Oxaprozin coupled cis-platinum compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and discloses an oxaprozin coupled cis-platinum compound as well as a preparation method and application thereof. The oxaprozin coupled cis-platinum compound disclosed by the invention is tetravalent platinum prepared by coupling cis-platinum and oxaprozin which is a drug with carboxyl; the oxaprozin coupled cis-platinum compound disclosed by the invention can improve the toxic and side effects of cis-platinum and increase novel axial functional groups, and has the technical advantages of excellent anti-tumor activity, stability, safety and low cost.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to an oxaprazin-coupled cisplatin compound, its preparation method and application. Background Technology

[0002] Cisplatin (cPt) is a chemotherapy drug that targets cancer cell DNA and remains the gold standard for cancer treatment. However, its toxicity, low bioavailability, and drug resistance limit its application and efficacy. Pt(IV) prodrugs, due to their higher stability and axial ligand modification space, offer the potential to overcome the shortcomings of Pt(II) drugs. Therefore, many cisplatin-based Pt(IV) prodrugs have been synthesized, such as isopropylplatin and saxaplatin. Compared with Pt(II), Pt(IV) prodrugs are more inert and more stable in the biological environment, avoiding the inactivation of proteins or other biomolecules, thereby reducing toxicity. Tetravalent platinum prodrugs are also easy to functionalize, and the bioactivity of platinum compounds can be improved by introducing functional groups and pharmacophores. Furthermore, by using axial active groups, cisplatin resistance can be overcome, potentially leading to the development of new platinum-based anticancer drugs.

[0003] To increase tumor sensitivity to cisplatin, cisplatin is often used in combination with other chemotherapy drugs or immune checkpoint inhibitors in clinical practice. However, many patients do not benefit from these combination strategies. Furthermore, tetravalent platinum drugs that have been developed are not yet available for clinical use. Therefore, developing new strategies for treating tumors using cisplatin-derived drugs is an urgent problem to be solved. Summary of the Invention

[0004] The purpose of this invention is to provide a two-step method for synthesizing novel cisplatin-oxaprazine conjugates, which reduces costs while preparing a tetravalent platinum prodrug with excellent anticancer activity, stability and safety for the efficient killing of tumor cells.

[0005] In a first aspect, the present invention provides an oxapezine-coupled cisplatin compound, the chemical structural formula of which is shown in formula (Ⅰ) below:

[0006]

[0007] The oxaprazin-coupled cisplatin compound shown in formula (Ⅰ) of this invention belongs to a class of cisplatin-derived tetravalent platinum compounds.

[0008] Secondly, the present invention also provides a method for preparing the above-mentioned oxaprazine-coupled cisplatin compound, i.e., compound (I), as shown in the reaction route below. Compound (I) can be prepared by reacting compound (II) with oxaprazine of formula (III), including the following steps:

[0009]

[0010] Cisplatin was oxidized with hydrogen peroxide to obtain tetravalent cisplatin, i.e., compound (II). Oxaprazine (III), the tetravalent cisplatin, TBTU, and TEA were mixed in a solvent and subjected to a condensation reaction to obtain the oxaprazine-coupled cisplatin compound. It should be noted that TBTU refers to O-benzotriazole-N,N,N′,N′-tetramethylurea tetrafluoroborate, and TEA refers to triethylamine.

[0011] As a solvent for the above reaction, a solvent that does not adversely affect the reaction and can dissolve compound (II) and oxapazine (III) can be used, preferably dimethylformamide (DMF) and dimethyl sulfoxide (DMSO).

[0012] Preferably, since cisplatin, a raw material in this reaction, is expensive, while oxaprozin of formula (III) is relatively inexpensive, an excess of oxaprozin of formula (III) is used to meet the requirements in order to save costs and maintain reaction efficiency. The molar ratio of compound (II) to oxaprozin of formula (III) is 1:(2.2-2.5), and the molar ratio of oxaprozin of formula (III) to the coupling agent TBTU is 1:(1-1.2).

[0013] Preferably, in the reaction, compound (I) can be prepared by adding a suitable basic reagent, namely triethylamine, in the presence of a solvent, with the molar ratio of oxapazine of formula (III) to triethylamine being 1:(1-1.2), and stirring the compound of formula (II) and oxapazine of formula (III) in a nitrogen atmosphere for 12-24 hours in the dark under a gradually increasing temperature condition from room temperature (25°C to 70°C).

[0014] Preferably, after the reaction is complete, the final product (I) compound and the impurities are separated and purified using silica gel column chromatography based on the difference in polarity. Alternatively, the product and impurities can be purified by slurrying or filtration using appropriate solvents based on their differences in solubility in different solvents.

[0015] Thirdly, the present invention provides the use of the oxaprazin-coupled cisplatin compound described in the first aspect above or the oxaprazin-coupled cisplatin compound obtained by the preparation method described in the second aspect above in the preparation of antitumor drugs.

[0016] Technical Effects: The oxaprozin-conjugated cisplatin compound provided in this application is a tetravalent platinum prepared by conjugating cisplatin with oxaprozin, a drug containing a carboxyl group. This compound improves the toxic side effects of cisplatin and adds novel axial functional groups, exhibiting excellent antitumor activity, stability, safety, and low cost. Testing shows that the purity of the oxaprozin-conjugated cisplatin compound as a tetravalent platinum prodrug can reach 96.01%, and the yield can reach 68.70%. This tetravalent platinum prodrug shows good killing effects on various tumor cells, including human hepatocellular carcinoma cells HepG2, murine breast cancer cells 4T1, and murine melanoma cells B16-F10. The oxapezil-conjugated cisplatin compound provided in this application, compared with cisplatin, exhibits a half-maximal inhibitory concentration (IC50) of approximately 3.1-fold reduced for HepG2, approximately 1.7-fold reduced for 4T1, and approximately 4.3-fold reduced for B16-F10. Furthermore, the tetravalent platinum prodrug demonstrates good safety in normal breast epithelial cells MCF-10A, significantly outperforming cisplatin in terms of safety. Attached Figure Description

[0017] Figure 1 The infrared absorption spectrum of cPt-OH prepared in Example 1;

[0018] Figure 2 The infrared absorption spectrum of cPt-Oxa obtained after purification in Example 3 is shown.

[0019] Figure 3 The high-resolution mass spectrometry results of cPt-Oxa obtained after purification in Example 3 are shown.

[0020] Figure 4 The results of the 1H NMR spectrum of cPt-Oxa obtained after purification in Example 3 are shown.

[0021] Figure 5 Cell survival rates of different cell lines under different treatments. Detailed Implementation

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0023] Main raw materials and reagent sources:

[0024] Cisplatin, O-benzotriazole-N,N,N′,N′-tetramethylurea tetrafluoroborate (TBTU) and triethylamine (TEA) were all purchased from Shanghai Adamas Reagent Co., Ltd.

[0025] Osapqin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0026] The human hepatocellular carcinoma cells HepG2, mouse breast cancer cells 4T1, mouse melanoma cells B16-F10, and normal breast epithelial cells MCF-10A used for cell experiments were all purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0027] Example 1: Preparation of cPt-OH

[0028] Cisplatin (cPt, 500 mg, 1.67 mmol) was suspended in H₂O₂ (30% w / v, 40 mL). The mixture was stirred at 55 °C for 4 h, then heated to 100 °C until the solution became clear. After cooling to room temperature, it was incubated overnight at -4 °C, precipitating a large amount of yellow-green needle-like crystals. After filtration, the precipitate was washed successively with cold water, cold ethanol, and diethyl ether, and finally dried under vacuum to give yellow-green crystals cPt-OH (yield 88.5%).

[0029] Example 2: Synthesis of the tetravalent platinum prodrug cPt-Oxa

[0030] Dissolve cPt-OH (167 mg, 0.5 mmol) in 10 mL of anhydrous DMF, then add oxaprazine (352 mg, 1.2 mmol) and TBTU (386 mg, 1.2 mmol), stir at room temperature (25 °C) for 10 min, then add TEA (168 μL, 1.2 mmol); subsequently, heat to 60 °C under a nitrogen atmosphere, stir continuously, and react in the dark for 24 h.

[0031] Example 3: Purification of cPt-Oxa

[0032] The crude product was passed through a column chromatography column. After evaporating the DMF to dryness, methanol / dichloromethane was added to dissolve the crude product. Silica gel powder was then added and mixed thoroughly, and the sample was loaded onto the column using a dry method. The initial eluent was petroleum ether:ethyl acetate at a ratio of 3:1 to remove less polar impurities; subsequently, a petroleum ether:ethyl acetate ratio of 1:1 was used to allow the product to extend in the column. Finally, dichloromethane:methanol at a ratio of 50:1 was used. The appearance of the product spot was detected by TLC, and the product was collected and dried. The purity reached 96.01%, and the yield was approximately 68.7%.

[0033] Example 4: Characterization of cPt-Oxa and its intermediates

[0034] The synthesized tetravalent platinum prodrug and its intermediates were characterized by high-resolution mass spectrometry, proton nuclear magnetic resonance (HMR) spectroscopy, and infrared spectroscopy. The results are as follows: Figures 1-4 As shown. Figure 1 The infrared absorption spectrum of cPt-OH shows the characteristic absorption peak of the hydroxyl group. Figure 2The infrared absorption spectrum of cPt-Oxa shows characteristic absorption peaks of the carbonyl group. Figure 3 The high-resolution mass spectrometry results for cPt-Oxa show proton peaks that match the molecular weight of cPt-Oxa, and fragment peaks that match the molecular weight of oxapazine. Figure 4 The results are the 1H NMR spectra of cPt-Oxa. 1 HNMR (300MHz, DMSO-d6) δppm: 2.76-2.84(4H,m), 2.99-3.04(4H,m), 6.54(6H,s), 7.35-7.46(12H,m), 7.53-7.59(8H,m). HR-FAB-MSm / z:885(M+1) + .

[0035] Example 5: Safety and anticancer activity of cPt-Oxa

[0036] MCF-10A cells were loaded at 1×10 4 Cells were seeded at a density of 100 cells / well in 96-well plates, cultured in DMEM complete medium containing 10% FBS, and incubated in a CO2 incubator (37°C, 5% CO2, saturated humidity) for 24 hours. When the cell density was approximately 80%, the drug was administered.

[0037] cPt-Oxa was diluted with DMEM medium (serum-free) to concentrations of 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μg / mL. The original culture medium in the wells was removed. 100 μL of culture medium was added to the negative control group, and 100 μL of the above-mentioned drug-containing culture medium was added to the experimental wells. After incubation for 24 h, the original culture medium was discarded, and the plates were washed once with PBS (200 μL). 100 μL of MTT solution (0.5 mg / mL) was added to each well, and the plates were incubated in a CO2 incubator for approximately 4 h. A cell-free MTT-added group served as a blank control. After 4 h, the culture plates were removed from the cell culture chamber, the supernatant yellow liquid was discarded (avoiding the aspiration of purple crystals), and 150 μL of DMSO solution was added to each well. The plates were incubated for 10 min with a tremor / shake. Finally, the absorbance of each well at 490 nm was measured using a microplate reader, and cell viability was calculated. (n=3).

[0038] HepG2, 4T1, and B16-F10 cells were cultured at a rate of 1×10⁻⁶. 4 Cells were seeded at a density of 100 cells / well in 96-well plates, cultured in DMEM complete medium containing 10% FBS, and incubated in a CO2 incubator (37°C, 5% CO2, saturated humidity) for 24 hours. When the cell density was approximately 80%, the drug was administered.

[0039] cPt-Oxa was diluted to different concentrations in DMEM medium (serum-free) before administration. After 24 h of incubation, the original medium in the plate was discarded, and the plate was washed once with PBS (200 μL). MTT solution (0.5 mg / mL, 100 μL) was added to each well, and the plate was incubated in a CO2 incubator for about 4 h. The cell-free MTT group served as a blank control. After 4 h, the culture plate was removed from the cell chamber, the supernatant yellow liquid was discarded, and the purple crystals were carefully removed. DMSO solution (150 μL) was added to each well, and the plate was incubated for 10 min with a tremor / shake. Finally, the absorbance of each well at 490 nm was measured using a microplate reader, and the cell viability was calculated (n=3).

[0040] Comparative Example 1

[0041] The only difference between Comparative Example 1 and Example 5 is that cPt with the same gradient dosage was used instead of cPt-Oxa in Example 5; all other reagents and operations were the same.

[0042] Comparative Example 2

[0043] The only difference between Comparative Example 2 and Example 5 is that Oxa with the same gradient dosage was used instead of cPt-Oxa in Example 5; all other reagents and operations were the same.

[0044] Comparative Example 3

[0045] The only difference between Comparative Example 3 and Example 5 is that the cPt+Oxa combined drug administration method with the same gradient total dosage setting is used instead of the cPt-Oxa single drug administration in Example 5, wherein the molar ratio of cPt and Oxa is 1:2; all other reagents and operations are the same.

[0046] like Figure 5 As shown in Figure a and Table 1, cPt-Oxa exhibits good safety against MCF-10A within a concentration range of 0.0625-32 μg / mL.

[0047] like Figure 5 As shown in Figures b, c, and d and Table 1, cPt-Oxa has a good killing effect on HepG2, 4T1, and B16-F10 cells, and its effect is significantly better than that of the cPt treatment group, Oxa treatment group, and cPt+Oxa treatment group.

[0048] Table 1 IC50 values ​​of different cell lines under different treatments

[0049]

Claims

1. An oxapezil-coupled cisplatin compound, the chemical structural formula of which is shown in formula (Ⅰ) below:

2. The method for preparing the oxapezine-coupled cisplatin compound according to claim 1, characterized in that, The reaction route of the preparation method is shown below, and the preparation method includes the following steps: Cisplatin and hydrogen peroxide were oxidized to obtain tetravalent cisplatin, i.e., compound (II); oxaprazine (III), the tetravalent cisplatin, TBTU, and TEA were mixed in a solvent and subjected to a condensation reaction to obtain the oxaprazine-coupled cisplatin compound.

3. The preparation method according to claim 2, characterized in that, The solvent is dimethylformamide (DMF) or dimethyl sulfoxide (DMSO).

4. The preparation method according to claim 2, characterized in that, The molar ratio of compound (II) to oxapazine (III) is 1:(2.2 to 2.5).

5. The preparation method according to claim 2, characterized in that, The molar ratio of oxaprin to TBTU in formula (III) is 1:(1-1.2).

6. The preparation method according to claim 2, characterized in that, The molar ratio of oxaprazine (Formula III) to triethylamine TEA is 1:(1-1.2).

7. The preparation method according to claim 6, characterized in that, Compound (I) was prepared by stirring compound (II) and oxaprazine (III) under a nitrogen atmosphere in the dark for 12–24 hours under gradually increasing temperature conditions from 25°C to 70°C.

8. The use of the oxaprazin-conjugated cisplatin compound of claim 1 or the oxaprazin-conjugated cisplatin compound obtained by the preparation method of claim 2 in the preparation of antitumor drugs.

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