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Article

Discovery of Cinnamic Acid Derivatives as Potent Anti-H. pylori Agents

by
Yonglian Li
1,2,
Kun Zhao
1,
Zhidi Wu
1,
Yujun Zheng
1,
Jialin Yu
3,
Sikun Wu
1,
Vincent Kam Wai Wong
4,
Min Chen
3,
Wenfeng Liu
3,* and
Suqing Zhao
1,*
1
Department of Pharmaceutical Engineering, School of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou 510006, China
2
School of Eco-Environment Technology, Guangdong Industry Polytechnic University, Guangzhou 510300, China
3
School of Pharmacy and Food Engineering, Wuyi University, Jiangmen 529020, China
4
Neher’s Biophysics Laboratory for Innovative Drug Discovery, State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau 999078, China
*
Authors to whom correspondence should be addressed.
Molecules 2024, 29(19), 4548; https://doi.org/10.3390/molecules29194548
Submission received: 9 August 2024 / Revised: 12 September 2024 / Accepted: 19 September 2024 / Published: 25 September 2024
(This article belongs to the Section Medicinal Chemistry)
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Abstract

:
Antibiotics are currently used for the treatment of Helicobacter pylori (H. pylori), which is confirmed to be the major cause of gastric disorders. However, the long-term consumption of antibiotics has already caused antibiotic resistance and side effects in vivo. Therefore, there is an emerging need for searching for safe and effective anti-H. pylori agents. Inspired by the excellent bioactivities of cinnamic acid, a series of cinnamic acid derivatives (compounds 1–30) were synthesized and determined for H. pylori inhibition. The initial screening revealed that compound 23, a 2,4-dinitro cinnamic acid derivative containing 4-methoxyphenol, showed excellent H. pylori inhibition with an MIC value of 4 μM. Further studies indicated that compound 23 showed anti-bacterial activity and had a bactericidal effect on H. pylori due to the destruction of the bacterial structure. Molecular docking analysis revealed that the 2,4-dinitro groups in cinnamic acid moiety formed hydrogen bonding with amino acid residues in an active pocket of H. pylori protein. Interestingly, the ester moiety fitted into the hydrophobic pocket, attaining additional stability to compound 23. Above all, the present study reveals that compound 23 could be considered a promising anti-H. pylori agent to treat H. pylori causing gastritis.

1. Introduction

Helicobacter pylori (H. pylori), colonized in the gastric mucosa of humans, is a Gram-negative bacillary spiral-shaped bacterium [1]. It is revealed that H. pylori was closely associated with a series of stomach-related diseases, such as chronic gastritis and gastric ulcer [2,3]. Notably, H. pylori has been officially categorized as a class-I carcinogen, which is associated with gastric cancer risk [4]. Recent studies demonstrate that eradication of H. pylori could reduce the risk of gastric cancer and improve the healing of peptic ulcers [5,6]. Currently, non-antibiotic drugs combined with one/two antibiotics and antibiotic-based therapies are used for the contemporary therapeutic strategies to treat H. pylori infection [7]. In addition, antibiotic-based therapies are primarily used to treat H. pylori infection, including standard triple therapy and quadruple therapy [8]. However, the increasing antibiotic resistance in H. pylori is receiving considerable attention in antibiotic-based therapy [9]. Additionally, the overuse of antibiotics for H. pylori infection has caused side effects, such as nausea, constipation, diarrhea and appetite disorder [10]. These disadvantages are the key challenges for treating H. pylori infection. Herein, developing novel strategies are urgently needed to prevent and treat H. pylori infection.
Since antibiotics are becoming less effective, natural phytochemicals that have antimicrobial activity are attractive candidates [11,12,13]. Several natural phytochemicals are reported to exhibit fewer side effects, low toxicity and good anti-bacterial activity, indicating that they can serve as potent anti-bacterial drugs [14]. Therefore, discovering anti-bacterial drugs from nature-based compounds is an important strategy for the treatment of H. pylori infection [15]. Cinnamon, obtained from the inner bark of several trees from the genus Cinnamomum, is widely used as a tropical Asian spice in daily diet [16]. It possesses anti-bacterial, anti-diabetic, anti-inflammatory and anticancer activities [17,18,19,20]. Cinnamic acid, a secondary metabolite of Cinnamon, is responsible for its anti-bacterial activity [21]. Furthermore, it has low toxicity [22,23]. In recent years, it has been recognized that cinnamic acid and its derivatives have significantly contributed to anti-H. pylori infection [24,25,26]. It is reported that a series of N-substituted cinnamamides were synthesized and screened for anti-H. pylori activity. The results showed that three 4-chloro derivatives exhibited excellent anti-H. pylori activities with minimum inhibitory concentration (MIC) values in the range of 7.5–10 μg/mL, suggesting that they were the most promising compounds to treat H. pylori infection. (Figure 1 (1a–1c)) [24]. Prenyloxy cinnamic acid derivatives, found in Boronia pinnata (Fam. Rutaceae), were determined for the growth inhibition of H. pylori. Furthermore, compound 1d had significant inhibition against H. pylori (MIC value of 1.62 mg/mL) [25]. It is revealed that cinnamic acid derivatives were synthesized and tested for anti-H. pylori activity. The obtained MIC values for compounds 1e and 1f were 250 μg/mL, which were four times less than that of caffeic acid [26]. Nevertheless, these observations indicate that there was no systemic research in cinnamic acid-based anti-H. pylori agents, especially for the anti-H. pylori mechanism. Thus, it is strongly needed to develop novel anti-H. pylori agents and exploring the mechanism of action.
Aiming to obtain novel cinnamic acid-based compounds with inhibitory effects on H. pylori, a series of cinnamic acid derivatives are synthesized and screened for their anti-H. pylori activity. Moreover, the MIC/minimum bactericidal concentration (MBC) and kinetic studies of the most potent compound are explored. The present study can accelerate follow-up research on cinnamic acid derivatives as a potential therapy in the treatment of H. pylori infection.

2. Results and Discussion

2.1. Chemistry

It is indicated that cinnamic acid derivatives had an inhibitory effect on H. pylori [23]. In addition, phenol esters were confirmed to be potent H. pylori inhibitors [27]. Herein, compounds having both these structural features might possess anti-H. pylori activity more efficiently than that of cinnamic acid or phenol esters alone. With this hypothesis, cinnamic acid derivatives containing 4-methoxyphenol were designed for synthesis.
As shown in Scheme 1, various cinnamic acids and 4-methoxyphenol were taken as the starting materials and then esterified into their aralkylacrylate in the presence of 4-dimethylaminopyridine (DMAP) as a catalyst. Firstly, the ester formation was monitored by thin-layer chromatographic (TLC) analysis. Secondly, the chemical structures of compounds 1–30 were confirmed by nuclear magnetic resonance (NMR) spectroscopy.
In all 1H-NMR spectrums, the olefinic protons appeared as a doublet with coupling constant J = 15.7–16.2 Hz. The results confirmed that the double bond in compound 1–30 was a trans double bond. In addition, the CH3O proton signals were detected as doublets with the range of 3.94–3.81 ppm. In all 13C-NMR spectrum, the O=C-O signals were observed between 166.75 and 163.80 ppm. The CH3O signals were detected as singlets between 55.64 and 55.61 ppm in the 13C-NMR spectrum for all compounds.

2.2. Inhibitory Effect of All Compounds against H. pylori

As presented in Table 1, the results indicated that twenty-four compounds that had different O-substituted ester moieties exhibited more potent inhibition than that of cinnamic acid at 32 μM (57.30%). The initial screening revealed that seven compounds (compounds 2, 16, 19, 21, 23, 24 and 25) showed the most H. pylori inhibitory activity at 32 μM (up to 90% inhibition). Additionally, compounds 2 and 5, where phenyl was ortho-substituted with CH3 and OCH3, had higher inhibition compared to compounds 3, 4, 6 and 7 with CH3 and OCH3 at meta or para position, respectively. The inhibitory effect of 8, 11, 14, 17 and 20, where phenyl was p-substituted with F, Cl, Br, CF3 and NO2, was weaker than that of compounds at meta or para position. Interestingly, the change in the benzyl group into naphthalene caused a loss of anti-H. pylori activity. The compounds (24, 25, 28 and 29) possessing heterocyclic group showed more potency than that of compounds 26 and 27, which had benzene rings, indicating that O, S and N atoms in aromatic rings enhanced H. pylori inhibition.
According to the MIC values, cinnamic acid alone did not have any effect on H. pylori activity. Twenty-nine compounds exhibited significant inhibition with an MIC in the range of 4 μM to 64 μM, indicating that they were more potent than cinnamic acid (MIC > 64 μM). Compound 23 with NO2 at ortho and para position and compound 25 with a quinoline ring exhibited profound inhibition against H. pylori. Importantly, compound 25 presented MIC values (8 μM) comparable to metronidazole (8 μM) against H. pylori. Compound 23 showed higher H. pylori inhibitory activity with a MIC value of 4 μM compared to metronidazole (8 μM).
Taken together, it is concluded that the increased hydrophobicity of esters might lead to a major structural modification of the in vitro anti-H. pylori activity. Importantly, compounds possessing electron-donating groups (CH3 and OCH3) at ortho-position enhanced the anti-H. pylori activity. Conversely, compounds possessing electron-withdrawing groups (F, Cl, Br, CF3 and NO2) at ortho-position attenuated the anti-H. pylori activity. Considering these results, the promising compound 23 with a MIC value of 4 μM was selected for the experiments.

2.3. MIC and MBC

In order to investigate the anti-bacterial activity of compound 23 against H. pylori, the microbroth dilution method for the determination of its MIC and MBC was used in the present study. As shown in Table 2, the MIC value of compound 23 against H. pylori was 4. The obtained results indicated that the MBC value of compound 23 against H. pylori was 8 μM. Importantly, the MBC/MIC ratio of compound 23 was 2, indicating that it showed bacteriostatic and bactericidal activity against H. pylori.

2.4. Inhibiting Kinetics and Killing Kinetics of Compound 23 against H. pylori

As exhibited in Figure 2A,B, the optical density at 600 nm (OD600) of H. pylori solution was significantly increased after 60 h without drug treatment. Compound 23 dose- and time-dependently inhibited and killed the H. pylori. Moreover, compound 23 showed potent in vitro inhibition of H. pylori growth at a concentration as low as 2 μM (1/2 MIC). Compound 23 completely killed H. pylori at 8–32 μM (2 MIC to 8 MIC) after 12–36 h, which suggested a dramatic decrease in the number of bacteria in comparison to the initial inoculation (Figure 2B). These data revealed that compound 23 showed anti-bacterial activity and had a bactericidal effect on H. pylori, indicating that they may serve as potential anti-H. pylori agents.

2.5. Effect of Compound 23 on the Morphology of H. pylori

To explore the effect of compound 23 at the concentration of MIC on H. pylori ultrastructure, the samples were tested via scanning electron microscopy (SEM). As described in Figure 3A,B, the H. pylori in the control group showed a smooth, uniform and curved rod-like shape biofilm structure. After treatment with compound 23, the biofilm structure of H. pylori was damaged and shrunk in the compound 23 treated group (Figure 3C,D). They could alter the morphology of H. pylori. These results indicated that compound 23 could destroy the structure of H. pylori to cause the loss of H. pylori activity.

2.6. Molecular Docking Analysis

To unravel the molecular interactions of compound 23 with H. pylori, molecular docking was used to infer the binding capacity of compound 23 to H. pylori (PDB code: 4TSD). It was indicated that HP1029 belonged to the DUF386 family, which was implicated in the bacterial biofilm formation. Additionally, metronidazole, which had an influence on bacterial biofilm formation, has been currently used to treat H. pylori infection. Therefore, metronidazole was used as a positive control. The results indicated that docking and fitting (metronidazole and compound 23) calculated a binding energy value of −7.9 and −5.2 kcal/mol, respectively. The root mean square distance (RMSD) values of metronidazole and compound 23 between the docking pose and the binding configuration in the crystallographic model were 2.120 and 0.625, respectively. It was revealed that compound 23 had a higher affinity for the H. pylori protein than that of metronidazole. In addition, the docking program predicted a good pose of metronidazole and compound 23 in the binding pocket. These findings further assisted in screening cinnamic acid derivatives for lead identification and optimization.
As presented in Figure 4A,B, the result indicated that metronidazole and compound 23 were fitted well into the binding pocket of the H. pylori protein, respectively. Furthermore, the nitrogen of the nitro group in metronidazole built hydrogen bonds with LYS-168, GLU-79 and HIS-81. The hydroxyl moiety formed hydrogen bonding with HIS145 and LEU-147. The nitrogen of the imidazole ring built hydrogen bonds with GLU-63 and GLN-88. It is demonstrated that the interactions of metronidazole with amino-acid residues could result in the inhibition of the active sites in H. pylori. Figure 4B indicated that the nitro group of compound 23 formed hydrogen bonding with LYS-168, HIS-81, HIS145, GLN-88, TYR-65, LYS-164 and ARG-148. Interestingly, the phenyl ring and ester moiety fitted snugly into the hydrophobic pocket, attaining additional stability to compound 23 in the active pocket. Taken together, it is suggested that the good binding affinity of compound 23 with the active site residues would enhance its anti-H. pylori activity.

3. Materials and Methods

3.1. Materials

Cinnamic acid, p-methoxyphenol, metronidazole and dimethyl sulfoxide (DMSO) were obtained from Aladin Scientific Corp. (Shanghai, China). Cinnamic acids were synthesized in our laboratory. The melting points of all compounds were measured on a micro melting point apparatus (Shanghai, China). 1H and 13C NMR spectra of all compounds were measured on a Bruker 500 NMR spectrometer (Bruker, MA, USA). High-resolution EI mass spectra were recorded on a Thermo Scientific liquid chromatography–mass spectrometer (LC-MS, LCQTM, Thermo Scientific, Waltham, MA, USA) with an ESI source. The H. pylori ATCC SS1 strain was obtained from Professor Ye Chen’s laboratory at Southern Medical University. Columbia agar base and brain heart infusion (BHI) were acquired from HuanKai Microbial Ltd., (Guangzhou, Guangdong, China). Fetal bovine serum (FBS) was purchased from TianHang Biotech (Hangzhou, Zhejiang, China). Sterile-defibrinated sheep blood was obtained from Ruite Biotechnology Co., Ltd., (Guangzhou, Guangdong, China). Unless otherwise indicated, all solvents, reagents and the other materials were directly used as obtained from the indicated commercial sources without further purification.

3.2. General Procedure for the Preparation of Compound 1–30

The procedure for the preparation of compound 1–30 is as follows: a mixture of cinnamic acids (1.0 mmol), p-methoxyphenol (1.0 mol), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI, 1.2 mmol)) and DMAP (0.1 mmol) was first dissolved in CH2Cl2. The reaction was carried out by continuous stirring at room temperature for 1 h. TLC was used to monitor these reactions. After completion of the chemical reaction, the mixture was washed twice with saturated NaHCO3 solution and water. Subsequently, the organic phase was collected, dried on anhydrous Na2SO4 and concentrated under vacuum. Finally, purification through column chromatography afforded the target compound 1–30.
4-methoxyphenyl cinnamate (1): White solid, 76% yield, mp 125.3–126.0 °C; 1H NMR (500 MHz, CDCl3) δ 7.87 (d, J = 16.0 Hz, 1H, Ph-H), 7.62–7.56 (m, 2H, Ph-H), 7.46–7.40 (m, 3H, 3 × Ph-H), 7.12–7.06 (m, 2H, 2 × Ph-H), 6.96–6.91 (m, 2H, Ph-H and -CH=C-), 6.63 (d, J = 16.0 Hz, 1H, -CH=C-), 3.82 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 165.84 (-C=O), 157.26 (Ph-C-OCH3), 146.43 (-CH=CH), 144.29 (Ph-C-O), 134.24 (Ph-C-CH), 130.69 (Ph-CH), 129.02 (Ph-CH), 128.31 (Ph-CH), 122.41 (Ph-CH), 117.38 (=CH-C=O), 114.51 (Ph-CH), 55.63 (-O-CH3). HRMS m/z: 255.0943 [M + H]+, found 255.0957.
4-methoxyphenyl (E)-3-(o-tolyl)acrylate (2): White solid, 85% yield, mp 108.9–109.2 °C; 1H NMR (500 MHz, CDCl3) δ 8.18 (d, J = 15.9 Hz, 1H, Ph-H), 7.66 (dd, J = 7.7, 1.4 Hz, 1H, Ph-H), 7.34 (td, J = 7.4, 1.4 Hz, 1H, Ph-H), 7.27 (dd, J = 7.6, 3.7 Hz, 2H, 2 × Ph-H), 7.15–6.93 (m, 4H, 3 × Ph-H and -CH=C-), 6.58 (d, J = 15.9 Hz, 1H, -CH=C-), 3.85 (s, 3H, -OCH3), 2.51 (s, 3H, -CH3). 13C NMR (126 MHz, CDCl3) δ 165.89 (-C=O), 157.25 (Ph-C-OCH3), 144.33 (-CH=CH), 144.08 (Ph-C-O), 137.96 (Ph-C-CH3), 133.19 (Ph-C-CH), 130.93 (Ph-CH), 130.42 (Ph-CH), 126.59 (Ph-CH), 126.46 (Ph-CH), 122.42 (Ph-CH), 118.36 (=CH-C=O), 114.48 (Ph-CH), 55.62 (-O-CH3), 19.87 (-CH3). HRMS m/z: 269.1099 [M + H]+, found 269.1107.
4-methoxyphenyl (E)-3-(m-tolyl)acrylate (3): White solid, 94% yield, mp 144.0–145.1 °C; 1H NMR (500 MHz, CDCl3) δ 7.86 (d, J = 15.9 Hz, 1H, Ph-H), 7.42 (dd, J = 6.6, 1.5 Hz, 2H, 2 × Ph-H), 7.37–7.31 (m, 1H, Ph-H), 7.27 (d, J = 7.6 Hz, 1H, Ph-H), 7.15–7.10 (m, 2H, 2 × Ph-H), 6.97–6.93 (m, 2H, Ph-H and -CH=C), 6.64 (d, J = 16.0 Hz, 1H, -CH=C), 3.84 (s, 3H, -OCH3), 2.42 (s, 3H, -CH3). 13C NMR (126 MHz, CDCl3) δ 165.90 (-C=O), 157.24 (Ph-C-OCH3), 146.62 (Ph-C-O), 144.32 (-CH=CH), 138.69 (Ph-C-CH3), 134.19 (Ph-C-CH), 131.53 (Ph-CH), 128.98 (Ph-CH), 128.91 (Ph-CH), 125.50 (Ph-CH), 121.43 (Ph-CH), 117.12 (=CH-C=O), 114.49 (Ph-CH), 55.62 (-O-CH3), 21.36 (-CH3). HRMS m/z: 269.1099 [M + H]+, found 269.1107.
4-methoxyphenyl (E)-3-(p-tolyl)acrylate (4): White solid, 78% yield, mp 156.2–157.0 °C; 1H NMR (500 MHz, CDCl3) δ 7.84 (d, J = 16.0 Hz, 1H, Ph-H), 7.49 (d, J = 8.1 Hz, 2H, 2 × Ph-H), 7.23 (d, J = 8.0 Hz, 2H, 2 × Ph-H), 7.12–7.06 (m, 2H, 2 × Ph-H), 6.96–6.88 (m, 2H, Ph-H and -CH=C-), 6.59 (d, J = 16.0 Hz, 1H, -CH=C-), 3.82 (s, 3H, -OCH3), 2.40 (s, 3H, -CH3). 13C NMR (126 MHz, CDCl3) δ 166.05 (-C=O), 157.22 (Ph-C-OCH3), 146.47 (Ph-C-O), 144.35 (-CH=CH), 141.21 (Ph-C-CH3), 131.52 (Ph-C-CH), 129.76 (Ph-CH), 128.33 (=CH-C=O), 122.44 (Ph-CH), 116.23 (Ph-CH), 114.49 (Ph-CH), 55.62 (-O-CH3), 21.56 (-CH3). HRMS m/z: 269.1099 [M + H]+, found 269.1104.
4-methoxyphenyl (E)-3-(2-methoxyphenyl)acrylate (5): White solid, 82% yield, mp 114.7–115.2 °C; 1H NMR (500 MHz, CDCl3) δ 8.19 (d, J = 16.1 Hz, 1H, Ph-H), 7.59 (dd, J = 7.8, 1.7 Hz, 1H, Ph-H), 7.41 (ddd, J = 8.5, 7.4, 1.7 Hz, 1H, Ph-H), 7.15–7.09 (m, 2H, 2 × Ph-H), 7.02 (t, J = 7.5 Hz, 1H, Ph-H), 6.99–6.92 (m, 3H, 2 × Ph-H and -CH=C-), 6.75 (d, J = 16.1 Hz, 1H, -CH=C-), 3.94 (s, 3H, -OCH3), 3.84 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 166.36 (-C=O), 158.57 (Ph-C-OCH3), 157.16 (Ph-C-OCH3), 144.44 (-CH=CH), 141.92 (Ph-C-O), 131.90 (Ph-C-CH), 129.33 (Ph-CH), 123.93 (Ph-CH), 122.48 (Ph-CH), 120.7 (Ph-CH), 117.89 (=CH-C=O), 114.45 (Ph-CH), 111.22 (Ph-CH), 55.61 (-O-CH3), 55.52 (-O-CH3). HRMS m/z: 285.1049 [M + H]+, found 285.1057.
4-methoxyphenyl (E)-3-(3-methoxyphenyl)acrylate (6): White solid, 56% yield, mp 120.5–121.3 °C; 1H NMR (500 MHz, CDCl3) δ 7.85 (d, J = 15.9 Hz, 1H, Ph-H), 7.36 (t, J = 7.9 Hz, 1H, Ph-H), 7.23–7.18 (m, 1H, Ph-H), 7.14–7.09 (m, 3H, 3 × Ph-H), 7.00 (dd, J = 8.3, 2.5 Hz, 1H, Ph-H), 6.97–6.92 (m, 2H, Ph-H and -CH=C-), 6.64 (d, J = 15.9 Hz, 1H, -CH=C-), 3.87 (s, 3H, -OCH3), 3.84 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 165.77 (-C=O), 159.96 (Ph-C-OCH3), 157.26 (Ph-C-OCH3), 146.34 (Ph-C-O), 144.27 (-CH=CH), 135.58 (Ph-C-CH), 130.02 (Ph-CH), 122.40 (Ph-CH), 121.00 (Ph-CH), 117.66 (=CH-C=O), 116.57 (Ph-CH), 114.50 (Ph-CH), 113.10 (Ph-CH), 55.61 (-O-CH3), 55.34 (-O-CH3). HRMS m/z: 285.1049 [M + H]+, found 285.1055.
4-methoxyphenyl (E)-3-(4-methoxyphenyl)acrylate (7): White solid, 97% yield, mp 108.3–109.6 °C; 1H NMR (500 MHz, CDCl3) δ 7.82 (d, J = 15.9 Hz, 1H, Ph-H), 7.54 (d, J = 8.7 Hz, 2H, 2 × Ph-H), 7.11–7.05 (m, 2H, 2 × Ph-H), 6.92 (dd, J = 12.1, 5.3 Hz, 4H, 3 × Ph-H and -CH=C-), 6.49 (d, J = 15.9 Hz, 1H, -CH=C-), 3.85 (s, 3H, -OCH3), 3.81 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 166.18 (-C=O), 161.71 (Ph-C-OCH3), 157.18 (Ph-C-OCH3), 146.11 (Ph-C-O), 144.39 (-CH=CH), 130.03 (Ph-C-CH), 126.97 (Ph-CH), 122.46 (Ph-CH), 114.73 (=CH-C=O), 114.46 (Ph-CH), 55.62 (-O-CH3), 55.44 (-O-CH3). HRMS m/z: 285.1049 [M + H]+, found 285.1057.
4-methoxyphenyl (E)-3-(2-fluorophenyl)acrylate (8): White solid, 62% yield, mp 140.2–141.2 °C; 1H NMR (500 MHz, CDCl3) δ 8.01 (d, J = 16.2 Hz, 1H, Ph-H), 7.62 (td, J = 7.6, 1.7 Hz, 1H, Ph-H), 7.42 (tdd, J = 7.4, 5.1, 1.7 Hz, 1H, Ph-H), 7.22 (td, J = 7.6, 1.1 Hz, 1H, Ph-H), 7.18–7.10 (m, 3H, 3 × Ph-H), 6.97–6.92 (m, 2H, Ph-H and -CH=C-), 6.76 (d, J = 16.2 Hz, 1H, -CH=C-), 3.84 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 165.67 (-C=O), 162.51 (Ph-CF), 160.49 (Ph-C-OCH3), 157.28 (Ph-C-O), 144.25 (-CH=CH), 139.02 (d, J = 2.6 Hz) (Ph-C-CF), 132.12 (d, J = 8.6 Hz) (Ph-C-CH), 129.33 (d, J = 2.7 Hz) (Ph-CH), 124.58 (d, J = 3.5 Hz) (Ph-CH), 122.33 (d, J = 11.4 Hz) (Ph-CH), 120.00 (d, J = 7.0 Hz) (Ph-CH), 116.41 (=CH-C=O), 116.24 (Ph-CH), 114.50 (Ph-CH), 55.61 (-O-CH3). HRMS m/z: 273.0849 [M + H]+, found 273.0861.
4-methoxyphenyl (E)-3-(3-fluorophenyl)acrylate (9): White solid, 67% yield, mp 153.9–154.4 °C; 1H NMR (500 MHz, CDCl3) δ 8.01 (d, J = 16.2 Hz, 1H, Ph-H), 7.62 (td, J = 7.6, 1.7 Hz, 1H, Ph-H), 7.42 (tdd, J = 7.5, 5.1, 1.7 Hz, 1H, Ph-H), 7.22 (td, J = 7.7, 1.1 Hz, 1H, Ph-H), 7.19–7.09 (m, 3H, 3 × Ph-H), 6.97–6.92 (m, 2H, Ph-H and -CH=C-), 6.75 (d, J = 16.2 Hz, 1H, -CH=C-), 3.84 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 165.66 (-C=O), 162.51 (Ph-C-OCH3), 160.49 (Ph-C-O), 157.28 (Ph-CF), 144.25 (-CH=CH), 139.02 (d, J = 2.6 Hz) (Ph-C-CF), 132.11 (d, J = 8.9 Hz) (Ph-C-CH), 129.32 (d, J = 2.7 Hz) (Ph-CH), 124.57 (d, J = 3.6 Hz) (Ph-CH), 122.37 (Ph-CH), 120.00 (d, J = 7.0 Hz) (Ph-CH), 116.41 (=CH-C=O), 116.24 (Ph-CH), 114.50 (Ph-CH), 55.62 (-O-CH3). HRMS m/z: 273.0849 [M + H]+, found 273.0859.
4-methoxyphenyl (E)-3-(4-fluorophenyl)acrylate (10): White solid, 84% yield, mp 120.5–122.0 °C; 1H NMR (500 MHz, CDCl3) δ 7.82 (d, J = 16.0 Hz, 1H, Ph-H), 7.60–7.55 (m, 2H, 2 × Ph-H), 7.15–7.05 (m, 4H, 4×Ph-H), 6.95–6.90 (m, 2H, Ph-H and -CH=C-), 6.55 (d, J = 16.0 Hz, 1H, -CH=C-), 3.82 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 165.75 (-C=O), 165.14 (Ph-C-OCH3), 163.14 (Ph-C-O), 157.28 (Ph-CF), 145.11 (Ph-CH), 144.23 (-CH=CH), 130.52 (Ph-C-CF), 130.47 (Ph-C-CH), 130.26 (Ph-CH), 130.21 (Ph-CH), 122.38 (Ph-CH), 117.10 (=CH-C=O), 116.29 (Ph-CH), 116.12 (Ph-CH), 116.03 (Ph-CH), 114.79 (Ph-CH), 114.51 (Ph-CH), 55.77, 55.62 (-O-CH3). HRMS m/z: 273.0849 [M + H]+, found 273.0859.
4-methoxyphenyl (E)-3-(2-chlorophenyl)acrylate (11): White solid, 74% yield, mp 132.5–132.8 °C; 1H NMR (500 MHz, CDCl3) δ 8.30 (d, J = 16.0 Hz, 1H, Ph-H), 7.71 (dd, J = 7.4, 2.0 Hz, 1H, Ph-H), 7.47 (dd, J = 7.7, 1.6 Hz, 1H, Ph-H), 7.36 (dtd, J = 16.1, 7.3, 1.7 Hz, 2H, 2 × Ph-H), 7.15–7.10 (m, 2H, 2 × Ph-H), 6.98–6.92 (m, 2H, Ph-H and -CH=C-), 6.64 (d, J = 16.0 Hz, 1H, -CH=C-), 3.84 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 165.31 (-C=O), 157.30 (Ph-C-OCH3), 144.23 (-CH=CH), 142.10 (Ph-C-O), 135.17 (Ph-C-Cl), 132.51 (Ph-C-CH), 131.41 (Ph-CH), 130.30 (Ph-CH), 127.81 (Ph-CH), 127.20 (Ph-CH), 122.37 (Ph-CH), 120.06 (Ph-CH), 114.50 (=CH-C=O), 55.62 (-O-CH3). HRMS m/z: 289.0553 [M + H]+, found 289.0560.
4-methoxyphenyl (E)-3-(3-chlorophenyl)acrylate (12): White solid, 70% yield, mp 135.8–136.2 °C; 1H NMR (500 MHz, CDCl3) δ 7.81 (d, J = 16.0 Hz, 1H, Ph-H), 7.59 (t, J = 1.9 Hz, 1H, Ph-H), 7.47 (dt, J = 7.4, 1.6 Hz, 1H, Ph-H), 7.43–7.35 (m, 2H, 2 × Ph-H), 7.14–7.09 (m, 2H, 2 × Ph-H), 6.97–6.92 (m, 2H, Ph-H and -CH=C-), 6.64 (d, J = 16.0 Hz, 1H, -CH=C-), 3.84 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 165.40 (-C=O), 157.32 (Ph-C-OCH3), 144.73 (-CH=CH), 144.17 (Ph-C-O), 136.02 (Ph-C-Cl), 135.04 (Ph-C-CH), 130.52 (Ph-CH), 130.26 (Ph-CH), 128.01 (Ph-CH), 126.45 (Ph-CH), 122.33 (Ph-CH), 118.88 (=CH-C=O), 114.51 (Ph-CH), 55.62 (-O-CH3). HRMS m/z: 289.0553 [M + H]+, found 289.0561.
4-methoxyphenyl (E)-3-(4-chlorophenyl)acrylate (13): White solid, 81% yield, mp 120.1–121.7 °C; 1H NMR (500 MHz, CDCl3) δ 7.80 (d, J = 16.0 Hz, 1H, Ph-H), 7.51 (d, J = 8.5 Hz, 2H, 2 × Ph-H), 7.39 (d, J = 8.5 Hz, 2H, 2 × Ph-H), 7.11–7.05 (m, 2H, 2 × Ph-H), 6.95–6.89 (m, 2H, Ph-H and -CH=C-), 6.59 (d, J = 16.0 Hz, 1H, -CH=C-), 3.81 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 165.63 (-C=O), 157.29 (Ph-C-OCH3), 144.96 (-CH=CH), 144.17 (Ph-C-O), 136.62 (Ph-C-Cl), 132.69 (Ph-C-CH), 129.39 (Ph-CH), 122.37 (Ph-CH), 117.92 (=CH-C=O), 114.51 (Ph-CH), 55.63 (-O-CH3). HRMS m/z: 289.0553 [M + H]+, found 289.0560.
4-methoxyphenyl (E)-3-(2-bromophenyl)acrylate (14): White solid, 76% yield, mp 114.0–114.2 °C; 1H NMR (500 MHz, CDCl3) δ 7.82–7.73 (m, 2H, 2 × Ph-H), 7.60–7.49 (m, 2H, 2 × Ph-H), 7.32 (t, J = 7.9 Hz, 1H, 2 × Ph-H), 7.14–7.08 (m, 2H, 2 × Ph-H), 6.98–6.91 (m, 2H, Ph-H and -CH=C-), 6.64 (d, J = 16.0 Hz, 1H, -CH=C-), 3.84 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 165.37 (-C=O), 157.32 (Ph-C-OCH3), 144.63 (-CH=CH), 144.17 (Ph-C-O), 136.29 (Ph-C-Br), 133.42 (Ph-C-CH), 130.96 (Ph-CH), 130.51 (Ph-CH), 126.87 (Ph-CH), 123.75 (Ph-CH), 122.33 (Ph-CH), 118.90 (=CH-C=O), 114.51 (Ph-CH), 55.63 (-O-CH3). HRMS m/z: 333.0048 [M + H]+, found 333.0054.
4-methoxyphenyl (E)-3-(3-bromophenyl)acrylate (15): White solid, 74% yield, mp 121.6–122.3 °C; 1H NMR (500 MHz, CDCl3) δ 8.25 (d, J = 15.9 Hz, 1H, Ph-H), 7.68 (ddd, J = 16.7, 7.9, 1.5 Hz, 2H, 2 × Ph-H), 7.42–7.36 (m, 1H, Ph-H), 7.30 (d, J = 1.7 Hz, 1H, Ph-H), 7.27 (d, J = 1.6 Hz, 1H, Ph-H), 7.15–7.11 (m, 2H, 2 × Ph-H), 6.97–6.93 (m, 2H, Ph-H and -CH=C-), 6.60 (d, J = 15.9 Hz, 1H, -CH=C-), 3.84 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 165.22 (-C=O), 157.30 (Ph-C-OCH3), 144.66 (-CH=CH), 144.23 (Ph-C-O), 134.30 (Ph-C-Br), 133.57 (Ph-C-CH), 131.57 (Ph-CH), 127.94 (Ph-CH), 127.83 (Ph-CH), 125.54 (Ph-CH), 122.37 (Ph-CH), 120.24 (Ph-CH), 114.49 (=CH-C=O), 55.63 (-O-CH3). HRMS m/z: 333.0048 [M + H]+, found 333.0053.
4-methoxyphenyl (E)-3-(4-bromophenyl)acrylate (16): White solid, 72% yield, mp 148.7–149.5 °C; 1H NMR (500 MHz, CDCl3) δ 7.81 (d, J = 16.0 Hz, 1H, Ph-H), 7.60–7.56 (m, 2H, 2 × Ph-H), 7.49–7.44 (m, 2H, 2 × Ph-H), 7.14–7.07 (m, 2H, 2 × Ph-H), 6.98–6.92 (m, 2H, Ph-H and -CH=C-), 6.63 (d, J = 16.0 Hz, 1H, -CH=C-), 3.84 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 165.57 (-C=O), 157.30 (Ph-C-OCH3), 144.99 (-CH=CH), 144.20 (Ph-C-O), 133.13 (Ph-C-Br), 132.28 (Ph-C-CH), 129.65 (Ph-CH), 125.00 (Ph-CH), 122.35 (Ph-CH), 118.07 (=CH-C=O), 114.51 (Ph-CH), 55.62 (-O-CH3). HRMS m/z: 333.0048 [M + H]+, found 333.0051.
4-methoxyphenyl (E)-3-(2-(trifluoromethyl)phenyl)acrylate (17): White solid, 74% yield, mp 102.6–103.5 °C; 1H NMR (500 MHz, CDCl3) δ 8.26 (dq, J = 15.7, 2.2 Hz, 1H, Ph-H), 7.83–7.74 (m, 2H, 2 × Ph-H), 7.63 (t, J = 7.6 Hz, 1H, Ph-H), 7.54 (t, J = 7.7 Hz, 1H, Ph-H), 7.16–7.11 (m, 2H, 2 × Ph-H), 6.98–6.92 (m, 2H, Ph-H and -CH=C-), 6.62 (d, J = 15.7 Hz, 1H, -CH=C-), 3.84 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 164.90 (-C=O), 157.34 (Ph-C-OCH3), 144.17 (-CH=CH), 141.77 (Ph-C-O), 133.14 (Ph-C-CH), 132.21 (Ph-CH), 129.94 (Ph-CH), 129.15 (Ph-CH), 128.90 (Ph-CH), 128.04 (Ph-CH), 126.28 (d, J = 5.5 Hz) (Ph-C-CF3), 125.00 (Ph-CH), 122.82 (Ph-CF3), 122.33 (Ph-CH), 121.77 (Ph-CH), 114.49 (=CH-C=O), 55.62 (-O-CH3). HRMS m/z: 323.0817 [M + H]+, found 323.0824.
4-methoxyphenyl (E)-3-(3-(trifluoromethyl)phenyl)acrylate (18): White solid, 82% yield, mp 110.3–111.9 °C; 1H NMR (500 MHz, CDCl3) δ 7.89 (d, J = 16.0 Hz, 1H, Ph-H), 7.85 (s, 1H, Ph-H), 7.77 (d, J = 7.8 Hz, 1H, Ph-H), 7.70 (d, J = 7.8 Hz, 1H, Ph-H), 7.58 (t, J = 7.8 Hz, 1H, Ph-H), 7.14–7.10 (m, 2H, 2× Ph-H), 6.97–6.92 (m, 2H, Ph-H and -CH=C-), 6.72 (d, J = 16.0 Hz, 1H, -CH=C-), 3.84 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 165.28 (-C=O), 157.35 (Ph-C-OCH3), 144.50 (-CH=CH), 144.14 (Ph-C-O), 135.00 (Ph-C-CH), 131.69 (Ph-CH), 131.43 (Ph-CH), 131.25 (Ph-CH), 129.59 (Ph-CH), 127.02 (d, J = 3.5 Hz) (Ph-C-CF3), 124.82 (d, J = 3.9 Hz) (Ph-C-CF3), 122.69 (Ph-CF3), 122.31 (Ph-CH), 119.40 (Ph-CH), 114.53 (=CH-C=O), 55.61 (-O-CH3). HRMS m/z: 323.0817 [M + H]+, found 323.0820.
4-methoxyphenyl (E)-3-(4-(trifluoromethyl)phenyl)acrylate (19): White solid, 79% yield, mp 131.7–132.1 °C; 1H NMR (500 MHz, CDCl3) δ 7.87 (d, J = 16.0 Hz, 1H, Ph-H), 7.68 (s, 4H, 4×Ph-H), 7.14–7.05 (m, 2H, 2 × Ph-H), 6.95–6.89 (m, 2H, Ph-H and -CH=C-), 6.70 (d, J = 16.0 Hz, 1H, -CH=C-), 3.82 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 165.29 (-C=O), 157.38 (Ph-C-OCH3), 144.47 (-CH=CH), 144.12 (Ph-C-O), 137.56 (Ph-C-CH), 132.22 (Ph-CH), 131.96 (Ph-CH), 128.41 (Ph-CH), 125.98 (Ph-CH), 124.89 (Ph-C-CF3), 122.72 (Ph-CF3), 122.31 (Ph-CH), 119.98 (Ph-CH), 116.02 (=CH-C=O), 114.79 (Ph-CH), 114.54 (Ph-CH), 55.62 (-O-CH3). HRMS m/z: 323.0817 [M + H]+, found 323.0823.
4-methoxyphenyl (E)-3-(2-nitrophenyl)acrylate (20): White solid, 82% yield, mp 11.9–112.4 °C; 1H NMR (500 MHz, CDCl3) δ 7.88 (d, J = 16.0 Hz, 1H, Ph-H), 7.63–7.57 (m, 2H, 2 × Ph-H), 7.44 (dd, J = 6.5, 3.4 Hz, 3H, 3 × Ph-H), 7.22 (d, J = 8.4 Hz, 2H, 2 × Ph-H), 7.07 (d, J = 8.4 Hz, 2H, Ph-H and -CH=C-), 6.64 (d, J = 16.0 Hz, 1H, -CH=C-), 2.37 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 164.61 (-C=O), 157.37 (Ph-C-OCH3), 148.34 (Ph-C-O), 144.08 (-CH=CH), 141.71 (Ph-C-NO2), 133.71 (Ph-C-CH), 130.64 (Ph-CH), 130.44 (Ph-CH), 129.30 (Ph-CH), 125.06 (Ph-CH), 122.48 (Ph-CH), 122.32 (Ph-CH), 114.51 (=CH-C=O), 55.63 (-O-CH3). HRMS m/z: 300.0794 [M + H]+, found 300.0799.
4-methoxyphenyl (E)-3-(3-nitrophenyl)acrylate (21): White solid, 77% yield, mp 120.4–121.5 °C; 1H NMR (500 MHz, CDCl3) δ 8.43 (t, J = 1.7 Hz, 1H, Ph-H), 8.26 (dd, J = 8.2, 2.0 Hz, 1H, Ph-H), 7.88 (dd, J = 12.0, 3.9 Hz, 2H, 2 × Ph-H), 7.61 (t, J = 8.0 Hz, 1H, Ph-H), 7.13–7.04 (m, 2H, 2 × Ph-H), 6.96–6.89 (m, 2H, Ph-H and -CH=C-), 6.77–6.71 (m, 1H, -CH=C-), 3.81 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 165.00 (-C=O), 157.40 (Ph-C-OCH3), 148.73 (Ph-C-O), 144.05 (-CH=CH), 143.42 (Ph-C-NO2), 135.92 (Ph-C-CH), 133.82 (Ph-CH), 130.12 (Ph-CH), 124.88 (Ph-CH), 122.64 (Ph-CH), 122.26 (Ph-CH), 120.61 (Ph-CH), 114.54 (=CH-C=O), 55.63 (-O-CH3). HRMS m/z: 300.0794 [M + H]+, found 300.0801.
4-methoxyphenyl (E)-3-(4-nitrophenyl)acrylate (22): White solid, 83% yield, mp 156.0–156.4 °C; 1H NMR (500 MHz, CDCl3) δ 7.88 (d, J = 16.0 Hz, 1H, Ph-H), 7.63–7.57 (m, 2H, 2 × Ph-H), 7.44 (dd, J = 6.5, 3.4 Hz, 3H, 3 × Ph-H), 7.22 (d, J = 8.4 Hz, 2H, 2 × Ph-H), 7.07 (d, J = 8.4 Hz, 2H, Ph-H and -CH=C-), 6.64 (d, J = 16.0 Hz, 1H, -CH=C-), 2.37 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 164.90 (-C=O), 157.44 (Ph-C-OCH3), 148.70 (Ph-C-O), 144.02 (-CH=CH), 143.33 (Ph-C-NO2), 140.25 (Ph-C-CH), 128.88 (Ph-CH), 124.28 (Ph-CH), 122.24 (Ph-CH), 121.72 (Ph-CH), 114.56 (=CH-C=O), 55.63 (-O-CH3). HRMS m/z: 300.0794 [M + H]+, found 300.0807.
4-methoxyphenyl (E)-3-(2,4-dinitrophenyl)acrylate (23): White solid, 50% yield, mp 114.6–115.2 °C; 1H NMR (500 MHz, CDCl3) δ 8.94 (d, J = 2.3 Hz, 1H, Ph-H), 8.54 (dd, J = 8.6, 2.3 Hz, 1H, Ph-H), 8.31 (d, J = 15.8 Hz, 1H, Ph-H), 7.93 (d, J = 8.5 Hz, 1H, Ph-H), 7.14–7.09 (m, 2H, 2 × Ph-H), 6.96–6.92 (m, 2H, Ph-H and -CH=C-), 6.67 (d, J = 15.8 Hz, 1H, -CH=C-), 3.84 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 163.80 (-C=O), 157.53 (Ph-C-OCH3), 148.21 (Ph-C-O), 148.14 (Ph-C-NO2), 143.85 (Ph-C-NO2), 139.38 (-CH=CH), 136.22 (Ph-C-CH), 131.53 (Ph-CH), 127.81 (Ph-CH), 125.72 (Ph-CH), 122.15 (Ph-CH), 120.68 (Ph-CH), 114.55 (=CH-C=O), 55.64 (-O-CH3). HRMS m/z: 345.0645 [M + H]+, found 345.0655.
4-methoxyphenyl (E)-3-(pyridin-3-yl)acrylate (24): White solid, 64% yield, mp 107.8–108.3 °C; 1H NMR (500 MHz, CDCl3) δ 8.72–8.69 (m, 2H, 2 × Ph-H), 7.78 (d, J = 16.0 Hz, 2H, 2 × Ph-H), 7.45–7.41 (m, 2H, 2 × Pyridine-H), 7.14–7.08 (m, 2H, 2 × Pyridine-H), 6.97–6.91 (m, 1H, -CH=C-), 6.80 (d, J = 16.1 Hz, 1H, -CH=C-), 3.83 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 164.87 (-C=O), 157.43 (Ph-C-OCH3), 150.71 (Pyridine-CH), 144.01 (Ph-C-O), 143.40 (-CH=CH), 141.34 (Pyridine-C-CH), 122.24 (Pyridine-CH), 122.09 (Ph-CH), 121.90 (Ph-CH), 114.55 (=CH-C=O), 55.62 (-O-CH3). HRMS m/z: 256.0895 [M + H]+, found 256.0902.
4-methoxyphenyl (E)-3-(quinolin-4-yl)acrylate (25): White solid, 70% yield, mp 119.2–120.5 °C; 1H NMR (500 MHz, CDCl3) δ 8.99 (d, J = 4.5 Hz, 1H, Ph-H), 8.59 (d, J = 15.9 Hz, 2H, 2 × Ph-H), 8.22–8.17 (m, 2H, 2 × Quinolin-H), 7.80 (ddd, J = 8.5, 6.8, 1.3 Hz, 2H, 2 × Quinolin-H), 7.18–7.13 (m, 2H, 2 × Quinolin-H), 6.99–6.93 (m, 2H, Ph-H and -CH=C-), 6.85 (d, J = 15.8 Hz, 1H, -CH=C-), 3.84 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 164.83 (-C=O), 157.45 (Ph-C-OCH3), 150.15 (Ph-C-O), 148.73 (Quinolin-CH), 144.07 (-CH=CH), 140.79 (Quinolin-C-CH), 139.62 (Quinolin-C-C), 130.31 (Quinolin-C-C), 129.89 (Quinolin-CH), 127.49 (Quinolin-CH), 125.96 (Quinolin-CH), 123.89 (Quinolin-CH), 123.28 (Quinolin-CH), 122.28 (Ph-CH), 118.30 (Ph-CH), 114.57 (=CH-C=O), 55.63 (-O-CH3). HRMS m/z: 306.1052 [M + H]+, found 306.1058.
4-methoxyphenyl (E)-3-(naphthalen-2-yl)acrylate (26): White solid, 79% yield, mp 126.0–127.3 °C; 1H NMR (500 MHz, CDCl3) δ 8.74 (d, J = 15.7 Hz, 1H, Ph-H), 8.27 (d, J = 8.5 Hz, 1H, Nap-H), 8.00–7.90 (m, 2H, 2 × Nap-H), 7.87 (d, J = 7.2 Hz, 1H, Nap-H), 7.66–7.52 (m, 3H, 3 × Nap-H), 7.21–7.15 (m, 2H, 2 × Ph-H), 7.01–6.95 (m, 2H, Ph-H and -CH=C-), 6.79–6.72 (m, 1H, -CH=C-), 3.86 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 165.73 (-C=O), 157.31 (Ph-C-OCH3), 144.35 (-CH=CH), 143.39 (Ph-C-O), 133.73 (Nap-C-C), 131.53 (Nap-C-C), 131.46 (Nap-C-C), 128.83 (Nap-C-CH), 127.07 (Nap-C-CH), 126.37 (Nap-C-CH), 125.53 (Nap-C-CH), 125.32 (Nap-C-CH), 124.73 (Nap-C-CH), 122.46 (Ph-CH), 119.94 (Ph-CH), 114.54 (=CH-C=O), 55.64 (-O-CH3). HRMS m/z: 305.1099 [M + H]+, found 305.1107.
4-methoxyphenyl (E)-3-(6-methoxynaphthalen-2-yl)acrylate (27): White solid, 81% yield, mp 116.7–117.8 °C; 1H NMR (500 MHz, CDCl3) δ 8.02 (d, J = 15.9 Hz, 1H, Ph-H), 7.95–7.93 (m, 1H, Nap-H), 7.82–7.76 (m, 2H, 2 × Nap-H), 7.72 (dd, J = 8.5, 1.8 Hz, 1H, Ph-H), 7.21 (dd, J = 8.9, 2.5 Hz, 1H, Ph-H), 7.17 (d, J = 2.5 Hz, 1H, Nap-H), 7.15–7.11 (m, 2H, 2 × Nap-H), 6.98–6.93 (m, 2H, Ph-H and -CH=C-), 6.70 (d, J = 15.9 Hz, 1H, -CH=C-), 3.97 (s, 3H, -OCH3), 3.85 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 166.62 (-C=O), 159.44 (Ph-C-OCH3), 157.39 (Ph-C-OCH3), 146.69 (Ph-C-O), 144.41 (-CH=CH), 136.34 (Nap-C-C), 130.26 (Nap-C-C), 129.62 (Nap-C-C), 128.91 (Nap-C-CH), 127.63 (Nap-C-CH), 124.20 (Nap-C-CH), 122.44 (Nap-C-CH), 119.59 (Nap-C-CH), 116.24 (=CH-C=O), 114.49 (Ph-CH), 106.01 (Ph-CH), 55.63 (-O-CH3), 55.43 (-O-CH3). HRMS m/z: 335.1205 [M + H]+, found 335.1212.
4-methoxyphenyl (E)-3-(thiophen-2-yl)acrylate (28): White solid, 77% yield, mp 100.3–101.4 °C; 1H NMR (500 MHz, CDCl3) δ 7.87 (d, J = 15.9 Hz, 1H, Ph-H), 7.59 (dd, J = 2.9, 1.3 Hz, 1H, Thiophen-H), 7.39 (qd, J = 5.1, 2.0 Hz, 2H, 2 × Ph-H), 7.13–7.08 (m, 2H, 2 × Thiophen-H), 6.97–6.91 (m, 2H, Ph-H and -CH=C-), 6.47 (d, J = 15.9 Hz, 1H, -CH=C-), 3.84 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 166.10 (-C=O), 157.23 (Ph-C-OCH3), 144.29 (-CH=CH), 139.82 (Ph-C-O), 137.43 (Thiophen-C-CH), 128.87 (Thiophen-C-CH), 127.21 (Thiophen-C-CH), 125.18 (Thiophen-C-CH), 122.41 (Ph-CH), 116.96 (=CH-C=O), 114.49 (Ph-CH), 55.62 (-O-CH3). HRMS m/z: 261.0507 [M + H]+, found 261.0513.
4-methoxyphenyl (E)-3-(furan-2-yl)acrylate (29): White solid, 72% yield, mp 143.5–144.1 °C; 1H NMR (500 MHz, CDCl3) δ 7.62 (d, J = 15.7 Hz, 1H, Ph-H), 7.55 (d, J = 1.8 Hz, 1H, Furan-H), 7.13–7.07 (m, 2H, 2 × Ph-H), 6.96–6.91 (m, 2H, 2 × Furan-H), 6.71 (d, J = 3.5 Hz, 1H, -CH=C-), 6.55–6.49 (m, 2H, Ph-H and -CH=C-), 3.83 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 165.9 (-C=O), 157.21 (Ph-C-OCH3), 150.80 (Ph-C-O), 144.76 (-CH=CH), 143.98 (Furan-C-CH), 132.52 (Furan-CH), 122.40 (Furan-CH), 115.62 (=CH-C=O), 114.89 (Furan-CH), 114.46 (Ph-CH), 112.49 (Ph-CH), 55.61 (-O-CH3). HRMS m/z: 245.0736 [M + H]+, found 245.0741.
4-methoxyphenyl (E)-3-(3,4,5-trimethoxyphenyl)acrylate (30): White solid, 74% yield, mp 150.7–151.0 °C; 1H NMR (500 MHz, CDCl3) δ 7.77 (d, J = 15.9 Hz, 1H, Ph-H), 7.12–7.04 (m, 2H, 2 × Ph-H), 6.94–6.90 (m, 2H, 2 × Ph-H), 6.81 (s, 2H, Ph-H and -CH=C-), 6.53 (d, J = 15.9 Hz, 1H, -CH=C-), 3.90 (d, J = 1.8 Hz, 9H, -OCH3), 3.81 (s, 3H, -OCH3). 13C NMR (126 MHz, CDCl3) δ 165.81 (-C=O), 157.26 (Ph-C-OCH3), 153.50 (Ph-C-OCH3), 146.38 (Ph-C-O), 144.26 (-CH=CH), 140.40 (Ph-C-CH), 129.70 (Ph-CH), 122.38 (Ph-CH), 116.56 (=CH-C=O), 114.50 (Ph-CH), 105.41 (Ph-CH), 61.03 (-O-CH3), 56.19 (-O-CH3), 55.62 (-O-CH3). HRMS m/z: 345.1260 [M + H]+, found 345.1267.

3.3. Bacterial Culture

H. pylori was grown and cultured in BHI containing 10% FBS in the presence of oxygen and 100% humidity conditions at 37 °C. The growth of the H. pylori ATCC SS1 strain was evaluated using visual inspection after 3 to 5 days.

3.4. Anti-Helicobacter pylori Activity

As described in the previous literature [28], we used the modified broth microdilution assay to determine the MIC and the MBC of the target compounds. For the MIC assay, the bacteria were exposed to the target compounds at different concentrations in culture with BHI containing 10% FBS for 72 h. The MICs of all target compounds were tested based on the lowest concentration of the nine compounds, resulting in Helicobacter pylori growth inhibition. For the MBC assay, 100 μL solution that contains the target compounds at 1×, 2×, 4× or 8× MICs was removed from the 96-well plate and cultured in a microaerophilic atmosphere at 37 ℃ for 72 h. The MBC was defined as the concentration that killed 99.9% of the initial bacterial population [29].

3.5. Inhibiting Kinetics and Killing Kinetics Assays

In order to investigate the effect of compounds 23 and 25 against H. pylori ATCC SS1 strain, the inhibiting kinetics and killing kinetics assays were used to evaluate the present study. For inhibiting kinetics assays, the ATCC SS1 strain was first exposed to water, compounds 23 or 25, at low MICs in BHI containing 10% FBS. Then, the bacteria in different groups were shaken at 150 rpm in a tri-gas incubator. Subsequently, 100 μL solution of each group at different times (0, 12, 24, 36, 48, 72 h) were pipetted for absorbance measurement at 600 nm, respectively. Finally, the inhibiting kinetics curves of water, compounds 23 or 25, were measured and drawn. For killing kinetics assays, the ATCC SS1 strain was firstly exposed to water, compounds 23 or 25, at high MICs in BHI containing 10% FBS. Secondly, the bacteria in different groups were shaken at 150rpm in a tri-gas incubator and incubated at different times (0, 12, 24, 36, 48, 72 h). Then, 50 μL solution of each group was pipetted for a series of 10-fold dilutions. Subsequently, 100 μL dilutions from different groups were plated on solid agar for several days to form single colonies. Finally, a counter was used to count the number of colonies in different groups. In addition, the results were expressed as Log (CFU/mL).

3.6. SEM

As described previously, the effect of compounds 23 and 25 on H. pylori morphology was examined using SEM [30]. Typically, the H. pylori ATCC SS1 strain was first incubated in BHI containing 10% FBS with or without compound 23 at the concentration of MIC. They were cultured in a microaerophilic atmosphere at 37 ℃ for 8h. Secondly, after 8 h, the bacteria, treated with compounds 23 and 25, were collected from the bacterial suspension by centrifugation and washed twice with PBS. The tested samples were subsequently fixed with 2.5% glutaraldehyde and dehydrated through a graduated ethanol series. Finally, the specimens derived from the collected bacteria were treated by metal spraying and observed using a scanning electron microscope.

3.7. Molecular Docking

It was indicated that Helicobacter pylori HP1029 (PDB ID: 4TSD) was a homodimer, in which each monomer comprised a molecular core formed by 12 antiparallel β-strands arranged in two β-sheets flanked by helices [31]. Moreover, it belonged to the DUF386 family, which was implicated in the bacterial biofilm formation [32]. Therefore, Helicobacter pylori HP1029 (PDB ID: 4TSD) was selected for the molecular docking.
As described previously, AutoDock Vina was used to simulate the interaction between metronidazole or compound 23 with HP1029 protein [33]. Firstly, the ligands (metronidazole and compound 23) were drawn by the ChemDraw 2014 software and then optimized by AutoDock Vina 1.5 software. The hydrogen atoms were added to metronidazole and compound 23 using Babel mode, and partial charges of metronidazole and compound 23 were generated using MOPAC. After that, the hydrogen bonding energy of metronidazole and compound 23 was minimized by using the Lennard-Jones 6/12 potential mode and optimized with iterative minimization. Secondly, a maximum of 300 iterations was used to optimize the energy gradient, and a total of 200 docking runs were performed in each cycle of the iterative minimization. Before docking, the crystal structure of Helicobacter pylori HP1029 (PDB ID: 4TSD) was available from the Protein Data Bank and prepared in AutoDock Vina. After all water molecules were removed, the hydrogen atoms were added to make Helicobacter pylori HP1029 protein protonation. The active pocket in Helicobacter pylori HP1029 protein was simulated by the automatic mode. In addition, other settings were used in the default settings. After the docking analysis was completed, the files were exported from AutoDock software and drawn using the PyMol software.

3.8. Data and Statistical Analysis

Data were presented as mean ± standard error (SD). The results were analyzed using GraphPad Prism 8.0.2 software (GraphPad Software, San Diego, CA, USA). All the statistical analyses were performed by Student’s t-test. p < 0.05 exhibited a statistically significant difference.

4. Conclusions

In summary, the present study describes our efforts to discover a series of cinnamic acid derivatives as potent anti-H. pylori agents. Most of the cinnamic acid derivatives exhibited different antimicrobial potency against H. pylori activity. Compound 23 emerged as the most potent anti-H. pylori agents among the series. Further studies indicated that compound 23 showed anti-bacterial activity and had a bactericidal effect on H. pylori due to the destruction of the bacterial structure. Molecular docking analysis revealed the binding pattern of compound 23 interacting with the amino acid residues in the active site. These findings provided a promising rationale for discovering the novel anti-H. pylori agents.

Author Contributions

W.L. and S.Z. designed the study and revised the manuscript. Y.L., K.Z. and Z.W. performed the in vitro experiment and drafted the manuscript. Y.L., Y.Z., S.W. and J.Y. synthesized the target compounds. M.C. carried out part of the in vitro experiment. W.L. and Y.L. co-wrote the manuscript. W.L., S.Z. and V.K.W.W. advised on experimental design and provided technical assistance. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Joint Fund of Wuyi University-Macau (No. 2019WGALH01) and the College Students’ Innovation and Entrepreneurship Training Program (No. 111).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are also reported in the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Molecules 29 04548 g001
Figure 1. The chemical structures of reported cinnamic acid derivatives (1a–1f).
Figure 1. The chemical structures of reported cinnamic acid derivatives (1a–1f).
Molecules 29 04548 g001
Molecules 29 04548 sch001
Scheme 1. Reagents and conditions: 4-methoxyphenol, EDCI, DMAP, CH2Cl2, 25 °C, 2 h.
Scheme 1. Reagents and conditions: 4-methoxyphenol, EDCI, DMAP, CH2Cl2, 25 °C, 2 h.
Molecules 29 04548 sch001
Molecules 29 04548 g002
Figure 2. In vitro anti-H. pylori activity of compound 23: (A) inhibiting kinetics curves of compound 23 against H. pylori; (B) killing kinetics curves of compound 23 against H. pylori.
Figure 2. In vitro anti-H. pylori activity of compound 23: (A) inhibiting kinetics curves of compound 23 against H. pylori; (B) killing kinetics curves of compound 23 against H. pylori.
Molecules 29 04548 g002
Molecules 29 04548 g003aMolecules 29 04548 g003b
Figure 3. Effect of compound 23 on the biofilm of H. pylori. (A) SEM images of ATCC SS1 (20 kV); (B) SEM images of ATCC SS1 (40 kV); (C) SEM images of ATCC SS1 after treated by compound 23 at MIC (20 kV); (D) SEM images of ATCC SS1 after treated by compound 23 at MIC (40 kV). Arrows represent morphological changes of H. pylori cells on SEM; yellow square represents local site amplification images of A and C.
Figure 3. Effect of compound 23 on the biofilm of H. pylori. (A) SEM images of ATCC SS1 (20 kV); (B) SEM images of ATCC SS1 (40 kV); (C) SEM images of ATCC SS1 after treated by compound 23 at MIC (20 kV); (D) SEM images of ATCC SS1 after treated by compound 23 at MIC (40 kV). Arrows represent morphological changes of H. pylori cells on SEM; yellow square represents local site amplification images of A and C.
Molecules 29 04548 g003aMolecules 29 04548 g003b
Molecules 29 04548 g004
Figure 4. Molecular docking study on the binding of metronidazole (A) and compound 23 (B) with H. pylori as analyzed using Pymol 3.7 software.
Figure 4. Molecular docking study on the binding of metronidazole (A) and compound 23 (B) with H. pylori as analyzed using Pymol 3.7 software.
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Table 1. Inhibitory values for cinnamic acid and its derivatives against H. pylori.
Table 1. Inhibitory values for cinnamic acid and its derivatives against H. pylori.
CompoundRInhibition (%) at 32 μM aMIC (μM) a
Metronidazole--99.92 ± 0.108
Cinnamic acid--57.30 ± 10.38>64
1Ph73.29 ± 1.2064
22-CH3-Ph98.16 ± 0.7332
33-CH3-Ph76.44 ± 4.5764
44-CH3-Ph77.60 ± 0.2364
52-CH3O-Ph82.03 ± 4.3064
63-CH3O-Ph50.58 ± 3.8264
74-CH3O-Ph83.16 ± 1.1864
82-F-Ph67.92 ± 3.3764
93-F-Ph69.00 ± 0.5264
104-F-Ph86.33 ± 1.3264
112-Cl-Ph55.91 ± 8.6464
123-Cl-Ph81.11 ± 2.7164
134-Cl-Ph18.52 ± 10.7364
142-Br-Ph64.67 ± 1.2164
153-Br-Ph62.24 ± 2.0532
164-Br-Ph100.55 ± 0.3016
172-CF3-Ph62.23 ± 0.6664
183-CF3-Ph65.30 ± 3.5564
194-CF3-Ph98.68 ± 0.1916
202-NO2-Ph49.26 ± 2.0564
213-NO2-Ph96.34 ± 0.5932
224-NO2-Ph87.10 ± 0.3264
232,4-diNO2-Ph95.70 ± 1.074
24pyridin96.48 ± 0.5432
25quinolin97.95 ± 0.418
26naphthalen40.43 ± 3.1264
276-CH3O-naphthalen54.48 ± 1.03>64
28thiophen82.17 ± 1.6964
29furan71.53 ± 5.9064
303,4,5-triCH3O-Ph74.98 ± 1.2764
a Data shown as mean ± SD of three independent experiments.
Table 2. MIC and MBC value of compound 23 against H. pylori.
Table 2. MIC and MBC value of compound 23 against H. pylori.
CompoundMIC (μM) aMBC (μM)MBC/MIC
Metronidazole8324
1–23482
a Data shown in Table 1.
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Li, Y.; Zhao, K.; Wu, Z.; Zheng, Y.; Yu, J.; Wu, S.; Wong, V.K.W.; Chen, M.; Liu, W.; Zhao, S. Discovery of Cinnamic Acid Derivatives as Potent Anti-H. pylori Agents. Molecules 2024, 29, 4548. https://doi.org/10.3390/molecules29194548

AMA Style

Li Y, Zhao K, Wu Z, Zheng Y, Yu J, Wu S, Wong VKW, Chen M, Liu W, Zhao S. Discovery of Cinnamic Acid Derivatives as Potent Anti-H. pylori Agents. Molecules. 2024; 29(19):4548. https://doi.org/10.3390/molecules29194548

Chicago/Turabian Style

Li, Yonglian, Kun Zhao, Zhidi Wu, Yujun Zheng, Jialin Yu, Sikun Wu, Vincent Kam Wai Wong, Min Chen, Wenfeng Liu, and Suqing Zhao. 2024. "Discovery of Cinnamic Acid Derivatives as Potent Anti-H. pylori Agents" Molecules 29, no. 19: 4548. https://doi.org/10.3390/molecules29194548

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