Synthesis and biological evaluation of pentacyclo[5.4.0.02,6.03,10.05,9]undecane derivatives as potential therapeutic agents in Parkinson's disease

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Abstract

In previous studies, the polycyclic cage amine 8-benzylamino-8,11-oxapentacyclo[5.4.0.02,6.03,10.05,9]undecane (NGP1-01) and a number of its derivatives showed positive effects in neuroprotection studies with MPTP, in vivo. In view of these findings, we examined these compounds for their effects on [3H]dopamine ([3H]DA) release and uptake inhibition in murine striatal synaptosomes, as well as for inhibition of baboon liver monoamine oxidase (MAO) B. In order to assess specificity, initial experiments focused on compounds that blocked dopamine uptake without causing appreciable release (<40% at 100 μM) of the transmitter. NGP1-01 blocked the uptake of [3H]DA with an IC50 of 57 μM, while another compound, 8-phenylethyl-8,11-oxapentacyclo[5.4.0.02,6.03,10.05,9]undecane, blocked uptake at an IC50 value of 23 μM. These values were comparable to that of another polycyclic cage amine, amantadine (IC50; 82 μ), that is used in parkinsonian therapy. Structure–activity relationships of this series of compounds support the importance of geometric and steric, rather than electronic effects, in determining biological activity. MAO-B inhibition for this group was weak, with less than 50% inhibition at 300 μM for any of the compounds in the series. The present study suggests that blockage of the dopamine transporter may underlie, at least in part, their neuroprotective effects against MPTP-induced parkinsonism. These compounds may be considered as potential lead compounds for Parkinson's Disease therapy.

Introduction

Parkinson's disease (PD) is a terminal, late-onset neurodegenerative disorder characterized by a progressive and relatively selective degeneration of dopaminergic neurons in the substantia nigra.1 To date, most therapies for PD are symptomatic in nature, with no neuroprotective therapies currently approved for prevention of this chronic disease. Some therapeutic agents used in the early treatment of PD have, however, been shown to have neuroprotective activity in addition to their symptomatic effects, such as the dopamine agonist, pramipexole.2

Amongst several drug therapies used in the symptomatic treatment of PD patients is the polycyclic cage amine, amantadine (Fig. 1). Amantadine expresses its antiparkinsonian activity, at least in part, by increasing extracellular dopamine levels via re-uptake inhibition3 or dopamine (DA) release.4 Electrophysiological studies further indicated that amantadine binds to the phencyclidine (PCP) or MK-801 (dizocilpine) binding site located within the NMDA receptor/ion channel complex.5 In addition, amantadine and its dimethyl derivative memantine have been shown to be neuroprotective.5, 6

Early interest in the in vivo biological activities of polycyclic cage amines was raised when Oliver et al.7, 8 reported antagonism of reserpine-induced catatonia as well as reduction of oxotremorine-induced tremor and salivation in rats by novel pentacycloundecylamines and trishomocubanes, at doses comparable to those of amantadine. These authors suggested that the polycyclic cage amines might be a new class of possible antiparkinsonian agents due to anticataleptic and mild to weak anticholinergic activities.

l-type calcium channel antagonism has been reported by our laboratory for pentacycloundecylamines, in particular for the prototypical compound 8-benzylamino-8,11-oxapentacyclo[5.4.0.02,6.03,10.05,9]undecane (Fig. 1, Scheme 1, Figure 1, NGP1-01).9, 10, 11, 12, 13 Recently, we reported an in vivo pilot evaluation of a small series of pentacycloundecylamines in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) parkinsonian mouse model and found that one of the compounds, 8-phenylethyl-8,11-oxapentacyclo[5.4.0.02,6.03,10.05,9]undecane, tested positively as a neuroprotective agent.14 MPTP is a protoxin that requires activation by means of monoamine oxidase (MAO) B to form the neurotoxic pyridinium species, 1-methyl-4-phenyl-pyridinium (MPP+).15 MPP+ is then transported into dopaminergic neurons via the dopamine transporter (DAT), where it inhibits mitochondrial complex 1.16, 17 Excitotoxicity is also implicated in the MPTP model of neuronal cell death, and involves the over-stimulation of the N-methyl-d-aspartate (NMDA) receptor by glutamate, ultimately resulting in excess intracellular calcium and cell death.18, 19

On the basis of this information, we have chosen to evaluate a series of polycyclic cage amines for activity in the dopamine system as part of an ongoing investigation into the therapeutic potential of novel pentacycloundecylamines. We are hoping to develop drugs that are dual neuroprotective and symptomatic therapy for neurodegenerative diseases such as PD. Since the DAT and MAO-B are key elements in the DA depletion seen in the MPTP mouse model, we investigated the pharmacology of the compounds in this study for interaction with the DAT and MAO-B in vitro, aiming to select compounds for future in vivo studies. Moreover, because our future plans include analysis of effects on the NMDA receptor and MPTP/C57 mouse model, we included MK-801 as an established NMDA antagonist in the present study.

Section snippets

Chemistry

Van der Schyf et al.9, 10, 11, 12 postulated that the cage-like pentacycloundecane skeleton mostly served as a bulk contributor to the calcium channel blocking activity of the parent compound, Scheme 1, Figure 1. Structure–activity studies were combined with synthetic efforts to investigate the effects of different aromatic amine side chains as well as gaining insight into the effects of the polycyclic skeleton.

Synthesis of compounds Figure 1, Scheme 1 and 9 was conducted as previously described

Pharmacology

Release and uptake inhibition of [3H]DA was carried out in murine striatal synaptosomes as described earlier.21 Compounds were screened at 100 μM, except for GBR-12909 ([3H]DA release, 10 μM; inhibition of [3H]DA uptake, 2 μM). Initial experiments identified compounds that blocked [3H]DA uptake without causing appreciable release (≤40% at 100 μM), in order to ensure specificity. IC50 values for inhibiting [3H]DA uptake were then determined for selected compounds. Inhibition of MAO-B was carried

Molecular modeling

Molecular modelling to obtain a reasonable minimum energy conformation for each structure was performed by optimizing the structures with the Chemplus™ extension of Hyperchem® modeling software (Release 4.5 for Windows 1994, Hypercube Inc. Ontario, Canada) using MM+ and AM1 with the Polak-Ribieré minimization procedure.23 The lowest energy conformer found for compounds 3b and 4b was used in superpositioning and 9 was used to calculate its physicochemical properties, and to assist in estimating

Results and discussion

Initial screening studies determined the compounds’ ability to release DA, and compounds with low activity were selected for further evaluation (Table 1). The compounds fell into three groups with respect to causing DA release; those that were able to elicit no more than 0–20% release of label (Scheme 1, Figure 1, 2, 3a, 6, and 7), those that elicited 20–40% release of label (3c, 4a, c, 5a, b, 8, 9, memantine, and MK-801), and those that released >60% of label (3b and 4b). Because these studies

Conclusions

In this report, we describe the syntheses and in vitro biological studies (interaction with the DAT and the enzyme MAO-B) of a novel group of polycyclic cage amines, in order to identify promising compounds for future neuroprotection studies in the well known MPTP parkinsonian mouse model. The cage compounds proved to be effective inhibitors of dopamine uptake, with IC50 values comparable to that of amantadine. The most active compound (9) had an IC50 value of 23 μM. In structure–activity

Chemistry

All melting points were determined on a Gallenkamp melting point apparatus and are uncorrected. The 1H and 13C spectra were recorded on a Varian VXR 300 spectrometer. Samples were dissolved in a deuterated solvent (CDCl3) with tetramethylsilane (Me4Si) as an internal standard. Infrared spectra were recorded as a neat film on KBr plates with a Shimadzu FT IR 4200 spectrophotometer. Mass spectra and HR-MS were recorded at 70 eV (EI) on a VG 7070E spectrometer. Elemental analyses were performed on

Animals

ICR male mice (20–26 g, 6–8 weeks old) were obtained from Harlan, Dublin, VA. All animal care and experimental protocols were approved by the Virginia Tech Institutional Animal Care and Use Committee in accordance with guidelines established by the United States Public Health Service and Virginia Tech.

Preparation of synaptosomes

Male ICR mice were sacrificed by cervical dislocation and the striata were rapidly removed. The striatal tissue was homogenized in 4 mL of sucrose buffer (0.32 M sucrose, 2 mM HEPES, pH 7.4). The

Acknowledgements

This study was funded in part by the National Research Foundation (South Africa). Opinions expressed and conclusions arrived at, are those of the authors and not necessarily to be attributed to the NRF.

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