CN110092902A - A kind of tanshinone IIA high-molecular compound and its preparation and application - Google Patents

A kind of tanshinone IIA high-molecular compound and its preparation and application Download PDF

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CN110092902A
CN110092902A CN201910379039.1A CN201910379039A CN110092902A CN 110092902 A CN110092902 A CN 110092902A CN 201910379039 A CN201910379039 A CN 201910379039A CN 110092902 A CN110092902 A CN 110092902A
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compound
peg
tanshinone iia
wgzb
acid
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王进欣
赵丽
宫志博
丁黎
尤启冬
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China Pharmaceutical University
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China Pharmaceutical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/06—Antiarrhythmics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/331—Polymers modified by chemical after-treatment with organic compounds containing oxygen
    • C08G65/332—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof
    • C08G65/3328—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof heterocyclic
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33396—Polymers modified by chemical after-treatment with organic compounds containing nitrogen having oxygen in addition to nitrogen

Abstract

Present invention relates particularly to a kind of Tanshinone I I A high-molecular compound and its applications in pharmacy, belong to pharmaceutical technology field；A kind of tanshinone IIA high-molecular compound and intermediate, it is characterized in that shown in the structure such as formula (II) of the tanshinone IIA high-molecular compound and intermediate: tanshinone IIA high-molecular compound of the invention has good water-soluble, significant pharmacological activity, can be used for preparing prevention or the therapeutic agent of following disease: the cardiovascular and cerebrovascular diseases such as atherosclerosis, hyperlipidemia, cerebral ischemia, apoplexy, myocardial infarction, arrhythmia cordis, angina pectoris, cardiomyopathy, myocardial hypertrophy；The nervous system diseases such as Alzheimer's disease, early senile dementia；The liver diseases such as virus hepatitis, cirrhosis；The cancers such as breast cancer, leukaemia, liver cancer, colon cancer, melanoma；

Description

A kind of tanshinone IIA high-molecular compound and its preparation and application
The present invention is 201610479054.X, the divisional application of a kind of tanshinone IIA high-molecular compound and its preparation and application
Technical field
Present invention relates particularly to a kind of tanshinone IIA high-molecular compound and its applications in pharmacy, belong to medical skill Art field.
Background technique
Salviamiltiorrhizabung is clinically widely used in the treatment of coronary heart diseases and angina pectoris.As Radix Salviae Miltiorrhizae it is most be extracted into point it One, tanshinone IIA has anti-inflammatory, anti-oxidant and cytotoxic activity and neuroprotective.However, the cause of tanshinone IIA Ordering defect is that oral administration biaavailability is low, to find out its cause, first is that this is close with its design feature since poorly water-soluble leads to absorption difference It is inseparable.Tanshinone IIA is the very strong planar molecule of hydrophobicity, polarity very little, in water as a diterpene-kind compound In it is almost insoluble, solubility is also little in methanol, ethyl alcohol, only in some weakly polar organic solvents such as chloroform, ethyl acetate, second Ether is medium preferable solubility, this feature significantly limits its clinical application.The second is by oral administration due to tanshinone IIA There are serious first pass effect after administration, cause extremely low into haemoconcentration.
The most drugs clinically used are small-molecule drugs, this makes it quickly be distributed to the tissue of health, finally It is distributed to whole body.As a result relatively small number of drug reaches target spot, and over the course for the treatment of along with serious secondary anti- It answers.These side reactions, which are often dose dependent, cannot reach effective treatment.Polymer drug have it is long-acting, efficient, less toxic, The advantages that sustained release, selectivity good, Small side effects.In general high-molecular compound is synthesized by covalent bond for parent drug Molecule is connected on suitable macromolecule.This high-molecular compound in human body can be by hydrolyzing or digesting as parent medicine Object molecule and macromolecule.On the one hand high-molecular compound can efficiently reduce side effects of pharmaceutical drugs, on the other hand can extend drug Release time, increase dissolubility.
Polyethylene glycol (PEG) is linear structure, has good water-soluble and biocompatibility, nontoxic, disimmune, nothing Teratogenesis and no antigen are one of the medicinal synthetic polymers for obtaining FDA approval.The present invention is carrier with polyethylene glycol, is led to It crosses different spacer group and tanshinone IIA and carries out covalent bond, obtain covalent conjunct agent and increase the water-soluble of tanshinone IIA Property, improve its bioavilability.
Summary of the invention
The purpose of the present invention is to provide a kind of tanshinone IIA high-molecular compound and its intermediate that medical value is high, The high-molecular compound and intermediate structure such as formula (I), (II), shown in (III):
X is various amino acid (glycine, alanine, lysine, threonine, proline, cysteine, cystine, color ammonia Acid, phenylalanine, leucine, isoleucine, arginine, histidine, serine, tyrosine, aspartic acid, glutamic acid), and The dipeptides of two kinds of amino acid composition；(C=O)-R1(C=O)-, wherein R1For C1-C4Linear saturation alkyl, C1-C4Straight chain insatiable hunger With alkyl, C1-C4Branch saturated alkyl, C1-C4Branch unsaturated alkyl.
Y1For-NH2,-OH；Y2For mono methoxy PEG, average molecular weight 550,750,1000,2000,5000, 8000；Y3For PEG, carboxyl PEG, wherein the average molecular weight of PEG be 600,800,1000,1500,2000,4000,6000, 8000,10000,20000；The average molecular weight of carboxyl PEG be 600,800,1000,1500,2000,4000,6000,8000, 10000、20000。
Dipeptides is glycine, alanine, lysine, threonine, proline, cysteine, cystine, tryptophan, phenylpropyl alcohol Propylhomoserin, leucine, isoleucine, arginine, histidine, serine, tyrosine, aspartic acid, any two kinds of ammonia in glutamic acid The dipeptides of base acid composition.
When X is amino acid, Y1For-NH2；When X is-(C=O)-R1When (C=O)-, Y1For-OH.
When X is amino acid, Y3For carboxyl PEG；When X is-(C=O)-R1When (C=O)-, Y3For PEG.
Another object of the present invention is to provide the preparation methods of tanshinone IIA high-molecular compound and its intermediate, close It is as follows at route:
Route one: X is-(C=O)-R1(C=O)-
Route two: X is amino acid
Tanshinone IIA high-molecular compound of the invention has good water-soluble, significant pharmacological activity, can be used for making The prevention of standby following disease or therapeutic agent: atherosclerosis, hyperlipidemia, cerebral ischemia, apoplexy, myocardial infarction, the rhythm of the heart lose Often, the cardiovascular and cerebrovascular diseases such as angina pectoris, cardiomyopathy, myocardial hypertrophy；The nervous systems diseases such as Alzheimer's disease, early senile dementia Disease；The liver diseases such as virus hepatitis, cirrhosis；The cancers such as breast cancer, leukaemia, liver cancer, colon cancer, melanoma.
The present invention is further illustrated by following embodiment, but should be noted that the scope of the present invention not by these embodiments Any restrictions.
Detailed description of the invention
Fig. 1 is the external cerebral ischemia active testing result of tanshinone IIA high-molecular compound
Specific embodiment
The preparation of embodiment 1:4- carboxyl-butyl ester tanshinone IIA (WGZB-24)
Weigh 5g (17mmol) tanshinone IIA, 5.6g (51mmol) SeO2In the mono- neck bottle of 1L, 700~800 ml are added Acetonitrile as solvents, 5~6h of back flow reaction stop heating and being cooled to room temperature, are evaporated under reduced pressure to solid, methylene chloride is molten by solid It filters after solution, is washed filtrate 2~3 times with saturated ammonium chloride solution, anhydrous sodium sulfate is dry, and it is red to obtain 1.32g for column chromatographic purifying Color solid chemical compound 2, yield 25%.
Weigh 620mg (2mmol) compound 2,300mg (3mmol) succinic anhydride in the mono- neck bottle of 100ml, with 30ml without After the dissolution of water methylene chloride, 0.83ml (6mmol) triethylamine is added, simultaneously nitrogen protection is vacuumized, after reacting at room temperature 30~40min Stop reaction, with saturated ammonium chloride solution washing reaction liquid 2~3 times, 800mg red is evaporated under reduced pressure to after anhydrous sodium sulfate is dry Solid chemical compound 4- carboxyl-butyl ester tanshinone IIA, yield 97.56%.
Obtaining spacer group with legal system is C1-C4Straight chain unsaturated alkyl；C1-C4Branch saturated alkyl；C1-C4Branch is unsaturated The formula (I) of alkyl.
Embodiment 2:4- carboxyl-butyl ester tanshinone IIA-mPEG preparation
100mg (0.24mmol) 4- carboxyl-butyl ester tanshinone IIA is weighed, 220mg (0.29mmol) molecular weight is 750 MPEG, 74mg (0.36mmol) DCC, 44mg (0.36mmol) DMAP are in the mono- neck bottle of 100ml, with 40 ml anhydrous methylene chlorides After dissolution, simultaneously nitrogen protection is vacuumized, is reacted for 24 hours at 25~45 DEG C, suction filtration removes DCU, washs filter with saturated ammonium chloride solution Liquid 2~3 times, anhydrous sodium sulfate is dry, and red oil is obtained after vacuum distillation, is dissolved grease with fraction of methylene chloride Afterwards, a large amount of ether are added under condition of ice bath, generates precipitating, filters and obtain red powder solid chemical compound 4- carboxylic after drying Base-butyl ester tanshinone IIA-mPEG.
Obtaining spacer group with legal system is C1-C4Straight chain unsaturated alkyl；C1-C4Branch saturated alkyl；C1-C4Branch is unsaturated Alkyl, 4- carboxyl-butyl ester tanshinone IIA-mPEG that mPEG average molecular weight is 550,1000,2000,5000,8000.
Embodiment 3:4- carboxyl-butyl ester tanshinone IIA-PEG preparation
100mg (0.24mmol) 4- carboxyl-butyl ester tanshinone IIA is weighed, 720mg (0.36mmol) molecular weight is 2000 PEG, 74mg (0.36mmol) DCC, 44mg (0.36mmol) DMAP are in the mono- neck bottle of 100ml, with 45ml anhydrous methylene chloride After dissolution, simultaneously nitrogen protection is vacuumized, is reacted for 24 hours at 25~45 DEG C, suction filtration removes DCU, washs filter with saturated ammonium chloride solution Liquid 2~3 times, anhydrous sodium sulfate is dry, and red oil is obtained after vacuum distillation, is dissolved grease with fraction of methylene chloride Afterwards, a large amount of ether are added under condition of ice bath, generates precipitating, filters and obtain red powder solid chemical compound 4- carboxylic after drying Base-butyl ester tanshinone IIA-PEG.
Obtaining spacer group with legal system is C1-C4Straight chain unsaturated alkyl；C1-C4Branch saturated alkyl；C1-C4Branch is unsaturated Alkyl, PEG average molecular weight are 600,800,1000,1500,4000,6000,8000 (WGZB-01), 10000,20000 4- carboxyl-butyl ester tanshinone IIA-PEG.
Embodiment 4: the preparation of glycine-tanshinone IIA (WGZB-02-1)
Weigh 310mg (1mmol) compound 2,210mg (1.2mmol) Boc- glycine, 248mg (1.2mmol) DCC, 122mg (1mmol) DMAP after the dissolution of 45ml anhydrous methylene chloride, is vacuumized and nitrogen protection, 25 in the mono- neck bottle of 100ml Stop reaction after reacting 30~40min at~45 DEG C, suction filtration removes DCU, washs filtrate 2~3 times with saturated ammonium chloride solution, nothing Aqueous sodium persulfate is dry, and column chromatographic purifying obtains 305mg red solid compound Boc- glycine-tanshinone IIA, yield 65.3%.
1H NMR (300MHz, CDCl3): δ 1.25 (9H, s ,-CH3× 3), 1.38 (6H, s ,-CH of δ3× 2), δ 1.74 (2H, M, CH2), δ 1.83 (2H, m, CH2), δ 3.19 (2H, t, J=6.45Hz, CH2), δ 3.90 (2H, d, J=5.73Hz, CH2), δ 5.24 (2H, s, CH2), δ 7.44 (1H, s, Ar), δ 7.52 (1H, d, J=8.16Hz, Ar), δ 7.60 (1H, d, J=8.16Hz, Ar)；ESI-MS (m/z): 490 [M+Na]+.
292mg (0.62mmol) compound 9 is weighed in the mono- neck bottle of 50ml, with 8ml methylene chloride by Boc- glycine-pellet Join ketone IIA dissolution, 4ml trifluoroacetic acid (V is addedMethylene chloride∶VTrifluoroacetic acid=2: 1), stopping reaction after reacting at room temperature 30min, decompression is steamed Methylene chloride and trifluoroacetic acid are removed in distillation, obtain red oil, and chloroform is added and makes it dissolve, is washed with saturated sodium bicarbonate solution Organic phase 2 times, the dry organic phase of anhydrous sodium sulfate, column chromatographic purifying obtains 140mg red solid compound glycine-tanshinone IIA, yield 61.1%.
Obtaining spacer group with legal system is alanine (WGZB-06-1), lysine, threonine, proline, cysteine, Guang Propylhomoserin, tryptophan, phenylalanine (WGZB-10-1), leucine, isoleucine, arginine, histidine, serine, tyrosine, The formula (I) of aspartic acid, glutamic acid.
1H NMR (300MHz, CDCl3): δ 1.37 (6H, s ,-CH3× 2), 1.63 (2H, m, CH of δ2), δ 1.75 (2H, m, CH2), δ 3.10 (2H, t, J=6.45Hz, CH2), δ 3.57 (2H, s, CH2), δ 5.24 (2H, s, CH2), δ 7.44 (1H, s, Ar), δ 7.52 (1H, d, J=8.16Hz, Ar), δ 7.60 (1H, d, J=8.16Hz, Ar)；ESI-MS (m/z): 368 [M+H]+.
Embodiment 5: glycine-tanshinone IIA-carboxyl PEG preparation
Weigh 50mg (0.14mmol) glycine-tanshinone IIA, the carboxyl that 340mg (0.17mmol) molecular weight is 2000 PEG, 35mg (0.17mmol) DCC, 20mg (0.14mmol) DMAP are molten with 40ml anhydrous methylene chloride in the mono- neck bottle of 100ml Xie Hou vacuumizes simultaneously nitrogen protection, reacts for 24 hours at 25~45 DEG C, and suction filtration removes DCU, washs filtrate 2 with saturated ammonium chloride solution ~3 times, anhydrous sodium sulfate is dry, and red oil, after being dissolved grease with fraction of methylene chloride, ice are obtained after vacuum distillation A large amount of ether are added under the conditions of bath, generates precipitating, filters and obtain powdered red solid compound glycine-Radix Salviae Miltiorrhizae after drying Ketone IIA- carboxyl PEG (WGZB-02).
Obtaining carboxyl PEG average molecular weight with legal system is 600,800,1000,1500,4000 (WGZB-03), 6000,8000 (WGZB-04), glycine-tanshinone IIA-carboxyl PEG of 10000,20000 (WGZB-05).
With legal system obtain spacer group be alanine, lysine, threonine, proline, cysteine, cystine, tryptophan, Phenylalanine, leucine, isoleucine, arginine, histidine, serine, tyrosine, aspartic acid, glutamic acid, carboxyl PEG Tanshinone IIA-carboxylic that average molecular weight is 600,800,1000,1500,2000,4000,6000,8000,10000,20000 Base PEG.Wherein alanine-tanshinone IIA-carboxyl PEG product is WGZB-06, WGZB-07, WGZB-08, WGZB-09；Phenylpropyl alcohol Propylhomoserin-tanshinone IIA-carboxyl PEG product is WGZB-10, WGZB-11, WGZB-12, WGZB-13；Glutamic acid-tanshinone IIA- carboxyl PEG product is WGZB-14, WGZB-15, WGZB-16, WGZB-17；Aspartic acid-tanshinone IIA-carboxyl PEG Product is WGZB-18, WGZB-19, WGZB-20, WGZB-21；
Embodiment 6: tanshinone IIA high-molecular compound plasma stability experiment
Chromatographic condition: C18Chromatographic column: 150L × 4.6 Shimadzu VP-ODS；Mobile phase: A:0.03% phosphate aqueous solution B: acetonitrile；Flow velocity: 1mL/min；Detection wavelength: 270nm；Column temperature: 25 DEG C；Sample introduction: 20 μ L.
The preparation of solution: weighing 25mg sample in 10mL volumetric flask respectively, is made into the molten of 2.5mg/mL with ethyl alcohol dissolution Liquid (needs ultrasonic dissolution assisting).The preparation of contrast solution: precision weighs tanshinone IIA 10mg, is dissolved with ethyl alcohol, is made into 40 μ g/mL Contrast solution.
The processing of sample solution: taking above-mentioned each solution 1mL in the EP pipe of 2mL respectively, after volatilizing solvent, respectively plus 1.5ml Blank plasma, be vortexed mix, be placed under 37 DEG C of water-baths and cultivate 0,0.5,2,4,6,8,12h, take respectively at every point of time Acetonitrile-methanol (1: 1) protein precipitation of 2 times of volumes (400 μ l), vortex mixed 2min, 12000rpm is added in the blood plasma of 200 μ L It is centrifuged 5min, takes 100 μ L of supernatant, taking 20 μ L, sample introduction is analyzed.The contrast solution 0.5mL for taking TS after volatilizing solvent, is added The blank plasma of 0.5mL is vortexed and mixes, and acetonitrile-methanol (1: 1) protein precipitation of 2 times of volumes (400 μ l) is added, and is vortexed and mixes 2min, 12000rpm are centrifuged 5min, take 100 μ L of supernatant, taking 20 μ L, sample introduction is analyzed.
Blank plasma: taking 200 μ L blank plasmas, and after ibid handling protein precipitation, 12000rpm is centrifuged 5min, takes supernatant 100 μ L, taking 20 μ L, sample introduction is analyzed.
Experimental result: under the same conditions, PEG molecular weight releases more greatly the amount of parent drug molecule more to spacer group It is few, extend the half-life period of drug.
The tanshinone IIA high-molecular compound of 1 different molecular weight different interval group of table releases parent medicine in blood plasma Object molecular percentage ratio
Embodiment 7: tanshinone IIA high-molecular compound anti tumor activity in vitro experiment
Material: human breast cancer cell line Bcap-37
Test method: the tumour cell to test in cell log growth period is inoculated with by certain cell concentration In in 96 well culture plates, sieved sample (can directly add after suspension cell fishplate bar) is added in culture afterwards for 24 hours, and cell is at 37 DEG C 5% CO2Under the conditions of continue cultivate 48h after, be added MTT continue culture 4h hours, be dissolved under microplate reader and detected with DMSO.
Experimental result: under the same conditions, molecular weight is bigger, and anti-tumor activity is stronger for spacer group, wherein WGZB-01, The activity of WGZB-04, WGZB-05, WGZB-07, WGZB-09, WGZB-15 are better than tanshinone IIA, and tanshinone IIA system It is standby at still retaining anti-tumor activity after Macromolecule Prodrug
2 tanshinone IIA high-molecular compound extracorporeal anti-tumor the selection result of table
MCF-7 IC50(μM)
WGZB-01 0.2198
WGZB-02 45.62
WGZB-03 42.21
WGZB-04 12.41
WGZB-05 6.706
WGZB-06 48.53
WGZB-07 19.41
WGZB-08 92.37
WGZB-09 6.887
WGZB-11 932.8
WGZB-13 329
WGZB-15 10.98
WGZB-17 121.9
WGZB-19 486.7
WGZB-21 280.5
WGZB-24 28.46
WGZB-02-1 29.86
WGZB-06-1 8.69
WGZB-10-1 13.43
Tanshinone IIA 41.79
Embodiment 8: the external cerebral ischemia activity experiment of tanshinone IIA high-molecular compound
Experimental method: the sodium dithionite (Na being added in low sugar DMEM culture medium2S2O4) dry powder sufficiently dissolves, make Final concentration 20mM, uses NaHCO3Adjusting pH is 7.2, as anoxic liquid.Primary cortical neurons cell is in 24 orifice plate tissue culture plates Middle culture 7-10 days is chosen the good cell of growth conditions and is tested.The culture medium in 24 orifice plates is gently sucked before administration, is used After PBS buffer solution is washed 2 times, continue to cultivate with pastille culture medium.Experiment includes blank control group, model control group, WGZB- altogether 02、WGZB-03、WGZB-04、WGZB-05、WGZB-06、WGZB-07、WGZB-08、 WGZB-09、WGZB-02-1、WGZB- 06-1, Lead compound sample sets.After for 24 hours.The culture medium for removing drug containing, is replaced as anoxic liquid, is added in blank control group Isometric maintenance culture medium (Neurobasal Media49 ml+B27-Supplement 1ml).By culture medium in incubator Supernatant is completely removed after middle incubation 2h, is added after being washed twice with PBS buffer solution and maintains culture medium.It is placed again into 37 DEG C, 5% CO2Continue after cultivating 2h in incubator, modeling is completed, and neuronal cell can be used for testing.Tissue culture plate is placed in down after modeling Set the growth and cellular damage situation of microscopically observation cortical neuronal cells.The culture medium in 24 orifice plates is removed, dimension is added Every hole adds 50 μ L of MTT solution (5 mg/ml) after holding 450 μ L of culture medium, is put into 37 DEG C of incubation 4h.Abandon the culture medium containing MTT Later, 200 μ L of DMSO liquid is added in every hole, is placed on shaking table and shakes 10min.It is moved in 96 orifice plates from 150 μ L are taken out in every hole, The OD value in each hole is measured at microplate reader 490nm, with the survival condition of OD value reflection cell.Experiment is repeated 3 times.
Experimental result: as shown in Figure 1, after neuronal cell anoxia/reoxygenation injury, model group neuronal cell survival rate and Blank group reduces (P < 0.01) compared to extremely significant property.Compared with model group, compound WGZB-02, WGZB-03, WGZB-04, WGZB-05, WGZB-06, WGZB-07, WGZB-08, WGZB-09, WGZB-02-1 and Lead compound can extremely significant property liters The survival rate of neuronal cell after high modeling and WGZB-06-1 grams be significant to increase the survival rate of neuronal cell after modeling.
The above is only optimization experiment mode of the invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Claims (4)

1. a kind of tanshinone IIA high-molecular compound and intermediate, it is characterised in that the tanshinone IIA high-molecular compound and in Shown in the structure of mesosome such as formula (II):
Wherein X be following amino acid any one: glycine, alanine, lysine, threonine, proline, cysteine, Guang Propylhomoserin, tryptophan, phenylalanine, leucine, isoleucine, arginine, histidine, serine, tyrosine, aspartic acid and paddy Propylhomoserin；X is dipeptides, the dipeptides be glycine, alanine, lysine, threonine, proline, cysteine, cystine, Tryptophan, phenylalanine, leucine, isoleucine, arginine, histidine, serine, tyrosine, aspartic acid, in glutamic acid The dipeptides of any two kinds of amino acid composition；
Y2 is mono methoxy PEG, average molecular weight 550,750,1000,2000,5000,8000；Y3 is PEG, carboxyl PEG, wherein the average molecular weight of PEG is 600,800,1000,1500,2000,4000,6000,8000,10000,20000；Carboxylic The average molecular weight of base PEG is 600,800,1000,1500,2000,4000,6000,8000,10000,20000.
2. tanshinone IIA high-molecular compound according to claim 1 and intermediate, synthetic route are as follows:
(1) route one: X is-(C=O)-R1- (C=O)-
Specific step is as follows:
I. compound 1 is aoxidized through oxidant SeO2, back flow reaction 5~6 hours, obtains compound 2, solvent for use is acetonitrile, two Six ring of oxygen, acetic acid, DMF or DMSO, compound 1 therein and molar ratio=1 SeO2: 3~5；
Ii. compound 2 is alkali in Et3N, and anhydrous methylene chloride, anhydrous chloroform make solvent, is reacted under room temperature with compound 3 Compound I is obtained, wherein molar ratio=1 of compound 2 and Et3N: 3~5, molar ratio=1 of compound 2 and compound 3: 1.5 ~3；
Iii. under the action of catalyst and condensing agent, compound I and PEG or mPEG react at 25~45 DEG C and obtain chemical combination Object II, compound III；Wherein molar ratio=1 of compound I and PEG: 1.5~2, molar ratio=1 of compound I and mPEG: 1.2~2；Solvent is anhydrous methylene chloride, anhydrous chloroform and anhydrous DMF；Used catalyst and condensing agent: 2- (7- azo benzo Triazole)-N, N, N ' N '-tetramethylurea hexafluorophosphoric acid ester, O- benzotriazole-tetramethylurea hexafluorophosphoric acid ester, O- benzo three Nitrogen azoles-N, N, N ', N '-tetramethylurea tetrafluoro boric acid, n,N-diisopropylethylamine, N, N '-dicyclohexylcarbodiimide, 4- diformazan Aminopyridine, N- dimethylamino-propyl-N- ethyl-carbodiimide hydrochloride, 1- hydroxy benzo triazole, N- hydroxysuccinimidyl acyl are sub- Amine；Its R1 be C1-C4 linear saturation alkyl, C1-C4 straight chain unsaturated alkyl, C1-C4 branch saturated alkyl, C1-C4 branch not Saturated alkyl；
(2) route two: X is amino acid
Specific step is as follows:
I. the method prepare compound 2 according to route one；
Under the conditions of ii.25~45 DEG C, under the action of catalyst and condensing agent compound 2 and compound 4 in anhydrous methylene chloride, Compound 5 is obtained in anhydrous chloroform；Used catalyst and condensing agent: 2- (7- azo benzotriazole)-N, N, N ' N '-tetramethyl Urea hexafluorophosphoric acid ester, O- benzotriazole-tetramethylurea hexafluorophosphoric acid ester, O- benzotriazole-N, N, N ', N '-tetramethylurea Tetrafluoro boric acid, n,N-diisopropylethylamine, N, N '-dicyclohexylcarbodiimide, 4-dimethylaminopyridine, N- dimethylamino third Base-N- ethyl-carbodiimide hydrochloride, 1- hydroxy benzo triazole, n-hydroxysuccinimide；Compound 2 and compound 4 Molar ratio=1: 1.2~2；The compound 4 be glycine, alanine, lysine, threonine, proline, cysteine, Cystine, tryptophan, phenylalanine, leucine, isoleucine, arginine, histidine, serine, tyrosine, aspartic acid or Glutamic acid, R2 are the substituent group on above-mentioned amino acid α carbon, and R3 is amino-substituent；
Iii. compound 5 obtains compound I under the conditions of deamination protecting group；Deamination protecting group: tertbutyloxycarbonyl removes item Part: trifluoroacetic acid, HCl/EA or HCl/CH3OH, benzyloxycarbonyl group remove condition: hydrogen bromide, hydrogen reducing or Na/NH3 liquid, 2- connection Phenyl -2- propylene carbonyl oxygen removes condition: trifluoroacetic acid or hydrogen bromide, and p-toluenesulfonyl removes condition: sodium or ammonia；
Under the conditions of iv.25~45 DEG C, in the action compound I and carboxyl PEG of catalyst and condensing agent in anhydrous methylene chloride, nothing Compound III is obtained in water chloroform and anhydrous DMF；Wherein molar ratio=1 of compound I and carboxyl PEG: 1.2~2；It is used to urge Agent and condensing agent: 2- (7- azo benzotriazole)-N, N, N ' N '-tetramethylurea hexafluorophosphoric acid ester, O- benzotriazole-four Methylurea hexafluorophosphoric acid ester, O- benzotriazole-N, N, N ', N '-tetramethylurea tetrafluoro boric acid, n,N-diisopropylethylamine, N, N '-dicyclohexylcarbodiimide, 4-dimethylaminopyridine, N- dimethylamino-propyl-N- ethyl-carbodiimide hydrochloride, 1- hydroxyl Benzotriazole, n-hydroxysuccinimide.
A kind of exist by the tanshinone IIA high-molecular compound and intermediate of carrier of macromolecule 3. according to claim 1 Preparation protects it to the application in the prevention or treatment in following disease medicament: cardiovascular and cerebrovascular disease, the nervous system disease, liver Disease and cancer.
4. application according to claim 3, it is characterised in that the cardiovascular and cerebrovascular disease includes atherosclerosis, height Pionemia, cerebral ischemia, apoplexy, myocardial infarction, arrhythmia cordis, angina pectoris, cardiomyopathy, myocardial hypertrophy；The nervous system disease includes Alzheimer's disease, early senile dementia；Liver disease includes virus hepatitis, cirrhosis；Cancer include breast cancer, leukaemia, Liver cancer, colon cancer, melanoma.
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