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Cholinesterase Inhibition Activity and Molecular Docking Study of Eugenol Derivatives

The study was conducted to explore the anticholinesterase inhibition property of eugenol derived molecules. Ten eugenol derivatives were synthesized and evaluated for the inhibitory activities against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) by Ellman’s method. Most of the tested derivatives showed higher inhibition on BChE than AChE, however, their overall inhibitory activity was weak. In contrast, three derivatives (compounds 3,6,9) showed higher and good AChE inhibitory activity of more than 50% inhibition at 10 μg/mL. Among them, compound 9 bearing a ethyl substituent at para position of the benzoyl ring showed the most potent AChE inhibition, with IC50 of 5.64 μg/mL. Ligand-protein docking simulation was also performed for the most active derived molecules (compounds 3,6,9).


INTRODUCTION
Alzheimer disease (AD) is an irreversible, chronic and progressive neurodegenerative disease characterized by memory loss and cognitive impairment (Thompson et al. 2012). According to the cholinesterase hypothesis, AD is associated with loss of cholinergic neurons in the brain and resulting a decrease inacetylcholine, which is a neurotransmitter (Lane et al. 2006). Cholinesterase inhibitors have been widely recognised as a gold standard for the management of AD. To date, six drugs have been approved by U.S. Food and Drug administration including galanthamine, donepezil, memantine, memantine combined with donepezil, tacrine (discontinued) and rivastigmine to treat mild to moderate AD. Due to the fact that the commercially available cholinesterase compounds were reported to possesses serious side effects (Ali et al. 2015), more studies are needed to discover potential compounds to treat AD. Eugenol or 4-ally-2-methoxyphenol (1) ( Figure  1) is one of the phenylpropanoids available in nature. It is the main constituent isolated from cloves, Syzygiumaromaticum, an aromatic plant belonging to family of Myrtaceae (Fichi et al. 2007). Eugenol and its derivatives have been shown to possess medicinal properties such as local antiseptic and analgesic (Markowitz et al. 1992), anesthetic (Goulet et al. 2010;Jirovetz et al. 2006), anti-spasmodic (Wagner et al. 1979), antipyretic (Feng & Lipton 1987), anti-bacterial (da Silva et al 2018; Devi et al. 2010;Johny et al. 2010;Tippayatum & Chonhenchob 2007), anti-inflammatory (Maurya et al. 2018), antifungal (Olea et al. 2019) and antioxidant (Alqareer et al. 2006;da Silva et al. 2018;Jirovetz et al. 2006;Nassar et al. 2007) activities. Besides, eugenol also has vast applications in industrial products such as perfumes and flavoring agents. Eugenol also has repellent action (Kang et al. 2009;Zeringóta et al. 2013) and has been used as astabilizer (Li et al. 2015;Milczarek & Ciszewski 2012).
Hence, in continuation of our previous work on this molecule (Rahim et al. 2017 General synthetic routes for eugenol derivatives (2-11) are shown in Scheme 1. Synthesis methods, spectroscopy analyses were described in detail in the previous report (Rahim et al. 2017 The cholinesterase inhibitory activity of eugenol derivatives was evaluated following Ellman's method as described previously (Khaw et al. 2014). In brief, for AChE inhibitory assay, 140 μL of 0.1 M sodium phosphate buffer (pH 8) was first added to the 96-well microplate followed by 20 μL of the test sample (in 10 % methanol), 20 μL of 0.09 unit/mL AChE or BChE, 10 μL of 10 mM 5,5′-dithiobis(2-nitrobenzoic acid) was added into each well followed by 10 μL of 14 mM acetylthiocholine iodide or S-butyrylthiocholine chloride. The absorbance of the colored end-product was measured at 412 nm at designated intervals for 30 min after the initiation of enzymatic reaction by Tecan Infinite 200 ProMicroplate Spectrometer (Switzerland). Physostigmine was used as reference to compare the differences between the sample and standard drug. Each sample test was conducted in triplicate. Absorbance of the test sample was corrected by subtracting the absorbance of its respective blank. A set of five concentrations was used to estimate the 50% inhibitory concentration (IC 50 ) for the active compounds that showed more than 50% inhibition at 10 µg/mL. Data were analyzed by one-way analysis of variance (ANOVA) followed by tukey post-hoc test for the determination of statistically significant between samples and standard. P values of 0.05 or less were considered significant.

SYNTHESIS AND STRUCTURAL ELUCIDATION OF EUGENOL DERIVATIVES
Ten eugenol derivatives (compounds 2-11) were synthesized, and their structures were confirmed with NMR, FTIR and MS, as reported previously (Rahim et al. 2017).

CHOLINESTERASE INHIBITION ASSAY
All compounds were examined for their AChE and BChE inhibitory activities by Ellman's assay. The cholinesterase inhibitory activity of the synthesized compounds is summarized in Table 1. All compounds were initially tested at 10 µg/mL on AChE and BChE enzymes. The eugenol derivatives showed inhibitory activity against the AChE in the range of 12.79 to 71.53%, while showing much weaker inhibition against BChE enzyme, in the range of 10.0 to 38.41%. Among them, compounds 4, 2, 7, 8, 10 and 11 had higher inhibition against BChE, while compounds 3, 6, 9 had higher inhibition against AChE. The substituents at the hyroxyl group of eugenols had variable effects on cholinesterase inhibition. Attachment of a benzoyl group resulted in better inhibitory activity as compared to aliphatic substituent (compound 4). The para substituent of the benzoyl ring also affects the overall inhibitory activity. For instance, among the halogens, para substituted bromo derivative (compound 6) had much higher AChE inhibitory activity as compared to fluorine and chlorine. Para substituted nitro and methoxy derivatives had relatively weaker inhibitory activity against AChE, while para substituted ethyl derivative had higher AChE inhibition than the para substituted methyl derivative. DETERMINATION OF IC 50 Three eugenol derivatives (compounds 3, 6, 9) demonstrated more than 50% inhibition on AChE were subjected for IC 50 determination. The results are summarized in Table 2. Compound 9 showed most promising AChE inhibitory activity among the compounds tested with IC 50 values of 5.64 ug/mL. Compounds 3, 6, 9 were statistical less significant than standard drug, physostigmine. to reproduce the experimentally observed conformation with a rmsd of 0.9 A. Three most potent AChE inhibitors (compound 9; IC 50 = 5.64 ± 1.12, compound 6; IC 50 = 13.12 ± 1.33, and compound 3 IC 50 = 12.23 ± 0.76) were selected for docking studies. Figure 2 shows most favorable docked conformation of compound 9. The carbonyl oxygen was found to be making a hydrogen bond with Tyr124. One of the phenyl rings was making a Pi-Pi T-shaped interaction with Tyr337. The other phenyl group was making a pi-pi staked interaction with Ty341. The alkyl and allyl substituents on both phenyl rings were found to be making pi-alkyl interactions surrounding amino acids His447, Tro286, Phe295 and Val294.
The most favorable docked conformation of second most potent inhibitor in the series (compound 6), is given in Figure 3. Although compound 9 binds at the same place, it was found to have a slightly different binding conformation as compared to compound 9, which may be due to the difference in bromo and bulky ethyl substituent on the phenyl ring. The carbonyl oxygen was making a hydrogen bond with Thr75. Additionally, pi-pi stacked interactions were observed between one of the phenyl rings and Trp286. The other phenyl ring was making a pi-alkyl interaction with Leu76. The docked conformation of compound 3 is given in Figure 4. The compound binds in the same binding site as that of co-crystallized ligand. A hydrogen bond was observed between the amino group of Phe295 and the fluorine atom of CF 3 group. The phenyl ring containing the CF 3 group was making pi-pi T-shaped and pi-pi stacked interactions with amino acids Phe297 and Tyr341. The other phenyl ring was making pi-pi T-shaped interaction with His447. Pi-alkyl interactions were observed for allyl group with amino acids His447 and Trp86. Pi-alkyl interactions were also observed between the CF 3 group and Trp286 and Val294.

CONCLUSION
In this preliminary study, ten eugenol derivatives were prepared and evaluated for acetylcholinesterase and butyrylcholinesterase inhibition. Three derivatives (compounds 3, 6, 9) showed higher and good AChE inhibitory activity of more than 50% inhibition at 10 µg/ mL. Compound 9 which bore an ethyl substituent at para position of the benzoyl ring exhibited the strongest AChE inhibition with IC 50 values of 5.64 ug/mL. However, these derivatives (3, 6, 9) were statistical less significant than standard drug, physostigmine. Further studies are necessary to investigate the potential of eugenol derived molecules with different substituents against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) for the development of new and effective synthetic anti-Alzheimer compounds to treat AD.