Quadrivalent platinum rhein conjugate and preparation method thereof

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a tetravalent platinum rhein conjugate and a preparation method thereof, and the tetravalent platinum rhein conjugate has a structure as shown in a formula I or a formula II. The preparation method comprises the following steps: oxidizing a bivalent platinum drug with H2O2 to obtain an oxidation intermediate; under inert atmosphere protection and mild reaction conditions, the oxidation intermediate and rhein react in a solvent in the presence of a condensing agent and organic alkali, and separation and purification are performed to obtain the tetravalent platinum rhein conjugate. The tetravalent platinum rhein conjugate is a novel asymmetric monosubstituted conjugate with a clear structure, and the preparation method is mild in condition, high in selectivity, simple and convenient to operate and suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a tetravalent platinum rhein conjugate and its preparation method. Background Technology

[0002] Platinum-based drugs are among the most widely used anticancer drugs in clinical practice. Currently, the five most commonly used platinum-based drugs in clinical practice are cisplatin, carboplatin, nedaplatin, oxaliplatin, and lobaplatin. However, all of the above-mentioned platinum-based drugs are divalent platinum (Pt(II)) compounds, which are chemically reactive in vivo and readily bind non-specifically to biomolecules such as plasma proteins, leading to strong toxic side effects (such as nephrotoxicity and neurotoxicity), and long-term use can easily lead to drug resistance.

[0003] To overcome the drawbacks of traditional divalent platinum drugs, such as strong toxicity and easy development of drug resistance, the development of tetravalent platinum (Pt(IV)) prodrugs with octahedral configuration and stable kinetic properties has become a research hotspot. By introducing bioactive ligands at two axial positions at the center of Pt(IV), synergistic anticancer drugs with multiple pharmacological activities can be constructed.

[0004] Rhein, a natural product with antitumor activity, has been used to construct Pt(IV) prodrugs. For example, Chinese invention patent CN110950915B discloses a novel rhein-platinum(IV) precursor anticancer complex and its synthesis method and application. The patent's technical solution involves reacting hydroxylated cisplatin with two molar amounts of rhein in an acetic acid system under high temperature (e.g., 120°C) for an extended period to prepare a symmetrically substituted conjugate with rhein substituted at both axial positions. This disubstituted conjugate has been shown to have good inhibitory activity against cisplatin-resistant lung adenocarcinoma cells. However, the preparation method disclosed in this patent is a high-temperature thermocondensation method, with relatively harsh reaction conditions, and is mainly suitable for preparing symmetrical disubstituted products. It does not provide any technical guidance on how to selectively obtain other possible substitution forms under mild conditions. Based on the above, this invention proposes a tetravalent platinum-rhein conjugate and its preparation method. Summary of the Invention

[0005] To address the problems of existing technologies that use high-temperature thermal shrinkage to prepare tetravalent platinum-rhein couplings, which involve harsh reaction conditions and are mainly applicable to the preparation of symmetrical disubstituted products, thus failing to yield other possible substitution forms, this invention proposes a tetravalent platinum-rhein coupling and its preparation method.

[0006] In a first aspect, the present invention provides a tetravalent platinum rhein coupling compound, which adopts the following technical solution: A tetravalent platinum rhein coupling compound having the structure shown in Formula I or Formula II: , .

[0007] Secondly, this invention provides a method for preparing a tetravalent platinum rhein coupling compound, employing the following technical solution: A method for preparing a tetravalent platinum rhein coupling compound includes the following steps: S1. Oxidize the divalent platinum drug with H2O2 to obtain an oxidation intermediate; S2. Under an inert atmosphere, the oxidized intermediate and rhein react in a solvent in the presence of a condensing agent and an organic base, and after separation and purification, a tetravalent platinum rhein conjugate is obtained.

[0008] Preferably, the divalent platinum drug in step S1 is oxaliplatin or cisplatin.

[0009] Preferably, in step S1, the mass ratio of divalent platinum drug to H2O2 is 1:15-25.

[0010] Preferably, the oxidation temperature in step S1 is 60-75℃, and the oxidation time is 5-12h.

[0011] Preferably, the condensing agent in step S2 is O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU).

[0012] Preferably, the organic base triethylamine is used in step S2.

[0013] Preferably, the solvent in step S2 is N,N-dimethylformamide or dimethyl sulfoxide.

[0014] Preferably, the reaction temperature in step S2 is 10-40℃ and the reaction time is 12-48h.

[0015] Preferably, the molar ratio of the oxidized intermediate to rhein in step S2 is 1:1.2-1.8.

[0016] Preferably, the molar ratio of the oxidized intermediate to triethylamine in step S2 is 1:1.5-2.

[0017] Preferably, the molar ratio of the oxidized intermediate to TBTU in step S2 is 1:1.5-2.

[0018] Preferably, the separation and purification step in step S2 is as follows: precipitate the reaction solution with diethyl ether, filter and collect precipitate I; dissolve precipitate I in saturated sodium bicarbonate solution and wash, filter and collect precipitate II; perform silica gel column chromatography on precipitate II to obtain the target product.

[0019] Preferably, the separation and purification step in step S2 is as follows: 5-6 times the volume of diethyl ether is added to the reaction solution and stirred, precipitate I is collected by filtration, precipitate I is added to 2 times the volume of saturated NaHCO3 solution of the reaction solution and stirred, filtered, washed with water, and precipitate II is collected; precipitate II is subjected to silica gel column chromatography with a mixture of dichloromethane and methanol as the eluent, and the eluent is removed to obtain the target product.

[0020] Preferably, in step S2, the volume ratio of dichloromethane (DCM) to methanol (MeOH) in the eluent for obtaining the target product as compound I is 20:1; and the volume ratio of dichloromethane (DCM) to methanol (MeOH) in the eluent for obtaining the target product as compound II is 30:1.

[0021] In summary, the present invention has the following beneficial effects: 1. This invention provides an asymmetric tetravalent platinum conjugate with an axially positioned hydroxyl group and a rhein ligand, offering a new molecular entity for the development of novel platinum-based prodrugs.

[0022] 2. The preparation method employed in this invention achieves highly efficient coupling under mild conditions (e.g., 10-40°C) by introducing a specific reaction system composed of a condensing agent (such as TBTU) and an organic base (such as triethylamine). Compared with the high-temperature thermal shrinkage method at 120°C in the prior art, this invention effectively avoids the decomposition of raw materials and non-selective reactions caused by high temperatures, protects the structural integrity of drug molecules, and demonstrates significant technological progress.

[0023] 3. By precisely controlling the feed ratio of the oxidation intermediate to rhein and employing a highly efficient condensation system, this invention can selectively generate the target asymmetric monosubstituted product, effectively suppress the generation of byproducts such as symmetric disubstituted products, simplify the subsequent purification process, and significantly improve the yield and purity of the target product, making it suitable for industrial production. Attached Figure Description

[0024] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0025] Figure 1 For the OPR in Embodiment 1 of the present invention 1 H NMR spectrum (d6-DMSO); Figure 2 For the OPR in Embodiment 1 of the present invention 13 C{ 1 H} NMR spectrum (d6-DMSO); Figure 3 For the OPR in Embodiment 1 of the present invention 195 Pt NMR spectrum (d6-DMSO); Figure 4 This is a high-resolution mass spectrum of OPR in Example 1 of the present invention; Figure 5 For CPR in Embodiment 2 of the present invention 1 H NMR spectrum (d6-DMSO); Figure 6 For CPR in Embodiment 2 of the present invention 13 C{ 1 H} NMR spectrum (d6-DMSO); Figure 7 For CPR in Embodiment 2 of the present invention 195 Pt NMR spectrum (d6-DMSO); Figure 8 This is a high-resolution mass spectrum of CPR in Example 2 of the present invention. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the embodiments.

[0027] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0028] Example 1: Preparation of Oxaliplatin-Rhein Conjugate (OPR) S1, Preparation of Oxaliplatin Oxide Hydrogen peroxide (30 wt%, 18 mL) was slowly added dropwise to oxaliplatin (300 mg) over 30 minutes. The reaction system was heated to 75 °C and magnetically stirred at 400 rpm for 5 hours. After the reaction was complete, the reaction solution was cooled to room temperature (25 °C). Using a rotary evaporator, the reaction solution was concentrated to 5 mL under reduced pressure at a water bath temperature of 45 °C and a vacuum degree of -0.09 MPa. 30 mL of anhydrous ethanol was added to the concentrated solution, and a white precipitate was formed. The precipitate was collected by vacuum filtration, and the white solid product was dried in a vacuum drying oven at 40 °C to constant weight to obtain oxaliplatin oxide (212 mg, yield 65%).

[0029] Synthesis of S2, Oxaliplatin-Rhein Conjugate (OPR) Under a nitrogen atmosphere, 150 mg of oxaliplatin oxide prepared in S1 was dissolved in 5 mL of N,N-dimethylformamide (DMF), and rhein (148.3 mg), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU, 223.4 mg), and triethylamine (NEt3, 97 μL) were added. The mixture was magnetically stirred at 500 rpm for 48 h at room temperature (25 °C). After the reaction was complete, 30 mL of diethyl ether was added to the reaction solution, and stirring was continued for 5 min to allow the product to precipitate completely. The product was collected by vacuum filtration and the brownish-black solid was collected. The brownish-black solid was dissolved in 10 mL of saturated NaHCO3 solution and washed with magnetic stirring for 15 min, at which point the system became a wine-red suspension. The product was then filtered under reduced pressure, and the filter cake was washed three times with deionized water to obtain a brownish-yellow powder as the crude product. The crude product was dried in a vacuum drying oven at 40℃ for 4 hours, and then purified by silica gel column chromatography (stationary phase: 200-300 mesh silica gel; eluent: DCM:MeOH = 20:1, v / v). The fraction containing the target product was collected, and after removing the eluent by rotary evaporation, it was dried in a vacuum drying oven at 40℃ to constant weight to obtain a yellow solid product OPR (102 mg, yield 42%).

[0030] The product 1 H, 13 C 195 Pt NMR and high-resolution mass spectrometry, such as Figure 1-4 As shown, the results indicate that the structure of the product is: This is consistent with the expected results of the synthetic route.

[0031] Example 2 Preparation of cisplatin-rheic acid conjugate (CPR) S1, Preparation of cisplatin oxide Hydrogen peroxide (30 wt%, 15 mL) was slowly added dropwise to cisplatin (250 mg) over a period of 30 minutes. The reaction mixture was heated to 60 °C and magnetically stirred at 400 rpm for 12 h. After the reaction was complete, the reaction solution was cooled to room temperature, and a pale yellow solid precipitated. The solid was filtered under reduced pressure, washed three times with cold water at 4 °C, and the pale yellow solid product was collected and dried in a vacuum drying oven at 40 °C to constant weight to obtain cisplatin oxide (156 mg, yield 56%).

[0032] Synthesis of S2, cisplatin-rheic acid conjugate (CPR) Under a nitrogen atmosphere, 100 mg of cisplatin oxide prepared in S1 was dissolved in 5 mL of dimethyl sulfoxide. Rhein (127.6 mg), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU, 192.2 mg), and triethylamine (NEt3, 83 μL) were added. The mixture was magnetically stirred at 500 rpm for 12 h at room temperature (25 °C). After the reaction was complete, 30 mL of diethyl ether was added to the reaction solution, and stirring was continued for 5 min to allow the product to precipitate completely. The product was collected by vacuum filtration and was a brownish-black solid. The brownish-black solid was dissolved in 10 mL of saturated NaHCO3 solution and washed with magnetic stirring for 15 min, resulting in a wine-red suspension. The product was then filtered under reduced pressure, and the filter cake was washed three times with deionized water to obtain a brownish-yellow powder as the crude product. The crude product was dried in a vacuum drying oven at 40℃ for 4 hours, and then purified by silica gel column chromatography (stationary phase: 200-300 mesh silica gel; eluent: DCM:MeOH = 30:1, v / v). The fraction containing the target product was collected, and after removing the eluent by rotary evaporation, it was dried in a vacuum drying oven at 40℃ to constant weight to obtain a yellow solid product CPR (83 mg, yield 45%).

[0033] The product 1 H, 13 C 195 Pt NMR and high-resolution mass spectrometry, such as Figure 5-8 As shown, the results indicate that the structure of the product is: This is consistent with the expected results of the synthetic route.

[0034] Example 3 Preparation of Oxaliplatin-Rhein Conjugate (OPR) S1, Preparation of Oxaliplatin Oxide Hydrogen peroxide (30 wt%, 18 mL) was slowly added dropwise to oxaliplatin (300 mg) over 30 minutes. The reaction system was heated to 75 °C and magnetically stirred at 400 rpm for 5 hours. After the reaction was complete, the reaction solution was cooled to room temperature (25 °C). Using a rotary evaporator, the reaction solution was concentrated to 5 mL under reduced pressure at a water bath temperature of 45 °C and a vacuum degree of -0.09 MPa. 30 mL of anhydrous ethanol was added to the concentrated solution, and a white precipitate was formed. The precipitate was collected by vacuum filtration, and the white solid product was dried in a vacuum drying oven at 40 °C to constant weight to obtain oxaliplatin oxide (212 mg, yield 65%).

[0035] Synthesis of S2, Oxaliplatin-Rhein Conjugate (OPR) Under a nitrogen atmosphere, 150 mg of oxaliplatin oxide prepared in S1 was dissolved in 5 mL of N,N-dimethylformamide (DMF), and rhein (148.3 mg), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU, 223.4 mg), and triethylamine (NEt3, 97 μL) were added. The mixture was magnetically stirred at 500 rpm for 24 h at 40 °C. After the reaction was complete, 30 mL of diethyl ether was added to the reaction solution, and stirring was continued for 5 min to allow the product to precipitate completely. The product was collected by vacuum filtration and the brownish-black solid was collected. The brownish-black solid was dissolved in 10 mL of saturated NaHCO3 solution and washed with magnetic stirring for 15 min, at which point the system became a wine-red suspension. The product was then filtered under reduced pressure, and the filter cake was washed three times with deionized water to obtain a brownish-yellow powder as the crude product. The crude product was dried in a vacuum drying oven at 40℃ for 4 hours, and then purified by silica gel column chromatography (stationary phase: 200-300 mesh silica gel; eluent: DCM:MeOH = 20:1, v / v). The fraction containing the target product was collected, and after removing the eluent by rotary evaporation, it was dried in a vacuum drying oven at 40℃ to constant weight to obtain a yellow solid product OPR (125 mg, yield 51%).

[0036] Characterized by 1H NMR, 13C NMR, 195Pt NMR and high-resolution mass spectrometry (HRMS), the structure of the obtained product was consistent with that of Example 1.

[0037] Comparative Example 1: Preparation of OPR using the high-temperature heat shrinkage method S1, Preparation of Oxaliplatin Oxide Hydrogen peroxide (30 wt%, 18 mL) was slowly added dropwise to oxaliplatin (300 mg) over 30 minutes. The reaction system was heated to 75 °C and magnetically stirred at 400 rpm for 5 hours. After the reaction was complete, the reaction solution was cooled to room temperature (25 °C). Using a rotary evaporator, the reaction solution was concentrated to 5 mL under reduced pressure at a water bath temperature of 45 °C and a vacuum degree of -0.09 MPa. 30 mL of anhydrous ethanol was added to the concentrated solution, and a white precipitate was formed. The precipitate was collected by vacuum filtration, and the white solid product was dried in a vacuum drying oven at 40 °C to constant weight to obtain oxaliplatin oxide (212 mg, yield 65%).

[0038] S2. Under a nitrogen atmosphere, 150 mg of oxaliplatin oxide prepared in S1 and 148.3 mg of rhein were dissolved together in 5 mL of acetic acid. The mixture was heated to 120 °C and magnetically stirred at 500 rpm for 24 h. After the reaction was completed, the reaction solution was cooled to room temperature and the reaction system was analyzed by high performance liquid chromatography (HPLC).

[0039] The results showed that the reaction system was extremely complex, mainly consisting of degradation peaks of the raw materials and a large number of unknown impurities. The target product OPR (compound of formula I) was not detected, and a small amount of symmetrical disubstituted products and a large amount of coking substances were detected. This result indicates that the high-temperature thermal shrinkage method in the background art is too harsh in preparing the asymmetric monosubstituted coupling compound of the present invention, leading to the decomposition of the raw materials, extremely poor selectivity, and failure to obtain the target product.

[0040] Comparative Example 2: No condensing agent TBTU was added during the reaction. S1, Preparation of Oxaliplatin Oxide Hydrogen peroxide (30 wt%, 18 mL) was slowly added dropwise to oxaliplatin (300 mg) over 30 minutes. The reaction system was heated to 75 °C and magnetically stirred at 400 rpm for 5 hours. After the reaction was complete, the reaction solution was cooled to room temperature (25 °C). Using a rotary evaporator, the reaction solution was concentrated to 5 mL under reduced pressure at a water bath temperature of 45 °C and a vacuum degree of -0.09 MPa. 30 mL of anhydrous ethanol was added to the concentrated solution, and a white precipitate was formed. The precipitate was collected by vacuum filtration, and the white solid product was dried in a vacuum drying oven at 40 °C to constant weight to obtain oxaliplatin oxide (212 mg, yield 65%).

[0041] Synthesis of S2, Oxaliplatin-Rhein Conjugate (OPR) Under a nitrogen atmosphere, 150 mg of oxaliplatin oxide prepared by S1 was dissolved in 5 mL of N,N-dimethylformamide (DMF), and rhein (148.3 mg) and triethylamine (NEt3, 97 μL) were added. The mixture was then magnetically stirred at 500 rpm for 48 h at room temperature (25 °C).

[0042] After the reaction was completed, the post-processing method of Example 1 was followed. HPLC analysis revealed only trace amounts of the target product OPR, with a yield of less than 2%, indicating that the majority of the product was unreacted raw material. This result demonstrates that even under mild conditions and prolonged reaction in the absence of the condensing agent TBTU, the axial hydroxyl group of oxaliplatin oxide hardly undergoes esterification with the carboxyl group of rhein.

[0043] Comparative Example 3: No organic base, triethylamine, was added during the reaction. S1, Preparation of Oxaliplatin Oxide Hydrogen peroxide (30 wt%, 18 mL) was slowly added dropwise to oxaliplatin (300 mg) over 30 minutes. The reaction system was heated to 75 °C and magnetically stirred at 400 rpm for 5 hours. After the reaction was complete, the reaction solution was cooled to room temperature (25 °C). Using a rotary evaporator, the reaction solution was concentrated to 5 mL under reduced pressure at a water bath temperature of 45 °C and a vacuum degree of -0.09 MPa. 30 mL of anhydrous ethanol was added to the concentrated solution, and a white precipitate was formed. The precipitate was collected by vacuum filtration, and the white solid product was dried in a vacuum drying oven at 40 °C to constant weight to obtain oxaliplatin oxide (212 mg, yield 65%).

[0044] Synthesis of S2, Oxaliplatin-Rhein Conjugate (OPR) Under a nitrogen atmosphere, 150 mg of oxaliplatin oxide prepared by S1 was dissolved in 5 mL of N,N-dimethylformamide (DMF), and rhein (148.3 mg) and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea tetrafluoroborate (TBTU, 223.4 mg) were added. The mixture was then magnetically stirred at 500 rpm for 48 h at room temperature (25 °C).

[0045] After the reaction was completed, the post-processing method of Example 1 was followed, and the product was purified to obtain a yellow solid, OPR (21 mg, yield of only 8.7%). This result indicates that although the reaction can occur in the absence of the organic base triethylamine, its efficiency is extremely low. This is because the acidic substances generated during the reaction inhibit its progress. Therefore, the organic base triethylamine, as an acid binder, is essential for ensuring the smooth and efficient conduct of the reaction.

[0046] Comparing Example 1 and Comparative Example 1, it is evident that the high-temperature thermal shrinkage method (120°C) in the prior art is completely unsuitable for the preparation of the asymmetric monosubstituted coupling compound of this invention, leading to severe decomposition of the raw materials and failure to obtain the target product. In contrast, the mild conditions (25°C) combined with a specific condensation system employed in this invention successfully prepare the target product OPR with a yield of 42%. This demonstrates that the method of this invention represents a significant advancement and unexpected technical effect compared to the prior art.

[0047] Comparing Example 1 and Comparative Examples 2-3, it can be seen that without TBTU, the reaction hardly occurs (yield <2%); without triethylamine, the reaction efficiency is significantly reduced (yield is only 8.7%). The technical features selected in this invention are combined to form the complete technical solution of this invention. The synergistic effect of these technical features enables the reaction to proceed efficiently under mild conditions.

[0048] In summary, the method provided by this invention is mild, highly selective, and easy to operate. By introducing a specific condensation system, it successfully solves the technical problem that existing technologies cannot prepare asymmetric monosubstituted tetravalent platinum-rheic acid conjugates under mild conditions, demonstrating significant inventiveness.

[0049] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A tetravalent platinum rhein coupling compound, characterized in that, The tetravalent platinum rhein conjugate has the structure shown in Formula I or Formula II: , .

2. A method for preparing the tetravalent platinum rhein coupling compound according to claim 1, characterized in that, Includes the following steps: S1. Oxidize the divalent platinum drug with H2O2 to obtain an oxidation intermediate; S2. Under an inert atmosphere, the oxidized intermediate and rhein react in a solvent in the presence of a condensing agent and an organic base, and after separation and purification, a tetravalent platinum rhein conjugate is obtained.

3. The method for preparing the tetravalent platinum rhein coupling compound according to claim 2, characterized in that, In step S1, the divalent platinum drug is oxaliplatin or cisplatin.

4. The method for preparing the tetravalent platinum rhein coupling compound according to claim 3, characterized in that, In step S1, the mass ratio of divalent platinum drug to H2O2 is 1:15-25.

5. The method for preparing the tetravalent platinum rhein coupling compound according to claim 2, characterized in that, The oxidation temperature in step S1 is 60-75℃, and the oxidation time is 5-12h.

6. The method for preparing the tetravalent platinum rhein coupling compound according to claim 2, characterized in that, In step S2, the condensing agent is TBTU; the organic base is triethylamine; and the solvent is N,N-dimethylformamide or dimethyl sulfoxide.

7. The method for preparing the tetravalent platinum rhein coupling compound according to claim 2, characterized in that, In step S2, the reaction temperature is 10-40℃ and the reaction time is 12-48h.

8. The method for preparing the tetravalent platinum rhein coupling compound according to claim 6, characterized in that, In step S2, the molar ratio of the oxidized intermediate to rhein is 1:1.2-1.8; the molar ratio of the oxidized intermediate to triethylamine is 1:1.5-2; and the molar ratio of the oxidized intermediate to TBTU is 1:1.5-2.

9. The method for preparing the tetravalent platinum rhein coupling compound according to claim 3, characterized in that, The separation and purification steps in step S2 are as follows: precipitate the reaction solution with diethyl ether, filter and collect precipitate I; dissolve precipitate I in saturated sodium bicarbonate solution and wash, filter and collect precipitate II; Precipitate II was subjected to silica gel column chromatography to obtain the target product.

10. The method for preparing the tetravalent platinum rhein coupling compound according to claim 9, characterized in that, In step S2, the volume ratio of dichloromethane to methanol in the eluent for obtaining the target product as compound I is 20:1; and the volume ratio of dichloromethane to methanol in the eluent for obtaining the target product as compound II is 30:1.

Images