An organic small molecule which can be activated by alkaline phosphatase and visible light, and a preparation method and application thereof

Abstract

The application discloses an organic small molecule which can be activated by alkaline phosphatase and visible light, and a preparation method and application thereof. The structural formula of the organic small molecule is shown as formula I. The organic small molecule and cis-trans isomers thereof exist in a stable cis or trans configuration under dark conditions and have no cytotoxicity. Under visible light irradiation, the organic small molecule undergoes cis-trans isomerization, and the isomerization efficiency is regulated by the concentration of serum albumin in the environment. Based on this, the good biocompatibility of visible light and the wide existence of albumin in vivo can be used as a double regulation means, so that the organic small molecule has no toxic side effects under high serum albumin physiological conditions. In a tumor cell environment, through visible light irradiation and albumin concentration removal, the organic small molecule is in a trans configuration, and through alkaline phosphatase reaction, the organic small molecule has a high tumor cell killing ability, achieves time and space control of drug activity, and has application prospects in on-demand precise treatment of tumors.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an organic small molecule that can be activated by alkaline phosphatase and visible light, its preparation method, and its application. Background Technology

[0002] Drug therapy is currently the primary treatment for most diseases, including malignant tumors. However, traditional drugs often suffer from severe side effects and poor efficacy due to a lack of selectivity, low absorption rates at the lesion site, and drug resistance, sometimes even leading to treatment failure. The root cause of poor selectivity lies in the uncontrolled temporal and spatial nature of drug action; that is, drugs remain active at all times outside the organism or at non-target sites within the body, but their concentration decreases and activity diminishes upon reaching the target site. To address these issues, designing novel molecules with dynamically controllable drug activity is an effective approach.

[0003] Photopharmacology is an emerging medical approach that utilizes light to manipulate the activation or temporary inactivation of pharmacological activity by introducing photo-switching molecules into pharmacophores, enabling targeted and precise therapy. Light, as a non-invasive, remotely controlled parameter, is highly orthogonal to most biochemical reactions within organisms, does not pollute the biological environment, and, more importantly, possesses high spatial and temporal precision, giving it a unique advantage in controlling the function of bioactive molecules. Currently, photo-switching molecular structures commonly used to "turn on" and "off" drug activity include azobenzene, spiropyran, or diarylethylene. However, the photoresponsive regions of these molecular structures are located in the short-wavelength ultraviolet light band. Ultraviolet light suffers from biotoxicity and poor tissue penetration, severely limiting its practical application in living systems. Furthermore, existing photo-switching molecules themselves lack therapeutic effects and must be covalently coupled with known pharmacophores, but chemical modification reactions may lead to a decrease or even loss of pharmacophore activity. Therefore, designing novel molecules that combine drug activity and photo-switching properties is of great significance for the development of highly efficient and selective drug candidates. Summary of the Invention

[0004] To overcome the problems of existing photoswitching molecules having short response wavelengths, limited modulation methods, and lack of inherent pharmacological effects, this invention provides an organic small molecule that can be activated by alkaline phosphatase and visible light, along with its preparation method and applications.

[0005] In a first aspect, the present invention protects a small organic molecule that can be activated by alkaline phosphatase and visible light, which is a compound represented by Formula I.

[0006]

[0007] in, Selected from cis isomers (cis TP), trans isomers (trans TP), or mixtures of cis and trans isomers (TP).

[0008] The compound shown in Formula I is a cis-trans isomer with visible light response as a novel photo-switching bioactive molecule. The photo-switching bioactive molecule is an organic small molecule that produces cis-trans isomers when exposed to visible light and has the ability to recognize alkaline phosphatase and albumin.

[0009] The structural formulas of the cis isomer and the trans isomer are as follows:

[0010]

[0011] The organic small molecule is stable in the cis (cis TP) or trans (trans TP) configuration under dark conditions and is non-toxic. It undergoes cis-trans isomerization under visible light irradiation.

[0012] The organic small molecule is a substrate of alkaline phosphatase, and after hydrolysis, it generates product Th. The reaction between the organic small molecule and alkaline phosphatase is configuration-selective, with the trans (trans TP) configuration reacting with alkaline phosphatase at a faster rate.

[0013] The organic small molecule has the ability to bind to albumin. The binding force between the organic small molecule and albumin is configuration-selective, with the cis (cis TP) configuration having a stronger binding force to albumin.

[0014] In a second aspect, the present invention provides a method for preparing the compound shown in Formula I, comprising the following steps:

[0015] 1)(1-(4-bromophenyl)-2-(4-methoxyphenyl)ethylene-1,2-diyl)diphenyl and N,N-dimethylbenzamide were synthesized via a bovoaldehyde reaction to obtain the compound shown in Formula II;

[0016]

[0017] 2) The compound shown in Formula II reacts with malondicyanate via the McMurray reaction to give the compound shown in Formula III;

[0018]

[0019] 3) The compound shown in Formula III undergoes demethylation under the action of boron tribromide to obtain the compound shown in Formula IV;

[0020]

[0021] 4) The compound shown in Formula IV is reacted with diethoxyphosphoryl chloride by esterification to give the compound shown in Formula V;

[0022]

[0023] 5) The compound shown in Formula V undergoes ethyl removal under the action of trimethylsilyl bromide to obtain the compound shown in Formula I with mixed cis and trans configurations;

[0024] 6) The compound of Formula I with mixed cis and trans configurations was separated and purified by high performance liquid chromatography to obtain the compound of Formula I with cis configuration and the compound of Formula I with trans configuration.

[0025] Further, step 1) is performed as follows: the (1-(4-bromophenyl)-2-(4-methoxyphenyl)ethylene-1,2-diyl)diphenyl is dissolved in an organic solvent, n-butyllithium is added under nitrogen protection at -78°C, and the reaction is carried out at -78°C for 2 to 4 hours (e.g., 2 hours). Then the N,N-dimethylbenzamide is added, and the reaction is carried out for 1 hour. The temperature is then slowly raised to room temperature, quenched with 10% hydrochloric acid solution, and the reaction is continued for 0.5 to 1 hour (e.g., 0.5 hours).

[0026] Furthermore, the organic solvent mentioned in step 1) can be tetrahydrofuran; the molar ratio of (1-(4-bromophenyl)-2-(4-methoxyphenyl)ethylene-1,2-diyl)diphenyl to n-butyllithium can be 1:1.2 to 1.5, specifically 1:1.2; the molar ratio of (1-(4-bromophenyl)-2-(4-methoxyphenyl)ethylene-1,2-diyl)diphenyl to N,N-dimethylbenzamide can be 1:1.2 to 2.0, specifically 1:1.2;

[0027] Further, step 2) is performed as follows: the compound shown in Formula II is mixed with the malononitrile and dissolved in an anhydrous organic solvent. Titanium tetrachloride is slowly added under nitrogen protection at zero degrees Celsius and reacted for 30 minutes. Then pyridine is added and reacted at 40 degrees Celsius for 4 to 8 hours (e.g., 4 hours). The reaction is then quenched with water.

[0028] Furthermore, the organic solvent in step 2) can be dichloromethane; the molar ratio of the compound represented by formula II to the malononitrile can be 1:1.5 to 2, specifically 1:1.5; the molar ratio of the compound represented by formula II to the titanium tetrachloride can be 1:3 to 5, specifically 1:3; the molar ratio of the compound represented by formula II to the pyridine can be 1:3 to 5, specifically 1:3.

[0029] Further, step 3) is performed as follows: the compound shown in Formula III is dissolved in an organic solvent, boron tribromide is added under nitrogen protection, and the reaction is carried out at room temperature for 4 to 6 hours (e.g., 4 hours);

[0030] Furthermore, the organic solvent mentioned in step 3) can be dichloromethane, and the molar ratio of the compound shown in Formula III to the boron tribromide can be 1:1.7 to 2.2, specifically 1:1.7;

[0031] Further, step 4) is performed as follows: under nitrogen protection, the compound shown in formula IV and sodium hydride are mixed and dissolved in an anhydrous organic solvent, and then the diethoxyphosphoryl chloride is added. The mixture is stirred at room temperature for 24 hours.

[0032] Furthermore, the organic solvent in step 4) can be tetrahydrofuran, and the molar ratio of the compound shown in Formula IV to the sodium hydride can be 1:2 to 3, specifically 1:2; the molar ratio of the compound shown in Formula IV to the diethoxyphosphoryl chloride can be 1:2 to 4, specifically 1:2.

[0033] Further, step 5) is performed as follows: the compound shown in formula V is dissolved in an anhydrous organic solvent, and under nitrogen protection at zero degrees, the trimethylsilane is added and stirred at room temperature for 4 h to 12 h (e.g., 4 h), then methanol is added and stirred at room temperature for 0.5 h to 2 h (e.g., 0.5 h).

[0034] Furthermore, the organic solvent mentioned in step 5) can be dichloromethane, and the molar ratio of the compound shown in formula V to the trimethylsilane can be 1:2 to 4, specifically 1:2;

[0035] Further, step 6) is performed as follows: the compound of formula I with cis and trans configurations is dissolved in a solvent and injected into a high-performance liquid chromatograph. Under gradient elution conditions, it is separated and purified by a reversed-phase column to obtain the compound of formula I with cis configuration and the compound of formula I with trans configuration.

[0036] Furthermore, the solvent mentioned in step 6) can be dimethyl sulfoxide, the stationary phase is a C18 packed column, the mobile phase is water containing 0.1% trifluoroacetic acid by volume as mobile phase A, and the mobile phase is acetonitrile containing 0.1% trifluoroacetic acid by volume as mobile phase B. The gradient elution program is as follows: 0-3 min, the volume fraction of mobile phase B is 25%; 3-28 min, the volume fraction of mobile phase increases from 25% to 100% (i.e. 0-3-28 min, 25%B-25%B-100%B).

[0037] Among them, the components with retention times of 18.3 min and 19.2 min correspond to the trans and cis configurations of compound I, respectively.

[0038] In this invention, the room temperature is 15–40°C, preferably 20–30°C, and more preferably 25°C.

[0039] Thirdly, the present invention protects a method for regulating the cis-trans isomer ratio in a compound of formula I, comprising the following steps: irradiating a solution containing the compound of formula I with visible light to obtain a mixture of cis-trans isomers in the compound of formula I with a molar ratio of trans to cis configuration of 53:47; or,

[0040] Irradiating a solution containing the compound shown in Formula I and serum albumin with visible light yields a mixture of cis-trans isomers in which the molar ratio of the trans and cis configurations of the compound shown in Formula I is 18:82.

[0041] The visible light can specifically be a light source with a wavelength of 450 nm.

[0042] Fourthly, the use of the compound of formula I of the present invention in at least one of the following A1)-A3):

[0043] A1) Prepare drugs for targeted cancer therapy targeting alkaline phosphatase;

[0044] A2) Prepare tumor-targeting drugs that utilize visible light and / or serum albumin to control the cis-trans isomer ratio, thereby controlling the antitumor activity of the drug and targeting alkaline phosphatase.

[0045] A3) Prepare a tumor-targeting therapy drug that utilizes binding to serum albumin to eliminate toxicity at non-target sites and targets alkaline phosphatase.

[0046] The tumor is cancer; specifically, it is cancer that highly expresses alkaline phosphatase, such as liver cancer, cervical cancer, or breast cancer.

[0047] Fifthly, the present invention protects the compound shown in Formula IV,

[0048]

[0049] In formula IV, Selected from cis isomers, trans isomers, or mixtures of cis and trans isomers.

[0050] Sixthly, the method for preparing the compound of Protective Formula IV of the present invention includes the following steps:

[0051] The compound shown in Formula I is hydrolyzed with alkaline phosphatase to give the compound shown in Formula IV.

[0052] In a seventh aspect, the use of the compound of Protective Formula IV of the present invention in the preparation of a medicament for tumor treatment or in the in vitro non-therapeutic purpose of promoting tumor cell apoptosis.

[0053] The tumor is cancer; specifically, it is cancer that highly expresses alkaline phosphatase, such as liver cancer, cervical cancer, or breast cancer.

[0054] The tumor cells may specifically be human liver cancer cells (HepG2), human cervical cancer cells (HeLa), and human breast cancer cells (MCF-7).

[0055] Compared with the prior art, the present invention has the following technical effects:

[0056] The organic small molecule and its cis-trans isomers disclosed in this invention exist stably in cis or trans configurations under dark conditions and are non-cytotoxic. This organic small molecule can serve as a substrate for alkaline phosphatase, and after hydrolysis, it generates a product (structure as shown in Formula IV) with tumor cell apoptosis-promoting activity. The trans configuration exhibits high alkaline phosphatase reactivity, thus demonstrating high tumor cell apoptosis-promoting activity; the cis configuration, however, has a stronger binding affinity to serum albumin and lower apoptosis-promoting activity. Under visible light irradiation, the organic small molecule undergoes cis-trans isomerization, and the efficiency of isomerization is regulated by the concentration of serum albumin in the environment. Based on this, biocompatible visible light and widely present albumin in the body can be used as dual regulatory mechanisms to ensure that the organic small molecule has no toxic side effects under physiological conditions of high serum albumin. In the tumor cell environment, through visible light irradiation and albumin removal, the organic small molecule is placed in the trans configuration, and after alkaline phosphatase reaction, it exerts a highly efficient tumor cell killing ability, achieving temporal and spatial control of drug activity, which has promising applications in on-demand precision tumor treatment. Attached Figure Description

[0057] Figure 1 This is a synthetic route diagram of the compound shown in Formula I in Example 1 of the present invention.

[0058] Figure 2 The high-performance liquid chromatography (HPLC) separation spectrum of the isomer of the compound shown in Formula I in Example 2 of this invention.

[0059] Figure 3 The diagram shows the reaction of the compound of Formula I in Example 3 of this invention with alkaline phosphatase, the fluorescence spectrum of the compound isomer after reaction with alkaline phosphatase or serum albumin, the HPLC chromatograms of the compound isomer before and after reaction with alkaline phosphatase, and the Michaelis constant of the reaction of the compound isomer with alkaline phosphatase.

[0060] Figure 4 The cis-trans isomerism of the compound of Formula I in Example 4 of this invention after visible light irradiation in different solvents.

[0061] Figure 5 The images shown are confocal laser scanning microscope images and HPLC separation diagrams of the cell lysate after the isomers of the compound shown in Formula I in Example 5 of this invention were incubated with liver cancer cells (HepG2) and normal embryonic kidney cells (HEK293).

[0062] Figure 6The images show flow cytometry analysis of the apoptosis behavior of the compound isomers shown in Formula I in Example 6 of this invention, as well as the MTT assay for the toxicity of the compound isomers to different types of tumor cells and normal cells.

[0063] Figure 7 This study investigated the anti-degradation properties of the compound shown in Formula I in Example 7 of the present invention in rat blood, and determined by flow cytometry the antitumor activity of the cis configuration under visible light irradiation and albumin clearance conditions. Detailed Implementation

[0064] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0065] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the materials and reagents used are commercially available unless otherwise specified.

[0066] Example 1: Synthesis of the compound shown in Formula I:

[0067] Chemical reaction flow chart as follows Figure 1 As shown, the specific reaction conditions are as follows:

[0068] 1) Synthesis of the compound shown in Formula II

[0069] (1-(4-bromophenyl)-2-(4-methoxyphenyl)ethylene-1,2-diyl)diphenyl (2 mmol) was added to a two-necked flask and dissolved in redistilled tetrahydrofuran. Under nitrogen protection, n-butyllithium (2.4 mmol) was added, and the reaction was continued at -78 °C for 2 h. Then, N,N-dimethylbenzamide (2.4 mmol) was added, and the reaction was continued for 1 h. The temperature was then slowly raised to room temperature, quenched with 10% hydrochloric acid solution, and the reaction was continued for 30 min. After the reaction was complete, the mixture was extracted with dichloromethane. The organic phase was dried, filtered, and purified by rotary evaporation to remove the solvent. The eluent was petroleum ether / dichloromethane (2:1, v / v), yielding compound II (383 mg, yield 41.1%).

[0070] The structural verification data is as follows:

[0071] 1 H NMR (400MHz, CDCl3) δ: 7.77-7.72 (t, 2H), 7.60-7.42 (m, 5H), 7.16-6.92 (m, 14H), 6.67-6.63 (t, 2H), 3.75 (s, 3H); 13C NMR (100MHz, CDCl3) δ: 158.5, 143.4, 137.8, 132.6, 132.2, 131.4, 131.2, 129.9, 12 9.8, 129.6, 128.4, 128.2, 127.9, 127.8, 127.7, 126.9, 126.6, 113.4, 113.1, 55.1;

[0072] Mass spectrometry: Calculated value is C 32 H 26 O2[M] + :466.6, mass spectrometry peak position: 466.0;

[0073] As can be seen from the above, the structure of the product is correct.

[0074] 2) Synthesis of the compound shown in Formula III

[0075] The compound shown in Formula II (0.4 mmol) and malononitrile (0.6 mmol) were mixed and dissolved in anhydrous dichloromethane under nitrogen protection. Titanium tetrachloride (1.2 mmol) was slowly injected at 0 °C. After reacting for 30 min, pyridine (1.2 mmol) was injected, and the reaction was continued at 40 °C for 4 h. After the reaction was completed, the mixture was quenched with water, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and purified by column chromatography using petroleum ether / dichloromethane (2:1, v / v) as the eluent to give an orange compound III solid (171 mg, yield 83.4%).

[0076] The structural verification data is as follows:

[0077] 1 H NMR (400MHz, CDCl3) δ: 7.61-7.35 (m, 5H), 7.21-7.01 (m, 14H), 6.94-6.90 (m, 2H), 6.68-6.65 (t, 2H), 3.76 (s, 3H); 13 C NMR (100MHz, CDCl3) δ: 158.8, 149.4, 143.2, 142.9, 140.8, 138.7, 136.2, 135.3, 133.6, 132.6, 13 2.5, 131.6, 131.4, 131.3, 130.4, 130.1, 128.8, 128.0, 127.7, 126.9, 126.8, 114.2, 114.0, 55.2;

[0078] Mass spectrometry: Calculated value is C 37 H 26 N2O[M] - 513.6, mass spectrometry peak position: 513.4;

[0079] As can be seen from the above, the structure of the product is correct.

[0080] 3) Synthesis of the compound shown in Formula IV

[0081] The compound shown in Formula III (0.3 mmol) was dissolved in dichloromethane in a two-necked flask under nitrogen protection, and then boron tribromide (0.5 mmol) was slowly added. After reacting at room temperature (25 °C) for 4 h, the solvent was removed by rotary evaporation, and the mixture was separated by column chromatography with petroleum ether / ethyl acetate (3:1, v / v) as the eluent to give compound IV (140 mg, yield 93.5%).

[0082] The structural verification data is as follows:

[0083] 1 H NMR (400MHz, DMSO-d6) δ: 9.45 (s, 1H), 7.64-7.52 (m, 3H), 7.44-7.42 (m, 2H), 7.29-7.11(m, 10H), 7.01-6.95(m, 4H), 6.78-6.72(t, 2H), 6.55-6.50(m, 2H); 13 C NMR (100MHz, DMSO-d6) δ: 156.8, 149.1, 143.7, 143.1, 138.6, 136.5, 134.1, 132.5, 132.4, 132.3, 131. 4, 131.2, 131.1, 130.3, 130.0, 129.9, 128.7, 128.0, 127.9, 127.7, 126.7, 126.7, 126.6, 114.7, 114.6;

[0084] Mass spectrometry: Calculated value is C 36 H 24 N2O[M] - :499.6, mass spectrometry peak position: 499.2;

[0085] As can be seen from the above, the structure of the product is correct.

[0086] 4) Synthesis of the compound shown in formula V

[0087] Compound IV (0.25 mmol) was added to 20 mL of anhydrous tetrahydrofuran under nitrogen protection along with sodium hydride (0.5 mmol), followed by the addition of diethoxyphosphoryl chloride (0.5 mmol). The mixture was stirred at room temperature (25 °C) for 24 h. After the reaction was complete, the mixture was quenched with 5 mL of water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The mixture was purified by column chromatography using dichloromethane / methanol (50:1, v / v) as the eluent, yielding an orange compound V solid (127 mg, 77.4% yield).

[0088] The structural verification data is as follows:

[0089] 1 H NMR (400MHz, CDCl3) δ: 7.59-7.48 (m, 3H), 7.46-7.43 (m, 2H), 7.38-7.36 (m, 2H ), 7.21-7.18(m, 8H), 7.14-6.93(m, 8H), 4.24-4.14(t, 4H), 1.35-1.30(t, 6H); 13 C NMR (100MHz, CDCl3) δ: 156.7, 149.3, 143.8, 143.2, 138.7, 136.6, 134.3, 132.4, 132.3, 132.2, 131.3, 131. 1, 131.0, 130.2, 130.1, 129.9, 128.8, 128.0, 127.8, 127.7, 126.8, 126.6, 126.4, 114.6, 114.5, 68.5, 20.1;

[0090] Mass spectrometry: Calculated value is C 40 H 34 N₂O₄P[M+H] + :637.2251, mass spectrometry peak position: 637.2247;

[0091] As can be seen from the above, the structure of the product is correct.

[0092] 5) Synthesis of the compound shown in Formula I

[0093] The compound shown in Formula V (0.1 mmol) was placed in a two-necked flask and dissolved in 10 mL of anhydrous dichloromethane. Under vacuum (N2) protection, trimethylsilane (0.2 mmol) was added using a syringe in an ice bath at 0 °C, and the reaction was stirred at room temperature (25 °C) for 4 h. Then, 1 mL of methanol was added, and stirring continued at room temperature for 30 min. After the reaction was complete, the solvent was removed by rotary evaporation. The crude product was purified by column chromatography using dichloromethane / methanol (4:1, v / v) as the eluent to give an orange-red powder (35.2 mg, yield 58%).

[0094] Example 2: Separation and characterization of the cis and trans configurations of the compound shown in Formula I

[0095] The compound of formula I obtained by column chromatography was dissolved in dimethyl sulfoxide, and isomers were separated using a C18 column in a high-performance liquid chromatography (HPLC) system. The mobile phase system was: phase A: water containing 0.1% trifluoroacetic acid; phase B: acetonitrile containing 0.1% trifluoroacetic acid; the linear gradient was: 0-3-28 min, 25% B-25% B-100% B. The HPLC separation chromatogram is shown below. Figure 2 As shown. The components with retention times of 18.3 min and 19.2 min correspond to the trans and cis configurations of compound I, respectively.

[0096] The following are the confirmatory data for the cis TP structure:

[0097] 1 H NMR (600MHz, DMSO-d6) δ: 7.67 (t, J = 3.0Hz, 1H), 7.60-7.57 (m, 2H), 7.44-7.43 (m, 2H), 7.29 -7.24(m,4H),7.21-7.19(m,4H),7.16-7.14(m,2H),7.09-7.08(m,2H),7.02-6.98(m,6H); 13 C NMR(150MHz, DMSO-d6):174.40,151.27,151.23,148.76,143.55,142.62,140.25,138.90,136.85,134.84,133.40,132 .76,131.83,131.54,131.10,131.02,129.77,128.98,128.81,127.85,127.77,120.22,120.19,115.29,115.23,82.23.

[0098] High-resolution mass spectrometry: Calculated value is C 36 H 25 N₂NaO₄P[M-H+Na] + :603.1444, mass spectrometry peak position: 603.1447;

[0099] As can be seen from the above, the product has the correct structure and is in the cis configuration of the compound.

[0100] The data confirming the trans TP structure are as follows:

[0101] 1 H NMR (600MHz, DMSO-d6) δ: 7.67 (t, J = 3.0Hz, 1H), 7.62-7.59 (m, 2H), 7.46-7.45 (m, 2H),7.34-7.32(m,2H),7.22-7.16(m,8H),7.06-7.03(m,4H),7.00-6.95(m,4H); 13C NMR(150MHz, DMSO-d6):174.31,148.78,143.46,143.23,142.67,140.24,138.99,136.85,134.76,133.38, 132.67,131.82,131.63,131.56,131.04,130.85,129.73,129.11,128.82,127.95,127.86,120.20,82.28.

[0102] High-resolution mass spectrometry: Calculated value is C 36 H 25 N₂NaO₄P[M-H+Na] + :603.1444, mass spectrometry peak position: 603.1447;

[0103] As can be seen from the above, the product has the correct structure and is the trans configuration of the compound.

[0104] Example 3: Recognition and stereoselectivity of the compound shown in Formula I with alkaline phosphatase and serum albumin.

[0105] Stock solutions of the isomers of compound TP shown in Formula I (trans TP and cis TP) were prepared at 1 mM using dimethyl sulfoxide as solvent. 10 μL of each stock solution was diluted with 890 μL of tris (10 mM, pH = 8.0) tris (hydroxymethyl)aminomethane (Tris) buffer, and then 100 μL of alkaline phosphatase (purchased from Sigma-Aldrich, product name: alkaline phosphatase (derived from bovine intestinal mucosa), product catalog number: P7640) was added to each (the enzyme activity of the stock solution was 1000 mU / mL). The solutions were incubated at room temperature for 90 min, and the fluorescence intensity was measured using a fluorescence spectrometer. The components after the enzymatic hydrolysis reaction were analyzed by HPLC.

[0106] Take 10 μL of each trans TP and cis TP stock solution, dilute with 980 μL of Tris buffer, and then add 10 μL of serum albumin stock solution (1 mM) to each. Incubate at room temperature for 90 min, and measure the fluorescence intensity of the solution using a fluorescence spectrometer.

[0107] The compound TP shown in Formula I of this invention has a tetraphenylethylene structure and exhibits aggregation-induced emission properties. For example... Figure 3 As shown in Figure a, the water-soluble TP undergoes an enzyme-catalyzed hydrolysis reaction with alkaline phosphatase (ALP) to generate the poorly water-soluble product Th. Th then aggregates and produces fluorescence (the structural confirmation data of Th is as follows: 1H NMR (400MHz, DMSO-d6) δ: 9.45 (s, 1H), 7.64-7.52 (m, 3H), 7.44-7.42 (m, 2H), 7.29-7.11(m, 10H), 7.01-6.95(m, 4H), 6.78-6.72(t, 2H), 6.55-6.50(m, 2H); 13 C10 NMR (100MHz, DMSO-d6) δ: 156.8, 149.1, 143.7, 143.1, 138.6, 136.5, 134.1, 132.5, 132.4, 132.3, 131.4, 131.2, 131.1, 130.3, 130.0, 129.9, 128.7, 128.0, 127.9, 127.7, 126.7, 126.7, 126.6, 114.7, 114.6; Mass spectrometry: calculated values ​​are C10 NMR values. 36 H 24 N2O[M] - :499.6, mass spectrum peak position: 499.7). The changes in fluorescence spectra after the reaction of transTP and cisTP with ALP are as follows: Figure 3 As shown in b. The solutions before and after the reaction were analyzed using HPLC, and the results are as follows. Figure 3 As shown in Figure c, cis TP and trans TP were hydrolyzed by ALP, and the corresponding chromatographic peaks essentially disappeared. New chromatographic peaks were generated at retention times of 22.4 min and 21.8 min, respectively, representing the hydrolysis products cis Th and trans Th. The enzymatic hydrolysis process was monitored and the kinetics of the enzymatic hydrolysis reaction were calculated according to the Michaelis-Menten equation, and the results are shown below. Figure 3 As shown in d, trans TP exhibits stronger reactivity with ALP, while cis TP shows lower reactivity with ALP, indicating the stereoselectivity of the enzymatic hydrolysis reaction.

[0108] The compound TP, represented by Formula I of this invention, has a tetraphenylethylene structure and exhibits aggregation-induced emission properties. When TP interacts with proteins, such as binding to a protein's hydrophobic pocket, it also generates a fluorescent signal due to the restricted rotation of intramolecular single bonds. Figure 3 As shown in b, cis TP and trans TP produced significant fluorescence signals after incubation with serum albumin (purchased from Beijing Solarbio Science & Technology Co., Ltd., product name: human serum albumin, product catalog number: A8230), indicating that the compound represented by Formula I has a specific interaction with serum albumin. Based on the magnitude of the fluorescence signal enhancement, cis TP was determined to have a stronger binding affinity to serum albumin, while trans TP had a lower binding affinity.

[0109] Example 4: Photoisomerization behavior of the compound shown in Formula I and its regulatory effect on serum albumin.

[0110] Take 10 μL of each of the trans TP and cis TP stock solutions and dilute them separately with 990 μL of standard phosphate buffered saline (PBS). Immerse the diluted solutions in a light source with a wavelength of 450 nm (24 mW / cm²). 2 Irradiation for 5 minutes was followed by HPLC analysis to investigate cis-trans isomerization. Results are as follows: Figure 4 As shown in a, regardless of whether cis TP or trans TP is the initial configuration, the final ratio of isomers in the solution after light irradiation is consistent, which is trans:cis = 53:47, with the trans configuration slightly dominant.

[0111] Take 10 μL of transTP and cisTP stock solutions respectively, dilute them with 890 μL of standard phosphate buffered saline (PBS), and then add 100 μL of serum albumin stock solution (stock solution concentration 40 mg / mL) to each. Examine the above solutions using a light source with a wavelength of 450 nm (24 mW / cm²). 2 After irradiation for 5 minutes, HPLC analysis was performed to investigate the effect of serum albumin on photoinduced cis-trans isomerization. Results are as follows: Figure 4 As shown in b, when serum albumin is present in the solution, regardless of whether cis TP or trans TP is the initial configuration, the final ratio of isomers in the solution after light irradiation is consistent, which is trans:cis = 18:82, with the cis configuration being the majority. This indicates that serum albumin has a regulatory effect on the photoinduced cis-trans isomerization behavior of compound I.

[0112] Example 5: The compound shown in Formula I was selectively taken up by tumor cells targeting alkaline phosphatase.

[0113] HepG2 liver cancer cells and normal embryonic kidney cells (HEK293) were seeded at the same density into confocal microscopy-specific culture dishes and cultured overnight in a cell culture incubator to allow cell adhesion. 10 μL of each of the trans TP and cis TP stock solutions were diluted with 990 μL of standard cell culture medium. The culture medium for HepG2 and HEK293 cells was removed, and the diluted trans TP and cis TP solutions were added, respectively, and incubated for 1 hour. The uptake of TP by tumor cells and normal cells was observed using a confocal laser scanning microscope under the following conditions: 405 nm laser, fluorescence receiving wavelength range 550-650 nm. Results are as follows: Figure 5As shown in Figure a, bright fluorescence was observed in HepG2 liver cancer cells after incubation with cis TP and trans TP, while no fluorescence signal was observed in HEK293 cells treated with the same method. This indicates that trans TP and cis TP can selectively enter liver cancer cells instead of normal cells.

[0114] HepG2 liver cancer cells and normal embryonic kidney cells (HEK293) were seeded at the same density into 6-well plates and cultured overnight in a cell culture incubator to allow cell adhesion. 20 μL of transTP and cisTP stock solutions were diluted with 980 μL of standard cell culture medium, respectively. The culture medium for HepG2 and HEK293 cells was removed, and the diluted transTP and cisTP solutions were added to each well, respectively. The cells were incubated for 2 h. After incubation, the solutions were removed, and the cells were washed once with PBS. 200 μL of cell lysis buffer was added to each well, and the cells were lysed on ice for 10 min. The cell lysates were collected and analyzed by HPLC to determine the TP taken up by the cells. The results are as follows: Figure 5 As shown in b, the enzymatic hydrolysis product Th of the compound after reacting with alkaline phosphatase was detected in HepG2 cells after incubation with trans TP and cis TP, respectively. The content of trans TP hydrolysis product in HepG2 cells was higher than that of cis TP hydrolysis product, indicating that trans TP and cis TP achieve selective uptake by tumor cells by recognizing alkaline phosphatase highly expressed in tumor cells. Since trans TP has stronger reactivity with alkaline phosphatase, the uptake and hydrolysis efficiency of trans TP in tumor cells is higher.

[0115] Example 6: The compound shown in Formula I achieves tumor suppression by promoting tumor cell apoptosis.

[0116] 10 μL of each trans TP and cis TP stock solution were diluted with 990 μL of standard cell culture medium. HepG2 liver cancer cells were collected in suspension, centrifuged, and the culture medium was discarded. Diluted trans TP and cis TP solutions were added, and the cells were resuspended by pipetting. The cells were incubated in an incubator for 3 h. After incubation, the solutions were removed by centrifugation, and the apoptosis status was determined using a commercially available apoptosis assay kit. The steps were as follows: HepG2 cells were resuspended in Binding Buffer (195 μL) from the kit, and Annexin V-FITC (5 μL) from the kit was added. The mixture was thoroughly mixed and incubated at room temperature in the dark for 10 min. After centrifugation, the supernatant was discarded, and the cells were washed once with Binding Buffer (200 μL). The cells were then resuspended in Binding Buffer (200 μL), and 10 μL of acridine iodide (PI, 20 μg / mL) was added. The apoptosis status of the treated HepG2 liver cancer cells was determined using flow cytometry. The results are as follows: Figure 6 As shown in Figure a, the Annexin V-FITC channel fluorescence signal was enhanced in HepG2 cells treated with TP, indicating that the apoptosis marker phosphatidylserine was everted and thus bound by Annexin V-FITC, suggesting that both trans TP and cis TP could induce apoptosis in HepG2 cells. Specifically, the apoptosis rate in HepG2 cells incubated with cis TP was 45%, while the apoptosis rate in HepG2 cells incubated with trans TP was 100% (98% early apoptosis and 2% late apoptosis), indicating that trans TP has a stronger ability to promote apoptosis.

[0117] The antiproliferative effects of the compound shown in Formula I and its cis-trans isomers on human hepatocellular carcinoma cells (HepG2), human cervical cancer cells (HeLa), and human breast cancer cells (MCF-7) were determined using the MTT assay. The experiment was conducted in the dark. MTT, or thiazolyl blue, is a tetrazolium salt. In living cells, mitochondrial succinate dehydrogenase reduces MTT to a blue-violet product soluble in dimethyl sulfoxide—methazine. This product absorbs at 570 nm, so the absorbance at 570 nm can be used to analyze cell proliferation. Specifically, cells can be incubated at 5 × 10⁻⁶ cells / day. 4Cells were seeded at a density of 100 μL / well in 96-well plates and incubated overnight to allow cell adhesion. The stock solutions of trans TP and cis TP were diluted to 10 μM with cell culture medium, replacing the original medium in each well. The 96-well plates were returned to the cell culture incubator and incubated for another 24 hours. Afterward, the solution in each well was replaced with 0.5 mg / mL MTT solution (100 μL / well), and the plates were returned to the incubator. After 4 hours of incubation, the MTT solution was completely replaced with dimethyl sulfoxide (150 μL / well). After thorough dissolution of formazan, the absorbance of each well was read at 570 nm using a microplate reader. Cell viability (VR) was calculated using the following formula: VR = (A / A0) × 100%, where A is the absorbance of the experimental group, and A0 is the absorbance of cells in the culture medium without any treatment. The cis-trans isomers of the compound shown in Formula I exhibited different cytotoxic effects on different tumor cells. Figure 6 As shown in b, both trans TP and cis TP can inhibit the proliferation of different types of tumor cells. The killing effect of trans TP on HepG2, HeLa and MCF-7 is higher than that of cis TP, which is consistent with the stronger ability of trans TP to promote tumor cell apoptosis.

[0118] Example 7: Targeted antitumor activity of compounds activated by visible light and proteins.

[0119] Drug molecule decomposition during blood delivery can reduce efficacy at the tumor site and is a major cause of toxic side effects in non-lesion tissues. TP binds to serum albumin, thus utilizing albumin as a protective barrier against degradation by alkaline phosphatase in the blood, improving stability and thereby enhancing efficacy at the tumor site while reducing toxic side effects. 40 μL of trans TP stock solution was directly injected into 960 μL of rat blood. Blood samples were collected at 0 h and 4 h for centrifugation, and plasma was collected. Acetonitrile was added to the plasma, and after centrifugation, the supernatant was collected for HPLC analysis. The results are as follows: Figure 7 As shown in Figure a, after 4 hours in the blood, transTP remained intact, and its chromatographic peak intensity did not decrease compared to the initial value (0 h), indicating that transTP can be protected by serum albumin in the blood and will not undergo hydrolysis to produce the toxic compound Th. Upon reaching tumor cells, transTP is hydrolyzed by alkaline phosphatase highly expressed on the tumor cell membrane, activating its antitumor activity.

[0120] The compound shown in Formula I can have its antitumor activity activated by visible light and albumin removal. The specific steps are as follows: Take two 10 μL portions of cis TP stock solution and dilute them with 990 μL of standard cell culture medium (containing albumin) and 990 μL of DMEM cell culture medium (without albumin), respectively. Collect suspended HepG2 liver cancer cells, divide them into two equal portions, centrifuge and discard the culture medium, add the two diluted cis TP solutions to each portion, and resuspend the cells by pipetting. Expose the cell suspensions to a light source with a wavelength of 450 nm (24 mW / cm²). 2 Irradiation for 5 minutes, followed by incubation in an incubator for 3 hours. After incubation, the solution was removed by centrifugation, and the apoptosis status of the cells was determined using a commercially available apoptosis assay kit. HepG2 cells were resuspended in Binding Buffer (195 μL) from the kit, and Annexin V-FITC (5 μL) from the kit was added. After mixing thoroughly, the cells were kept at room temperature in the dark for 10 minutes. After centrifugation, the supernatant was discarded, and the cells were washed once with Binding Buffer (200 μL). The cells were then resuspended in Binding Buffer (200 μL), and 10 μL of acridine iodide (PI, 20 μg / mL) was added. The apoptosis status of the treated HepG2 cells was determined by flow cytometry. The results are as follows: Figure 7 As shown in b, after HepG2 cells were irradiated with visible light in a cisTP solution containing albumin, the proportion of cells entering apoptosis was 63% (61% early apoptosis and 2% late apoptosis), which was higher than the results of cisTP in HepG2 cells without light treatment (apoptosis rate 45%). Figure 6 a) This indicates that photoinduced cis→trans isomerization can enhance antitumor activity. In albumin-free cis TP solution, HepG2 cells irradiated with visible light showed an apoptosis rate of 97% (92% early apoptosis, 5% late apoptosis), with a further significant increase in the apoptosis rate. This suggests that visible light and albumin can be used to control cis→trans isomerization, thereby significantly activating the pro-apoptotic and antitumor activities of cis TP. The unique visible light response and protein recognition properties of compound I have great application potential in improving the efficacy of drugs at tumor sites and eliminating toxic side effects at non-target sites.

[0121] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The compound shown in Formula I, Formula I In Formula I, Selected from cis isomers, trans isomers, or mixtures of cis and trans isomers.

2. A method for preparing the compound of formula I according to claim 1, comprising the following steps: 1) (1-(4-bromophenyl)-2-(4-methoxyphenyl)ethylene-1,2-diyl)diphenyl reacts with N,N-dimethylbenzamide via a bovoaldehyde synthesis reaction to obtain the compound shown in Formula II; Formula II 2) The compound shown in Formula II reacts with malononitrile via a McMurray reaction to give the compound shown in Formula III; Formula III 3) The compound shown in Formula III undergoes demethylation under the action of boron tribromide to obtain the compound shown in Formula IV; Formula IV 4) The compound shown in Formula IV is esterified with diethoxyphosphoryl chloride to give the compound shown in Formula V; Formula V 5) The compound shown in Formula V undergoes ethyl removal under the action of trimethylsilyl bromide to obtain the compound shown in Formula I with mixed cis and trans configurations; 6) The compound of Formula I with mixed cis and trans configurations was separated and purified by high performance liquid chromatography to obtain the compound of Formula I with cis configuration and the compound of Formula I with trans configuration.

3. The method according to claim 2, characterized in that: Step 1) is performed as follows: The (1-(4-bromophenyl)-2-(4-methoxyphenyl)ethylene-1,2-diyl)diphenyl is dissolved in an organic solvent at -78°C. o Add n-butyllithium under nitrogen protection, -78 o After reacting at C for 2-4 hours, N,N-dimethylbenzamide is added, and the reaction is continued for 1 hour. The mixture is then slowly brought to room temperature, quenched with 10% hydrochloric acid solution, and the reaction is continued for another 0.5-1 hour. Step 2) is performed as follows: The compound shown in Formula II is mixed with the malononitrile and dissolved in an anhydrous organic solvent. Titanium tetrachloride is slowly added under nitrogen protection at 0°C, and the reaction is carried out for 30 min. Then pyridine is added, and the reaction is carried out at 40°C. o C reacts for 4 to 8 hours, and is then quenched with water. Step 3) is performed as follows: Dissolve the compound shown in Formula III in an organic solvent, add the boron tribromide under nitrogen protection, and react at room temperature for 4 h to 6 h; Step 4) is performed as follows: Under nitrogen protection, the compound shown in Formula IV and sodium hydride are mixed and dissolved in an anhydrous organic solvent, and then the diethoxyphosphoryl chloride is added. The mixture is stirred at room temperature for 24 h. Step 5) is performed as follows: Dissolve the compound shown in Formula V in an anhydrous organic solvent, add the trimethylsilane under nitrogen protection at zero degrees, stir and react at room temperature for 4 h to 12 h, then add methanol and stir and react at room temperature for 0.5 h to 2 h. Step 6) is performed as follows: The compound of Formula I with cis and trans configurations is dissolved in a solvent and injected into a high-performance liquid chromatograph. Under gradient elution conditions, it is separated and purified by a reversed-phase column to obtain the compound of Formula I with cis configuration and the compound of Formula I with trans configuration.

4. The method according to claim 3, characterized in that: The organic solvent mentioned in step 1) is tetrahydrofuran; the molar ratio of (1-(4-bromophenyl)-2-(4-methoxyphenyl)ethylene-1,2-diyl)diphenyl to n-butyllithium is 1:1.2~1.5; the molar ratio of (1-(4-bromophenyl)-2-(4-methoxyphenyl)ethylene-1,2-diyl)diphenyl to N,N-dimethylbenzamide is 1:1.2~2.0; The organic solvent mentioned in step 2) is dichloromethane; the molar ratio of the compound shown in Formula II to the malononitrile is 1:1.5~2, the molar ratio of the compound shown in Formula II to the titanium tetrachloride is 1:3~5, and the molar ratio of the compound shown in Formula II to the pyridine is 1:3~5; The organic solvent mentioned in step 3) is dichloromethane, and the molar ratio of the compound shown in Formula III to the boron tribromide is 1:1.7~2.2; The organic solvent mentioned in step 4) is tetrahydrofuran, the molar ratio of the compound shown in formula IV to the sodium hydride is 1:2~3, and the molar ratio of the compound shown in formula IV to the diethoxyphosphoryl chloride is 1:2~4. The organic solvent mentioned in step 5) is dichloromethane, and the molar ratio of the compound shown in formula V to the trimethylsilane is 1:2~4; The solvent mentioned in step 6) is dimethyl sulfoxide, the stationary phase is a C18 packed column, the mobile phase is water containing 0.1% trifluoroacetic acid by volume A, and the mobile phase is acetonitrile containing 0.1% trifluoroacetic acid by volume B. The gradient elution program is as follows: 0~3 min, the volume fraction of mobile phase B is 25%; 3~28 min, the volume fraction of mobile phase increases from 25% to 100%.

5. A method for controlling the cis-trans isomer ratio in the compound of formula I according to claim 1, comprising the following steps: irradiating a system containing the compound of formula I with visible light to obtain a mixture of cis-trans isomers in which the molar ratio of the trans configuration to the cis configuration in the compound of formula I is 53:47; or, Irradiating a system containing the compound shown in Formula I and serum albumin with visible light yields a mixture of cis-trans isomers in which the molar ratio of the trans and cis configurations in the compound shown in Formula I is 18:

82.

6. The use of the compound of Formula I according to claim 1 in the preparation of a drug for targeted tumor therapy targeting alkaline phosphatase.

7. The application according to claim 6, characterized in that, The application is any one of the following A2)-A4): A2) The application of the compound shown in Formula I in the preparation of a tumor-targeting therapy drug that uses visible light to control the cis-trans isomer ratio and thus control the antitumor activity of the drug, targeting alkaline phosphatase. The application of the compound shown in Formula I (A3) in the preparation of a tumor-targeting therapy drug that uses visible light and serum albumin to control the cis-trans isomer ratio and thus control the antitumor activity of the drug, targeting alkaline phosphatase. The application of the compound shown in Formula I (A4) in the preparation of a drug for tumor-targeted therapy that utilizes binding to serum albumin to reduce toxicity at non-target sites and targets alkaline phosphatase.

8. The application according to claim 6 or 7, characterized in that: The tumor is cancer; specifically, it may be liver cancer, cervical cancer, or breast cancer.

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