Racemic Resolution

One of our clients approached us with a request to develop a racemic resolution method for a promising preclinical candidate. The main features of the compound were an aryl group introduced through an organometallic coupling, and a dimethylamino moiety introduced by means of an aminative reduction of a ketone. The compound presented a chiral centre in that position, and initial assays showed that the enantiomer S presented better activity than the R. Chirality was therefore a critical issue.

We were provided with the details of a straightforward route involving two steps; the necessary building block for the coupling was also supplied. A method for the enantioselective synthesis of the compound was being developed, but a classic racemic resolution was required to cover intellectual property claims and allow for faster delivery of bigger batches of enantiomerically pure compound for use in upcoming trials.

We considered three possible approaches to the problem: Approach 1, resolution of the final compound; Approach 2, resolution of the intermediate dimethylamine generated prior to the coupling reaction; and Approach 3, resolution of another intermediate amine suitable for the preparation of the desired compound.

Each approach had its advantages and disadvantages. Approach 1 would be faster since the route was established and the client had developed a chiral HPLC method for the ee determination. However, Approaches 2 and 3 would allow the introduction of an expensive building block in a later stage, therefore reducing costs. Nevertheless, synthetic steps needed to be tested and chiral HPLC methods developed.

We started by exploring Approach 1. First, a fast optimisation of the coupling reaction was performed whilst increasing the scale to grams. The subsequent aminative reduction provided us with the final compound in enough quantities for our study. Applying the usual DoE principles, a short screening was designed using three solvents and five common and inexpensive resolution reagents under standard conditions.

Of the fifteen parallel experiments, nine showed no crystals. In the remaining six experiments, crystals were isolated from the solution and assayed using a chiral HPLC method. One result was promising, with a 73% ee. And although not spectacular in and of itself, it offered us a good starting point.

A further crystallization of the enriched mixture allowed us to produce the compound with an ee higher than 99%. A comparison between the isolated enantiomer and some samples obtained by preparative chiral HPLC revealed that we had separated the incorrect enantiomer. We reproduced the resolution with the resolution agent’s enantiomer, and the conditions, results, ee values and identity of the obtained product were all checked successfully.

Though these results were excellent and obtained in only a few weeks, we pursued the other two approaches with a mind to both containing costs and reducing the mass introduced in the resolution.

Approaches 2 and 3 involved swapping the synthetic steps, with the aminative reduction first. The starting material’s aminative reduction with dimethylamine was troublesome. The reaction was incomplete, and needed harsh conditions and long reaction times. We turned to microwave conditions in order to expedite the reaction and provide enough product for screening. However, development of the chiral HPLC method was also troublesome, and when the samples of a screening study were assayed the results were disappointing. Approach 2 was obviously pushed to the background.

We had been working simultaneously on Approach 3, which involved an aminative reduction with a protected amine to yield a product suitable for the resolution. The reductive amination was carried out using a multigram scale, resulting in excellent yields and purities that provided the desired compound. A chiral HPLC method was promptly developed and the protected amine submitted for a screening study. Taking Approach 1 results into account, only one solvent was chosen and the pool of chiral reagents was expanded to eight.

Of the eight parallel experiments only two revealed an absence of crystals. The remaining six crystals were isolated from the solution and assayed by chiral HPLC. Only one result was promising, however, with a 94% ee. A further crystallization of the enriched mixture allowed us to achieve an ee greater than 99%. The pure enantiomeric product was submitted to a sequence of four reactions to give the final product in a 25% yield and >99% ee.

Since the sequence of reactions from the resolved amine to the final compound was too long, we introduced a small change in the sequence. This allowed us to reduce the process by one step and produce the final compound in a 68% yield and >99% ee. We applied DoE principles, parallelisation techniques and analytical skills to the problem, and offered our client not one, but two solutions to the problem, with a broader range of conditions from which to make a choice. Furthermore, the development of these classic resolution methods addressed intellectual property issues that might arise at the regulatory phase, and also helped to block possible competitor challenges.