A preparation method and application of anticancer prodrug based on cascade reaction

Abstract

A method for preparing an anticancer prodrug based on a cascade reaction and its application. The present invention belongs to the field of medical technology, and discloses a method for preparing a prodrug molecule that can be activated by hydrogen peroxide and its application. The present invention connects podophyllotoxin with a fluorophore precursor via an ester bond to obtain a visualized prodrug that releases podophyllotoxin and a fluorophore after activation with hydrogen peroxide. The present invention monitors its fluorescence release by an ultraviolet-visible spectrometer and a fluorescence spectrometer, and at the same time, takes the prodrug molecule as the research object and human liver cancer cell HepG2 as the test cell line to carry out its in vitro anticancer activity research, laying a foundation for the application of prodrug molecules in diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the fields of organic synthesis and molecular medicine, and in particular to a preparation method of an anticancer prodrug based on a cascade reaction and an application thereof. Background Art

[0002] The development of diagnostic and therapeutic strategies can simultaneously be used to diagnose early-stage cancer, deliver therapeutic drugs to tumors and visualize the stage of cancer, providing more effective and personalized treatment for cancer patients. The tumor microenvironment (TME) is the cellular environment in which tumors develop and thrive.

[0003] Different from normal cells, various tumors and cancer cells overproduce reactive oxygen species (ROS), such as hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (·OH) and superoxide anions (O 2 ·- ), higher levels of oxidative stress have been observed. Based on this, we designed and synthesized a H 2 O 2 After activation, the prodrug releases the fluorophore and active drug molecule to achieve the purpose of targeted drug delivery to cancer cells and simultaneous diagnosis. Summary of the invention

[0004] The first purpose of the present invention is to provide an anticancer prodrug molecule based on a new strategy. The prodrug molecule has a simple structure, is easy to obtain, is convenient to synthesize, has a good tumor treatment effect, and has a high application value.

[0005] The second purpose of the present invention is to provide a method for preparing an anticancer prodrug molecule. The method connects the anticancer active molecule with the coumarin precursor through a simple reaction, and has a high reaction yield and good purity.

[0006] The third object of the present invention is to provide an anticancer prodrug molecule.

[0007] The embodiment of the present invention is achieved as follows:

[0008] An anticancer prodrug molecule, the structural formula of which is:

[0009]

[0010] A method for preparing an anticancer prodrug molecule, comprising:

[0011] The anticancer prodrug molecule is obtained by a simple condensation reaction of a coumarin precursor with a carboxyl group and an anticancer active molecule with a hydroxyl group.

[0012] An anticancer prodrug molecule is prepared by the above preparation method.

[0013] The beneficial effects of the embodiments of the present invention are:

[0014] The present invention provides an anticancer prodrug molecule, and the fluorescence release of the prodrug molecule is monitored by a UV-visible spectrometer and a fluorescence spectrometer. At the same time, the prodrug molecule is used as a research object, and human liver cancer cell HepG2 is used as a test cell line to carry out its in vitro anticancer activity research, which lays a foundation for the application of prodrug molecules in diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 The prodrug molecule CM-PPT provided in Experimental Example 1 of the present invention and hydrogen peroxide (H 2 O 2 )'s ultraviolet absorption spectrum action result diagram;

[0017] Figure 2 The prodrug molecule CM-PPT provided in Experimental Example 1 of the present invention and H 2 O 2 The fluorescence spectrum time dependence result diagram;

[0018] Figure 3 The prodrug molecule CM-PPT provided in Experimental Example 1 of the present invention and H 2 O 2 The fluorescence spectrum concentration dependence result diagram;

[0019] Figure 4 This is a diagram showing the selectivity test results of the prodrug molecule CM-PPT provided in Experimental Example 1 of the present invention;

[0020] Figure 5 The prodrug molecule CM-PPT provided in Experimental Example 1 of the present invention and H 2 O 2 The effect results under different pH conditions;

[0021] Figure 6 The prodrug molecule CM-PPT and H 2 O 2 The corresponding HPLC action result graph, standard curve and release curve of the anticancer active molecule podophyllotoxin (PPT);

[0022] Figure 7This is a graph showing the toxicity results of the prodrug molecule CM-PPT provided in Experimental Example 1 of the present invention on HepG22 cells;

[0023] Figure 8 This is a cell imaging image of the prodrug molecule CM-PPT provided in Experimental Example 1 of the present invention in HepG2 cells;

[0024] Fig. 9 This is a diagram of cell apoptosis of the prodrug molecule CM-PPT and the anticancer active molecule PPT in HepG2 cells provided in Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0026] The following is a detailed description of an anticancer prodrug molecule and a preparation method thereof and a targeting compound according to an embodiment of the present invention.

[0027] Reactive oxygen species (ROS) are a type of 2 Highly reactive oxygen-containing intermediates produced by continuous single-electron reduction play an important role in regulating cell growth, proliferation, differentiation, apoptosis, and participating in biosynthetic pathways. Redox dynamic balance is achieved by regulating the generation and catabolism of ROS. When ROS are overproduced or the antioxidant system is insufficient, cells are placed in a state of oxidative stress, leading to cell damage. Studies have shown that oxidative stress is associated with the development of many diseases, including cancer, autoimmune diseases, inflammation, cardiovascular diseases, and neurodegenerative diseases including Alzheimer's and Parkinson's disease. Hydrogen peroxide (H 2 O 2 ) is unique among endogenous ROS. 2 O 2 It plays important roles in the body, including immune responses and cell signaling. 2 O 2 When it is activated, it will cause oxidative damage to cells. Studies have shown that H 2 O 2 The concentration will be significantly higher than that of H in normal tissues. 2 O 2 This physiological level of concentration difference is based on the design of a specific H 2 O 2Activated prodrug molecules. Studies have found that aryl borate esters can be specifically activated by H 2 O 2 Activation. The coumarin precursor with aryl boronate is connected to the anticancer active molecule to obtain a prodrug compound; the prodrug compound can specifically activate H overexpressed in cancer cells. 2 O 2 Activated, it releases anticancer active molecules and fluorophores.

[0028] The present invention provides a method for preparing an anticancer prodrug molecule, comprising:

[0029] The coumarin precursor with aryl borate ester is connected to the hydroxyl group of the anticancer active molecule to obtain the anticancer prodrug molecule.

[0030] Furthermore, the active group is a hydroxyl group, and the preparation method comprises:

[0031] The -OH group of the anticancer active molecule is condensed with the coumarin precursor through an ester condensation reaction to obtain a prodrug molecule.

[0032] The structural formula of the above anticancer active molecule is:

[0033]

[0034] The embodiment of the present invention provides an anticancer prodrug molecule, which is prepared by the above-mentioned preparation method.

[0035] Furthermore, the above anticancer prodrug molecule has the structural formula:

[0036]

[0037] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0038] Example 1

[0039] This embodiment provides an anticancer prodrug molecule (named CM-PPT), whose structural formula is:

[0040] The reaction formula for preparing the anticancer prodrug molecule (CM-PPT) is:

[0041]

[0042] Reagentsandconditions:a.4-(Bromomethyl)benzeneboronicacidpinacolester,K 2 CO 3 ,CH 3CN, 65℃; b.

[0043] Cyanoaceticacid,Piperidine,AcOH,EtOH,50℃;c.Podophyllotoxin,EDCI,DMAP,DCM,rt;d.Ethylcyanoacetate,

[0044] Piperidine, AcOH, EtOH, 90℃.

[0045] The preparation method is as follows:

[0046] S1. Weigh 4-diethylamino salicylaldehyde (0.966 g, 5 mmol), 4-bromomethylphenylboronic acid pinacol ester (1.485 g, 3 mmol), K 2 CO 3 (2.073 g, 15 mmol) was dissolved in 30 mL CH 3 CN, and stirred the mixture at 65°C for 10 h under the protection of Ar gas. After the reaction, the solvent was dried and the product (1.534 g, 75%) was obtained by column chromatography (petroleum ether:ethyl acetate=5:1).

[0047] S2. Weigh compound 1 (309 mg, 0.75 mmol) and cyanoacetic acid (128 mg, 1.50 mmol) and dissolve them in 30 mL of ethanol, then add 0.3 mL of piperidine and 0.3 mL of glacial acetic acid. The mixture is reacted at 50 °C for 12 h under Ar gas protection. After the reaction, the ethanol is dried and the product is obtained by column chromatography (dichloromethane: methanol = 60:1) as a yellow solid (286 mg, 80%).

[0048] S3. Compound 2 (48 mg, 0.1 mmol) was weighed and dissolved in 5 mL of anhydrous DCM, EDCI (21 mg, 0.11 mmol) and DMAP (1.2 mg, 0.01 mmol) were added thereto, and the reaction system was stirred at room temperature for 5 min, and compound B (41 mg, 0.1

[0049] mmol) was added thereto, and the mixture was stirred at room temperature overnight. After the reaction, the solvent was dried by spin drying, and the product was obtained as a yellow solid (20 mg, 45%) after column chromatography (petroleum ether: dichloromethane: ethyl acetate = 2:1:1).

[0050] S4. Dissolve 4-(diethylamino)-salicylaldehyde (387 mg, 2 mmol) in 20 ml of ethanol, and then add ethyl cyanoacetate (319 μL, 3 mmol), piperidine (792 μL, 8 mmol) and acetic acid (10 μL). Reflux the reaction solution at 90°C for 1 h and pour it into ice water to precipitate a yellow solid. The residue was purified by column chromatography (petroleum ether: acetone = 2:1) to obtain the product (242 mg, yield 50%) as a yellow solid.

[0051] It should be noted that, in this embodiment, the anticancer drug podophyllotoxin PPT is preferably used as the anticancer active molecule, and the anticancer active molecule may also be other anticancer drugs containing active groups such as amino and / or hydroxyl groups.

[0052] Experimental Example 2

[0053] This experimental example provides a prodrug molecule provided in Example 1 and H 2 O 2 Investigate the reaction situation.

[0054] This experimental example provides an anticancer prodrug molecule (named CM-PPT) provided in Example 1 and H 2 O 2 interaction.

[0055] The reaction of the prodrug molecule CM-PPT with H was monitored by UV-visible spectrometer and fluorescence spectrometer. 2 O 2 Interaction process.

[0056] Prodrug molecules (10 μM) and H 2 O 2 (10eq) effect

[0057] UV absorption and fluorescence spectra were measured by UV and fluorescence spectrophotometer in DMSO / PBS buffer solution (pH = 7.4, V / V = 5 / 5). 2 O 2 (100μM) UV absorption and fluorescence spectra before and after the reaction.

[0058] Prodrug molecule CM-PPT and H 2 O 2 Reaction: CM-PPT and HO were added to a final concentration of 10 μM at 37°C. 2 O 2 (100 μM) was mixed in 2 mL of PBS (10 mM, pH = 7.4) buffer solution and incubated for 8 h. The UV absorption was tested by UV spectrophotometer, and the following results were obtained: Figure 1 Spectrum shown. DMSO content is 50%.

[0059] like Figure 1 As shown, no H was added 2 O 2 Previously, the maximum UV absorption was at 455 nm, but after adding H 2 O 2 After incubation, the maximum UV absorption blue shifted to 431 nm. The experimental results show that the prodrug CM-PPT has an effect on H 2 O 2 There is a certain response.

[0060] Experimental Example 3

[0061] This experimental example verifies that H 2 O 2 The interaction with the prodrug molecule CM-PPT changes with time and different concentrations of H 2 O 2 Interaction with the prodrug molecule CM-PPT.

[0062] The excitation wavelength of the fluorescence spectrum test was 431 nm, the excitation slit width was 5 nm, the emission slit width was 10 nm, and the test voltage was 590 V. 2 O 2 (100 μM) and the prodrug molecule CM-PPT (10 μM) and different concentrations of H 2 O 2 The test was performed at 37°C.

[0063] like Figure 2 As shown, the prodrug CM-PPT reacts with H 2 O 2 After the reaction, the fluorescence intensity of the system at 473 nm gradually increased with time and reached equilibrium at 4 h. Figure 3 As shown, when H 2 O 2 When the concentration was 200 μM, the fluorescence change rate slowed down, and when the concentration was 300 μM, the fluorescence signal tended to be stable. 2 O 2 The reaction is concentration-dependent, and the fluorescence intensity is related to H 2 O 2 The concentration has a good linear relationship in the range of 0-10eq, R 2 The above experimental results show that CM-PPT has the potential to release PPT and fluorophores in a time- and dose-dependent manner.

[0064] Experimental Example 4

[0065] This experimental example provides the interaction between the anticancer prodrug molecule (named CM-PPT) synthesized in Example 1 and other interfering substances and the interaction with H under different pH conditions. 2 O 2 reaction situation.

[0066] After the prodrug molecule CM-PPT (10 μM) was incubated with other interfering substances for a certain period of time, its fluorescence signal was tested and the following results were obtained: Figure 4 The results are shown. 2 O 2 In addition, other ROS and amino acid interfering substances did not respond well to CM-PPT. 2 O 2 It has good selectivity, which lays a good foundation for its application in organisms. Figure 5 As shown, no H 2 O 2 Previously, the prodrug molecule CM-PPT had good stability in the pH range of 4-10. 2 O 2 After incubation for 4 h, the prodrug molecule CM-PPT and H 2 O 2 The reaction systems all showed strong fluorescence intensity, and the prodrugs had good stability under strong acid and strong alkaline conditions. The experimental results showed that the prodrug molecule CM-PPT can react with H under physiological conditions. 2 O 2 It has a good response and can be applied to cell imaging and tumor treatment.

[0067] Experimental Example 5

[0068] With H 2 O 2 After the interaction, the anti-cancer active molecule PPT is released.

[0069] The HPLC detection method was used; the final concentration of CM-PPT was 10 μM and the final concentration of H 2 O 2 Mix in 20mL PBS buffer solution with 1% DMSO. Then incubate at 37°C for 1h, 2h, 3h, 4h, 5h, 6h, 7h and 8h. After incubation, analyze the PPT content in the solution by HPLC. The mobile phase is methanol / water = 75 / 25, and the flow rate is 0.6mL / min.

[0070] A standard curve of PPT concentration was established by HPLC.

[0071] Concentration (μM) 1 2 5 10 15 20 Retention time (min) 4.278 4.284 4.272 4.281 4.274 4.269 Peak area 3826 6990 17344 35468 53243 68974

[0072] like Figure 6 As shown, the standard curve of PPT is: y = 3748.00x + 215.84, R 2 =0.9995. HPLC determination of CM-PPT and H 2 O 2 The concentration of PPT released after interaction is shown in Figure 6 c, As the incubation time increases, the released PPT also increases, indicating that CM-PPT can be released through H 2 O 2 Achieve a sustained release effect.

[0073] Experimental Example 6

[0074] This experimental example determines the toxicity of CM-PPT synthesized in Example 1 to cells.

[0075] In this experiment, human liver cancer cell line HepG2 cells were selected to evaluate the biological activity of the prodrug molecules. HepG2 cells were cultured in a prepared cell culture medium (DMEM) and maintained at 5% CO 2 concentration and placed in a cell culture incubator at 37°C. 4 HepG2 cells and different concentrations of prodrug molecules CM-PPT were mixed and planted in 96-well plates (final volume of 100 μL), and cells were treated with DMSO as a control. After the prodrug CM-PPT and the prototype drug PPT acted on the cells for 44 hours, 100 μL of MTT (5 mg / mL) was added to each well and incubated for 4 hours. Then 100 μL of triple reagent (10% SDS, 5% isobutanol, 0.1% HCl) was added to the 96-well plate, placed in a cell culture incubator overnight, and then its absorption at 570nm was measured using an ELISA reader.

[0076] The results are as follows Figure 7 As shown, the experimental results show that CM-PPT has less cytotoxicity to cancer cells. In cancer cells, the time for CM-PPT to exert its efficacy is significantly longer than that of PPT, indicating that CM-PPT has a sustained release effect.

[0077] Experimental Example 7

[0078] This experimental example measured the cell imaging of CM-PPT synthesized in Example 1.

[0079] HepG2 cells were cultured at 1×10 4 Cells were inoculated in 12-well plates in 0.5 mL of culture medium and cultured at 37°C for 24 h. The cells were then divided into three groups. The first group of cells was incubated at 37°C for 1 h and 4 h with only prodrug CM-PPT (2 μM). The second group of cells was incubated with H 2 O 2(200μM) pretreatment for 30min, the cells were washed 3 times with PBS, and the prodrug CM-PPT (2μM) was added, and the cells were incubated at 37°C for 1h and 4h. Studies have shown that stimulating cells with lipopolysaccharide (LPS) can further increase the level of endogenous ROS. Therefore, the third group of cells were pretreated with LPS (1μg / mL) for 24h, and the prodrug CM-PPT (2μM) was added after washing the cells 3 times with PBS, and the cells were incubated at 37°C for 1h and 4h. After washing the cells 3 times with PBS, the cells were photographed using the Floid cell imaging workstation.

[0080] The results are as follows Figure 8 As shown in a, the control group had almost no fluorescence. The cells incubated with only the prodrug CM-PPT showed only weak green fluorescence after 4 hours. The fluorescence intensity of the second group of cells pretreated with H2O2 was stronger after 4 hours of CM-PPT. The fluorescence intensity of the third group of cells pretreated with LPS after 4 hours of CM-PPT was stronger than that of the first group, and was not much different from that of the second group. This result can also be obtained from the fluorescence quantitative graphs of 8b and 8c. The experimental results of cell imaging show that the prodrug CM-PPT can be oxidized by endogenous H 2 O 2 The drug release was monitored by the release of fluorophores after activation. 2 O 2 Alternatively, LPS-stimulated increase in endogenous ROS can promote the activation of the prodrug.

[0081] Experimental Example 8

[0082] This experimental example measured the cell apoptosis of CM-PPT synthesized in Example 1.

[0083] HepG2 cells were cultured at 1 × 10 cells per well in 1.0 mL of culture medium. 4 Cells were inoculated in a 6-well plate and cultured at 37°C for 24 hours. The cells were divided into two groups. PPT (5μM, 10μM) and prodrug CM-PPT (5μM, 10μM) were added to the two groups of cells for 48 hours, and the amount of DMSO introduced was 0.5%. The experimental results are shown in Fig. 9 As shown, CM-PPT can induce more cell apoptosis than PPT, and the number of apoptotic cells increases with the increase of concentration. The experimental results show that the prodrug molecule CM-PPT can better induce cell apoptosis, laying the foundation for its application in tumor treatment in mice.

[0084] In summary, the present invention provides an anticancer prodrug molecule designed and synthesized based on a cascade-intramolecular cyclization reaction, which can be activated by H overexpressed in tumor cells. 2 O 2Selective recognition; A method for preparing an anticancer prodrug molecule, wherein the aforementioned coumarin precursor is connected to the hydroxyl group of an anticancer active molecule to prepare an anticancer prodrug molecule; after the prodrug molecule is stained by H 2 O 2 After selective recognition, anti-cancer active molecules are released, directly acting on cancer cells to achieve the purpose of targeted treatment.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An anticancer prodrug molecule, the structural formula of which is:

Images