Phenotypic screen identifies calcineurin-sparing FK506 analogs as BMP potentiators for treatment of acute kidney injury.

Acute kidney injury (AKI) is a life-threatening disease with no known curative or preventive therapies. Data from multiple animal models and human studies have linked dysregulation of bone morphogenetic protein (BMP) signaling to AKI. Small molecules that potentiate endogenous BMP signaling should have a beneficial effect in AKI. We performed a high-throughput phenotypic screen and identified a series of FK506 analogs that act as potent BMP potentiators by sequestering FKBP12 from BMP type I receptors. We further showed that calcineurin inhibition was not required for this activity. We identified a calcineurin-sparing FK506 analog oxtFK through late-stage functionalization and structure-guided design. OxtFK demonstrated an improved safety profile in vivo relative to FK506. OxtFK stimulated BMP signaling in vitro and in vivo and protected the kidneys in an AKI mouse model, making it a promising candidate for future development as a first-in-class therapeutic for diseases with dysregulated BMP signaling.

Discovery and Optimization of a Novel Series of Highly Selective JAK1 Kinase Inhibitors.

Janus kinases (JAKs) have been demonstrated to be critical in cytokine signaling and have thus been implicated in both cancer and inflammatory diseases. The JAK family consists of four highly homologous members: JAK1-3 and TYK2. The development of small-molecule inhibitors that are selective for a specific family member would represent highly desirable tools for deconvoluting the intricacies of JAK family biology. Herein, we report the discovery of a potent JAK1 inhibitor, 24, which displays ∼1000-fold selectivity over the other highly homologous JAK family members (determined by biochemical assays), while also possessing good selectivity over other kinases (determined by panel screening). Moreover, this compound was demonstrated to be orally bioavailable and possesses acceptable pharmacokinetic parameters. In an in vivo study, the compound was observed to dose dependently modulate the phosphorylation of STAT3 (a downstream marker of JAK1 inhibition).

Structure guided design of a series of selective pyrrolopyrimidinone MARK inhibitors.

The initial structure activity relationships around an isoindoline uHTS hit will be described. Information gleaned from ligand co-crystal structures allowed for rapid refinements in both MARK potency and kinase selectivity. These efforts allowed for the identification of a compound with properties suitable for use as an in vitro tool compound for validation studies on MARK as a viable target for Alzheimer's disease.

MARK inhibitors: Declaring a No-Go decision on a chemical series based on extensive DMPK experimentation.

Attempts to optimize pharmacokinetic properties in a promising series of pyrrolopyrimidinone MARK inhibitors for the treatment of Alzheimer's disease are described. A focus on physical properties and ligand efficiency while prosecuting this series afforded key tool compounds that revealed a large discrepancy in the rat in vitro-in vivo DMPK (Drug Metabolism/Pharmacokinetics) correlation. These differences prompted an in vivo rat disposition study employing a radiolabeled representative of the series, and the results from this experiment justified the termination of any further optimization efforts.

Delayed and Prolonged Histone Hyperacetylation with a Selective HDAC1/HDAC2 Inhibitor.

The identification and in vitro and in vivo characterization of a potent SHI-1:2 are described. Kinetic analysis indicated that biaryl inhibitors exhibit slow binding kinetics in isolated HDAC1 and HDAC2 preparations. Delayed histone hyperacetylation and gene expression changes were also observed in cell culture, and histone acetylation was observed in vivo beyond disappearance of drug from plasma. In vivo studies further demonstrated that continuous target inhibition was well tolerated and efficacious in tumor-bearing mice, leading to tumor growth inhibition with either once-daily or intermittent administration.

Direct determination of the ratio of unbound fraction in plasma to unbound fraction in microsomal system (fu p/fu mic) for refined prediction of phase I mediated metabolic hepatic clearance.

At the drug discovery stage, in vivo metabolic hepatic clearance (CL(hep)) is commonly predicted using in vitro parent compound disappearance data generated in liver microsomes or hepatocytes. Correction for the unbound fraction of a compound in the in vitro system and in plasma/serum is known to be critical for the accuracy of metabolic clearance predictions. Discrete generation of these required experimental parameters can be laborious. Herein, we describe a straightforward and direct approach to obtain the ratio of unbound fraction in plasma (fu(p)) to unbound fraction in the microsomal system (fu(mic)) of a small molecule compound using equilibrium dialysis. Experimental conditions were optimized with respect to incubation time, temperature, and plate shaking speed. Results obtained from this system were validated for a set of test compounds by comparison to individually measured fu(p) and fu(mic) data using ultracentrifugation. The correlation for fu(p)/fu(mic) between the two methods for a set of 23 data points was very good with R(2) of 0.94, slope of 1.05 and an intercept of 0.007. The impact of microsomal binding on predicted CL(hep) was illustrated for a tightly bound compound using a series of incubations with increasing concentration of monkey liver microsomal protein. Alteration of this experimental parameter profoundly affected calculated CL(hep) using the well-stirred model. Significant differences were observed in the prediction when the model was corrected for fu(p) only; in contrast, the model corrected for plasma protein and microsomal protein binding predicted clearance values independent of the microsomal protein concentration.

Exploring the pharmacokinetic properties of phosphorus-containing selective HDAC 1 and 2 inhibitors (SHI-1:2).

We report the preparation and structure-activity relationships of phosphorus-containing histone deacetylase inhibitors. A strong trend between decreasing phosphorus functional group size and superior mouse pharmacokinetic properties was identified. In addition, optimized candidates showed tumor growth inhibition in xenograft studies.

Parallel medicinal chemistry approaches to selective HDAC1/HDAC2 inhibitor (SHI-1:2) optimization.

The successful application of both solid and solution phase library synthesis, combined with tight integration into the medicinal chemistry effort, resulted in the efficient optimization of a novel structural series of selective HDAC1/HDAC2 inhibitors by the MRL-Boston Parallel Medicinal Chemistry group. An initial lead from a small parallel library was found to be potent and selective in biochemical assays. Advanced compounds were the culmination of iterative library design and possess excellent biochemical and cellular potency, as well as acceptable PK and efficacy in animal models.

SAR profiles of spirocyclic nicotinamide derived selective HDAC1/HDAC2 inhibitors (SHI-1:2).

A potent family of spirocyclic nicotinyl aminobenzamide selective HDAC1/HDAC2 inhibitors (SHI-1:2) is profiled. The incorporation of a biaryl zinc-binding motif into a nicotinyl scaffold resulted in enhanced potency and selectivity versus HDAC3, but also imparted hERG activity. It was discovered that increasing polar surface area about the spirocycle attenuates this liability. Compound 12 induced a 4-fold increase in acetylated histone H2B in an HCT-116 xenograft model study with acute exposure, and inhibited tumor growth in a 21-day efficacy study with qd dosing.

Phenylglycine and phenylalanine derivatives as potent and selective HDAC1 inhibitors (SHI-1).

An HTS screening campaign identified a series of low molecular weight phenols that showed excellent selectivity (>100-fold) for HDAC1/HDAC2 over other Class I and Class II HDACs. Evolution and optimization of this HTS hit series provided HDAC1-selective (SHI-1) compounds with excellent anti-proliferative activity and improved physical properties. Dose-dependent efficacy in a mouse HCT116 xenograft model was demonstrated with a phenylglycine SHI-1 analog.

Identification of novel metabolites of colchicine in rat bile facilitated by enhanced online radiometric detection.

Three novel conjugation metabolites of colchicine were identified in rat bile facilitated by enhanced on-line liquid chromatography-accurate radioisotope counting. The known 2- and 3-demethylcolchicines (DMCs) underwent O-sulfate conjugation in addition to the previously described O-glucuronidation. 2-DMC was preferably O-glucuronidated, whereas 3-DMC predominantly yielded O-sulfation conjugates, indicating phase II conjugation regiopreferences. Moreover, M1 was identified as a novel glutathione conjugate and a possible biotransformation pathway for its formation was proposed. The known 2-DMC (M6), 3-DMC (M7), 2-DMC glucuronide (M4), and novel 3-DMC sulfate (M3) were confirmed as the major metabolites. Radiometric data were acquired by the XFlow liquid chromatography-accurate radioisotope counting (XFlow LC-ARC) system, a novel technology for dynamic control of both on-column and postcolumn high-performance liquid chromatography flow rates to maximize sensitivity and resolution of radiochromatograms. A comparative evaluation was also performed between the XFlow LC-ARC system and a conventional flow radiometric detection system using bile samples from an in vivo disposition study of colchicine in male Sprague-Dawley rats. Results demonstrated a 20-fold sensitivity improvement of the XFlow LC-ARC system in comparison with radioactivity detection by conventional flow scintillation analyzers. The dynamic flow mode also provided the best chromatographic resolution. Unambiguous metabolite identification was performed by high-resolution mass spectrometry and nuclear magnetic resonance analysis.

Exploration of the internal cavity of histone deacetylase (HDAC) with selective HDAC1/HDAC2 inhibitors (SHI-1:2).

We report herein the initial exploration of novel selective HDAC1/HDAC2 inhibitors (SHI-1:2). Optimized SHI-1:2 structures exhibit enhanced intrinsic activity against HDAC1 and HDAC2, and are greater than 100-fold selective versus other HDACs, including HDAC3. Based on the SAR of these agents and our current understanding of the HDAC active site, we postulate that the SHI-1:2 extend the existing HDAC inhibitor pharmacophore to include an internal binding domain.

The discovery of 6-amino nicotinamides as potent and selective histone deacetylase inhibitors.

This communication highlights the development of a nicotinamide series of histone deacetylase inhibitors within the benzamide structural class. Extensive exploration around the nicotinamide core led to the discovery of a class I selective HDAC inhibitor that possesses excellent intrinsic and cell-based potency, acceptable ancillary pharmacology, favorable pharmacokinetics, sustained pharmacodynamics in vitro, and achieves in vivo efficacy in an HCT116 xenograft model.

Design of novel histone deacetylase inhibitors.

Histone deacetylase (HDAC) inhibitors that target Class I and Class II HDACs are of synthetic and therapeutic interest and ongoing clinical studies indicate that they show great promise for the treatment of cancer. Moreover, Zolinza (vorinostat) was recently approved by the FDA for the treatment of the cutaneous manifestations of cutaneous T-cell lymphoma [Nat. Rev. Drug Disc. 2007, 6, 21]. As part of a broader effort to more fully explore the structure-activity relationships (SAR) of HDAC inhibitors, we sought to identify novel HDAC inhibitor structures through iterative design by utilizing low affinity ligands as synthetic starting points for SAR development. Novel and potent HDAC inhibitors have been identified using this approach and herein we report the optimization of the recognition elements of a novel series of malonyl-derived HDAC inhibitors.

A 13C NMR approach to categorizing potential limitations of alpha,beta-unsaturated carbonyl systems in drug-like molecules.

Compounds that contain an alpha,beta-unsaturated carbonyl moiety are often flagged as potential Michael acceptors. All alpha,beta-unsaturated carbonyl moieties are not equivalent, however, and we sought to better understand this system and its potential implications in drug-like molecules. Measurement of the (13)C NMR shift of the beta-carbon and correlation to in vitro results allowed compounds in our collection to be categorized as potential Michael acceptors, potential substrates for NADPH, or as photoisomerizable.