Lupeol derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of synthesis of antitumor drugs, and particularly relates to lupeol derivatives as well as a preparation method and application thereof. According to the present invention, lupeol is adopted as a POI ligand, pomalidomide is adopted as an E3 ligand, and a linker selects a PEG chain, such that the preparation method of the PROTACs drug using the lupeol pomalidomide derivative, and the application of the PROTACs drug in anti-tumor and neuroprotection aspects are provided. The lupeol derivative provided by the invention has the following structure: # imgabs0 #.

Description

Technical Field

[0001] The invention belongs to the technical field of anti-tumor drug synthesis, and specifically relates to a lupeol derivative and a preparation method and application thereof. Background Art

[0002] Cancer, or malignant tumor, is a major disease that endangers human health due to multi-gene mutations, multi-factor triggering, and multi-stage development. In recent years, with the increase of factors such as population aging and environmental pollution, the incidence and mortality of cancer have accelerated, and it has become a major cause of human death. The latest data show that in 2018, there were nearly 20 million new cancer cases and 10 million cancer deaths worldwide. It is estimated that by 2025, the number of new cancer cases worldwide will reach 19.3 million. Therefore, actively carrying out various work to prevent and treat cancer, how to effectively treat cancer has become a top priority. Faced with the challenges brought by malignant tumors, the existing anti-tumor drugs are far from meeting the needs, and there is an urgent need to develop new anti-tumor drugs with low toxicity and high efficiency.

[0003] The natural product Lupeol (LUP) is a pentacyclic triterpenoid compound with significant biological activity derived from plants such as lupine, mango, Euphorbia chamaejasme and dandelion. Lupeol molecular formula C 30 H 50 O, molecular weight 426.7174, studies have found that it has significant pharmacological effects such as anti-inflammatory, antioxidant, cardioprotective and immune regulation. In addition, studies have shown that lupeol is a potential anti-tumor drug. Experimental results have found that it can significantly inhibit the growth of various tumor cells both in vitro and in vivo, including colorectal cancer, prostate cancer, liver cancer, lung cancer, bladder cancer, etc., involving the regulation of multiple intracellular signaling pathways. However, since lupeol is insoluble in water, diffuses slowly in the cytoplasm and has low selectivity, its research on anti-tumor is limited, and its development into clinical drugs is even more hindered.

[0004] Existing anti-tumor drugs are far from meeting the demand, and there is an urgent need to develop new anti-tumor drugs with low toxicity and high efficiency. Summary of the invention

[0005] The present invention selects lupeol to be prepared into a protein degradation targeting chimera, wherein the protein degradation targeting chimera (Proteolysis-Targeting Chimeras, PROTACs) is a new type of drug, and its structure is mainly composed of three parts, including E3 ligase ligand (E3 ligand), target protein ligand (POI ligand) and a linker connecting the two. One end of the PROTACs molecule binds to the target protein, and the other end binds to the E3 ubiquitin ligase. The E3 ubiquitin ligase can "stick" a small protein called ubiquitin to the target protein, marking the target protein as a defective or damaged protein, and then using the cell's intrinsic proteolytic enzyme (i.e., 26S proteasome) to specifically recognize and degrade the marked target protein. E3 ligands can recruit E3 ligases, and POI ligands can recognize and bind to target proteins. Therefore, PROTACs molecules will form a ternary complex of POI:PROTAC:E3 ligase in cells. In the patient's body, the POI ligand of PROTACs binds to the target protein, and the E3 ligand binds to the substrate binding region of the E3 ubiquitin ligase in the cell. The target protein can be "pulled" to the side of the E3 ubiquitin ligase through the Linker, thereby achieving degradation of the target protein by the ubiquitin-proteasome (UPS) system.

[0006] The present invention uses lupeol as a POI ligand, pomalidomide as an E3 ligand, and a PEG chain as a linker to synthesize PROTACs drugs with lupeol pomalidomide derivatives.

[0007] Pomalidomide is an important E3 ubiquitin ligand in PROTAC molecules. The modification is mainly through targeting a ubiquitin E3 ligase CRBN. However, pomalidomide and lupeol are both fat-soluble compounds. Direct connection may lead to poor water solubility, affecting bioavailability and efficacy. At the same time, due to the large structural differences between the two, there is a risk of premature breakage in the body. Therefore, the present invention specifically selects the linker PEG chain to improve the water solubility of the compound, improve solubility and biocompatibility. At the same time, the flexibility of the PEG chain balances the rigidity and flexibility of the molecule and enhances the binding ability with the target. Pomalidomide is an E3 ubiquitin ligase ligand, and lupeol is a target protein ligand. They are connected by PEG chains to form a ternary complex, and the target protein is selectively degraded by the ubiquitin-proteasome system.

[0008] The present invention provides a lupeol pomalidomide derivative or a pharmacologically or physiologically acceptable salt thereof:

[0009] wherein the E3 ligase is selected from pomalidomide; Linker is selected from PEG chain.

[0010] Preferably, the PEG chain is selected from -NH-(CH 2 -CH 2 -O) n -CH 2 CH 2 -, where n represents a natural number 2 or 3.

[0011] Preferably, the PROTACs compound of lupeol or its pharmacologically or physiologically acceptable salt has the chemical formula C 54 H 78 N 6 O 6 , its molecular weight is 939.2520.

[0012]

[0013] The method for preparing the lupeol pomalidomide derivative according to a specific embodiment of the present invention comprises the following steps: Pomalidomide and PEG chain are reacted at 85-95° C. Then DIPEA is dissolved in DMF, cooled to 0° C. in an ice bath, HATU is added, and then reacted at room temperature for 1-5 hours to synthesize intermediate 1, and finally intermediate 1 is amidated with intermediate 2 of N3-labeled lupeol, and then extracted, concentrated, and purified to prepare the lupeol pomalidomide derivative.

[0014] The present invention utilizes click chemistry synthesis to efficiently construct a stable triazole linker through a copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC), thereby improving synthesis efficiency and product purity.

[0015] Preferably, the structural formula of lupeol and pomalidomide is:

[0016] Preferably, the structural formula of the intermediate product 1 and the intermediate product 2 is:

[0017] The preparation method of the lupeol pomalidomide derivative of the present invention is as follows:

[0018] Specifically, LUP, PEG chain and pomalidomide were used as raw materials, and DMF solvent was added. After stirring for a few minutes, DIPEA and HATU were added in sequence, and the reaction was carried out at room temperature for 24 hours to obtain the intermediate:

[0019] Then, it was dissolved in DCM under ice bath, anisole was added and stirred evenly, TFA was added, and then reacted at room temperature for 12 hours. After dilution with water, it was extracted with dichloromethane three times, the organic layers were combined and washed three times with saturated brine, dried with anhydrous sodium sulfate, filtered and concentrated to obtain the intermediate:

[0020] The obtained product was directly used for the next step reaction. After adding lupeol and DMF, the mixture was stirred for a few minutes, and then DIPEA and HATU were added in sequence. After reacting for 24 hours at room temperature, the final products with different PEG lengths were obtained:

[0021] Formula (I).

[0022] The present invention provides the use of the lupeol derivative or its pharmacologically or physiologically acceptable salt in the preparation of anti-tumor drugs.

[0023] Preferably, the tumor includes at least one of lung cancer, liver cancer, human brain glioma, skin cancer, prostate cancer, breast cancer, and gastric cancer.

[0024] The present invention provides the use of the lupeol pomalidomide derivative in the preparation of a neuroprotective product, wherein the neuroprotective product includes a health food, a health drink, and the like.

[0025] The present invention provides the use of the lupeol pomalidomide derivative in preparing medicines for treating and / or improving Parkinson's disease.

[0026] Beneficial effects of the present invention: The present invention comprehensively considers the solubility, stability, targeting and toxicity issues required when pomalidomide is linked to lupeol, and can effectively solve the above challenges and improve the efficacy and safety of anti-tumor drugs through strategies such as structural modification (such as PEG chain) and PROTAC technology.

[0027] Compared with lupeol, the compound of the present invention has the following advantages: (1) The water solubility is significantly improved. By introducing the hydrophilic groups (such as amino groups) and flexible linkers (such as PEG chains) of pomalidomide, the water solubility of lupeol is improved, making it easily soluble in water, ethanol and DMSO, and the bioavailability is significantly improved.

[0028] (2) Enhanced anti-tumor activity: The immunomodulatory effect of pomalidomide and the pro-apoptotic properties of lupeol work synergistically, and the inhibition rate of various tumor cells such as liver cancer, lung cancer, and breast cancer is significantly better than lupeol.

[0029] (3) Targeted optimization: Pomalidomide, as an E3 ubiquitin ligase ligand, can form a ternary complex through linker-mediated degradation of tumor-related proteins (such as KDM5A and BRD4), thereby enhancing selectivity for tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 This is the H NMR spectrum of the intermediate product prepared in Example 1.

[0032] Figure 2 This is the H NMR spectrum of the target compound prepared in Example 2.

[0033] Figure 3 This is the ultimate experimental behavioral test of the target compound of Example 5 on the PD mouse model.

[0034] Figure 4 This is a behavioral test of the target compound of Example 5 on the PD mouse model using a suspension experiment.

[0035] Figure 5 This is a rotating column behavioral test of the target compound of Example 5 on a PD mouse model. DETAILED DESCRIPTION

[0036] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0037] Example 1 Preparation of intermediate product Synthesis of pomalidomide intermediates containing linker Pomalidomide: Linker reagent: HATU: DIPEA = 1: 1.5: 1.2: 2 (molar ratio), reaction conditions: stirring at room temperature for 24 hours, TLC monitoring. Ice quenching, ethyl acetate extraction, column chromatography purification (developing solvent: dichloromethane: methanol = 20: 1).

[0038] DIPEA (2 eq) and NH 2-PEG3-N 3 (1 eq). Stir at 90 °C for 4 h. After completion, EtOAc and brine were added. The organic layer was washed with NH 4 Cl solution and brine washing, anhydrous Na 2 SO 4 Dry, filter, evaporate, and purify on silica gel CC to obtain the intermediate product as a yellow-green solid with a yield of 32%. The NMR results are as follows: Figure 1 shown.

[0039] 1 H NMR (400 MHz, CDCl 3 ) δ 7.99 (s, 1H), 7.51-7.47 (m, J = 7.49 Hz, 1H),7.12-7.10 (d, J = 7.11 Hz, 1H), 6.94-6.92 (d, J = 6.93 Hz, 1H), 6.50-6.47 (t, J =6.49 Hz, 1H), 4.93-4.89 (dd, J = 4.91 Hz, 1H), 3.72-3.66 (m, J = 3.68 Hz, 12H),3.48-3.47 (q, J = 3.47 Hz, 2H), 3.39 - 3.38 (m, J = 3.38 Hz, 2H), 2.88 - 2.75 (m, J = 2.81 Hz, 3H), 2.14 - 2.10 (m, J = 2.12 Hz, 1H). Example 2 Preparation of Lupeol Pomalidomide Derivatives For deprotection and connection with lupeol, reactants: Boc-protected pomalidomide-Linker intermediate, lupeol (Lupeol), deprotection reagent: TFA (trifluoroacetic acid), condensation agent: HATU, base: DIPEA, solvent: DCM / MeOH (dichloromethane / methanol, volume ratio 3:1).

[0040] Reaction conditions: Deprotection: The intermediate was dissolved in DCM / MeOH under ice bath, TFA was added dropwise, and the reaction was carried out at room temperature for 12 hours, intermediate: TFA = 1:2.

[0041] Connecting reaction: After deprotection, no purification is required, and lupeol, HATU, and DIPEA are directly added. The reaction is carried out at room temperature for 24 hours. Lupeol: intermediate: HATU: DIPEA = 1:1.5:1.2:3 (molar ratio).

[0042] Post-treatment: ice quenching, dichloromethane extraction, column chromatography purification (developing solvent: petroleum ether: ethyl acetate = 10:1).

[0043] TLC detection (petroleum ether: ethyl acetate (V: V) = 1:3) showed that the reaction was completely stopped, and water was added, extracted with ethyl acetate, dried, and concentrated in vacuo. A mixture of petroleum ether and ethyl acetate in a volume ratio of 1:1 was used as the eluent, and the product was purified by chromatography on a polyamide column. The collected eluate containing the product was concentrated and dried to obtain a yellow-green solid with a yield of 29%.

[0044] At 254nm wavelength, the purity of the liquid phase test sample is 100%. Positive ion mode, molecular weight 939.8. Corresponding to M+H; molecular weight 469.9 corresponds to [M+2H] / 2. HRMS m / z[M+H]+calcd for C 54 H 78 N 6 O 6 , Exact Mass: 938.5881, Molecular Weight: 939.2520.

[0045] Example 3 Antitumor Effects of Lupeol Pomalidomide Derivatives In order to explore the inhibitory ability of lupeol pomalidomide derivatives on tumor cells, CCK-8 method was used for detection. A variety of tumor cells and other non-tumor cells were revived and cultured, including human lung cancer cells (A549), human liver cancer cells (HepG2), human glioma cells (BT325), human skin cancer cells (B16F10), human prostate cancer cells (LNCaP), human breast cancer cells (MCF7), and human gastric cancer cells (MKN-45).

[0046] When the cells grow to 80% to 90% of the culture dish, digest them and resuspend them in fresh DMEM / 1640 complete medium. Count them under a microscope, then dilute the cell suspension to 50 cells / μL, 100 μL per well, and spread 100 μL of the diluted cell suspension in each well of the remaining wells. Incubate at 37°C with 5% CO 2After 24 hours, the medium in the 96-well plate was removed, and the drug was diluted with fresh complete medium in a certain concentration gradient (0, 3.90625, 7.8125, 15.626, 31.25, 62.5, 125, 250, 500, 1000 μM), and then added to the 96-well plate in sequence. After 48 hours, the medium containing the drug was removed, and 10 μL of CCK-8 was added and placed at 37°C, 5% CO 2 The cells were cultured in an incubator for 30-40 minutes, and the absorbance was measured at OD 450 on a microplate reader. The inhibition rate was calculated based on the absorbance at OD 450. The half-maximal inhibitory concentration (IC) was calculated using SPSS software. 50 .

[0047] The calculation formula is as follows: Inhibition rate = [(Ac-As) / (Ac-Ab)] × 100%; As: absorbance of experimental wells (containing cells, culture medium, CCK-8 solution, and drug solution); Ac: absorbance of control well (containing cells, culture medium, CCK-8 solution, but no drug); Ab: absorbance of blank wells (containing culture medium and CCK-8 solution, but not cells or drugs).

[0048] Experimental results: The in vitro antitumor activity of the target compound was further evaluated, and the test results are shown in the following table.

[0049]

[0050] Overall, under the same treatment conditions, the lupeol pomalidomide derivative has a broader and stronger anti-tumor activity than lupeol, and its tumor inhibition ability is significantly better than that of the control group.

[0051] Example 4 Neuroprotective Effects of Lupeol Pomalidomide Derivatives In order to explore the neuroprotective effect of lupeol pomalidomide derivatives on glial cells, CCK8 was used for detection. The CCK8 method was the same as in Example 3.

[0052] The effects of MPP+ (50uM), a classic Parkinson's disease modeling drug, and pomalidomide derivative of lupeol (50uM) on the activity of human neuroblastoma cell line SH-SY5Y, mouse hippocampal neuron cell line HT22, and rat pheochromocytoma cell line PC12 were respectively applied. The test results are shown in the following table.

[0053]

[0054] Example 5 Therapeutic effect of lupeol pomalidomide derivatives in Parkinson's disease model In order to explore the therapeutic effect of lupine alcohol pomalidomide derivatives on Parkinson's disease model, MPTP (25 mg / kg / d) was intraperitoneally injected for 7 days to induce PD mouse model, followed by oral gavage treatment with lupine alcohol pomalidomide derivatives at low doses (10 mg / kg / d), medium doses (50 mg / kg / d), and high doses (100 mg / kg / d) for 21 consecutive days, and levodopa (20 mg / kg / d) was used as a positive drug for 21 days. Seven days after the end of treatment, pole test, hanging score and rotating column test were performed on all mice to evaluate the motor function of mice.

[0055] The results are as follows Figure 3 , Figure 4 and Figure 5 As shown, PD mice showed a significant increase in pole time, a decrease in hanging score, and a decrease in the time spent on the rotating column. After treatment, PD mice in the low, medium, and high dose groups of the lupeol pomalidomide derivative showed a significant decrease in pole time, an increase in hanging score, and a prolonged time spent on the rotating column.

[0056] In summary, the present invention provides a lupine alcohol pomalidomide derivative and a preparation method thereof, and for the first time discovered that the derivative can effectively inhibit the survival and growth of multiple tumor cell lines, and the inhibitory activity is stronger than that of lupine alcohol. At the same time, the lupine alcohol pomalidomide derivative has a neuroprotective effect and can improve the motor function of Parkinson's disease, indicating that it has research and development potential and application prospects in clinical tumor treatment and neuroprotection.

[0057] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A lupeol derivative, characterized in that: Its structural formula is shown in formula (I): Formula (I) Wherein, n is selected from 2 or 3.

2. The method for preparing the lupeol derivative according to claim 1, characterized in that: The preparation method comprises the following steps: Pomalidomide and PEG chain were reacted at 85-95°C; DIPEA was then dissolved in DMF, cooled to 0°C in an ice bath, HATU was added, and then reacted at room temperature for 1-5h to obtain intermediate 1; N3 labeled lupeol to obtain intermediate 2; The intermediate product 1 and the intermediate product 2 are subjected to amidation reaction, and then extracted, concentrated and purified to obtain the lupeol derivative.

3. The method for preparing the lupeol derivative according to claim 2, characterized in that: The PEG chain is selected from -NH-(CH2-CH2-O) n -CH2CH2-, wherein n is 2 or 3.

4. Use of the lupeol derivative or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for treating and / or preventing tumors.

5. A drug for treating and / or preventing tumors, characterized in that: The medicine contains the lupeol derivative according to claim 1.

6. The drug according to claim 1, characterized in that The drug contains a pharmaceutically acceptable carrier.

7. The drug according to claim 5 or 6, characterized in that The tumor includes at least one of lung cancer, liver cancer, human brain glioma, skin cancer, prostate cancer, breast cancer or gastric cancer.

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