CN117945842A - A β-elemene aryl derivative and its preparation method and application - Google Patents

Abstract

The invention relates to the technical field of beta-elemene aryl derivatives, in particular to a beta-elemene aryl derivative, a preparation method and application thereof. The beta-elemene aryl derivative provided by the invention has better inhibition activity on bladder cancer cells T24, colorectal cancer cells SW480, glioma cancer cells U251 and glioma cancer cells U87, and has stronger application potential and economic value in the field of biological medicines.

Description

A β-elemene aryl derivative and its preparation method and application

Technical Field

The invention relates to the technical field of beta-elemene aryl derivatives, and in particular to a beta-elemene aryl derivative and a preparation method and application thereof.

Background technique

β-elemene is a national Class II new anti-tumor drug. It is a sesquiterpenoid broad-spectrum, low-toxic anti-cancer active ingredient extracted from the ginger plant Curcuma aromatica. It has good application prospects in the clinical treatment of lung cancer, liver cancer, breast cancer, etc. However, due to the poor water solubility and low bioavailability of β-elemene, its clinical application is greatly limited.

In order to improve the water solubility, bioavailability and anti-tumor activity of β-elemene, some synthetic methods have been developed to improve the administration type and structural modification of β-elemene. Among them, the structural modification of β-elemene is more important for improving anti-tumor activity and bioavailability. It can not only change the solubility of β-elemene derivatives by introducing hydrophilic groups and improve their bioavailability, but also change the conformation of the molecule, enhance the orientation, increase the target of action or improve the anti-tumor activity through synergistic effects. At present, the structural modification site of β-elemene is mainly at the allylic position. Under the premise that the β-elemene skeleton and its double bonds are not damaged, the active groups amino and hydroxyl groups are introduced at the allylic position, and then other structural fragments are connected with hydroxyl and amino groups as connection points to improve its water solubility and bioavailability, increase the affinity of the drug to the receptor, and thus obtain better anti-tumor activity. However, the modification of other sites of elemene has not been reported.

Summary of the invention

In view of this, the purpose of the present invention is to provide a β-elemene aryl derivative and its preparation method and application. The present invention provides a new β-elemene olefin site aryl derivatization method, and the β-elemene aryl derivative has good anti-tumor (bladder cancer cell T24, colorectal cancer cell SW480, brain glioma cancer cell U251 and brain glioma cancer cell U87) activity.

In order to solve the above problem, the present invention provides a technical solution.

The present invention provides a β-elemene aryl derivative having a structure shown in Formula I:

In the formula I, Includes/>

R ₁ , R ₂ , and R ₃ are independently phenyl, alkoxy, phenylalkenyl, substituted phenylalkenyl, or

R ₄ is an alkylphenyl group or a substituted alkylphenyl group; R ₅ and R ₆ are independently hydroxyl or alkoxy;

_R7 is hydroxyl and/or phenyl;

_R9 includes phenyl or substituted phenyl.

Preferably, when R ₉ is a substituted phenyl group, the substituted phenyl group includes an alkoxy group, a substituted phenyl group or an alkene substituted phenyl group.

Preferably, the compounds including the structures shown in 2a to 2j are:

The present invention also provides a method for preparing the above-mentioned β-elemene aryl derivative, comprising the following steps:

Dissolving β-elemene and an aromatic compound having a structure shown in Formula I-1, and performing a coupling reaction under alkaline conditions and a catalyst to obtain the β-elemene aromatic derivative;

_R8 is -I, -Br or -OTf.

Preferably, the molar ratio of the β-elemene to the aromatic compound having the structure represented by formula I-1 is 1 to 1.5:1.

Preferably, the coupling reaction is carried out at a temperature of 60 to 150° C. and for 24 hours.

Preferably, the catalyst comprises 4-(anthracen-9-yl)-3-(tert-butyl)-2,3-dihydrobenzo[d][1,3]oxaphosphole and Pd(OAc) ₂ .

Preferably, the molar ratio of 4-(anthracen-9-yl)-3-(tert-butyl)-2,3-dihydrobenzo[d][1,3]oxaphosphole to Pd(OAc) ₂ is 2:1.

Preferably, the molar ratio of the aromatic compound having the structure shown in Formula I-1 to Pd(OAc) ₂ is 25:1.

The present invention also provides an application of a compound having a 2c structure in the preparation of drugs for resisting bladder cancer cells T24, resisting colorectal cancer cells SW480, and resisting brain glioma cancer cells U251 and U81.

The present invention provides a β-elemene aryl derivative having a structure shown in Formula I. By introducing a hydrophilic group at the olefin terminal of β-elemene, the solubility of the β-elemene derivative is changed, the bioavailability thereof is improved, and the conformation of the molecule can be changed, the orientation is enhanced, the action targets are increased, or the anti-tumor activity is improved through synergistic effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a hydrogen nuclear magnetic resonance spectrum of compound 2a;

FIG2 is a carbon NMR spectrum of compound 2a;

FIG3 is a hydrogen nuclear magnetic resonance spectrum of compound 2b;

FIG4 is a carbon NMR spectrum of compound 2b;

FIG5 is a hydrogen nuclear magnetic resonance spectrum of compound 2c;

FIG6 is a carbon NMR spectrum of compound 2c;

FIG7 is a hydrogen nuclear magnetic resonance spectrum of compound 2d;

FIG8 is a carbon NMR spectrum of compound 2d;

FIG9 is a hydrogen nuclear magnetic resonance spectrum of compound 2e;

FIG10 is a carbon NMR spectrum of compound 2e;

FIG11 is a hydrogen NMR spectrum of compound 2f;

FIG12 is a carbon NMR spectrum of compound 2f;

FIG13 is a hydrogen nuclear magnetic resonance spectrum of compound 2g;

FIG14 is a carbon NMR spectrum of compound 2g;

FIG15 is a hydrogen NMR spectrum of compound 2h;

FIG16 is a carbon NMR spectrum of compound 2h;

FIG17 is a hydrogen NMR spectrum of compound 2i;

FIG18 is a carbon NMR spectrum of compound 2i;

FIG19 is a hydrogen NMR spectrum of compound 2j;

FIG20 is a carbon NMR spectrum of compound 2j;

FIG21 is a test result of the relative activity of β-elemene and its derivatives against glioma cells;

FIG22 shows the morphological changes of U251 cells observed under a microscope after treatment of β-elemene, compound 2c and compound 2b for 48 hours;

FIG23 shows the morphological changes and relative mortality rates of U251 cells observed under a microscope after treatment of β-elemene, compound 2c and compound 2b for 48 hours;

FIG24 shows the morphological changes and relative mortality rates of U87 cells observed under a microscope after treatment with β-elemene, compound 2c and compound 2b for 48 hours;

FIG25 is the DNA proliferation rate of glioma cells after treatment with compound 2c;

FIG26 shows the changes in the glioma cell cycle after treatment with compound 2c;

FIG27 shows the molecular changes in glioma cells after treatment with compound 2c;

FIG28 shows the apoptosis of glioma cells after treatment with compound 2c;

FIG29 is the effect of compound 2c on the phosphorylation level of glioma cells;

FIG30 is the result of compound 2c treating mouse U87 cell transplant tumors;

FIG31 is the H&E staining result of U87 cell transplant tumor;

Figure 32 shows the results of tissue immunofluorescence (IF) staining of PI3K, phosphorylated PI3K (p-PI3K), AKT, phosphorylated AKT (p-AKT), Ki67, GSK3β, HMOX1, p62, Bcl-2, Bax and other molecules in U87 cell transplant tumors.

Detailed ways

The present invention provides a β-elemene aryl derivative having a structure shown in Formula I:

In the formula I, Includes/>

R ₁ , R ₂ , and R ₃ are independently phenyl, alkoxy, phenylalkenyl, substituted phenylalkenyl, or R ₄ is alkylbenzene or substituted alkylbenzene; R ₅ and R ₆ are independently hydroxyl or alkoxyl; R ₇ is hydroxyl and/or phenyl; R ₉ includes phenyl or substituted phenyl.

In the present invention, the substituent in the substituent in the substituted phenylalkenyl group is preferably a methoxy group;

In the present invention, in R ₄ , the alkylbenzene is preferably ethylbenzene; the substituted alkylbenzene is preferably substituted ethylbenzene; the substituent of the substituted alkylbenzene preferably includes an alkoxy group; the alkoxy group is preferably a methoxy group. In R ₅ and R ₆ , the alkoxy group is preferably a methoxy group.

In the present invention, when R ₉ is a substituted phenyl group, the substituent of the substituted phenyl group preferably includes an alkoxy group or an alkene group, wherein the alkoxy group is preferably a methoxy group; the alkene group is preferably

In the present invention, the β-elemene aryl derivative preferably has structures shown in 2a to 2j:

The present invention also provides a method for preparing the above-mentioned β-elemene aryl derivative, comprising the following steps:

After dissolving β-elemene and an aromatic compound having a structure shown in Formula I-1, a coupling reaction is carried out under alkaline conditions and a catalyst to obtain the β-elemene aromatic derivative;

_R8 is -I, -Br or -OTf.

In the present invention, the catalyst comprises 4-(anthracen-9-yl)-3-(tert-butyl)-2,3-dihydrobenzo[d][1,3]oxaphosphole (rac-AntPhos) and Pd(OAc) ₂ . In the present invention, the alkaline condition is preferably provided by potassium carbonate. In the present invention, the dissolving solvent is preferably DMF. In the present invention, the structural formula of rac-AntPhos is:

In the present invention, the molar ratio of the β-elemene to the aromatic compound having the structure shown in Formula I-1 is preferably 1 to 1.5:1, more preferably 1.2:1. In the present invention, the molar ratio of the 4-(anthracen-9-yl)-3-(tert-butyl)-2,3-dihydrobenzo[d][1,3]oxaphospholene to Pd(OAc) ₂ is 2:1. In the present invention, the molar ratio of the aromatic compound to potassium carbonate is preferably 1:1.5 to 1:2, more preferably 1:1.7 to 1.8. In the present invention, the amount ratio of the aromatic compound to the solvent is 0.1 mmol:0.5 to 2 mL, more preferably 0.1 mmol:1 mL. In the present invention, the molar ratio of the aromatic compound having the structure shown in Formula I-1 to Pd(OAc) ₂ is 25:1.

In the present invention, the coupling reaction temperature is preferably 60-150° C., more preferably 100° C.; the reaction time is preferably 24 h.

In the present invention, the equation of the coupling reaction is:

In the present invention, when the coupling reaction is finished, the reaction is preferably quenched with water. In the present invention, after the coupling reaction, it is preferred to further include extracting the product obtained from the coupling reaction with ethyl acetate, washing the obtained ethyl acetate phase with saturated brine, drying with a desiccant, filtering and concentrating, and separating the concentrated organic phase by silica gel column chromatography. In the present invention, the desiccant is preferably sodium sulfate. In the present invention, the concentration method is preferably reduced pressure distillation. In the present invention, the eluent for silica gel column chromatography separation is preferably a mixture of petroleum ether and ethyl acetate; the volume ratio of petroleum ether to ethyl acetate in the eluent is preferably 95:5 or 98:2.

The invention discloses a beta-elemene aryl derivative prepared by using a palladium catalyst. The method has mild reaction conditions, high yield, good universality of functional groups and low catalyst usage.

The present invention also provides the use of the compound with 2c structure in the preparation of drugs against bladder cancer cell T24, colorectal cancer cell SW480, and brain glioma cancer cell U251 and U81.

It has good inhibitory activity against bladder cancer cells T24, colorectal cancer cells SW480, glioma cancer cells U251 and glioma cancer cells U87, and has strong application potential and economic value in the field of biomedicine.

The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1

Compound 2a: Under nitrogen protection, 28 mg (0.1 mmol) of 4-iodobiphenyl, 0.9 mg (0.04 mmol) of palladium acetate, 3 mg (0.008 mmol) of rac-rac-AntPhos, 24.5 mg (0.12 mmol) of β-elemene and 21 mg of potassium carbonate (0.15 mmol) were added to an 8 mL screw-capped reaction bottle, and then 1 mL of DMF was added to the reaction bottle, the bottle mouth was sealed, and stirred at 100°C for 24 h. After the reaction was completed, 5 mL of water was added to quench the reaction, and 10 mL of ethyl acetate was used each time for extraction for 3 times, the organic phases were combined, the organic phases were washed with saturated brine, the organic phases were separated, the organic phases were dried with an appropriate amount of anhydrous sodium sulfate for 2 hours, filtered, and the solvent was recovered by vacuum distillation. The organic phases were combined, and the residue was purified and concentrated by silica gel column chromatography (petroleum ether/ethyl acetate = 98:2) to obtain a colorless oil 2a (25 mg, 82% yield).

Figure 1 is the H NMR spectrum of compound 2a. It can be seen from Figure 1 that:

¹ H NMR (500 MHz, CDCl ₃ ) δ 7.62 (d, J = 7.7 Hz, 2H), 7.57 (d, J = 7.9 Hz, 2H), 7.50-7.41 (m, 4H), 7.35 (t, J = 7.1 Hz, 1H), 6.40-6.24 (m, 2H), 4.94-4.58 (m, 4H), 2.21-2.10 (m, 1H), 2.05-1.99 (m, 1H), 1.80 (s, 3H), 1.76 (s, 3H), 1.73-1.46 (m, 7H), 1.16 (s, 3H).

Figure 2 is the carbon NMR spectrum of compound 2a. It can be seen from Figure 2 that:

¹³ C NMR (151 MHz, CDCl ₃ ) δ 150.32, 147.62, 142.87, 140.87, 139.47, 137.23, 128.72, 127.19, 127.10, 126.86, 126.37, 124.57, 112.31, 108.32, 53.04, 45.69, 40.01, 39.47, 32.85, 26.80, 25.02, 21.09, 17.14. HRMS (ESI) calculated for [M+H, C ₂₇ H ₃₃ ] ⁺ : 357.2582; found: 357.2584.

Example 2

Compound 2b: Under nitrogen protection, 22 mg (0.1 mmol) 1-bromo-3,5-dimethoxybenzene, 0.9 mg (0.04 mmol) palladium acetate, 3 mg (0.008 mmol) rac-rac-AntPhos, 24.5 mg (0.12 mmol) β-elemene and 21 mg potassium carbonate (0.15 mmol) were added to an 8 mL screw-capped reaction bottle, and then 1 mL DMF was added to the reaction bottle, the bottle mouth was sealed, and stirred at 100 ° C for 24 hours. After the reaction was completed, 5 mL of water was added to quench the reaction, and 10 mL of ethyl acetate was used each time for extraction 3 times, the organic phases were combined, the organic phases were washed with saturated brine, the organic phases were separated, the organic phases were dried with an appropriate amount of anhydrous sodium sulfate for 2 hours, filtered, and the solvent was recovered by vacuum distillation. The organic phases were combined, and the residue was purified and concentrated by silica gel column chromatography (petroleum ether/ethyl acetate = 98:2) to obtain a colorless oil 2b (24.5 mg, 82% yield).

FIG3 is the H NMR spectrum of compound 2a. It can be seen from FIG3 that:

¹ H NMR (500 MHz, CDCl ₃ ) δ7.47 (d, J=8.2 Hz, 2H), 7.37 (d, J=8.2 Hz, 2H), 7.12-6.99 (m, 2H), 6.69 (d, J=2.1 Hz, 2H), 6.42 (t, J=2.0 Hz, 1H), 6.29 (s, 2H), 4.82 (d, J=51.0 Hz, 2H), 4.71 (d, J=44.3 Hz, 2H), 3.85 (s, 6H), 2.19-2.11 (m, 1H), 2.05-1.96 (m, 1H), 1.80 (s, 3H), 1.74 (s, 3H), 1.70-1.43 (m, 6H), 1.15 (s, 3H).

FIG4 is the carbon NMR spectrum of compound 2b. From FIG4, we can see that: ¹³ C NMR (126MHz, CDCl3) δ160.94, 150.32, 147.60, 142.86, 139.44, 137.69, 135.58, 128.93, 127.97, 126.76, 126.26, 124.67, 112.29, 108.31, 104.47, 99.88, 55.34, 53.02, 45.66, 39.96, 39.48, 32.83, 26.78, 25.00, 21.09, 17.10. HRMS (ESI) calculated for [M+H, C23H33O2] ⁺ :341.2481; found:341.2480.

Example 3

Compound 2c: Under nitrogen protection, 25 mg (0.1 mmol) 1-bromo-3,4,5-trimethoxybenzene, 0.9 mg (0.04 mmol) palladium acetate, 3 mg (0.008 mmol) rac-rac-AntPhos, 24.5 mg (0.12 mmol) β-elemene and 21 mg potassium carbonate (0.15 mmol) were added to an 8 mL screw-capped reaction bottle, and then 1 mL DMF was added to the reaction bottle, the bottle mouth was sealed, and stirred at 100 ° C for 24 h. After the reaction was completed, 5 mL of water was added to quench the reaction, and 10 mL of ethyl acetate was used each time for extraction, and the extraction was performed 3 times. The organic phases were combined, washed with saturated brine, separated, and dried with an appropriate amount of anhydrous sodium sulfate for 2 hours, filtered, and the solvent was recovered by vacuum distillation. The organic phases were combined, and the residue was purified and concentrated by silica gel column chromatography (petroleum ether/ethyl acetate = 95:5) to obtain a white solid 2c (31.5 mg, 85% yield).

FIG5 is the H NMR spectrum of compound 2c. It can be seen from FIG5 that:

¹ H NMR (500 MHz, CDCl ₃ ) δ 6.58 (s, 2H), 6.20 (d, J = 16.2 Hz, 1H), 6.13 (d, J = 16.1 Hz, 1H), 4.85 (s, 1H), 4.73 (d, J = 9.6 Hz, 2H), 4.64 (s, 1H), 3.88 (s, 6H), 3.83 (s, 3H), 2.16-2.09 (m, 1H), 2.03-1.94 (m, 1H), 1.77 (s, 3H), 1.72 (s, 3H), 1.68-1.43 (m, 6H), 1.12 (s, 3H).

FIG6 is the carbon NMR spectrum of compound 2c. It can be seen from FIG6 that:

¹³ C NMR (126 MHz, CDCl ₃ ) δ 153.30, 150.26, 147.58, 142.17, 137.20, 133.97, 125.02, 112.35, 108.37, 103.02, 60.91, 56.05, 53.05, 45.71, 40.07, 39.39, 32.85, 26.81, 24.99, 21.10, 17.19. HRMS (ESI) calculated for [M+H, C ₂₄ H ₃₅ O ₃ ] ⁺ : 371.2586; found: 371.2588.

Example 4

Compound 2d: Under nitrogen protection, 21 mg (0.1 mmol) of 6-bromoquinoline, 0.9 mg (0.04 mmol) of palladium acetate, 3 mg (0.008 mmol) of rac-rac-AntPhos, 24.5 mg (0.12 mmol) of β-elemene and 21 mg of potassium carbonate (0.15 mmol) were added to an 8 mL screw-capped reaction bottle, and then 1 mL of DMF was added to the reaction bottle, the bottle mouth was sealed, and stirred at 100 ° C for 24 hours. After the reaction was completed, 5 mL of water was added to quench the reaction, and 10 mL of ethyl acetate was used each time for extraction, and the extraction was performed 3 times. The organic phases were combined, washed with saturated brine, separated, and dried with an appropriate amount of anhydrous sodium sulfate for 2 hours, filtered, and the solvent was recovered by vacuum distillation. The organic phases were combined, and the residue was purified and concentrated by silica gel column chromatography (petroleum ether/ethyl acetate = 95:5) to obtain a colorless oil 2d (26 mg, 78% yield).

FIG7 is the H NMR spectrum of compound 2d. It can be seen from FIG7 that:

¹ H NMR (500 MHz, CDCl ₃ ) δ 8.97-8.84 (m, 1H), 8.42 (d, J = 8.5 Hz, 1H), 8.00 (d, J = 8.3 Hz, 1H), 7.66 (t, J = 7.8Hz,1H),7.59(d,J＝7.1Hz,1H),7.39(dd,J＝8.5,4.1Hz,1H),6.92(d,J＝16.0Hz,1H),6 .25 (d, J = 16.0 Hz, 1H), 4.83 (d, J = 62.9 Hz, 2H), 4.71 (d, J = 26.3 Hz, 2H), 2.23-2.12 (m, 1H), 2.08-1.94 ( m, 1H), 1.78(s, 3H), 1.74(s, 3H), 1.71-1.51(m, 6H), 1.23(s, 3H).

FIG8 is the carbon NMR spectrum of compound 2d. It can be seen from FIG8 that:

¹³ C NMR (126 MHz, CDCl ₃ ) δ 150.14, 150.04, 148.38, 147.39, 147.13, 136.46, 132.32, 129.20, 128.31, 126.28, 123.55, 120.98, 120.60, 112.55, 108.38, 52.92, 45.58, 40.17, 40.01, 32.74, 26.72, 24.75, 21.06, 17.26. HRMS (ESI) calculated for [M+H, C ₂₄ H ₃₀ N] ⁺ : 332.2378; found: 332.2377.

Example 5

Compound 2e: Under nitrogen protection, 20 mg (0.1 mmol) of 5-bromoindole, 0.9 mg (0.04 mmol) of palladium acetate, 3 mg (0.008 mmol) of rac-rac-AntPhos, 24.5 mg (0.12 mmol) of β-elemene and 21 mg of potassium carbonate (0.15 mmol) were added to an 8 mL screw-capped reaction bottle, and then 1 mL of DMF was added to the reaction bottle, the bottle mouth was sealed, and stirred at 100 ° C for 24 hours. After the reaction was completed, 5 mL of water was added to quench the reaction, and 10 mL of ethyl acetate was used each time for extraction, and the organic phases were combined, washed with saturated brine, separated, and dried with an appropriate amount of anhydrous sodium sulfate for 2 hours, filtered, and the solvent was recovered by distillation under reduced pressure. The organic phases were combined, and the residue was purified and concentrated by silica gel column chromatography (petroleum ether/ethyl acetate = 95:5) to obtain a colorless oil 2e (24 mg, 74% yield).

FIG9 is the H NMR spectrum of compound 2e. It can be seen from FIG9 that:

¹ H NMR (500 MHz, CDCl ₃ ) δ 8.08 (s, 1H), 7.62 (s, 1H), 7.32 (q, J = 8.5 Hz, 2H), 7.19 (t, J = 2.6 Hz, 1H), 6.54 (s, 1H), 6.40 (d, J = 16.2 Hz, 1H), 6.20 (d, J = 16.2 Hz, 1H), 4.82 (d, J = 47.3 Hz, 2H), 4.71 (d, J = 39.3 Hz, 2H), 2.22-2.08 (m, 1H), 2.07-1.95 (m, 1H), 1.78 (d, J = 19.2 Hz, 6H), 1.70-1.52 (m, 6H), 1.16 (s, 3H).

FIG10 is the carbon NMR spectrum of compound 2d. It can be seen from FIG10 that:

¹³ C NMR (126 MHz, Chloroform-d) δ 161.49, 150.56, 148.01, 140.12, 135.11, 130.42, 128.20, 125.83, 124.48, 120.48, 118.35, 112.07, 111.01, 108.25, 53.16, 45.80, 40.23, 39.28, 33.02, 26.94, 25.27, 21.14, 17.27. HRMS (ESI) calculated for [M+H, C ₂₃ H ₃₀ N] ⁺ : 320.2378; found: 320.2388.

Example 6

Compound 2f: Under nitrogen protection, 39 mg (0.1 mmol) (E)-4-(3,5-dimethoxyphenyl)phenyl trifluoromethanesulfonate, 0.9 mg (0.04 mmol) palladium acetate, 3 mg (0.008 mmol) rac-rac-AntPhos, 24.5 mg (0.12 mmol) β-elemene and 21 mg potassium carbonate (0.15 mmol) were added to an 8 mL screw-capped reaction bottle, and then 1 mL DMF was added to the reaction bottle, the bottle mouth was sealed, and stirred at 100 ° C for 24 hours. After the reaction was completed, 5 mL of water was added to quench the reaction, and 10 mL of ethyl acetate was used each time for extraction 3 times, the organic phases were combined, the organic phases were washed with saturated brine, the organic phases were separated, the organic phases were dried with an appropriate amount of anhydrous sodium sulfate for 2 hours, filtered, and the solvent was recovered by vacuum distillation. The organic phases were combined, and the residue was purified and concentrated by silica gel column chromatography (petroleum ether/ethyl acetate = 95:5) to obtain a colorless oil 2f (36 mg, 82% yield).

FIG11 is the H NMR spectrum of compound 2f. It can be seen from FIG11 that:

¹ H NMR (500 MHz, CDCl ₃ ) δ7.47 (d, J=8.2 Hz, 2H), 7.37 (d, J=8.2 Hz, 2H), 7.12-6.99 (m, 2H), 6.69 (d, J=2.1 Hz, 2H), 6.42 (t, J=2.0 Hz, 1H), 6.29 (s, 2H), 4.82 (d, J=51.0 Hz, 2H), 4.71 (d, J=44.3 Hz, 2H), 3.85 (s, 6H), 2.19-2.11 (m, 1H), 2.05-1.96 (m, 1H), 1.80 (s, 3H), 1.74 (s, 3H), 1.70-1.43 (m, 6H), 1.15 (s, 3H).

FIG12 is the carbon NMR spectrum of compound 2d. It can be seen from FIG12 that:

¹³ C NMR (126 MHz, Chloroform-d) δ 160.94, 150.32, 147.60, 142.86, 139.44, 137.69, 135.58, 128.93, 127.97, 126.76, 126.26, 124.67, 112.29, 108.31, 104.47, 99.88, 55.34, 53.02, 45.66, 39.96, 39.48, 32.83, 26.78, 25.00, 21.09, 17.10. HRMS (ESI) calculated for [M+H, C ₃₁ H ₃₉ O ₂ ] ⁺ : 443.2950; found: 443.2949.

Example 7

Compound 2g: Under nitrogen protection, 52 mg (0.1 mmol) of the compound, 0.9 mg (0.04 mmol) of palladium acetate, 3 mg (0.008 mmol) of rac-rac-AntPhos, 24.5 mg (0.12 mmol) of β-elemene and 21 mg of potassium carbonate (0.15 mmol) were added to an 8 mL screw-capped reaction bottle, and then 1 mL of DMF was added to the reaction bottle, the bottle mouth was sealed, and stirred at 100 ° C for 24 hours. After the reaction was completed, 5 mL of water was added to quench the reaction, and 10 mL of ethyl acetate was used each time for extraction, and the extraction was performed 3 times. The organic phases were combined, washed with saturated brine, separated, and dried with an appropriate amount of anhydrous sodium sulfate for 2 hours, filtered, and the solvent was recovered by vacuum distillation. The organic phases were combined, and the residue was purified and concentrated by silica gel column chromatography (petroleum ether/ethyl acetate = 95:5) to obtain 2 g (14 mg, 23% yield) of colorless oil.

FIG13 is a hydrogen nuclear magnetic resonance spectrum of compound 2g. It can be seen from FIG13 that:

¹ H NMR (600 MHz, Chloroform-d) δ8.22 (d, J = 8.2 Hz, 1H), 7.99 (s, 1H), 7.52 (d, J = 8.2 Hz, 2H), 7.43 (t, J = 7.8 Hz, 2H), 7.37(s, 1H), 7.26(s, 1H), 6.46(d, J＝16.2Hz, 1H), 6.36(d, J＝16.3Hz, 1H), 6.33- 6.22 (m, 2H), 4.86 (d, J = 12.0 Hz, 2H), 4.80-4.69 (m, 4H), 4.65 (d, J = 11.7 Hz, 2H), 2.21-2.07 (m, 2H), 2.05 -1.96(m,2H),1.78(s,6H),1.71(s,6H),1.66-1.46(m,12H),1.16(s,3H),1.13(s,3H).

FIG14 is a carbon NMR spectrum of compound 2g. It can be seen from FIG14 that:

¹³ C NMR (151 MHz, Chloroform-d) δ 175.97, 156.66, 152.68, 150.36, 150.11, 147.62, 147.19, 147.05, 143.98, 143.25, 138.13, 130.27, 129.00, 126.52, 126.06, 125.16, 124.68, 123.97, 123.05, 12 3.00,114.70,112.71,112.30,108.47,108.30,53.06,53.01,45.72,45.62,40.01,39.92,39.79,39.50,32.89,32.73,26.82,26.69,25.00,24.72,21.08,17.12,17.08.HRMS(ESI) calculated for [M+H, C ₄₅ H ₅₅ O ₂ ] ⁺ : 627.4202; found: 627.4204.

Example 8

Compound 2h: Under nitrogen protection, 40 mg (0.1 mmol) of the compound, 0.9 mg (0.04 mmol) of palladium acetate, 3 mg (0.008 mmol) of rac-rac-AntPhos, 24.5 mg (0.12 mmol) of β-elemene and 21 mg of potassium carbonate (0.15 mmol) were added to an 8 mL screw-capped reaction bottle, and then 1 mL of DMF was added to the reaction bottle, the bottle mouth was sealed, and stirred at 100 ° C for 24 h. After the reaction was completed, 5 mL of water was added to quench the reaction, and 10 mL of ethyl acetate was used each time for extraction, and the extraction was performed 3 times. The organic phases were combined, washed with saturated brine, separated, and dried with an appropriate amount of anhydrous sodium sulfate for 2 h, filtered, and the solvent was recovered by vacuum distillation. The organic phases were combined, and the residue was purified and concentrated by silica gel column chromatography (petroleum ether/ethyl acetate = 95:5) to obtain a colorless oil 2h (22 mg, 48% yield).

FIG15 is a hydrogen nuclear magnetic resonance spectrum of compound 2h. It can be seen from FIG15 that:

¹ H NMR (600 MHz, Chloroform-d) δ8.21 (d, J = 8.2 Hz, 1H), 7.95 (s, 1H), 7.51 (d, J = 8.5 Hz, 2H), 7.43 (d, J = 7.9 Hz, 1H), 7.37(s, 1H), 6.97(d, J＝8.5Hz, 2H), 6.45(d, J＝16.2Hz, 1H), 6.35(d, J＝16 .2Hz,1H),4.81(d,J＝68.1Hz,2H),4.70(d,J＝49.0Hz,2H),3.84(s,3H),2.15(dd,J＝10.9,4.6Hz,1H) ,2.00(t,J＝11.1Hz,1H),1.78(s,3H),1.71(s,3H),1.69-1.43(m,6H),1.16(s,3H).

FIG16 is the carbon NMR spectrum of compound 2h. It can be seen from FIG16 that:

¹³ C NMR (151 MHz, Chloroform-d) δ 176.14, 159.58, 156.66, 152.37, 150.10, 147.18, 146.98, 143.89, 130.08, 126.46, 124.95, 124.23, 123.95, 122.96, 122.93, 114.69, 113.96, 112.68, 108.45, 55.31, 52.97, 45.59, 39.89, 39.75, 32.70, 26.66, 24.72, 21.06, 17.04. HRMS (ESI) calculated for [M+H, C ₃₁ H ₃₅ O ₃ ] ⁺ :455.2586; found:455.2588.

Example 9

Compound 2i: Under nitrogen protection, 46 mg (0.1 mmol) of the compound, 0.9 mg (0.04 mmol) of palladium acetate, 3 mg (0.008 mmol) of rac-rac-AntPhos, 24.5 mg (0.12 mmol) of β-elemene and 21 mg of potassium carbonate (0.15 mmol) were added to an 8 mL screw-capped reaction bottle, and then 1 mL of DMF was added to the reaction bottle, the bottle mouth was sealed, and stirred at 100 ° C for 24 hours. After the reaction was completed, 5 mL of water was added to quench the reaction, and 10 mL of ethyl acetate was used each time for extraction, and the extraction was performed 3 times, the organic phases were combined, the organic phases were washed with saturated brine, the organic phases were separated, the organic phases were dried with an appropriate amount of anhydrous sodium sulfate for 2 hours, filtered, and the solvent was recovered by vacuum distillation. The organic phases were combined, and the residue was purified and concentrated by silica gel column chromatography (petroleum ether/ethyl acetate = 95:5) to obtain a colorless oil 2i (40 mg, 77% yield).

FIG17 is a hydrogen nuclear magnetic resonance spectrum of compound 2i. It can be seen from FIG17 that:

¹ H NMR (600 MHz, Chloroform-d) δ7.54 (d, J = 7.8 Hz, 2H), 7.37 (t, J = 7.6 Hz, 2H), 7.28 (t, J = 7.3 Hz, 1H), 7.20 ( d, J = 7.9 Hz, 2H), 7.13 (d, J = 7.9 Hz, 2H), 7.00 (d, J = 7.9 Hz, 2H), 6.91 (d, J = 8.0 Hz, 1H), 6.82 (t, J＝7.4Hz,1H),6.21(d,J＝16.3Hz,1H),6.16(d,J＝16.3Hz,1H),4.78(d,J＝ 57.8 Hz, 2H), 4.67 (d, J = 56.4 Hz, 2H), 4.22 (d, J = 11.2 Hz, 1H), 4.03 (d, J = 11.2 Hz, 1H), 3.76 (d, J = 7.5 Hz ,1H),3.16(d,J＝15.2Hz,1H),2.78(dd,J＝15.3,8.4Hz,1H),2.57(s,1H),2.14-2.05(m,1H),2.01-1.92( m, 1H), 1.76(s, 3H), 1.70(s, 3H), 1.67-1.39(m, 6H), 1.10(s, 3H).

FIG18 is the carbon NMR spectrum of compound 2i. It can be seen from FIG18 that:

¹³ C NMR (151 MHz, Chloroform-d) δ 152.87, 150.31, 147.64, 142.14, 142.05, 140.12, 135.90, 129.29, 128.75, 128.53, 127.67, 127.51, 126.08, 125.99, 125.44, 124.67, 121.46, 116.71, 112.24, 108.30, 73.65, 72.28, 53.06, 45.74, 45.70, 40.02, 39.32, 35.93, 32.87, 26.82, 25.00, 21.06, 17.15. HRMS (ESI) calculated for [M+H, C ₃₇ H ₄₃ O ₂ ] ⁺ : 519.3263; found: 519.3265.

Example 10

Compound 2j: Under nitrogen protection, 43 mg (0.1 mmol) of the compound, 0.9 mg (0.004 mmol) of palladium acetate, 3 mg (0.008 mmol) of rac-rac-AntPhos, 24.5 mg (0.12 mmol) of β-elemene and 21 mg of potassium carbonate (0.15 mmol) were added to an 8 mL screw-capped reaction bottle, and then 1 mL of DMF was added to the reaction bottle, the bottle mouth was sealed, and stirred at 100 ° C for 24 hours. After the reaction was completed, 5 mL of water was added to quench the reaction, and 10 mL of ethyl acetate was used each time for extraction, and the organic phases were combined, washed with saturated brine, separated, and dried with an appropriate amount of anhydrous sodium sulfate for 2 hours, filtered, and the solvent was recovered by vacuum distillation. The organic phases were combined, and the residue was purified and concentrated by silica gel column chromatography (petroleum ether/ethyl acetate = 95:5) to obtain a colorless oil (18 mg, 37% yield).

FIG19 is a hydrogen nuclear magnetic resonance spectrum of compound 2j. It can be seen from FIG19 that:

¹ H NMR (600 MHz, Chloroform-d) δ 14.04 (s, 1H), 7.32 (d, J = 7.9 Hz, 2H), 7.20 (d, J = 10.5 Hz, 2H), 6.24 (q, J = 16.3 Hz, 2H), 6.10(s, 1H), 5.95(s, 1H), 4.81(d, J＝57.3Hz, 2H), 4.70(d, J＝5 3.7Hz, 2H), 3.85(d, J＝6.2Hz, 6H), 3.33(q, J＝7.8Hz, 2H), 3.00(q, J＝7.1Hz, 2H), 2.19-2.06(m, 1H) ,2.06-1.95(m,1H),1.79(s,3H),1.74(s,3H),1.64-1.43(m,6H),1.13(s,3H).

FIG20 is the carbon NMR spectrum of compound 2j. It can be seen from FIG20 that:

¹³ C NMR (151 MHz, Chloroform-d) δ 204.51, 167.71, 165.96, 162.73, 150.39, 147.71, 142.07, 140.27, 136.02, 128.62, 126.05, 124.76, 112.24, 108.29, 105.78, 93.70, 90.85, 55.59, 55.54, 53.11, 45.74, 40.10, 39.35, 32.92, 30.39, 26.86, 25.01, 21.08, 18.45, 17.21. HRMS (ESI) calculated for [M+H, C ₃₂ H ₄₁ O ₄ ] ⁺ :489.3005; found:489.3003.

Test Example 1:

Cell culture and main reagents: Glioma U87 (HTB-14, ATCC, USA) cells were obtained from ATCC cell bank. Glioma U251 (BNCC100123, BNCC, China) cells were obtained from BeNa Culture Collection. U251 and U87 cells were cultured in DMEM (06-1055-57-1ACS, BI, Israel) containing 10% fetal bovine serum (10099-141, Gibco, Australia) and 1% penicillin and streptomycin (UB89609, Bioodin, China). All cells were cultured in a humidified incubator at 37°C in 5% CO2.

Calcein AM fluorescent probe was used to label living cells for cell activity detection. CCK8 and CCK-F kits were used to test the compounds. According to the instructions, the half-inhibitory concentration IC50 of the above-mentioned elemene aryl derivatives on human bladder cancer T24 cells, human colorectal cancer SW480 cells, human brain glioma U251 and U87 cells, and human normal embryonic kidney 293T cells was determined, as shown in Table 1.

Table 1 IC50 test results

Compound T24 SW480 U251 U87 293T β-elemene ＞150μM ＞150μM 175μM 154μM ＞150μM Compound 2a ＞300μM ＞300μM ＞300μM ＞300μM ＞300μM Compound 2f ＞300μM ＞300μM ＞300μM ＞300μM ＞300μM Compound 2c 47μM 42μM 45μM 95μM 185μM Compound 2b 81μM 62μM 68μM 134μM 271μM Compound 2d 40uM 41uM 42uM 44uM 128μM Compound 2e 41uM 54uM 41uM 86uM 132μM

As can be seen from Table 1: Anti-tumor activity test: The results show that the IC50 of compound 2c against human bladder cancer cell T24, colorectal cancer cell SW480, brain glioma cells U251 and U87, and human normal embryonic kidney cell 293T are 47μM, 42μM, 45μM, 95μM, and 185μM, respectively. It has good anti-tumor cell biological activity, and has good biological safety while inhibiting tumor cells; compound 2c and β-elemene have significant improvements in inhibiting brain glioma cells U251 and U87, and have low toxicity.

Test Example 2

Biological activity and mechanism of action of β-elemene derivatives on brain glioma cells

In the in vitro cell model, the cytotoxicity, cell morphology and killing ability of compounds 2a, 2f and 2b on glioma cells were inferior to those of compound 2c. Therefore, compound 2c was selected to study its molecular mechanism of inhibiting glioma cells.

In Figures 21 to 32 of the present invention, P values were obtained by two-tailed t-test; *p<0.05; **p<0.01; ***p<0.001; ns, not significant.

(1) Anti-glioma (U251 and U87) activity of β-elemene and its derivatives

Figure 21 is the relative activity test result of β-elemene and its derivatives against glioma cells. As shown in Figure 21, the IC50 values of β-elemene, compound 2a, compound 2f, compound 2c and compound 2b against U251 and U87 cells are 175μM and 155μM, >300μM and >300μM, >300μM and >300μM, 45μM and 95μM, 76μM and 138μM, respectively. Compared with β-elemene, compound 2a and compound 2f have weaker inhibitory activity against the two glioma cells. Compared with β-elemene and compound 2b, compound 2c has stronger inhibitory activity against the activity of the two glioma cells, all of which are statistically significant.

(2) Effects of β-elemene and its derivatives on morphological changes of glioma U251 cells

Figure 22 shows that U251 cells were treated with β-elemene, compound 2c and compound 2b for 48 hours, and the cell morphology changes were observed under a microscope. The results (Figure 22A) show that β-elemene, compound 2c and compound 2b can shrink cells (as shown by the blue arrow), and even non-adherent floating cells appear (as shown by the yellow arrow). Quantification was performed using ImageJ software, and the bar graph shows the quantitative statistical results. The proportions of cell shrinkage (Figure 22B) and non-adherent cells (Figure 22C) of U251 cells in the three groups were significantly different from those in the control groups of each group, and were statistically significant. In addition, the difference between the compound 2c treatment group and the control group was more obvious.

(3) Cytotoxicity of β-elemene and its derivatives on glioma U251 cells

Figure 23 shows that β-elemene, compound 2c and compound 2b were used to treat U251 cells for 48 hours, and the cell morphological changes and relative mortality rates were observed under a microscope. The fluorescence results of using β-elemene, compound 2c and compound 2b to treat U251 cells showed that with the increase of treatment concentration, the staining of live cells and dead cells in the three treatment groups showed a trend of gradual decrease (Figure 23A). Quantification was performed using ImageJ software, and the bar graph showed the quantitative statistical results. The relative cell activity (Figure 23B) and relative death ratio (Figure 23C) of U251 cells in the three groups were significantly different from those in the control groups of each group. In addition, the killing of U251 cells by the compound 2c treatment group was more obvious.

(4) Inhibitory effect of compound 2c on glioma U87 cells

After verification of the above experimental results, it was shown that compound 2c has more excellent anticancer activity. In order to further verify the universality of the inhibitory killing activity of compound 2c on glioma cells, compound 2c was used to treat another type of glioma U87 cells. Quantification was performed by ImageJ software, and the bar graph showed the quantitative statistical results. The results showed that the U87 cells treated with compound 2c also showed atrophic cells and non-adherent cells (Figure 24A). As shown in Figure 24C, with the increase of treatment concentration, the number of morphological changes in U87 cells increased. Quantification was performed by ImageJ software, and the bar graph showed the quantitative statistical results. The ratio of cell atrophy and non-adherent cells of U87 cells in the compound 2c treatment group was significantly different from that in the control group, which was statistically significant. In addition, the staining of live and dead cells in the compound 2c treatment group showed a gradually decreasing trend (Figure 24B). Quantitative statistical comparison showed that the relative cell activity and relative death ratio of U87 cells in the compound 2c treatment group were significantly different from those in the control group (Figure 24D), which was statistically significant.

(5) Compound 2c inhibits glioma cell DNA proliferation

In order to study the effect of compound 2c on DNA proliferation of brain glioma cells, EdU (5-ethynyl-2'-deoxyuridine) method was used to detect DNA replication, and quantification was performed using ImageJ software. The bar graph showed the quantitative statistical results.

Specifically, the brain glioma cell samples treated with compound 2c were co-incubated with EdU at a final concentration of 10 μM for 30 minutes, washed once with PBS, and 1 mL of 4% paraformaldehyde (BL539A, Biosharp, China) solution was added to each sample to fix the cells, and incubated at room temperature for 15 minutes; the paraformaldehyde was discarded and washed once with PBS, and 1 mL of 0.3% Triton-X-100 (ST795, Beyotime, China) solution was added to each sample to permeabilize the cells, and incubated at room temperature for 15 minutes; Triton-X-100 was discarded, and 1 mL of 3% BSA (ST023-1000g, Beyotime, China) solution was added to each sample to wash twice; Click reaction solution was prepared according to the instructions of the EdU cell proliferation detection kit (C0078S, Beyotime, China) to perform Click reaction on the cell samples in the dark; 3% BSA was used to wash twice, and Hoechst 33342 stain solution, incubate at room temperature away from light to color the cell nucleus; wash twice with PBS, observe and photograph under an inverted fluorescence microscope in a PBS environment.

As shown in Figure 25, the red fluorescence signal of EdU gradually decreased, indicating that with the treatment of compound 2c, the DNA proliferation of brain glioma cells became less and less active. Quantitative statistical results showed that the DNA proliferation rate (EdU/H33342) of U251 and U87 cells was significantly reduced after treatment with compound 2c. There was no significant difference with the control group at 50μM, and at 100μM and 200μM, the treatment group showed significant differences compared with the control group.

(6) Compound 2c blocks the cell cycle of glioma cells

Glioma cells treated with compound 2c were digested and collected, and 1 mL of 70% cold ethanol was added to each sample for resuspending. The cell samples were fixed in an ice bath for 30 minutes and left to stand at 4°C overnight. The samples were centrifuged at 2500 rpm for 10 minutes, 70% cold ethanol was discarded, and the samples were washed twice with PBS. Glioma cells treated with compound 2c were collected with 0.25% trypsin (25200-056, Gibco, USA). A mixed stain of 5 μg/mL propidium iodide (KGA214, KeyGEN, China), 0.2% Triton X-100, and 100 μg/mL RNase A (ST578, Beyotime, China) was added to each sample, and the cell samples were incubated at room temperature in the dark. Flow cytometry (Beckman Coulter, Brea, CA, USA) was used for analysis, and ImageJ software was used for quantification. The bar graph shows the quantitative statistical results. The results showed that compound 2c significantly reduced the G2 phase proportion of glioma cells, which may mean that compound 2c blocked the transition of glioma cells from G0/G1 phase to S and G2 phase (Figures 26A and 26B).

(7) Compound 2c affects the molecular expression of glioma cells

In order to clarify the effect of compound 2c on the level of ROS in glioma cells. Glioma cells were treated with different concentrations of compound 2c, and the culture medium was replaced with serum-free culture medium containing 2μM H2DCFDA (HY-D0940, MCE, China), and incubated in a 37°C incubator in the dark for 30 minutes. Glioma cell samples treated with different concentrations of compound 2c were digested with 0.25% trypsin, and the samples were collected by centrifugation at 1000rpm for 4 minutes. Pure DMSO culture medium was added to resuspend the samples for flow cytometry analysis and detection, and quantification was performed using ImageJ software, and the bar graph showed the quantitative statistical results. The results showed that the ROS level of U251 cells was significantly higher than that of the control group when the concentration of compound 2c was 100μM (Figure 27A).

In order to analyze whether compound 2c changes the protein level of glioma cells, the present invention uses protein immunoblotting to analyze the expression of protein molecules in glioma cells treated with compound 2c. The primary antibodies used were rabbit anti-CDK2 (1:1500, WL01543, Wanleibio, China), rabbit anti-HMOX1 (1:3000, 10701-1-AP, Proteintech, China), mouse anti-SQSTM1/p62 (1:200, sc-28359, Santa, USA), rabbit anti-PI3K (1:500, bs-10657R, Bioss, China), rabbit anti-p-PI3K (1:500, bs-3332R, Bioss, China), mouse anti-AKT (1:5000, 60203-2-Ig, Proteintech, China), rabbit anti-p-AKT (1:5000, 80455-1-RR, Proteintech, China), rabbit anti-GSK3β(1:1000,22104-1-AP,Proteintech,China), rabbit anti-Bcl-2(1:500,WL01556,Wanleibio,China), rabbit anti-Bax(1:500,WL01637,Wanleibio,China), rabbit anti-Cleaved caspase-1(1:1000,4199,Cell Signaling Technology,USA), rabbit anti-Cleaved gasdermin D(1:1000,36425,Cell Signaling Technology,USA), rabbit anti-p-MLKL(1:1000,91689,Cell Signaling Technology,USA), rabbit anti-p-RIP3(1:1000,93654,Cell Signaling Technology,USA), mouse Anti-GAPDH (1:2000, TA802519, Origene, USA). Horseradish peroxidase (HRP) was used for the secondary antibody, such as HRP Goat anti-Mouse IgG (H+L) (1:3000, AS003, ABclonal, China), HRP Goat anti-Rabbit IgG (H+L) (1:3000, AS014, Abclonal, China). Enhanced chemiluminescence detection solution ECL (P0018AS, Beyotime, China) was used to expose the signal.

WB results confirmed the above experimental results. Compound 2c increased HMOX1 and p62 molecules in glioma cells. The increase of these two molecules will cause the increase of intracellular ROS levels and induce cellular oxidative stress. In addition, compound 2c reduced CDK2 molecules in glioma cells, which also echoed the previous cell cycle arrest results (Figure 27B).

In summary: Compound 2c inhibited the expression of CDK2 molecules to block the cell cycle of glioma cells and thus inhibited proliferation, while promoting the expression of HMOX1 and p62 molecules to increase the level of ROS in glioma cells and induce cell apoptosis.

(8) Compound 2c induces apoptosis of glioma cells

In order to find out whether compound 2c can induce apoptosis of glioma cells. Glioma cells treated with compound 2c were digested using 0.25% Trypsin (T1350, Solarbio, China) without EDTA, and the cells were collected by centrifugation at 1000 rpm for 4.00 min and washed once with PBS. The staining solution was prepared using the Annexin V-FITC apoptosis detection kit (C1062M, Beyotime, China) according to the instructions. The staining solution was added to each sample and incubated at room temperature for 20 minutes in the dark. The cell samples were detected by flow cytometry.

After treatment with compound 2c, glioma cells showed apoptosis ( FIGS. 28A and 28B ), and as the treatment concentration increased, the proportion of apoptotic cells gradually increased ( FIGS. 28C and 28D ).

(9) Compound 2c affects the Wnt/β-catenin, p53, PI3K/AKT and other pathways in brain glioma cells

Phosphorylation kinase chip was used to detect the effect of compound 2c on the phosphorylation level of glioma cells. The experiment was performed using human phosphorylation kinase array reagent (ARY003C, R&D Systems, USA). Cell samples were operated according to the instructions of the manual. Signal molecules were incubated with the developer of the kit and exposed to light in the luminescence detection system.

As shown in Figure 29A, compared with the control group, the phosphorylation levels of AKT, GSK3β, p53, HSP60, RSK1/2/3 and other molecules in the compound 2c treatment group were reduced to varying degrees. This suggests that compound 2c may affect the classic Wnt/β-catenin, p53, PI3K/AKT and other pathways to inhibit glioma cells. The results are easily confirmed, as shown in Figure 29B, the molecular levels of PI3K, AKT and GSK3β are reduced. At the same time, Bcl-2 molecules are reduced and Bax molecules are increased, further proving that compound 2c induces apoptosis in glioma cells (Figure 29B). We tried to explore the cause of non-apoptotic cell death, and the results showed that the pyroptosis indicator molecule Cleavedcaspase-1 molecules were reduced, and the Cleaved Gasdermin D molecules were almost unchanged, indicating that compound 2c did not induce pyroptosis in glioma cells. Changes in the phosphorylation molecules of necroptosis indicator molecules such as MLKL and RIP3 do not point to necroptosis (Figure 29C). As shown in Figure 29D, when U87 cells were treated with 0.06 μM pyroptosis inhibitor, the relative activity of the cells was statistically different, indicating that this concentration of pyroptosis inhibitor had produced a certain cytotoxicity to U87 cells. Therefore, 0.01-0.06 μM pyroptosis inhibitor was used to rescue the cell activity of U87 cells treated with compound 2c. The results showed that the pyroptosis inhibitor targeting Gasdermin D was ineffective in rescuing the cytotoxicity of compound 2c. This once again verified that the non-apoptotic reason for compound 2c to kill glioma cells was not pyroptosis.

Test Example 3

β-elemene derivative compound 2c inhibits the growth of brain glioma transplanted tumors in mice

U87 glioma cell transplantation model in female Balb/c nude mice

1.1×10 ⁷ U87 cells were implanted subcutaneously into 8 Balb/c female nude mice (5 weeks old) from Jinan Pengyue Experimental Animal Breeding Co., Ltd. When the subcutaneous tumor volume reached about 60 mm ³ , the nude mice were randomly divided into two groups, with 4 mice in each group. The compound 2c-treated group was intraperitoneally injected with 27.8 mg/kg of compound 2c (dissolved in dimethyl sulfoxide (DMSO) and then diluted with corn oil for administration), and the control group was given an equal dose of solvent. The mice were injected once every 2 days, and the changes in body weight and tumor volume were monitored at the same time. The mice were euthanized in carbon dioxide, and the transplanted tumors were dissected to remove the transplanted tumors, and the transplanted tumors were photographed and weighed.

Hematoxylin-eosin (H&E) staining of transplanted tumor tissue

The obtained transplanted tumor tissue was dehydrated, embedded and sliced, and then baked in a 68°C oven for 2 hours. Dewaxed in the order of xylene, xylene, anhydrous ethanol, anhydrous ethanol, 95% ethanol, and 80% ethanol for 5 minutes. Hydrated in tap water and ultrapure water for 5 minutes. Stained using a hematoxylin-eosin staining kit (G1120, Solarbio, China). Soaked in hematoxylin stain for 2 minutes, rinsed twice with ultrapure water, differentiated in differentiation solution for 3 minutes, rinsed twice with ultrapure water, stained in eosin stain for 1 minute, and extracted in ultrapure water for 3 times. Soaked in the order of 80% ethanol, 95% ethanol, anhydrous ethanol, anhydrous ethanol, xylene, and xylene for 5 minutes for dehydration. Seal the slides with neutral resin glue (10004160, Sinopharm Group, China) and dry naturally. Scan the tissue sections and observe them on a pathology slide scanner (3DHISTECH, Hungary).

Immunohistochemistry (IHC) staining of transplanted tumors

The obtained transplanted tumor tissue was dehydrated, embedded and sliced, and then baked in a 68℃ oven for 2h. Dewaxed in the order of xylene, xylene, anhydrous ethanol, anhydrous ethanol, 95% ethanol, and 80% ethanol for 5min. Hydrated in tap water and ultrapure water for 5min. Improved sodium citrate antigen retrieval solution (50×) (P0083, Beyotime, China) was diluted to 1× in ultrapure water. The hydrated tissue sections were immersed in antigen retrieval solution (1×), heated to 95℃-100℃ in a water bath, maintained for 15min for antigen retrieval, and left to cool naturally at room temperature. Washed once in PBS, circled the tissue with an immunohistochemistry pen, and added an appropriate amount of ready-to-use 3% H ₂ O ₂ in the ready-to-use rabbit/mouse IgG-two-step immunohistochemistry kit (SV0004, Boster, China) to inactivate endogenous tissue peroxidase, and inactivated at room temperature for 30min in a dark place. The 3% H ₂ O ₂ was discarded and the cells were washed twice in PBS. An appropriate amount of ready-to-use normal goat serum (AR0009, Boster, China) was added for blocking and the cells were left to stand at room temperature for 30 min. The blocking solution was discarded, and appropriate amounts of pre-prepared rabbit anti-PI3K (1:100, bs-10657R, Bioss, China), rabbit anti-p-PI3K (1:100, bs-3332R, Bioss, China), mouse anti-AKT (1:100, 60203-2-Ig, Proteintech, China), rabbit anti-GSK3β (1:100, 22104-1-AP, Proteintech, China), rabbit anti-HMOX1 (1:200, 10701-1-AP, Proteintech, China), mouse anti-SQSTM1/p62 (1:50, sc-28359, Santa Clara, USA), rabbit anti-Bcl-2 (1:100, WL01556, Wanleibio, China), rabbit Anti-Bax (1:50, A0207, Abclonal, China) primary antibody dilution was incubated at 4°C overnight. Washed 4 times in PBST, added an appropriate amount of polymerized HRP secondary antibody in the immunohistochemistry kit, and incubated at room temperature for 30 minutes. Discarded the secondary antibody, washed twice in PBST, added an appropriate amount of pre-prepared DAB color reagent (AR1022, Boster, China) for color development, and immersed in ultrapure water to stop color development after color development. Add an appropriate amount of Mayor's hematoxylin (AR0005, Boster, China) for counterstaining for 2 minutes, washed in ultrapure water, and immersed in alkaline PBS for blueing for 1 minute. Dehydrated by soaking in 80% ethanol, 95% ethanol, anhydrous ethanol, anhydrous ethanol, xylene, and xylene for 5 minutes respectively. Seal the slides with neutral resin glue and dry them naturally. Scan the tissue sections on a pathology section scanner and observe them.

Immunofluorescence (IF) staining of transplanted tumor tissue

After dehydrating, embedding and slicing the obtained transplanted tumor tissue, bake the slices in a 68°C oven for 2 hours. Dewax by soaking in xylene, xylene, anhydrous ethanol, anhydrous ethanol, 95% ethanol, and 80% ethanol for 5 minutes respectively. Hydrate by soaking in tap water and ultrapure water for 5 minutes respectively. Soak the tissue slices in the pre-prepared improved sodium citrate antigen repair solution (1×), heat to 95°C-100°C in a water bath, keep for 15 minutes for antigen repair, and let it cool naturally at room temperature. Wash once in PBS, circle the tissue with an immunohistochemistry pen, add an appropriate amount of ready-to-use normal goat serum for blocking, and let it stand at room temperature for 30 minutes. The blocking solution was discarded, and appropriate amounts of pre-prepared rabbit anti-PI3K (1:150, bs-10657R, Bioss, China), rabbit anti-p-PI3K (1:100, bs-3332R, Bioss, China), mouse anti-AKT (1:100, 60203-2-Ig, Proteintech, China), rabbit anti-p-AKT (1:100, 80455-1-RR, Proteintech, China), rabbit anti-Ki67 (1:800, AB15580, Abcam, UK), rabbit anti-GSK3β (1:1000, 22104-1-AP, Proteintech, China), rabbit anti-HMOX1 (1:200, 10701-1-AP, Proteintech, China), mouse Anti-SQSTM1/p62 (1:50, sc-28359, Santa, USA), rabbit anti-Bcl-2 (1:200, WL01556, Wanleibio, China), rabbit anti-Bax (1:200, A0207, Abclonal, China) primary antibody diluent was incubated overnight at 4°C. After washing 4 times in PBST, appropriate amount of pre-prepared CoraLite 594-conjugated Goat anti-rabbit IgG (H+L) (1:250, SA00013-4, Proteintech, China) and CoraLite 488-conjugated Goat anti-mouse IgG (H+L) (1:250, SA00013-1, Proteintech, China) fluorescent secondary antibodies were added dropwise and incubated at room temperature for 2 hours in the dark. Discard the fluorescent secondary antibody, wash once in PBST, add an appropriate amount of pre-prepared DAPI (1:1000, C1002, Beyotime, China) in the dark, and incubate at room temperature for 10 min in the dark. After washing once in PBST and PBS respectively, use anti-fluorescence quenching sealing solution (P0126, Beyotime, China) to seal the slices. Observe the tissue sections under an inverted fluorescence microscope and take fluorescence photos.

(1) Evaluation of compound 2c’s ability to inhibit the growth of U87 transplanted tumors in mice

The effect of compound 2c on subcutaneous glioma in vivo was monitored using a brain glioma U87 cell Balb/c female nude mouse xenograft model, and the graph was plotted using GraphPad Prism software. There was no significant difference in the appearance and body weight of mice in the compound 2c-treated group compared with the control group, indicating that the biosafety of compound 2c is reliable (Figure 30A). Obviously, the transplanted tumors in the compound 2c-treated group were smaller than those in the control group, and there was a statistical difference in the weight of the transplanted tumors in the two groups, indicating that compound 2c can inhibit the growth of gliomas in mice (Figure 30B).

(2) H&E staining and immunohistochemistry (IHC) techniques were used to verify the molecular changes of compound 2c in U87 transplanted tumors in mice

After dehydration, embedding and sectioning of the transplanted tumor tissue obtained from the transplanted tumor animal model, the U87 transplanted tumor tissue was pathologically examined using H&E staining technology. As shown in Figure 31A, the U87Vehicle control group showed a more vigorous cell proliferation phenomenon, and it was found that more cell nuclei were in the division phase. In contrast, the transplanted tumor cells in the compound 2c treatment group tended not to divide or divide less, indicating that the treatment of compound 2c can inhibit the malignant proliferation of brain glioma cells in the animal model. At the same time, in order to further verify this, the transplanted tumor tissue was subjected to immunohistochemistry (IHC) staining of key molecules such as PI3K, phosphorylated PI3K (p-PI3K), AKT, GSK3β, HMOX1, p62, Bcl-2, and Bax. As shown in Figure 31B, the results of protein immunoblotting in the second part of the cell model are the same. After treatment with compound 2c, the expression of PI3K, phosphorylated PI3K (p-PI3K), AKT, GSK3β, Bcl-2 and other molecules in U87 transplanted tumor cells showed different degrees of decrease, and the expression of HMOX1, p62, Bax and other molecules showed different degrees of increase. This further proves that compound 2c has good inhibitory and killing ability on malignant brain glioma at the cellular level and animal level.

(3) Tissue immunofluorescence (IF) technology was used to verify the molecular changes of compound 2c in U87 transplanted tumors in mice

In order to make the research results more convincing, the effect of compound 2c on key molecules in U87 transplanted tumor tissue was verified by tissue immunofluorescence (IF) technology. As shown in Figure 32, the expression of key molecules such as PI3K, phosphorylated PI3K (p-PI3K), AKT, phosphorylated AKT (p-AKT), Ki67, GSK3β, Bcl-2, etc. showed varying degrees of decrease, and the expression of molecules such as HMOX1, p62, Bax, etc. showed varying degrees of increase. This is completely consistent with the previous WB and IHC experimental results, and more effectively verifies the good inhibitory and killing ability of compound 2c on malignant brain glioma at the animal level.

In vitro cell model and animal model studies have shown that compound 2c can block the G0/G1 phase of the cell cycle to inhibit cell proliferation and induce cell apoptosis. Compound 2c has excellent anti-proliferative effects and anti-tumor molecular mechanisms at the animal and cell levels, and still has good inhibitory and killing capabilities against malignant gliomas at the animal level. Therefore, compound 2c is a potential β-elemene-derived anti-tumor drug with great application prospects.

It should be pointed out that a person skilled in the art can make several improvements without departing from the principles of the present invention, and these improvements should also be considered as within the protection scope of the present invention.

Claims (10)

1. A beta-elemene aryl derivative is characterized by having a structure shown in a formula I:

In the formula I of the present description, Includes/>

R ₁、R₂、R₃ is independently phenyl, alkoxy, alkenyl, substituted alkenyl or

R ₄ is alkylphenyl or substituted alkylphenyl; r ₅、R₆ is independently hydroxy or alkoxy;

r ₇ is hydroxy and/or phenyl;

R ₉ includes phenyl or substituted phenyl.

2. The β -elemene aryl derivative according to claim 1, wherein when R ₉ is a substituted phenyl group, the substituted phenyl group comprises an alkoxy-substituted phenyl group or an alkenyl-substituted phenyl group.

3. The beta-elemene aryl derivative according to claim 1, comprising a compound having a structure represented by formulae 2a to 2 j:

4. A process for the preparation of a β -elemene aryl derivative as claimed in any one of claims 1 to 3 comprising the steps of:

dissolving beta-elemene and aryl compound with a structure shown in a formula I-1, and carrying out coupling reaction under the conditions of alkalinity and catalyst to obtain the beta-elemene aryl derivative;

R ₈ is-I, -Br or-OTf.

5. The method according to claim 4, wherein the molar ratio of the beta-elemene to the aryl compound having the structure represented by formula I-1 is 1 to 1.5:1.

6. The method according to claim 4 or 5, wherein the coupling reaction is carried out at a temperature of 60 to 150 ℃ for 24 hours.

7. The method of claim 4, wherein the catalyst comprises 4- (anthracene-9-yl) -3- (tert-butyl) -2, 3-dihydrobenzo [ d ] [1,3] oxaphosphole and Pd (OAc) ₂.

8. The method according to claim 4, wherein the molar ratio of 4- (anthracene-9-yl) -3- (tert-butyl) -2, 3-dihydrobenzo [ d ] [1,3] oxaphosphole to Pd (OAc) ₂ is 2:1.

9. The method according to claim 4 or 7, wherein the molar ratio of the aryl compound having the structure represented by formula I-1 to Pd (OAc) ₂ is 25:1.

10. Application of a compound with a 2c structure in preparing medicines for resisting bladder cancer cells T24, colorectal cancer cells SW480 and brain glioma cancer cells U251 and U81.