In situ click chemistry generation of cyclooxygenase-2 inhibitors — TEI-tagged view

Atul BhardwajpersName, Jatinder KaurpersName, Melinda WuestpersName, Frank WuestpersName (2017date). “In situ click chemistry generation of cyclooxygenase-2 inhibitors.” Nature Communicationstitle·j.

Object of study — COX-2-templated in situ click chemistryterm and the resulting selective inhibitors compounds 18 and 21

Research question — whether the COX-2term binding pocket can select complementary azide and alkyne building blocksterm·var and template potent, selective inhibitor formation

Contribution — an experimentally controlled demonstration that COX-2term can template regioselective formation of compounds 18 and 21, with biochemical, cellular, animal, calorimetric, SAR, and docking characterization

Full text — tagged

In situ click chemistryterm generation of cyclooxygenase-2 inhibitors Abstract Cyclooxygenase-2 isozymeterm 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 studiesterm confirmed that the cyclooxygenase-2 isozymeterm 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 synthesisterm, also termed as in situ click chemistryterm, we describe the discovery of two highly potent and selective cyclooxygenase-2 isozymeterm 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.

Introduction Cyclooxygenase (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-1term, COX-2term, and COX-3) 5 – 7 . COX-1term and COX-2term isoforms are of primary interest, as they are involved in physiological as well as pathological processes. COX-1term is a constitutively expressed house-keeping isozyme responsible for the basal production of essential PGs 8 . These PGs mediates homoeostatic functions in the gastrointestinal and cardiovascular system. COX-3 (a splice variant of COX-1term) is expressed only in specific parts of the brain and spinal cord and its exact functions are still unclear 9 . In contrast, COX-2term isozyme is expressed at very low levels under normal conditions. However, COX-2term 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-2term 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-2term inhibitors have recently been used for molecular imaging 17 – 19 and therapy 20 – 22 of cancer. Hence, the development of selective COX-2term 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-1term and COX-2term isoforms; and their use is limited due to associated ulcerogenic and gastrointestinal side effects. Discovered in the late 1990’s, COX-2term selective inhibitors (the COXIBs: celecoxibterm, rofecoxib) are diarylheterocycles possessing a SO 2 NH 2 or SO 2 Me group as COX-2 pharmacophoreterm·def, 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 . As a result, COXIBs such as rofecoxib and valdecoxib were withdrawn from the market thereby leaving a demand for the synthesis and screening of novel COX-2term inhibitors.

Development of compounds that selectively inhibit COX-2term over COX-1term 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 difference between the COX-1term and COX-2term isozyme active site is the exchange of isoleucine in COX-1term for valine in COX-2term at positions 434 and 523. The difference in the amino-acid sequence make the COX-2 substrate-binding siteterm more flexible and approximately 25% larger by creating a distinct secondary-binding pocketterm 3 , 30 . Many COX-2term selective inhibitors explicitly bind to this secondary-binding pocketterm (lined by H90, R513, and V523) resulting in the specific inhibition of COX-2term activity. Another important region in the COX-2 active siteterm is the hydrophobic pocketterm (lined by W387, Y385, F518, F381, L352), and a recent mutational study described the involvement of hydrophobic pocketterm residues in the proper positioning of fatty acid substrates for oxygenation 31 . Therefore, highly potent and selective COX-2term 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-2term.

Here, we deviated from conventional drug discovery approaches involving the laborious synthesis and screening of a range of compounds, and envisioned to explore the utility of in situ click chemistryterm for the discovery of specific and high-affinity COX-2term inhibitors. Click chemistry 32 , 33 , including 1,3-dipolar cycloadditionterm between alkyne and azide (Huisgen cycloadditionterm), 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 targeting 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 synthesisterm (TGS), also termed as in situ click chemistryterm 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 blocksterm·var ( 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-1term/COX-2 inhibitory potencyterm, various 5-azido-pyrazolesterm ( 5, 14, 27, and 31 ) and aryl acetylenes as click chemistry building blocksterm were incubated in pairs with human recombinant COX-2term isozyme to test the capability of COX-2term for assembling its own highly potent inhibitors. Identification of compounds 18 and 21 as highly potent and selective COX-2term inhibitors demonstrated the feasibility of the in situ click chemistryterm 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 studiesterm confirmed that COX-2term can selectively group most appropriate building blocks from a pool of compounds to construct highly potent COX-2term inhibitors. The thermodynamic-binding signatures calculated from isothermal titration calorimetryterm (ITC) confirmed that binding of click chemistry building blockterm 5-azido-pyrazoleterm ( 14 ) to COX-2term involves a favorable change in free energy (ΔG = −36.20 kJ mol −1 ), which was mainly based on H-bondingterm and van der Waals interactionsterm. Moreover, comprehensive computational analysis including structure activity relationshipterm (SAR) and molecular dockingterm indicated that the size and type of the COX-2 pharmacophoreterm, and the orientation of the clickable building blocks inside the binding site of the target protein collectively contribute to the in situ constructionterm of highly potent and selective COX-2term inhibitor.

Results Design of clickable building blocks Based on the structural features of the COX-2 active siteterm, 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 pharmacophoreterm to facilitate its tight bindingterm into the secondary-binding pocketterm of the COX-2term 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 siteterm, allowing the in situ click chemistryterm formation of a potent and selective inhibitor large enough to block the hydrophobic channel of the COX-2term isozyme (Fig. 1 ).

Design of building blocks for in situ click chemistryterm reaction in the COX-2term isozyme. Structure of selective COX-2term inhibitor celecoxibterm ( 1 ) and rofecoxib ( 2 ), target compounds ( 7–12 , 16–25 , 28 , 29 , 32, and 33 ) and illustration of in situ click chemistryterm reaction principle inside the COX-2-binding pocket Hence, to optimize these parameters we synthesized a range of 5-azido-pyrazoleterm derivatives with variable sizes containing a COX-2 pharmacophoreterm·def (SO 2 CH 3 ) and evaluated the influence of the chemical structure on the success of in situ click chemistryterm reaction. We selected a pyrazole motif as the central scaffold for the azide building blocksterm·var, as pyrazole-containing compounds are quite prevalent in many anti-inflammatory drugs. The design of click chemistry building blocksterm and target compounds is illustrated in Fig. 1 , where the central pyrazole motif was adapted from approved drug celecoxibterm ( 1 ). The SO 2 CH 3 COX-2 pharmacophoreterm·var is located at the para position of one of the phenyl ring which was introduced to facilitate binding of 5-azido-pyrazoleterm ( 14 ) into the COX-2term secondary-binding pocketterm. 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 pharmacophoresterm·def for selective binding to the COX-2term 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 blocksterm·var selected for this study was based on structures frequently present in various NSAIDs.

Chemical synthesis of target compounds The synthetic methodologies used to prepare 5-azido-pyrazolesterm ( 5 , 14 , 27 , 31 ) are illustrated in Fig. 2 . Briefly, precursor compounds 2,5-diphenyl-2 H -pyrazol-3-ylamine ( 4 ) and 2-(4-methane-sulfonyl-phenyl)-5-methyl-2 H -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-pyrazolesterm ( 5 , 14 ) by diazotization and subsequent treatment with sodium azide. 5-azido-pyrazolesterm ( 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 pharmacophoreterm·var in selective COX-2term inhibition, we synthesized compounds 28 , 29 , 32, and 33 according to synthetic methods shown in Fig. 2 .

Synthesis scheme of target compounds. Synthesis of various 5-azido-pyrazolesterm ( 5 , 14 , 27, and 31 ) and reference compounds ( 7–12 , 16–25, 28 , 29 , 32, and 33 ) In order to investigate the COX-2term isozyme-mediated in situ click chemistryterm reaction, 5-azido-pyrazoleterm 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-2term isozyme (95 µl COX-2term) in 1 µl of 1M Tris-HCl, pH 8.0 for 24 h at room temperature. After 3, 6, 9, 12, 15, 18, 21, and 24 h each sample was analyzed in triplicate by injecting (10 µl) into the liquid chromatography–mass spectrometry (LC/MS) instrument with selected-ion-monitoring (SIM) mode. In library 1, where 5-azido-1,3-diphenyl-1 H -pyrazole ( 5 ) was incubated in the presence with various alkynes ( 6a – 6f , 15a , and 15b Table 1 , library 1), in situ click chemistryterm formation of corresponding triazole compounds could not be detected. After refinement of the structure of 5-azido-1,3-diphenyl-1 H -pyrazole ( 5 ,V molecular = 332.9 Å 3 ) into a smaller sized 5-azido-1-(4-methanesulfonyl-phenyl)-3-methyl-1 H -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 pharmacophoreterm·def (SO 2 CH 3 ) was incorporated at C-4 position of one of the phenyl rings.

In situ click chemistryterm building blocks and corresponding hit compounds LC/MS-SIMterm analysis of 11 different reactions ( 6a – 6f, 15a – 15e Table 1 , library 2) revealed two combinations where in situ click chemistryterm formation of two compounds, 4-(4-fluorophenyl)-1-[2-(4-methanesulfonyl-phenyl)-5-methyl-2 H -pyrazol-3-yl]-1H [1,2,3]triazole ( 18 ) and 4-{1-[2-(4-methanesulfonyl-phenyl)-5-methyl-2 H -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 signalterm·var (an indicator for the amount of triazole product formed) was noticed up to 15 and 12 h, respectively.

Monitoring of COX-2term isozyme-mediated in situ click chemistryterm reaction. In situ click chemistryterm formation of compounds 18 ( a ) and 21 ( b ) as determined by LC/MS-SIMterm Likewise, three control experimentsterm·mth 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-2term isozyme(b) presence of bovine serum albumin (BSAterm, 1 mg ml −1 ) as model protein instead of COX-2term isozyme(c) or in the presence COX-2term isozyme and known COX-2term selective inhibitor celecoxibterm (1 µl of celecoxibterm, 100 µM final concentration). LC/MS-SIMterm 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 formed when a suitable 5-azido-pyrazoleterm and alkyne combination can undergo in situ click chemistryterm reaction in the presence of an accessible COX-2-binding pocketterm. In line with other known in situ click chemistryterm examples 42 , 43 , our results confirm that the COX-2-binding site can also serve as a reaction vessel for 1,3-dipolar cycloadditionterm reactions, and this innovative methodology can facilitate the discovery and quick screening of novel selective COX-2term inhibitors.

The 1,4/1,5 triazole regiochemistry of triazole products ( 18 and 21 ) formed during the COX-2term mediated in situ click chemistryterm reaction was determined through LC/MS analysis comparing the retention time of 1,4-triazole regioisomersterm (compound 18term and 21 ) with respective 1,4/1,5 triazole regioisomer mixtures of compounds 18 and 21 (Supplementary Figs 1 and 2 ). Results from this study demonstrated that in situ click chemistryterm reaction in the presence of the COX-2-binding site was highly regioselective, yielding the desired 1,4-triazole regioisomersterm exclusively.

Furthermore, the influence of the size and the role of the SO 2 CH 3 COX-2 pharmacophoreterm·var of the 5-azido-pyrazoleterm building blocks upon in situ click chemistryterm reaction was studied with two newly synthesized 5-azido-pyrazoleterm building blocks 27 and 31 (Fig. 2 ). In contrast to 5-azido-pyrazoleterm ( 14 ), the new building block 31 lacks the SO 2 CH 3 COX-2 pharmacophoreterm·var. Upon incubating 5-azido-pyrazoleterm ( 31 ) with alkynes ( 6c or 15a ) in the presence of COX-2term isozyme, no triazole product formation was detected even after 36 h. This experimentterm·mth demonstrated that (for library 3) the presence of the SO 2 CH 3 COX-2 pharmacophoreterm·var is an important structural requirement for the appropriate positioning of 5-azido-pyrazoleterm building block ( 14 ) inside the COX-2-binding site to achieve a suitable steric orientation of the azide building blockterm·var needed for the COX-2term mediated in situ click chemistryterm reaction. Another set of experimentsterm·mth involved the incubation of phenyl group-containing 5-azido-pyrazolesterm 27 (with SO 2 CH 3 COX-2 pharmacophoreterm·var) and 5 (without SO 2 CH 3 COX-2 pharmacophoreterm·var) as examples for larger size azide building blocksterm·var with the alkyne building blocksterm·var ( 6c or 15a ) in the presence of COX-2term isozyme. And in none of the reagent combinations, no in situ generation of expected triazole product was detected. These results led to the following important two conclusions: ( i ) the presence of the SO 2 CH 3 COX-2 pharmacophoreterm·var is the defining factor to advance the in situ click chemistryterm reaction between azides and alkynes inside the COX-2-binding site; ( ii ) a larger sized (additional steric bulk)5-azido-pyrazoleterm building block like compound ( 27 ) greatly reduces the possibility to achieve an in situ click chemistryterm reaction in the COX-2-binding site. The results of in situ click chemistryterm reaction with various azides ( 5, 14, 27, and 31 ) and alkynes ( 6a-f , 15a-e ) in the presence of the COX-2term isozyme are summarized in Fig. 4 .

Multicomponent in situ click chemistryterm reaction. Comparison of triazole compounds formed after the incubation of all azide and alkyne building blocksterm·var in the presence of the COX-2term isozyme We also tested whether COX-2term isozyme is capable of selecting the most suitable compounds from a combined mixture of different azides and alkynes building blocks for assembling the “best-fit inhibitorterm.” For this purpose, each azide ( 5, 14, 27, and 31, 1 µl of 3 mM DMSO solution) was mixed with eleven alkynes ( 6a–6f and 15a–15e, 1 µl of 20 mM DMSO solution) in the presence of COX-2term isozyme (95 µl COX-2term) in 1 µl of 1 M Tris-HCl, pH 8.0 and incubated for 24 h at room temperature. After incubation for 24 h, only expected triazole products ( 18 , 21 ) were detected, demonstrating that COX-2term isozyme can sense small structural differencesterm of the building blocks enabling selection of suitable building blocks for the in situ click chemistryterm formation of high affinity and selective COX-2term inhibitors (Fig. 4 ). The amount of triazole product 21 formed seemed to be higher compared to triazole 18 (estimated from the mass response), although the yield of both triazole products ( 18 , 21 ) was lower (almost 50% reduced) compared to previously performed in situ click chemistryterm reaction when both building blocked were used as reaction pairs. The decrease in yield could be attributed to a reduction in the concentration of click chemistry building blocksterm and/or an increase in the competition for the COX-2-binding site.

In parallel to the COX-2term isozyme mediated in situ click chemistryterm reactions, similar experimentsterm·mth were also performed using COX-1term isozyme. However, no in situ click chemistryterm reaction to form the desired triazole products was not observed in any of the used reagent combinations. As demonstrated in a later in vitro COX inhibitory-binding assay, none of the compounds displayed inhibitory potencies in the studied concentration range towards COX-1term isozyme. Thus, in situ click chemistryterm can also be used for the identification of highly selective COX-2term inhibitors. Compound 5 , 14, 27, and 31 were also studied for their stability in human and rat serum at 37 °C, and no significant degradation was observed up to 30 h (see Supplementary Fig. 3 ). The stability of hit compounds ( 18 and 21 ) was tested, and the percentage of intact compound at various time points is given in Supplementary Fig. 3 .

Isothermal titration calorimetryterm The success of identifying 5-azido-pyrazoleterm ( 14 ) as suitable building block for in situ click chemistryterm reaction prompted us to determine the protein–ligand-binding stoichiometry ( N ), change in thermodynamic parameters, and association constantterm ( K a ) for the binding of 5-azido-pyrazoleterm ( 14 ) with pure COX-2term protein under physiological conditions using ITC. The determined binding stoichiometryterm for compound 14term ( N = 1.02 ± 0.0259) indicated that one molecule of the 5-azido-pyrazoleterm ( 14 ) was bound per COX-2term isozyme. The high association constantterm ( K a = 2.20 × 10 −6 M −1 ) and favourable free energy changeterm (ΔG < 0; ΔG = −36.20 kJ mol −1 ) clearly supports the assumption of tight bindingterm of compound 14term to the COX-2term protein (Supplementary Table 1 ). The ITC results summarized in Fig. 5 demonstrate that protein–ligand interactionsterm are thermodynamically favored and that they are mainly driven by the enthalpy factorterm. The binding of compound 14term to COX-2term isozyme involves an exothermic reaction (ΔH < 0; ΔH = −34.51 kJ mol −1 ) which is indicative of a combination of H-bondingterm, π - π stacking and ion–dipole interactions. Interestingly, docking studies have also supported the contribution of H-bondingterm in protein–ligand binding. The determined favorable small entropic change (TΔS > 0; TΔS = 1.69 kJ mol −1 ) also reflects the little contributions of electrostatic contact, conformational freedom, and solvent reorganization.

Thermodynamic analyses for the binding of 5-azido-pyrazoleterm (14) to COX-2term. a Raw ITC data obtained upon titration of compound 14term with COX-2term protein at room temperature (298.15 K); b Plot of the integrated heat signal as a function of the molar ratio of ligand to the protein; c Thermodynamic parameters for the binding of compound 14term with COX-2term ( n = 3, ±s.e.m.) In vitro COX-1term/COX-2term inhibition assay All representative triazole compounds ( 7–12 , 16–25, 28 , 29 , 32, and 33 ) as well as 5-azido-pyrazoleterm click chemistry building blocksterm ( 5 , 14 , 27, and 31 ) were tested for in vitro COX-1term/COX-2term inhibitory activity. All compounds displayed higher inhibitory potencies for the COX-2term isozyme (IC 50 range: 0.05 to 26.0 µM) compared to the COX-1term isozyme (IC 50 range: 45.5 to >100 µM) (Table 2 ). Interestingly, both in situ click chemistryterm hit compounds ( 18 , 21 ) showed particularly high COX-2 inhibitory potencyterm (COX-2 IC 50term·var = 0.09 µM and 0.05 µM, respectively) and COX-2term selectivity (COX-2 selectivity indexterm (SI) = 1,111 and 2,000, respectively). Moreover, compound 21term exhibited better COX-2term inhibitory activity and SI than widely used COX-2term inhibitor celecoxibterm 1 (COX-2 IC 50term·var = 0.07 µM, SI = 1,428).

In vitro COX-1term and COX-2term inhibition data, COX-2 selectivity indexterm (SI), molecular volume, and calculated log P data of compounds 1 , 5 , 14 , 7–12 , 16–25 , 28, 29 , 32, and 33 The relative inhibitory potency and selectivity profile were observed in the following order: 21 (R 1 = NH 2 ), > 9 , 18 (R 1 = F) > 8 , 17 (R 1 = Cl), which shows that the para substituentterm (R 1 ) on the C-1 phenyl ring of triazole is crucial for both COX-2 inhibitory potencyterm and high COX-2term selectivity. However, compounds 23–25 containing heterocyclic or aliphatic (ibuprofen like) motifs on C1 position of triazole ring displayed only low inhibitory potencies towards COX-2term. Comparison of COX-2 inhibitory potencyterm profiles of compounds 28, 29, 32, and 33 with 7–12 and 16–25 confirmed the importance of the SO 2 CH 3 COX-2 pharmacophoreterm·var as an important structural requirement for high COX-2term inhibition. Among all tested compounds, triazoles 18 and 21 were identified as the most potent and selective COX-2term inhibitors. Thus, obtained in vitro COX-1term/2 enzyme inhibitory data evidently demonstrate that in situ click chemistryterm reaction can also be employed to identify highly potent and selective COX-2term inhibitors. However, compounds 8 (COX-2 IC 50term·var = 0.16 µM), 9 (COX-2 IC 50term·var = 0.14 µM), and 22 (COX-2 IC 50term·var = 0.22 µM) also showed appreciable COX-2term inhibitory potencies in the in vitro COX-binding assay albeit nor formation of compounds 8 , 9, and 22 was detected in the COX-2term mediated in situ click chemistryterm reaction. Based on the in vitro COX-2term inhibitory data, seventeen compounds (Table 2 ) were selected and further evaluated for their cellular COX-2term inhibitory activity in COX-2term expressing colorectal cancer cell line HCA-7term. Both in situ click chemistryterm hit compounds ( 18 , 21 ) also displayed highest cell-based COX-2term inhibitory potencies. Compounds 18 and 21 displayed a better COX-2term inhibitory profile in HCA-7term cells (COX-2 IC 50term·var = 0.06 µM and 0.08 µM, respectively) compared to celecoxibterm (COX-2 IC 50term·var = 0.09 µM).

In vivo anti-inflammatory activity The two in situ click chemistryterm hit compounds 18 and 21 were further studied for their in vivo anti-inflammatory potency. Both compounds proved to be highly potent anti-inflammatory agents. ED 50term values were determined in a carrageenan-induced rat paw edema assayterm at 3 and 5 h time points after treatment with 0.3, 1.0, 3.0, and 10 mg kg −1 p.o. of compounds 18 and 21 (Fig. 6 , Supplementary Figs 4 and 5 ). Compound 18term showed high anti-inflammatory activity at 3 h (ED 50term = 0.44 mg kg −1 ) and 5 h (ED 50term = 0.99 mg kg −1 ) time points. An ever-higher anti-inflammatory activity at 3 h (ED 50term = 0.12 mg kg −1 ) and 5 h (ED 50term = 0.34 mg kg −1 ) time points was found for compound 21term . However, both compounds were found to be >25 times more potent than widely used COX-2term inhibitor celecoxibterm (ED 50term = 10.8 mg kg −1 at 3 h). To the best of our knowledge, compounds 18 and 21 are the most potent known anti-inflammatory agents. The determined very high COX-2 inhibitory potencyterm and selectivity of in situ click chemistryterm hit compounds 18 and 21 clearly demonstrates the power of innovative in situ click chemistryterm for the identification of novel highly potent and selective COX-2term inhibitors.

In vivo anti-inflammatory activity profile of in situ click chemistryterm hits. a , b Effective dose (ED 50term ) calculated at 3 and 5 h time after treatment of compound 18term ( n = 4, ±s.e.m.) and 21 ( n = 5, ±s.e.m.); c , e Control; d , f Images showing the in vivo effect at 3 h after treatment with 0.3 mg kg −1 p.o. of compounds 18 (left paw) and 21 (right paw), respectively Molecular dockingterm studies To further support our experimental results and to investigate the mode of ligand–protein molecular interactions, all click chemistry building blocksterm ( 6, 14, 27, 31, 6a–6f, and 15a–15e ) and final triazole compounds ( 7–12, 16–25, 28, 29, 32, and 33 ) were docked into the COX-2 active siteterm. It was found that the para SO 2 CH 3 group in 5-azido-pyrazoleterm compound ( 14 ) was deeply inserted into the secondary-binding pocketterm region of the COX-2 active siteterm (Fig. 7 and Supplementary Fig. 6 ). The O -atoms of the SO 2 CH 3 group showed H-bondingterm interaction with one of the terminal amino group of R513 (distance O---NH 2 = 2.25 Å) and with the nitrogen atom of the imidazole ring of the H90 residue (distance O---N = 1.92 Å). Interestingly, the other 5-azido-pyrazolesterm ( 5, 27, and 31 ) were located away from the secondary pocket region, and did not indicate any H-bondingterm interactions (Supplementary Fig. 7 ), except for compound 27 where one of the O -atoms of the SO 2 CH 3 group was H-bonded to the-NH 2 group of R120 (distance O---NH 2 = 1.96 Å). Alkynes possessing either a-F or -NH 2 group ( 6c and 15a ) were positioned in the vicinity of azide group of compound 14term (Fig. 5 and Supplementary Fig. 9 ), whereas the other three alkynes ( 6b , R 1 = Cl; 6d , R 1 = OCH 3 ; and 15b R 1 = CF 3 ) were clustered together near Y385; alkynes with para CH 3 or para C 2 H 5 groups and heterocyclic rings ( 6e, 6f, and 15c – 15e ) were also docked far away from the azide group of pyrazole compounds ( 6,14, 27, and 31 ). Molecular dockingterm indicated that 5-azido-pyrazoleterm ( 14 ) and alkynes ( 6c and 15a ) were favorably oriented in such a way that the azide group (-N 3 ) is present in proximity of 4-ethynyl group (-C≡CH) of both alkynes; and the calculated distance between the terminal carbon atom of the ethynyl groups in 6c ( R 1 = F) and 15a ( R 1 = NH 2 ) and one of the nitrogen atoms of the azide group in compound 14term was 1.78 and 2.85 Å, respectively (Fig. 7 ). Therefore, the observed formation of in situ click chemistry productsterm ( 18 and 21 ) can be attributed to the close and special orientationterm of reactive functional groups of the complementary click chemistry building blocksterm ( 14 , 6c, and 15a ) in the COX-2-binding site.

Computational analysis of click chemistry building blocksterm in the COX-2 active siteterm. Molecular dockingterm of 5-azido-pyrazoleterm compounds ( 5 , 14 , 27, and 31 ) and alkynes ( 6a–6f and 15a–15e ) in the binding site of COX-2term isozyme (PDB ID: 6COX). Hydrogen atoms of amino-acid residues are omitted for clarity Further molecular dockingterm studies with in situ click chemistryterm products ( 18 , 21 ) demonstrated that both compounds fit favorably into the COX-2-binding sites ( E intermolecular = −15.9 and −16.8 kcal mol −1 respectively) displaying favorable electrostatic interactions with the secondary pocket residues (Supplementary Figs 8 and 9 ). For compound 18term , the O -atoms of the para SO 2 CH 3 group showed H-bondingterm with R513 (distance O---N = 2.90 Å) and the H90 residue (distance O---N = 2.37 Å); and two of the N-atoms of the pyrazole ring were also H-bonded to A527 (Supplementary Fig. 9 ). In the case of compound 21term, the O -atoms of the para SO 2 CH 3 group indicated H-bondingterm with Y355 (distance O---HO = 2.39 Å) which is considered as one of the important catalytic amino acid for COX reaction of enzyme 57 , with the R513 residue (distance O---N = 2.08 Å); and the NH 2 group in compound 21term showed H-bondingterm with Q192 (distance N---O = 2.64 Å) in the COX-2 active siteterm (Supplementary Fig. 9 ). In contrast, upon docking of all azide compounds ( 5 , 14, 27, and 31 ) and alkynes ( 6a–6f and 15a–15e ) into the COX-1term active site, it was found that none of the azide compounds were located near any of the alkyne compounds, indicating less opportunity for an in situ click chemistryterm reaction in the COX-1term active site. The preference of the COX-2 isozymeterm for in situ click chemistryterm reaction over the COX-1term isozyme can be explained by considering the structural differencesterm of both enzymes. The size of the COX-2term isozyme active site (volume = 394 Å 3 ) is about 25% larger than the COX-1term isozyme-binding site (volume = 316 Å 3 ). Moreover, the COX-2term isozyme possesses an additional secondary pocket region. Because of these two distinct structural differencesterm, the COX-2term isozyme can conjointly hold two reactive/more bulky ligands more appropriately than the COX-1term isozyme. The energy of intermolecular interactions ( E intermolecular ) between the ligand and the enzyme residues obtained after computational studies (docking) for all compounds within the COX-1term and COX-2 active siteterm are summarized in Supplementary Table 2 and Supplementary Fig. 8 . Interestingly, compound 18term and 21 occupy an orientation that is very similar to the binding mode of crystallized COX-2term inhibitor SC558 in the COX-2term isozyme (Fig. 8 ). Overall, the computational results suggest that similar to the orientation of parent azide click chemistry building blockterm 14 , the 4-methanesulfonyl-phenyl motif of in situ click chemistryterm hit compounds 18 and 21 occupy conformational spaces in the secondary-binding pocketterm region of the COX-2-binding site, and the fluoro/amino-phenyl ring portion is embedded into the hydrophobic pocketterm; H90, R120, Q192, Y355, Y 385, R513, and F518 are the key amino-acid residues, which considerably contributed in the strong protein–ligand interactionsterm. All these observations agree with the experimental results and support the success of in situ click generation of 18 and 21 from clickable building blocks 14 (azide) and 6c / 15a (alkyne) in the COX-2-binding pocket.

Comparison of the binding conformation of in situ click chemistryterm reaction hit compounds with crystalized SC558. a In COX-2term isozyme (PDB:6COX) the compound 18term ( cyan carbon) and SC558 ( green carbon) are displayed in stick mode and surface of compound 18term is highlighted ( grey ) color; b compound 21term ( magenta carbon) and SC558 ( green carbon) are displayed in stick mode and surface of compound 21term is highlighted ( grey ) color Discussion We have, for the first time, demonstrated the use of in situ click chemistryterm for the identification and synthesis of highly potent and selective COX-2term isozyme inhibitors. In situ click chemistryterm with COX-2term isozyme as molecular template was highly regioselective and products are exclusively formed as 1,4-regioisomers through[3,2]-cycloaddition reaction inside the COX-2-binding pocket. Our comprehensive SAR and computational studies suggest that size, presence of a SO 2 Me COX-2 pharmacophoreterm, proper orientation of the azide click chemistry building blockterm inside the COX-2-binding site, H-bondingterm, and hydrophobic interactionsterm of building blocks collectively contribute to the in situ click chemistryterm synthesis of highly potent and selective COX-2term isozyme inhibitors. Moreover, we demonstrated that the COX-2term isozyme is capable of finding and selecting the most favourable complementary azide and alkyne building blockterm·var pairs from a pool of reactive precursors to generate its own ‘best fit’ inhibitor. Results of this study provide an efficient tool for economical and fast screening of potential drug candidates. Based on the extremely high in vivo activity of identified and characterized in situ click chemistryterm hit compounds, we are currently working to expand this strategy for the detection of COX-2term expression at the cellular and in vivo level.

Methods Chemical synthesis Detailed procedures for the synthesis of all new compounds and their characterization are provided in the Supplementary Methods (For 1 H and 13 C spectra of compounds 14 , 18, and 21 , see Supplementary Figs 10 , 11 , 12 , 13 , 14 , and 15 ).

In situ click chemistryterm screening procedure for binary reagent mixtures To investigate the COX-2term isozyme templated synthesis, each 5-azido-pyraozle ( 5, 14, 27, and 31, 1 µl of 3 mM DMSO solution) and alkyne ( 6a – 6f, 15a – 15e , 1 µl of 20 mM DMSO solution) were pairwise mixed with human recombinant COX-2term isozyme (95 µl COX-2term) in 1 µl of 1 M Tris-HCl, pH 8.0. The each reaction mixture was vortexed for 1 min, and then incubated at room temperature (For temperature dependency of COX-2term enzyme activity, see Supplementary Fig. 16 ). Final reagent concentrations were as follows: COX-2term (7 µM), azide (30 µM) alkyne (200 µM). After 3, 6, 9, 12, 15, 18, 21, and 24 h each sample was analyzed in triplicate by injecting (10 µl) into the LC/MS instrument with SIM mode (Water’s Micromass ZQTM 4000 LC−MS instrument, operating in the ESI-positive mode, equipped with a Water’s 2795 separation module). Calibration curve for hit compounds 18 and 21 is given in Supplementary Fig. 17 . Summaries of all LC/MS data are presented in Supplementary Tables 3 – 7 . Separations were performed in triplicate using a Kromasil 100-5-C18 (100 μm pore size, 5 μm particle size) reverse phase column (2.1 mm diameter × 50 mm length), preceded by a Kromasil 100-5-C18 2.1 × guard column. Separations were effected using a gradient MeCN/H 2 O (0.05% trifluoroacetic acid (TFA))/MeOH in 40/30/30, v/v/v over 15 min at flow rate 0.25 ml min −1 . Operating parameters were as follows: capillary voltage = 3.5 kV; cone voltage = 20 V; source temperature = 140 °C; sesolvation temperature = 250 °C; cone nitrogen gas flow = 100 l h −1 ; desolvation nitrogen gas flow = 550 l h −1 . The identities of triazole products (retention time of 6.73 min for 18 ), (retention time of 4.56 min for 21 ), and the internal standard (retention time of 10.89 min) were confirmed by molecular weight and comparison of the retention times of the authentic products formed from copper catalyzed reactions. Control experimentsterm·mth in the presence of BSAterm (1 mg mL −1 ) instead of the COX-2term enzyme as well as in the absence of COX-2term enzyme and the known COX-2term selective inhibitor (1 µl of celecoxibterm, 100 µM final concentration) were run as described above. For multicomponent in situ click chemistryterm reactions, each azide ( 5, 14, 27, and 31, 1 µL of 3 mM DMSO solution) and eleven alkynes ( 6a – 6f and 15a – 15e, 1 µl of 20 mM DMSO solution) were thoroughly mixed together in the presence of COX-2term isozyme (95 µl COX-2term) in 1 µl of 1 M Tris-HCl, pH 8.0 and incubated at room temperature. After 24 h each sample was analyzed in triplicate by injecting (10 µl) into the LC/MS instrument by following the procedure described above, except the ions are monitored for all possible masses. The cyclo addition products were identified by their molecular weights and by comparison of the retention times of authentic products prepared through Cu-catalyzed reactions. Control experimentsterm·mth using BSAterm (1 mg ml −1 ) in place of COX-2term isozyme and in the absence of COX-2term isozyme were run consecutively.

Isothermal titration calorimetryterm The thermodynamics of the precursor 5-azido-pyrazoleterm ( 14 ) binding with pure human COX-2term protein studied with a high sensitivity ITC instrument (VP-ITC, MicroCal). All titrations were performed by following VP-ITC general procedure described in manual. Protein (20 µM) and ligand (200 µM) solution were prepared in PBS buffer, pH 7.4. Prior to each ITC run, all solutions were filtered using membrane filters (pore size 0.45 μm) and thoroughly stirred and degassed using Thermovac accessory for 20 min to remove any air bubbles at 25 °C. In a typical experimentterm·mth, aliquots (6 µl) of ligand solution (prepared at 10 times the COX-2term protein concentration) were injected with a computer-controlled stirrer-syringe into a reaction cell containing COX-2term protein. Titrations were carried out with a stirring speed of 400 rpm and 240 s intervals between injections. All experimentsterm·mth were conducted at 25 °C. Control experimentsterm·mth, including titration of the ligand into buffer alone or COX-2term protein into buffer alone, were carried to determine the heat due to dilution and subtracting during the original experimentterm·mth. Calorimetric data analysis was performed with ORIGIN software provided by MicroCal. All results, including the binding constant ( K a ), stoichiometry of binding ( N ), and thermodynamics of binding (Δ H and TΔ S ) were determined by fitting the experimental binding isotherms. Each ITC experimentterm·mth was repeated three times and showed good reproducibility.

COX inhibition assay The ability of known COX-2term selective inhibitor celecoxibterm ( 1 ), 5-azido-pyraozles ( 5 and 14 ) and new triazole products ( 7–12 , 16–25, 28 , 29 , 32, and 33 ) to inhibit ovine COX-1term and recombinant human COX-2term was determined using a COX inhibitor assayterm (Cayman Chemical, Ann Arbor, USA; item number: 700100) following the manufacturer’s protocol. Each compound was assayed in concentration range of 10 −9 M to 10 −3 M, in triplicate. PRISM5 software was used to calculate IC 50 values. In addition to celecoxibterm, both Dup-697 (potent COX-2term inhibitor) and SC-560 (potent COX-1term inhibitor) were used as internal controls during screening test compounds.

In vivo anti-inflammatory activity The two lead compounds 18 and 21 were advanced for in vivo anti-inflammatory activity study and celecoxibterm ( 1 ) was used as reference drug. In vivo anti-inflammatory activity was measured using a carrageenan-induced rat paw edema assayterm. In brief, three to five male Sprague–Dawley rats, 8–11-weeks-old, weighing 180–200 g (Charles-River Canada) were used in each group. Animals were randomized into different treatment groups based on similar paw size and body weight. Test compounds 18 and 21 suspended in water containing 1% methyl cellulose were administered orally for a minimum of four different doses (0.3. 1, 5, 10 mg kg −1 ) 1 h prior to a 0.05 ml subcutaneous injection of fresh 1% carrageenan in 0.9% NaCl solution under the plantar skin of the hind paw. Control experimentsterm·mth were identical, except that the vehicleterm·var did not contain a test compound. The volume of the injected paw was measured at 0, 3, and 5 h using a UGO Basile 7141 Plethysmometer (series no. 43201), each value is mean of 10 measurements. A dose–response curve was constructed using GraphPad Prism 5.0 and ED 50term were calculated. No unusual change in behavior and toxic effects was noticed in all animals. In vivo anti-inflammatory assays were carried using a protocol approved by the Health Sciences Animal Welfare Committee, University of Alberta, Edmonton, Canada.

Molecular dockingterm procedure The detailed procedure for the molecular dockingterm is provided in the Supplementary Information .

Statistical analysis Unless specified, all data were obtained from at least triplicate samples and represent at least three independent experimentsterm·mth and presented as mean ± s.e.m. Graphs were constructed using GraphPad Prism 4.0 (GraphPad Software). Where applicable, statistical differences were tested by unpaired Student’s t -testterm·var and were considered significant for P < 0.05.

Data availability The authors declare that all data supporting the findings of this study are available in the article and in the supplementary information file. Additional information are available from the corresponding author upon request.

Electronic Supplementary Material Supplementary Figures, Supplementary Tables, Supplementary Methods and Supplementary References.

Acknowledgements We dedicate this work to Prof. Dr Edward E. Knaus for the occasion of his 74th birthday. We gratefully acknowledge the Dianne and Irving Kipnes Foundation, the Canadian Institute for Health Research (CIHR) and the National Science and Engineering Research Council of Canada (NSERC) for supporting this work. F.W. and J.K. thank the Alberta Cancer Foundation (ACF) for a postdoctoral fellowship. We thank Alison Marshall for help in cell-based assays, Ingrit Hamann for her support with all animal experimentsterm·mth, and Vishwa Somayaji for his help with LC/MS analyses.

Competing financial interests The authors declare no competing financial interests.

Author contributions A.B. and J.K. designed the experimentsterm·mth. A.B. and J.K. synthesized the compounds. A.B. designed and performed in situ click chemistryterm reactions, LC/MS analysis, ITC experimentsterm·mth, stability analysis, in vitro, and cellular biological experimentsterm·mth. A.B. and J.K. performed SAR and computational studies. M.W. and A.B. designed in vivo studies, and M.W. performed the experimentsterm·mth. A.B. and J.K. wrote manuscript and compiled the supporting information. A.B., J.K., and F.W. analyzed the data, discussed the results, and contributed to editing of manuscript.

Footnotes References Associated Data Supplementary Materials Supplementary Figures, Supplementary Tables, Supplementary Methods and Supplementary References.

Data Availability Statement The authors declare that all data supporting the findings of this study are available in the article and in the supplementary information file. Additional information are available from the corresponding author upon request.