Preparation method and use of a thiol-activated lenalidomide anticancer diagnosis and treatment prodrug

Abstract

This invention belongs to the field of pharmaceutical technology and discloses a method for preparing and using a biothiol-activatable anticancer prodrug molecule. By linking a BH derivative backbone with a fluorophore to lenalidomide, the invention produces a prodrug that releases the lenalidomide drug and the fluorophore upon biothiol activation. The disclosed prodrug molecule reacts with highly expressed biothiol groups in tumor cells to release the anticancer drug molecule, achieving targeted cancer therapy.

Description

Technical Field

[0001] The present invention relates to the fields of organic synthesis and molecular medicine, and in particular to a preparation method and use of a thiol-activated lenalidomide anti-cancer diagnostic and therapeutic prodrug. Background Art

[0002] The development of diagnostic and therapeutic strategies can be used simultaneously to diagnose early-stage cancer, provide therapeutic drugs for tumors and visualize the stage of cancer, and provide more effective and personalized treatment for cancer patients. Diagnostic and therapeutic prodrugs usually consist of four parts, including: drug molecules, signal output units, trigger response units, and connection sites. The activation of small molecule diagnostic and therapeutic reagents is generally achieved by breaking chemical bonds due to the differences between the tumor tissue microenvironment and normal tissue, releasing the original drug molecules and fluorescent imaging molecules, so as to achieve the purpose of exerting diagnostic and therapeutic effects. The tumor microenvironment mainly includes high concentrations of biological thiol groups, high levels of reactive oxygen species, low pH values, unique enzyme activity, etc. Therefore, stimulus-responsive drugs induced by the tumor microenvironment can significantly enhance the efficacy of anticancer drugs by increasing the drug concentration at the tumor site. Based on this, the present invention discloses a new thiol-activated diagnostic and therapeutic prodrug based on lenalidomide to achieve the purpose of targeted cancer therapy. Summary of the Invention

[0003] The present invention discloses a novel strategy for providing an anticancer prodrug molecule with a simple structure, easy availability, and convenient synthesis. The prodrug exhibits excellent tumor therapeutic efficacy and possesses high application value. Furthermore, the present invention provides a method for preparing the anticancer prodrug molecule by linking the anticancer active molecule to a BH derivative backbone, resulting in high reaction yield and excellent purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Figure 1 This is a graph showing the UV absorption spectrum of the prodrug molecule Lena-green provided in Experimental Example 1 of the present invention after reacting with rBSA;

[0005] Figure 2 This is a graph showing the time-dependent fluorescence spectrum of the prodrug molecule Lena-green provided in Experimental Example 1 of the present invention after the reaction with rBSA;

[0006] Figure 3 This is a diagram showing the selectivity test results of the prodrug molecule Lena-green provided in Experimental Example 1 of the present invention;

[0007] Figure 4 This is a graph showing the HPLC results of the reaction between the prodrug molecule Lena-green and rBSA in Experimental Example 1 of the present invention;

[0008] Figure 5This is a graph showing the toxicity results of the prodrug molecule Lena-green and the parent drug molecule (Lena) on HeLa cells provided in Experimental Example 1 of the present invention;

[0009] Figure 6 This is a cell fluorescence imaging image of the prodrug molecule Lena-green provided in Experimental Example 1 of the present invention in HeLa cells;

[0010] Example 1

[0011] The present invention discloses a novel diagnostic and therapeutic prodrug molecule based on lenalidomide, the structural formula of which is:

[0012]

[0013] The synthetic route of the novel diagnostic and therapeutic prodrug molecule based on lenalidomide described in the present invention is as follows:

[0014]

[0015] The method for preparing the novel diagnostic and therapeutic prodrug molecule based on lenalidomide of the present invention is as follows:

[0016] Synthesis of compound S1: 4-diethylaminosalicylaldehyde (5 g, 26 mmol) was dissolved in 100 mL of n-butanol, and ethyl nitroacetate (3.0 mL, 26 mmol) was added, followed by piperidine (0.5 mL) and acetic acid (1.1 mL). The reaction solution was refluxed at 120°C for 24 hours, then cooled in an ice-water bath for crystallization, and the precipitate was collected by filtration. The filter cake was washed with icy petroleum ether and dried to obtain compound S1 as an orange-red needle-shaped solid.

[0017] Synthesis of Compound S2: Compound S1 (5 g, 19 mmol) and stannous chloride (25.5 g, 113 mmol) were added to a 250 mL round-bottom flask. 90 mL of ethanol was added, and concentrated hydrochloric acid (45 mL) was slowly added dropwise over an ice-water bath. Stir at room temperature and monitor the reaction by TLC. After the reaction, 200 mL of ice water was prepared in three separate batches, and NaOH powder was added. The reaction solution was slowly added in three separate batches. After standing for a period of time, a yellow precipitate formed, which was filtered and dried. Purification by column chromatography afforded Compound S2 as a yellow solid.

[0018] Synthesis of Compound S3: Compound S2 (192 mg, 0.83 mmol) and triethylamine (0.12 mL, 0.86 mmol) were dissolved in dry dichloromethane (10 mL) at room temperature. Acryloyl chloride (0.14 mL, 1.66 mmol) was then added at 0°C under argon. After stirring at room temperature for 6 hours, the reaction mixture was diluted with dichloromethane. The mixture was washed with saturated aqueous NaHCO₃, dried over anhydrous Na₂SO₄, and concentrated to remove the solvent. Purification by chromatography (PE / EA = 4 / 1) afforded Compound S3 as a light yellow solid.

[0019] Synthesis of compound S4: Under argon protection, a mixed solution of compound S3 (60 mg, 0.2 mmol), paraformaldehyde (44 mL, 0.6 mmol), 1,4-diazabicyclo[2.2.2]octane (DABCO) (12 mg, 0.1 mmol) and 9 mL of acetonitrile was stirred at 50 ° C for 24 hours. The mixture was then diluted with 150 mL of water, neutralized with solid NaHCO3, extracted with ethyl acetate, and separated. The organic layer was dried over Na2SO4, filtered, and the filtrate was evaporated under reduced pressure. The residue was purified by column chromatography (PE / EA=3 / 1) to obtain compound S4 as a yellow solid.

[0020] Synthesis of Lena-green: Dissolve triphosgene (30 mg, 0.1 mmol), lenalidomide (65 mg, 0.25 mmol), and triethylamine (41 mL, 0.3 mmol) in 4 mL of dichloromethane. Heat the reaction mixture under reflux at 45°C for 2 hours to obtain the intermediate. Compound S4 (63 mg, 0.2 mmol) and triethylamine (41 mL, 0.3 mmol) were then added and reacted for 20 minutes. The product was collected by column chromatography (DCM / MeOH = 200 / 1 to 50 / 1) to obtain Lena-green as a yellow solid. NMR (400MHz, CDCl3)d:9.05(s,1H),8.57(s,1H),8.45(s,1H),7.84(d,J=8.0Hz,1H),7.64(d,J=7.5Hz ,1H),7.55(s,1H),7.46(t,J=7.8Hz,1H),7.28(d,J=8.8Hz,1H),6.60(d,J=8.3Hz,1H),6.46(s,1H),6. 28(s,1H),5.81(s,1H),5.21(dd,J=13.2,5.2Hz,1H),5.00(d,J=4.7Hz,2H),4.45(d,J=4.2Hz,2H),3. 40(q,J=7.1Hz,4H),2.85(s,2H),2.40(dd,J=12.9,5.3Hz,1H),2.22(s,1H),1.20(t,J=7.0Hz,6H).13C NMR(101MHz, CDCl3)d:171.31,169.75,169.00,164.10,160.09,152.78,149.77,137.55,132.62,132.57,129.36,128 .93,126.90,120.21,118.45,109.87,108.06,97.20,65.16,51.84,50.88,45.92,44.77,31.54,29.71,23.32,12.47.

[0021] Experimental Example 2

[0022] This experimental example provides the UV absorption spectrum results of the prodrug molecule Lena-green after reacting with rBSA;

[0023] Experimental method: The stock solution of prodrug Lena-green was prepared by DMSO, and the final test compound concentration was 2μM. The UV absorption of Lena-green (2μM) and rBSA (2μM) before and after the reaction was measured by UV spectrophotometer, and the following results were obtained: Figure 1 The results shown.

[0024] Experimental results: Figure 1 As shown, the maximum absorption wavelength of the prodrug Lena-green is 415 nm. After adding 2 mM rBSA and reacting with the prodrug for 2 hours, the maximum absorption wavelength shifts to 400 nm. This experimental result shows that the prodrug Lena-green has a certain response to rBSA.

[0025] Experimental Example 3

[0026] This experimental example provides the time-dependent results of the fluorescence spectrum after the prodrug molecule Lena-green interacts with rBSA;

[0027] Experimental method: The maximum excitation wavelength of the prodrug molecule Lena-green is 410nm. The prodrug molecule Lena-green (2μM) and rBSA (2μM) were tested for time-dependent fluorescence spectroscopy at 37°C using a fluorescence spectrophotometer.

[0028] Experimental results: Figure 2 As shown in the figure, under the excitation wavelength of 410 nm, Lena-green has a weak fluorescence intensity at 490 nm. After adding 2 mM rBSA, the fluorescence intensity gradually increases with the action time.

[0029] Experimental Example 4

[0030] This experimental example provides the results of the selective testing of the prodrug molecule Lena-green;

[0031] Experimental method: The prodrug molecule Lena-green (2μM) was incubated with other interfering substances for a certain period of time and then the fluorescence signal was tested ( Figure 3 The interfering substances given in the table are 1, blank; 2, GSH; 3, Hcy; 4, Cys; 5, E-Trx; 6, reduced E-Trx; 7, lysozyme; 8, reduced lysozyme; 9, BSA; 10, rBSA). The specific experimental results are shown in the table below. Figure 3 shown.

[0032] Experimental results: When the prodrug Lena-green was incubated with a 100-fold concentration of small thiols for 90 minutes, virtually no fluorescence was released. Incubation with oxidized proteins that lacked vicinal dithiols for 90 minutes also showed no significant fluorescence enhancement. However, when Lena-green was incubated with proteins containing vicinal dithiols for 90 minutes, the fluorescence intensity increased significantly by more than 20-fold, demonstrating that the prodrug Lena-green specifically responds to proteins with vicinal dithiols and is unaffected by other biological thiols.

[0033] Experimental Example 5

[0034] This experimental example provides the HPLC results of the reaction between the prodrug molecule Lena-green and rBSA;

[0035] Experimental method: Lena-green (20mM), Lena (20mM), and rBSA (20mM) were all tested in PBS buffer at 37°C. Lena-green and rBSA were incubated in PBS buffer for a specified time and then analyzed by HPLC for the content of Lena in the solution. The results are as follows: Figure 4 shown.

[0036] Experimental results: When rBSA (20 mM) was added to the prodrug Lena-green (20 mM) and incubated, the Lena-green peak decreased significantly with increasing incubation time, and a Lena peak appeared around retention time 4.7 minutes. After incubation of the visualized prodrug Lena-green with rBSA for 60 minutes, the drug release efficiency reached 88%. After 120 minutes of incubation, the drug release efficiency reached as high as 98%.

[0037] Experimental Example 6

[0038] This experimental example provides the toxicity results of the prodrug molecule Lena-green and the parent drug molecule (Lena) on HeLa cells;

[0039] Experimental method: HeLa cells were evenly seeded into 96-well plates, and 2.5×10 3 After cells were attached overnight, different concentrations of Lena and Lena-green were added. The cells were incubated in a cell culture incubator for 72 hours. Then, 10 mL of CCK-8 solution was added to each well and incubated in a cell culture incubator for another 4 hours. The absorbance at 450 nm was measured using a microplate reader.

[0040] Experimental results: From Figure 5 As can be seen, the prodrug Lena-green exhibits similar cytotoxicity to Lena at low concentrations. However, when its concentration is increased from 12.5mM to 25mM, cell viability decreases significantly, from 70% to approximately 30%. Subsequent high concentrations also exhibit a strong inhibitory effect on cell viability.

[0041] Experimental Example 7

[0042] This experimental example provides cell fluorescence imaging of the prodrug molecule Lena-green in HeLa cells;

[0043] Experimental method: HeLa cells were evenly seeded into 12-well plates, and 2×104 The cells were cultured overnight in a 37°C, 5% CO2 incubator until the cells were completely attached. 30 mM PAO inhibitor (a commonly used protein vicinal dithiol blocking agent) or 10 mM Lena-green were then added and incubated for the corresponding time. After washing three times with PBS, fluorescence imaging was performed using a fluorescence microscope.

[0044] Experimental results: Figure 6 As shown, the control group showed almost no fluorescence. When HeLa cells were incubated with the prodrug molecule Lena-green, bright green fluorescence was observed under a fluorescence microscope. However, after using PAO to inhibit the content of intracellular reduced vicinal dithiol proteins and then incubating them with Lena-green, a significant decrease in intracellular fluorescence was observed, demonstrating that Lena-green is specifically activated by endogenous vicinal dithiol proteins.

Claims

1. An anticancer diagnostic and therapeutic prodrug molecule, characterized in that: Its structural formula is: .

2. Use of the prodrug molecule according to claim 1 in the preparation of anticancer prodrugs.

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