ABSTRACT
Polymer-assisted solution-phase (PASP) parallel library synthesis was used to discover a piperazinyl glutamate pyridine as a P2Y(12) antagonist. Exploitation of this lead provided compounds with excellent inhibition of platelet aggregation as measured in a human platelet rich plasma (PRP) assay. Pharmacokinetic and physiochemical properties were optimized through modifications at the 4-position of the pyridine ring and the terminal nitrogen of the piperazine ring, leading to compound (4S)-4-[({4-[4-(methoxymethyl)piperidin-1-yl]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic acid 47s with good human PRP potency, selectivity, in vivo efficacy, and oral bioavailability. Compound 47s was selected for further preclinical evaluations.
Subject(s)
Piperazines/pharmacokinetics , Platelet Aggregation Inhibitors/pharmacokinetics , Purinergic P2 Receptor Antagonists , Pyridines/pharmacokinetics , Administration, Oral , Adolescent , Adult , Aged , Animals , Biological Availability , CHO Cells , Cricetinae , Cricetulus , Female , Glutamates/chemical synthesis , Glutamates/pharmacokinetics , Humans , Magnetic Resonance Spectroscopy , Male , Mass Spectrometry , Middle Aged , Piperazines/chemical synthesis , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/chemical synthesis , Pyridines/chemical synthesis , Rats , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2Y12 , Structure-Activity Relationship , Young AdultABSTRACT
Efforts to refine the SAR of the piperazinyl-glutamate-pyridines for more potent analogs with improved pharmacokinetic profiles are described. Exploring substituted piperidines and other ring systems at the 4-pyridyl position led to compounds with improved potency and pharmacokinetic properties over candidate I. In particular, compounds 4t and 5t were discovered with a 10-fold improvement over potency and improved pharmacokinetic profiles in both the rat and dog.
Subject(s)
Fibrinolytic Agents/pharmacology , Glutamic Acid/chemical synthesis , Piperidines/chemical synthesis , Platelet Aggregation/drug effects , Purinergic P2 Receptor Antagonists , Pyridines/chemical synthesis , Pyridines/pharmacology , Administration, Oral , Animals , CHO Cells , Cricetinae , Cricetulus , Dogs , Fibrinolytic Agents/chemical synthesis , Fibrinolytic Agents/chemistry , Glutamic Acid/chemistry , Glutamic Acid/pharmacology , Humans , Inhibitory Concentration 50 , Molecular Structure , Piperidines/chemistry , Piperidines/pharmacology , Pyridines/chemistry , Rats , Receptors, Purinergic P2Y12 , Structure-Activity RelationshipABSTRACT
Piperazinyl-glutamate-pyrimidines were prepared with oxygen, nitrogen, and sulfur substitution at the 4-position of the pyrimidine leading to highly potent P2Y12 antagonists. In particular, 4-substituted piperidine-4-pyrimidines provided compounds with exceptional potency. Pharmacokinetic and physicochemical properties were fine-tuned through modifications at the 4-position of the piperidine ring leading to compounds with good human PRP potency, selectivity, clearance and oral bioavailability.
Subject(s)
Fibrinolytic Agents/chemistry , Glutamic Acid/chemistry , Piperidines/chemistry , Platelet Aggregation/drug effects , Purinergic P2 Receptor Antagonists , Pyrimidines/chemistry , Animals , Fibrinolytic Agents/chemical synthesis , Fibrinolytic Agents/pharmacokinetics , Humans , Male , Pyrimidines/chemical synthesis , Pyrimidines/pharmacokinetics , Rats , Rats, Sprague-Dawley , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2Y12 , Structure-Activity RelationshipABSTRACT
Polymer-assisted solution-phase (PASP) parallel library synthesis was used to discover a piperazinyl-glutamate-pyridine as a P2Y(12) antagonist. Exploitation of this lead provided compounds with excellent inhibition of platelet aggregation as measured in a human platelet rich plasma (PRP) assay. Pharmacokinetic and physiochemical properties were optimized leading to compound (4S)-4-[({4-[4-(methoxymethyl)piperidin-1-yl]-6-phenylpyridin-2-yl}carbonyl)amino]-5-oxo-5-{4-[(pentyloxy)carbonyl]piperazin-1-yl}pentanoic acid 22J with good human PRP potency, selectivity, in vivo efficacy and oral bioavailability.
Subject(s)
Glutamic Acid/chemistry , Piperazines/chemistry , Platelet Aggregation Inhibitors/chemistry , Platelet Aggregation/drug effects , Purinergic P2 Receptor Antagonists , Pyridines/chemistry , Administration, Oral , Animals , Biological Availability , Humans , Male , Platelet Aggregation Inhibitors/pharmacokinetics , Platelet Aggregation Inhibitors/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2Y12 , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Structure-Activity RelationshipABSTRACT
3T3-L1 adipocytes have proven difficult to transfect with plasmid-encoded cDNAs or even infect with virally-derived cDNAs. We have developed and characterized a 3T3-L1 adipocyte cell line stably expressing the truncated receptor for coxsackievirus and adenovirus receptor (CAR) for its ability to be infected with adenoviruses at a low multiplicity of infection (m.o.i.). Using green fluorescent protein driven by the cytomegalovirus promoter in adenovirus fiber type 5 we compared infection efficiencies of CAR adipocytes versus the parental 3T3-L1 adipocytes. As assessed by immunofluorescence, CAR adipocytes were infected at approximately 100-fold greater efficiency than regular 3T3-L1 adipocytes. The efficiency of transduction for the CAR adipocytes was >90% at multiplicities of infection of 50 whereas standard adipocytes were poorly transduced even at an m.o.i. of 2000. Since many investigators studying insulin action use 3T3-L1 adipocytes, we compared CAR adipocytes versus regular adipocytes and showed that the two cell lines were similar with respect to insulin stimulation of insulin receptor, MAPK, and Akt phosphorylation and basal- and insulin-stimulated glucose transport. In addition, CAR adipocytes accumulated GLUT4 and SCD1 proteins during the adipogenesis program with the same time course as regular 3T3-L1 adipocytes. Lastly, CAR adipocytes produced and secreted the adipose-specific hormone Acrp30. These data suggest 3T3-L1CARDelta1 adipocytes are virtually indistinguishable from their parental cells, but demonstrate a significant advantage with improved efficiency of adenoviral transduction for gain or deletion of function studies.
Subject(s)
Adenoviridae/genetics , Adipocytes/virology , Receptors, Virus/metabolism , 3T3 Cells , Adipocytes/metabolism , Animals , Cells, Cultured , Coxsackie and Adenovirus Receptor-Like Membrane Protein , Mice , Transduction, GeneticABSTRACT
Anorexia and weight loss are frequent complications of acute and chronic infections and result from induction of cytokines, prostaglandins, and other inflammatory mediators that are critical for pathogen elimination. Selective attenuation of the hypophagic response to infection and maintenance of the production of factors essential for infection control would be a useful addition to antimicrobial therapy in the treatment of human disease. Here, we evaluate the relative contribution of cyclooxygenase (COX)-1- and COX-2-derived prostaglandins to anorexia and weight loss precipitated by systemic immune activation by lipopolysaccharide (LPS). Using COX isoform-selective pharmacological inhibitors and gene knockout mice, we found that COX-2 inhibition during LPS-induced inflammation results in preserved food intake and maintenance of body weight, whereas COX-1 inhibition results in augmented and prolonged weight loss. Regulation of neuropeptide Y, corticotropin-releasing hormone, leptin, and interleukin-6 does not change as a function of COX-2 inhibition after LPS administration. Our data implicate COX-2 inhibition as a therapeutic target to maintain nutritional status while still allowing a normal cytokine response during infection.