Celecoxib

To investigate the COX-2 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-2 isozyme (95 µl COX-2) 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-2 enzyme activity, see Supplementary Fig. 16). Final reagent concentrations were as follows: COX-2 (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/H₂O (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 experiments in the presence of BSA (1 mg mL⁻¹) instead of the COX-2 enzyme as well as in the absence of COX-2 enzyme and the known COX-2 selective inhibitor (1 µl of celecoxib, 100 µM final concentration) were run as described above. For multicomponent in situ click chemistry 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-2 isozyme (95 µl COX-2) 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 experiments using BSA (1 mg ml⁻¹) in place of COX-2 isozyme and in the absence of COX-2 isozyme were run consecutively.

The two lead compounds 18 and 21 were advanced for in vivo anti-inflammatory activity study and celecoxib (1) was used as reference drug. In vivo anti-inflammatory activity was measured using a carrageenan-induced rat paw edema assay. 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 experiments were identical, except that the vehicle 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 ED₅₀were 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.

The ability of known COX-2 selective inhibitor celecoxib (1), 5-azido-pyraozles (5 and 14) and new triazole products (7–12, 16–25, 28, 29, 32, and 33) to inhibit ovine COX-1 and recombinant human COX-2 was determined using a COX inhibitor assay (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 IC₅₀ values. In addition to celecoxib, both Dup-697 (potent COX-2 inhibitor) and SC-560 (potent COX-1 inhibitor) were used as internal controls during screening test compounds.