Synthesis and Characterization of Tetrahydroquinolines as acetylcholinesterase inhibitors

A serie of tetrahydroquinolines were synthesized in good yields using the Povarov reaction and evaluated as inhibitors of Acetilcholinesterase in vitro. The most active compounds showed an IC50 of 193μg/mL, the analysis of result suggest modification in the patters of substitution to increassed activity.


Introduction
Quinoline is a heterocyclic scaffold of paramount importance to human race. Compounds containing quinoline structure are widely used as antiasthmatic, antimalarials, anti-viral, anti-inflammatory, antibacterials, antifungals, and anticancer (1). Quinoline derivatives have been used in the synthesis of fungicides, virucides, biocides, alkaloids, rubber chemicals and flavoring agents (2). The importance as substructures in a broad range of products, significant efforts continue to be directed into the development of new quinoline-based structures.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder associated with cognitive, functional and behavioral impairments. The current therapies for AD, yet symptomatic and palliative, rely mainly on the restoration of acetylcholine levels (9) and on the partial antagonism of NMDA receptor (10). Since cholinergic transmission is involved in a variety of physiological systems, AChE is a pursued pharmacological target for treatment of several pathologies besides AD. AChE inhibitors (AChEIs) have therapeutic application in surgical anesthesia, in the treatment of neuromuscular blockade, in myasthenia gravis and in glaucoma (11).
We reported a serie of THQs synthetic as potencials AChE inhibitors that could be employed in the design of new drugs for AD treatments.

Materials
All chemical reactive for synthesis was obtained in Sigma-Aldrich, solvents were reagent grade and, in most cases, dried and distilled before use according to standard procedures. Enzymes and substrates were purchased from Aldrich Co.

Measurements
All compounds was characterized by 1 H NMR with on Bruker AM-400 spectrometer (400 MHz), using CDCl 3 as solvent and TMS was used as an internal standard. Melting point (uncorrected) was measured on a Fischer-Johns melting point apparatus. ESI-MS(/MS) data were collected using a high resolution hybrid quadrupole (Q) and orthogonal time-of-flight (TOF) mass spectrometer (Q-Tof, Micromass UK) with constant nebulizer to 100 ºC. The equipment were operated in the positive ion mode, and the cone and extractor potentials were set to 40 and 5 V, respectively, with a scan range of m/z 80-1000. Samples were infused into the ESI source at flow rates of 5µL/min via a microsyringe pump.

Synthesis
General Procedure for the Three-Component Reaction of alquene with Aldehydes and Anilines.
The Lewis acid BiCl 3 (20% mol) was added to imine obtained from a mixture of a solution of aldehyde (3.4 mmol) and amine (1 mmol) in dry CH 3 CN (15 mL), this mixture was stirred at room temperature. A solution of alquene (1 mmol) in dry CH 3 CN (15 mL) was then added, and the resulting suspension was stirred at room temperature under N 2 atmosphere for 18-24 h (Scheme 1). The reaction progress was monitored by means of TLC using Merck Kiesel-gel 60 (230-240 mesh). A saturated aqueous NaHCO 3 solution was added, and the resulting mixture was extracted with EtOAc. The organic phase was dried (Na 2 SO 4 ), filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (SiO 2 , hexane-EtOAc) to give the desired product (compounds 1-7). All compounds was purified against chromatographic technical and characterized for spectroscopic methods. The spectroscopic data of compounds 1-7 was describe previously (18). Scheme 1. Synthesis of tetrahydroquinolines 1-7 using Povarov Reaction.

Acetylcholinesterase inhibitors
To assess AChE inhibition, an adapted version of the Ellmann (19) assay in 96 well plates was used (20).The enzyme activity was calculated as a percentage compared to a control using only the buffer and enzyme solution. The compounds were assayed in the dilution interval of 500 -15 μg/mL, and the alkaloid galanthamine was used as the reference compound. Each assay was run in triplicate and each reaction was repeated at least three independent times. The IC 50 values were calculated by means of regression analysis. The values were obtained and expressed in µM ±SD.

Results and Discussion
Seven THQs were synthesized by imino Diels-Alder cycloaddition between different substituted anilines, 2-furaldehyde, and N-Vinylpyrrolidin-2-one used as alkene, with acetonitrile as solvent in the presence of 20 mol% of bismuth trichloride (III) as catalyst (Scheme 1). It must be taken into consideration that bismuth compounds have attracted attention due to their low toxicity, low cost, water tolerant catalyst and good stability in several reactions such as the imino Diels-Alder (Povarov) reaction obtaining good yield with almost no by-products. The substituents employed are show in table 1. All THQs were purified by SiO 2 column chromatography and obtained as solids and exclusively as the cis-diastereoisomers. The cis configuration of the substituents was determined by measurement of the relevant H-H coupling constants in their 1 H NMR spectra.

-7
Chemical data All compounds were evaluated as potential AChE inhibitors; compounds 1, 2, and 5-7 were not actives against AChE with values of IC 50 over 1000 µM. The compound with the highest activity was 3 with IC 50 of 805± 3.2 µM and 4, with IC 50 of 618± 4.7µM, the ppor activity showed for the compounds is not comparable with galanthamine used as reference (IC 50 =0.54±0.07µM).

Conclusions
Our results suggest that synthetic heterocyclic tetrahydroquinolines obtained using the Povarov reaction, may possess anticholinesterasic effects. The analysis of results suggests new modifications in the patters of substitution for increased activity. These findings encourage us to continue the efforts towards the optimization of the pharmacological profile of these structural types as important scaffolds in the neurodegenerative diseases.