Synthesis of novel epibatidine-related derivatives through 1,3-dipolar cycloaddition of pyridinenitrile oxides

The ∆ 2 -isoxazoline derivatives 3a-c and 4a-c , structurally related to epibatidine, and the simplified analogues 5a-c were synthesized by means of a 1,3-dipolar cycloaddition of regioisomeric pyridinenitrile oxides to suitable dipolarophiles. Target compounds were assayed at α 4 β 2 and α 7 neuronal acetylcholine receptor (nAChR) subtypes. Competition binding experiments at α 4 β 2 nAChRs showed an overall significant reduction in affinity for the compounds under study in comparison to the reference radioligand [ 3 H]-epibatidine. On the other hand, compounds 3b , 3c , and 4b exhibited a noticeable affinity for the α 7 receptors and 3c showed also a slight degree of α 7 over α 4 β 2 selectivity.


Introduction
The nicotinic acetylcholine receptors (nAChRs) are a superfamily of ligand-gated ion channels, which also includes GABA A,C , glycine, and 5-HT 3 receptors. 1,2Neuronal nAChRs are widely distributed in the human brain, where they are frequently associated with modulatory events and, to a lesser extent, mediate synaptic transmission. 34][15][16][17] Research efforts have mainly concerned ligands selectively acting at the α4β2 and α7 receptor subtypes, 18 the two major populations of nAChRs found in the brain.
1][22] Epibatidine has potent nAChR mediated analgesic activity since it agonizes neuronal α4β2 subtypes with a binding affinity that is 30 times higher than that of nicotine. 235][26] Nevertheless, epibatidine is a reference compound for all investigations on nAChRs and a model structure for the design of novel, high affinity and subtype-selective nAChR ligands.Epibatidine-related derivatives have been synthesized by varying the heteroaryl moiety, [27][28][29] or functionalizing and expanding the alicyclic skeleton, [30][31][32][33] or modifying the substituent and/or the position of the epimino nitrogen atom. 34,35n the light of the above discussed evidences we designed a group of novel compounds in which the two structural elements featuring epibatidine (i.e. the 7-azabicyclo[2.2.1]heptane system and the pyridine ring) were distanced by the insertion of a ∆ 2 -isoxazoline moiety, either spiro-condensed (derivatives 3a-c) or fused (derivatives 4a-c) with the azanorbornane core (Figure 1).In addition, we synthesized and tested compounds 5a-c (Figure 1), in which the ethylene bridge of the bicyclic system of 4a-c was removed.The aim of this study was to investigate the effect of the variation of both the epimino-N/pyridine-N distance and the conformational profile on the affinity/selectivity for the nAChR subtypes.Since the chlorine atom located on the pyridine ring makes a minor contribution to the affinity of epibatidine, 36 we decided to synthesize the unsubstituted pyridinyl regioisomers only.Moreover, the novel chiral derivatives 3a-c and 4a-c were prepared as racemates by taking into account the similar receptor binding efficiencies of the epibatidine enantiomers. 28,37,38This paper reports the synthesis of compounds 3-5 and the evaluation of their binding affinity at α4β2 and α7 nAChR subtypes.The present results represent a further application of the cycloaddition strategy to the synthesis of biologically active heterocycles, which has characterized our research in the recent years with a focus on chiral ∆ 2 -isoxazoline derivatives selectively acting at the different glutamic acid receptors and transporters. 39
As expected, pyridinenitrile oxides attack the less hindered face of bicyclic olefin 10 yielding exclusively the exo-cycloadducts 11a-c.According to the literature, [46][47][48] the observed syn facial selectivity is dictated by the anti pyramidalization of the olefinic hydrogens in the dipolarophile.The structural assignment to adducts 11a-c was unequivocally established by 1 H-NMR spectroscopy, since the absence of vicinal coupling constants between protons H-1 and H-2 as well as between protons H-3 and H-4 (Scheme 1) is a clear indication of their exo configuration. 49,50By taking into account the outcome of reactions carried out on olefins structurally related to 8, 51,52 we assigned the exo configuration even to spirocyclic intermediates 9a-c, which were the only isomers isolated in each cycloaddition step.Finally, the pericyclic reaction of pyridinenitrile oxides with olefin 12 yielded bicyclic cycloadducts 13a-c.Treatment of cycloadducts 9a-c, 11a-c, and 13a-c with trifluoroacetic acid provided the desired epibatidine analogues as the free bases 3a-c, 4a-c, and 5a-c, respectively.The latter compounds were converted into the corresponding fumarates by treatment with fumaric acid in methanol and the salts obtained were submitted to biological testing.The fumarates of the target compounds 3a-c, 4a-c, and 5a-c were assessed for their binding affinity to α4β2 and α7 rat nicotinic receptor subtypes using [ 3 H]-epibatidine and [ 125 I]-αbungarotoxin as radioligands, respectively.The K i values were calculated from the competition curves by means of the LIGAND program. 53On the whole, the insertion of the ∆ 2 -isoxazoline moiety between the azanorbornane system and the pyridine ring greatly reduced or abolished the binding affinity for the α4β2 nAChR subtype, since the K i value of the reference radioligand falls within the 20-40 pM concentration range.Among the studied compounds, only derivative 5b (K i = 72 µM) and the fused analogues 4b (K i = 86 µM) and 4c (K i = 68 µM), which are characterized by the pyridine nitrogen in position 3' and 4' respectively, retained a residual and comparable affinity for the α4β2 nAChRs.Conversely, the two spirocyclic analogues 3b (K i = 41 µM) and 3c (K i = 28 µM), in which the pyridine nitrogen atoms are located in position 3' and 4' respectively, showed the highest affinity for the α7 nAChR subtype, thus behaving quite similarly to the fused analogue 4b (Ki = 32 µM).In addition, derivative 3c evidenced a slight degree of selectivity for α7 over α4β2 receptor subtypes (K i values of 28 vs 400 µM, respectively).
The above discussed results on the group of novel epibatidine-related derivatives will be examined by taking into account both the ligand-based pharmacophore models reported in the literature and a recently proposed molecular model of the α4β2 receptor subtype. 54n conclusion, homologation of the structure of epibatidine, a prototype α4β2 selective agonist of nAChRs, led to the appearance of an unexpected, non negligible affinity for the α7 receptors.Moreover, among the compounds under study, the spirocyclic derivative 3c showed a degree of α7 vs α4β2 selectivity, a result which could be exploited in designing novel selective agonists of this nAChR subtype.

Figure 1 .
Figure 1.Structure of model and target ligands for neuronal nAChR subtypes.