Semi-synthetic analogs of 17-hydroxycativic acid and their biological evaluation

A new series of analogs of the naturally occurring 17-hydroxycativic acid (1) were synthesized and evaluated as cholinesterase inhibitors. All derivatives have been fully characterized by monoand bidimensional NMR spectroscopy. The butyrylcholinesterase (BChE) inhibitory activity of the analogs was of the same order of magnitude as that of compound 1 (IC50 = 171.1 M), while the acetylcholinesterase (AChE) inhibition was not improved by these derivatizations, being 1 the most potent AChE inhibitor of the series (IC50 =21.1 M). These results suggest that both carboxylic group (C15) and allylic alcohol (C17) could be important for AChE inhibitory activity.


Introduction
Alzheimer´s disease (AD) is a progressive neurodegenerative disorder associated with memory impairment and cognitive deficit.Although many factors have been implicated in AD, its etiology and pathogenesis remains unclear.The cholinergic hypothesis postulates that at least some of the cognitive decline results from the 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 followed to treat AD.Recent reports suggest that drug development would be targeted not only to increasing AChE inhibition but also selectivity for AChE over butyrylcholinesterase (BuChE) 1 , which activity decreases in synapses of AD patients.
In the course of our ongoing studies of natural products from our regional flora with AChE inhibitory activity, a bioassay-guided fractionation of the ethanolic extract of Grindelia ventanesis Bartola & Tortosa (Asteraceae) resulted in the isolation of a labdane diterpene of the normal series identified as 17-hydroxycativic acid (1).Taking into account that 1 showed a significant inhibition of AChE (IC 50 = 21.1 M), selectivity over BuChE (IC 50 = 171.1 M) and that it was easily isolated from the plant extract in a very good yield (150 mg/ 100 g of aerial parts), we decided to obtain semisynthetic analogs of this natural diterpene through simple structural modifications of two of the three hot spots of this molecule: the carboxylic group (C15) and the primary allylic alcohol (C17) (Figure 1).

Experimental
General.NMR measurements were carried out at 22 °C from DMSO-d 6 or CDCl 3 solutions, on a Bruker ARX300 spectrometer operated at 300 and 75 MHz for hydrogen and carbon, respectively.Chemical shifts are given in ppm () with TMS as an internal standard.Silica gel 60 (Merck, 200 -425 mesh) was used for column chromatography.Analytical TLC was performed on Silicagel 60 F 254 sheets (0.2 mm thickness, Merck).The p-anisaldehyde-acetic acid spray reagent was used for detection.UV spectra were recorded on a JASCO V-630BIO spectrophotometer.Microwave assisted reactions were carried out in a CEM Discover reactor.Acetylcholinesterase from electric eel (type VI-S), 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB), acetylthiocholine iodide (ATCI), butyrylthiocholine iodide (BTCI) and tacrine were purchased from Sigma.Butyrylcholinesterase (horse serum) was purchased from MP Biomedicals.

Synthesis of 4.
Compound 1 (0.1 mmol) was added to a mixture of Ac 2 O (31.7 mmol) and pyridine (37.7 mmol) and left at room temperature for 24 h.The reaction mixture was poured onto distilled water (20 ml) and partitioned with CH 2 Cl 2 (3×8 mL).The organic layer was washed with water, dried over CaCl 2 and purified via silicagel column chromatography with CH 2 Cl 2 /MeOH to obtain by elution with 99:1 compound 4 (19.0 mg, 45.3 %).For 1 H-NMR and 13 C-NMR data see tables 1 and 2.
NMR data for semisynthetic derivatives.

Cholinesterase inhibition assay
Electric eel (Torpedo californica) AChE and horse serum BuChE were used as source of both the cholinesterases.AChE and BuChE inhibitory activities were measured in vitro by the spectrophotometric method developed by Ellman with slight modification 7 .The lyophilized enzyme, 500U AChE/300U BuChE, was prepared in buffer A (8 mM K 2 HPO 4 , 2.3 mM NaH 2 PO 4 ) to obtain 5/3 U/mL stock solution.Further enzyme dilution was carried out with buffer B (8 mM K 2 HPO 4 , 2.3 mM NaH 2 PO 4 , 0.15 M NaCl, 0.05% Tween 20, pH 7.6) to produce 0.126/0.06U/mL enzyme solution.Samples were dissolved in buffer B using 2.5% of MeOH as cosolvent.Enzyme solution (300 μL) and 300 μL of sample solution were mixed in a test tube and incubated for 60/120 min at room temperature.The reaction was started by adding 600 μL of the substrate solution (0.5 mM DTNB, 0.6 mM ATCI/BTCI, 0.1 M Na 2 HPO 4 , pH 7.5).The absorbance was read at 405 nm for 180 s at 27ºC.Enzyme activity was calculated by comparing reaction rates for the sample to the blank.All the reactions were performed in triplicate.IC 50 values were determined with GraphPad Prism 5. Tacrine (99%) was used as the reference AChE/BuChE inhibitor.

Results and discussion
In the present work four derivatives of 17-hydroxycativic acid isolated from G. ventanensis were prepared as depicted in Scheme.Compound 2 was obtained by sulfation with trimethylaminesulfur trioxide complex of the hydroxyl group; reduction of the carboxylic acid to alcohol using LiAlH 4 afforded diol 3 ; acetylation with Ac 2 O/pyridine of the hydroxyl group gave 4; using K 2 CO 3 and CH 3 I, compound 5 was synthesized.These derivatives are new diterpenes and have been fully characterized by mono-and bidimensional NMR spectroscopy (Tables 1 and 2).

Conclusions
These preliminary results suggest that both carboxylic group (C15) and allylic alcohol (C17) could be important functional groups for AChE inhibitory activity of this type of labdane diterpenes.Structural modifications achieved until now have not enhanced AChE inhibition, while compounds 3-5 showed selectivity for BuChE over AChE.
a Registered in DMSO-d 6 , b Registered in CDCl 3