CN110003304A - A kind of water solubility triptolide derivative and its preparation method and application - Google Patents
A kind of water solubility triptolide derivative and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of water-soluble triptolide derivative and its preparation method and application, the chemical structural formula of the water solubility triptolide derivative is as follows:Wherein: n=2,3,4 ..., n are the positive integer greater than 1.The present invention improves the water solubility of compound, reduces the toxic side effect of triptolide under the premise of keeping triptolide bioactivity, and triptolide is enable safely and effectively to be applied to clinic.
Description
Technical field
The present invention relates to pharmaceutical technology fields more particularly to a kind of water-soluble triptolide derivative and preparation method thereof
And purposes.
Background technique
Triptolide is also known as triptolide, is a kind of epoxy separated from Celastraceae tripterygium plant
Diterpenic lactone is the main active of traditional Chinese medicine plant tripterygium wilfordii, have anti-inflammatory, immunosupress, antifertility and
The effects of antitumor.Triptolide antitumor action attracts wide attention.(~nM), tripterygium wilfordii first under low consistency conditions
Element can not only inhibit tumour growth, and can also enhance the anti-tumor effect of Treated with Chemotherapeutic Drugs object.Triptolide poorly water-soluble,
Therapeutic window is narrow, has biggish toxic side effect to digestive system, urogenital system and hematological system etc., limits it clinically
Research and development.
Summary of the invention
The present invention introduces after quinone propiono spreading out of obtaining by principle of pro-drug on 19 free hydroxyl groups of triptolide
Biology.Carboxyl is introduced on compound structure, increases the solubility of compound in water.Additionally due to quinone aoxidizes in tumor tissues
Reductase (NAD (P) H:quinone oxidoreductase-1, NQO1) is compared to normal tissue great expression.Therefore drug
After absorbing distribution, the choosing of drug can be improved at triptolide by spontaneous hydrolysis after NOQ1 is restored in tumor tissues
Selecting property, to reduce the toxicity of triptolide normal tissue.Has good clinical development prospect.
(as follows):
The technical solution adopted by the present invention is that:
Shown in the chemical structural formula such as following formula (I) of water-soluble triptolide derivative of the invention:
Wherein: n=2,3,4 ..., n are the positive integer greater than 1.
The synthetic route of the preparation method of water-soluble triptolide derivative of the invention is as follows:
Specific step is as follows for the method:
(1) synthesis of compound 1 (coumarin derivative): using hydroquinone compound as reaction raw materials, molal quantity, which is added, is
1-2 times of acrylic acid derivative reacted under conditions of 50-100 DEG C, and the reaction time is 2-24 hours, is reacted and is used
Solvent is methane sulfonic acid.After completion of the reaction, it is successively extracted with ethyl acetate, pure water, saturated sodium bicarbonate aqueous solution and saturation
Salt water washing, anhydrous sodium sulfate is dry, and evaporated under reduced pressure is recrystallized to give purpose compound with chloroform-n-hexane.
(2) synthesis of compound 2: the coumarin derivative reacted using the first step is added 1-3 times of molal quantity as raw material
Bromine water, reacted 24 hours under conditions of 25 DEG C, reaction dissolvent is acetic acid.Evaporated under reduced pressure after completion of the reaction, residue pass through
Methylene chloride is extracted, washing, and anhydrous sodium sulfate is dry, and evaporated under reduced pressure obtains purpose compound.
(3) synthesis of compound 3: using second step product and methanol as raw material, in the dichloro of 0 DEG C of 1.5-3 times of addition mole
Sulfoxide reacts 1-3 hours under the conditions of 25 DEG C.It is that stationary phase is isolated and purified with adsorbent, eluent is recovered dry
To compound 3.
(4) in the methanol solution of compound 3, mole 2.5-5 times of nitrine measured the synthesis of compound 4: is added several times
Change sodium water solution, evaporated under reduced pressure after room temperature reaction 24 hours, residue is extracted by methylene chloride, is washed, anhydrous slufuric acid
Sodium is dry, is that stationary phase is isolated and purified with adsorbent, eluent is recovered to be dried to obtain compound 4.
(5) synthesis of compound 5: compound 4 is dissolved in methylene chloride, and the triphenylphosphine that equimolar amounts is added several times is anti-
It answers 1 hour, after solvent is removed under reduced pressure, the aqueous solution that the tetrahydrofuran containing acetic acid is added is heated to reflux evaporated under reduced pressure after 4 hours, residual
It stays object to be extracted by ethyl acetate, is saturated NaHCO3With salt water washing, it is stationary phase with adsorbent that anhydrous sodium sulfate is dry
It is isolated and purified, eluent is recovered to be dried to obtain compound 5.
(6) synthesis of compound 6: the lithium hydroxide of 2-4 times of addition mole in the methanol aqueous solution of compound 5, in room
After temperature reaction 24 hours, methanol is removed under reduced pressure, adjusts aqueous solution pH to 2.0, filtering obtains compound 6.
(7) synthesis of compound 7: the Boc acid anhydrides of 2 times of addition mole in the dichloromethane solution of compound 6, in room temperature
After reaction 4 hours, methylene chloride is removed under reduced pressure, is that stationary phase is isolated and purified with adsorbent, eluent is recovered dry
To compound 7.
(8) using triptolide as raw material, compound 7 and 1-4 that molal quantity is 1.5-2 times the synthesis of compound 8: is added
The dicyclohexylcarbodiimide (DCC) of times molar ratio and the 4-dimethylaminopyridine (DMAP) for being catalyzed equivalent, in 20-60 DEG C of item
It being reacted under part, evaporated under reduced pressure after reaction 48 hours, residue is extracted by ethyl acetate, is washed, and anhydrous sodium sulfate is dry,
It is that stationary phase is isolated and purified with adsorbent, eluent is recovered to be dried to obtain compound 8.
(9) trifluoroacetic acid (TFA) synthesis of compound 9: is added in the dichloromethane solution of compound 8 in room temperature reaction
It is that stationary phase is isolated and purified with adsorbent after 4-8 hours, eluent is recovered to be dried to obtain compound 9.
(10) biosynthesis of compound 10: alkane diacid is slowly added dropwise in the dichloromethane solution of compound 9, in 65
DEG C after reaction 2-4 hour, it is removed under reduced pressure methylene chloride, adjusts aqueous solution pH to 2.0, filter, obtain compound 10.
Formula (II).
Further, above-mentioned technical proposal, step (1) hydroquinone compound are 2,3,5 quilt-CH3, NH2, OCH3,
X etc. substituted hydroquinone compounds.
Further, above-mentioned technical proposal, step (1) acrylic acid derivative are the hydrogen atom on the position β by 1-2 first
The alkene acid compounds that base or amino replace, preferably 3- methyl-2-butenoic acid or acrylic acid.
Further, above-mentioned technical proposal, step (10) the alkane diacid are malonic anhydride, succinic anhydride, glutaric acid
Any one of acid anhydride, adipic anhydride, pimelic acid acid anhydride or suberic anhydride etc..Amino on hydroquinone compound described in the step
Amido bond is formed with the alkane diacid.
Water-soluble triptolide derivative of the invention can be used for preparing anti-tumor drug.
A kind of anti-tumor drug, the anti-tumor drug include that the present invention water-soluble triptolide described above is derivative
Object.
The positive effect of the present invention is as follows:
The present invention on the hydroxyl that triptolide dissociates by introducing quinone propionic acid structure.Due to containing on compound structure
One free carboxyl, facilitates compound at salt, improves the water solubility of compound.Quinone oxidoreductase in tumor tissues simultaneously
(NAD (P) H:quinone oxidoreductase-1, NQO1) is compared to normal tissue great expression.Therefore drug is through absorbing
After distribution, spontaneous hydrolysis improves the selectivity of drug at triptolide after can restoring in tumor tissues, to reduce
The toxicity of triptolide normal tissue.The present invention reduces tripterygium wilfordii under the premise of keeping triptolide bioactivity
The toxic side effect of A prime enables triptolide to be safely and effectively applied to clinic.
Detailed description of the invention
Fig. 1 (A) TP is to the pharmacological activity of 2 cell of HepG and to the toxicity of normal liver cell, and (B) CX-94 is to HepG 2
The pharmacological activity of cell and toxicity to normal liver cell.(n=6)
Fig. 2 .CX-94 and 2 cell homogenates liquid temperature of HepG incubate 0h (A) and 1h (B) chromatogram afterwards.(C) CX-94 metabolic conversion
Generate XC-01 and TP.
Metabolic stability (n=3) of Fig. 3 .CX-94 in people's hepatomicrosome (A) and human plasma (B).
Tail vein injection gives triptolide and CX-94 after 1.5mg/kg CX-94 and is averaged blood in Fig. 4 .ICR Mice Body
Concentration-time graph (n=6).
Specific embodiment
The following examples are a further detailed description of the invention.
Embodiment 1
The preparation method of the water-soluble triptolide derivative of the present embodiment specifically comprises the following steps:
(1) 1.0g (6.58mmol) quinhydrones is disposably added to 10mL first after mixing with 0.54g (7.50mmol) acrylic acid
Alkyl sulfonic acid detects fully reacting in 70 DEG C of heating stirrings 18h, TLC, stops stirring, 125mL water is added, successively use acetic acid second
Ester extraction, pure water, saturated sodium bicarbonate aqueous solution and saturated brine washing, anhydrous sodium sulfate is dry, evaporated under reduced pressure.Use chlorine
Imitative-n-hexane recrystallization, obtaining yellow solid is compound XC-01, and yield is about 67%.[M+H]+:221.0；1H NMR,
600MHz(CDCl3) δ 6.45 (q, 1H), 3.04 (s, 2H), 1.96 (m, 3H), 1.92 (m, 3H), 1.41 (s, 6H).
(2) compound of 1.1g (5mmol) step (1) is dissolved in 40mL acetic acid solution；Take 0.57mL (11mmol) bromine water
It is added dropwise in above-mentioned solution after reaction being stirred at room temperature 24 hours, evaporated under reduced pressure, residue is extracted by methylene chloride, water
It washes, anhydrous sodium sulfate is dry, and evaporated under reduced pressure obtains yellow solid compound.Yield is about 70%.[M-H]-:313.0；1H NMR
(600MHz,CDCl3)δ3.04(s,2H),2.19(s,3H),2.16(s,3H),1.44(s,6H)。
(3) yellow solid compound that every 1.2g (3.9mmol) step (2) obtains is dissolved in 12mL methanol, in ice bath
0.56mL (8mmol) thionyl chloride is slowly added dropwise, the evaporated under reduced pressure after room temperature continues stirring 3 hours is added dropwise, residue is logical
Crossing adsorbent is that stationary phase is isolated and purified, and eluent is recovered to be dried to obtain yellow solid compound.Yield is about 64%.
[M+H]+:329；1H NMR(600MHz,CDCl3)δ3.56(s,3H),2.98(s,2H),2.14(s,3H),2.14(s,3H),
1.43(s,6H)。
(4) yellow solid compound that every 1.44g (4.36mmol) step (3) obtains is dissolved in 15mL methanol solution；It takes
0.85g (13.1mmol) sodium azide compound is dissolved in 5mL water, is added in above-mentioned solution several times in room temperature reaction 24 hours
Evaporated under reduced pressure afterwards, residue are extracted by methylene chloride, and washing, anhydrous sodium sulfate is dry, with adsorbent be stationary phase into
Row isolates and purifies, and eluent is recovered to be dried to obtain yellow solid compound.Yield is about 85%.[M+H]+:292.0；1H
NMR(500MHz,CDCl3)δ3.63(s,3H),2.99(s,2H),2.16(s,3H),1.89(s,3H),1.41(s,6H)。
(5) compound that every 1.22g (4.17mmol) step (4) obtains is dissolved in 25mL methylene chloride；It is added several times
Solvent is removed under reduced pressure after room temperature reaction 1 hour in 1.1g (4.17mmol) triphenylphosphine.Acetic acid containing 15ml, 15mL tetrahydro is added
The aqueous solution of furans is heated to reflux evaporated under reduced pressure after 4 hours, and residue is extracted by ethyl acetate, is saturated NaHCO3And salt
Water washing, it is that stationary phase is isolated and purified with adsorbent that anhydrous sodium sulfate is dry, and eluent is recovered to be dried to obtain red admittedly
Body compound.Yield is about 60%.[M+H]+:266.0；1H NMR(600MHz,CDCl3)δ4.61(s,2H),3.56(s,3H),
2.94(s,2H),2.19(s,3H),1.84(s,3H),1.43(s,6H)。
(6) red solid compound that every 1.3g (4.9mmol) step (5) obtains is dissolved in the mixed of 16mL methanol and 4mL water
It closes in solution；By the way that 360mg (15mmol) lithium hydroxide is added after room temperature reaction 24 hours, methanol is removed under reduced pressure, adjusts water
For pH value of solution to 2.0, filtering obtains violet solid compound.Yield is about 90%.[M+H]+:252.0；1H NMR,600MHz,
(CDCl3)δ11.98(s,1H),6.32(s,2H),2.80(s,2H),2.07(s,3H),1.72(s,3H),1.35(s,6H)。
(7) every 1.26g (5.0mmol) violet solid compound is dissolved in 15mL methylene chloride；By the way that 2.18g is added dropwise
(Boc) of (10.0mmol)2Methylene chloride is removed under reduced pressure after room temperature reaction 4 hours in O acid anhydrides, with adsorbent be stationary phase into
Row isolates and purifies, and eluent is recovered to be dried to obtain violet solid compound.Yield is about 82%.[M+H]+:352.0；1H
NMR,600MHz,(CDCl3)δ11.98(s,1H),6.31(s,1H),2.82(s,2H),2.07(s,3H),1.72(s,3H),
1.44 (s, 9H), 1.37 (s, 6H).
(8) the violet solid counterpart and 360mg (1mmol) tripterygium wilfordii first that every 527mg (1.5mmol) step (7) obtains
Element is dissolved in 15mL methylene chloride, is stirred at 60 DEG C by the way that 824mg (4mmol) DCC and 127mg (1mmol) DMAP is added
48h is filtered after completion of the reaction, collects filtrate, and filtrate volatilizes, and ethyl acetate redissolves, and filtering, filtrate volatilizes to obtain violet solid
Close object.Yield is about 30%.[M+H]+:695.0；1H NMR,600MHz(CDCl3)δ6.39(s,1H),5.30(s,1H),4.97
(s,1H),4.19(d,2H),3.84(d,1H),3.56(d,1H),3.49(d,1H),3.02(s,2H),2.65(m,1H),2.24
(s, 3H), 2.12 (m, 2H), 1.99 (s, 3H), 1.97 (s, 3H), 1.86 (m, 2H), 1.55 (m, 1H), 1.49 (s, 3H), 1.46
(s, 9H), 1.22 (m, 1H), 1.01 (s, 3H), 0.90 (d, 3H), 0.80 (d, 3H).
(9) the violet solid counterpart that every 694mg (1mmol) step (8) obtains is dissolved in 15mL methylene chloride, by adding
Enter 5mLTFA to be stirred at room temperature 4-8 hours, methylene chloride is removed under reduced pressure, be that stationary phase is isolated and purified with adsorbent, elutes
Liquid is recovered to be dried to obtain violet solid compound.Yield is about 67%.[M+H]+:594.0；1H NMR,600MHz(CDCl3)δ
6.34(s,2H),5.30(s,1H),4.97(s,1H),4.19(d,2H),3.84(d,1H),3.56(d,1H),3.49(d,1H),
3.02(s,2H),2.65(m,1H),2.16(s,3H),2.12(m,2H),1.96(s,3H),1.94(s,3H),1.86(m,2H),
1.55 (m, 1H), 1.49 (s, 3H), 1.22 (m, 1H), 1.01 (s, 3H), 0.90 (d, 3H), 0.80 (d, 3H).
(10) the violet solid counterpart that every 594mg (1mmol) step (9) obtains is dissolved in 15mL methylene chloride, is passed through
5mL is added, 150mg (1.5mmol) succinic anhydride is slowly added dropwise, after reaction 2-4 hours, methylene chloride is removed under reduced pressure in 65 DEG C, use
Adsorbent is that stationary phase is isolated and purified, and eluent is recovered to be dried to obtain violet solid compound.Yield is about 67%.
[M-H]-:692.0；1H NMR,600MHz(CDCl3)δ6.38(s,1H),5.31(s,1H),4.99(s,1H),4.19(d,2H),
3.84 (d, 1H), 3.57 (d, 1H), 3.49 (d, 1H), 3.12 (s, 2H), 2.64 (m, 1H), 2.42 (s, 4H), 2.19 (s, 3H),
2.14 (m, 2H), 1.96 (s, 3H), 1.91 (s, 3H), 1.86 (m, 2H), 1.54 (m, 1H), 1.49 (s, 3H), 1.22 (m, 1H),
1.15(s,3H),0.90(d,3H),0.81(d,3H)。
(11) the violet solid counterpart that every 594mg (1mmol) step (9) obtains is dissolved in 15mL methylene chloride, is passed through
5mL is added and is slowly added to 171mg (1.5mmol) glutaric anhydride, after reaction 2-4 hours, methylene chloride is removed under reduced pressure in 65 DEG C, use
Adsorbent is that stationary phase is isolated and purified, and eluent is recovered to be dried to obtain violet solid compound.Yield is about 67%.
[M-H]-:706.0；1H NMR,600MHz(CDCl3)δ6.39(s,1H),5.34(s,1H),4.97(s,1H),4.29(d,2H),
3.86 (d, 1H), 3.59 (d, 1H), 3.49 (d, 1H), 3.12 (s, 2H), 2.65 (m, 1H), 2.46 (s, 4H), 2.18 (s, 3H),
2.12 (m, 2H), 2.06 (s, 3H), 1.97 (s, 3H), 1.92 (s, 2H), 1.86 (m, 2H), 1.55 (m, 1H), 1.49 (s, 3H),
1.24(m,1H),1.21(s,3H),0.92(d,3H),0.84(d,3H)。
Embodiment 2
The preparation method of the water-soluble triptolide derivative of the present embodiment specifically comprises the following steps:
(1) every 1.0g (6.58mmol) quinhydrones correspondence is primary after mixing with 0.85g (7.50mmol) 3- methyl-2-butenoic acid
Property be added to 10mL methyl sulfonic acid, in 70 DEG C of heating stirring 4h, TLC detects fully reacting, stops stirring, 125mL water is added,
It is successively extracted with ethyl acetate, water, saturated sodium bicarbonate aqueous solution and saturated brine washing, anhydrous sodium sulfate is dry, decompression
It is evaporated, is recrystallized with chloroform-n-hexane, obtain yellow solid.[M+H]+:221.0；1H NMR,600MHz(CDCl3)δ6.45
(q, 1H), 3.04 (s, 2H), 1.96 (m, 3H), 1.92 (m, 3H), 1.41 (s, 6H).
(2) compound of 6.6g (30mmol) step (1) is dissolved in 200mL acetic acid solution；Take 3.4mL (66mmol) bromine water
It is added dropwise in above-mentioned solution after reaction being stirred at room temperature 24 hours, evaporated under reduced pressure, residue is extracted by methylene chloride, water
It washes, anhydrous sodium sulfate is dry, and evaporated under reduced pressure obtains yellow solid compound.[M-H]-:313.0；1H NMR(600MHz,CDCl3)
δ3.04(s,2H),2.19(s,3H),2.16(s,3H),1.44(s,6H)。
(3) yellow solid compound that every 4.88g (15.5mmol) step (2) obtains is dissolved in 50mL methanol, in ice bath
In 2.25mL (31mmol) thionyl chloride is slowly added dropwise, be added dropwise in room temperature continue stirring 3 hours after evaporated under reduced pressure, residue
It is that stationary phase is isolated and purified by adsorbent, eluent is recovered to be dried to obtain yellow solid compound.[M+H]+:329；
1H NMR(600MHz,CDCl3)δ3.56(s,3H),2.98(s,2H),2.14(s,3H),2.14(s,3H),1.43(s,6H)。
(4) yellow solid compound that every 2.87g (8.72mmol) step (3) obtains is dissolved in 30mL methanol solution；It takes
1.7g (26.2mmol) sodium azide compound is dissolved in 10mL water, is added in above-mentioned solution several times in room temperature reaction 24 hours
Evaporated under reduced pressure afterwards, residue are extracted by methylene chloride, and washing, anhydrous sodium sulfate is dry, with adsorbent be stationary phase into
Row isolates and purifies, and eluent is recovered to be dried to obtain yellow solid compound.[M+H]+:292.0；1H NMR(500MHz,
CDCl3)δ3.63(s,3H),2.99(s,2H),2.16(s,3H),1.89(s,3H),1.41(s,6H)。
(5) compound that every 2.43g (8.35mmol) step (4) obtains is dissolved in 50mL methylene chloride；It is added several times
Solvent is removed under reduced pressure after room temperature reaction 1 hour in 2.19g (8.35mmol) triphenylphosphine.Acetic acid containing 30ml, 30mL tetrahydro is added
The aqueous solution of furans is heated to reflux evaporated under reduced pressure after 4 hours, and residue is extracted by ethyl acetate, is saturated NaHCO3And salt
Water washing, it is that stationary phase is isolated and purified with adsorbent that anhydrous sodium sulfate is dry, and eluent is recovered to be dried to obtain red admittedly
Body compound.[M+H]+:266.0；1H NMR(600MHz,CDCl3)δ4.61(s,2H),3.56(s,3H),2.94(s,2H),
2.19(s,3H),1.84(s,3H),1.43(s,6H)。
(6) red solid compound that every 1.3g (4.9mmol) step (5) obtains is dissolved in the mixed of 16mL methanol and 4mL water
It closes in solution；By the way that 360mg (15mmol) lithium hydroxide is added after room temperature reaction 24 hours, methanol is removed under reduced pressure, adjusts water
For pH value of solution to 2.0, filtering obtains violet solid compound.[M+H]+:252.0；1H NMR,600MHz,(CDCl3)δ11.98
(s,1H),6.32(s,2H),2.80(s,2H),2.07(s,3H),1.72(s,3H),1.35(s,6H)。
(7) every 1.26g (5.0mmol) violet solid compound is dissolved in 15mL methylene chloride；By the way that 2.18g is added dropwise
The Boc acid anhydrides of (10.0mmol) is removed under reduced pressure methylene chloride, is that stationary phase is divided with adsorbent after room temperature reaction 4 hours
From purifying, eluent is recovered to be dried to obtain violet solid compound.[M+H]+:352.0；1H NMR,600MHz,(CDCl3)δ
11.98 (s, 1H), 6.31 (s, 1H), 2.82 (s, 2H), 2.07 (s, 3H), 1.72 (s, 3H), 1.44 (s, 9H), 1.37 (s,
6H)。
(8) the violet solid counterpart and 360mg (1mmol) tripterygium wilfordii first that every 527mg (1.5mmol) step (7) obtains
Element is dissolved in 15mL methylene chloride, is stirred at 60 DEG C by the way that 824mg (4mmol) DCC and 127mg (1mmol) DMAP is added
48h is filtered after completion of the reaction, collects filtrate, and filtrate volatilizes, and ethyl acetate redissolves, and filtering, filtrate volatilizes to obtain violet solid
Close object.[M+H]+:695.0；1H NMR,600MHz(CDCl3)δ6.39(s,1H),5.30(s,1H),4.97(s,1H),4.19
(d,2H),3.84(d,1H),3.56(d,1H),3.49(d,1H),3.02(s,2H),2.65(m,1H),2.24(s,3H),2.12
(m, 2H), 1.99 (s, 3H), 1.97 (s, 3H), 1.86 (m, 2H), 1.55 (m, 1H), 1.49 (s, 3H), 1.46 (s, 9H), 1.22
(m,1H),1.01(s,3H),0.90(d,3H),0.80(d,3H)。
(9) the violet solid counterpart that every 694mg (1mmol) step (8) obtains is dissolved in 15mL methylene chloride, by adding
Enter 5mLTFA to be stirred at room temperature 4 hours, methylene chloride is removed under reduced pressure, is that stationary phase is isolated and purified with adsorbent, eluent
It is recovered to be dried to obtain violet solid compound.[M+H]+:594.0；1H NMR,600MHz(CDCl3)δ6.34(s,2H),5.30
(s,1H),4.97(s,1H),4.19(d,2H),3.84(d,1H),3.56(d,1H),3.49(d,1H),3.02(s,2H),2.65
(m,1H),2.16(s,3H),2.12(m,2H),1.96(s,3H),1.94(s,3H),1.86(m,2H),1.55(m,1H),1.49
(s, 3H), 1.22 (m, 1H), 1.01 (s, 3H), 0.90 (d, 3H), 0.80 (d, 3H).
(10) the violet solid counterpart that every 594mg (1mmol) step (9) obtains is dissolved in 15mL methylene chloride, is passed through
5mL is added, 150mg (1.5mmol) succinic anhydride is slowly added dropwise, after reaction 2-4 hours, methylene chloride is removed under reduced pressure in 65 DEG C, use
Adsorbent is that stationary phase is isolated and purified, and eluent is recovered to be dried to obtain violet solid compound.[M-H]-:692.0；1H
NMR,600MHz(CDCl3)δ6.38(s,1H),5.31(s,1H),4.99(s,1H),4.19(d,2H),3.84(d,1H),3.57
(d, 1H), 3.49 (d, 1H), 3.12 (s, 2H), 2.64 (m, 1H), 2.42 (s, 4H), 2.19 (s, 3H), 2.14 (m, 2H), 1.96
(s, 3H), 1.91 (s, 3H), 1.86 (m, 2H), 1.54 (m, 1H), 1.49 (s, 3H), 1.22 (m, 1H), 1.15 (s, 3H), 0.90
(d,3H),0.81(d,3H)。
The detection of product properties made from embodiment 1 is as follows:
The detection of 1.CX-94 water solubility
A certain amount of CX-94 is weighed, a series of CX-94 solution of concentration gradients is dissolved and be diluted to acetonitrile, passes through LC-
The content of MS/MS detection CX-94 simultaneously prepares standard curve；A certain amount of CX-94 is separately taken to be prepared into the supersaturated molten of CX-94 with water
Liquid, 0.22 μm of membrane filtration of the saturated solution, collects filtrate, using the concentration of LC-MS/MS detection CX-94.The result shows that
The solubility of CX-94 in water is 4.52mg/mL, and the solubility of comparison TP in water is only 17 μ g/mL.The water of purpose compound
Dissolubility improves 265 times.
2. external pharmacological activity and toxicity detection
The HepG2 cell frozen and normal liver cell are taken respectively, is recovered, and adjustment cell density is by every hole 1x105Carefully
Born of the same parents are inoculated in 96 orifice plates respectively, when cell fusion is up to 60%~70%, changes not serum-containing medium culture and make at synchronization for 24 hours
Reason.Then, cell is divided negative control group at random, positive controls (0.01,0.02,0.04,0.08,0.16,0.31,
0.625,1.25,2.5 μM of triptolide handles cell), CX-94 (0.01,0.02,0.04,0.08,0.16,0.31,
0.625,1.25,2.5 μM) administration group.Every hole is added 20 μ 1 of 5mg/ml MTT and cultivates 4h after 48h, discards supernatant liquid, every hole point
Not Jia Ru 150 μ l of dimethyl sulfoxide, shake 15min at room temperature, after yellow crystal dissolution after, with microplate reader measurement 570nm at extinction
Angle value (OD).
Normal liver cell administration after the result is shown in Figure 1: various concentration triptolide (Figure 1A), CX-94 (Figure 1B) respectively with
Normal liver cell and liver cancer cells temperature incubate after to the toxicity of cell.Triptolide (IC50=38.9nM) is compared as the result is shown,
CX-94 (IC50=1250nM) reduces 32 times to normal liver cell toxicity respectively.Show through this method to triptolide
Structural modification can reduce triptolide to the toxicity of normal liver cell.The CX-94 and triptolide pair of same concentrations
The IC50 of HepG2 is respectively 38.4nM and 36.9nM, shows the change obtained by structural modification of this method to triptolide
Triptolide can be retained to the pharmacological activity of HepG2 liver cancer cells by closing object CX-94.
The conversion of 3.HepG2 cell pyrolysis liquid In vitro metabolism
Human liver cancer cell HepG2 cell uses the DMEM culture containing 10% fetal calf serum to be based on 37 DEG C, 5%CO2And saturation
Culture in the carbon dioxide incubator of humidity.Logarithmic growth phase cell to be collected, is homogenized after PBS washing, homogenate is in 8000 × g, and 4
Supernatant is collected after DEG C centrifugation 20min, and BCA method surveys cell pyrolysis liquid protein concentration, in -80 DEG C of refrigerator storages.
Total volume is compound CX-94,1mg/mL the HepG2 cell cracking in 100 μ L reaction systems comprising 10 μ g/mL
Liquid, 1mM NADPH, 50mM PBS buffer solution.After the external temperature of sample incubates 1h, reaction is terminated by being added on 2 times of acetonitriles.Sample passes through
12000 × g takes supernatant sample introduction after 4 DEG C of centrifugation 20min, and the generation of triptolide is detected by UPLC.As a result see figure
2.CX-94 can be metabolized in HepG2 lysate and be converted into TP and XC-01.
4. metabolic stability in vitro is studied
People's hepatomicrosome is total to temperature with CX-94 to incubate, final concentration of 10 μM of CX-94, it is 100 μ L, including phosphoric acid that temperature, which incubates system,
Salt buffer (50mM, pH 7.4), hepatomicrosome (1mg/mL), MgCl2(5mM).It is 20mM's by the way that 10 μ L concentration are added
The shake of 200 μ L acetonitriles is added eventually after incubating 0,5,10,20,30,40 and 60min in the reaction of NADPH (final concentration of 1mM) initial, 37 DEG C of temperature
Only react.The concussion of sample whirlpool, which is mixed, is centrifuged 10min under 4 DEG C, 12000rpm, takes supernatant, sample introduction is analyzed by LC-MS/MS.
CX-94 (final concentration of 10 μM) and human plasma are added after in 37 DEG C, temperature incubates 0,5,10,20,30,40 and 60min respectively
Reaction is terminated after the acetonitrile of two volumes, the concussion of sample whirlpool, which is mixed, is centrifuged 10min under 4 DEG C, 12000rpm, takes supernatant, LC-
Sample introduction is analyzed by MS/MS.As a result see Fig. 3 .CX-94 in blood plasma and hepatomicrosome in metabolic stability, the metabolic half life phase be greater than 1
Hour.
5. pharmacokinetic in Mice Body
Mouse tail vein injection is administered after (1.5mg/kg CX-94) respectively at 2,5,10,20,40min, 1,2,3,4,8,
18 hours heart extracting bloods.Respectively at 10,30min, 1,2,3 after taking another group of Mouse oral stomach-filling to give the CX-94 of 1.5mg/kg,
4,5,6,8,24 hours heart extracting bloods.The blood plasma of collection is placed in the 1.5mLEP pipe of test tube of hepari, in 1000g, 4 DEG C of centrifugations
10min takes supernatant up to blood plasma.It is added after 2 times of acetonitrile vortex mixed concussion by volume in 12000g, 4 DEG C of centrifugations
10min, takes supernatant sample introduction, and LC-MS/MS method detects CX-94 content in blood plasma.Using compound concentration in blood plasma as ordinate, with
Take the blood time for abscissa mapping.And calculate pharmacokinetic parameter.Result of study shows that CX-94 is small as a prodrug
It can be metabolized quickly in mouse body and be converted into TP.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (9)
1. a kind of water solubility triptolide derivative, it is characterised in that: the chemistry of the water solubility triptolide derivative
Shown in structural formula such as following formula (I):
Wherein: n=2,3,4 ..., n are the positive integer greater than 1.
2. water solubility triptolide derivative as described in claim 1, it is characterised in that: the water solubility triptolide
The chemical structural formula of derivative is as follows:
3. a method of prepare water-soluble triptolide derivative as claimed in claim 2, it is characterised in that: the side
The synthetic route of method is as follows:
4. the method as claimed in claim 3 for preparing water-soluble triptolide derivative, it is characterised in that: the method
Specific step is as follows:
(1) using hydroquinone compound as reaction raw materials, it is derivative that the acrylic acid that molal quantity is 1-2 times the synthesis of compound 1: is added
Object is reacted under conditions of 50-100 DEG C, and the reaction time is 2-24 hours, and reacting the solvent used is methane sulfonic acid；Instead
It after answering, is successively extracted with ethyl acetate, pure water, saturated sodium bicarbonate aqueous solution and saturated brine washing, anhydrous slufuric acid
Sodium is dry, and evaporated under reduced pressure is recrystallized to give purpose compound with chloroform-n-hexane；
(2) synthesis of compound 2: 1-3 times of molal quantity of bromine water is added for raw material in the compound 1 reacted with the first step,
It is reacted 24 hours under conditions of 25 DEG C, reaction dissolvent is acetic acid；Evaporated under reduced pressure after completion of the reaction, residue by methylene chloride into
Row extraction, washing, anhydrous sodium sulfate is dry, and evaporated under reduced pressure obtains purpose compound；
(3) synthesis of compound 3: using second step product and methanol as raw material, mole 1.5-3 times of thionyl chloride being added in 0 DEG C,
It is reacted 1-3 hours under the conditions of 25 DEG C；It is that stationary phase is isolated and purified with adsorbent, eluent is recovered to be dried to obtain chemical combination
Object 3；
(4) in the methanol solution of compound 3, mole 2.5-5 times of sodium azide measured the synthesis of compound 4: is added several times
Aqueous solution, evaporated under reduced pressure after room temperature reaction 24 hours, residue are extracted by methylene chloride, are washed, and anhydrous sodium sulfate is dry
It is dry, it is that stationary phase is isolated and purified with adsorbent, eluent is recovered to be dried to obtain compound 4；
(5) synthesis of compound 5: compound 4 is dissolved in methylene chloride, and the triphenylphosphine reaction 1 of equimolar amounts is added several times
Hour, after solvent is removed under reduced pressure, the aqueous solution that the tetrahydrofuran containing acetic acid is added is heated to reflux evaporated under reduced pressure after 4 hours, remains
Object is extracted by ethyl acetate, is saturated NaHCO3With salt water washing, anhydrous sodium sulfate is dry, with adsorbent be stationary phase into
Row isolates and purifies, and eluent is recovered to be dried to obtain compound 5；
(6) synthesis of compound 6: the lithium hydroxide of 2-4 times of addition mole in the methanol aqueous solution of compound 5, it is anti-in room temperature
After answering 24 hours, methanol is removed under reduced pressure, adjusts aqueous solution pH to 2.0, filtering obtains compound 6；
(7) synthesis of compound 7: the Boc acid anhydrides of 2 times of addition mole in the dichloromethane solution of compound 6, in room temperature reaction
After 4 hours, methylene chloride is removed under reduced pressure, is that stationary phase is isolated and purified with adsorbent, recovered being dried to obtain of eluent
Close object 7；
(8) synthesis of compound 8: using triptolide as raw material, molal quantity is added and rubs for 1.5-2 times of compound 7 and 1-4 times
The dicyclohexylcarbodiimide (DCC) of your ratio and the 4-dimethylaminopyridine (DMAP) for being catalyzed equivalent, under the conditions of 20-60 DEG C
Reaction, evaporated under reduced pressure after reaction 48 hours, residue are extracted by ethyl acetate, are washed, and anhydrous sodium sulfate is dry, with suction
Attached dose is isolated and purified for stationary phase, and eluent is recovered to be dried to obtain compound 8；
(9) trifluoroacetic acid (TFA) synthesis of compound 9: is added in the dichloromethane solution of compound 8 in room temperature reaction 4-8
It is that stationary phase is isolated and purified with adsorbent, eluent is recovered to be dried to obtain compound 9 after hour；
(10) biosynthesis of compound 10: being slowly added dropwise alkane diacid in the dichloromethane solution of compound 9, anti-in 65 DEG C
After answering 2-4 hours, methylene chloride is removed under reduced pressure, adjusts aqueous solution pH to 2.0, filtering obtains compound 10.
5. the method as claimed in claim 4 for preparing water-soluble triptolide derivative, it is characterised in that: step (1) institute
Stating hydroquinone compound is 2,3,5 quilt-CH3, NH2, OCH3Or the hydroquinone compound that X replaces.
6. the method as claimed in claim 4 for preparing water-soluble triptolide derivative, it is characterised in that: step (1) institute
Stating acrylic acid derivative is the alkene acid compounds that the hydrogen atom on the position β is replaced by 1-2 methyl or amino.
7. the method as claimed in claim 4 for preparing water-soluble triptolide derivative, it is characterised in that: step (10) institute
Stating alkane diacid is any in malonic anhydride, succinic anhydride, glutaric anhydride, adipic anhydride, pimelic acid acid anhydride or suberic anhydride
Kind.
8. water solubility triptolide derivative as claimed in claim 1 or 2 is used to prepare the purposes in anti-tumor drug.
9. a kind of anti-tumor drug, it is characterised in that: the anti-tumor drug includes water-soluble thunder of any of claims 1 or 2
Public rattan A prime derivative.
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| CN111518157B (en) * | 2020-06-11 | 2021-02-23 | 山东大学 | Triptolide derivative and preparation method and application thereof |
| WO2022040487A1 (en) | 2020-08-21 | 2022-02-24 | Reyoung Corporation | Triptolide conjugates and uses thereof |
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| WO2022040487A1 (en) | 2020-08-21 | 2022-02-24 | Reyoung Corporation | Triptolide conjugates and uses thereof |
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