(60) Continuation-in-part of application No. 10/634,483, filed on Aug. 5, 2003. Continuation-in-part of application No. 10/078,949, filed on Feb. 20, 2002, which is a continuation of application No. 09/479,783, filed on Jan. 7, 2000, which is a division of application No. 08/870,608, filed on Jun. 6, 1997, now Pat. No. 6,107,094, which is a continuation-in-part of application No. 08/659, 440, filed on Jun. 6, 1996, now Pat. No. 5,898,031.

Potent small molecule antagonists of the urotensin receptor are described. These inhibitors were derived via systematically deconstructing a literature inhibitor to understand the basic pharmacophore and key molecular features required to inhibit the protein receptor. The series of benzylamine and benzylsulfone antagonists herein reported display a combination of nanomolar molecular and cellular potency as well as acceptable in vitro permeability and metabolic stability.

Sodium-hydrogen exchanger isoform 1 (NHE1) is a ubiquitously expressed transmembrane ion channel responsible for intracellular pH regulation. During myocardial ischemia, low pH activates NHE1 and causes increased intracellular calcium levels and aberrant cellular processes, leading to myocardial stunning, arrhythmias, and ultimately cell damage and death. The role of NHE1 in cardiac injury has prompted interest in the development of NHE1 inhibitors for the treatment of heart failure. This report outlines our efforts to identify a compound suitable for once daily, oral administration with low drug-drug interaction potential starting from NHE1 inhibitor sabiporide. Substitution of a piperidine for the piperazine of sabiporide followed by replacement of the pyrrole moiety and subsequent optimization to improve potency and eliminate off-target activities resulted in the identification of N-[4-(1-acetyl-piperidin-4-yl)-3-trifluoromethyl-benzoyl]-guanidine (60). Pharmacological evaluation of 60 revealed a remarkable ability to prevent ischemic damage in an ex vivo model of ischemia reperfusion injury in isolated rat hearts.

A 270-membered library of trisubstituted ureas was synthesized and evaluated for inhibition of soluble epoxide hydrolase. Library design and reagent selection was guided by the use of a pharmacophore model and synthesis of the array was enabled with a general solid-phase method. This array approach facilitated multi-dimensional SAR around this series and identified functionality responsible for binding affinity, as well as opportunities for modulating the overall in vitro profiles of this class of soluble epoxide hydrolase inhibitors.

A series of potent nicotinamide inhibitors of soluble epoxides hydrolase (sEH) is disclosed. This series was designed using structure-based deconstruction and a combination of two HTS hit series, resulting in hybrid analogs that retained the optimal potency from one series, and acceptable in vitro metabolic stability from the other. Structure-guided optimization of these analogs gave rise to nanomolar inhibitors of human sEH that had acceptable plasma exposure to qualify them as probes to determine the in vivo phenotypic consequences of sEH inhibition.

Inhibition of sEH is hypothesized to lead to an increase in epoxyeicosatrienoic acids resulting in the potentiation of their anti-inflammatory and vasodilatory effects. In an effort to explore sEH inhibition as an avenue for the development of vasodilatory and cardio- or renal-protective agents, a lead identified through high-throughput screening was optimized, guided by the determination of a solid state co-structure with sEH. Replacement of potential toxicophores was followed by optimization of cell-based potency and ADME properties to provide a new class of functionally potent sEH inhibitors with attractive in vitro metabolic profiles and high and sustained plasma exposures after oral administration in the rat.

Inhibition of soluble epoxide hydrolase (sEH) is hypothesized to lead to an increase in circulating levels of epoxyeicosatrienoic acids, resulting in the potentiation of their in vivo pharmacological properties. As part of an effort to identify inhibitors of sEH with high and sustained plasma exposure, we recently performed a high throughput screen of our compound collection. The screen identified N-(3,3-diphenyl-propyl)-nicotinamide as a potent inhibitor of sEH. Further profiling of this lead revealed short metabolic half-lives in microsomes and rapid clearance in the rat. Consistent with these observations, the determination of the in vitro metabolic profile of N-(3,3-diphenyl-propyl)-nicotinamide in rat liver microsomes revealed extensive oxidative metabolism and a propensity for metabolite switching. Lead optimization, guided by the analysis of the solid-state costructure of N-(3,3-diphenyl-propyl)-nicotinamide bound to human sEH, led to the identification of a class of potent and selective inhibitors. An inhibitor from this class displayed an attractive in vitro metabolic profile and high and sustained plasma exposure in the rat after oral administration.

Soluble epoxide hydrolase (sEH) has been demonstrated to play a role in a number of physiological processes through its ability to convert epoxy fatty acids such as the highly active epoxyeicosatrienoic acids (EETs) into their less active vicinal diols. Because EETs exhibit potentially beneficial effects in various pathologies such as inflammation and hypertension, an sEH inhibitor could exert a therapeutic benefit by preventing their hydrolysis. We have developed a competitive binding fluorescence polarization screening assay for sEH inhibitors which utilizes a di‐alkylurea active site inhibitor linked to a rhodamine probe. The probe binds to various mammalian sEH enzymes with Kd values in the low nanomolar range. Inhibitor compounds were initially fit to yield IC50 values. However, using that simple fitting paradigm it was not possible to distinguish the affinities of more potent compounds. To address this, a sophisticated fitting algorithm was developed which enabled the use of more information from the binding curves and the calculation of Kd values for the inhibitors.

Nucleosides have been widely used in the treatment of viral diseases, but relatively few have been identified as inhibitors of hepatitis C virus (HCV). The modified ribonucleosides, 2'-C-methyl-adenosine and 2'-O-methyl-cytidine, are potent inhibitors of HCV replication which specifically target the NS5B polymerase. Herein, a more extensive characterization of the effect of these compounds upon HCV replication in subgenomic replicons is reported. A highly selective antireplicative effect induced by the nucleosides in replicon-containing cell lines was maintained during an exponential growth period with potencies which paralleled the reduction of both positive- and negative-strand RNA replication. Moreover, the inhibitory effect closely correlated with the intrinsic metabolic properties of differing replicon clonal lines. Interestingly, while 2'-C-methyl-adenosine elicited similar inhibitory potencies in different cell lines, 2'-O-methyl-cytidine was found to be inactive in one replicon cell line tested, although the corresponding triphosphates comparably inhibited the in vitro activity of replication complexes isolated from these cells and the activity of NS5B polymerase using synthetic templates. The lack of antireplicative effect, attributed to poor intracellular conversion of the 2'-O-methyl-cytidine nucleoside to the active 5'-triphosphate, was reversed using a monophosphate prodrug. Thus, although replicon cells are useful for evaluating the effect of inhibitors upon HCV replication, these findings have important implications for their use in the identification and characterization of nucleosides and other chemotherapeutic agents requiring cellular metabolism.

Improving cellular uptake and biodistribution remains one of the major obstacles for a successful and broad application of peptide nucleic acids (PNAs) as antisense therapeutics. Recently, we reported the identification and functional characterization of an antisense PNA, which redirects splicing of murine CD40 pre-mRNA. In this context, it was discovered that a simple octa(l-lysine) peptide covalently linked to the PNA is capable of promoting free uptake of the conjugate into BCL1 cells as well as primary murine macrophages. On the basis of this peptide motif, the present study aimed at identifying the structural features, which define effective peptide carriers for cellular delivery of PNA. While the structure-activity relationship study revealed some clear correlations, only a few modifications actually led to an overall improvement as compared to the parent octa(l-lysine) conjugate. In a preliminary PK/tissue distribution study in healthy mice, the parent conjugate exhibited relatively broad tissue distribution and only modest elimination via excretion within the time frame of the study.