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. In the present invention, parthenium is used as the initial main raw material, and 11 parthenolide derivatives are synthesized through a series of chemical reactions, and these derivatives have certain anti-proliferative activity on glioblastoma cells. The present invention also confirms that the deuterium-containing derivative DMAPT-D6 can significantly induce the accumulation of reactive oxygen species in glioblastoma cells, thereby causing DNA damage in glioblastoma cells. Furthermore, DMAPT‑D6 promotes exogenous apoptosis mediated by caspase-dependent death receptors, suggesting that DNA damage induced by DMAPT‑D6 induces apoptosis in glioblastoma cells. Taken together, ROS accumulation induced by DMAPT‑D6 treatment leads to DNA damage followed by death receptor-mediated apoptosis, suggesting that DMAPT‑D6 with novel components has therapeutic potential for the treatment of glioblastoma, which may The invention is used in 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 technique

Reactive oxygen species (ROS) are a class of short-half-life, highly reactive, oxygen-containing by-products of aerobic metabolism, including superoxide, hydroxyl radicals, and hydroxides. Cellular reactive oxygen species are mainly produced intracellularly by mitochondria, NADPH oxidase, peroxisomes and endoplasmic reticulum. New data suggest that reactive oxygen species act as a double-edged sword in cells. Low levels of ROS are necessary for cell survival and proliferation; conversely, excess ROS can cause oxidative stress, leading to DNA damage, apoptosis, and necrosis. DNA damage is caused by chemical and physical factors, and it can cause a series of complex processes, including cell cycle arrest, DNA repair, regulation of cellular checkpoints, initiation of apoptosis, etc. For this reason, enhancing cellular ROS production to induce DNA damage and cell death is a well-known and effective anticancer strategy.

Parthenolide (PTL), a sesquiterpene lactone (SL) isolated from the buds of parthenium, has received particular attention for its antitumor activity in a variety of human cancer cell lines. PTL induces cytotoxicity in a variety of solid tumors, including colorectal cancer, melanoma, pancreatic cancer, breast cancer, prostate cancer, and glioblastoma, but is ineffective in normal tissues. The lack of stability and poor solubility of PTL under acidic and alkaline conditions and in medium containing 0.5% serum are the main factors limiting its pharmacological application.

Gliomas are the most aggressive primary intracranial malignant tumors of the central nervous system, posing a fatal threat to human health. Currently, the World Health Organization (WHO) classifies gliomas into four grades (I-IV) based on their prognostic histopathological features, among which grade IV glioblastoma (GBM) is the most lethal. Despite tremendous advances in glioma research, including surgery, postoperative adjuvant radiation therapy, and chemotherapy, the 5-year survival rate for glioma patients remains below 5% after diagnosis. Therefore, new treatment options, such as access to new drugs targeting proliferating tumor cells, are needed to prolong the survival of patients with brain tumors, especially those with GBM. Despite great progress in the treatment of glioblastoma, it has a poor prognosis and is associated with excessive morbidity and mortality due to resistance to radiotherapy and chemotherapy. There is an urgent need to develop new drugs that inhibit the proliferation and growth of glioblastoma cells and reverse the multidrug resistance of glioblastoma cells.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide parthenolide derivatives and preparation methods and applications thereof, and to synthesize PTL derivatives by improving the solubility of PTL, and these PTL derivatives have certain cell activity on glioblastoma. The inhibitory effect of DMAPT-D6, especially the deuterium-containing DMAPT-D6, can cause DNA damage by inducing excessive ROS accumulation, thereby inhibiting the growth of glioblastoma cells, and can be used in the preparation of drugs for the treatment of glioblastoma.

The present invention solves the above-mentioned technical problems through the following technical means:

One aspect of the present invention is to provide a parthenolide derivative, the general structural formula of the derivative is any one of formula (I), formula (II), formula (III) and formula (IV), The structural formulas of the formula (I), formula (II), formula (III) and formula (IV) are as follows:

Wherein, the structural formula of R is =CH ₂

Preferably, the derivative is one of the following compounds:

Another aspect of the present invention is to provide the preparation method of above-mentioned parthenolide derivative, and described preparation method is as follows:

As preferably, the preparation method is as follows:

Compound PTL and p-toluenesulfonic acid were stirred and reacted in dichloromethane, and the reaction was quenched with saturated NaHCO ₃ , the obtained organic layer was washed with saturated brine, dried over anhydrous Na ₂ SO ₄ , concentrated under reduced pressure, and recrystallized with acetone to obtain compound MCL;

Compound MCL and m-chloroperoxybenzoic acid were stirred in dichloromethane to carry out epoxidation reaction, the obtained reactant was washed successively with Na ₂ SO ₄ , NaHCO ₃ and saturated brine, dried over anhydrous Na ₂ SO ₄ , and reduced pressure. Concentrated and recrystallized with acetone to obtain compound 1;

Compound 1 and phosphorus oxychloride were stirred and reacted in pyridine, ether was added, the resulting reaction mixture was washed with NaHCO ₃ and saturated brine successively, dried over anhydrous Na ₂ SO ₄ , concentrated under reduced pressure, and separated and purified on a silica gel column to obtain Compound Arglabin;

Compounds PTL, MCL, Compound 1 and Arglabin were treated with dimethylamine in tetrahydrofuran under basic conditions, respectively. The resulting reaction mixture was added with dichloromethane, washed with saturated brine, dried over anhydrous Na ₂ SO ₄ , and concentrated under reduced pressure. , respectively obtain compound DMAPT, compound 2, compound 3 and compound 4;

Under basic conditions, compounds PTL, MCL, compound 1 and Arglabin were treated with dimethyl-d6-amine hydrochloride in tetrahydrofuran, respectively, and dichloromethane was added to the resulting reaction mixture, washed with saturated brine, and anhydrous Na ₂ Dry over SO ₄ and concentrate under reduced pressure to obtain compound DMAPT-D6, compound 5, compound 6 and compound 7, respectively.

Preferably, the preparation method of the compound DMAPT-D6 is as follows:

20 mg of PTL was dissolved in 2 mL of tetrahydrofuran, then 50 mg of K ₂ CO ₃ and 20 mg of dimethyl-d6-amine hydrochloride were added and stirred overnight, 20 mL of dichloromethane was added, washed with saturated brine, and the obtained organic layer was washed with anhydrous Na ₂ SO ₄ was dried and concentrated under reduced pressure to obtain compound DMAPT-D6.

In addition, the present invention also provides the application of the above parthenolide derivatives, the application of the derivatives in the preparation of drugs for treating glioblastoma.

Preferably, the compound DMAPT-D6 is used in the preparation of a medicament for treating glioblastoma.

Preferably, the application of the compound DMAPT-D6 in the preparation of a drug for treating glioblastoma is to induce glioblastoma cells by accumulating intracellular reactive oxygen species, causing DNA damage in glioblastoma cells apoptosis.

Preferably, the drug for treating glioblastoma comprises parthenolide derivatives or pharmaceutically acceptable salts, hydrates or combinations thereof and excipients.

In the present invention, PTL is used as the main raw material, and 11 parthenolide derivatives are synthesized through a series of chemical reactions. These derivatives have certain anti-proliferative activity on glioblastoma cells, especially the deuterium-containing derivatives. The compound DMAPT-D6 has strong activity on both U87 and LN229 cells. The present invention also confirms that DMAPT-D6 can significantly induce the accumulation of reactive oxygen species (ROS) in glioblastoma cells, thereby causing DNA damage in glioblastoma cells. Furthermore, DMAPT-D6 promotes exogenous apoptosis mediated by caspase-dependent death receptors, suggesting that DNA damage induced by DMAPT-D6 induces apoptosis in glioblastoma cells. Taken together, the present data suggest that ROS accumulation caused by DMAPT-D6 treatment leads to DNA damage followed by death receptor-mediated apoptosis, suggesting that DMAPT-D6 with a novel component has the potential to treat glioblastoma therapeutic potential.

Description of drawings

Fig. 1 is the cell growth curve diagram of compound DMAPT-D6 treating U87 cells and LN229 cells;

Figure 2 is a graph showing the cell growth of U87 cells and LN229 cells treated with different concentrations of compound DMAPT-D6 for 24h, 48h and 72h;

Fig. 3 is the result graph of colony formation test;

Fig. 4 is the EdU staining test result graph;

Fig. 5 is a flow cytometry analysis result of cell cycle;

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

Figure 7 is a graph showing the effect of different concentrations of compound DMAPT-D6 on intracellular reactive oxygen species;

Figure 8 is a graph showing the effect of different concentrations of compound DMAPT-D6 on DNA damage;

Figure 9 is a graph showing the effect of different concentrations of compound DMAPT-D6 on oxidative damage and DNA repair signaling pathway-related proteins NRF2, γH2AX, 53BP1, and DNA LIG IV;

Figure 10 is a graph showing the effect of different concentrations of compound DMAPT-D6 on cell death;

Figure 11 is a graph showing the results of Annexin V-FITC/PI combined with flow cytometry to analyze the apoptosis of glioma cells;

Figure 12 is a graph showing the effect of different concentrations of compound DMAPT-D6 on the expression of cell death receptor signaling pathway-related proteins;

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

Figure 14 is a graph of the results of using the inhibitor Z-VAD-FMK on the expression of related pathway proteins.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

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

Wherein, the structural formula of R is =CH ₂

The synthetic route of above-mentioned parthenolide derivative is as follows:

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

The product detection conditions in the following examples are as follows: ¹ H and ¹³ C NMR were recorded on a 400 MHz solid-state nuclear magnetic resonance spectrometer (Bruker AVANCE III 400 MHz) with tetramethylsilicon (TMS) as an internal standard. ¹ H NMR data are reported as follows: chemical shift, in ppm (δ), multiplicity (s=singlet, d=doublet, t=triplet, m=multiplet), coupling constant (Hz), relative intensity ; ¹³ C NMR data are reported as follows: chemical shift (ppm).

Example 1

The structural formula of compound MCL is as follows:

The preparation method of compound MCL is as follows: add 86 mg of p-toluenesulfonic acid (0.5 mmol) to 100 mL of CH ₂ Cl ₂ to prepare a p-toluene sulfonic acid solution, and add 3.5 g of parthenolide (14 mmol) to 20 mL of CH ₂ Cl ₂ to prepare The parthenolide solution was obtained, then at room temperature, the parthenolide solution was added dropwise to the p-toluenesulfonic acid solution, stirred at room temperature overnight, quenched with 20 mL of saturated NaHCO ₃ , and the obtained organic layer was washed with saturated brine (2× 20 mL) was washed, dried over anhydrous Na ₂ SO ₄ , and concentrated under reduced pressure. The obtained crude residue was recrystallized with acetone to obtain 3.2 g of a pale yellow crystalline solid, namely compound MCL, with a calculated yield of 91%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.22 (d, J=3.3 Hz, 1H), 5.51 (d, J=3.0 Hz, 1H), 3.82 (t, J=10.3 Hz, 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). ¹³ C NMR (101MHz, CDCl ₃ )δ169.71,

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 compound 1 is as follows:

At room temperature, 1.75 g of compound MCL (mmol) prepared in Example 1 and 1.8 g of m-chloroperoxybenzoic acid (10.5 mmol) were added to 50 mL of CH ₂ Cl ₂ and stirred overnight, and the resulting reaction mixture was sequentially added with Na ₂ SO ₄ (2 x 30 mL), NaHCO ₃ (2 x 50 mL) and saturated brine (2 x 30 mL), dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure, the resulting crude residue was recrystallized from acetone to give 1.3 g of crystalline solid , namely compound 1, the calculated yield is 70%.

¹ H NMR (400MHz, CDCl ₃ )δ6.20(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). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 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 compound Arglabin is as follows:

Under the condition that the temperature is 0 °C, 264 mg of compound 1 (1.0 mmol) prepared in Example 2 was added to 5 mL of pyridine and stirred, and then 300 μL of phosphorus oxychloride was added to the obtained solution with stirring, stirred for 2 hours, and 30 mL of ether was added to obtain The organic layer was washed successively with NaHCO ₃ and saturated brine, dried over anhydrous Na ₂ SO ₄ , and concentrated under reduced pressure to obtain a crude residue, which was separated and purified on a silica gel column to obtain 112 mg of compound Arglabin, with a calculated yield of 45 %.

¹ H NMR (400 MHz, CDCl ₃ ) δ 6.15 (d, J=3.3 Hz, 1H), 5.58 (s, 1H), 5.42 (d, J=3.1 Hz, 1H), 4.01 (t, J=10.2 Hz) , 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=7.9 Hz, 3H), 1.88–1.82 (m, 1H), 1.55–1.45 (m, 1H), 1.35 (d, J=6.4Hz, 3H). ¹³ C NMR (101MHz, CDCl ₃ )δ170.43, 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 structural formula of the compound DMAPT is as follows:

The compound DMAPT was prepared as follows: 20 mg of parthenolide (PTL) was dissolved in 2 mL of tetrahydrofuran (THF), then 10 mg of K ₂ CO ₃ and 0.5 mL of dimethylamine (40 wt % aqueous solution) were added respectively, and the resulting mixture was at room temperature It was stirred overnight under low pressure, 20 mL of CH ₂ Cl ₂ was added, washed with saturated brine (2×20 mL), the obtained organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to obtain 22 mg of yellow solid, namely compound DMAPT, whose yield was calculated by calculation. The rate is 93%.

¹ H NMR (400MHz, CDCl ₃ )δ5.25-5.16(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). ¹³ C NMR (101MHz, CDCl ₃ )δ176.21,134.68,125.12,82.29,66.38,61.56,57.31,48.21,46.15,45.77,41.09,36.68,29.81,24.12,17.23,16.93.HRMS(ESI)m/ z calcd for C ₁₇ H ₂₈ NO ₃ ⁺ (M+H) ⁺ 294.20637, found 294.20624.

Example 5

The structural formula of compound 2 is as follows:

The preparation method of compound 2 is as follows: take 20 mg of compound MCL prepared in Example 1 and dissolve it in 2 mL of tetrahydrofuran (THF), then add 10 mg of K ₂ CO ₃ and 0.5 mL of dimethylamine (40 wt % aqueous solution) respectively, and the resulting mixture is in Stir overnight at room temperature, add 20 mL of CH ₂ Cl ₂ , wash with saturated brine (2×20 mL), the obtained organic layer is dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to obtain 23 mg of a yellow solid, namely compound 2, which is calculated as The yield was 97%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 3.76 (t, J=10.3 Hz, 1H), 2.67 (dd, J=12.9, 5.0 Hz, 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). ¹³ C NMR (101MHz, CDCl ₃ )δ176.00, 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 C ₁₇ H ₂₈ NO ₃ ⁺ (M+H) ⁺ 294.20637,found 294.20630.

Example 6

The structural formula of compound 3 is as follows:

The preparation method of compound 3 is as follows: take 20 mg of compound 1 prepared in Example 2 and dissolve it in 2 mL of tetrahydrofuran (THF), then add 10 mg of K ₂ CO ₃ and 0.5 mL of dimethylamine (40 wt % aqueous solution) respectively, and the resulting mixture is in Stir overnight at room temperature, add 20 mL of CH ₂ Cl ₂ , wash with saturated brine (2×20 mL), the obtained organic layer is dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to obtain 22 mg of a yellow solid, namely compound 3, which is calculated as The yield was 96%.

¹ H NMR (400MHz, CDCl ₃ ) δ 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). ¹³ C NMR (101MHz, CDCl ₃ )δ175.94,80.60,78.67,68.84,61.23,56.89,54.27,49.39,44.93, 43.22,36.42,32.57,28.37,22.20.HRMS(ESI)m/z calcd for C ₁₇ H ₂₈ NO ₄ ⁺ (M+H) ⁺ 310.20128,found310.20114.

Example 7

The structural formula of compound 4 is as follows:

The preparation method of compound 4 is as follows: Dissolve 20 mg of the compound Arglabin prepared in Example 3 in 2 mL of tetrahydrofuran (THF), then add 10 mg of K ₂ CO ₃ and 0.5 mL of dimethylamine (40 wt % aqueous solution) respectively, and the resulting mixture is in Stir overnight at room temperature, add 20 mL of CH ₂ Cl ₂ , wash with saturated brine (2×20 mL), the obtained organic layer is dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to obtain 23 mg of a yellow solid, namely compound 4. The yield was 92%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.49 (s, 1H), 3.94 (t, J=10.2 Hz, 1H), 2.68 (ddd, J=21.9, 17.7, 7.6 Hz, 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). ¹³ C NMR (101MHz, CDCl ₃ )δ176.72, 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 C ₁₇ H ₂₆ NO ₃ ⁺ (M+H) ⁺ 292.19072,found 292.19067.

Example 8

The structural formula of compound DMAPT-D6 is as follows:

The preparation method of compound DMAPT-D6 is as follows: take 20 mg parthenolide (PTL) and dissolve it in 2 mL of tetrahydrofuran (THF), then add 50 mg K ₂ CO ₃ and 20 mg dimethylamine-d6-amine hydrochloride, respectively, to obtain a mixture Stir overnight at room temperature, add 20 mL of CH ₂ Cl ₂ , wash with saturated brine (2×20 mL), the obtained organic layer is dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to obtain 20 mg of yellow solid, namely compound DMAPT-D6 , the calculated yield is 83%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.14 (d, J=9.9 Hz, 1H), 3.76 (t, J=9.0 Hz, 1H), 2.68 (dt, J=6.7, 3.9 Hz, 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) _a ^_ ,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 forC ₁₇ H ₂₂ D ₆ NO ₃ ⁺ (M+H) ⁺ 300.24403,found 300.24438.

Example 9

The structural formula of compound 5 is as follows:

The preparation method of compound 5 is as follows: take 20 mg of MCL prepared in Example 1 and dissolve it in 2 mL of tetrahydrofuran (THF), then add 50 mg _of K2CO3 and ₂₀ mg of dimethylamine-d6-amine hydrochloride, respectively, and the resulting mixture is at room temperature. It was stirred overnight under low pressure, 20 mL of CH ₂ Cl ₂ was added, washed with saturated brine (2×20 mL), the obtained organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to obtain 23 mg of a yellow solid, namely compound 5, the yield of which was calculated. The rate is 95%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 3.75 (t, J=10.3 Hz, 1H), 2.70-2.45 (m, 4H), 2.31 (dt, J=11.6, 5.3 Hz, 2H), 2.21-2.04 ( m, 4H), 1.97 (dd, J=22.6, 11.1 Hz, 1H), 1.78–1.67 (m, 2H), 1.61 (s, 3H), 1.23 (s, 3H), 1.21–1.15 (m, 1H) . ¹³ C NMR (101MHz, CDCl ₃ )δ176.06,130.86,130.32,83.11,79.33,57.36,57.10,49.94,43.76,37.41,34.39,29.00,26.36,22.75,21.81.HRMS(ESI)m/z calcd ₁₇ H ₂₂ D ₆ NO ₃ ⁺ (M+H) ⁺ 300.24403, found 300.24490.

Example 10

The structural formula of compound 6 is as follows:

The preparation method of compound 6 is as follows: take 20 mg of compound 1 prepared in Example 2 and dissolve it in 2 mL of tetrahydrofuran (THF), then add 50 mg of K ₂ CO ₃ and 20 mg of dimethylamine-d6-amine hydrochloride respectively, and the resulting mixture is in Stir overnight at room temperature, add 20 mL of CH ₂ Cl ₂ , wash with saturated brine (2×20 mL), the obtained organic layer is dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to obtain 21 mg of a yellow solid, namely compound 6, which is calculated as The yield was 90%.

¹ H NMR (400MHz, CDCl ₃ ) δ 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). ¹³ C NMR (101MHz, CDCl ₃ )δ176.00, 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 C ₁₇ H ₂₂ D ₆ NO ₄ ⁺ (M+H) ⁺ 316.23895,found316.23895.

Example 11

The structural formula of compound 7 is as follows:

The preparation method of compound 7 is as follows: take 20 mg of the compound Arglabin prepared in Example 3 and dissolve it in 2 mL of tetrahydrofuran (THF), then add 50 mg of K ₂ CO ₃ and 20 mg of dimethylamine-d6-amine hydrochloride respectively, and the obtained mixture is in Stir overnight at room temperature, add 20 mL of CH ₂ Cl ₂ , wash with saturated brine (2×20 mL), the obtained organic layer is dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to obtain 23 mg of a yellow solid, namely compound 7, which is calculated as The yield was 91%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 5.56 (s, 1H), 4.01 (t, J=10.2 Hz, 1H), 2.86-2.67 (m, 3H), 2.57 (dd, J=13.1, 6.0 Hz, 1H), 2.33–2.00(m, 5H), 1.93(s, 3H), 1.46(dd, J=18.6, 6.3Hz, 2H), 1.33(s, 3H). ¹³ C NMR (101MHz, CDCl ₃ )δ177 .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 ₂ C ₁₇ HSI) m/z calcd D ₆ NO ₃ ⁺ (M+H) ⁺ 298.22838, found 298.22818.

Example 12

In this example, the effects of the parthenolide derivatives prepared in Examples 1-11 on glioblastoma were studied, and the details are as follows.

The human glioblastoma cell lines U87 and LN229 used in this example were purchased from Cobioer, both cell lines were free of mycoplasma and had been identified by STR detection. Both U87 and LN229 cell lines were cultured in DMEM medium purchased from HyClone containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (100 U/ ml Penicillium and 100 μg/ml streptomycin), and the cells were cultured at 37 °C in a constant temperature incubator with 5% _CO . The cell culture steps are as follows:

(1) After counting U87 cells with an automatic cell counter, dilute U87 cells and LN229 with DMEM medium to 5×10 ³ cells/mL to obtain a cell suspension;

(2) Add 100 μL of cell suspension to each well of the 96-well plate, pipetting and mixing, and incubate in a 37°C incubator for 24 hours;

(3) PTL and the compounds prepared in Examples 1-11 were diluted with dimethyl sulfoxide to a concentration of 0.5 μM, 1.0 μM, 1.5 μM, and 2.0 μM, respectively, and added to different cultured cell lines. Incubate for 48h in an incubator;

(4) Add MTT solution (20 μL/well) and incubate in an incubator at 37°C for 4 hours;

(5) Remove the supernatant, dissolve the formed crystals in DMSO (200 μL/well), then measure the OD value (OD570) at 570 nm with a microplate reader (Bio-Tek, VT, USA), and measure the OD value (OD570) at 570 nm by GraphPad Prism 7.0 It is analyzed, the influence of each compound on the viability of U87 cells is shown in Table 1, the data in Table 1 shows that the parthenolide derivatives prepared by the present invention have different degrees of influence on the viability of U87 cells, among which the compound DMAPT-D6 The _IC50 value in U87 cells was 15.5 μM.

Table 1

The effect of the compound DMAPT-D6 on the viability of U87 cells and LN229 cells is shown in Figure 1. The data in Figure 1 show that the _IC50 values of DMAPT-D6 in U87 cells and LN229 cells are 15.5 μM and 11.15 μM, respectively. In order to further observe the inhibitory effect of the compound DMAPT-D6 on glioblastoma, the glioblastoma cells were treated with concentrations of 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM for 24 h, 48 h, and 72 h, and the cell growth and exposure time were observed. The relationship between the dose and the growth curve is shown in Figure 2. The growth curve diagram in Figure 2 shows that the compound DMAPT-D6 reduced the proliferation ability of U87 and LN229 cells, indicating that the compound has a dose and a dose-dependent effect on glioblastoma cells. Time-dependent cytotoxicity.

In order to further confirm the growth inhibitory effect of compound DMAPT-D6 on glioblastoma cells, a colony formation test was carried out in this example, specifically: human glioblastoma cells U87 and LN229 were divided according to the cell concentration of 1×10 ³ cells /cells/well were seeded on six-well plates, cultured overnight, treated cells with DMAPT-D6 at concentrations of 0 μM, 5 μM, 10 μM, and 20 μM for 48 h, and the treated cells were cultured in new medium for 15 days. After fixation 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 with at least 50 cells was counted under a 4x microscope. The results are shown in Figure 3. Figure 3 shows that DMAPT-D6 has a significant inhibitory effect on the growth of glioblastoma cells in a dose-dependent manner. The growth of tumor cells was significantly inhibited. The EdU staining test was performed, and the test results are shown in Figure 4. Figure 4 shows that in the EdU staining test, after treatment with the compound DMAPT-D6, the number of cells was significantly reduced in a dose-dependent manner, indicating that the compound DMAPT-D6 was effective on U87 and DMAPT-D6. The proliferation of LN229 cells was significantly inhibited.

In order to further understand the cytotoxic mechanism of compound DMAPT-D6 on glioblastoma cells, cell cycle analysis was also performed on U87 and LN229 cells treated with compound DMAPT-D6 or without DMAPT-D6 in this example. Specifically, glioblastoma cells in logarithmic growth phase were collected and transferred to six-well plates with a cell density of 30% in each well, and were treated with the compound DMAPT-D6 at concentrations of 0 μM, 5 μM, 10 μM, and 20 μM, respectively. After 48 h of cells, the cells were collected and used for flow cytometry analysis. For cell cycle determination, the collected cells were fixed with 70% ethanol at 4 °C for 24 h, and then washed with PBS for 3 times. /ml propidium iodide and 100mg/ml RNase in PBS were incubated at 37°C for 0.5 hours, and finally, the stained cells were analyzed by a BD-AccuriTM-C6 flow cytometer and the results were statistically compared using FlowJo Visualization was performed automatically by the software; for apoptosis detection, cells were harvested and processed using the Annexin V-FITC/PI Apoptosis Assay Kit within 1 hour using BD-AccuriTM-C6 flow cytometry according to the manufacturer's instructions The fluorescence-activated cells were analyzed by the instrument, and the apoptosis rate was analyzed by FlowJo software. The results are shown in Figure 5. Figure 5 shows that the compound DMAPT-D6 has a significant dose-dependent induction of cell cycle arrest on U87 and LN229 cells, arresting the cell cycle in S phase.

On the basis of the above experiments, immunoblot analysis was also performed in this example. Specifically, the collected glioblastoma cells were lysed in RIPA buffer (Beyotime, Shanghai, China) containing Halt ^TM protease and phosphatase inhibitors. For 30 min, after quantifying total protein concentration using BCA protein assay kit (Beyotime, Shanghai, China), protein samples (30 μg/lane) were separated by electrophoresis and transferred to PVDF membrane (Millipore, Billerica, MA, USA), Membranes were then blocked with 5% BSA for 2 hours at room temperature, and the primary antibodies were incubated overnight at 4°C with the corresponding IRDye 800CW goat anti-mouse IgG (h+L) or IRDye 680LT donkey anti-rabbit IgG (h+L) secondary After antibody incubation, images were imaged using an Odyssey dual-color infrared fluorescence imaging system (Li-cor, NE, USA), and β-actin was used as a control, and the results are shown in Figure 6. Figure 6 shows that the immunoblotting results showed that the compound DMAPT-D6 significantly reduced the protein levels of cyclin B, cyclin E, CDK1 and CDK2, while the expression of p27 in U87 and LN229 cell lines was dose-dependent with the increase of the compound DMAPT-D6 concentration Sex increases.

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

To further understand the mechanism of DMAPT-D6-induced cell proliferation inhibition, the production of reactive oxygen species (ROS) in U87 and LN229 cells was observed by fluorescence microscopy. Specifically, a ROS detection kit (Beyotime, Shanghai, China) was used to detect the formation of ROS, and glioblastoma cells treated with different concentrations of compound DMAPT-D6 were collected and centrifuged at 800 rpm for 5 minutes. DCFH-DA (10 μM) was resuspended in serum-free DMEM and incubated for 20 minutes. 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). The results are as follows As shown in Figure 7, the levels of reactive oxygen species produced by different concentrations of DMAPT-D6 treatment were significantly higher than those of the control group without DMAPT-D6 treatment.

Several studies have shown that the accumulation of intracellular ROS can induce DNA damage and lead to DNA damage response. To evaluate whether DMAPT-D6 can initiate the DNA damage process caused by excessive intracellular ROS, U87 and LN229 were detected by immunofluorescence and immunoblotting. Phosphorylated histone γH2AX in the cell line, the operation steps of immunofluorescence are as follows: Glioblastoma cells were grown in 24-well plates, and then treated with different concentrations of DMAPT-D6 for 48 hours, washed with PBS for 3 times, the cells were After fixing in 4% paraformaldehyde for 30 min, permeabilizing in 0.1% Triton X-100 for 10 min, and blocking for 30 min in immunostaining blocking buffer (Beyotime, Shanghai) at 37°C, cells were separately treated with anti-γH2AX (1:250) antibody was incubated overnight at 4°C, then stained cells were washed 3 times with PBS and incubated with Alexa Fluor-conjugated secondary antibody (1:2000) for 1 hour at room temperature using 1 mg/mL DAPI Nuclei were labeled for 30 minutes, and images were captured with a fluorescence microscope (Olympus IX53/DP80, Japan). The immunofluorescence results are shown in FIG. 8 . The results shown in Figure 8 show that, in U87 and LN229 cells, the green signal representing γH2AX was significantly accumulated in the nucleus in a dose-dependent manner, and γH2AX was significantly up-regulated, indicating DNA double-strand breaks in TNBC cells compared to the control group. To test whether DMAPT-D6 disrupts the DNA repair (DR) process, the expression of DR-related proteins such as p53-binding protein 1 (53BP1) and DNA ligase IV (LIG IV) in glioblastoma cells was detected by western blotting level, and the results of immunoblotting assay are shown in Figure 9. Figure 9 shows that after DMAPT-D6 treatment, the expression of NRF2 was up-regulated in a dose-dependent manner with the increase of DMAPT-D6 concentration, implying excessive accumulation of ROS in cells. In addition, as shown in Figure 9, DMAPT-D6 significantly reduced 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 extrinsic 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 explores whether DMAPT-D6 can promote the initiation of apoptosis. Notably, PI staining analysis showed that the percentage of PI-positive cells in U87 and LN229 cell lines significantly increased with increasing DMAPT-D6 concentration, as shown in Figure 10, indicating that DMAPT-D6 induced cell death. To further confirm the effect of DMAPT-D6 on apoptosis, Annexin V-FITC/PI analysis by flow cytometry was performed after U87 and LN229 cells were treated with DMAPT-D6 for 48 hours, as shown in Fig. At a low dose of 5 μM, 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 20 μM, the percentage of apoptosis increased, respectively to 46.9% and 34.7%. Next, the expression of external apoptosis signaling pathway-related proteins was analyzed to determine whether cell death receptors were involved in the apoptotic response to DMAPT-D6. Consistent with the above results, cell death receptor signaling pathway-related proteins such as DR3, DR5, FADD, TRADD were significantly up-regulated with increasing DMAPT-D6 concentration, as shown in Figure 12. Subsequently, due to the increase of FADD and TRADD, the caspase precursor (Procaspase) 8 is cleaved to produce the active enzyme form of caspase 8 (Caspase 8). Furthermore, downstream caspase 3 and PARP were spliced-activated in U87 and LN229 cells, implying that it mediated exogenous apoptosis through death receptors, as shown in Figure 12. To further confirm DMAPT-D6-induced cell death receptor-mediated apoptosis, a caspase inhibitor, Z-VAD-FMK, was used to examine the recovery effect of DMAPT-D6-induced apoptosis, such as As shown in Figure 13, Z-VAD-FMK can significantly rescue apoptosis induced by DMAPT-D6, and when treated with Z-VAD-FMK, can partially restore the proteins of DNA Lig IV, DR3, Caspase8, 3 and PARP levels, as shown in Figure 14, indicate that DMAPT-D6-induced death receptor-mediated apoptosis is caspase-dependent.

In conclusion, the compound of the present invention, DMAPT-D6, significantly up-regulated proteins related to death receptor signaling pathway, such as DR3, DR5, FADD, TRADD, and the activities of cysteine proteases 3, 8 and PARP, indicating that death receptors The mediated exogenous apoptosis was induced after DMAPT-D6 treatment; the above results showed that U87 and LN229 cells treated with the compound DMAPT-D6 induced excessive ROS accumulation leading to DNA damage, which further induced cell cycle S It inhibits the growth of glioblastoma cells and exerts anticancer effects on glioblastoma cells.

Therefore, the compound DMAPT-D6 is a potential anticancer drug with the ability to regulate reactive oxygen species, and can be used as a lead compound in the preparation of drugs for the treatment of glioblastoma, especially in the preparation of drugs for the treatment of glioblastoma, The medicine for treating glioblastoma comprises the above 11 parthenolide derivatives or pharmaceutically acceptable salts, hydrates or combinations thereof and adjuvants.

The above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced. Without departing from the spirit and scope of the technical solutions of the present invention, all of them should be included in the scope of the claims of the present invention. The technology, shape, and structural part that are not described in detail in the present invention are all well-known technologies.

Claims (9)

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

Wherein, the structural formula of R is = _CH or

2. parthenolide derivative according to claim 1, is characterized in that, described derivative is a kind of in following compound:

3. the preparation method of parthenolide derivative, is characterized in that, described preparation method is as follows:

4. the preparation method of parthenolide derivative according to claim 3, is characterized in that, described preparation method is as follows: Compound PTL and p-toluenesulfonic acid were stirred and reacted in dichloromethane, and the reaction was quenched with saturated NaHCO ₃ , the obtained organic layer was washed with saturated brine, dried over anhydrous Na ₂ SO ₄ , concentrated under reduced pressure, and recrystallized with acetone to obtain compound MCL; Compound MCL and m-chloroperoxybenzoic acid were stirred in dichloromethane to carry out epoxidation reaction, the obtained reactant was washed successively with Na ₂ SO ₄ , NaHCO ₃ and saturated brine, dried over anhydrous Na ₂ SO ₄ , and reduced pressure. Concentrated and recrystallized with acetone to obtain compound 1; Compound 1 and phosphorus oxychloride were stirred and reacted in pyridine, ether was added, and the resulting reaction mixture was washed with NaHCO ₃ and saturated brine successively, dried over anhydrous Na ₂ SO ₄ , concentrated under reduced pressure, and purified and separated on a silica gel column to obtain Compound Arglabin; Compounds PTL, MCL, Compound 1 and Arglabin were treated with dimethylamine in tetrahydrofuran under basic conditions, respectively. The resulting reaction mixture was added with dichloromethane, washed with saturated brine, dried over anhydrous Na ₂ SO ₄ , and concentrated under reduced pressure. , respectively obtain compound DMAPT, compound 2, compound 3 and compound 4; Under basic conditions, compounds PTL, MCL, compound 1 and Arglabin were treated with dimethyl-d6-amine hydrochloride in tetrahydrofuran, respectively, and dichloromethane was added to the resulting reaction mixture, washed with saturated brine, and anhydrous Na ₂ Dry over SO ₄ and concentrate under reduced pressure to obtain compound DMAPT-D6, compound 5, compound 6 and compound 7, respectively. 5. parthenolide derivative according to claim 4, is characterized in that, the preparation method of described compound DMAPT-D6 is specifically as follows: 20 mg of PTL was dissolved in 2 mL of tetrahydrofuran, then 50 mg of K ₂ CO ₃ and 20 mg of dimethyl-d6-amine hydrochloride were added and stirred overnight, 20 mL of dichloromethane was added, washed with saturated brine, and the obtained organic layer was washed with anhydrous Na ₂ SO ₄ was dried and concentrated under reduced pressure to obtain compound DMAPT-D6. 6 . The application of the parthenolide derivative according to claim 1 or 2 , wherein the application of the derivative in the preparation of a drug for treating glioblastoma. 7 . 7 . The application of the parthenolide derivative according to claim 6 , wherein the compound DMAPT-D6 is used in the preparation of a drug for treating glioblastoma. 8 . 8. The application of the parthenolide derivative according to claim 7, wherein the application of the compound DMAPT-D6 in the preparation of a drug for the treatment of glioblastoma is by accumulating intracellular reactive oxygen species, resulting in a DNA damage in plasmoblastoma cells, thereby inducing apoptosis in glioblastoma cells. 9. The application of the parthenolide derivative according to claim 8, wherein the medicine for treating glioblastoma comprises parthenolide derivative or a pharmaceutically acceptable salt, hydrate or Its combination and accessories.

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