Cinnamic acid derivatives, processes for their preparation and use thereof

Abstract

This invention belongs to the field of pharmaceutical technology and discloses a cinnamic acid derivative, its preparation method, and its application. The structural formula of the cinnamic acid derivative is shown in Formula 1A or Formula 1B, wherein there are 0 to 5 substituents R; the R at different substitution sites are independently selected from: fluorine, chlorine, bromine, iodine, trifluoromethyl, oxotrifluoromethyl, 2,3-phenyl, methyl, methoxy, ester, carboxyl, hydroxyl, and allyloxy. This invention improves the chemical structure of cinnamic acid derivatives and their preparation methods, resulting in novel cinnamic acid derivatives that can be used to prepare drugs. For example, they can exert therapeutic effects by inhibiting the MAPK signaling pathway, by inhibiting ferroptosis, and by activating UDP-glucuronyl transferase, and are particularly useful for treating liver injury.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and more specifically, relates to a cinnamic acid derivative, its preparation method, and its application. Background Technology

[0002] Cinnamon is a traditional Chinese medicinal herb, consistently listed as a superior-grade herb in ancient herbal texts. According to the 2015 edition of the Chinese Pharmacopoeia, the National Compendium of Traditional Chinese Medicine Standards, and the Ministry of Health's Drug Standards, there are 565 medicinal products in China that use cinnamon. Cinnamic acid is one of the main active monomeric components of cinnamon, and it has been proven to possess various physiological activities, including antioxidant, anticancer, anti-inflammatory, antibacterial, anti-allergic, and antithrombotic effects. Its effects on acute liver injury have also been reported in limited numbers. Cinnamic acid is also a crucial intermediate in fine organic synthesis, and its derivatives have wide applications in food, medicine, and daily chemicals. It can be used to synthesize important drugs for treating coronary heart disease, osteomodulators, local anesthetics, bactericides, and hemostatic agents. Furthermore, many cinnamic acid derivatives, especially those containing phenolic hydroxyl groups, are recognized antioxidants with strong free radical scavenging capabilities. Muhammad et al. reported that the scavenging activity of the cinnamic acid derivative 2,4-dihydroxy5-methoxycinnamic acid against DPPH free radicals in the concentration range of 2-50 μg / mL was similar to that of the antioxidants α-tocopherol and vitamin C; in anti-glycation experiments, this compound exhibited moderate activity similar to the standard drug rutin. Based on its antioxidant potential, cinnamic acid derivatives, such as ethylhexyl methoxycinnamate (cinnamic acid ester), isoamyl p-methoxycinnamate (amino acid ester), octoline, and cinnamic acid ester, are also widely used in cosmetics. In addition, some natural and synthetic derivatives of cinnamic acid also have skin whitening and anti-aging properties, especially 4-hydroxycinnamic acid, which is currently listed as a candidate new drug for the treatment of pigmentation. Cinnamic acid and its derivatives have also been widely reported in the prevention and treatment of diabetes and its complications, with mechanisms including stimulating insulin secretion, improving pancreatic β-cell function, and inhibiting hepatic gluconeogenesis. According to the latest review, several reported cinnamic acid derivatives have demonstrated superior efficacy compared to positive controls or clinical treatments in in vitro studies, including antitumor, antituberculosis (cinnamic acid itself has been used to treat pulmonary tuberculosis), anti-Staphylococcus aureus, antimalarial, and dual inhibition of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBuChE) (potential therapeutic effects for Alzheimer's disease). Furthermore, toxicological studies have confirmed the safety of these derivatives, indicating potential for further development.

[0003] On the other hand, acetaminophen (APAP) is an effective antipyretic and analgesic, but APAP overdose is the most significant cause of drug poisoning and acute liver failure. APAP overdose depletes glutathione (GSH), leading to the excessive accumulation of the intermediate metabolite N-acetyl-p-benzoquinone imine (NAPQI). NAPQI covalently binds to thiol groups in cellular proteins, especially mitochondrial proteins, resulting in hepatocellular damage and liver necrosis. Oxaliplatin (OXA) is a third-generation platinum-based anticancer drug. Liver injury is a common and serious adverse reaction of OXA, mainly manifested clinically as splenomegaly, thrombocytopenia, abnormal liver function, and portal hypertension. Its mechanism is closely related to OXA-induced activation of hepatic DNA damage repair pathways and oxidative stress, causing damage to hepatic sinusoidal endothelial cells, which in turn leads to platelet aggregation and vascular obstruction within the hepatic sinusoids. Summary of the Invention

[0004] In view of the above-mentioned defects or improvement needs of the prior art, the purpose of this invention is to provide a cinnamic acid derivative, its preparation method and application. By improving the chemical structure of the cinnamic acid derivative and its preparation method, a novel cinnamic acid derivative is designed that can be used to prepare drugs, for example, to exert therapeutic effects by inhibiting the MAPK signaling pathway, to exert therapeutic effects by inhibiting ferroptosis, and to exert detoxification effects by activating UDP-glucuronyltransferase, especially for the treatment of liver injury.

[0005] To achieve the above objectives, according to one aspect of the present invention, a cinnamic acid derivative is provided, characterized in that its structural formula is shown in Formula 1A or Formula 1B:

[0006]

[0007] And Exclusion Equation 18:

[0008]

[0009] For equation 1A or equation 1B:

[0010] The number of substituents R is 0 to 5; when the number of substituents R is 1 to 5, the substitution sites correspond to any 1 to all 5 of the positions 2, 3, 4, 5, and 6 of the benzene ring; the R at different substitution sites is independently selected from: fluorine, chlorine, bromine, iodine, trifluoromethyl, oxotrifluoromethyl, 2,3-phenyl, methyl, methoxy, ester, carboxyl, hydroxyl, and allyloxy.

[0011] The dashed and solid lines represent carbon-carbon single bonds or carbon-carbon double bonds.

[0012] As a further preferred embodiment of the present invention, the structural formula of the cinnamic acid derivative is specifically Formula 5 to 5.

[0013] Any one of Equations 17, 19 to 30:

[0014]

[0015] According to another aspect of the present invention, the present invention provides a method for synthesizing the above-mentioned cinnamic acid derivative, characterized by comprising the following steps:

[0016] (1) Using cinnamic acid as shown in Formula 3A as a reaction substrate, oxaloyl chloride undergoes a substitution reaction with a catalytic amount of DMF to obtain cinnamicoyl chloride as shown in Formula 4A;

[0017]

[0018] In Formula 3A, there are 0 to 5 substituents R1; when there are 1 to 5 substituents R1, the substitution sites correspond to any 1 to all 5 of the positions 2, 3, 4, 5, and 6 of the benzene ring; R1 at different substitution sites is independently selected from: fluorine, chlorine, bromine, iodine, trifluoromethyl, oxotrifluoromethyl, 2,3-phenyl, methyl, methoxy, and ester groups;

[0019] The dashed and solid lines represent carbon-carbon single bonds or carbon-carbon double bonds;

[0020] (2) The cinnamoyl chloride obtained in step (1) as shown in Formula 4A undergoes a substitution reaction with compound KD to generate a corresponding cinnamic acid derivative, the structural formula of which satisfies Formula 1A; or, the cinnamoyl chloride obtained in step (1) as shown in Formula 4A undergoes a substitution reaction with compound MDG to generate a corresponding cinnamic acid derivative, the structural formula of which satisfies Formula 1A.

[0021] Formula 1B;

[0022]

[0023] According to another aspect of the present invention, the present invention provides a method for synthesizing the above-mentioned cinnamic acid derivative, characterized by comprising the following steps:

[0024] S1: Cinnamic acid, as shown in Formula Cinnamic acids, undergoes a substitution reaction with allyl bromide under alkaline conditions to generate cinnamic ester as shown in Formula 2; then, the cinnamic ester as shown in Formula 2 is hydrolyzed under alkaline conditions and then acidified to obtain cinnamic acid as shown in Formula 3B.

[0025]

[0026] In the formula Cinnamic acids, there are 1 to 5 hydroxyl substituents, with substitution sites corresponding to any one to all five of the positions 2, 3, 4, 5, and 6 of the benzene ring; there are 0 to 4 substituents R2, and the sum of the number of hydroxyl substituents and substituents R2 does not exceed 5; when there are non-zero substituents R2, the substitution sites of substituents R2 correspond to any one or several of the positions 2, 3, 4, 5, and 6 of the benzene ring, excluding the hydroxyl substitution site; and R2 at different substitution sites is independently selected from: fluorine, chlorine, bromine, iodine, trifluoromethyl, oxotrifluoromethyl, 2,3-phenyl, methyl, methoxy, and ester groups;

[0027] The dashed and solid lines represent carbon-carbon single bonds or carbon-carbon double bonds;

[0028] S2: Using cinnamic acid as shown in Formula 3B obtained in step S1 as a reaction substrate, and oxaloyl chloride undergoing a substitution reaction with a catalytic amount of DMF to obtain cinnamicoyl chloride as shown in Formula 4B.

[0029]

[0030] S3: The cinnamoyl chloride obtained in step S2, as shown in Formula 4B, undergoes a substitution reaction with compound KD to generate the corresponding cinnamic acid derivative, the structural formula of which satisfies Formula 1A; or, the cinnamoyl chloride obtained in step S2, as shown in Formula 4B, undergoes a substitution reaction with compound MDG to generate the corresponding cinnamic acid derivative, the structural formula of which satisfies Formula 1B.

[0031]

[0032] According to another aspect of the present invention, the present invention provides the use of the above-mentioned cinnamic acid derivative in the preparation of a drug for treating liver injury.

[0033] As a further preferred embodiment of the present invention, the drug for treating liver injury is a drug for treating acute liver injury.

[0034] According to a final aspect of the invention, the invention also provides various applications of a cinnamic acid derivative, including formula 18, the structural formula of which is shown in formula 1A or formula 1B:

[0035]

[0036] For equation 1A or equation 1B:

[0037] The number of substituents R is 0 to 5; when the number of substituents R is 1 to 5, the substitution sites correspond to any 1 to all 5 of the positions 2, 3, 4, 5, and 6 of the benzene ring; the R at different substitution sites is independently selected from: fluorine, chlorine, bromine, iodine, trifluoromethyl, oxotrifluoromethyl, 2,3-phenyl, methyl, methoxy, ester, carboxyl, hydroxyl, and allyloxy.

[0038] The dashed and solid lines represent carbon-carbon single bonds or carbon-carbon double bonds;

[0039] in:

[0040] The first application is in the preparation of drugs that exert therapeutic effects by inhibiting the MAPK signaling pathway;

[0041] The second application is the use of this cinnamic acid derivative in the preparation of drugs that exert therapeutic effects by inhibiting ferroptosis;

[0042] The third application is the use of this cinnamic acid derivative in the preparation of drugs that exert therapeutic effects by activating UDP-glucuronyltransferase.

[0043] Compared with the prior art, the present invention, through the above-described technical solutions, integrates drug design concepts such as structural assembly and bioelectronic isochoricization to design and synthesize multiple cinnamic acid derivatives, which have the chemical structures shown in general formulas 1A and 1B. The synthesized novel cinnamic acid derivatives are structurally stable and non-cytotoxic within the effective concentration range (as illustrated in the examples below, the examples tested cinnamic acid derivatives at concentrations of 10 μM to 1000 μM, and it has been verified that a concentration of 1 mM is non-toxic and effective, and a concentration of 10 μM is also effective).

[0044] Specifically, the present invention can achieve the following beneficial effects:

[0045] (1) This invention provides novel cinnamic acid derivatives with specific chemical structures, which satisfy general formulas 1A and 1B. The following embodiments of this invention utilize high-resolution mass spectrometry, proton nuclear magnetic resonance (HMR) spectroscopy, and carbon nuclear magnetic resonance (CMR) spectroscopy to comprehensively analyze the synthesized cinnamic acid derivatives and determine their chemical structures. The chemical structures of some cinnamic acid derivatives (e.g., compounds 5–17 and 19–30) are shown below:

[0046]

[0047] (2) In view of the above-mentioned cinnamic acid derivatives with specific chemical structures, the present invention also provides two synthetic routes based on whether the raw materials are commercially available, which are as follows.

[0048]

[0049] Both R1 and R2 can be 0.

[0050] Route (a) uses a compound of formula 3A as its starting material, which can be obtained directly through commercial channels. R1 is any one or more groups (≥2, combinations of the same or different groups) selected from fluorine, chlorine, bromine, iodine, trifluoromethyl, oxotrifluoromethyl, 2,3-phenyl, methyl, methoxy, and ester groups, with substitution sites at positions 2, 3, 4, 5, and 6. Route (b) uses a compound of formula Cinnamic. Acids compounds (where there is ≥1 hydroxyl group, and the corresponding substitution sites are any one or more of positions 2, 3, 4, 5, and 6; R2 at different substitution sites is independently selected from fluorine, chlorine, bromine, iodine, trifluoromethyl, oxotrifluoromethyl, 2,3-phenyl, methyl, methoxy, and ester groups; the substitution sites are the remaining positions of positions 2, 3, 4, 5, and 6; and when there are ≥2 R2 groups, they can be combinations of the same or different groups) are first prepared as intermediate compound 3B (compound 3B is not currently commercially available). Compounds 3A and 3B can be used as reaction substrates to undergo substitution reactions with oxaloyl chloride under the action of a catalytic amount of DMF to obtain compounds 4A and 4B. They further undergo substitution reactions with KD to generate the corresponding cinnamic acid derivatives satisfying formula 1A; if they undergo substitution reactions with MDG, they generate the corresponding cinnamic acid derivatives satisfying formula 1B.

[0051] Taking route (b) as an example (steps three and four below are common to route (a) and will be explained together), step one: Cinnamic acid containing hydroxyl substituents (≥1 hydroxyl group) on the benzene ring undergoes a substitution reaction with allyl bromide under alkaline conditions (i.e., reaction i in the synthetic route) to generate cinnamate ester 2; step two: Cinnamate ester 2 obtained from step one undergoes a hydrolysis reaction under alkaline conditions (i.e., reaction ii in the synthetic route), followed by acidification (i.e., reaction iii in the synthetic route) to obtain a benzene ring containing allyloxy group. Substituent cinnamic acid 3B; Step 3: Using commercially available cinnamic acid 3A with or without substituents, or cinnamic acid 3B with an allyloxy substituent (≥1 allyloxy group) on the benzene ring, as the reaction substrate, cinnamic acid 3A with or without substituents, as the reaction substrate, and reacting with oxaloyl chloride in the presence of a catalytic amount of DMF (i.e., reaction iv in the synthetic route); Step 4: Using KD or MDG as the reaction substrate, reacting with cinnamoyl chloride 4 as the reaction substrate, and reacting with cinnamoyl chloride 4 as the reaction substrate (i.e., reaction v in the synthetic route), to generate the corresponding cinnamic acid derivatives 5–30.

[0052] In this invention, cinnamoyl chloride is used as one of the reaction substrates, and pyridine is used as a base to undergo substitution reactions with KD or MDG, respectively, successfully yielding the target cinnamic acid derivative. The above synthetic route ensures the preparation of the target cinnamic acid derivative having the structure shown in general formulas 1A and 1B. As illustrated in the comparative examples below, the inventors also attempted DCC or EDCI-promoted ester condensation reactions during the research and development process, but neither of these successfully provided the target cinnamic acid derivative.

[0053] (3) This invention used APAP-induced acute liver injury (ALI) cell models and mouse models for preliminary screening and found that these cinnamic acid derivatives have anti-ALI effects. Simultaneously, an OXA-induced ALI model was constructed to further verify the anti-ALI effects of these cinnamic acid derivatives. For example, in the following examples, compounds 16 and 28 showed stronger anti-liver injury effects than the positive control drug N-acetyl-L-cysteine ​​(NAC).

[0054] NAC is currently the only antidote used clinically for APAP-induced ALI, but it is only effective for patients within 8 hours of APAP poisoning. It is ineffective for patients in the late stages of APAP poisoning or with severe liver damage, and its large-scale use can prolong the patient's liver recovery and regeneration time. The cinnamic acid derivative developed in this invention has a stronger effect than the positive control NAC.

[0055] In addition, among the common enzymes involved in drug metabolism, UGT enzyme (uridine diphosphate glucuronide transferase) accounts for the second largest proportion and is one of the most important enzymes in phase II metabolism in the human body. UGT enzyme is a membrane protein bound to the endoplasmic reticulum, which plays a catalytic role in the transfer of glucuronic acid from UDP-glucuronic acid (UDPGA) to other molecules (usually hydrophobic molecules). UGT enzyme uses glucuronic acid as a glycosyl donor and widely catalyzes the binding reactions of endogenous (such as bilirubin, fatty acids, steroid hormones, food compounds, drugs, environmental pollutants, etc.) and exogenous chemical substances. Usually, the compounds formed after the binding reaction are more water-soluble and easily excreted from the body, thus playing a detoxification role and participating in the metabolic clearance process of many drugs. Therefore, the novel cinnamic acid derivative discovered in this invention, which can be used for diseases related to MAPK signaling pathway activation, ferroptosis response, and liver detoxification mechanisms, is of great significance for the development of related new drugs.

[0056] As illustrated in the following examples, this invention uses APAP-induced ALI cell and mouse models to demonstrate that multiple cinnamic acid derivatives possess anti-ALI activity, which is stronger than the positive control NAC. Simultaneously, OXA-induced cell and mouse ALI models were constructed to further verify the anti-ALI activity of the aforementioned compounds (i.e., cinnamic acid derivatives satisfying general formulas 1A and 1B, represented by compounds 16 and 28). Further mechanistic studies indicate that the cinnamic acid derivatives of this invention exert their effects by inhibiting MAPK signaling pathway activation and ferroptosis in vivo, and enhancing UDP-glucuronyltransferase activity. This invention discovers novel cinnamic acid derivatives related to the preparation of drugs for treating diseases involving MAPK signaling pathway activation, ferroptosis, and UDP-glucuronyltransferase activation, which is of great significance for the development of related new drugs.

[0057] For the past 50 years, researchers have been working to develop drugs that may be effective in treating ALI caused by APAP. Promising compounds reported so far include 4-methylpyrazole (4MP, a CYP450 enzyme and JNK inhibitor), calmangafodipir (CMFP, an SOD mimic), metformin, and methylene blue. However, these active molecules have not yet been marketed as drugs. Providing more active anti-ALI candidates would undoubtedly enrich preclinical research on anti-ALI drugs and greatly promote their development. Meanwhile, many other molecules and natural products have been reported to be effective in ALI models, but most studies have significant flaws in experimental design; for example, pretreating mice with compounds before APAP modeling and then determining efficacy, some even pretreating them for a month, is completely inconsistent with clinical practice and hinders the further development of these compounds. Therefore, more than 50 years after NAC was approved for clinical treatment of ALI, no other drugs are available on the market.

[0058] In this invention, as illustrated in the following embodiments, we first establish an experimental model of APAP poisoning 1 hour prior (at which point liver damage has already occurred), and then administer the test compound, similar to a patient undergoing clinical treatment. The hepatoprotective effect of the compound is then evaluated based on this. Simultaneously, we establish a high-dose APAP-induced acute liver failure (ALF) mouse model, which can simulate ALF patients caused by excessive APAP intake or late-stage patients with ordinary-dose APAP poisoning in clinical settings. These patients are essentially incurable clinically, even with NAC treatment. Taking compounds of formulas 16 and 28 as examples, survival experiments based on the ALF mouse model show that treatment with CK16 and CG28 significantly prolongs the survival time of mice, while NAC does not. These results demonstrate that CK16 and CG28 have stronger hepatoprotective effects than NAC. From a mechanistic perspective, the novel cinnamic acid derivatives CK16 and CG28 alleviate APAP hepatotoxicity by activating UDP-glycosyltransferase and enhancing APAP uronic acidification, while simultaneously inhibiting MAPK signaling pathway activation and ferroptosis. Compared to NAC, they are more effective and have completely different mechanisms, possessing certain advantages and novelty. CK16 and CG28 are also the only two UGT activators discovered so far, and no compounds with similar mechanisms have been reported. Attached Figure Description

[0059] Figure 1 Screening for the cytotoxic activity of cinnamic acid derivatives. In the figure, each gray-filled bar corresponds to the experimental results using different concentrations of KD, MDG, and compounds 5 through 30; the first blank bar corresponds to the negative control group treated with DMSO (<0.1%); for the remaining gray bars, "-" indicates that the corresponding compound was not applied (e.g., KD, compound 5, compound 9, NAC, etc.; the same applies below); "+" indicates that the corresponding compound was applied; the specific value indicates that the corresponding compound was applied and the compound concentration met the specified value.

[0060] Figure 2 This study aimed to screen mouse models for the anti-APAP-induced liver injury activity of cinnamic acid derivatives. The figure shows the injection of multiple test compounds one hour after APAP injection, followed by sample collection 23 hours later to detect serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in each experimental group. In the figure, the "Control" group represents normal mice, the "APAP" group represents the model group, and "NAC" represents the positive control group.

[0061] Figure 3 The cinnamic acid derivatives CK16 and CG28 exhibited anti-APAP-induced liver injury activity. Figure 3 In the diagram, A represents the manipulation and treatment of mice over time. Figure 3In this context, B represents the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice treated with NAC (50, 100, 200, 300 mg / kg). Figure 3 In this context, C represents the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice treated with CK16, CG28, or NAC. Figure 3 D in the image represents a representative liver photograph and a liver section stained with H&E. Figure 3 E in the figure represents the quantitative result of liver necrosis area in different groups; Figure 3 F in the image represents a representative image of liver TUNEL, HMGB1, and p-MLKL staining; Figure 3 In this context, G represents mice that received an intraperitoneal injection of APAP (600 mg / kg) for 1 hour, followed by treatment with CK16, CG28, or NAC. Survival rates within 24 hours were recorded for each group. Figure 3 Taking A as an example, it involves injecting multiple test compounds one hour after the APAP injection, and collecting samples to detect the results 23 hours later. Figure 3 The data in B through F also follow this experimental design.

[0062] Figure 4 The cinnamic acid derivatives CK16 and CG28 exhibited anti-oxaliplatin-induced liver injury activity. Figure 4 In the figure, A represents the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice in different treatment groups during in vivo animal experiments with oxaliplatin. Figure 4 In this context, B represents the activity assay of CK16, CG28, or NAC in an in vitro oxaliplatin-induced acute liver injury cell model.

[0063] Figure 5 Cinnamic acid derivatives CK16 and CG28 inhibit the activation of the MAPK signaling pathway in vivo. Figure 5 Western blot analysis was performed on the expression of p-P38, p38, p-ERK1 / 2, ERK1 / 2, p-JNK and JNK in the liver of control mice, APAP group mice and CK16 (or CG28) treated mice 24 hours after APAP treatment. β-actin was used as an internal reference for protein expression.

[0064] Figure 6 The cinnamic acid derivatives CK16 and CG28 inhibit ferroptosis in vivo. Figure 6 In the figure, A represents the expression of 4-HNE in liver tissue as shown by immunohistochemical staining (scale bar: 80 μm); Figure 6 In the image, B represents immunohistochemical staining showing that DAB enhances the expression of Prussian blue in liver tissue (scale bar: 200 μm). Figure 6C in the figure represents the expression of GPX4, FSP1, SLC7A11, TfR1, FTL, FTH1, FPN and FABP4 in the liver of mice in the control group, APAP group and CK16 or CG28 group 24 hours after APAP treatment. β-actin is the internal reference for protein expression. Figure 6 D in the figure represents the mRNA expression of FGF21, DHODH, FTH1, GPX4, SLC7A11, DHFR, TfR1, FPN, FTL and GCH1 in mouse liver detected by RT-PCR.

[0065] Figure 7 To increase the UDP-glucuronyl transferase activity of cinnamic acid derivatives CK16 and CG28. Figure 7 In this study, A represents the reaction kinetics of glucuronidation of the fluorescent substrate in mouse donor liver microsomes (MLMs, 0.05 mg / mL) at 37 °C, and the inhibition of UGT activity in MLMs by CK16 or CG28 and the non-selective UGT ligand diclofenac. Under blank reaction conditions, the dose-response curves of UGT activation in MLMs using the carrier (assay buffer) instead of the cofactor UDPGA, CK16, or CG28 were compared with the reaction activity of the carrier-only formulation, and the percentage of activity at each concentration was calculated. Figure 7 In this context, B represents the levels of APAP-Cys, APAP-Gluc, and APAP-Sulf in mouse serum. Detailed Implementation

[0066] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0067] Compound KD used in Example 1 below was extracted and isolated from the natural plant *Anoectochilus roxburghii* using existing techniques (Journal of Ethnopharmacology 2007, 114, 141–145). Compound MDG was purchased from a reagent store; its Chinese name is methyl-α-D-glucopyranoside, and its CAS number is 97-30-3. The reactant compound 3A and the reactant cinnamic acids were also purchased from a reagent store.

[0068] Example 1: Synthesis and Structural Identification of Cinnamic Acid Derivatives (I) The synthesis route of cinnamic acid derivatives is shown below:

[0069]

[0070] The synthetic routes and key reactants used in Formulas 5–30 of this invention are shown in the table below:

[0071]

[0072] Taking cinnamic acid derivative 28 as an example, the substrate raw materials and synthetic intermediates required for its synthesis are as follows:

[0073]

[0074] The reactions in the synthetic route were carried out in a refined manner as follows:

[0075] (i-iii) Synthesis of cinnamic acid 3B (with ≥1 allyloxy substituent on the benzene ring)

[0076]

[0077] Add magnetic flux, cinnamic acid (30 mmol, 1.0 equiv.), and potassium carbonate (x mmol. Assuming a compound has one hydroxyl and one carboxyl group, x = 180 mmol = 6.0 equiv.; assuming a compound has two hydroxyl and one carboxyl group, x = 270 mmol = 9.0 equiv.; taking the preparation of cinnamic acid derivative 28 as an example, cinnamic acid derivative 28 has one allyloxy group in its structure, and the raw material cinnamic acid for the synthesis of 28 contains one hydroxyl and one carboxyl group, then 180 mmol of potassium carbonate, i.e., 6.0 equiv., should be added). Add 120 mL of acetonitrile as the reaction solvent, and slowly add allyl bromide (x mmol, equivalent to the amount of potassium carbonate; dissolved in an appropriate amount of acetonitrile solvent) in batches by injection under stirring. Then, transfer the reaction flask to an 80°C oil bath and heat under reflux overnight. The following day, the reaction flask was moved to room temperature to cool, and the reaction was monitored by TLC. After the reaction was complete, the reaction solution was filtered, the residue was washed with ethyl acetate, and the filtrate (a mixture of acetonitrile and ethyl acetate) was collected. The filtrate was concentrated under rotary evaporation conditions, and the concentrate was dissolved in ethyl acetate. The solution was extracted three times by two-phase extraction with ethyl acetate and water. The ethyl acetate layer was collected, dried with anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation to obtain cinnamate 2. No further purification was required, and it could be used directly in the next step.

[0078] The cinnamate 2 (25 mmol, 1.0 equiv.) obtained in the previous step was added to a dry reaction flask. A magnetic stir bar was added, followed by a mixture of methanol and water (40 mL: 40 mL). The mixture was stirred at room temperature. Sodium hydroxide (100 mmol, 4.0 equiv.) was slowly added to the reaction system in portions. After the addition was complete, the reaction flask was transferred to a 60°C oil bath and heated under reflux overnight. The following day, the reaction flask was moved to room temperature to cool, and the reaction was monitored by TLC. After the reaction was complete, preliminary concentration was carried out under rotary evaporation conditions to remove methanol solvent. Subsequently, the concentrate was dissolved in dichloromethane and extracted three times using dichloromethane and water as two phases. The aqueous layer was collected and placed in a separatory funnel. 1M HCl aqueous solution was added to adjust the pH to 1-2. An appropriate amount of ethyl acetate was added, and the mixture was extracted three times. The ethyl acetate layer was collected, and TLC was performed on the aqueous layer to observe whether the organic matter in the aqueous layer had been completely extracted by ethyl acetate. Anhydrous sodium sulfate was added to the collected ethyl acetate layer for drying, and the mixture was filtered and concentrated by rotary evaporation to obtain cinnamic acid 3B. No further purification treatment was required, and it could be used directly in the next step.

[0079] (iv) Synthesize cinnamyl chloride 4A or 4B (with ≥1 allyloxy substituent on the benzene ring)

[0080]

[0081] Cinnamyl acid 3A (15 mmol, 1.0 equiv.) obtained commercially or cinnamyl acid 3B (15 mmol, 1.0 equiv.) prepared via the above steps were added to dry reaction flasks. A magnetic stir bar was added, followed by 40 mL of anhydrous dichloromethane and a catalytic amount of N,N-dimethylformamide (0.75 mmol, 0.05 equiv.). Oxaloyl chloride (45 mmol, 3.0 equiv.) was then slowly added dropwise via injection. The reaction mixture was stirred at room temperature for 2–6 h. Once the reaction was complete, it was ready for further processing via TLC. Note that gas is generated in this reaction system; therefore, gas balance should be maintained during the addition of reactants. After the reaction was complete, the mixture was concentrated using a rotary evaporator to remove the solvent, yielding cinnamyl chloride 4A or 4B. No further purification is required; it can be used directly in the next step.

[0082] (v) Synthetic cinnamic acid derivatives 5–30

[0083]

[0084] Add KD (10 mmol, 1.0 equiv.) or MDG (10 mmol, 1.0 equiv.) to a dry reaction flask, add a magnetic stir bar, add dry anhydrous pyridine (100 mmol, 10.0 equiv.), and add an appropriate amount of anhydrous dichloromethane as a solvent. Stir the reaction flask at -10°C. Dissolve cinnamyl chloride 4A (11 mmol, 1.1 equiv.) or 4B (11 mmol, 1.1 equiv.) prepared in the above steps in anhydrous dichloromethane, and slowly add them dropwise to the reaction flask by injection. Then, move the reaction flask to room temperature and continue stirring overnight. The next day, perform TLC to monitor the reaction completion. Quench the reaction system with a small amount of methanol, concentrate the solution using a rotary evaporator, and transfer the concentrate to a separatory funnel after dissolving it in dichloromethane. Add 1M... The pH of the HCl aqueous solution was adjusted to 5-6, and the mixture was extracted approximately six times using dichloromethane and water as a two-phase extraction process until the dichloromethane layer no longer contained the target compound. The dichloromethane layer was collected, dried with anhydrous sodium sulfate, filtered, and concentrated using a rotary evaporator to obtain the crude product. The crude product was dissolved in an appropriate amount of dichloromethane, and 100-200 mesh silica gel was added for mixing. Column chromatography was then performed (200-300 mesh silica gel, petroleum ether: ethyl acetate = 3:1–1:4) to obtain cinnamic acid derivatives 5–30.

[0085] (II) Structural identification of cinnamic acid derivatives 5–30

[0086] The structure of the cinnamic acid derivative 5–30 was determined by comprehensive analysis of high-resolution mass spectrometry, proton NMR, and carbon NMR data. The results are as follows:

[0087] ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((R)-5-oxotetrahydrofuran-3yl)oxy)tetrahydro-2H-pyran-2-yl)methyl cinnamate(5)

[0088] 1H),3.42–3.34(m,2H),3.25–3.19(m,1H),2.85(dd,J=18.0,6.5Hz,1H),2.64(ddd,J=18.0,2.1,0.9Hz,1H). 13C NMR(150MHz,MeOD)δ178.4,168.4,146.6,135.7,131.6,130.0,129.3,118.6,104.0,77.7,76.7,76.1,75.5,74.8,71.5,64.6,36.0.HRMS(ESI):calcd.ForC 19 H 22 O9Na[M+Na] + :417.1156,found 417.1163.

[0089] ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((R)-5-oxotetrahydrofuran-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl 3-phenylpropanoate(6)

[0090] 8.9Hz,1H),3.17(dd,J=9.2,7.8Hz,1H),2.94(t,J=7.6Hz,2H),2.83(dd,J=18.0,6.6Hz,1H),2.68(t,J=7.6Hz,2H),2.62(ddd,J=18.0,2.1,0.9Hz,1H). 13 C NMR(150MHz,MeOD)δ177.0,173.0,140.6,128.1,128.0,125.9,102.6,76.3,75.3,74.7,74.0,73.3,70.0,63.1,35.4,34.7,30.5.HRMS(ESI):calcd.for C 19 H 24 O9Na[M+Na] + :419.1313,found 419.1312.

[0091] ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((R)-5-oxotetrahydrofuran-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl(E)-3-(4-fluorophenyl)acrylate(7)

[0092] 3.23–3.19(m,1H),2.85(dd,J=18.0,6.6Hz,1H),2.63(ddd,J=18.1,2.1,0.9Hz,1H). 13 CNMR(150MHz,MeOD)δ178.4,168.3,165.4(d,J=249Hz),145.3,132.2(d,J=3Hz),131.5(d,J=9Hz),118.5(d,J=3Hz),116.9(d,J=22.5Hz),104.0,77.7,76.7,76.1,75.5,74.8,71.5,64.6,36.0.HRMS(ESI):calcd.for C 19 H 21 FO9Na[M+Na] + :435.1062,found 435.1059.

[0093] ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((R)-5-oxotetrahydrofuran-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl(E)-3-(4-chlorophenyl)acrylate(8)

[0094] 3.42–3.33(m,2H),3.25–3.17(m,1H),2.85(dd,J=18.0,6.5Hz,1H),2.63(dd,J=17.9,2.1Hz,1H). 13 C NMR(100MHz,MeOD)δ178.5,168.2,145.2,137.5,134.6,130.9,130.3,119.6,104.2,77.8,76.8,76.2,75.6,74.9,71.6,64.8,36.2.HRMS(ESI):calcd.forC 19 H 21 ClO9Na[M+Na] + :451.0766,found 451.0769,453.0742.

[0095] ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((R)-5-oxotetrahydrofuran-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl(E)-3-(4-bromophenyl)acrylate(9)

[0096] =8.3Hz,1H),2.85(dd,J=18.1,6.5Hz,1H),2.63(dd,J=18.1,1.9Hz,1H). 13 C NMR(150MHz,MeOD)δ178.4,168.1,145.1,134.8,133.2,130.9,125.6,119.6,104.0,77.7,76.7,76.1,75.5,74.8,71.5,64.7,36.0.HRMS(ESI):calcd.for C 19 H 21 BrO9Na[M+Na] + :495.0261,found 495.0264,497.0243.

[0097] ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((R)-5-oxotetrahydrofuran-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl(E)-3-(4-(trifluoromethyl)phenyl)acrylate(10)

[0098] 4.6Hz,1H),4.42(d,J=7.8Hz,1H),4.38(dd,J=11.9,5.9Hz,1H),3.57(ddd,J=8.6,5.8,2.2Hz,1H),3.41–3.36(m,2H),3.22(t,J=8.2Hz,1H),2.85(dd,J=18.0,6.5Hz,1H),2.64(dd,J=18.1,2.0Hz,1H). 13C NMR(150MHz,MeOD)δ178.4,167.8,144.5,139.5,132.8(q,J=31.5Hz),129.8,126.9(q,J=4.5Hz),125.4(d,J=270Hz),121.6,104.0,77.9,76.7,76.1,75.5,74.8,71.5,64.8,36.0.HRMS(ESI):calcd.for C 20 H 21 F3O9Na[M+Na] + :485.1030,found485.1042.

[0099] ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((R)-5-oxotetrahydrofuran-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl(E)-3-(naphthalen-1-yl)acrylate(11)

[0100] Hz,1H),4.52(ddd,J=10.3,1.8,0.9Hz,1H),4.45(d,J=4.8Hz,1H),4.46–4.39(m,2H),3.60(ddd,J=9.6,6.0,2.3Hz,1H),3.43–3.38(m,2H),3.27–3.21(m,1H),2.84(dd,J=18.1,6.6Hz,1H),2.64(ddd,J=18.1,2.1,0.9Hz,1H).HRMS(ESI):calcd.for C 23 H 24 O9Na[M+Na] + :467.1313,found 467.1308.

[0101] ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((R)-5-oxotetrahydrofuran-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl(E)-3-(p-tolyl)acrylate(12)

[0102] 3.37–3.25(m,2H),3.20–3.10(m,1H),2.76(dd,J=18.0,6.5Hz,1H),2.55(dd,J=18.0,2.0Hz,1H),2.26(s,3H). 13 C NMR(100MHz,MeOD)δ178.4,168.6,146.6,142.3,132.9,130.7,129.3,117.5,104.0,77.7,76.6,76.1,75.5,74.7,71.5,64.6,36.0,21.5.HRMS(ESI):calcd.for C 20 H 24 O9Na[M+Na] + :431.1313,found 431.1310.

[0103] ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((R)-5-oxotetrahydrofuran-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl(E)-3-(4-methoxyphenyl)acrylate(13)

[0104] J=8.1,3.6,2.2Hz,1H),3.40–3.33(m,2H),3.21(td,J=7.7,1.5Hz,1H),2.85(dd,J=18.1,6.6Hz,1H),2.63(ddd,J=18.0,2.0,0.8Hz,1H). 13 C NMR(150MHz,MeOD)δ178.4,168.8,163.2,150.0,146.4,131.0,128.2,115.8,115.4,104.0,77.7,76.6,76.1,75.5,74.7,71.5,64.5,55.9,36.0.HRMS(ESI):calcd.for C 20 H 24 O 10 Na[M+Na] + :447.1262,found447.1258.

[0105] ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((R)-5-oxotetrahydrofuran-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl(E)-3-(4-(allyloxy)phenyl)acrylate(14)

[0106] 4.54(dd,J=11.9,2.2Hz,1H),4.49(dd,J=10.3,1.9Hz,1H),4.47–4.40(m,1H),4.41(d,J=7.8Hz,1H),4.35(dd,J=11.9,6.1Hz,1H),3.56(ddd,J=8.7,6.2,2.2Hz,1H),3.44–3.31(m,2H),3.22(t,J=8.3Hz,1H),2.85(dd,J=18.0,6.5Hz,1H),2.63(dd,J=17.8,2.0Hz,1H). 13 C NMR(100MHz,MeOD)δ178.4,168.8,162.1,146.4,134.5,131.0,128.4,117.8,116.2,115.9,104.0,77.9,76.6,76.1,75.5,74.7,71.5,69.8,64.5,36.0.HRMS(ESI):calcd.for C 22 H 26 O 10 Na[M+Na] + :473.1418,found 473.1413.

[0107] ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((R)-5-oxotetrahydrofuran-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl(E)-3-(3-(allyloxy)-4-methoxyphenyl)acrylate(15)

[0108] 2.1Hz,1H),4.60(dt,J=5.4,1.6Hz,2H),4.54(dd,J=11.9,2.2Hz,1H),4.49(dd,J=10.2,1.9Hz,1H),4.45(d,J=4.7Hz,1H),4.40(d,J=7.8Hz,1H),4.35(dd,J=11.9,6.0Hz,1H),3.86(s,3H),3.55(ddd,J=8.6,6.0,2.3Hz,1H),3.44–3.34(m,2H),3.26–3.17(m,1H),2.85(dd,J=18.0,6.5Hz,1H),2.63(dd,J=17.9,2.1Hz,1H). 13 C NMR(100MHz,MeOD)δ178.3,168.7,153.3,149.6,146.6,134.7,128.6,124.3,117.9,116.2,113.7,112.9,104.0,77.7,76.6,76.1,75.5,74.8,71.5,70.9,64.5,56.5,36.0.HRMS(ESI):calcd.for C 23 H 28 O 11 Na[M+Na] + :503.1524,found 503.1521.

[0109] ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((R)-5-oxotetrahydrofuran-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl(E)-3-(4-(allyloxy)-3,5-dimethoxyphenyl)acrylate(16)

[0110] (dd,J=12.0,6.0Hz,1H),3.86(s,6H),3.55(ddd,J=8.6,5.9,2.3Hz,1H),3.42–3.33(m,2H),3.24–3.18(m,1H),2.85(dd,J=18.1,6.6Hz,1H),2.63(dd,J=18.0,2.0Hz,1H). 13CNMR(150MHz,MeOD)δ178.3,168.5,155.0,146.7,139.9,135.6,131.5,118.1,117.9,106.7,104.1,77.7,76.7,76.1,75.5,75.1,74.8,71.5,64.5,56.7,36.0.HRMS(ESI):calcd.forC 24 H 30 O 12 Na[M+Na] + :533.1629,found 533.1630.

[0111] ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((R)-5-oxotetrahydrofuran-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl(E)-3-(3,4-bis(allyloxy)phenyl)acrylate(17)

[0112] J=11.9,2.3Hz,1H),4.49(ddd,J=10.4,1.9,0.9Hz,1H),4.44(dd,J=10.4,4.8Hz,1H),4.40(d,J=7.8Hz,1H),4.35(dd,J=11.9,6.1Hz,1H),3.58–3.52(m,1H),3.42–3.33(m,2H),3.24–3.18(m,1H),2.85(dd,J=18.1,6.6Hz,1H),2.63(ddd,J=18.0,2.2,0.9Hz,1H). 13 C NMR(150MHz,MeOD)δ178.3,168.7,152.3,150.0,146.6,134.8,134.5,128.9,124.1,117.8,117.7,116.3,114.8,114.2,104.0,77.7,76.7,76.1,75.5,74.8,71.5,71.0,70.7,64.5,36.0.HRMS(ESI):calcd.for C 25 H 30 O 11 Na[M+Na] + :529.1680,found 529.1684.

[0113] ((2R,3S,4S,5R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl cinnamate(18)

[0114] 131.6,130.0,129.3,118.6,101.3,75.0,73.5,71.9,71.1,64.9,55.6.HRMS(ESI):calcd.for C 16 H 20 O7Na[M+Na] + :347.1101,found 347.1104.

[0115] ((2R,3S,4S,5R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl(E)-3-(4-fluorophenyl)acrylate(19)

[0116] 248Hz),145.1,132.2(d,J=3Hz),131.5(d,J=9Hz),118.5(d,J=2Hz),116.9(d,J=22Hz),101.3,75.0,73.5,71.9,71.1,65.0,55.6.HRMS(ESI):calcd.for C 16 H 19 FO7Na[M+Na] + :365.1007,found 365.1016.

[0117] ((2R,3S,4S,5R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl(E)-3-(4-chlorophenyl)acrylate(20)

[0118] 168.2,144.9,137.3,134.4,130.7,130.2,119.5,101.3,75.0,73.5,71.9,71.1,65.0,55.6.HRMS(ESI):calcd.for C 16 H 19 ClO7Na[M+Na] +:381.0712,found 381.0709,383.0677.

[0119] ((2R,3S,4S,5R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl(E)-3-(4-bromophenyl)acrylate(21)

[0120] 145.0,134.8,133.2,130.9,125.5,119.6,101.3,75.0,73.5,71.9,71.1,65.0,55.6.HRMS(ESI):calcd.for C 16 H 19 BrO7Na[M+Na] + :425.0206,found 425.0201,427.0181.

[0121] ((2R,3S,4S,5R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl(E)-3-(4-(trifluoromethyl)phenyl)acrylate(22)

[0122] MeOD)δ167.8,144.4,139.5,132.7(q,J=31.5Hz),129.7,126.9(q,J=4.5Hz),125.4(d,J=270Hz),121.6,101.3,75.0,73.5,71.9,71.1,65.1,55.6.HRMS(ESI):calcd.forC 17 H 19 F3O7Na[M+Na] + :415.0975,found 415.0982.

[0123] ((2R,3S,4S,5R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl(E)-3-(naphthalen-1-yl)acrylate(23)

[0124] (m,1H),3.67(dd,J=9.7,8.9Hz,1H),3.48–3.44(m,1H),3.44(s,3H),3.40(dd,J=10.1,8.8Hz,1H). 13 C NMR(150MHz,MeOD)δ167.0,141.7,133.8,131.3,131.1,130.5,128.5,126.7,125.9,125.2,124.8,122.6,119.7,99.94,73.7,72.1,70.6,69.8,63.7,54.3.HRMS(ESI):calcd.for C 20 H 22 O7Na[M+Na] + :397.1258,found 397.1261.

[0125] ((2R,3S,4S,5R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl(E)-3-(p-tolyl)acrylate(24)

[0126] (m,1H),2.34(s,3H). 13 C NMR(100MHz,MeOD)δ168.6,146.5,142.2,132.9,130.7,129.3,117.5,101.2,75.0,73.4,71.9,71.1,64.9,55.6,21.5.HRMS(ESI):calcd.for C 17 H 22 O7Na[M+Na] + :361.1258,found 361.1254.

[0127] ((2R,3S,4S,5R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl(E)-3-(4-methoxyphenyl)acrylate(25)

[0128] (m,1H). 13C NMR(150MHz,MeOD)δ168.9,163.2,146.3,131.0,128.2,115.9,115.4,101.3,75.0,73.5,71.9,71.1,64.8,55.9,55.6.HRMS(ESI):calcd.for C 17 H 22 O8Na[M+Na] + :377.1207,found 377.1207.

[0129] ((2R,3S,4S,5R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl(E)-3-(4-(allyloxy)phenyl)acrylate(26)

[0130] (dd,J=11.9,2.2Hz,1H),4.32(dd,J=11.9,5.9Hz,1H),3.78(ddd,J=10.1,5.9,2.2Hz,1H),3.64(t,J=9.2Hz,1H),3.44(d,J=3.7Hz,1H),3.41(s,3H),3.35(dd,J=10.1,8.9Hz,1H). 13 C NMR(100MHz,MeOD)δ168.9,162.1,146.3,134.5,131.0,128.4,117.8,116.2,116.0,101.3,75.1,73.5,71.9,71.1,69.8,64.8,55.6.HRMS(ESI):calcd.for C 19 H 24 O8Na[M+Na] + :403.1363,found 403.1357.

[0131] ((2R,3S,4S,5R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl(E)-3-(3-(allyloxy)-4-methoxyphenyl)acrylate(27)

[0132] 11.8,5.9Hz,1H),3.87(s,3H),3.78(ddd,J=10.1,5.9,2.2Hz,1H),3.64(t,J=9.3Hz,1H),3.46–3.41(m,1H),3.42(s,3H),3.36(dd,J=10.0,8.9Hz,1H). 13 C NMR(150MHz,MeOD)δ167.5,152.0,148.2,145.2,133.4,127.2,123.0,116.5,114.8,112.3,111.5,99.9,73.7,72.1,70.5,69.8,69.6,63.4,55.0,54.2.HRMS(ESI):calcd.for C 20 H 26 O9Na[M+Na] + :433.1469,found 433.1471.

[0133] ((2R,3S,4S,5R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl(E)-3-(4-(allyloxy)-3,5-dimethoxyphenyl)acrylate(28)

[0134] 1H),3.42(s,3H),3.36(dd,J=10.1,8.9Hz,1H). 13 C NMR(150MHz,MeOD)δ167.2,153.6,145.2,138.5,134.2,130.2,116.7,116.5,105.3,99.9,73.7,73.6,72.1,70.5,69.7,63.5,55.3,54.3.HRMS(ESI):calcd.for C 21 H 28 O 10 Na[M+Na] + :463.1575,found 463.1580.

[0135] ((2R,3S,4S,5R)-3,4,5-trihydroxy-6-methoxytetrahydro-2H-pyran-2-yl)methyl(E)-3-(3,4-bis(allyloxy)phenyl)acrylate(29)

[0136] 3.78 (ddd, J = 10.1, 5.8, 2.2 Hz, 1H), 3.64 (dd, J = 9.6, 8.9 Hz, 1H), 3.45–3.42 (m, 1H), 3.42 (s, 3H), 3.36 (dd, J = 10.1, 8.9 Hz, 1H). 13 C NMR (150 MHz, MeOD) δ 167.5, 150.9, 148.6, 145.1, 133.4, 133.1, 127.5, 122.8, 116.5, 116.3, 114.9, 113.4, 112.8, 99.9, 73.7, 72.1, 70.5, 69.8, 69.6, 69.3, 63.4, 54.3. HRMS (ESI): calcd. for C 22 H 28 O9Na [M+Na] + : 459.1626, found 459.1624.

[0137] ((2R,3S,4S,5R,6R)-3,4,5-trihydroxy-6-(((R)-5-oxotetrahydrofuran-3-yl)oxy)tetrahydro-2H-pyran-2-yl)methyl (E)-3-(3-fluorophenyl)acrylate (30)

[0138] 2.1 Hz, 1H), 3.41–3.33 (m, 2H), 3.25–3.18 (m, 1H), 2.86 (dd, J = 18.0, 6.5 Hz, 1H), 2.63 (dd, J = 18.4, 2.1 Hz, 1H). 13 C NMR (100 MHz, MeOD) δ 178.4, 168.0, 164.5 (d, J = 244 Hz), 145.1 (d, J = 3 Hz), 138.1 (d, J = 8 Hz), 131.8 (d, J = 8 Hz), 125.5 (d, J = 3 Hz), 120.3, 118.2 (d, J = 21 Hz), 115.3 (d, J = 22 Hz), 104.0, 77.7, 76.7, 76.1, 75.5, 74.7, 71.5, 64.7, 36.0. HRMS (ESI): calcd. for C 19 H 21 FO9Na [M+Na] + : 435.1062, found 435.1068.

[0139] Comparative example

[0140] In addition to the preparation method described in Example 1 above, the inventors also attempted two common ester condensation methods—DCC- and EDCI-promoted ester condensation reactions—to prepare cinnamic acid derivatives 5–30, but neither method successfully yielded the target cinnamic acid derivatives. Taking compound 5 as an example:

[0141] Method 1: EDCI-promoted ester condensation reaction: EDCI (1-ethyl-3-(3-dimethylpropylamine)carbodiimide, 0.34 mmol, 1.7 equiv.) and 4-DMAP (4-dimethylaminopyridine, 0.02 mmol, 0.1 equiv.) were added to a dry reaction flask, along with anhydrous dichloromethane (4 mL) as a solvent. The reaction flask was moved to 0°C, and triethylamine (0.4 mmol, 2.0 equiv.) was slowly added. Subsequently, cinnamic acid (0.3 mmol, 1.5 equiv.) was slowly added in portions. The mixture was stirred at 0°C for 15 minutes, and MDG (0.2 mmol, 1.0 equiv.) was added. The reaction flask was moved to room temperature and stirred overnight. The next day, TLC was performed, and the target product 5 was not detected.

[0142] Method 2: DCC-promoted ester condensation reaction: Cinnamic acid (0.24 mmol, 1.2 equiv.) was added to a dry reaction flask, a magnetic stir bar was added, and anhydrous dichloromethane solvent (4 mL) was added as a solvent. The mixture was stirred at 0 °C, and MDG (0.2 mmol, 1.0 equiv.) was added. Subsequently, DMAP (0.02 mmol, 0.1 equiv.) and DCC (dicyclohexylcarbodiimide, 0.3 mmol, 1.5 equiv.) were added to the reaction flask in sequence. The reaction flask was moved to room temperature and stirred overnight. The next day, TLC was performed, and the target product 5 was not found.

[0143] Example 2: Determination of the cytotoxic and anti-hepatotoxic activities of cinnamic acid derivatives

[0144] (1) The cytotoxic effects of cinnamic acid derivatives were first evaluated using cell viability assay (CCK-8 kit). Primary immortalized mouse normal hepatocyte line AML12 was used as the target cell line. AML12 cells were treated with cinnamic acid derivatives (10, 100, 1000 μM) for 48 hours, and then the proportion of viable cells was detected using the CCK-8 kit. The results showed that, except for compounds 21, 22, and 23, whose cell viability was less than 50% at a concentration of 1000 μM, the other compounds had no cytotoxic effect on cells at all concentrations, indicating that these compounds are generally safe. Figure 1 ).

[0145] (2) An acute liver injury model was established in C57BL / 6J mice by intraperitoneal injection of 300 mg / kg APAP. One hour after APAP injection, mice were injected intraperitoneally with different cinnamic acid derivatives (200 mg / kg) and NAC (300 mg / kg). The experiment was terminated after 24 hours of APAP treatment, and relevant mouse samples were collected for relevant tests. The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels of mice were significantly increased 24 hours after APAP injection. The increased ALT activity indicates the degree of liver inflammation, while AST indicates that hepatocytes have died and is a recognized marker of severe liver injury. The AST results showed that compounds 5, 12, 13, 14, 15, 16, 18, 21, 22, 27, 28, 29, and 30 reduced the elevated AST levels caused by APAP treatment to varying degrees, while compounds 12, 16, 22, 28, and 29 reduced the elevated ALT levels caused by APAP treatment to varying degrees. Compounds 16 and 28, in particular, showed a strong antagonistic effect against APAP-induced acute liver injury, as evidenced by the simultaneous reduction in both ALT and AST levels, even approaching the effect of the positive control NAC. This strongly demonstrates the therapeutic potential of compounds 16 and 28 in antagonizing APAP-induced acute liver injury. Figure 2 In the figure, the "Control" group represents normal mice, the "APAP" group represents the model group, and the "NAC" group represents the positive control group.

[0146] The inventors of this invention previously reported on cinnamic acid derivatives (compound MCGP) as shown in Formula 18, which are mainly used for drug-induced liver injury induced by acetaminophen (APAP) and chemical liver injury induced by carbon tetrachloride. Its mechanism may be related to inhibiting oxidative stress, inhibiting hepatocyte apoptosis, and promoting liver regeneration (Reference: Front. Pharmacol. 2022, 13, 873938.). The administration method in that paper differs significantly from that of this invention: that paper used MCGP pre-administered for 10 days before initiating ALI mouse modeling, while this invention administers cinnamic acid derivatives (Formulas 1A–1B) after ALI modeling has occurred in mice, and only after liver injury has already occurred. This experimental protocol in this invention is more in line with clinical practice, because most ALI patients in clinical practice cannot predict that they will develop ALI beforehand and will not take medication in advance. More importantly, in the experiments of this invention, MCGP showed weak anti-hepatotoxic effects, while several compounds described in this invention (such as compounds 5, 12, 13, 14, 15, 16, 21, 22, 27, 28, 29, 30) exhibited stronger anti-hepatotoxic effects than MCGP.

[0147] Example 3: Determination of the anti-hepatic effects of representative compounds 16 (denoted as CK16) and 28 (denoted as CG28) by inhibiting MAPK signaling pathway activation, inhibiting ferroptosis, and activating UDP-glucuronyltransferase.

[0148] (1) To confirm the effects of CK16 and CG28 on APAP-induced acute liver injury, mice were intraperitoneally injected with 300 mg / kg APAP for 1 h and then treated with different concentrations of CK16 and CG28 (100, 200, 300 mg / kg) (see [link to relevant documentation]). Figure 3 (A) The solvent was 2% DMSO and 98% corn oil (volume ratio: 2:98), used as a control. An investigation was conducted on the dosage of NAC, and it was found that only at 300 mg / kg did NAC significantly alleviate the increase in serum ALT and AST induced by APAP. Therefore, in subsequent experiments, we used 300 mg / kg NAC as a positive reference (see section A). Figure 3 (B in the text). Intraperitoneal injection of CK16 and CG28 1 hour after APAP injection significantly reduced the APAP-induced increase in ALT and AST levels in mice (see [reference]). Figure 3 (C in the text). Histological examination with H&E staining showed that CK16 and CG28 significantly improved APAP-induced liver hemorrhage, steatosis, hepatocyte swelling, and necrosis (see C). Figure 3 (D and E in the text). Furthermore, immunofluorescence staining of liver specimens showed that the number of TUNEL-positive cells in mice treated with CK16 and CG28 was significantly lower than in the APAP-treated group, and CK16 and CG28 treatment significantly inhibited the translocation of HMGB1 to the cytoplasm in APAP-treated mice. Immunochemical staining results of mouse liver tissue showed that, compared with the APAP model group, the expression level of p-MLKL was significantly reduced after CK16 and CG28 administration (see D and E in the text). Figure 3 (F in the original text). We also examined the effects of CK16 and CG28 on APAP-induced mortality by intraperitoneal injection of a lethal dose of APAP (600 mg / kg). Mice survival was monitored every 4 hours until 24 hours post-administration. During this observation period, CK16 and CG28 administration significantly improved mouse survival, and the survival rate of mice treated with CK16 and CG28 was significantly higher than that of the NAC group, indicating that CK16 and CG28 mice had a stronger ability to alleviate APAP-induced liver injury than NAC (see F in the original text). Figure 3 (G in the text). In summary, these results indicate that CK16 and CG28 can effectively ameliorate APAP-induced acute liver injury in mice.

[0149] (2) Further, the anti-hepatic injury effects of CK16 and CG28 were investigated using an oxaliplatin (OXA)-induced liver injury model. OXA was dissolved in 5% glucose solution (mass percentage) and injected intraperitoneally at 8 mg / kg into mice to induce an acute liver injury model. CK16 and CG28 were dissolved in 2% DMSO and 98% corn oil (volume ratio: 2:98) and administered intraperitoneally at a dose of 100 mg / kg one hour after OXA treatment. NAC was dissolved in physiological saline and administered intraperitoneally at a dose of 300 mg / kg one hour after OXA treatment. The experiment lasted for three days, with administration once daily. After three days of OXA treatment, liver and blood samples were collected for further experiments. The results showed that intraperitoneal injection of CK16 and CG28 significantly reduced the OXA-induced increase in ALT and AST levels in mice (see...). Figure 4 The results (A) indicate that CK16 and CG28 have a certain protective effect against OXA-induced acute liver injury in mice. An AML12 cell line was used to construct a liver injury cell model by treating AML12 cells with OXA (35 μM). Simultaneously, AML12 cells were treated with CK16 and CG28 (6.25, 12.5, 25, 50, 100 μM) for 24 hours. The NAC (10 mM) treatment group served as a positive control. The proportion of viable cells was then detected using CCK-8 reagent. The results showed that CK16 and CG28 exhibited a certain ameliorative effect on the OXA-induced in vitro cell-based liver injury model (see A). Figure 4 (B in the text). These results show that cinnamic acid derivatives CK16 and CG28 have anti-oxaliplatin-induced liver injury activity.

[0150] (3) An APAP-induced liver injury model was used to evaluate the inhibitory effects of cinnamic acid derivatives CK16 and CG28 on MAPK signaling pathway activation and ferroptosis in vivo.

[0151] In the APAP model group mice, 24 hours after APAP administration, significant phosphorylation of MAPK family proteins (P38, ERK1 / 2, and JNK) was observed in the liver tissue, indicating activation of this pathway. In contrast, phosphorylation of MAPK family proteins was significantly inhibited in the CK16 or CG28 treatment groups (see [link to APAP model group]). Figure 5 ).

[0152] Immunohistochemical analysis showed that 4-HNE was significantly upregulated in the APAP model group, while CK16 or CG28 could significantly prevent this upregulation (see [link to relevant documentation]). Figure 6 (A) This indicates that CK16 and CG28 can reduce lipid accumulation and ferroptosis in the liver of APAP-treated mice. Furthermore, CK16 or CG28 can significantly reduce iron accumulation (see A). Figure 6(See section B). Expression of ferroptosis-related genes was detected by qRT-PCR. CK16 and CG28 significantly altered the expression of these genes. Specifically, they increased the expression of FTH1, DHODH, FGF21, SLC7A11, FTL, DHFR, FPN, and GCH1, which are negative regulators of iron oxidation (see section B). Figure 6 (C) Immunoblotting results showed that CK16 and CG28 increased the expression levels of GPX4 and FSP1 compared to the APAP model group, while the expression levels of these two proteins were essentially similar to those in the control group. Consistent with the results of qRT-PCR, CK16 and CG28 also significantly increased the expression levels of other proteins negatively associated with ferroptosis in the liver, such as SLC7A11, FTL, FTH1, FPN, and CD71 (see C). Figure 6 (D in the text). These results indicate that CK16 and CG28 can alleviate APAP-induced hepatocyte ferroptosis and improve liver injury.

[0153] (4) The activation effect of cinnamic acid derivatives CK16 and CG28 on UGT enzyme activity was detected using a UGT enzyme activity assay kit. This invention first analyzed the APAP metabolites of each experimental group, including APAP-Cys, APAP-Sulf, and APAP-Gluc, using HPLC-MS / MS. This assay was performed by a CRO company (Wuhan Hongren Biopharmaceutical Co., Ltd.) specializing in drug development technology services. UDP-glycosyltransferase detection is currently difficult; this invention used a UGT activity assay kit (Abcam, UK, ab273331) to determine the effect of compounds on UGT activity. The results showed that CK16 and CG28 could enhance UGT enzyme activity in vitro (see...). Figure 7 (A) We collected blood from mice 2 hours after APAP injection (1 hour after compound injection) to detect changes in APAP metabolites. Results showed that APAP-cys levels in the CK16 or CG28 treatment groups were significantly lower than in the APAP model group, while APAP-cys levels were higher in the NAC treatment group. Simultaneously, CK16 or CG28 treatment significantly increased APAP-gluc levels in mice, but had little effect on APAP-sulf. NAC treatment had no effect on APAP-gluc levels in mice, but significantly reduced APAP-sulf levels (see A). Figure 7 (B in the text). The APAP-induced liver injury model confirmed that cinnamic acid derivatives CK16 and CG28 can activate UGT enzyme activity in vivo.

[0154] Results and Analysis:

[0155] The novel cinnamic acid derivatives synthesized in this invention have stable structures and are non-cytotoxic within the effective concentration range. In one cell and two mouse acute liver injury models, multiple cinnamic acid derivatives showed strong effects in alleviating acute liver injury. Among them, the representative compounds CK16 and CG28 showed stronger anti-liver injury effects than the positive control drug N-acetyl-L-cysteine ​​(NAC). The representative compounds CK16 and CG28 exert their effects by inhibiting the activation of the MAPK signaling pathway and ferroptosis response in vivo, and by activating UDP-glucuronyltransferase.

[0156] Although Example 3 verifies the anti-hepatotoxic effects of representative compounds CK16 and CG28 by inhibiting MAPK signaling pathway activation, inhibiting ferroptosis, and activating UDP-glucuronyltransferase, it is not limited to the treatment of hepatotoxicity. This invention can also be applied to other diseases that utilize similar mechanisms of action (either alone or in combination with other drugs). This is because MAPK signaling pathway activation and ferroptosis are common stages in many diseases, playing a role in the development and treatment of many diseases. UDP-glucuronyltransferase is one of the most important enzymes in phase II metabolism in the human body, responsible for approximately 40-70% of endogenous and heterologous reactions. It makes the compounds bound by the reaction more water-soluble and easier to excrete, participating in the metabolic clearance of many drugs, thus playing a detoxification role. The associated physiological and pathological processes are also quite extensive.

[0157] Furthermore, although Example 3 only focuses on representative compounds CK16 and CG28, other cinnamic acid derivatives satisfying general formulas 1A and 1B can also alleviate APAP-induced liver injury to varying degrees. This is because they are structurally similar to CK16 and CG28, containing the same glycoside-cinnamyl structural backbone. The hydroxyl group in the glycoside structure and the carbonyl group in the cinnamyl structure are key structures for these compounds to exert their anti-ALI effects. In addition, reducing the interval between APAP induction and administration can enhance their therapeutic effect on liver injury.

[0158] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of a cinnamic acid derivative in the preparation of a medicament that exerts therapeutic effects by inhibiting the MAPK signaling pathway, by inhibiting ferroptosis, or by activating UDP-glucuronyltransferase, characterized in that, The structural formulas of the cinnamic acid derivatives are shown in Formula 12, Formula 16, Formula 22, Formula 28 or Formula 29: 。

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