Purpose: Norditerpenoid alkaloids are also known as C18- and C19-diterpenoid alkaloids. These natural products are the main pharmacological and toxicological components of Aconitum and Delphinium plants. Aconitum alkaloids are well-known for their potent activity on sodium or (and) potassium channels. Aconitine, the first alkaloid isolated from Aconitum, is lethal as it is a strong activator of Na+ channels keeping it in the open state, but another alkaloid from Aconitum, lappaconitine can block Na+ channels and has been introduced into clinical use as an antiarrhythmic agent. Delphinium alkaloid methyllycaconitine (MLA) 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.
It is important to gain a better understanding of the conformations of these natural products as part of a structure-activity relationships (SAR) study. This is essential as the shape of a compound is an important determinant of its bioactivity. The position of the important functional groups in the pharmacophore is influenced by the conformation of the molecules. To gain such comprehensive 3D structural information, detailed NMR spectroscopic and single crystal diffraction studies were used to analyse the pharmacophore conformations of the A/E-rings in selected norditerpenoid alkaloids.
Methods: Aconitine, crassicauline A, lappaconitine, lycoctonine, and MLA were studied by detailed NMR spectroscopy. Their free bases were also recrystallized, as well as aconitine HCl, lappaconitine HBr, and MLA HClO4. NMR spectra including 1H, 13C, COSY, HSQC, HMBC, NOESY, 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: All the selected natural products showed a small difference between axial and equatorial δ2-H (Fig. 1), which is distinct from the large separation between the two δ7-H of synthetic [3.3.1]azabicycles where 7-H correlates with 2-H in the C18- and C19-diterpenoid alkaloids. The large difference between axial and equatorial δ7-H was proven to be the effect of steric compression.
A/E-rings in the free bases of aconitine, crassicauline A, lappaconitine, and lycoctonine are in the twisted-chair/chair conformation with the N-ethyl group occupying the equatorial position as displayed in their crystal structures (Fig. 2), where the N-atom lone-pair electrons do not overlap with axial 2-H, thus there is no steric compression. Moreover, that the conformation of the A/E-bicycle in MLA free base is also twisted-chair/chair is supported by the NMR NOESY spectrum.
As the alkaloids become charged, the A-ring converts into a boat conformation. H-bonds displayed in the crystals of lappaconitine HBr and MLA HClO4 twist the A-ring boat conformations. However, this H-bond does not exist in the crystal structure of aconitine HCl and its A-ring is a more typical boat conformer (Fig. 3).
Conclusion: A twisted chair-chair conformation of A/E-rings is observed in the norditerpenoid alkaloids, and the twisted A-ring means that 2-H and the N-atom lone-pair electrons are not overlapped. Significantly, for biological activity, these A-rings are converted into twisted boat conformers as the alkaloids become protonated. 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