John J. Venit

An integrated approach to the selection of optimal salt form for a new drug candidate

Abstract A general method was developed to select the optimal salt form for BMS-180431, a novel HMG-CoA reductase inhibitor and a candidate for oral dosage form development, in an expeditious manner at the onset of the drug development process. The physicochemical properties such as hygroscopicity, physical stability of crystal forms at different humidity conditions, aqueous solubility, and chemical stability of seven salts e.g., sodium, potassium, calcium, zinc, magnesium, arginine and lysine, were studied using a multi-tier approach. The progression of studies among different tiers was such that the least time-consuming experiments were conducted earlier, thus saving time and effort. A ‘go/no go’ decision was made after each tier of testing the salts, thus avoiding generation of extensive data on all available salt forms. The hygroscopicities of all BMS-180431 salts were evaluated at tier 1 and four salts (sodium, potassium, calcium and zinc) were dropped from consideration due to excessive moisture uptake within the expected humidity range of pharmaceutical manufacturing plants (30–50% R.H. at ambient temperature). The remaining three salts were subjected to the tier 2 evaluation for any change in their crystal structures with respect to humidity and the determination of their aqueous solubilities in the gastrointestinal pH range. The magnesium salt was dropped from further consideration due to humidity-dependent changes in its crystal structure and low solubility in water (3.7 mg/ml at room temperature). Arginine and lysine salts, which were resistant to any change in their crystalline structures under extremes of humidity conditions (6 and 75% R.H.) and had high aqueous solubilities (> 200 mg/ml), were elevated to tier 3 for the determination of their chemical stability. Based on solid state stability of these two salts under accelerated conditions (temperature, humidity, and presence of excipients), consideration of ease of synthesis, ease of analysis, potential impurities, etc., and input from the marketing group with respect to its preference of counter ion species, the arginine salt was selected for further development. The number of tiers necessary to reach a decision on the optimal salt form of a compound may depend on the physicochemical properties studied and the number of salts available. This salt selection process can be completed within 4–6 weeks and be easily adopted in the drug development program.

Synthesis of allysine ethylene acetal using phenylalanine dehydrogenase from Thermoactinomyces intermedius.

Allysine ethylene acetal [(S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (2)] was prepared from the corresponding keto acid by reductive amination using phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedius ATCC 33205. Glutamate, alanine, and leucine dehydrogenases, and PDH from Sporosarcina species (listed in order of increasing effectiveness) also gave the desired amino acid but were less effective. The reaction requires ammonia and NADH. NAD produced during the reaction was recyled to NADH by the oxidation of formate to CO(2) using formate dehydrogenase (FDH). PDH was produced by growth of T. intermedius ATCC 33205 or by growth of recombinant Escherichia coli or Pichia pastoris expressing the Thermoactinomyces enzyme. Using heat-dried T. intermedius as a source of PDH and heat-dried Candida boidinii SC13822 as a source of FDH,98%, but production of T. intermedius could not be scaled up. Using heat-dried recombinant E. coli as a source of PDH and heat-dried Candida boidinii 98%. In a third generation process, heat-dried methanol-grown P. pastoris expressing endogenous FDH and recombinant Thermoactinomyces98% ee.

Biocatalytic preparation of a chiral synthon for a vasopeptidase inhibitor: enzymatic conversion of N2-[N-Phenylmethoxy)carbonyl] L-homocysteinyl]- L-lysine (1- > 1′)-disulfide to [4S-(4I,7I,10aJ)] 1-octahydro-5-oxo-4-[phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester by a novel L-lysine ϵ-aminotransferase

[4S-(4I,7I,10aJ)]1-Octahydro-5-oxo-4-[phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester (BMS-199541-01) is a key chiral intermediate for the synthesis of Omapatrilat (BMS-186716), a new vasopeptidease inhibitor under development. By using a selective enrichment culture technique we have isolated a strain of Sphingomonas paucimobilis SC 16113, which contains a novel L-lysine ϵ-aminotransferase. This enzyme catalyzed the oxidation of the ϵ-amino group of lysine in the dipeptide dimer N2-[N[phenyl-methoxy)-carbonyl] L-homocysteinyl] L-lysine)1,1-disulphide (BMS-201391-01) to produce BMS-199541-01. The aminotransferase reaction required α-ketoglutarate as the amino acceptor. Glutamate formed during this reaction was recycled back to α-ketoglutarate by glutamate oxidase from Streptomyces noursei SC 6007. Fermentation processes were developed for growth of S. paucimobilis SC 16113 and S. noursei SC 6007 for the production of L-lysine ϵ-amino transferase and glutamate oxidase, respectively. L-lysine ϵ-aminotransferase was purified to homogeneity and N-terminal and internal peptides sequences of the purified protein were determined. The mol wt of L-lysine ϵ-aminotransferase is 81 000 Da and subunit size is 40 000 Da. L-lysine ϵ-aminotransferase gene (lat gene) from S. paucimobilis SC 16113 was cloned and overexpressed in Escherichia coli. Glutamate oxidase was purified to homogeneity from S. noursei SC 6003. The mol wt of glutamate oxidase is 125 000 Da and subunit size is 60 000 Da. The glutamate oxiadase gene from S. noursei SC 6003 was cloned and expressed in Streptomyces lividans. The biotransformation process was developed for the conversion of BMS-201391-01 to BMS-199541-01 by using L-lysine ϵ-aminotransferase expressed in E. coli. In the biotransformation process, for conversion of BMS-201391-01 (CBZ protecting group) to BMS-199541-01, a reaction yield of 65–70 M% was obtained depending upon reaction conditions used in the process. Phenylacetyl or phenoxyacetyl protected analogues of BMS-201391-01 also served as substrates for L-lysine ϵ-aminotransferase giving reaction yields of 70 M% for the corresponding BMS-199541-01 analogs. Two other dipeptides N-[N[(phenylmethoxy)carbonyl]-L-methionyl]-L-lysine (BMS-203528) and N,2-[S-acetyl-N-[(phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine (BMS-204556) were also substrates for L-lysine ϵ-aminotransferase. N-α-protected (CBZ or BOC)-L-lysine were also oxidized by L-lysine ϵ-aminotransferase.

A chemoselective, acid mediated conversion of amide acetal to oxazole: The key step in the synthesis of cardiovascular drug, ifetroban sodium

Abstract The cyclization of acetal amide was carried out with trimethylsilyl trifluoromethanesulfonate, followed by elimination using sodium methoxide to give 2,5-disubstituted oxazole, thus completing a new route to the cardivascular drug ifetroban sodium.

REGIO- AND STEREOSELECTIVE RING OPENING OF EPOXIDE WITH CYANOGUANIDINE DIANIONS, A FACILE SYNTHESIS OF THE KATP OPENER BMS-180448

Abstract BMS-180448, (3S,4R)- 2 , was prepared in 63% yield via a facile method which involved a new regio- and stereoselective ring opening of epoxide (3S,4S)- 12 with the potassium dianion of cyanoguanidine 11 .

Integrating a Raman Microscope into the Workflow of a High-Throughput Crystallization Laboratory

Optimizing the choice of polymorphs of an active pharmaceutical ingredient (API) has become a significant step in the drug development process. High-throughput combinatorial techniques have been developed to reduce the time required to identify and select the best form of an API. A very important part of the high-throughput crystallization (HTC) workflow is reliable integration of information produced by various analytical instruments and easy access to the information by multiple scientists. Raman spectroscopy has become one of the key analytic techniques used to differentiate between the polymorphs and salts of the API. A Raman microscope has been integrated into an HTC workflow analyzing samples in a 96-well microtiter plate. The control software on the Raman instrument has been modified to open a text file prepared by the HTC software suite to initiate semiautomated analysis of the samples in a specified well plate. All of the data acquisition and spectral analysis is performed by the Raman instrument including various chemometric techniques for classifying and clustering the samples based on their Raman spectra. The final analytical results and spectra are then formatted and saved for easy entry into the central database (SQL LIMS). An HTC software suite has been developed in-house for the HTC laboratory, which includes routines for display and manipulation of the combined information.