CN103755562B - The preparation method of a kind of preparation method of lovastatin acid compound, composition, composition and application thereof - Google Patents

The preparation method of a kind of preparation method of lovastatin acid compound, composition, composition and application thereof Download PDF

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CN103755562B
CN103755562B CN201310722852.7A CN201310722852A CN103755562B CN 103755562 B CN103755562 B CN 103755562B CN 201310722852 A CN201310722852 A CN 201310722852A CN 103755562 B CN103755562 B CN 103755562B
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lovastatin
acid compound
preparation
compound
simvastatin
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CN103755562A (en
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胡昌勤
张斗胜
黄权华
佟军威
苏军权
何婷
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Shenzhen China Resources Gosun Pharmaceutical Co Ltd
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Shenzhen China Resources Gosun Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/28—Preparation of carboxylic acid esters by modifying the hydroxylic moiety of the ester, such modification not being an introduction of an ester group
    • C07C67/297—Preparation of carboxylic acid esters by modifying the hydroxylic moiety of the ester, such modification not being an introduction of an ester group by splitting-off hydrogen or functional groups; by hydrogenolysis of functional groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C231/00—Preparation of carboxylic acid amides
    • C07C231/10—Preparation of carboxylic acid amides from compounds not provided for in groups C07C231/02 - C07C231/08

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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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Abstract

The invention discloses a kind of preparation method of lovastatin acid compound, this preparation method is simple, raw material is easy to get, and is conducive to suitability for industrialized production; The invention also discloses a kind of composition of lovastatin acid compound, each auxiliary material component in said composition is easy to get, and is conducive to industrialization; The invention also discloses a kind of preparation method of composition of lovastatin acid compound, this preparation method requires that processing condition are not high, easy to operate; The invention also discloses the application of a kind of lovastatin acid compound in the medicine for the preparation for the treatment of hyperlipidemia, this lovastatin acid compound toxicity significantly reduces, and does not need metabolism to be in vivo used for the treatment of hyperlipidemia.

Description

The preparation method of a kind of preparation method of lovastatin acid compound, composition, composition and application thereof
Technical field
The present invention relates to technical field of pharmaceuticals, particularly relate to a kind of preparation method of lovastatin acid compound, composition, the preparation method of composition and application thereof.
Background technology
Statins is HMG-CoA reductase inhibitor, this type of medicine prepares rate-limiting enzyme HMG-CoA reductase by competitive inhibition endogenous cholesterol, block hydroxyl first valeric acid pathways metabolism in cell, prepared by intracellular cholesteryl reduce, thus surperficial low-density lipoprotein (LDL) acceptor quantity of feedback irritation cell film and activity increase, make serum cholesterol remove increase, level reduces; Statins also can suppress liver to prepare Apolipoprotein B-100, thus reduces and be rich in triglyceride level AV, the preparation method of lipoprotein and secretion.
Lovastatin is the medicine for the treatment of hyperlipidemia conventional at present, clinical study shows, lovastatin is the prodrug of inactive lactone structure, must metabolism in vivo, hydrolysis, forming the lovastatin acid compound with active structure could suppress HMG-CoA reductase; The structure of lovastatin acid compound is extremely similar to Pravastatin sodium, and Pravastatin sodium is marketed drug, has good DEVELOPMENT PROSPECT; And lovastatin acid compound only controls as related substances in the research of lovastatin drug quality at present, there is not the research being directly used in treatment hyperlipidemia aspect.
Summary of the invention
For solving problems of the prior art, the invention provides a kind of preparation method of lovastatin acid compound, composition, the preparation method of composition and application thereof.
The invention provides a kind of preparation method such as formula the compound lovastatin acid compound shown in C, comprise the following steps:
1) preparation is such as formula the compound lovastatin ammonia propyl ester shown in B
Get such as formula the compound lovastatin shown in A, add ethanol and propylamine carries out ring-opening reaction, obtained such as formula the compound lovastatin ammonia propyl ester shown in B;
2) preparation is such as formula the compound lovastatin acid compound shown in C
Get that step 1) obtains such as formula the compound lovastatin ammonia propyl ester shown in B, add hydrochloric acid and carry out acidification hydrolization reaction, recrystallization, obtained such as formula the compound lovastatin acid compound shown in C.
Preferably, under 30 DEG C ~ 50 DEG C conditions, add propylamine in described step 1) and carry out ring-opening reaction.
Preferably, propylamine reaction 1h ~ 5h is added in described step 1).
Preferably, described step 2) in use re-crystallizing in ethyl acetate.
In above-mentioned preparation method, such as formula the compound lovastatin shown in A, its chemistry 8-(2-(4-hydroxyl-6-oxygen-tetrahydrochysene-2H-pyrans-2-base) ethyl)-7-methyl isophthalic acid by name, 2,3,7,8,8a-hexahydro naphthalene-1-base-2-methylbutyl butenoate, its English be expressed as 8-(2-(4-hydroxy-6-oxo-tetrahydro-2H-pyran-2-yl) ethyl)-7-methyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl2-methylbutanoate; Such as formula the compound lovastatin ammonia propyl ester shown in B, its chemistry 8-by name (3,5-dihydroxyl-7-oxygen-7-(propylcarbamic) heptyl)-7-methyl isophthalic acid, 2,3,7,8,8a-hexahydro naphthalene-1-base 2-methylbutyl butenoate, its English is expressed as: 8-(3,5-dihydroxy-7-oxo-7-(propylamino) heptyl)-7-methyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl2-methylbutanoate; Such as formula the compound lovastatin acid compound shown in C; its chemistry by name 3; 5-dihydroxyl-7-(2-methyl-8-(2-methylbutyryl base oxygen base)-1,2,6,7,8,8a-hexahydro naphthalene-1-base) enanthic acid; its English is expressed as: 3,5-dihydroxy-7-(2-methyl-8-(2-methylbutanoyloxy)-1,2,6,7,8,8a-hexahydronaphthalen-1-yl) heptanoicacid; this is those skilled in the art is easy and way that is that generally adopt, and those of ordinary skill in the art can grasp completely.
Present invention also offers a kind of composition of lovastatin acid compound, by weight, comprise following component:
Preferably, the composition of described lovastatin acid compound, by weight, comprises following component:
Preferably, the composition of described lovastatin acid compound, by weight, comprises following component:
Preferably, the composition of described lovastatin acid compound, by weight, comprises following component:
Present invention also offers a kind of preparation method of composition of lovastatin acid compound, comprise the following steps:
1) lovastatin acid compound, Polysorbate 80, hydroxypropylcellulose, croscarmellose sodium, N.F,USP MANNITOL and Microcrystalline Cellulose is taken, wet granulation;
2) Magnesium Stearate is taken, compressing tablet;
3) Opadry film coating pre-mix dose is taken, film coating.
In above-mentioned preparation method, Polysorbate 80 is tensio-active agent, adds with the form of tackiness agent; Opadry film coating pre-mix dose composition comprises hypromellose, polyoxyethylene glycol, titanium dioxide and talcum powder etc., use commercially available commodity, the consumption of Opadry film coating pre-mix dose holds according to the experience in pharmaceutics, be that those of ordinary skill in the art can grasp, be generally weightening finish 2% ~ 4%.
Present invention also offers the application of a kind of lovastatin acid compound in the medicine for the preparation for the treatment of hyperlipidemia.
Lovastatin is the prodrug of inactive lactone structure, must metabolism in vivo, hydrolysis, and forming the lovastatin acid compound with active structure could suppress HMG-CoA reductase; And lovastatin acid compound provided by the invention, do not need metabolism to be in vivo used for the treatment of hyperlipidemia.
The invention provides a kind of preparation method of lovastatin acid compound, its beneficial effect is as follows:
1. preparation method of the present invention is simple, and raw material is easy to get, and is conducive to suitability for industrialized production;
2. the present invention uses reagent toxicity lower, is conducive to environment protection;
3. yield of the present invention is higher, reaches more than 97%.
Present invention also offers a kind of composition of lovastatin acid compound, its beneficial effect is as follows:
1. composition of the present invention, does not need prior metabolism in vivo, is directly used in treatment hyperlipidemia;
2. each auxiliary material component in the present invention is cheap and easy to get, is conducive to industrialization.
Present invention also offers a kind of preparation method of composition of lovastatin acid compound, its beneficial effect is as follows:
1. preparation method of the present invention is simple, easy to operate;
2. preparation method of the present invention requires that processing condition are not high, is beneficial to industrialized production.
Present invention also offers the application of a kind of lovastatin acid compound in the medicine for the preparation for the treatment of hyperlipidemia, its beneficial effect is as follows:
1. toxicity of the present invention significantly reduces;
2. lovastatin acid compound of the present invention does not need metabolism to be in vivo used for the treatment of hyperlipidemia.
Accompanying drawing explanation
Fig. 1 lovastatin acid compound structural formula;
Fig. 2 lovastatin acid compound synthetic route chart;
Fig. 3 lovastatin acid compound 3D schemes;
Fig. 4 Simvastatin alcohol acid compound 3D schemes;
Fig. 5 Pravastatin sodium 3D schemes;
Figure 61 dqa3D schemes;
Fig. 7 lovastatin acid compound docks result with 1dqa;
Fig. 8 Simvastatin alcohol acid compound docks result with 1dqa;
Fig. 9 Pravastatin sodium docks result with 1dqa;
Figure 10 lovastatin acid compound and 1dqa combination;
Figure 11 Simvastatin alcohol acid compound and 1dqa combination;
Figure 12 Pravastatin sodium and 1dqa combination;
Figure 13 Pravastatin sodium (the 50%-outsourcing phase-48hpf administration) toxicity (3dpf) to zebrafish embryo;
Figure 14 lovastatin administration (the 50%-outsourcing phase-48hpf administration) toxicity (3dpf) to zebrafish embryo;
Figure 15 Simvastatin administration (the 50%-outsourcing phase-48hpf administration) toxicity (3dpf) to zebrafish embryo;
Figure 16 lovastatin places 0h Solution H PLC color atlas;
Figure 17 lovastatin places 24h Solution H PLC color atlas;
Figure 18 Pravastatin sodium 500ug/ml3dpf acts on the deformity of zebrafish embryo;
Figure 19 Pravastatin sodium 500ug/ml3dpf acts on the deformity of zebrafish embryo;
Figure 20 Pravastatin sodium 1000ug/ml3dpf acts on the deformity of zebrafish embryo;
Figure 21 Pravastatin sodium 1000ug/ml3dpf acts on the deformity of zebrafish embryo;
Figure 22 Pravastatin sodium 1000ug/ml3dpf acts on the deformity of zebrafish embryo;
Figure 23 Pravastatin sodium wild type control (Wt) 3dpf acts on the deformity of zebrafish embryo;
Figure 24 Pravastatin sodium 1500ug/ml2dpf acts on the deformity of zebrafish embryo;
Figure 25 Pravastatin sodium Wt3dpf acts on the deformity of zebrafish embryo;
Figure 26 Pravastatin sodium Wt2dpf acts on the deformity of zebrafish embryo;
Figure 27 lovastatin 0.01ug/ml1dpf acts on the deformity of zebrafish embryo;
Figure 28 lovastatin 0.005ug/ml1dpf acts on the deformity of zebrafish embryo;
Figure 29 lovastatin 0.0005ug/ml3dpf acts on the deformity of zebrafish embryo;
Figure 30 lovastatin 0.001ug/ml2dpf acts on the deformity of zebrafish embryo;
Figure 31 lovastatin 0.001ug/ml3dpf acts on the deformity of zebrafish embryo;
Figure 32 lovastatin Wt2dpf acts on the deformity of zebrafish embryo;
Figure 33 lovastatin Wt3dpf acts on the deformity of zebrafish embryo;
Figure 34 Simvastatin 0.005ug/ml3dpf acts on the deformity of zebrafish embryo;
Figure 35 Simvastatin 0.03ug/ml3dpf acts on the deformity of zebrafish embryo;
Figure 36 Simvastatin 0.05ug/ml1dpf acts on the deformity of zebrafish embryo;
Figure 37 Simvastatin 0.01ug/ml3dpf acts on the deformity of zebrafish embryo;
Figure 38 Simvastatin 0.03ug/ml3dpf acts on the deformity of zebrafish embryo;
Figure 39 Simvastatin 0.01ug/ml2dpf acts on the deformity of zebrafish embryo;
Figure 40 Simvastatin Wt3dpf acts on the deformity of zebrafish embryo.
Embodiment
Embodiment one
Such as formula a preparation method for the compound lovastatin acid compound shown in C, comprise the following steps:
1) preparation is such as formula the compound lovastatin ammonia propyl ester shown in B
Get such as formula the compound lovastatin 40.4g shown in A, add 400ml ethanol, add propylamine 29.5g under 30 DEG C of conditions and carry out ring-opening reaction 1h, TLC detects to reacting completely, frozen water drops to 10 DEG C and terminates reaction, and decompression steams ethanol, obtained such as formula the compound lovastatin ammonia propyl ester shown in B;
2) preparation is such as formula the compound lovastatin acid compound shown in C
Get that step 1) obtains such as formula the compound lovastatin ammonia propyl ester shown in B, add 2mol/L hydrochloric acid soln 300ml stirring at room temperature 1h and carry out acidification hydrolization reaction, by re-crystallizing in ethyl acetate, obtained such as formula the compound lovastatin acid compound 38.1g shown in C, yield 94.3%(is in the compound lovastatin shown in formula A).
Embodiment two
Such as formula a preparation method for the compound lovastatin acid compound shown in C, comprise the following steps:
1) preparation is such as formula the compound lovastatin ammonia propyl ester shown in B
Get such as formula the compound lovastatin 40.4g shown in A, add 400ml ethanol, add propylamine 29.5g under 40 DEG C of conditions and carry out ring-opening reaction 5h, TLC detects to reacting completely, frozen water drops to 10 DEG C and terminates reaction, and decompression steams ethanol, obtained such as formula the compound lovastatin ammonia propyl ester shown in B;
2) preparation is such as formula the compound lovastatin acid compound shown in C
Get that step 1) obtains such as formula the compound lovastatin ammonia propyl ester shown in B, add 2mol/L hydrochloric acid soln 300ml stirring at room temperature 1h and carry out acidification hydrolization reaction, by re-crystallizing in ethyl acetate, obtained such as formula the compound lovastatin acid compound 38.7g shown in C, yield 95.6%(is in the compound lovastatin shown in formula A).
Embodiment three
Such as formula a preparation method for the compound lovastatin acid compound shown in C, comprise the following steps:
1) preparation is such as formula the compound lovastatin ammonia propyl ester shown in B
Get such as formula the compound lovastatin 40.4g shown in A, add 400ml ethanol, add propylamine 29.5g under 50 DEG C of conditions and carry out ring-opening reaction 3h, TLC detects to reacting completely, frozen water drops to 10 DEG C and terminates reaction, and decompression steams ethanol, obtained such as formula the compound lovastatin ammonia propyl ester shown in B;
2) preparation is such as formula the compound lovastatin acid compound shown in C
Get that step 1) obtains such as formula the compound lovastatin ammonia propyl ester shown in B, add 2mol/L hydrochloric acid soln 300ml stirring at room temperature 1h and carry out acidification hydrolization reaction, by re-crystallizing in ethyl acetate, obtained such as formula the compound lovastatin acid compound 39.3g shown in C, yield 97.3%(is in the compound lovastatin shown in formula A).
Embodiment four
A composition for lovastatin acid compound, by weight, comprises following component:
Table 1 embodiment Four composition proportioning
Component Weight (part)
Lovastatin acid compound 5
Polysorbate 80 0.3
Hydroxypropylcellulose 3
Croscarmellose sodium 9
N.F,USP MANNITOL 40
Microcrystalline Cellulose 30
Magnesium Stearate 0.6
Opadry film coating pre-mix dose In right amount
A preparation method for the composition of lovastatin acid compound, comprises the following steps (each amounts of components is by described in table 1):
1) lovastatin acid compound, Polysorbate 80, hydroxypropylcellulose, croscarmellose sodium, N.F,USP MANNITOL and Microcrystalline Cellulose is taken, wet granulation;
2) Magnesium Stearate is taken, compressing tablet;
3) Opadry film coating pre-mix dose is taken, film coating.
Embodiment five
A composition for lovastatin acid compound, by weight, comprises following component:
Table 2 embodiment five component proportion
Component Weight (part)
Lovastatin acid compound 20
Polysorbate 80 0.3
Hydroxypropylcellulose 3
Croscarmellose sodium 9
N.F,USP MANNITOL 40
Microcrystalline Cellulose 30
Magnesium Stearate 0.6
Opadry film coating pre-mix dose In right amount
A preparation method for the composition of lovastatin acid compound, comprises the following steps (each amounts of components is by described in table 2):
1) lovastatin acid compound, Polysorbate 80, hydroxypropylcellulose, croscarmellose sodium, N.F,USP MANNITOL and Microcrystalline Cellulose is taken, wet granulation;
2) Magnesium Stearate is taken, compressing tablet;
3) Opadry film coating pre-mix dose is taken, film coating.
Embodiment six
A composition for lovastatin acid compound, by weight, comprises following component:
Table 3 embodiment six component proportion
Component Weight (part)
Lovastatin acid compound 10
Polysorbate 80 0.3
Hydroxypropylcellulose 3
Croscarmellose sodium 9
N.F,USP MANNITOL 40
Microcrystalline Cellulose 30
Magnesium Stearate 0.6
Opadry film coating pre-mix dose In right amount
A preparation method for the composition of lovastatin acid compound, comprises the following steps (each amounts of components is by described in table 3):
1) lovastatin acid compound, Polysorbate 80, hydroxypropylcellulose, croscarmellose sodium, N.F,USP MANNITOL and Microcrystalline Cellulose is taken, wet granulation;
2) Magnesium Stearate is taken, compressing tablet;
3) Opadry film coating pre-mix dose is taken, film coating.
One, the comparison in drug effect
Lovastatin acid compound, Simvastatin alcohol acid compound are respectively the activity in vivo product of lovastatin and Simvastatin, the two is HMG-CoA reductase inhibitor, do not need through metabolism in vivo, can be directly used in treatment hyperlipidemia, its drug effect compares in table 4, table 5.
Table 4 lovastatin compares with lovastatin acid compound drug effect
As can be seen from Table 4, compared with lovastatin, lovastatin acid compound has more excellent bioavailability, stronger hypolipemic function and more long-acting therapeutic action.
Table 5 Simvastatin compares with Simvastatin alcohol acid compound drug effect
As can be seen from Table 5, compared with Simvastatin, Simvastatin hydroxy acid compound has more excellent bioavailability, stronger hypolipemic function and more long-acting therapeutic action.
Two, utilize docking assay lovastatin acid compound, Simvastatin alcohol acid compound and Pravastatin sodium to the restraining effect of HMG-CoA reductase albumen (1dqa).
1, part is selected
PUMBEDLINE searches lovastatin acid compound, Simvastatin alcohol acid compound, Pravastatin sodium information are shown in Fig. 3, Fig. 4, Fig. 5 and table 6.
Table 6 part information
Remarks: 1. CID is compound information code, to be numbered according to data message date created by PUMBEDLINE and to obtain.
2. XLogP3 represents hydrophobic parameter calculated value, is used for evaluating the fat water partition coefficients of medicine, have employed the legal conversion of atom adding.
2, acceptor selection
By the information of inquiring about in PDB protein pool in table 7, delivered 8 kinds of data at present, respectively corresponding inhomogeneous statin and substrate binding site, according to this research purpose, select 1dqa as acceptor, the 3D figure of 1dqa is shown in Fig. 6.
Table 7PDB protein pool Query Information table
3, docking prepares
First optimal treatment is carried out to the conformation of 1dqa, CHARm molecular field is applied to 1dqa, adopt MM calculation optimization 1dqa energy; After carrying out hydrogenation to 1dqa, removing invalid conformation, residue, select No. 1 avtive spot as docking pocket, its radius is 15.0 dusts; According to the character of part and 1dqa, DockLigands program is adopted to carry out half flexible docking.
4, result
Docking the results are shown in Figure 7, Fig. 8 and Fig. 9; The combination of part and 1dqa is shown in Figure 10, Figure 11, Figure 12, and visible lovastatin acid compound, Simvastatin alcohol acid compound have similar combination with Pravastatin sodium to 1dqa; The combination of Pravastatin sodium, lovastatin acid compound and Simvastatin alcohol acid compound and 1dqa avtive spot 1 mainly relies on the hydrogen atom in part to form hydrogen bond with LYS606, HIS635, SER637 amino acid in 1dqa respectively; Another Pravastatin sodium and Simvastatin alcohol acid compound also can form Pi-Pi conjugated link(age) with amino acid LYS62; The matching degree of assay lovastatin acid compound, Simvastatin alcohol acid compound and Pravastatin sodium and 1dqa, thus evaluate it further to the rejection ability of HMG-CoA reductase, the results are shown in Table 8.
The matching degree of table 8 three kinds of compounds and 1dqa
Index Number -PLP1 -PLP2 PMF RMSD
2 Lovastatin acid compound 105.880 109.980 161.920 1.101990
26 Simvastatin alcohol acid compound 82.330 91.070 149.000 1.092800
12 Pravastatin sodium 114.250 123.460 157.340 1.148210
Remarks: 1. Index: index.
2. Number: number, title.
3. PLP:(Piecewiselinearpotential) adopt piecewise linearity potential energy add and calculate; Score higher expression part and receptor affinity stronger.Whether participate according to hydrogen bond in docking operation, can be divided into PLP1 and PLP2 two kinds of functions, PLP1 does not consider the impact that hydrogen bond docks; PLP2 considers the impact of hydrogen bond on docking.
4. PMF:(Potentialofmeanforce), adopt adding and calculating of the average energy of intermolecular distance; Score higher expression part and receptor affinity stronger.
5. RMSD: the docking conformation of compound and the root-mean-square deviation of protocrystal conformation, characterize the difference degree on position and conformation that to predict the outcome with actual value, unit is dust.
In molecular docking process, the normal scoring functions that adopts is used for evaluating in docking procedure (LigandFit), the power of compound and receptor protein associativity.For specific protein research system, generally often need multiple different scoring functions to check docking result; To avoid single scoring functions by the Preference of the training set molecule selected when it is set up, lack the shortcoming of universality; As can be seen from above-mentioned table 8, lovastatin acid compound, Simvastatin alcohol acid compound and Pravastatin sodium are docked in conformation with receptor protein 1dqa's, and the score value of PLP1, PLP2 and PMF is all higher, and between each compound, difference is less; RMSD is all less than 2.0 dusts simultaneously, represents that LigandFit docking result is accurate; The above results shows: lovastatin acid compound, Simvastatin alcohol acid compound and Pravastatin sodium suppress HMG-C othe effect of A reductase enzyme is similar, and namely they have similar biological activity.
Three, statins toxicity ratio comparatively
In toxicologic study, zebra fish is usually used in the acute and chronic toxicity test of various medicine, and the transparent feature of its embryo is more conducive to the Structure and form change observing embryo's different steps; In its juvenile stage, each tissue system (neural system, cardiovascular systems, blood system, muscle skeleton motor system, secretion, absorption and Excretory system etc.) of body, close to ripe and perfect, start the physiological function of bringing into normal play, is similar to adult.Therefore, zebrafish embryo stage and juvenile stage can be used for studying the fetal development toxicity of compound and the target toxicity of each histoorgan fast.This research and utilization zebra fish model animal comparative studies toxicity of multiple statins to fetal development, especially on the impact that muscle tissue is grown.
1, the medication of zebrafish embryo
Treat the state of fetal development to the 50%-outsourcing phase after collecting embryo, select normotrophic embryo by often organizing 30 groupings, be placed in the statin liquid of different concns, wild control group is all established in each test.
Lovastatin, Simvastatin dimethyl sulfoxide (DMSO) prepare mother liquor, and diluting with artificial seawater before using is desired concn, and because mother liquor is dimethyl sulfoxide (DMSO) configuration, for avoiding dimethyl sulfoxide (DMSO) to have an impact to embryo, mother liquor at least dilutes more than 100 times; Test fluid artificial seawater dilutes, and after administration, every day observes the developmental condition of embryo, number dead and lopsided in statistics embryo, and takes pictures.After embryo's contamination, every day changes a liquid, administration process continues to the embryo 48dpf phase, and (48hpf is from the outsourcing phase to hatching administration in 48 hours, other are by that analogy), by liquid sucking-off, rinse embryo and be again placed in artificial seawater for several times and make it to continue to grow.
2, experimental result
1) comparison of lovastatin, Simvastatin and Pravastatin sodium embryotoxicity
Lovastatin and Simvastatin are the prodrug of inactive lactone structure, must distinguish metabolism open loop in vivo and form lovastatin acid compound and Simvastatin alcohol acid compound and could suppress HMG-CoA reductase; Pravastatin sodium then exists with the form of alcohol acid compound; Pravastatin sodium, lovastatin, Simvastatin act on zebrafish embryo (50%-outsourcing phase-48hpf) at 3dpf teratogenesis lethal range as shown in Figure 13, Figure 14, Figure 15 (3dpf for from the outsourcing phase to hatching administration in 3 days, other by that analogy).
Table 9 three kinds of compound impacts on zebrafish embryo are compared
Three kinds of statinses are that concentration dependent rises gradually to embryo's teratogenesis within the scope of respective concentration; From the concentration range that teratogenesis is lethal, the a little higher than lovastatin of activity of Simvastatin, the activity of Pravastatin sodium is then far above Simvastatin and lovastatin, namely the toxicity of Pravastatin sodium is far below Simvastatin and lovastatin, thus lovastatin acid compound can be described, the drug toxicity of Simvastatin alcohol acid compound is less than the prodrug of inactive lactone structure.
By table 9, 10 can find out, from the lethal severity display of teratogenesis that three kinds of statinses act on zebrafish embryo in early days, lovastatin is the most serious, next is Simvastatin, least seriously Pravastatin sodium, and the difference of concentration is fairly obvious when different classes of statins reaches identical teratogenesis or lethal effect to zebrafish embryo, such as 100% lethal dose of 100% lethal dose of the maximum lovastatin of the early stage zebrafish embryo toxicity Pravastatin sodium minimum with toxicity is differed and reach 100,000 times, and Pravastatin sodium, lovastatin, the molecular weight differences of Simvastatin three kinds of medicines is apart from less, three kinds of statinses recommendation dosage clinically also differs less, but but differ greatly in the toxicity acting on early stage zebrafish embryo.
Table 10. six kinds of statinses act on zebrafish embryo teratogenesis lethal concentration and compare
Chemically structure compares Pravastatin sodium, lovastatin, Simvastatin, its relative molecular mass closely; Pravastatin sodium exists with tool activated alcohol acid compound structure, but lovastatin and Simvastatin are the existence of inactive lactone structure formation; Pravastatin sodium to zebrafish embryo teratogenesis lethal effect much smaller than lovastatin and Simvastatin; Its plasma protein binding ratio of Pravastatin sodium, much smaller than lovastatin and Simvastatin, is about 50% of lovastatin and Simvastatin, show its main medicine existence in a free form in vivo, but toxicity is far smaller than lovastatin and Simvastatin; The lovastatin acid compound formed after the above results display open loop, the toxicity of Simvastatin alcohol acid compound are compared with the inactive lactone structure of Simvastatin with lovastatin respectively, and its toxic action obviously alleviates.
2) embryotoxicity of lovastatin acid compound, Simvastatin alcohol acid compound compares
Carry out HPLC after lovastatin, Simvastatin solution 24h and detect analysis, as can be seen from detected result, all basic after lovastatin, Simvastatin 24h exist with lovastatin acid compound, Simvastatin alcohol acid solvate forms.
Table 11 lovastatin and Simvastatin comparative result
Remarks: WT is wild contrast type, namely the embryo not adding medicine in contrast.
As can be seen from Table 11, when administration concentration increases to 0.01ug/ml, lovastatin and Simvastatin 0, the normal chick embryo of 24h digital display work of surviving declines, when concentration increases to 0.1ug/ml, total survival of lovastatin and Simvastatin and normal chick embryo number of surviving is 0; Result shows that administration concentration reaches more than 0.01ug/ml, and lovastatin and Simvastatin all show embryotoxicity.
3) statins acts on the teratogenecity of zebrafish embryo
After statins is contaminated to zebra fish, every day observes once, and the record main lopsided form of statistics is also taken pictures; Although can find out that the concentration range that these three kinds of statinses of Pravastatin sodium, lovastatin, Simvastatin act on early stage zebrafish embryo differs greatly by the picture of Figure 18 ~ Figure 39, but the phenotype form affecting the early development of embryo is comparatively similar, the toxic reaction phenotype caused to lovastatin acid compound, Simvastatin alcohol acid compound is also similar, mainly concentrates on the following aspects:
1. the overall body of embryo is long shortens, and yolk sac extension shortens thick, and caudal style bends towards side, left and right; High density group motor capacity reduces, responsive not as Normal group with headband touching reaction;
2. in embryo development procedure, pigment is influenced, and entirety weakens;
3. embryo has extravasated blood to occur, occurs that the position of extravasated blood mainly concentrates on the caudal style position of head, trunk back and yolk sac extending end;
4. zebrafish embryo all finds pericardium enlargement phenomenon under finite concentration, occurs the banded deformity of heart simultaneously;
5. all there is exception in treatment group embryo trunk place muscle tissue, and body segment is unintelligible, has the trend of parallelization at the maximum concentration group body segment of each group.
3, conclusion is analyzed
1) statins acts on zebrafish embryo teratogenesis lethal concentration scope: the six kinds of statinses giving different concns concentration at zebrafish embryo early development 50%-outsourcing phase-48hpf act on, result shows that the concentration range that six kinds of statins effect body early embryo teratogenesis are lethal is widely different, and what difference was maximum reaches up to ten thousand times; The lethal severity of teratogenesis that three kinds of statinses act on zebrafish embryo is followed successively by: lovastatin is the most serious, and being secondly Simvastatin, is thirdly the alcohol acid compound of lovastatin acid compound, Simvastatin, least seriously Pravastatin sodium.
2) toxicity: lovastatin and the open loop of SV key form lovastatin acid compound, Simvastatin alcohol acid compound respectively, and toxicity at least reduces 50 times; But owing to also creating other related substanceses after degraded, therefore the toxicity ratio of lovastatin acid compound and Simvastatin alcohol acid compound mensuration is lower.
3) deformity: although statins to act on early stage zebrafish embryo concentration difference very large, the deformity caused is consistent substantially, mainly contains that the overall body of embryo longly shortens, yolk sac extends thick, the caudal style of shortening and bend; Pigment weakens; The caudal style position extravasated blood of head, trunk back and yolk sac extending end; Pericardium enlargement phenomenon, there is the banded deformity of heart simultaneously; All there is exception in embryo's trunk place muscle tissue, has the trend of parallelization at the maximum concentration group body segment of each group.
More than detailed description of the invention in conjunction with embodiment; only for technical conceive of the present invention and feature are described; its object is to allow person skilled in the art understand content of the present invention and to be implemented; protection scope of the present invention can not be limited with this; all equivalences done according to spirit of the present invention change or modify, and all should be encompassed in protection scope of the present invention.

Claims (4)

1., such as formula a preparation method for the compound lovastatin acid compound shown in C, comprise the following steps:
1) preparation is such as formula the compound lovastatin ammonia propyl ester shown in B
Get such as formula the compound lovastatin shown in A, add ethanol and propylamine carries out ring-opening reaction, obtained such as formula the compound lovastatin ammonia propyl ester shown in B;
2) preparation is such as formula the compound lovastatin acid compound shown in C
Get step 1) obtained such as formula the compound lovastatin ammonia propyl ester shown in B, add hydrochloric acid and carry out acidification hydrolization reaction, recrystallization, obtained such as formula the compound lovastatin acid compound shown in C
2. preparation method according to claim 1, is characterized in that: described step 1) under 30 DEG C ~ 50 DEG C condition, add propylamine carry out ring-opening reaction.
3. preparation method according to claim 1, is characterized in that: described step 1) in add propylamine reaction 1h ~ 5h.
4. preparation method according to claim 1, is characterized in that: described step 2) in use re-crystallizing in ethyl acetate.
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