This dataset contains the replication data for the study "Photoswitchable COX-2-Selective Inhibitors as Light-Regulated Anti-Inflammatory Agents", covering computational, synthetic, photochemical, biochemical, and in vivo experimental data generated between 2020 and 2026. The computational data includes molecular docking (GOLD), molecular dynamics simulations (AMBER), MM-PBSA binding free energies, and TD-DFT calculations (Gaussian) for seven photocoxib (PC1–PC7) analogues derived from celecoxib by azologization. Synthetic and characterization data comprise raw and processed Bruker NMR spectra (1H, 13C, COSY, HSQC, HMBC, NOESY), IR spectra, and high-resolution mass spectrometry (HRMS) reports for the three synthesized candidates (PC4, PC5, PC6). Photochemical data include UV-vis absorption spectra, photoisomerization quantum yields, thermal half-lives, fatigue resistance profiles, and glutathione stability assays documenting the reversible trans–cis photoswitching behavior of the compounds under UV (365 nm) and visible light (405 or 445 nm). Biochemical and cell biology data encompass fluorometric COX-1 and COX-2 enzyme inhibition assays, ELISA-based prostaglandin E2 (PGE2) production measurements in J774 murine macrophage cultures, and MTT cell viability assays comparing trans and cis isomers. In vivo data consist of Sudan Black-stained fluorescence microscopy images and leukocyte count spreadsheets from a zebrafish tailfin amputation model of acute inflammation, organized across multiple treatment conditions and experimental batches. Data are provided in ten file formats including .pdb, .inpcrd/.prmtop, .fid (Bruker NMR), .dat/.log, .xlsx, .tif, .jpg, and .pdf.

This dataset supports the study of photoswitchable nonsteroidal anti-inflammatory drugs (NSAIDs) derived from celecoxib (CEL), the most widely used COX-2-selective NSAID. A library of seven azoaromatic photocoxib (PC) analogues (PC1–PC7) was computationally designed by azologization of the celecoxib scaffold. Three candidates (PC4, PC5, and PC6) were synthesized and fully characterized. These compounds undergo efficient and reversible trans–cis photoisomerization upon UV (365 nm) and visible light irradiation (405 or 445 nm). The cis-enriched photostationary states show enhanced, selective COX-2 inhibitory activity relative to the dark-adapted trans isomers, demonstrated in fluorometric enzyme assays, in J774 murine macrophage cell cultures (COX-1 and COX-2 PGE2 production), and in vivo in a zebrafish tailfin amputation model of acute inflammation. PC6 is the best-performing candidate, displaying up to 5-fold potency enhancement in macrophage assays and complete abolition of leukocyte recruitment at the wound site when administered as its photoinduced cis form.

GraphPad Prism, 9

Universitat Autònoma de Barcelona. Grup d’Electroquímica, Fotoquímica i Reactivitat Orgànica (GEFRO). Description of Methods Used for Collection-Generation of Data: Computational data: The human COX-2 (hCOX-2) crystal structure was taken from the Protein Data Bank (PDB code: 5F1A). Molecular docking was performed with GOLD software (100 poses per ligand, 26 Å sphere centred at Arg513, ChemScore function). Molecular dynamics (MD) simulations were carried out using AMBER20 (GPU-accelerated pmemd.cuda) with the AMBER force field; systems of ~100,000 atoms were solvated with TIP3P water and neutralised with Na+/Cl- ions. Each simulation consisted of energy minimisation (22,000 steps), NPT heating (200 ps), NPT equilibration (1 ns), NVT equilibration (10 ns), and 200 ns NVT production. Binding free energies were estimated using MM-PBSA (single-trajectory approach, last 50 ns of each trajectory) with entropy contributions calculated via the interaction entropy method. TD-DFT absorption spectra were calculated at the M06-2X/6-31+G(d) level with implicit SMD solvation (water) using Gaussian 16. Synthesis: Photocoxibs PC4, PC5, and PC6 were synthesised using Mills reaction (for arylazopyrazole PC4) and Mills reaction with mCPBA oxidation (for azobenzene-based PC5 and PC6), combined with Chan–Lam N-arylation (PC4) or Suzuki–Miyaura cross-coupling (PC5, PC6). All compounds were characterised by 1H, 13C{1H}, 19F NMR (including COSY, HSQCed, HMBC, NOESY), IR (ATR), and HRMS (ESI). Photochemical characterisation: UV-vis absorption spectra were recorded in DMSO:H2O mixtures on an Agilent HP 8453 spectrophotometer (10 mm quartz cells, room temperature). Trans–cis and cis–trans photoisomerization were induced using a Hg lamp (365 nm), Nd:YAG laser third harmonic (355 nm), LEDs (365 or 405 nm), and CW diode lasers (405 or 445 nm). Photostationary state compositions were determined by 1H NMR in methanol-d4 and DMSO-d6. Photoisomerization quantum yields were measured by an actinometric method using 1,2-bis(2-methyl-5-phenyl-3-thienyl)perfluorocyclopentene in hexane as reference. Thermal cis-to-trans half-lives were obtained from monoexponential fits of time-dependent absorbance decays in the dark at 25 °C. Enzyme inhibition assays: COX-1 and COX-2 inhibitory activity was measured using Abcam fluorometric COX Inhibitor Screening Kits (COX-1: ab204698; COX-2: ab283401), following the manufacturer's protocol. Compounds were tested at 45 µM under dark (trans) and UV-irradiated (365 nm, 3 min, PSSt-c) conditions. Fluorescence was monitored kinetically for 10 min (Ex/Em = 535/587 nm) on a BioTek Synergy H1 reader. Macrophage cell culture: J774A.1 murine macrophages were maintained in DMEM without phenol red (supplemented with L-glutamine, HEPES, penicillin/streptomycin, FBS, and sodium pyruvate) at 37 °C, 5% CO2. Cell viability was assessed by MTT assay. COX-1 inhibition was evaluated by AA stimulation (15 µM, 30 min) after 15-min preincubation with compounds (10 µM). COX-2 inhibition was evaluated by LPS stimulation (10 µg mL-1, 24 h) in the presence of compounds (0.1–10 µM). PGE2 levels in supernatants were quantified by ELISA (Cayman Chemical). IC50 values were calculated using a sigmoidal dose-response equation (variable slope) in GraphPad Prism 9. Statistical analysis: one-way ANOVA with Bonferroni post hoc correction. Zebrafish experiments: Zebrafish (Danio rerio) larvae were maintained at the University of Milan according to EU Directive 2010/63/EU and Italian Decree No. 26/2014. Acute inflammation was induced by tailfin amputation at 2 days post-fertilisation. Larvae were treated for 6 h post-amputation with DMSO (vehicle), CEL, or photocoxibs (10, 50, or 100 µM) under dark (trans) or UV-irradiated (365 nm, PSSt-c) conditions. Leukocyte recruitment was assessed by Sudan Black staining and manual counting. Statistical analysis: one-way ANOVA with Tukey post hoc correction using GraphPad Prism. Methods for Processing the Data: Computational: MD trajectories were analysed for RMSD and key residue–ligand distances using AMBER analytical tools. MM-PBSA energies were computed with the AmberTools module, using the interaction entropy method (code from Cruz & Pérez-Sánchez, Zenodo 2023) for entropic contributions. Photochemical: UV-vis spectra of cis isomers were estimated by linear deconvolution of photostationary state spectra using the trans-isomer spectra and NMR-derived PSS compositions. Quantum yields were calculated following the actinometric procedure of Lees (Anal. Chem. 1996). Biological: Fluorescence traces from COX enzyme assays were blank-subtracted and averaged across technical replicates. Endpoint RFU values were used to calculate % inhibition (equation 4 of Supporting Information); propagated errors were calculated using equation 5. PGE2 ELISA data were fitted with GraphPad Prism (sigmoidal dose-response, variable slope) to obtain IC50 values. Instruments, Calibration, and Standards Information: NMR spectra were referenced to residual solvent signals: CDCl3 (1H: 7.26 ppm; 13C: 77.16 ppm) and acetone-d6 (1H: 2.05 ppm; 13C: 29.9 ppm). HRMS was performed by ESI on a Q-TOF instrument. UV-vis photoisomerization quantum yields were calibrated against 1,2-bis(2-methyl-5-phenyl-3-thienyl)perfluorocyclopentene in hexane (ring-closing Φ = 0.59; ring-opening Φ = 0.013). Melting points were measured on a LLG-Melting point meter MPM-H2 (uncorrected). IR spectra were recorded with a Golden Gate Single Refraction Diamond ATR accessory. Environmental or Experimental Conditions: UV-vis measurements were conducted at ambient temperature (~25 °C) in DMSO:H2O mixtures (75:25 for PC4; 50:50 for PC5; 40:60 for PC6, v/v) in 10 mm quartz cells. Cell culture experiments were conducted at 37 °C, 5% CO2. Zebrafish larvae were maintained at 28 °C in E3 medium. Thermal cis-to-trans back-isomerization half-lives were determined at 25 °C in the respective DMSO:H2O mixtures. Quality-Assurance Procedures Performed on the Data: All synthesised compounds were characterised by at least 1H and 13C NMR, IR, and HRMS before use in biological studies. NMR purity was assessed from integrated spectra. Enzyme inhibition assays included blank wells (no enzyme, no inhibitor) and enzyme-only controls on every plate; reference inhibitors (SC-560 for COX-1, celecoxib for COX-2) were run in parallel to validate each assay. COX fluorescence data are reported as the mean of two technical replicates. Cell culture PGE2 data represent the mean ± SEM of three independent experiments run in triplicate wells. Zebrafish experiments were conducted in three independent experiments; statistical significance was assessed by one-way ANOVA with Tukey correction. MD simulations were run for 200 ns and stability was verified by RMSD and key distance analysis before MM-PBSA calculations were performed on the last 50 ns.