Molecular modelling insights into a physiologically favourable approach to eicosanoid biosynthesis inhibition through novel thieno[2,3-b]pyridine derivatives

Article abstract of DOI:10.1080/14756366.2018.1457657

In this research, we exploited derivatives of thieno[2,3-b]pyridine as dual inhibitors of the key enzymes in eicosanoid biosynthesis, cyclooxygenase (COX, subtypes 1 and 2) and 5-lipoxygensase (5-LOX). Testing these compounds in a rat paw oedema model revealed potency higher than ibuprofen. The most active compounds 7a, 7b, 8b, and 8c were screened against COX-1/2 and 5-LOX enzymes. Compound 7a was the most powerful inhibitor of 5-LOX with IC₅₀ = 0.15 μM, while its p-chloro analogue 7b was more active against COX-2 (IC₅₀ = 7.5 μM). The less desirable target COX-1 was inhibited more potently by 8c with IC₅₀ = 7.7 μM. Surflex docking programme predicted that the more stable anti- conformer of compound (7a) formed a favourable complex with the active site of 5-LOX but not COX-1. This is in contrast to the binding mode of 8c, which resembles the syn-conformer of series 7 and binds favourably to COX-1.

Full text of DOI:10.1080/14756366.2018.1457657

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Molecular modelling insights into a physiologically
favourable approach to eicosanoid biosynthesis
inhibition through novel thieno[2,3-b]pyridine
derivatives
Mosaad S. Mohamed, Yara E. Mansour, Hatem K. Amin & Moustafa E. El-
Araby
To cite this article: Mosaad S. Mohamed, Yara E. Mansour, Hatem K. Amin & Moustafa E. El-
Araby (2018) Molecular modelling insights into a physiologically favourable approach to eicosanoid
biosynthesis inhibition through novel thieno[2,3-b]pyridine derivatives, Journal of Enzyme Inhibition
and Medicinal Chemistry, 33:1, 755-767, DOI: 10.1080/14756366.2018.1457657
To link to this article: https://doi.org/10.1080/14756366.2018.1457657
© 2018 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2018
VOL. 33, NO. 1, 755–767
https://doi.org/10.1080/14756366.2018.1457657
RESEARCH PAPER
Molecular modelling insights into a physiologically favourable approach to
eicosanoid biosynthesis inhibition through novel thieno[2,3-b]pyridine derivatives
a
a
b
a
ꢀ
Mosaad S. Mohamed , Yara E. Mansour , Hatem K. Amin and Moustafa E. El-Araby
aDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Helwan University, Cairo, Egypt; Department of Biochemistry and
b
Molecular Biology, Faculty of Pharmacy, Helwan University, Cairo, Egypt
ABSTRACT
ARTICLE HISTORY
Received 12 February 2018
Revised 16 March 2018
Accepted 21 March 2018
In this research, we exploited derivatives of thieno[2,3-b]pyridine as dual inhibitors of the key enzymes in
eicosanoid biosynthesis, cyclooxygenase (COX, subtypes 1 and 2) and 5-lipoxygensase (5-LOX). Testing
these compounds in a rat paw oedema model revealed potency higher than ibuprofen. The most active
compounds 7a, 7b, 8b, and 8c were screened against COX-1/2 and 5-LOX enzymes. Compound 7a was
the most powerful inhibitor of 5-LOX with IC50 ¼ 0.15 mM, while its p-chloro analogue 7b was more active
against COX-2 (IC50 ¼ 7.5 mM). The less desirable target COX-1 was inhibited more potently by 8c with
IC50 ¼ 7.7 mM. Surflex docking programme predicted that the more stable anti- conformer of compound
(7a) formed a favourable complex with the active site of 5-LOX but not COX-1. This is in contrast to the
binding mode of 8c, which resembles the syn-conformer of series 7 and binds favourably to COX-1.
KEYWORDS
Anti-inflammatory; COX-2;
5-LOX; COX-1;
thieno[2,3-b]pyridine
Introduction
Before withdrawals, some valuable studies placed selective COX-2
inhibitors under scrutiny as evidences accumulated about COX-1
and COX-2 overlapping functions. COX-1’s role was proved not to
be limited to “housekeeping” as it was widely claimed in litera-
ture10. COX-1 was found to contribute considerably to some inflam-
matory responses11 leading to decreased efficacy of some COX-2
selective inhibitors unless they exert a minimum of COX-1 inhibition
at the therapeutic dose12. Moreover, COX-2 is constitutively
expressed in certain tissues to catalyse the biosynthesis of some
beneficial PGs such as PGI2, a critically needed prostaglandin for
homeostasis of the cardiovascular system13. Therefore, the clinical
use of COX1/2 inhibitors will remain associated with some undesir-
able effects regardless being a selective coxib or a non-selective
NSAID14. Moreover, shutting down the COX1/2 pathway causes a
shunting of AA to the other proinflammatory route, the lipoxyge-
nase (LOX) pathway. This possibly results in decreased efficacy15 as
well as aggravation of GI side effect of NSAIDs16. Encoding gene of
5-lipooxygensase (5-LOX), the key enzyme for biosynthesis of the
inflammatory mediators LTs, is almost undetectable in healthy indi-
vidual17 but activated during various inflammatory events18.
Inhibitors of 5-LOX (e.g. Zileuton, Figure 1) are compassionately pre-
scribed for controlling some inflammatory disorders such as bron-
chial asthma19, but it is much less efficacious in OA20 regardless the
Eicosanoids are arachidonic acid (AA) metabolites that comprise
prostaglandins (PGs) and leukotrienes (LTs), the principal inflam-
matory mediators in osteoarthritis (OA)1,2. PGs and LTs play key
roles in triggering the failure of innate homeostasis of chondro-
cytes that contribute to pain, swelling, joint damage, and other
symptoms of OA3,4. PGs’ biosynthesis starts when cyclooxygenases
(COXs) catalyse two reactions, first the bis-peroxidation of AA to
PGG2 and second, the reduction of the acyclic peroxide group in
PGG2 to form the key precursor PGH2. PGH2 metabolises, accord-
ing to elicited biological needs, to responsive PGs under catalysis
of various prostaglandin synthases5.
The “mostly” inducible subtype cyclooxygenase-2 (COX-2) is
greatly upregulated during the inflammatory processes while the
constitutive subtype COX-1 is “mostly” assigned to the regulation
of PGs in healthy individuals3. In 1990s, the approach of selective
inhibition of COX-2 by coxibs was sought as the imminent strat-
egy to control OA, taking the advantage of less side effects
than the classic non-steroidal anti-inflammatory drugs (NSAIDs,
Figure 1)6. NSAIDs (non-selective inhibitors of COX 1&2), though
widely used as analgesics and anti-inflammatory drugs to date, are
frequently implicated in a variety of adverse actions. Patients
under NSAIDs frequently report GI inflammation, bleeding, ulcers,
in addition to hepatic problems, renal toxicities, and others2.
However, only few years after the first coxib approval, rofecoxib,
and valdecoxib (Figure 1) were withdrawn from world markets
subsequent to findings of links to increased cardiovascular events
such as atherosclerosis and infarction7,8. Other coxibs and some
NSAIDs had to add warnings to their labels and limitation of uses
proven involvement of 5-LOX in arthritic progression21,22
.
Based on undisputable findings, dual inhibitors for both COX1/
2 and 5-LOX pathways should provide superior efficacies in con-
trolling inflammation and pain in patients suffering from OA with
less adverse effects23. For instance, dual COX1/2–5LOX inhibitors
demonstrated excellent analgesic and anti-inflammatory activities
in certain patients with risks of cardiovascular complications9. with lower gastric irritation, bleeding and ulcerogenic side effects.
CONTACT Moustafa E. El-Araby
madaoud@kau.edu.sa
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Helwan University, Cairo
11790, Egypt
ꢀ
Current address: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia.
Supplemental data for this article can be accessed here.
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.

756
M. S. MOHAMMED ET AL.
Figure 1. Structures of reported inhibitors of arachidonic acid biosynthesis.
In our quest to finding novel anti-inflammatory agents, we
relied on a screening model which primarily started with measur-
ing the in vivo anti-inflammatory activities28–30. On the other hand,
we sought 3-aminothieno[2,3-b]pyridine as promising molecular
entities to develop novel anti-inflammatory agents with possible
dual inhibitory mechanism against COX1/2 and 5-LOX. This hetero-
bicyclic scaffold has been studied for its versatile activities against
inflammatory disorders, albeit for other subcellular targets31–33
Thienopyridine derivatives have also been reported for their
potent anti-proliferative activities34,35
Moreover, our target
.
.
thieno[2,3-b]pyridine is a close congener to benzothiophene, a
nucleus recently tested for inhibition of COX1/2 and 5-LOX36
(Figure 2).
Figure 2. Approach to develop target compounds with dual COX1/2 and 5-LOX
inhibition from previously reported compounds.
Experimental
Chemistry
In addition, dual inhibition of COX1/2 and 5-LOX provided cardio- General methods
protective activity in a noteworthy contrast to COX-2 inhibitors24
All melting points were uncorrected and measured using Electro-
.
Efforts for developing clinically useful dual COX1/2–5LOX inhibitors thermal IA 9100 apparatus (Shimadzu, Japan); IR spectra were
are still active area in drug discovery, since no approval has been recorded as potassium bromide pellets on a Perkin-Elmer 1650
granted yet for the first drug in this class2. Licofelone, the first spectrophotometer (USA), Faculty of Science, Cairo University,
clinically studied dual inhibitor has completed phase III clinical tri- Cairo, Egypt. 1H-NMR spectra were determined on a Varian
als but it is not clear why it is not marketed yet2. The potential Mercury (500 MHz) spectrometer (Varian UK) and chemical shifts
benefits of dual COX1/2–5LOX inhibitors extend beyond OA. For were expressed as ppm against TMS as an internal reference
instance, they demonstrated anticonvulsant25 as well as anticancer (Faculty of Science, CairoUniversity, Cairo, Egypt). Mass spectra
activities26,27
.
were recorded on 70 eV (EI Ms-QP 1000 EX, Shimadzu, Japan),

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
757
Faculty of Science, CairoUniversity, Cairo, Egypt. Microanalyses CH3), 2.65 (s, 3H, CH3), 3.96 (s, 3H, S-CH3), 6.9 (s, 1H, pyr-H). Anal.
were operated using Vario, Elmentar apparatus (Shimadzu, Japan), Calcd. for C9H10N2S (178.25): C, 60.64; H, 5.65; N, 15.72%. Found: C,
Organic Microanalysis Unit, Faculty of Science, Cairo University, 60.41; H, 5.84; N, 15.96%.
Cairo, Egypt. Column Chromatography was performed on (Merck)
Silica gel 60 (particle size 0.06–0.20 mm).
General procedure for preparation of compounds 4a–c. An excess
of aqueous 37% fomaldehyde (5–6 ml) was added to a suspension
of pyridinethione 1 (1.64 g, 0.01 mol) in EtOH (15 ml). The mixture
was heated with stirring to complete dissolution and then the
appropriate primary amine (0.01 mol) was added in one portion.
The resulting mixture was refluxed with vigorous stirring for 3 min
and then stirred at 20 ꢁC for 4 h. The precipitate formed was fil-
tered off and crystallised from ethanol to afford 4a–c.
Synthesis
1,2-Dihydro-4,6-dimethyl-2-thioxopyridine-3-carbonitrile (1). A mix-
ture of 4,6-dimethyl-3-cyano-2-chloropyridine (1.66 g, 0.01 mol) and
thiourea (1.52 g, 0.02 mol) was refluxed in dry ethanol (20 ml) for
6 h37. Then evaporated under reduced pressure and the residue
was recrystallised from methanol to give 1. Yield 68%, m.p. 183 ꢁC
(Lit. m.p. 180 ꢁC24).
( )3-Aryl-2,3,4,9a-tetrahydro-6,8-dimethylpyrido[2,1-b][1,3,5]thia-
diazine-9-carbonitrile (4a). Yield: 53%; m.p.: 180–182 ꢁC; IR (KBr) t
General procedure for preparation of compounds (2a–c). A mix-
ture of the pyridinethione 1 (1.64 g, 0.01 mol), chloroacetic acid
(2.8 g, 0.03 mol), appropriate aldehyde (0.05 mol), and fused
sodium acetate (2.0 g) in acetic acid/acetic anhydride (30 ml, 1:1)
was heated under reflux for 8 h and left to cool. The solid formed
was filtered off and washed with water and recrystallised from
ethanol/H2O to afford 2a–c.
1
(cmꢂ1): 2230 (CꢃN). MS (EI) m/z: 283 [Mþ] (69%). H NMR (DMSO-
d6, 500 MHz) d (ppm): 2.30 (s, 3H, CH3), 2.58 (s, 3H, CH3), 4.5 (s, 2H,
S-CH2), 4.9 (s, 2H, N-CH2), 5.2 (s, 1H, CH), 6.8 (s, 1H, pyr-H), 6.9–7.5
(m, 5H, Ar-H). Anal. Calcd. for C16H17N3S (283): C, 67.81; H, 6.05; N,
14.83%. Found: C, 67.65; H, 6.31; N, 14.62%.
( )3-Aryl-2,3,4,9a-tetrahydro-6,8-dimethylpyrido[2,1-b][1,3,5]thia-
diazine-9-carbonitrile (4b). Yield: 68%; m.p.: 115–117 ꢁC; IR (KBr) t
( )
3-Benzylidene-3,
8a-dihydro-5,7-dimethyl-2-oxo-2H-thia-
1
(cmꢂ1): 2235 (CꢃN). MS (EI) m/z: 297 [Mþ] (79%). H NMR (DMSO-
zolo[3,2-a]pyridine-8-carbonitrile (2a). Yield: 77%; m.p.: 105–107 ꢁC;
d6, 500 MHz) d (ppm): 2.1 (s, 3H, CH3), 2.3 (s, 3H, CH3), 2.8 (s, 3H,
CH3), 4.6 (s, 2H, S-CH2), 4.8 (s, 2H, N-CH2), 5.1 (s, 1H, CH), 6.2 (s,
1H, pyr-H), 7.0–7.5 (m, 4H, Ar-H). Anal. Calcd. for C17H19N3S (297):
C, 68.65; H, 6.44; N, 14.13%. Found: C, 68.31; H, 6.25; N, 14.39%.
IR (KBr) t (cmꢂ1): 1725 (C¼O), 2256 (CꢃN). MS (EI) m/z: 294 [Mþ]
1
(85%). H NMR (DMSO-d6, 500 MHz) d (ppm): 2.41 (s, 3H, CH3), 2.75
(s, 3H, CH3), 5.4 (s, 1H, CH), 6.5 (s, 1H, pyr-H), 6.9 (s, H, ¼CH),
7.1–7.6 (m, 5H, Ar-H).; Anal. Calcd. for C17H14N2OS (294.08): C,
69.36; H, 4.79; N, 9.52%. Found: C, 69.67; H, 4.42; N, 9.79%.
( )3-Aryl-2,3,4,9a-tetrahydro-6,8-dimethylpyrido[2,1-b][1,3,5]thia-
diazine-9-carbonitrile (4c). Yield: 59%; m.p.: 125–127 ꢁC; IR (KBr) t
( )
3-Furan-2-ylmethylene-3,8a-dihydro-5,7-dimethyl-2-oxo-2H-
1
(cmꢂ1): 2210 (CꢃN). MS (EI) m/z: 297 [Mþ] (72%). H NMR (DMSO-
thiazolo[3,2-a]pyridine-8-carbonitrile (2b). Yield: 69%; m.p.:
100–102 ꢁC; IR (KBr) t (cmꢂ1): 1730 (C¼O), 2220 (CꢃN). MS (EI) m/z:
d6, 500 MHz) d (ppm): 2.30 (s, 3H, CH3), 2.58 (s, 3H, CH3), 4.5 (s, 2H,
S-CH2), 4.7 (s, 2H, CH2), 5.2 (s, 2H, N-CH2), 5.9 (s, 1H, CH), 6.8 (s,
1H, pyr-H), 7.2–7.6 (m, 5H, Ar-H). Anal. Calcd. for C17H19N3S (297):
C, 68.65; H, 6.44; N, 14.13%. Found: C, 68.88; H, 6.71; N, 14.00%.
1
284 [Mþ] (85%). H NMR (DMSO-d6, 500 MHz) d (ppm): 2.12 (s, 3H,
CH3), 2.3 (s, 3H, CH3), 5.5 (s, 1H, CH), 6.4 (s, 1H, pyr-H), 7.0 (s, H,
¼CH), 7.1–7.4 (m, 3H, Ar-H).; Anal. Calcd. for C15H12N2O2S (284.06):
C, 63.36; H, 4.25; N, 9.85%. Found: C, 63.14; H, 4.59; N, 9.61%.
Ethyl-3-amino-4,6-dimethylthieno[2,3-b]pyridine-2-carboxylate (5).
This compound was prepared by two methods.
( )3-(3-Phenyl-allylidene)-3,8a-dihydro-5,7-dimethyl-2-oxo-2H-
thiazolo[3,2-a]pyridine-8-carbonitrile (2c). Yield: 76%; m.p.:
80–82 ꢁC; IR (KBr) t (cmꢂ1): 1580 (C ¼ C), 1750 (C¼O), 2260 (CꢃN).
Method 1: A mixture of pyridinethione 1 (1.64 g, 0.01 mol) and
ethyl bromoacetate (3.34 g, 0.02 mol) in dry ethanol (50 ml) con-
taining sodium metal (1 g) was stirred for 2 h, and then was
refluxed with stirring for 4 h. The reaction mixture was cooled and
poured into ice water. The solid formed was filtered off, dried, and
recrystallised from methanol to afford 5.
Method 2: A mixture of ethyl 2-(3-cyano-4,6-dimethylpyridin-2-
ylthio)acetate 3b (2.5 g, 0.01 mol) and dry ethanol (50 ml) containing
sodium ethoxide (2%) was refluxed with stirring for 4 h. The reaction
mixture was cooled and poured into ice water. The solid formed
was filtered off, dried, and recrystallised from ethanol to afford 5.
Yield 70%, m.p. 138 ꢁC (Lit. m.p. 135 ꢁC24).
1
MS (EI) m/z: 320 [Mþ] (85%). H NMR (DMSO-d6, 500 MHz) d (ppm):
2.2 (s, 3H, CH3), 2.4 (s, 3H, CH3), 5.4 (s, 1H, CH), 6.2 (s, 1H, pyr-H),
6.5 (s, H, ¼CH), 6.7 (s, H, ¼CH), 7.1 (s, H, ¼CH), 7.3–7.6 (m, 5H, Ar-
H). Anal. Calcd. for C19H16N2OS (320.1): C, 71.22; H, 5.03; N, 8.74%.
Found: C, 71.53; H, 5.28; N, 8.45%.
General procedure for preparation of compounds 3a–b. To an
ethanolic sodium hydroxide solution [sodium hydroxide (0.01 mol)
in ethanol (20 ml)] pyridinethione 1 (1.64 g, 0.01 mol) was added,
with stirring for 30 min, and then the appropriate alkyl halide
(0.02 mol) was added drop-wise with vigorous shaking. The reac-
tion mixture was stirred for 2 h at r.t., poured onto cold water
(100 ml) and neutralised with dilute HCl. The solid precipitate was
3-Amino-4,6-dimethylthieno[2,3-b]pyridine-2-carbazide (6). A mix-
ture of thienopyridine-2-carboxylate 5 (2.5 g, 0.01 mol) and hydra-
zine hydrate (0.05 mol, 99%) in absolute ethanol (15 ml); was
filtered off and washed with water and recrystallised from metha- heated under reflux for 6 h. The reaction mixture was concentrated,
nol to afford 3a–b. The reported compound 3b was prepared in cooled, and the precipitate formed was filtered and crystallised
70% yield and m.p. 93 ꢁC (Lit. m.p. 90 ꢁC)37.
from ethanol to afford 6. Yield 70%, m.p. 200 ꢁC (Lit. m.p. 198 ꢁC)37.
S-Methyl-4, 6-dimethyl-2-thiopyridine-3-carbonitrile (3a). Yield: 72%; General procedure for preparation of compounds 7a–c. A mixture
m.p.: 80–82 ꢁC; IR (KBr) t (cmꢂ1): 2218 (CꢃN). MS (EI) m/z: 178 of thienopyridine-2-carbazide (2.36 g, 0.01 mol) and the appropri-
[Mþ] (91%). 1H NMR (DMSO-d6, 500 MHz) d (ppm): 2.59 (s, 3H, ate aldehyde (0.02 mol) in pyridine:ethanol (15:20 ml) was heated

758
M. S. MOHAMMED ET AL.
1
under reflux for 2 h. The reaction mixture was cooled and poured (C–O). MS (EI) m/z: 365 [Mþ] (56%), H NMR (DMSO-d6, 500 MHz) d
into ice water. The solid formed was filtered off, dried, and crystal- (ppm): 2.32 (s, 3H, CH3), 2.61 (s, 3H, CH3), 3.5 (s, 3H, CH3), 7.0 (s,
lised from the acetic acid to afford 7a–c.
1H, pyr-H), 7.2–7.6 (dd, 4H, Ar-H), 8.6 (s, 1H, CH). Anal. Calcd. for
C18H15N5O2S (365): C, 59.16; H, 4.14; N, 19.17%. Found: C, 59.42; H,
4.41; N, 19.54%.
3-Amino-4,6-dimethyl-thieno [2,3-b] pyridine-2-carboxylic acid
benzylidene-hyrazide (7a). Yield: 65%; m.p.: 250–252 ꢁC; IR (KBr) t
(cmꢂ1): 3100, 3260, 3310 (NH)(NH2), 1690 (C¼O), 1230 (C¼N). MS
(EI) m/z: 324 [Mþ] (76%). 1H NMR (DMSO-d6, 500 MHz) d (ppm):
2.32 (s, 3H, CH3), 2.71 (s, 3H, CH3), 7.0 (s, 1H, pyr-H), 6.9 (s, 2H,
NH2, D2O exchangeable), 7.3–7.6 (dd, 5H, Ar-H), 8.2 (s, 1H, NH, D2O
exchangeable), 8.5 (s, 1H, CH). Anal. Calcd. for C17H16N4OS (324): C,
62.94; H, 4.97; N, 17.27%. Found: C, 62.97; H, 4.62; N, 17.62%.
General procedure for preparation of compounds 9a–b. A mixture
of thienopyridine-2-carbazide 6 (2.36 g, 0.01 mol) and ethyl acetoa-
cetate (1.3 g, 0.01 mol) or acetylacetone (1 g, 0.01 mol) in glycial
acetic acid (30 ml) was heated under reflux for 5 h. The excess
solvent was evaporated under reduced pressure to one-third of
the solution and the reaction mixture was allowed to cool. The
precipitate formed was collected and crystallised from the acetic
acid to afford 9a and 9b.
3-Amino-4,6-dimethyl-thieno [2,3-b] pyridine-2-carboxylic acid (4-
chloro-benzylidene)-hyrazide (7b). Yield: 72%; m.p.: 280–282 ꢁC; IR
(KBr) t (cmꢂ1): 3150, 3290, 3310 (NH)(NH2), 1685 (C¼O), 1245
3-Amino-2-[carbonyl (3-methyl-4,5-dihydro-5H-pyrazol-5-on-1-yl)]-
4,6-dimethylthieno [2,3-b] pyridine (9a). Yield: 59%; m.p.:
140–142 ꢁC; IR (KBr) t (cmꢂ1): 3425, 3265 (NH2), 1686 (C¼O). MS
(EI) m/z: 302 [Mþ] (71%), 1H NMR (DMSO-d6, 500 MHz) d (ppm):
2.00 (s, 3H, CH3), 2.29 (s, 3H, CH3), 2.65 (s, 3H, CH3), 2.9 (s, 2H,CH2),
5.58 (s,2H,NH2, D2O exchangeable), 6.9 (s, 1H, pyr-H).; Anal. Calcd.
for C14H14N4O2S (302.35): C, 55.61; H, 4.67; N, 18.53%. Found: C,
55.39; H, 4.90; N, 18.81%.
(C¼N). MS (EI) m/z: 358 [Mþ] 35Cl (66%), 360 [M þ 2] 37Cl (22%), H
1
NMR (DMSO-d6, 500 MHz) d (ppm): 2.45 (s, 3H, CH3), 2.62 (s, 3H,
CH3), 6.9 (s, 1H, pyr-H), 7.0 (s, 2H, NH2, D2O exchangeable), 7.2–7.5
(dd, 4H, Ar-H), 8.3 (s, 1H, NH, D2O exchangeable), 8.6 (s, 1H, CH).
Anal. Calcd. for C17H15ClN4OS (358): C, 56.90; H, 4.21; N, 15.61%.
Found: C, 56.46; H, 4.49; N, 15.93%.
3-Amino-4, 6-dimethyl-thieno [2,3-b] pyridine-2-carboxylic acid (4-
methoxy-benzylidene)-hyrazide (7c). Yield: 74%; m.p.: 270–272 ꢁC;
IR (KBr) t (cmꢂ1): 3180, 3240, 3320 (NH) (NH2), 1680 (C¼O), 1220
3-Amino-2-[carbonyl
(3,5-dimethyl-pyrazol-1-yl)]-4,6-dimethylth-
ieno[2,3-b] pyridine (9b). Yield: 67%; m.p.: 150–152 ꢁC; IR (KBr) t
(cmꢂ1): 3425, 3265 (NH2), 1686 (C¼O). MS (EI) m/z: 300 [Mþ]
1
(C¼N), 1150 (C–O). MS (EI) m/z: 354 [Mþ](58%). H NMR (DMSO-d6,
1
(62%), H NMR (DMSO-d6, 500 MHz) d (ppm): 2.38 (s, 3H, CH3), 2.65
500 MHz) d (ppm): 2.32 (s, 3H, CH3), 2.61 (s, 3H, CH3), 3.5 (s, 3H,
CH3), 6.8 (s, 1H, pyr-H), 7.0 (s, 2H, NH2, D2O exchangeable), 7.2–7.6
(dd, 4H, Ar-H), 8.0 (s, 1H, NH, D2O exchangeable), 8.6 (s, 1H, CH).
Anal. Calcd. for C18H18N4O2S (354): C, 61.00; H, 5.12; N, 15.81%.
Found: C, 61.33; H, 5.45; N, 15.56%.
(s, 3H, CH3), 2.79 (s, 6H, two CH3), 5.2 (s, 2H, NH2, D2O exchange-
able), 7.1 (s, 1H, pyr-H), 8.2 (s, 1H,4H-pyrazole). Anal. Calcd. for
C15H16N4OS (300.38): C, 59.98; H, 5.37; N, 18.65%. Found: C, 59.68;
H, 5.68; N, 18.40%.
General procedure for preparation of compounds 10a–b. A mix-
ture of thienopyridine-2-carbazide 6 (0.01 mol) in formic acid
(15 ml) or acetic anhydride (15 ml) was heated under reflux for 6 h.
The excess solvent was evaporated in vacuo (to one-third of the
solution) and cooled; the solid was collected by filtration, dried,
General procedure for preparation of compounds 8a–c. To an ice-
cooled solution of the corresponding thienopyridine-2-carboxylic
acid aryldine-hydrazide 7a–c (0.01 mol) in AcOH (20 ml) and conc.
HCl (3 ml) a cold solution of sodium nitrite (0.23 g in 5 ml of water)
was added drop-wise over 5 min. The solution was stirred at room
temperature for 3 h at 0–5 ꢁC; where a solid was formed, collected and crystallised from acetic acid to afford 10a–b.
by filtration, dried, and crystallised from dioxane to afford 8a–c.
7,9-Dimethyl-3-fomylaminopyrido [3',2':4,5]thieno[3,2-d]pyrimidin-
4(3H)-one (10a). Yield: 48%; m.p.: 220–222 ꢁC; IR (KBr) t (cmꢂ1):
7,9-Dimethyl-3-[(phenylmethylene)amino]pyrido[30,20:4,5]-
thieno[3,2-d][1,2,3]triazin-4(3H)-one (8a). Yield: 72%; m.p.: 1715, 1760 (two C¼O). MS (EI) m/z: 274 [Mþ] (71%), 1H NMR
300–302 ꢁC; IR (KBr) t (cmꢂ1): 1690 (C¼O), 1250 (C¼N). MS (EI) m/ (DMSO-d6, 500 MHz) d (ppm): 2.4 (s, 3H, CH3), 2.8 (s, 3H, CH3), 7.1
z: 335 [Mþ] (45%), 1H NMR (DMSO-d6, 500 MHz) d (ppm): 2.35 (s, (s, 1H, pyr-H), 7.9 (s, 1H, CH pyrimidine), 8.5 (s, 1H, H-C¼O), 9.5 (s,
3H, CH3), 2.75 (s, 3H, CH3), 6.8 (s, 1H, pyr-H), 7.3–7.6 (m, 5H, Ar-H), 1H, NH). Anal. Calcd. for C12H10N4O2S (274.3): C, 52.54; H, 3.67; N,
8.2 (s, 1H, CH). Anal. Calcd. for C17H13N5OS (335): C, 60.88; H, 3.91; 20.43%. Found: C, 52.81; H, 3.41; N, 20.69%.
N, 20.88%. Found: C, 60.47; H, 3.64; N, 20.57%.
3-Diacetylamino-2,7,9-trimethylpyrido [3',2':4,5]thieno[3,2-d]pyrimi-
din-4(3H)-one (10b). Yield: 65%; m.p.: 133–135 ꢁC; IR (KBr) t (cmꢂ1):
7,9-Dimethyl-3-[(4-chloro-phenyl-methylene)amino]pyrido[3’,2’:4,5]
thieno [3,2-d][1,2,3]triazin-4(3H)-one (8b). Yield: 74%; m.p.: 1720, 1745, 1780 (three C¼O). MS (EI) m/z: 344 [Mþ] (65%), 1H
255–257 ꢁC; IR (KBr) t (cmꢂ1): 1685 (C¼O), 1270 (C¼N). MS (EI) m/ NMR (DMSO-d6, 500 MHz) d (ppm): 1.9 (s, 3H, CH3), 2.2 (s, 3H, CH3),
z: 369 [Mþ] 35Cl (60%), 371 [M þ 2] 37Cl (20%). 1H NMR (DMSO-d6,
2.5 (s, 3H, CH3), 2.7 (s, 6H, 2CH3), 7.2 (s, 1H, pyr-H). Anal. Calcd. for
C16H16N4O3S (344.09): C, 55.80; H, 4.68; N, 16.27%. Found: C, 56.00;
H, 4.91; N, 16.50%.
500 MHz) d (ppm): 2.45 (s, 3H, CH3), 2.62 (s, 3H, CH3), 6.9 (s, 1H,
pyr-H), 7.2–7.5 (dd, 4H, Ar-H), 8.3 (s, 1H, CH). Anal. Calcd. for
C17H12ClN5OS (369): C, 55.21; H, 3.27; N, 18.94%. Found: C, 55.49;
H, 3.51; N, 18.72%.
Biological assay
7,9-Dimethyl-3-[(4-methoxy-phenyl-methylene)amino]pyri-
Animals
do[3’,2’:4,5]thieno [3,2-d][1,2,3]triazin-4(3H)-one (8c). Yield: 65%; To define the minimum total sample size and the average number
m.p.: 240–242 ꢁC; IR (KBr) t (cmꢂ1): 1680 (C¼O), 1240 (C¼N), 1150 for each group the a priori test with the following parameters was

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
759
conducted: anticipated effect size (Cohen's d): 0.7; desired statis-
tical power level: 0.8; probability level: 0.05. The a priori test calcu-
lation returned the following values: minimum total sample size
(one-tailed hypothesis)38; minimum sample size per group (one-
tailed hypothesis)39.
The Ligand Structures Library was built on Chemsketch41 and
saved as .sdf files. The structures were converted to 3D and opti-
mised using SYBYL-X’s Concord embedded in “Prepare
Ligands” tools.
According to a priori test results, experiments were conducted
on young adult male Sprague–Dawley rats (5 rats per group),
weighing 140–170 g. The animals were housed at cages in a tem-
perature controlled (25 1 ꢁC) environment and were fed ad libi-
tum with standard laboratory chow and allowed free access to
water. This investigation conforms to the ethical Guide for the
Care and Use of Laboratory Animals published by the US National
Institutes of Health (NIH Publication No. 85–23, revised in 1996).
The animal protocol is in accordance with the Animal Ethical Care
regulations in the Faculty of Pharmacy, Helwan University.
Surflex docking
Docking was performed using Surflex programme embedded in
“Dock Ligand” tools. First, the target previously prepared protein
was selected and underwent final preparation for docking. Surflex
docking was performed after protomol generation on lig-
and mode.
Manual docking
Ligand preparation. The manual docking was used in case no lig-
and was present to guide Surflex automatic docking procedure.
This protocol was used only for docking experiments of syn-7a,
anti-7a, and 8c to the active site of 5-LOX crystal structure (PDB
Code 3O8Y) because it contains no ligand to guide a Surflex auto-
matic docking. In this regards, ligands prepared above we further
optimised to the global minimum conformation by energy mini-
misation tools until the global minimum is reached
(Termination: Gradient).
Assessment of anti-inflammatory activity
Compounds (equimolar to the reference drug) were dissolved in
DMSO and administrated subcutaneously. One hour later, paw
oedema was induced by subplantar injection of 0.1 ml of 1% carra-
geenan (Sigma-Aldrich, St. Louis, MO) into the right hind paw.
Paw volume was measured using a water plethysmometer (Basile,
Comerio, Italy). The difference between the right and left paw vol-
ume was measured at 1, 2, 3, and 4 h after induction of
inflammation.
Docking. We used three-step, visually-guided procedure (Place-
Merge-MD) as follows.
The control group (five rats per group) received DMSO subcuta-
neously and carrageenan in the subplantar region. Results were
expressed as percentage inhibition of inflammation. Ibuprofen
1. Place. Prepared protein structure (as described above) and the
intended ligand were imported to two different SYBYL-X screen
levels. The ligand was moved until it was visually entered the
active site. Careful inspection of features in the ligand and resi-
dues around to ensure maximum positive interactions with the
least clashes with the active site residues.
(70 mg kgꢂ1) was used as the reference drug40
.
Biochemical assay
Drugs capacities to inhibit COX-1, COX-2, and 5-LOX enzymes
were assessed using ELISA kits; COX-1 (human) Inhibitor Screening
Assay Kit (Item @ 701070), COX-2 (human) Inhibitor Screening
Assay Kit (Item @ 701080), and 5-LOX Inhibitor Screening Assay
Kit (Item @ 760700) from Cayman (Ann Arbor, MI). The used
protocol was according to the manufacturer protocol guide and
instructions using ELISA plate reader.
2. Merge. When convinced that the ligand is situated in the best
docking position, the ligand was merged into the protein screen.
To confirm that the ligand has no serious clash with the active
site residues, AAs with 5 Å sphere distance around the docked lig-
and were unhidden. If a clash existed, another round of moving
the ligand was performed and the procedure is repeated until the
least possible clash is reached. After merge, the energy of the
complex it is measured and compared to uncomplexed protein.
The process is repeated several times with different docking
modes. The best docking complexes pose according to visual
inspection of clashes and energy computation were retained and
compared. The poses ranked according to their energies (first pri-
ority) and positive interactions of the ligand with the active site.
Statistics. All assays results are expressed as mean standard error
of the mean (SEM). The comparisons between the control and the
treatment groups were carried out using One-way ANOVA using a
statistical package (SPSS version 17.0). A p value of <0.05 was con-
sidered significant.
Molecular modelling
3. Molecular dynamics (MD). Molecular dynamics was performed to
check the stability of the complex generated by manual docking
according to the following protocol with time length 1000 fs at
300 K and 0 atm pressure. Snapshots every 5 fs were taken and
inspected visually. The best snapshot in energy is considered pro-
All molecular modelling work was performed using SYBYL-X pack-
age (www.certara.com) installed with licence to the Faculty of
Pharmacy, King Abdulaziz University on Windows 7-operated com-
puter, equipped with Samsung SyncMaster 2233RZ 120 Hz LCD
DisplayTM (3D ready) and Nvidia Geforce 3D Vision Glasses KitTM
.
vided that
a minimum movement of the protein loops
are observed.
Preparation of protein and ligands
Crystal structures were downloaded as .pdb files from Protein
Databank Website (www.rcsb.org). The initial biopolymers were
simplified by deleting all but one monomer in the quaternary
structures and then prepared for docking. The previous two steps
were performed using “Biopolymer” Tools.
Results
Chemical synthesis
Thienopyridine synthesis has been previously reported42. Synthesis
of target anti-inflammatory compounds was accomplished via the

760
M. S. MOHAMMED ET AL.
Scheme 1. Utilisation of compound 1 to produce series 2–4 (a ¼ ClCH2COOH, Ac2O, AcOH, NaOAc along with an aldehyde RCHO, b ¼ RX where X ¼ Br or I, c ¼ RNH2
and HCHO).
key intermediate pyridin-2-thione (compound 1), which was pre- ibuprofen, while those of series 8 showed a much slower onset
pared according to the reported method37. Compound 1 was then
(Supplementary Table S1).
utilised in different reactions to yield three different series of com-
pounds2–4. Condensation of 1 with chloroacetic acid and certain
aldehydes in a mixture of acetic acid and acetic anhydride gave
the thiazolopyridine derivatives 2a–c43. Compound 1 was also
treated with different alkylating agents to afford the correspond-
ing 2-alkylthio analogues 3a–b. Condensation of 1 with formalin
or certain primary amines afforded the pyridothiadiazine deriva-
tives 4a–c (Scheme 1).
In vitro inhibition assays of 5LOX, COX-2, COX-1 enzymes
The compounds belonging to series 7 and 8 were subjected to
enzyme assay investigations against 5-LOX, COX-1, and COX-2.
Those compounds were chosen based on the demonstrated pro-
found anti-inflammatory activity in the above in vivo test.
Compound 3b was subsequently converted to the thienopyri-
dine derivatives 5 via reaction with NaOEt/EtOH. Compound 5 was
treated with NH2NH2 to yield the corresponding hydrazide 6
(Scheme 2)37.
Discussion
The phenotypic screening preceding isolated enzyme assays pro-
vided an opportunity to shift focus towards series of compounds
that exhibited higher potential therapeutic benefit. The structur-
e–activity relationship (SAR) of screened compounds for in vivo
anti-inflammatory activities favoured thieno[2,3-b]pyridines over
other scaffolds such as 2 and 4. The 5-LOX inhibition assay, how-
ever, favoured the hydrazides 7 as the most potent compound,
7a, is 35 and 87 times higher than the tricyclic derivatives 8a and
8c, respectively. A similar result was noticed in the COX-2 inhib-
ition assay, albeit to a lower extent. In this regard, 7a inhibited
COX-2 at 3.6 and 1.8 times higher than 8a and 8c, respectively.
Results, displayed in Table 1, were interesting since potency
level, as well as selectivity between the two series 7 and 8,
explained the potential of these compounds. The in vitro enzyme
inhibition assays revealed that compounds 7a and 7b realised
higher potency and selectivity towards 5-LOX and COX-2 over
COX-1. They are also higher than the compounds 8b and 8c as
well as ibuprofen45 in their 5-LOX and COX-2 inhibition potency.
Compounds 7a and 7b are also more selective than the other
three compounds towards COX-2 than COX-1 with selectivity
index COX-2/COX-1 ¼ 4.6 and 4.2 for 7a and 7b, respectively.
Compound 8b and 8c demonstrated approximately 2-fold more
affinity towards COX-1 over COX-2.
The intermediate compound 6 was used as a diversity precur-
sor to prepare
a .
number of target compounds’ series7–11
Compound 6 was reacted with certain aldehydes to give the cor-
responding arylidene hydrazide 7a–c which were subsequently
reacted with NaNO2/HCl to afford the pyrido[30,20:4,5]thieno[3,2-
d][1,2,3]triazin-4(3H)-one 8a–c44. In addition, condensation of com-
pound 6, with ethyl acetoacetate, acetylacetone or carbon disul-
phide to afford the derivatives 9a–c respectively. Finally, the
reaction of 6, separately with HCOOH and acetic anhydride
afforded the pyridothienodiazine analogues 10a and 10b, respect-
ively (Scheme 3).
Biological screening
In vivo anti-inflammatory assay
The obtained compounds were tested for their anti-inflammatory
activity using the carrageenan-induced rat paw oedema assay.
Compound potency and duration of action were compared with
those of the reference compound ibuprofen. The in vivo anti-
inflammatory activity is shown in Figure 3.
Compounds 7a, 7b, 8b, and 8c induced a satisfactory anti-
inflammatory activity, exceeding that of ibuprofen at the 4 h time
interval. The compounds produced 73.5, 73.2, 78.2, and 79.4%
inhibition respectively, compared to 69.5% inhibition for ibupro-
fen. The time–activity profiles of these compounds are commen-
surate to that of ibuprofen, varying only in the onset of action.
The lack of proportionality between in vitro and in vivo read-
outs can be attributed to several common factors, mainly phar-
macokinetic parameters (absorption, distribution, metabolism,
and excretion) that account for discriminations in drug
Compounds of series
7 showed a
more rapid onset than efficacies46.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
761
Scheme 2. Conversion of 3b to thienopyridine derivatives (a ¼ NaOEt/EtOH, b ¼ NH2NH2).
Scheme 3. Various reactions of compound 6 to afford the target compound series 7–10 (a ¼ RCHO, b ¼ NaNO2, HCl, c ¼ ethyl acetoacetate, d ¼ acetylacetone, e ¼ CS2,
f ¼ HCOOH or Ac2O).
Figure 3. In vivo anti-inflammatory assay of thieno[2,3-b]pyridine derivatives. The anti-inflammatory potency is proportional to the height of the bar (inhibition %). The
height of the bars (inhibition%) ¼ (swell.drug/swell.control)100. Swell ¼ mean difference in rat paw volume between right and left paw. RF ¼ ibuprofen (reference
drug). For more details, refer Supplementary Table S1.
A structure-based analysis by docking selected compounds in org) were downloaded and employed in the study, accession
the active site of 5-LOX, COX-2, and COX-1 was conducted to pro- codes: 3O8Y for 5-LOX47, 4PH9 for COX-248 and 1EQG for COX-138.
pose an understanding of the enzyme inhibition assay results. All the molecular modelling work was carried out using SYBLYL-X
Crystal structures from the Protein Databank website (www.rcsb. v.2.1 package licenced to King Abdulaziz University according to

762
M. S. MOHAMMED ET AL.
Table 1. Enzyme inhibition assay results for COX-1, COX-2, and 5-LOX along with per cent inflammation
reduction at the 4 h time interval in the rat paw oedema assay.
Compound
5-LOX IC50 (mM)
COX-2 IC50 (mM)
COX-1 IC50 (mM)
inflammation reduction % (4h)
7a
7b
8b
8c
0.154
0.279
13.520
5.419
0.436
9.988
7.524
35.990
18.992
43.628
45.869
31.797
19.902
7.663
73.54
73.02
78.23
79.36
69.52
Ibuprofen
31.945
All standard error of means (SEM) are lower than 10%.
methodologies and protocols described in the “Experimental” sec- generally able to maintain the major polar and non-polar interac-
tion and detailed in Supplementary Material. tions with the active site residues. However, 8b approached Ile-
Among all crystal structures of 5-LOX, only three entries belong 415 and Leu-368 side chains inconveniently, thus making possible
to H. sapiens 5-LOX (Entries: 3O8Y, 3V98, and 3V99). All of these clashes with the active site in this docking mode (Supplementary
crystal structures are mutated by replacing three lysines (K653KK635
)
Material). To be accommodated in the active site, significant shifts
into Glutamic acid–Aspargine–Leucine (ENL). The latter two entries in the primary backbone protein loops have to be made after the
had additional mutation S663D which resulted in the loss of 5-LOX MD step. Therefore, it should be challenging to predict the actual
activity (converted to 15-LOX-like activity)49. Therefore, we settled binding mode of this compound in this less flexible closed
with 3O8Y as our target for docking experiments.
active site.
According to this model, it is noted that the 4,6-dimethylth-
The active site of 5-LOX is framed by two a-helices, the a2-helix
and the arched helix, forming an elongated U-shaped hydrophobic ieno[2,3-b]pyridine scaffold has a good physicochemical balance in
cavity that accommodates the substrate AA, a 20-carbon polyun- the molecular properties as it rests in a dominantly lipophilic
saturated fatty acid50. There is a catalytic iron nearby the active pocket. The two methyl groups bolstered the aromatic ring hydro-
site but access to this iron is regulated by the side chains of Phe- phobic attractions with Leu-368 and Leu-373 side chains (residues
177 and Tyr-181 (the FY cork), which also caps the cavity once the of a2 helix, Figure 4) while the pyridine nucleus conferred a much
substrate enters the active site47. The defined conformation of this needed polar character to our compounds.
active site helped in understanding the clearly higher 5-LOX
inhibitory activities of 7a and 7b (IC50 are at nanomolar level) over virtual screening model using Surflex docking application
the cyclised analogues 8b and 8c (IC50 are at micromolar level).
embedded in SYBYL-X package and the Surflex docking scores
Lead compounds’ affinity towards COX-2 were investigated in a
To rationalise the higher activity of 7a and 7b against 5-LOX, a were compared51. For this purpose, we initially chose a crystal
study of different 7a conformers was carried out. The anti-con- structure of COX-2 containing ibuprofen (Code: 4PH9), the refer-
former is the calculated global minimum and it was found to be ence drug for both in vivo and in vitro screenings. Our analysis
3.8 kcal/mol less energy than the syn-conformer. This anti-con- suggests possible binding modes of the tricyclic analogue 8c rea-
former can make an intramolecular hydrogen bonding between sonably. The active site of COX-2 is composed of four main
the 3-amino and the carbonyl oxygen, more efficiently than that regions according to the crystal structures; the main hydrophobic
of the syn-conformer (Supplementary Material). It is therefore pro- channel, the polar mouth area (mainly side chains of Arg-120 and
posed that the anti-conformer of 7a attains a higher population.
Tyr-355), the selectivity side pocket and another smaller hydropho-
Interestingly, the pyrido[3',2':4,5]thieno[3,2-d][1,2,3]triazin-4(3H)- bic pocket near the mouth area52. Therefore, in a previous model,
one derivatives8 attain a global minimum conformation that well we reported that a pyridylurea derivative of naproxen binds pos-
overlaps with the syn-conformation of 7a. For docking experi- sibly by inserting the naphthalene moiety properly deep in the
ments into 5-LOX crystal structure, we used visually guided man- main channel. Meanwhile, the urea group makes several hydrogen
ual docking followed by molecular dynamics (MD), since the active bonding with the polar area near the active site mouth28. In our
site contained no ligand to guide the automatic docking process present work, compound 8c was predicted to insert the bulky
by Surflex51. All three conformers (anti-7a, syn-7a and 8c) could thieno[2,3-b]pyridine moiety into the hydrophobic channel and
rest inside the active site of the human 5-LOX in a hydrophobic make hydrophobic contacts with side chains of Val-344, Tyr-348,
tunnel between the a-2 helix and the arched helix considering the Val-349, Phe-385, Trp-387, Leu-531, and Leu-534 (Figure 5). The
least steric clashes with surrounding residues (Figure 4). Moreover, triazine ring and imine group form a network of hydrogen bond-
according to the proposed model, our compounds established ing with the guanidine side chains of Arg-120 and the phenol
several hydrophobic contacts within convenient van der Waals dis- group of Tyr-355 (similar to ibuprofen’s carboxylate interactions).
tances with hydrophobic residues lining the active site. The anti- The terminal aryl group of Schiff base was directed to the outside
conformer of the most active compound 7a made attractions with of the protein surface possibly because this tricyclic ligand does
side chains of Leu-373, Leu-368, Ile-406, Leu-414, and Leu-607. In not enjoy much flexibility.
addition, anti-7a is theorised to form a crucial hydrogen bonding
A limitation of this model is that it was not successful in
interaction through the hydrazide NH to Asn-407, an important describing the good binding of 7a, from our perspective. The top-
residue of the arched helix that contributes to the shape of the scoring pose (Table 2) of this bicyclic compound, being more flex-
active site. Surprisingly, the pyridine nitrogen of within the ible than 8c, was placed by Surflex as an syn-conformer while
thieno[2,3-b]pyridine nucleus formed what is possibly a very directing its thieno[2,3-b]pyridine moiety towards the hydrophobic
important ionic interaction with Glu-376 side chain carboxylate channel. It also forced the terminal phenyl group of the Schiff
with all three docked structures (anti-, syn-7a as well as 8a).
base to an eclipsed torsion that penetrated the characteristic side
Compound 8b was approximately 90 times less potent than 7a pocket of COX-2. We had some concerns about this suggested
in inhibiting 5-LOX in enzyme inhibition assays. This can be model of 7a because it was predicted to be half the potency of
explained using the structure-based analysis of an affinity model 8c according to Surflex scores (Table 2). In addition, the docking
with 5-LOX active site. As mentioned above, both 8b and 8c conformation, though filled the side pocket, did not make any sig-
resemble the syn-conformation of 7a. These entities were nificant polar interactions (cf. 8c) with the active site polar region.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
763
Figure 4. (A) Compound 7a (coloured green) docked into the active site of 5-LOX. The a2 helix is seen to the left and the arched helix is located to the right (both
coloured magenta). The hydrophobic residues interacting with the ligand Leu-368, Leu-373 (of a2 helix), Ile-406, and Leu-414 (of arched helix) are shown. The FY cork
residues Phe-177 and Tyr-181 are seen to the top of the image. The graphics of this image was generated using Pymol free software (https://pymol.org/2/). (B) 2D rep-
resentation of the interactions of 7a with the active site of 5-LOX generated using Maestro visualiser.
This limitation highlights that this model is merely a speculative 3OLU) that contained arachidonic acid glyceryl ester as a co-crys-
trial to explain the enzyme inhibition activities of the tallised ligand of larger size53. The larger ligand gave rise to a sig-
reported compounds.
nificantly longer but narrower protomol in the main channel
We suspected that the protomol formation did not help the pocket (Supplementary Material). The docking experiments of the
model prediction efficiency because the process is known to be four compounds to the active site returned different predictions of
affected by the co-crystallised ligand’s size and shape51. In add- binding modes (Figure 6(B)). The Schiff base group was inserted
ition, the main channel of the active site of this crystal structure is inside the main hydrophobic channel (reverse mode to the previ-
seemingly shrunk tightly around the small isobutyl tail of ibupro- ous model on 4PH9). Both ligands (7a and 8c) made hydrogen
fen, the co-crystallised ligand. Therefore, we decided to perform bonding with Ser-530 side chain. It is worthy to mention that Ser-
the modelling work on another COX-2 crystal structure (PDB Code: 530 is a residue involved in the catalytic peroxidation of AA

764
M. S. MOHAMMED ET AL.
Figure 5. Docking of 7a and 8b to COX-2 crystal structures. (A) 2D representation of the binding modes of compounds 7a (left) and compound 8c (right) with the
active site of COX-2 generated using Maestro visualiser. (B) Model of docking of 7a (violet) and 8c (Orange) on COX-2 crystal structure (Code: 3OLU). The active site
pocket is coloured green as determined by Surflex’s protomol except the side pocket, which was given an orange surface. Surface of the active site’s main hydrophobic
channel residues are coloured coffee-brown. Ser-530 is given a blue surface.
Table 2. Surflex total scores of docked com-
pounds into COX-2 (Code 4PH9 and 3OLU) and
COX-1 (1EQG).
conformation (ꢄ17 kcal/mol lower than that of the first docking
model). The bulkier thieno[2,3-b]pyridine moiety was oriented
towards the outside of the active site with shallow penetration of
the selectivity pocket. Based on these assumptions, the model also
Surflex docking score
Compound
4PH9
3OLU
1EQG
helped explain the slight selectivity of 7a to COX-2 over COX-1
(Will be discussed later in further details). In our analysis, this
model had improved predictions but it is not free from concerns.
The much lipophilic dimethyl pyridyl ends are projecting outside
the active site to the aqueous medium. In addition, the impact of
the substituents on the Schiff base phenyl groups on the experi-
mental activities are not supported by this model. Finally, the cal-
culated scores in this model still rank 8b and 8c higher than 7a
and 7b but the differences in the total Surflex scores are not as
drastic as the first model (for the 4PH9 structure) (Table 2). Since
the experimental differences between the ligands are not drastic
Ibu
8d
8b
7a
7.5
7.7
5.7
4.0
2.3
6.8
7.2
6.3
5.7
6.2
6.5
4.6
3.9
1.3
1.2
7b
Surflex docking score ¼ Calculated –log Kd
substrate, forming a hydrogen bond with the carboxylic group of
diclofenac, and being acetylated by acetylsalicylic acid as proven
by structural biology reports54. Compound 7a in this model
existed in the previously predicted more probable anti- either, the model might be considered qualitatively aligned with

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
765
Figure 6. (A) 8c (violet) and 7a (magenta) in COX-1 active site as determined by Surflex Graphics of this image was generated using Pymol free software (https://
pymol.org/2/). (B) 2D diagram showing the putative model of binding mode of compound 8c with COX-1 active site (PDB Code: 1EQG).
the experimental results to provide a possible explanation of the hydrophobic bottom of the main channel and was adjusted to
relative compounds’ activities. We expect that the scoring, as well form a hydrogen bond with Ser-530. The terminal triazine ring
as the binding modes, may vary if we use different crystal struc- formed a bifurcate hydrogen bonding with Tyr-355. The rigid
ture entries among the many available in the PDB website.
nature of 8c added convincing evidence for a high probability of
Modelling the COX-1 inhibition was performed on a crystal its postulated binding mode. Meanwhile, Surflex placed the flex-
structure that also contained ibuprofen as a ligand (PDB Code: ible Schiff base end of 7a to the bottom of the active site and
1EQG)38. It is established that there are several common features twisted the imine side chain to fill this pocket. This way, the anti-
between COX-1 and COX-2 active sites, the main hydrophobic conformer of the ligand was totally immersed inside the active
channel, the catalytic Ser-530 and the mouth having polar residues site assuring that the dimethylpyridyl end is exposed to a hydro-
such as Arg-120. However, COX-1 is characterised by the absence phobic microenvironment. No effective polar contacts were
of the side pocket and by a narrower main hydrophobic channel55. observed for the ligand (Figure 6). We checked if the linear global
In docking experiments, Surflex successfully granted 8c the high- energy minimum would give a better binding quality by a manual
est scoring on COX-1, which aligns with experimental results docking experiment but found serious steric clash with Val-518
(Tables 1 and 2). The dimethyl pyridine moiety reached the (Supplementary Material).

766
M. S. MOHAMMED ET AL.
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and improved our understanding of the experimental results
because it reasonably aligned these calculations with experimental
results within a plausible margin of deviation.
ꢀ
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Acknowledgment
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Disclosure statement
Authors declare no conflict of interest in this work with any poten-
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This work was partially supported by two grants from Helwan
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second grant was given to the Department of Pharmaceutical
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