CN105859777B - Suppress aloe-emodin quaternary alkylphosphonium salt of group and preparation method thereof containing glycolysis - Google Patents

Abstract

The invention discloses an aloe-emodin quaternary phosphonium salt dichloroacetate containing a glycolysis inhibiting group and a preparation method thereof; React with tri-n-octylphosphine to generate aloe-emodin quaternary phosphonium salt, and finally react with dichloroacetyl chloride to obtain aloe-emodin quaternary phosphonium salt dichloroacetate: . The in vitro cancer cell inhibition test of aloe-emodin quaternary phosphonium salt dichloroacetate shows that it has inhibitory activity on various cancer cells, and has great application prospects in the preparation of antitumor drugs.

Description

Aloe-emodin quaternary phosphonium salt containing glycolysis inhibitory group and preparation method thereof

technical field

The invention belongs to the field of preparation of anticancer drugs, in particular to an aloe-emodin quaternary phosphonium salt dichloroacetate containing a glycolysis inhibiting group and a preparation method thereof.

Background technique

Cancer cells have "Warburg effect", that is, cancer cells still tend to glycolysis even under the condition of sufficient oxygen supply. Cancer drug design ideas. At present, medical scientists generally use the combination of chemotherapy drugs and glycolysis inhibitors to study the anti-cancer mechanism of drugs.

Aloe-emodin (AE) is the antibacterial active ingredient of rhubarb, which can be extracted from aloe vera, and has antitumor activity, antibacterial activity and immunosuppressive effect. However, the activity of aloe-emodin itself is relatively weak. Therefore, structural modification of aloe-emodin to improve its activity has great application prospects. Early studies of our research group have shown that aloe-emodin quaternary phosphonium salt (AP) has good anticancer activity. Its combination with glycolysis inhibitor 2-DG can inhibit the mitochondrial function of cancer cells by reducing the mitochondrial membrane potential of cancer cells, and enhance the anticancer activity of emodin quaternary phosphonium salts. However, in the process of drug combination, it is found that the dosage of 2-DG is too large, which is easy to produce toxic and side effects; at the same time, it is difficult to ensure that the drug can reach the cancer site at the same time, and it is also difficult to ensure that the drug can produce a synergistic anti-cancer effect at an appropriate concentration.

Dichloroacetic acid (DCA) is a well-known glycolysis inhibitor, which can activate the kinase activity of pyruvate dehydrogenase, convert pyruvate into acetyl coenzyme and enter the aerobic metabolic pathway, thereby inhibiting the anaerobic metabolic pathway (ie, the glycolytic pathway) . The present invention couples emodin quaternary phosphonium salt with dichloroacetic acid through esterification reaction to form aloe-emodin quaternary phosphonium salt dichloroacetate (AP - DCA) . On the one hand, it enhances the anticancer activity of the quaternary phosphonium salt of aloe-emodin; on the other hand, it reduces the dosage of glycolysis inhibitors and reduces toxic and side effects.

Contents of the invention

The object of the present invention is to provide a kind of aloe-emodin quaternary phosphonium salt dichloroacetate containing glycolysis inhibitory group and preparation method thereof. By introducing a long carbon chain quaternary phosphonium salt on aloe-emodin as a lipophilic cation to target mitochondria, and then introducing a dichloroacetate to produce dichloroacetic acid as a glycolysis inhibitor after hydrolysis, thereby greatly improving the aloe-emodin anticancer activity.

To achieve the above object, the present invention adopts the following technical solutions:

A kind of aloe-emodin quaternary phosphonium salt dichloroacetate containing glycolysis inhibitory group, its structural formula is as follows:

.

The preparation method of described aloe-emodin quaternary phosphonium salt dichloroacetate, concrete steps are:

1) Synthesis of brominated aloe-emodin: 3.70 mmol aloe-emodin was dissolved in 100 mLCCl ₄ , 10.8 mmol PBr ₃ was added at room temperature, the temperature was raised to 65 °C, and the reaction was stirred under reflux for 24 h; the solvent was removed by rotary evaporation, and the residue Purified by silica gel column chromatography to obtain brominated aloe-emodin;

2) Synthesis of quaternary phosphonium salt of aloe-emodin: Dissolve 1.00 mmol of bromoaloe-emodin in 15 mL of acetone, add 1.12 mmol of tri-n-octylphosphine at room temperature, raise the temperature to 50 °C, and react for 6 h. Spin dry, load the sample with dichloromethane wet method, and obtain aloe-emodin quaternary phosphonium salt through gradient elution of silica gel column chromatography;

3) Synthesis of aloe-emodin quaternary phosphonium salt dichloroacetate: 0.14 mmol of aloe-emodin quaternary phosphonium salt and 1.09 mmol of potassium carbonate were dissolved in 20 mL of acetone in a three-necked flask, heated to 37 °C and stirred for 0.5 h, then added 1.05 mmol dichloroacetyl chloride, stirred for 3 h; after the reaction was stopped, the solvent was removed with a rotary evaporator, the sample was wet-loaded with dichloromethane, and the column chromatography was purified by gradient elution to obtain aloe-emodin quaternary phosphonium salt dichloroacetic acid ester.

The eluent used in the silica gel column chromatography purification in step 1) is CH ₂ Cl ₂ .

The sequence of gradient elution in step 2) is: V (CH ₂ Cl ₂ ): V (C ₂ H ₅ OH)= 40:1→30:1→25:1→20:1.

The elution order of gradient elution in step 3) is: V (CH ₂ Cl ₂ ): V (C ₂ H ₅ OH)=40:1→30:1→25:1→20:1.

The application of the above-mentioned aloe-emodin quaternary phosphonium salt dichloroacetate containing glycolysis inhibiting group in the preparation of medicines for treating cancer; the cancer includes lung cancer and liver cancer.

Significant advantage of the present invention is:

In the present invention, the glycolysis inhibitor is introduced into the aloe-emodin quaternary phosphonium salt through a chemical bonding method, and the high activity of the hydrolase of cancer cells can be used to ensure that the chemotherapeutic drug aloe-emodin quaternary phosphonium salt and the glycolysis inhibitor dichloroacetic acid have the same effect. At the same time, it reaches the cancer cell site and produces a synergistic anticancer effect; the anticancer activity test shows that the drug has good anticancer activity, and it has a proliferation inhibitory effect on a variety of cancer cells, and it is expected to be developed as a broad-spectrum anticancer drug.

Description of drawings

The synthetic route of Fig. 1 aloe-emodin quaternary phosphonium salt dichloroacetate;

Figure 2 The inhibitory activity of dichloroacetic acid, aloe-emodin and its derivatives on lung cancer cell A549 (IC ₅₀ , μM);

Fig. 3 Inhibitory activity of dichloroacetic acid, aloe-emodin and its derivatives on hepatoma cell HepG2 (IC ₅₀ , μM).

detailed description

In order to make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited thereto.

Example 1

A kind of preparation method of aloe-emodin quaternary phosphonium salt dichloroacetate, concrete steps are:

1) Synthesis of brominated aloe-emodin

Dissolve 3.70 mmol of aloe-emodin in 100 mLCCl ₄ , add 10.8 mmol of PBr ₃ at room temperature, raise the temperature to 65 °C, and stir under reflux for 24 h; after the solvent is removed by rotary evaporation, the residue is purified by silica gel column chromatography (elution Agent CH ₂ Cl ₂ ) to obtain orange-yellow solid brominated aloe-emodin; product characterization data are as follows:

Yield 72.1%; mp 72-73°C; R _f (CH ₂ Cl ₂ )=0.74; ¹ H NMR (400 MHz, CDCl ₃ ) δ: 12.09 (s, 1H, ArOH), 12.06 (s, 1H, ArOH ), 7.89 (d, J =5.2 H z , 1H, ArH), 7.88(s, 1H, ArH), 7.73 (t, J =7.6 H z , 1H, ArH), 7.36 (s, 1H, ArH), 7.35 (d, J =6.4H z , 1H, ArH), 4.51 (s, 2H, ArCH ₂ Br); ESI-MS m/z 331.1 (MH) ^- ; HRMS m/z330.9619, theoretical (MH) ^- 330.9611.

2) Synthesis of quaternary phosphonium salt of aloe-emodin

Dissolve 1.00 mmol of brominated aloe-emodin synthesized in step 1) in 15 mL of acetone, add 1.12 mmol of tri-n-octylphosphine at room temperature, raise the temperature to 50 °C, and react for 6 h. Methane was wet-loaded, and aloe-emodin quaternary phosphonium salt was obtained by gradient elution of silica gel column chromatography; the characterization data are as follows:

Yield 51.2%; mp 54-56°C; R _f (CH ₂ Cl ₂ :C ₂ H ₅ OH=20:1) = 0.21; ¹ H NMR (400 MHz, CDCl ₃ ) δ : 11.75 (br, 2H, OH), 7.63 (t, J =7.6Hz, 1H, Ar-H), 7.58 (m, 1H, Ar-H), 7.57 (m, 2H, Ar-H), 7.17 (m, 1H, Ar-H ), 4.67 [s, 1H, ArC H ₂ P ⁺ (C ₈ H ₁₇ ) ₃ ], 4.63[s, 1H, ArC H ₂ P ⁺ (C ₈ H ₁₇ ) ₃ ], 2.38 (m, 6H, P ⁺ (C H ₂ C ₇ H ₁₅ ) ₃ ), 1.49-1.21 [m, 36H, 3×CH ₂ (C ₆ H ₁₂ CH ₃ ) ₃ ], 0.81 (t, 9H, CH ₃ ); ¹³ C NMR (400 MHz, CDCL ₃ ) Δ : Δ :1, 180.4,162.7, 162.5, 140.5, 137.4, 133.8, 132.9, 126.0, 120.7, 120.1, 115.3, 115.1, 31.6, 31.2, 30.8, 29.0, 28.9.9.9 , 28.8, 22.6, 22.5,21.9, 21.6, 19.5, 19.0, 14.0; LC-MS(ESI) m/z : 623.67(M-Br) ⁺ .

3) Synthesis of aloe-emodin quaternary phosphonium salt dichloroacetate

Dissolve 0.14 mmol of aloe-emodin quaternary phosphonium salt and 1.09 mmol of potassium carbonate synthesized in step 2) in a three-neck flask with 20 mL of acetone, heat up to 37 °C and stir for 0.5 h, add 1.05 mmol of dichloroacetyl chloride, and stir for 3 h; After stopping the reaction, remove the solvent with a rotary evaporator, load the sample with dichloromethane wet method, and carry out column chromatography purification with gradient elution to obtain a reddish-brown viscous solid, that is, aloe-emodin quaternary phosphonium salt dichloroacetate; product characterization The data is:

Yield 36.6%; mp 46-48°C; R _f (CH ₂ Cl ₂ :C ₂ H ₅ OH=20:1) = 0.20; ¹ H NMR (400 MHz, CDCl ₃ ) δ : 7.70 (d, J = 7.6Hz, 1H, Ar-H), 7.60 (s, 1H, Ar-H), 7.53 (s, 1H, Ar-H), 7.37 (s, 1H, Ar-H), 7.20 (s, 1H, Ar -H), 6.06 (s, 1H, COC H Cl ₂ ), 4.32 [s,1H, ArC H ₂ P ⁺ (C ₈ H ₁₇ ) ₃ ], 4.30 [s, 1H, ArC H ₂ P ⁺ (C ₈ H ₁₇ ) ₃ ], 2.24 [m, 6H, ArCH ₂ P ⁺ (CH ₂ C ₇ H ₁₅ ) ₃ ] , 1.60-1.50 [m, 6H, 3×P ⁺ (CH ₂ CH ₂ C ₆ H ₁₃ ) ₃ ], 1.46-1.24 [m, 30H, 3×P ⁺ (C ₂ H ₄ C ₅ H ₁₀ CH ₃ ) ₃ ], 0.86(t, J =7.2Hz, 9H, CH ₃ ); ¹³ C NMR ( 400 MHz, CDCL ₃ ) Δ : 192.3,181.1, 167.4, 162.9, 162.7, 138.7, 137.7, 134.4, 133.1, 125.5, 120.51,120.47, 115.6, 37.1, 31.7, 31.7, 31.0, 31.0, 31.0, 31.7 30.8, 30.6, 30.0,29.7, 29.4, 29.0, 28.9, 28.8, 22.7, 22.6, 21.6, 21.3, 19.1, 18.6,14.1; LC-MS(ESI) m/z : (M-Br) ⁺ 733.67.

Application Example 1

Inhibitory experiment of aloe-emodin quaternary phosphonium salt dichloroacetate on the proliferation of lung cancer cell A549

Digest the A549 cells in the logarithmic phase with trypsin, count the cells, and prepare the cell concentration of 5×10 ⁴ -1×10 ⁵ /ml, inoculate 100 μL of the prepared cell suspension into a 96-well plate , placed in a 37 ℃, 5% CO ₂ incubator for 24 h, discarded the old medium, added different concentrations of aloe-emodin quaternary phosphonium salt dichloroacetate drugs, acted for 24 h respectively, and absorbed Discard the drug-containing medium, add 100 μL of 0.5 mg/ml MTT solution to each well, and continue to incubate for 4 h, then terminate the culture; carefully discard the supernatant in the wells of the 96-well plate, add 100 μL DSMO to each well, shake for 10 min, The light absorption value (OD value) of each well was measured on a microplate reader at a wavelength of 570 nm, and the cell survival rate (%) was calculated = (average OD value of the medication group/average OD value of the blank control group) × 100%. The GraphPad Prism software was used for data processing and the _IC50 value of the half maximal inhibitory concentration was calculated, and the results are shown in Figure 2.

Application Example 2

Inhibitory experiment of aloe-emodin quaternary phosphonium salt dichloroacetate on the proliferation of liver cancer cells HepG2

Digest the HepG2 cells in the logarithmic phase with trypsin, count the cells, and prepare the cell concentration of 5×10 ⁴ -1×10 ⁵ /ml, inoculate the prepared cell suspension, 100 μL per well, into a 96-well plate , placed in a 37°C, 5% CO ₂ incubator for 24 h, discarded the old medium, added different concentrations of aloe-emodin quaternary phosphonium salt dichloroacetate drugs, acted for 24 h respectively, and absorbed Discard the drug-containing medium, add 100 μL of a solution containing 0.5 mg/ml MTT to each well, continue to incubate for 4 h, and then terminate the culture; carefully discard the supernatant in the wells of the 96-well plate, add 100 μL DSMO to each well, shake for 10 The light absorption value (OD value) of each well was measured on a microplate reader at a wavelength of 570 nm, and the cell survival rate (%) was calculated = (average OD value of the medication group/average OD value of the blank control group) × 100%. GraphPad Prism software was used for data processing and the half inhibitory concentration IC ₅₀ value was calculated. The result is shown in Figure 3.

Table 1 IC ₅₀ values (μM) of dichloroacetic acid, aloe-emodin and their derivatives on lung cancer cell A549 and liver cancer cell HepG2.

The above experimental results show that aloe-emodin quaternary phosphonium salt dichloroacetate can produce aloe-emodin quaternary phosphonium salt and glycolysis inhibitor dichloroacetic acid after hydrolysis, which can simultaneously destroy the mitochondrial energy supply and glycolysis of cancer cells It provides energy and shows good anticancer activity against various tumor cells such as leukemia cell HL60, lung cancer cell A549 and liver cancer cell HepG2. This result shows that the covalent combination of dichloroacetic acid and aloe-emodin quaternary phosphonium salt is a good idea for the design of anticancer drugs.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims (8)

1. a kind of aloe-emodin quaternary phosphonium salt containing glycolysis inhibitory group, is characterized in that: its structural formula is as follows: . 2. a kind of preparation method of the aloe-emodin quaternary phosphonium salt containing glycolysis inhibiting group as claimed in claim 1, is characterized in that: aloe-emodin is reacted with excessive phosphorus tribromide, obtains brominated aloe-emodin brominated aloe-emodin and tri-n-octylphosphine for nucleophilic substitution reaction to obtain aloe-emodin quaternary phosphonium salt; aloe-emodin quaternary phosphonium salt and dichloroacetyl chloride for esterification to obtain Chloroacetate. 3. the preparation method of the aloe-emodin quaternary phosphonium salt containing glycolysis inhibitory group according to claim 2, is characterized in that: specifically comprise the following steps: 1) Synthesis of brominated aloe-emodin: 3.70 mmol aloe-emodin was dissolved in 100 mLCCl ₄ , 10.8 mmol PBr ₃ was added at room temperature, the temperature was raised to 65 °C, and the reaction was stirred under reflux for 24 h; the solvent was removed by rotary evaporation, and the residue Purified by silica gel column chromatography to obtain brominated aloe-emodin; 2) Synthesis of quaternary phosphonium salt of aloe-emodin: Dissolve 1.00 mmol of bromoaloe-emodin in 15 mL of acetone, add 1.12 mmol of tri-n-octylphosphine at room temperature, raise the temperature to 50 °C, and react for 6 h. Spin dry, load the sample with dichloromethane wet method, and obtain aloe-emodin quaternary phosphonium salt through gradient elution of silica gel column chromatography; 3) Synthesis of aloe-emodin quaternary phosphonium salt dichloroacetate: 0.14 mmol of aloe-emodin quaternary phosphonium salt and 1.09 mmol of potassium carbonate were dissolved in 20 mL of acetone in a three-neck flask, heated to 37 °C and stirred for 0.5 h, then added 1.05 mmol dichloroacetyl chloride, stirred for 3 h; after the reaction was stopped, the solvent was removed by a rotary evaporator, the sample was wet-loaded with dichloromethane, and the gradient elution was carried out for column chromatography purification to obtain aloe-emodin quaternary phosphonium salt dichloroacetic acid ester. 4. The preparation method of aloe-emodin quaternary phosphonium salt containing glycolysis inhibitory group according to claim 3, characterized in that: the eluent used in the silica gel column chromatography purification in step ₁ ) is _CH2Cl2 . 5. The preparation method of aloe-emodin quaternary phosphonium salt containing glycolysis inhibitory group according to claim 3, characterized in that: the order of gradient elution in step 2) is: V(CH ₂ Cl ₂ ): V(C ₂ H ₅ OH)= 40:1→30:1→25:1→20:1. 6. The preparation method of aloe-emodin quaternary phosphonium salt containing glycolysis inhibitory group according to claim 3, characterized in that: the elution order of gradient elution in step 3) is: V(CH ₂ Cl ₂ ): V(C ₂ H ₅ OH)=40:1→30:1→25:1→20:1. 7. an application of the aloe-emodin quaternary phosphonium salt containing glycolysis inhibitory group as claimed in claim 1 in the preparation of a medicine for treating cancer. 8. The application of the aloe-emodin quaternary phosphonium salt containing glycolysis inhibiting group in the preparation of cancer treatment medicine according to claim 7, characterized in that: said cancer comprises lung cancer and liver cancer.