Hydrazinonaphthalene and azonaphthalene thrombopoietin mimics are nonpeptidyl promoters of megakaryocytopoiesis.

High-throughput screening for the induction of a luciferase reporter gene in a thrombopoietin (TPO)-responsive cell line resulted in the identification of 4-diazo-3-hydroxy-1-naphthalenesulfonic acids as TPO mimics. Modification of the core structure and adjustment of unwanted functionality resulted in the development of (5-oxo-1,5-dihydropyrazol-4-ylidene)hydrazines which exhibited efficacies equivalent to those of TPO in several cell-based assays designed to measure thrombopoietic activity. Furthermore, these compounds elicited biochemical responses in TPO-receptor-expressing cells similar to those in TPO itself, including kinase activation and protein phosphorylation. Potencies for the best compounds were high for such low molecular weight compounds (MW < 500) with EC(50) values in the region of 1-20 nM.

Discovery of potent and selective phenylalanine derived CCR3 antagonists. Part 1.

The discovery of a series of phenylalanine derived CCR3 antagonists is reported. Parallel, solution-phase library synthesis has been utilized to delineate the structure-activity relationship leading to the synthesis of highly potent, CCR3-selective antagonists.

Discovery of potent and selective phenylalanine derived CCR3 receptor antagonists. Part 2.

Highly potent CCR3 antagonists have been developed from a previously reported series of phenylalanine ester-based leads. Solution-phase, parallel synthesis optimization was utilized to identify highly potent, functional CCR3 antagonists.

Metal mediated protease inhibition: design and synthesis of inhibitors of the human cytomegalovirus (hCMV) protease.

A versatile synthetic route to a novel series of bis-imidazolemethanes designed to inhibit the hCMV protease has been developed and a series of potential metal binding inhibitors has been identified. In selectivity assays, the compounds were highly specific for CMV protease and showed no inhibition (IC50 > 100 microM) of other prototypical serine proteases such as trypsin, elastase, and chymotrypsin. Although the presence of free zinc ions was found to be an absolute requirement for the in vitro biological activity of this class of inhibitor, the potency of the inhibitors could not be improved beyond the micromolar level.

Preclinical pharmacokinetics and interspecies scaling of a novel vitronectin receptor antagonist.

Allometric scaling may be used in drug development to predict the pharmacokinetics of xenobiotics in humans from animal data. Although allometry may be successful for compounds that are excreted unchanged or that are oxidatively metabolized (with corrections for metabolic capacity), it has been more challenging for compounds excreted primarily as conjugates in bile. (S)-10, 11-Dihydro-3-[3-(pyridin-2-ylamino)-1-propyloxy]-5H-dibenzo[ a, d]cycloheptene-10-acetic acid (SB-265123) is a novel alphavbeta3 ("vitronectin receptor") antagonist. In this study, the in vivo pharmacokinetics and in vitro plasma protein binding of SB-265123 were examined in four species: mice, rats, dogs, and monkeys. In monkeys and dogs, SB-265123 exhibited moderate clearance, whereas low clearance (<20% hepatic blood flow) was observed in the rat, and high clearance (>70% hepatic blood flow) was seen in the mouse. The concentration-time profiles indicated the possibility of enterohepatic recirculation; subsequent studies in bile duct-cannulated rats demonstrated extensive biliary excretion of an acyl-glucuronide of SB-265123. In allometric scaling to predict the disposition of SB-265123 in humans, various standard correction factors were applied, including protein binding, maximum lifespan potential, and brain weight; each failed to produce adequate interspecies scaling of clearance (r(2) < 0.72). Consequently, a novel correction factor incorporating bile flow and microsomal UDP-glucuronosyltransferase activity in each species was applied, demonstrating substantial improvement in the correlation of the allometric plot (r(2) = 0.96). This study demonstrates a novel allometric correction that may be applicable to compounds that undergo conjugation and biliary excretion.

Design and synthesis of diaminopyrrolidinone inhibitors of human osteoclast cathepsin K.

The structure-based design and synthesis of lactam-constrained azapeptide inhibitors of human cathepsin K are described. Enhanced stability to proteolytic cleavage over acyclic analogues is discussed.

Orally bioavailable nonpeptide vitronectin receptor antagonists containing 2-aminopyridine arginine mimetics.

A peptide RGD analog containing a novel 2-aminopyridine arginine mimetic was discovered to have good affinity and selectivity for the vitronectin receptor. Incorporation of the 2-aminopyridine arginine mimetic into the 3-oxo-1,4-benzodiazepine-2-acetic acid integrin antagonist series led to novel and potent nonpeptide vitronectin receptor antagonists with promising levels of oral bioavailability.

Orally bioavailable nonpeptide vitronectin receptor antagonists with efficacy in an osteoporosis model.

A new series of potent nonpeptide vitronectin receptor antagonists, based on a novel carbocyclic Gly-Asp mimetic, has been discovered. A representative of this series, SB 265123 (4), has 100% oral bioavailability in rats, and is orally active in vivo in the ovariectomized rat model of osteoporosis.

Conformational preferences in a benzodiazepine series of potent nonpeptide fibrinogen receptor antagonists.

Previously, we reported the direct design of highly potent nonpeptide 3-oxo-1,4-benzodiazepine fibrinogen receptor antagonists from a constrained, RGD-containing cyclic semipeptide. The critical features incorporated into the design of these nonpeptides were the exocyclic amide at the 8-position which overlaid the Arg carbonyl, the phenyl ring which maintained an extended Gly conformation, and the diazepine ring which mimicked the gamma-turn at Asp. In this paper, we investigate conformational preferences of the 8-substituted benzodiazepine analogues by examining structural modifications to both the exocyclic amide and the seven-membered diazepine ring and by studying the conformation of the benzodiazepine ring using molecular modeling, X-ray crystallography, and NMR. We found that the directionality of the amide at the 8-position had little effect on activity and the (E)-olefin analogue retained significant potency, indicating that the trans orientation of the amide, and not the carbonyl or NH groups, made the largest contribution to the observed activity. For the diazepine ring, with the exception of the closely analogous 3-oxo-2-benzazepine ring system described previously, all of the modifications led to a significant reduction in activity compared to the potent 3-oxo-1, 4-benzodiazepine parent ring system, implicating this particular type of ring system as a desirable structural feature for high potency. Energy minimizations of a number of the modified analogues revealed that none could adopt the same low-energy conformation as the one shared by the active (S)-isomer of the 3-oxo-1, 4-benzodiazepines and 3-oxo-2-benzazepines. The overall data suggest that the features contributing to the observed high potency in this series are the orientation of the 3-4 amide and the conformational constraint imposed by the seven-membered ring, both of which position the key acidic and basic groups in the proper spatial relationship.

Benzimidazole derivatives as arginine mimetics in 1,4-benzodiazepine nonpeptide vitronectin receptor (alpha v beta 3) antagonists.

In a 3-oxo-1,4-benzodiazepine-2-acetic acid series of vitronectin receptor (alpha v beta 3) antagonists containing a benzimidazole as a novel arginine mimetic, we examined the effects of benzimidazole modifications and amide substitutions on both activity and pharmacokinetics.

Discovery of an imidazopyridine-containing 1,4-benzodiazepine nonpeptide vitronectin receptor (alpha v beta 3) antagonist with efficacy in a restenosis model.

In the 3-oxo-1,4-benzodiazepine-2-acetic acid series of vitronectin receptor (alpha v beta 3) antagonists, a compound containing an imidazopyridine arginine mimetic was discovered which had sufficient potency and i.v. pharmacokinetics for demonstration of efficacy in a rat restenosis model.

Benzothiopyran-4-one based reversible inhibitors of the human cytomegalovirus (HCMV) protease.

A novel class of CMV protease inhibitors based on a benzothiopyran-S,S-dioxide nucleus has been discovered. Enzyme kinetic data supports a reversible mode of inhibition for a representative member of this class, 2-(3-pyridyl-N-oxide)benzothiopyran-4-one-S,S-dioxide, 1. Experiments in the presence and absence of the disulfide reducing agent DTT suggest that the inhibition by 1 is not due to oxidative inactivation of the enzyme. Also presented are results of some SAR studies of the benzothiopyranone ring system.

Sweat testing for heroin, cocaine, and metabolites.

Although a variety of drugs have been detected in sweat, little information is available on the characteristics of drug excretion in sweat under controlled-dosing conditions. A series of clinical studies were designed to determine the identity, concentration, time course, dose dependency, and variability of drug and metabolite excretion in sweat following administration of single doses of cocaine and heroin to human subjects. Sweat was collected by means of a sweat patch that could be worn for a period of several days to several weeks at a time, resulting in accumulation of drug in the patch. Sweat patches were removed at specified times and frozen until analyzed by gas chromatography--mass spectrometry. Cocaine and heroin were the major analytes excreted in sweat following their administration. Smaller amounts of cocaine metabolites were also detected following cocaine administration. 6-Acetylmorphine appeared rapidly after heroin administration and continued to increase while heroin content decreased, suggesting that heroin was undergoing hydrolysis in the sweat patch. Cocaine appeared in sweat within 1-2 hours and peaked within 24 hours in an apparent dose-dependent manner. Analysis of duplicate adjacent patches from individual subjects who had been administered cocaine provided similar quantitative results, suggesting that intrasubject variability was relatively low, whereas intersubject variability was high. These observations regarding the excretion of cocaine and heroin analytes in sweat have important forensic implications to other fields such as hair analysis. Sweat excretion could be an important mechanism by which drugs enter hair. These data also suggest that the sweat patch could serve as a useful monitoring device in surveillance of individuals in treatment and probation programs.

Pharmacokinetics and pharmacodynamics of smoked heroin.

Despite the current popularity of smoking as a route of drug self-administration, there have been few human studies characterizing the pharmacokinetics and pharmacodynamics of smoked drugs of abuse. A variety of technological difficulties are encountered in the design of smoking studies, such as delivering reproducible doses and limiting the amount of pyrolysis of parent drug. As part of a concerted research effort to deliver precise, smoked doses of drug, a computer-assisted smoking device was utilized that delivered single puffs of heroin vapor to human subjects under controlled clinical conditions. Recovery studies indicated that the smoking device delivered approximately 89% of parent heroin to subjects. Although only two qualified heroin smokers could be identified as eligible volunteers, their participation provided the unique opportunity to study the pharmacokinetics and pharmacodynamics of smoked heroin. The two subjects were administered four smoked heroin doses in ascending order. In addition, four intravenous doses of heroin were administered for comparison of effects and estimation of bioavailability. Concurrent physiological, behavioral, and performance measures were collected along with blood samples. Blood was analyzed for heroin, 6-acetylmorphine, and morphine by solid-phase extraction gas chromatography-mass spectrometry. Heroin appeared rapidly in blood after administration and peaked 1-5 minutes after smoking, which is similar to that observed following intravenous administration. Heroin concentrations declined rapidly to the limit of detection (1.0 ng/mL) by 30 minutes. 6-Acetylmorphine blood concentrations also peaked and declined rapidly after smoked heroin with peak concentrations occurring at 1-2 minutes after smoking. Morphine levels rose and decayed more slowly. Mean elimination half-lives for heroin, 6-acetylmorphine, and morphine were 3.3 min, 5.4 min, and 18.8 min, respectively, by the smoked route. The bioavailability of smoked heroin was highly variable. Physiological measures such as pupil diameter demonstrated a counterclockwise hysteresis compared with heroin blood levels. The rapid onset of pharmacological effects together with the early appearance of heroin and metabolites in blood following smoked heroin demonstrated the effectiveness of this route of drug administration. It is evident that the smoking route enables individuals to obtain similar pharmacological effects as are produced by intravenous administration of heroin.

Design of a potent and orally active nonpeptide platelet fibrinogen receptor (GPIIb/IIIa) antagonist.

The direct design of the potent nonpeptide platelet fibrinogen receptor (GPIIb/IIIa) antagonist, 8-[[[4- (aminoiminomethyl)phenyl]amino]carbonyl]-2,3,4,5-tetrahydro-3-oxo- 4- (2-phenylethyl)-1H-1,4-benzodiazepine-2-acetic acid, (3) (SB 207448), based on the structure and conformation of the potent and highly constrained cyclic peptide antagonist SK&F 107260 (2), has been reported [Ku et al., J. Am. Chem. Soc. 1993, 115, 8861]. While 3 displayed in vivo activity in the conscious dog following intravenous administration, it was not active following intraduodenal administration; activity was measured with an ex vivo platelet aggregation assay. The secondary amide in 3 was N-methylated in the expectation of increased absorption and bioavailability. The resulting tertiary amide, 4 (SB 208651), also showed high binding affinity for human GPIIb/IIIa and potent antiaggregatory activity in human platelet-rich plasma. Most importantly, 4 was active in vivo following intravenous and intraduodenal administration. Comparison of the iv and id inhibition curves suggests an apparent bioavailability of approximately 10%. Thus, 4 represents the first orally active compound in this series of potent, nonpeptide fibrinogen receptor antagonists.

Pharmacodynamic effects of cotinine in abstinent cigarette smokers.

The nicotine metabolite cotinine was administered to abstinent cigarette smokers to determine whether it has pharmacologic activity as assessed by various physiologic and subjective measurements. By means of a randomized, double-blind, placebo-controlled counterbalanced-order design, subjects received cotinine base (30 mg) intravenously after 48 hours of abstinence from cigarette smoking. Serum cotinine concentrations increased to levels commonly achieved during daily cigarette smoking, whereas no change in serum nicotine concentration was observed. Cotinine compared with placebo produced subjective differences in self-reported ratings of restlessness, anxiety and tension, insomnia, sedation, and pleasantness. Cotinine had minimal effects on cardiovascular measurements. These findings indicate that cotinine is behaviorally active in the setting of cigarette abstinence at blood concentrations similar to those commonly achieved through daily cigarette smoking.