ARTICLE
In situ click chemistry generation of
cyclooxygenase-2 inhibitors
Atul Bhardwaj
1,2
, Jatinder Kaur
1,2
, Melinda Wuest
1
& Frank Wuest
1,2
Cyclooxygenase-2 isozyme is a promising anti-inflammatory drug target, and overexpression
of this enzyme is also associated with several cancers and neurodegenerative diseases. The
amino-acid sequence and structural similarity between inducible cyclooxygenase-2 and
housekeeping cyclooxygenase-1 isoforms present a significant challenge to design selective
cyclooxygenase-2 inhibitors. Herein, we describe the use of the cyclooxygenase-2 active site
as a reaction vessel for the in situ generation of its own highly specific inhibitors.
Multi-component competitive-binding studies confirmed that the cyclooxygenase-2 isozyme
can judiciously select most appropriate chemical building blocks from a pool of chemicals to
build its own highly potent inhibitor. Herein, with the use of kinetic target-guided synthesis,
also termed as in situ click chemistry, we describe the discovery of two highly potent and
selective cyclooxygenase-2 isozyme inhibitors. The in vivo anti-inflammatory activity of these
two novel small molecules is significantly higher than that of widely used selective
cyclooxygenase-2 inhibitors.
DOI: 10.1038/s41467-016-0009-6
OPEN
1
Department of Oncology, University of Alberta, 11560 University Avenue, Edmonton, Alberta, Canada T6G 1Z2.
2
Faculty of Pharmacy and Pharmaceutical
Sciences University of Alberta, 8613-114 Street, Edmonton, Alberta, Canada T6G 2H7. Correspondence and requests for materials should be addressed to
F.W. (email: wuest@ualberta.ca)
NATURE COMMUNICATIONS
|
8: 1
|
DOI: 10.1038/s41467-016-0009-6
|
www.nature.com/naturecom munications 1
C
yclooxygenase (COX) enzymes catalyze the metabolic
conversion of arachidonic acid to prostanoids including
prostaglandins (PGs), prostacyclin, and thromboxane,
which play important roles in human physiology and various
pathological conditions
1–4
. Despite several known side effects like
myocardial infarction and atherothrombotic events, drugs aimed
at COXs inhibition is a billion dollar industry, inspiring scientists
to search constantly for novel COX inhibitors. COX exists in
three isoforms: cyclooxygenase-1, 2, and 3 (COX-1, COX-2, and
COX-3)
5–7
. COX-1 and COX-2 isoforms are of primary interest,
as they are involved in physiological as well as pathological
processes. COX-1 is a constitutively expressed house-keeping
isozyme responsible for the basal production of essential PGs
8
.
These PGs mediates homoeostatic functions in the gastro-
intestinal and cardiovascular system. COX-3 (a splice variant of
COX-1) is expressed only in specific parts of the brain and spinal
cord and its exact functions are still unclear
9
. In contrast, COX-2
isozyme is expressed at very low levels under normal conditions.
However, COX-2 expression is rapidly upregulated in the
immediate response to diverse pro-inflammatory and pathogenic
stimuli. There is accumulating evidence for the critical
involvement of COX-2 in various pathologies that include
inflammation
10,11
, cancer
12–14
, neurodegenerative diseases
15
and
multidrug resistance
16
. Therefore, beyond their traditional use as
anti-inflammatory agents, COX-2 inhibitors have recently been
used for molecular imaging
17–19
and therapy
20–22
of cancer.
Hence, the development of selective COX-2 inhibitors as anti-
inflammatory and anti-tumor drugs is a major direction in
academic research and pharmaceutical industry
23–25
. Traditional
nonsteroidal anti-inflammatory drugs (NSAIDs) (aspirin,
ibuprofen, naproxen) inhibit both COX-1 and COX-2 isoforms;
and their use is limited due to associated ulcerogenic and
gastrointestinal side effects. Discovered in the late 1990’s, COX-2
selective inhibitors (the COXIBs: celecoxib, rofecoxib) are dia-
rylheterocycles possessing a SO
2
NH
2
or SO
2
Me group as COX-2
pharmacophore, which exert similar anti-inflammatory and
antipyretic properties as traditional NSAIDs but are devoid of
gastrointestinal toxicity
4
. However, COXIBs are also under
scrutiny since several studies have demonstrated that chronic use
of COXIBs can elevate the risk of myocardial infarction and
other thrombotic events by stalling the biosynthesis of anti-
aggregatory prostacyclin (PGI
2
) while leaving the biosynthesis of
pro-thrombotic thromboxane A
2
(TxA
2
) unaffected
26–29
.Asa
result, COXIBs such as rofecoxib and valdecoxib were withdrawn
from the market thereby leaving a demand for the synthesis and
screening of novel COX-2 inhibitors.
Development of compounds that selectively inhibit COX-2
over COX-1 is a substantial challenge as both isoforms share
similar cellular expression locations, molecular weight, and
amino-acid composition. In addition, the both isoforms share
more than 60% sequence homology and their three-dimensional
structures are almost superimposable. However, the key differ-
ence between the COX-1 and COX-2 isozyme active site is the
exchange of isoleucine in COX-1 for valine in COX-2 at positions
434 and 523. The difference in the amino-acid sequence make the
COX-2 substrate-binding site more flexible and approximately
25% larger by creating a distinct secondary-binding pocket
3,30
.
Many COX-2 selective inhibitors explicitly bind to this
secondary-binding pocket (lined by H90, R513, and V523)
resulting in the specific inhibition of COX-2 activity. Another
important region in the COX-2 active site is the hydrophobic
pocket (lined by W387, Y385, F518, F381, L352), and a recent
mutational study described the involvement of hydrophobic
pocket residues in the proper positioning of fatty acid substrates
for oxygenation
31
. Therefore, highly potent and selective COX-2
inhibitors should possess a pharmacophore which can selectively
bind in the secondary pocket and deliver sufficient steric bulk to
block the hydrophobic channel of COX-2.
Here, we deviated from conventional drug discovery approa-
ches involving the laborious synthesis and screening of a range of
compounds, and envisioned to explore the utility of in situ click
chemistry for the discovery of specific and high-affinity COX-2
inhibitors. Click chemistry
32,33
, including 1,3-dipolar cycloaddi-
tion between alkyne and azide (Huisgen cycloaddition), have
attracted much attention because of their remarkable efficiency,
simplicity, and its ability to be employed for the synthesis of a
wide range of compounds such as molecular imaging agents
34,35
and drugs
36
, protein modification
37,38
, DNA and RNA target-
ing
39,40
, and glycan imaging
41,42
. Considering its versatility, click
chemistry has found numerous synthesis applications not only
performed in traditional reaction vessels, but also in living
systems. Kinetic target-guided synthesis (TGS), also termed as
in situ click chemistry is an innovative synthesis process where a
biological target assembles its own inhibitor through target-
guided selection of appropriate building blocks
43,44
. TGS was
elegantly used for the preparation of high affinity inhibitors of
enzymes like acetylcholine esterase
45–47
, HIV protease
48
, bovine
carbonic anhydrase II
49,50
, protein tyrosine phosphatases
51
,
metalloproteases
52,53
, nicotinic acetylcholine receptors
54
, and
chitinase
55
.
Herein, we demonstrate the use of the COX-2-binding site as a
reaction vessel for generating its own highly potent and selective
inhibitors.We designed and synthesized a range of pyrazole-based
azide building blocks (5, 14, 27, and 31) and a collection of
corresponding triazole-containing biheterocyclic compounds
(7–12, 16–25, 28, 29, 32, and 33). After screening for their
COX-1/COX-2 inhibitory potency, various 5-azido-pyrazoles
(5, 14, 27, and 31) and aryl acetylenes as click chemistry build-
ing blocks were incubated in pairs with human recombinant
COX-2 isozyme to test the capability of COX-2 for assembling its
own highly potent inhibitors. Identification of compounds 18 and
21 as highly potent and selective COX-2 inhibitors demonstrated
the feasibility of the in situ click chemistry approach. Compounds
18 and 21 displayed a superior in vivo anti-inflammatory activity
profile compare to clinically used anti-inflammatory drugs.
Multi-component competitive-binding studies confirmed that
COX-2 can selectively group most appropriate building blocks
from a pool of compounds to construct highly potent COX-2
inhibitors. The thermodynamic-binding signatures calculated
from isothermal titration calorimetry (ITC) confirmed that
binding of click chemistry building block 5-azido-pyrazole (14)to
COX-2 involves a favorable change in free energy (ΔG = −36.20
kJ mol
−1
), which was mainly based on H-bonding and van der
Waals interactions. Moreover, comprehensive computational
analysis including structure activity relationship (SAR) and
molecular docking indicated that the size and type of the COX-2
pharmacophore, and the orientation of the clickable building
blocks inside the binding site of the target protein collectively
contribute to the in situ construction of highly potent and
selective COX-2 inhibitor.
Results
Design of clickable building blocks. Based on the structural
features of the COX-2 active site, we concluded that suitable
clickable building blocks should meet two criteria: (i) at least one
of the building blocks (in our case, the azide component) should
possess a SO
2
Me COX-2 pharmacophore to facilitate its tight
binding into the secondary-binding pocket of the COX-2
isozyme; (ii) the azide component should have a proper size
and orientation that will not interfere with the entry of the alkyne
component into the COX-2 active site, allowing the in situ click
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-016-0009-6
2 NATURE COMMUNICATIONS
|
8: 1
|
DOI: 10.1038/s41467-016-0009-6
|
www.nature.com/naturecom munications
chemistry formation of a potent and selective inhibitor large
enough to block the hydrophobic channel of the COX-2 isozyme
(Fig.
1).
Hence, to optimize these parameters we synthesized a range of
5-azido-pyrazole derivatives with variable sizes containing a
COX-2 pharmacophore (SO
2
CH
3
) and evaluated the influence of
the chemical structure on the success of in situ click chemistry
reaction. We selected a pyrazole motif as the central scaffold for
the azide building blocks, as pyrazole-containing compounds are
quite prevalent in many anti-inflammatory drugs. The design of
click chemistry building blocks and target compounds is
illustrated in Fig.
1, where the central pyrazole motif was adapted
from approved drug celecoxib (1). The SO
2
CH
3
COX-2
pharmacophore is located at the para position of one of the
phenyl ring which was introduced to facilitate binding of 5-azido-
pyrazole (14) into the COX-2 secondary-binding pocket.
Extensive SAR data is available in the literature, which describes
the importance of SO
2
CH
3
and SO
2
NH
2
groups as COX-2
pharmacophores for selective binding to the COX-2 isozyme.
However, SO
2
NH
2
groups are also found in many drugs
inhibiting members of the carbonic anhydrase family
56
.
Substitution pattern on the alkyne building blocks selected for
this study was based on structures frequently present in various
NSAIDs.
Chemical synthesis of target compounds. The synthetic meth-
odologies used to prepare 5-azido-pyrazoles (5, 14, 27, 31) are
illustrated in Fig.
2. Briefly, precursor compounds 2,5-diphenyl-
2H-pyrazol-3-ylamine (4) and 2-(4-methane-sulfonyl-phenyl)-
5-methyl-2H-pyrazol-3-ylamine (13) were synthesized in high
yields according to known procedures involving the reaction of 3-
amino-3-phenyl-acrylonitrile (3) or 3-amino-but-2-ene-nitrile
with the appropriate arylhydrazine. Subsequently, compounds 4
and 13 were converted into corresponding 5-azido-pyrazoles
(5, 14) by diazotization and subsequent treatment with sodium
azide. 5-azido-pyrazoles (5, 14) were reacted with various alkynes
(6a–6f, 15a–15e) using standard Cu(I)-catalyzed azide-alkyne
cycloaddition (CuAAC) reaction conditions. Respective triazole
products (7–12, 16–25) were isolated in high yields (Fig.
2). To
illustrate the role of SO
2
CH
3
COX-2 pharmacophore in selective
COX-2 inhibition, we synthesized compounds 28, 29, 32, and 33
according to synthetic methods shown in Fig.
2.
In order to investigate the COX-2 isozyme-mediated in situ
click chemistry reaction, 5-azido-pyrazole building blocks (5, 14,
27, and 31, 1 µl of 3 mM dimethyl sulfoxide (DMSO) solution)
and alkynes (6a–6f, 15a–15e, 1 µl of 20 mM DMSO solution)
were incubated as pairs in the presence of the human
recombinant COX-2 isozyme (95 µl COX-2) in 1 µlof1M
Tris-HCl, pH 8.0 for 24 h at room temperature. After 3, 6, 9, 12,
Y
F
3
C
H
3
C
H
3
C
H
3
C
H
3
C
N
N
N
N
N
N
3
N
3
N
3
N
3
N
3
N
N
N
N
N
N
N
7, R
1
= H
16, R
1
= H
17, R
1
= CI
18, R
1
= F
19, R
1
= OCH
3
20, R
1
= CH
3
21, R
1
= NH
2
22, R
1
= CF
3
29, R
1
= NH
2
28, R
1
= F
32, R
1
= F
33, R
1
= NH
2
8, R
1
= CI
9, R
1
= F
10, R
1
= OCH
3
11, R
1
= CH
3
12, R
1
= CH
2
CH
3
N
N
N
N
N
O
N
NN
H
3
C
CH
3
CH
3
CH
3
, NH
2
, CF
3
,
CH
3
CH
3
CH
3
,,
N
N
O
N
3
R
1
R
1
R
1
23, R
1 =
24, R
1 =
25, R
1 =
R
2
R
1
R
1
= H, CI, F, OCH
3
, CH
3
,
R
1
27
31
N
3
N
3
N
3
5
14
SO
2
NH
2
Celecoxib (1, Celebrex)
SO
2
CH
3
SO
2
CH
3
SO
2
CH
3
SO
2
CH
3
SO
2
CH
3
SO
2
CH
3
CH
3
O
O
2
Rofecoxib (2, Vioxx)
Selective COX-2 inhibitors
Designed clickable alkyne
building blocks
Synthesized target compounds
N
N
N
N
N
N
N
Adapted from
Celecoxib
N
N
N
N
N
N
N
N
N
N
N
N
N
Most of selective COX-2 inhibitors:
- are diaryheterocycles or carbocycles
- bear a methylsulfone or sulfonamide
group on one of the phenyl ring
(Y= SO
2
CH
3
/SO
2
NH
2
)
Y
Fig. 1 Design of building blocks for in situ click chemistry reaction in the COX-2 isozyme. Structure of selective COX-2 inhibitor celecoxib (1) and
rofecoxib (2), target compounds (7–12, 16–25, 28, 29, 32, and 33) and illustration of in situ click chemistry reaction principle inside the COX-2-binding
pocket
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-016-0009-6 ARTICLE
NATURE COMMUNICATIONS
|
8: 1
|
DOI: 10.1038/s41467-016-0009-6
|
www.nature.com/naturecom munications 3
15, 18, 21, and 24 h each sample was analyzed in triplicate by
injecting (10 µl) into the liquid chromatography–mass spectro-
metry (LC/MS) instrument with selected-ion-monitoring (SIM)
mode. In library 1, where 5-azido-1,3-diphenyl-1H-pyrazole (5)
was incubated in the presence with various alkynes (6a–6f, 15a,
and 15b Table 1, library 1), in situ click chemistry formation of
corresponding triazole compounds could not be detected. After
refinement of the structure of 5-azido-1,3-diphenyl-1H-pyrazole
(5,V
molecular
= 332.9 Å
3
) into a smaller sized 5-azido-1-(4-metha-
nesulfonyl-phenyl)-3-methyl-1H-pyrazole (14,V
molecular
= 326.1
Å
3
), where one of the phenyl ring present at C-5 position of
the pyrazole ring was replaced with a CH
3
group. In addition,
a COX-2 pharmacophore (SO
2
CH
3
) was incorporated at C-4
position of one of the phenyl rings.
LC/MS-SIM analysis of 11 different reactions (6a–6f, 15a–15e
Table 1, library 2) revealed two combinations where in situ click
CN
CH
3
CN
NH
2
NHNH
2
.HCI
NH
2
NaN
3
25 °C, 3 h
NaNO
2
TFA-H
2
O
N
N
N
N
N
N
N
N
N
N
3
R
1
R
1
(6a-6f)
7–12
6a, 7, R
1
= H
16, R
1
= H
17, R
1
= CI
18, R
1
= F
6b, 8, R
1
= CI
6c, 9, R
1
= F
6d, 10, R
1
= OCH
3
6f, 12, R
1
= CH
2
CH
3
6e, 11, R
1
= CH
3
19, R
1
= OCH
3
15a, 21, R
1
= NH
2
15b, 22, R
1
= CF
3
CH
3
H
3
C
H
3
C
H
3
C
H
3
C
H
3
C
H
3
C
NH
2
H
3
C
H
2
N
CH
3
CN
+
NHNH
2
.HCI
NHNH
2
.HCI
NHNH
2
.HCI
SO
2
CH
3
SO
2
CH
3
SO
2
CH
3
SO
2
CH
3
CH
3
SO
2
CH
3
SO
2
CH
3
SO
2
CH
3
SO
2
CH
3
SO
2
CH
3
13
14
15c-15e
6c, 15a
6c, 15a
NaN
3
, NaNO
2
TFA-H
2
O
CuSO
4
.5 H
2
O
Sodium ascorbate
EtOH: H
2
O, 3–5 h
CuSO
4
.5 H
2
O
Sodium ascorbate
EtOH: H
2
O, 3–5 h
CuSO
4
.
5 H
2
O
Sodium ascorbate
EtOH: H
2
O, 3–5 h
CuSO
4
.5 H
2
O
Sodium ascorbate
EtOH: H
2
O, 3–5 h
23–25
16–22
(6a-6e, 15a, 15b)
CH
3
15c, 23, R
1
=
15d, 24, R
1
=
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
HCI, 110 °C
30 min
HCI, 110 °C,
20 min
HCI, 110 °C
30 min
N
O
15e, 25, R
1
=
20, R
1
= CH
3
CuSO
4
.
5 H
2
O
Sodium ascorbate
EtOH: H
2
O, 3–5 h
CN
HCI, 110 °C,
20 min
3
3
C
6
H
6
, 25 °C
KOBu
t
N
3
25 °C, 3.5 h
R
1
5
4
R
1
R
1
R
1
N
N
N
N
N
N
N
28, R
1
= F
29, R
1
= NH
2
32, R
1
= F
33, R
1
= NH
2
N
N
N
R
1
R
1
NaN
3
NaNO
2
NaN
3
, NaNO
2
N
N
N
3
N
N
N
+
N
N
3
TFA-H
2
O
25 °C, 3 h
TFA-H
2
O
25 °C, 3.5 h
NH
2
N
26
30
27
31
N
NH
2
NH
2
CN
H
2
N
CN
R
1
R
1
Fig. 2 Synthesis scheme of target compounds. Synthesis of various 5-azido-pyrazoles (5, 14, 27, and 31) and reference compounds (7–12, 16–25, 28, 29,
32, and 33)
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-016-0009-6
4 NATURE COMMUNICATIONS
|
8: 1
|
DOI: 10.1038/s41467-016-0009-6
|
www.nature.com/naturecom munications
chemistry formation of two compounds, 4-(4-fluorophenyl)-
1-[2-(4-methanesulfonyl-phenyl)-5-methyl-2H-pyrazol-3-yl]-1H
[1,2,3]triazole (18) and 4-{1-[2-(4-methanesulfonyl-phenyl)-
5-methyl-2H-pyrazol-3-yl]-1H-[1,2,3]triazol-4-yl}-phenylamine
(21) was detected (Fig.
3). Formation of compounds 18 and 21
was first detected at a time interval of 6 h, and continuous
elevation in the LC/MS response signal (an indicator for the
amount of triazole product formed) was noticed up to 15 and
12 h, respectively.
Likewise, three control experiments were performed under the
same experimental conditions to test false-positive results: each
azide/ alkyne combination was incubated either in the (a)
absence of COX-2 isozym e(b) presence of bovine ser um albumin
(BSA, 1 mg ml
−1
) as model protein instead of COX-2 isozyme(c)
or in the presen ce COX-2 is ozyme and known C OX-2 selective
inhibitor celecoxib (1 µl of celecoxib, 100 µM final con centra-
tion). LC/MS-SIM analysis of each reaction mixture revealed that
none of the reagent combinations led to the formation of triazole
products. These results demonstrated that desired triazole
product 18 and 21 were only form ed when a suitable 5-azido-
pyraz ole and alkyne combination can undergo in situ click
chemistry reaction in the presence of an accessible CO X-2-
binding pocket. In line with other known in situ click chemistry
examples
42,43
, our results confirm that the COX-2-binding site
can also serv e as a reaction vessel for 1,3-d ipolar cycloadd ition
reactions, and th is innovative methodology can facilitate th e
discovery and quick screening of novel selective COX-2
inhibitors.
Table 1 In situ click chemistry building blocks and corresponding hit compounds
Library Azide Alkyne In situ Hit
l
N
N
N
3
5
H
Cl
F
OCH
3
CH
3
C
2
H
5
NH
2
CF
3
No hits
2
N
N
N
3
H
3
C
SO
2
CH
3
14
H
Cl
OCH
3
F
CH
3
C
2
H
5
NH
2
CF
3
Two hits
N
N
N N
N
F
H
3
C
SO
2
CH
3
18
N
N
N N
N
NH
2
H
3
C
SO
2
CH
3
21
H
3
CCH
3
O
N
N
3
N
N
N
3
SO
2
CH
3
27
F
NH
2
No hits
4
N
N
N
3
H
3
C
31
F
NH
2
No hits
NATURE COMMUNICATIONS | DOI: 10.1038/s41467-016-0009-6 ARTICLE
NATURE COMMUNICATIONS
|
8: 1
|
DOI: 10.1038/s41467-016-0009-6
|
www.nature.com/naturecom munications 5