Purpose: Plants of Aconitum and Delphinium families are used as herbal medicines in several traditional medicinal systems, even though they are highly toxic. The biologically active components of these plants are C18- and C19-diterpenoid alkaloids. Among these natural products, methyllycaconitine (MLA, Fig. 1) is highlighted because it is one of the most potent competitive antagonists of α7 nAChR with highly selective targeting of the snake venom toxin α-bungarotoxin (αBgTx) binding site. Functional group substituents on cyclohexane (A) and piperidine (E) rings in the hexacyclic norditerpenoids isolated from Aconitum and Delphinium are key to the biological activities of these alkaloids. This study is on detailed NMR spectroscopy and X-ray single crystal diffraction of the selected norditerpenoid alkaloids from Aconitum and Delphinium, especially the synthetic analogues of MLA. Studying the conformations of these [3.3.1]azabicycles will afford a better understanding of the structure-activity relationships (SAR) of these A/E-bicycles which bear key functional groups in the norditerpenoid alkaloids as the shape of the compound determines its bioactivities.
Methods: Double-Mannich reactions were used to synthesize the azabicycles and then derivatives, 2,4-DNP, HCl and MeI, were prepared. The mono-Mannich reaction product was also synthesized.
NMR spectra including: 1H, 13C, COSY, HSQC, HMBC, NOESY, and 15N-HMBC were recorded on a Bruker Avance III 500 MHz spectrometer at 25 oC. Data for X-ray single crystal structures were collected on a RIGAKU SuperNova at 150K.
Results: 1H NMR spectroscopy of [3.3.1]azabicyclic analogue 1 showed large differences between equatorial and axial δ7-H (Δδ7-H = 1.32 ppm) that is significantly different from (the equivalent proton) δ2-H in MLA (Δδ2-H = 0.10 ppm). To aid in the assignment of these signals, 7-iPr- and two 7-Me-[3.3.1]azabicycles (2, 3a,b, Et and Me esters) were synthesized whose axial δ7-H resonances are 3.02, 3.40, and 3.41 ppm, respectively. NOESY correlation data were obtained (Fig 2). 2,4-DNP derivatives were also prepared from these ketones in order to obtain crystal structures (Fig 3). These showed A/E chair-chair conformations displayed in the [3.3.1]azabicyclic derivatives 4-6.
In the [3.3.1]azabicyclic analogues 1-6, the significant downfield shift of axial δ7-H is due to steric compression from the lone-pair electrons of the N-atom pushing electron density away from axial 7-H. This was proven in a mono-Mannich product 7 (δ5-H = 1.65 and 1.77 ppm, Δδ5-H = 0.12 ppm). On acidification of 1, hydrochloride salt 8 also showed only a small distance between δ7-H signals; its crystal structure exhibits a typical boat-chair shape, with the hydrated ketal. N-Methylation (quaternisation) of 1 produced isomers 9a,b where N-ethyl occupied both the axial and equatorial positions, and both N-methyl and N-ethyl do sterically compress 7-Hax. Furthermore, [3.4.1]- and [3.2.1]azabicycles 10, 11 were also synthesized in order to investigate the influence of ring size on the effect of steric comparison. As the 7-Hax and 8-Hax in [3.4.1]bicycle 10 are not overlapped with the nitrogen lone-pair electrons, the scale of the steric compression effect decreased. As in compounds 7 and 8, this effect was not displayed in [3.2.1]azabicycle 11 as both 6-Hendo and 7-Hendo are remote from the nitrogen lone-pair electrons. The four 2,4-DNP derivatives of 4, 5, 6, and 12, as well as the HCl salt 8, have been recrystallized (Fig. 3).
Conclusion: Free base synthetic [3.3.1]azabicycles were proven to exist in typical chair-chair conformations while the conformation converts into boat-chair on tertiary amine protonation, not quaternisation. A rare NMR effect of steric compression has been clearly demonstrated, and its existence is related to the angle between the nitrogen lone-pair electrons and the compressed proton. This steric compression effect is not only caused by nitrogen lone-pair electrons, but also by intramolecular alkyl groups, e.g., Me, Et, that are close to the compressed proton. This work is funded (in part) by the University of Bath.
Ian Blagbrough– Senior lecturer in the department of pharmacy and pharmacology, University of Bath
Michael Rowan– Visiting Lecturer, Department of Pharmacy and Pharmacology, University of Bath