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The preclinical pharmacodynamic and pharmacokinetic properties of 4-methylbenzyl (3S, 4R)-3-fluoro-4-[(Pyrimidin-2-ylamino) methyl] piperidine-1-carboxylate (CERC-301), an orally bioavailable selective N-methyl-D-aspartate (NMDA) receptor subunit 2B (GluN2B) antagonist, were characterized to develop a translational approach based on receptor occupancy (RO) to guide CERC-301 dose selection in clinical trials of major depressive disorder. CERC-301 demonstrated high-binding affinity (K i, 8.1 nmol L(-1)) specific to GluN2B with an IC 50 of 3.6 nmol L(-1) and no off-target activity. CERC-301 efficacy was demonstrated in the forced swim test with an efficacy dose (ED 50) of 0.3-0.7 mg kg(-1) (RO, 30-50%); increase in locomotor activity was observed at ED 50 of 2 mg kg(-1), corresponding to an RO of 75%. The predicted 50% RO concentration (Occ50) in humans was 400 nmol L(-1), similar to that predicted for rat, dog, and monkey (300, 200, and 400 nmol L(-1), respectively). Safety pharmacology and neurotoxicity studies raised no specific safety concerns. A first-in-human study in healthy males demonstrated a dose-proportional pharmacokinetic profile, with T max of ~1 h and t 1/2 of 12-17 h. Based on the preclinical and pharmacodynamic data, doses of ≥8 mg in humans are hypothesized to have an acceptable safety profile and result in clinically relevant peak plasma exposure.

We have automated in vitro dose-inhibition assays to evaluate newly synthesized chemical entities and to rapidly produce and disseminate the results with minimal personnel. A variety of assay methods were automated using the Tecan Genesis Workstation to produce ten-point titration curves and generate reproducible KI values. Our integrated software package provides the unique ability to simultaneously inventory and schedule compounds into various in vitro assays. A worklist generator, in combination with a Gemini script automatically makes variable pre-dilutions of compounds, retrieves incubation plates from the Carousel using the RoMa Arm, performs serial dilutions of compounds within the final 96 well incubation plate using disposable tips, and adds essential assay reagents. Our “Expert System for Data Analysis” automatically retrieves data from a reader (e.g. TopCount, FLIPR or VIPR), analyzes the data based on a set of rules, and provides value added reports. An overview of the basic workflow and the integrated software for support of the drug discovery process is described. The ability of the Tecan Robot to produce dose-inhibition profiles is illustrated and validated by using a radioligand binding assay and a DMSO solubilized fluorescein dye method.

We have automated in vitro dose-inhibition assays to evaluate newly synthesized chemical entities and to rapidly produce and disseminate the results with minimal personnel. A variety of assay metho...

A novel series of benzamidines was synthesized and shown to exhibit NR2B-subtype selective NMDA antagonist activity. Compound 31 is orally active in a carrageenan-induced rat hyperalgesia model of pain and shows no motor coordination side effects.

The synthesis and pharmacological evaluation of 5 (L-738, 167), a potent, selective non-peptide fibrinogen receptor antagonist is reported. Compound 5 inhibited the aggregation of human gel-filtered platelets with an IC50 value of 8 nM and was found to be33000-fold less effective at inhibiting the attachment of human endothelial cells to fibrinogen, fibronectin, and vitronectin than it was at inhibiting platelet aggregation. Ex vivo platelet aggregation was inhibited by85% 24 h after the oral administration of 5 to dogs at 100 micrograms/kg. The extended pharmacodynamic profile exhibited by 5 appears to be a consequence of its high-affinity binding to GPIIb/IIIa on circulating platelets and suggests that 5 is suitable for once-a-day dosing.

Potency enhancing features of two series of fibrinogen receptor antagonists were combined to give analogs with improved potency and oral activity. Antagonists containing either alkyl or aryl sulfonamides and a central isoindolinone structural constraint demonstrate high affinity for both activated and unactivated platelet receptors.

The physiologic role of several transglutaminases could be more precisely defined with the development of specific inhibitors for these enzymes. In addition, specific plasma transglutaminase (fXIIIa) inhibitors may have therapeutic utility in the treatment of thrombosis. For these purposes, the inactivation of fXIIIa and human erythrocyte transglutaminase (HET) by 2-[(2-oxopropyl)thio]imidazolium derivatives, which comprise a novel class of transglutaminase inactivators, was studied. As a specific example, 1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride (III) inactivated fXIIIa with an apparent second-order rate constant (specificity constant of inactivation) of 6.3 x 10(4) M-1 s-1, corresponding to a rate 4 x 10(7) times greater than its reaction rate with glutathione (GSH). The mechanism of fXIIIa inactivation by this class of compounds was investigated utilizing two [14C]-isotopic regioisomers of 1,3-dimethyl-2-[(2-oxopropyl)thio]imidazolium iodide (II). Structural analyses demonstrated that acetonylation of the active site cysteinyl residue of fXIIIa occurred along with the stoichiometric release of the complementary fragment of the inactivator as the corresponding thione. Kinetic analysis of the inactivation of fXIIIa by nonquarternary analogs of II and III indicated the formation of a reversible complex between the inactivator and fXIIIa prior to irreversible modification of the enzyme. At 1 mM, III displayed no detectable levels of inhibition or inactivation with several serine proteases and thiol reagent-sensitive enzymes. 2-[(2-Oxopropyl)thio]imidazolium derivatives and the related molecule 2-(1-acetonylthio)-5-methylthiazolo-[2,3]-1,3,4-thiadiazo lium perchlorate (I), when present at the time of clot formation at 1-10 microM, enhanced the rates of tissue plasminogen activator catalyzed clot lysis in vitro. These inactivators prevented the fXIIIa-catalyzed covalent incorporation of alpha 2-antiplasmin into the alpha chain of fibrin and the formation of high molecular weight fibrin alpha chain polymers, providing the basis for the observed enhancements in clot lysis rates.

A novel series of annulated tricyclic compounds was synthesized and evaluated as NMDA/NR2B antagonists. Structure-activity development was directed towards in vitro optimization of NR2B activity and selectivity over the hERG K(+) channel. Preferred compounds were subsequently evaluated for selectivity in an alpha(1)-adrenergic receptor binding counter-screen and a cell-based assay of NR2B activity.

A novel series of cyclic benzamidines was synthesized and shown to exhibit NR2B-subtype selective NMDA antagonist activity. Compound 29 is orally active in a carrageenan-induced rat hyperalgesia model of pain.

The discovery of a novel series of NR2B subtype selective N-methyl-d-aspartate (NMDA) antagonists is reported. Initial optimization of a high-throughput screening lead afforded an aminopyridine derivative 13 with significant NR2B antagonist potency but limited selectivity over hERG-channel and other off-target activities. Further structure−activity studies on the aminoheterocycle moiety and optimization of the carbamate led to the highly potent 2-aminopyrimidine derivative 20j with a significantly improved off-target activity profile and oral bioavailability in multiple species coupled with good brain penetration. Compound 20j demonstrated efficacy in in vivo rodent models of antinociception, allodynia, and Parkinson's disease.

Two classes of 5-substituted benzimidazoles were identified as potent antagonists of the NR2B subtype of the N-methyl-d-aspartate (NMDA) receptor. Selected compounds show very good selectivity versus the NR2A, NR2C, and NR2D subtypes of the NMDA receptor as well as versus hERG-channel activity and alpha(1)-adrenergic binding. Benzimidazole 37a shows excellent activity in the carrageenan-induced mechanical hyperalgesia assay in rats as well as good pharmacokinetic behavior in dogs.

The synthesis and biological evaluation of a series of nonpeptidic small molecule antagonists of the human platelet thrombin receptor (PAR-1) are described. Optimization of the 5-amino-3-arylisoxazole lead resulted in an approximate 100-fold increase in potency. The most potent of these compounds (54) inhibits platelet activation with IC50s of 90 nM against the thrombin receptor agonist peptide (TRAP) and 510 nM against thrombin as the agonist. Further, antagonist 54 fully blocks platelet aggregation stimulated by 1 nM thrombin for 10 min.

Thrombin is the most potent agonist of platelet activation, and its effects are predominantly mediated by platelet thrombin receptors. Therefore, antagonists of the thrombin receptor have potential utility for the treatment of thrombotic disorders. Screening of combinatorial libraries revealed 2 to be a potent antagonist of the thrombin receptor. Modifications of this structure produced 11k, which inhibits thrombin receptor stimulated secretion and aggregation of platelets.

The synthesis and pharmacology of 4, a potent thienothiophene non-peptide fibrinogen receptor antagonist, are reported. Compound 4 inhibited the aggregation of human gel-filtered platelets with an IC50 of 8 nM and demonstrated an 8-fold improvement in affinity for isolated GPIIb/IIIa receptors over analogues possessing an isoindolinone backbone. Flow cytometry studies revealed that the binding of 4 to resting platelets is a diffusion-controlled process (kon = 3.3 x 10(6) M-1 s-1) and that 4 binds to dog and human platelets with comparable affinity (Kd = 0.04 and 0.07 nM, respectively). Ex vivo platelet aggregation in dogs was completely inhibited by an iv dose of 5 microg/kg [corrected], and an oral dose of 50-90 microg/kg [corrected] followed by low daily doses of 10 microg/kg [corrected] was sufficient to maintain approximately 80% inhibition of ex vivo platelet aggregation over several days. Inhibition of ADP-induced platelet aggregation in anesthetized dogs at 77 +/- 7% resulted in a moderate 2.5-fold increase in bleeding time, while complete inhibition (100%) resulted in an approximately 10-min bleeding time. Additional doses were required to increase the bleeding time to the maximum time allowed in the protocol (15 min), thus indicating a potentially useful and safe separation of efficacy and bleeding time.

Hydrochlorothiazide, administered at 1, 3 and 9 mg/kg/day p.o. for 4 days, produced a dose-dependent lowering of plasma potassium and an elevation in plasma renin activity in unanesthetized dogs. When plasma renin activity was suppressed in diuretic-treated dogs by the potent β-adrenergic receptor-blocking drug, timolol (0.5, 2 mg/kg/day p.o. for 4 days), the hypokalemia and hyperreninemia were significantly less pronounced. The data suggest that β-adrenergic blocking drugs can be used to antagonize these side effects of diuretics.