CN104311434B - Schuttgelb double-strand bi-quaternary ammonium salt with antitumour activity and preparation method thereof - Google Patents

Abstract

The invention discloses an emodin double-chain diquaternary ammonium salt with anticancer activity and a preparation method thereof, which is a mixture of emodin 1,3-position diquaternary ammonium salt and emodin 3,8-position diquaternary ammonium salt; It is the Williamson etherification reaction of emodin and p-dibenzyl bromide in the presence of K ₂ CO ₃ to generate a mixture of 1,3-dibromobenzyl emodin and 3,8-dibromobenzyl emodin, and then The dibromomethylbenzyl-emodin mixture is reacted with a tertiary amine to obtain a mixture of the 1,3-position diquaternary ammonium salt of emodin and the 3,8-position diquaternary ammonium salt of emodin, and the two diquaternary ammonium salts are isomers , cannot be separated on the chromatographic column. The evaluation of anticancer activity shows that the activity of emodin double-chain diquaternary ammonium salt obtained in the present invention is higher than that of emodin monoquaternary ammonium salt, and can be used as a drug application in the treatment of malignant tumors, especially for the treatment of liver cancer, and has the effect on normal cells simultaneously. Small inhibitory effect has great application prospects.

Description

Emodin double-chain diquaternary ammonium salt with anticancer activity and preparation method thereof

technical field

The invention specifically relates to an emodin double-chain diquaternary ammonium salt with anticancer activity and a preparation method and application thereof.

Background technique

Emodin (1,3,8-trihydroxy-6-methylanthraquinone, emodin) is a natural anthraquinone derivative isolated from Polygonaceae plants, and its structural formula is: . Modern medicine has confirmed that emodin has spectrum anti-cancer activity, and has inhibitory effect on dozens of cancer cells such as liver cancer and gastric cancer. However, it still has some disadvantages, such as poor water solubility and insufficient anticancer activity, which cannot meet the requirements of direct medicine. Therefore, chemical modification of emodin to improve its water solubility and anticancer activity is the main research direction to develop it into a new anticancer drug. Wang Conghui et al. (Wang Conghui, Zhang Fengsen, Du Huadong, etc., "Synthesis and anticancer activity of double long-chain emodin quaternary ammonium salt derivatives", Journal of Fuzhou University (Natural Science Edition), 2011, 39 (3)) has It was found that the introduction of long carbon chain quaternary ammonium salt on emodin can significantly improve the anticancer activity of emodin, indicating that long carbon chain quaternary ammonium salt is a good pharmacophore of emodin.

Contents of the invention

The object of the present invention is to provide an emodin double-chain diquaternary ammonium salt with anticancer activity and a preparation method thereof, by introducing two long chains of quaternary ammonium salt on the emodin to further improve the anticancer activity of emodin.

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

An emodin double-chain diquaternary ammonium salt with anticancer activity, which is a mixture of emodin 1,3-position diquaternary ammonium salt and emodin 3,8-position diquaternary ammonium salt;

Described emodin 1, the structural formula of 3 double quaternary ammonium salts is:

;

The structural formula of the emodin 3,8-position diquaternary ammonium salt is:

;

Wherein, R ₁ is C ₉ H ₁₉ or C ₁₀ H ₂₁ , and R ₂ is C ₁₀ H ₂₁ .

The preparation method of the emodin double-chain diquaternary ammonium salt with anticancer activity is to react the Williamson etherification of emodin and p-dibenzyl bromide in the presence of K ₂ CO ₃ to generate a mixture of dibromomethylbenzyl emodin , then reacting the dibromomethylbenzyl-emodin mixture with a tertiary amine to obtain a mixture of emodin 1,3-position diquaternary ammonium salt and emodin 3,8-position diquaternary ammonium salt;

Its synthetic route is shown in the following formula:

.

Its preparation method specifically comprises the following steps:

1) Synthesis of dibromomethylbenzyl-emodin mixture: dissolve emodin in acetone, add two equivalents of K ₂ CO ₃ , heat and reflux at 65°C for 20 minutes, then add two equivalents of p-dibenzyl bromide, and react for 2 hours Cool to room temperature; add dilute HCl to the solution to adjust pH=6, add water to precipitate a large amount of precipitate; after suction filtration, the resulting precipitate is eluted and separated by silica gel column chromatography to obtain 1,3-dibromobenzyl-emodin ( 1 ) and 3,8-dibromomethylbenzyl emodin ( 2 ) mixture;

2) Synthesis of emodin double-chain diquaternary ammonium salt mixture: 1,3-dibromomethylbenzyl-emodin ( 1 ) and 3,8-position dibromomethylbenzyl-emodin ( 2 ) prepared in step 1) Add the mixture of CHCl 3 into CHCl ₃ , add two equivalents of tertiary amine to the mixture after heating to dissolve, reflux the reaction for 12 hours under stirring, and cool to room temperature; after the solvent is removed by rotary evaporation, the solid obtained is eluted and separated by silica gel column chromatography to obtain an orange solid, which is rhubarb A mixture of emodin 1,3-position diquaternary ammonium salts ( 3a-3b ) and emodin 3,8-position diquaternary ammonium salts ( 4a-4b ).

The eluent used in step 1) is dichloromethane; the eluent used in step 2) is prepared by dichloromethane and ethanol at a volume ratio of 20:1.

The obtained emodin double-chain diquaternary ammonium salt can be used to prepare cancer treatment drugs; the cancer includes liver cancer HepG2.

The remarkable advantage of the present invention is that: the anticancer activity of the emodin double-chain diquaternary ammonium salt prepared by the present invention is better than that of the emodin monoquaternary ammonium salt. In vitro cancer cell inhibition experiments show that the obtained emodin double-chain diquaternary ammonium salt can effectively kill liver cancer HepG2 cells, and has less toxicity to normal cells (HELF, lung fibroblasts), and has good anticancer activity. Tumor therapy has a good application prospect.

Detailed ways

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.

Embodiment 1: the synthesis of dibromomethylbenzyl emodin 1 and 2

Put 100mg (0.37mmol) emodin, 100mg (0.74mmol) K ₂ CO ₃ and 100ml acetone into a 250ml three-neck flask, control the temperature of the oil bath at 65°C, heat and reflux for 20 minutes, then add 195mg p-dibenzyl bromide ( 0.74mmol), reacted for 2h and cooled to room temperature, added dilute hydrochloric acid solution to the solution to adjust pH = 6, added 200mL water to precipitate a large amount of yellow solid, filtered with suction, dried and dissolved the obtained solid, applied dry method, passed through a silica gel column Chromatography, using dichloromethane as eluent, isolated an orange solid. It was determined that it was the compound 6-methyl-8-hydroxyl-1,3-bis(p-bromomethylbenzyloxy)-9,10-anthraquinone ( 1 ) and 6-methyl-1-hydroxyl-3 , a mixture of 8-bis(p-bromomethylbenzyloxy)-9,10-anthraquinone ( 2 ), abbreviated as 1 + 2 , the molar ratio of the two is about 2:1, R _f =0.87. Product characterization data are as follows:

Compound 1 + 2 , yield 45%; m.p: 218-221°C. ¹ HNMR (500MHz, CDCl ₃ ) δ: 13.24 (s, 0.5H, OH), 13.21 (s, 1.0H, OH), 7.81 (d, J =1.0Hz, 0.5H, Ar-H), 7.61 (m , 4.0H, ArO C ₆ H ₄ Br), 7.58 (d, J =2.5Hz, 1.0H, Ar-H), 7.50 (t, J =2.5Hz, 2.0H, ArO C ₆ H ₄ Br), 7.48 (d, J =2.5Hz, 2.0H, ArO C ₆ H ₄ Br), 7.47 (m, 4.0H, ArO C ₆ H ₄ Br), 7.44 (s, 1.0H, Ar-H), 7.41 (d, J =2.5Hz, 0.5H, Ar-H), 7.19 (s, 0.5H, Ar-H), 7.13 (d, J =0.5Hz, 1.0H, Ar-H), 6.90 (d, J =2.5Hz , 1.0H, Ar-H), 6.80 (d, J =2.5Hz, 0.5H, Ar-H), 5.30 (s, 2.0H, ArO CH ₂ C ₆ H ₄ CH ₂ Br), 5.23 (s, 2.0 _H , ArOCH2C6H4CH2Br ₎ , _{5.21 ( s} , 1.0H , _{ArOCH2C6H4CH2Br )} , 5.15 _{( s} , _{1.0H , ArOCH2C6H4CH2Br} ), 4.55 (s, 2.0H, ArOCH ₂ C ₆ H ₄ CH ₂ Br), 4.54 (ArOCH ₂ C ₆ H ₄ CH ₂ Br), 2.50 (s, 1.5H, Ar CH ₃ ), 2.46 (s, 3 .OH, ArCH3 ₎ .

Embodiment 2: 1, the synthesis of 3 double quaternary ammonium salts and 3,8 double quaternary ammonium salts

Take 100mg (0.16mmol) of dibromomethylbenzyl-emodin ( 1 + 2) obtained in Example 1 and add 20ml of chloroform into a 100ml three-necked flask, heat and dissolve under reflux, add 0.32mmol of tertiary amine, reflux under stirring for 12h, cool After reaching room temperature, the solvent was removed by rotary evaporation, and the obtained solid was subjected to silica gel column chromatography, using dichloromethane:ethanol=20:1 (v/v) as the eluent, and an orange solid was isolated. It was determined that it was a mixture of emodin 1,3-position diquaternary ammonium salt ( 3a-3b ) and emodin 3,8-position diquaternary ammonium salt ( 4a-4b ), abbreviated as 3a-3b + 4a-4b . With the different tertiary amines, the molar ratios of compounds 3 and 4 are different, and the product characterization data are as follows:

Compound 3a + 4a (molar ratio about 5:1), yield 41%; m.p. 128-130℃. ¹ H NMR (400Hz, CDCl ₃ ) δ: 13.21 (s, 1.0H, OH), 13.13 (s, 0.2H, OH), 7.74 (s, 1.0H, ArOCH ₂ C ₆ H ₄ -), 7.72 (s , 1.0H, ArOCH ₂ C ₆ H ₄ -), 7.67-7.65 (m, 2.0H, ArOCH ₂ C ₆ H ₄ -), 7.64 (s, 0.4H, ArOCH ₂ C ₆ H ₄ ), 7.61-7.59 ( m, 2.0H, ArOCH ₂ C ₆ H ₄ ), 7.58 (s, 0.4H, ArOCH ₂ C ₆ H ₄ ), 7.52 (s, 1.2H, Ar-H), 7.36 (s, 0.2H, Ar-H ), 7.35 (m, 0.4H, ArOCH ₂ C ₆ H ₄ ), 7.33 (s, 1.0H, ArOCH ₂ C ₆ H ₄ ), 7.31 (s, 1.0H, ArOCH ₂ C ₆ H ₄ ), 7.19 (s , 0.2H, ArOCH ₂ C ₆ H ₄ ), 7.17 (s, 0.2H, ArOCH ₂ C ₆ H ₄ ), 7.11 (s, 0.2H, Ar-H), 7.09 (d, J =2.4Hz, 1.0H , Ar-H), 7.02 (s, 1.0H, Ar-H), 6.61 (d, J =2.4Hz, 1.0H, Ar-H), 6.44 (d, J =2.0Hz, 0.2H, Ar-H ), 5.37 (s, 0.4H, ArOC H ₂ C ₆ H ₄ ), 5.29-5.26 (m, 4.4H, ArOC H ₂ C ₆ H ₄ ), 5.21 (s, 0.4H, -C ₆ H ₄ CH ₂ N ⁺ ), 5.11 (s, 0.4H, -C ₆ H ₄ CH ₂ N ⁺ ), 5.08 (s, 2.0H, -C ₆ H ₄ CH ₂ N ⁺ ), 4.94 (s, 2.0H, -C ₆ H ₄ CH ₂ N ⁺ ), 3.40 (m, 9.6H, 2× CH ₂ N ⁺ CH ₂ ), 3.18 (s, 6.0H, 2×N ⁺ CH ₃ ), 3.14(s, 1.2H, 2×N ⁺ CH ₃ ), 2.45 (s, 0.6H, ArCH ₃ ), 2.39 (s, 3.0H, ArCH ₃ ), 1.92-1.82[m, 9.6H, 2×N ⁺ (CH ₂ C H ₂ -) ₂ ], 1.39-1.28 (m, 62.4H, 2×N ⁺ C ₂ H ₄ (CH ₂ ) ₆ CH ₃ +2×N ⁺ C ₂ H ₄ (CH ₂ ) ₇ CH ₃ ), 0.89 (m, 14.4H, 4×RC H ₃ ). ESI-MS, m/z : 535.75 [M-2Br ⁻ ] ²⁺ . Anal. Calcd for C ₇₁ H ₁₁₀ Br ₂ N ₂ O ₅ ·0.5H ₂ O: C 68.75, H 9.02, N 2.26, Found: C 68.83, H 9.31, N 2.35.

Compound 3b + 4b (molar ratio greater than 20:1), yield 43%; m.p. 217-221°C. ¹ H NMR (400Hz, CDCl ₃ ) δ: 13.17 (s, 1H, OH), 7.79-7.77 (m, 2H, ArOCH ₂ C ₆ H ₄ ), 7.72-7.70 (m, 2H, ArOCH ₂ C ₆ H ₄ ), 7.64-7.62 (m, 2H, ArOCH ₂ C ₆ H ₄ ), 7.51 (s, 1H, Ar-H), 7.40-7.38 (m, 2H, ArOCH ₂ C ₆ H ₄ ), 7.06 (d, J =2.0Hz, 1H, Ar-H), 7.01 (s, 1H, Ar-H), 6.56 (d, J =2.0Hz, 1H, Ar-H), 5.26 (s, 2H, ArOC H ₂ C ₆ H ₄ ), 5.24 (s, 2H, ArOCH ₂ C ₆ H ₄ ), 5.08 (s, 2H, C ₆ H ₄ CH ₂ N ⁺ ), 4.98 (s, 2H, C ₆ H ₄ CH ₂ N ⁺ ), 3.69-3.54 (m, 8H, 2× CH ₂ N ⁺ CH ₂ ), 3.28 (s, 6H, 2×N ⁺ CH ₃ ), 2.39 (s, 3H, ArCH ₃ ), 1.85-1.78[ m, 8H, 2×N ⁺ (CH ₂ CH ₂ -) ₂ ], 1.26 (m, 56H, 4×N ⁺ C ₂ H ₄ (CH ₂ ) ₇ CH ₃ ), 0.89 (m, 12H, 4 ×N ⁺ C ₉ H ₁₈ CH ₃ ). ESI-MS, m/z : 549.67 [M-2Br ⁻ ] ²⁺ . Anal. Calcd for C ₇₃ H ₁₁₈ Br ₂ N ₂ O ₅ ·2H ₂ O: C 67.68, H 9.18, N 2.16, Found: C 67.55, H 9.49, N 2.21.

Embodiment 3: Cancer cell proliferation inhibition experiment

Emodin 1,3 and 3,8 bis-quaternary ammonium salts ( 3a+4a ) and ( 3b+4b ) were used as test drugs, and the drugs were diluted with medium; liver cancer cells Hep2 and normal cells HELF were taken, and their The density was adjusted to 1×10 ⁵ cells/ml, inoculated in 96-well plates, 100 μl per well, and cultured in a 37°C, 5% CO ₂ incubator for 24 h; the old medium was removed, and the test drug was added, every 100 μl wells, and a blank control group and an emodin group were set up, and 3 replicate wells were set up in each group. After 24 hours of drug action, discard the drug-containing medium, add 100 μl of serum-free and phenol red-free 1640 medium to each well, then add 10 μl of MTT solution, continue to incubate for 4 hours, and terminate the culture; To the supernatant, add 100 μl DSMO to each well, shake for 10 minutes, measure the light absorption value (OD value) of each well on a microplate reader at a wavelength of 570 nm, and calculate the half inhibitory concentration IC ₅₀ . The results are shown in Table 1.

Table 2 The activity of cancer cells and normal cells after treatment (IC ₅₀ , μmol/L)

The experimental results showed that emodin 1,3 and 3,8 bis-quaternary ammonium salts ( 3a+4a and 3b+4b ) all showed good anticancer activity on liver cancer cells, although they showed greater anticancer activity on normal cells. Toxicity, but the combination of drugs can often reduce the side effects of drugs, so it has a good application prospect in the treatment of cancer.

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 (4)

1. there is a Schuttgelb double-strand bi-quaternary ammonium salt for antitumour activity, it is characterized in that: described Schuttgelb double-strand bi-quaternary ammonium salt is Schuttgelb 1, the mixture of 3 bi-quaternary ammonium salts and Schuttgelb 3,8 bi-quaternary ammonium salts;

The structural formula of described Schuttgelb 1,3 bi-quaternary ammonium salts is:

；

The structural formula of described Schuttgelb 3,8 bi-quaternary ammonium salts is:

；

Wherein, R ₁for C ₉h ₁₉or C ₁₀h ₂₁, R ₂for C ₁₀h ₂₁.

2. there is a preparation method for the Schuttgelb double-strand bi-quaternary ammonium salt of antitumour activity as claimed in claim 1, it is characterized in that: by Schuttgelb with to dibenzyl bromine at K ₂cO ₃there is lower generation Williamson etherification reaction, generate dibromo xylyl Schuttgelb mixture, then by dibromo xylyl Schuttgelb mixture and reactive tertiary amine, obtain Schuttgelb 1, the mixture of 3 bi-quaternary ammonium salts and Schuttgelb 3,8 bi-quaternary ammonium salts.

3. there is the preparation method of the Schuttgelb double-strand bi-quaternary ammonium salt of antitumour activity according to claim 2, it is characterized in that: specifically comprise the following steps:

1) synthesis of dibromo xylyl Schuttgelb mixture: Schuttgelb is dissolved in acetone, adds the K of two equivalents ₂cO ₃, add again after 65 DEG C of reflux 20 min two equivalents to dibenzyl bromine, reaction 2h after be cooled to room temperature; In solution, add rare HCl adjust pH=6, the more a large amount of precipitation of the precipitation that adds water; After suction filtration, gained precipitation is separated through silica gel column chromatography wash-out, obtains 1, the mixture of 3 dibromo xylyl Schuttgelbs and 3,8 dibromo xylyl Schuttgelbs;

2) synthesis of Schuttgelb double-strand bi-quaternary ammonium salt mixture: 1,3 the dibromo xylyl Schuttgelbs and 3 obtained by step 1), the mixture of 8 dibromo xylyl Schuttgelbs adds CHCl ₃in, add the tertiary amine of mixture two equivalent after heating for dissolving, stir lower back flow reaction 12h, be cooled to room temperature; Revolve and steam except gained solid after desolventizing obtains orange solids through the separation of silica gel column chromatography wash-out, be Schuttgelb 1, the mixture of 3 bi-quaternary ammonium salts and Schuttgelb 3,8 bi-quaternary ammonium salts.

4. there is the preparation method of the Schuttgelb double-strand bi-quaternary ammonium salt of antitumour activity according to claim 3, it is characterized in that: step 1) eluent used is methylene dichloride; Step 2) eluent used is that 20:1 is formulated by volume for methylene dichloride and ethanol.