Quadrivalent platinum prodrug formed by coupling polyunsaturated fatty acid with cisplatin and preparation method thereof

Abstract

A tetravalent platinum prodrug formed by coupling a polyunsaturated fatty acid with cisplatin and a preparation method thereof, wherein the tetravalent platinum prodrug has a structure shown in formula (I): #imgabs0# wherein R is independently selected from H or #imgabs1# and is not H at the same time; R 1 It is an n-3 or n-6 type polyunsaturated fatty chain with a carbon number of 17 to 23. The present invention effectively solves the drug resistance problem of platinum drugs and can effectively kill common cancer cells and cisplatin-resistant cells.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a tetravalent platinum prodrug formed by coupling a polyunsaturated fatty acid with cisplatin and a preparation method thereof. Technical Background

[0002] Chemotherapy is currently one of the mainstays of cancer treatment. Since its approval for cancer treatment in 1978, cisplatin has been widely used as a first-line chemotherapy drug for a variety of cancers, including non-small cell lung cancer, testicular cancer, cervical cancer, ovarian cancer, and head and neck cancer. However, with long-term cisplatin use, tumor cells can develop resistance to the platinum, leading to tumor recurrence and metastasis, and ultimately treatment failure. Therefore, overcoming cisplatin-resistant cells is a pressing clinical challenge.

[0003] There are many mechanisms for cisplatin resistance, including: (1) reduced cisplatin uptake by resistant cells, resulting in increased drug output; (2) cisplatin interacts with other biological molecules before entering cancer cells, rendering cisplatin ineffective; (3) cisplatin binds to DNA after entering cells to form Pt-DNA adducts, inducing cell death, but the DNA damage repair capacity of resistant cells is enhanced; (4) cisplatin-resistant cells express abnormal proteins that regulate apoptosis, leading to apoptotic escape of cancer cells. Currently, it is still necessary to design new platinum drug molecules or new cisplatin use strategies that can overcome cisplatin resistance based on these resistance mechanisms. Summary of the Invention

[0004] In view of this, one of the main objectives of the present invention is to propose a tetravalent platinum prodrug formed by coupling polyunsaturated fatty acids with cisplatin and a preparation method, which can improve the uptake of drugs by tumor cells and effectively kill common cancer cells and cisplatin-resistant cells.

[0005] In order to achieve the above objectives, as one aspect of the present invention, a tetravalent platinum prodrug is provided, having a structure shown in formula (I):

[0006]

[0007] wherein R is independently selected from H or And not H; R 1 It is an n-3 or n-6 polyunsaturated fatty chain with 17 to 21 carbon atoms.

[0008] As another aspect of the present invention, there is provided a method for preparing the tetravalent platinum prodrug as described above, comprising the following steps:

[0009] Reacting cisplatin with hydrogen peroxide to obtain dichlorodihydroxydiammineplatinum represented by formula (III);

[0010]

[0011] reacting an n-3 or n-6 polyunsaturated fatty acid with N-hydroxysuccinimide to obtain a compound represented by formula (IV);

[0012]

[0013] The dichlorodihydroxydiammineplatinum is reacted with the compound represented by formula (IV) to obtain a tetravalent platinum prodrug represented by formula (I).

[0014] As another aspect of the present invention, there is provided a use of the tetravalent platinum prodrug as described above in the preparation of an anti-tumor drug.

[0015] Based on the above technical solutions, it can be seen that the tetravalent platinum prodrug formed by coupling polyunsaturated fatty acids with cisplatin and the preparation method of the present invention have one or part of the following beneficial effects:

[0016] The present invention utilizes polyunsaturated fatty acids and cisplatin to form a tetravalent platinum prodrug. On the one hand, it has high lipid solubility to improve the drug uptake by tumor cells; on the other hand, it is reduced to cisplatin and polyunsaturated fatty acids in the reducing environment of cancer cells. While inducing DNA damage through cisplatin molecules, it also synergizes with polyunsaturated fatty acids with anti-cancer effects to effectively block the energy source of cancer cells, ultimately efficiently killing ordinary cancer cells and cisplatin-resistant cells, effectively overcoming cisplatin resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the cisplatin-docosahexaenoic acid prodrug in Example 1 of the present invention;

[0018] Figure 2 This is a high-resolution mass spectrum of the cisplatin-docosahexaenoic acid prodrug in Example 1 of the present invention;

[0019] Figure 3 This is a high performance liquid chromatogram of the reduction of the cisplatin-docosahexaenoic acid prodrug by ascorbic acid in Example 1 of the present invention, wherein A is cisplatin-docosahexaenoic acid, B is docosahexaenoic acid, and C is the reduction process of cisplatin-docosahexaenoic acid;

[0020] Figure 4 This is a diagram showing the uptake of cisplatin-docosahexaenoic acid prodrug by tumor cells in Example 3 of the present invention;

[0021] Figure 5 This is a graph showing the inhibition of cancer cell energy metabolism by cisplatin-docosahexaenoic acid prodrug in Example 5 of the present invention, wherein A is the oxygen consumption rate change curve, and B is the extracellular acidification rate change curve. DETAILED DESCRIPTION

[0022] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0023] In the platinum drug related technology, divalent platinum (Pt II ) drug-derived tetravalent platinum (Pt IV ) complex has an octahedral coordination structure. Compared with its corresponding divalent platinum matrix, Pt IV The complex is more stable under physiological conditions. IV After entering the cancer cell, it is reduced to Pt in the reducing environment of the cancer cell. II It is toxic only when the drug is injected into the body, thereby reducing the binding of the drug with other biological molecules in the body before it acts on the target cancer cells, effectively reducing toxic side effects and preventing failure before acting on the target.

[0024] On the other hand, Pt IV The axial ligand is modifiable and can be modified by changing the Pt IV Axial ligands can be used to enhance the lipid solubility of drugs to increase cellular uptake, and Pt can be changed by changing the axial ligands. IV In addition, many functional molecules with anticancer activity can be coupled to Pt through chemical reactions. IV Then, after reduction in tumor cells, the cells react with Pt II They are released together to fight tumors synergistically and overcome platinum drug resistance.

[0025] n-3 and n-6 ​​polyunsaturated fatty acids are essential fatty acids for the human body and have anti-tumor properties. n-3 polyunsaturated fatty acids are widely found in deep-sea fish oils and include α-linolenic acid (ALA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and docosapentaenoic acid (DPA). Numerous studies have shown that n-3 polyunsaturated fatty acids can effectively inhibit tumor proliferation, metastasis, and invasion, reduce tumor angiogenesis, and enhance the efficacy of certain anticancer drugs. n-6 polyunsaturated fatty acids are primarily derived from plants and include linoleic acid (LA) and arachidonic acid (AA).

[0026] In the process of realizing the present invention, it was found that by coupling polyunsaturated fatty acids with cisplatin to form a tetravalent platinum prodrug, the drug uptake by tumor cells was significantly improved by enhancing the lipid solubility of cisplatin; and, in the reducing environment of cancer cells, it was reduced to cisplatin and polyunsaturated fatty acids. The synergistic effect of the two can efficiently kill ordinary cancer cells and cisplatin-resistant cells, effectively overcoming the drug resistance of cisplatin.

[0027] Specifically, according to some embodiments of the present invention, a tetravalent platinum prodrug formed by coupling a polyunsaturated fatty acid with cisplatin is provided, having a structure shown in formula (I):

[0028]

[0029] wherein R is independently selected from H or And not H; R 1 It is an n-3 or n-6 polyunsaturated fatty chain with 17 to 21 carbon atoms.

[0030] More specifically, the tetravalent platinum prodrug represented by the general formula (I) has a structure represented by formula (Ia) or formula (Ib):

[0031]

[0032] According to an embodiment of the present invention, polyunsaturated fatty acids are used as Pt IV Axial ligands can improve the lipid solubility and anticancer activity of drugs and overcome the problem of cisplatin resistance.

[0033] According to an embodiment of the present invention, R 1 It has the structure shown in formula (II):

[0034]

[0035] Wherein, m is 1 or 4, n is a positive integer from 3 to 6, and p is a positive integer from 1 to 6.

[0036] Furthermore, R 1 Preferably, it is selected from one of the following structures:

[0037]

[0038] According to an embodiment of the present invention, R having the above structure 1 Since the methylene group connecting each two double bonds has oxidative activity, the tetravalent platinum prodrug constructed based on this polyunsaturated fatty chain exhibits significant cytotoxicity in the cancer cell environment and has an excellent effect in inhibiting tumor proliferation.

[0039] According to some embodiments of the present invention, a method for preparing the tetravalent platinum prodrug as described above is also provided, comprising steps 1 to 3 below.

[0040] In step 1, cisplatin is reacted with hydrogen peroxide to obtain dichlorodihydroxydiammineplatinum represented by formula (III).

[0041]

[0042] In step 2, an n-3 or n-6 polyunsaturated fatty acid is reacted with N-hydroxysuccinimide to obtain a compound represented by formula (IV);

[0043]

[0044] In step 3, dichlorodihydroxydiammineplatinum is reacted with the compound represented by formula (IV) to obtain the tetravalent platinum prodrug represented by formula (I).

[0045] According to an embodiment of the present invention, step 1 specifically includes: reacting cisplatin and 30 wt % hydrogen peroxide in a dark environment at 70-90° C. for 5 to 8 hours.

[0046] According to an embodiment of the present invention, in step 1, the molar ratio of cisplatin to hydrogen peroxide is preferably 1:(5-100), for example, 1:10, 1:20, 1:50, 1:80, etc.

[0047] According to an embodiment of the present invention, step 1 further includes post-processing the product obtained by the reaction to purify the product. Specifically:

[0048] After the reaction is completed, water is removed by rotary evaporation at 60-70° C. to obtain light yellow crystals. A small amount of distilled water is then added to the light yellow crystals, and the mixture is heated at 120° C. for 10-20 minutes. After cooling, the mixture is washed three times with distilled water, methanol, and ether, and dried to obtain pure dichlorodihydroxydiammineplatinum.

[0049] According to an embodiment of the present invention, step 2 specifically includes: dissolving the polyunsaturated fatty acid and N-hydroxysuccinimide in a first organic solvent, adding a condensing agent, and reacting under a protective atmosphere at 20-30° C. for 8-12 hours.

[0050] According to an embodiment of the present invention, in step 2, the molar ratio of the polyunsaturated fatty acid, N-hydroxysuccinimide and the condensing agent is preferably 1:(1-1.2):(1.5-2.5), for example, it can be 1:1:2.

[0051] According to an embodiment of the present invention, in step 2, N,N'-dicyclohexylcarbodiimide (DCC) is preferably used as a condensation agent to facilitate esterification of polyunsaturated fatty acids and N-hydroxysuccinimide.

[0052] According to an embodiment of the present invention, in step 2, the first organic solvent may be dichloromethane or chloroform.

[0053] According to an embodiment of the present invention, in step 2, the protective atmosphere may be nitrogen.

[0054] According to an embodiment of the present invention, step 2 further includes post-processing the product obtained by the reaction to purify the product. Specifically,

[0055] After the reaction is completed, the reaction product is separated and purified by silica gel column chromatography to obtain the compound represented by formula (IV).

[0056] According to an embodiment of the present invention, step 3 specifically includes: dissolving dichlorodihydroxydiammineplatinum and the compound represented by formula (IV) in a second organic solvent, and reacting them in a light-proof, protective atmosphere at 50-60°C.

[0057] According to an embodiment of the present invention, in step 3, the molar ratio of dichlorodihydroxydiammineplatinum to the compound represented by formula (IV) is preferably 1:(1-2), for example, 1:1, 1:1.5, or 1:2. The reaction product may include compounds represented by both formula (Ia) and formula (Ib).

[0058] According to an embodiment of the present invention, in step 3, the second organic solvent may be dimethyl sulfoxide.

[0059] According to an embodiment of the present invention, in step 3, the protective atmosphere may be nitrogen.

[0060] According to an embodiment of the present invention, step 3 further includes post-processing the product obtained by the reaction to purify the product. Specifically,

[0061] After the reaction is completed, the second organic solvent is removed by rotary evaporation, and the reaction product is separated and purified by silica gel column chromatography to obtain the tetravalent platinum prodrug represented by formula (I).

[0062] According to the embodiments of the present invention, the reaction conditions in the above preparation method are mild, and the tetravalent platinum prodrug represented by formula (I) can be successfully prepared.

[0063] According to some embodiments of the present invention, there is also provided a use of the tetravalent platinum prodrug as described above in the preparation of an anti-tumor drug.

[0064] According to an embodiment of the present invention, the tumor includes non-small cell lung cancer, breast cancer, liver cancer or melanoma.

[0065] The technical solutions of the present invention are further described below by means of specific examples in conjunction with the accompanying drawings. It should be noted that the following specific examples are merely illustrative and the scope of protection of the present invention is not limited thereto. The drugs or reagents used in the following examples are all commercially available or prepared in-house by known preparation methods.

[0066] Example 1 Synthesis of Cisplatin-Docosahexaenoic Acid Prodrug

[0067] The present invention synthesizes cisplatin-docosahexaenoic acid prodrug according to the following route, specifically comprising:

[0068]

[0069] Step 1: Weigh 1.0 g of cisplatin (3.3 mmol) into a 100 mL round-bottom flask. Add 50 mL of 30% hydrogen peroxide solution (15.0 g, 132.3 mmol) and heat with stirring at 80°C in the dark for 8 hours. After the reaction, remove the water by rotary evaporation to obtain a yellow solid, the crude product of dichlorodihydroxydiammineplatinum. Add a small amount of distilled water to the yellow solid and boil for 10 minutes in the dark. Finally, wash the solid three times with water, methanol, and ether, then dry it in a 60°C oven to obtain pure dichlorodihydroxydiammineplatinum.

[0070] Step 2: Weigh N-hydroxysuccinimide (0.35 g, 3.04 mmol) into a 150 mL three-necked flask. Add 70 mL of anhydrous chloroform as the solvent. Dissolve the N-hydroxysuccinimide by ultrasonication. Then, add docosahexaenoic acid (1.0 g, 3.0 mmol) and N,N-dicyclohexylcarbodiimide (1.3 g, 6.0 mmol) sequentially. N2 protection is introduced and the mixture is stirred at room temperature for 12 h. After the reaction, the precipitate is removed by filtration, and the chloroform is removed by rotary evaporation. The resulting mixture is purified by silica gel column (eluting with chloroform) to obtain N-succinimidyldocosahexaenoic acid.

[0071] Step 3: Weigh the dichlorodihydroxydiammineplatinum (0.19 g, 0.57 mmol) obtained in step 1 and the N-succinimidyldocosahexaenoic acid (0.6 g, 1.4 mmol) obtained in step 2, place them in a glass tube, add 5 mL of dry dimethyl sulfoxide, use an oil pump to remove the air in the tube, seal the tube with fire, and stir at 55 ° C in the dark until the reaction solution is clear. After the reaction is completed, centrifuge at a speed of 5000 rpm to remove unreacted dichlorodihydroxydiammineplatinum, and use an oil pump to rotary evaporate the supernatant to remove dimethyl sulfoxide. The resulting mixture is purified by silica gel column (with V 二氯甲烷 :V 甲醇 =10:1 as eluent) to obtain a yellow solid, which is the final product, cisplatin-docosahexaenoic acid prodrug.

[0072] Structural characterization and results:

[0073] 1. H NMR spectrum of cisplatin-docosahexaenoic acid prodrug Figure 1 H NMR (400 MHz, DMSO) Figure 1 As shown, the H atoms in the structure are characterized by the H NMR spectrum. The results show that the cisplatin-docosahexaenoic acid prodrug was successfully synthesized.

[0074] 2. High resolution mass spectrometry (ESI-MS) of cisplatin-docosahexaenoic acid prodrug Figure 2 As shown in FIG, there is a molecular ion peak of cisplatin-docosahexaenoic acid prodrug. The results show that cisplatin-docosahexaenoic acid prodrug was successfully synthesized.

[0075] 3. The HPLC chromatogram of cisplatin-docosahexaenoic acid prodrug is as follows: Figure 3 As shown in Figure A, the following steps were performed to obtain cisplatin-docosahexaenoic acid: cisplatin-docosahexaenoic acid was dissolved in chromatographic grade methanol, filtered through a 220 nm filter membrane, and detected by an Agilent liquid chromatograph using a C18 high performance liquid chromatography column and a mobile phase of V 甲醇 :V 水 =9:1, 0.2% formic acid in water, a flow rate of 1 mL / min, a detection wavelength of 220 nm, and a reference wavelength of 365 nm. The results showed that the synthesized cisplatin-docosahexaenoic acid prodrug had a purity of up to 98%.

[0076] Example 2: Reduction of Cisplatin-Docosahexaenoic Acid Prodrug by Ascorbic Acid

[0077] Cisplatin-docosahexaenoic acid was dissolved in methanol (chromatographic grade) to a final concentration of 100 μM, and then ascorbic acid, a reducing agent, was added to a final concentration of 500 μM. The mixture was shaken on a shaker at 37°C. At different time points (0, 1.5, 3, and 4.5 h), 10 μL of the mixture was sampled and its HPLC chromatogram was measured. A C18 HPLC column was used, and the mobile phase was V 甲醇 :V 水 =9:1, water contains 0.2% formic acid, the flow rate is 1 mL / min, the detection wavelength is 220 nm, and the reference wavelength is 365 nm.

[0078] The restoration process is as follows Figure 3 As shown, Figures A and B are HPLC chromatograms of cisplatin-docosahexaenoic acid, respectively, and Figure C is the HPLC chromatogram of the reaction between cisplatin-docosahexaenoic acid and ascorbic acid. Over time, the signal peak for cisplatin-docosahexaenoic acid gradually weakened and then disappeared, while the signal peak for docosahexaenoic acid gradually strengthened, indicating that cisplatin-docosahexaenoic acid can be reduced by ascorbic acid to release cisplatin and docosahexaenoic acid.

[0079] Example 3 Cellular Uptake

[0080] Put 10 6Mouse melanoma B16F10 cells were seeded into 6-cm-diameter cell culture dishes and incubated in a cell culture incubator for 24 hours. The old medium was then aspirated and discarded. Medium containing 10 μM cisplatin or 10 μM cisplatin-docosahexaenoic acid was then added and incubated for an additional 12 hours. The drug-containing medium was then aspirated and the cells were washed thoroughly with PBS multiple times. 1 mL of cell lysis buffer was then added and the cells were lysed at 4°C for 20 minutes. Finally, 10 μL of the lysed liquid was aspirated for protein determination using the BCA protein assay. The remaining 990 μL of lysate was evaporated to dryness and then digested at 120°C with 5 mL of concentrated nitric acid. After 24 hours of digestion, the nitric acid was evaporated to dryness, and 3 mL of ultrapure water was added to the volume. Pt content was determined by ICP-MS. Intracellular platinum content was normalized using protein concentration.

[0081] like Figure 4 The results showed that the cellular uptake of cisplatin-docosahexaenoic acid prodrug was much higher than the cellular uptake of cisplatin, which is one of the key ways to effectively overcome cisplatin resistance.

[0082] Example 4 Cytotoxic activity detection

[0083] Mouse colon cancer cells CT26, mouse melanoma cells B16F10, human breast cancer cells MDA-MB-231, human non-small cell lung cancer cells A549, and cisplatin-resistant human non-small cell lung cancer cells A549cisR were seeded at a density of 3000 cells per well in 96-well plates. After 24 hours of incubation in a cell culture incubator, the culture medium was removed and fresh culture medium containing varying concentrations of cisplatin, cisplatin-docosahexaenoic acid, and a 1:1 molar ratio of cisplatin and docosahexaenoic acid was added. The cells were cultured for an additional 72 hours. After 72 hours, the culture medium was removed and 100 μL of fresh culture medium containing 0.5 mg / mL MTT was added to each well. After 4 hours of incubation in a cell culture incubator, the culture medium was removed and 100 μL of dimethyl sulfoxide was added to dissolve the generated crystal violet. The absorbance at 570 nm was measured using a microplate reader to calculate cell viability. Cell viability (%) = (absorbance of drug-treated group / absorbance of control group) × 100%. Graphpad Prism 8 software was used to process the data and calculate the half-inhibitory concentration (IC 50 ).

[0084] IC values ​​of cisplatin, cisplatin-docosahexaenoic acid prodrug, and cisplatin and docosahexaenoic acid mixture on various cells after 72 h of treatment 50The values ​​are shown in Table 1. The results show that compared with cisplatin, cisplatin-docosahexaenoic acid prodrug has a more excellent anti-tumor effect, and B16F10 cells, MDA-MB-231 cells and A549cisR cells that are insensitive to cisplatin also show good anti-proliferative activity, successfully overcoming the problem of cisplatin resistance.

[0085] Table 1 Cytotoxicity assay results of cisplatin-docosahexaenoic acid prodrug

[0086]

[0087] Example 5: Cisplatin-Docosahexaenoic Acid Inhibits Cancer Cell Energy Metabolism

[0088] After cisplatin-docosahexaenoic acid is taken up by cells, it is reduced to cisplatin and docosahexaenoic acid under the action of intracellular reducing agents. Cisplatin binds not only to nuclear DNA but also to mitochondrial DNA, causing mitochondrial damage. Docosahexaenoic acid effectively reduces levels of the hypoxia-inducible factor (HIF-1α), thereby reducing glycolysis in cancer cells. Ultimately, by reducing mitochondrial respiration and glycolysis, cisplatin-docosahexaenoic acid effectively blocks the cancer cells' energy source and induces cancer cell death.

[0089] The "oxygen consumption rate," representing mitochondrial respiratory capacity, and the "extracellular acidification rate," representing glycolysis, were measured using a Seahorse XF 96 Cell Metabolism Analyzer. MDA-MB-231 cells were seeded at a density of 10,000 cells per well in a Seahorse XF96 cell culture plate and cultured at 37°C in a 5% CO2 atmosphere for 24 hours. The cells were then divided into a control group, a cisplatin group, and a cisplatin-docosahexaenoic acid group. The old culture medium was removed, and 200 μL of fresh culture medium was added to the control group. The remaining two groups were incubated with fresh culture medium containing 1.0 μM cisplatin and 1.0 μM cisplatin-docosahexaenoic acid, respectively, for 12 hours.

[0090] Next, for cells to be tested for oxygen consumption, the old culture medium was removed and the cells were washed twice with XF assay medium containing 25 mM glucose, 1 mM pyruvate, and 2 mM glutamine. Subsequently, 180 μL of XF assay medium was added to each well, and the culture plate was incubated at 37°C in the absence of CO2 for 1 hour before the assay. The culture plate was then placed on a Seahorse XF96 cell metabolic analyzer for testing. During the assay, oligomycin, carbonyl cyanide-4-trifluoromethoxyphenylhydrazone, antimycin A, and rotenone were added to the culture plate in sequence, each at a final concentration of 1.0 μM.

[0091] For cells tested for the "Extracellular Acidification Rate" assay, the protocol is similar to that for the "Oxygen Consumption Rate" assay. However, the XF Assay Medium for the "Extracellular Acidification Rate" assay contains only 2 mM glutamine. For assays performed on the Seahorse XF 96 Cell Metabolic Analyzer, glucose (10 mM), oligomycin (1.0 μM), and 2-deoxy-D-glucose (50 mM) are added to the culture plate sequentially during the assay.

[0092] like Figure 5 As shown, cisplatin-docosahexaenoic acid prodrug can effectively reduce the "oxygen consumption rate" of mitochondrial respiration and the "extracellular acidification rate" of glycolysis, indicating that cisplatin-docosahexaenoic acid prodrug effectively blocks the energy source of cancer cells and effectively overcomes cisplatin-resistant cells.

[0093] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of a tetravalent platinum compound in the preparation of an anti-tumor drug, wherein: The tumor is breast cancer; the tetravalent platinum compound has the structure shown below: 。

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