Glycyrrhetinic acid-platinum compound as well as preparation method and application thereof

Abstract

The invention provides a glycyrrhetinic acid-platinum compound as shown in a structural formula as well as a preparation method and application thereof, and belongs to the technical field of medicines. The preparation method comprises the following steps: taking glycyrrhetinic acid as a raw material, coupling the glycyrrhetinic acid with dimethyl 3-amino-1, 1-cyclobutanedicarboxylate through an amido bond, converting the glycyrrhetinic acid into a sodium salt, and carrying out coordination substitution reaction on the sodium salt and a hydrate cis-[PtA2 (H2O) 2] (NO3) 2 of a platinum drug on the market to prepare a compound 1; the compound provided by the invention has inhibitory activity on human hepatoma cell lines HepG2, Huh7 and SK-Hep-1, can form a pharmaceutical composition with a pharmaceutically acceptable carrier, and can be used for preparing anti-hepatoma drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, in particular, the present application relates to a new glycyrrhetinic acid-platinum complex, a preparation method thereof, a pharmaceutical composition for treating liver cancer with the complex as an active ingredient, and an application of the compound and the pharmaceutical composition thereof in preparing an anti-liver cancer drug. BACKGROUND

[0002] Liver cancer includes primary liver cancer and secondary liver cancer, among which hepatocellular carcinoma (HCC) accounts for more than 90%. Liver cancer has the characteristics of long incubation period, occult onset, strong invasiveness, easy metastasis, and high malignancy. For patients with unresectable or advanced disease, the 5-year survival rate is only 13%. The treatment of liver cancer depends on its stage and type, and the main treatment methods include surgical treatment (surgical resection of lesions or liver transplantation), ablation therapy, transarterial chemoembolization, radiotherapy and systemic anti-tumor therapy (targeted therapy, immunotherapy and chemotherapy).

[0003] Targeted drugs mainly treat cancer-causing mutations and can play an anti-liver cancer role by blocking abnormal protein kinases caused by gene mutations in patients. The five approved targeted drugs on the market, sorafenib, regorafenib, cabozantinib, ramucirumab and lenvatinib, are all tyrosine kinase inhibitors. Immunotherapy can more effectively identify and kill cancer cells by affecting the patient's own immune system. The FDA-approved immunotherapy drugs for liver cancer, pembrolizumab and nivolumab, are both immune checkpoint inhibitors, which are PD-1 inhibitors. They activate T cells to kill tumors through the human immune system and are mainly used for patients who have failed chemotherapy. The approved avelumab by the State Drug Administration can also inhibit the expression of PD-L1, etc.

[0004] Targeted drugs and immunotherapy drugs are not suitable for many patients, while platinum-based anticancer drugs (including cisplatin, carboplatin, oxaliplatin, etc.) have strong anticancer effects and are the most commonly used drugs for liver cancer, used for hepatic arterial infusion chemotherapy and hepatic arterial interventional embolization chemotherapy. Platinum drugs mainly cause cell apoptosis by damaging DNA, cell cycle arrest, inhibiting DNA replication and transcription processes, and apoptosis and necrosis. As cytotoxic drugs, platinum not only inhibits the growth of cancer cells, but also has a great harm to normal cells that grow vigorously, leading to serious toxic side effects such as myelosuppression, gastrointestinal adverse reactions, and neurotoxicity.

[0005] Targeted drug delivery can selectively deliver drugs to cancer cells or cancer tissues, thereby reducing the accumulation in normal tissue cells while increasing the local concentration of drugs at the lesion, which is one of the most effective methods to reduce the toxicity of anticancer drugs and improve efficacy. Professor Liu Weiping's team of Kunming Institute of Precious Metals in the previous stage used endogenous cholic acid as a carrier, used the leaving group 1,1-cyclobutane dicarboxylic acid of carboplatin as a linker, and coupled the pharmacodynamic group of oxaliplatin to design and synthesize a liver-targeting oxaliplatin prodrug LLC-202 with strong in vivo and in vitro anticancer activity, better safety and liver targeting (201410636868.0).

[0006] The triterpenoid active ingredient glycyrrhizic acid (Glycyrrhetinic acid, GA) in Chinese medicine licorice has good hepatoprotective and detoxification effects and anti-tumor effects, and can promote cancer cell apoptosis. Domestic and foreign scholars have found that the liver cell membrane components contain a large number of glycyrrhizic acid specific binding sites, therefore, glycyrrhizic acid and its derivatives show in-depth research prospects in new anticancer drugs and as liver-targeting carriers for liver disease treatment drugs. According to the drug binding splicing principle, glycyrrhizic acid and platinum drugs are coupled to obtain platinum drugs with high liver targeting and strong anticancer effect.

[0007] There is no report on the synthesis of glycyrrhizic acid-oxaliplatin complex in the prior art, and there is no report on the drug composition of the effective ingredient, and there is no report on the application of the drug composition containing the same in the preparation of liver cancer treatment drugs. SUMMARY

[0008] The purpose of the present application is to provide a new glycyrrhizic acid-platinum complex and its pharmaceutical composition and its preparation method and application, the preparation method of the present application uses glycyrrhizic acid and 3-hydroxy-1,1-cyclobutane dicarboxylic acid ethyl ester as raw materials, which is easy to obtain and easy to operate, and is suitable for industrial production.

[0009] In order to achieve the above purpose of the present application, the present application provides the following technical scheme:

[0010] The present application provides a glycyrrhizic acid-platinum complex 1 as shown in the following structural formula,

[0011] .

[0012] The application also provides a preparation method of the glycyrrhetinic acid-platinum complex 1 shown in the structural formula, which mainly comprises the following three key reaction steps: glycyrrhetinic acid is condensed with 3-amino-1,1-cyclobutanedicarboxylic acid dimethyl ester under the action of DCC, HOBt and NMM to obtain 3-glycyrrhetinic acid amido-1,1-cyclobutanedicarboxylic acid dimethyl ester; 3-glycyrrhetinic acid amido-1,1-cyclobutanedicarboxylic acid dimethyl ester is hydrolyzed under the action of lithium hydroxide to obtain 3-glycyrrhetinic acid amido-1,1-cyclobutanedicarboxylic acid; 3-glycyrrhetinic acid amido-1,1-cyclobutanedicarboxylic acid is subjected to a coordination substitution reaction with potassium hydroxide under alkaline conditions to form 3-glycyrrhetinic acid amido-1,1-cyclobutanedicarboxylic acid potassium, and a hydrate cis-[PtA2 (H2O)2] (NO3)2 of a marketed platinum drug to obtain the target compound glycyrrhetinic acid-platinum complex 1,

[0013]

[0014] The application also provides application of the new glycyrrhetinic acid-platinum complex 1 in preparation of a liver cancer resistant drug.

[0015] The application also provides a pharmaceutical composition comprising the glycyrrhetinic acid-platinum complex 1 shown in the structural formula and a pharmaceutically acceptable carrier.

[0016] The application of the pharmaceutical composition in preparation of a liver cancer resistant drug.

[0017] The pharmaceutical composition provided by the application comprises a pharmaceutically acceptable carrier of the glycyrrhetinic acid-platinum complex 1. In the application, the pharmaceutically acceptable carrier is preferably a solid, semi-solid or liquid diluent, a filler and a pharmaceutical product adjuvant. The application does not have special limitations on the pharmaceutically acceptable carrier, and a pharmaceutically acceptable carrier well known in the art and inert to humans and animals can be selected.

[0018] The preparation method of the pharmaceutical composition comprises the following steps: obtaining the glycyrrhetinic acid-platinum complex 1 by the above method for preparing the glycyrrhetinic acid-platinum complex 1, and then adding a pharmaceutically acceptable carrier.

[0019] The application does not have special limitations on the preparation method of the pharmaceutical composition, and the glycyrrhetinic acid-platinum complex can be mixed with the pharmaceutically acceptable carrier directly. The application does not have special limitations on the mixing process, and a process well known in the art can be selected to obtain the pharmaceutical composition.

[0020] The application provides application of the pharmaceutical composition in the above technical solution in preparation of a liver cancer resistant drug. The application does not have special limitations on the application method, and a method well known in the art can be selected.

[0021] In the present application, when the pharmaceutical composition is used for preparing an anti-liver cancer drug, the content of the composition in the drug is preferably 0.1-99%; in the pharmaceutical composition, the content of the glycyrrhetinic acid-platinum complex 1 in the pharmaceutical composition is preferably 0.5-90%. The pharmaceutical composition of the present application is preferably used in the form of a unit dose. In the present application, the prepared drug can be administered in two forms of injection (intravenous injection, intramuscular injection) and oral administration.

[0022] Compared with the prior art, the present application has the following advantages:

[0023] 1. The present application provides a new glycyrrhetinic acid-platinum complex 1.

[0024] 2. The present application provides a preparation method of the glycyrrhetinic acid-platinum complex 1, which has readily available raw materials, simple process and easy operation.

[0025] 3. The present application provides a pharmaceutical composition taking the glycyrrhetinic acid-platinum complex 1 as an effective component, which provides a new drug with good medicinal effect for a new anti-liver cancer drug.

[0026] 4. The glycyrrhetinic acid-platinum complex 1 of the present application has strong activity on the cytotoxic activity of three liver cancer cells (HepG2, SK-Hep-1 and Huh7), and the IC 50 values are 25.4 (HepG2), 26.9 (Huh7) and 27.7 μM (SK-Hep-1), respectively.

[0027] 5. The synthesized glycyrrhetinic acid-platinum complex 1 has potential clinical application prospects and can be used as a drug for treating liver cancer-related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The present application is a schematic diagram of the structural formula of the glycyrrhetinic acid-platinum complex. DETAILED DESCRIPTION

[0029] In order to better understand the present application, the following embodiments of the present application are further described in conjunction with the drawings, but the present application is not limited thereto.

[0030] Example 1: Synthesis method of glycyrrhetinic acid-platinum complex.

[0031] (a) Preparation of 3-glycyrrhetinic acid amido-1,1-cyclobutanedicarboxylic acid dimethyl ester.

[0032]

[0033] Glycyrrhetinic acid (9.41 g, 20 mmol, 1.0 eq.) was dissolved in 100 mL of tetrahydrofuran. After stirring and cooling in an ice-salt bath, 1-hydroxybenzotriazole (HOBt, 2.70 g, 20 mmol, 1 eq.) and N-methylmorpholine (NMM, 2.02 g, 20 mmol) were added sequentially. Then, a tetrahydrofuran solution of dicyclohexylcarbodiimide (DCC, 4.33 g, 21 mmol, 1.05 eq.) was slowly added dropwise. The reaction was allowed to proceed for 2 h, then allowed to rise naturally to room temperature overnight. The white insoluble solid was filtered off, and the filtrate was concentrated under reduced pressure to obtain a yellow viscous liquid. After dissolving in dichloromethane, the liquid was washed sequentially with saturated sodium bicarbonate solution, 10% citric acid solution, saturated sodium bicarbonate solution, and saturated brine. The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the product was separated by silica gel column chromatography to obtain dimethyl 3-glycyrrhetinic acid-1,1-cyclobutanedicarboxylic acid, with a yield of 62%.

[0034] (b) Preparation of 3-glycyrrhetinic acid-1,1-cyclobutanedicarboxylic acid.

[0035]

[0036] In a 10 mL reaction flask, dimethyl 3-glycyrrhetinic acid amamido-1,1-cyclobutanedicarboxylate (128 mg, 0.2 mmol, 1.0 eq.) was dissolved in a mixture of tetrahydrofuran (3 mL) and water (1 mL), and the mixture was cooled to 0 °C in an ice bath. o After step C, lithium hydroxide (19.2 mg, 0.8 mmol, 4.0 eq) was added, and the mixture was allowed to rise naturally to room temperature for 6 h. The pH was adjusted to 1 with dilute hydrochloric acid, and the mixture was extracted with ethyl acetate (3 × 10 mL). The organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a white product. This product was then separated by silica gel column chromatography to give 3-glycyrrhetinic acid amamido-1,1-cyclobutanedicarboxylic acid in 86% yield.

[0037] (c) Preparation of glycyrrhetinic acid-platinum complex 1.

[0038]

[0039] The 3-glycyrrhetinic acid amido-1,1-cyclobutanedicarboxylic acid ligand was added to a proper amount of water to make a slurry, and then a potassium hydroxide solution was slowly added dropwise until the solution was clear (pH between 7 and 8). After filtration, the filtrate was left at room temperature for standby. The diiodocyclohexanediamine platinum was added to a proper amount of water to make a slurry, and then a proper amount of silver nitrate solution was added. After stirring at 45°C for 4 hours, a colorless transparent solution and part of the precipitate were obtained. After filtration, the filtrate was reserved. The 3-glycyrrhetinic acid amido-1,1-cyclobutanedicarboxylic acid potassium solution was slowly added to the filtrate. After stirring at 45°C for 2 hours, a large amount of white solid was generated. After cooling, filtration, washing with a large amount of ice water, and drying, a white solid was obtained, with a yield of 69%.

[0040] Structural parameters:

[0041] 13C NMR (CD3OD, 100 MHz): δ 38.5 (C-1), 27.0 (CH2, C-2), 76.6 (CH,C-3), 39.1 (C, C-4), 54.1 (CH, C-5), 17.2 (CH2, C-6), 32.2 (CH2, C-7), 42.7(C, C-8), 61.2 (CH, C-9), 36.7 (C, C-10), 199.2 (C, C-11), 127.4 (CH, C-12),170.0 (C, C-13), 44.9 (C, C-14), 26.1 (CH2, C-15), 25.9 (CH2, C-16), 31.4 (C,C-17), 47.9 (CH, C-18), 38.3 (CH2, C-19), 42.9 (C, C-20), 30.4 (CH2, C-21),37.3 (CH2, C-22), 16.0 (CH3, C-23), 16.2 (CH3, C-24), 28.2 (CH3, C-25), 18.3(CH3, C-26), 23.0 (CH3, C-27), 28.5 (CH3, C-28), 28.6 (CH3, C-29), 174.5 (C,C-30), 38.8 (CH, C-1'), 32.2 (CH2, C-2'), 50.2 (C, C-3'),31.5 (CH2, C-4'),176.7(C, C-5'), 177.2 (C, C-6'), 62.3(CH, C-7'), 31.4(CH2, C-8'), 24.1(CH2, C-9'), 24.1(CH2, C-10'), 31.5(CH2, C-11'), 61.9 (CH, C-12').

[0042] [M+H] + , HRESIMS: calcd for C 42 H 66 N3O7Pt [M+H] + 919.4549, found 919.4540.

[0043] Example 2:

[0044] The inhibitory activity of glycyrrhetinic acid-platinum complex 1 on hepatoma cell lines was evaluated.

[0045] 1. Materials and Methods

[0046] 1.1 Materials

[0047] Hepatoma cell lines (HepG2, Huh7 and SK-Hep-1) were purchased from Shanghai Jinin Biotechnology Co., Ltd.; culture medium (Dulbecco's Modified Eagle Medium, DMEM) was purchased from Thermo Fisher Scientific (Suzhou, China); serum (fetal bovine serum, FBS) was purchased from Life Technologies (NY, USA); RPMI-1640 was purchased from Thermo Fisher Biochemical Products (Beijing, China).

[0048] 1.2 Instruments

[0049] Flex Station 3 benchtop multifunctional enzyme label instrument (Bio-RAD 680, USA); analytical balance (AG135, Metler Toledo, China); thermostat (DHP-9082, Shanghai).

[0050] 1.3 Experimental process

[0051] 1). Take the logarithmic phase growth of hepatoma cells, discard the old culture medium, wash twice with PBS, and discard the PBS;

[0052] 2). Digest the cells with 0.25% trypsin, and when the cell outline is deepened and has a tendency to round under the microscope, quickly aspirate the trypsin;

[0053] 3) Stop digestion with DMEM complete medium containing 10% FBS and resuspend the cells, take 10 μL of cell suspension, count with a cell counter, and adjust the cell concentration to 1 × 10 4 / mL with medium, inoculate in a 96-well plate, add 100 μL of cell suspension per well, incubate in a 37 ℃, 5% CO2 incubator for 24 h, and make the cells adhere;

[0054] 4). Aspirate the culture medium, add the diluted sample to the plate, add 100 μL per well, set 3 replicate wells for each concentration, and continue incubation in the incubator for 48 h;

[0055] 5). Aspirate the culture medium, add the prepared MTT solution (1 mg / mL), 100 μL per well, and incubate in the incubator for 4 h;

[0056] 6). The MTT solution was removed, DMSO was added, 100 μL per well, incubated in the incubator for 10 min;

[0057] 7). The absorbance value was measured at 490 nm wavelength using a microplate reader, the cell inhibition rate was calculated by the formula: inhibition rate = (negative - experimental group) / (negative - blank group) x 100%, and the IC 50 was calculated by statistical software GraphPad prism 5, and the experiment was repeated 3 times.

[0058] 2. Results

[0059]

[0060] Glycyrrhetinic acid-platinum complex 1 has strong activity on the proliferation of three liver cancer cells (HepG2, SK-Hep-1 and Huh7), and the IC 50 values are 25.4 (HepG2), 26.9 (Huh7) and 27.7 μM (SK-Hep-1), which are comparable to oxaliplatin (29.5, 30.1 and 32.3 μM).

[0061] Preparation examples 1-7:

[0062] In the following preparation examples, conventional reagents are selected, and preparation is carried out according to existing conventional methods, and the application examples only embody that the compound 1 described in the application can be prepared into different preparations, and specific reagents and operations are not specifically limited:

[0063] 1. Glycyrrhetinic acid-platinum complex 1 is dissolved in DMSO, and then water for injection is added according to the conventional method, and the injection solution is prepared by precision filtration, filling and sterilization, and the concentration of the injection solution is 0.5-5 mg / mL.

[0064] 2. Glycyrrhetinic acid-platinum complex 1 is dissolved in DMSO, and then dissolved in sterile water for injection, stirred to dissolve, filtered with a sterile suction filter funnel, and then sterilely filtered, and then packaged in ampoules, and then sterilely sealed after low-temperature freeze-drying, to obtain a powder injection.

[0065] 3. Glycyrrhetinic acid-platinum complex 1 is added to the excipient at a mass ratio of 9:1 to prepare a powder.

[0066] 4. Glycyrrhetinic acid-platinum complex 1 is added to the excipient at a mass ratio of 5:1 to prepare granules for tabletting.

[0067] 5. Glycyrrhetinic acid-platinum complex 1 is prepared into an oral solution according to the conventional oral solution preparation method.

[0068] 6. The glycyrrhetinic acid-platinum complex 1 is added into the excipient at a mass ratio of 5:1, and a capsule is prepared.

[0069] 7. The glycyrrhetinic acid-platinum complex 1 is added into the excipient at a mass ratio of 5:1, and a granule is prepared.

[0070] It can be seen from the above examples that the present application provides a glycyrrhetinic acid-platinum complex, a preparation method and application thereof, and a pharmaceutical composition containing the glycyrrhetinic acid-platinum complex 1 and application thereof. The new glycyrrhetinic acid-platinum complex 1 provided by the present application has significant inhibitory activity on liver cancer cell proliferation, can be used to prepare an anti-liver cancer drug.

[0071] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. The glycyrrhetinic acid-platinum complex 1 shown in the following structural formula, 。 2. The method for preparing the glycyrrhetinic acid-platinum complex 1 shown in claim 1, characterized in that: Using glycyrrhetinic acid as a raw material, it was coupled with dimethyl 3-amino-1,1-cyclobutanedicarboxylate to obtain dimethyl 3-glycyrrhetinic acid amamide-1,1-cyclobutanedicarboxylate. The ester group was hydrolyzed to convert it into a potassium salt, which underwent a coordination substitution reaction with cis-[PtA2(H2O)2](NO3)2 to prepare complex 1. 。 3. The use of the glycyrrhetinic acid-platinum complex 1 with the structural formula shown in claim 1 in the preparation of an anti-hepatocellular carcinoma drug.

4. A pharmaceutical composition comprising the glycyrrhetinic acid-platinum complex 1 as shown in claim 1 and a pharmaceutically acceptable carrier.

5. The use of the pharmaceutical composition of claim 4 in the preparation of an anti-liver cancer drug.

6. The method for preparing the pharmaceutical composition according to claim 4, characterized in that... The method includes the following steps: first, obtaining glycyrrhetinic acid-platinum complex 1 by the method of claim 2, and then adding a pharmaceutically acceptable carrier.

Images