α-Glucosidase inhibitory activities of phenolic acid amides with L-amino acid moiety

Abstract

α-Glucosidase inhibitors can effectively control postprandial hyperglycemia. In this study, a series of phenolic acids with the L-amino acid moiety were synthesized and their inhibitory activities against α-glucosidase from Saccharomyces cerevisiae (EC 3.2.1.20) were evaluated. The results suggested that all these compounds showed strong α-glucosidase inhibitory activities. In particular, N-(4-hydroxyl-phenylpropenoyl)-L-alanine (c2) and N-(4-hydroxyl-phenylpropenoyl)-L-methionine (c8) exhibited much higher potency (IC₅₀ values 0.04 mM) than the positive control, acarbose (IC₅₀ values 1.70 mM). Three-dimensional quantitative structure–activity relationship (3D-QSAR) model for comparative molecular field analysis (CoMFA) was generated and the result showed that bulk groups and high electron density groups on the amino acid residues were a benefit to their activities. Moreover, the substituents with low electron density and little steric hindrance on the para position of the benzene ring were helpful in improving the activities. Kinetic analysis indicated that compound (c2) acted as a mixed-type inhibitor with a K_i value of 0.0124 mM. Docking analysis showed that they could bind to α-glucosidase at the catalytic site via hydrogen bonds and a π–π stacking.

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1. Introduction

Diabetes mellitus (DM) is a common chronic metabolic disease that is characterized by elevating plasma glucose levels.^1,2 The type II diabetes mellitus (TIIDM) caused by insulin resistance accounts for more than 90 percent of DM³ and has become a worldwide health concern due to its constantly growing incidence and serious complications.^4,5 Currently, glucose-lowering agents include sulfonylureas, glinides, biguanides and α-glucosidase inhibitors.^6–8 Among them, α-glucosidase inhibitors can effectively control postprandial hyperglycemia by inhibiting the catalytic activity of α-glucosidase, a key enzyme that catalyzes the final step in the digestive process of carbohydrates.^9,10 Sugar analogue α-glucosidase inhibitors, such as acarbose, miglitol, and voglibose are commonly used in clinics. However, most of them have gastrointestinal side effects.^11–13 Many natural non-glycosidic compounds present in plants have been reported to exhibit good α-glucosidase inhibition.^14–16 This may provide a valuable approach for the development of safe and effective α-glucosidase inhibitors.

Polyphenols are widespread in plants and exhibit various medicinal properties.^17,18 As an important type of polyphenol, phenolic acid amides with the L-amino acid moiety are widely distributed in plants such as cocoa (Theobroma cacao),^19,20 Angelica archanglica root, Sambucus nigra flowers, and Coriandrum sativum fruit.²¹ Research has demonstrated that the cocoa extract or cocoa products could well control the glucose level in diabetic-induced rats and genetically inherited diabetic rats.^22,23 Abbe Maleyki et al. (2008) showed that the polyphenol extract from cocoa has an effect on postprandial glucose control.²⁴ Apart from polyphenol, are there any other α-glucosidase inhibitors in cocoa products? Exploring this problem would be valuable for developing food-based glucose-lowering agents. Considering the large family of phenolic acid amides with the L-amino acid moiety present in cocoa, we speculated that they may act as α-glucosidase inhibitors and have a hypoglycemic effect induced by cocoa. To validate this hypothesis, 42 such phenolic acid amides were synthesized and some of them have already been isolated from cocoa or other natural products. Their α-glucosidase inhibitory activities in vitro were investigated.

2. Results and discussion

2.1. Chemistry

The synthetic method was illustrated in Fig. 1 and ferulic acid derivative was exemplified. Forty two N-phenylpropenoyl-L-amino acids were synthesized. The structures are illustrated in Table 1. All the synthesized compounds were confirmed by ¹H NMR and MS techniques. All spectral data were in accordance with the assumed structures.

Fig. 1 Synthetic route of N-[4-hydroxy-3-methoxyl-(E)-cinnamoyl]-L-amino acid.

Table 1 In vitro α-glucosidase inhibition activities of the compounds^a

Compound	R	IC50 (mM)	Compound	R	IC50 (mM)	Compound	R	IC50 (mM)
a IC 50 (acarbose) = 1.70 mM.
a1	Gly	34.43	b1	Gly	3.53	c1	Gly	0.72
a2	Ala	23.07	b2	Ala	9.63	c2	Ala	0.04
a3	Leu	31.50	b3	Leu	6.43	c3	Leu	0.07
a4	Ile	17.76	b4	Ile	10.60	c4	Ile	0.27
a5	Val	22.16	b5	Val	15.29	c5	Val	0.40
a6	Pro	2.16	b6	Pro	29.42	c6	Pro	0.21
a7	Phe	2.13	b7	Phe	1.17	c7	Phe	0.30
a8	Met	19.27	b8	Met	11.16	c8	Met	0.04
a9	Trp	1.84	b9	Trp	3.95	c9	Trp	0.68
a10	Ser	1.10	b10	Ser	1.20	c10	Ser	3.42
a11	Gln	4.31	b11	Gln	2.93	c11	Gln	1.42
a12	Thr	3.25	b12	Thr	3.80	c12	Thr	0.23
a13	Glu	2.10	b13	Glu	1.95	c13	Glu	0.12
a14	Asp	1.91	b14	Asp	1.53	c14	Asp	0.13

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2.2. Inhibitory activities against α-glucosidase and 3D QSAR model development

All the synthesized compounds were evaluated for their inhibitory activities against α-glucosidase. The IC₅₀ values are summarized in Table 1. Some compounds had better activities than acarbose (IC₅₀ value of 1.70 mM). Among them, compounds c2, c3, and c8 showed much stronger activities than acarbose with IC₅₀ values of 0.04, 0.07 and 0.04 mM, respectively, indicating that they are much more active than acarbose. Compounds a9, a13, a14, c9, c13, and c14, which were natural present in cocoa beans,²⁵ showed similar or better activities than acarbose. This result indicated that the phenolic acid amides in cocoa might play partial roles in the hypoglycemic effects of cocoa beans.

The statistical parameters of CoMFA are shown in Table 2. An optimal number of principal components (ONC = 10) was recommended based on the LOO cross-validated run with a q² value of 0.561. The subsequent non-cross-validated correlation coefficients r² value was 0.977, which was greater than the value required for a good model. The CoMFA model contour map included 51.2% of the electrostatic field descriptor and 48.8% of the contributing steric field, indicating a greater influence of the electrostatic field. To validate the prediction capabilities of the 3D QSAR model further, the predicted activity and the residuals between the experimental and predicted activity of the training set and test set were measured, respectively (see ESI Table S1‡). The linear regression analysis plot between predicted pIC₅₀ against observed pIC₅₀ of the training set (Fig. 2(A)) showed that the predicted pIC₅₀ values were almost in accordance with the experimental values with a slope of 0.980. The predicted pIC₅₀ values of the test set well suited the experiment data (Fig. 2(B)). The result indicated that the CoMFA model had good predictability.

Table 2 Statistical results of the CoMFA model (q² was leave-one-out cross-validated correlation coefficient; ONC, optimal number of principal components; r², non-cross-validated correlation coefficients; SEE, standard error of the estimate; F, the ratio between explained and unexplained variance)

LOO			NV		Relative contributor
q^2	ONC	r^2	SEE	F	Steric	Electrostatic
0.561	10	0.977	0.143	101.655	0.488	0.512

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Fig. 2 Predicted versus the observed pIC₅₀ of the (A) training set and (B) test set.

2.3. Interpretation of the inhibitory activity and 3D QSAR model

The steric and electrostatic contour map obtained via the CoMFA model is shown in Fig. 3. In the steric field, bulky groups (the green area distributed on the amino acid residual chain, such as aromatic rings in compound a7, a9, b7 and b9) were responsible for the high inhibitory activities, whereas compounds with small groups (a1–a5 and a8, b1–b5 and b8) had much lower activities in the a and b series compounds. However, on the para position of the benzene rings (the yellow area), bulk groups decreased the inhibitory activities; thus, the activities of b series compounds with a methoxy group on the para of benzene rings were weaker than that of the c series. In the electrostatic field, groups with high electron density (red areas on the residual part of amino acids) increased the activities. For example, compounds with negative electron groups, such as hydroxyl (a10, a12, b10 and b12), amino (a11 and b11), and carboxyl (a13, a14, b13 and b14), on their amino acid side chains performed better inhibitory activities among the a and b series compounds. Substituents with a low electron density (blue) regions on the benzene ring of cinnamic acid were expected to increase the activities. Among the three series, the electron density of substituents on the cinnamic acid had the following order: 4-hydroxy-3-methoxy > 4-methoxy > 4-hydroxy and the 4-hydroxy substituted compounds (c series) exhibited much stronger activities than the a and b series. Overall, the electrostatic field had more influence than the steric field on the activities.

Fig. 3 CoMFA contour map for α-glucosidase. Steric areas: bulk groups increased (green) or decreased (yellow) potency. Electrostatic areas: high electron density (red) and low electron density (blue) regions are expected to increase the potency.

2.4. Kinetic analysis

To further explore the inhibitory characteristics of these compounds, compound c2 was chosen as a representative to study the inhibition mode. Kinetic analysis was performed using Lineweaver–Burk (Fig. 4(A)) and Dixon plots (Fig. 4(B)). Compounds c2 showed a mixed inhibitory type against α-glucosidase from S. cerevisiae. The inhibitory constant (K_i) was 0.0124 mM. The result indicated that the inhibitory type was different from acarbose, which was reported to be a competitive inhibitor.²⁶

Fig. 4 Lineweaver–Burk (A) and Dixon (B) plots of α-glucosidase inhibition at different concentrations of pNPG and compound c2.

2.5. Docking study

The Ramachandran plot (see ESI Fig. S1‡) analysis by PROCHECK confirmed the quality of the modelling 3D structure with 87.6% of residues lied in the most favored regions, 11.6% in the additional allowed regions, 0.2% in the generously allowed regions. Considering the inhibitory type was a mixed one, blind docking was first conducted using c2 as a ligand model; interestingly, the binding sites were coincident with the catalytic pocket of α-glucosidase from Saccharomyces cerevisiae (Fig. 5(A)).

Fig. 5 Docking model predicted structural details of compound c2 (A and B), a2 (C), a14 (D) green: target inhibitors; yellow: native ligand maltose; blue: bonding residues of inhibitors.

As showed in Fig. 5(A), the amino acid terminal of compound c2 was oriented towards the core of the suit. In addition, c2 had a close interaction with the active sites, Asp214, of the pocket. Moreover, the hydroxyl group on the benzene ring of c2 interacted with the residues of Asp349 and Arg439. Furthermore, π–π stacking between the benzene ring and Phe157 also contributed to the binding. In all, as shown in Fig. 5(B), c2 had a very similar location to the native ligand maltose (Fig. 5(B)). Thus, c2 could well block the active center and exert its inhibitory actions. However, it was different in the case of compound a2 (Fig. 5(C)). The introduction of 3-methoxyl to the benzene ring caused the reverse of the binding locations. Moreover, the phenyl group entered the pocket in a sideways because of the narrow pore of the pocket. When the hydrophilic property of the amino acid terminal was increased by the introduction of a carboxyl to compound a14 (Fig. 5(D)), it could enter the pocket in a perfect way by binding to Asp214, Glu276, Asp349, Asp68, Arg212, and Arg439 via hydrogen bond. Therefore, the binding types were not totally the same among these compounds; both substituents on the phenyl ring and amino acid residues had a great influence on the bonding position.

3. Experimental

3.1. General

α-Glucosidase (EC 3.2.1.20) from Saccharomyces cerevisiae was purchased from Sigma. The p-nitrophenyl-α-D-glucopyranoside (pNPG), phenolic acids and amino acids were purchased from Aladdin Co. Ltd (Shanghai, China) and all the chemicals were of analytical reagent grade. The melting points (mp) were measured on a Taike digital micro melting point apparatus X-4 (Beijing, China). ¹H NMR spectroscopy was performed using Bruker AM spectrometers on 600 MHz or 400 Hz in DMSO. The MS (ESI) spectra were obtained on an Agilent 6100 Single Quad spectrometer.

3.2. Syntheses of N-phenylpropenoyl-L-amino acids

The synthetic method was based on the reported method with modifications.²⁷ The experimental procedure was as follows using ferulic acid derivative as an example: (E)-ferulic acid (10 mmol each) was added to an aqueous solution (15 mL) of sodium hydroxide (64 mmol) below 15 °C. After the solid dissolved completely, acetic anhydride (26 mmol) was added dropwise to the solution in an ice bath. After stirring for 30 min at room temperature, the reaction mixture was acidified to pH 1.0 with hydrochloric acid (2 mol L⁻¹) to precipitate. The suspension was filtered. The filtered cake was washed three times with water and dried in a vacuum. The solid was used in the next step without further purification. Thionyl chloride (2 mmol) and a catalytic amount of DMF were added to the suspension of the abovementioned acetylated phenylpropenoic acid derivative in 20 mL chloroform. The mixture was heated for 7–8 h at 55 °C. The remaining thionyl chloride and solvent were removed by evaporation to give a pale yellow oily liquid.

A solution of the abovementioned acyl chlorides (10 mmol) in 20 mL acetone and 10 mL hydroxide sodium (10 mmol) solution were added dropwise simultaneously to a solution of sodium salts of L-amino acid in 10 mL acetone and water (1 : 1) in an ice bath. This was followed by 2 h of stirring, two more equivalents of sodium hydroxide solid (20 mmol) was added to the reaction mixture. The mixture was stirred for an additional 3–4 h at room temperature. The acetone was then evaporated. The residual aqueous phase was adjusted to pH 2 with aqueous hydrochloric acid and then extracted with ethyl acetate (20 mL × 3). The combined organic phase was washed with water, dried over MgSO₄, and concentrated to afford the crude product, which was purified by column chromatography on silica gel (ethyl acetate/petroleum ether 1 : 2 (v/v)) to obtain the final product. The spectra data and physical properties of the compounds are showed as follows:

N-[4-Hydroxy-3-methoxy-(E)-cinnamoyl]-L-glycine acid a1, a light yellow solid (1.63 g, 65% yield); mp 224–226 °C; ¹H NMR (400 MHz, d₆-DMSO) δ 12.56 (s, 1H, –COOH̲), 9.44 (s, 1H, –OH̲), 8.23 (t, J = 5.9 Hz, 1H, –NH̲), 7.34 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH), 7.15 (d, J = 1.8 Hz, 1H, Ph–H̲), 7.01 (dd, J = 8.2, 1.8 Hz, 1H, Ph–H̲), 6.79 (d, J = 8.1 Hz, 1H, Ph–H̲), 6.54 (d, J = 15.7 Hz, 1H, –CH=CH̲–), 3.87 (d, J = 5.9 Hz, 2H, –CH₂), 3.81 (s, 3H, –CH̲₃); MS m/z 252.2 [M + H]⁺.

N-[4-Hydroxy-3-methoxy-(E)-cinnamoyl]-L-alanine acid a2, a yellow amorphous solid (1.59 g, 60% yield); mp 95–97 °C; ¹H NMR (400 MHz, d₆-DMSO) δ 12.84 (s, 1H, –COOH̲), 9.45 (s, 1H, –OH̲), 8.23 (d, J = 7.4 Hz, 1H, –NH̲), 7.33 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 7.13 (d, J = 1.7 Hz, 1H, Ph–H̲), 7.00 (dd, J = 8.2, 1.7 Hz, 1H, Ph–H̲), 6.79 (d, J = 8.1 Hz, 1H, Ph–H̲), 6.52 (d, J = 15.7 Hz, 1H, Ph–CH=CH̲–), 4.32 (q, J = 7.8 Hz, 1H, –CH̲–), 3.80 (s, 3H, –OCH̲₃), 1.31 (d, J = 7.3 Hz, 3H, –CH̲₃); MS m/z 266.2 [M + H]⁺.

N-[4-Hydroxy-3-methoxy-(E)-cinnamoyl]-L-leucine acid a3, a light yellow amorphous solid (1.68 g, 55% yield); mp 96–98 °C; ¹H NMR (400 MHz, d₆-DMSO) δ 12.56 (s, 1H, –COOH̲), 9.45 (s, 1H, –OH), 8.18 (d, J = 8.1 Hz, 1H, –NH̲), 7.33 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH), 7.13 (d, J = 1.6 Hz, 1H, Ph–H̲), 6.99 (dd, J = 8.2, 1.7 Hz, 1H, Ph–H̲), 6.79 (d, J = 8.1 Hz, 1H, Ph–H̲), 6.53 (d, J = 15.7 Hz, 1H, –CH=CH̲), 4.34 (dt, J = 8.9, 5.6 Hz, 1H, –CH̲–), 3.81 (s, 3H, –OCH̲₃), 1.75–1.61 (m, 1H, –CH̲–), 1.61–1.48 (m, 2H, –CH̲₂–), 0.91 (d, J = 6.5 Hz, 3H, –CH̲₃–), 0.86 (d, J = 6.5 Hz, 3H, –CH̲₃–). MS m/z 308.4 [M + H]⁺.

N-[4-Hydroxy-3-methoxy-(E)-cinnamoyl]-L-isoleucine acid a4, a white amorphous solid (1.53 g, 50% yield); mp 94–97 °C; ¹H NMR (400 MHz, d₆-DMSO) δ 12.58 (s, 1H, –COOH̲), 9.45 (s, 1H, –OH̲), 8.05 (d, J = 8.5 Hz, 1H, –NH̲), 7.33 (d, J = 15.7 Hz, 1H, Ar-CH̲=CH), 7.13 (d, J = 1.6 Hz, 1H, Ar-H̲), 6.99 (dd, J = 8.2, 1.6 Hz, 1H, Ar-H̲), 6.81 (t, J = 12.4 Hz, 1H, Ar-H̲), 6.67 (d, J = 15.7 Hz, 1H –CH=CH̲), 4.32 (dd, J = 8.4, 5.9 Hz, 1H, –NH–CH̲–), 3.81 (s, 3H, –OCH̲₃), 1.82 (dd, J = 7.5, 5.6 Hz, 1H, –CH̲–), 1.44–1.16 (m, 2H, –CH̲₂), 0.92–0.85 (m, 6H, –(CH̲₃)₂); MS m/z 308.4 [M + H]⁺.

N-[4-Hydroxy-3-methoxy-(E)-cinnamoyl]-L-valine acid a5, a white amorphous solid (1.61 g, 55% yield); mp 90–92 °C; ¹H NMR (400 MHz, d₆-DMSO) δ 12.53 (s, 1H, –COOH̲), 9.45 (s, 1H, –OH̲), 8.03 (d, J = 8.6 Hz, 1H, –NH̲), 7.32 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 7.13 (d, J = 1.7 Hz, 1H, Ph–H̲), 6.99 (dd, J = 8.2, 1.7 Hz, 1H, Ph–H̲), 6.79 (d, J = 8.1 Hz, 1H, Ph–H̲), 6.68 (d, J = 15.7 Hz, 1H, Ph–CH=CH̲–), 4.28 (dd, J = 8.5, 5.6 Hz, 1H, –NH–CH̲–), 3.81 (s, 3H, –OCH̲₃), 2.14–2.06 (m, 1H, –CH̲–), 0.97–0.85 (m, 6H, –CH(CH̲₃)₂); MS m/z 294.3 [M + H]⁺.

N-[4-Hydroxy-3-methoxy-(E)-cinnamoyl]-L-proline acid a6, a white solid (1.90 g, 65% yield); mp 171–173 °C; ¹H NMR (400 MHz, d₆-DMSO) δ 12.39 (s, 1H, –COOH̲), 9.46 (s, 1H, –OH̲), 7.38 (d, J = 15.4 Hz, 1H, Ph–CH̲=CH–), 7.29 (d, J = 1.6 Hz, 1H, Ph–H̲), 7.10 (d, J = 8.2, 1H, Ph–H̲), 6.81 (d, J = 15.8 Hz, 1H, Ph–CH=CH̲–), 6.78 (d, J = 8.3 Hz, 1H, Ph–H̲), 4.33 (dd, J = 8.7, 3.9 Hz, 1H, –N–CH̲–), 3.82 (s, 3H, –OCH̲₃), 3.77–3.61 (m, 1H, –CH̲₂–), 2.22–1.81 (m, 3H, –CH̲₂–, –CH̲₂–); MS m/z 292.4 [M + H]⁺.

N-[4-Hydroxy-3-methoxy-(E)-cinnamoyl]-L-phenylalanine acid a7, a white amorphous solid (2.11 g, 62% yield); mp 82–85 °C; ¹H NMR (400 MHz, d₆-DMSO) δ 12.67 (s, 1H, –COOH̲), 9.45 (s, 2H, –OH̲), 8.23 (d, J = 8.1 Hz, 2H, –NH̲), 7.24 (m, 6H, Ph–H, Ph–CH̲=CH–), 7.11 (d, J = 1.7 Hz, 1H, Ph–H̲), 6.98 (dd, J = 8.2, 1.7 Hz, 1H, Ph–H̲), 6.78 (d, J = 8.1 Hz, 1H, Ph–H̲), 6.52 (d, J = 15.7 Hz, 1H, Ph–CH=CH̲–), 4.56 (td, J = 9.1, 4.8 Hz, 1H), 3.80 (s, 3H, –OCH̲₃), 3.11 (dd, J = 13.9, 4.8 Hz, 1H, –CH̲₂–), 2.92 (dd, J = 13.9, 9.5 Hz, 1H, –CH̲₂–); MS m/z 342.3 [M + H]⁺.

N-[4-Hydroxy-3-methoxy-(E)-cinnamoyl]-L-methionine acid a8, a yellow amorphous solid (1.85 g, 57% yield); mp 92–94 °C; ¹H NMR (400 MHz, d₆-DMSO) δ 9.47 (s, 1H, –OH̲), 8.27 (d, J = 7.9 Hz, 1H, –NH̲), 7.37 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 7.15 (d, J = 1.6 Hz, 1H, Ph–H̲), 7.02 (dd, J = 8.2, 1.6 Hz, 1H, Ph–H), 6.81 (d, J = 8.1 Hz, 1H, Ph–H̲), 6.55 (d, J = 15.7 Hz, 1H, Ph–CH=CH̲–), 4.46 (td, J = 8.7, 4.6 Hz, 1H, –CH̲–), 3.82 (s, 3H, –OCH̲₃), 2.66–2.31 (m, 2H, –CH₂–CH̲₂–), 2.10–1.98 (m, 5H, –CH̲₂–CH₂–S–CH̲₃); MS m/z 326.3 [M + H]⁺.

N-[4-Hydroxy-3-methoxy-(E)-cinnamoyl]-L-tryptophane acid a9, a white solid (1.93 g, 51% yield); mp 85–87 °C; ¹H NMR (400 MHz, d₆-DMSO) δ 10.83 (s, 1H, –OH̲), 9.44 (s, 1H, –NH̲), 8.18 (d, J = 7.9 Hz, 1H, –NH̲), 7.54 (d, J = 7.8 Hz, 1H, Ar-H), 7.32 (dd, J = 15.6, 11.9 Hz, 2H, Ph–CH̲=CH–, Ar-H̲), 7.16 (d, J = 2.1 Hz, 1H, Ph–H), 7.12 (d, J = 1.6 Hz, 1H, Ar-H̲), 7.07 (t, J = 7.2 Hz, 1H, Ar-H), 7.02–6.94 (m, 2H, Ar-H, Ph–H̲), 6.79 (d, J = 8.1 Hz, 1H, Ph–H̲), 6.56 (d, J = 15.7 Hz, 1H, Ph–CH=CH̲–), 4.63 (td, J = 8.3, 5.0 Hz, 1H, –CH̲–), 3.80 (s, 3H, –OCH̲₃), 3.23 (dd, J = 14.7, 4.9 Hz, 1H, –CH̲₂–), 3.07 (dd, J = 14.7, 8.7 Hz, 1H, –CH̲₂–); MS m/z 381.3 [M + 1]⁺.

N-[4-Hydroxy-3-methoxy-(E)-cinnamoyl]-L-serine acid a10, a light yellow solid (1.32 g, 47% yield); mp 184–187 °C; ¹H NMR (400 MHz, d₆-DMSO) δ 12.51 (s, 1H, –COOH̲) 9.44 (s, 1H, Ph–OH̲), 8.04 (d, J = 8.0 Hz, 1H, –NH̲), 7.32 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 7.15 (d, J = 1.7 Hz, 1H, Ph–H̲), 7.00 (dd, J = 8.2, 1.7 Hz, 1H, Ph–H), 6.79 (d, J = 8.1 Hz, 1H, Ph–H̲), 6.69 (d, J = 15.7 Hz, 1H, Ph–CH=CH̲–), 5.06 (s, 1H, –OH), 4.42–4.39 (m, 1H, –CH̲–), 3.75 (dd, J = 10.8, 5.0 Hz, 1H, –CH̲₂), 3.66 (dd, J = 10.8, 4.1 Hz, 1H, –CH̲₂). MS m/z 282.3 [M + H]⁺.

N-[4-Hydroxy-3-methoxy-(E)-cinnamoyl]-L-glutamine acid a11, a white amorphous solid (1.61 g, 50% yield); mp 96–99 °C; ¹H NMR (400 MHz, d₆-DMSO) δ 12.40 (s, 1H, –COOH̲), 9.46 (s, 1H, Ph–OH̲), 8.21 (d, J = 7.9 Hz, 1H, –NH̲), 7.33 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 7.13 (d, J = 1.7 Hz, 1H, Ph–H̲), 7.00 (dd, J = 8.2, 1.7 Hz, 1H, Ph–H̲), 6.80 (d, J = 8.1 Hz, 1H, Ph–H̲), 6.53 (d, J = 15.7 Hz, 1H, Ph–CH=CH̲–), 4.33 (td, J = 8.7, 5.1 Hz, 1H, –CH̲–), 3.81 (s, 3H, –OCH̲₃–), 2.41–2.20 (m, 2H, –CH̲₂–), 2.09–1.93 (m, 1H, –CH̲₂–), 1.94–1.75 (m, 1H, –CH̲₂–); MS m/z 324.3 [M + H]⁺.

N-[4-Hydroxy-3-methoxy-(E)-cinnamoyl]-L-threonine acid a12, a white solid (1.36 g, 46% yield); mp 192–194 °C; ¹H NMR (400 MHz, d₆-DMSO) δ 12.92 (s, 1H, –COOH̲), 9.44 (s, 1H, Ph–OH̲), 7.77 (d, J = 8.7 Hz, 1H, –NH̲), 7.31 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 7.17 (d, J = 1.6 Hz, 1H, Ph–H̲), 7.00 (dd, J = 8.2, 1.6 Hz, 1H, Ph–H̲), 6.81 (d, J = 8.1 Hz, 1H, Ph–H̲), 6.80 (d, 1H, Ph–CH=CH̲–), 4.33 (dd, J = 8.7, 3.1 Hz, 1H, –CH̲–), 4.16 (dd, J = 6.3, 3.2 Hz, 1H, –CH̲–), 3.81 (s, 4H, –OCH̲₃), 1.07 (d, J = 6.3 Hz, 3H, –CH̲₃); MS m/z 296.3 [M + H]⁺.

N-[4-Hydroxy-3-methoxy-(E)-cinnamoyl]-L-glutamic acid a13, a light yellow amorphous solid (1.97 g, 61%); mp 108–110 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.41 (s, 2H, –COOH̲), 9.46 (s, 1H), 8.22 (d, J = 7.9 Hz, 1H, –NH̲), 7.33 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 7.14 (d, J = 1.8 Hz, 1H, Ph–H̲), 7.00 (dd, J = 8.2, 1.8 Hz, 1H, Ph–H), 6.79 (d, J = 8.1 Hz, 1H, Ph–H), 6.53 (d, J = 15.7 Hz, 1H, Ph–CH=CH̲–), 4.37–4.28 (m, 1H, –CH–), 3.81 (s, 3H, –OCH̲₃), 2.38–2.22 (m, 2H, –CH̲₂–), 2.06–1.97 (m, 1H, –CH̲₂–), 1.85–1.178 (m, 1H, –CH̲₂–); MS m/z 324.2 [M + H]⁺.

N-[4-Hydroxy-3-methoxy-(E)-cinnamoyl]-L-aspartic acid a14, a yellow solid (1.76 g, 57% yield); mp 187–189 °C; ¹H NMR (400 MHz, d₆-DMSO) δ 12.49 (s, 2H, –COOH̲), 9.45 (s, 1H, –OH̲), 8.22 (d, J = 8.0 Hz, 1H, –NH̲), 7.33 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 7.14 (d, J = 1.6 Hz, 1H, Ph–H̲), 6.99 (dd, J = 8.2, 1.6 Hz, 1H, Ph–H̲), 6.79 (d, J = 8.1 Hz, 1H, Ph–H̲), 6.58 (d, J = 15.7 Hz, 1H, Ph–CH=CH̲–), 4.66 (dd, J = 13.3, 7.2 Hz, 1H, –CH̲–), 3.81 (s, 3H, –OCH̲₃), 2.70 (qd, J = 16.6, 6.3 Hz, 2H, –CH̲₂–); MS m/z 310.3 [M + H]⁺.

N-[4-Methoxy-(E)-cinnamoyl]-L-glycine acid b1, a white solid (1.90 g, 81% yield); mp 165–167 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.58 (s, 1H, –COOH̲), 8.32 (t, J = 5.9 Hz, 1H, –NH̲), 7.53 (d, J = 8.6 Hz, 2H, Ph–H̲), 7.40 (d, J = 15.8 Hz, 1H, Ph–CH̲=CH–), 6.98 (d, J = 8.6 Hz, 2H, Ph–H̲), 6.57 (d, J = 15.8 Hz, 1H, Ph–CH=CH̲–), 3.88 (d, J = 5.9 Hz, 2H, –CH̲₂–), 3.78 (s, 3H, –OCH̲₃); MS m/z 236.3 [M + H]⁺.

N-[4-Methoxy-(E)-cinnamoyl]-L-alanine acid b2, a white solid (1.99 g, 80% yield); mp 162–164 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.57 (s, 1H, –COOH̲), 8.32 (d, J = 7.3 Hz, 1H, –NH̲), 7.51 (d, J = 8.7 Hz, 2H, Ph–H̲), 7.38 (d, J = 15.8 Hz, 1H, Ph–CH̲=CH–), 6.98 (d, J = 8.7 Hz, 2H, Ph–H̲), 6.56 (d, J = 15.8 Hz, 1H, Ph–CH=CH̲–), 4.32 (t, J = 7.3 Hz, 1H, –CH̲–), 3.78 (s, 3H, –OCH̲₃), 1.32 (d, J = 7.3 Hz, 3H, –CH̲₃); MS m/z 250.1 [M + H]⁺.

N-[4-Methoxy-(E)-cinnamoyl]-L-leucine acid b3, a white amorphous solid (2.15 g, 74% yield); mp 85–87 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.57 (s, 1H, –COOH), 8.25 (d, J = 8.1 Hz, 1H, –NH̲), 7.51 (d, J = 8.8 Hz, 2H, Ph–H̲), 7.38 (d, J = 15.8 Hz, 1H, Ph–CH̲=CH–), 7.03–6.93 (m, 2H, Ph–H̲), 6.57 (d, J = 15.8 Hz, 1H, Ph–CH̲=CH–), 4.36 (ddd, J = 9.8, 8.1, 5.2 Hz, 1H, NH–CH̲–), 3.79 (s, 1H, –OCH̲₃), 1.71–1.62 (m, 1H, –CH̲–), 1.62–1.50 (m, 2H, –CH̲₂–), 0.91 (d, J = 6.6 Hz, 3H, –CH̲₃), 0.87 (d, J = 6.5 Hz, 3H, –CH̲₃); MS m/z 292.3 [M + H]⁺.

N-[4-Methoxy-(E)-cinnamoyl]-L-isoleucine acid b4, a white amorphous solid (2.03 g, 70% yield); mp 83–85 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.61 (s, 1H, –COOH̲), 8.12 (d, J = 8.3 Hz, 1H, –NH̲), 7.51 (d, J = 7.9 Hz, 2H, Ph–H̲), 7.37 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 6.98 (d, J = 8.0 Hz, 2H, Ph–H̲), 6.70 (d, J = 15.7 Hz, 1H, Ph–CH=CH̲–), 4.32 (t, J = 6.9 Hz, 1H, –CH̲–), 3.79 (s, 3H, –OCH̲₃), 1.83 (m, 1H, –CH̲–), 1.45–1.42 (m, 1H, –CH̲₂–), 1.26–1.98 (m, 1H, –CH̲₂–), 0.89–0.85 (m, 6H, –CH̲₃); MS m/z 292.3 [M + H]⁺.

N-[4-Methoxy-(E)-cinnamoyl]-L-valine acid b5, a white amorphous solid (1.52 g, 55% yield); mp 70–73 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.62 (s, 1H, –COOH̲), 8.11 (d, J = 8.6 Hz, 1H, –NH̲), 7.51 (d, J = 8.3 Hz, 2H, Ph–H̲), 7.38 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 6.99 (d, J = 8.3 Hz, 2H, Ph–H̲), 6.72 (d, J = 15.8 Hz, 1H, Ph–CH=CH̲–), 4.29 (dd, J = 8.3, 5.9 Hz, 1H, –CH̲–), 3.79 (s, 3H, –OCH̲₃), 2.10 (dd, J = 13.2, 6.6 Hz, 1H, –CH̲(CH₃)₂), 0.92 (dd, J = 6.5, 4.0 Hz, 6H, –CH̲(CH₃)₂); MS m/z 278.2 [M + H]⁺.

N-[4-Methoxy-(E)-cinnamoyl]-L-proline acid b6, a white solid (1.81 g, 66% yield); mp 197–199 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.51 (s, 1H, –COOH̲), 7.66 (d, J = 7.9 Hz, 2H, Ph–H̲), 7.43 (d, J = 15.4 HZ, 1H, Ph–CH̲=CH–), 6.97 (d, J = 8.0 Hz, 2H, Ph–H̲), 6.87 (d, J = 15.5 Hz, 1H, Ph–CH=CH̲–), 4.34 (dd, J = 8.6, 3.7 Hz, 1H, –CH–), 3.79 (s, 3H, –OCH̲₃), 3.77–3.67 (m, 2H, –NH–CH̲₂–), 2.32–2.06 (m, 1H, NH–CH₂–CH̲₂–), 2.01–1.79 (m, 3H, NH–CH̲₂–CH̲₂–); MS m/z 276.2 [M + H]⁺.

N-[4-Methoxy-(E)-cinnamoyl]-L-phenylalanine acid b7, a white solid (2.43 g, 75% yield); mp 161–165 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.76 (s, 1H, –COOH̲), 8.31 (d, J = 8.1 Hz, 1H, –NH̲), 7.49 (d, J = 8.4 Hz, 2H, Ph–H̲), 7.33 (d, J = 15.8 Hz, 1H, Ph–CH̲=CH–), 7.30–7.24 (m, 4H, Ph–H̲), 7.20 (dd, J = 10.8, 4.1 Hz, 1H, Ph–H̲), 6.97 (d, J = 8.4 Hz, 2H, Ph–H̲), 6.55 (d, J = 15.8 Hz, 1H, Ph–CH=CH̲–), 4.57 (td, J = 8.7, 5.0 Hz, 1H, –CH̲–), 3.78 (s, 3H, –OCH̲₃), 3.12 (dd, J = 13.9, 4.7 Hz, 1H, –CH̲₂), 2.99–2.85 (m, 1H, –CH̲₂–); MS m/z 326.3 [M + H]⁺.

N-[4-Methoxy-(E)-cinnamoyl]-L-methionine acid b8, a white solid (1.91 g, 62% yield); mp 149–152 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.71 (s, 1H, –COOH̲), 8.33 (d, J = 7.9 Hz, 1H, –NH̲), 7.52 (d, J = 8.7 Hz, 2H, Ph–H̲), 7.39 (d, J = 15.8 Hz, 1H, Ph–CH̲=CH–), 6.99 (d, J = 8.7 Hz, 2H, Ph–H̲), 6.57 (d, J = 15.8 Hz, 1H, Ph–CH=CH̲–), 4.50–4.38 (m, 1H, –CH̲–), 3.79 (s, 3H, –OCH̲₃), 2.51–2.49 (m, 2H, –CH̲₂–S–), 2.05 (s, 3H, –CH̲₃), 2.03–1.90 (m, 2H, –CH̲₂–); MS m/z 310.2 [M + H]⁺.

N-[4-Methoxy-(E)-cinnamoyl]-L-tryptophane acid b9, a light yellow amorphous solid (2.00 g, 55%); mp 70–72 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.71 (s, 1H, –COOH̲), 10.85 (d, J = 1.4 Hz, 1H, –NH̲–), 8.29 (d, J = 7.9 Hz, 1H, –NH̲–COOH), 7.55 (d, J = 7.9 Hz, 1H, Ar-H̲), 7.49 (d, J = 8.8 Hz, 2H, Ph–H), 7.34 (d, J = 15.7, 1H, Ph–CH̲=CH–), 7.32 (d, J = 8.0 Hz, 1H, Ar-H̲), 7.16 (d, J = 2.2 Hz, 1H, Ar-H̲), 7.10–7.02 (m, 1H, Ar-H̲), 7.01–6.93 (m, 3H, Ar-H̲, 2Ph–H̲), 6.59 (d, J = 15.8 Hz, 1H, Ph–CH=CH̲–), 4.62 (td, J = 8.3, 5.0 Hz, 1H, –CH̲–), 3.78 (s, 3H, –OCH̲₃), 3.23 (dd, J = 14.7, 4.9 Hz, 1H, –CH̲₂–), 3.07 (dd, J = 14.7, 8.8 Hz, 1H, –CH̲₂–); MS m/z 365.2 [M + H]⁺.

N-[4-Methoxy-(E)-cinnamoyl]-L-serine acid b10, a white solid (1.59 g, 60% yield); mp 183–184 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.66 (s, 1H, –COOH̲), 8.17 (d, J = 8.0 Hz, 1H, –NH̲–), 7.52 (d, J = 8.7 Hz, 2H, Ph–H̲), 7.38 (d, J = 15.8 Hz, 1H, Ph–CH̲=CH–), 6.98 (d, J = 8.8 Hz, 2H, Ph–H̲), 6.73 (d, J = 15.8 Hz, 1H, Ph–CH=CH̲–), 4.50–4.36 (m, 1H, –CH̲–), 3.79 (s, 3H, –OCH̲₃), 3.76 (dd, J = 10.9, 5.0 Hz, 1H, –CH̲₂–), 3.67 (dd, J = 10.9, 4.0 Hz, 1H, –CH̲₂–), 2.55–2.43 (m, 1H, –OH); MS m/z 266.3 [M + H]⁺.

N-[4-Methoxy-(E)-cinnamoyl]-L-glutamine acid b11, a white solid (1.74 g, 57% yield); mp 165–168 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.79 (s, 1H, –COOH̲), 8.28 (d, J = 6.6 Hz, 1H, –NH̲–), 7.51 (d, J = 8.6 Hz, 2H, Ph–H̲), 7.37 (d, J = 15.9 Hz, 1H, Ph–CH̲=CH), 7.35 (s, 1H, –NH̲₂), 6.98 (d, J = 8.6 Hz, 2H, Ph–H̲), 6.79 (s, 1H, –NH̲₂), 6.65 (d, J = 15.8 Hz, 1H, Ph–CH=CH̲–), 4.27 (m, 1H, –CH̲–), 3.79 (s, 3H, –OCH̲₃), 2.23–2.11 (m, 2H, –CH̲₂–), 2.01 (m, 1H, –CH̲₂–), 1.81 (m, 1H, –CH̲₂–); MS m/z 307.3 [M + H]⁺.

N-[4-Methoxy-(E)-cinnamoyl]-L-threonine acid b12, a white solid (1.08 g, 43% yield); mp 153–156 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.57 (s, 1H, –COOH̲), 7.93 (d, J = 8.8 Hz, 1H, –NH̲–), 7.52 (d, J = 8.7 Hz, 2H, Ph–H̲), 7.37 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 6.98 (d, J = 8.7 Hz, 2H, Ph–H̲), 6.83 (d, J = 15.8 Hz, 1H, Ph–CH=CH̲–), 4.99 (s, 1H, –OH̲), 4.35 (dd, J = 8.8, 3.1 Hz, 1H, –NH–CH̲), 4.23–4.12 (m, 1H, –CH̲), 3.79 (s, 3H, –OCH̲₃), 1.08 (d, J = 6.4 Hz, 3H, –CH̲₃); MS m/z 254.2 [M + H]⁺.

N-[4-Methoxy-(E)-cinnamoyl]-L-glutamic acid b13, a white amorphous solid (1.68 g, 55% yield); mp 85–87 °C; ¹H NMR (600 MHz, DMSO) δ 12.44 (s, 1H), 8.30 (d, J = 7.9 Hz, 1H), 8.30 (d, J = 7.9 Hz, 1H), 7.58–7.47 (m, 1H), 7.39 (d, J = 15.8 Hz, 1H), 6.99 (d, J = 8.8 Hz, 1H), 6.58 (d, J = 15.8 Hz, 1H), 4.35 (td, J = 8.9, 5.1 Hz, 1H), 3.79 (s, 2H), 2.42–2.22 (m, 1H), 2.11–1.99 (m, 1H), 1.83 (dtd, J = 14.7, 8.9, 6.0 Hz, 1H); MS m/z 308.3 [M + H]⁺.

N-[4-Methoxy-(E)-cinnamoyl]-L-aspartic acid b14, a white solid (1.81 g, 62% yield); mp 160–163 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.60 (s, 2H, –COOH̲), 8.34 (d, J = 8.0 Hz, 1H, –NH̲–), 7.53 (d, J = 8.7 Hz, 2H, Ph–H̲), 7.39 (d, J = 15.8 Hz, 1H, Ph–CH̲=CH–), 6.98 (d, J = 8.8 Hz, 2H, Ph–H̲), 6.60 (d, J = 15.8 Hz, 1H, Ph–CH=CH̲–), 4.66 (m, 1H, –NH–CH̲), 3.79 (s, 3H, –OCH̲₃), 2.75 (dd, J = 16.6, 5.4 Hz, 1H, –CH̲₂), 2.66 (dd, J = 16.6, 7.2 Hz, 1H, –CH̲₂); MS m/z 294.3 [M + H]⁺.

N-[4-Hydroxy-(E)-cinnamoyl]-L-glycine acid c1, a white solid (1.65 g, 75% yield); mp 232–233 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.49 (s, 1H, –COOH̲), 10.00 (s, 1H, –OH̲), 8.26 (t, J = 5.9 Hz, 1H, –NH̲), 7.41 (d, J = 8.6 Hz, 2H, Ph–H̲), 7.34 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 6.79 (d, J = 8.6 Hz, 2H, Ph–H̲), 6.50 (d, J = 15.8 Hz, 1H, Ph–CH=CH̲–), 3.86 (d, J = 5.9 Hz, 2H, –CH̲₂–); MS m/z 222.3 [M + H]⁺.

N-[4-Hydroxy-(E)-cinnamoyl]-L-alanine acid c2, a white solid (1.59, 68% yield); mp 205–206 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.55 (s, 1H, –COOH̲), 9.85 (s, 1H, –OH̲), 8.27 (d, J = 7.4 Hz, 1H, –NH̲), 7.39 (d, J = 8.6 Hz, 2H, Ph–H̲), 7.33 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 6.79 (d, J = 8.6 Hz, 2H, Ph–H̲), 6.48 (d, J = 15.8 Hz, 1H, Ph–CH=CH̲–), 4.31 (m, 1H, –CH̲–), 1.31 (d, J = 7.3 Hz, 3H, –CH̲₃); MS m/z 236.1 [M + H]⁺.

N-[4-Hydroxy-(E)-cinnamoyl]-L-leucine acid c3, a white solid (1.77 g, 64% yield); mp 182–184 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.54 (s, 1H, –COOH̲), 9.85 (s, 1H, –OH̲), 8.21 (d, J = 8.1 Hz, 1H, –NH̲), 7.39 (d, J = 8.6 Hz, 2H, Ph–H̲), 7.33 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 6.80 (d, J = 8.6 Hz, 2H, Ph–H̲), 6.49 (d, J = 15.8 Hz, 1H, Ph–CH=CH̲–), 4.34 (ddd, J = 9.8, 8.1, 5.2 Hz, 1H, –NH–CH̲–), 1.73–1.59 (m, 1H, –CH̲–), 1.62–1.44 (m, 2H, –CH̲₂), 0.91 (d, J = 6.6 Hz, 3H, –CH̲₃), 0.86 (d, J = 6.5 Hz, 3H, –CH̲₃); MS m/z 278.3 [M + H]⁺.

N-[4-Hydroxy-(E)-cinnamoyl]-L-isoleucine acid c4, a white amorphous solid (1.66 g, 60% yield); mp 108–110 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.61 (s, 1H, –COOH), 9.85 (s, 1H, –OH), 8.10 (d, J = 8.5 Hz, 1H, –NH), 7.39 (d, J = 8.6 Hz, 2H, Ph–H), 7.33 (d, J = 15.7 Hz, 1H, Ph–CH=CH–), 6.80 (d, J = 8.6 Hz, 2H, Ph–H), 6.63 (d, J = 15.7 Hz, 1H, Ph–CH=CH–), 4.31 (dd, J = 8.5, 6.0 Hz, 1H, NH–CH–), 1.90–1.75 (m, 1H, –CH–), 1.46–1.40 (m, 1H, –CH₂–), 1.25–1.15 (m, 1H, –CH₂–), 0.89 (d, J = 6.84, 3H, –CH₃) 0.86 (t, J = 7.38, 3H, –CH₃); MS m/z 278.2 [M + H]⁺.

N-[4-Hydroxy-(E)-cinnamoyl]-L-valine acid c5, a white amorphous solid (1.23 g, 47% yield); mp 108–107 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.61 (s, 1H, –COOH̲), 9.85 (s, 1H, –OH̲), 8.08 (d, J = 8.6 Hz, 1H, –NH̲), 7.40 (d, J = 8.6 Hz, 2H, Ph–H̲), 7.33 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 6.80 (d, J = 8.6 Hz, 2H, Ph–H̲), 6.65 (d, J = 15.7 Hz, 1H, Ph–CH=CH̲–), 4.28 (dd, J = 8.6, 5.7 Hz, 1H, –NH–CH̲–), 2.10 (dd, J = 12.8, 6.7 Hz, 1H, –CH̲–), 0.91 (d, J = 4.0 Hz, 3H, –CH̲₃), 0.90 (d, J = 3.9 Hz, 3H, –CH̲₃); MS m/z 264.2 [M + H]⁺.

N-[4-Hydroxy-(E)-cinnamoyl]-L-proline acid, a white solid (1.59 g, 61% yield); mp 235–236 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.61 (s, 1H, –COOH̲), 9.85 (s, 1H, –OH̲), 8.08 (d, J = 8.6 Hz, 1H, –NH̲), 7.39 (d, J = 8.6 Hz, 2H, Ph–H̲), 7.33 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 6.80 (d, J = 8.6 Hz, 2H, Ph–H̲), 6.64 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 4.28 (dd, J = 8.6, 5.7 Hz, 1H, –N–CH̲–), 2.09 (dd, J = 12.8, 6.7 Hz, 1H, –CH̲₂–), 0.91 (dd, J = 6.8, 4.0 Hz, 5H, –CH̲₂–); MS m/z 262.2 [M + H]⁺

N-[4-Hydroxy-(E)-cinnamoyl]-L-phenylalanine acid c7, a white solid (2.14, 69% yield); mp 235–236 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.77 (s, 1H, –COOH̲), 9.87 (s, 1H, –OH̲), 8.30 (d, J = 8.1 Hz, 1H, –NH̲), 7.38 (d, J = 8.6 Hz, 2H, Ph–H), 7.32–7.23 (m, 5H, Ph–CH̲=CH–, Ph–H), 7.21–7.18 (m, 1H, Ph–H), 6.80 (d, J = 8.6 Hz, 2H, Ph–H), 6.49 (d, J = 15.8 Hz, 1H, Ph–CH=CH̲–), 4.57 (ddd, J = 9.5, 8.3, 4.8 Hz, 1H, –CH–), 3.11 (dd, J = 13.9, 4.8 Hz, 1H, –CH̲₂–), 2.92 (dd, J = 13.9, 9.6 Hz, 1H, –CH₂–); MS m/z 312.3 [M + H]⁺.

N-[4-Hydroxy-(E)-cinnamoyl]-L-methionine acid c8, a white solid (1.68 g, 57% yield); mp 169–172 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.66 (s, 1H, –COOH̲), 9.86 (s, 1H, –OH̲), 8.28 (d, J = 7.9 Hz, 1H, –NH̲), 7.40 (d, J = 8.6 Hz, 2H, Ph–H̲), 7.34 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 6.80 (d, J = 8.6 Hz, 2H, Ph–H̲), 6.49 (d, J = 15.8 Hz, 1H, Ph–CH=CH̲–), 4.45–4.41 (m, 1H, –CH̲–), 2.54–2.50 (m, 2H, –CH̲₂–), 2.05 (s, 3H, –CH̲₃), 2.02–1.88 (m, 2H, –CH̲₂–); MS m/z 296.2 [M + H]⁺.

N-[4-Hydroxy-(E)-cinnamoyl]-L-tryptophane acid c9, a white solid (1.75 g, 50% yield); mp 171–173 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.68 (s, 1H, –COOH̲), 10.85 (d, J = 1.4 Hz, 1H, –OH̲), 9.85 (s, 1H, –NH̲), 8.25 (d, J = 7.9 Hz, 1H, –CO–NH̲–), 7.56 (d, J = 7.9 Hz, 1H, Ar-H), 7.38 (d, J = 8.6 Hz, 2H, Ph–H), 7.33 (d, J = 8.1 Hz, 1H, Ar-H), 7.30 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 7.16 (d, J = 2.2 Hz, 1H, Ar-H), 7.11–7.03 (m, 1H, Ar-H), 7.02–6.93 (m, 1H, Ar-H), 6.79 (d, J = 8.6 Hz, 2H, Ph–H̲), 6.52 (d, J = 15.7 Hz, 1H, Ph–CH=CH̲–), 4.62 (td, J = 8.3, 5.1 Hz, 1H, –CH–), 3.23 (dd, J = 14.7, 4.9 Hz, 1H, –CH̲₂–), 3.07 (dd, J = 14.7, 8.8 Hz, 1H, –CH̲₂–); MS m/z 351.2 [M + H]⁺.

N-[4-Hydroxy-(E)-cinnamoyl]-L-serine acid c10, a light yellow solid (1.12 g, 45% yield); mp 185–189 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.62 (s, 1H, –COOH̲), 9.85 (s, 1H, Ph–OH̲), 8.12 (d, J = 8.0 Hz, 1H, NH̲–), 7.40 (d, J = 8.6 Hz, 2H, Ph–H̲), 7.33 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 6.80 (d, J = 8.6 Hz, 2H, Ph–H̲), 6.64 (d, J = 15.7 Hz, 1H, Ph–CH=CH̲–), 5.04 (s, 1H, –OH̲), 4.41 (dt, J = 8.2, 4.7 Hz, 1H, –CH̲–), 3.75 (dd, J = 10.9, 5.1 Hz, 1H, –CH̲₂–), 3.66 (dd, J = 10.9, 4.2 Hz, 1H, –CH̲₂–); MS m/z 252.3 [M + H]⁺.

N-[4-Hydroxy-(E)-cinnamoyl]-L-glutamine acid c11, a white solid (1.54 g, 53% yield); mp 196–198 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.59 (s, 1H, –COOH̲), 9.89 (s, 1H, Ph–OH̲), 8.27 (d, J = 7.7 Hz, 1H, NH̲–), 7.39 (d, J = 8.6 Hz, 2H, Ph–H̲), 7.33 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH–), 7.31 (s, 1H, –NH̲₂), 6.79 (d, J = 8.1 Hz, 2H, Ph–H̲), 6.78 (s, 1H, –NH̲₂), 6.50 (d, J = 15.8 Hz, 1H, Ph–CH=CH̲–), 4.28 (td, J = 8.8, 5.0 Hz, 1H, –CH̲–), 2.16–2.13 (m, 2H, –CH̲₂–), 2.10–1.90 (m, 1H, –CH̲₂–), 1.79 (m, 1H, –CH̲₂–); MS m/z 293.2 [M + H]⁺.

N-[4-Hydroxy-(E)-cinnamoyl]-L-threonine acid c12, a white solid (1.08 g, 41% yield); mp 201–204 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.54 (s, 1H, –COOH̲), 9.84 (s, 1H, Ph–OH̲), 7.89 (d, J = 8.8 Hz, 1H, –NH̲), 7.41 (d, J = 8.6 Hz, 2H, Ph–H̲), 7.33 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH), 6.80 (d, J = 8.6 Hz, 2H, Ph–H̲), 6.75 (d, J = 15.7 Hz, 1H, Ph–CH=CH̲), 4.97 (s, 1H, –OH̲), 4.35 (dd, J = 8.8, 3.2 Hz, 1H, –CH), 4.18 (qd, J = 6.3, 3.3 Hz, 1H, –CH̲–), 1.08 (d, J = 6.4 Hz, 3H, –CH̲₃); MS m/z 266.2 [M + H]⁺.

N-[4-Hydroxy-(E)-cinnamoyl]-L-glutamate acid c13, a white amorphous solid (1.63 g, 56%); mp 201–204 °C; ¹H NMR (600 MHz, DMSO) δ 12.41 (s, 1H, –COOH̲), 9.86 (s, 1H, –OH̲), 8.24 (d, J = 7.9 Hz, 1H, –NH̲), 7.40 (d, J = 8.6 Hz, 1H, Ph–H̲), 7.34 (d, J = 15.7 Hz, 1H, Ph–CH̲=CH), 6.80 (d, J = 8.6 Hz, 1H, Ph–H̲), 6.49 (d, J = 15.8 Hz, 1H, Ph–CH=CH̲), 4.58–4.11 (m, 1H, –CH̲–), 2.37–2.24 (m, 2H, –CH₂–), 2.07–1.97 (m, 1H, –CH̲₂–), 1.82–1.184 (m, 1H, –CH̲₂–); MS m/z 294.3 [M + H]⁺.

N-[4-Hydroxy-(E)-cinnamoyl]-L-asparagic acid c14, a white solid (1.72 g, 62% yield); mp 214–216 °C; ¹H NMR (600 MHz, d₆-DMSO) δ 12.59 (s, 2H, –COOH̲), 9.87 (s, 1H, –OH̲), 8.30 (d, J = 8.0 Hz, 1H, –NH̲), 7.41 (d, J = 8.6 Hz, 2H, Ph–H), 7.34 (d, J = 15.7 Hz, 1H, –Ph–CH̲₂=CH₂), 6.79 (t, J = 5.6 Hz, 2H, Ph–H̲), 6.52 (d, J = 15.7 Hz, 1H, Ph–CH₂=CH̲₂–), 4.65 (td, J = 7.4, 5.6 Hz, 1H, –CH̲–), 2.74 (dd, J = 16.6, 5.5 Hz, 1H, –CH̲₂–), 2.65 (dd, J = 16.6, 7.2 Hz, 1H, –CH̲₂–); MS m/z 280.2 [M + H]⁺.

3.3. The inhibitory activity against α-glucosidase

The α-glucosidase inhibitory activities were evaluated using the reported method.²⁸ A 500 μL aqueous solution of sodium salt of the test sample was incubated at 37 °C for 15 min with 100 μL α-glucosidase (0.5 U mL⁻¹ in 100 mM solution of sodium phosphate buffer) in 500 μL phosphate buffer (pH 6.8). 0.5 mL of pNPG (2.5 mM) was then added and incubated for another 15 min. The reaction was terminated by the addition of 1 mL of a 1 M sodium carbonate solution. The inhibitory activities were quantified by measuring the absorbance at 405 nm. Acarbose was used as the positive control. The inhibition of the test sample was calculated according to the following equation:
where a (%) is the percentage of inhibition of the tested sample or acarbose, A_sample is the tested absorbance after reaction, A_background is the initial absorbance of the test sample. A_blank is the absorbance of enzyme solution with pNPG.

Lineweaver–Burk plot analysis was performed to determine the mode of inhibition of α-glucosidase. The reaction was carried out with increasing concentration of inhibitors and varying concentrations of pNPG as the substrate. All the data were analyzed using a computer program for nonlinear regression (OriginPro 8.0).

3.4. 3D QSAR model development and validation

The QSAR study was performed in SYBYL 7.3. In our study, 85% of the synthesized compounds and their pIC₅₀ (−log IC₅₀) was selected for training set to develop the 3D QSAR model and other 15% for test set to evaluate the model based on their molecular properties (see ESI Table S1‡). Prior to the form of the model, compound c1 was selected as the template molecule and optimized to derive the low energy conformation. The other molecules were optimized and superimposed based on the template using Database Alignment. Least-Squares Analysis (LSA) method was used to obtain the CoMFA model. The model was validated using the test set correlation and leave-one-out (LOO) cross validation.

3.5. Docking studies

The crystal 3D structure of α-glucosidase from S. cerevisiae was predicted by homology modelling. The amino acid sequence and the active sites were retrieved from the UniProt protein resource data bank with the accessing code P53341 (http://www.uniprot.org).²⁹ A similarity search and homology modelling were conducted in SWISS MODEL server (http://www.swissmodel.expasy.org). The crystallographic structure of S. cerevisiae isomaltase (PDB code 3AXH) was selected as the template with a 72.51% sequence identity with the target protein. The final structure generated from homology modelling was evaluated using PROCHECK. The docking study was run by Surflex-dock in SYBYL 7.3 and the results were visualized using PyMol.

4. Conclusions

The synthesized N-phenylpropenoyl-L-amino acids exhibited strong α-glucosidase inhibitory activities and some of them were even more active than the traditional anti-diabetic drug, acarbose, in the in vitro test. This study provided a proof of the hypoglycemic activities of cocoa beans and other related plant medicine. They may be developed further as a new type of potential food-based hypoglycemic agents. Considering the low toxicity of their natural property, these compounds have a bright future as medicinal lead compounds. However, in vivo studies of their anti-diabetic properties need to be performed.

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Footnotes

† The authors declare no competing interests.

‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra08330g

§ These authors contributed equally to this work.

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