CN111518065A - Parthenolide derivative and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of organic synthesis, in particular to a parthenolide derivative and a preparation method and application thereof. The invention takes the feverfew as the initial main raw material, and synthesizes 11 feverfew lactone derivatives through a series of chemical reactions, and the derivatives have certain antiproliferative activity on glioblastoma cells. The invention also proves that the derivative DMAPT-D6 containing deuterium can remarkably induce the accumulation of active oxygen of glioblastoma cells, thereby causing DNA damage in the glioblastoma cells. In addition, DMAPT-D6 promoted caspase-dependent death receptor-mediated exogenous apoptosis, suggesting that DNA damage induced by DMAPT-D6 could induce glioblastoma cell apoptosis. The two are combined, and the ROS accumulation caused by the treatment of DMAPT-D6 causes DNA damage and then causes death receptor mediated apoptosis, which shows that the DMAPT-D6 with the novel component has the treatment potential for treating glioblastoma and can be applied to the preparation of drugs for treating glioblastoma.

Description

Parthenolide derivative and preparation method and application thereof

Technical Field

The invention relates to the technical field of organic synthesis, in particular to a parthenolide derivative and a preparation method and application thereof.

Background

Reactive Oxygen Species (ROS) are a class of short half-life, highly Reactive, oxygen-containing, aerobic metabolic byproducts, including superoxide, hydroxyl radicals, and hydroxide. Cellular reactive oxygen species are produced intracellularly mainly by mitochondria, NADPH oxidase, peroxisomes and endoplasmic reticulum. New data indicate that reactive oxygen species act as double-edged swords in cells. Low levels of ROS are necessary for cell survival and proliferation; in contrast, excess ROS can cause oxidative stress, resulting in DNA damage, apoptosis, and necrosis. DNA damage is caused by chemical and physical factors that can lead to a complex series of processes including cell cycle arrest, DNA repair, modulation of cellular checkpoints, initiation of apoptosis, etc. For this reason, increasing cellular ROS production to induce DNA damage and cell death is a well-known and effective anti-cancer strategy.

Parthenolide (PTL), a Sesquiterpene Lactone (SL) isolated from the buds of feverfew, has received particular attention for its anti-tumor activity in a variety of human cancer cell lines. PTL induces cytotoxicity in a variety of solid tumors, including colorectal, melanoma, pancreatic, breast, prostate, and glioblastoma, but is ineffective in normal tissues. The lack of stability and poor solubility of PTL under acidic and alkaline conditions and in media containing 0.5% serum is a major factor limiting its pharmacological applications.

Gliomas are the most aggressive primary intracranial malignancies of the central nervous system, posing a fatal threat to human health. Currently, the World Health Organization (WHO) classifies gliomas into grades four (I-IV) according to their prognostic histopathological features, with grade IV Glioblastoma (GBM) being the most fatal. Despite tremendous advances in glioma research, including surgery, postoperative adjuvant radiation therapy, and chemotherapy, glioma patients consistently have less than 5% survival 5 years after diagnosis. Therefore, new therapeutic regimens are needed, such as obtaining new drugs that target proliferative tumor cells, to prolong the survival of brain tumor patients, especially GBM patients. Despite the great progress currently made in the treatment of glioblastoma, the prognosis is poor due to resistance to radiotherapy and chemotherapy, with an excessive incidence of morbidity and mortality. There is an urgent need to develop new drugs for inhibiting the proliferation and growth of glioblastoma cells and reversing the multidrug resistance of glioblastoma cells.

Disclosure of Invention

In view of the above, the present invention aims to provide parthenolide derivatives, and a preparation method and applications thereof, wherein the PTL derivatives are synthesized by increasing the solubility of PTL, have a certain inhibitory effect on cell activity of glioblastoma, and in particular, DMAPT-D6 containing deuterium can inhibit the growth of glioblastoma cells by inducing DNA damage due to excessive ROS accumulation, so that the growth of the glioblastoma cells can be inhibited, and the PTL derivatives can be used for preparing a drug for treating glioblastoma.

The invention solves the technical problems by the following technical means:

the invention provides a parthenolide derivative, wherein the structural general formula of the derivative is any one of formula (I), formula (II), formula (III) and formula (IV), and the structural formulas of the formula (I), the formula (II), the formula (III) and the formula (IV) are as follows:

wherein, the structural formula of R is ═ CH₂

Preferably, the derivative is one of the following compounds:

another aspect of the present invention provides a method for preparing the above parthenolide derivative, wherein the method comprises the following steps:

preferably, the preparation method specifically comprises the following steps:

compound PTL and p-toluenesulfonic acid were stirred for reaction in dichloromethane and saturated NaHCO was used₃The reaction was quenched, and the resulting organic layer was washed with saturated brine, anhydrous Na₂SO₄Drying, concentrating under reduced pressure, and recrystallizing with acetone to obtain compound MCL;

stirring compound MCL and m-chloroperoxybenzoic acid in dichloromethane for epoxidation reaction, and sequentially adding Na to the obtained reactant₂SO₄、NaHCO₃And saturated brine, anhydrous Na₂SO₄Drying, concentrating under reduced pressure, and recrystallizing with acetone to obtain compound 1;

stirring compound 1 and phosphorus oxychloride in pyridine for reaction, adding diethyl ether, and sequentially using NaHCO to obtain reaction mixture₃And saturated brine, anhydrous Na₂SO₄Drying, concentrating under reduced pressure, and separating and purifying on silica gel column to obtain Arglabin;

separating compound PTL, MCL, compound 1 and Arglabin with dimethylamine in tetrahydrofuran under alkaline condition, adding dichloromethane into the obtained reaction mixture, washing with saturated saline, and adding anhydrous Na₂SO₄Drying, and concentrating under reduced pressure to respectively obtain a compound DMAPT, a compound 2, a compound 3 and a compound 4;

separating compound PTL, MCL, compound 1 and Arglabin in tetrahydrofuran with dimethyl-d 6-amine hydrochloride under alkaline condition, adding dichloromethane into the obtained reaction mixture, washing with saturated saline, and adding anhydrous Na₂SO₄Drying, concentrating under reduced pressure to obtain compounds DMAPT-D6, 5, 6 and 6And (7) a compound.

Preferably, the compound DMAPT-D6 is prepared by the following specific method:

20mgPTL was dissolved in 2mL tetrahydrofuran, followed by addition of 50mgK₂CO₃And 20mg of dimethyl-d 6-amine hydrochloride were stirred overnight, 20mL of dichloromethane was added, the mixture was washed with saturated brine, and the resulting organic layer was purified over anhydrous Na₂SO₄Drying and concentrating under reduced pressure to obtain the compound DMAPT-D6.

In addition, the invention also provides application of the parthenolide derivative, and application of the derivative in preparation of a medicine for treating glioblastoma.

Preferably, the compound DMAPT-D6 is used for preparing a medicine for treating glioblastoma.

Preferably, the compound DMAPT-D6 is used for preparing a medicine for treating glioblastoma, and the compound DMAPT-D6 is used for accumulating intracellular active oxygen to cause DNA damage in glioblastoma cells so as to induce the apoptosis of the glioblastoma cells.

Preferably, the drug for treating glioblastoma comprises a parthenolide derivative or a pharmaceutically acceptable salt, a hydrate or a combination thereof and an auxiliary material.

The invention takes PTL as the initial main raw material, and synthesizes 11 parthenolide derivatives through a series of chemical reactions, and the derivatives have certain antiproliferative activity on glioblastoma cells, in particular, deuterium-containing compound DMAPT-D6 has stronger activity on U87 and LN229 cells. The present invention also demonstrates that DMAPT-D6 significantly induces the accumulation of Reactive Oxygen Species (ROS) in glioblastoma cells, leading to DNA damage in glioblastoma cells. In addition, DMAPT-D6 promoted caspase-dependent death receptor-mediated exogenous apoptosis, suggesting that DNA damage induced by DMAPT-D6 could induce glioblastoma cell apoptosis. Taken together, the data of the present invention indicate that ROS accumulation resulting from DMAPT-D6 treatment leads to DNA damage and then to death receptor-mediated apoptosis, suggesting that DMAPT-D6 with a novel composition has therapeutic potential for the treatment of glioblastoma.

Drawings

FIG. 1 is a graph of cell growth curves of U87 cells and LN229 cells treated with compound DMAPT-D6;

FIG. 2 is a graph of cell growth curves for 24h, 48h, 72h of U87 cells and LN229 cells treated with various concentrations of compound DMAPT-D6;

FIG. 3 is a graph showing the results of a colony formation test;

FIG. 4 is a graph showing the results of the EdU staining test;

FIG. 5 is a graph of flow cytometry analysis cell cycle results;

FIG. 6 is a graph showing the effect of different concentrations of DMAPT-D6 treatment on cell cycle-related proteins P27, CDK1, CDK2, CylinB and Cylin E;

FIG. 7 is a graph showing the effect of treatment with DMAPT-D6 compound at various concentrations on intracellular reactive oxygen species;

FIG. 8 is a graph showing the effect of treatment with the compound DMAPT-D6 at various concentrations on DNA damage;

FIG. 9 is a graph showing the effect of different concentrations of the compound DMAPT-D6 on oxidative damage and DNA repair signal pathway related proteins NRF2, gamma H2AX, 53BP1, DNA LIG IV;

FIG. 10 is a graph showing the effect of treatment with the compound DMAPT-D6 at various concentrations on cell death;

FIG. 11 is a graph showing the results of the analysis of glioma cell apoptosis by Annexin V-FITC/PI in combination with flow cytometry;

FIG. 12 is a graph showing the effect of treatment with DMAPT-D6 on the expression of proteins involved in the cell death receptor signaling pathway at various concentrations;

FIG. 13 is a graph showing the results of recovery of cell death using the caspase inhibitor Z-VAD-FMK;

FIG. 14 is a graph showing the results of the effect of using the inhibitor Z-VAD-FMK on expression of relevant pathway proteins.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The parthenolide derivatives are prepared from the main raw materials of Parthenolide (PTL) through chemical reaction, and the parthenolide used in the invention is synthesized by adopting the prior art and purchased. The structural general formula of the parthenolide derivative is any one of formula (I), formula (II), formula (III) and formula (IV), and the structural formulas of formula (I), formula (II), formula (III) and formula (IV) are as follows:

wherein, the structural formula of R is ═ CH₂

The synthetic route of the parthenolide derivative is as follows:

specifically, the specific preparation method of each derivative is as follows:

the product testing conditions in the following examples are as follows: on a 400MHz solid nuclear magnetic resonance spectrometer (Bruker AVANCEIII400MHz), recording by taking Tetramethylsilicon (TMS) as an internal standard¹H and¹³C NMR。¹h NMR data are reported below: chemical shift, in ppm (), multiplicity (s ═ singlet, d ═ doublet, t ═ triplet, m ═ multiplet), coupling constant (Hz), relative intensity;¹³the C NMR data are reported below: chemical shift (ppm).

Example 1

The structural formula of compound MCL is as follows:

the preparation method of compound MCL is as follows: to 100mLCH₂Cl₂To this solution was added 86mg of p-toluenesulfonic acid (0.5mmol) to prepare a p-toluenesulfonic acid solution, and the solution was stirred at 20mL CH₂Cl₂Adding parthenolide (14mmol) 3.5g to obtain parthenolide solution, adding parthenolide solution dropwise into p-toluenesulfonic acid solution at room temperature, stirring at room temperature overnight, and adding 20mL saturated NaHCO₃The reaction was quenched, and the resulting organic layer was washed with saturated brine (2 × 20mL), anhydrous Na₂SO₄Drying and concentration under reduced pressure gave a crude residue which was recrystallized from acetone to give 3.2g of a pale yellow crystalline solid, Compound MCL, in a yield of 91% by calculation.

¹H NMR(400MHz,CDCl₃)6.22(d,J＝3.3Hz,1H),5.51(d,J＝3.0Hz,1H),3.82(t,J＝10.3Hz,1H),2.80–2.59(m,3H),2.40(dd,J＝16.3,8.4Hz,1H),2.30–2.14(m,3H),2.14–2.04(m,

1H),1.84–1.74(m,2H),1.69(s,3H),1.40–1.28(m,4H).¹³C NMR(101MHz,CDCl₃)169.71,

138.88,131.87,130.88,119.39,84.45,80.25,58.69,49.62,38.38,34.97,30.08,25.81,23.88,22.73.

Example 2

The structural formula of compound 1 is as follows:

the preparation method of the compound 1 comprises the following steps:

1.75g of the compound MCL (mmol) prepared in example 1 and 1.8g of m-chloroperoxybenzoic acid (10.5mmol) are added to 50mL of CH at room temperature₂Cl₂Stirring overnight, the resulting reaction mixture was then washed with Na₂SO₄(2×30mL)、NaHCO₃(2 × 50mL) and saturated brine (2 × 30mL), anhydrous Na₂SO₄Drying and concentration under reduced pressure gave a crude residue which was recrystallized from acetone to give 1.3g of a crystalline solid, Compound 1, in a calculated yield of 70%.

¹H NMR(400MHz,CDCl₃)6.20(d,J＝3.3Hz,1H),5.48(d,J＝3.0Hz,1H),4.05(t,J＝10.4Hz,1H),2.81(s,1H),2.38–2.19(m,4H),2.04–1.82(m,4H),1.70–1.61(m,1H),1.48(s,3H),

1.46–1.37(m,1H),1.30(s,3H).¹³C NMR(101MHz,CDCl₃)169.62,138.16,119.55,81.83,79.71,69.89,62.19,55.62,49.45,37.37,33.41,29.53,23.26,21.97.

Example 3

The structural formula of the compound Arglabin is as follows:

the preparation method of the compound Arglabin comprises the following steps:

264mg of the compound 1(1.0mmol) prepared in example 2 are added to 5mL of pyridine at 0 ℃ and stirred, 300. mu.L of phosphorus oxychloride is further added to the obtained solution under stirring, the mixture is stirred for 2 hours, 30mL of diethyl ether is added, and the obtained organic layer is sequentially treated with NaHCO₃And saturated brine, anhydrous Na₂SO₄Drying and concentration under reduced pressure gave a crude residue which was isolated and purified on a silica gel column to give 112mg of the compound Arglabin in a calculated yield of 45%.

¹H NMR(400MHz,CDCl₃)6.15(d,J＝3.3Hz,1H),5.58(s,1H),5.42(d,J＝3.1Hz,1H),4.01(t,J＝10.2Hz,1H),2.94(d,J＝10.7Hz,1H),2.83–2.74(m,1H),2.29–2.11(m,3H),2.07–2.01(m,1H),1.99(d,J＝7.9Hz,3H),1.88–1.82(m,1H),1.55–1.45(m,1H),1.35(d,J＝6.4Hz,3H).¹³C NMR(101MHz,CDCl₃)170.43,140.57,139.14,124.91,118.27,82.89,72.52,62.68,52.85,51.05,39.71,33.48,22.79,21.45,18.25.

Example 4

The compound DMAPT has the following structural formula:

the compound DMAPT was prepared as follows: 20mg of Parthenolide (PTL) was dissolved in 2mL of tetrahydroFuran (THF), then adding 10mgK respectively₂CO₃And 0.5mL dimethylamine (40 wt% in water), the resulting mixture was stirred at room temperature overnight, and 20mL CH was added₂Cl₂Washed with saturated brine (2 × 20mL), and the resulting organic layer was purified over anhydrous Na₂SO₄Drying and concentration under reduced pressure gave 22mg of a yellow solid, compound DMAPT, in a yield of 93%.

¹H NMR(400MHz,CDCl₃)5.25–5.16(m,1H),3.86(t,J＝9.1Hz,1H),2.89–2.82(m,1H),2.78–2.70(m,2H),2.57(d,J＝11.2Hz,1H),2.39(s,6H),2.33–2.02(m,7H),1.74–1.62(m,4H),1.30(s,3H),1.26–1.20(m,1H).¹³C NMR(101MHz,CDCl₃)176.21,134.68,125.12,82.29,66.38,61.56,57.31,48.21,46.15,45.77,41.09,36.68,29.81,24.12,17.23,16.93.HRMS(ESI)m/z calcd for C₁₇H₂₈NO₃ ⁺(M+H)⁺294.20637,found 294.20624.

Example 5

The structural formula of compound 2 is as follows:

the preparation method of the compound 2 comprises the following steps: 20mg of the compound MCL prepared in example 1 was dissolved in 2mL of Tetrahydrofuran (THF), and 10mgK was added thereto₂CO₃And 0.5mL dimethylamine (40 wt% in water), the resulting mixture was stirred at room temperature overnight, and 20mL CH was added₂Cl₂Washed with saturated brine (2 × 20mL), and the resulting organic layer was purified over anhydrous Na₂SO₄Drying and concentration under reduced pressure gave 23mg of a yellow solid, Compound 2, in a 97% yield.

¹H NMR(400MHz,CDCl₃)3.76(t,J＝10.3Hz,1H),2.67(dd,J＝12.9,5.0Hz,1H),2.60–2.49(m,2H),2.38–2.28(m,2H),2.20(s,6H),2.14–2.03(m,4H),1.96(d,J＝11.4Hz,1H),1.72(dd,J＝15.1,8.3Hz,2H),1.60(s,3H),1.21(d,J＝17.0Hz,4H).¹³C NMR(101MHz,CDCl₃)176.00,130.84,130.34,83.13,79.34,57.34,57.11,49.97,44.93,43.68,37.43,34.37,29.00,26.32,22.75,21.81.HRMS(ESI)m/z calcd for C₁₇H₂₈NO₃ ⁺(M+H)⁺294.20637,found 294.20630.

Example 6

The structural formula of compound 3 is as follows:

the preparation method of the compound 3 comprises the following steps: 20mg of Compound 1 prepared in example 2 was dissolved in 2mL of Tetrahydrofuran (THF), and 10mgK was added thereto₂CO₃And 0.5mL dimethylamine (40 wt% in water), the resulting mixture was stirred at room temperature overnight, and 20mL CH was added₂Cl₂Washed with saturated brine (2 × 20mL), and the resulting organic layer was purified over anhydrous Na₂SO₄Drying and concentration under reduced pressure gave 22mg of a yellow solid, Compound 3, in a calculated yield of 96%.

¹H NMR(400MHz,CDCl₃)4.00(t,J＝10.4Hz,1H),2.63(dd,J＝13.0,5.0Hz,1H),2.51–2.44(m,1H),2.31–2.24(m,1H),2.17(s,6H),2.09–2.03(m,1H),1.88–1.81(m,3H),1.80–1.70(m,2H),1.66–1.53(m,3H),1.39(s,3H),1.34–1.27(m,1H),1.21(s,3H).¹³C NMR(101MHz,CDCl₃)175.94,80.60,78.67,68.84,61.23,56.89,54.27,49.39,44.93,43.22,36.42,32.57,28.37,22.20.HRMS(ESI)m/z calcd for C₁₇H₂₈NO₄ ⁺(M+H)⁺310.20128,found310.20114.

Example 7

The structural formula of compound 4 is as follows:

the preparation method of the compound 4 comprises the following steps: 20mg of Arglabin, a compound prepared in example 3, was dissolved in 2mL of Tetrahydrofuran (THF), and 10mgK was added thereto₂CO₃And 0.5mL dimethylamine (40 wt% in water), the resulting mixture was stirred at room temperatureAt night, 20mL of CH was added₂Cl₂Washed with saturated brine (2 × 20mL), and the resulting organic layer was purified over anhydrous Na₂SO₄Drying and concentration under reduced pressure gave 23mg of a yellow solid, Compound 4, in a calculated yield of 92%.

¹H NMR(400MHz,CDCl₃)5.49(s,1H),3.94(t,J＝10.2Hz,1H),2.68(ddd,J＝21.9,17.7,7.6Hz,3H),2.49(dd,J＝13.0,6.1Hz,1H),2.23(dd,J＝11.8,5.5Hz,1H),2.17(s,6H),2.10–2.00(m,2H),1.86(s,4H),1.56(dd,J＝22.8,10.8Hz,2H),1.39(d,J＝12.5Hz,1H),1.26(s,3H).¹³C NMR(101MHz,CDCl₃)176.72,139.74,123.71,81.53,71.49,61.65,56.99,51.43,50.87,45.04,43.55,38.56,32.66,21.83,21.70,17.27.HRMS(ESI)m/zcalcd for C₁₇H₂₆NO₃ ⁺(M+H)⁺292.19072,found 292.19067.

Example 8

The compound DMAPT-D6 has the following structural formula:

the compound DMAPT-D6 was prepared as follows: 20mg of Parthenolide (PTL) was dissolved in 2mL of Tetrahydrofuran (THF), and 50mg K was added₂CO₃And 20mg of dimethylamine-d 6-amine hydrochloride, the resulting mixture was stirred at room temperature overnight and 20mL of CH was added₂Cl₂Washed with saturated brine (2 × 20mL), and the resulting organic layer was purified over anhydrous Na₂SO₄Drying and concentration under reduced pressure gave 20mg of a yellow solid, compound DMAPT-D6, in 83% yield.

¹H NMR(400MHz,CDCl₃)5.14(d,J＝9.9Hz,1H),3.76(t,J＝9.0Hz,1H),2.68(dt,J＝6.7,3.9Hz,2H),2.56(dd,J＝13.2,4.7Hz,1H),2.35–2.27(m,2H),2.25–2.16(m,2H),2.12–1.96(m,4H),1.62(d,J＝6.1Hz,3H),1.60–1.50(m,1H),1.23(s,3H),1.17(dd,J＝13.1,5.6Hz,1H).¹³C NMR(101MHz,CDCl₃)175.44,133.64,124.10,81.10,65.50,60.43,56.57,46.92,45.57,40.12,35.67,28.98,23.11,16.23,15.92.HRMS(ESI)m/z calcd forC₁₇H₂₂D₆NO₃ ⁺(M+H)⁺300.24403,found 300.24438.

Example 9

The structural formula of compound 5 is as follows:

the preparation method of the compound 5 comprises the following steps: 20mg of MCL prepared in example 1 was dissolved in 2mL of Tetrahydrofuran (THF), and 50mgK was added thereto₂CO₃And 20mg of dimethylamine-d 6-amine hydrochloride, the resulting mixture was stirred at room temperature overnight and 20mL of CH was added₂Cl₂Washed with saturated brine (2 × 20mL), and the resulting organic layer was purified over anhydrous Na₂SO₄Drying and concentration under reduced pressure gave 23mg of a yellow solid, compound 5, in a calculated yield of 95%.

¹H NMR(400MHz,CDCl₃)3.75(t,J＝10.3Hz,1H),2.70–2.45(m,4H),2.31(dt,J＝11.6,5.3Hz,2H),2.21–2.04(m,4H),1.97(dd,J＝22.6,11.1Hz,1H),1.78–1.67(m,2H),1.61(s,3H),1.23(s,3H),1.21–1.15(m,1H).¹³C NMR(101MHz,CDCl₃)176.06,130.86,130.32,83.11,79.33,57.36,57.10,49.94,43.76,37.41,34.39,29.00,26.36,22.75,21.81.HRMS(ESI)m/z calcd for C₁₇H₂₂D₆NO₃ ⁺(M+H)⁺300.24403,found 300.24490.

Example 10

The structural formula of compound 6 is as follows:

the preparation method of the compound 6 comprises the following steps: 20mg of Compound 1 prepared in example 2 was dissolved in 2mL of Tetrahydrofuran (THF), and 50mgK was added thereto₂CO₃And 20mg of dimethylamine-d 6-amine hydrochloride, the resulting mixture was stirred at room temperature overnight and 20mL of CH was added₂Cl₂Washed with saturated brine (2 × 20mL), and the resulting organic layer was purified over anhydrous Na₂SO₄Drying and concentration under reduced pressure gave 21mg of a yellow solid, compound 6, in a calculated yield of 90%.

¹H NMR(400MHz,CDCl₃)4.00(t,J＝10.4Hz,1H),2.61(dd,J＝13.0,5.0Hz,1H),2.47(dd,J＝13.0,6.2Hz,1H),2.28–2.12(m,3H),2.09–2.02(m,1H),1.84(dd,J＝11.6,6.2Hz,3H),1.79–1.73(m,1H),1.65–1.55(m,2H),1.39(s,3H),1.30(d,J＝11.8Hz,1H),1.21(s,3H).¹³C NMR(101MHz,CDCl₃)176.00,80.56,78.66,68.85,61.23,56.84,54.27,49.38,43.27,36.42,32.58,28.37,22.20.HRMS(ESI)m/z calcd for C₁₇H₂₂D₆NO₄ ⁺(M+H)⁺316.23895,found316.23895.

Example 11

The structural formula of compound 7 is as follows:

the preparation method of the compound 7 comprises the following steps: 20mg of Arglabin, a compound prepared in example 3, was dissolved in 2mL of Tetrahydrofuran (THF), and 50mgK was added thereto₂CO₃And 20mg of dimethylamine-d 6-amine hydrochloride, the resulting mixture was stirred at room temperature overnight and 20mL of CH was added₂Cl₂Washed with saturated brine (2 × 20mL), and the resulting organic layer was purified over anhydrous Na₂SO₄Drying and concentration under reduced pressure gave 23mg of a yellow solid, Compound 7, in a 91% yield.

¹H NMR(400MHz,CDCl₃)5.56(s,1H),4.01(t,J＝10.2Hz,1H),2.86–2.67(m,3H),2.57(dd,J＝13.1,6.0Hz,1H),2.33–2.00(m,5H),1.93(s,3H),1.46(dd,J＝18.6,6.3Hz,2H),1.33(s,3H).¹³C NMR(101MHz,CDCl₃)177.74,140.75,124.75,82.58,72.51,62.68,57.78,52.46,51.84,44.57,39.58,33.67,29.71,29.33,27.23,22.86,22.73,18.29,14.11.HRMS(ESI)m/z calcd for C₁₇H₂₀D₆NO₃ ⁺(M+H)⁺298.22838,found 298.22818.

Example 12

In this example, the effect of the parthenolide derivatives prepared in examples 1 to 11 on glioblastoma was examined, specifically as follows.

The human glioblastoma cell lines U87 and LN229 used in this example were purchased from Cobioer, both of which were mycoplasma free and have been identified by STR testing. Both the U87 and LN229 cell lines were cultured in DMEM medium, purchased from HyClone, containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin solution (100U/ml Penicillium and 100. mu.g/ml streptomycin) at 37 ℃ in 5% CO₂Culturing in a constant temperature incubator. The cell culture steps are as follows:

(1) after counting U87 cells with a fully automatic cell counter, U87 cells and LN229 were diluted to 5 × 10 with DMEM medium³Obtaining cell suspension per mL;

(2) adding 100 mu L of cell suspension into each hole of a 96-hole plate, blowing, uniformly mixing, and culturing in an incubator at 37 ℃ for 24 hours;

(3) PTL and the compounds prepared in examples 1 to 11 were diluted with dimethyl sulfoxide to concentrations of 0.5. mu.M, 1.0. mu.M, 1.5. mu.M, and 2.0. mu.M, respectively, and added to the respective cultured cell lines, followed by incubation at 37 ℃ in an incubator for 48 hours;

(4) MTT solution (20. mu.L/well) was added and incubated at 37 ℃ for 4h in an incubator;

(5) the supernatant was removed, the crystals formed were dissolved in DMSO (200. mu.L/well) and then assayed for OD570 nm (OD570) using a microplate reader (Bio-Tek, VT, USA) and analyzed by GraphPad Prism 7.0, the effect of each compound on U87 cell viability being shown in Table 1, the data in Table 1 showing that the parthenolide derivatives prepared according to the invention all have different degrees of effect on U87 cell viability, with the IC of compound DMAPT-D6 in U87 cells₅₀The value was 15.5. mu.M.

TABLE 1

Compound DMAPT-D6 vs U87 cellsAnd LN229 cell viability as shown in FIG. 1, the data of FIG. 1 indicate the IC of DMAPT-D6 in U87 cells and LN229 cells₅₀The values were 15.5. mu.M and 11.15. mu.M, respectively. To further observe the inhibitory effect of compound DMAPT-D6 on glioblastoma, glioblastoma cells were treated at concentrations of 2.5. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, and 40. mu.M for 24h, 48h, and 72h, respectively, and the relationship between cell growth and exposure time and dose was observed, the growth profile of which is shown in FIG. 2, and the growth profile of FIG. 2 shows that compound DMAPT-D6 decreased the proliferative capacity of U87 and LN229 cells, indicating that the compound has dose-and time-dependent cytotoxicity on glioblastoma cells.

To further confirm the growth inhibitory effect of DMAPT-D6 on glioblastoma cells, colony formation assays were performed in this example, using human glioblastoma cells U87 and LN229 at a cell concentration of 1 × 10³The cells/cell/well were inoculated on a six-well plate, cultured overnight, and after treating the cells with the compound DMAPT-D6 at concentrations of 0. mu.M, 5. mu.M, 10. mu.M, and 20. mu.M for 48 hours, the treated cells were cultured with a fresh medium for 15 days, fixed with 4% paraformaldehyde solution for 30 minutes, the cells were stained with 1% crystal violet for 30 minutes to identify colonies, and the number of colonies of at least 50 cells was counted under a 4-fold microscope, the results are shown in FIG. 3. FIG. 3 shows that DMAPT-D6 has significant inhibitory effect on the growth of glioblastoma cells, and is dose-dependent, and DMAPT-D6 has significant inhibitory effect on the growth of glioblastoma cells compared to the control group without DMAPT-D6. The EdU staining test was performed, and the test results are shown in fig. 4, and fig. 4 shows that the cell number was significantly reduced in a dose-dependent manner after treatment with compound DMAPT-D6 in the EdU staining test, indicating that compound DMAPT-D6 has a significant inhibitory effect on the proliferation of U87 and LN229 cells.

To further understand the mechanism of cytotoxic effect of the compound DMAPT-D6 on glioblastoma cells, this example also performed cell cycle analysis on U87 and LN229 cells treated with the compound DMAPT-D6 or without DMAPT-D6. Specifically, glioblastoma cells in a logarithmic growth phase were collected and transferred to a six-well plate, the cell density in each well was 30%, the cells were treated with the compound DMAPT-D6 at concentrations of 0 μ M, 5 μ M, 10 μ M, and 20 μ M for 48 hours, collected and used for flow cytometry analysis, for cell cycle determination, the collected cells were fixed with 70% ethanol at 4 ℃ for 24 hours, and then washed 3 times with PBS, and then, the cells were incubated with PBS containing 50mg/ml propidium iodide and 100mg/ml RNase at 37 ℃ for 0.5 hour, and finally, the stained cells were analyzed by BD-accuri-C6 flow cytometry, and the results were statistically compared and visualized automatically using FlowJo software; for apoptosis detection, cells were harvested and processed using Annexin V-FITC/PI apoptosis assay kit according to the manufacturer's instructions, fluorescence activated cells were analyzed within 1 hour using a BD-AccuriTM-C6 flow cytometer, and the apoptosis rate was analyzed by FlowJo software, with the results shown in FIG. 5. FIG. 5 shows that compound DMAPT-D6 has significant dose-dependent induced cell cycle arrest on both U87 and LN229 cells, arresting the cell cycle in S phase.

Based on the above experiments, immunoblot analysis was also performed in this example, and specifically, collected glioblastoma cells were analyzed in the presence of Halt^TMThe protein samples (30 μ g/lane) were separated by electrophoresis after 30min of lysis in RIPA buffer of protease and phosphatase inhibitors (Beyotime, shanghai, china), and after quantification of total protein concentration using BCA protein assay kit (Beyotime, shanghai, china), transferred to PVDF membrane (Millipore, Billerica, MA, usa), followed by blocking the membrane with 5% BSA for 2h at room temperature, incubation of one antibody overnight at 4 ℃, followed by incubation with the corresponding IRDye 800 CW-goat anti-mouse IgG (h + L) or IRDye 800-LT donkey anti-rabbit IgG (h + L) secondary antibody, followed by imaging using Odyssey infrared fluorescence imaging system (Li-cor, NE, usa), and using β -actin supernatant as a control, as shown in fig. 6, the immunoblot results show that the compound DMAPT-D6 significantly reduced the protein levels of cyclin B, cyclin E, 1 and CDK 34, while the protein levels of CDK 56 and pt 39229 in cell line increased with increasing the dose of the compound dmaln-46 6.

The above data indicate that compound DMAPT-D6 may inhibit cell proliferation by inducing cell cycle arrest in the S phase of glioblastoma cells.

To further understand the mechanism by which DMAPT-D6 induces inhibition of cell proliferation, the production of Reactive Oxygen Species (ROS) in U87 and LN229 cells was observed using a fluorescence microscope. Specifically, the formation of ROS was detected using an ROS detection kit (Beyotime, shanghai, china), glioblastoma cells treated with various concentrations of the compound DMAPT-D6 were collected, centrifuged at 800rpm for 5 minutes, the resulting precipitate after centrifugation was resuspended in serum-free DMEM containing DCFH-DA (10 μ M) and incubated for 20 minutes, and finally, the cell suspension was centrifuged and washed 3 times with serum-free DMEM, and then observed by a fluorescence microscope (Olympus IX53/DP80, Japan), and as a result, the reactive oxygen species level generated by the treatment with various concentrations of DMAPT-D6 was significantly higher than that of the control group which had not been treated with DMAPT-D6, as shown in fig. 7.

Several studies have shown that intracellular ROS accumulation can induce DNA damage and lead to DNA damage responses, and in order to evaluate whether DMAPT-D6 can initiate the DNA damage process caused by excessive intracellular ROS, phosphohistone γ H2AX was detected in U87 and LN229 cell lines by immunofluorescence and immunoblotting, the procedure for immunofluorescence was as follows: glioblastoma cells were grown in 24-well plates and then treated with varying concentrations of DMAPT-D6 for 48 hours, washed 3 times with PBS, the cells were fixed in 4% paraformaldehyde for 30 minutes, permeabilized in 0.1% Triton X-100 for 10 minutes, and blocked in immunostaining blocking buffer (Beyotime, shanghai) at 37 ℃ for 30 minutes, the cells were incubated with anti- γ H2AX (1: 250) antibody, respectively, at 4 ℃ overnight, then the stained cells were washed 3 times with PBS and incubated with Alexa Fluor-coupled secondary antibody (1: 2000) for 1 hour at room temperature, the nuclei were labeled with 1mg/mL DAPI for 30 minutes, images were captured with a fluorescence microscope (Olympus IX53/DP80, japan), and the immunofluorescence results are shown in figure 8. The results shown in fig. 8 indicate that in U87 and LN229 cells, the green signal representing γ H2AX was significantly accumulated in the nucleus in a dose-dependent manner and γ H2AX was significantly upregulated compared to the control, indicating DNA double strand breaks in TNBC cells. In order to detect whether the DMAPT-D6 destroys the DNA Repair (DR) process, the expression level of DR-related proteins such as p53 binding protein 1(53BP1) and DNA ligase IV (LIG IV) in glioblastoma cells was detected by immunoblotting, and the results of immunoblotting are shown in FIG. 9. FIG. 9 shows that expression of NRF2 is dose-dependently upregulated with increasing DMAPT-D6 concentration after DMAPT-D6 treatment, suggesting that too much ROS accumulate in the cells. Furthermore, as shown in fig. 9, DMAPT-D6 significantly caused a reduction in the expression of both 53BP1 and DNA LIG IV proteins in a dose-dependent manner, showing an inhibitory effect on DR.

Disproportionate increases in ROS and severe DNA damage can induce endogenous and exogenous apoptotic pathways, mediated by mitochondrial and cell death receptor signaling, respectively. Based on the effect of DMAPT-D6 on ROS induction and subsequent DNA damage, the present invention also discusses whether DMAPT-D6 can promote the initiation of apoptosis. Notably, PI staining analysis showed a significant increase in the percentage of PI positive cells in U87 and LN229 cell lines with increasing concentrations of DMAPT-D6, as shown in fig. 10, indicating that DMAPT-D6 induced cell death. To further confirm the effect of DMAPT-D6 on apoptosis, Annexin V-FITC/PI analysis was performed by flow cytometry 48 hours after U87 and LN229 cells were treated with DMAPT-D6, as shown in fig. 11, DMAPT-D6 induced 7.28% and 10.7% late apoptosis (Annexin V-FITC and PI positive cells) in U87 and LN229 cell lines, respectively, at low doses of 5 μ M; at a dose of 20 μ M, the percentage of apoptosis rose to 46.9% and 34.7%, respectively. Next, the expression of external apoptosis signaling pathway-related proteins was analyzed to determine whether cell death receptors participate in the apoptotic response to DMAPT-D6, and consistent with the above results, cell death receptor signaling pathway-related proteins such as DR3, DR5, FADD, TRADD were significantly up-regulated with increasing concentrations of DMAPT-D6, as shown in fig. 12. Subsequently, Caspase precursor (Procaspase)8 is cleaved to produce the active enzyme form of Caspase 8(Caspase 8) due to the increase of FADD and TRADD. In addition, downstream caspase 3 and PARP are splice-activated in U87 and LN229 cells, meaning that it mediates exogenous apoptosis through death receptors, as shown in fig. 12. To further demonstrate DMAPT-D6-induced apoptosis mediated by cell death receptors, a Caspase inhibitor, Z-VAD-FMK, was used to test the recovery effect of DMAPT-D6-induced apoptosis, as shown in FIG. 13, which can significantly rescue DMAPT-D6-induced apoptosis and partially restore the protein levels of DNA Lig IV, DR3, Caspase8, 3 and PARP when treated with Z-VAD-FMK, as shown in FIG. 14, indicating that DMAPT-D6-induced apoptosis mediated by death receptors is dependent on caspases.

Taken together, the compound DMAPT-D6 of the present invention significantly up-regulated the activity of proteins associated with death receptor signaling pathways, such as DR3, DR5, FADD, TRADD, and cysteine proteases 3, 8, and PARP, indicating that death receptor-mediated exogenous apoptosis is induced following DMAPT-D6 treatment; the results show that U87 and LN229 cells after being treated with compound DMAPT-D6 induce excessive ROS accumulation to cause DNA damage, and further induce cell cycle S phase block and cell death receptor mediated cell external apoptosis signal pathway, thereby inhibiting growth of glioblastoma cells and playing an anti-cancer role on the glioblastoma cells.

Therefore, the compound DMAPT-D6 is a potential anticancer drug with active oxygen regulation capacity, can be used as a lead compound to be applied to the preparation of drugs for treating glioblastoma, and particularly to the preparation of drugs for treating glioblastoma, wherein the drugs for treating glioblastoma comprise the 11 parthenolide derivatives or pharmaceutically acceptable salts, hydrates or combinations and auxiliary materials thereof.

Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims. The techniques, shapes, and configurations not described in detail in the present invention are all known techniques.

Claims (9)

1. The parthenolide derivative is characterized in that the structural general formula of the derivative is any one of a formula (I), a formula (II), a formula (III) and a formula (IV), and the structural formulas of the formula (I), the formula (II), the formula (III) and the formula (IV) are as follows:

wherein, the structural formula of R is ═ CH₂Or

2. The parthenolide derivative according to claim 1, wherein the derivative is one of the following compounds:

3. the preparation method of the parthenolide derivative is characterized by comprising the following steps:

4. the method for preparing a parthenolide derivative according to claim 3, wherein the method specifically comprises the following steps:

compound PTL and p-toluenesulfonic acid were stirred for reaction in dichloromethane and saturated NaHCO was used₃The reaction was quenched, and the resulting organic layer was washed with saturated brine, anhydrous Na₂SO₄Drying, concentrating under reduced pressure, and recrystallizing with acetone to obtain compound MCL;

stirring the compound MCL and m-chloroperoxybenzoic acid in dichloromethane for epoxidation reaction to obtainTo the reactant is sequentially added with Na₂SO₄、NaHCO₃And saturated brine, anhydrous Na₂SO₄Drying, concentrating under reduced pressure, and recrystallizing with acetone to obtain compound 1;

stirring compound 1 and phosphorus oxychloride in pyridine for reaction, adding diethyl ether, and sequentially using NaHCO to obtain reaction mixture₃And saturated brine, anhydrous Na₂SO₄Drying, concentrating under reduced pressure, and purifying and separating on silica gel column to obtain Arglabin;

separating compound PTL, MCL, compound 1 and Arglabin with dimethylamine in tetrahydrofuran under alkaline condition, adding dichloromethane into the obtained reaction mixture, washing with saturated saline, and adding anhydrous Na₂SO₄Drying, and concentrating under reduced pressure to respectively obtain a compound DMAPT, a compound 2, a compound 3 and a compound 4;

separating compound PTL, MCL, compound 1 and Arglabin in tetrahydrofuran with dimethyl-d 6-amine hydrochloride under alkaline condition, adding dichloromethane into the obtained reaction mixture, washing with saturated saline, and adding anhydrous Na₂SO₄Drying and concentrating under reduced pressure to obtain the compound DMAPT-D6, the compound 5, the compound 6 and the compound 7 respectively.

5. The parthenolide derivative according to claim 4, wherein the compound DMAPT-D6 is prepared by the following method:

20mgPTL was dissolved in 2mL tetrahydrofuran, followed by addition of 50mgK₂CO₃And 20mg of dimethyl-d 6-amine hydrochloride were stirred overnight, 20mL of dichloromethane was added, the mixture was washed with saturated brine, and the resulting organic layer was purified over anhydrous Na₂SO₄Drying and concentrating under reduced pressure to obtain the compound DMAPT-D6.

6. Use of a parthenolide derivative according to claim 1 or 2 in the manufacture of a medicament for the treatment of glioblastoma.

7. The use of a parthenolide derivative according to claim 6, wherein compound DMAPT-D6 is used in the preparation of a medicament for the treatment of glioblastoma.

8. The use of the parthenolide derivative according to claim 7, wherein the compound DMAPT-D6 is used for preparing a drug for treating glioblastoma through accumulation of intracellular reactive oxygen species, resulting in DNA damage in glioblastoma cells, thereby inducing glioblastoma cell apoptosis.

9. The use of a parthenolide derivative according to claim 8, wherein the medicament for treating glioblastoma comprises a parthenolide derivative or a pharmaceutically acceptable salt, hydrate or combination thereof and an adjuvant.

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