Part I. The First Enantiospecific, Stereospecific Total Synthesis of the Indole Alkaloid Ervincidine. Part II. The Synthesis of Alpha 5 Subtype Selective Ligands for Gaba(a) /Benzodiazepine Receptors

Part I. The first enantiospecific, stereospecific total synthesis of ervincidine 89 has been accomplished from commercially available D-(+)-tryptophan 37 which has served both as the chiral auxiliary and the starting material. Moreover, this is the first synthesis which unequivocally sets the stereochemistry of the hydroxyl group at C-6 in sterospecific fashion, as well as the C-16 hydroxy methyl group. The stereospecific conversion of D-(+)-tryptophan 37 into the key template (−)-Na-H, Nb-benzyl tetracyclic ketone 49 via the asymmetric Pictet-Spengler reaction (600 gram scale) and Dieckmann cyclization on multi-hundred gram scale was reduced to only two reaction vessels. The optically active tetracyclic ketone 49 was converted into the core pentacyclic framework 56 using the intramolecular palladium-mediated enolate cross coupling reaction which was developed here in Milwaukee to afford the core pentacylic framework 56. This robust reaction could be scaled up to multigram scale in this series. Important to success here were the sequence of chemical reactions which included a Wittig reaction, a regioselective hydroboration and protection/deprotection steps in order to provide regiospecific oxidation at C-6. The IBX mediated oxidation and the Luche reduction using CeCl3. 7H20 in the presence of NaBH4 afforded the first enantiospecific, stereospecific total synthesis of ervincidine 89. The indole alkaloid ervincidine 89 could be prepared from D-(+)-tryptophan 37 in 13 reaction vessels in 19.2% overall yield. Another important experiment was the epimerization of the C-6 alcohol with 0.2N HCl which indicated that care must be employed in isolation of these alkaloids which contain a benzylic hydroxyl group. This research process developed here also provides a general entry to the C-6 hydroxy substituted indole alkaloids of either alpha or beta stereochemistry. Two other diasteromers were made to rule them out as potential structures. This research corrects the errors in Glasby\u27s book and Lousnamma\u27s review and clarifies the work of Yunusov et al. as well as providing the correct absolute configuration of the C-6 hydroxyl function in ervincidine 89. Part.II..GABAA/BzR chloride ion channels comprise the major inhibitory neurotransmitter system in the CNS. This central role carries with it a direct influence on many diseases of the CNS. Inverse agonists acting at α5 subunits containing GABAA receptors are thought to act as cognitive enhancers while eliminating unwanted side effects associated with non-selective compounds. From the recent work of Rowlett, Cook et.al. it was demonstrated that novel alpha5 selective inverse agonist PWZ-029 was evaluated as a cognitive enhancer in rhesus monkeys in the CANTAB paradigm. This ligand had the ability to reverse cholinergic deficits in performance induced by the antimuscarinic scopolamine under mixed trial conditions. In the ORD task, PWZ-029 showed only a modest trend for enhancement of performance, but when task difficulty was increased by testing with difficult trials only, PWZ-029 robustly increased performance. This enhancement was reversed by administration of the alpha 5 GABA (A) subtype selective antagonist XLi-093 and this antagonism in turn was reversed by increasing the dose of PWZ-029. In addition, PWZ-029 enhanced performance in the DNMS task using the 10 minute delay with distracters. This ligand also exhibited anxiolytic activity in some primates and was an orally active anticonvulsant in rats. These findings are consistent with a key role for alpha5 GABAA receptors in the treatment of age-associated memory impairment and Alzheimer\u27s disease

Antagonism of triazolam self-administration in rhesus monkeys responding under a progressive-ratio schedule: In vivo apparent pA2 analysis.

BACKGROUND: Conventional benzodiazepines bind non-selectively to GABAA receptors containing α1, α2, α3, and α5 subunits (α1GABAA, α2GABAA, α3GABAA, and α5GABAA receptors, respectively), and the role of these different GABAA receptor subtypes in the reinforcing effects of benzodiazepines has not been characterized fully. We used a pharmacological antagonist approach with available subtype-selective ligands to evaluate the role of GABAA receptor subtypes in the reinforcing effects of the non-selective conventional benzodiazepine, triazolam. METHODS: Rhesus monkeys (n=4) were trained under a progressive-ratio schedule of intravenous midazolam delivery and dose-response functions were determined for triazolam, in the absence and presence of flumazenil (non-selective antagonist), βCCT and 3-PBC (α1GABAA-preferring antagonists), and XLi-093 (α5GABAA-selective antagonist). RESULTS: Flumazenil, βCCT and 3-PBC shifted the dose-response functions for triazolam to the right in a surmountable fashion, whereas XLi-093 was ineffective. Schild analyses revealed rank orders of potencies of flumazenil=βCCT\u3e3-PBC. Comparison of potencies between self-administration and previous binding studies with human cloned GABAA receptor subtypes suggested that the potencies for βCCT and 3-PBC were most consistent with binding at α2GABAA and α3GABAA receptors, but not α1GABAA or α5GABAA receptor subtypes. CONCLUSIONS: Our findings were not entirely consistent with blockade of α1GABAA receptors and are consistent with the possibility of α2GABAA and/or α3GABAA subtype involvement in antagonism of the reinforcing effects of triazolam. The α5GABAA receptor subtype likely does not play a substantial role in self-administration under these conditions

A Review of the Updated Pharmacophore for the Alpha 5 GABA(A) Benzodiazepine Receptor Model

An updated model of the GABA(A) benzodiazepine receptor pharmacophore of the alpha 5-BzR/GABA(A) subtype has been constructed prompted by the synthesis of subtype selective ligands in light of the recent developments in both ligand synthesis, behavioral studies, and molecular modeling studies of the binding site itself. A number of BzR/GABA(A) alpha 5 subtype selective compounds were synthesized, notably alpha 5-subtype selective inverse agonist PWZ-029 (1) which is active in enhancing cognition in both rodents and primates. In addition, a chiral positive allosteric modulator (PAM), SH-053-2'F-R-CH3 (2), has been shown to reverse the deleterious effects in the MAM-model of schizophrenia as well as alleviate constriction in airway smooth muscle. Presented here is an updated model of the pharmacophore for alpha 5 beta 2 gamma 2 Bz/GABA(A) receptors, including a rendering of PWZ-029 docked within the alpha 5-binding pocket showing specific interactions of the molecule with the receptor. Differences in the included volume as compared to alpha 1 beta 2 gamma 2, alpha 2 beta 2 gamma 2, and alpha 3 beta 2 gamma 2 will be illustrated for clarity. These new models enhance the ability to understand structural characteristics of ligands which act as agonists, antagonists, or inverse agonists at the Bz BS of GABA(A) receptors

Search for α3β2/3γ2 subtype selective ligands that are stable on human liver microsomes

Selective modulation of specific benzodiazepine receptor (BzR) gamma amino butyric acid-A (GABAA) receptor ion channels has been identified as an important method for separating out the variety of pharmacological effects elicited by BzR-related drugs. Importantly, it has been demonstrated that both α2β(2/3)γ2 (α2BzR) and α3BzR (and/or α2/α3) BzR subtype selective ligands exhibit anxiolytic effects with little or no sedation. Previously we have identified several such ligands; however, three of our parent ligands exhibited significant metabolic liability in rodents in the form of a labile ester group. Here eight analogs are reported which were designed to circumvent this liability by utilizing a rational replacement of the ester moiety based on medicinal chemistry precedents. In a metabolic stability study using human liver microsomes, four compounds were found to undergo slower metabolic transformation, as compared to their corresponding ester analogs. These compounds were also evaluated in in vitro binding as well as efficacy assays. Additionally, bioisostere 11 was evaluated in a rodent model of anxiety. It exhibited anxiolytic activity at doses of 10 and 100 mg/kg and was devoid of sedative properties

Expression Quantitative Trait Loci and Receptor Pharmacology Implicate Arg1 and the GABA-A Receptor as Therapeutic Targets in Neuroblastoma

SummaryThe development of targeted therapeutics for neuroblastoma, the third most common tumor in children, has been limited by a poor understanding of growth signaling mechanisms unique to the peripheral nerve precursors from which tumors arise. In this study, we combined genetics with gene-expression analysis in the peripheral sympathetic nervous system to implicate arginase 1 and GABA signaling in tumor formation in vivo. In human neuroblastoma cells, either blockade of ARG1 or benzodiazepine-mediated activation of GABA-A receptors induced apoptosis and inhibited mitogenic signaling through AKT and MAPK. These results suggest that ARG1 and GABA influence both neural development and neuroblastoma and that benzodiazepines in clinical use may have potential applications for neuroblastoma therapy

A study of the structure–activity relationship of GABAA–benzodiazepine receptor bivalent ligands by conformational analysis with low temperature NMR and X-ray analysis

The stable conformations of GABAA-benzodiazepine receptor bivalent ligands were determined by low temperature NMR spectroscopy and confirmed by single crystal X-ray analysis. The stable conformations in solution correlated well with those in the solid state. The linear conformation was important for these dimers to access the binding site and exhibit potent in vitro affinity and was illustrated for α5 subtype selective ligands. Bivalent ligands with an oxygen-containing linker folded back upon themselves both in solution and the solid state. Dimers which are folded do not bind to Bz receptors

Completion of the Total Synthesis of Several Bioactive Sarpagine/Macroline Alkaloids including the Important NF-κB Inhibitor <i>N</i>₄-Methyltalpinine

The unification of the general synthetic strategy regarding the important and emerging group of C-19 methyl-substituted sarpagine/macroline alkaloids has culminated in the completion of the total synthesis of several bioactive alkaloids. Key transformations include an ACE-Cl mediated late-stage N(4)-demethylation and an anhydrous acid-mediated intramolecular quaternary hemiaminal formation between a tertiary amine and an aldehyde function to allow efficient access to several biologically important alkaloids from this group. Herein, the enantiospecific total synthesis of the first known sarpagine/macroline alkaloid with NF-κB inhibitory activity, N(4)-methyltalpinine (as a chloride salt), as well as the anticancer alkaloids talpinine, O-acetyltalpinine, and macrocarpines F–G, are described

Ethanol, not detectably metabolized in brain, significantly reduces brain metabolism, probably via action at specific GABA(A) receptors and has measureable metabolic effects at very low concentrations

Ethanol is a known neuromodulatory agent with reported actions at a range of neurotransmitter receptors. Here, we measured the effect of alcohol on metabolism of [3-C-13]pyruvate in the adult Guinea pig brain cortical tissue slice and compared the outcomes to those from a library of ligands active in the GABAergic system as well as studying the metabolic fate of [1,2-C-13]ethanol. Analyses of metabolic profile clusters suggest that the significant reductions in metabolism induced by ethanol (10, 30 and 60mM) are via action at neurotransmitter receptors, particularly 43 receptors, whereas very low concentrations of ethanol may produce metabolic responses owing to release of GABA via GABA transporter 1 (GAT1) and the subsequent interaction of this GABA with local 5- or 1-containing GABA(A)R. There was no measureable metabolism of [1,2-C-13]ethanol with no significant incorporation of C-13 from [1,2-C-13]ethanol into any measured metabolite above natural abundance, although there were measurable effects on total metabolite sizes similar to those seen with unlabelled ethanol

Stereospecific Total Synthesis of the Indole Alkaloid Ervincidine. Establishment of the C‑6 Hydroxyl Stereochemistry

The total synthesis of the indole alkaloid ervincidine (<b>3</b>) is reported. This research provides a general entry into C-6 hydroxy-substituted indole alkaloids with either an α or a β configuration. This study corrects the errors in Glasby’s book (Glasby, J. S. Encyclopedia of the Alkaloids; Plenum Press: New York, 1975) and Lounasmaa et al.’s review (Lounasmaa, M.; Hanhinen, P.; Westersund, M. In The Alkaloids; Cordell, G. A., Ed.; Academic Press: San Diego, CA, 1999; Vol. 52, pp 103–195) as well as clarifies the work of Yunusov et al. (Malikov, V. M.; Sharipov, M. R.; Yunusov, S. Yu. Khim. Prir. Soedin. 1972, 8, 760−761. Rakhimov, D. A.; Sharipov, M. R.; Aripov, Kh. N.; Malikov, V. M.; Shakirov, T. T.; Yunusov, S. Yu. Khim. Prir. Soedin. 1970, 6, 724–725). It establishes the correct absolute configuration of the C-6 hydroxyl function in ervincidine. This serves as a structure proof and corrects the misassigned structure reported in the literature