The kinetic deuterium isotope effect as applied to metabolic deactivation of imatinib to the des-methyl metabolite, CGP74588

There has recently been a burgeoning interest in impeding drug metabolism by replacing hydrogen atoms with deuterium to invoke a kinetic isotope effect. Imatinib, a front-line therapy for both chronic myeloid leukemia and of gastrointestinal stromal tumours, is often substantially metabolised via N-demethylation to the less active CGP74588. Since deuterium-carbon bonds are stronger than hydrogen-carbon bonds, we hypothesised that the N-trideuteromethyl analogue of imatinib might be subject to a reduced metabolic turnover as compared to imatinib and lead to different pharmacokinetic properties, and hence improved efficacy, in vivo. Consequently, we investigated whether the N-trideuteromethyl analogue would maintain target inhibition and show a reduced propensity for N-demethylation in both in vitro assays with liver microsomes and following oral administration to rats. The N-trideuteromethyl compound exhibited similar activity as a tyrosine kinase inhibitor as imatinib and similar efficacy as an antiproliferative in cellular assays. In comparison to imatinib, the trideuterated analogue also showed reduced N-demethylation upon incubation with both rat and human liver microsomes, consistent with a deuterium isotope effect. However, the reduced in vitro metabolism did not translate into increased exposure of the N-trideuteromethyl analogue following intravenous administration of the compound to rats and no significant difference was observed for the formation of the N-desmethyl metabolite from either parent drug

Enantioselective esterification of racemic cyanohydrins and enantioselective hydrolysis or transesterification of cyanohydrin esters by lipases (Enzyme-catalyzed reactions ; 7)

In der vorliegenden Arbeit berichten wir zusammenfassend über die enantioselektive Hydrolyse oder enantioselektive Umesterung racemischer Cyanhydrinester und über die enantioselektive Veresterung racemischer Cyanhydrine mittels Lipasen.Pure cyanohydrin enantiomers (S)-1/(R)-1 and their O-acyl derivatives (R)-3/(S)-3 are obtained from three different lipasecatalyzed reactions: i) enantioselective hydrolysis of aliphatic and aromatic racemic cyanohydrin esters 3, ii) enantioselective acylation of racemic cyanohydrins 1, iii) enantioselective transesterification of 3 with primary alcohols. Both the cyanohydrin esters and the free cyanohydrins (which are prone to racemization) are isolated as enantiomers with high optical purity on a preparative scale. Hydrolysis of the racemic butyrates 3b, e with Candida cylindracea lipase and pseudomonas sp. lipase, respectively, for example, affords the free (S)-cyanohydrins (S)-2-hydroxypentanenitril [(S)-1a] and (S)-mandelonitrile [(S)-1b)] in high yield with 97 and 96% ee, respectively. (S)-1a is obtained with the same optical purity by candida sp. lipase-catalyzed transesterification of 3b with 1-octanol

Dipeptide nitrile inhibitors of cathepsin K.

A series of dipeptidyl nitriles as inhibitors of cathepsin K have been explored starting from lead structure 1 (Cbz-Leu-NH-CH2-CN, IC50 = 39 nM). Attachment of non-natural amino acid side chains in P1 and modification of the P3 subunit led to inhibitors with higher potency and improved pharmacokinetic properties

Exploring subtype selectivity and metabolic stability of a novel series of ligands for the benzodiazepine binding site of the GABAA receptor

A novel series of agonists at the benzodiazepine binding site of the GABAA receptor was prepared by functionalizing a known template. Adding substituents to the pyrazolone-oxygen of CGS-9896 led to a number of compounds with selectivities for either α2- or α1-containing GABAA receptor subtypes offering an entry into indications such as anxiety and insomnia. In this communication, structure-activity relationship and efforts to increase in vitro stabilities are discussed

On the importance of metabolic stability to achieve high oral exposures for cyclic peptides

Following up on a previous publication in which we reported a high liver first pass effect in rats for the cyclic peptide (1) [Ala-Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro], we decided to investigate the type of metabolites formed and to suggest solutions to this problem. As a result of a bile duct cannulation study in rats and subsequent derivatization of this peptide by an isolated Cyp-enzyme, several hydroxylated variants were identified. Cyclopropyl-Ala (Cpa) residues as surrogates for Leu alleviated metabolism at these particular side chains. Significant progress was achieved, when in addition the D-Pro residue was exchanged by 4,4 difluoro-D-Pro (DiF-D-Pro). Albeit the Ala was kept constant in this process, in the corresponding in-vivo studies in rats, peptide (6) [Ala-Cpa-NMe-D-Cpa-NMe-Cpa-Cpa-4,4 difluro-D-Pro] exhibited M exposures at 3mg/kg and an absolute oral bioavailability of > 90%. Thus, we conclude the Cpa- and DiF-D-Pro residues are metabolically stable isosteres for Leu, and D-Pro respectively

Biocatalytic synthesis and structure elucidation of cyclized metabolites of the deacetylase inhibitor Panobinostat (LBH589)

Panobinostat (LBH589) is a novel pan-deacetylase inhibitor that is currently being evaluated in clinical phase III trials for treatment of Hodgkin’s lymphoma and multiple myeloma. Under catalysis of recombinant human cytochrome P450 3A4 and 2D6 co-expressed with human P450 reductase in E. coli JM109, five metabolites of panobinostat were produced via whole cell biotransformation. The structures of the metabolites were elucidated with the spectroscopic methods MS and NMR and revealed an oxidative cyclization of the ethyl-amino-group to the methylindole moiety. The MS2 spectrum of a cyclized metabolite showed a base peak, where the closed ring is reopened and that – taken as sole base for structure proposals - would have lead to wrong conclusions. The metabolites were substantially less potent deacetylase inhibitors than the parent compound

Increasing metabolic stability via the deuterium kinetic isotope effect: an example from a proline-amide-urea aminothiazole series of phosphatidylinositol-3 kinase alpha inhibitors

In vitro metabolic identification studies with a PI3K-α inhbitor lead molecule 1 identified a single predominant site of oxidative metabolism to be occuring within a tert.butyl moiety. Modification of the tert.butyl group within the lead molecule 1, to the corresponing d9-tert.butyl analogue 2, led to an increase in both the in vitro and in vivo metabolic stability. This increase in metabolic stability resulted in a 2-fold increase in the oral bioavailability measured in the rat, and a 3-fold increase in potency in a chronic in vivo study in the mouse, for 2 when compared to 1

Micropreparative isolation and NMR structure elucidation of metabolites of a drug candidate from rat bile and urine

LC-MS based drug metabolism studies are effective in the optimization stage of drug discovery for rapid partial structure identification of metabolites. However, these studies usually do not provide unambiguous structural characterization of all metabolites, due to the limitations of MS-based structure identification. LC-MS-SPE-NMR is a more powerful approach that allows complete structure identification of compounds from mixtures. LC-MS-SPE-NMR of compounds in complex in vivo samples (such as bile samples from drug metabolism studies) is difficult due to the presence of high concentrations of interfering endogenous components, and potentially also dosage vehicle components (e.g. polyethylene glycols). Here, we describe the isolation and structure characterization of seven metabolites of a drug development candidate in bile and urine samples from a routine low dose bile-duct cannulated rat metabolism study. The structure of all seven compounds could be determined, resulting in much higher information content than common LC-MS structure characterization studies

Discovery and profiling of a selective and efficacious Syk inhibitor

We describe the discovery of the potent and selective Syk inhibitor 11 which showed favorable PK profiles in rat and dog and was found to be active in a collagen induced arthritis model in rats. Compound 11 was selected for further profiling but, unfortunately, in GLP toxicological studies it showed liver findings in rat and dog. Consequently, compound 11 had to be abandoned. Nevertheless, 11 might be a valuable tool compound to study the biology of Syk in vitro and in vivo

Discovery and Characterization of the Topical Soft JAK Inhibitor CEE321 for Atopic Dermatitis.

The JAK kinases JAK1, JAK2, JAK3, and TYK2 play key roles in cytokine signaling. Activation of the JAK/STAT pathways is linked to many diseases involving the immune system, including atopic dermatitis. As systemic JAK inhibitor pharmacology is associated with side effects, topical administration to the skin has been considered to locally restrict the site of action. Several orally bioavailable JAK inhibitors repurposed for topical use have been recently approved or are in clinical development. Here, we disclose our clinical candidate CEE321, which is a potent pan JAK inhibitor in enzyme and cellular assays. In contrast to repurposed oral drugs, CEE321 does not display high potency in blood and has a high clearance in vivo. Therefore, we consider CEE321 to be a "soft drug". When applied topically to human skin that was stimulated with the cytokines IL4 and IL13 ex vivo, CEE321 potently inhibited biomarkers relevant to atopic dermatitis