A styrene acetate compound, a preparation method and application thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a styrene acetate compound, a preparation method and application thereof. The application provides a styrene acetate compound with a structure shown in formula 1. The styrene acetate compound with the structure shown in formula 1 provided by the application can play a role of inhibiting RAC1 activity by being combined with RAC1 protein, and is suitable for treating diseases related to RAC1 protein, including Alzheimer's disease, atherosclerosis, tumor cell migration and tumor cell invasion, etc. The results of the examples also show that the styrene acetate compound with the structure shown in formula 1 provided by the application can significantly inhibit the formation of cytoskeleton of tumor cells such as breast cancer and gastric cancer, thereby playing a role of inhibiting the migration and invasion activity of tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a styrene acetate compound, its preparation method, and its application. Background Technology

[0002] Cancer is one of the leading causes of death worldwide. With the rapid development of medical technology and improved healthcare, cancer prevention, screening, and treatment have received significant attention, and the detection rate of many types of cancer is increasing. For many years, cancer has posed a persistent threat to human life, and humanity's exploration of cancer treatments and drugs has never ceased. The most fundamental biological characteristics of cancer are the uncontrolled proliferation, unlimited replication, and abnormal differentiation of tumor cells. The main cause of death in cancer patients is the metastasis and invasion of cancer cells. Many tumor cells detach from cell-cell adhesion and achieve invasion and metastasis through a multi-step process involving migration and chemotactic stimulation leading to the formation of membrane protrusions. This process is a complex, multi-factor-driven tumor cell migration process. In invasive and metastatic cancer cells, the formation of actin cytoskeletons such as platelets is crucial for cell migration. Studies have shown that Rho GTPases, a class of GTP-binding proteins in the Ras superfamily, are key molecules regulating cell migration initiation. They participate in processes such as the extension of cell lamellar pseudopodia, the establishment of new adhesions, and cell tail contraction. Furthermore, they trigger signaling cascades that induce tumor cell motility, migration, and invasion by initiating downstream effector molecules. Abundant evidence suggests that Rho GTPases are associated with cancer, making them an attractive therapeutic target at the molecular level. Rac1, also known as Ras-associated C3 botulinum toxin subunit 1, is derived from the Ras GTPases superfamily.

[0003] As research into the Rac1 protein deepens, it has been discovered that abnormal Rac1 activity and expression are closely related to tumor occurrence, development, metastasis, and invasion. Rac1 participates in various cellular processes and plays a crucial role in the body's life processes, including cytoskeleton reorganization, cell proliferation, metabolism, reactive oxygen species (ROS) production, and inflammatory responses. Aberrant Rac1 expression has been reported in various tumors, including liver cancer, ovarian cancer, breast cancer, and pancreatic cancer. Recent cancer genome sequencing results indicate that Rac1 mutations are associated with melanocyte proliferation and motility. As a key regulatory molecule in the activation and regulation of multiple signaling pathways, Rac1's abnormal expression, leading to uncontrolled proliferation, invasion, and metastasis, is a hallmark of cancer.

[0004] Due to the crucial role of Rho GTPases in the development and progression of human cancer, they have gradually become highly attractive targets for anti-tumor therapy. However, the discovery of their inhibitors faces many obstacles, primarily due to their lack of selectivity and specificity in blocking RhoGTPase function. To date, reported inhibitors mainly function through two mechanisms. One example is the small molecule NSC23766, which specifically prevents the conversion of Rac1 from its inactive to its activated state by competitively blocking the binding of Rac1 to GEF. EHT1864 is a Rac1-specific inhibitor that effectively blocks the binding of Rho GTPases to GTP, rendering Rho GTPases inactive and inhibiting multiple downstream signaling pathways associated with Rac1, including its binding to the downstream effector PAK1. Studies have shown that the tight binding of EHT1864 to Rac1 can effectively inhibit Rac1-mediated changes in cell morphology. However, later studies have found that the inhibitors NSC23766 and EHT1864 have certain off-target effects in mammalian cells. The lack of specificity limits their development as anti-tumor therapies.

[0005] The lack of active sites on the surface of the Rac1 protein that facilitate the binding of small molecule drugs has hampered the development of small molecule inhibitors. Therefore, continuous exploration to discover novel small molecule inhibitors targeting Rac1 is of great significance for the treatment of diseases such as cancer. Summary of the Invention

[0006] The purpose of this invention is to provide a styrene acetate compound, its preparation method, and its application. The styrene acetate compound provided by this invention can significantly inhibit the formation of the cytoskeleton in tumor cells such as breast cancer and gastric cancer, thereby inhibiting the migration and invasion activities of tumor cells and providing a new option for anti-tumor drugs.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a styrene acetate compound having the structure shown in Formula 1.

[0009]

[0010] In Formula 1: R1 is a five-membered heterocycle containing nitrogen atoms or a six-membered heterocycle containing nitrogen atoms, and R2 is a hydroxyl, alkoxy, haloalkyl or hydrogen.

[0011] Preferably, the number of nitrogen atoms in the five-membered heterocycle containing nitrogen atoms is 1; the number of nitrogen atoms in the six-membered heterocycle containing nitrogen atoms is 1 or 2.

[0012] Preferably, R1 is

[0013] Preferably, R2 is a hydroxyl group, C 1～3 Alkoxy, C 1～3 Halogenated alkyl or hydrogen.

[0014] Preferably, R2 is hydroxyl, methoxy, CF3 or hydrogen.

[0015] Preferably, it has any one of the following structures:

[0016]

[0017] This invention provides a method for preparing the styrene acetate compounds described in the above technical solution, comprising the following steps:

[0018] The compound with the structure shown in Formula 2, the compound with the structure shown in Formula 3, the condensing agent and the catalyst were mixed in an organic solvent and subjected to an esterification reaction to obtain a styrene acetate compound with the structure shown in Formula 1.

[0019]

[0020] Preferably, the mass ratio of the compound with the structure shown in Formula 2 to the compound with the structure shown in Formula 3 is 1:1;

[0021] The catalyst is 4-dimethylaminopyridine, and the condensing agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; the mass ratio of the compound with the structure shown in Formula 2 to the catalyst is 1:0.5; the mass ratio of the compound with the structure shown in Formula 2 to the condensing agent is 1:2.

[0022] This invention provides the application of the styrene acetate compounds described in the above-described technical solutions, their pharmaceutically acceptable salts, solvates, hydrates, prodrug compounds, polymorphs or enantiomers, or styrene acetate compounds obtained by the preparation methods described in the above-described technical solutions, in the preparation of RAC1 inhibitors.

[0023] This invention provides the application of the styrene acetate compounds described in the above-described technical solutions, their pharmaceutically acceptable salts, solvates, hydrates, prodrug compounds, polymorphs or enantiomers, or styrene acetate compounds obtained by the preparation methods described in the above-described technical solutions, in the preparation of antitumor drugs.

[0024] This invention provides a styrene acetate compound having the structure shown in Formula 1. The styrene acetate compound of Formula 1 provided by this invention can inhibit RAC1 activity by binding to the RAC1 protein, and is suitable for treating diseases related to the RAC1 protein, including Alzheimer's disease, atherosclerosis, tumor cell migration, and tumor cell invasion. The results of the embodiments also show that the styrene acetate compound of Formula 1 of this invention can significantly inhibit the formation of the cytoskeleton in tumor cells such as breast cancer and gastric cancer, thereby inhibiting the migration and invasive activities of tumor cells.

[0025] This invention provides a method for preparing the styrene acetate compounds described in the above-mentioned technical solution, comprising the following steps: mixing a compound with the structure shown in Formula 2, a compound with the structure shown in Formula 3, a condensing agent, and a catalyst in an organic solvent to carry out an esterification reaction, thereby obtaining a styrene acetate compound with the structure shown in Formula 1. The preparation method provided by this invention involves esterifying an alcohol with the structure shown in Formula 2 and an acid with the structure shown in Formula 3 under the conditions of a condensing agent and a catalyst to obtain the target product. The preparation method provided by this invention has simple steps and is suitable for industrial production. Detailed Implementation

[0026] This invention provides a styrene acetate compound having the structure shown in Formula 1.

[0027]

[0028] In Formula 1: R1 is a five-membered heterocycle containing nitrogen atoms or a six-membered heterocycle containing nitrogen atoms, and R2 is a hydroxyl, alkoxy, haloalkyl or hydrogen.

[0029] In this invention, the number of nitrogen atoms in the nitrogen-containing five-membered heterocycle is preferably one. The number of nitrogen atoms in the nitrogen-containing six-membered heterocycle is preferably one or two.

[0030] In a specific embodiment of the present invention, R1 is preferably...

[0031] In this invention, R2 is preferably a hydroxyl group or a C group. 1～3 Alkoxy, C 1～3 Halogenated alkyl or hydrogen, more preferably hydroxyl, methoxy, CF3 or hydrogen.

[0032] In this invention, the styrene acetate compounds with the structure shown in Formula 1 have any one of the following structures:

[0033]

[0034] This invention provides a method for preparing the styrene acetate compounds described in the above technical solution, comprising the following steps:

[0035] The compound with the structure shown in Formula 2, the compound with the structure shown in Formula 3, the condensing agent and the catalyst were mixed in an organic solvent and subjected to an esterification reaction to obtain a styrene acetate compound with the structure shown in Formula 1.

[0036]

[0037] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0038] In this invention, R1 in the compound structure shown in Formula 2 is a five-membered heterocycle containing nitrogen atoms or a six-membered heterocycle containing nitrogen atoms. Preferably, the five-membered heterocycle containing nitrogen atoms has one nitrogen atom. Preferably, the six-membered heterocycle containing nitrogen atoms has one or two nitrogen atoms.

[0039] In a specific embodiment of the present invention, R1 in the compound with the structure shown in Formula 2 is preferably...

[0040]

[0041] In this invention, the compound with the structure shown in Formula 2 is preferably any one of the following structures:

[0042]

[0043] In this invention, R2 in the compound with the structure shown in Formula 3 is a hydroxyl group, an alkoxy group, a haloalkyl group, or hydrogen, preferably a hydroxyl group or a C group. 1～3 Alkoxy, C 1～3 Halogenated alkyl or hydrogen, more preferably hydroxyl, methoxy, CF3 or hydrogen.

[0044] In this invention, the compound with the structure shown in Formula 3 preferably has any one of the following structures:

[0045]

[0046] This invention does not have any special requirements regarding the source of the compounds with the structures shown in Formula 2 and Formula 3; commercially available products or self-made products may be used. This invention also does not have any special requirements regarding the preparation methods of the compounds with the structures shown in Formula 2 and Formula 3; methods well known to those skilled in the art may be used.

[0047] In this invention, the catalyst is preferably 4-dimethylaminopyridine (DMAP), and the condensing agent is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl). The organic solvent is preferably dichloromethane.

[0048] In this invention, the preferred mass ratio of the compound shown in Formula 2 to the compound shown in Formula 3 is 1:1; the preferred mass ratio of the compound shown in Formula 2 to the catalyst is 1:0.5; and the preferred mass ratio of the compound shown in Formula 2 to the condensing agent is 1:2. This invention does not have special requirements regarding the amount of organic solvent used, as long as the esterification reaction proceeds smoothly. This invention does not have special requirements regarding the specific implementation process of the mixing; simply dissolving the compound shown in Formula 2, the compound shown in Formula 3, the condensing agent, and the catalyst in the organic solvent is sufficient. The preferred temperature for the esterification reaction is room temperature. The esterification reaction is preferably monitored using thin-layer TLC (Thin-Layer Chromatography) spotting plate. The preferred developing solvent used in the TLC spotting plate is a mixed solvent of CHCl3 and CH3OH, with a preferred volume ratio of CHCl3 to CH3OH of 5:1. The esterification reaction ends when the compound shown in Formula 2 disappears as detected by the TLC spotting plate.

[0049] After the esterification reaction is completed, an esterification reaction solution is obtained. Preferably, the esterification reaction solution is mixed with water for extraction to obtain an organic phase. The organic phase is then mixed with methanol and successively dried and concentrated to obtain a concentrate. The concentrate is dissolved in methanol to obtain a concentrated solution. The concentrated solution is purified by column chromatography to obtain a pure styrene acetate compound with the structure shown in Formula 1. The extraction is preferably performed twice. The water used for extraction is preferably purified water. Before mixing the water with the esterification reaction solution, preferably, 2 drops of concentrated hydrochloric acid are added to the water, and the mass content of the concentrated hydrochloric acid is preferably 37%. The volume ratio of the water to the volume of the reaction solution before extraction is preferably 1:1. Before drying, preferably, methanol is used to dissolve the insoluble matter in the extracted organic phase. The reagent used for drying is preferably anhydrous sodium sulfate. The specific implementation process of the concentration is not particularly required. Preferably, methanol is used to dissolve the concentrate until it becomes clear. The column chromatography purification is preferably carried out in a medium-high pressure preparation system. The purification column used is preferably a reversed-phase C18 column, the detector is preferably a UV 254nm detector, and the detection wavelength is preferably 365nm. The mobile phase used for column chromatography purification is preferably methanol and water, with a volume ratio of methanol to water preferably 90%:10%. The elution program for column chromatography purification is preferably isocratic elution, with an elution time preferably 30 min. This invention preferably uses thin-layer TLC (Thin-Layer Chromatography) to monitor the eluted sample obtained from column chromatography purification. The developing solvent used for TLC is preferably a mixed solvent of CHCl3 and CH3OH, with a volume ratio of CHCl3 to CH3OH preferably 5:1. This invention preferably collects the product when a single spot is detected by TLC to obtain a pure styrene acetate compound with the structure shown in Formula 1.

[0050] This invention provides the use of the styrene acetate compounds described in the above-described technical solutions, their pharmaceutically acceptable salts, solvates, hydrates, prodrug compounds, polymorphs, or enantiomers, or the styrene acetate compounds obtained by the preparation methods described in the above-described technical solutions, in the preparation of RAC1 inhibitors. In this invention, the diseases associated with the RAC1 protein include Alzheimer's disease, atherosclerosis, tumor cell migration, or tumor cell invasion.

[0051] This invention provides the use of the styrene acetate compounds described in the above-described technical solutions, their pharmaceutically acceptable salts, solvates, hydrates, prodrug compounds, polymorphs or enantiomers, or styrene acetate compounds obtained by the preparation methods described in the above-described technical solutions, in the preparation of medicaments for treating Alzheimer's disease.

[0052] This invention provides the use of the styrene acetate compounds described in the above-described technical solutions, their pharmaceutically acceptable salts, solvates, hydrates, prodrug compounds, polymorphs or enantiomers, or styrene acetate compounds obtained by the preparation methods described in the above-described technical solutions, in the preparation of medicaments for treating atherosclerosis.

[0053] This invention provides the use of the styrene acetate compounds described in the above-described technical solutions, their pharmaceutically acceptable salts, solvates, hydrates, prodrug compounds, polymorphs or enantiomers, or styrene acetate compounds obtained by the preparation methods described in the above-described technical solutions, in the preparation of drugs for treating tumor cell migration.

[0054] This invention provides the use of the styrene acetate compounds described in the above-described technical solutions, their pharmaceutically acceptable salts, solvates, hydrates, prodrug compounds, polymorphs or enantiomers, or styrene acetate compounds obtained by the preparation methods described in the above-described technical solutions, in the preparation of drugs for treating tumor cell invasion.

[0055] This invention provides the application of the styrene acetate compounds described in the above-described technical solutions, or the styrene acetate compounds obtained by the preparation methods described in the above-described technical solutions, in the preparation of antitumor drugs. In this invention, the antitumor drug is preferably an anti-breast cancer drug and / or an anti-gastric cancer drug.

[0056] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0057] Example 1

[0058] Weigh 30.00 mg of 1-methyl-4-(hydroxyethyl)piperidine, 30.00 mg of (3E)-4-(4-methoxyphenyl)but-3-enoic acid, 60.00 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 15.00 mg of 4-dimethylaminopyridine (DMAP) and place them in a beaker. Add 60 mL of CH2Cl2 and stir for 20 h. Observe the reaction by TLC to see if the 1-methyl-4-(hydroxyethyl)piperidine has reacted completely. The developing solvent is CHCl3:CH3OH = 5:1. After the reaction is complete, the esterification product is obtained.

[0059] Extraction was performed on the esterification product by adding an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), followed by separation of the layers. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. The organic phase was then extracted again by adding an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), followed by separation of the layers. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. After extraction, the organic phase was still turbid, so CH3OH was added until the organic phase solution became clear. Then, anhydrous sodium sulfate was added to dry the solution and remove water. The solution was concentrated and then dissolved in 90% methanol. A medium-high pressure preparation system was used, with a reversed-phase C18 column and a mobile phase of 90% methanol:10% water. The solution was eluted isocratically for 30 min. The detector was UV 254 nm and the detection wavelength was 365 nm. The eluted sample was spotted on a TLC plate using CHCl3:CH3OH = 5:1. When a single spot was observed, the product was collected to obtain compound ZSD0001, 30.25 mg, with a yield of 61.21%. The compounds were identified using nuclear magnetic resonance (NMR) (dissolved in deuterated chloroform).

[0060] Example 2

[0061] Weigh 30.00 mg of 1-methyl-4-(hydroxyethyl)piperidine, 30.00 mg of (3E)-4-(3-methoxyphenyl)but-3-enoic acid, 60.00 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 15.00 mg of 4-dimethylaminopyridine (DMAP) and place them in a beaker. Add 60 mL of CH2Cl2 and stir for 20 h. Observe the reaction by TLC, using CHCl3:CH3OH = 5:1 as the developing solvent. After the reaction is complete, the esterification product is obtained.

[0062] Extraction was performed on the esterification product by adding an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), followed by separation of the layers. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. The organic phase was then extracted again by adding an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), followed by separation of the layers. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. After extraction, the organic phase was still turbid, so CH3OH was added until the organic phase solution became clear. Then, anhydrous sodium sulfate was added to dry the solution and remove water. The solution was concentrated and then dissolved in 90% methanol. A medium-high pressure preparation system was used, with a reversed-phase C18 column and a mobile phase of 90% methanol:10% water. The solution was eluted isocratically for 30 min. The detector was UV 254 nm and the detection wavelength was 365 nm. The eluted sample was spotted using CHCl3:CH3OH = 5:1 to determine if it was a single spot and the product was collected. Compound ZSD0002, 30.28 mg, was obtained, with a yield of 62.55%. The compounds were identified using nuclear magnetic resonance (NMR) (dissolved in deuterated chloroform).

[0063] Example 3

[0064] Weigh 30.00 mg of 1-methyl-4-(hydroxyethyl)piperidine, 30.00 mg of (3E)-4-(4-hydroxyphenyl)but-3-enoic acid, 60.00 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 15.00 mg of 4-dimethylaminopyridine (DMAP) and place them in a beaker. Add 60 mL of CH2Cl2 and stir for 20 h. Observe the reaction by TLC to see if the reaction is complete. Use CHCl3:CH3OH = 5:1 as the developing solvent to observe whether 1-methyl-4-(hydroxyethyl)piperidine has reacted completely. After the reaction is complete, the esterification product is obtained.

[0065] Extraction was performed on the esterification product by adding an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), followed by separation of the layers. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. The obtained organic phase was then extracted again by adding an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), followed by separation of the layers. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. After extraction, the organic phase was still turbid, so CH3OH was added until the organic phase solution became clear. Then, anhydrous sodium sulfate was added to dry and remove water. The solution was concentrated and then dissolved in 90% methanol. A medium-high pressure preparation system was used, with a reversed-phase C18 column and a mobile phase of 90% methanol:10% water. The solution was eluted isocratically for 30 min. The detector was UV 254 nm and the detection wavelength was 365 nm. The eluted sample was spotted using CHCl3:CH3OH = 5:1 to determine if it was a single spot and the product was collected. Compound ZSD0003 was obtained, 38.63 mg, with a yield of 68.29%. The compounds were identified using nuclear magnetic resonance (NMR) (dissolved in deuterated chloroform).

[0066] Example 4

[0067] Weigh 30.00 mg of 1-methyl-4-(hydroxyethyl)piperazine, 30.00 mg of (3E)-4-(3-hydroxyphenyl)but-3-enoic acid, 60.00 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 15.00 mg of 4-dimethylaminopyridine (DMAP) and place them in a beaker. Add 60 mL of CH2Cl2 and stir for 20 h. Observe the reaction by TLC, using CHCl3:CH3OH = 5:1 as the developing solvent. After the reaction is complete, the esterification product is obtained.

[0068] Extraction was performed on the esterification product by adding an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), followed by separation of the layers. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. The organic phase was then extracted again by adding an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), followed by separation of the layers. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. After extraction, the organic phase was still turbid, so CH3OH was added until the organic phase solution became clear. Then, anhydrous sodium sulfate was added to dry the solution and remove water. The solution was concentrated and then dissolved in 90% methanol. A medium-high pressure preparation system was used, with a reversed-phase C18 column and a mobile phase of 90% methanol:10% water. The solution was eluted isocratically for 30 min. The detector was UV 254 nm and the detection wavelength was 365 nm. The eluted sample was spotted using CHCl3:CH3OH = 5:1 to determine if it was a single spot and the product was collected. Compound ZSD0004, 40.05 mg, was obtained, with a yield of 69.24%. The compounds were identified using nuclear magnetic resonance (with deuterated chloroform).

[0069] Example 5

[0070] Weigh 30.00 mg of 1-methyl-4-(hydroxyethyl)piperazine, 30.00 mg of (3E)-4-(4-trifluoromethylphenyl)but-3-enoic acid, 60.00 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 15.00 mg of 4-dimethylaminopyridine (DMAP) and place them in a beaker. Add 60 mL of CH2Cl2 and stir for 20 h. Observe the reaction by TLC, using CHCl3:CH3OH = 5:1 as the developing solvent. After the reaction is complete, the esterification product is obtained.

[0071] Extraction was performed on the esterification product by adding an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), followed by separation of the layers. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. The obtained organic phase was then extracted again by adding an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), followed by separation of the layers. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. After extraction, the organic phase was still turbid, so CH3OH was added until the organic phase solution became clear. Then, anhydrous sodium sulfate was added to dry the solution and remove water. The solution was concentrated and then dissolved in 90% methanol. A medium-high pressure preparation system was used, with a reversed-phase C18 column and a mobile phase of 90% methanol:10% water. The solution was eluted isocratically for 30 min. The detector was UV 254 nm and the detection wavelength was 365 nm. The eluted sample was spotted using CHCl3:CH3OH = 5:1 to determine if it was a single spot and the product was collected. Compound ZSD0005, 329.22 mg, was obtained, with a yield of 60.13%. The compounds were identified using nuclear magnetic resonance (NMR) (dissolved in CD3OH).

[0072] Example 6

[0073] Weigh 30.00 mg of 1-methyl-3-(hydroxyethyl)pyridine, 30.00 mg of (3E)-4-phenylbut-3-enoic acid, 60.00 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 15.00 mg of 4-dimethylaminopyridine (DMAP) and place them in a beaker. Add 60 mL of CH2Cl2 and stir for 20 h. Observe the reaction by TLC, using CHCl3:CH3OH = 5:1 as the developing solvent. After the reaction is complete, the esterification product is obtained.

[0074] The esterification product was extracted with an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), and the layers were separated. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. The obtained organic phase was then extracted again with an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), and the layers were separated. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. After extraction, the organic phase was still turbid, so CH3OH was added until the organic phase solution became clear. Then, anhydrous sodium sulfate was added to dry and remove water. The solution was concentrated and then dissolved in 90% methanol. A medium-high pressure preparation system was used, a reversed-phase C18 column was used, and the mobile phase was 90% methanol:10% water. The solution was eluted isocratically for 30 min. The detector was UV 254 nm and the detection wavelength was 365 nm. The eluted sample was spotted using CHCl3:CH3OH = 5:1 to determine whether it was a single spot and the product was collected. Compound ZSD0006, 30.15 mg, was obtained, with a yield of 62.08%. The compounds were identified using nuclear magnetic resonance (NMR) (dissolved in CD3OH).

[0075] Example 7

[0076] Weigh 30.00 mg of 2-(4-methylpiperazin-1-yl)ethanol, 30.00 mg of (3E)-4-phenylbut-3-enoic acid, 60.00 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 15.00 mg of 4-dimethylaminopyridine (DMAP) and place them in a beaker. Add 60 mL of CH2Cl2 and stir for 20 h. Observe the reaction by TLC, using CHCl3:CH3OH = 5:1 as the developing solvent. After the reaction is complete, the esterification product is obtained.

[0077] Extraction was performed on the esterification product by adding an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), followed by separation of the layers. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. The organic phase was then extracted again by adding an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), followed by separation of the layers. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. After extraction, the organic phase was still turbid, so CH3OH was added until the organic phase solution became clear. Then, anhydrous sodium sulfate was added to dry the solution and remove water. The solution was concentrated and then dissolved in 90% methanol. A medium-high pressure preparation system was used, with a reversed-phase C18 column and a mobile phase of 90% methanol:10% water. The solution was eluted isocratically for 30 min. The detector was UV 254 nm and the detection wavelength was 365 nm. The eluted sample was spotted using CHCl3:CH3OH = 5:1 to determine if it was a single spot and the product was collected. Compound ZSD0007 was obtained, 35.51 mg, with a yield of 64.09%. The compounds were identified using nuclear magnetic resonance (NMR) (dissolved in CD3OH).

[0078] Example 8

[0079] Weigh 30.00 mg of 2-(4-methylpiperazin-1-yl)ethanol, 30.00 mg of (3E)-4-(4-hydroxyphenyl)but-3-enoic acid, 60.00 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 15.00 mg of 4-dimethylaminopyridine (DMAP) and place them in a beaker. Add 60 mL of CH2Cl2 and stir for 20 h. Observe the reaction by TLC, using CHCl3:CH3OH = 5:1 as the developing solvent. After the reaction is complete, the esterification product is obtained.

[0080] The esterification product was extracted with an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), and the layers were separated. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. The obtained organic phase was then extracted again with an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), and the layers were separated. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. After extraction, the organic phase was still turbid, so CH3OH was added until the organic phase solution became clear. Then, anhydrous sodium sulfate was added to dry and remove water. The solution was concentrated and then dissolved in 90% methanol. A medium-high pressure preparation system was used, a reversed-phase C18 column was used, and the mobile phase was a 90% methanol:10% water system with isocratic elution for 30 min. The detector was UV 254 nm and the detection wavelength was 365 nm. The eluted sample was spotted using CHCl3:CH3OH = 5:1 to determine whether it was a single spot and the product was collected. Compound ZSD0008, 38.22 mg, was obtained, with a yield of 66.57%. The compounds were identified using nuclear magnetic resonance (NMR) (dissolved in CD3OH).

[0081] Example 9

[0082] Weigh 30.00 mg of 2-(4-methylpiperazin-1-yl)ethanol, 30.00 mg of (3E)-4-(4-methoxyphenyl)but-3-enoic acid, 60.00 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 15.00 mg of 4-dimethylaminopyridine (DMAP) and place them in a beaker. Add 60 mL of CH2Cl2 and stir for 20 h. Observe the reaction by TLC, using CHCl3:CH3OH = 5:1 as the developing solvent. After the reaction is complete, the esterification product is obtained.

[0083] Extraction was performed on the esterification product by adding an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), followed by separation of the layers. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. The organic phase was then extracted again by adding an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), followed by separation of the layers. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. After extraction, the organic phase was still turbid, so CH3OH was added until the organic phase solution became clear. Then, anhydrous sodium sulfate was added to dry the solution and remove water. The solution was concentrated and then dissolved in 90% methanol. A medium-high pressure preparation system was used, with a reversed-phase C18 column and a mobile phase of 90% methanol:10% water. The solution was eluted isocratically for 30 min. The detector was UV 254 nm and the detection wavelength was 365 nm. The eluted sample was spotted using CHCl3:CH3OH = 5:1 to determine if it was a single spot and the product was collected. Compound ZSD0009 was obtained in 29.88 mg, with a yield of 60.17%. The compounds were identified using nuclear magnetic resonance (NMR) (dissolved in CD3OH).

[0084] Example 10

[0085] Weigh 30.00 mg of 2-(1-methyltetrahydro-1H-pyrrolo-3-yl)ethanol-1-ol, 30.00 mg of (3E)-4-phenylbut-3-enoic acid, 60.00 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 15.00 mg of 4-dimethylaminopyridine (DMAP) and place them in a beaker. Add 60 mL of CH2Cl2 and stir for 20 h. Observe the reaction by TLC, using CHCl3:CH3OH = 5:1 as the developing solvent. After the reaction is complete, the esterification product is obtained.

[0086] The esterification product was extracted with an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), and the layers were separated. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. The obtained organic phase was then extracted again with an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), and the layers were separated. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. After extraction, the organic phase was still turbid, so CH3OH was added until the organic phase solution became clear. Then, anhydrous sodium sulfate was added to dry and remove water. The solution was concentrated and then dissolved in 90% methanol. A medium-high pressure preparation system was used, a reversed-phase C18 column was used, and the mobile phase was a 90% methanol:10% water system with isocratic elution for 30 min. The detector was UV 254 nm and the detection wavelength was 365 nm. The eluted sample was spotted using CHCl3:CH3OH = 5:1 to determine whether it was a single spot and the product was collected. Compound ZSD00010, 28.32 mg, was obtained, with a yield of 59.21%. The compounds were identified using nuclear magnetic resonance (NMR) (dissolved in CD3OH).

[0087] Example 11

[0088] Weigh 30.00 mg of 2-(1-methyltetrahydro-1H-pyrrolo-3-yl)ethanol-1-ol, 30.00 mg of (3E)-4-(4-hydroxyphenyl)but-3-enoic acid, 60.00 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 15.00 mg of 4-dimethylaminopyridine (DMAP) and place them in a beaker. Add 60 mL of CH2Cl2 and stir for 20 h. Observe the reaction by TLC, using CHCl3:CH3OH = 5:1 as the developing solvent. After the reaction is complete, the esterification product is obtained.

[0089] Extraction was performed on the esterification product by adding an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), followed by separation of the layers. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. The organic phase was then extracted again by adding an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), followed by separation of the layers. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. After extraction, the organic phase was still turbid, so CH3OH was added until the organic phase solution became clear. Then, anhydrous sodium sulfate was added to dry the solution and remove water. The solution was concentrated and then dissolved in 90% methanol. A medium-high pressure preparation system was used, with a reversed-phase C18 column and a mobile phase of 90% methanol:10% water. The solution was eluted isocratically for 30 min. The detector was UV 254 nm and the detection wavelength was 365 nm. The eluted sample was spotted using CHCl3:CH3OH = 5:1 to determine if it was a single spot and the product was collected. Compound ZSD00011, 35.45 mg, was obtained, with a yield of 66.05%. The compounds were identified using nuclear magnetic resonance (NMR) (dissolved in CD3OH).

[0090] Example 12

[0091] Weigh 30.00 mg of 2-(1-methyltetrahydro-1H-pyrrolo-3-yl)ethanol-1-ol, 30.00 mg of (3E)-4-(4-methoxyphenyl)but-3-enoic acid, 60.00 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 15.00 mg of 4-dimethylaminopyridine (DMAP) and place them in a beaker. Add 60 mL of CH2Cl2 and stir for 20 h. Observe the reaction by TLC, using CHCl3:CH3OH = 5:1 as the developing solvent. After the reaction is complete, the esterification product is obtained.

[0092] Extraction was performed on the esterification product by adding an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), followed by separation of the layers. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. The organic phase was then extracted again by adding an equal volume of purified water (2 drops of concentrated HCl, 37% in 100 mL of water), followed by separation of the layers. The upper layer was the aqueous phase, which was discarded; the lower layer was the organic phase, which was retained. After extraction, the organic phase was still turbid, so CH3OH was added until the organic phase solution became clear. Then, anhydrous sodium sulfate was added to dry the solution and remove water. The solution was concentrated and then dissolved in 90% methanol. A medium-high pressure preparation system was used, with a reversed-phase C18 column and a mobile phase of 90% methanol:10% water. The solution was eluted isocratically for 30 min. The detector was UV 254 nm and the detection wavelength was 365 nm. The eluted sample was spotted using CHCl3:CH3OH = 5:1 to determine if it was a single spot and the product was collected. Compound ZSD00011, 26.47 mg, was obtained, with a yield of 56.27%. The compounds were identified using nuclear magnetic resonance (NMR) (dissolved in CD3OH).

[0093] The structural determination results of the compounds prepared in Examples 1-12 are shown in Table 1.

[0094] Table 1. NMR characterization results of the compounds prepared in Examples 1-12

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] Test case

[0102] (1) Cytoskeleton inhibition rate

[0103] Experimental methods:

[0104] Place a slide specifically designed for climbing experiments in the center of a 6-well plate, resuspend logarithmic growth phase MDA-MB-231 breast cancer cells, and seed 500 μL (2 × 10⁶ cells) into each well. 4 Add cells to a glass slide, and once the cells have adhered, add culture medium to a final volume of 2 mL. After overnight culture, discard the old culture medium and add 3 mL of fresh culture medium containing 10 μM inhibitor to each well. Continue culturing for another 24 hours. After co-incubation, the slides were washed with PBS, and 1 mL of 4% paraformaldehyde was added to each well for fixation at room temperature for 15 min. After PBS washing, 1 mL of Triton X-100 immunostaining permeabilization buffer was added to each well, and the slides were incubated at room temperature for 10 min. The coverslips were removed with sterile metal forceps and washed three times with PBS containing 0.1% Triton X-100. 200 μL of Actin-Tracker Green diluted 1:100 was added to each slide, and the slides were incubated at room temperature in the dark for 30 min. After washing three times with PBS containing 0.1% Triton X-100, 200 μL of DAPI staining solution was added to each slide, and the slides were incubated at room temperature in the dark for 5 min. After PBS washing, the slides were placed on slides with 50 μL of anti-fluorescence quenching mounting solution, and the changes in the cytoskeleton were observed under a confocal microscope, and the area was calculated. The results are shown in Table 2.

[0105] Table 2. Results of the inhibitory rate of the compounds prepared in Examples 1-12 on the breast cancer cytoskeleton.

[0106] ZSD0001 85.22±3.08％ ZSD0002 80.68±2.44％ ZSD0003 66.25±1.72％ ZSD0004 65.51±2.92％ ZSD0005 83.20±5.11％ ZSD0006 74.51±4.23％ ZSD0007 80.09±2.77％ ZSD0008 79.19±4.18％ ZSD0009 82.35±3.66％ ZSD00010 75.30±4.24％ ZSD00011 81.31±2.28％ ZSD00012 84.58±2.64％

[0107] (2) RAC1 protein activity inhibition rate

[0108] Experimental methods:

[0109] Add 10 μL of 0.5 M EDTA (pH 8.0) to an EP tube containing 500 μg Rac1 protein solution (1 mg / mL) and vortex to mix. Add 5 μL of 10 mM GMPPNP activated protein and divide the sample into two groups. Add 2.5 μL of each inhibitor (5 mM) to the experimental group, while no further inhibitors are added to the blank total protein group. Incubate at 30 °C with stirring for 15 min. Place the sample on ice and add MgCl2 to terminate the reaction by vortexing.

[0110] Label the spin cups; resuspend glutathione agarose beads and transfer 100 μL into the spin cups, centrifuge at 6000g for 30 s; add 400 μL of Lysis / Binding / Wash buffer, centrifuge again at 6000g for 30 s; add 20 μg of expressed GST-PAK1-PBD (1 mg / mL) to the spin cups containing glutathione resin, immediately transfer to an EP tube containing Rac1 protein, and mix the mixture at low speed in a rotary mixer at 4℃ for 1 h; after incubation, centrifuge at 6000g for 30 s, add 400 μL of Lysis / Binding / Wash buffer and wash twice; replace the spin cups with new collection tubes and place them on ice; add 50 μL of Reducing Sample to each spin cup, vortex the sample, incubate at room temperature for 2 min; centrifuge at 6000g for 2 min, and collect the eluted sample. The protein sample was diluted 5-fold, and the loading volume was set at 3 μL. The primary antibody was diluted with Antibody Diluent II antibody diluent, and the secondary antibody was ready for immediate use. The sample was loaded according to the instructions. Correction and comparison were performed using the internal control protein normalization method. The test results are shown in Table 3.

[0111] Table 3. Results of RAC1 protein activity inhibition rate tests of the compounds prepared in Examples 1-12.

[0112] ZSD0001 88.3±2.09％ ZSD0002 79.1±3.10％ ZSD0003 80.9±6.62％ ZSD0004 75.2±1.57％ ZSD0005 81.5±2.41％ ZSD0006 78.9±3.74％ ZSD0007 79.2±4.02％ ZSD0008 77.2±3.22％ ZSD0009 70.3±2.93％ ZSD00010 72.6±1.04％ ZSD00011 73.8±3.66％ ZSD00012 79.9±1.22％

[0113] (3) Inhibitory activity against breast cancer cell solid tumors

[0114] Experimental methods:

[0115] Female BALB / c-nude mice (13-15g, 4 weeks old) were first acclimatized for one week under specific pathogen-free conditions. Once the mice had adapted to the diet and lifestyle and were in good mental and physical condition, an in situ nude mouse model of breast cancer was established.

[0116] MDA-MB-231 cells in logarithmic growth phase were harvested and adjusted to a cell density of 2 × 10⁶ cells / year using PBS and Matrigel gel (1:1). 7 Cells were stored at a density of 0.1 mL and placed on ice to reduce metabolism and maintain viability. In the SPF laboratory, the inoculation site of nude mice was disinfected with povidone-iodine. After the cells were mixed, 0.1 mL of suspension was injected into the penultimate pair of mammary fat pads of nude mice using a disposable sterile syringe to establish an orthotopic model of mammary breast cancer in nude mice.

[0117] When the tumor volume approaches 100mm 3 During the experiment, participants were randomly divided into groups according to tumor volume. The control group was administered physiological saline by gavage, while each compound group was treated with 10 mg / kg by gavage in a volume of 0.5 mL, administered daily for 4 consecutive weeks. Tumor volume was measured every three days using calipers, and the tumor inhibition effect was statistically analyzed at the end of the experiment. The test results are shown in Table 4.

[0118] Table 4. Results of the inhibitory activity test of the compounds prepared in Examples 1-12 against breast cancer cell solid tumors.

[0119] control group 949.83±28.69 ZSD0001 552.61±33.21 ZSD0002 408.79±20.15 ZSD0003 439.78±35.64 ZSD0004 508.62±27.64 ZSD0005 403.55±44.17 ZSD0006 413.91±22.49 ZSD0007 514.78±40.06 ZSD0008 554.67±20.87 ZSD0009 409.55±31.82 ZSD00010 672.59±30.27 ZSD00011 551.06±64.10 ZSD00012 447.60±35.74

[0120] As demonstrated by the above embodiments, the styrene acetate compounds of Formula 1 provided by this invention can significantly inhibit the formation of the cytoskeleton in tumor cells such as breast cancer and gastric cancer, thereby inhibiting the migration and invasion of tumor cells. Further research revealed that these compounds exert their effects by binding to the RAC1 protein, and in vivo activity studies have shown excellent anti-tumor activity. This invention can provide a new option for anti-tumor drugs.

[0121] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of a styrene acetate compound, a pharmaceutically acceptable salt thereof, in the preparation of a RAC1 inhibitor, characterized in that, The styrene acetate compounds have any one of the following structures: 、 、 、 、 、 、 、 、 、 、 、 。

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