Back to the future, the need for early stage green optimization of drug candidates: PIGAs as a case study

The increasing environmental impact of pharmaceutical manufacturing scheme highlights the need for greener synthetic strategies in the development of active pharmaceutical ingredients. N , N -disubstituted-2-arylindol-3-ylglyoxylamides, known as PIGAs, represent a promising class of TSPO ligands with potential therapeutic relevance in inflammatory and neurodegenerative disorders. In this work, a multilevel approach for green optimization of PIGAs synthesis is presented. The Chem21 toolkit in the first-pass mode was used to identify critical issues in the reported synthesis. Instead, the Chem21 solvent selection guide was updated with relevant recent information, and cyclopentyl methyl ether was selected as a safer and more sustainable alternative to diethyl ether and toluene, while a telescopic approach was developed to avoid isolation of the intermediate. For the model PIGA compound 1 , the optimized procedure allowed us to obtain the target product in higher yield (54%) reducing at the same time the overall process mass intensity (PMI) by 98%. Extension of the method to compounds 2 and 3 showed that compound-specific optimization was required. For compound 2 , reduction of 22% in total PMI and 44% in hazardous solvent were achieved, while compound 3 was obtained in 65% yield, with 81% reduction in total PMI and a decrease of the most impactful parameters of the Chem21 toolkit, marked as red flags. In terms of PMI, for the tested compounds the new approach compares favourably to the literature one even using the “PMI calculator” app. Given the emerging role of TSPO as a therapeutic target for metabolic syndrome, compound 2 was evaluated in Caco-2 cells exposed to palmitate and lipopolysaccharide as an in vitro model of early metabolic alteration. In this context, 2 counteracted ZO-1 reduction and attenuated NLRP3 inflammasome activation, as shown by the reduction in ASC and caspase-1 expression as well as IL-1β release. Overall, these findings demonstrate that Chem21 help guide multilevel optimization approaches, improving the sustainability of PIGA synthesis and suggest that TSPO modulation may represent a promising strategy to counteract intestinal barrier impairment and enteric inflammation.