Synthesis of 3,16,30-trioxolup-20(29)-ene, a selective butyrylcholinesterase inhibitor, from a natural triterpene

Six new lupanes type triterpenoids derivatives have been synthesized using lup-20(29)-en-3β,16βdiol (1) and 30-oxolup-20(29)-en-3β,16β-diol (2) as starting material. Ketones 3 (lup-20(29)-en3,16-dione) and 4 (3,16,30-trioxolup-20(29)-ene) were obtained by Jone ́s oxidation of compounds 1 and 2, respectively. Further reaction of 3 and 4 with hydroxylamine hydrochloride provided compounds 5 (lup-20(29)-en-16-one-3-oxime), 6 (lup-20(29)-en-3,16-dioxime), 7 (lup-20(29)-en16-one-3,30-dioxime) and 8 (lup-20(29)-en-3,16,30-trioxime). Derivatives 3-8 were obtained in moderate good yields. Their structures were confirmed by analysis of their 1 H and 13 C NMR and ESI-MS spectra. The complete assignation of the signals was achieved with the aid of 2D NMR experiments (COSY, HSQC, HMBC, NOESY). All of the new derivatives were evaluated as potential in vitro butyrylcholinesterase (BChE) inhibitors by the Ellman ́s colorimetric method. Their anti acetylcholinesterase (AChE) activity was evaluated in order to determine the BChE/AChE selectivity of compounds 3-8. All of them failed to inhibit AChE, but we found that the best BChE inhibition was observed for 3,16,30-trioxolup20(29)-ene (4) with an IC50 value of 21.5 M, which elicited a selective inhibitor profile.


Introduction
The chemistry of lupane-type triterpenoids has been actively explored due to and their biological and pharmacological properties which include anti-inflammatory 1 , anti-oedematous 2 , antitumor 3 and anti-HIV activities 4 .Abundant in many plants, these metabolites are valuable natural raw materials to perform chemical modifications and obtain semisynthetic analogs for structureactivity relationship studies.
According to the cholinergic hypothesis, the inhibition of cholinesterase increases the levels of acetylcholine in the brain, thus improving cholinergic functions in Alzheimer´s Disease (AD) patients.Butyrylcholinesterase (BChE) is one of the enzymes involved in the metabolic degradation of acetylcholine, together with acetylcholinesterase (AChE).BChE activity increases as AD progresses, which suggests that BChE may play an important role at the latter stages of AD.Therefore, selective BChE inhibitors attract interest nowadays.
In our continuing search of new anti-cholinesterase agents, we found that calenduladiol (1), obtained from ethanolic extract of Chuquiraga erinacea (Asteraceae) in good yields, showed a moderate cholinesterase inhibition 5 .That result encouraged us synthesize derivatives of this natural compound.Calenduladiol (1) (lup-20(29)-en-3β,16β-diol) is a pentacyclic lupane-type triterpene with the interesting feature of being hydroxylated at C-16.Previous reports indicate that this position is essential for a biological activity.In the present work we report the synthesis of several new derivatives from natural calenduladiol and their evaluation as potential cholinesterase inhibitors.

Results and Discussion
We report here the synthesis of six new lupanes (3-8).These compounds have been obtained by sequential oxidations and reaction with NH 2 OH of the starting compounds 1 and 2 (Figure 1).Their structures were characterized by 1 H and 13 C NMR spectroscopy.Compounds 1-8 were screened for AChE and BChE inhibition using the Ellman's assay 7 .Enzyme activity was calculated by comparing reaction rates for the samples to the blank previously reported 9 .None of the new derivatives (3-8) inhibited the enzyme AChE.On the other hand, BChE inhibition was observed for compounds 1-8 (Table 1).These results suggest that these type of compounds have selectivity towards BChE.3,16,30-trioxolup-20(29)-ene (4) resulted to be the most potent BChE inhibitor with an IC value of 21,5 M.

Conclusions
We have obtained a series of natural and semi-synthesized lupane-type triterpenes, by oxidation at C-3, C-16 and C-30, followed by treatment with NH 2 OH in order to obtain the corresponding oximes.Our results on BChE inhibition of these analogs indicate that 3,16,30trioxolup-20(29)-ene (4) could be a promising leader compound to develop a strategy for the enhancement of pharmacological properties of this type of BChE inhibitor.

Table 1 -
In vitro BChE inhibitory activity.a Samples were dissolved in buffer with MeOH as cosolvent (final conc.2,5%).Values represent the mean of three replicates ± standard deviation.b Sample concentration 200 M.
a c Positive control.