 13C NMR spectrum (DMSO-d6), d,
ppm: 23.62, 32.27, 55.71, 79.71, 112.04, 114.40, 116.94, 121.41,
127.78, 128.08, 128.94, 131.13, 146.82, 151.58. IR spectrum, ꢀ,
cmꢂ1: 3445, 2964, 2220, 1630, 1510, 1265, 810. C24H22N2O3 MS: m/
z 387 (Mþþ1), 386 (Mþ).
The colorimetric COX-2 inhibition assay was performed in accord-
ance with the manufacturer’s instructions (Kit 560101, Cayman
Chemical, Ann Arbour, MI)40–42
.
In vitro TNF-a inhibition assay
The concentration of TNF-a was measured by human-specific
sandwich enzyme-linked immunosorbent assay (ELISA) in accord-
ance with the manufacturer’s instructions (no. 589201, Cayman
Chemical, Ann Arbour, MI)43,44
.
4-(3-(cyclopentyloxy)-4-methoxyphenyl)-2-oxo-6-(p-tolyl)-1,2-
dihydropyridine-3-carbonitrile (12b)
Yield, 84%; MP > 300 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
1.56–1.57 (2H, t, J ¼ 4.0 Hz), 1.70–1.76 (4H, m), 1.88–1.92 (2H, m),
2.36 (3H, s), 3.81 (3H, s), 4.86–4.89 (1H, m), 6.74 (1H, s), 7.10–7.12
(1H, d, J ¼ 8.5 Hz), 7.28–7.30 (2H, q, J ¼ 4.0 Hz), 7.31 (1H, s),
7.33–7.34 (2H, d, J ¼ 8.0 Hz), 7.77–7.79 (2H, d, J ¼ 7.0 Hz). 13C NMR
spectrum (DMSO-d6), d, ppm: 20.92, 23.59, 32.24, 55.69, 79.69,
112.02, 114.39, 116.98, 121.34, 127.63, 128.14, 129.49, 141.27,
146.77, 151.52. IR spectrum, ꢀ, cmꢂ1: 3447, 2959, 2216, 1629,
1514, 1263, 807; C25H24N2O3 MS: m/z 401 (Mþþ1), 400 (Mþ).
Docking methodology
The molecular docking technique was performed by using MOE
2008.10, from the Chemical Computing Group Inc.45 in accordance
with previously established methods18,40–42
.
Results and discussion
Chemistry
The synthetic strategies used to obtain the target compounds are
presented in Schemes 1–3. The O-alkylation of isovanillin (1) with
bromocyclopentane was successively conducted in the presence
of K2CO3 and a phase transfer catalyst tetrabutylammonium brom-
ide (TBAB) in THF to obtain the key intermediate 3-cyclopenty-
loxy-4-methoxybenzaldehyde (2) that provided the core structure
of phosphodiesterase-4 inhibitors37. Tetrabutylammonium bromide
successively exhibited the character of phase transfer catalyst in
an environmentally friendly procedure under mild conditions37.
4-(3-(Cyclopentyloxy)-4-methoxyphenyl)-6-(3,4-dichlorophenyl)-2-
oxo-1,2-dihydropyridine-3-carbonitrile (12c)
Yield, 81%; MP > 300 ꢁC. 1H NMR spectrum (DMSO-d6), d, ppm:
1.52–1.57 (2H, m), 1.69–1.76 (4H, m), 1.89–1.94 (2H, m), 3.82 (3H,
s), 4.86–4.89 (1H, m), 7.11–7.13 (2H, d, J ¼ 8.0 Hz), 7.30–7.31 (2H, d,
J ¼ 2.5 Hz), 7.31–7.32 (1H, d, J ¼ 2.5 Hz), 7.33–7.34 (1H, d,
J ¼ 2.5 Hz), 7.79–7.81 (2H, d, J ¼ 9.0 Hz). IR spectrum, ꢀ, cmꢂ1
:
3443, 2964, 2222, 1635, 1508, 1268, 808. C24H20Cl2N2O3 MS: m/z
456 (Mþþ2), 454 (Mþ).
Synthesis of compounds 3–6
Synthesis of compound 14
First, the cyclocondensation of 3-cyclopentyloxy-4-methoxybenzal-
A mixture of compound 13 (1 mmol, 0.34 g), chloroacetic acid dehyde (2)37 with cyclic ketones in the ethanolic solution of
(1 mmol, 0.10 g), anhydrous sodium acetate (4 mmol, 0.33 g) in sodium hydroxide afforded chalcones 3a–c and 4a,b in good
acetic anhydride (2 ml), and glacial acetic acid (10 ml) was heated yields (Scheme 1). In addition, the one-pot cyclocondensation
under reflux for 24 h. The reaction mixture was cooled to room reaction of 2 with the cyclic ketone (cyclohexanone/cyclohepta-
temperature and poured into crushed ice. The obtained solid was none/dimedone) and urea or thiourea in ethanol containing few
filtered, washed with water, and crystallised from methanol drops of concentrated hydrochloric acid yielded the quinazoline
(Scheme 3).
derivatives 5a,b and 6a,b46, as shown in Scheme 1.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
751
Synthesis of compounds 7–11
with chloroacetic acid in the presence of acetic anhydride and
anhydrous sodium acetate in glacial acetic acid to yield thia-
zolo[3,2-a]pyrimidine-3,5-dione derivative 1451 (Scheme 3).
The synthesis of imidazole via multicomponent reactions (MCRs)
was achieved through the cyclocondensation of 1,2-diketone, an
aldehyde, and ammonium acetate using a catalytic amount of ceric
ammonium sulphate (CAS) or molecular iodine47,48 (Scheme 2).
Thus, a one-pot synthesis achieved phenanthroimidazole derivatives
7a–e in good yield via the cyclocondensation of 9,10-phenanthra-
quinone, 3-cyclopentyloxy-4-methoxybenzaldehyde (2), and ammo-
nium acetate in the presence of 5% mole of iodine or CAS.
Furthermore, acridinedione 8 was prepared by a one-pot, three-
component cyclocondensation reaction of 3-cyclopentyloxy-4-
methoxybenzaldehyde (2), 1,3-dicarbonyl compound (dimedone),
and ammonium acetate in the presence of a catalytic amount of
5% CAS using polyethylene glycol (PEG) as a solvent49. Thioamides
9a–c, 10, and 11 were synthesised50 by the reaction of elemental
sulphur (S8), 3-cyclopentyloxy-4-methoxybenzaldehyde (2), and sec-
ondary amines, such as piperidine, morpholine, N-Boc-piperazine,
and dimethylamine, or primary amines, such as 4-fluoroaniline in
dimethylformamide (DMF), under heating condition.
Biological evaluation
Antitumor evaluation using the MTT assay
Compounds 3a–c, 4a,b, 5a,b, 6a,b, 7a–e, 8, 9a–c, 10, 11, 12a–c,
13, and 14 were screened for their in vitro antitumor activity by
using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT) assay against five human cancers: HePG2,
HCT-116, MCF-7, PC3, and HeLa cell lines39. The antitumor activ-
ities of the synthesised compounds 3–14 and the reference drugs,
celecoxib, afatinib, and doxorubicin, are shown in Table 18–10
.
Compounds 3a–c, incorporating the cycloalkanone core, pos-
sessed strong to weak antitumor activity against some of the
investigated cell lines (IC50 ffi 19.34–95.96 lM). Interestingly, the
replacement of the cycloalkanone moieties, such as in compounds
3a–c, with a piperidin-4-one fragment, such as compound 4a,b,
resulted in
(IC50 ffi 4.38–14.32 lM) against all of the investigated five cell lines,
compared with the reference drug, celecoxib
a
sharp increase in antitumor activity
Synthesis of compounds 12–14
(IC50 ffi 25.6–36.08 lM), afatinib (IC50 values of 5.4–11.4 lM), and
The MCRs of 3-cyclopentyloxy-4-methoxybenzaldehyde (2), ethyl
cyanoacetate, an appropriate acetophenone, and ammonium acet-
ate in EtOH at reflux temperature gave pyridine-3-carbonitrile
derivatives 12a–c in good yield. In contrast, the reaction of
3-cyclopentyloxy-4-methoxybenzaldehyde (2) with ethyl cyanoace-
tate and thiourea in an ethanolic solution of K2CO3 afforded 6-(3-
(cyclopentyloxy)-4-methoxyphenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahy-
dropyrimidine-5-carbonitrile (13)18,38. Compound 13 was cyclised
doxorubicin (IC50 ffi 4.17–8.87 lM).
Moreover, the introduction of quinazoline-2-thione or pyrimi-
dine-2-thione moieties, instead of a piperidin-4-one moiety, as in
compounds 5a,b, resulted in a sharp decrease in antitumor activ-
ity against all the investigated five cancer cell lines, with IC50 val-
ues in the range 46.29–92.37 lM. In contrast, the replacement of
the quinazoline-2-thione fragment, as in compound 5a, with qui-
nazoline-2,5-dione and 2-thioxo-quinazolin-5-one fragments at the
same position, such as compounds 6a and 6b, resulted in a sharp
increase in antitumor activity against all the investigated cancer
cell lines, for HePG2 (IC50 values of 18.53 and 16.05 lM, respect-
ively), HCT-116 (IC50 values of 30.49 and 25.41 lM, respectively),
MCF-7 (IC50 values of 28.62 and 10.27 lM, respectively), PC3 (IC50
values of 27.44 and 17.95 lM, respectively), and HeLa (IC50 values
of 19.12 and 13.49 lM, respectively), compared with celecoxib
(IC50 values of 25.6, 29.54, 31.28, 30.69, and 36.08 lM, respect-
ively), afatinib (IC50 values of 5.4, 11.4, 7.1, 7.7, and 6.2 lM,
respectively), and doxorubicin (IC50 values of 4.50, 5.23, 4.17, 8.87,
and 5.57 lM, respectively).
Table 1. In vitro antitumor activity of the designed compounds, celecoxib, afati-
nib, and doxorubicin against human tumour cells.
IC50 (mM)a
Compound no.
HePG2
HCT-116
MCF-7
PC3
HeLa
3a
3b
95.96 5.2
>100
56.14 2.6 51.43 3.0 59.12 3.8
53.87 3.7 80.56 3.9 23.81 1.5 19.34 1.8 26.11 1.9
3c
4a
86.90 4.5 93.46 5.1
>100
5.13 0.3
7.18 0.8 14.32 1.2
>100
9.18 0.8
81.65 4.7
7.24 0.7
8.56 0.9
6.04 0.5
4.38 0.4
4b
5a
5b
6a
10.96 1.1
9.48 0.8
73.41 3.7 66.48 3.8 92.37 5.2 78.95 4.1 84.26 4.6
59.08 3.5 61.13 3.6 81.20 4.3 55.17 3.1 46.29 3.0
18.53 1.7 30.49 1.8 28.62 1.6 27.44 2.1 19.12 1.7
16.05 1.4 25.41 1.7 10.27 1.1 17.95 1.6 13.49 1.4
Moreover, weak antitumor activity against some of the tested
cancer cell lines was exhibited by some polycyclic derivatives
incorporating imidazole and quinoline ring systems, such as com-
pounds 7a and 7c (IC50 ffi 53.18–90.34 lM), whereas compounds
7e and 8 showed moderate antitumor activity against some
selected cancer cell lines (IC50 ffi 29.8–46.97 lM). Unexpectedly,
derivative 7b showed a sharp increase in antitumor activity com-
pared with the structural analogues 7a, c, d, and 8, with IC50 val-
ues of 13.68, 19.67, 11.85, 22.89, and 17.18 lM against HeG2, HCT-
116, MCF-7, PC3, and HeLa cancer cell lines, respectively.
6b
7a
7b
7c
7e
8
9a
9b
19c
10
78.21 4.4 90.34 4.9 89.79 4.3
>100
77.64 4.6
13.68 1.2 19.67 1.4 11.85 1.3 22.89 1.9 17.18 1.5
57.08 3.9 81.19 4.2 65.32 3.4 68.06 3.5 53.18 3.7
29.89 2.1 44.82 2.3 42.41 2.2 46.97 2.7 38.05 2.5
41.82 3.0 70.52 3.5 60.48 2.8 55.82 3.2 43.47 2.9
32.87 2.3 48.13 2.4 35.17 1.9 29.23 2.3 37.50 2.5
24.85 1.9 39.07 2.2 37.09 2.0 31.50 2.4 28.37 2.3
36.27 2.5 52.87 2.7 48.93 2.3 33.39 2.6 40.61 2.8
49.86 3.5 79.12 3.8 64.10 3.1 47.32 2.9 52.50 3.7
91.23 4.8 96.79 5.5 94.27 4.7 88.63 5.0 90.89 4.9
45.24 3.4 76.05 3.6 71.63 3.9 79.83 4.0 65.72 4.1
38.14 2.8 67.74 3.5 58.28 2.7 61.45 3.3 45.69 3.2
59.63 4.0 83.42 4.3 66.07 3.7 73.48 3.8 62.76 3.9
11
12a
12b
12c
13
In contrast, the introduction of thioamide fragments in the 2-
cyclopentyloxyanisole scaffold resulted in variable antitumor activ-
ity against the tested cancer cell lines; for example, compounds
9a–c showed strong to moderate antitumor activity
8.71 0.7
20.11 1.8 34.93 1.9
25.6 2.3 29.54 2.1 31.28 2.5 30.69 2.7 36.08 2.8
7.66 0.6
6.93 0.5 11.45 1.1
9.62 0.9 15.31 1.3 12.48 1.2
5.86 0.6
14
(IC50
(IC50
ffi
24.85–48.93 lM) in comparison with thioamide 10
47.32–79.12 lM) and 11 (IC50 88.63–96.79 lM).
Celecoxib
Afatinib
DOX
5.4 0.25 11.4 1.26
4.50 0.2 5.23 0.3
7.1 0.49
4.17 0.2
7.7 0.57
8.87 0.6
6.2 0.67
5.57 0.4
ffi
ffi
Furthermore, replacement of the thioamide moiety with a pyridine
fragment, such as in compounds 12a–c, retained the antitumor
activity against all cancer cell lines, as indicated by their IC50 val-
ues in the range 38.14–83.42 lM. In contrast, the 2-cyclopentylox-
yanisole scaffold bearing the pyrimidine ring system, such as
DOX: doxorubicin.
aIC50, compound concentration required to inhibit tumour cell proliferation by
50% (mean SD, n ¼ 3). IC50, (lM): 1–10 (very strong), 11–25 (strong), 26–50
(moderate), 51–100 (weak), and above 100 (non-cytotoxic). Compound 7d had
an IC50 of >100 mM.

752
W. M. EL-HUSSEINY ET AL.
Table 2. In vitro inhibitory effects of COX-2, PDE-4B, and TNF-a of the antitumor
compounds 4a, 4b, 7b, and 13.a
compounds 13 and 14, exhibited strong antitumor activities
against the cancer cell lines tested (IC50 ffi 5.86–20.11 lM). In brief,
the compounds 4a, 4b, 7b, and 13 exhibited the strongest antitu-
mor activities among the designed compounds against the HeG2,
HCT-116, MCF-7, PC3, and HeLa cancer cell lines
(IC50 ffi 4.38–22.89 lM).
IC50 (mM)a
Compound no.
COX-2 inhibition
PDE-4B inhibition
TNFa inhibition
4a
4b
7b
13
3.34
1.08
24.02
1.88
–
5.62
11.62
5.65
3.98
1.55
–
2.012
17.67
13.94
6.72
–
Roflumilast
Celecoxib
Structure–activity relationship of antitumor activity
0.68
6.44
According to the aforementioned antitumor activity, the SARs for
the designed compounds indicated the following. (i) N-
Methylpiperidin-4-one derivative 4a and N-ethylpiperidin-4-one
aIC50 value is the compound concentration required to produce 50% inhibition.
derivative
4b
exhibited
higher
antitumor
activity
the four compounds (4a, 4b, 7b, and 13) that exhibited the great-
est antitumor activity, as well as celecoxib (used as the reference
drug) were subjected to colorimetric COX-2 inhibition assays by
using a COX-2 assay kit (catalogue no. 560101, Cayman Chemicals
Inc., Ann Arbour, MI). The measured IC50 (lM) values are shown in
Table 2, and are expressed as the means of three acquired deter-
minations40–42. The IC50 values of celecoxib for COX-2 inhibition
are found to be 0.68 lM. It is clear that compounds 4b and 13
were found to be the most active inhibitors of COX-2, with IC50
values of 1.08 and 1.88 lM, respectively, whereas compound 4a
exhibited lower COX-2 inhibitory effect with an IC50 value of
3.34 lM. In contrast, compound 7b showed a very low inhibitory
effect, with an IC50 value for COX-2 inhibition of 24.02 lM. Briefly,
a small heterocyclic substituent on the 2-cyclopentyloxyanisole
core, such as the piperidine ring in compounds 4a and 4b and
the pyrimidine ring in compound 13, exhibited higher COX-2
inhibition in comparison with the polycyclic 1H-phenanthro[9,10-
d]imidazole in compound 7b. The reduced inhibitory effect of
compound 7b on COX-2 may be attributed to the bulkiness of
the polycyclic system, which interferes with the COX-2 binding
interactions.
(IC50 ffi 4.38–14.32 lM) than the corresponding cycloalkanones
3a–c (IC50 ffi 19.34 to >100 lM). It was clear that the derivative
with N-methylpiperidin-4-one 4a had greater antitumor activity
against all tested cancer cell lines (IC50 ffi 4.38–9.18 lM) than the
N-ethylpiperidin-4-one derivative 4b (IC50 ffi 7.18–14.32 lM). (ii)
Similarly, cyclohexanone derivative 3b exhibited greater antitumor
activity against MCF-7 (IC50¼23.81 lM), PC3 (IC50¼19.34 lM), and
HeLa (IC50¼26.11 lM) cancer cells than cyclopentanone derivative
3a (IC50 ffi 51.43 to >100 lM), and cycloheptanone derivative 3c
(IC50 ffi 81.65 to >100 lM). (iii) Compounds incorporating a quina-
zoline fragment, such as quinazoline-2,5(1H,3H)-dione derivative
6a (IC50
ffi
18.53–30.49 lM) and 2-thioxoquinazolin-5(1H)-one
derivative 6b (IC50 ffi 10.27–25.41 lM) showed higher antitumor
activity than the corresponding derivatives quinazoline-2(1H)-thi-
one 5a, and pyrimidine-2-thione 5b (IC50 ffi 46.29–92.37 lM). (iv)
The 2-cyclopentyloxyanisole scaffold bearing the bulky polycyclic
1H-phenanthro[9,10-d]imidazoles 7a,c,d,e (IC50
ffi
29.89 to
>100 lM), and acridine-1,8(2H,5H)-dione 8 (IC50 ffi 41.82–70.52 lM)
showed lower antitumor activity than the corresponding 2-cyclo-
pentyloxyanisole scaffold bearing quinazoline moiety 6a,b
(IC50 ffi 10.27–30.49 lM). Interestingly, the derivative 7b with the
phenyl ring at position 1 of 1H-phenanthro[9,10-d]imidazole core
structure (IC50 ffi 11.85–22.89 lM) showed a sharp increase in anti-
tumor activity in comparison with derivatives 7a,c,d,e and had
PDE-4B enzyme assay
Compounds that inhibit PDE4 were recently shown to possess
effective antitumor activities owing to the overexpression of PDE4
in cancer and its role in cell proliferation and tumour cell
growth18–27. The compounds that were the most active antitumor
agents, such as compounds 4a, 4b, 7b, and 13, were subjected to
a PDE4B inhibition assay using roflumilast as a reference drug; the
IC50 values are presented in Table 2. Compound 13 showed the
highest inhibition against PDE4B, with an IC50 value of 3.98 lM
comparable to that of the reference drug roflumilast
(IC50¼1.55 lM), whereas compounds 4a and 7b were found have
moderate activity, with IC50 values of 5.62 and 5.65 lM, respect-
ively. Compound 4b possessed the lowest activity against PDE4B,
with an IC50 value of 11.62 lM. From the structural study of the
tested derivatives, including 4a, 4b, 7b, and 13, we concluded
that the 2-cyclopentyloxyanisole scaffold bearing a cyanopyrimi-
dine fragment, such as compound 13, increased the PDE4B inhibi-
tory activity in comparison with other heterocyclic derivatives.
approximately
similar
activity
with
compound
6b
(IC50 ffi 10.27–25.41 lM). (v) The antitumor activities of the 2-cyclo-
pentyloxyanisole scaffold bearing a methanethione fragment, such
as
N-(4-fluorophenyl)benzothioamide
derivative
10
(IC50 ffi 47.32–79.12 lM) and N,N-dimethylbenzothioamide deriva-
tive 11 (IC50 ffi 88.63–96.79 lM), were less potent than derivatives
that
contained
morpholinomethanethione
derivative
9a
(IC50 ffi 29.23–48.13 lM), piperidin-1-ylmethanethione derivative
9b (IC50 ffi 24.85–39.07 lM), and tert-butyl piperazine-1-carboxylate
derivative 9c (IC50 ffi 33.39–52.87 lM). (vi) The pyrimidine deriva-
tives,
4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile
derivative 13 and 3,5-dioxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimi-
dine-6-carbonitrile derivative 14, had potent antitumor activities
(IC50 ffi 5.86–20.11 lM) compared with that of the pyridine deriva-
tives, 6-aryl-2-oxo-1,2-dihydropyridine-3-carbonitriles 12a–c, which
have moderate to weak antitumor activity (IC50 ffi 38.14–83.42 lM)
against all tested cancer cells. Briefly, the structure–activity correl-
ation of antitumor activity revealed that compounds 4a, 4b, 6b,
7b, 13, and 14 were the most active compounds, whereas com-
pound 7d was the only derivative that had no antitumor activity
against any of the tested cancer cell lines.
TNF-a inhibition assay
TNF-a has been reported as a target for cancer treatment; pres-
ently, TNF antagonists are under clinical investigation in phase I
and II trials as single agents for cancer therapy29–32. Accordingly,
compounds 4a, 4b, 7b, and 13, which are the most active antitu-
mor agents, were subjected to the TNF-a inhibition assay using
celecoxib as a reference drug43; the IC50 values are presented in
COX-2 inhibition assay
Several compounds that possess COX-2 inhibition activity have
shown potent antitumor activities that may be attributable to the
role of the COX-2 enzyme in cell proliferation8,14–17. Accordingly, Table 2. Compound 4a possessed potent TNF-a inhibitory effect,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
753
with an IC50 value of 2.01 mM, comparable with the reference hydrophobic interactions with its inhibitors, consists of key amino
acid residues, such as Arg510, Gln192, Arg120, Tyr355, His90,
drug celecoxib (IC50¼6.44 mM), whereas compound 13 was found
Val523, Ser353, and Ile517. The docking procedure was validated
by including the bound inhibitor SC-558 for a one-ligand run
docking calculation.
to be an effective inhibitor, with an IC50 value of 6.72 mM, similar
to the TNF-a inhibitory effect of the reference drug celecoxib
(IC50¼6.44 mM). In contrast, compounds 4b and 7b were the least
active derivatives, with IC50 values of 17.67 and 13.94 mM,
respectively.
The bound ligand SC-558 exhibited two types of hydrogen
bonds, classical and non-classical hydrogen bonds. Four classical
hydrogen bonding interactions were observed with Arg513, His90,
Arg120, and Tyr355. In addition, three non-classical hydrogen
bonds connected the amino acids Tyr385, Phe518, and Ala516,
and the benzenesulfonamide and 4-bromophenyl fragments of
SC-558 through CH–O and CH–Br interactions (Figure 2,
upper panel).
Interestingly, compounds 4b and 13, which were the most
active COX-2 inhibitors, were placed in the same binding site of
the inhibitor SC-558 (Figure 2). Compound 4b, which has nearly
similar COX-2 inhibition activity as celecoxib, accommodated an
orientation within the COX-2 binding site (Figure 2, left lower
panel), in which the N-ethylpiperdine-4-one fragment was located
towards the secondary pocket of the COX-2 isoenzyme and inter-
acted with the amino acid residues of Arg513, His90, Leu352, and
Gln192. In general, when compound 4b was docked into the
enzyme pocket, nine hydrogen bonds were formed with the sur-
rounding amino acids lining the pocket. One of these interactions
was a classical hydrogen bond between the carbonyl (C¼O) group
of the N-ethylpiperdine-4-one fragment and the OH group of the
Molecular modelling analysis
Molecular modelling and docking analysis is an important tech-
nique used to establish the theoretical interaction between the
bioactive molecules and the target enzyme and receptor to under-
stand their binding mode52,53. Therefore, a molecular docking ana-
lysis was performed by using MOE 2008.10 software and viewer
utility (Chemical Computing Group Inc., Montreal, Canada) in
accordance with the standard MOE procedure45.
Docking with the COX-2 isoenzyme
The molecular interaction of the most active compounds, 4b and
13, with the COX-2 isoenzyme was studied by molecular docking.
The crystal structure of the COX-2 isoenzyme interacting with its
inhibitor SC-558 was obtained from the RSC Protein Data Bank
(PDB code: 1CX2)54. The putative binding site of the COX-2 isoen-
zyme (Figure 2), which is responsible for the hydrogen bonds and Tyr355 residue (3.06 Å). Moreover, eight non-bonding interactions,
Figure 2. Three-dimensional (3D) orientation of the docked ligand SC-558 (upper left panel); docked compounds 4b (lower left panel), and 13 (lower right panel) in
the active pocket of the COX-2 enzyme (H bond interactions are shown as green lines). Upper right panel showed the alignment of SC-558, 4b, and 13 in the active
pocket of the COX-2 enzyme.

754
W. M. EL-HUSSEINY ET AL.
Figure 3. Three-dimensional (3D) orientation of the docked roflumilast (upper left panel); docked compound 13 (upper right panel), in the active pocket of the PDE4B
enzyme (H bond interactions are shown as green lines). Lower left panel showed near picture of compound 13 in the active pocket of the PDE4B enzyme. Lower right
panel showed the hydrophobic interactions of compound 13 in the active pocket of the PDE4B enzyme.
namely non-classical hydrogen bonds were formed, among the with amino acid residues Arg513 and His90 through two classical
two bonds of the OH of the Tyr355 residue, and the C¼O of the hydrogen bonds (2.81 Å and 3.11 Å, respectively). The final inter-
action was the hydrophobic interaction between Tyr348 and the
methoxyl moiety of anisole through a CH2–p bond, with a non-
bonding distance of 3.46 Å.
Leu352 residue with the CH2 of the piperdine-4-one moiety
(3.44 Å, and 2.85 Å, respectively), and among two more bonds
among the C¼O fragments of the Gln192 and Ser353 residues
and the CH3 moiety of N-ethylpiperdine-4-one (3.18 Å and 3.08 Å,
respectively). The amino acid residues Arg513 and His90 formed
additional two bonds between their HN groups and the CH2 of
the piperdine-4-one ring (3.52 Å and 3.00 Å, respectively). Finally,
the amino acid residues Arg120 and Ser530 formed two non-clas-
sical hydrogen bonds with the cyclopentyl and methoxyl moieties
of the anisole core structure (NH–CH2, 2.87 Å; and CH2–OCH3,
3.22 Å, respectively). The overall outcome of the molecular dock-
ing of compound 4b, with respect to non-classical hydrogen
bonds, showed that compound 4b had more hydrophobic interac-
tions with the protein than the bound ligand SC-558.
The molecular docking analysis of compound 13 showed that
the 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile moi-
ety was the main fragment responsible for COX-2 activity, which
interacted with the surrounding amino acid residues of the active
pocket of the COX-2 isoenzyme, such as Arg513, His90, Tyr348,
Tyr355, and Arg120 (Figure 2, right lower panel). Four classical
and one non-classical hydrogen bonding interactions were formed
between the abovementioned amino acid residues and compound
13. The nitrile group (CN) of compound 13 formed two classical
Docking with the PDE4B enzyme
The binding mode of the most active compound, 13, within the
PDE4B enzyme was analysed by using molecular docking. The
crystal structure of the PDE4B enzyme bound with its inhibitor
roflumilast was obtained from the RSC Protein Data Bank (PDB
code: 1XMU)55. The binding site of the PDE4B enzyme (Figure 3),
which is responsible for the formation of coordination bonds,
hydrogen bonds, and hydrophobic interactions with its inhibitor
roflumilast, has three main sites for interaction: the solvent-filled
metal coordination pocket, including both zinc and magnesium;
the conserved residue Gln443; and the hydrophobic pocket. The
amino acid residues Phe414, Ile410, Phe446, and Ile450 were the
key residues that formed the tunnel, and were responsible for the
accommodation of the hydrophobic interaction with the bound
inhibitor, roflumilast. The molecular docking procedure was vali-
dated by performing a one-ligand run docking calculation for the
bound inhibitor roflumilast. The results of the docking calculation
of compound 13 are presented in Figure 3 (upper right panel).
hydrogen bonds with Arg120 (3.01 Å) and Tyr355 (3.24 Å), From the docking results, it was clear that the 2-cyclopentyloxya-
whereas the 4-oxo-tetrahydropyrimidine ring system interacted nisole scaffold and the pyrimidine ring adapted for hydrophobic

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
755
recognition at the binding cavity lining with the amino acid resi-
dues Phe414, Ile410, Phe446, and Ile450 (Figure 3, lower right
panel), similar to the bound inhibitor roflumilast (Figure 3, upper
left panel). In contrast, the methoxyl group of the 2-cyclopentylox-
yanisole scaffold formed a non-classical hydrogen bond with
Ser442 (2.94 Å), whereas the conserved residue Gln443 interacted
with the pyrimidine ring system through the nitrile moiety by the
formation of hydrogen bond with a distance of 3.36 Å (Figure 3,
lower left panel). Moreover, the pyrimidine ring projected towards
the metal-coordinating site filled with water molecules.
Accordingly, the thione (C¼S) moiety of the pyrimidine ring is
coordinated with Zn and Mg ions, mediated by HOH2009, and
formed a hydrogen bond with the amino acid residue His234.
Meanwhile, the carbonyl oxygen (C¼O) of the pyrimidine formed
one hydrogen bond with Tyr233 (2.90 Å) and another two hydro-
gen bonds with the amino acid residues Asn395 and Asp392,
mediated by HOH18. Finally, the internal NH group of pyrimidine
ring was adapted to form a hydrogen bond with Asp392 medi-
ated by HOH18.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
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.
ORCID
Magda A.-A. El-Sayed
Adel S. El-Azab http://orcid.org/0000-0001-7197-1515
Alaa A.-M. Abdel-Aziz
http://orcid.org/0000-0001-9599-9248
http://orcid.org/0000-0002-3362-9337
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tor roflumilast, both compounds accommodated approximately
similar interactions at the hydrophobic clamp site (Phe414, Ile410,
Phe446, and Ile450) and the metal coordination site.
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was
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roflumilast
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