Pharmacodynamic Relationships between Duration of Action of JDTic-like Kappa-Opioid Receptor Antagonists and Their Brain and Plasma Pharmacokinetics in Rats

JDTic is a potent and selective κ-opioid receptor (KOR) antagonist that reverses U50,488-induced diuresis in rats. It partitions into brain with a duration of action lasting for weeks. In a search for KOR antagonists that do not accumulate in the brain, we compared single doses of five methylated JDTic analogs (RTI-97, -194, -212, -240, and -241) for reversal of U50,488 diuresis and pharmacokinetic (PK) properties. All six compounds showed potent and selective KOR antagonism in a [³⁵S]­GTPγS binding assay. Plasma half-lives ranged from 24 to 41 h and brain half-lives from 24 to 76 h. JDTic and RTI-194 showed increasing brain to plasma ratios over time, indicating increasing partitioning into brain and a longer duration of action for reversal of diuresis than did RTI-97. RTI-240 did not show significant brain accumulation. RTI-212 showed no substantive difference between brain and plasma levels and was inactive against diuresis. RTI-241, with a lower brain to plasma ratio than JDTic and RTI-194, formed JDTic as a metabolite, which still reduced diuresis after 9 weeks. The fact that the duration of action was correlated with the brain to blood plasma ratios and area under the concentration–time curves suggests that PK properties could help to predict safety and acceptable duration of action for KOR antagonists

Synthesis and Nicotinic Acetylcholine Receptor in Vitro and in Vivo Pharmacological Properties of 2′-Fluoro-3′-(substituted phenyl)deschloroepibatidine Analogues of 2′-Fluoro-3′-(4-nitrophenyl)deschloroepibatidine

Herein, we report the synthesis and nicotinic acetylcholine receptor (nAChR) in vitro and in vivo pharmacological properties of 2′-fluoro-3′-(substituted phenyl)­deschloroepibatidines <b>5b</b>–<b>g</b>, analogues of 3′-(4-nitrophenyl) compound <b>5a</b>. All compounds had high affinity for α4β2-nAChR and low affinity for α7-nAChR. Initial electrophysiological studies showed that all analogues were antagonists at α4β2-, α3β4-, and α7-nAChRs. The 4-carbamoylphenyl analogue <b>5g</b> was highly selective for α4β2-nAChR over α3β4- and α7-nAChRs. All the analogues were antagonists of nicotine-induced antinociception in the tail-flick test. Molecular modeling docking studies using the agonist-bound form of the X-ray crystal structure of the acetylcholine binding protein suggested several different binding modes for epibatidine, varenicline, and <b>5a</b>–<b>g</b>. In particular, a unique binding mode for <b>5g</b> was suggested by these docking simulations. The high binding affinity, in vitro efficacy, and selectivity of <b>5g</b> for α4β2-nAChR combined with its nAChR functional antagonist properties suggest that <b>5g</b> will be a valuable pharmacological tool for studying the nAChR and may have potential as a pharmacotherapy for addiction and other central nervous system disorders

Synthesis and Evaluation of Metabotropic Glutamate Receptor Subtype 5 Antagonists Based on Fenobam

In an effort to discover potent and selective metabotropic glutamate receptor subtype 5 (mGluR5) antagonists, 15 tetrahydropyrimidinone analogues of 1-(3-chlorophenyl)-3-(1-methyl-4-oxo-4,5-dihydro-1<i>H</i>-imidazol-2-yl)-urea (fenobam) were synthesized. These compounds were evaluated for antagonism of glutamate-mediated mobilization of internal calcium in an mGluR5 in vitro efficacy assay. The IC₅₀ value for 1-(3-chlorophenyl)-3-(1-methyl-4-oxo-1,4,5,6-tetrahydropyridine)­urea (<b>4g</b>) was essentially identical to that of fenobam

Synthesis, Nicotinic Acetylcholine Receptor Binding, and Antinociceptive Properties of 2′-Fluoro-3′-(substituted pyridinyl)-7-deschloroepibatidine Analogues

2′-Fluoro-3-(substituted pyridine)­epibatidine analogues <b>7a</b>–<b>e</b> and <b>8a</b>–<b>e</b> were synthesized, and their in vitro and in vivo nAChR properties were determined. 2′-Fluoro-3′-(4″-pyridinyl)­deschloroepibatidine (<b>7a</b>) and 2′-fluoro-3′-(3″-pyridinyl)­deschloroepibatidine (<b>8a</b>) were synthesized as bioisosteres of the 4′-nitrophenyl lead compounds <b>5a</b> and <b>5g</b>. Comparison of the in vitro nAChR properties of <b>7a</b> and <b>8a</b> to those of <b>5a</b> and <b>5g</b> showed that <b>7a</b> and <b>8a</b> had in vitro nAChR properties similar to those of <b>5a</b> and <b>5g</b> but both were more selective for the α4β2-nAChR relative to the α3β4- and α7-nAChRs than <b>5a</b> and <b>5g</b>. The in vivo nAChR properties in mice of <b>7a</b> were similar to those of <b>5a</b>. In contrast, <b>8a</b> was an agonist in all four mouse acute tests, whereas <b>5g</b> was active only in a spontaneous activity test. In addition, <b>5g</b> was a nicotine antagonist in both the tail-flick and hot-plate tests, whereas <b>8a</b> was an antagonist only in the tail-flick test

4β-Methyl-5-(3-hydroxyphenyl)morphan Opioid Agonist and Partial Agonist Derived from a 4β-Methyl-5-(3-hydroxyphenyl)morphan Pure Antagonist

In previous studies we reported that addition of 7α-acylamino groups to <i>N</i>-phenylpropyl-4β-methyl-5-(3-hydroxyphenyl)­morphan (<b>4</b>) led to compounds that were pure opioid receptor antagonists. In contrast to these findings we report in this study that addition of a 7α-amino (<b>5a</b>), 7α-alkylamino (<b>5b</b>–<b>e</b>), or 7α-dialkylamino (<b>5f</b>–<b>h</b>) group to <b>4</b> leads to opioid receptor ligands with varying degrees of agonist/antagonist activity. The 7α-amino and 7α-methylamino analogues were full agonists at the μ and δ receptors and antagonists at the κ receptor. The 7α-cyclopropylmethylamino analogue <b>5h</b> was a full agonist at the μ receptor with weaker agonist activity at the δ and κ receptors. Whereas the addition of a 7α-acylamino group to the pure nonselective opioid receptor antagonist <i>N</i>-phenylpropyl-4β-methyl-5-(3-hydroxyphenyl)­morphan (<b>4</b>) led to κ selective pure opioid receptor antagonist, the addition of a 7α-amino, 7α-alkylamino, or 7α-dialkylamino group to <b>4</b> leads to opioid ligands that are largely μ or δ agonist with mixed agonist/antagonist properties

Design, Synthesis, and Biological Evaluation of Structurally Rigid Analogues of 4‑(3-Hydroxy­phenyl)­piperidine Opioid Receptor Antagonists

In order to gain additional information concerning the active conformation of the <i>N</i>-substituted <i>trans</i>-3,4-dimethyl-4-(3-hydroxy­phenyl)­piperidine (<b>1</b>) class of opioid receptor antagonists, procedures were developed for the synthesis of structurally rigid <i>N</i>-substituted-6-(3-hydroxy­phenyl)­3-azabicyclo­[3.1.0]­hexane and 3-methyl-4-(3-hydroxy­phenyl)-4-azabicyclo­[4.1.0]­heptanes. Evaluation of the conformationally constrained series in a [³⁵S]­GTPγS assay showed that structural rigid compounds having the 3-hydroxyphenyl group locked in the piperidine equatorial orientation had potencies equal to or better than similar compounds having more flexible structures similar to <b>1</b>. The studies of the rigid compounds also suggested that the 3-methyl group present in compound <b>1</b> type antagonists may not be necessary for their pure opioid antagonist properties

Simple Radiometric Method for Accurately Quantitating Epitope Densities of Hapten–Protein Conjugates with Sulfhydryl Linkages

Control of small molecule hapten epitope densities on antigenic carrier proteins is essential for development and testing of optimal conditions for vaccines. Yet, accurate determination of epitope density can be extremely difficult to accomplish, especially with the use of small haptens, large molecular weight carrier proteins, and limited amounts of protein. Here we report a simple radiometric method that uses ¹⁴C-labeled cystine to measure hapten epitope densities during sulfhydryl conjugation of haptens to maleimide activated carrier proteins. The method was developed using a (+)-methamphetamine (METH)-like hapten with a sulfhydryl terminus, and two prototype maleimide activated carrier proteins, bovine serum albumin (BSA) and immunocyanin monomers of keyhole limpet hemocyanin. The method was validated by immunochemical analysis of the hapten–BSA conjugates, and least-squares linear regression analysis of epitope density values determined by the new radiometric method versus values determined by matrix-assisted laser desorption/ionization mass spectrometry. Results showed that radiometric epitope density values correlated extremely well with the mass spectrometrically derived values (<i>r</i>² = 0.98, <i>y</i> = 0.98<i>x</i> + 0.91). This convenient and simple method could be useful during several stages of vaccine development including the optimization and monitoring of conditions for hapten–protein conjugations, and choosing the most effective epitope densities for conjugate vaccines

Caged Naloxone: Synthesis, Characterization, and Stability of 3‑<i>O</i>‑(4,5-Dimethoxy-2-nitrophenyl)carboxymethyl Naloxone (CNV-NLX)

The photolabile analogue of the broad-spectrum opioid antagonist naloxone, 3-<i>O</i>-(4,5-dimethoxy-2-nitrophenyl)­carboxymethyl naloxone (also referred to as “caged naloxone”, 3-<i>O</i>-(α-carboxy-6-nitroveratryl)­naloxone, CNV-NLX), has been found to be a valuable biochemical probe. While the synthesis of CNV-NLX is simple, its characterization is complicated by the fact that it is produced as a mixture of α<i>R</i>,5<i>R</i>,9<i>R</i>,13<i>S</i>,14<i>S</i> and α<i>S</i>,5<i>R</i>,9<i>R</i>,13<i>S</i>,14<i>S</i> diastereomers. Using long-range and heteronuclear NMR correlations, the ¹H NMR and ¹³C NMR resonances of both diastereomers have been fully assigned, confirming the structures. Monitoring of solutions of CNV-NLX in saline buffer, in methanol, and in DMSO has shown CNV-NLX to be stable for over a week under fluorescent laboratory lights at room temperature. Exposure of such solutions to λ 365 nm from a hand-held UV lamp led to the formation of naloxone and CNV-related breakdown products

Combining Active Immunization with Monoclonal Antibody Therapy To Facilitate Early Initiation of a Long-Acting Anti-Methamphetamine Antibody Response

We hypothesized that an anti-METH mAb could be used in combination with a METH-conjugate vaccine (MCV) to safely improve the overall quality and magnitude of the anti-METH immune response. The benefits would include immediate onset of action (from the mAb), timely increases in the immune responses (from the combined therapy) and duration of antibody response that could last for months (from the MCV). A novel METH-like hapten (METH-SSOO9) was synthesized and then conjugated to immunocyanin monomers of keyhole limpet hemocyanin (IC_KLH) to create the MCV IC_KLH-SOO9. The vaccine, in combination with previously discovered anti-METH mAb7F9, was then tested in rats for safety and potential efficacy. The combination antibody therapy allowed safe achievement of an early high anti-METH antibody response, which persisted throughout the study. Indeed, even after 4 months the METH vaccine antibodies still had the capacity to significantly reduce METH brain concentrations resulting from a 0.56 mg/kg METH dose

Discovery of <i>N</i>‑{4-[(3-Hydroxyphenyl)-3-methylpiperazin-1-yl]methyl-2-methylpropyl}-4-phenoxybenzamide Analogues as Selective Kappa Opioid Receptor Antagonists

There is continuing interest in the discovery and development of new κ opioid receptor antagonists. We recently reported that N-substituted 3-methyl-4-(3-hydroxyphenyl)­piperazines were a new class of opioid receptor antagonists. In this study, we report the syntheses of two piperazine JDTic-like analogues. Evaluation of the two compounds in an in vitro [³⁵S]­GTPγS binding assay showed that neither compound showed the high potency and κ opioid receptor selectivity of JDTic. A library of compounds using the core scaffold <b>21</b> was synthesized and tested for their ability to inhibit [³⁵S]­GTPγS binding stimulated by the selective κ opioid agonist U69,593. These studies led to <i>N</i>-[(1<i>S</i>)-1-{[(3<i>S</i>)-4-(3-hydroxyphenyl)-3-methylpiperazin-1-yl]­methyl}-2-methylpropyl]-4-phenoxybenzamide (<b>11a</b>), a compound that showed good κ opioid receptor antagonist properties. An SAR study based on <b>11a</b> provided 28 novel analogues. Evaluation of these 28 compounds in the [³⁵S]­GTPγS binding assay showed that several of the analogues were potent and selective κ opioid receptor antagonists