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Incorporation of a spacer group between the central scaffold and the aryl ring resulted in a new cyclooxygenase-2 (COX-2) selective inhibitor core structure, 3-[4-(methylsulfonyl)phenyl]-5-(trifluoromethyl)(2-pyridyl) phenyl ketone (20), with COX-2 IC50 = 0.25 μM and COX-1 IC50 = 14 μM (human whole blood assay). Compound 20 was orally active in the rat air pouch model of inflammation, inhibiting white blood cell infiltration and COX-2-derived PG production. Our data support the identification of a novel COX-2 selective inhibitor core structure exemplified by 20.

The combination of a nitric oxide (NO) donor and a paclitaxel−NO donor conjugate coated on a vascular stent was tested in a rabbit iliac artery model of stenosis as a potential therapy for restenosis. Paclitaxel was conjugated with a NO donor at the 7-position to give compound 7. An adamantane-based NO donor 14 was synthesized and combined with 7 to provide a burst of NO in the first few critical hours following injury to the vessel wall. Both 7 and 14 demonstrated antiproliferative activity (IC50 = 20 nM and 15 μM, respectively) and antiplatelet activity (IC50 = 10 and 1 μM, respectively). Stents were coated with a layer of a polymer containing test compounds. The total amount of NO eluted from the stents after a 6 h implantation in the rabbit iliac artery was 35%, 95%, and 69% of the original content for the stents coated with 7, 14, and the combination of 7 and 14, respectively. The antistenotic activity of 7 and 14 was determined in a 28-day rabbit model with two control groups (uncoated stents and polymer-coated stents) and two study groups (paclitaxel-coated stents and stents coated with the combination of 7 and 14). Polymer-coated stents caused inflammation and increased stenosis by 39% when compared to the uncoated stents. The stents coated with 7 plus 14 were as good as the uncoated stents, 41% better than the polymer-coated stents and 34% better than the paclitaxel-coated stents. These data indicate a beneficial effect of adding NO to an antiproliferative agent (paclitaxel) and suggest a potential therapeutic combination for the treatment of stenotic vessel disease.

The synthesis of a series of novel pyrazoles containing a nitrate (ONO2) moiety as a nitric oxide (NO)-donor functionality is reported. Their COX-1 and COX-2 inhibitory activities in human whole blood are profiled. Our data demonstrate that pyrazole ring substituents play an important role in COX-2 selective inhibition, such that a cycloalkyl pyrazole (6b) was found to be a potent and selective COX-2 inhibitor. Other modifications at the 3 position of the central pyrazole ring (17b, 23b, 26b-I) enhanced COX-2 inhibitory potency. Among the pyrazoles synthesized, the oxime (23b) was identified as the most potent COX-2 selective inhibitor. Accordingly, 23b was profiled pharmacologically in the rat after oral administration and shown to possess potent antiinflammatory activity in the carrageenan-induced air-pouch model and less gastric toxicity than a standard COX-2 inhibitor when administered with background aspirin treatment. We suggest that the enhanced gastric tolerance of an NO-donor COX-2 selective inhibitor has the potential to augment the clinical profile of this drug class.

A novel series of benzo-1,3-dioxolane metharyl derivatives was synthesized and evaluated for cyclooxygenase-2 (COX-2) and cyclooxygenase-1 (COX-1) inhibition in human whole blood (HWB). In the present study, structure−activity relationships (SAR) in the metharyl analogues were investigated. The spacer group and substitutions in the spacer group were found to be quite important for potent COX-2 inhibition. Compounds in which a methylene group (8a−c), carbonyl group (12a−c), or methylidene group (7a−c) connected cycloalkyl groups to the central benzo-1,3-dioxolane template were found to be potent and selective COX-2 inhibitors. Aryl-substituted compounds linked to the central ring by either a methylene or a carbonyl spacer resulted in potent, highly selective COX-2 inhibitors. In this series of substituted-(2H-benzo[3,4-d]1,3-dioxolan-5-yl))-1-(methylsulfonyl)benzene compounds, SAR studies demonstrated that substitution at the 3-position of the aryl group optimized COX-2 selectivity and potency, whereas substitution at the 4-position attenuated COX-2 inhibition. Mono- or difluoro substitution at meta position(s), as in 22c and 22h, was advantageous for both in vitro COX-2 potency and selectivity (e.g., COX-2 IC50 for 22c = 1 μM and COX-1 IC50 for 22c = 20 μM in HWB assay). Several novel compounds in the (2H-benzo[3,4-d]1,3-dioxolan-5-yl))-1-(methylsulfonyl)benzene series, as shown in structures 7c, 8a, 12a, 21c, 22c, 22e, and 22h, selectively inhibited COX-2 activity by 40−50% at a test concentration of 1 μM in an in vitro HWB assay.

Despite its widespread use, diclofenac has gastrointestinal liabilities common to nonsteroidal antiinflammatory drugs (NSAIDs) that might be reduced by concomitant administration of a gastrointestinal cytoprotectant such as nitric oxide (NO). A series of novel diclofenac esters containing a nitrosothiol (−S-NO) moiety as a NO donor functionality has been synthesized and evaluated in vivo for bioavailability, pharmacological activity, and gastric irritation. All S-NO-diclofenac derivatives acted as orally bioavailable prodrugs, producing significant levels of diclofenac in plasma within 15 min after oral administration to mice. At equimolar oral doses, S-NO-diclofenac derivatives (20a−21b) displayed rat antiinflammatory and analgesic activities comparable to those of diclofenac in the carrageenan-induced paw edema test and the mouse phenylbenzoquinone-induced writhing test, respectively. All tested S-NO-diclofenac derivatives (20a−21b) were gastric-sparing in that they elicited markedly fewer stomach lesions as compared to the stomach lesions caused by a high equimolar dose of diclofenac in the rat. Nitrosothiol esters of diclofenac comprise a novel class of NO-donating compounds having therapeutic potential as nonsteroidal antiinflammatory agents with an enhanced gastric safety profile.

We designed and evaluated a new class of molecules, nitrosylated alpha-adrenergic receptor antagonists, as potential agents for the treatment of impotence. In in vitro studies with human and rabbit corpus cavernosum strips in organ chambers, the alpha-adrenergic receptor antagonists (alpha-ARAs) moxisylyte and yohimbine and their corresponding nitrosylated compounds, SNO-moxisylyte (NMI-221) and SNO-yohimbine (NMI-187), concentration-dependently relaxed endothelin-induced contraction. The nitrosylated compounds were significantly more potent than the parent alpha-ARA. In human tissues, the specific phosphodiesterase type 5 inhibitor zaprinast potentiated the relaxing effects of the nitrosylated compounds. Only nitrosylated compounds induced accumulation of cyclic GMP in rabbit corpus cavernosum strips. Yohimbine and NMI-187 demonstrated a potent alpha2-blocking activity, with no significant differences in pA2 values (8.9 versus 8.2, respectively). Moxisylyte and NMI-221 showed moderate potency in antagonizing phenylephrine contraction, with comparable pA2 values for both molecules (6.5 versus 6.6, respectively). alpha-Adrenergic receptor-binding studies showed similar binding affinities for the alpha-ARA and their corresponding nitrosylated compounds. In vivo, intracavernosal injection of nitrosylated molecules caused greater increases in intracavernosal pressure (NMI-221 versus moxisylyte) that were more long lasting than those of moxisylyte or yohimbine. There were no significant differences between nitrosylated and non-nitrosylated compounds in the magnitude of systemic mean arterial pressure decrease after intracavernosal injection. alpha-ARA and the nitrosylated compounds showed no pain-inducing activity as evaluated with the paw-lick model in mice. In summary, nitrosylated alpha-ARA have the dual functionalities of nitric oxide donors and alpha-ARA. These drugs induced penile erection in animals, suggesting their possible therapeutic value as agents for the local pharmacological treatment of impotence.

Although nitrosothiols have been suggested to act as regulators of cell (patho)physiology, little is known about the pharmacology of nitrosylated proteins as nitric oxide (NO.) congeners. We describe the molecular consequences of nitrosylating bovine serum albumin (BSA) at multiple specific sites and demonstrate that the product S-nitrosoproteins exert NO.-like activity. The content of nucleophilic nitrosylation sites (i.e., free sulfhydryl groups) in native BSA was increased by either reduction with dithiothreitol or thiolation with N-acetylhomocysteine. Fourteen moles of nitrogen monoxide (NO)/mol BSA equivalent were then selectively positioned on either the endogenous sulfhydryl groups of reduced BSA or the homocysteine moieties of thiolated BSA, respectively. Each resulting S-nitrosoprotein adduct was an oligomeric mixture across the >2000 kDa to approximately 66 kDa molecular mass range. The BSA-derived S-nitrosoproteins were immunoreactive with antibodies against native BSA but evidenced compromised long-chain fatty acid binding. Both types of BSA-derived S-nitrosoproteins suppressed human coronary artery smooth muscle cell proliferation to a similar degree (IC50 approximately 70 microM NO. equivalents) and were significantly more effective antiproliferative agents than a standard NO. donor, DETA NONOate. Antiproliferative bioactivity reflected the NO functionalities carried by each protein, but was independent of molecular mass of the nitrosylated BSA adducts. These data exemplify the rational design and characterization of protein-based S-nitrosothiols as NO. congeners and suggest that such agents could have therapeutic potential as NO delivery systems.

To study the effects of chronic maternal hypoxia on the growth and functional development of foetal and neonatal adrenal glands, Long–Evans rats were acclimatized to high altitude (3800 m) before mating and were maintained at this height throughout gestation. The body growth of the progeny at high altitude was essentially normal during the perinatal period, but adrenal weight and adrenocortical function showed marked differences from those of control rats maintained at sea level. The adrenal glands were larger in foetuses but smaller in neonates, compared with the adrenal glands of control animals maintained at sea level. Differences in the protein content of the adrenal glands between the two groups paralleled differences in adrenal weight. The concentration and content of corticosterone in the adrenal glands of both foetuses and neonates kept at high altitude were markedly lower than values in animals kept at sea level. The lower adrenal corticosterone content was not reflected in the concentration of the hormone in the peripheral plasma, since this was essentially the same at high altitude and at sea level in both mothers and perinatal animals. The reduction in the adrenal corticosterone content was accompanied by and may have resulted from, a reduction in the concentration of cytochrome P-450 in the adrenal tissue of foetuses maintained at high altitude. Possible explanations for the dichotomous results are discussed.

2-Methyl-3-(2(S)-pyrrolidinylmethoxy)pyridine, ABT-089 (S-4), a member of the 3-pyridyl ether class of nicotinic acetylcholine receptor (nAChR) ligands, shows positive effects in rodent and primate models of cognitive enhancement and a rodent model of anxiolytic activity and possesses a reduced propensity to activate peripheral ganglionic type receptors. The profiles of S-4, its N-methyl analogue, and the corresponding enantiomers across several measures of cholinergic channel function in vitro and in vivo are presented, together with in vitro metabolism and in vivo bioavailability data. On the basis of its biological activities and favorable oral bioavailability, S-4 is an attractive candidate for further evaluation as a treatment for cognitive disorders.

Recent evidence indicating the therapeutic potential of cholinergic channel modulators for the treatment of central nervous system (CNS) disorders as well as the diversity of brain neuronal nicotinic acetylcholine receptors (nAChRs) have suggested an opportunity to develop subtype-selective nAChR ligands for the treatment of specific CNS disorders with reduced side effect liabilities. We report a novel series of 3-pyridyl ether compounds which possess subnanomolar affinity for brain nAChRs and differentially activate subtypes of neuronal nAChRs. The synthesis and structure−activity relationships for the leading members of the series are described, including A-85380 (4a), which possesses ca. 50 pM affinity for rat brain [³H]-(−)-cytisine binding sites and 163% efficacy compared to nicotine to stimulate ion flux at human α4β2 nAChR subtype, and A-84543 (2a), which exhibits 84-fold selectivity to stimulate ion flux at human α4β2 nAChR subtype compared to human ganglionic type nAChRs. Computational studies indicate that a reasonable superposition of a low energy conformer of 4a with (S)-nicotine and (−)-epibatidine can be achieved.

As part of a comprehensive simulation of a prototype locomotive propulsion system, a detailed model has been developed that predicts the dynamic response of an experimental two-stroke, turbocharged and intercooled diesel engine. Engine fueling and brake torque are computed from regression equations derived from an extensive data base. Corrections are applied to the calculated steady-state torque to account for dynamic deviations of in-cylinder trapped air-fuel ratio from the steady-state value. The engine simulation accurately represents the operation of the turbocharger, which is gear-driven at low turbocharger speeds, and freewheels through an overrunning clutch when exhaust energy accelerates the turbocharger beyond its geared speed. Engine fueling level, i.e., rack, is determined from a dynamic simulation of an electrohydraulic governor, which responds to the difference between the desired and the actual engine speeds. The governor representation includes: (1) finite rate of change of engine set speed; (2) load regulator feedback for control of applied engine loads; and (3) fuel limiting under conditions of excessively high load demand. The fundamentals of the engine/governor model are given in the paper along with examples that emphasize the dynamic operation of these particular components.

A diversity of nicotinic acetylcholine receptor (nAChR) subtypes has been identified in mammalian brain using recombinant DNA technology. Alterations in the activity of these acetylcholinegated ion channels have been implicated in a number of central nervous system disorders including Alzheimer's disease (AD). The potential therapeutic usefulness of (-)-nicotine [(S)-3-(1-methyl-2-pyrrolidinyl) pyridine], the prototypic agonist at nAChRs, is severely limited by side effects that are the result of activation of both cholinergic and noncholinergic pathways in the central and peripheral nervous systems. This study sought to determine the in vitro selectivity of (S)-3-methyl-5-(1methyl-2-pyrrolidinyl)isoxazole (ABT 418), a novel analog of (-)-nicotine in which the pyridine ring was replaced with an isoxazole bioisotere, to activate nAChRs. ABT 418 was a potent inhibitor of [3H]-cytisine binding to nAChR in rat brain (Ki = 3 nM) but was inactive (Ki > 10,000 nM) in 37 other receptor/neurotransmitter-uptake/enzyme/transduction system binding assays, including those for alpha-bungarotoxin, muscarinic and 5-hydroxytryptamine3 receptors. In PC12 cells, patch-clamp studies indicated that ABT 418 was an agonist with an EC50 value of 209 microM, a potency to activate cholinergic channel currents some 4-fold less than that of (-)-nicotine (52 microM). Channel current responses elicited by ABT 418 were prevented by the cholinergic channel blocker, mecamylamine. ABT 418 was also approximately 10-fold less potent (EC50 value = 380 nM) than (-)-nicotine (40 nM) in increasing [3H]-dopamine release from rat striatal slices, an effect that was blocked by the nAChR antagonist, dihydro-beta-erythroidine (10 microM).2+ In contrast, ABT 418 appeared equipotent with (-)-nicotine in enhancing 86Rb+ flux from mouse thalamic synaptosomes. ABT 418 demonstrated an in vitro pharmacological profile of cholinergic channel activation that was robust at some nAChR, but not others. The reasons for this are unclear. However, a nAChR subtype selectivity may account for the in vitro potency differences of ABT 418 on various neurotransmitter systems, and the substantial separation between the cognitive enhancement/anxiolytic benefits, and the reduced central nervous system side-effect liabilities seen in vivo. ABT 418 represents the first neuronal nAChR ligand that differentiates the toxicities/liabilities and other negative aspects normally associated with liabilities and other negative aspects normally associated with (-)-nicotine from the potential pharmacological benefits of selective cholinergic channel activation.

S-Nitrosylated tissue plasminogen activator (tPA) is formed by S-nitrosylation of the clinically important agent tPA by nitric oxide, thus conferring nitric oxide donor properties to the molecule. Cats were subjected to 90 min of myocardial ischemia and 270 min of reperfusion and were treated with either tPA or S-nitrosylated tPA 10 min before reperfusion. S-Nitrosylated tPA-treated cats demonstrated marked attenuation of cardiac necrosis after myocardial ischemia/reperfusion, compared with cats receiving only tPA (13 +/- 3% vs. 28 +/- 3%, P < .01). Relaxation of ischemic/reperfused left anterior descending coronary artery rings in response to the endothelium-dependent dilators acetylcholine and A23187 was greater in the S-nitrosylated tPA-treated group, compared with the cats receiving only tPA, indicating that coronary vascular endothelial function was preserved by S-nitrosylated tPA. S-Nitrosylated tPA also resulted in markedly reduced adherence of neutrophils to the coronary vascular endothelium, compared with nonnitrosylated tPA (P < .01). Immunohistochemical localization of P-selectin in the ischemic region was also significantly reduced by S-nitrosylated tPA, compared with the control group (P < .01). These data indicate that S-nitrosylated tPA is a cardioprotective agent, likely exerting its effect by site-specific nitric oxide donation resulting in inhibition of neutrophil-endothelium interaction via a P-selectin-dependent mechanism.

The partial differential equations describing distributed parameter systems may often be reduced to transcendental transfer functions with the aid of appropriate boundary conditions. In the analysis and synthesis of closed loop systems, the transcendental transfer functions have to be approximated in a suitable manner. In this paper, discrete-time model of distributed parameter systems is obtained. The model employs a sample and hold circuit in the loop. The response of the model system is compared with the response obtained by approximating the transcendental transfer function by root factor and other approximations. The stability of linear and nonlinear systems with distributed parameters is investigated by employing the Mikhailov stability criterion.