# Trimethoxycinnamates and Their Cholinesterase Inhibitory Activity
## 3 Results and Discussion
### In Vitro Evaluation of AChE- and BChE-Inhibiting Activity
The dependences of the BCHE inhibition of the investigated compounds expressed as log(1/IC\({}_{50}\) (M)) on lipophilicity expressed as log \(P\), electronic \(\sigma\) parameters and bulkiness of the individual ester substituents are illustrated in Figure 3A-C. In contrast to AChE inhibition, a bilinear dependence of BCHE inhibition on log \(P\) values can be observed in Figure 3A, with a maximum ca. log \(P\) = 3.7. From the dependence shown in Figure 3B, a linear dependence is clearly seen, where the activity increases with the electron-withdrawing properties of the ester substituents. The bilinear trend (increasing BCHE inhibition to MV = 92 cm\({}^{3}\) (7, R = 2-CI-Ph) followed by decreasing activity with increasing ester bulkiness) can also be traced to Figure 3C. It is important to specify that while for AChE inhibition, the position of the substituent on the phenyl ring does not appear to be absolutely crucial (_para_ substituted isomers have only slightly better activity; compare _para_ vs. _ortho_ position of the substituents of compounds **5** and **7** or **4** and **6**), for BChE inhibition, it is absolutely the fundamental position of the substituent in the _ortho_ position. The same observations were found recently, e.g., in [53; 56; 64; 65; 66; 67; 68]. Figure 2: Dependences of AChE-inhibiting activity log(1/IC\({}_{50}\) (M)) of all compounds **1–13** on lipophilicity expressed as log \(P\) (**A**), electronic \(\sigma\) parameters and bulkiness expressed as molar volume (MV) of whole O-part of individual esters (**B**,**C**). IPMP = 2-isopropyl-5-methylphenyl, TMP = 4-(2,4,4-trimethylpentan-2-yl)phenyl, PPXE = 2-proposythyl. The reversible inhibitors are usually divided into four groups: competitive, noncompetitive, uncompetitive and mixed. The mechanism of action of the inhibitor can be determined using the Lineweaver-Burk (L-B) plot and comparing the kinetic parameters--Michaelis constant (\(K_{\text{M}}\)) and maximum velocity (\(V_{\text{m}}\)) of the uninhibited and inhibited reaction. The competitive inhibitor increases \(K_{\text{M}}\) by interfering with the active site of the enzyme but does not affect \(V_{\text{m}}\). All lines in the L-B plot intersect at the same point on the \(y\)-axis. The non-competitive inhibitor does not change \(K_{\text{M}}\) but decreases \(V_{\text{m}}\). All lines in the L-B plot intersect at the same point on the \(x\)-axis. The uncompetitive inhibitor decreases \(K_{\text{M}}\) and \(V_{\text{m}}\), but their ratio is the same as for the uninhibited reaction. All lines in the L-B plot are parallel. The inhibitor acting via the mixed type of inhibition changes \(K_{\text{M}}\), \(V_{\text{m}}\) and their ratio compared to the reaction in the absence of the inhibitor. All lines in the L-B plot intersect in quadrant II or III [54]. L-B plots for the most effective compound 7. inhibiting AChE and BChE, are depicted on Figures 4A and 5A. Based on the L-B plots, ester 7 acts via the mixed type of inhibition. Moreover, the inhibition constant \(K_{\text{i}}\) (i.e., dissociation constant of complex enzyme-inhibitor) was calculated using the regression equation of the dependence of the slope (L-B plot) vs. the concentration of the inhibitor (Figures 4B and 5B).