Isoform-Selective and Stereoselective Inhibition of Hypoxia Inducible Factor-2.

Hypoxia inducible factor (HIF) transcription factors reside at the center of signaling pathways used by mammalian cells to sense and respond to low oxygen levels. While essential to maintain oxygen homeostasis, misregulation of HIF protein activity correlates with tumor development and metastasis. To provide artificial routes to target misregulated HIF activity, we identified small molecule antagonists of the HIF-2 transcription factor that bind an internal cavity within the C-terminal PAS domain of the HIF-2α subunit. Here we describe a new class of chiral small molecule ligands that provide the highest affinity binding, the most effective, isoform-selective inhibition of HIF-2 in cells, and trigger the largest protein conformation changes reported to date. The current results further illuminate the molecular mechanism of HIF-2 antagonism and suggest additional routes to develop higher affinity and potency HIF-2 antagonists.

Electrolytic macrocyclizations: scalable synthesis of a diazonamide-based drug development candidate.

An electrochemical method to synthesize the core macrolactam of diazonamides is described. Large ring-forming dehydrogenation is initiated by anodic oxidation at a graphite surface. The reaction requires no tailoring of the substrate and occurs at ambient temperature in aqueous DMF in an undivided cell open to air. This unique chemistry has enabled a concise, scalable preparation of DZ-2384; a refined analog of diazonamide A slated for clinical development as a cancer therapeutic.

Synthesis and evaluation of [(1R)-1-amino-2-(2,5-difluorophenyl)ethyl]cyclohexanes and 4-[(1R)-1-amino-2-(2,5-difluorophenyl)ethyl]piperidines as DPP-4 inhibitors.

A series of 4-amino cyclohexanes and 4-substituted piperidines were prepared and evaluated for inhibition of DPP-4. Analog 20q displayed both good DPP-4 potency and selectivity against other proteases, while derivative 20k displayed long half life and modest oral bioavailability in rat. The most potent analog, 3-(5-aminocarbonylpyridyl piperidine 53j, displayed excellent DPP-4 activity with good selectivity versus other proline enzymes.

Imidazopiperidine amides as dipeptidyl peptidase IV inhibitors for the treatment of diabetes.

A series of substituted imidazopiperidine amides has been prepared and evaluated for inhibition of dipeptidyl peptidase IV (DPP-4). Substitution at the 1- and 3-positions produced increased selectivity for DPP-4 relative to DPP-8 and DPP-9. Compounds in this series had IC(50) values as low as 5.8 nM for inhibition of DPP-4.

Metabolic activation of fluoropyrrolidine dipeptidyl peptidase-IV inhibitors by rat liver microsomes.

The current study evaluated the potential for two dipeptidyl peptidase-IV (DPP-IV) inhibitor analogs (1S)-1-(trans-4-([(4-trifluoromethoxyphenyl)sulfonyl]amino)cyclohexyl)-2-[(3S)-3-fluoropyrrolidin-1-yl]-2-oxoethanaminium chloride and (1S)-1-(trans-4-([(2,4-difluorophenyl)sulfonyl]amino)cyclohexyl)-2-[(3S)-3-fluoropyrrolidin-1-yl]-2-oxoethanaminium chloride (MRL-A and MRL-B), containing a fluoropyrrolidine moiety in the structure, to undergo metabolic activation. The irreversible binding of these tritium-labeled compounds to rat liver microsomal protein was time- and NADPH-dependent and was attenuated by the addition of reduced glutathione (GSH) or N-acetylcysteine (NAC) to the incubation, indicating that chemically reactive intermediates were formed and trapped by these nucleophiles. Mass spectrometric analyses and further trapping experiments with semicarbazide indicated that the fluoropyrrolidine ring had undergone sequential oxidation and defluorination events resulting in the formation of GSH or NAC conjugates of the pyrrolidine moiety. The bioactivation of MRL-A was catalyzed primarily by rat recombinant CYP3A1 and CYP3A2. Pretreatment of rats with prototypic CYP3A1 and 3A2 inducers (pregnenolone-16alpha-carbonitrile and dexamethasone) enhanced the extent of bioactivation which, in turn, led to a higher degree of in vitro irreversible binding to microsomal proteins (5- and 9-fold increase, respectively). Herein, we describe studies that demonstrate that the fluoropyrrolidine ring is prone to metabolic activation and that GSH or NAC can trap the reactive intermediates to form adducts that provide insight into the mechanisms of bioactivation.

Synthesis of analogs of (1,4)-3- and 5-imino oxazepane, thiazepane, and diazepane as inhibitors of nitric oxide synthases.

A series of 3- and 5-imino analogs from oxazepane, thiazepane, and diazepane was prepared and evaluated as inhibitors of human nitric oxide synthesis (NOS). The most potent iNOS inhibitor was the thiazepane analog 25 (IC(50) = 0.19 microM).

Syntheses and biological evaluation of 5-(piperidin-1-yl)-3-phenyl-pentylsulfones as CCR5 antagonists.

Cellular proliferation of HIV-1 requires the cooperative assistance of both the CCR5 and CD4 receptors. Our medicinal chemistry efforts in this area have resulted in the identification of N-alkyl piperidine sulfones as CCR5 antagonists. These compounds display potent binding and show antiviral properties in HIV-1 spread cell-based assays.

Fluoropyrrolidine amides as dipeptidyl peptidase IV inhibitors.

Amides derived from fluorinated pyrrolidines and 4-substituted cyclohexylglycine analogues have been prepared and evaluated as inhibitors of dipeptidyl dipeptidase IV (DP-IV). Analogues which incorporated (S)-3-fluoropyrrolidine showed good selectivity for DP-IV over quiescent cell proline dipeptidase (QPP). Compound 48 had good pharmacokinetic properties and was orally active in an oral glucose tolerance test in lean mice.

CCR5 antagonists: 3-(pyrrolidin-1-yl)propionic acid analogues with potent anti-HIV activity.

[reaction: see text] A novel approach to alpha,alpha-disubstituted-beta-amino acids (beta(2,2)-amino acids) was employed in the synthesis of a series of 3-(pyrrolidin-1-yl)propionic acids possessing high affinity for the CCR5 receptor and potent anti-HIV activity. The rat pharmacokinetics for these new analogues featured higher bioavailabilities and lower rates of clearance as compared to cyclopentane 1.

Binding of 2-aryl-4-(piperidin-1-yl)butanamines and 1,3,4-trisubstituted pyrrolidines to human CCR5: a molecular modeling-guided mutagenesis study of the binding pocket.

The results of investigations in these laboratories of 2-aryl-4-(piperidin-1-yl)butanamines and 1,3,4-trisubstituted pyrrolidines as human CCR5 antagonists have recently been disclosed. To facilitate further development of these antagonists, we have developed a pharmacophore model based on the structure-activity relationships (SAR) and a human CCR5 receptor docking model using the crystal structure of rhodopsin as a template [Palczewski, K., et al. (2000) Science 289, 739-745]. Guided by the receptor docking model, we have mapped the compounds' site of interaction with CCR5 using site-directed mutagenesis experiments. Our results are consistent with a binding site for the two series that is located within a cavity near the extracellular surface formed by transmembrane helices 2, 3, 6, and 7. This site is overlapping yet distinct from that reported for another antiviral agent which binds to CCR5 [Dragic, T., et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 5639-5644].

1,3,4-Trisubstituted pyrrolidine CCR5 receptor antagonists: modifications of the arylpropylpiperidine side chains.

The 4-(3-phenylprop-1-yl)piperidine moiety of the 1,3,4-trisubstituted pyrrolidine CCR5 antagonist 1 was modified with electron deficient aromatics as well as replacement of the benzylic methylene with sulfones, gem-difluoromethylenes and alcohols in an effort to balance the antiviral potency with reasonable pharmacokinetics.