# Trimethoxycinnamates and Their Cholinesterase Inhibitory Activity
## 3 Results and Discussion
### In Vitro Evaluation of AChE- and BChE-Inhibiting Activity  
All the esters were evaluated for their ability to inhibit AChE and BChE. This activity was compared with parent TMCA and the clinically used drugs rivastigmine (RIV) and galantamine (GLT). RIV (Figure 1) is a carbamate derivative structurally derived from physostigmine. The exact mechanism of RIV has not been fully established, but RIV is thought to be a classical acylation pseudo-reversible carbamate cholinesterase inhibitor that inhibits both AChE and BChE, thereby preventing hydrolysis of acetylcholine, leading to increased acetylcholine concentrations at cholinergic synapses. The anticholinesterase activity of rivastigmine is relatively specific for brain AChE and BChE compared to activities in peripheral tissues [62]. GAL (Figure 1) is a tertiary alkaloid extracted from _Galanthus nivalis_ (_Amaryllidaceae_). It is a centrally and peripherally acting non-acylated competitive reversible inhibitor of muscle and brain AChE, thereby increasing cholinergic tone. In addition, it also acts as a positive allosteric modulator of neuronal nicotinic acetylcholine receptors, which may also contribute to ameliorating AD symptoms [63]. This activity was expressed as IC\({}_{50}\) (\(\upmu\)M) (concentration of inhibitor that was required for 50% inhibition of the enzymes). Based on the results summarized in Table 1, it can be stated that cholinesterase's inhibitory activity is moderate, but several of the most active compounds showed comparable inhibition of AChE with RIV. Compounds **5** (R = 4-Br-Ph), **7** (R = 2-Cl-Ph) and **9** (R = IPMP) showed IC\({}_{50}\) ca. 46 \(\upmu\)M and compound **11** (R = Bn) had IC\({}_{50}\) = 49.44 \(\upmu\)M. On the other hand, only two compounds demonstrated more significant BChE inhibition: ester 7 (R = 2-Cl-Ph, IC\({}_{50}\) = 32.46 \(\upmu\)M) and **6** (R = 2-F-Ph, IC\({}_{50}\) = 44.41 \(\upmu\)M). Moreover, it should be noted that the selectivity indices (increased BChE inhibition in comparison with AChE \begin{table}
\begin{tabular}{c c c c c c c c c c} \hline \hline \multirow{2}{*}{**Comp.**} & \multirow{2}{*}{**R**} & \multirow{2}{*}{**log**\(P\)\({}^{a}\)**} & \multirow{2}{*}{**\(\sigma_{(\text{RB})}\)\({}^{a}\)**} & \multirow{2}{*}{**MV\({}_{(\text{RB})}\)\({}^{a}\)**} & \multirow{2}{*}{**AChE**} & \multirow{2}{*}{**BChE**} & & \multicolumn{3}{c}{**Tox IC\({}_{50}\) (\(\upmu\)M)**} \\ \cline{6-11}  & & & & & & & & & **(72 h)** & **(24 h)** \\ \hline
**11** & & & 3.17 & 0.23 & 91.49 & 49.44 \(\pm\) 0.73 & 118.41 \(\pm\) 0.22 & 0.42 & \textgreater{}30 & \textgreater{}30 & \textgreater{}60 \\ \hline
**12** & & & & & & & & & & \\ \hline
**TMCA** & \(\upmu\)M & 1.61 & – & – & 102.13 \(\pm\) 0.62 & 325.65 \(\pm\) 20.53 & 0.31 & – & – & \textgreater{}40 \\ \hline
**RIV** & – & – & – & – & 50.10 \(\pm\) 3.08 & 19.95 \(\pm\) 0.31 & **2.51** & – & – & – \\ \hline
**GLT** & – & – & – & – & 4.0 \(\pm\) 0.13 & 7.96 \(\pm\) 0.59 & 0.50 & – & – & – \\ \hline \hline \end{tabular}  
* calculated using ACD/Percepta 2012; \({}^{b}\) SI (index selectivity) = IC\({}_{50}\) (AChE)/IC\({}_{50}\) (BChE). A higher selectivity to BChE is indicated in bold.  
\end{table} 
Table 1: _Cont._  
Figure 1: Structures of approved drugs used as standards in this work. inhibition) for these two compounds are 1.42 and 1.71, respectively, which makes these two compounds valuable, as mentioned above. The dependences of the AChE inhibition of the compounds expressed as log(1/IC\({}_{50}\) (M)) on lipophilicity; expressed as log \(P\), electronic \(\sigma\) parameters and bulkiness of the individual ester substituents; and expressed as MV are illustrated in Figure 2A-C. While it is evident that the effect of \(\sigma\) parameters on AChE inhibition is negligible (see Figure 2B), it can be seen that lipophilicity in the log \(P\) 4-5 range is advantageous for higher activity (Figure 2A), similar to the bulkiness of ester substituents in the MV range from 94 to 146 cm\({}^{3}\) (Figure 2C).