Mitochondria-targeted cholic acid derivative as well as preparation method and application thereof

Abstract

The invention discloses a mitochondria-targeted cholic acid derivative as well as a preparation method and application thereof. The cholic acid-triphenylphosphine bromide compound is simply and conveniently synthesized by adopting a two-step synthesis method of firstly forming ester and introducing a brominated carbon chain and then introducing triphenylphosphine cations. Compared with the existing similar compounds, the method has the characteristics of simplicity in operation and high yield. The compound can be used in the field of liver cancer resistance. In-vivo and in-vitro experiments prove that the compound not only has excellent anti-hepatoma activity, but also can increase infiltration of immune cells in tumors to activate an immune system, and a promising way is provided for research and development of anti-hepatoma drugs, especially immunotherapy drugs for hepatoma.

Description

Technical Field

[0001] The invention belongs to the field of small molecule compounds and relates to mitochondrial-targeted bile acid derivatives and preparation methods and applications thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is a primary liver cancer with a high mortality rate. It is the most common malignant tumor worldwide. Although surgery and liver transplantation are effective, only about 20% of patients can undergo surgery. Due to the difficulty of early diagnosis and rapid progression of the disease, most patients are already in the middle and late stages when diagnosed, and treatment options are limited. Conventional radiotherapy and chemotherapy cannot cure solid tumors, and have significant toxic side effects, greatly reducing the patient's immunity, and patients are often in unbearable pain, which seriously reduces the patient's quality of life. At present, systemic treatment based on small molecule multi-target receptor tyrosine kinase inhibitors (including sorafenib and lenvatinib) has been clinically verified to have side effects caused by off-target, drug resistance and limited efficacy; the effectiveness of immune checkpoint inhibitors in immunotherapy of liver cancer is only about 10-20%, mainly because most HCCs are "cold tumors" with few tumor-infiltrating T cells (TILs) in the tumor microenvironment, especially TILs are inhibited, and macrophages in tumors are often affected by the microenvironment and converted from M1 to M2, which is conducive to tumor immune escape and weak tumor immunogenicity. Immune checkpoint inhibitors only relieve the immunosuppressive effect of HCC cells on T cells, allowing T cells to re-recognize HCC cells, and do not affect the number and type of immune cells in the tumor microenvironment. In addition, this type of therapy can also have unexpected side effects, especially damage to liver function, which will further aggravate the condition of liver cancer. Therefore, it is urgent to develop drugs or treatments with new mechanisms of action to treat liver cancer.

[0003] Farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily. Its dysregulation is associated with a variety of diseases, including metabolic disorders, inflammatory diseases, immune disorders, etc. It is mainly distributed in the liver and intestine (Comput Struct Biotechnol J. 2021, 19: 2148-2159). Increasing evidence shows that FXR can affect the development and progression of tumors by regulating related carcinogenic and anti-cancer signaling pathways. For example, FXR inhibits HCC progression by directly inhibiting the Wnt / β-catenin signaling pathway (Oncotarget. 2015, 6(6): 4226-38; J Pharmacol ExpTher. 2011, 338(1): 12-21; Nutr Metab (Lond). 2022, 19(1): 57). In addition, there are literature reports that FXR plays an important role in regulating the components of the tumor microenvironment (TME) (Int J Mol Sci. 2023, 25(1):6). At the same time, FXR can also regulate mitochondrial function (Arch Pharm Res. 2016, 39(8):1062-74). As early as 1994, Reichen discovered that lipophilic bile acids (deoxycholate, deoxycholate and lithocholate) would impair the function of the mitochondrial electron transport chain enzyme complex in rat liver (Hepatology. 1994, 19(2): 471-9.). Cholic acid and deoxycholic acid can cause mitochondrial dysfunction, impaired biogenesis and reduced autophagic flux in skeletal muscle cells, as well as produce more mitochondrial reactive oxygen species (ROS) (Biol Res. 2023, 56(1): 30). Deoxycholic acid and deoxycholic acid induce apoptosis of human colon adenocarcinoma cells through oxidative stress (increased ROS generation) (Apoptosis. 2011, 16(10): 1054-67.). HS-1200, a deoxycholic acid derivative synthesized by Hui Liu and colleagues, has been shown to induce apoptosis of BEL7402 human liver cancer cells through the mitochondrial pathway (Cancer Lett. 2008, 270(2): 242-9). Deoxycholic acid bound to mitochondria can cause mitochondrial morphological damage, including swelling and shrinking of cristae, decreased mitochondrial membrane potential, and increased mitochondrial calcium levels, thereby generating excessive reactive oxygen species (ROS) (Redox Biol. 2022, 56: 102452.). In addition, it is worth noting that bile acid compounds can also target the liver by binding to liver bile acid binding proteins (L-BABPs).Taking advantage of this property, bile acid is used as a liver targeting carrier to improve the targeting of drugs to the liver (Sci Rep. 2024, 14 (1): 22514; Molecules. 2023, 28 (5): 2358; Polymers (Basel). 2021, 13 (17): 2939. Pharmazie. 2016 Mar; 71 (3): 139-45). In addition, bile acid compounds may have endoplasmic reticulum targeting ability due to their hydrophobic structure (Angew Chem Int Ed Engl. 2021, 60 (20): 11158-11162.).

[0004] The patent (CN111944005B) approved by the State Patent Office on April 8, 2023 by Li Li et al. introduces a triphenylphosphine group on the carboxyl group of bile acid, which has a complex structure, a long reaction route, and does not involve the influence of the immune system. The present invention directly connects the triphenylphosphine group to the carboxyl group of bile acid compounds (hyodeoxycholic acid, cholic acid, dehydrocholic acid, deoxycholic acid, chenodeoxycholic acid and ursodeoxycholic acid, etc.) with an ester group, and the reaction route is simple and the yield is high; the superior compound T3 successfully activates the immune system of C57 / 6 mice bearing mouse-derived liver cancer Hep1-6 cells, and the number of immune cells such as professional antigen presenting cells dendritic cells (DC) cells and T cells that can directly kill tumor cells in the tumor is significantly higher than that of the blank group. In summary, the present invention provides candidate compounds for the immunotherapy of liver cancer. Summary of the invention

[0005] The object of the present invention is to provide a bile acid-triphenylphosphonium bromide cation compound in view of the above-mentioned deficiencies in the prior art.

[0006] Another object of the present invention is to provide a method for preparing the bile acid-triphenylphosphonium bromide cation compound.

[0007] Another object of the present invention is to provide the application of bile acid-triphenylphosphonium bromide cation compounds.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] Bile acid-triphenylphosphonium bromide cation compound, the structure is shown below:

[0010]

[0011] Wherein, R1, R2, R3 and R4 are independently selected from H, -OH or =O.

[0012] As a preferred embodiment of the present invention, the bile acid-triphenylphosphonium bromide cation compound is selected from any one of the following compounds:

[0013]

[0014] The compounds described in the present invention may exist in one or more specific geometric, optical, enantiomeric, diastereomeric, epimeric, stereoisomeric, tautomeric, conformational or anomer forms, including but not limited to cis and trans forms; E- and Z- forms; c-, t- and r- forms; endo- and exo- forms; R-, S- and meso- forms; D- and L- forms; d- and l- forms; (+) and (-) forms; keto, enol and enolate forms; syn- and anti- forms; syn- and anti- forms; α- and β- forms; axial- and flat- forms; boat-, chair-, twist-, envelope- and semi-chair- forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomer forms"). It is worth noting that the term "isomer" specifically includes compounds having one or more isotopic substitutions. For example, H can be in any isotopic form, including 1 H. 2 H(D), 3 H(T). Similarly, C can be in any isotopic form, including 12 C. 13 C. 14 C; O includes 16 O. 18 O; etc.

[0015] If a compound is in crystalline form, it may exist in a number of different polymorphic forms.

[0016] Unless otherwise specified, references to compounds of the invention include all such isomeric forms, including (completely or partially) racemic and other mixtures thereof. Methods for preparing and separating such isomers are known in the art or are readily obtained by adapting the methods taught herein or known methods in a known manner.

[0017] Unless otherwise specified, a reference to a particular compound also includes its ionic, salt, solvate and protected forms as well as its different polymorphs.

[0018] The preparation method of the bile acid-triphenylphosphonium bromide cationic compound of the present invention has the following reaction scheme:

[0019]

[0020] As a preferred embodiment of the present invention, the S is selected from hyodeoxycholic acid, cholic acid, dehydrocholic acid and deoxycholic acid.

[0021] As a preferred embodiment of the present invention, the preparation method comprises the following steps:

[0022] (1) Compound S is dissolved in N,N-dimethylformamide, potassium carbonate is added, and 1,5-dibromopentane is added after 0-45 minutes. The mixed solution is stirred for reaction. After the reaction is complete, dilute hydrochloric acid is added to adjust the solution to neutrality, and the solution is extracted with ethyl acetate, concentrated, and purified by column chromatography to obtain an intermediate;

[0023] (2) The intermediate obtained in the previous step is dissolved in acetonitrile, and triphenylphosphine is added to form a mixed solution, which is heated and stirred for reaction. After the reaction is complete, the mixture is concentrated and purified by column chromatography to obtain the final product.

[0024] As a preferred embodiment of the present invention, in the step (1), the concentration of compound S in N,N-dimethylformamide is 0.01 mmol / mL to 1.0 mmol / mL; the molar ratio of compound S to potassium carbonate is 1:1 to 1:10; the molar ratio of compound S to 1,5-dibromopentane is 1:1 to 1:10; the temperature for stirring the mixed solution for reaction is -10 to 180°C; the stirring reaction time is 1 to 24 hours; and the eluent for purification by silica gel column chromatography is a mixed solution of ethyl acetate and petroleum ether for gradient elution.

[0025] As a further preferred embodiment of the present invention, in the step (1), the concentration of compound S in N,N-dimethylformamide is 0.1 mmol / mL; the molar ratio of compound S to potassium carbonate is 1:1 to 1:5; the molar ratio of compound S to 1,5-dibromopentane is 1:1 to 1:5; the temperature for stirring the mixed solution for reaction is 0 to 100°C; the stirring reaction time is 1 to 24h; the eluent for purification by silica gel column chromatography is a mixed solution of ethyl acetate and petroleum ether for gradient elution, and the volume ratio of ethyl acetate to petroleum ether is 1:1 to 1:10.

[0026] As a preferred embodiment of the present invention, in the step (2), the molar ratio of intermediate I to triphenylphosphine is 1:1 to 1:100; the solid-liquid ratio of intermediate I to acetonitrile is 0.01 mmol / mL to 1.0 mmol / mL; and the temperature of the mixed solution for stirring the reaction is -10 to 100°C.

[0027] As a further preference of the present invention, in the step (2), the molar ratio of intermediate I to triphenylphosphine is 1:1 to 1:100, preferably 1:10; the solid-liquid ratio of intermediate I to acetonitrile is 0.01 mmol / mL to 1.0 mmol / mL, preferably 0.1 mmol / mL; and the temperature of the mixed solution for stirring the reaction is -10 to 100°C, preferably 82°C.

[0028] The bile acid-triphenylphosphonium bromide cation compound of the present invention is used in the preparation of a drug for treating liver cancer, preferably in the preparation of an anti-liver cancer immunotherapy drug.

[0029] In vivo experiments showed that: on the one hand, the superior compounds of the present invention have excellent tumor inhibition effects, and the tumor inhibition effects of some superior compounds in high-dose groups are stronger than those of the positive drug oxaliplatin; on the other hand, the superior compounds successfully activated the immune system of C57 / 6 mice, and the number of immune cells such as DC cells and T cells in the tumor was significantly higher than that in the blank group.

[0030] A pharmaceutical composition for treating liver cancer comprises the bile acid-triphenylphosphonium bromide cation compound and a pharmaceutically acceptable carrier or diluent.

[0031] As a preferred embodiment of the present invention, the bile acid-triphenylphosphonium bromide cationic compound is selected from its anionic form-O - , its salt or solvent form or a conventionally protected form of the hydroxyl group.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] Firstly, the mitochondria-targeted bile acid derivatives of the present invention are simple to synthesize, the synthetic route has only two steps, the operation is simple, and the product yield is high;

[0034] Secondly, the bile acid derivatives of the present invention have good anti-tumor activity, can inhibit the growth of liver cancer cells in vitro and in vivo, promote their apoptosis, and damage liver cancer cell mitochondria, promoting the leakage of ROS and mitochondrial DNA that can cause immune response; thirdly, the bile acid derivatives of the present invention can activate the immune system and reshape the immune microenvironment of liver cancer;

[0035] Fourthly, the compounds of the present invention have good targeting properties to liver cancer cells, can effectively reduce the occurrence of toxic and side effects, have the potential to be developed into anti-liver cancer immunotherapy drugs, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Chemical structures of bile acid derivatives I1~I6 and T1~T6.

[0037] Figure 2 The inhibition rate of bile acid derivatives S1-S6 and T1-T6 on HepG2 cells.

[0038] Figure 3 Compound T3 stimulates DC maturation and T cell activation.

[0039] Compound T3 stimulates DC cell maturation and T cell activation: (A) Activated CD4 + T cell ratio; (B) activated CD8 + T cell ratio; (C) mature DC cell ratio in each drug-treated group. Among them: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3.

[0040] Figure 4 A schematic diagram of the mechanism of action of compound T3 in activating the immune response of liver cancer DETAILED DESCRIPTION

[0041] The present invention will be described in detail below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description progresses. However, it should be understood that the embodiments are exemplary only, are only used as examples, and do not constitute any limitation to the scope of the present invention. At the same time, the advantages of the present invention will become clearer and easier to understand by explaining them. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0042] Example 1: Preparation of (5-(((R)-4-((3R,5R,6S,8S,9S,10R,13R,14S,17R)-3,6-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)oxy)pentyl)triphenylphosphonium bromide (T1)

[0043]

[0044] Preparation of intermediate I1: Weigh hyodeoxycholic acid (S1, 1.00 g, 2.55 mmol) and dissolve it in N, N-dimethylformamide (20.0 mL), add potassium carbonate (0.53 g, 3.83 mmol), stir at room temperature for 30 minutes, then add 1,5-dibromopentane (2.93 g, 12.74 mmol), and stir the mixture at room temperature for 16 hours. After the reaction is complete, add an appropriate amount of 1N dilute hydrochloric acid to neutralize the solution. Extract with ethyl acetate (50 ml) three times, combine the organic phases, rinse with saturated brine (20 ml) three times, dry with anhydrous sodium sulfate, concentrate, and purify by silica gel column chromatography (silica gel: 200-300 mesh, the polarity of the eluent is gradually increased from ethyl acetate / petroleum ether = 1:10 (v:v) to 1:3 (v:v)) to obtain 1.28 g of light yellow oily intermediate I1, yield: 92.8%. 1H NMR (500MHz, CDCl3) δ4.15–4.03(m,3H),3.67–3.59(m,1H),3.43(t,J=6.7Hz,2H),2. 36(ddd,J=15.2,10.0,5.1Hz,1H),2.27–2.18(m,1H),1.98(d,J=11.0Hz,1H),1.94–1. 87(m,4H),1.81(dd,J=17.4,5.8Hz,3H),1.73–1.57(m,7H),1.56–1.50(m,2H),1.48–1 .40(m,4H),1.39–1.26(m,4H),1.22–1.03(m,7H),0.93(d,J=8.8Hz,6H),0.65(s,3H).

[0045] Preparation of product T1: Dissolve intermediate I1 (1.00 g, 1.85 mmol) in anhydrous acetonitrile (50.0 mL), add triphenylphosphine solid (4.85 g, 18.49 mmol), add cooling circulation device, and heat to 82 ° C in nitrogen atmosphere for reaction. Monitor after about 10 hours of reaction, remove the heating device after the reaction is complete, and cool naturally to room temperature. Directly concentrate and purify by silica gel column chromatography (silica gel: 200-300 mesh, elution phase is dichloromethane / methanol = 1:10 (v:v)) to obtain 1.42 g of white solid, yield: 95.9%. 1 H NMR(500MHz, CDCl3)δ7.87–7.78(m,9H),7.71(td,J=7.7,3.2Hz,6H),4.06–4.01(m,1H),3.98(t,J=6.4Hz,2 H),3.81(t,J=14.1Hz,2H),3.65–3.55(m,1H),2.40(s,1H),2.27(ddd,J=15.3,10.1,5.2Hz,1H),2.18–2.10( m,2H),1.93(t,J=10.3Hz,2H),1.85–1.79(m,1H),1.77–1.71(m,4H),1.69–1.53(m,8H),1.45–1.32(m,6H), 1.29(dd,J=11.6,7.8Hz,1H),1.19(d,J=14.8Hz,1H),1.16–0.98(m,7H),0.87(d,J=5.9Hz,6H),0.60(s,3H); 13C NMR (126MHz, CDCl3) δ174.32,135.06,135.04,133.73,133.65,130.57,130.47,11 8.62,117.94,71.43,67.90,63.63,56.15,55.84,48.39,42.81,39.94,39.79,35. 93,35.60,35.30,34.96,34.81,31.16,30.90,30.20,29.24,28.14,26.76,26.63, 24.19,23.51,22.92,22.52,22.30,22.26,20.74,18.27,12.03; m / z:724.07[M-Br] + ,725.49[M-Br+H] + .

[0046] Example 2: Preparation of (5-(((R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)oxy)pentyl)triphenylphosphonium bromide (T2) Preparation of intermediate I2: Replace the hyodeoxycholic acid in the preparation of intermediate I1 in Example 1 with cholic acid (S2) to obtain 1.25 g of colorless oily liquid with a yield of 91.9%. 1 H NMR(500MHz, CDCl3)δ4.09(t,J=6.5Hz,2H),3.99(s,1H),3.87(s,1H),3.55–3.38(m,3H),2.39(dd d,J=14.6,9.7,4.6Hz,1H),2.23(ddd,J=17.2,13.5,6.7Hz,6H),2.00–1.87(m,5H),1.84–1.73(m, 4H),1.67(dt,J=19.8,6.1Hz,4H),1.61(s,1H),1.58–1.48(m,5H),1.42(d,J=13.2Hz,3H),1.37–1 .26(m,2H),1.13(ddd,J=18.2,12.1,6.2Hz,1H),0.99(t,J=10.2Hz,4H),0.91(s,3H),0.70(s,3H).

[0047] Preparation of product T2: Replace I1 with I2 in the preparation of product T1 in Example 1 to obtain 1.32 g of a white solid with a yield of 89.8%. 1H NMR (500MHz, CDCl3) δ7.82(dd,J=12.9,7.7Hz,9H),7.73(td,J=7.6,3.4Hz,6H),3.98(t,J=5.9Hz,2H),3.87(s,1H),3.81(s,1H),3.72( dd,J=14.7,7.2Hz,2H),3.55(s,1H),3.34(s,1H),2.37–2.22(m,3H),2.14(dt,J=15.1,7.5Hz,1H),1.93(dd,J=18.8,11.6Hz,1H),1.82 (d,J=9.2Hz,3H),1.64(ddd,J=53.5,25.6,14.0Hz,14H),1.47(dd,J=22.9,11.6Hz,3H),1.38–1.28(m,3H),1.25(dd,J=15.4,8.7Hz,1H ),1.17(dd,J=21.1,10.7Hz,1H),1.00(dd,J=11.9,5.2Hz,1H),0.94(d,J=5.8Hz,3H),0.88(d,J=13.6Hz,1H),0.84(s,3H),0.62(s,3H); 13 C NMR (126MHz, CDCl3) δ174.48,135.20,135.18,133.66,133.58,130.66,130.56,118.5 3,117.84,73.25,71.97,68.43,63.60,53.46,46.56,46.42,41.73,41.49,39.63,39. 50,35.45,35.28,34.90,34.57,31.10,30.93,30.35,28.14,28.07,27.53,27.04,26. 91,26.36,23.35,22.93,22.53,22.43,22.30,22.27,17.36,12.48; m / z:739.64[M-Br] + ,740.69[M-Br+H] + .

[0048] Example 3: Preparation of (5-(((R)-4-((5S,8R,9S,10S,13R,14S,17R)-10,13-dimethyl-3,7,12-trioxohexadecahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)oxy)pentyl)triphenylphosphonium bromide (T3) Preparation of intermediate I3: Replace the hyodeoxycholic acid in the preparation of intermediate I1 in Example 1 with dehydrocholic acid (S3) to obtain 1.30 g of colorless oily liquid, yield: 94.8%.1 H NMR(500MHz, CDCl3)δ4.08(t,J=6.5Hz,2H),3.43(t,J=6.7Hz,2H),2.96–2.91(m,1H ),2.87(dd,J=24.4,12.0Hz,2H),2.43–2.37(m,1H),2.37–2.32(m,2H),2.30(d,J=4. 3Hz,1H),2.26(dd,J=15.5,6.7Hz,2H),2.21(d,J=4.5Hz,1H),2.16(d,J=5.2Hz,1H) ,2.14(d,J=3.9Hz,1H),2.09(d,J=14.9Hz,1H),2.05(d,J=5.2Hz,1H),2.02(d,J=7.2 Hz,1H),1.98(dd,J=14.4,3.2Hz,1H),1.93–1.90(m,1H),1.89(s,1H),1.87(d,J=3. 3Hz,1H),1.85–1.81(m,1H),1.69–1.65(m,2H),1.64–1.59(m,1H),1.53(d,J=7.0Hz, 1H),1.51–1.42(m,1H),1.41(s,3H),1.38(dd,J=9.0,5.3Hz,1H),1.34(dd,J=8.8,5 .6Hz,1H),1.32–1.28(m,1H),1.28–1.20(m,1H),1.08(s,3H),0.86(d,J=6.6Hz,3H).

[0049] Preparation of product T3: Replace I1 with I3 in the preparation of product T1 in Example 1, and obtain 1.36 g of a white solid with a yield of 92.2%. 1H NMR (500MHz, CDCl3) δ7.88–7.77(m,9H),7.70(td,J=7.7,3.3Hz,6H),3.97(t,J=6.4Hz,2H),3.81(dd,J=15.3 ,13.0Hz,2H),2.97–2.88(m,2H),2.85(t,J=12.7Hz,1H),2.35–2.24(m,4H),2.20(dd,J=15.6,6.8Hz,3H),2. 10(dd,J=18.4,10.6Hz,2H),2.02(d,J=3.1Hz,2H),2.00–1.95(m,2H),1.95–1.91(m,1H),1.85–1.70(m,4H), 1.68–1.54(m,5H),1.40(s,3H),1.34–1.25(m,3H),1.23(d,J=7.1Hz,1H),1.06(s,3H),0.80(d,J=6.4Hz,3H); 13 C NMR (126MHz, CDCl3) δ212.13,209.13,208.89,174.06,135.07,135.04,133.71,133 .63,130.56,130.46,118.59,117.91,63.66,56.89,51.83,48.95,46.78,45.64,45 .00,42.76,38.65,36.47,36.00,35.45,35.20,31.47,30.40,28.13,27.60,26.81, 26.68,25.10,22.95,22.55,22.31,22.28,21.95,18.66,11.92; m / z:733.96[M-Br] + ,735.40[M-Br+2H] + .

[0050] Example 4: Preparation of (5-(((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)oxy)pentyl)triphenylphosphonium bromide (T4) Preparation of intermediate I4: Replace the hyodeoxycholic acid in the preparation of intermediate I1 in Example 1 with deoxycholic acid (S4) to obtain 1.33 g of colorless oily liquid, yield: 96.3%. 1H NMR(500MHz, CDCl3)δ4.09(t,J=6.5Hz,2H),3.99(s,1H),3.69–3.55(m,1H), 3.43(t,J=6.7Hz,2H),2.38(ddd,J=14.9,9.8,4.9Hz,1H),2.29–2.20(m,1H), 1.94–1.87(m,3H),1.87–1.84(m,1H),1.83(d,J=3.6Hz,1H),1.81(s,1H),1.7 8(d,J=5.1Hz,1H),1.77–1.73(m,1H),1.71(s,1H),1.69(d,J=2.8Hz,1H),1.6 7(s,1H),1.66(s,1H),1.65–1.62(m,1H),1.59(s,1H),1.56(s,1H),1.54(s,2 H),1.53(d,J=3.0Hz,2H),1.51(t,J=5.2Hz,2H),1.45(d,J=9.4Hz,1H),1.42( s,2H),1.41–1.37(m,2H),1.37–1.32(m,1H),1.29(d,J=4.2Hz,1H),1.27(d,J =6.8Hz,1H),1.19–1.04(m,2H),1.03–0.96(m,4H),0.92(s,3H),0.69(s,3H).

[0051] Preparation of product T4: Replace I1 with I4 in the preparation of product T1 in Example 1 to obtain 1.12 g of a white solid with a yield of 75.5%. 1 H NMR (500MHz, CDCl3) δ7.88–7.78(m,9H),7.71(td,J=7.5,3.1Hz,6H),3.98(t,J=6.3Hz,2H),3.94(s,1H ),3.88–3.78(m,2H),3.64–3.54(m,1H),2.28(ddd,J=14.8,9.6,5.0Hz,1H),2.20–2.13(m,1H),2.09(s ,1H),1.82(dd,J=23.6,12.7Hz,5H),1.77–1.60(m,10H),1.59–1.46(m,5H),1.45–1.29(m,6H),1.23(d ,J=4.2Hz,2H),1.19–1.10(m,1H),1.10–1.01(m,1H),0.95(t,J=9.3Hz,4H),0.89(s,3H),0.65(s,3H); 13C NMR (126MHz, CDCl3) δ174.38,135.04,135.02,133.73,133.65,130.57,130.47,118 .65,117.97,73.07,71.67,63.61,48.19,47.05,46.48,42.10,36.36,36.01,35.31, 35.12,34.12,33.57,31.14,30.83,30.44,28.66,28.15,27.46,27.13,26.82,26.69 ,26.12,23.68,23.11,22.92,22.53,22.32,22.29,17.35,12.75; m / z:724.01[M-Br] + ,725.42[M-Br+H] + .

[0052] Example 5: Preparation of (5-(((4R)-4-((3R,7R,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)oxy)pentyl)triphenylphosphonium bromide (T5) Preparation of intermediate I5: Replace the hyodeoxycholic acid in the preparation of intermediate I1 in Example 1 with chenodeoxycholic acid (S5) to obtain 1.21 g of colorless oily liquid with a yield of 87.7%. 1 H NMR (500MHz, CDCl3) δ4.10(t,J=6.5Hz,2H),3.61(s,2H),3.44(t,J=6.7Hz,2H),2.37(ddd,J=15.2,10.0,5.1 Hz,1H),2.28–2.21(m,1H),2.02(d,J=12.6Hz,1H),1.98–1.88(m,3H),1.87–1.77(m,4H),1.73–1.65(m,4H), 1.65–1.61(m,1H),1.59(s,2H),1.57–1.53(m,2H),1.52–1.50(m,1H),1.49–1.40(m,6H),1.40–1.33(m,2H), 1.32–1.23(m,3H),1.20–1.13(m,1H),1.07(ddd,J=22.0,16.8,10.5Hz,2H),1.00–0.89(m,6H),0.70(s,3H).

[0053] Preparation of product T5: Replace I1 with I5 in the preparation of product T1 in Example 1, and obtain 1.29 g of a white solid with a yield of 86.9%. 1H NMR (500MHz, CDCl3) δ7.88–7.78(m,9H),7.71(td,J=7.6,3.2Hz,6H),3.98(t,J=6.3Hz,2H),3.86(d,J=20.1Hz,3H),3.50–3.40(m,1H) ,2.27(ddd,J=15.9,10.5,5.5Hz,1H),2.23–2.11(m,2H),1.99–1.91(m,2H),1.89–1.79(m,3H),1.77–1.60(m,11H),1.53(s,1H),1.51– 1.47(m,1H),1.45(d,J=8.1Hz,1H),1.38(dd,J=11.6,6.9Hz,3H),1.33(d,J=14.4Hz,1H),1.29(d,J=3.2Hz,1H),1.28–1.23(m,3H),1. 16(dd,J=13.2,3.7Hz,1H),1.12(d,J=9.3Hz,1H),1.10–1.03(m,1H),0.96(td,J=14.1,2.7Hz,1H),0.90(d,J=4.1Hz,6H),0.63(s,3H); 13 C NMR (126MHz, CDCl3) δ174.34,135.05,135.03,133.73,133.65,130.56,130.46,11 8.62,117.94,71.90,68.38,63.63,55.68,50.44,42.63,41.48,39.83,39.62,39. 39,35.31,35.03,34.64,32.81,31.08,30.93,30.64,28.18,28.15,26.77,26.64, 23.69,22.91,22.79,22.51,22.30,22.27,20.57,18.30,11.78; m / z:724.03[M-Br] + ,725.43[M-Br+H] + .

[0054] Example 6: Preparation of (5-(((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexahydro-1H-cyclopenta[a]phenanthrene-17-yl)pentanoyl)oxy)pentyl)triphenylphosphonium bromide (T6) Preparation of intermediate I6: Replace the hyodeoxycholic acid in the preparation of intermediate I1 in Example 1 with ursodeoxycholic acid (S5) to obtain 1.33 g of colorless oily liquid, yield: 96.4%. 1H NMR (500MHz, CDCl3) δ4.09(t,J=6.5Hz,2H),3.60(dd,J=10.2,5.2Hz,2H),3.44(t,J=6.7Hz,2H),2.37(dd d,J=15.2,10.0,5.1Hz,1H),2.29–2.18(m,1H),2.02(d,J=12.6Hz,1H),1.97–1.88(m,3H),1.87–1.77(m,4 H),1.69(dd,J=13.6,6.4Hz,4H),1.65–1.56(m,3H),1.56–1.49(m,4H),1.48–1.40(m,5H),1.40–1.30(m, 3H), 1.26 (dd, J=16.8, 11.8Hz, 2H), 1.20–1.13 (m, 1H), 1.12–1.00 (m, 2H), 1.00–0.90 (m, 6H), 0.69 (s, 3H).

[0055] Preparation of product T6: Replace I1 with I6 in the preparation of product T1 in Example 1, and obtain 1.21 g of a white solid with a yield of 81.8%. 1 H NMR (500MHz, CDCl3) δ7.88–7.78(m,9H),7.71(td,J=7.7,3.3Hz,6H),3.98(t,J=6.4Hz,2H),3.83(dd,J=15. 5,12.9Hz,2H),3.58(dd,J=10.0,5.5Hz,2H),2.28(ddd,J=15.3,10.1,5.1Hz,1H),2.19–2.10(m,1H),2.03–1 .92(m,3H),1.89–1.71(m,7H),1.69–1.52(m,9H),1.50–1.34(m,6H),1.33–1.26(m,3H),1.21(dd,J=14.4,7 .3Hz,1H),1.12(td,J=12.8,3.2Hz,1H),1.07–0.96(m,2H),0.93(s,3H),0.89(d,J=6.5Hz,3H),0.64(s,3H); 13C NMR (126MHz, CDCl3) δ174.32,135.07,135.04,133.72,133.64,130.57,130.47,11 8.59,117.91,71.27,71.25,63.63,55.72,54.81,43.71,42.45,40.11,39.15,37. 28,36.94,35.22,34.97,34.06,31.16,30.96,30.31,28.64,28.15,26.90,26.77, 26.64,23.41,22.91,22.51,22.30,22.26,21.16,18.40,12.15; m / z:724.02[M-Br] + ,725.43[M-Br+H] + .

[0056] Example 7: In order to evaluate the anti-liver cancer activity of the cholic acid derivatives before and after structural modification, the inhibition rate on human liver cancer cells HepG2 was determined using the following method.

[0057] The MTT method was used to determine the inhibition rate of the test samples and the positive control oxaliplatin in HepG2 tumor cells. Each compound was set at the same concentration (20 μM) and 3 replicates were used. The specific operation was as follows: human liver cancer cells HepG2 in the logarithmic growth period were digested with trypsin and centrifuged (centrifugation conditions were 1000 rpm, 5 min), the supernatant was discarded, the complete culture medium was blown evenly and counted, and the cell density was adjusted to 2×10 4 / mL, add 100μL to each well of the 96-well plate, shake evenly, after the cells adhere, dissolve the compound in DMSO, dilute with complete culture medium to make the compound concentration 40μM, add 100μL to the original 96-well plate, that is, the final concentration is 20μM, put it back into the cell culture incubator and continue to culture for 72h, then add 20μL MTT solution (5mg / mL, dissolved in PBS) to each well of the 96-well plate, incubate for 4h, discard the supernatant, add 200μL DMSO to dissolve the blue-purple formazan crystals, shake on a shaker at a constant speed for 10min, measure the absorbance at 490nm with an enzyme reader, calculate the cell viability (Cell viability,%), and use GraphPad Prism 7 software to analyze and process the result data.

[0058] Cell viability (%) = (OD d –OD0) / (OD c –OD0)×100%

[0059] OD d is the absorbance value of the drug-treated group, OD0 is the absorbance value of the zero-adjustment well, and OD cis the absorbance value of the blank group.

[0060] Cell inhibition rate (%) = 1-cell viability (%)

[0061] The results of the determination of each compound are as follows:

[0062]

[0063] Note: Oxa: oxaliplatin, inhibition rate is expressed as M±SD, n=3.

[0064] The average cell viability of compounds T1, T3 and T4 on HepG2 cells were 90.37±1.51%, 96.48±0.28% and 94.71±0.76% respectively. Preferably, T3 was selected as the dominant compound of the series of compounds for in vivo anti-liver cancer experiments.

[0065] Example 8: Evaluation of the anti-liver cancer activity of compound T3 in vivo.

[0066] Here’s how:

[0067] Male C57BL / 6 mice (18-22 g) were kept at 25°C and 55% relative humidity with free access to food and water. The experiment began after one week of adaptive feeding of mice. Mouse hepatocellular carcinoma cells Hepa1-6 inoculated in a 10 cm dish were digested and collected, resuspended in serum-free cell culture medium, and counted. The cell density was adjusted to 1×10 6 / 100μL / mouse, inoculated in the left armpit of the mouse. After the tumor animal model was established, it was randomly divided into 4 groups, with 5 mice in each group, divided into a blank control group (only given the same volume of normal saline), a positive drug oxaliplatin group (5mg / kg), and a compound T3 group (divided into low and high doses, 2.5mg / kg and 5mg / kg respectively). The drug was administered intraperitoneally once every 2 days, and the body weight of tumor-bearing mice was recorded every day and the tumor volume was measured by vernier caliper. Two weeks later, the mice were killed, and the tumors and major organs were removed for subsequent experiments. The tumor tissue was taken for photographing, and after the tumor tissue was digested, the activation of immune cells such as DC cells and T cells was analyzed by flow cytometry.

[0068] The tumor volume calculation formula is: V (cm 3 )=a 2 ×b / 2, (a: short diameter, b: long diameter, V: tumor volume)

[0069] Tumor inhibition rate % = (average tumor weight of the control group - average tumor weight of the drug group) / average tumor weight of the control group × 100%

[0070] The results are as follows:

[0071]

[0072] Note: Tumor weight is expressed as M±SD, n=5, NS: blank control group, Oxa: oxaliplatin-positive group.

[0073] Example 9: Evaluation of compound T3's ability to activate the mouse immune system.

[0074] Here’s how:

[0075] Freshly harvested mouse spleen and tumor tissue were added to the lysis buffer, ground into fragments and placed on a shaker at room temperature for 2 hours, then the complete culture medium was terminated, filtered and centrifuged (2000 rpm, 10 min), the supernatant was discarded, PBS was added for washing twice (800 rpm, 3 min), cell staining buffer was added for resuspending, antibodies were added for staining at room temperature in the dark for 15 minutes, and then analyzed by flow cytometry.

[0076] The results are as follows:

[0077]

[0078] Note: All data in the table are expressed as M±SD, n=3, NS: saline blank control group, Oxa: oxaliplatin positive drug group. Statistical methods: independent sample T test and variance analysis.

[0079] result( Figure 3 ) Analysis: Compared with the blank control group, the positive drug oxaliplatin and compound T3 can activate the immune system in vivo: CD4 + and CD8 + The proportion of activated T cells increased significantly, and the proportion of mature DC (CD80-CD86 cells) cells increased significantly. More importantly, the high-dose T3 group had a better immune activation ability than the group with the same dose of oxaliplatin (a clinically used anticancer drug that is recognized to activate the immune system through immunogenic cell death).

Claims

1. A bile acid-triphenylphosphonium bromide cation compound, characterized in that: The structure is as follows: Wherein, R1, R2, R3 and R4 are independently selected from H, -OH or =O.

2. The bile acid-triphenylphosphonium bromide cation compound according to claim 1, characterized in that: Select any one of the following compounds:

3. The method for preparing the bile acid-triphenylphosphonium bromide cation compound according to any one of claims 1 to 2, characterized in that: The reaction route is as follows:

4. The preparation method according to claim 3, characterized in that: The S is selected from hyodeoxycholic acid, cholic acid, dehydrocholic acid and deoxycholic acid.

5. The preparation method according to claim 3 or 4, characterized in that: The following steps are involved: (1) Compound S is dissolved in N,N-dimethylformamide, potassium carbonate is added, and 1,5-dibromopentane is added after 0-45 minutes. The mixed solution is stirred for reaction. After the reaction is complete, dilute hydrochloric acid is added to adjust the solution to neutrality, and the solution is extracted with ethyl acetate, concentrated, and purified by column chromatography to obtain intermediate I; (2) The intermediate I obtained in the previous step is dissolved in acetonitrile, and triphenylphosphine is added to form a mixed solution, which is stirred and heated to react. After the reaction is complete, the mixture is concentrated and purified by column chromatography to obtain the final product.

6. The preparation method according to claim 5, characterized in that: In the step (1), the concentration of compound S in N,N-dimethylformamide is 0.01 mmol / mL to 1.0 mmol / mL, preferably 0.1 mmol / mL; the molar ratio of compound S to potassium carbonate is 1:1 to 1:10, preferably 1:1.5; the molar ratio of compound S to 1,5-dibromopentane is 1:1 to 1:10, preferably 1:5; the temperature for stirring the mixed solution for reaction is -10 to 180°C; the stirring reaction time is 12 to 20 hours, preferably 14 to 16 hours; the eluent for silica gel column chromatography purification is a mixed solution of ethyl acetate and petroleum ether for gradient elution, and the volume ratio of ethyl acetate to petroleum ether is 1:1 to 1:

10.

7. The preparation method according to claim 5, characterized in that: In the step (2), the molar ratio of intermediate I to triphenylphosphine is 1:1 to 1:100, preferably 1:10; the solid-liquid ratio of intermediate I to acetonitrile is 0.01 mmol / mL to 1.0 mmol / mL, preferably 0.1 mmol / mL; and the temperature of the mixed solution for stirring the reaction is -10 to 100°C, preferably 82°C.

8. Use of the bile acid-triphenylphosphonium bromide cation compound according to any one of claims 1 to 2 in the preparation of a drug for treating liver cancer, preferably in the preparation of an anti-liver cancer immunotherapy drug.

9. A pharmaceutical composition for treating liver cancer, characterized in that: The invention comprises the bile acid-triphenylphosphonium bromide cation compound according to any one of claims 1 to 2 and a pharmaceutically acceptable carrier or diluent.

10. The pharmaceutical composition according to claim 9, characterized in that The bile acid-triphenylphosphonium bromide cationic compound is selected from its anionic form-O - , its salt or solvent form or a conventionally protected form of the hydroxyl group.

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