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

Abstract

The present 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 chromene derivative with camptothecin, the prodrug is produced, which releases the camptothecin anticancer drug and a fluorophore upon biothiol activation. The prodrug molecule interacts with highly expressed biothiol groups in tumor cells, releasing the anticancer drug, thereby 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 camptothecin 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 to tumors, and visualize the stage of cancer, providing 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 through the differences between the tumor tissue microenvironment and normal tissue, releasing the original drug molecules and fluorescent imaging molecules to achieve the purpose of diagnostic and therapeutic effects. The tumor microenvironment mainly includes high concentrations of biological thiol groups, high levels of reactive oxygen species, low pH values, and unique enzyme activity. Therefore, stimuli-responsive drugs induced by the tumor microenvironment can significantly enhance the efficacy of anticancer drugs by increasing the drug concentration at the tumor site.

[0003] Camptothecin is a natural anticancer drug isolated from the plant Camptotheca acuminata, which is unique to southwest China. It has a good therapeutic effect on liver cancer and head and neck cancer. As an alkaloid, it can destroy the structure of DNA by binding to DNA, and at the same time inhibit DNA polymerase in the S phase of DNA self-replication, thereby leading to cell apoptosis. However, due to the poor water solubility and strong toxicity of camptothecin, its clinical manifestations are: bone marrow suppression leading to a decrease in white blood cell count; loss of appetite, vomiting, nausea and hair loss, etc., all of which inhibit its clinical application. Based on this, the present invention discloses a new diagnostic and therapeutic prodrug based on thiol-activated camptothecin to achieve the purpose of targeted cancer treatment. Summary of the Invention

[0004] The present invention discloses a novel strategy to provide 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 an anticancer active molecule with a chromene derivative, resulting in a high reaction yield and excellent purity. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0006] Figure 2 This is a graph showing the concentration dependence of the fluorescence spectrum of the prodrug molecule Chromene-CPT provided in Experimental Example 1 of the present invention after the reaction with GSH;

[0007] Figure 3 This is a diagram showing the selectivity test results of the prodrug molecule Chromene-CPT provided in Experimental Example 1 of the present invention;

[0008] Example 1

[0009] The present invention discloses a novel diagnostic and therapeutic prodrug molecule based on camptothecin, the structural formula of which is:

[0010]

[0011] The synthetic route of the novel camptothecin-based prodrug molecule of the present invention is as follows:

[0012]

[0013] The method for preparing the novel camptothecin-based diagnostic and therapeutic prodrug molecule of the present invention is as follows:

[0014] Synthesis of Compound Chromene-1: Salicylaldehyde (5.3 mL, 141 mmol) was placed in a 250 mL round-bottom flask. 50 mL of concentrated hydrochloric acid and 3.7 mL of aqueous formaldehyde were added and stirred at room temperature for 24 h. A purple solid was produced, which was filtered and washed with cold water. Copper sulfate pentahydrate (28.5 g, 114 mmol) was added, followed by 45 mL of DMSO / H₂O (v:v = 2:1). The mixture was stirred at 110°C for 2 h and then cooled to room temperature. The mixture was then extracted with DCM and washed with saturated brine. The organic phase was dried over anhydrous Na₂SO₄ and purified by column chromatography (PE:EA = 12:1 to 6:1) to afford Compound Chromene-1 as a white solid.

[0015] Synthesis of Chromene-2: Chromene-1 (300 mg, 2 mmol), 2-cyclopentenone (320 mg, 4 mmol), and imidazole (280 mg, 4 mmol) were placed in a round-bottom flask. 20 mL of THF / H₂O (1:1) was added and stirred at room temperature for 72 h. After TLC monitoring, the reaction was stopped and a small amount of dilute hydrochloric acid was added. The mixture was extracted with EA and washed with saturated brine. The organic phase was dried over anhydrous Na₂SO₄ and purified by column chromatography (PE:DCM:EA = 1:1:1) to afford Chromene-2 ​​as a yellow solid.

[0016] Synthesis of Chromene-CPT: Camptothecin (105 mg, 0.3 mmol) and DMAP (110 mg, 0.9 mmol) were placed in a round-bottom flask. Anhydrous DCM was added, and triphosgene (36 mg, 0.12 mmol) was added under argon. After stirring at room temperature for 30 minutes, a solution of Chromene-2 ​​(65 mg, 0.3 mmol) in anhydrous DCM was slowly added dropwise. Stirring at room temperature was continued, and the reaction was monitored by TLC. After completion of the reaction, the mixture was extracted with DCM, and the organic phase was dried over anhydrous NaSO and purified by column chromatography (DCM:MeOH = 100:1) to afford Chromene-CPT as a light yellow solid. 1 H NMR (600MHz, CDCl3): δ8.39 (s, 1H), 8.19 (dd, J = 11.0Hz, 8.8Hz, 1H), 7.94 (d, J = 8.0Hz, 1H), 7.86-7.83 (m, 1H), 7 .69-7.66(m,1H),7.27(dd,J=8.4Hz,2.4Hz,1H),7.25-7.23(m,1H),7.21(dd,J=7.8Hz,2.0Hz,1H),6.94(dd,J= 41.4Hz,2.4Hz,1H),6.86-6.81(m,1H),5.72(dd,J=16.0Hz,1.6Hz,1H),5.39(dd,J=16.0Hz,2.4Hz,1H),5.31-5 .30(m,2H),5.11-5.00(m,3H),2.58-2.44(m,2H),2.30-2.10(m,2H),2.04-1.82(m,2H),1.00(q,J=7.4Hz,3H). 13 C NMR (400MHz, CDCl3): δ200.86,167.30,148.90,146.43,133.05,132.89, 131.89,131.77,131.22,130.90,130.78,130.69,129.82,128.61,128.4 6,128.21,128.17,128.08,126.88,116.75,116.67,95.87,78.04,75.68 ,69.72,67.02,50.02,36.92,36.87,27.78,7.63.HRMS(ESI,m / z):Calcd forC 34 H 21 N2O8([M+H] + )591.1758,found:591.1762.

[0017] Experimental Example 2

[0018] This experimental example provides the time-dependent results of the fluorescence spectrum after the prodrug molecule Chromene-CPT interacts with GSH;

[0019] Experimental method: The stock solution of prodrug Chromene-CPT was prepared in DMSO, and the final test compound concentration was 5 μM. The time-dependent spectra of Chromene-CPT (5 μM) and GSH (20 μM) were tested by fluorescence spectrophotometry at 37°C, and the following results were obtained: Figure 1 The results shown.

[0020] Experimental results: Figure 1 As shown in the figure, under the excitation wavelength of 365 nm, Chromene-CPT has a weak fluorescence intensity at 445 nm. After adding 10 μM GSH, the fluorescence intensity gradually increases with the action time.

[0021] Experimental Example 3

[0022] This experimental example provides the concentration-dependent results of the fluorescence spectrum of the prodrug molecule Chromene-CPT after interaction with GSH;

[0023] Experimental method: The maximum excitation wavelength of the prodrug molecule Chromene-CPT is 365 nm. A fluorescence spectrophotometer was used to perform concentration-dependent fluorescence spectroscopy tests on the prodrug molecule Chromene-CPT (5 μM) and different concentrations of GSH at 37°C.

[0024] Experimental results: Figure 2 As shown in the figure, Chromene-CPT responded to GSH in a concentration-dependent manner, and the fluorescence intensity gradually increased with the action time.

[0025] Experimental Example 4

[0026] This experimental example provides the results of the selective testing of the prodrug molecule Chromene-CPT;

[0027] Experimental method: The prodrug molecule Chromene-CPT (5μM) was incubated with other interfering substances for a certain period of time and then its fluorescence signal was tested to obtain the following results: Figure 3 The results shown.

[0028] Experimental results: Chromene-CPT, a prodrug, showed little fluorescence after incubation with amino acids for 10 minutes. However, after incubation with sulfhydryl-containing amino acids for 10 minutes, the fluorescence intensity increased significantly, demonstrating the prodrug's selectivity for sulfhydryl amino acids.

Claims

1. An anticancer diagnostic and therapeutic prodrug molecule, characterized in that: Its structural formula is: 。 2. Use of the anticancer diagnostic and therapeutic prodrug molecule as claimed in claim 1 in the preparation of anticancer drugs.

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