CN104892668B - Prodrug and preparation method thereof before combretastatin analog water solublity - Google Patents

Abstract

The invention relates to a pro-prodrug of a combretastatin analogue and a preparation method thereof. The structural formula of this pro-prodrug is: The pro-prodrug improves its transport performance, and is made into a prodrug that has good transport properties in vivo and can effectively reach the target site of action.

Description

Combretastatin analog water-soluble pro-prodrug and preparation method thereof

technical field

The invention relates to a water-soluble pro-prodrug of a combretastatin analog (Combretastatin A-4 analogue) and a preparation method thereof.

Background technique

Combretastatin analogues are a class of small molecule tumor vascular-targeted blocking agents (Vascular Disrupting Agents, VDA). It can specifically target and block tumor blood vessels and destroy the generated tumor blood vessels, so that the tumor will not receive oxygen and nutrients until the tumor "starves to death". Therefore, combretastatin analogues can kill cancer cells at the core of tumors that cannot be easily killed by existing chemotherapy and radiotherapy ( British Journal of Cancer 110, 2170-2177 (29 April 2014 )).

Combretastatin analogues are a class of cis-1,2-stilbene arylamine derivatives. Due to its very low water solubility, its clinical application is limited. Currently, the water-soluble prodrug of serine hydrochloride (AVE8062), a combretastatin analogue, is being developed by Sanofi-Aventis Pharmaceuticals and has entered phase II clinical research. However, there are still problems such as poor hydrolytic stability, poor in vivo transport properties, and high toxicity. (Lancet Oncol. 2015 Apr 8.)

Prodrug (prodrug) design has become one of the important effective means in the field of drug design. Many patents have been applied for and obtained on the concept and application of prodrugs. However, such prodrugs often have some problems. For example, prodrugs designed according to the specific target cleavage mechanism (such as enzyme activity) often fail because they cannot reach the target tissue; predrugs designed to improve water solubility Medicine, because of its instability, easy to decompose in solution and can not be used. To overcome the above-mentioned shortcomings of prodrugs and improve the properties of prodrugs, it seems that a more promising method is to adopt the cascade latentiation method, that is, to design double prodrugs (Double prodrugs) or pro-prodrugs (Pro-prodrugs) ) (Bundgaard H. The doubleprodrug concept and its applications. Adv Drug Delivery Rev 1999; 3: 39). For example: the prodrug that is cleaved at a specific site is further latentized to improve its transport performance, and it is made into a prodrug that has good transport properties in the body and can effectively reach the target site of action. Another example: using tandem latentization, the prodrug has more ideal properties, that is, it has sufficient stability in vitro and high sensitivity of regenerating the parent drug in vivo. The general idea is to make the double prodrug stable enough in vitro to be enzymatically hydrolyzed into prodrug in vivo, and then to release the parent drug rapidly through enzymatic or non-enzymatic decomposition, so as to exert the drug effect. Further studies have found that: double prodrugs or pre-prodrugs are enzymatically hydrolyzed to generate prodrugs in the body, and then the parent drug can be released rapidly through enzymatic or non-enzymatic chemical action to exert the drug effect. Previous studies have shown that the amide form of the compound 3-(2'-hydroxy-4', 6'-dimethylphenyl)-3,3-dimethylpropanoic acid has rapid reactivity in the pH environment of the human body, Can meet the requirements of double prodrug design. Milstien and Cohen's group (J. Am. Chem. Soc.1972, 94 (26), 9158-9165) and Caswell and Schmir's group (J. Am. Chem. Soc.1980, 102 (14), 4815 -4821) all indicate that the dimethyl on the 3 and 3 positions and the methyl on the 6' position form a so-called "trimethyl lock" effect, which makes the molecule have rapid lactonization. Because of this feature, it can be well used to design prodrugs of amine compounds.

Contents of the invention

One of the objects of the present invention is to provide a combretastatin analog pro-prodrug.

The second object of the present invention is to provide a preparation method of the pro-prodrug.

The present invention creatively uses the concept of "sodium phosphate salt" water-soluble prodrug and "'trimethyl lock effect' of hydroxyl-amide compounds to improve in vivo transport performance" to design and synthesize the pro-pro drug of combretastatin analogues. As a drug, compound 2c with "trimethyl lock effect" was constructed first. Using 3,5-dimethylphenol and methyl isopentenoate to reflux reaction in methanesulfonic acid solution, 4,4,5,7-tetramethyl-3,4-dihydrocoumarin (2a ). Then 2a was reduced to 3-(2'-hydroxy-4', 6'-dimethylphenyl)-3,3-dimethylpropane under the condition of lithium aluminum hydride as reducing agent and anhydrous tetrahydrofuran as solvent. Alcohol (2b). Compound 2b was dissolved in dichloromethane, added a protective reagent tert-butyldimethylsilyl chloride, and reacted at room temperature for 24 hours under the action of an acid-binding agent triethylamine to obtain 1-O-tert-butyldimethylsilyl- 3-(2'-Hydroxy-4',6'-dimethylphenyl)-3,3-dimethylpropanol (2c). Phosphorylation is then performed to form a water-soluble disodium phosphate prodrug. Compound 2c was dissolved in anhydrous tetrahydrofuran, under the action of potassium tert-butoxide and tetrabenzyl pyrophosphate, reacted at 60°C for 1 hour to obtain 1-O-tert-butyldimethylsilyl-3-[2'-oxo (Dibenzyl phosphate)-4', 6'-dimethylphenyl]-3, 3-dimethylpropanol (2d), compound 2d was dissolved in acetone, after adding potassium fluoride, drop into The Jones reagent was newly prepared, and after the reaction, 3-[2'-oxo(dibenzylphosphoryl)-4', 6'-dimethylphenyl]-3, 3-dimethylpropionic acid (2e) was obtained. In tetrahydrofuran solvent, compound 2e was dissolved under stirring, then added freshly distilled thionyl chloride, heated to reflux for 4 hours, removed the solvent and excess thionyl chloride under reduced pressure, then added anhydrous tetrahydrofuran again to dissolve the reaction residue, added cis-1, 2-(3, 4, 4' , 5-tetramethoxy-3'-amino) stilbene (combretastatin analogue; Combretastatin A-4 analogue), overnight reaction at room temperature, column chromatography After separation and purification, the amidated prodrug of combretastatin analogue (2f) was obtained. Combretastatin analog amidated prodrug (2f) was dissolved in dry acetonitrile, trimethylbromosilane was added dropwise in ice-water bath, and after the reaction was completed by thin-plate chromatography (TCL), sodium methoxide/methanol solution was added , under stirring at room temperature, when a large amount of precipitation occurs, add acetone and stir overnight until the precipitation is complete to obtain 2 g of the water-soluble pro-prodrug of combretastatin analogs. The synthetic route of this method is:

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

A water-soluble pro-prodrug of combretastatin analogs, characterized in that its structural formula is as follows:

.

A method for preparing the water-soluble pro-prodrug of the above-mentioned combretastatin analog, characterized in that the specific steps of the method are:

a. Dissolve 3,5-dimethylphenol and methyl methacrylate in methanesulfonic acid at a molar ratio of 1:1.1 to 1:1.5, and reflux under stirring until the end of the TLC tracking reaction; Diluted with water, extracted with ethyl acetate, the organic layer was washed with 5% _NaHCO3 by mass percentage concentration, washed with saturated NaCl solution, dried with anhydrous _MgSO4 , filtered, the filtrate was rotary evaporated, and then purified to obtain a yellow solid, namely It is 4, 4, 5, 7-tetramethyl-3, 4-dihydrocoumarin, its structural formula is: ;

b. Under the protection of an inert atmosphere, reduce the 4,4,5,7-tetramethyl-3,4-dihydrocoumarin obtained in step a to 3- (2'-hydroxy-4',6'-dimethylphenyl)-3,3-dimethylpropanol, which has the structural formula: ;

c. Under an inert atmosphere, mix 3-(2'-hydroxyl-4',6'-dimethylphenyl)-3,3-dimethylpropanol and the protective reagent tert-butyldimethyl chloride obtained in step b Silane was dissolved in dichloromethane at a molar ratio of 1:1.1 to 1:1.5, and under the action of the acid-binding agent triethylamine, the reaction was stirred until the reaction was followed by TLC; Dissolved, washed with water, dried the organic phase with anhydrous MgSO ₄ , filtered, and the filtrate was rotary evaporated, separated and purified to obtain 1-O-tert-butyldimethylsilyl-3-(2'-hydroxyl-4', 6' -Dimethylphenyl)-3,3-dimethylpropanol, its structural formula is: ;

d. Combine 1-O-tert-butyldimethylsilyl-3-(2'-hydroxy-4',6'-dimethylphenyl)-3,3-dimethylpropanol and tert- Potassium butoxide was dissolved in tetrahydrofuran at a molar ratio of 1:1.1 to 1:1.5, and refluxed until the reaction solution changed from blue to yellow; then tetrabenzyl pyrophosphate was added, and the stirring and reflux reaction was continued until the reaction was followed by TLC; cooled to Add petroleum ether after room temperature, filter, remove the solvent from the filtrate, and obtain 1-O-tert-butyldimethylsilyl-3-[2'-oxo(dibenzylphosphate)-4', 6 '-dimethylphenyl]-3,3-dimethylpropanol, its structural formula is: ; The tetrabenzyl pyrophosphate and 1-O-tert-butyldimethylsilyl-3-(2'-hydroxyl-4', 6'-dimethylphenyl)-3,3-dimethyl The molar ratio of propanol is: 1:1.1～1:1.5;

e. In an ice-water bath, the 1-O-tert-butyldimethylsilyl-3-(2'-hydroxy-4', 6'-dimethylphenyl)-3,3-di Dissolve methyl propanol and potassium fluoride in acetone at a molar ratio of 1:4 to 1:7, slowly add the amount of Jones reagent as a catalyst dropwise, and continue to stir the reaction until the end of the TLC tracking reaction; add isopropyl alcohol dropwise to the reaction solution Continue stirring until the reaction solution turns from yellow-green to earth green; remove the solvent to obtain a dark green solid, dissolve it in ethyl acetate, wash with water, extract with ethyl acetate, and wash the organic phase with saturated NaCl solution. Dry, filter, remove the solvent, and then purify to obtain a white solid, which is 3-[2'-oxo(dibenzylphosphoryl)-4', 6'-dimethylphenyl]-3, 3-di Methylpropionic acid, its structural formula is: ;

f. Prepare 3-[2'-oxo(dibenzylphosphonyl)-4', 6'-dimethylphenyl]-3, 3-dimethylpropionic acid and thionyl chloride obtained in step e by 1 : 2 ~ 1:5 molar ratio dissolved in tetrahydrofuran solvent, reflux reaction for 4 hours, remove the solvent and excess thionyl chloride, then add anhydrous tetrahydrofuran again to dissolve the reaction residue, then add cis-1, 2-( 3, 4, 4, 5-tetramethoxy-3'-amino) stilbene, reacted at room temperature until the reaction was followed by TLC, after separation and purification, a combretastatin analog amidated prodrug was obtained, and its structural formula was: ; The 3-[2'-oxo(dibenzylphosphonyl)-4', 6'-dimethylphenyl]-3,3-dimethylpropionic acid and cis-1, 2-( The molar ratio of 3,4,4,5-tetramethoxy-3'-amino)stilbene is: 1:0.5～1:1;

g. Dissolve the amidated prodrug of the combretastatin analog obtained in step f in acetonitrile, add trimethylbromosilane dropwise under ice-water bath conditions, continue to stir the reaction until the TLC tracking reaction is complete, and then add methanol solution of sodium methoxide , Stirring at room temperature reacts with a white precipitate; add a large amount of acetone solution and stir overnight, and filter to obtain a white powder solid, which is the water-soluble pro-prodrug of combretastatin analogues, and its structural formula is: .

The water-soluble pro-prodrug of the combretastatin analog involved in the present invention is a novel controlled-release prodrug. The National Shanghai New Drug Safety Evaluation Research Center conducts the test according to the standard of acute toxicology test. The mouse acute toxicity test data of Viagra, an analogue of the test substance, showed that when the dose reached 1000 mg/kg, the animals died. Therefore, the doses of this experiment were initially planned to be 300, 500, and 1000 mg/kg, and the 2000 mg/kg dose group was added on the fourth day after administration, and the toxicity and death of animals after a single administration were observed. Its acute toxicity test showed that the MTD was 2000mg/kg.

In the invention, tetrabenzyl pyrophosphate is used to phosphorylate phenolic hydroxyl groups to form sodium phosphate salt soluble prodrugs. Use 3-(2'-hydroxy-4', 6'-dimethylphenyl)-3, 3-dimethylpropanoic acid to construct the "trimethyl lock" effect to form a pro-prodrug, which is rapidly in vivo Esterification targets the release of the parent drug. The pro-prodrug improves its transport performance, has good transport properties in vivo, and can effectively reach the target site of action. The process of releasing the parent drug is shown in the following formula:

.

detailed description

Each of the following examples is a preparation method of a water-soluble pro-prodrug of a combretastatin analogue, and they are only used to illustrate the present invention, not to limit the present invention.

Example 1: Preparation of 4, 4, 5, 7-tetramethyl-3, 4-dihydrocoumarin (2a)

Weigh 3, 5-dimethylphenol (5.002 g 41 mmol) into a 50 ml eggplant-shaped bottle containing 5 ml methanesulfonic acid solution, and then add methyl methacrylate (5.161 g 45.2 mmol) under stirring , the solution was reddish brown after the addition. Install a spherical condenser tube and a drying tube in turn on the eggplant-shaped bottle, heat the oil bath to 80°C with magnetic stirring, condense and reflux, track with TLC (petroleum ether: ethyl acetate = 1:1), and the reaction ends (about 12 hours). Dilute the reaction solution with 800 ml of water, extract with ethyl acetate (200 ml×2), combine the organic layers, wash with 5% NaHCO ₃ (200 ml×3), then wash with 100 ml saturated NaCl solution, anhydrous MgSO ₄ Dry, filter, and rotary evaporate the filtrate to obtain a yellow solid, recrystallize from ethyl acetate-petroleum ether, filter to obtain a white crystalline solid, vacuum-dry (40°C), weigh 7.344 g, yield: 87.8%; mp 89- 90°C;

Compound 2a: ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.741 (1H, d, J = 0.5 Hz); 6.711 (1H, d, J = 0.5Hz); 2.589 (2H, s); 2.457 (3H, s ); 2.270 (3H,s); 1.434 (6H,s).

Example 2: Preparation of 3-(2'-hydroxy-4', 6'-dimethylphenyl)-3, 3-dimethylpropanol (2b)

Weigh LAH (1 equiv 0.951 g) dissolved in 40 ml of freshly distilled tetrahydrofuran solution in a 100 ml three-neck flask, stir under ice bath to form a gray suspension, then weigh compound 2a (5.109 g 25 mmol) and dissolve in 18 ml of freshly distilled The tetrahydrofuran solution was slowly dropped into the tetrahydrofuran solution of LAH under the protection of argon, and gas was generated. After dropping, the temperature was naturally raised to room temperature, and the reaction was stirred for 1 hour, and the reaction was followed by TLC (petroleum ether: ethyl acetate = 2:1). Slowly add saturated NH ₄ Cl solution dropwise to the above reaction solution to quench until no more bubbling, and the solution turns into a white suspension. Filtrate (add a layer of diatomaceous earth for filter aid and adsorption), mix the filtrate with 100 ml of water, extract with ether (50 ml×3), combine the organic phases, dry over anhydrous MgSO ₄ , filter, and remove the solvent by rotary evaporation of the filtrate to obtain Color viscous liquid, recrystallized with ethyl acetate-n-hexane, filtered to obtain a white shiny solid, dried in vacuum (40°C), weighed to obtain 4.655 g, yield: 89.4%; mp 112-114°C;

Compound 2b: ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.487 (1H, d, J = 0.5Hz), 6.334 (1H, d, J = 0.5Hz), 5.867 (1H, br), 3.624 (2H, t , J = 7.0 Hz), 2.471 (3H, s), 2.233 (2H, t, J = 7.0 Hz), 2.166 (3H, s), 1.548 (6H, s).

Example 3: Preparation of 1-O-tert-butyldimethylsilyl-3-(2'-hydroxyl-4',6'-dimethylphenyl)-3, 3-dimethylpropanol (2c )

Weigh compound 2b (1.165 g 6.5 mmol) into a 100 ml three-neck flask, then add tert-butyldimethylsilyl chloride (0.932 g 7.15 mmol), and dissolve it in 20 ml of dichloromethane under the protection of argon, in an ice bath, Stir for about 10 minutes. Add 6 ml of a dichloromethane solution of triethylamine (3.4 ml) dropwise into a three-necked flask through a constant-pressure dropping funnel at a rate of 1d/(6~7)S. After the dropwise addition, the ice bath was removed, and the temperature was naturally raised to room temperature, and the reaction system was light brown. Stir the reaction for 24 h, the reaction solution becomes clear, TLC traces the end of the reaction (petroleum ether: ethyl acetate = 1:1), and the reaction stops. The reaction solution was filtered, and the filtrate was spin-dried, then added 25 ml of dichloromethane to dissolve it, washed with water (10 ml×4), combined the organic phases, dried over anhydrous MgSO ₄ , filtered, and the filtrate was rotary-evaporated to obtain a white solid, washed with ethyl acetate -Recrystallized from n-hexane, filtered to obtain a colorless crystalline solid, dried in vacuum (40°C), weighed to obtain 2.064g, yield: 98.6%;

Compound 2c: ¹ H NMR (500 MHz, CDCl ₃ ) δ 6.487 (1H, d, J = 0.5Hz), 6.409 (1H, d, J = 0.5Hz), 5.622 (1H, br), 3.590 (2H, t , J = 7.0 Hz), 2.449 (3H, s), 2.183 (3H, s), 2.108 (2H, t, J = 7.0 Hz), 1.545 (6H, s), 0.868 (9H, s), 0.013 (6H , s).

Example 4: Preparation of 1-O-tert-butyldimethylsilyl-3-[2'-oxo(dibenzylphosphoryl)-4', 6'-dimethylphenyl]-3, 3 -Dimethylpropanol (2d)

Weigh compound 2c (0.252 g 0.78 mmol) and dissolve it in 20 ml of freshly distilled THF solution in a 100 ml three-neck flask, and then add potassium tert-butoxide (0.1 g 0.86 mmol) into the bottle while stirring, the solution turns blue. The oil bath was heated to 60°C, and the solution changed from blue to yellow after reflux for about 5 minutes. Add tetrabenzyl pyrophosphate (0.463 g 0.86 mmol) while hot, and continue to stir, a milky white precipitate forms. Continue the reaction under reflux for 1 hour, TLC tracking the end of the reaction (petroleum ether: ethyl acetate = 3:2), stop heating the reaction, add 30 ml petroleum ether after cooling to room temperature, filter, remove insoluble matter, the filtrate is light yellow clear solution, and the solvent was removed under reduced pressure to obtain a yellow oil. Separation and purification by column chromatography (eluent: ethyl acetate:petroleum ether=1:3), solvent removal by rotary evaporation, to obtain a light yellow oil, vacuum drying (40°C), weighing 0.394g, yield: 87.2%.

Compound 2d: ¹ H NMR (500 MHz, CDCl ₃ ) δ 7.135 (10H, m), 6.838 (1H, d, J =0.5Hz), 6.654 (1H, d, J = 0.5Hz), 5.171 (4H, d , J = 8.0 Hz), 3.562 (2H, t, J =7.0 Hz), 2.462 (3H, s), 2.149 (3H, s), 2.096 (2H, t, J = 7.0 Hz), 1.598 (6H,s ), 0.921(9H, s), 0.021(6H, s).

Example 5: Preparation of 3-[2'-oxo(dibenzylphosphoryl)-4', 6'-dimethylphenyl]-3, 3-dimethylpropionic acid (2e)

Weigh compound 2d (0.394 g 0.68 mmol) and dissolve it in 5 ml of acetone in a 25 ml three-neck flask, put it in an ice bath, then add potassium fluoride (0.040 g 2.72 mmol) into the bottle, and stir for 5 minutes. Slowly add 0.6 ml of freshly prepared Jones reagent into the reaction flask dropwise through the dropping funnel, the solution turns brown and precipitates form, continue to stir for 2 hours, and TLC traces the end of the reaction (petroleum ether: ethyl acetate = 3: 2). 2.7 ml of isopropanol was added dropwise into the reaction flask, the solution changed from yellow-green to earth green, and the stirring was continued for 20 minutes, the solution turned blue-green. The reaction solution was transferred to a 50 ml one-necked bottle, and the solvent was removed by rotary evaporation to obtain a dark green solid. The dark green solid was dissolved with 12 ml ethyl acetate, washed with 15 ml H ₂ O, and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (10 ml×3), and the organic phases were combined. Wash with saturated NaCl solution (10 ml×2), dry over anhydrous MgSO ₄ , filter, and remove the solvent by rotary evaporation of the filtrate to obtain a yellow-green oil, recrystallize with ether-n-hexane, filter to obtain a white solid, and dry in vacuo (40°C) , weighing 0.247g, yield: 75.4%;

Compound 2e: ¹ H NMR (500 MHz, DMSO-d6) δ 11.804 (1H, br), 7.367 (10H,m), 6.929 (1H,d, J = 0.5Hz), 6.735 (1H, d, J = 0.5 Hz), 5.141 (4H, d, J = 8.0Hz), 2.795 (2H, s), 2.475 (3H, s), 2.103 (3H, s), 1.512 (6H, s).

Example 6: Preparation of combretastatin analogs and the amidation product (2f) of 2e

Weigh compound 2e (0.531 g 1.1 mmol) into a 50 ml eggplant-shaped bottle, add 40 ml freshly steamed THF, stir to dissolve, then add SOCl ₂ (0.17 ml; 2.3 mmol), and install a spherical condenser with a drying tube , heated to reflux for 4 hours. After the reaction, the heating was stopped, and after cooling slightly, the solvent and excess SOCl ₂ were evaporated under reduced pressure to obtain a light yellow oil. The oil was dissolved in 40 ml of freshly distilled THF, and a combretastatin analogue (0.182 g 0.55 mmol) was added with stirring, and the reaction was stirred at room temperature overnight, and the reaction was completed by TLC tracking. The solvent was removed under reduced pressure, the oil was separated and collected by column chromatography, and the solvent was removed by rotary evaporation to obtain 0.402 g of a brown oil, with a yield of 90%.

Compound 2f: ¹ H NMR (500 MHz, CDCl ₃ ) δ 8.238 (1H, br), 7.670 (1H, s), 7.291 (10H, m), 7.114 (1H, s), 6.931 (1H, d, J = 0.5Hz), 6.920 (1H, q, J =8.5Hz), 6.722 (1H, d, J = 0.5Hz), 6.708 (1H, s), 6.582 (1H, d, J = 8.5Hz), 6.465 (1H , d, J = 12.5Hz), 6.386 (1H, d, J = 12.5Hz), 5.108 (4H, d, J = 8Hz), 3.825 (3H, s), 3.772 (3H, s), 3.645 (6H, s), 2.879 (2H, s), 2.467 (3H, s), 2.158 (3H, s), 1.647 (6H, s).

Example 7: Preparation of Combretastatin Analog Water-Soluble Pro-Prodrug (2g)

Weigh compound 2f (1.427g 1.8mmol) into a 50ml round bottom flask, add 9ml of dried acetonitrile, stir and dissolve under ice bath. Trimethylbromosilane (0.6ml 4.5mmol) was added dropwise into the constant pressure dropping funnel, the solution changed from colorless to purple, and the color of the solution gradually became lighter when stirring was continued, and the reaction was tracked by TLC. After the reaction was complete, sodium methoxide (195mg 3.6mmol) was added ) and methanol solution 18ml, stirred at room temperature, a white precipitate formed. Add a large amount of acetone solution and stir overnight, filter to obtain a white powder solid, vacuum dry (40°C), weigh 0.8972 g, yield: 75.8%;

Compound 2g: ¹ H NMR (500 MHz, D ₂ O) δ 8.226 (1H, br), 7.645 (1H, s), 7.117(1H, s), 6.923 (1H, d, J = 0.5Hz), 6.878 ( 1H, q, J = 8.5Hz), 6.767 (1H, d, J =0.5Hz), 6.718 (1H, s), 6.585 (1H, d, J = 8.5Hz), 6.457 (1H, d, J = 12Hz ), 6.382 (1H, d, J = 12.5Hz), 3.834 (3H, s), 3.766 (3H, s), 3.638 (6H, s), 2.855(2H, s), 2.439 (3H, s), 2.143 (3H, s), 1.623 (6H, s).

Claims (1)

1. a kind of method of before water solublity for preparing combretastatin analog-prodrug, it is characterised in that the concrete steps of the method For：

A. 3,5- xylenols are pressed into 1 with senecioic acid methyl ester:1.1～1:1.5 mol ratio is dissolved in pyrovinic acid, under stirring Back flow reaction terminates to TLC tracking reactions；By reactant liquor dilute with water, it is extracted with ethyl acetate, organic layer mass percent Concentration is 5% NaHCO₃Washing, then washed with saturation NaCl solution, anhydrous MgSO₄It is dried, filters, filtrate rotary evaporation, then Jing Purify to obtain yellow solid, as 4,4,5,7- tetramethyl -3,4- dihydrocoumarin, its structural formula is：；

B., under inert atmosphere protection, by obtained by step a 4,4,5,7- tetramethyl -3,4- dihydrocoumarin is anhydrous in lithium aluminium hydride reduction Under conditions of tetrahydrofuran makees solvent, 3- is reduced into（2 '-hydroxyl -4 ', 6 ' -3,5-dimethylphenyl）- 3,3- dimethyl propanol, Its structural formula is：；

C. under an inert atmosphere, by 3- obtained by step b（2 '-hydroxyl -4 ', 6 ' -3,5-dimethylphenyl）- 3,3- dimethyl propanol and guarantor Shield reagent tert-butyl chloro-silicane presses 1:1.1～1:1.5 mol ratio is dissolved in dichloromethane, in the work of acid binding agent triethylamine With under, stirring reaction terminates to TLC tracking reactions；Reacting liquid filtering, after filtrate is spin-dried for, is dissolved with dichloromethane, and water washing has Machine mutually uses anhydrous MgSO₄It is dried, filters, filtrate rotary evaporation, separated purification obtains 1-O- t-Butyldimethylsilyl -3- (2 '-hydroxyl -4 ', 6 ' -3,5-dimethylphenyl) -3,3- dimethyl propanol, its structural formula is：；

D. by 1-O- t-Butyldimethylsilyls -3- obtained by step c (2 '-hydroxyl -4 ', 6 ' -3,5-dimethylphenyl) -3,3- dimethyl Propanol and potassium tert-butoxide press 1:1.1～1:1.5 mol ratio is dissolved in tetrahydrofuran, is back to reactant liquor and is changed into yellow from blueness Color；Four benzyl ester of pyrophosphoric acid is added, is continued to be stirred at reflux to react to TLC tracking reactions and is terminated；Petroleum ether is added after being cooled to room temperature, Filter, filtrate removes solvent, separated purification obtains 1-O- t-Butyldimethylsilyl -3- [2 '-oxos（Dibenzyl phosphate Base）- 4 ', 6 ' -3,5-dimethylphenyl] -3,3- dimethyl propanol, its structural formula is：；It is described Four benzyl ester of pyrophosphoric acid and 1-O- t-Butyldimethylsilyl -3- (2 '-hydroxyl -4 ', 6 ' -3,5-dimethylphenyl) -3,3- dimethyl The mol ratio of propanol is：1:1.1～1:1.5；

E. in ice-water bath, by 1-O- t-Butyldimethylsilyls -3- obtained by step d (2 '-hydroxyl -4 ', 6 '-dimethyl benzene Base) -3,3- dimethyl propanol and potassium fluoride press 1:4～1:7 mol ratio is dissolved in acetone, is being slowly added dropwise catalyst amount Jones reagents, continue stirring reaction and terminate to TLC tracking reactions；To in reactant liquor Deca isopropanol to reactant liquor by yellow green Become native green, it is in aeruginouss to continue stirring to reactant liquor；Remove solvent and obtain dark green solid, dissolved with ethyl acetate, washing, It is extracted with ethyl acetate, organic faciess saturation NaCl solution is washed, is dried, filter, removes solvent, then purified white solid, As 3- [2 '-oxos（Dibenzyl phosphoric acid base）- 4 ', 6 ' -3,5-dimethylphenyl] -3,3- neopentanoic acids, its structural formula is：；

F. by 3- [2 '-oxos obtained by step e（Dibenzyl phosphoric acid base）- 4 ', 6 ' -3,5-dimethylphenyl] -3,3- dimethyl propylenes Acid and thionyl chloride press 1:2～1:5 mol ratio is dissolved in tetrahydrofuran solvent, and back flow reaction 4 as a child, removes solvent and mistake The thionyl chloride of amount, subsequently adds the remaining thing of anhydrous tetrahydro furan dissolving reaction again, adds cis-1,2-（3, 4, 4, 5- -3 '-amino of tetramethoxy）Stilbene, room temperature reaction are complete to TLC tracking reactions, it is separated after purification, obtain Kao Buta Spit of fland analog amidatioon prodrug, its structural formula is：；Described 3- [2 '-oxo（Dibenzyl phosphoric acid base）- 4 ', 6 ' -3,5-dimethylphenyl] -3,3- neopentanoic acids and cis-1,2-（3, 4, 4,5- -3 '-amino of tetramethoxy）The mol ratio of stilbene is：1:0.5～1:1；

G. combretastatin analog amidatioon prodrug obtained by step f is dissolved in acetonitrile, under the conditions of ice-water bath, Deca front three Bromide silane, continues stirring reaction and reacts complete to TLC tracking, add the methanol solution of Feldalat NM, and reaction is stirred at room temperature to be had White precipitate is generated；A large amount of acetone solns are added to be stirred overnight, sucking filtration obtains white powder solid, as combretastatin analog Before water solublity-prodrug, its structural formula is：。