Proton and Repare Therapeutics publish a paper in OPRD, demonstrating the strength of synthesis process development and optimization
Recently, Repare Therapeutics and Porton Pharma Solutions Ltd. published the paper "Scalable Synthesis of Lunresertib, a Selective PKMYT1 Inhibitor" in the authoritative journal "Organic Process Research & Development" (OPRD) of the American Chemical Society (ACS). As a top academic platform in the field of process chemistry, OPRD's inclusion once again confirms Porton's cutting-edge strength in the field of drug synthesis technology - up to now, Porton has had many innovative results published in heavyweight journals in the industry.
As a result of this research, researchers from both parties jointly developed an efficient, robust and scalable synthesis route for Lunresertib (RP-6306), which can support the rapid mass production of this API. The synthetic route includes two palladium-catalyzed coupling reactions, a novel chiral isomer resolution, a one-pot hydration/demethylation sequence, and a recrystallization process to improve intrinsic purity. It systematically demonstrates Porton's outstanding capabilities in process optimization and technology transfer.
PKMYT1 is a regulator of CDK1 phosphorylation. Due to its synthetic lethal relationship with CCNE1 amplification, it has become an important therapeutic target for the treatment of certain DNA damage response types of cancer. Lunresertib is a selective and orally bioavailable PKMYT1 inhibitor currently in Phase 1 clinical trials for the treatment of genetically selected malignancies. Lunresertib drug substance RP-6306 contains a rotation-resistant chiral axis caused by restricted rotation about the C-N bond between the azaindole and dimethylphenol moieties. Only the S-isomer has PKMYT1 activity, while the corresponding R-isomer is inactive. In the early stages of the project, the Porton process R&D team and the Supercritical Fluid Chromatography (SFC) technical team followed the following route and used SFC splitting to provide customers with gram-level RP-6306 for preclinical research. Although this route quickly provides customers with APIs that meet quality requirements for early research, the splitting efficiency of this process is low, the cycle is long, and the cost is high. Therefore, there is an urgent need to re-develop a new process before scaling up the preparation of RP-6306.
It is known that compound 7 has low solubility in common solvents for SFC chromatography, which results in low SFC resolution efficiency. Therefore, in the early stage of process development, Porton Process R&D combined with the new technology platform to conduct Boc derivatization of compound 7 to improve the solubility, thereby more effectively separating the S-enantiomer and deprotecting it to obtain RP-6306. However, this method adds synthesis steps and has limited improvements in splitting efficiency, failing to fundamentally solve the problems of long cycle time and high cost.
Since intermediate 2 contains a basic amino group, we focused our attention on the feasibility of classic chiral salt resolution. After screening about 150 salt/solvent combinations, Porton J-STAR's R&D team finally determined the most promising resolution reagent, chiral acid (R)-CSA, and resolution solvent, anisole. The Porton process R&D team then inspected the crystallization temperature, solvent amount, crystallization aging time, and crystallization times, and confirmed the process parameter range for salt-forming chiral separation.
After identifying a scalable chiral purity improvement method, the Porton Process R&D team explored other steps to further reduce raw material costs. As shown in the original synthetic route, intermediate 2 was obtained via Buchwald–Hartwig coupling of dibromide 3 with aniline 4 followed by cyclization with malononitrile. These two-step reactions can proceed smoothly, and the isolation yield is >70%. The optimization results of the first step are summarized in the following table. Direct substitution methods have been attempted before to synthesize compound 5, but the results were not as effective as palladium-catalyzed reactions. When Pd2(dba)3 is replaced by Pd(OAc)2, the catalyst loading can be reduced to 0.01 equivalents and the amount of base can be reduced to 1.5 equivalents without affecting the reaction conversion rate.
For the second step of the reaction, the Porton Catalyst Screening Platform initially explored alternative catalytic reaction condition screening using copper instead of palladium for azaindole synthesis, but the results were unsatisfactory. Therefore, the Porton process R&D team focuses on the optimization of palladium catalytic reaction conditions. Interestingly, directly reducing the catalyst loading results in incomplete conversion; but when lower amounts of malononitrile are used, the reaction can be completed at lower catalyst equivalents. Since Pd(dppf)Cl2·CH2Cl2 is more stable than PdCl2, the conditions of entry 9 in the following table were finally selected for this step.
For the preparation of RP-6306 from compound 8, the Porton Process R&D team initially carried out a two-step reaction because the final API prepared by the one-pot method only had an HPLC purity of about 96%. In the stepwise method, compound 8 was heated in methanesulfonic acid and water at 40°C for 1 hour and was almost quantitatively converted to compound 1 in 100% yield. Compound 1 was demethylated with DL-methionine in methanesulfonic acid to obtain RP-6306 in 92% yield and 98.8% HPLC purity. However, both reactions require a large amount of methanesulfonic acid, and the quenching process produces a large amount of waste liquid.
In order to reduce the output of waste liquid, the Porton Process R&D team re-explored a one-pot reaction without separating compound 1. First, it was confirmed that the low purity of the product obtained by the one-pot method was caused by dimer 10 and decarboxylation impurity 12. Decarboxylation may occur during demethylation; while dimer 10 is poorly soluble, it cannot be effectively removed from crude RP-6306 by recrystallization. After a series of optimizations, the levels of these two impurities during the reaction were reduced and effectively controlled.
The final process of RP-6306 is shown in the figure below. First, a Buchwald-Hartwig amination reaction, followed by an intramolecular cyclization reaction, affords azaindole 2. On a scale of greater than 100kg of compound 2, (R)-CSA was used for chiral resolution, followed by one round of recrystallization in anisole, to obtain compound 8 in 42% yield and 97.4% chiral purity. Crude RP-6306 was prepared through an optimized one-pot process, and then decolorized and crystallized in methanol to improve the quality of the final API to >99.5% chiral purity and >99% HPLC purity, with an API output of >30 kg.
As the world's leading CDMO company, Porton and Repare Therapeutics jointly published research results in OPRD this time, thanks to its deep accumulation in the field of small molecule drug services for many years - the R&D team's continuous optimization of the process, the in-depth empowerment of new technologies such as metal catalysis and SFC, and the efficient collaboration between multinational teams, jointly promoted the successful large-scale implementation of this process at Porton's production base. This not only demonstrates Porton's full-chain technological advantages from R&D to production, but also confirms its core value in global pharmaceutical cooperation: In the future, we will continue to help customers break through the bottlenecks of new drug research and development through innovative and reliable CMC solutions, so that good drugs can benefit the public earlier.