Discovery of spirofused piperazine and diazepane amides as selective histamine-3 antagonists with in vivo efficacy in a mouse model of cognition.

A new series of potent and selective histamine-3 receptor (H3R) antagonists was identified on the basis of an azaspiro[2.5]octane carboxamide scaffold. Many scaffold modifications were largely tolerated, resulting in nanomolar-potent compounds in the H3R functional assay. Exemplar compound 6s demonstrated a selective profile against a panel of 144 secondary pharmacological receptors, with activity at only σ2 (62% at 10 µM). Compound 6s demonstrated free-plasma exposures above the IC50 (∼50×) with a brain-to-plasma ratio of ∼3 following intravenous dosing in mice. At three doses tested in the mouse novel object recognition model (1, 3, and 10 mg/kg s.c.), 6s demonstrated a statistically significant response compared with the control group. This series represents a new scaffold of H3 receptor antagonists that demonstrates in vivo exposure and efficacy in an animal model of cognition.

Elucidation of a novel bioactivation pathway of a 3,4-unsubstituted isoxazole in human liver microsomes: formation of a glutathione adduct of a cyanoacrolein derivative after isoxazole ring opening.

Studies on the biotransformation of isoxazole rings have shown that molecules containing a C3-substituted isoxazole or a 1,2-benzisoxazole can undergo a two-electron reductive ring cleavage to form an imine. In the absence of a C3 substituent, the isoxazole ring opens via deprotonation of the C3 proton followed by N-O bond cleavage to yield an α-cyanoenol analog. We report the identification of a novel bioactivation pathway of a 3,4-unsubstituted isoxazole in human liver microsomes. After the enzyme-catalyzed cleavage of the 3,4-unsubstituted isoxazole ring of N-((2-isopropyl-7-methyl-1-oxoisoindolin-5-yl)methyl)isoxazole-5-carboxamide (P) in human liver microsomes, the formed α-cyanoenol (M1) condenses with formaldehyde to generate an α,ß-unsaturated Michael acceptor intermediate (a cyanoacrolein derivative, VII), which further reacts with the cysteinyl thiol of glutathione to yield a GSH adduct of a cyanoacrolein derivative (M3). The same adduct also is formed when M1, generated in 0.1 N NaOH aqueous solution, reacts with formaldehyde and GSH. (13)C-labeled methanol was used to confirm that methanol from the drug stock solution was oxidized by liver microsomal enzymes to formaldehyde and the carbon atom from methanol was finally incorporated in the corresponding GSH adduct. The formation of isoxazole ring-opened products (M1 and M2) in human liver microsomes is NADPH-dependent. M1 and M2 were found in human liver microsomes preincubated with 1-aminobenzotriazole (1 mM) and NADPH (5 mM) at ∼ 10% of the levels found in the samples in the absence of 1-aminobenzotriazole, suggesting that this biotransformation pathway is primarily catalyzed by cytochrome P450. The formation of M3 also was inhibited by 1-aminobenzotriazole at a similar level.

Discovery of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides as selective antagonists of the kappa opioid receptor. Part 1.

Initial high throughput screening efforts identified highly potent and selective kappa opioid receptor antagonist 3 (κ IC(50)=77 nM; µ:κ and δ:κ IC(50) ratios>400) which lacked CNS exposure in vivo. Modification of this scaffold resulted in development of a series of 8-azabicyclo[3.2.1]octan-3-yloxy-benzamides showing potent and selectivity κ antagonism as well as good brain exposure. Analog 6c (κ IC(50)=20 nM; µ:κ=36, δ:κ=415) was also shown to reverse κ-agonist induced rat diuresis in vivo.

Novel small molecule inhibitors of caspase-3 block cellular and biochemical features of apoptosis.

Caspase-3 is an intracellular cysteine protease, activated as part of the apoptotic response to cell injury. Its interest as a therapeutic target has led many to pursue the development of inhibitors. To date, only one series of nonpeptidic inhibitors have been described, and these have limited selectivity within the caspase family. Here we report the properties of a series of anilinoquinazolines (AQZs) as potent small molecule inhibitors of caspase-3. The AQZs inhibit human caspase-3 with Ki values in the 90 to 800 nM range. A subset of AQZs are equipotent against caspase-6, although most lack activity against this isoform and caspase-1, -2, -7, and -8. The AQZs inhibit endogenous caspase-3 activity toward a cell permeable, exogenously added substrate in staurosporine-treated SH-SY5Y cells. The AQZs reduce biochemical and cellular features of apoptosis that are thought to be a consequence of caspase-3 activation including DNA fragmentation, TUNEL staining, and the various morphological features that define the terminal stages of apoptotic cell death. Moreover, the AQZs also inhibit apoptosis induced by nerve growth factor withdrawal from differentiated PC12 cells. Thus, the AQZs represent a new and structurally novel class of inhibitors, some of which selectively inhibit caspase-3 and will thereby allow evaluation of the role of caspase-3 activity in various cellular models of apoptosis.