Medicinal chemistry
An- Najah National University
By
Anwar Khanfa
Taqwa Kabaha
Sara Diab
Instructor
Mohyeddin Assali

1,4-Diaryl-substituted triazoles
as cyclooxygenase-2 inhibitor
: synthesis, biological evaluation and
molecular modeling studies

Introduction
• Over the last 100 years, nonsteroidal anti-inflammatory drugs (NSAIDs)
were among the most prominent and frequently used drugs in medicine
for the treatment of pain, fever, inflammation and arthritis-associated
disorders.
• The main therapeutic mechanism of NSAIDs action involves inhibition of
cyclooxygenase-mediated biosynthesis of prostaglandins (PGs).
• Cyclooxygenase (prostaglandin endoperoxide synthase or COX) enzyme
exists in at least two closely related isoforms1–4 viz. COX-1 and COX-
2. COX-1 is consecutively expressed in most tissues.
• COX-1 is involved in homeostasis of cytoprotective PGs in the
gastrointestinal tract, homeostasis of proaggregatory thromboxane A2
(TXA2) in blood platelets and in overall vascular homeostasis.

• In contrast, inducible COX-2 isoform is absent in most organs and
tissues, except for brain, kidney, pancreas, uterus, ovary, and
macrophages.
• However, upon inflammatory or carcinogenic stimulation, COX-2
expression levels are elevated, and COX-2 is responsible for the
production of inflammatory PGs.
• Moreover, high levels of COX-2 were also found in various human
cancers like gastric, breast, lung, colon, oesophageal, prostate, and
hepatocellular carcinomas.
• Classical NSAIDs (aspirin, ibuprofen, meclofenamic acid and
indomethacin) block the activity of both COX isoforms non-selectively
to exhibit their anti-inflammatory effects, which leads to unwanted side
effects by the inhibition of COX-1

• Inhibition of consecutively expressed COX-1 isoform and/or downfall in
the production of physiological PGs by classical NSAIDs is considered
as the basis of life-threatening side effects such as gastrointestinal
irritation, ulcer, hepatic and renal toxicity.
• In this context, selective inhibition of COX-2 was proposed to be a safer
therapeutical approach to reduce deleterious side effects of classical
NSAIDs.
• As a result, a variety of selective COX-2 inhibitors (COXIBs) such as
celecoxib (1), rofecoxib (2), valdecoxib (3), etoricoxib (4) and lumiracoxib
(5) have been developed (fig1)

• Selective COX-2 inhibitors have widely been used for the
treatment of inflammation and a variety of diseases such as
rheumatoid arthritis, osteoarthritis, Alzheimer’s disease (AD) and
Parkinson’s disease.
• Besides these therapeutic applications, NSAIDs have also been
found to play an important role in the prevention of several types
of human cancer such as colon, lung, breast and prostate
cancers.
• Over the last two decades, numerous studies have been
carried out to improve the COX-2 selectivity and safety profile.

• However, various selective COX-2 inhibitors came under scrutiny
due to an increased risk of adverse cardiovascular side effects.
• This development led to the withdrawal of rofecoxib and
valdecoxib from the market and stimulated the development of
novel classes of selective and safe COX-2 inhibitors with reduced
cardiovascular side effects.
• In this line, several nitric oxide releasing NSAIDs and dual
COX/LOX inhibitors have been developed recently.

• Among the different classes of COXIBs, several vicinal diaryl
heterocycles bearing a p-methylsulfonyl- or p-aminosulfonyl-aryl
substituent rings represent the most important class of selective COX-
2 inhibitors.
• Structure–activity relationship (SAR) data indicate that the nature and
position of substituents on two vicinal aryl rings is an important
determinant.
• It has been observed that compounds containing RSO2 (R = NH2, CH3,)
groups effectively interact with amino acid residues of the COX-2
secondary pocket resulting in a generally high COX-2 selectivity and
potency profile.
• Recently, our group has reported the synthesis of various 1,4 and 1,5-
diaryl substituted 1,2,3-triazoles (6–11, Fig. 1) as COX-2 inhibitors.
• .

• From this study it was concluded that compounds containing a vicinal
diaryl substitution pattern display higher COX-2 inhibition potency
compared to their corresponding 1,4- diaryl-substituted counterparts.
Both series of compounds showed moderate COX-2 selectivity profiles.
• As an extension to this work, we now describe the synthesis of a novel
class of 1,4 diaryl-substituted triazoles as potential selective COX-2
inhibitors.
• Novel compounds were designed based on Cu(I)-mediated click
chemistry between azides containing the SO2NH2 COX-2
pharmacophore attached to an aryl ring and various aryl acetylenes
containing different substitutents (H, F, Cl, CH3 or OCH3) attached to
the para position of the aryl ring

• . Furthermore, the size of the molecule was increased through the
introduction of an alkyl linker to enhance flexibility of the compound to
facilitate COX-2 binding. The COX-1/COX-2 inhibitory activities of
these compounds were determined in a COX binding assay, and
plausible binding interactions within the COX-1/COX-2 active sites
were explored using molecular docking studies

Chemistry
There is three categories of compounds which is 1,4
-
elozairt detutitsbuslyraid
:
1. Category 14
_
18
2. Category 21
_
25
3. Category 28
_
32
They are all di substituted triazole but they have a linker
(n= 0,1,2
HC,lC,F,H=R dna (
3
HCO,
3
All contain triazole ring as the name implies and two aromatic
rings.

General structure

Point of view
To discuss the effect of chain length, size and flexibility of the
molecule on the COX- 1
XOC/
-
2 inhibitory potency and selectivity
(SI).
The chemical approach used here is popular click chemistry Cu+
mediated [ 2+3
ediza citamora eerht fo noitcaer noitiddaolcyc ]
) erutcurts enelyteca lyra detutitsbus suoirav htiw
4
-
, H
4
-
,F
4
-
,lC
4
-
HC
3,4
-
HCO
3
ekam ot (
1,4
-
elozairt detutitsbus lyraid
.
Equimolar amount are used in precence of ETOH/H2O ,Cu+and
triethylamine at 60
°
rof C
14
_
16 hour.
Moderate to good chemical yeild 54
_
89% after purification.

Results and Discussion

• The COX-1/2 inhibitory potency and selectivity profile of
compounds 14
–
18
,
21
–
25
dna ,
28
–
32 was determined in
triplicate using a fluorescent COX- 1
XOC/
-
2 inhibitor screening
assay kit.
• In vitro study showed that all classes exhibit potency on COX-
2 over COX-1.
• Range of IC50 of COX-2 = 0.17-28 Mm
• Range of IC50 of COX-1 = 21 to >100 mM

• Synthesis of compounds 14, 15, 17 and 18 as
potential inhibitors of carbonic anhydrases VA and
VB was reported recently.

Category I (14-18)
N=0 R=H,F,Cl,CH3,OCH3
Para-phenyl sulfonamide attached directly to
N-1 of the triazole ring.

• N= 0, R=H
• Most potent and selective cox-2 inhibitor.
• COX-1 IC50 = >100mM
• COX-2 IC50 = 1.3mM
• SI= >77

• Has satisfactory cox-2 inhibitor potency but
less selectivity.
• COX-2 IC50 = 1.3mM.
• SI= 16.1

Category II 25
-
21
( )
N=1, R=H,F,Cl,CH3,OCH3

• MOST POTENT and SELECTIVE inhibitor of COX-2 .
• COX-1 IC50 = > 100mM.
• COX-2 IC50 = 1.2mM.
• SI = 83

• displayed comparable COX-2 inhibitory potency but COX-2
selectivity index of compound 22 was smaller than that of
compound 24.
• COX-2 IC50 = 1.5mM.
• SI = 13.3

Category III )
32
-
28
(
• N=2, R=H,F,Cl,CH3,OCH3
Compound 30 the most potent cox-2
inhibitor.
• COX-2 IC50 = 0.17mM.
•SI = >588

• COX-1 IC50 = >100mM.
• COX-2 IC50 = 1.1mM.
• SI = 91

• COX-1 IC50 = >100mM.
• COX-2 IC50 = 1mM.
Compounds 28,29 have low potency toward cox-
2 but high cox-2 selectivity.

What can be used as lead
compound ?
The most effective compound is 30 because it
display high potency and selectivity.
So it can be a lead compound.

Molecular modeling(docking)
studies
Structure of COX1 & COX2 enzyme :

Molecular modeling (docking) studies were carried out for compounds
(16, 23, 30) to gain more insights into the probable nature of their
respective binding interactions within the active site of COX-1 and COX-2
isozymes. Docking experiments were performed using X-ray crystal
structure data for COX-1 and COX-2 obtained from the protein data bank.

Compound 16, when docked into the COX-2 binding site , comfortably
enters into the COX-2 active site and places its para-Cl-phenyl
substituent near S530, L531residues, while one of the nitrogen atom of the
triazole ring indicates hydrogen bonding interactions with V523 residue of
COX-2 active site (NO = 1.55 Å). The SO2NH2 group as prominent and
effective COX-2 pharmacophore) is pointed near the COX-2 isozyme
secondary pocket amino acid residues (A516, F518, R513, Q192 and H90).

In a comparable manner, compound 23 displays a favorable orientation in
the COX-2 active site and the SO2NH2 group is oriented at the entrance
of the secondary pocket. This places the SO2NH2 group near the R513,
L352 residues. The 4-chlorophenyl substituent was moved near to the
L359 amino acid of the COX-2 binding site.

A similar molecular docking simulation for compound 30 within the COX-2
active site, showed noticeable interactions between compound 30 and active
binding site residues .The SO2NH2 group in compound 30 deeply enters into
the secondary pocket region of the COX-2 active site, where it is oriented
towards Q192, R513, H90, and S353 residues. The nitrogen atom of the
SO2NH2 group indicates hydrogen bonding interactions with the oxygen of
carbonyl group of Q192 ,and one of the oxygen atom of SO2NH2 group is
hydrogen bonded to the nitrogen atom of H90 residue.

However, in contrast to binding interactions of compound 16
and 23, the docking results of compound 30 indicate that upon
increasing the size of the linker chain (n = 2), there is an
increase in the flexibility of the molecule which facilitates the
deeper insertion of the SO2NH2 group into the secondary binding
pocket and more significant intermolecular binding interactions.
This favorable orientation and electrostatic binding interactions of
compound 30 with COX-2 active site residues are consistent
with experimentally observed high COX-2 inhibitory potency
(COX-2 IC50 = 0.17 lM). A comparison of the binding mode of
compounds 16, 23 and 30 in the COX-2 active site using a
molecular docking structure containing all three compounds (16,
23 and 30) docked in the same COX-2 active site is given in the
Supplementary section.

Alternatively, docking results for compound 16, 23 and 30 in
COX-1 active site show that compound 16 comfortably enters
into the COX-1 active site and its p-Cl-phenyl substituent has
moved into the hydrophobic subpocket of COX-1isozyme
constituted by W387, Y385 and F381 residues.

However, during docking studies of compound 23 and 30 into
COX-1, compounds were not able to enter into the COX-1 active
site completely .This inability is likely due to the increase in their
size or/and due to the steric conflict with side chains of amino acid
residues of the active site. This body of data evidently supports the
experimentally observed weak COX-1inhibitory activity of
compound 23 and 30.
23 30

Conclusions
In conclusion, a new class of 1,4-diaryl-substituted triazoles was
synthesized using high yielding Cu(I)-mediated click chemistry. In vitro
COX-1/COX-2 inhibition studies showed that some compounds of this
novel class of 1,4-diaryl-substituted triazoles are able to inhibit COX-2
with high otency and selectivity. Especially compounds (28–30) were
identified as highly potent (IC50 range 0.17–1.1Mm) and selective novel
COX-2 inhibitors (SI >91 to >588). Among all investigated compounds, a
‘lead compound’ 4-{2-[4-(4-Chloro-phenyl)-[1,2,3]triazol-1-yl]-ethyl}-
benzenesulfon-amide was identified that exhibited appreciable COX-2
inhibitory potency and selectivity (COX-1 IC50 >100 lM, COX-2 IC50 =
0.17 lM; SI >588).

Molecular docking analyses for compounds 16, 23 and 30 on the
COX-1/COX-2 isozymes complement the experimental results. From
both the experimental structure– activity data and the molecular
docking results it can be concluded that an increase of the length of
the linker chain is accompanied with an enhancement in molecular
interactions between functional groups of the compounds and amino
acid residues of the secondary pocket of the COX-2 active site.

The End