A licorice chalcone nrf2 small molecule agonist and preparation and application thereof

Abstract

The application discloses a licorice chalcone NRF2 small molecule agonist and a preparation and application thereof, a structure of the licorice chalcone NRF2 small molecule agonist is shown as formula (I), the compound can be used for preparing a drug for treating tumors, and experimental results show that the compound has better Nrf2 activation activity than licorice chalcone E and has higher anticancer activity.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, and more specifically, it relates to glycyrrhizin chalcone compounds with antitumor activity. Furthermore, this invention also relates to the antitumor uses of such compounds. Background Technology

[0002] With the increasing aging of the global population, the socioeconomic burden of malignant tumors is also increasing year by year. In my country, according to surveys, an average of 8,550 people die from cancer every day. Current cancer treatments mainly include chemotherapy, radiotherapy, surgery, targeted drug therapy, and the currently popular immunotherapy. Traditional treatments such as chemotherapy and radiotherapy are too harmful to cancer patients, easily causing symptoms such as weakened immunity and hair loss. Although targeted drugs are widely used, they cannot achieve a radical cure for some specific types of cancer, such as lung cancer, breast cancer, and pancreatic cancer. In fact, some targeted drugs can lead to drug resistance after prolonged use. Therefore, developing new anti-cancer drugs remains an important task.

[0003] External stimuli (such as drugs, ultraviolet radiation, and ionizing radiation) and endogenous free radicals and reactive oxygen species (ROS) can directly or indirectly damage cellular components such as proteins, lipids, and DNA. To counteract these adverse effects, the body has developed a complex oxidative stress response system to alleviate cellular damage. Nrf2 (NFE2 P45-related factor 2), as a key transcription factor regulating antioxidative stress, plays an important role in inducing the body's antioxidant response, such as regulating redox balance, drug metabolism and excretion, energy metabolism, iron metabolism, amino acid metabolism, survival, proliferation, autophagy, proteasome degradation, DNA repair, and mitochondrial physiological functions. Furthermore, the Keap1-Nrf2 system has become an important therapeutic target for cancer, neurodegenerative diseases, and many autoimmune and inflammatory diseases. Therefore, developing novel Nrf2 small molecule agonists has become a new strategy for cancer treatment. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a new glycyrrhizin chalcone antitumor compound and its drug.

[0005] Specifically, the present invention provides a compound of formula (MZY029) or a pharmaceutically acceptable salt thereof:

[0006]

[0007] Compound (MZY029) is:

[0008] (E)-1-(4-ethoxyphenyl)-3-[4-hydroxy-3-(3-methyl-3-buten-2-yl)phenyl]-2-propen-1-one

[0009] (E)-1-(4-ethoxyphenyl)-3-[4-hydroxy-3-(3-methylbut-3-en-2-yl)phenyl]prop-2-en-1-one

[0010] One of the objectives of this invention is to provide a novel glycyrrhizin chalcone-based antitumor small molecule compound.

[0011] The second objective of this invention is to provide a method for preparing a novel antitumor small molecule compound with glycyrrhizin chalcone as the parent nucleus and its antitumor activity.

[0012] In this invention, we first synthesized a novel glycyrrhizin chalcone compound, MZY029, using a chemical method (see Example 1 for details). The Nrf2 activation activity of this compound was determined using an Nrf2-dual-luciferase reporter gene assay. Compound MZY029 was found to have better Nrf2 activation activity than glycyrrhizin E. (See Example 2 for details). Cellular level experiments evaluated the effect of compound MZY029 on the survival rate and IC50 of human cholangiocarcinoma RBE cells. 50 The determination of the value revealed that compound MZY029 had a better killing effect on human cholangiocarcinoma cells (RBE cells) than glycyrrhizin E, and its IC50 value on RBE cells was higher. 50 The value is approximately 10 μM (see Example 3 for details).

[0013] The present invention also provides a method for preparing the chalcone compounds described above, comprising the following steps:

[0014] (1) In the presence of alkali and sodium iodide, p-hydroxybenzaldehyde was reacted with 3,3-dimethylallyl bromide in acetone to give intermediate 2[4-((3-methyl-2-butenyl)oxy)benzaldehyde];

[0015] (2) Intermediate 2 [4-((3-methyl-2-butenyl)oxy)benzaldehyde] was dissolved in N,N-diethylaniline and stirred under reflux for rearrangement reaction. Then it was dissolved in dichloromethane, and 3,4-dihydro-2H-pyran and a catalytic amount of pyridine p-toluenesulfonate were added to continue the reaction to obtain intermediate 3 [3-(3-methyl-2-butenyl)-4-((4-hydro-2H-pyran-2-yl)oxy)benzaldehyde];

[0016] (3) The intermediate 3[3-(3-methyl-2-butenyl)-4-((4-hydro-2H-pyran-2-yl)oxy)benzaldehyde] was dissolved in a mixed solvent of ethanol and water, and then potassium hydroxide was added to react first, followed by extraction and then hydrochloric acid was added to react to obtain the chalcone compound. Attached Figure Description

[0017] Figure 1 Describe the synthesis route and structure of the compound MZY029;

[0018] Figure 2 Comparison of Nrf2 activation activities between compound MZY029 and glycyrrhizin E;

[0019] Figure 3 Effects of compound MZY029 on the survival rate and IC50 of human cholangiocarcinoma RBE cells 50 value; Detailed Implementation

[0020] The compounds listed herein are for the purpose of better illustrating the types and structures of compounds in this invention and are not intended to limit the invention.

[0021] The invention will be described in more detail in the following non-limiting embodiments.

[0022] Example 1: Preparation of compound MZY029:

[0023] Preparation of intermediate 2: 10g of p-hydroxybenzaldehyde (1) was weighed into a reaction flask, and an appropriate amount of acetone was added to dissolve the raw material. Then, 10g of potassium carbonate was weighed, and 15ml of 3,3-dimethylallyl bromide was slowly added under oil bath conditions. Finally, 11g of potassium carbonate and 3g of sodium iodide solid were slowly added. The mixture was stirred at room temperature, and the reaction was monitored by TLC. After the reaction was completed, the acetone was evaporated, the residue was dissolved in water, and the product was extracted with ethyl acetate. The ethyl acetate layer was evaporated, and the column was packed by wet method. The column was filled with petroleum ether: ethyl acetate = 10:1 to obtain colorless and transparent raw material intermediate 2 [4-((3-methyl-2-butenyl)oxy)benzaldehyde].

[0024] Preparation of intermediate 3: 3g of intermediate 2 was weighed and added to a reaction flask, followed by 5mL of N,N-diethylaniline for dissolution. The reaction apparatus was vacuum filtered for 5 minutes to remove dissolved oxygen. The reaction system was stirred and refluxed for 4 hours under sand bath conditions (200℃). After the reaction was completed, the reaction system was cooled to room temperature and directly packed into a column using a wet method. The product was purified by silica gel column chromatography and then evaporated to dryness to obtain the rearranged product. The rearranged product was redissolved in 10mL of dichloromethane, and 1.5mL of 3,4-dihydro-2H-pyran and a catalytic amount of pyridine p-toluenesulfonate were added. The reaction was stirred at room temperature and monitored by TLC. After the reaction was completed, water was added to quench the reaction, and the product was extracted with ethyl acetate. The ethyl acetate layer was evaporated to dryness, packed into a column using a wet method, and passed through a column with petroleum ether:ethyl acetate = 10:1 to obtain colorless and transparent intermediate 3 [3-(3-methyl-2-butenyl)-4-((4-hydro-2H-pyran-2-yl)oxy)benzaldehyde].

[0025] Preparation of compound MZY029: 100 mg of intermediate 3 and an equimolar amount of p-ethoxyacetophenone were dissolved in 10 mL of a 2:1 mixture of ethanol and water. 0.05 g of potassium hydroxide was added to the reaction flask, and the mixture was stirred at room temperature for 8 hours. After the reaction was complete, water was added to quench the reaction, and the product was extracted with ethyl acetate. The ethyl acetate layer was evaporated to dryness, and the residue was dissolved in 5 mL of methanol. 6 N hydrochloric acid was added dropwise, and the mixture was stirred at room temperature for 1 hour. After the reaction was complete, water was added to quench the reaction, and the product was extracted with ethyl acetate. The ethyl acetate layer was evaporated to dryness, and the mixture was packed into a column using a wet packing method. The column chromatography ratio of petroleum ether to ethyl acetate was 5:1 to obtain the final product MZY029 as a yellow solid powder (overall yield approximately 25%). (See the overall synthetic route for details.) Figure 1 ).

[0026] The basic information and NMR spectrum data of compound MZY029 are as follows:

[0027] (E)-1-(4-ethoxyphenyl)-3-[4-hydroxy-3-(3-methylbut-3-en-2-yl)phenyl]prop-2-en-1-one

[0028] (E)-1-(4-ethoxyphenyl)-3-[4-hydroxy-3-(3-methyl-3-buten-2-yl)phenyl]-2-propen-1-one

[0029] Yellow power.Yield:25.4%.mp:139.1-141.2℃. 1H NMR (CDCl3, 300MHz): δ8.02 (d, 2H, J = 9.0Hz), 7.76 (d, 1H, J = 15.6Hz), 7.46 (dd, 1H, J1 = 7.4H z,J2=2.4Hz),7.41(d,1H,J=2.1Hz),7.40(d,1H,J=15.6Hz),6.97(d,2H,J=9.0Hz),6.85(d ,1H,J=7.4Hz),6.13(s,1H),5.10[d,2H,J=3.3Hz],4.12(q,2H,J=6.9Hz),3.62[q,1H,J=6. 6Hz,],1.69[s,3H],1.60(t,3H,J=6.9Hz),1.44[d,3H,J=6.9Hz].ESI-MS,m / z:337.3[M+H] - .calcdfor C 22 H 24 O3:336.1

[0030] Example 2: Comparison of Nrf2 activation activities of compound MZY029 and glycyrrhizin E

[0031] After removing the 293T cells from the cryovials stored in liquid nitrogen, they were quickly thawed in a 37°C water bath. The cryovials were then sterilized with 75% alcohol. The cells were transferred to 1.5mL centrifuge tubes and centrifuged at 800 rpm at 25°C, discarding the supernatant. 1mL of complete culture medium was added, gently mixed, and the cells were seeded into culture dishes and incubated in a 5% CO2 incubator at 37°C. Next, 293T cells that were in good condition and had reached approximately 80% confluence were collected. The original culture medium was aspirated, and the cells were washed twice with PBS buffer. They were then digested with 0.25% trypsin for 2 minutes. Under a microscope, the cells were observed to shrink, round out, and slightly detach from the cell wall. 3mL of culture medium was added to stop the digestion, and the cells were gently mixed and centrifuged again at 800 rpm at 25°C, discarding the supernatant. The cells were resuspended in the culture medium and transferred to new culture dishes, then incubated in a 5% CO2 incubator at 37°C.

[0032] Log-grown 293T cells were seeded into 96-well plates and cultured in DMEM medium containing 10% fetal bovine serum and 1% antibiotics for 12 h. The 293T cells were then transfected with expression plasmids for 24 h. The transfection system consisted of 3.2 μg Gal4-Nrf2-TKrLUC, 3.2 μg the Gal4 reporter vector 9×GAL4-UAS-promoter-fLuc, 0.3 μg Renillareporter plasmids, and 6 μL FuGENE transfection reagent. Subsequently, positive control drugs (licochalcone E, 50 μmol·L⁻¹) and self-synthesized MZY029 (25 μmol·L⁻¹) were added, while a blank control group was added with 1‰ DMSO. After culturing in a cell incubator for 24 h, luciferase activity was detected according to the instructions of the "Dual Luciferase Reporter Gene Detection Kit". The experiment was repeated three times for verification. Nrf2 agonist activity was expressed as the ratio of "RLU-induced / RLU-uninduced", while all relative luciferase activities (RLU) were expressed as the ratio of "Firefly RLUs / Renilla RLUs". Data were statistically analyzed using GraphPadPrism software. Quantitative data were expressed as mean ± standard error (mean ± SEM). The t-test was used for comparisons between groups, and P < 0.05 was considered statistically significant.

[0033] The activity results of compounds MZY029 and glycyrrhizin E are as follows: Figure 2 As shown.

[0034] Example 3: Verification of the antitumor activity of compound MZY029 at the cellular level

[0035] Cell culture and drug delivery

[0036] Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C with 5% CO2. Compound MZY029 was dissolved in DMSO to prepare a 50 μg / ml stock solution, which was stored at -20°C. Dosage was controlled at four concentrations: 0, 5, 7.5, and 10 μM. For each working solution concentration, a final solution with a DMSO concentration of less than 0.1% was prepared using basal medium. Cells without the compound served as a negative control, and cells given 20 μM curcumin served as a positive control.

[0037] Assay of compound MZY029 against human cholangiocarcinoma cell proliferation

[0038] RBE cells were cultured in 96-well plates (5000 cells / well) in RPMI 1640 medium containing 10% FBS. Different concentrations of the compound were administered after 24 hours of cell growth. Fresh MTT (5 mg / ml) solution prepared in PBS was added to each well. Formazan crystals formed in live cells and were dissolved in 150 μL of DMSO solution. The absorbance of the compound was measured at 490 nm using a microplate reader. The IC50 of the compound was... 50 The values ​​can be calculated using Graphpad Prism 5.0 software (see cell experiment results). Figure 3 ).

Claims

1. Use of a chalcone compound or a pharmaceutically acceptable salt thereof, characterized in that, The chalcone compound structure is shown in general formula (I): (Ⅰ) The chalcone compound is used for preparing a medicine for treating tumors. The tumor is cholangiocarcinoma.

2. The chalcone compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein The chalcone compound is an NRF2 small molecule agonist.

3. The chalcone compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein The medicine comprises an effective component and pharmaceutical adjuvants, and the effective component comprises the chalcone compound or a pharmaceutically acceptable salt thereof.

4. The chalcone compound or a pharmaceutically acceptable salt thereof according to claim 3, wherein The preparation of the medicine is any one of injection, tablet, capsule, aerosol, suppository, film, dropping pill, ointment, controlled release preparation, sustained release preparation or nano preparation.

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