P3 optimization of functional potency, in vivo efficacy and oral bioavailability in 3-aminopyrazinone thrombin inhibitors bearing non-charged groups at the P1 position.

Although the S3 pocket of the thrombin active site is lined with lipophilic amino acid residues, the accommodation of polarity within the lipophilic P3 moiety of small molecule inhibitors is possible provided that the polar functionality is capable of pointing away from the binding pocket outwards toward solvent while simultaneously allowing the lipophilic portion of the P3 ligand to interact with the S3 amino acid residues. Manipulation of this motif provided the means to effect optimization of functional potency, in vivo antithrombotic efficacy and oral bioavailability in a series of 3-aminopyrazinone thrombin inhibitors which contained non-charged groups at the P1 position.

The Dose Proportionality of Telcagepant after Administration of Single Oral and Intravenous Doses in Healthy Adult Subjects.

INTRODUCTION: Telcagepant (MK-0974) is a novel, orally active and selective CGRP receptor antagonist being investigated for acute treatment of migraine. Early clinical data suggested greater than dose proportional increases in exposure following oral administration. The aim of the present studies was to definitively characterize the oral and IV dose proportionality of telcagepant.

The identification of potent, orally bioavailable tricyclic CGRP receptor antagonists.

A series of tricyclic CGRP receptor antagonists was optimized in order to improve oral bioavailability. Attenuation of polar surface area and incorporation of a weakly basic indoline nitrogen led to compound 5, a potent antagonist with good oral bioavailability in three species.

Hydrophilic interaction chromatography/tandem mass spectrometry for the simultaneous determination of three polar non-structurally related compounds, imipenem, cilastatin and an investigational beta-lactamase inhibitor, MK-4698, in biological matrices.

A method coupling hydrophilic interaction chromatography (HILIC) with tandem mass spectrometry (MS/MS) has been developed for the simultaneous determination of three polar non-structurally related compounds--a carbapenem antibiotic, imipenem (IMP), a renal dehydropeptidase inhibitor, cilastatin (CIL), and an investigational beta-lactamase inhibitor, MK-4698 (BLI), in rat plasma, monkey plasma and mouse blood. The analytes were extracted through protein precipitation, chromatographed on a Waters Atlantis HILIC column, and detected on a Sciex API4000 mass spectrometer using a Turbo-Ion Spray ion source in positive ionization mode following multiple-reaction monitoring (MRM). The assay dynamic range was 0.

Elimination of diastereomer interference to determine Telcagepant (MK-0974) in human plasma using on-line turbulent-flow technology and off-line solid-phase extraction coupled with liquid chromatography/tandem mass spectrometry.

To eliminate the diastereomer interference on Telcagepant (MK-0974) determination during clinical study support, on-line high turbulent-flow liquid chromatography (HTLC) methods, HTLC-A and HTLC-B that covered dynamic range of 0.5-500 nM and 5-5000 nM, respectively, were developed. To meet the requirement of rapid assay transfer among multiple laboratories and analysts, a solid-phase extraction (SPE) assay was derived from the existing HTLC-B assay under the same dynamic range.

A flexible and high throughput liquid chromatography-tandem mass spectrometric assay for the quantitation of telcagepant in human plasma.

Telcagepant (MK-0974) is a novel oral calcitonin gene-related peptide (CGRP) receptor antagonist and is currently under clinical development. Results from phases II and III clinical trials have suggested that telcagepant is effective for migraine treatment. A reliable and high throughput protein precipitation (PPT) method for determination of telcagepant in human plasma using liquid chromatography coupled with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry has been developed.

Calcitonin gene-related peptide (CGRP) receptor antagonists: investigations of a pyridinone template.

In our effort to find potent, orally bioavailable CGRP receptor antagonists for the treatment of migraine, a novel series based on a pyridinone template was investigated. After optimizing the privileged structure and the placement of the attached phenyl ring, systematic SAR was carried out on both the N-alkyl and C-5 aryl substituents. Several analogs with good potency and pharmacokinetic profiles were identified.

Potent, orally bioavailable calcitonin gene-related peptide receptor antagonists for the treatment of migraine: discovery of N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1- (2,2,2-trifluoroethyl)azepan-3-yl]-4- (2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin- 1-yl)piperidine-1-carboxamide (MK-0974).

Calcitonin gene-related peptide (CGRP) has been implicated in the pathogenesis of migraine. Herein we describe optimization of CGRP receptor antagonists based on an earlier lead structure containing a (3R)-amino-(6S)-phenylcaprolactam core. Replacement of the phenylimidazolinone with an azabenzimidazolone gave stable derivatives with lowered serum shifts.

Identification of novel, orally bioavailable spirohydantoin CGRP receptor antagonists.

A rapid analogue approach to identification of spirohydantoin-based CGRP antagonists provided novel, low molecular weight leads. Modification of these leads afforded a series of nanomolar benzimidazolinone-based CGRP receptor antagonists. The oral bioavailability of these antagonists was inversely correlated with polar surface area, suggesting that membrane permeability was a key limitation to absorption.

Potent 2-[(pyrimidin-4-yl)amine}-1,3-thiazole-5-carbonitrile-based inhibitors of VEGFR-2 (KDR) kinase.

Pyrimidino-thiazolyl carbonitriles were prepared that are potent VEGFR-2 (KDR) kinase inhibitors. The modification of lead structures resulted in 3m which exhibited the best overall profile in KDR inhibitory activity, iv/po pharmacokinetics, and reduced hERG affinity.

P2 pyridine N-oxide thrombin inhibitors: a novel peptidomimetic scaffold.

In this study, we have demonstrated that the critical hydrogen bonding motif of the established 3-aminopyrazinone thrombin inhibitors can be effectively mimicked by a 2-aminopyridine N-oxide. As this peptidomimetic core is more resistant toward oxidative metabolism, it also overcomes the metabolic liability associated with the pyrazinones. An optimization study of the P(1) benzylamide delivered the potent thrombin inhibitor 21 (K(i) = 3.

9-hydroxyazafluorenes and their use in thrombin inhibitors.

Optimization of a previously reported thrombin inhibitor, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-trans-4-aminocyclohexylmethylamide (1), by replacing the aminocyclohexyl P1 group provided a new lead structure, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (2), with improved potency (K(i) = 0.49 nM for human thrombin, 2x APTT = 0.37 microM in human plasma) and pharmacokinetic properties (F = 39%, iv T(1/2) = 13 h in dogs).

Potent N-(1,3-thiazol-2-yl)pyridin-2-amine vascular endothelial growth factor receptor tyrosine kinase inhibitors with excellent pharmacokinetics and low affinity for the hERG ion channel.

A series of N-(1,3-thiazol-2-yl)pyridin-2-amine KDR kinase inhibitors have been developed that possess optimal properties. Compounds have been discovered that exhibit excellent in vivo potency. The particular challenges of overcoming hERG binding activity and QTc increases in vivo in addition to achieving good pharmacokinetics have been acomplished by discovering a unique class of amine substituents.

Low molecular weight thrombin inhibitors with excellent potency, metabolic stability, and oral bioavailability.

Modification of lead compound 1 by reducing lipophilicity in the P3 group produced a series of low molecular weight thrombin inhibitors with excellent potency in functional assays, metabolic stability, and oral bioavailability. These modifications led to the identification of two optimized compounds, 14 and 16.

Discovery and evaluation of potent P1 aryl heterocycle-based thrombin inhibitors.

In an effort to discover potent, clinically useful thrombin inhibitors, a rapid analogue synthetic approach was used to explore the P(1) region. Various benzylamines were coupled to a pyridine/pyrazinone P(2)-P(3) template. One compound with an o-thiadiazole benzylic substitution was found to have a thrombin K(i) of 0.

Synthesis and evaluation of imidazole acetic acid inhibitors of activated thrombin-activatable fibrinolysis inhibitor as novel antithrombotics.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is an important regulator of fibrinolysis, and inhibitors of this enzyme have potential use in antithrombotic and thrombolytic therapy. Appropriately substituted imidazole acetic acids such as 10j were found to be potent inhibitors of activated TAFI and selective versus the related carboxypeptidases CPA, CPN, and CPM but not CPB. Further, 10j accelerated clot lysis in vitro and was shown to be efficacious in a primate model of thrombosis.

Pharmacokinetic optimization of 3-amino-6-chloropyrazinone acetamide thrombin inhibitors. Implementation of P3 pyridine N-oxides to deliver an orally bioavailable series containing P1 N-benzylamides.

In this manuscript we demonstrate that a modification principally directed toward the improvement of the aqueous solubility (i.e., introduction a P3 pyridine N-oxide) of the previous lead compound afforded a new series of potent orally bioavailable P1 N-benzylamide thrombin inhibitors.

Design and synthesis of a pro-drug of vinblastine targeted at treatment of prostate cancer with enhanced efficacy and reduced systemic toxicity.

Chemotherapy of prostate cancer with antimitotic agents such as vinblastine and doxorubicin is only marginally effective, due to dose-limiting systemic toxicity. Herein we report the development of peptidyl conjugate 5 of the cytotoxic agent vinblastine (1), along with the results of its in vitro and in vivo evaluation as a pro-drug targeted at prostate cancer cells. Prostate-derived tumors are known to produce significant amounts of prostate specific antigen (PSA), a serine protease with chymotrypsin-like properties.

Determination of chiral sulfoxides in plasma by normal-phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

A sensitive and specific assay has been developed and validated for the separation of a chiral sulfoxide drug candidate with simultaneous determination of the corresponding sulfide and sulfone in plasma by normal-phase LC-MS-MS. Separation was achieved on a Chiralpak AD (100 x 2.1 mm) column with a 2-propanol-hexane (80:20) mobile phase within 7 min.

Description and validation of a staggered parallel high performance liquid chromatography system for good laboratory practice level quantitative analysis by liquid chromatography/tandem mass spectrometry.

The Aria LX4 staggered parallel high performance liquid chromatography (HPLC) system is evaluated for application to good laboratory practice (GLP) level quantitative analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS). This system consists of four fully independent binary HPLC pumps, a modified autosampler, and a series of switching and selector valves all controlled by a single computer program. The system improves sample throughput without sacrificing chromatographic separation or data quality.