Abstract:The non-proteinogenic amino acid 2-(3-hydroxy-1-adamantyl)-(2S)-aminoethanoic acid [2, (S)-3-hydroxyadamantylglycine], is a key intermediate required for the synthesis of Saxagliptin, a dipeptidyl peptidase IV inhibitor under development for treatment of type 2 diabetes mellitus. Keto acid 2-(3-hydroxy-1-adamantyl)-2-oxoethanoic acid (1) was converted to (S)-3-hydroxyadamantylglycine by reductive amination using a phenylalanine dehydrogenase from Thermoactinomyces intermedius expressed in a modified form in Pichia pastoris or Escherichia coli. NAD (nicotinamide adenine dinucleotide) produced during the reaction was recycled to NADH (reduced form of nicotinamide adenine dinucleotide) using formate dehydrogenase. Pichia pastoris produces an endogenous formate dehydrogenase when grown on methanol, and the corresponding gene was cloned and expressed in E. coli. The modified phenylalanine dehydrogenase contains two amino acid changes at the C-terminus and a 12-amino acid extension of the C-terminus. The modified enzyme is more effective with keto acid 1 than the wild-type enzyme, but less effective with the natural substrate, phenylpyruvate. Production of multi-kg batches was originally carried out with extracts of Pichia pastoris expressing the modified phenylalanine dehydrogenase from Thermoactinomyces intermedius and endogenous formate dehydrogenase, and further scaled up using a preparation of the two enzymes expressed in E. coli.
The commercial-scale synthesis of the DPP-IV inhibitor, saxagliptin (1), is described from the two unnatural amino acid derivatives 2 and 3. After the deprotection of 3, the core of 1 is formed by the amide coupling of amino acid 2 and methanoprolinamide 4. Subsequent dehydration of the primary amide and deprotection of the amine affords saxagliptin, 1. While acid salts of saxagliptin have proven to be stable in solution, synthesis of the desired free base monohydrate was challenging due to the thermodynamically favorable conversion of the free amine to the six-membered cyclic amidine 9. Significant process modifications were made late in development to enhance process robustness in preparation for the transition to commercial manufacturing. The impetus and rationale for those changes are explained herein.
A practical synthesis of the antiviral agent lobucavir, [1R-(1r,2β,3r)]-2-amino-9-[2,3-bis(hydroxymethyl)cyclobutyl]-6Hpurin-6-one (BMS-180194), is described. The key chiral intermediate, [1S-(1r,2β,3r)]-3-hydroxy-1,2-cyclobutanedimethanol, dibenzoate ester, was made by an asymmetric [2 + 2] cycloaddition of dimenthyl fumarate with ketene dimethyl acetal followed by sequential diester reduction, benzoylation, deketalization, and stereoselective ketone reduction. Regioselective N9-alkylation of the tetra-n-butylammonium salt of 2-amino-6-iodopurine with the derived cyclobutyltriflate furnished the purinecyclobutyl dibenzoate. Methanolysis followed by acid hydrolysis produced lobucavir in a 35% overall yield with an ee > 99%.
S)-N-(tert-Butoxycarbonyl)-3-hydroxymethylpiperidine 1 was made from (R,S)-3-hydroxymethylpiperidine 2 via fractional crystallization of the corresponding L(-)-dibenzoyl tartarate salt 3 followed by hydrolysis and acylation. Lipase from Pseudomonas cepacia was found to be the best enzyme for the stereospecific resolution of (R,S)-N-(tert-butoxycarbonyl)-3-hydroxymethylpiperidine 4. (S)-N-(tert-Butoxycarbonyl)-3-hydroxymethylpiperidine 1 was obtained in 16% yield and >95% enantiomeric excess (ee) by hydrolysis of (R,S)-acetate 5 by lipase PS from Pseudomonas cepacia. Lipase PS-catalyzed esterification of the (R,S)-N-(tert-butoxycarbonyl)-3-hydroxymethylpiperidine 4 with succinic anhydride provided the Shemisuccinate ester 6, which could be easily separated and hydrolyzed by base to the (S)-N-(tert-butoxycarbonyl)-3-hydroxymethylpiperidine 1. The yield and ee could be improved greatly by repetition of the process. Using the repeated esterification procedure (S)-N-(tert-butoxycarbonyl)-3-hydroxymethylpiperidine 1 was obtained in 32% yield (maximum theoretical yield 50%) and 98.9% ee.
Two well-known methodologies, the Jacobsen asymmetric epoxidation (AE) and the Sharpless asymmetric dihydroxylation (AD) followed by epoxidation, were evaluated for the large-scale preparation of a chiral dihydrobenzofuran epoxide. The AE method was improved by substituting ethanol for dichloromethane for the dissolution of meta-chloroperbenzoic acid (m-CPBA). This change in solvent had a significant impact on scaleability of the AE procedure by preventing crystallization of the m-CPBA during addition to the cold reaction mixture. Factors affecting the enantiomeric excess and yield of the chiral epoxide resulting from AD followed by epoxidation were studied. The Sharpless AD reaction provided the intermediate chiral diol as a solid with high ee (>98.5%). The Sharpless−Kolb conversion of the chiral diol to a chiral epoxide was modified to potassium tert-butoxide/tetrahydrofuran to obtain the product in good yield (74−84%) and high ee (>98%). Both the AE and AD processes were scaled up to prepare large quantities of the chiral epoxide.
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