OPTIMIZING SYNTHESIS AND IN VIVO EVALUATION OF MEDS433: ADVANCING TO PRECLINICAL DEVELOPMENT IN ACUTE MYELOID LEUKEMIA

In recent years, Drug Discovery and Clinic s.r.l., a spin-off at UniTo, developed a novel class of human Dihydroorotate Dehydrogenase (hDHODH) inhibitors based on hydroxypyrazolo[1,5-a]pyridine scaffold and MEDS433 is emerged as a potent pro-apoptotic agent in AML cells, since the inhibition of hDHODH can restore differentiation of leukemic cells independent of their oncogenic driver.1 The MEDS433 high potency on isolated enzyme (IC50hDHODH:1.2 nM) is translated into powerful effects at the cellular level on leukemic cells.2 Considering the encouraging results obtained, MEDS433 stands out as the most favourable candidate for advancement into the non-clinical development phase as investigational-new-drug (IND). AIM Considering the necessity for pivotal non-clinical study methodologies to accurately reflect materials intended for First-in-Human clinical trial submission, and recognizing the significant impact of synthesis of a compound on cost, scalability, and environmental impact, the current phase offers an ideal window for enhancing the synthetic route. This refinement will also serve in the next pre-clinical development steps, as a guidance for Contract-Manufacturing-Organizations (CMOs) for the Good-Manufacturing-Practice synthesis of the IND. In addressing these challenges, we aimed to optimize the synthetic route and overcome obstacles hindering scalability and efficiency. METHODS The first synthetic scheme for the synthesis of the drug candidate, showed in Scheme 1, consists of nine steps with an overall yield of 5%. While suitable for small-scale production, this method presented limitations for large-scale manufacturability. We evalueted the synthetic scheme in terms of cost-effectiveness and production time. Our objective was to explore novel reactions aimed at improving yields and adopting purification techniques that minimize the process waste, by-products and reduce the number of chromatography steps promoting product isolation through precipitation or crystallization. RESULTS The primary drawback included the challenging four-step process to synthesize the 2-hydroxypyrazolo[1,5-a]pyridine core 1, often yielding low conversion rates (14% over 4 steps). Its isolation required a large amount of solvent and two different purification steps by flash chromatography, rendering the process also time-consuming. Another critical point of the scheme is the amide bond formation reaction that posed a critical challenge in the synthesis process. The poor nucleophilicity of the used aniline necessitated the use of AlMe3 as the activation reagent, leading to a concomitant migration of the protecting group from the O- to the endocyclic -N1 position. This results in a major difficulty in the final deprotection step. Additionally, the final purification step was optimized by replacing the hydrogenation step and facilitating product isolation through precipitation. Notably, the elimination of palladium usage is significant, as its inclusion in the certified synthesis's final stage would necessitate supplementary purification steps and verification that residual palladium levels have been quantified and kept within acceptable limits (the Permitted Daily Exposure is 100 μg/day).3 The optimization of these steps, led to a more practical synthesis of MEDS433 in multi-gram quantities. This optimization involved the removal of three chromatographic steps and scaling up each reaction step by a factor of 5, ultimately increasing the overall yield of the scheme from 5% to 14%. As a result, approximately 10 grams of compound could be synthesized within approximately three weeks of work. This advancement has permitted significant in vivo studies. We assessed the anti-tumour ability of MEDS433 in two AML xenograft models yielding promising results comparable to other hDHODH inhibitors currently in clinical trials. Additionally, a pharmacokinetic study was conducted to evaluate the compound's main pharmacokinetic proprieties. Furthermore, acute and sub-acute toxicity studies were carried out, revealing no obvious signs of distress (inappetence, social isolation, antalgic postures, ect.) or significant alterations in body weight for the three doses analysed up to 1000 mg/Kg, suggesting a favourable safety profile of the drug candidate. CONCLUSION Through process improvements and optimization of various steps in the synthetic scheme, a more practical and cost-effective synthesis of MEDS433 in multi-gram quantities has been achieved. The new generation scheme allowed the production of 10 grams of product needed to perform in vivo efficacy, pharmacokinetics and toxicity studies. Moving forward, our focus will be on industrial-scale synthesis development in compliance with Good Manufacturing Practices (GMP), alongside toxicity tests in compliance with Good Laboratory Practices (GLP), to progress along the preclinical development pathway.