Lupane triterpenoids , selective butyrylcholinesterase inhibitors

Alzheimer ́s disease (AD) is a progressive neurodegenerative disorder associated with memory impairment and cognitive deficit. It is characterized by low levels of the neurotransmitter acetylcholine (ACh) in the brain of AD patients. The inhibition of acetylcholinesterase (AChE), the enzyme that catalyzes ACh hydrolysis, is the main therapeutic strategy used to treat AD. In the healthy brain, another enzyme, namely butyrylcholinesterase (BChE), is involved in the metabolic degradation of ACh. 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. The chemistry of lupane-type triterpenoids has been actively explored due to their biological and pharmacological properties. Abundant in many plants, these metabolites are valuable natural raw materials to perform chemical modifications. In the present work, we aimed to evaluate natural and semisynthetic lupanes as potential in vitro cholinesterase inhibitors. Lupeol (1) (lup-20(29)-en-3βol) and calenduladiol (2) (lup-20(29)-en-3β,16β-diol) have been isolated from Chuquiraga erinacea subsp. erinacea (Asteraceae), an endemic species growing wildly in our region. Semisynthetic lupanes 3-17 have been prepared from them. All of them failed to inhibit AChE, but we found that most of calenduladiol derivatives exhibited BChE inhibition. The best BChE inhibitors were 3βhydroxylup-20(29)-en-16-one (5) and 3,16-dioxolup-20(29)-en-30-al (15) with IC50 values of 28.9 and 21.5 μM, respectively, an interesting result considering that the role of BChE is more relevant as the disease progresses.


Introduction
Neurodegenerative diseases gain more and more of importance in ageing societies.Especially, the number of patients suffering from Alzheimer´s disease (AD) increased continuously during the last decades.
One characteristic feature of AD is a decreased level of the neurotransmitter acetylcholine (ACh).This results in a decline in memory and recognition.Usually, the level of ACh is controlled by the enzyme acetylcholinesterase (AChE), which cleaves the neurotransmitter in the postsynaptic area.Another enzyme, butyrylcholinesterase (BChE) is also able to hydrolyze ACh, although it doesn´t possess the same affinity for the neurotransmitter as AChE does.Drugs currently used for the treatment of AD inhibit the two ACh controlling enzymes, AChE as well as BChE.However the AChE inhibitors retard the progress of AD only in a very early stage; during the progress of AD, the loss of AChE-activity is compensated by BChE. 1 Therefore, the searching of new selective BChE inhibitors should provide additional benefits in the treatment of AD.
Triterpenoids are naturally occurring compounds with ubiquitous distribution and a wide range of biological activities. 2-4Pentacyclic triterpenoids provide privileged structures for further modifications and structure activity relationship (SAR) studies. 5-7Lupanes in particular, have attracted attention due to the broad range of biological and pharmacological properties that they exhibit such as anticancer, antitumor, anti-inflammatory, anti-HIV, anticholinesterase, insecticidal and antimalarial activities. 3,4,8-15    Our interest in bioactive triterpenes, prompted us to synthesize a series of derivatives from natural lupeol (1) and calenduladiol (2), isolated from Chuquiraga erinacea D. Don.subsp.erinacea (Asteraceae). 14-16Lupeol (1) and calenduladiol (2) are pentacyclic triterpenes belonging to the lupane family.In a previous work, we observed the enhancement of the inhibitory activity against BChE of 2 by the introduction of keto groups at C-3, C-16 and C-30. 15In this paper we report the preparation of 15 lupane derivatives from compound 1 and 2, and their ability as potential cholinesterase inhibitors.
These results suggest that the keto group at C-16 may be responsible for the antiBChE activity for this type of molecules.

Conclusions
We have obtained a series of natural and semi-synthesized lupane-type triterpenes, by oxidation or sequential oxidations, and by reaction with hydroxylamine hydrochloride.Our results on BChE inhibition of calenduladiol analogs which have been oxidized at the C-16 position indicate that they could be promising leader compounds to develop a strategy for the enhancement of pharmacological properties of this type of BChE inhibitors.

Table 1 .
Inhibition of AChE and BChE activity and selectivity index.AChE from electric eel; b BChE from horse serum; c Selectivity Index = IC 50 (AChE)/IC 50 (BChE); d n.i.no inhibition. a