Modulation of the 2-(1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)acetic acid scaffold to generate COXTRANs: a new class of dual cyclooxygenase inhibitors/thromboxane receptor antagonists

Background: Chronic inflammation-related diseases contribute to over 50% of mortality worldwide, driving the progression of cardiovascular and oncologic disorders.1,2 Both Nonsteroidal anti-inflammatory drugs (NSAIDs) and coxibs are effective in treating inflammation and pain, inhibiting cyclooxygenase (COX)-mediated prostanoid synthesis. Unfortunately, their long-term administration is often associated with cardiovascular risks, especially due to unopposed thromboxane A₂ (TXA₂) activity.3 A dual-target approach inhibiting simultaneously COX and thromboxane receptor (TP) may overcome these limitations and represent a promising strategy for the treatment of inflammation-associated pathologies. Aims: The aim of the project was to design, synthesize, and evaluate novel compounds with dual COX-2 inhibitory and TP receptor antagonistic activity, namely COXTRANs. This project wants to develop safer, and more effective anti-inflammatory agents with balanced in vitro and in vivo activity, improved pharmacokinetics, and reduced risk of gastrointestinal and cardiovascular toxicity. Methods: Starting from the 2-(1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)acetic acid scaffold of etodolac, an FDA-approved NSAID, over 50 analogues were synthesized by modifying positions 1, 6, 7, and 8. Compounds were tested in vitro for their ability to inhibit TP receptor activation and COX-1 and COX-2 activity. The TP receptor antagonism was tested by measuring human platelet aggregation induced by U-46619, while COX-1 and COX-2 inhibition was assessed by quantifying TXB2 and PGE2 in human washed platelets and in isolated lymphomonocyte, respectively. Finally, one selected compound was tested in vivo to investigate the antinociceptive effect in a murine model of CFA-induced inflammatory pain. Results: Biological data from the first-generation compounds (Series I), showed that the substitution pattern greatly influenced the dual activity. The introduction of a fluorine atom at position 6 combined with an ethyl group at position 1 optimized COX-2 inhibition, while lipophilic groups at position 8 enhanced TP receptor antagonism. Compound 20, for example, demonstrated 32-fold stronger TP antagonism than etodolac, with comparable potency and COX-2 selectivity; however, there was a concomitant loss of selectivity toward COX-2. Subsequently, in Series II, aromatic and heteroaromatic moieties were introduced at position 8 to exploit additional interactions. Interestingly, increasing steric hindrance through the introduction of substituted phenyl rings at position 8 furnished more balanced compounds. Specifically, compound 21, demonstrated an IC₅₀ of 0.070 µM for COX-2 and 0.086 µM for TP antagonism, thus resulting in a better balance between the activities. In Series III, a linker was interposed between the indole scaffold and the lipophilic substituent at position 8 to enhance molecular flexibility. Among the synthesized compounds belonging to this series, compound 51 (CXT29), bearing a benzylic substituent, emerged as a lead molecule due to its potent, selective and balanced dual activity. Compound 51 maintained high plasma protein binding (99.6%), moderate aqueous solubility (0.53 mg/mL) and showed an adequate pharmacokinetic profile. After oral administration in mice, compound 51 significantly reduced TXA₂-mediated platelet aggregation in platelet-rich plasma withdrawn 1 hour after treatment, confirming in vivo TP antagonism. In addition, its antinociceptive effect was determined in a mouse model of inflammatory pain induced by intraplantar injection of CFA, compared to the reference diclofenac. In the reported figure, paw withdrawal latencies increased after oral administration of 51 (50 mg/kg) over a period of 1 and 2 hours, compared to latencies 24 h post-CFA injection similarly to what was observed with the reference drug. Conclusion: This study identified a novel class of dual COX-2 inhibitors/TP receptor antagonists (COXTRANs). Compound 51 emerged as the most promising candidate with in vitro and in vivo efficacy, suppressing TXA₂-mediated platelet aggregation and inflammatory pain in mice. The identification of a multitarget drug able to dampen these processes may offer new therapeutic options in cardiovascular pathologies, fibrosis and cancer metastatisation.2,4 Studies aimed at evaluating the cardiovascular safety of COXTRANs and their antimetastatic potential in preclinical models are in progress. References: 1) GBD 2017 Causes of Death Collaborators. Global, regional, and national age-sex-specific mortality for 282 causes of death in 195 countries and territories, 1980-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet 2018, 392, 1736–1788. 2) Lucotti, S.; Cerutti, C.; Soyer, M.; Gil-Bernabé, A. M.; Gomes, A. L.; Allen, P. D.; Smart, S.; Markelc, B.; Watson, K.; Armstrong, P. C.; Mitchell, J. A.; Warner, T. D.; Ridley, A. J.; Muschel, R. J. Aspirin blocks formation of metastatic intravascular niches by inhibiting platelet-derived COX-1/thromboxane A2. J. Clin. Invest. 2019, 129(5), 1845-1862. 3) Patrignani, P.; Patrono, C. Cyclooxygenase inhibitors: From pharmacology to clinical read-outs. Biochim. Biophys. Acta. 2015, 1851(4), 422-432. 4) Wang, Y.; Jia, J.; Wang, F.; Fang, Y.; Yang, Y.; Zhou, Q.; Yuan, W.; Gu, X.; Hu, J.; Yang, S. Pre-Metastatic Niche: Formation, Characteristics and Therapeutic Implication. Signal Transduct Target Ther 2024, 9 (1), 236.