Anthraquinone parent nucleus structural compound, and preparation method and application thereof

Abstract

The application discloses an anthraquinone parent nucleus structural compound and a preparation method and application thereof. The compound is 1,8-dibenzyl-oxy-9,10-anthraquinone-N-(4-phenylethanol)-3-formamide. The preparation method is as follows: 1,8-dibenzyl-oxy-rhein, 4-aminophenylethanol, EDCI, HOBT and anhydrous dichloromethane are mixed to react, the organic layer is washed with pure water, the solvent is removed under reduced pressure after drying, and the product is prepared after purification. The application is the use of the anthraquinone parent nucleus structural compound in preparation of a medicine for treating liver cancer. The synthesis condition of the compound is simple, the yield of the compound is high, the compound can effectively inhibit the expression of MCL-1, can significantly kill liver cancer cells with high expression of MCL-1 in vivo and in vitro, and no obvious toxic effect of the compound on the heart is observed, so that a new safe medication approach for treating liver cancer is provided.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical technology, and in particular relates to an anthraquinone core structure compound, its preparation method and application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a rapidly progressing malignant tumor with a high incidence and mortality rate. The early detection rate of HCC is less than 20%, and for unresectable or advanced HCC, the 5-year survival rate is only 13%. Chemotherapy, targeted therapy, and immunotherapy are the main treatments for advanced HCC. However, chemotherapy alone offers very little clinical benefit to patients with advanced HCC. Even with the combination of chemotherapy and the small-molecule targeted drug sorafenib, the efficacy rate for advanced HCC patients is only 33%, and more than 90% of patients develop resistance within 6 months. Chemotherapy resistance in liver cancer severely impacts treatment efficacy and patient prognosis, necessitating the discovery of new second-line therapies.

[0003] Myeloid Cell Leukemia-1 (MCL-1) gene, located on chromosome lq21, is an anti-apoptotic member of the BCL-2 family and a key protein in the mitochondrial apoptosis pathway. Recent studies show that MCL-1 is highly expressed in liver cancer, and this high expression is associated with drug resistance and recurrence. Downregulating MCL-1 protein expression can promote tumor cell apoptosis and improve sensitivity to chemotherapeutic drugs, such as enhancing the chemosensitivity of gemcitabine in pancreatic cancer and strengthening the anti-invasive effect of dasatinib in pancreatic cancer.

[0004] The development of MCL-1 inhibitors has been a research hotspot both domestically and internationally. In the past decade, several compounds targeting MCL-1 ligands have entered clinical trials; however, the results have been unsatisfactory. For example, the MCL-1 inhibitors AZD5991 and AMG397 were forced to terminate their Phase 1 clinical trials due to cardiac and other toxic side effects. S63845 also showed poor efficacy in clinical trials. Although studies have confirmed that MCL-1 inhibitors have strong anti-tumor effects in malignant tumors with high MCL-1 expression, they often pose a potential cardiotoxicity risk because they cannot specifically recognize the cardiac MCL-1 binding site. Therefore, to address the cardiotoxicity risk of MCL-1 inhibitors, researchers are exploring two approaches: firstly, seeking breakthroughs at the tumor / cardiac MCL-1 specific binding sites to avoid drug cardiotoxicity; and secondly, exploring other signaling pathways that regulate MCL-1 to find alternative treatments. Summary of the Invention

[0005] The purpose of this application is to provide an anthraquinone core structure compound, its preparation method, and its application. This compound significantly inhibits the growth of MCL-1-expressing liver cancer cells both in vitro and in vivo, without cardiotoxicity, thus providing a novel candidate compound for the treatment of MCL-1-expressing liver cancer. To this end, the following technical solution is adopted in this application:

[0006] In a first aspect, this application provides an anthraquinone core structure compound (code name KA-5j), the specific structure of which is as follows:

[0007]

[0008] The full name of the compound is: 1,8-dibenzyloxy-9,10-anthraquinone-N-(4-phenylethanol)-3-carboxamide.

[0009] Secondly, this application also provides a method for preparing the above-mentioned compound, comprising the following steps:

[0010] The raw materials were mixed and reacted at 30-40℃ for 5-20 hours. The raw materials included 1,8-dibenzyloxyrheic acid, 4-aminophenylethanol, EDCI [1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride], HOBT [1-hydroxybenzotriazole] and anhydrous dichloromethane. The organic layer was washed with pure water, dried, and the solvent was removed under reduced pressure. The product was obtained after purification.

[0011] The proportions of the raw materials are as follows: 60-100 mg of 1,8-dibenzyloxyrheic acid, 70-110 mg of 4-aminophenylethanol, 40-80 mg of EDCI, 20-60 mg of HOBT, and 1-20 mL of anhydrous dichloromethane.

[0012] In at least one embodiment, the proportions of the raw materials are as follows: 80 mg of 1,8-dibenzyloxyrheic acid, 90 mg of 4-aminophenylethanol, 56 mg of EDCI, 39 mg of HOBT, and 10 mL of anhydrous dichloromethane.

[0013] In at least one embodiment, the mixing reaction is carried out at a temperature of 33°C for 10 hours.

[0014] In at least one embodiment, the drying is performed using anhydrous sodium sulfate.

[0015] In at least one embodiment, the purification method is silica gel column chromatography, with gradient elution using dichloromethane and methanol.

[0016] Thirdly, this application also provides the use of the above-mentioned anthraquinone core structure compound in the preparation of a drug for treating liver cancer.

[0017] The drug comprises the anthraquinone nucleus compound and pharmaceutically acceptable excipients.

[0018] The dosage form of the drug may include granules, powders, tablets, capsules, pills, oral liquids, or injections.

[0019] Compared with the prior art, this application achieves at least the following beneficial effects:

[0020] This application provides an anthraquinone core structure compound, its preparation method, and its applications. The compound KA-5j has simple synthetic conditions, high yield, and is easily obtained. KA-5j effectively inhibits MCL-1 expression and significantly kills hepatocellular carcinoma cells expressing high MCL-1 both in vitro and in vivo, without observing significant cardiotoxicity, demonstrating a certain degree of in vivo safety. Compound KA-5j provides a novel, non-cardiotoxic MCL-1 inhibitor core structure, laying the research foundation for the development of novel, highly effective, and low-toxicity MCL-1 inhibitors, and also providing a new compound for the treatment of hepatocellular carcinoma cells expressing high MCL-1. Attached Figure Description

[0021] Figure 1 MCL-1 expression in different hepatocellular carcinoma cell lines and MTT results of the compound. Where: A: MCL-1 expression in different hepatocellular carcinoma cell lines; B: MTT results of the compound; *P<0.05, **P<0.01, ***P<0.001, n=3.

[0022] Figure 2 Construction and validation of a Huh7 hepatocellular carcinoma model with high MCL-1 expression. A: Cell fluorescence image after lentiviral transfection (200x); B: Western blot results after transfection; *P<0.05, **P<0.01, ***P<0.001, n=3.

[0023] Figure 3 In vivo results of compound KA-5j against hepatocellular carcinoma with high MCL-1 expression. AC: statistical analysis of tumor growth inhibition rate in mice; *P<0.05, **P<0.01, n=4.

[0024] Figure 4 Effects of compound KA-5j on MCL-1 expression in HepG2 and Hep3b cells. *P<0.05, **P<0.01, ***P<0.001, n=3.

[0025] Figure 5 Pathological changes in the hearts of mice in each group. Detailed Implementation

[0026] The present application will now be described in detail with reference to exemplary embodiments or experimental examples in the accompanying drawings. However, it should be understood that the present application may be implemented in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided herein to make the disclosure of the present application more complete and to fully convey the concept of the present application to those skilled in the art.

[0027] The inventors of this application have developed a new compound KA-5j [1,8-dibenzyloxy-9,10-anthraquinone-N-(4-phenylethanol)-3-carboxamide], the specific chemical structural formula of which is shown below:

[0028]

[0029] Furthermore, the inventors have developed a method for preparing the synthetic compound KA-5j, which has simple synthesis conditions and a high yield of the compound. The method includes the following specific steps:

[0030] The raw materials, including 1,8-dibenzyloxyrhein, 4-aminophenylethanol, EDCI [1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride], HOBT [1-hydroxybenzotriazole], and anhydrous dichloromethane, were mixed and reacted at 30–40 °C for 5–20 h. The organic layer was washed with pure water, dried, and the solvent was removed under reduced pressure. The product was then purified. The proportions of the raw materials were as follows: 60–100 mg of 1,8-dibenzyloxyrhein, 70–110 mg of 4-aminophenylethanol, 40–80 mg of EDCI, 20–60 mg of HOBT, and 1–20 mL of anhydrous dichloromethane.

[0031] Furthermore, the steps of a preferred embodiment of this preparation method are as follows:

[0032] The raw materials were mixed and reacted at 33℃ for 10 h. The raw materials and their amounts were: 80 mg of 1,8-dibenzyloxyrheic acid, 90 mg of 4-aminophenylethanol, 56 mg of EDCI, 39 mg of HOBT, and 10 mL of anhydrous dichloromethane. The organic layer was washed 2-3 times with pure water, dried with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was purified by silica gel column chromatography (eluting with a gradient of dichloromethane and methanol). The final product was a yellow solid (63 mg, 0.11 mmol, yield 63%, MP: 219.5-220.6℃).

[0033] On the one hand, the inventors of this application conducted a series of preliminary studies on compound KA-5j and found that compound KA-5j has strong inhibitory activity and therapeutic selectivity against liver cancer cells with high MCL-1 expression in vivo and in vitro, and its inhibitory efficiency is better than that of commonly used liver cancer chemotherapy drugs.

[0034] On the other hand, addressing the cardiotoxicity risk of existing MCL-1 inhibitors, the inventors of this application constructed a Huh7 hepatocellular carcinoma model overexpressing MCL-1 to conduct in vivo toxicity studies. Histopathological observation revealed that compound KA-5j had no significant toxic effect on mouse cardiomyocytes. Compound KA-5j could provide a new direction and candidate compound for research on highly effective and low-toxicity anti-MCL-1-expressing hepatocellular carcinoma.

[0035] Therefore, the aforementioned anthraquinone core structure compounds can be used to prepare drugs for treating liver cancer, for example, in the form of solid, semi-solid, or liquid pharmaceutical formulations. These formulations contain the compound KA-5j of this application as the active ingredient, and may also be mixed with inorganic or organic excipients and excipients. Possibly, the active ingredient can be compounded with commonly used non-toxic pharmaceutical excipients to form granules, powders, tablets, capsules, pills, oral liquids, or injections, and any other usable dosage form. The excipients or excipients of this application may include talc, water, glucose, honey, lactose, gum arabic, gelatin, mannitol, starch, magnesium trisilicate, keratin, colloidal silica, and any carrier capable of preparing solid, semi-solid, or liquid formulations. Additionally, auxiliary, stabilizing, thickening, coloring, and flavoring agents may also be used.

[0036] To illustrate in detail the inhibitory effect of the compound KA-5j on hepatocellular carcinoma cells that highly express MCL-1 and its lack of cardiotoxicity, this application also provides the following experimental examples.

[0037] Example 1: MTT assay was used to detect the inhibitory effect of compound KA-5j on the proliferation of liver cancer cells with different MCL-1 expression levels.

[0038] Cell culture:

[0039] HepG2 and Hep3B cells were cultured in MEM medium, and Huh7 cells were cultured in DMEM high-glucose medium (containing 10% fetal bovine serum and 1% penicillin-dextrose antibodies). Cells were passaged in T25 cell culture flasks and then incubated at 37°C in a 5% CO2 incubator. Cells used in the experiment were passaged three times after resuscitation.

[0040] 1. Western Blot Experiment:

[0041] HepG2, Hep3B, and Huh7 cells were seeded in their respective culture dishes. Once the cells had adhered and grown to confluence, RIPA lysis buffer and a protease phosphatase inhibitor (100:1) were added and the cells were placed on ice for 10 min to lyse. The cells were scraped off and centrifuged at 12000×g and 4℃ for 30 min. The supernatant was collected after centrifugation.

[0042] Protein quantification was performed according to the BCA quantitative kit instructions. The total protein quantification amount was 100 μg. An appropriate amount of loading buffer was added, and the tissue protein lysis buffer was brought to the top. The mixture was then vortexed to mix thoroughly. The protein sample denaturation conditions were: boiling water bath at 100°C for 5 min. An appropriate separating gel (10%) was selected based on the required protein molecular weight. The rapid gel preparation agent and coagulant were mixed according to the instructions, and an appropriate amount of gel was poured along the glass plate. The gel was ready for use after solidification.

[0043] Sample loading: Thaw the cooked protein samples on ice, centrifuge quickly to mix, and then add them sequentially and in equal volumes to the wells. Perform electrophoresis using a constant voltage method: first, electrophoresis at 79V for approximately 30 minutes, then at 119V for approximately 1.5 hours. Electrophoresis conditions: constant voltage 96V in an ice-water bath for 100 minutes. After electrophoresis, block the NC membrane in 5% skim milk powder for 1 hour.

[0044] Prepare the primary antibody working solution according to the proportions provided in the antibody instructions. After cutting the NC membrane into bands, place each band into a corresponding primary antibody incubation tube and incubate overnight on ice with low-speed shaking. Wash three times with TBST for 5 minutes each time, then incubate with secondary antibody (1:30,000) at room temperature on a shaker for 1 hour, followed by three washes with TBST in the dark for 5 minutes each time.

[0045] The membrane was scanned using an Odessey far-infrared membrane scanner, and the grayscale values ​​of the strips were analyzed using ImageJ.

[0046] 2. Cell proliferation inhibition activity test:

[0047] Grouping: Doxorubicin (ADM), Etoposide (VP-16), Sorafenib, Oxaliplatin, and compound KA-5j.

[0048] HepG2 and Hep3B cells in the logarithmic growth phase were harvested at a concentration of 1×10⁻⁶. 4 Each well was seeded with a specific concentration of dexamethasone (positive control) and the test compound. After 24 hours of incubation, different concentrations of dexamethasone (positive control) and the test compound were added. After another 48 hours of incubation, 10 μL of CCK-8 was added to each well, and the plates were incubated for another 2 hours. The absorbance of each well was then measured at 450 nm using a microplate reader. The cell inhibition rate was calculated. The experiment was repeated three times.

[0049] Western blot (WB) results showed that HepG2 and Hep3B cells expressed relatively high levels of MCL-1, while Huh7 cells expressed relatively low levels. MTT assays revealed that the hepatocellular carcinoma cell line Hep3b, which highly expressed MCL-1, was significantly less sensitive to first-line anti-hepatocellular carcinoma chemotherapy drugs doxorubicin (ADM) and etoposide (VP-16) than the hepatocellular carcinoma cell line Huh7, which expressed low levels of MCL-1 (P<0.05). Interestingly, compound KA-5j showed a significantly stronger inhibitory effect on the proliferation of Hep3b cells, which highly expressed MCL-1, than on Huh7 cells, which expressed low levels of MCL-1. This indicates that compound KA-5j exhibits a certain selectivity in its action against hepatocellular carcinoma cells that highly express MCL-1, effectively inhibiting their growth. (See attached results). Figure 1 .

[0050] Experimental Example 2: Construction and Validation of a Huh7 Hepatocellular Carcinoma Cell Model with High MCL-1 Expression

[0051] 1. Lentiviral construction and packaging: The lentivirus was constructed and packaged by Sangon Biotech (Shanghai) Co., Ltd. The negative control lentivirus titer was 6.44 × 10⁻⁶. 9 TU / mL; human Mcl-1 overexpressing lentivirus titer was 6.46 × 10⁻⁶. 9 TU / mL.

[0052] 2. Human hepatocellular carcinoma Huh7 cells were transfected with lentivirus and seeded into 96-well plates at a density of 4000 cells / well. After 24 hours of cell adhesion, the supernatant was removed, and the cells were infected with virus at an MOI of 100 and an appropriate amount of transfection aid. 24 hours after infection, the virus-containing medium was removed and replaced with complete medium for further culture. 72 hours after infection, fluorescence expression and infection efficiency were confirmed using the RFP channel of a fluorescence microscope.

[0053] 3. Puromycin selection: Transfected Huh7 cells were selected using 4 μg / mL puromycin (at this concentration, the inhibition rate on parental cells was approximately 70%) and cultured stably for one week. After one week, the puromycin concentration was halved for maintenance culture. Mcl-1 expression was detected by Western blot after transfection using the same method as before.

[0054] To further clarify the effect of compound KA-5j on hepatocellular carcinoma cells overexpressing MCL-1, a hepatocellular carcinoma cell model overexpressing MCL-1 was constructed using Huh7 cells. After transfection, fluorescence microscopy and Western blot analysis confirmed the successful construction of the Huh7 hepatocellular carcinoma cell model overexpressing MCL-1. The results are shown in the table below. Figure 2 .

[0055] Experimental Example 3: In vivo activity results of compound KA-5j against hepatocellular carcinoma with high MCL-1 expression.

[0056] Animal experiments:

[0057] The cell types were Huh7 cells transfected with Mcl-1 lentivirus (Huh7). MCL-1 ) and negative control virus Huh7 cells (Huh7 NC );

[0058] When cells reached the logarithmic growth phase, they were digested, washed twice with pre-chilled PBS, centrifuged at 800 rpm for 5 min, resuspended, counted, and the cell concentration was adjusted to 8 × 10⁶ cells / year. 6 Cells / mL were mixed with 100 μL of cell suspension and injected into the skin of the right axilla of nude mice. Each nude mouse injection site contained 8 × 10⁶ live cells. 5 indivual.

[0059] When the tumor volume reaches 60-100 mm 3 After tumor formation, nude mice were randomly divided into four groups: solvent control group, oxaliplatin 10 mg / kg treatment group, 5 mg / kg oxaliplatin 2.5 mg / kg treatment group, and 5 mg / kg oxaliplatin 5 mg / kg treatment group, with four mice in each group. The mice were administered the drug via intraperitoneal injection every other day, with an injection volume of 100 μL. After 10 days of continuous observation, the mice were sacrificed by cervical dislocation, and the tumor tissue was dissected and measured.

[0060] This experiment constructed Huh7 MCL-1 A nude mouse xenograft model of liver cancer was established, using Oxa, a first-line chemotherapy drug for liver cancer, as a positive control. The inhibitory effect of the compound KA-5j on tumor growth in mice was observed. It was found that compound KA-5j, at a concentration lower than that of Oxa (5 mg / kg), effectively inhibited Huh7 growth. MCL-1 The growth of xenografts in nude mice indicated that compound KA-5j exhibited superior in vivo activity against hepatocellular carcinoma cells with high MCL-1 expression compared to Oxa. (See results below.) Figure 3 .

[0061] Experiment Example 4: Effect of compound KA-5j on MCL-1 expression in liver cancer cells.

[0062] Western blot was used to detect the expression of MCL-1 and Tubulin proteins, using the same method as described above.

[0063] To understand the effect of compound KA-5j on MCL-1 protein expression, Western blot analysis was performed to detect MCL-1 expression. The results showed that 2 μmol / L of compound KA-5j significantly inhibited MCL-1 expression in HepG2 and Hep3b cells (P < 0.05). (See attached table). Figure 4 .

[0064] Experimental Example 5: Effects of compound KA-5j on mouse heart

[0065] Animal experiments:

[0066] The cell types were Huh7 cells (Huh7MCL-1) transfected with Mcl-1 lentivirus and Huh7 cells (Huh7NC) with negative control virus, respectively.

[0067] When cells reached the logarithmic growth phase, they were digested, washed twice with pre-chilled PBS, centrifuged at 800 rpm for 5 min, resuspended, counted, and the cell concentration was adjusted to 8 × 10⁶ cells / year. 6 Cells / mL were mixed with 100 μL of cell suspension and injected into the skin of the right axilla of nude mice. Each nude mouse injection site contained 8 × 10⁶ live cells. 5 indivual.

[0068] When the tumor volume reaches 60-100 mm 3 Tumors formed in mice. After tumor formation, nude mice were randomly divided into four groups: solvent control group, oxaliplatin 10 mg / kg treatment group, 5 mg / kg oxaliplatin 2.5 mg / kg treatment group, and 5 mg / kg oxaliplatin 5 mg / kg treatment group, with four mice in each group. The mice were administered the drug via intraperitoneal injection every other day, with an injection volume of 100 μL. After 10 days of continuous observation, the mice were sacrificed by cervical dislocation, and their hearts were dissected for HE staining.

[0069] HE staining:

[0070] Heart tissue was collected and fixed in 5 times its volume of 4% paraformaldehyde, then routinely embedded in paraffin and sectioned at 4 μm. Sections were routinely dewaxed with xylene, followed by washing with various amounts of ethanol and water: xylene (1) 15 min, xylene (2) 15 min, anhydrous ethanol 10 min, 95% ethanol 10 min, 75% ethanol 10 min, 50% ethanol 10 min, pure water 5 min, PBS 5 min. Hematoxylin staining was performed for 5 min, followed by rinsing with tap water. Eosin staining was performed for 5 min, followed by rinsing with tap water. After drying, the sections were mounted with neutral resin and observed and photographed under a microscope.

[0071] HE staining of mouse heart tissue revealed no significant pathological changes in cardiomyocytes compared to the control group. Results are shown below. Figure 5 .

[0072] The above experimental examples show that KA-5j can effectively inhibit the expression of MCL-1, and can significantly kill liver cancer cells that highly express MCL-1 both in vivo and in vitro. No obvious toxic effects on the heart were observed, indicating that it has a certain degree of in vivo safety.

Claims

1. A compound with an anthraquinone core structure, characterized in that, It has the following chemical structural formula: 。 2. The method for preparing the anthraquinone core structure compound according to claim 1, characterized in that, The process includes the following steps: raw materials are mixed and reacted at 30-40℃ for 5-20 hours. The raw materials include 1,8-dibenzyloxyrheic acid, 4-aminophenylethanol, EDCI, HOBT and anhydrous dichloromethane. The organic layer is washed with pure water, dried and the solvent is removed under reduced pressure. The product is obtained after purification.

3. The preparation method according to claim 2, characterized in that, The proportions of the raw materials are as follows: 60-100 mg of 1,8-dibenzyloxyrheic acid, 70-110 mg of 4-aminophenylethanol, 40-80 mg of EDCI, 20-60 mg of HOBT, and 1-20 mL of anhydrous dichloromethane.

4. The preparation method according to claim 2, characterized in that, The proportions of the raw materials are as follows: 80 mg of 1,8-dibenzyloxyrheic acid, 90 mg of 4-aminophenylethanol, 56 mg of EDCI, 39 mg of HOBT, and 10 mL of anhydrous dichloromethane.

5. The preparation method according to claim 2, characterized in that, The mixing reaction was carried out at a temperature of 33°C for 10 hours.

6. The preparation method according to claim 2, characterized in that, The drying process is performed using anhydrous sodium sulfate.

7. The preparation method according to claim 2, characterized in that, The purification method is silica gel column chromatography, with gradient elution using dichloromethane and methanol.

8. The use of the anthraquinone core structure compound of claim 1 or the anthraquinone core structure compound prepared by any one of claims 2 to 7 in the preparation of a medicament for treating liver cancer.

9. The application according to claim 8, characterized in that, The drug comprises the anthraquinone nucleus compound and pharmaceutically acceptable excipients.

10. The application according to claim 8, characterized in that, The dosage forms of the drug include granules, powders, tablets, capsules, pills, oral liquids, or injections.

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