Effect of various pretreatments on the hypothermic activity of repin in naive rats.

Repin is a sesquiterpene lactone found in Centaurea solstitialis, a plant responsible for Parkinson-like disease in horses. Repin, on intraperitoneal (i.p.) injection, produces a dose-dependent and highly significant hypothermia in naive rats. The hypothermia is long-lasting with a peak 3 h after the injection and a return to normal temperature after more than 8 h. The effects of atropine sulfate, atropine methylbromide, propranolol, metergoline, ketanserin, diphenhydramine and apomorphine pretreatment on repin-induced hypothermia were investigated. None of the pretreatments directly antagonized repin's hypothermic effect. However, partial but significant reversals of hypothermia by atropine sulfate, metergoline, ketanserin, diphenhydramine and apomorphine were observed 2-4 h after the repin injection. These late-onset effects are probably due to secondary physiological mechanisms. On the other hand, propranolol, at 20 mg/kg i.p., clearly accentuated both the early-onset (30-90 min) and late-onset (2-4 h) hypothermic effects of repin.

Inhibition of N8-acetylspermidine deacetylase by active-site-directed metal coordinating inhibitors.

A number of substrate analogues of N8-acetylspermidine (N8-AcSpd) (16) and chemical modifying agents containing metal coordinating ligands were assayed as inhibitors of the cytoplasmic enzyme N8-AcSpd deacetylase from rat liver. The enzyme is inhibited by metal chelators, several omega-amino-substituted carboxylic acids, and some thiol reagents. Inhibition by diisopropyl fluorophosphate was observed only at high concentrations. These results suggest that the catalytic mechanism of the enzyme requires a transition state metal and free sulfhydryl groups for activity. The most potent inhibitor synthesized 6-[(3-aminopropyl)amino]-N-hydroxyhexanamide (15), has an apparent Ki of 0.001 microM. It binds to the target enzyme 11,000 times tighter than the substrate (apparent Km = 11 microM). These compounds and a previously reported series of compounds (Dredar, S. A.; Blankenship, J. W.; Marchant, P. E.; Manneh, V. A.; Fries, D. S. J. Med. Chem. 1989, 32, 984-989) are useful in mapping the active site and determining the physiological function of N8-AcSpd deacetylase.

Design and synthesis of inhibitors of N8-acetylspermidine deacetylase.

Analogues of N8-acetylspermidine (1) were synthesized as potential inhibitors of the cytoplasmic enzyme N8-acetylspermidine deacetylase. The compounds were assayed for their ability to inhibit the deacetylation of 1 in a cytosolic fraction from rat liver. The apparent Ki values were determined by Dixon plots. The apparent Km of 1 for this enzyme is 11.0 microM. It was found that compounds which lacked the N1 or the N4 of spermidine were less effective at competing for the enzyme than the substrate. All compounds with acyl substituents larger than acetyl were less potent inhibitors than the corresponding acetylated derivatives. Thus, the enzyme's selectivity as a deacetylase seems to be attributable to steric hindrance which occurs with larger acyl groups. The N8 of the substrate is not essential for its binding to the enzyme. Replacement of N8 with a CH2 group gives the ketone 14, which has an apparent Ki of 0.18 microM, 60-fold lower than the apparent Km of 1. The inhibitory potency of 14 is retained in compounds substituted at the N1 position. The N1,N1-dimethyl and the N1,N1-diethyl analogues (15 and 16) of 14 have apparent Ki values of 0.096 and 0.10 microM, respectively. These agents are the most potent inhibitors of N8-acetylspermidine deacetylase reported, and they are promising tools for use in determining the physiological function of N8-acetylspermidine deacetylation.

Synthesis and toxicity toward nigrostriatal dopamine neurons of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) analogues.

Six compounds having structural features in common with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were synthesized and tested in mice for the ability to produce a prolonged decrease in nigrostriatal dopamine (DA) and DA metabolites. The compounds that were prepared and tested include the ester elimination products of the analgetic drugs alpha-prodine and trimeperidine. None of the compounds in this study, except for MPTP, produced significant neurotoxic effects in the mouse model. The study shows that minor changes in the tetrahydropyridine ring of MPTP result in a marked decrease in neurotoxicity.

HPLC-assay with electrochemical detection for the neurotoxin MPTP, its metabolite MPP+ and MPTP-analogues in biological samples after purification over Sephadex G10.

A sensitive and selective method for assaying the neurotoxin MPTP and some MPTP-analogues in mouse brain and serum is described. The method is based on isolation of the compounds from biological samples on small Sephadex G10 columns followed by reverse phase HPLC with amperometric detection. HPLC separation was performed at pH 3, after which the pH was increased to 6.8 by mixing the column effluent with 0.5 M phosphate pH 9, to provide the conditions required for electrochemical detection. A metabolite of MPTP, MPP+, was determined as MPTP after reduction with NaBH4. This assay allows the determination of brain and serum concentrations in the pmol/g range of administered MPTP and MPTP-analogues and the effects of these substances on dopamine and its metabolites in the same tissue sample.

Narcotic antagonist-induced hypotension in the spontaneously hypertensive rat.

Intravenous naloxone or naltrexone produced transient, dose-related reductions in the mean arterial pressure (MAP) and heart rate (HR) of urethane-anesthetized spontaneously hypertensive rats (SHRs). Yet these same doses of narcotic antagonists reduced HR but not MAP of normotensive Wistar-Kyoto rats (WKYs). Such effects were not observed upon administration to SHRs of increasing doses of methylnaltrexone, which possesses no central activity. (+)-Naloxone, which does not block opiate receptors, reduced HR but not MAP of both SHRs and WKYs. These findings indicate that SHRs and WKYs differ in their MAP and HR responses to narcotic antagonists. The high doses required for effect plus the brevity of the responses suggest that these drug effects are perhaps not mu-opiate receptor-mediated; however, the methylnaltrexone and (+)-naloxone findings clearly implicate a central specificity of action. We conclude that narcotic antagonist-induced changes in MAP and HR in SHRs are possibly specific and central in origin yet not mediated by mu-opiate receptors.

Opioid receptor binding characteristics of the non-equilibrium mu antagonist, beta-funaltrexamine (beta-FNA).

beta-Funaltrexamine (beta-FNA) bound to mouse brain membranes in a reversible and an irreversible (not removed by washing of the membrane) manner, and a portion of each type of binding was opioid-specific. Addition of 100 mM NaCl to the incubating medium enhanced the binding of beta-FNA to membranes. Using membranes preincubated with beta-FNA (1 microM) and then washed three times, the maximum number of binding sites available to [3H]morphine was markedly diminished whereas the affinity of morphine for binding sites was not significantly altered. The binding of [3H]naltrexone was also reduced markedly by beta-FNA pretreatment. In similarly pretreated membranes, the binding of [3H]methionine enkephalin [3H][D-Ala2,D-Leu5]enkephalin (DADLE) or [3H]ethylketazocine was reduced to a smaller extent. Using brain membranes from mice pretreated with a single subcutaneous injection of beta-FNA (100 mg/kg) 48 h prior to use, the binding of [3H]methionine enkephalin was unaffected whereas the number of binding sites available to [3H]morphine was significantly reduced. The inhibition by various ligands of the reversible binding of [3H] beta-FNA resembled the relative ability of the same ligands to inhibit the binding of [3H]ethylketazocine. It was concluded that the irreversible portion of the binding of beta-FNA demonstrates a selectivity for mu over delta binding sites, and that the reversible portion of the binding of beta-FNA demonstrates a selectivity for kappa binding sites over mu or delta binding sites. As such, the binding characteristics of beta-FNA are consistent with its profile in vivo and in isolated tissue studies in vitro.

Narcotic antagonist potentiation of apomorphine drug effect: a stereospecific, centrally mediated drug action.

Systemic pretreatment with naloxone but not its (+)-enantiomer significantly potentiated apomorphine-induced stereotypic climbing activity in mice. Systemic or central pretreatment with naltrexone also significantly potentiated the apomorphine drug effect. The N-methyl derivative of naltrexone had no influence when administered systemically but significantly potentiated the apomorphine drug effect when administered centrally. These findings suggest that narcotic antagonist potentiation of apomorphine-induced stereotypic climbing in mice is a stereospecific phenomenon that originates in the central nervous system.

Importance of C-6 chirality in conferring irreversible opioid antagonism to naltrexone-derived affinity labels.

A series of five epimeric pairs of naltrexone derivatives that contain an electrophilic substituent at the 6 alpha- or 6 beta-position was synthesized and tested on the guinea pig ileal longitudinal muscle (GPI) and mouse vas deferens (MVD) preparations in order to determine if the orientation of the electrophile is important for covalent bonding to opioid receptors. In the GPI all compounds were pharmacologically active as reversible agonists, but only the 6 beta-isomers of the fumaramate ester 2b (beta-FNA) and isothiocyanate 6b exhibited covalent reactivity, involving a selective irreversible antagonism of the mu agonist, morphine, without affecting kappa agonists. The 6 alpha-isomer 2a (alpha-FNA) was itself nonalkylating but was able to protect the GPI against alkylation by its epimer, beta-FNA, indicating that the two epimers bind to the same receptor. These results suggest that the proper orientation of the electrophilic substituent is required for covalent bonding with a proximal nucleophile in the case of mu receptor blockade. Moreover, the lack of covalent bonding to kappa receptors by these or other ligands in this series indicates the possible absence of sufficiently reactive nucleophiles on this recognition site. In the MVD, 2b, but not 2a, irreversibly antagonized morphine (as in GPI), whereas neither epimer exhibited irreversible antagonism toward the delta agonist, [D-Ala2,D-Leu5]enkephalin (DADLE). In contrast, both of the isothiocyanate epimers (6a,b) irreversibly blocked mu and delta receptors. Evidence suggesting differences between mu receptors in the MVD and GPI was obtained with the beta-iodoacetamide 5b, which was an irreversible blocker of morphine only in the MVD. When analyzed together with those of previous studies with the nitrogen mustard analogues, alpha- and beta-chlornaltrexamine, the data suggest that the receptor-alkylating ability of each isomer in an epimeric pair differs most when the electrophile possesses a narrow spectrum of reactivity.

Aminotetralins as narcotic antagonists. 2. Synthesis and opiate-related activity of 1-phenyl-3-aminotetralins.

The synthesis and analgetic agonist and antagonist activities of several 3-[N-(cyclopropylmethyl)-N-methylamino]-1-phenyltetralins are reported. The design of these agents was based partially on the possibility of two aryl receptor binding sites on the opiate receptor. The agents lack the phenolic hydroxyl and quaternary carbon functionalities generally associated with opiate activity; yet both the cis- and trans-1-phenyl-3-aminotetralins displayed significant agonist and antagonist activity. In preliminary studies, the trans isomer neither suppressed nor precipitated withdrawal signs in addicted monkeys.

Stereochemical studies on medicinal agents. 25. Absolute configuration and analgetic potency of beta-1,2-dimethyl-2-phenyl-4-(propionyloxy)piperidine enantiomers.

Enantiomers of beta-1,2-dimethyl-4-phenyl-4-(propionyloxy)piperidine (4) were employed as probes to demonstrate that opioid receptors are capable of distinguishing between the enantiotopic edges (the Ogston effect) of the piperidine ring. These enantiomers, (-)- and (+)-4.HCl, were prepared by esterification of the corresponding alcohols, (+)- and (-)-4a. Single crystal X-ray studies of (-)-4a.HCl reveal that it possesses the 2R,4S absolute configuration. Analgetic testing in mice (hot-plate) and receptor binding studies indicate that (-)-(2S,4R)-4.HCl is approximately ten times more potent than its enantiomer. The results are consistent with the operation of the Ogston effect in the interaction of achiral 4-phenylpiperidines with opioid receptors. Additionally, it is suggested that the piperidine ring of these and other closely related 4-phenylpiperidines bind within a receptor subsite cleft whose dimensions exclude diequatorial 2,6- and 3,5-dimethyl-substituted ligands.

Aminotetralins as narcotic antagonists. Synthesis and opiate-related activity of 1-phenyl-2-aminotetralin derivatives.

The synthesis and the opiate agonist and antagonist activities of three derivatives of cis-2-[methyl(cyclopropanemethyl)amino]-1-phenyltetralin are reported. The compounds were obtained by synthetic modification from 2-amino-1-tetralone. The 1-propionoxy derivative 4c shows analgetic activity (ED50 = 17.8 mg/kg) one-half that of codeine, and the 1-methoxy derivative 4b has weak antagonist activity (AD50 = 33.5 mg/kg). The compounds showed no other significant opiate-related activity.

Synthesis and preliminary pharmacological activity of aminoalkoxy isosteres of glycolate ester anticholinergics.

A sides of 2-(N-substituted amino)alkoxy-1,1-diphenylethanols was synthesized and evaluated for anticholinergic activity. The compounds differ structurally from the glycolate ester-type anticholinergic compounds by the bioisosteric substitution of a methylene group for the ester carbonyl moiety. The ethers which result from this change have increased lipophilicity compared to their ability to inhibit perphenazine-induced catatonia in rats. Structure-activity relationships of the compounds are discussed.