Juglone derivative and application thereof

Abstract

The invention provides juglone derivatives and application thereof, and belongs to the technical field of medicines. A series of juglone derivatives are designed and synthesized, and the juglone derivatives can target the HuR protein, inhibit the binding activity of the HuR protein and mRNA and have an obvious inhibiting effect on brain glioma cells. Data of the embodiment shows that the juglone derivative disclosed by the invention has relatively good anti-glioma activity and relatively good safety, the activity of the juglone derivative disclosed by the invention is superior to that of a positive control drug, and the juglone derivative can be used for developing a novel anti-tumor drug and has a relatively good application prospect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a juglone derivative and its applications. Background Technology

[0002] Juglone, a 1,4-naphthoquinone natural product, is widely found in the roots, leaves, shells, and bark of the jujube tree. It possesses various biological activities, including antiviral, antibacterial, anti-inflammatory, antitumor, and cardiovascular protective effects. Studies have reported inhibitory activity against breast cancer cell lines, bladder cancer cell lines, liver cancer cell lines, gastric cancer cell lines, glioblastoma cell lines, and esophageal cancer cell lines. However, the antitumor activity of juglone is weak, its mechanism of action is not well understood, its target is unclear, and its poor solubility, low bioavailability, and structural instability limit its clinical application. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a juglone derivative and its application. The juglone derivative of the present invention can inhibit the binding of HuR protein to mRNA sequences and has antitumor activity.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a juglone derivative, comprising compounds or pharmaceutically acceptable salts with structures shown in Formula I or Formula II:

[0006]

[0007] R1 is an alkyl, aryl, or substituted aryl group;

[0008] The R2 is -H, aromatic heterocyclic thio group, phenyl ring thio group or substituted phenyl ring thio group;

[0009] R3 is an alkyl, aryl, or substituted aryl group;

[0010] X is N or O.

[0011] Preferably, R1 is p-nitrophenyl, p-methoxyphenyl, p-trifluoromethylphenyl, p-fluorophenyl, p-chlorophenyl, cyclohexyl, cyclopentyl, cyclopropyl, o-methylphenyl, or o-isopropylphenyl.

[0012] Preferably, R2 is p-nitrophenylthio, p-hydroxyphenylthio, p-methoxyphenylthio, p-aminophenylthio, p-trifluoromethylphenylthio, p-acetaminophenylthio, p-fluorophenylthio, p-chlorophenylthio, p-bromophenylthio, 4-pyridinethio, 2-pyrimidinethio, or 2-thiophenethio.

[0013] Preferably, R3 is p-nitrophenyl, p-methoxyphenyl, p-trifluoromethylphenyl, p-fluorophenyl, p-chlorophenyl, cyclohexyl, cyclopentyl, cyclopropyl, o-methylphenyl, or o-isopropylphenyl.

[0014] Preferably, the juglone derivative has the structure shown in any one of ZLCY-1~3, ZLCY-5~14, ZLCY-16~17, ZLCY-21~25, and ZLCY-27~45:

[0015]

[0016] The present invention also provides the application of the juglone derivatives described in the above technical solution in the preparation of antitumor drugs.

[0017] Preferably, the antitumor drug includes an anti-glioma drug.

[0018] Preferably, the antitumor drug comprises an effective dose of the juglone derivative and a pharmaceutically acceptable carrier.

[0019] Preferably, the administration route of the antitumor drug includes oral, intravenous, local, enteral, inhalation, spray, sublingual, transdermal, or rectal administration.

[0020] Preferably, the dosage form of the antitumor drug includes aerosol, cream, gel, pill, granule, capsule, tablet, syrup, transdermal patch, injection or nanoformulation.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] HuR protein is an mRNA-binding protein that stabilizes the structure of mRNA by binding to the ARE sequence, thereby promoting the expression of related genes. HuR protein is widely upregulated in various malignant tumor tissues, and malignant tumors associated with HuR protein expression have been shown to include breast cancer, liver cancer, lung cancer, colorectal cancer, cervical cancer, ovarian cancer, and glioma. This invention designs and synthesizes a series of juglone derivatives that can target HuR protein, inhibit its binding activity to mRNA, and have a significant inhibitory effect on glioma cells.

[0023] Data from the examples show that the juglone derivative of the present invention has good anti-glioma activity and good safety. The juglone derivative of the present invention has better activity than the positive control drug and can be used for the development of novel anti-tumor drugs, showing good application prospects. Attached Figure Description

[0024] Figure 1(a) shows a schematic diagram of the fitting of the 1,4-naphthoquinone structure to the pharmacophore model, and (b) shows a schematic diagram of the fitting of the 1,2-naphthoquinone structure to the pharmacophore model.

[0025] Figure 2 A schematic diagram of the synthetic route for intermediates 1a-c and 1e-m;

[0026] Figure 3 A schematic diagram of the synthetic routes for juglone derivatives ZLCY-1~3 and ZLCY-5~11;

[0027] Figure 4 A schematic diagram of the synthetic route for intermediates 2c~2e, 2g~2h, 2n~2o;

[0028] Figure 5 A schematic diagram of the synthetic routes for juglone derivatives ZLCY-12~14, ZLCY-16~17, and ZLCY-21~22;

[0029] Figure 6 A schematic diagram of the synthetic route for intermediates 3a-3c, 3e, 3f, 3m, and 3n;

[0030] Figure 7 A schematic diagram of the synthetic route for intermediates 3h, 3j, and 3l;

[0031] Figure 8 Schematic diagram of the synthesis routes for ZLCY-23~25 and ZLCY-27~33;

[0032] Figure 9 A schematic diagram of the synthetic route for the juglone derivatives ZLCY-34-45;

[0033] Figure 10 For HuR-ARE Vegf-a Screening of juglone analogs using a fluorescence polarization model (n=3);

[0034] Figure 11 Positive analogue for HuR-ARE Vegf-a Plot of inhibitory activity of interactions (n=3);

[0035] Figure 12 Figure 1 shows the in vitro inhibitory activity of ZLCY-9 and ZLCY-32 against the proliferation of glioma GL261 cells (n=6).

[0036] Figure 13The results of different treatment groups inhibiting the growth of GL261 subcutaneous gliomas (n=6) are shown in the following figures: (a) morphology of tumor tissue in each group; (b) mass of tumor tissue in each group (***P<0.001vs Ctrl); (c) changes in tumor volume in each group (***P<0.001vs Ctrl); and (d) changes in body weight of mice in each group. Detailed Implementation

[0037] This invention provides a juglone derivative, comprising compounds or pharmaceutically acceptable salts with structures shown in Formula I or Formula II:

[0038]

[0039] R1 is an alkyl, aryl, or substituted aryl group;

[0040] The R2 is -H, aromatic heterocyclic thio group, phenyl ring thio group or substituted phenyl ring thio group;

[0041] R3 is an alkyl, aryl, or substituted aryl group;

[0042] X is N or O.

[0043] In this invention, R1 is preferably p-nitrophenyl, p-methoxyphenyl, p-trifluoromethylphenyl, p-fluorophenyl, p-chlorophenyl, cyclohexyl, cyclopentyl, cyclopropyl, o-methylphenyl, or o-isopropylphenyl.

[0044] In this invention, R2 is preferably p-nitrophenylthio, p-hydroxyphenylthio, p-methoxyphenylthio, p-aminophenylthio, p-trifluoromethylphenylthio, p-acetaminophenylthio, p-fluorophenylthio, p-chlorophenylthio, p-bromophenylthio, 4-pyridinethio, 2-pyrimidinethio, or 2-thiophenethio.

[0045] In this invention, R3 is preferably p-nitrophenyl, p-methoxyphenyl, p-trifluoromethylphenyl, p-fluorophenyl, p-chlorophenyl, cyclohexyl, cyclopentyl, cyclopropyl, o-methylphenyl, or o-isopropylphenyl.

[0046] In this invention, the juglone derivative preferably has the structure shown in any one of ZLCY-1~3, ZLCY-5~14, ZLCY-16~17, ZLCY-21~25, and ZLCY-27~45:

[0047]

[0048] This invention employs a computer-aided drug design simulation process. First, a training set is constructed using positive compounds obtained from preliminary laboratory screening. Discovery Studio 2019 software is used to generate 3D-QSAR pharmacophore models containing hydrogen-bonded acceptors (HBA), hydrophobic (H), and cyclic aromatic (RA) chemical characteristics. The best pharmacophore model had a total cost of 69.393; the largest cost difference was Δcast, 83.203; the lowest root mean square deviation (RMSD) was 1.078; and the highest correlation coefficient (R²) was... 2 The maximum fit (Max.fit) was 10.0234, indicating that the pharmacophore model could perfectly and accurately predict the activity of compounds in the training set and had significant guiding significance for the structural modification of juglone. Subsequently, the lead compound juglone was matched with the obtained pharmacophore model. It was found that the carbonyl group at position 1 and the benzene ring could match a hydrogen bond acceptor and a cyclic aromatic chemical feature, respectively, but its fit value with the pharmacophore model was only 4.83448. Further optimization of the juglone structure was carried out to match more chemical features with the pharmacophore model. The 1,4-naphthoquinone structure was changed to the 1,2-naphthoquinone structure. The 1,2-naphthoquinone structure can match two hydrogen bond acceptors and a cyclic aromatic chemical feature of the pharmacophore model. After this change, the fit value of the compound with the pharmacophore model increased to 7.26865. Figure 1 As shown, (a) is a schematic diagram of the fitting of the 1,4-naphthoquinone structure to the pharmacophore model, and (b) is a schematic diagram of the fitting of the 1,2-naphthoquinone structure to the pharmacophore model.

[0049] Based on the above results, two strategies are provided for the structural modification of juglone: ​​First, using 1,2-naphthoquinone as the parent ring, hydrophobic groups, such as benzene rings and cycloalkanes, are introduced onto the benzene ring to match the hydrophobic chemical features that are not yet matched in the pharmacophore model; Second, the benzene ring in 1,2-naphthoquinone is replaced with aromatic furans and pyrroles, and hydrophobic benzene rings and cycloalkanes are introduced onto the furans and pyrroles to maximize the match with the pharmacophore model and increase the activity of the compound in inhibiting the binding of HuR protein to mRNA. Based on this, juglone derivatives ZLCY-1~3, ZLCY-5~14, ZLCY-16~17, ZLCY-21~25, and ZLCY-27~33 were designed and synthesized. Furthermore, by adding "hydrogen bond donor / acceptor surfaces" and "aromatic" surfaces near the binding site defined by the HuR protein, it was found that the amino acid residues near the binding site are mainly hydrogen bond donors, and several obvious aromatic centers exist. When modifying the structure, it is important to consider the addition of aromatic groups and hydrogen bond acceptors to improve binding activity. Based on this, juglone derivatives ZLCY-34-45 were designed and synthesized.

[0050] This invention provides a series of juglone derivatives, which are HuR inhibitors and can be used to prepare anti-glioma drugs. Among them, derivatives ZLCY-9 and ZLCY-32 have good in vitro and in vivo anti-glioma activity.

[0051] In this invention, the pharmaceutically acceptable salt refers to a substance that is suitable for human use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit-risk ratio.

[0052] In this invention, the pharmaceutically acceptable salt is preferably a salt formed by the juglone derivative and a pharmaceutically acceptable inorganic or organic acid. The inorganic acid may be selected from hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, sulfuric acid, or nitric acid. The organic acid may be selected from formic acid, acetic acid, propionic acid, trifluoroacetic acid, butyric acid, succinic acid, methanesulfonic acid, toluenesulfonic acid, oxalic acid, tartaric acid, fumaric acid, salicylic acid, succinic acid, oxalic acid, gluconic acid, nicotinic acid, ascorbic acid, citric acid, cinnamic acid, or amino acids.

[0053] The present invention also provides a method for preparing the juglone derivatives described in the above technical solution, wherein the raw materials used in the preparation method are all commercially available products in the art or compounds obtained by conventional methods in the art.

[0054] The preparation method of the compound with the structure shown in Formula I is described below.

[0055] In this invention, when R2 is -H, the method for preparing the compound with the structure shown in Formula I includes the following steps:

[0056] 6-bromo-2-naphthol, boric acid compound, potassium phosphate, 1,1-bis(diphenylphosphine)ferrocene palladium dichloride and a mixed solvent were mixed to carry out the first substitution reaction to obtain the substituted product;

[0057] The substituted product, oxidant, and organic solvent are mixed and subjected to a first oxidation reaction to obtain a compound with the structure shown in Formula I when R2 is -H.

[0058] In this invention, the molar equivalent ratio of 6-bromo-2-naphthol to the boric acid compound is preferably 1.0:1.2 to 1.5, specifically 1.0:1.2, 1.0:1.3, 1.0:1.4 or 1.0:1.5.

[0059] In this invention, the molar equivalent ratio of 6-bromo-2-naphthol to potassium phosphate is preferably 1.0:3 to 6, specifically 1.0:3, 1.0:4, 1.0:5 or 1.0:6.

[0060] In this invention, the mixed solvent preferably comprises 1,4-dioxane and water.

[0061] In this invention, the temperature of the first substitution reaction is preferably 90-110°C, specifically 90, 100 or 110°C, and the time is preferably 10-12 hours. The first substitution reaction is preferably carried out under nitrogen and reflux conditions.

[0062] In this invention, the molar equivalent ratio of the substituted product to the oxidant is preferably 1.0:1 to 1.5, specifically 1.0:1, 1.0:1.1, 1.0:1.2, 1.0:1.3, 1.0:1.4 or 1.0:1.5.

[0063] In this invention, the oxidant preferably includes 2-iodobenzoic acid.

[0064] In this invention, the organic solvent preferably includes anhydrous N,N-dimethylformamide.

[0065] In this invention, the first oxidation reaction is preferably carried out at room temperature for a time of 2 to 6 hours, and the oxidation reaction is preferably carried out under nitrogen protection.

[0066] In this invention, when R2 is an aromatic heterocyclic thio group, a benzene ring thio group, or a substituted benzene ring thio group, the preparation method of the compound with the structure shown in Formula I includes the following steps:

[0067] A second substitution reaction is carried out by mixing a compound with the structure of Formula I (where R2 is -H), an arylthiophenol, and an organic solvent to obtain a compound with R2 being an aromatic heterocyclic thio group, a benzene ring thio group, or a substituted benzene ring thio group and having the structure of Formula I.

[0068] In this invention, the organic solvent preferably includes methanol.

[0069] In this invention, the molar equivalent ratio of the compound of formula I with R2 being -H to the arylthiophenol is preferably 1.0:1.0 to 1.2, specifically 1.0:1, 1.0:1.1 or 1.0:1.2.

[0070] In this invention, the temperature of the second substitution reaction is preferably room temperature, the time is preferably 2 hours, and the second substitution reaction is preferably carried out under nitrogen conditions.

[0071] The preparation method of the compound with the structure shown in Formula II will be described below.

[0072] In this invention, when X is O, the method for preparing the compound with the structure shown in Formula II includes the following steps:

[0073] Intermediate 2b, borate compound, potassium phosphate, 1,1-bis(diphenylphosphine)ferrocene palladium dichloride, and a mixed solvent were mixed to carry out a third substitution reaction to obtain the substituted product; the structure of intermediate 2b is as follows:

[0074] Intermediate 2b;

[0075] The substituted product, oxidant, and organic solvent are mixed to carry out a second oxidation reaction to obtain a compound in which X is O and has the structure shown in Formula II.

[0076] In this invention, the molar equivalent ratio of intermediate 2b to boric acid compound is preferably 1.0:1.2 to 1.4, specifically 1.0:1.2, 1.0:1.3 or 1.0:1.4.

[0077] In this invention, the molar equivalent ratio of intermediate 2b to potassium phosphate is preferably 1.0:3 to 5, specifically 1.0:3, 1.0:4 or 1.0:5.

[0078] In this invention, the mixed solvent preferably comprises tetrahydrofuran and water.

[0079] In this invention, the temperature of the third substitution reaction is preferably 70-80°C, specifically 70, 75 or 110°C, and the time is preferably 12-14 hours. The third substitution reaction is preferably carried out under nitrogen and reflux conditions.

[0080] In this invention, the molar equivalent ratio of the substitution product to the oxidant is preferably 1.0:1 to 1.4, specifically 1.0:1, 1.0:1.1, 1.0:1.2, 1.0:1.3 or 1.0:1.4.

[0081] In this invention, the oxidant preferably includes 2-iodobenzoic acid.

[0082] In this invention, the organic solvent preferably includes anhydrous dimethyl sulfoxide.

[0083] In this invention, the second oxidation reaction is preferably carried out at room temperature for a time of 2 to 6 hours, and preferably under nitrogen protection.

[0084] In this invention, when X is N, the method for preparing the compound with the structure shown in Formula II includes the following steps:

[0085] Intermediate 3, an oxidant, and an organic solvent are mixed and subjected to a third oxidation reaction to obtain a compound in which X is N and has the structure shown in Formula II. The structure of intermediate 3 is as follows:

[0086] Intermediate 3,

[0087] R is an alkyl, aryl, or substituted aryl group.

[0088] In this invention, the molar equivalent ratio of the intermediate 3 and the oxidant is preferably 1.0:1.2 to 1.5, specifically 1.0:1.2, 1.0:1.3, 1.0:1.4 or 1.0:1.5.

[0089] In this invention, the oxidant preferably includes 2-iodobenzoic acid.

[0090] In this invention, the organic solvent preferably includes anhydrous dimethyl sulfoxide.

[0091] In this invention, the third oxidation reaction is preferably carried out at room temperature for a time of 2 to 6 hours, and preferably under nitrogen protection.

[0092] The present invention also provides the application of the juglone derivatives described in the above technical solution in the preparation of antitumor drugs.

[0093] In this invention, the antitumor drug preferably includes an anti-glioma drug.

[0094] The juglone derivative of this invention has inhibitory activity against glioma cells GL261.

[0095] In this invention, ZLCY-9 and ZLCY-32 exhibit good activity in inhibiting glioma growth in vivo. When ZLCY-9 and ZLCY-32 are administered via intraperitoneal injection, with temozolomide as the positive control, intervention with ZLCY-9 and ZLCY-32 can significantly inhibit glioma growth, and their activity is comparable to that of the positive control drug temozolomide.

[0096] In this invention, the antitumor drug preferably comprises an effective dose of the juglone derivative and a pharmaceutically acceptable carrier.

[0097] In this invention, the preferred routes of administration of the antitumor drug include oral, intravenous, local, enteral, inhalation, spray, sublingual, transdermal, or rectal administration.

[0098] In this invention, the dosage form of the antitumor drug preferably includes aerosol, cream, gel, pill, granule, capsule, tablet, syrup, transdermal patch, injection or nano-formulation.

[0099] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0100] In the embodiments of this invention, 10wt% and 20wt% refer to the proportion of the mass of the catalyst or ligand to the mass of the reactants.

[0101] Example 1

[0102] Synthesis of Juglone Derivatives ZLCY-1~3 and ZLCY-5~11

[0103] (1) Synthesis of intermediates 1a~c, 1e~m

[0104] Figure 2 This is a schematic diagram of the synthetic routes for intermediates 1a-c and 1e-m, as shown below. Figure 2 As shown, 6-bromo-2-naphthol (1.0 eq), borate compounds (1.2 eq), potassium phosphate (3 eq), and 1,1-bis(diphenylphosphine)ferrocene palladium dichloride [Pd(dppf)Cl2, 10 wt%] were added to a mixture of 1,4-dioxane / water and refluxed at 90 °C for 10 h under nitrogen protection. After the reaction was completed by TLC, 50 mL of water and 50 mL of ethyl acetate were added to the reaction system, and the mixture was extracted. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was purified by vacuum distillation to remove ethyl acetate. The remaining residue was purified by silica gel column chromatography.

[0105] Intermediates 1 g and 1 h (1.0 eq) were dissolved in methanol, respectively. Palladium on carbon (Pd / C) (10 wt%) was added, and H2 was bubbled through. The reaction was carried out at room temperature for 1 h. After the reaction was completed by TLC, the mixture was filtered. The methanol in the filtrate was removed by vacuum distillation. The remaining residue was purified by silica gel column chromatography to obtain intermediates 1i and 1j, respectively.

[0106] (2) Synthesis of juglone derivatives ZLCY-1~3 and ZLCY-5~11

[0107] Figure 3 The schematic diagram shows the synthetic routes for the juglone derivatives ZLCY-1~3 and ZLCY-5~11, as follows: Figure 3As shown, the intermediate (1.0 eq) from step (1) and the oxidant 2-iodobenzoic acid (1 eq) were added to anhydrous N,N-dimethylformamide and reacted at room temperature for 2 h under nitrogen protection. After the reaction was completed, 50 mL of water and 50 mL of ethyl acetate were added to the reaction system, and the mixture was extracted and the organic phase was collected. The organic phase was washed three times with saturated saline solution, collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure to remove ethyl acetate. The remaining residue was mixed with a small amount of ethyl acetate, ultrasonicated for 10 min, filtered, and the filter cake was the product.

[0108] Example 2

[0109] Synthesis of juglone derivatives ZLCY-12~14, ZLCY-16~17, and ZLCY-21~22

[0110] (1) Synthesis of intermediates 2c~2e, 2g~2h, 2n~2o

[0111] Figure 4 The schematic diagram shows the synthetic route of intermediates 2c-2e, 2g-2h, and 2n-2o. Triphenylphosphine (6 eq) was dissolved in dichloromethane and placed in an ice bath. Carbon tetrabromide (3 eq), triethylamine (4 eq), and 2-hydroxy-5-methoxybenzaldehyde (1 eq) were added sequentially, and the reaction was carried out at room temperature for 1 h. After the reaction was completed as detected by TLC, dichloromethane was removed by vacuum distillation, and the residue was dissolved in anhydrous ethanol. Cuprous iodide (0.05 eq) and potassium phosphate (2 eq) were added, and the reaction was carried out under nitrogen protection at 90 °C for 12 h. After the reaction was completed as detected by TLC, ethanol was removed by vacuum distillation, and the remaining residue was purified by silica gel column chromatography to obtain intermediate 2a.

[0112] Dissolve 2a in anhydrous dichloromethane, add a dichloromethane solution of boron tribromide dropwise at 0°C under nitrogen protection, and react at room temperature for 10 h. After the reaction is complete as detected by TLC, pour the reaction system into ice water, add sodium bicarbonate to neutralize until no more bubbles are generated, extract, collect the organic phase, dry with anhydrous sodium sulfate, filter, remove dichloromethane from the filtrate by vacuum distillation, and purify the residue by silica gel column chromatography to obtain intermediate 2b;

[0113] Intermediate 2b (1 eq), borate compounds (1.2 eq), potassium phosphate (3 eq), and 1,1-bis(diphenylphosphine)ferrocene palladium dichloride (10 wt%) were added to a tetrahydrofuran / water mixture and refluxed at 75 °C for 12 h under nitrogen protection. After the reaction was completed as detected by TLC, tetrahydrofuran was removed by vacuum distillation. The residue was dissolved in ethyl acetate, washed three times with saturated saline solution, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was purified by vacuum distillation to remove ethyl acetate. The residue was purified by silica gel column chromatography to give intermediates 2c–2o.

[0114] (2) Synthesis of juglone derivatives ZLCY-12~14, ZLCY-16~17, and ZLCY-21~22

[0115] Figure 5 The schematic diagram shows the synthetic route of juglone derivatives ZLCY-12~14, ZLCY-16~17, and ZLCY-21~22. The intermediate (1.0 eq) from step (1) and the oxidant 2-iodobenzoic acid (1.2 eq) were added to anhydrous dimethyl sulfoxide and reacted at room temperature for 2 h under nitrogen protection. After the reaction was completed, 50 mL of water and 50 mL of ethyl acetate were added to the reaction system, and the mixture was extracted and the organic phase was collected. The organic phase was washed three times with saturated saline solution, collected, dried over anhydrous sodium sulfate, filtered, and an equal volume of petroleum ether was added to the filtrate. After stirring, the mixture was purified by wet loading and silica gel column chromatography.

[0116] Example 3

[0117] Synthesis of Juglone Derivatives ZLCY-23~17 and ZLCY-21~22

[0118] (1) Synthesis of intermediates 3a~3c, 3e, 3f, 3m, and 3n

[0119] Figure 6 The schematic diagram shows the synthetic route for intermediates 3a–3c, 3e, 3f, 3m, and 3n. 5-hydroxyindole (1 eq), a borate compound (1.5 eq), palladium acetate (10 wt%), and ligand L6 (2,9-dimethyl-1,10-phenanthroline, 20 wt%) were added to 1,2-dichloroethane and refluxed at 90 °C for 10 h in air. After the reaction was complete as detected by TLC, 1,2-dichloroethane was removed by vacuum distillation. The residue was dissolved in ethyl acetate, washed three times with saturated saline solution, and the organic phase was collected. The solution was dried over anhydrous sodium sulfate, filtered, and the filtrate was purified by vacuum distillation to remove ethyl acetate. The residue was purified by silica gel column chromatography.

[0120] (2) Synthesis of intermediates 3h, 3j, and 3l

[0121] Figure 7The schematic diagram shows the synthetic route for intermediates 3h, 3j, and 3l. 4-Methoxy-2-methylaniline (1.0 eq) and potassium carbonate (2 eq) were added to acetone and stirred at 0°C for 15 min under nitrogen protection. Acyl chloride (1.2 eq) was dissolved in acetone and slowly added dropwise to the reaction system. The reaction system was then transferred to room temperature and reacted for 4 h. After the reaction was complete as detected by TLC, acetone was removed under reduced pressure, the residue was dissolved in ethyl acetate, washed three times with saturated saline solution, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was purified by removing ethyl acetate under reduced pressure. The residue was purified by slurrying with dichloromethane. The resulting solid (1.0 eq) was dissolved in anhydrous tetrahydrofuran, and n-butyllithium (1.05 eq) was added dropwise at 0°C under nitrogen protection. The reaction was carried out for 1 h, and then the reaction system was transferred to room temperature and reacted overnight. After the reaction was complete as detected by TLC, ethyl acetate was added to dilute the reaction system, and the reaction was quenched by adding saturated ammonium chloride solution. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was removed by vacuum distillation. The residue was purified by silica gel column chromatography to obtain intermediates 3g, 3i, and 3k.

[0122] Intermediates 3g, 3i, and 3k were dissolved in anhydrous dichloromethane, and a dichloromethane solution of boron tribromide was added dropwise under nitrogen protection at 0°C. The reaction was carried out at room temperature for 3 hours. After the reaction was completed by TLC, the reaction system was poured into ice water, and sodium bicarbonate was added to neutralize until no more bubbles were generated. The mixture was extracted, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was purified by vacuum distillation to remove dichloromethane. The residue was purified by silica gel column chromatography to obtain intermediates 3h, 3j, and 3l.

[0123] (2) Synthesis of ZLCY-23~25 and ZLCY-27~33

[0124] Figure 8 The schematic diagram shows the synthetic routes for ZLCY-23~25 and ZLCY-27~33. The intermediate (1 eq) obtained in step (1) and the oxidant 2-iodobenzoic acid (1.2 eq) were added to anhydrous dimethyl sulfoxide and reacted at room temperature for 2 h under nitrogen protection. After the reaction was completed, 50 mL of water and 50 mL of ethyl acetate were added to the reaction system, and the mixture was extracted and the organic phase was collected. The organic phase was washed three times with saturated saline solution, collected, dried over anhydrous sodium sulfate, filtered, and 1 / 4 volume of petroleum ether was added to the filtrate. After stirring, the mixture was purified by wet loading and silica gel column chromatography.

[0125] Example 4

[0126] Synthesis of Juglone Derivatives ZLCY-34-45

[0127] Figure 9The diagram shows the synthetic route for juglone derivatives ZLCY-34-45. Juglone derivative ZLCY-9 (1.0 eq) and arylthiophenol (1.0 eq) were dissolved in methanol and reacted at room temperature for 2 h under nitrogen protection. After the reaction was completed, the mixture was filtered, and the filter cake was washed with methanol to obtain the product.

[0128] The juglone derivatives obtained in the embodiments of the present invention were structurally characterized, and Table 1 shows the NMR data of the juglone derivatives.

[0129] Table 1. NMR data of juglone derivatives

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141] Activity assay at the molecular level of juglone derivatives ZLCY-11~3, ZLCY-5~14, ZLCY-16~17, ZLCY-21~25, and ZLCY-27~33

[0142] The activity of juglone derivatives in inhibiting the binding of HuR protein to AREVegf-a at the molecular level was determined using fluorescence polarization assays.

[0143] In HuR-ARE Vegf-a In the fluorescence polarization model, the HuR concentration is 600 nM, and the FAM-ARE concentration is... Vegf-a The concentration was 10 nM, the concentration of the juglone derivative was 40 μM, and the concentration of the positive control ELB (eltrombopag) was 40 μM. Figure 10 For HuR-ARE Vegf-aThe fluorescence polarization model for screening juglone analogs (n=3) shows that there are 11 juglone analogs with inhibition rates greater than 80%, namely ZLCY-8 (77.21±2.91), ZLCY-9 (80.62±3.31), ZLCY-12 (91.80±1.28), ZLCY-13 (94.52±3.72), ZLCY-14 (93.47±3.37), ZLCY-16 (88.76±1.74), ZLCY-17 (86.00±1.57), ZLCY-21 (86.97±0.44), ZLCY-22 (83.99±0.64), ZLCY-32 (85.81±0.63), and ZLCY-33 (87.42±1.71).

[0144] Eleven positive analogues were tested to inhibit HuR-ARE. Vegf-a The activity of the interaction was determined and the IC was calculated. 50 The values ​​are ZLCY-8 (7.13±0.33μM), ZLCY-9 (5.52±0.89μM), ZLCY-12 (2.23±0.04μM), ZLCY-13 (6.78±0.33μM), ZLCY-14 (4.33±0.50μM), ZLCY-16 (5.54±0.25μM), ZLCY-17 (6.59±0.79μM), ZLCY-21 (6.51±0.38μM), ZLCY-22 (3.86±0.29μM), ZLCY-32 (4.92±0.37μM), and ZLCY-33 (12.95±1.07μM). Figure 11 Positive analogue for HuR-ARE Vegf-a Diagram of the inhibitory activity of interactions (n=3).

[0145] Test of the inhibitory activity of juglone derivatives against glioma cells

[0146] The inhibitory activity of 11 positive analogues against the proliferation of glioma cells GL261 was evaluated using the MTT assay. Among the 11 positive analogues, ZLCY-9 showed similar inhibitory effects on GL261 cell proliferation as juglone (IC50). 50 The inhibitory effect was equivalent to 24.54 ± 1.49 μM, and its IC50 value was [missing value]. 50 The value was 22.89 ± 1.75 μM. The IC50 value of ZLCY-32 in inhibiting GL261 cell proliferation was... 50 The value was 33.78±2.07 μM, slightly weaker than juglone. Figure 12 Figure 1 shows the in vitro inhibitory activity of ZLCY-9 and ZLCY-32 against the proliferation of glioma GL261 cells (n=6).

[0147] Inhibitory activity of juglone derivatives against glioma in vivo

[0148] The in vivo tumor-inhibiting activity of juglone analogs ZLCY-9 and ZLCY-32 was evaluated using a GL261 subcutaneous tumor model. The mean tumor weights of the solvent control group, the 2 mg / kg ZLCY-9 group, the 2 mg / kg ZLCY-32 group, the 2 mg / kg juglone group, and the 20 mg / kg TMZ group (temozolomide) were 746.4, 294.3, 456.3, 329.1, and 406.8 mg, respectively. Figure 13 In (a) and (b) of the study, the mean tumor volumes on day 14 after drug administration were 457.1, 175.8, 269.3, 156.8, and 232.8 mm, respectively. 3 ( Figure 13 (c)). ZLCY-9 exhibited superior inhibitory activity compared to ZLCY-32 and TMZ, and was comparable to that of juglone. Furthermore, mice in the juglone, ZLCY-9, and ZLCY-32 treatment groups showed a slight increase in body weight, but the rate of increase was slower than that in the solvent control group and the TMZ group. Figure 13 (d)

[0149] In summary, this invention provides a series of juglone derivatives, which are HuR inhibitors that can target HuR protein, inhibit the binding activity of HuR protein to tumor-associated RNA, and have a significant inhibitory effect on glioma cells in vitro. These derivatives can be used to prepare anti-glioma drugs. Among them, derivatives ZLCY-9 and ZLCY-32 have good in vitro and in vivo anti-glioma activity and good safety, and can be used for the development of novel anti-tumor drugs, showing good application prospects.

[0150] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A juglone derivative comprising compounds of Formula I or Formula II, or pharmaceutically acceptable salts thereof: R1 is an alkyl, aryl, or substituted aryl group; The R2 is -H, aromatic heterocyclic thio group, phenyl ring thio group or substituted phenyl ring thio group; R3 is an alkyl, aryl, or substituted aryl group; X is N or O.

2. The juglone derivative according to claim 1, characterized in that, R1 is p-nitrophenyl, p-methoxyphenyl, p-trifluoromethylphenyl, p-fluorophenyl, p-chlorophenyl, cyclohexyl, cyclopentyl, cyclopropyl, o-methylphenyl, or o-isopropylphenyl.

3. The juglone derivative according to claim 1, characterized in that, R2 is p-nitrophenylthio, p-hydroxyphenylthio, p-methoxyphenylthio, p-aminophenylthio, p-trifluoromethylphenylthio, p-acetaminophenylthio, p-fluorophenylthio, p-chlorophenylthio, p-bromophenylthio, 4-pyridinethio, 2-pyrimidinethio, or 2-thiophenethio.

4. The juglone derivative according to claim 1, characterized in that, R3 is p-nitrophenyl, p-methoxyphenyl, p-trifluoromethylphenyl, p-fluorophenyl, p-chlorophenyl, cyclohexyl, cyclopentyl, cyclopropyl, o-methylphenyl, or o-isopropylphenyl.

5. The juglone derivative according to claim 1, characterized in that, The juglone derivative has the structure shown in any one of ZLCY-1~3, ZLCY-5~14, ZLCY-16~17, ZLCY-21~25, and ZLCY-27~45:

6. The use of the juglone derivative according to any one of claims 1 to 5 in the preparation of antitumor drugs.

7. The application according to claim 6, characterized in that, The anti-tumor drugs include anti-glioma drugs.

8. The application according to claim 6, characterized in that, The antitumor drug comprises an effective dose of the juglone derivative and a pharmaceutically acceptable carrier.

9. The application according to claim 6, characterized in that, The administration routes of the antitumor drugs include oral, intravenous, local, enteral, inhalation, spray, sublingual, transdermal, or rectal.

10. The application according to claim 6, characterized in that, The dosage forms of the antitumor drugs include aerosols, creams, gels, pills, granules, capsules, tablets, syrups, transdermal patches, injections, or nano-formulations.

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