Design , Synthesis , and Acetylcholinesterase Inhibition Assay of Novel 9-( 1-( Substituted-benzyl ) piperidin-4-yl )-2-chloro-9 H-purin-6-amine Derivatives

1 Department ofMedicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, Jinan, Shandong 250012, China 2Department of Pharmacology, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, Jinan, Shandong 250012, China


Introduction
Since a cholinergic hypothesis has been postulated as a crucial element in Alzheimer's disease (AD) symptoms etiology, catalytic acetylcholinesterase (AChE) inhibitors have constituted until today the main drugs used against AD [1].Reversible inhibition of brain AChE by anti-AD drugs such as tacrine [2], donepezil (Figure 1) [3], and huperzine [4] increases the AChE levels and improves neurotransmission in cholinergic synapses [5].Since AChE plays a proaggregating (noncatalytic) role to accelerating -amyloid peptide (A) aggregation and deposition into the fibrils, inhibition of AChE is still the most successful therapeutic strategy for the symptomatic treatment of AD and its progression [6].The search for novel AChE inhibitors with improved biological profiles continues to be of great interest to medicinal chemists.
The refinement of the central core in bioactive molecules combined with introducing privileged substituents is a common practice in medicinal chemistry to find proprietary and novel hits.Based on the considerable similarities of these lead compounds, a fragment-based design was employed to identify alternative chemotypes (Figure 2).Meanwhile, in current drug research field, substituted purines have attracted considerable attention from the medicinal chemists in recent years, owing to the high number of positive hits encountered with this heterocycle.In this paper, a novel

Cholinesterase Inhibition.
All the newly synthesized compounds (7a1-a13, 7b1-b13) were evaluated for their inhibitory activities toward AChE using an in vitro assay based on the reported protocol [7][8][9][10], in comparison with donepezil as standard drug.The anticholinesterase activities are summarized in Table 1.Preliminary results in Table 1 revealed that compound 7b9 with a 3-cyanobenzyl group in the benzo[d] [1,3]dioxol-5-yl series displayed the highest AChE inhibitory activity (15.42% at 100 M) among all the tested compounds.Besides, compounds 7a1, 7a7, and 7b10 with inhibitory percentage more than 10% at 100 M showed similar activities with compound 7b9 against AChE.Generally, all the compounds in both 3,4-dimethoxyphenyl and benzo[d] [1,3]dioxol-5-yl series demonstrated much lower inhibitory activity toward AChE than the reference drug donepezil (80.07%at 100 M).Obviously, the activity of the compounds depends upon the nature of the substituents attached with purine ring.Even small modifications in every series can decrease and even lost their activity against AChE.Therefore, the coherent structure-activity relationship will be afforded by further pharmacology investigation, which is currently underway in our lab.

Conclusion
In summary, a novel series of 9-(1-(substituted-benzyl) piperidin-4-yl)-2-chloro-9H-purin-6-amine derivatives were designed and synthesized, and their AChE inhibitory activities were preliminary examined.Biological assay demonstrated that four compounds showed moderate activities against AChE with inhibitory percentage more than 10% at 100 M.Further investigation of other bioactivities associated with AD of this series is ongoing work within our group and will be reported in due course.4.1.5.General Procedure for the Target Compounds 7a and 7b.Compound 6a (or 6b) was dissolved in anhydrous DMF (10 mL) in the presence of anhydrous K 2 CO 3 (1.2eq), followed by addition of appropriate substituted benzyl chloride (bromine, fluorine) (1.1 eq).The reaction mixture was stirred at room temperature overnight.The solvent was removed under reduced pressure, and then water (20 mL) was added.

Experimental Section
Extracted with ethyl acetate (3 × 10 mL), and the organic phase was washed with saturated sodium chloride (10 mL) then dried over anhydrous Na 2 SO 4 to give the corresponding crude product, which was purified by flash column chromatography to afford compounds 7a1-13 (or 7b1-13).

Acetylcholinesterase Inhibition Assay.
The ability of derivatives 7a1-7a13 and 7b1-7b13 to inhibit human acetylcholinesterase (hAChE) (Sigma-Aldrich, USA) was examined using the 5,5  -dithiobis-(2-nitrobenzoic acid) (DTNB) method (to generate a yellow chromophore (5-mercapto-2-nitrobenzoic acid) detectable at the 405-412 nm range) with the acetylcholinesterase assay kit (Keygen, China).The assay was carried out as a previously described protocol [10] using donepezil as controls ( = 4).Stock solutions of test compounds were dissolved in a minimum volume of DMSO (1%) and were diluted using saline.In 96-well plates, 50 L of AChE (0.22 U/mL diluted using saline) was incubated with 10 L of various concentrations of test compounds (1.0, 10.0, 100 M) at room temperature for 10 min followed by the addition of relative agents according to the kit instruction.The absorbance was measured at a wavelength of 405 nm with Thermo MK3 microplate reader.Percent inhibition was calculated by the comparison of compound-treated to various control incubations.