Cinnamic acid derivatives and processes for their preparation

Abstract

The application discloses a cinnamic acid derivative and a preparation method thereof, and aims to provide the cinnamic acid derivative which has better inhibiting and killing activities on helicobacter pylori and can be used for preparing helicobacter pylori resisting drugs, and relates to the technical field of medicines. The application discloses a cinnamic acid derivative, and the structural formula is shown in the following formula: wherein R1 is NO2, R2 is H or NO2, and R3 is -OCH2CH3 or Cl.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to cinnamic acid derivatives and their preparation methods. Background Technology

[0002] Helicobacter pylori (Hp) is closely associated with a variety of gastrointestinal diseases, such as chronic gastritis, gastric ulcers, duodenal ulcers, and even gastric cancer.

[0003] Currently, for Helicobacter pylori infection, antibiotics are the primary treatment for these patients. However, with the widespread use of antibiotics, bacterial resistance has increased significantly, and the effectiveness of simple Western medicine treatment is generally limited. Clinically, the main treatment for H. pylori is "triple therapy." Triple therapy consists of two antibiotics and a proton pump inhibitor or bismuth citrate, used in combination to achieve therapeutic goals. While current clinical treatment regimens can improve the eradication rate of H. pylori to some extent, they also have significant side effects, unstable efficacy against H. pylori, and poor patient compliance. No better solution has yet been found. In addition, the resistance rate of H. pylori to metronidazole and clarithromycin is increasing in clinical patients, with clarithromycin resistance rates ranging from 20% to 50% and metronidazole resistance rates from 40% to 70%. However, long-term use of antibiotics can disrupt the balance of intestinal flora to varying degrees, altering the types and abundance of intestinal bacteria, leading to a decrease in beneficial bacteria and an increase in harmful bacteria, causing homeostasis imbalance, and in severe cases, a series of adverse reactions such as diarrhea, abdominal pain, and constipation. Therefore, finding novel compounds with anti-Helicobacter pylori properties as lead drugs is of great significance for inhibiting and killing Helicobacter pylori, counteracting the side effects of long-term antibiotic use, and effectively treating digestive tract diseases caused by Helicobacter pylori.

[0004] Cinnamon, a homologous compound used in both food and medicine, was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) and has been documented in numerous herbal texts throughout history. Some scholars have extracted the active ingredients of cinnamon and confirmed their anti-Helicobacter pylori activity, but the inhibitory effect has been less than ideal. Cinnamic acid is one of the main active components of cinnamon, possessing advantages such as safety, low toxicity, and numerous biological activities. Some scholars have found that cinnamic acid has significant antibacterial effects against four pathogenic bacteria: Staphylococcus aureus, Escherichia coli, Salmonella, and Bacillus anthracis. However, there are very few reports on the application of cinnamic acid derivatives obtained through structural modification to combat Helicobacter pylori. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide a cinnamic acid derivative that exhibits good inhibitory and bactericidal activity against Helicobacter pylori and can be used to prepare anti-Helicobacter pylori drugs.

[0006] Therefore, the first technical solution provided by this invention is as follows:

[0007] Therefore, the first technical solution provided by the present invention is as follows: a cinnamic acid derivative, the structural formula of which is shown below:

[0008]

[0009] Wherein: R1 is NO2, R2 is H or NO2; R3 is -OCH2CH3 or Cl.

[0010] The second technical solution provided by the present invention is a method for preparing the above-mentioned cinnamic acid derivative, which includes the following steps in sequence: under nitrogen protection, cinnamic acid substituted product, phenol substituted product, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and organic base are reacted at room temperature for 0.5-1 h, the reaction solution is washed, the solvent phase is evaporated and then separated by column chromatography to obtain the cinnamic acid derivative.

[0011] The molar ratio of the cinnamic acid derivative, the phenol derivative, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 4-dimethylaminopyridine is 4-6:4-6:5-7:0.4-1.

[0012] Furthermore, in the above-mentioned method for preparing cinnamic acid derivatives, the molar ratio of the cinnamic acid substitute, phenol substitute, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 4-dimethylaminopyridine is 5:5:6:0.5.

[0013] Furthermore, in the above-mentioned method for preparing cinnamic acid derivatives, the cinnamic acid substitute is quinoline cinnamic acid, 4-nitrocinnamic acid, or 2,4-dinitrocinnamic acid.

[0014] Furthermore, in the above-mentioned method for preparing cinnamic acid derivatives, the phenol substitute is p-chlorophenol or p-ethoxyphenol.

[0015] Furthermore, in the above-mentioned method for preparing cinnamic acid derivatives, the organic base is 4-dimethylaminopyridine.

[0016] Furthermore, in the above-mentioned method for preparing cinnamic acid derivatives, the solvent is dichloromethane.

[0017] Furthermore, in the above-mentioned method for preparing cinnamic acid derivatives, the washing process uses a saturated sodium bicarbonate solution.

[0018] Compared with the prior art, the technical solution provided by the present invention has the following technical advantages:

[0019] The cinnamic acid derivative provided by this invention has been shown by activity tests and pharmacodynamic experiments to have a good inhibitory effect on multiple strains of Helicobacter pylori. It can be used to prepare anti-Helicobacter pylori drugs, providing a new type of drug for the clinical treatment of Helicobacter pylori infection. Attached Figure Description

[0020] Figure 1 It is compound I. 13 C-NMR spectrum;

[0021] Figure 2 It is compound I. 1 H-NMR spectrum;

[0022] Figure 3 It is compound II. 13 C-NMR spectrum;

[0023] Figure 4 It is compound II. 1 H-NMR spectrum;

[0024] Figure 5 It is compound III. 13 C-NMR spectrum;

[0025] Figure 6 It is compound III. 1 H-NMR spectrum;

[0026] Figure 7 This is the MBC result graph for compound I;

[0027] Figure 8 This is the MBC result graph for compound II;

[0028] Figure 9 This is the MBC result graph for compound IV;

[0029] Figure 10 This is the inhibition kinetics curve of compound I;

[0030] Figure 11 This is the inhibition kinetics curve of compound II;

[0031] Figure 12 This is the inhibition kinetics curve of compound IV;

[0032] Figure 13 This is the bactericidal curve of compound II;

[0033] Figure 14 This is the bactericidal curve of compound IV;

[0034] Figure 15 This is a SEM image showing the effect of compound II on the ultrastructure of Helicobacter pylori SS1.

[0035] Figure 16 This is a SEM image showing the effect of compound IV on the ultrastructure of Helicobacter pylori SS1.

[0036] Figure 17 This is a TEM image showing the effect of compound II on the ultrastructure of Helicobacter pylori SS1;

[0037] Figure 18 These are TEM images showing the effect of compound IV on the ultrastructure of Helicobacter pylori SS1.

[0038] Figure 19 This is a graph showing the results of the rapid urease experiment for compound II;

[0039] Figure 20 Figure showing the results of the rapid urease experiment for compound IV;

[0040] Figure 21 This is a graph showing the effect of compound II on the proliferation activity of GES-1 cells. Detailed Implementation

[0041] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0043] Example 1

[0044] Under nitrogen protection, 4 mmol of quinoline cinnamic acid, 4 mmol of p-ethoxyphenol, 5 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.4 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane and reacted at room temperature for 45 min. The reaction solution was washed three times with saturated sodium bicarbonate solution, and the dichloromethane phase was evaporated to dryness before separation by column chromatography to obtain compound I.

[0045] Molecular formula: C 20 H 17 NO3, molecular weight: 319.36. White solid, yield: 64.7%, melting point: 124.0℃. 1HNMR(500MHz, CDCl3)δ8.96(d,J=4.5Hz,1H),8.57(d,J=15.9Hz,1H),8.23–8.13(m,2H),7.78(ddd,J=8.3,6.9,1.2Hz,1H),7 .67–7.58(m,2H),7.16–7.08(m,2H),6.98–6.88(m,2H),6.83(d,J=15.8Hz,1H),4.03(q,J=7.0Hz,2H),1.42(t,J=7.0Hz,3H). 13 C NMR (126MHz, CDCl3) δ 164.85, 156.84, 150.15, 148.73, 143.94, 140.76, 139.66, 130.30, 129.91, 127.50, 125.97, 123.94, 123.29, 122.24, 118.31, 115.13, 63.87, 14.87. (NMR spectrum shown below) Figure 1 , 2 As shown.

[0046] Example 2

[0047] Under nitrogen protection, 4 mmol of 2,4-dinitrocinnamic acid, 4 mmol of p-ethoxyphenol, 5 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.4 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane and reacted at room temperature for 45 min. The reaction solution was washed three times with saturated sodium bicarbonate solution, and the dichloromethane phase was evaporated to dryness and separated by column chromatography to obtain compound II.

[0048] Molecular formula: C 17 H 14 N₂O₇, molecular weight: 358.306. Brown solid, yield: 45.8%, melting point: 155.7℃. 1HNMR(500MHz, CDCl3)δ8.94(d,J=2.3Hz,1H),8.54(dd,J=8.6,2.3Hz,1H),8.31(d,J=15.8Hz,1H),7 .93(d,J=8.5Hz,1H),7.14–7.09(m,2H),6.96–6.92(m,2H),6.67(d,J=15.8Hz,1H),3.84(s,3H).13C NMR (126MHz, CDCl3) δ 163.80, 157.53, 148.21, 148.14, 143.85, 139.38, 136.22, 131.53, 127.81, 125.72, 122.15, 120.68, 114.55, 55.64. (NMR spectrum shown below) Figure 3 , 4 As shown.

[0049] Example 3

[0050] Under nitrogen protection, 4.5 mmol of quinoline cinnamic acid, 4.5 mmol of p-ethoxyphenol, 5.5 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.45 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane and reacted at room temperature for 1 h. The reaction solution was washed three times with saturated sodium bicarbonate solution, and the dichloromethane phase was evaporated to dryness and separated by column chromatography to obtain compound I.

[0051] Molecular formula: C 20 H 17 NO3, molecular weight: 319.36. White solid, yield: 65.3%, melting point: 124.0℃. 1 HNMR(500MHz, CDCl3)δ8.96(d,J=4.5Hz,1H),8.57(d,J=15.9Hz,1H),8.23–8.13(m,2H),7.78(ddd,J=8.3,6.9,1.2Hz,1H),7 .67–7.58(m,2H),7.16–7.08(m,2H),6.98–6.88(m,2H),6.83(d,J=15.8Hz,1H),4.03(q,J=7.0Hz,2H),1.42(t,J=7.0Hz,3H). 13 C NMR (126MHz, CDCl3) δ 164.85, 156.84, 150.15, 148.73, 143.94, 140.76, 139.66, 130.30, 129.91, 127.50, 125.97, 123.94, 123.29, 122.24, 118.31, 115.13, 63.87, 14.87. (NMR spectrum shown below) Figure 1 , 2 As shown.

[0052] Example 4

[0053] Under nitrogen protection, 4.5 mmol of 2,4-dinitrocinnamic acid, 4.5 mmol of p-ethoxyphenol, 5.5 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.45 mmol of 4-dimethylaminopyridine were reacted at room temperature for 1 h. The reaction solution was washed three times with saturated sodium bicarbonate solution, and after evaporation of the dichloromethane phase to dryness, compound II was obtained by column chromatography.

[0054] Molecular formula: C 17 H 14 N₂O₇, molecular weight: 358.306. Brown solid, yield: 48.6%, melting point: 155.7℃. 1 HNMR(500MHz, CDCl3)δ8.94(d,J=2.3Hz,1H),8.54(dd,J=8.6,2.3Hz,1H),8.31(d,J=15.8Hz,1H),7 .93(d,J=8.5Hz,1H),7.14–7.09(m,2H),6.96–6.92(m,2H),6.67(d,J=15.8Hz,1H),3.84(s,3H).13C NMR (126MHz, CDCl3) δ 163.80, 157.53, 148.21, 148.14, 143.85, 139.38, 136.22, 131.53, 127.81, 125.72, 122.15, 120.68, 114.55, 55.64. (NMR spectrum shown below) Figure 3 , 4 As shown.

[0055] Example 5

[0056] Under nitrogen protection, 5 mmol of quinoline cinnamic acid, 5 mmol of p-ethoxyphenol, 6 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.5 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane and reacted at room temperature for 1 h. The reaction solution was washed three times with saturated sodium bicarbonate solution, and the dichloromethane phase was evaporated to dryness and separated by column chromatography to obtain compound I.

[0057] Molecular formula: C 20 H 17 NO3, molecular weight: 319.36. White solid, yield: 66.8%, melting point: 124.0℃. 1HNMR(500MHz, CDCl3)δ8.96(d,J=4.5Hz,1H),8.57(d,J=15.9Hz,1H),8.23–8.13(m,2H),7.78(ddd,J=8.3,6.9,1.2Hz,1H),7 .67–7.58(m,2H),7.16–7.08(m,2H),6.98–6.88(m,2H),6.83(d,J=15.8Hz,1H),4.03(q,J=7.0Hz,2H),1.42(t,J=7.0Hz,3H). 13 C NMR (126MHz, CDCl3) δ 164.85, 156.84, 150.15, 148.73, 143.94, 140.76, 139.66, 130.30, 129.91, 127.50, 125.97, 123.94, 123.29, 122.24, 118.31, 115.13, 63.87, 14.87. (NMR spectrum shown below) Figure 1 , 2 As shown.

[0058] Example 6

[0059] Under nitrogen protection, 5 mmol of 2,4-dinitrocinnamic acid, 5 mmol of p-ethoxyphenol, 6 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 0.5 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane and reacted at room temperature for 1 h. The reaction solution was washed three times with saturated sodium bicarbonate solution, and the dichloromethane phase was evaporated to dryness and separated by column chromatography to obtain compound II.

[0060] Molecular formula: C 17 H 14 N₂O₇, molecular weight: 358.306. Brown solid, yield: 51.0%, melting point: 155.7℃. 1HNMR(500MHz, CDCl3)δ8.94(d,J=2.3Hz,1H),8.54(dd,J=8.6,2.3Hz,1H),8.31(d,J=15.8Hz,1H),7 .93(d,J=8.5Hz,1H),7.14–7.09(m,2H),6.96–6.92(m,2H),6.67(d,J=15.8Hz,1H),3.84(s,3H).13C NMR (126MHz, CDCl3) δ 163.80, 157.53, 148.21, 148.14, 143.85, 139.38, 136.22, 131.53, 127.81, 125.72, 122.15, 120.68, 114.55, 55.64. (NMR spectrum shown below) Figure 3 , 4 As shown.

[0061] Example 7

[0062] Under nitrogen protection, 5.5 mmol of quinoline cinnamic acid, 5.5 mmol of p-ethoxyphenol, 6.5 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.75 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane and reacted at room temperature for 1.5 h. The reaction solution was washed three times with saturated sodium bicarbonate solution, and the dichloromethane phase was evaporated to dryness and separated by column chromatography to obtain compound I.

[0063] Molecular formula: C 20 H 17 NO3, molecular weight: 319.36. White solid, yield: 59.0%, melting point: 124.0℃. 1 HNMR(500MHz, CDCl3)δ8.96(d,J=4.5Hz,1H),8.57(d,J=15.9Hz,1H),8.23–8.13(m,2H),7.78(ddd,J=8.3,6.9,1.2Hz,1H),7 .67–7.58(m,2H),7.16–7.08(m,2H),6.98–6.88(m,2H),6.83(d,J=15.8Hz,1H),4.03(q,J=7.0Hz,2H),1.42(t,J=7.0Hz,3H). 13 C NMR (126MHz, CDCl3) δ 164.85, 156.84, 150.15, 148.73, 143.94, 140.76, 139.66, 130.30, 129.91, 127.50, 125.97, 123.94, 123.29, 122.24, 118.31, 115.13, 63.87, 14.87. (NMR spectrum shown below) Figure 1 , 2 As shown.

[0064] Example 8

[0065] Under nitrogen protection, 5.5 mmol of 2,4-dinitrocinnamic acid, 5.5 mmol of p-ethoxyphenol, 6.5 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.75 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane and reacted at room temperature for 1.5 h. The reaction solution was washed three times with saturated sodium bicarbonate solution, and the dichloromethane phase was evaporated to dryness and separated by column chromatography to obtain compound II.

[0066] Molecular formula: C 17 H 14 N₂O₇, molecular weight: 358.306. Brown solid, yield: 50.2%, melting point: 155.7℃. 1 HNMR(500MHz, CDCl3)δ8.94(d,J=2.3Hz,1H),8.54(dd,J=8.6,2.3Hz,1H),8.31(d,J=15.8Hz,1H),7 .93(d,J=8.5Hz,1H),7.14–7.09(m,2H),6.96–6.92(m,2H),6.67(d,J=15.8Hz,1H),3.84(s,3H).13C NMR (126MHz, CDCl3) δ 163.80, 157.53, 148.21, 148.14, 143.85, 139.38, 136.22, 131.53, 127.81, 125.72, 122.15, 120.68, 114.55, 55.64. (NMR spectrum shown below) Figure 3 , 4 As shown.

[0067] Example 9

[0068] Under nitrogen protection, 6 mmol of quinoline cinnamic acid, 6 mmol of p-ethoxyphenol, 7 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane and reacted at room temperature for 2 h. The reaction solution was washed three times with saturated sodium bicarbonate solution, and the dichloromethane phase was evaporated to dryness and separated by column chromatography to obtain compound I.

[0069] Molecular formula: C 20 H 17 NO3, molecular weight: 319.36. White solid, yield: 75.6%, melting point: 124.0℃. 1HNMR(500MHz, CDCl3)δ8.96(d,J=4.5Hz,1H),8.57(d,J=15.9Hz,1H),8.23–8.13(m,2H),7.78(ddd,J=8.3,6.9,1.2Hz,1H),7 .67–7.58(m,2H),7.16–7.08(m,2H),6.98–6.88(m,2H),6.83(d,J=15.8Hz,1H),4.03(q,J=7.0Hz,2H),1.42(t,J=7.0Hz,3H). 13 C NMR (126MHz, CDCl3) δ 164.85, 156.84, 150.15, 148.73, 143.94, 140.76, 139.66, 130.30, 129.91, 127.50, 125.97, 123.94, 123.29, 122.24, 118.31, 115.13, 63.87, 14.87. (NMR spectrum shown below) Figure 1 , 2 As shown.

[0070] Example 10

[0071] Under nitrogen protection, 6 mmol of 2,4-dinitrocinnamic acid, 6 mmol of p-ethoxyphenol, 7 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane and reacted at room temperature for 2 h. The reaction solution was washed three times with saturated sodium bicarbonate solution, and the dichloromethane phase was evaporated to dryness and separated by column chromatography to obtain compound II.

[0072] Molecular formula: C 17 H 14 N₂O₇, molecular weight: 358.306. Brown solid, yield: 50.9%, melting point: 155.7℃. 1 HNMR(500MHz, CDCl3)δ8.94(d,J=2.3Hz,1H),8.54(dd,J=8.6,2.3Hz,1H),8.31(d,J=15.8Hz,1H),7 .93(d,J=8.5Hz,1H),7.14–7.09(m,2H),6.96–6.92(m,2H),6.67(d,J=15.8Hz,1H),3.84(s,3H).13C NMR (126MHz, CDCl3) δ 163.80, 157.53, 148.21, 148.14, 143.85, 139.38, 136.22, 131.53, 127.81, 125.72, 122.15, 120.68, 114.55, 55.64. (NMR spectrum shown below) Figure 3 , 4 As shown.

[0073] Example 11

[0074] Under nitrogen protection, 4 mmol of 4-nitrocinnamic acid, 4 mmol of p-chlorophenol, 5 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 0.4 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane and reacted at room temperature for 45 min. The reaction solution was washed three times with saturated sodium bicarbonate solution, and the dichloromethane phase was evaporated to dryness before separation by column chromatography to obtain compound III.

[0075] Molecular formula: C 15 H 10 ClNO4, molecular weight: 303.698. White solid, yield 77.1%, melting point 133.8℃. 1 H NMR (500MHz, CDCl3) δ8.37–8.26(m,2H),7.89(d,J=16.1Hz,1H),7.74(d,J=8.7Hz,2H),7.47–7.36(m,2H),7.17–7.10(m,2H),6.74(d,J=16.0Hz,1H). 13 C NMR (126MHz, CDCl3) δ 164.26, 149.01, 148.82, 143.92, 140.02, 131.50, 129.63, 128.95, 124.32, 122.87, 121.22. (NMR spectrum shown below) Figure 5 , 6 As shown.

[0076] Example 12

[0077] Under nitrogen protection, 4 mmol of 2,4-dinitrocinnamic acid, 4 mmol of p-chlorophenol, 5 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.4 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane and reacted at room temperature for 45 min. The reaction solution was washed three times with saturated sodium bicarbonate solution, and the dichloromethane phase was evaporated to dryness before separation by column chromatography to obtain compound IV.

[0078] Molecular formula: C 15 H9ClN2O6, molecular weight: 348.695. Brown solid, yield 46.2%, melting point 157.6℃. 1H NMR (500MHz, CDCl3) δ8.96(d,J=2.3Hz,1H),8.56(dd,J=8.5,2.3Hz,1H),8.33(d,J=15.8Hz,1H),7.93(d, J=8.6Hz,1H),7.41(d,J=8.8Hz,1H),7.16(d,J=8.8Hz,2H),6.78(d,J=8.8Hz,1H),6.66(d,J=15.8Hz,1H). 13 C NMR (126MHz, CDCl3) δ140.01,130.81,129.65,127.85,125.20,122.77,120.73,116.79.

[0079] Example 13

[0080] Under nitrogen protection, 4.5 mmol of 4-nitrocinnamic acid, 4.5 mmol of p-chlorophenol, 5.5 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.45 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane and reacted at room temperature for 1 h. The reaction solution was washed three times with saturated sodium bicarbonate solution, and the dichloromethane phase was evaporated to dryness and separated by column chromatography to obtain compound III.

[0081] Molecular formula: C 15 H 10 ClNO4, molecular weight: 303.698. White solid, yield 74.9%, melting point 133.8℃. 1 H NMR (500MHz, CDCl3) δ8.37–8.26(m,2H),7.89(d,J=16.1Hz,1H),7.74(d,J=8.7Hz,2H),7.47–7.36(m,2H),7.17–7.10(m,2H),6.74(d,J=16.0Hz,1H). 13 C NMR (126MHz, CDCl3) δ 164.26, 149.01, 148.82, 143.92, 140.02, 131.50, 129.63, 128.95, 124.32, 122.87, 121.22. (NMR spectrum shown below) Figure 5 , 6 As shown.

[0082] Example 14

[0083] Under nitrogen protection, 4.5 mmol of 2,4-dinitrocinnamic acid, 4.5 mmol of p-chlorophenol, 5.5 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.45 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane and reacted at room temperature for 1 h. The reaction solution was washed three times with saturated sodium bicarbonate solution, and the dichloromethane phase was evaporated to dryness and separated by column chromatography to obtain compound IV.

[0084] Molecular formula: C 15 H9ClN2O6, molecular weight: 348.695. Brown solid, yield 47.9%, melting point 157.6℃. 1 H NMR (500MHz, CDCl3) δ8.96(d,J=2.3Hz,1H),8.56(dd,J=8.5,2.3Hz,1H),8.33(d,J=15.8Hz,1H),7.93(d, J=8.6Hz,1H),7.41(d,J=8.8Hz,1H),7.16(d,J=8.8Hz,2H),6.78(d,J=8.8Hz,1H),6.66(d,J=15.8Hz,1H). 13 C NMR (126MHz, CDCl3) δ140.01,130.81,129.65,127.85,125.20,122.77,120.73,116.79.

[0085] Example 15

[0086] Under nitrogen protection, 5 mmol of 4-nitrocinnamic acid, 5 mmol of p-chlorophenol, 6 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.5 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane and reacted at room temperature for 1 h. The reaction solution was washed three times with saturated sodium bicarbonate solution, and the dichloromethane phase was evaporated to dryness and separated by column chromatography to obtain compound III.

[0087] Molecular formula: C 15 H 10 ClNO4, molecular weight: 303.698. White solid, yield: 65.2%, melting point: 133.8℃. 1 H NMR (500MHz, CDCl3) δ8.37–8.26(m,2H),7.89(d,J=16.1Hz,1H),7.74(d,J=8.7Hz,2H),7.47–7.36(m,2H),7.17–7.10(m,2H),6.74(d,J=16.0Hz,1H). 13C NMR (126MHz, CDCl3) δ 164.26, 149.01, 148.82, 143.92, 140.02, 131.50, 129.63, 128.95, 124.32, 122.87, 121.22. (NMR spectrum shown below) Figure 5 , 6 As shown.

[0088] Example 16

[0089] Under nitrogen protection, 5 mmol of 2,4-dinitrocinnamic acid, 5 mmol of p-chlorophenol, 6 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.5 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane and reacted at room temperature for 1 h. The reaction solution was washed three times with saturated sodium bicarbonate solution, and the dichloromethane phase was evaporated to dryness and separated by column chromatography to obtain compound IV.

[0090] Molecular formula: C 15 H9ClN2O6, molecular weight: 348.695. Brown solid, yield 52.8%, melting point 157.6℃. 1 H NMR (500MHz, CDCl3) δ8.96(d,J=2.3Hz,1H),8.56(dd,J=8.5,2.3Hz,1H),8.33(d,J=15.8Hz,1H),7.93(d, J=8.6Hz,1H),7.41(d,J=8.8Hz,1H),7.16(d,J=8.8Hz,2H),6.78(d,J=8.8Hz,1H),6.66(d,J=15.8Hz,1H). 13 C NMR (126MHz, CDCl3) δ140.01,130.81,129.65,127.85,125.20,122.77,120.73,116.79.

[0091] Example 17

[0092] Under nitrogen protection, 5.5 mmol of 4-nitrocinnamic acid, 5.5 mmol of p-chlorophenol, 6.5 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.75 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane, and 0.75 mmol of triethylamine was slowly added dropwise. The reaction solution was washed three times with saturated sodium bicarbonate solution, and after evaporation of the dichloromethane phase to dryness, compound III was obtained by column chromatography.

[0093] Molecular formula: C 15 H 10ClNO4, molecular weight: 303.698. White solid, yield 63.0%, melting point 133.8℃. 1 HNMR (500MHz, CDCl3) δ8.37–8.26(m,2H),7.89(d,J=16.1Hz,1H),7.74(d,J=8.7Hz,2H),7.47–7.36(m,2H),7.17–7.10(m,2H),6.74(d,J=16.0Hz,1H). 13 C NMR (126MHz, CDCl3) δ 164.26, 149.01, 148.82, 143.92, 140.02, 131.50, 129.63, 128.95, 124.32, 122.87, 121.22. (NMR spectrum shown below) Figure 5 , 6 As shown.

[0094] Example 18

[0095] Under nitrogen protection, 5.5 mmol of 2,4-dinitrocinnamic acid, 5.5 mmol of p-chlorophenol, 6.5 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.75 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane and reacted at room temperature for 1.5 h. The reaction solution was washed three times with saturated sodium bicarbonate solution, and the dichloromethane phase was evaporated to dryness and separated by column chromatography to obtain compound IV.

[0096] Molecular formula: C 15 H9ClN2O6, molecular weight: 348.695. Brown solid, yield 53.1%, melting point 157.6℃. 1 H NMR (500MHz, CDCl3) δ8.96(d,J=2.3Hz,1H),8.56(dd,J=8.5,2.3Hz,1H),8.33(d,J=15.8Hz,1H),7.93(d, J=8.6Hz,1H),7.41(d,J=8.8Hz,1H),7.16(d,J=8.8Hz,2H),6.78(d,J=8.8Hz,1H),6.66(d,J=15.8Hz,1H). 13 C NMR (126MHz, CDCl3) δ140.01,130.81,129.65,127.85,125.20,122.77,120.73,116.79.

[0097] Example 19

[0098] Under nitrogen protection, 6 mmol of 4-nitrocinnamic acid, 6 mmol of p-chlorophenol, 7 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane and reacted at room temperature for 2 h. The reaction solution was washed three times with saturated sodium bicarbonate solution, and the dichloromethane phase was evaporated to dryness before separation by column chromatography to obtain compound III.

[0099] Molecular formula: C 15 H 10 ClNO4, molecular weight: 303.698. White solid, yield 68.1%, melting point 133.8℃. 1 H NMR (500MHz, CDCl3) δ8.37–8.26(m,2H),7.89(d,J=16.1Hz,1H),7.74(d,J=8.7Hz,2H),7.47–7.36(m,2H),7.17–7.10(m,2H),6.74(d,J=16.0Hz,1H). 13 C NMR (126MHz, CDCl3) δ 164.26, 149.01, 148.82, 143.92, 140.02, 131.50, 129.63, 128.95, 124.32, 122.87, 121.22. (NMR spectrum shown below) Figure 5 , 6 As shown.

[0100] Example 20

[0101] Under nitrogen protection, 6 mmol of 2,4-dinitrocinnamic acid, 6 mmol of p-chlorophenol, 7 mmol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1 mmol of 4-dimethylaminopyridine were dissolved in 20 mL of dichloromethane and reacted at room temperature for 2 h. The reaction solution was washed three times with saturated sodium bicarbonate solution, and the dichloromethane phase was evaporated to dryness and separated by column chromatography to obtain compound IV.

[0102] Molecular formula: C 15 H9ClN2O6, molecular weight: 348.695. Brown solid, yield 54.8%, melting point 157.6℃. 1 H NMR (500MHz, CDCl3) δ8.96(d,J=2.3Hz,1H),8.56(dd,J=8.5,2.3Hz,1H),8.33(d,J=15.8Hz,1H),7.93(d, J=8.6Hz,1H),7.41(d,J=8.8Hz,1H),7.16(d,J=8.8Hz,2H),6.78(d,J=8.8Hz,1H),6.66(d,J=15.8Hz,1H).13 C NMR (126MHz, CDCl3) δ140.01,130.81,129.65,127.85,125.20,122.77,120.73,116.79.

[0103] The structural formulas of compounds 1-4 are as follows:

[0104]

[0105] Experimental Example

[0106] To demonstrate the technical advantages of the technical solution provided in this application, the following is an experimental test of the anti-Helicobacter pylori activity of the cinnamic acid derivative provided in this application:

[0107] 1. Drug preparation

[0108] Cinnamic acid derivatives (compounds I, II, III, and IV) were dissolved in dimethyl sulfoxide (DMSO) before use and prepared into 9.6 mmol / L stock solutions, which were then stored at 4°C.

[0109] 2. Strains

[0110] Helicobacter pylori standard strain SS1, kindly provided by Professor Chen Ye of Southern Medical University.

[0111] 3. Experimental Methods

[0112] (1) Bacterial culture

[0113] The Helicobacter pylori strain frozen at -80°C was removed and revived on Columbia agar plates containing 5% sterile defibrinated sheep blood. It was then placed in an incubator and cultured at 37°C under a microaerophilic environment (approximately 85% N2, 10% CO2, and 5% O2) for 48–72 hours. After the bacteria had confluently grown onto the agar plates, they were scraped off using a disposable sterile spreader in a laminar flow hood and resuspended in 0.85% physiological saline. This resuspended bacteria was then inoculated onto fresh Columbia agar plates containing 5% sterile defibrinated sheep blood and cultured for another 48 hours before use in experiments.

[0114] (2) Determination of the minimum inhibitory concentration (MIC) of cinnamic acid derivatives by micro-broth dilution method

[0115] The minimum inhibitory concentration (MIC) of cinnamic acid derivative against Helicobacter pylori standard strain SS1 was determined using the microbroth dilution method. First, BHI complete culture medium was prepared by adding fetal bovine serum (FBS) to BHI to achieve a final FBS concentration of 10%. Experimental groups were set up as follows: blank control group (BHI culture medium), growth control group (BHI culture medium + Hp), negative control group (BHI culture medium + Hp + 1% DMSO), positive control group (BHI culture medium + Hp + antibiotic), and test compound group (BHI culture medium + Hp + cinnamic acid derivative). Serial dilutions of the cinnamic acid derivative were prepared in sterile EP tubes at concentrations of 2, 4, 8, 16, 32, 64, and 128 μmol / L. These were then pipetted into 96-well microplates at 50 μL per well to achieve final concentrations of 1, 2, 4, 8, 16, 32, and 64 μmol / L. Collect Helicobacter pylori inoculated on fresh Columbia solid medium containing 5% sterile defibrinated sheep blood, resuspend in 0.85% physiological saline, and adjust the bacterial concentration to 2 × 10⁻⁶. 6 CFU / mL, except for the blank control group, 50 μL of bacterial culture was added to each well (final volume 100 μL). The 96-well microplate was placed in an incubator and cultured at 37°C in a microaerophilic environment (approximately 85% N2, 10% CO2, and 5% O2) for 72 hours. The results were then observed. The experiment was repeated three times. After 72 hours, the 96-well plate was removed, and the results were observed from the bottom. The lowest concentration of antimicrobial agent that completely inhibits bacterial growth visible to the naked eye in the microdilution wells, i.e., the lowest concentration of antimicrobial agent where bacterial growth is not visible to the naked eye, is the MIC. The experiment was repeated three times. The results are shown in Table 1.

[0116] Table 1 shows the minimum inhibitory concentrations (MICs) of the cinnamic acid derivatives.

[0117]

[0118] (3) Determination of the minimum bactericidal concentration (MBC) of cinnamic acid derivatives

[0119] After the MIC experiment, 50 μL of culture medium from the microdilution wells, where bacterial growth was not visible to the naked eye, was inoculated onto Columbia agar plates containing 5% sterile defibrinated sheep blood. Results were observed after 72 hours of incubation. The experiment was repeated three times. MBC is defined as the minimum drug concentration that can reduce bacterial concentration by three orders of magnitude; that is, the minimum drug concentration that kills more than 99.9% of bacteria in the test sample compared to the untreated control group. Results are shown in Table 2 and... Figures 7-9According to CLSI standards, a test compound is considered a bactericide when MBC / MIC ≤ 2, and a bacteriostatic compound is considered a bacteriostatic compound when MBC / MIC ≥ 4. The results showed that compounds I, II, III, and IV acted in a bactericidal mode, exhibiting inhibitory and bactericidal effects against Helicobacter pylori SS1. The results are shown in Table 2.

[0120] Table 2. Minimum bactericidal concentrations (MBC) for compounds I-IV.

[0121]

[0122] (4) Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of four commonly used antibiotics

[0123] The minimum inhibitory concentration (MIC) of four commonly used antibiotics against the standard strain SS1 of Helicobacter pylori was tested using the micro-broth dilution method. The procedure was the same as above (2). The minimum bactericidal concentration (MBC) was determined using the same procedure as in (3). The results are shown in Table 3. The results show that the MIC and MBC values ​​of the synthesized compounds II and IV are lower than those of metronidazole, and they can be used to prepare drugs against Helicobacter pylori. The results are shown in Table 3.

[0124] Table 3. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Four Commonly Used Antibiotics

[0125]

[0126]

[0127] (5) Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of cinnamic acid derivatives and antibiotics under acidic conditions

[0128] Bacterial culture was performed as per procedure (1). Columbia solid culture medium containing 5% sterile defibrinated sheep blood was adjusted to a pH of approximately 5.0 with concentrated hydrochloric acid.

[0129] The method for determining the minimum inhibitory concentration (MIC) is the same as in procedure (2). The pH of the complete culture medium containing 10% BHI was adjusted to approximately 5.0 with concentrated hydrochloric acid.

[0130] The method for determining the minimum bactericidal concentration (MBC) is the same as in procedure (3). Columbia solid culture medium containing 5% sterile defibrinated sheep blood was adjusted to a pH of approximately 5.0 with concentrated hydrochloric acid.

[0131] It can be seen that: under acidic conditions, the MIC value of compound II is equal to that of the antibiotic metronidazole, while its MBC value is lower than that of the antibiotic metronidazole; the MIC and MBC values ​​of compound IV are lower than those of the antibiotic metronidazole, and it works in a bactericidal mode, exhibiting inhibitory and bactericidal effects against Helicobacter pylori SS1, as detailed in Table 4.

[0132] Table 4. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of compounds I-IV and four commonly used antibiotics under acidic conditions.

[0133]

[0134] (6) The antibacterial effects of compounds I, II, and IV on Helicobacter pylori SS1 were dynamically monitored based on the time-antibacterial curve.

[0135] The final concentrations of compounds I, II, and IV were determined at sub-inhibitory and inhibitory concentrations, respectively. The inhibition kinetics of Helicobacter pylori SS1 were plotted, with a bacterial suspension without the drug as the growth control group and BHI medium containing 10% FBS as the blank control. The bacteria were then incubated at 37°C under a microaerophilic environment (approximately 85% N2, 10% CO2, and 5% O2). At 0, 12, 24, 36, 48, 60, and 72 h, the mixture of drug and bacterial suspension or the bacterial suspension itself was taken out, and the absorbance was measured at 600 nm. The experiment was repeated three times. The absorbance (OD) was plotted on the x-axis as time (h). 600 Plot the inhibitory kinetics curves of compounds I, II, and IV on Helicobacter pylori SS1 with nm as the ordinate.

[0136] like Figure 10 As shown, compound I exhibits dose-dependent inhibitory activity against Helicobacter pylori SS1. It can inhibit the growth of the strain at a concentration of 4 μmol / L, and at a concentration of 8 μmol / L, it can basically inhibit the growth of Hp, thus demonstrating good Hp inhibitory activity.

[0137] like Figure 11 As shown, compound II exhibits dose-dependent inhibitory activity against Helicobacter pylori SS1. It can inhibit the growth of the strain at a concentration of 2 μmol / L, and at a concentration of 4 μmol / L, it can basically inhibit the growth of Hp, thus demonstrating good Hp inhibitory activity.

[0138] like Figure 12 As shown, compound IV exhibits dose-dependent inhibitory activity against Helicobacter pylori SS1. It can inhibit the growth of the strain at a concentration of 2 μmol / L, and completely inhibits the growth of Hp at a concentration of 4 μmol / L, demonstrating excellent Hp inhibitory activity.

[0139] (7) The bactericidal effects of compounds II and IV on Helicobacter pylori SS1 were dynamically monitored based on the time-bactericidal curve.

[0140] The bactericidal curves of Compound II and Compound IV at their final concentrations and inhibitory concentrations (1×, 2×, 4×, 8×MIC) against Helicobacter pylori SS1 were determined. A bacterial suspension without the drug was used as the growth control group, and BHI medium containing 10% FBS was used as the blank control. 2 mL of bacterial suspension: broth containing the drug / broth without the drug was inoculated into each well of a 6-well plate at a ratio of 1:1, and then incubated at 37°C under a microaerophilic environment (approximately 85% N2, 10% CO2, and 5% O2). At 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h, 50 μL of culture medium was serially diluted 10-fold (1:10–1:100000) and inoculated into Columbia agar medium containing 5% sterile defibrinated sheep blood. After 72 h of incubation, bacterial colony counts were performed, and the bactericidal curves at different drug concentrations were obtained by plotting. The results for Compound II are as follows: Figure 13 As shown, compound II inhibited the growth of Helicobacter pylori SS1 at a drug concentration of 4 μmol / L, killed all strains at a drug concentration of 8 μmol / L for 48 h, and killed all strains at a drug concentration of 16 μmol / L for 36 h, demonstrating strong bactericidal ability; the results of compound IV determination are as follows... Figure 14 As shown, compound IV can kill all strains at a drug concentration of 8 μmol / L for 12 h and at a drug concentration of 4 μmol / L for 48 h, demonstrating strong bactericidal ability.

[0141] (8) SEM experiments of compounds II and IV

[0142] The morphology of Helicobacter pylori SS1 was observed using scanning electron microscopy. First, Helicobacter pylori SS1 was treated with compounds II and IV at 1.0×MIC for 24 h, respectively, with untreated Helicobacter pylori SS1 as a control. The bacterial pellet was collected, washed three times with PBS, and fixed overnight at 4°C with 2.5% glutaraldehyde. The pellet was then washed three times with PBS for 15 min each time. Dehydration was performed with ethanol at concentrations of 30%, 50%, 70%, 90%, and 100% for 15 min each time, with two cycles of 100% ethanol dehydration. After dehydration, the pellet was replaced twice with tert-butanol for 20 min each time. The pellet was then freeze-dried under vacuum. Gold sputtering was performed, and the morphology was observed under a scanning electron microscope. The results showed that compound II at a concentration of 1.0×MIC significantly disrupted the structure of Helicobacter pylori SS1, as shown in the figure below. Figure 15 As shown in the figure. Compound IV significantly disrupted the structure of Helicobacter pylori SS1 at a concentration of 1.0 × MIC, as shown in the figure. Figure 16 As shown.

[0143] (9) TEM experiments of compounds II and IV

[0144] The morphology of Helicobacter pylori SS1 was observed using transmission electron microscopy. First, Helicobacter pylori SS1 was treated with 1.0×MIC compounds II and IV for 24 h, respectively, with untreated Helicobacter pylori SS1 as a control. The bacterial pellet was collected, washed three times with PBS, and fixed overnight with 2.5% glutaraldehyde at 4°C. The pellet was then washed three times with PBS. The samples were dehydrated using a gradient concentration of ethanol solutions (30%, 50%, 70%, 80%, 90%, and 95%) for 15 min at each concentration, followed by treatment with 100% ethanol for 20 min; finally, treatment with pure acetone was performed for 20 min. The samples were then treated with a mixture of embedding agent and acetone (V / V = 1 / 1) for 1 h; with a mixture of embedding agent and acetone (V / V = 3 / 1) for 3 h; and with pure embedding agent overnight. The permeation-treated samples were then embedded and heated overnight at 70°C to obtain the embedded samples. The samples were sectioned in an ultramicrotome to obtain sections with a size of 70–90 nm. The sections were stained with lead citrate solution and 50% ethanol saturated solution of uranium acetate for 5–10 min each, and then dried before being observed in a transmission electron microscope.

[0145] Helicobacter pylori exists in two forms: spiral and spherical. When the external environment is unfavorable for bacterial growth, most Helicobacter pylori will transform into spherical form, which is a form of self-protection. Figure 17 The results showed that the morphology of Helicobacter pylori SS1 in the control group was not altered. Figure 17 -A and 17-B); After Helicobacter pylori SS1 was treated with compound II at a concentration of 1.0×MIC for 24 hours, the normal structure was destroyed, and an increase in the number of spherical bacteria was clearly observed. Some bacterial cell walls were damaged, the bacteria swelled, the plasmolysis occurred, and various signs of necrosis were observed in the formation of vesicles inside the bacteria. Figure 17 -C and 17-D). Figure 18 The results showed that the morphology of Helicobacter pylori SS1 in the control group was not altered. Figure 18 -A and 18-B); After Helicobacter pylori SS1 was treated with compound IV at a concentration of 1.0×MIC for 24 hours, the normal structure was destroyed, and an increase in the number of spherical bacteria was clearly observed. Some bacterial cell walls were damaged, the bacteria swelled, the plasmolysis occurred, and various signs of necrosis were observed in the formation of vesicles inside the bacteria. Figure 18 -C and 18-D).

[0146] (10) Rapid urease test for compounds II and IV

[0147] Helicobacter pylori SS1 was treated with compounds II and IV at final concentrations at sub-inhibitory and inhibitory concentrations. The sample was incubated for 24 hours, and then rapid urease reagent was added. The absorbance at 560 nm was measured using a microplate reader.

[0148] Studies have shown that the survival of Helicobacter pylori after it enters the gastric mucosa and is subjected to acid shock (pH < 3) depends on the activity of its protein urease. This enzyme can convert urea in the human body into ammonia and bicarbonate, neutralizing gastric acid and thus promoting the colonization and survival of Helicobacter pylori in the acidic environment of the stomach. Research results on compounds II and IV of this invention regarding urease activity indicate that compounds II and IV can inhibit the growth of Helicobacter pylori by inhibiting the urease activity of SS1. Figure 19 As shown, under different concentrations of compound II, its urease activity decreased with increasing concentration, even though Compound II at high concentrations also inhibits urease activity. For example... Figure 20 As shown, under different concentrations of compound IV, its urease activity decreased with increasing concentration, even though Compound IV at certain concentrations also inhibits urease activity.

[0149] (11) Effects of compound II on cell proliferation activity

[0150] Experimental cells: human gastric mucosal epithelial cells GES-1.

[0151] Cell thawing: Remove the cryovials from the liquid nitrogen container and immediately place them in a 37°C water bath, agitating until completely thawed. Transfer the thawed cell suspension to a 15mL centrifuge tube and slowly add 5mL of complete culture medium. Centrifuge to remove the supernatant (1000rpm, 5min). Resuspend the cell pellet in 5mL of complete culture medium, then transfer the cells to a T25 cell culture flask and incubate at 37°C in a 5% CO2 incubator for 1-2 days.

[0152] Cell medium change: ① Remove the cells, aspirate the original culture medium in a clean bench, and wash the cells twice with 2 mL of PBS; ② Add 3-5 mL of complete culture medium and place them back in the cell culture incubator to continue culturing.

[0153] Cell seeding: ① Take GES-1 cells in good growth condition, wash with PBS, digest with trypsin, centrifuge, and adjust the cell concentration to 5×10⁻⁶. 4 ① Seeds per mL; ② Seed 100 μL per well into a 96-well plate, add PBS to the peripheral wells to avoid edge effects, and incubate in a cell culture incubator for 24 h.

[0154] Compound preparation: ① Compound II was prepared into a stock solution of 256 mmol / L and stored at -20℃. It is effective within 2 weeks. ② The stock solution of Compound II was diluted with complete culture medium to obtain concentrations of 4, 8, 16, 32, 64, 128, and 256 μmol / L.

[0155] Drug administration: ① In 3 to 10 groups of 96-well plates, each group was given 100 μL of compound II solution at concentrations of 0 (control group), 4, 8, 16, 32, 64, 128, and 256 μmol / L, respectively. ② Each group was divided into 6 replicates. PBS was added to the peripheral wells to avoid edge effects. The plates were then incubated in a cell culture incubator for 24 h, 48 h, and 72 h.

[0156] CCK-8 assay: ① After culturing for 24h, 48h, and 72h, remove the 96-well plate, add 10μL of CCK-8 solution to each well, and incubate at 37℃ for 1h; ② After 1h, measure the absorbance at 450nm for each well using a microplate reader; ③ Calculate the relative cell proliferation rate using the formula: Relative cell proliferation rate = (OD value of sample wells - OD value of blank wells) / (OD value of control wells - OD value of blank wells) × 100%; Plot the concentration of compound II on the x-axis and the proliferation rate on the y-axis, and fit a curve using GraphPad software, see [link to graph]. Figure 21 ((A)24H; (B)48H; (C)72H), through Figure 21 It can be seen that compound II showed over 100% cell viability against human gastric mucosal epithelial cells GES-1 at concentrations below 64 μM.

Claims

1. A cinnamic acid derivative, characterized in that, It has the following structural formula: 。 2. The method for preparing the cinnamic acid derivative according to claim 1, characterized in that, The process includes the following steps: under nitrogen protection, cinnamic acid derivative, phenol derivative, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine are reacted at room temperature for 0.5-1 h, the reaction solution is washed, the solvent phase is evaporated, and the cinnamic acid derivative is obtained by column chromatography. The molar ratio of the cinnamic acid derivative, the phenol derivative, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 4-dimethylaminopyridine is 4-6:4-6:5-7:0.4-1. The cinnamic acid substitute is 2,4-dinitrocinnamic acid; The phenol substitute is p-chlorophenol.

3. The method for preparing the cinnamic acid derivative according to claim 2, characterized in that, The molar ratio of the cinnamic acid derivative, the phenol derivative, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 4-dimethylaminopyridine is 5:5:6:0.

5.

4. The method for preparing the cinnamic acid derivative according to claim 2, characterized in that, The solvent is dichloromethane.

5. The method for preparing the cinnamic acid derivative according to claim 2, characterized in that, The washing process uses a saturated sodium bicarbonate solution.

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