Macrocycle-stabilization of its interaction with 14-3-3 increases plasma membrane localization and activity of CFTR.

Impaired activity of the chloride channel CFTR is the cause of cystic fibrosis. 14-3-3 proteins have been shown to stabilize CFTR and increase its biogenesis and activity. Here, we report the identification and mechanism of action of a macrocycle stabilizing the 14-3-3/CFTR complex. This molecule rescues plasma membrane localization and chloride transport of F508del-CFTR and works additively with the CFTR pharmacological chaperone corrector lumacaftor (VX-809) and the triple combination Trikafta®. This macrocycle is a useful tool to study the CFTR/14-3-3 interaction and the potential of molecular glues in cystic fibrosis therapeutics.

Design and synthesis of potent, isoxazole-containing renin inhibitors.

The design and optimization of a novel isoxazole S(1) linker for renin inhibitor is described herein. This effort culminated in the identification of compound 18, an orally bioavailable, sub-nanomolar renin inhibitor even in the presence of human plasma. When compound 18 was found to inhibit CYP3A4 in a time dependent manner, two strategies were pursued that successfully delivered equipotent compounds with minimal TDI potential.

Impact of passive permeability and gut efflux transport on the oral bioavailability of novel series of piperidine-based renin inhibitors in rodents.

An oral bioavailability issue encountered during the course of lead optimization in the renin program is described herein. The low F(po) of pyridone analogs was shown to be caused by a combination of poor passive permeability and gut efflux transport. Substitution of pyridone ring for a more lipophilic moiety (logD>1.7) had minimal effect on rMdr1a transport but led to increased passive permeability (P(app)>10 × 10(-6) cm/s), which contributed to overwhelm gut transporters and increase rat F(po). LogD and in vitro passive permeability determination were found to be key in guiding SAR and improve oral exposure of renin inhibitors.

Renin inhibitors for the treatment of hypertension: design and optimization of a novel series of tertiary alcohol-bearing piperidines.

The design and optimization of a novel series of renin inhibitor is described herein. Strategically, by committing the necessary resources to the development of synthetic sequences and scaffolds that were most amenable for late stage structural diversification, even as the focus of the SAR campaign moved from one end of the molecule to another, highly potent renin inhibitors could be rapidly identified and profiled.

Renin inhibitors for the treatment of hypertension: design and optimization of a novel series of pyridone-substituted piperidines.

An SAR campaign aimed at decreasing the overall lipophilicity of renin inhibitors such as 1 is described herein. It was found that replacement of the northern appendage in 1 with an N-methyl pyridone and subsequent re-optimization of the benzyl amide handle afforded compounds with in vitro and in vivo profiles suitable for further profiling. An unexpected CV toxicity in dogs observed with compound 20 led to the employment of a time and resource sparing rodent model for in vivo screening of key compounds. This culminated in the identification of compound 31 as an optimized renin inhibitor.

The discovery and synthesis of potent zwitterionic inhibitors of renin.

The incorporation of a carboxylic acid within in a series of 3-amido-4-aryl substituted piperidines (represented by general structure 32) led to the discovery of potent, zwitterionic, renin inhibitors with improved off-target profiles (CYP3A4 time-dependent inhibition and hERG affinity) relative to analogous non-zwitterionic inhibitors of the past (i.e., 3). Strategies to address the oral absorption of these zwitterions are also discussed within.

Discovery of MK-0952, a selective PDE4 inhibitor for the treatment of long-term memory loss and mild cognitive impairment.

The structure-activity relationship of a novel series of 8-biarylnaphthyridinones acting as type 4 phosphodiesterase (PDE4) inhibitors for the treatment of long-term memory loss and mild cognitive impairment is described herein. The manuscript describes a new paradigm for the development of PDE4 inhibitor targeting CNS indications. This effort led to the discovery of the clinical candidate MK-0952, an intrinsically potent inhibitor (IC(50)=0.6 nM) displaying limited whole blood activity (IC(50)=555 nM). Supporting in vivo results in two preclinical efficacy tests and one test assessing adverse effects are also reported. The comparative profiles of MK-0952 and two other Merck compounds are described to validate the proposed hypothesis.

Addressing time-dependent CYP 3A4 inhibition observed in a novel series of substituted amino propanamide renin inhibitors, a case study.

Time-dependent inhibitors of CYPs have the potential to perpetrate drug-drug interactions in the clinical setting. After finding that several leading compounds in a novel series of substituted amino propanamide renin inhibitors inactivated CYP3A4 in an NADPH-dependent and time-dependent manner, a search to identify the cause of this liability was initiated. Extensive SAR revealed that the amide bridge present in compound 1 as a possible culprit. Through the installation of a metabolic soft spot distal to this moiety, potent renin inhibitors with improved CYP profile were identified.

Design and optimization of a substituted amino propanamide series of renin inhibitors for the treatment of hypertension.

The discovery and SAR of a new series of substituted amino propanamide renin inhibitors are herein described. This work has led to the preparation of compounds with in vitro and in vivo profiles suitable for further development. Specifically, challenges pertaining to oral bioavailability, covalent binding and time-dependent CYP 3A4 inhibition were overcome thereby culminating in the identification of compound 50 as an optimized renin inhibitor with good efficacy in the hypertensive double-transgenic rat model.

Alkyl-bridged substituted 8-arylquinolines as highly potent PDE IV inhibitors.

Substituted 8-arylquinoline analogs bearing alkyl-linked side chain were identified as potent inhibitors of type 4 phophodiesterase. These compounds address the potential liabilities of the clinical candidate L-454560. The pharmacokinetic profile of the best analogs and the in vivo efficacy in an ovalbumin-induced bronchoconstriction assay in conscious guinea pigs are reported.

Substituted 2,2-bisaryl-bicycloheptanes as novel and potent inhibitors of 5-lipoxygenase activating protein.

The discovery and SAR of a novel series of substituted 2,2-bisaryl-bicycloheptane inhibitors of 5-lipoxygenase activating protein (FLAP) are herein described. SAR studies have shown that 2,5-substitution on the exo-aryl group is optimal for potency. The most potent compounds in this series have an ortho-nitrogen aryl linked with a methyleneoxy as the 5-substituent and a polar group such as a urethane as the 2-substituent. One of the most potent compounds identified is the 5-benzothiazolymethoxy-2-pyridinylcarbamate derivative 2 (FLAP IC(50)=2.8 nM) which blocks 89% of ragweed induced urinary LTE(4) production in dogs (at an I.V. dose of 2.5 microg/kg/min). This compound inhibits calcium ionophore stimulated LTB(4) production in both human polymorphonuclear (PMN) leukocytes and human whole blood (IC(50)=2.0 and 33 nM, respectively).

Design, synthesis, and biological evaluation of 8-biarylquinolines: a novel class of PDE4 inhibitors.

The structure-activity relationship of a novel series of 8-biarylquinolines acting as type 4 phosphodiesterase (PDE4) inhibitors is described herein. Prototypical compounds from this series are potent and non-selective inhibitors of the four distinct PDE4 (IC(50)<10 nM) isozymes (A-D). In a human whole blood in vitro assay, they inhibit (IC(50)<0.5 microM) the LPS-induced release of the cytokine TNF-alpha. Optimized inhibitors were evaluated in vivo for efficacy in an ovalbumin-induced bronchoconstriction model in conscious guinea pigs. Their propensity to produce an emetic response was evaluated by performing pharmacokinetic studies in squirrel monkeys. This work has led to the identification of several compounds with excellent in vitro and in vivo profiles, including a good therapeutic window of efficacy over emesis.

Interspecies in vitro metabolism of the phosphodiesterase-4 (PDE4) inhibitor L-454,560.

L-454,560 is a potent phosphodiesterase 4 (PDE4) inhibitor which was identified as a development candidate for the treatment of asthma and chronic obstructive pulmonary disease (COPD). As part of the discovery of this compound, interspecies in vitro metabolism data was generated using liver microsomes and hepatocytes in order to understand the metabolic fate of the compound. In microsomes, metabolism of the 3-methyl-1,2,4-oxadiazole ring was the predominant pathway observed, including ring cleavage. In rat hepatocytes, hydroxylation of the methyl group on the oxadiazole ring and double-bond isomerization were the most abundant metabolites observed. No major species differences were found in terms of microsomal metabolite profiles. The use of LC with UV and MS detection is highlighted, as well as information from tandem mass spectrometry and NMR.

Enantiomer discrimination illustrated by the high resolution crystal structures of type 4 phosphodiesterase.

Type 4 phosphodiesterase (PDE4) inhibitors are emerging as new treatments for a number of disorders including asthma and chronic obstructive pulmonary disease. Here we report the biochemical characterization on the second generation inhibitor (+)-1 (L-, IC50=0.4 nM) and its enantiomer (-)-1 (L-, IC50=43 nM) and their cocrystal structures with PDE4D at 2.0 A resolution. Despite the 107-fold affinity difference, both enantiomers interact with the same sets of residues in the rigid active site. The weaker (-)-1 adopts an unfavorable conformation to preserve the pivotal interactions between the Mg-bound waters and the N-oxide of pyridine. These structures support a model in which inhibitors are anchored by the invariant glutamine at one end and the metal-pocket residues at another end. This model provides explanations for most of the observed structure-activity relationship and the metal ion dependency of the catechol-ether based inhibitors and should facilitate their further design.

Nitrogen-bridged substituted 8-arylquinolines as potent PDE IV inhibitors.

Potent inhibitors of the human PDE IV enzyme are described. Substituted 8-arylquinoline analogs bearing nitrogen-linked side chain were identified as potent inhibitors based on the SAR described herein. The pharmacokinetic profile of the best analog and the in vivo efficacy in an ovalbumin-induced bronchoconstriction assay in conscious guinea pigs are reported.

Discovery of a substituted 8-arylquinoline series of PDE4 inhibitors: structure-activity relationship, optimization, and identification of a highly potent, well tolerated, PDE4 inhibitor.

The discovery and SAR of a new series of substituted 8-arylquinoline PDE4 inhibitors are herein described. This work has led to the identification of several compounds with excellent in vitro and in vivo profiles, including a good therapeutic window of emesis to efficacy in several animal models. Typical optimized compounds from this series are potent inhibitors of PDE4 (IC(50)<1nM) and also of LPS-induced TNF-alpha release in human whole blood (IC(50)<0.5microM). The same compounds are potent inhibitors of ovalbumin-induced bronchoconstriction in conscious guinea pigs (EC(50)<0.1mg/kg ip) but require a dose of about 10mg/kg po in the squirrel monkey to produce an emetic response. From this series of compounds, 23a (L-454,560) was identified as an optimized compound.

Deletion of phosphodiesterase 4D in mice shortens alpha(2)-adrenoceptor-mediated anesthesia, a behavioral correlate of emesis.

A combination of pharmacological and genetic approaches was used to determine the role of type 4 cAMP-specific cyclic nucleotide phosphodiesterase 4 (PDE4) in reversing alpha(2)-adrenoceptor-mediated anesthesia, a behavioral correlate of emesis in non-vomiting species. Among the family-specific PDE inhibitors, PDE4 inhibitors reduced the duration of xylazine/ketamine-induced anesthesia in mice, with no effect on pentobarbital-induced anesthesia. The rank order of the PDE4 inhibitors tested was 6-(4-pyridylmethyl)-8-(3-nitrophenyl)quinoline (PMNPQ) > (R)-rolipram > (S)-rolipram >> (R)-N-[4-[1-(3-cyclopentyloxy-4-methoxyphenyl)-2-(4-pyridyl)ethyl]phenyl]N'-ethylurea (CT-2450). The specific roles of PDE4B and PDE4D in this model were studied using mice deficient in either subtype. PDE4D-deficient mice, but not PDE4B-deficient mice, had a shorter sleeping time than their wild-type littermates under xylazine/ketamine-induced anesthesia, but not under that induced with pentobarbital. Concomitantly, rolipram-sensitive PDE activity in the brain stem was decreased only in PDE4D-deficient mice compared with their wild-type littermates. While PMNPQ significantly reduced the xylazine/ketamine-induced anesthesia period in wild-type mice and in PDE4B-null mice, it had no effect in PDE4D-deficient mice. These findings strongly support the hypothesis that inhibition of PDE4D is pivotal to the anesthesia-reversing effect of PMNPQ and is likely responsible for emesis induced by PDE4 inhibitors.