Synthesis and characterization of succinylcholine-d18 and succinylmonocholine-d3 designed for simultaneous use as internal standards in mass spectrometric analyses.

Succinylcholine (SUX) is a routinely used yet potentially lethal depolarizing muscle relaxant, the detection of which poses severe problems to the clinical or forensic analyst: within a few minutes after its in vivo administration, SUX is broken down via succinylmonocholine (SMC) to yield the endogenous substances succinic acid and choline. For quantification of SUX and SMC in biological matrices using mass spectrometric detection, appropriate internal standards, i.e. deuterated analogs of the above substances, are indispensable but not commercially available. Internal standards for both substances were hence tailored to fit the analytical needs. The two-step synthesis and subsequent characterization of SUX-d(18) and SMC-d(3) using a combination of nuclear magnetic resonance (NMR) spectroscopy, fast atom bombardment mass spectroscopy (FAB-MS) and high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) are described. SUX-d(18) was synthesized by reacting ethanolamine and iodomethane-d(3) in a first quaternization step to choline-d(9), which in turn was esterified with succinyldichloride to yield the final product. SMC-d(3) was produced by esterification of succinic acid anhydride with dimethylaminoethanol, yielding desmethyl-SMC as intermediate product. The latter was then reacted with iodomethane-d(3) to obtain SMC-d(3). (1)H- and (13)C-NMR data support the identity and purity as well as the designated deuteration of both preparations, findings which were further confirmed by FAB-MS as well as HPLC-MS/MS. Owing to a thoughtful design, the obtained substances SUX-d(18) and SMC-d(3) feature different deuteration patterns at their trimethylamine moieties, and thus finally offer the possibility to simultaneously quantify SUX and SMC in clinical as well as forensic samples using isotope dilution mass spectrometry.

Synthesis and evaluation of radiolabeled piperazine derivatives of vesamicol as SPECT agents for cholinergic neurons.

To diagnose and investigate neurodegenerative diseases affecting cholinergic neuron density, piperazine derivatives of vesamicol were synthesized and evaluated. Previously, we reported that trans-5-iodo-2-hydroxy-3-[4-phenylpiperazinyl] tetralin (DRC140, 1) possessed high selectivity for vesicular acetylcholine transporter (VAChT). In present study of the effect of alkyl substituents, we observed that the introduction of a methyl group into the ortho or meta positions of the phenyl group of 1 increased affinity for VAChT. trans-5-Iodo-2-hydroxy-3-[4-[2-methylphenyl] piperazinyl]tetralin (2) displayed high affinity and specificity for VAChT. The regional distributions of radioactivity in the rat brain correlated well with known patterns of central cholinergic innervation. [(123)I]2 is a potentially useful compound for SPECT imaging.

Preparation of difluoro analogs of CCGs and their pharmacological evaluations.

All the stereoisomers of 2-(2-carboxy-3,3-difluorocyclopropyl)glycines (F2CCGs) were synthesized in enantiomerically pure forms using (R)-2,3-O-isopropyl-ideneglyceraldehyde as a chiral precursor. L-F2CCG-I, one of the stereoisomers corresponding to an extended form of L-glutamate was found to be a potent agonist for metabotropic glutamate receptors (mGluRs).

N-hydroxyalkyl derivatives of 3 beta-phenyltropane and 1-methylspiro[1H-indoline-3,4'-piperidine]: vesamicol analogues with affinity for monoamine transporters.

As part of our ongoing structure-activity studies of the vesicular acetylcholine transporter ligand 2-(4-phenylpiperidino)cyclohexanol (vesamicol, 1), 22 N-hydroxy(phenyl)alkyl derivatives of 3 beta-phenyltropane, 6, and 1-methylspiro[1H-indoline-3,4'-piperidine], 7, were synthesized and tested for binding in vitro. Although a few compounds displayed moderately high affinity for the vesicular acetylcholine transporter, no compound was more potent than the prototypical vesicular acetylcholine transporter ligand vesamicol. However, a few derivatives of 6 displayed higher affinity for the dopamine transporter than cocaine. We conclude that modification of the piperidyl fragment of 1 will not lead to more potent vesicular acetylcholine transporter ligands.

Vesamicol receptor mapping of brain cholinergic neurons with radioiodine-labeled positional isomers of benzovesamicol.

UNLABELLED: Alzheimer's disease is characterized by progressive cerebral cholinergic neuronal degeneration. Radiotracer analogs of benzovesamicol, which bind with high affinity to the vesamicol receptor located on the uptake transporter of acetylcholine storage vesicles, may provide an in vivo marker of cholinergic neuronal integrity. Five positional isomers of racemic iodobenzovesamicol (4'-, 5-, 6-, 7-, and 8-IBVM) were synthesized, exchange-labeled with iodine-125, and evaluated as possible in vivo markers for central cholinergic neurons. Only two isomers, 5-IBVM (5) and 6-IBVM (10), gave distribution patterns in mouse brain consistent with cholinergic innervation: striatum >> hippocampus > or = cortex > hypothalamus >> cerebellum. The 24-h tissue-to-cerebellum concentration ratios for 5-IBVM (5) were 3-4-fold higher for striatum, cortex, and hippocampus than the respective ratios for 6-IBVM (10). Neither 8-IBVM (16) nor 4'-IBVM (17) exhibited selective retention in any of the brain regions examined. In the heart, only 5-IBVM (5) exhibited an atria-to-ventricles concentration ratio consistent with high peripheral cholinergic neuronal selectivity. The 7-IBVM (14) isomer exhibited an anomalous brain distribution pattern, marked by high and prolonged retention in the five brain regions, most notably the cerebellum. This isomer was screened for binding in a series of 26 different biological assays; 7-IBVM (14) exhibited affinity only for the delta-receptor with an IC50 of approximately 30 nM. Drug-blocking studies suggested that brain retention of 7-IBVM (14) reflects high-affinity binding to both vesamicol and delta-receptors. Competitive binding studies using rat cortical homogenates gave IC50 values for binding to the vesamicol receptor of 2.5 nM for 5-IBVM (5), 4.8 nM for 6-IBVM (10), and 3.5 nM for 7-IBVM (14). Ex vivo autoradiography of rat brain after injection of (-)-5-[125I]IBVM ((-)-[125I]5) clearly delineated small cholinergic-rich areas such as basolateral amygdala, interpeduncular nucleus, and facial nuclei. Except for cortex, regional brain levels of (-)-5-[123I]IBVM ((-)-[123I]5) at 4 h exhibited a linear correlation (r2 = 0.99) with endogenous levels of choline acetyltransferase. CONCLUSION: Vesamicol receptor mapping of cholinergic nerve terminals in murine brain can be achieved with 5-IBVM (5) and less robustly with 6-IBVM (10), whereas the brain localization of 7-IBVM (14) reflects high-affinity binding to both vesamicol and delta-receptors.

Nonsymmetrical bipiperidyls as inhibitors of vesicular acetylcholine storage.

Introduction of a nitrogen atom into the cyclohexane ring of 2-(4-phenylpiperidinyl)cyclohexanol (vesamicol, AH5183) yielded two positional isomers, 5-azavesamicol (5, prezamicol) and 4-azavesamicol (6, trozamicol). As inhibitors of vesicular acetylcholine transport, 5 and 6 were found to be 147 and 85 times less potent than vesamicol. N-Benzoylation of 5 (to yield 9a) increased the potency 3-fold. In contrast, 10a, a compound derived from N-benzoylation of 6, was 50 times more potent than the latter and almost equipotent with vesamicol, thereby suggesting a preference for the 4-azavesamicol series. Although (-)-vesamicol is more potent than its dextrorotary isomer, (+)-10a was found to be 3 times more potent than (-)-10a, suggesting a reversal of the sign of rotation in the azavesamicol series. Reduction of 9a and 10a (to yield the corresponding N-benzyl derivatives 11a and 12a) increased potency 20- and 2-fold, respectively, indicating a preference for a basic nitrogen. The reaction of 5 or 6 with substituted benzyl halides yielded several potent inhibitors of vesicular acetylcholine transport, including N-(p-fluorobenzyl)trozamicol, 12d, which is twice as potent as vesamicol. Thus the introduction of a nitrogen atom into the cyclohexane ring of vesamicol provides opportunities for developing a new class of anticholinergic agents.

Tetrahydropyrrolo[1,2-a]quinoxalines and tetrahydropyrrolo[1,2-a]pyrido[3,2-a]pyrazines: vascular smooth muscle relaxants and antihypertensive agents.

A series of tetrahydropyrrolo[1,2-a]quinoxalines and tetrahydropyrrolo[1,2-a]pyrido[3,2-a]pyrazines were synthesized and tested for their ability to relax K+-depolarized aortic smooth muscle and antihypertensive activity. It was shown that compounds producing the most relaxation of aortic smooth muscle (5-[2,6-dimethoxyphenyl)methyl]-1,2,3,3a-tetrahydropyrrolo[1,2-a] quinoxalin-4(5H)-one and 5-[(2,6-dimethoxyphenyl)methyl]-5,6,6a,7,8,9-hexahydropyrrolo[1,2- a] pyrazine, 10 and 19, respectively) demonstrated the least hypotensive activity. Those compounds that were the most effective hypotensive agents (6a,7,8,9-tetrahydro-5-(phenylmethyl)pyrido[3,2-a]pyrrolo[1,2-a]++ +pyrazin- 6(5H)-one and 6a,7,8,9-tetrahydro-5-(4-pyridinylmethyl)pyrido[3,2-e]pyrrolo [1,2-a]pyrazin-6(5H)-one, 12 and 13, respectively) displayed little vascular smooth muscle relaxant activity.