Reductions in log P improved protein binding and clearance predictions enabling the prospective design of cannabinoid receptor (CB1) antagonists with desired pharmacokinetic properties.

Several strategies have been employed to reduce the long in vivo half-life of our lead CB1 antagonist, triazolopyridazinone 3, to differentiate the pharmacokinetic profile versus the lead clinical compounds. An in vitro and in vivo clearance data set revealed a lack of correlation; however, when compounds with <5% free fraction were excluded, a more predictable correlation was observed. Compounds with log P between 3 and 4 were likely to have significant free fraction, so we designed compounds in this range to give more predictable clearance values. This strategy produced compounds with desirable in vivo half-lives, ultimately leading to the discovery of compound 46. The progression of compound 46 was halted due to the contemporaneous marketing and clinical withdrawal of other centrally acting CB1 antagonists; however, the design strategy successfully delivered a potent CB1 antagonist with the desired pharmacokinetic properties and a clean off-target profile.

Insight into the mechanism of inactivation of ribonucleotide reductase by gemcitabine 5'-diphosphate in the presence or absence of reductant.

Gemcitabine 5'-diphosphate (F(2)CDP) is a potent inhibitor of ribonucleotide reductases (RNRs), enzymes that convert nucleotides (NDPs) to deoxynucleotides and are essential for DNA replication and repair. The Escherichia coli RNR, an alpha2beta2 complex, when incubated with 1 equiv of F(2)CDP catalyzes the release of two fluorides and cytosine concomitant with enzyme inactivation. In the presence of reductant (thioredoxin/thioredoxin reductase/NADPH or DTT), the enzyme inactivation results from its covalent labeling of alpha with the sugar of F(2)CDP (one label/alpha2beta2). SDS-PAGE analysis of the inactivated RNR without boiling of the sample reveals that alpha migrates as an 87 and 110 kDa protein in a ratio of 0.6:0.4. When the reductant is omitted, RNR is inactivated by loss of the essential tyrosyl radical and formation of a new radical. Inactivation studies with C225S-alpha in the presence or absence of reductants, reveal it behaves like wt-RNR in the absence of reductant. Inactivated C225S-alpha migrates as an 87 kDa protein and is not covalently modified. C225 is one of the cysteines in RNR's active site that supplies reducing equivalents to make dNDPs. To identify the new radical formed, [1'-(2)H]-F(2)CDP was studied with wt- and C225S-RNR by 9 and 140 GHz EPR spectroscopy. These studies revealed that the new radical is a nucleotide derived with g values of g(x) 2.00738, g(y) 2.00592, and g(z) 2.00230 and with altered hyperfine interactions (apparent triplet collapsed to a doublet) relative to [1'-(1)H]-F(2)CDP. The EPR features are very similar to those we recently reported for the nucleotide radical generated with CDP and E441Q-RNR.

A selective small molecule agonist of the melanocortin-1 receptor inhibits lipopolysaccharide-induced cytokine accumulation and leukocyte infiltration in mice.

It is well established that melanocortins are peptides that have potent anti-inflammatory activity. Recent research has focused on understanding which of the known melanocortin receptors mediates the anti-inflammatory actions of the melanocortins. The aim of this study was to assess the anti-inflammatory activity of a synthetic MC-1R agonist. BMS-470539 is a potent, selective, full agonist of human and murine MC-1R with EC(50) values in a cAMP accumulation assay of 16.8 and 11.6 nM, respectively. BMS-470539 dose-dependently inhibited TNF-alpha-induced activation of a NF-kappaB transcriptional reporter in human melanoma cells, which endogenously express MC-1R. In vivo studies with BMS-470539 demonstrated that subcutaneous administration of BMS-470539 resulted in a dose-dependent inhibition of LPS-induced TNF-alpha production in BALB/c mice. In this model, the compound had an ED(50) of approximately 10 micromol/kg and a pharmacodynamic half-life of approximately 8 h. Pharmacokinetic analysis of the compound indicated that the compound had a t(1/2) of 1.7 h. In a model of lung inflammation, administration of 15 micromol/kg BMS-470539 resulted in a 45% reduction in LPS-induced leukocyte infiltration (an infiltrate comprised primarily of neutrophils). The compound was also effective in a model of delayed-type hypersensitivity, reducing paw swelling by 59%, comparable with that seen with 5 mg/kg dexamethasone. These studies demonstrate that a selective small molecule agonist of the melanocortin-1 receptor is a potent anti-inflammatory agent in vivo and provides compelling evidence for the involvement of this receptor in the modulation of inflammation.

beta-alanine dipeptides as MC4R agonists.

beta-Alanine derivative 2 (IC(50)=16 nM) and related compounds were found to be potent MC4R agonists.

Pre-steady-state and steady-state kinetic analysis of E. coli class I ribonucleotide reductase.

E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates (NDPs) to dNDPs and is composed of two homodimeric subunits: R1 and R2. R1 binds NDPs and contains binding sites for allosteric effectors that control substrate specificity and turnover rate. R2 contains a diiron-tyrosyl radical (Y(*)) cofactor that initiates nucleotide reduction. Pre-steady-state experiments with wild type R1 or C754S/C759S-R1 and R2 were carried out to determine which step(s) are rate-limiting and whether both active sites of R1 can catalyze nucleotide reduction. Rapid chemical quench experiments monitoring dCDP formation gave k(obs) of 9 +/- 4 s(-1) with an amplitude of 1.7 +/- 0.4 equiv. This amplitude, generated in experiments with pre-reduced R1 (3 or 15 microM) in the absence of reductant, indicates that both monomers of R1 are active. Stopped-flow UV-vis spectroscopy monitoring the concentration of the Y(*) failed to reveal any changes from 2 ms to seconds under similar conditions. These pre-steady-state experiments, in conjunction with the steady-state turnover numbers for dCDP formation of 2-14 s(-1) at RNR concentrations of 0.05-0.4 microM (typical assay conditions), reveal that the rate-determining step is a physical step prior to rapid nucleotide reduction and rapid tyrosine reoxidation to Y(*). Steady-state experiments conducted at RNR concentrations of 3 and 15 microM, typical of pre-steady-state conditions, suggest that, in addition to the slow conformational change(s) prior to chemistry, re-reduction of the active site disulfide to dithiol or a conformational change accompanying this process can also be rate-limiting.

Discovery of tyrosine-based potent and selective melanocortin-1 receptor small-molecule agonists with anti-inflammatory properties.

The melanocortin-1 receptor (MC-1R) is a G-protein-coupled receptor involved in inflammation and skin pigmentation. Compound 2 is the first highly potent and selective MC-1R small-molecule agonist reported. Compound 2 showed efficacy in an acute model of inflammation, which has demonstrated the role of MC-1R in modulation of inflammation.

Substituted pyrazolopyridopyridazines as orally bioavailable potent and selective PDE5 inhibitors: potential agents for treatment of erectile dysfunction.

Novel pyrazolopyridopyridazine derivatives have been prepared as potent and selective PDE5 inhibitors. Compound 6 has been identified as a more potent and selective PDE5 inhibitor than sildenafil (1). It is as efficacious as sildenafil in in vitro and in vivo PDE5 inhibition models, and it is orally bioavailable in rats and dogs. The superior isozyme selectivity of 6 is expected to exert less adverse effects in humans when used for erectile dysfunction treatment.