ABSTRACT
An enantioselective synthesis of the CGRP antagonist BMS-846372, amenable to large scale preparation, is presented. This new synthesis showcases a chemo- and enantioselective reduction of a cyclohepta[b]pyridine-5,9-dione as well as a Pd-catalyzed alpha-arylation reaction to form the key carbon-carbon bond and set the absolute and relative stereochemistry.
Subject(s)
Calcitonin Gene-Related Peptide/antagonists & inhibitors , Heterocyclic Compounds, 4 or More Rings/chemical synthesis , Heterocyclic Compounds, 4 or More Rings/pharmacology , Catalysis , Heterocyclic Compounds, 4 or More Rings/chemistry , Molecular Structure , StereoisomerismABSTRACT
Four synthetic strategies were evaluated towards the preparation of (-)-(3R,4R)-1-benzyl-4-(benzylamino)piperidin-3-ol (1), which was constructed with control over the relative and absolute stereochemistry of the 4,3-amino alcohol moiety. The first strategy employed a novel Rh(I) catalyzed asymmetric hydrogenation, while two other strategies exploited the existing stereochemistry in 2-deoxy-D-ribose, and the fourth explored both biocatalytic and classical resolution techniques as a means to impart enantioenrichment to racemic intermediates en route to targeted structure (-)-1.
Subject(s)
Piperidines/chemical synthesis , Hydrogenation , Models, Molecular , Molecular Structure , StereoisomerismABSTRACT
A continuously-fed crystallization chamber that allows for kinetic path control through the crystallization phase diagram (from labile/nucleation to metastable/growth) was fabricated and used to crystallize lysozyme. A lumped kinetic model was developed, and parameters for heterogeneous nucleation kinetics were determined. Heterogeneous nucleation was found to have faster nucleation kinetics and slower growth kinetics than homogeneous nucleation, as expected. The major contributions of the new device are (1) to allow better control of the chemical environment for studies of crystal nucleation and growth, and (2) to allow lumped-model analysis of those studies to extract kinetic parameters.
Subject(s)
Crystallization/instrumentation , Crystallization/methods , Proteins/chemistry , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methodsABSTRACT
Adenylyl cyclase (AC) superactivation is thought to play an important role in opioid tolerance, dependence, and withdrawal. In the present study, we investigated the involvement of protein kinases in chronic delta-opioid agonist-mediated AC superactivation in Chinese hamster ovary (CHO) cells stably expressing the human delta-opioid receptor (hDOR/CHO). Maximal forskolin-stimulated cAMP formation in hDOR/CHO cells increased by 472 +/- 91, 399 +/- 2, and 433 +/- 73% after chronic treatment with the delta-opioid agonists (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N,N-diethyl benzamide (SNC 80), [d-Pen2,d-Pen5]-enkephalin, and deltorphin II, respectively. Concurrently, chronic SNC 80 (1 micro M, 4-h) treatment augmented 32P incorporation into a 200-kDa protein immunoreactive with the ACV/VI antibody by 300 +/- 60% in hDOR/CHO cell lysates. The calmodulin antagonist calmidazolium significantly attenuated chronic deltorphin II-mediated AC superactivation. Tyrosine kinase (genistein) and protein kinase C (chelerythrine) inhibitors individually had minimal effect on chronic delta-opioid agonist-mediated AC superactivation. Conversely, simultaneous treatment with both genistein and chelerythrine significantly attenuated AC superactivation. Because we showed previously that the Raf-1 inhibitor 3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one (GW5074) attenuates AC superactivation, we hypothesize that parallel calmidazolium-, chelerythrine-, and genistein-sensitive pathways converge at Raf-1 to mediate AC superactivation by phosphorylating AC VI in hDOR/CHO cells.
Subject(s)
Adenylyl Cyclases/metabolism , Benzamides/pharmacology , Piperazines/pharmacology , Protein Kinases/metabolism , Receptors, Opioid, delta/agonists , Animals , CHO Cells , Colforsin/pharmacology , Cricetinae , Cyclic AMP/metabolism , Enzyme Activation/drug effects , Female , Humans , Phosphorylation , Receptors, Opioid, delta/genetics , TransfectionABSTRACT
Chronic delta-opioid receptor agonist treatment of Chinese hamster ovary (CHO) cells stably expressing the human delta-opioid receptor (hDOR/CHO) leads to increased cAMP formation after the removal of the agonist (adenylyl cyclase superactivation). We have previously found that at the same time, chronic delta-opioid receptor agonist treatment augments phosphorylation of the adenylyl cyclase VI isoenzyme. Since phosphorylation of adenylyl cyclase VI by Raf-1 protein kinase was recently shown, we tested the role of Raf-1 in adenylyl cyclase superactivation in hDOR/CHO cells. We found that pretreatment of the cells with the selective Raf-1 inhibitor GW5074 (3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one) (10 microM, 30 min) attenuates chronic deltorphin II-mediated increase in forskolin-stimulated cAMP formation by 40% (n = 6, P < 0.05). Better understanding of the molecular mechanism of adenylyl cyclase superactivation should aid in the development of analgesics that act longer and have fewer side effects.
