Methyl-substitution of an iminohydantoin spiropiperidine β-secretase (BACE-1) inhibitor has a profound effect on its potency.

The IC50 of a beta-secretase (BACE-1) lead compound was improved ∼200-fold from 11 μM to 55 nM through the addition of a single methyl group. Computational chemistry, small molecule NMR, and protein crystallography capabilities were used to compare the solution conformation of the ligand under varying pH conditions to its conformation when bound in the active site. Chemical modification then explored available binding pockets adjacent to the ligand.

Shaping suvorexant: application of experimental and theoretical methods for driving synthetic designs.

Dual Orexin Receptor Antagonists (DORA) bind to both the Orexin 1 and 2 receptors. High resolution crystal structures of the Orexin 1 and 2 receptors, both class A GPCRs, were not available at the time of this study, and thus, ligand-based analyses were invoked and successfully applied to the design of DORAs. Computational analysis, ligand based superposition, unbound small-molecule X-ray crystal structures and NMR analysis were utilized to understand the conformational preferences of key DORAs and excellent agreement between these orthogonal approaches was seen in the majority of compounds examined.

Direct evidence of 2-cyano-2-propoxy radical activity during AIBN-based oxidative stress testing in acetonitrile-water solvent systems.

Oxidative susceptibility testing was performed on a drug substance containing a methoxy-naphthalene moiety. 2,2'-azobisisobutyronitrile (AIBN) was employed to initiate peroxy radical oxidation to mimic autoxidation processes. In acetonitrile (ACN)-water solvents, three major degradation products are formed.

Mechanism of the solution oxidation of rofecoxib under alkaline conditions.

Purpose: The rapid oxidation of rofecoxib under alkaline conditions has been previously reported. The oxidation was reported to involve gamma-lactone ring opening to an alcohol, which further oxidized to a dicarboxyclic acid. The oxidation was suspected to be mediated by peroxy radicals.

Identification and quantitation of extractables from cellulose acetate butyrate (CAB) and estimation of their in vivo exposure levels.

The purpose of this study was to qualitatively and quantitatively determine potential cellulose acetate butyrate (CAB) extractables in a way to meaningfully predict the in vivo exposure resulting from clinical administration. Extractions of CAB-381-20 were performed in several solvent systems, consistently resulting in the detection of three extractables. The extractables have been identified as acetic acid, butyric acid, and E-2-ethyl-2-hexenoic acid (E-EHA) by LC/UV, LC/MS and NMR.

Bioactivation of the 3-amino-6-chloropyrazinone ring in a thrombin inhibitor leads to novel dihydro-imidazole and imidazolidine derivatives: structures and mechanism using 13C-labels, mass spectrometry, and NMR.

Thrombin is a serine protease that plays a key role in the blood coagulation cascade. Compound I [2-[6-chloro-3-[(2,2-difluoro-2-pyridin-2-ylethyl)amino]-2-oxopyrazin-1(2H)-yl]-N-[(3-fluoropyridin-2-yl)methyl]acetamide] is a potent, selective, and orally bioavailable thrombin inhibitor that is being studied as a possible anticoagulant. Biotransformation studies in rats revealed that 84% of an i.

Conformation of a novel tetrapeptide inhibitor NH2-D-Trp-D-Met-Phe(pCl)-Gla-NH2 bound to farnesyl-protein transferase.

Farnesyl-protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the C-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for membrane association and cell-transforming activities of ras. Inhibitors of FPTase have been demonstrated to display antitumor activity in both tissue culture and animal models, and thus represent a potential therapeutic strategy for the treatment of human cancers.

Design and synthesis of potent, selective, and orally bioavailable tetrasubstituted imidazole inhibitors of p38 mitogen-activated protein kinase.

Novel potent and selective diarylimidazole inhibitors of p38 MAP (mitogen-activated protein) kinase are described which have activity in both cell-based assays of tumor necrosis factor-alpha (TNF-alpha) release and an animal model of rheumatoid arthritis. The SAR leading to the development of selectivity against c-Raf and JNK2alpha1 kinases is presented, with key features being substitution of the 4-aryl ring with m-trifluoromethyl and substitution of the 5-heteroaryl ring with a 2-amino substituent. Cell-based activity was significantly enhanced by incorporation of a 4-piperidinyl moiety at the 2-position of the imidazole which also enhanced aqueous solubility.

Antibacterial activity of lonchocarpol A.

Lonchocarpol A, a flavanone, demonstrates in vitro inhibitory activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. This activity is antagonized by mouse plasma, which may account for its lack of in vivo activity. This compound demonstrates no differentiation with respect to the inhibition of RNA, DNA, cell wall, and protein synthesis.

High fatty acid content in rabbit serum is responsible for the differentiation of osteoblasts into adipocyte-like cells.

Osteoblasts and adipocytes originate from common mesenchymal precursors. With aging, there is a decrease in osteoprogenitor cells that parallels an increase of adipocytes in bone marrow. We observed that rabbit serum (RS) induces adipocyte-like differentiation in human osteosarcoma SaOS-2/B10 and MG-63 cell lines, in rat ROS17/2.

Characterization of a solid state reaction product from a lyophilized formulation of a cyclic heptapeptide. A novel example of an excipient-induced oxidation.

Purpose: To elucidate the structure of a degradation product arising from a lyophilized formulation of a cyclic heptapeptide, and to provide a mechanism to account for its formation.

Methods: Preparative HPLC was used to isolate the degradate in quantities sufficient for structural studies. A structure assignment was made on the basis of the compounds spectroscopic properties (UV, MS, NMR) and the results of amino acid analysis.

Identification of fatty acid methyl ester as naturally occurring transcriptional regulators of the members of the peroxisome proliferator-activated receptor family.

The nuclear hormone receptors NUC-1 (PPAR delta) and PPAR alpha are members of the peroxisome proliferator-activated receptor (PPAR) family. The members of this receptor family are activated by agents that stimulate peroxisome proliferation, free fatty acids, prostaglandin 12 metabolites, and agents considered for the therapy of insulin-independent diabetes mellitus. To identify putative physiological agents that activate NUC-1, we tested the ability of acetone extracts of various rat tissues to activate the transcription of an MMTV-luciferase reporter gene, via a GR/NUC-1 hybrid receptor.

Species differences in the metabolism of a potent HIV-1 reverse transcriptase inhibitor L-738,372. In vivo and in vitro studies in rats, dogs, monkeys, and human.

In vivo and in vitro metabolism of 6-chloro-4(S)-cyclopropyl-3,4-dihydro-4-((2-pyridyl) ethynyl)quinazolin-2(1H)-one (L-738,372), a potent human immunodeficiency virus-type 1 reverse transcriptase inhibitor, has been investigated in rats, dogs, and monkeys. Following 0.9 mg/kg iv and 9 mg/kg po doses, systemic blood clearance (CLB) and bioavailability (F) of L-738,372 were species-dependent and inversely related (CLB = 48, 15, and 3 ml/min/kg; F = 6, 62 and 94%, in dogs, rats, and monkeys, respectively).

Solution structure of the cytoplasmic domain of phopholamban: phosphorylation leads to a local perturbation in secondary structure.

Peptides representing the N-terminal domain (Ia) of the cardiac sarcoplasmic reticulum protein phospholamban (residues 1-25 [PLB(1-25)] and a phosphorylated form [pPLB(1-25)]) were synthesized and their conformations examined using circular dichroism and nuclear magnetic resonance spectroscopy. In aqueous solution, both PLB(1-25) and pPLB(1-25) adopt a primarily disordered conformation. In 30% trifluoroethanol/10 mM phosphate, PLB(1-25) exhibits a CD spectrum consistent with 60% helical structure.

NMR studies of novel inhibitors bound to farnesyl-protein transferase.

Farnesyl-protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the carboxyl-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for the membrane association and cell-transforming activities of ras. Inhibitors of FPTase have been demonstrated to inhibit ras-dependent cell transformation and thus represent a potential therapeutic strategy for the treatment of human cancers.

Metabolism of L-689,502 by rat liver slices to potent HIV-1 protease inhibitors.

L-689,502, N-[2(R)-hydroxy-1(S)-indanyl]-5(S)-(1,1-dimethylethoxy- carbonyl-amino)-4(S)-hydroxy-6-phenyl-2(R)-(4-[2(R)-(4-morpholinyl) ethoxy]phenyl)methylhexamide, is a potent and specific inhibitor of human immunodeficiency virus-type 1 (HIV-1) protease in vitro. Metabolism of this compound in rat liver slices produced four major and several minor metabolites. The major metabolites were identified as morpholin-2-one, 3'(S)-hydroxyindan and 4'-hydroxyindan analogs, and a 4-O-glucuronic acid conjugate of the parent compound.

Hepatic metabolism of spironolactone. Production of 3-hydroxy-thiomethyl metabolites.

Spironolactone (SP) is used clinically as a renal aldosterone antagonist and as an antiandrogen. It is known that the drug is extensively metabolized and that metabolites mediate its therapeutic actions, but hepatic metabolism of SP has not been comprehensively investigated. Hepatic disposition may also be important in the toxicity of SP, because the parent compound prevents the hepatocarcinogenic effects of its metabolite, canrenone (CAN).

Transglutaminase inhibition by 2-[(2-oxopropyl)thio]imidazolium derivatives: mechanism of factor XIIIa inactivation.

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.

Three-dimensional structure of echistatin and dynamics of the active site.

The snake venom protein echistatin contains the cell recognition sequence Arg-Gly-Asp and is a potent inhibitor of platelet aggregation. The three-dimensional structure of echistatin and the dynamics of the active RGD site are presented. A set of structures was determined using the Distance Geometry method and subsequently refined by Molecular Dynamics and energy minimization.

The solution conformation of Ac-Pen-Arg-Gly-Asp-Cys-OH, a potent fibrinogen receptor antagonist.

The solution conformation of Ac-Pen-Arg-Gly-Asp-Cys-OH, a potent fibrinogen receptor antagonist, was characterized in DMSO-d6 by the combination of nmr and molecular modeling. The conformational space available to the peptide was explored using a distance geometry algorithm with distance constraints derived from 1H-nmr spectra. The dynamics of the peptide were examined by relaxation time measurements and low temperature studies.

Biotransformation of 5-chloro-3-phenylthioindole-2-carboxamide (L-734,005) in rhesus monkeys and rat liver microsomes to a potent HIV-1 reverse transcriptase inhibitor.

Rhesus monkeys were dosed orally with 10 mg/kg 5-chloro-3-phenylthioindole-2-carboxamide (L-734,005), a nonnucleoside human immunodeficiency virus type 1 (HIV-1) reverse transcriptase inhibitor, in polyethylene glycol 300. Plasma samples from these monkeys demonstrated greater bioactivity in an HIV-1 reverse transcriptase inhibition assay than anticipated from the parent compound concentrations as determined by an HPLC-UV assay. One major and three minor metabolites, as well as the parent compound, were detected in the plasma.

Structure of synthetic peptide analogues of an eggshell protein of Schistosoma mansoni.

The peptide (Gly-L-Tyr-L-Asp-L-Lys-L-Tyr)6, referred to as F4-6, was synthesized as a model for a schistosome eggshell protein in which the Gly-Tyr-Asp-Lys-Tyr consensus sequence is repeated over 40 times. Analysis by CD, Fourier transform infrared spectroscopy, potentiometric and spectrophotomertric titrations, NMR, and molecular modeling suggests that F4-6 forms some type of left-handed structure. A likely possibility appears to be a left-handed alpha-helix stabilized by Lysi-Aspi +4 salt bridges and possibly Aspi-Tyri +4 hydrogen bonding and Tyr-Tyr interactions.

In vivo and in vitro metabolism studies on a class III antiarrhythmic agent.

The metabolism of L-691,121 (I), a class III antiarrhythmic agent, was studied in vivo in rats and dogs and in vitro by using liver S9 or slices from these species and humans. After oral doses of [14C]I to rats (5 mg/kg) and dogs (1 mg/kg), urinary recoveries of label were, respectively, 6% and 28%. Biliary excretion (0-24 hr) accounted for 68% of a 5 mg/kg, po dose in rats and 19% of a 10 mg/kg dose, po in dogs.

Metabolism of a new HIV-1 reverse transcriptase inhibitor, 3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-methylpyridin-2(1H)-one (L-696,229), in rat and liver slices.

L-696,229 is a potent and specific inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and is currently undergoing clinical evaluation. In vivo metabolism in rats was investigated using an intravenous bolus dose of 5 mg/kg [3H]L-696,229. The amount of radioactivity eliminated in bile and urine over a period of 6 hr was 60 and 22%, respectively.