Trimethoxycinnamates and Their Cholinesterase Inhibitory Activity

: A series of twelve nature-inspired 3,4,5-trimethoxycinnamates were prepared and characterized. All compounds, including the starting 3,4,5-trimethoxycinnamic acid, were tested for their ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in vitro; the selectivity index (SI) was also determined. 2-Fluororophenyl (2 E )-3-(3,4,5-trimethoxyphenyl)-prop-2-enoate demonstrated the highest SI (1.71) in favor of BChE inhibition. 2-Chlorophenyl (2 E )-3-(3,4,5-trimethoxyphenyl)prop-2-enoate showed the highest AChE-inhibiting (IC 50 = 46.18 µ M) as well as BChE-inhibiting (IC 50 = 32.46 µ M) activity with an SI of 1.42. The mechanism of action of the most potent compound was determined by the Lineweaver–Burk plot as a mixed type of inhibition. An in vitro cell viability assay conﬁrmed the insigniﬁcant cytotoxicity of the discussed compounds on the two cell lines. Trends between structure, physicochemical and activity were discussed.


Introduction
Nature is an inspirational and rich source of compounds with biological effects. More than a half of all small-molecule-approved drugs between 1981 and 2014 belong to groups of natural products or mimic natural products [1]. Many plant secondary metabolites [2] have also been used as model compounds for the subsequent development of synthetic drugs [3][4][5][6]. One such example is a cinnamic acid and its derivatives, e.g., coumaric acids, caffeic acid, ferulic acid or sinapic acid, which are widely distributed in many plants and assure their many physiological processes. They also serve as precursors for other compounds in plants such as lignols, (iso)flavonoids, coumarins, aurones, stilbenes, catechin, phenylpropanoids, etc. [7].
Natural cinnamic acid also serves as a precursor for new synthetic compounds. Synthetic derivatives of cinnamic acid showed interesting antimicrobial, anticancer, antiparasitic, neurological, antioxidant, and anti-inflammatory activities in vitro [8]. 3,4,5-Trimethoxycinnamic acid (TMCA) is an ingredient isolated from the root of Polygala tenuifolia Willd. (Polygalaceae), and its extracts have been used as traditional sedative drugs in

Evaluating In Vitro AChE and BChE-Inhibition Potencies
The ability of all the prepared compounds to inhibit AChE from electric eel (Electrophorus electricus) and BChE from equine serum (both purchased from Sigma, St. Louis, MO, USA) was determined in vitro using a modified Ellman's method, as described previously [49][50][51][52][53].

Measurement of Inhibition Type
Compound 7 was used for the determination of the type of inhibition. The measuring procedure was analogue to the determination of IC 50 . For the measurement, the following concentrations of the substrate were used: 20, 40, 60 and 80 µM. Methodology was performed as the method described previously [54,55].

In Vitro Viability Assay
The safety of the investigated compounds (effect on cell viability) was evaluated on two different cell lines: the adherent human synovial cell line SW982 (ATCC, Manassas, VA, USA) and suspension human genetically modified leukemia cell line THP1-Blue™ NF-κB (Invivogen, San Diego, CA, USA). Screening was performed as described previously [44,45].

Chemistry and Physicochemical Properties
All the investigated compounds 1-12 were synthesized according to Scheme 1. The carboxyl group of the starting TMCA was converted with thionyl chloride to the acyl chloride, which, upon subsequent reaction with the appropriate alcohol/phenol, yielded the targeted ester. Both reactions (conversion to the acid chloride and subsequent ester formation) were performed in dry toluene under reflux. All the compounds were recrystallized from ethanol.

Evaluating In Vitro AChE and BChE-Inhibition Potencies
The ability of all the prepared compounds to inhibit AChE from electric eel (Electrophorus electricus) and BChE from equine serum (both purchased from Sigma, St. Louis, MO, USA) was determined in vitro using a modified Ellman's method, as described previously [49][50][51][52][53].

Measurement of Inhibition Type
Compound 7 was used for the determination of the type of inhibition. The measuring procedure was analogue to the determination of IC50. For the measurement, the following concentrations of the substrate were used: 20, 40, 60 and 80 μM. Methodology was performed as the method described previously [54,55].

In Vitro Viability Assay
The safety of the investigated compounds (effect on cell viability) was evaluated on two different cell lines: the adherent human synovial cell line SW982 (ATCC, Manassas, VA, USA) and suspension human genetically modified leukemia cell line THP1-Blue™ NF-κB (Invivogen, San Diego, CA, USA). Screening was performed as described previously [44,45].

Chemistry and Physicochemical Properties
All the investigated compounds 1-12 were synthesized according to Scheme 1. The carboxyl group of the starting TMCA was converted with thionyl chloride to the acyl chloride, which, upon subsequent reaction with the appropriate alcohol/phenol, yielded the targeted ester. Both reactions (conversion to the acid chloride and subsequent ester formation) were performed in dry toluene under reflux. All the compounds were recrystallized from ethanol. Lipophilicity (expressed as log P values) of compounds 1-12, electronic σ parameters of individual ester substituents (-R) and molar volumes (MV (cm 3 )) of individual ester substituents (-R) were used as physicochemical descriptors characterizing individual investigated compounds. The program ACD/Percepta ver. 2012 was used for all predicted values (see Table 1). As expected, TMCA showed the lowest log P. Of the target esters, aliphatic derivative 12 (R = 2-propoxyethyl (PPXE), log P = 2.48) and compound 8 (R = 2,6-OCH 3 -Ph, log P = 2.90) were the least lipophilic compounds. Compound 10 (R = 4-(2,4,4-trimethylpentan-2-yl)phenyl (TMPP), log P = 6.43) showed the highest predicted lipophilicity. The fact that methoxyphenyl or benzyl substituents have lower lipophilicity than their "parent" phenyl substituent was described, for example, in [56][57][58][59][60][61]. The lowest electronic σ parameters for individual ester substituents were predicted for compound 8 (σ = 0.09); the highest electron-withdrawing effect was observed for compounds substituted in the ortho position of the phenyl ring by fluorine (6, σ = 1.02) and chlorine (7, σ = 1.05). Thus, it can be stated that most ester substituents possess electron-withdrawing properties in the range from approx. 0.2 to 1.05. The bulkiness of the individual ester substituents, expressed as molar volume, was logically the smallest for the unsubstituted phenyl ester 1 (MV = 80.88 cm 3 ); on the contrary, compound 10 (R = TMPP) has by far the largest volume MV = 213.31 cm 3 within the whole series. In addition, compound 9 (R = 2-isopropyl-5-methylphenyl (IPMP)) has the second largest volume within the investigated series of esters, with an MV value of 146.96 cm 3 . Table 1. Structures of 3,4,5-trimethoxycinnamic acid (TMCA) and its discussed ring-substituted esters 1-12; calculated values of log P, electronic σ parameters of R substituents and molar volume (MV (cm 3 )) of R substituents; in vitro AChE and BChE inhibition (IC 50 (µM)) compared with standards rivastigmine (RIV) and galantamine (GLT) and in vitro viability (SW982, THP1-Blue™ NF-κB) assays (IC 50 (µM) after 24 h and 72 h incubation) of investigated compounds. Cholinesterase inhibition is expressed as the mean ± SD (n = 3 experiments).

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 IC50 (μM) (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. . Moreover, it should be noted that the selectivity indices (increased BChE inhibition in comparison with AChE 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/IC50

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 IC50 (μM) (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. . Moreover, it should be noted that the selectivity indices (increased BChE inhibition in comparison with AChE 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/IC50 (M)) on lipophilicity; expressed as log P, electronic σ parameters and bulkiness of the

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 IC50 (μM) (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. . Moreover, it should be noted that the selectivity indices (increased BChE inhibition in comparison with AChE inhibition) for these two compounds are 1.42 and 1.71, respectively, which makes these two compounds valuable, as mentioned above.

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 (µM) (concentration of inhibitor that was required for 50% inhibition of the enzymes).

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 IC50 (μM) (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. . Moreover, it should be noted that the selectivity indices (increased BChE inhibition in comparison with AChE 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/IC50 (M)) on lipophilicity; expressed as log P, electronic σ parameters and bulkiness of the 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. 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 σ 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 σ 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).
The dependences of the BChE inhibition of the investigated compounds expressed as log(1/IC 50 (M)) on lipophilicity expressed as log P, electronic σ 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-Cl-Ph) followed by decreasing activity with increasing ester bulkiness) can also be traced to Figure 3C. individual ester substituents; and expressed as MV are illustrated in Figure 2A-C. While it is evident that the effect of σ 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). The dependences of the BChE inhibition of the investigated compounds expressed as log(1/IC50 (M)) on lipophilicity expressed as log P, electronic σ 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 electronwithdrawing properties of the ester substituents. The bilinear trend (increasing BChE inhibition to MV = 92 cm 3 (7, R = 2-Cl-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. 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].

Type of Inhibition
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 (KM) and maximum velocity (Vm) of the uninhibited and inhibited reaction. The competitive inhibitor increases KM by interfering with the active site of the enzyme but does not affect Vm. All lines in the L-B plot intersect at the same point on the y-axis. The non-competitive inhibitor does not change KM but decreases Vm. All lines in the L-B plot intersect at the same point on the x-axis. The uncompetitive inhibitor decreases KM and Vm, 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 KM, Vm 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 Ki (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).

Type of Inhibition
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 M ) and maximum velocity (V m ) of the uninhibited and inhibited reaction. The competitive inhibitor increases K M by interfering with the active site of the enzyme but does not affect V 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 M but decreases V m . All lines in the L-B plot intersect at the same point on the x-axis. The uncompetitive inhibitor decreases K M and V 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 M , V 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 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).

In Vitro Cell Viability
To exclude the potential cytotoxic effect of the tested compounds, their influence on cell viability was evaluated on two different cell lines-adherent human synovial cell line SW982 and suspension human genetically modified leukemia cell line THP1-Blue™ NF-κB. All of the used compounds slightly reduced cell viability (measured as mitochondrial metabolism) at the highest concentration of 30 µM after 24 and 72 h. However, the observed relative cell viability was >70% for both cell lines and all compounds. Moreover, the most active AChE and BChE inhibitors 6, 7, 9, and 11 did not decrease the relative viability of THP1-Blue™ NF-κB cells under 50% after 24 h incubation, when they were used at the concentration of 60 µM. The compound 5 with the most "cytotoxic" behavior had an IC 50 value for THP1-Blue™ NF-κB of~54 µM after 24 h incubation.

In Vitro Cell Viability
To exclude the potential cytotoxic effect of the tested compounds, their influence on cell viability was evaluated on two different cell lines-adherent human synovial cell line SW982 and suspension human genetically modified leukemia cell line THP1-Blue™ NF-κB. All of the used compounds slightly reduced cell viability (measured as mitochondrial metabolism) at the highest concentration of 30 µ M after 24 and 72 h. However, the observed relative cell viability was >70% for both cell lines and all compounds. Moreover, the most active AChE and BChE inhibitors 6, 7, 9, and 11 did not decrease the relative viability of THP1-Blue™ NF-κB cells under 50% after 24 h incubation, when they were used at the concentration of 60 µ M. The compound 5 with the most "cytotoxic" behavior had an IC50 value for THP1-Blue™ NF-κB of ~54 μM after 24 h incubation.

Conclusions
A series of twelve trimethoxycinnamic acid esters was prepared and characterized. The starting trimethoxycinnamic acid and all prepared esters were tested for their ability to inhibit in vitro AChE and BChE. 2-Chlorophenyl (2E)-3-(3,4,5-trimethoxyphenyl)-prop-2-enoate (7) showed the highest activity against both AChE (IC 50 = 46.18 µM) and BChE (IC 50 = 32.46 µM). However, (2E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoate (6) had the best BChE selectivity index (SI = 1.71). Based on the Lineweaver-Burk plot for compound 7, it was determined that it acts via a mixed type of inhibition for both enzymes. The position of the substituent on the phenyl ring does not appear to be essential for AChE inhibition, nor have significant effects for the lipophilicity, bulkiness or electronic parameters of the substituents on AChE activity been found. On the other hand, for BChE-inhibiting activity, the absolutely essential position of the substituent is in the ortho position. Additionally, for BChE inhibition, an increase in activity can be observed with an increasing electronwithdrawing effect of the substituent, which, at the same time, has lipophilicity expressed as log P ca. 3.7 and rather less bulkiness (ca. MV = 92 cm 3 ). All studied compounds were evaluated for their in vitro cytotoxicity on two human cell lines and the active compounds were found to be insignificantly cytotoxic, underlining the importance of studying these small molecules in the future.