Synthesis of 4-oxatricyclo[5.2.1.0 2,6 ]dec-8-ene-3,5-dione derivatives as lead scaffolds for neuroprotective agents

Neurodegenerative disorders are characterised by progressive loss of neuronal functions. Of the proposed mechanisms, excitotoxicity, mediated by prolonged glutamate activation and calcium overload, is prominent. NGP1-01, a polycyclic cage amine, and tricyclo[6.2.1.0 2,7 ]undec-9-ene-3,6-dione have been shown to display calcium-modulating properties. In this study, we synthesised structurally-related 4-oxatricyclo[5.2.1.0 2,6 ]dec-8-ene-3,5-dione as the base scaffold, and incorporated various functional moieties through aminolysis, to afford a series of imide derivatives. All final compounds were obtained in yields between 47-97% and their structures were confirmed by NMR, IR and MS. These structurally-related derivatives could potentially act as neuroprotective agents. Additionally, their synthetic versatilities could make them precursors, as lead compounds, to potential pharmacologically-active agents.


Introduction
Neurodegenerative disorders are diseases characterised by persistent and progressive loss of neuronal function that eventually lead to neuronal death and reduction of sensory, motor, cognition and memory functions. 1,2These disorders include Parkinson's disease (PD), Alzheimer's disease (AD), progressive supranuclear palsy, amyotrophic lateral sclerosis (ALS) and Huntington disease. 3These multifactorial diseases not only share many common features, such as a late appearance in life, neuronal loss and synaptic abnormalities, but also share common molecular mechanisms that include excitotoxicity, mitochondrial dysfunction, apoptosis, oxidative stress and impaired protein homeostasis. 4Of the proposed mechanisms, the influence of excitotoxicity is prominent.In excitotoxicty, extracellular glutamate levels are elevated to such an extent that they over-activate ionotropic receptors, N-methyl-D-aspartate (NMDA) receptors, and associated voltage-gated calcIum channels (VGCC) in particluar.1][12][13] Therefore, molecules capable of modulating calcium influx via the NMDA receptor and/or VGCC could provide protection against calcium overload mediated by prolonged glutamate activity.MK-801 has been shown to uncompetitively block NMDA receptors, but is marked by undesirable psychotomimetic side effects such as hallucination, psychosis, dysphoria and amnesia.This makes it unsuitable for therapeutic purpose. 14o date, no drug is available to stop the neurodegenerative process.Adamatanes such as amantadine and memantine (Figure 1), however, have been shown to offer symptomatic relief in patients with PD and AD, respectively, and have subsequently been approved by the FDA for these purposes.0][21][22][23][24][25][26] A very good example is NGP1-01, a multifuctional neuroprotective agent that displayed dual attenuation of calcium entry in neuronal cells by blocking NMDA receptors and VGCCs.8][29][30][31] The need for more structurally-related scaffolds with similar effects and good safety profiles, that could potentially halt the degenerative process of neuronal cells, is thus, clear.

Results and Discussion
Synthesis and structural elucidation Under reflux conditions, the Diels-Alder reaction between maleic anhydride and cyclopentadiene afforded compound 2, an open cage-like 4-oxatricyclo[5.2.1.0 2,6]dec-8-ene-3,5-dione molecule, 43 which was used to obtain compounds 3-13 in moderate-to-high yields (see Figure 4).The aminolysis of 2 with benzylamine afforded compound 3 and the byproduct 4 (dibenzylamide moiety; Figure 5) in yields of 83% and 6%, respectively.The presence of the norbornene scaffold in compound 4 justifies its inclusion in the series.Moreover, the replacement of the furandione moiety with a dibenzylamine moiety provides scope for additional structureactivity relationships within this series.Compounds 9 and 10 were obtained under similar reaction conditions involving compound 2 and ethylenediamine; however, they differed in their work-up.Extraction using ethyl acetate afforded 9 in a yield of 90%, while precipitation from ice-cold methanol produced compound 10 (10%; Figure 5).Compound 10 was included in the series due to its bis-4-oxatricyclo[5.2.1.0 2,6]dec-8-ene-3,5-dione structure.Amongst the diamines (9-13), the reaction of compound 2 and N,N-dimethylethylenediamine produced the highest yield, 96% (13).This was due to the dimethyl substitution of one of the amines which limited byproduct formation.The structures of compounds 2-13 were characterised by their respective 1 H, 13 C, IR and HRMS spectra.In the IR spectra, the characteristic C=O signals were observed in the 1654-1766 cm -1 region for all the compounds (2-13).The appearance of absorption bands at around 1654 cm -1 (C=O) and 3304 cm -1 (N-H) confirms the amide moieties observed in 4. The presence of characteristic medium and broad signals for aliphatic primary amines in the 3372-3386 cm -1 region indicate mono-substitution of the diamines, which correspond to the structure of compound 9, 11 and 12.The appearance of a strong, sharp absorption band at 3265 cm -1 supported the presence of an aliphatic alkyne in the structure of 6.In the 1 H NMR spectra, the bridge protons resonate in the δ = 1.55-1.81and δ = 1.45-1.59ppm range for all compounds.The aromatic protons of 3 and 4 were identified as multiplets in the δ = 7.18-7.31ppm region with integrations of five and ten hydrogens, respectively.The aromatic protons of 8 were observed as a doublet of doublets (δ = 7.20-7.24ppm; J 15.9, 7.6 Hz), a triplet (δ = 6.93-6.97ppm; J 7.4 Hz) and a doublet (δ = 6.75-6.76ppm; J 7.7 Hz).The piperidinyl protons of 5 were observed as a triplet (δ = 3.07-3.10ppm; 4H) and two multiplets (δ = 1.61-1.67ppm, 4H; δ = 1.37-1.43ppm, 2H).In compound 6, the doublet (δ 4.09 ppm; J 2.5 Hz; 2H) and triplet (δ 2.13 ppm; J 2.5 Hz; 1H) signals were assigned to the CH 2 and CH groups of the propargyl conjugate.The appearance of multiplet (δ = 1.41-1.51ppm; 2H) and triplet (δ = 0.84-0.87ppm; J 7.4 Hz; 3H) signals in the spectra of 7 indicates the presence of a propyl moiety.The presence of a triplet at δ = 2.72-2.75ppm (J 6.4 Hz; 2H), and a doublet at δ = 3.40 ppm (J 6.4 Hz; 2H), confirms the successful incorporation of the diamine conjugate (9).The disappearance of signals in the 2.00-3.00ppm region suggest disubstitution of the diamine to afford 10.Due to the symmetrical nature of the carbons adjacent to the nitrogen groups, the CH 2 protons were observed downfield as a multiplet (δ = 3.64 ppm; 4H).In comparison to 9, the appearance of a singlet (δ = 2.20 ppm; 6H) indicates the presence of the dimethyl substituent as observed in 13.The appearance of a doublet of triplet signal and a doublet of doublet signal at δ = 1.59-1.64ppm (J 6.7, 4.8 Hz; 1H) and δ = 1.65-1.71ppm (J 13.7, 5.1 Hz; 1H), respectively, indicates the presence of an additional CH 2 group when compared to compound 9, thus, confirming the formation of the diaminopropane conjugate (11).In comparison to 11, the additional CH 2 group of 12 was observed as a multiplet at δ = 1.34-1.41ppm with an integration of two protons, thus, confirming the presence of a diaminobutane conjugate.In general, all of the protons of compounds 9-13 were accounted for, and assigned according to their respective spectra.In the 13 C NMR spectra, the characteristic signals for carbonyl and aromatic moieties were observed in the δ = 171-178 ppm and δ = 115-138 ppm regions, respectively.The carbon signals in the spectra of all compounds (2-13) were identified, and they corresponded to their proposed structures.The HRMS showed the respective molecular ion peaks that correspond to the empirical formula of each compound.
Reaction conditions that led to the formation of 2 and 3 afforded the respective endo-isomers.4][45][46] Similar reaction conditions were used in the synthesis of the remaining compounds (5-13), and their respective isomers were derived in a similar manner.

NMDA and VGCC studies
The abilities of compounds 2-13 to block calcium influx via the NMDA receptor and VGCCs were evaluated using neuroblastoma cells, and the fluorescent ratiometric calcium indicator FURA-2AM, as previously described in detail by our group. 21,22,26The compounds were found to exhibit calcium modulating abilities at the NMDA receptor and VGCC within a similar activity range as NGP1-01 and 1 at a 10 µM concentration.These initial promising biological findings have now led to the further exploration of their calcium modulating effect, neuroprotective abilities and toxicity profiles.

Figure 5 .
Figure 5. Formation of compounds 4 and 10: a) Compound 4 was obtained as a by-product from the synthesis of 3; b) Compound 10 was obtained as a precipitate from the reaction between compound 2 and ethylenediamine in the synthesis of 9. Percentage yields are shown in brackets.