Synthesis of Some Phenylisoindoline-1,3-Diones and their Acetylcholinesterase and Butyryl Cholinesterase Inhibitory Activities

Aims: To synthesize some phthalimides derivatives and evaluate the compounds for their possible biological properties. Methods: The substituted phenylisoindoline-1,3-dione were synthesized from the reactions of Nphenyl phthalimide with different substituted aromatic aldehyde. The synthesized compounds were characterized using nuclear magnetic resonance spectroscopic analysis. The acetylcholinesterase and butyryl cholinesterase inhibitions were determined by Spectro photochemical analysis of acetylthiocholine and butyryl choline chloride. Results: Compounds 6 (IC50 = 30±3 μg/mL) and 4 (IC50 = 141±60 μg/mL) were found to be the most active inhibitors against acetylcholinesterase, while compounds 4 (IC50 = 102±10 μg/mL), 5 (IC50 = 105 ± 20 μg/mL) and 2 (IC50 = 190 ± 10 μg/mL), were found to be most active inhibitor against butyryl cholinesterase. Conclusion: The considerable acetylcholinesterase and butyryl cholinesterase inhibitory activities of the synthesized compounds makes them good candidates for the development of selective acetylcholinesterase and butyryl cholinesterase inhibitors. Original Research Article Taiwo et al.; JPRI, 33(51B): 194-201, 2021; Article no.JPRI.71132 195


EXPERIMENTAL
Melting points were determined with open capillary tube on a Gallenkamp (variable heater) melting point apparatus and were uncorrected. Infrared spectra were recorded as KBr pellets on a Bruker 2000 Spectrometer. The 1 H and 13 C NMR was run on a Bruker 600 MHz spectrometer ( in ppm relative to Me 4 Si) at the Department of Chemistry, Portland state University, Portland U.S.A. The purity of the compounds was routinely checked by TLC on silica gel G plates using n-hexane/ethyl acetate (1:1, v/v) solvent system and the developed plates were visualized by UV light. All reagents used were obtained from Sigma-Aldrich Chemical Ltd, except Glacial acetic acid, ethanol, oxalic acid and vanillin which were obtained from BDH Chemical Limited.

In vitro acetylcholinesterase and butyryl cholinesterase inhibitory assays
The anti-cholinesterase (acetylcholinesterase and butyryl cholinesterase) inhibiting activities of the synthesized compounds were determined by using modified method of Ellman et al. (1961) as described by Obuotor (2004). The synthesized compounds were prepared in a stock solution of DMSO in buffer and was used for the cholinesterase inhibition assay, while Eserine prepared in buffer was used as the reference compound (positive control).
The reaction was then initiated by the addition of 20µl of 25mM ATChI (1.042 mM final concentration). The rate of hydrolysis of ATChI was then determined spectrophotometrically by measuring the change in the absorbance per minute (∆A/min) due to the formation of the yellow 5-thio-2-nitrobenzoate anion at 412 nm over a period of 4min at 30s interval. A solution of buffer was used as negative control. The percentage inhibition (% I) of the synthesized compounds were obtained using the formula: Where: I (%) = Percentage inhibition V i = enzyme activity in the presence of synthesized compounds V 0 = enzyme activity in the absence of synthesized compounds

Chemistry
The respective chalcones were synthesized by reacting the phthalimide derivative 1, obtained from the reaction of 4-aminoacetophenone with phthalic anhydride in glacial acetic acid with different substituted aromatic aldehydes in the presence of ethanolic NaOH using the method described by Singh et. al,2010 (Scheme 1). All the desired chalcones (1-6) were obtained in high yields with the percentage yield between 66.30% -72.12%. The melting point of compounds (1-6) ranged between 155 and 238 0 C ( Table 1). The structures of the compounds were partially characterized using infrared, 1 H and 13 C NMR spectroscopic methods. The diagnostic bands for presence of chalcones HC=CH bonds in the infrared spectral data was observed at 1680 cm -1 . The results of the 1 H spectroscopic data showed the presence of the olefinic protons of the chalcones appearing downfield between 7.36 and 7.68 ppm for the compounds synthesized. The results of the 13C NMR spectroscopic data showed the presence of the olefinic carbons of the chalcones appearing at 145 ppm.

Acetylcholinesterase activity
The activity of the compounds ranged from 1.03±0.3 to 4.83±1.0 (Fig. 1). It was observed that compound 6 has the lowest IC 50 value. This study indicated that compound 6 having IC 50 value of 1.03±0.3 has the highest activity compared to other compounds and eserin which is a standard drug used as a positive control. Moreover, all the six compounds showed activity that is higher than the standard drug Eserine that was used. The activity of compound 6 could be as a result of the presence of an electron donating groups at the ortho (hydroxyl) and para (methoxy) of the chalcone phenyl ring. The lower activity exhibited by compound 3 may be due to the presence of a bulky indole moiety of the chalcone which may exhibit some steric hindrance.

Butylrylcholineterase activity
The activities observed ranged from 1.02±0.1 to 4.99±0.3 (Fig. 2). It was observed that compound 5 has the lowest IC 50 value of 1.02±0.1 which indicated that compound 5 has the highest Butylry lcholineterase inhibitory activity compared to rest of the compounds. Also, all the six compounds showed higher inhibitory properties than the standard drug Eserine used in this test. The higher inhibitory activity exhibited by compound 5 could be as a result of the presence of an electron withdrawing chlorogroup that has 7 electrons in his valence orbitals of the chalcone ring. Looking at the lower activity obtained for compound 3 (4.99 ± 0.3) this could be as a result of steric hindrance caused by a bulky indole ring attached to the chalcone.

CONCLUSION
It can be concluded that the synthesis of compounds 1-6 was successful and the workup stage was environmentally friendly. The bioactivities of all synthesized chalcones analogues 1-6 were tested in vitro by using the standard activity protocol. It was observed that this study identifies a new class of potential AChE, BuChE, nitric oxide and FRAP actives that could be developed into drugs.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.