A process using an engineered phenylalanine ammonia lyase (PAL) enzyme was developed as part of an alternative route to a key intermediate of olodanrigan (EMA401). In the first part of this report, the detailed results from a screening for the optimal reaction conditions are presented, followed by a discussion of several workup strategies investigated. In the PAL-catalyzed reaction, 70−80% conversion of a cinnamic acid derivative to the corresponding phenylalanine derivative could be achieved. The phenylalanine derivative was subsequently telescoped to a Pictet−Spengler reaction with formaldehyde, and the corresponding tetrahydroisoquinoline derivative was isolated in 60−70% yield with >99.9:0.1 er. On the basis of our screenings, carbonate/ carbamate-buffered ammonia at an NH 3 concentration of 9−10 M and pH 9.5−10.5 was found to be optimal. Enzyme loadings down to 2.5 wt % (E:S = 1:40 w/w) could be achieved, and substrate concentrations between 3−9 v/w (1.17−0.39 M) were found to be compatible with the reaction conditions. A temperature gradient was applied in the final process: a pre-equilibrium was established at 45 °C, before making use of the temperature dependence of the entropy term with subsequent cooling to 20 °C to achieve maximum conversion. This temperature gradient also allowed balancing of the enzyme stability (low at 45 °C, high at 20 °C) with the activity (high at 45 °C, low at 20 °C) in order to achieve optimal conversion (low at 45 °C, high at 20 °C). From the various workup operations investigated, a sequence consisting of denaturation of the enzyme, NH 3 /CO 2 removal by distillation, acidification, and telescoping to the subsequent Pictet−Spengler cyclization was our preferred approach. The process presented in this study is a more sustainable, shorter, and more cost-effective alternative to the previous process.

The development of the late-phase manufacturing process of the drug candidate ZPL389 and the strategies for the control of impurities are outlined in detail. Selective salt formation at several stages was pivotal to controlling the process impurities. The extensive optimization of the N-methylation of a Boc-protected amine with dimethyl sulfate and of a nucleophilic aromatic substitution without the use of metal catalysts led to a robust, scalable process. The process was demonstrated on a >100 kg scale. Overall, improved drug substance quality, higher yield, and reduction of the process mass intensity were achieved.