CN101904841A - Phthalide acid imide analog and the application in preparation treatment diabetic macular edema medicine thereof - Google Patents

Phthalide acid imide analog and the application in preparation treatment diabetic macular edema medicine thereof Download PDF

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CN101904841A
CN101904841A CN2010102389529A CN201010238952A CN101904841A CN 101904841 A CN101904841 A CN 101904841A CN 2010102389529 A CN2010102389529 A CN 2010102389529A CN 201010238952 A CN201010238952 A CN 201010238952A CN 101904841 A CN101904841 A CN 101904841A
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陈旦洋
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Abstract

The invention provides and use a kind of Phthalide acid imide analog and prevent and treat application in the relevant blind medicine of diabetic macular edema in preparation.In addition, this patent proposes to wrap up the drug effect that is used to prolong Phthalide acid imide analog by polylactic-co-glycolic acid copolymer nano granule.At last, this patent discloses a kind of synthetic (2, the 6-diisopropylbenzyl)-5-amino-1-hydrogen-iso-indoles-1,3-diketone (chemical compound 4, new method CLT-003).

Description

Phthalide acid imide analog and the application in preparation treatment diabetic macular edema medicine thereof
[related application reference]
In application on July 24th, 2009 United States Patent (USP), application number is 12/509,369 in the present invention.Now with the priority application Chinese patent.
[research that federation supports]
This research is by U.S.'s National Eye Institute fund assistance (NIH EY016627-01, EY017229-02, and EY017229-03)
[background technology]
Thalidomide is a kind of effective angiogenesis inhibitor medicine, and it and Phthalide acid imide are approved for treatment multiple myeloma and ENL.Discover, they can be blocked the increase of the VEGF in the STZ diabetes rat aqueous humor and suppress thickening of retinal capillary basement membrane, thereby be expected to become effective treatment diabetic retinopathy, comprise the potential drug of diabetic macular edema.
In addition, a kind of synthetic (2, the 6-diisopropylbenzyl)-5-amino-1-hydrogen-iso-indoles-1, (chemical compound 4, new method CLT-003) is public publish in this application for the 3-diketone.
[application summary]
This patent provides Phthalide acid imide analog to prevent and treat application in the relevant blind medicine of diabetic macular edema in preparation.In addition, this patent proposes to wrap up the drug effect that is used to prolong the administration of Phthalide acid imide analog by polylactic-co-glycolic acid copolymer nano granule.At last, this patent discloses a kind of synthetic (2, the 6-diisopropylbenzyl)-5-amino-1-hydrogen-iso-indoles-1,3-diketone (chemical compound 4, new method CLT-003).
Present patent application discloses a kind of medicine for the treatment of retinal edema, and this medicine is a Phthalide acid imide analog, contains following chemical constitution:
Figure BSA00000208007300021
Present patent application discloses a kind of ingredient for the treatment of retinal edema, and this ingredient comprises a kind of chemical compound that contains following chemical constitution:
Figure BSA00000208007300022
This chemical compound is wrapped in polylactic-co-glycolic acid copolymer nano granule.
Present patent application discloses a kind of method of drug effect of energy prolong drug composition, comprises the chemical compound that contains following chemical constitution in this ingredient:
Figure BSA00000208007300023
Be about to this chemical compound and be wrapped in polylactic-co-glycolic acid copolymer nano granule.This parcel capsule can continue to discharge compounds effective to prolong the therapeutical effect to diabetic macular edema.
Present patent application discloses a kind of synthetic method that contains the chemical compound of following chemical constitution:
The method is: make 4-nitrophthalic anhydride and 2 by refluxing in acetic acid, 6-diisopropyl aniline pairing condensation generates (2, the 6-diisopropylbenzyl)-5-nitro-1-hydrogen-iso-indoles-1, the 3-diketone; Again it is passed through catalytic hydrogenation or transform the hydrogenation hydrogenation.
[drawing explanation]
These characteristics of the present invention and data will be by more clear in conjunction with the following description and accompanying drawing, and accompanying drawing indicates with digital mode:
Fig. 1 shows the influence of Thalidomide and Phthalide acid imide analog on cell proliferation thereof for form.
Fig. 2 shows chemical compound 1, chemical compound 4 and the Thalidomide inhibitory action to endotheliocyte (HUVEC) migration.
Fig. 3 shows the influence that 4 pairs of tubuloses of chemical compound form.
Fig. 4 show chemical compound 4 in CAM experiment to angiopoietic influence.
Fig. 5 shows the influence of Phthalide acid imide analog to the HIF-1 alpha expression.
Fig. 6 shows chemical compound 4 downward modulation vegf expressions.
Fig. 7 shows the influence of Thalidomide, 1,2,4 pairs of OIR rat retina vascular leakages of chemical compound.
Fig. 8 shows the influence of Thalidomide, 1,2,4 pairs of STZ-diabetic retinal tissue in rat vascular leakages of chemical compound.
Fig. 9 shows intravitreal injection Thalidomide, the angiographic situation of chemical compound 1,4 back OIR rat retinas.
Figure 10 shows the infiltrative species variation of rat aorta in the OIR model.
Figure 11 shows the infiltrative species variation of STZ-diabetes model medium vessels.
Figure 12 shows the VEGF level of OIR BN and SD rat
Figure 13 shows inductive BN of STZ and SD diabetic retinal tissue in rat VEGF level.
Figure 14 for form show chemical compound 4 in the CAM experiment to angiopoietic influence.
Figure 15 shows the influence of 4 pairs of big rathole A ripples of chemical compound and B ripple for form.
Figure 16 shows the route map of chemical compound 4 synthetic.
Figure 17 shows the chemical constitution of Thalidomide and Phthalide acid imide analog.
Figure 18 shows chemical compound 4 processing back rat retina function and morphologic analyses.
Figure 19 shows 4 pairs of activated influences of HIF-1 α of chemical compound.
Figure 20 shows the influence that 4 pairs of ARPE-19 cell VEGEs of chemical compound and solubility ICAM-1 express.
Figure 21 shows the influence that chemical compound 4 pairs of OIR rat retinas VEGF and ICAM-1 express.
Figure 22 shows the influence of 4 couples of OIR of chemical compound and STZ-diabetic retinal tissue in rat vascular leakage.
Figure 23 shows the influence of 4 pairs of STZ-diabetic retinal tissue in rat of oral administration of compound vascular leakage.
Figure 24 shows the electron microscope scanning figure of chemical compound 4PLGA nano-particle.
Figure 25 shows the analysis of chemical compound 4PLGA nano-particle dynamic light scattering
Figure 26 shows the influence of chemical compound 4 nano-particle to STZ-diabetic retinal tissue in rat vascular leakage.
Figure 27 shows chemical compound 4 nano-particle and the inhibitory action of growing without 4 couples of BRCEC of the chemical compound that wraps up.
[detailed description]
In the detailed description of following disclosed example, accompanying drawing wherein indicates similar part for reference, shows existing published graphic example. The detailed description of these examples so that those skilled in the art implement, must be clear that, may use other examples, and in the scope of existing disclosure, logistics may be arranged, Mechanics of Machinery, electronics, function assessment and other change. So, just be used for explaining the present invention, and limitation of the present invention absolutely not. As said in existing disclosure, the term "or" should be appreciated that and be defined as logical option, and is not to refer to only option, unless specially marked " or ".
Term, lead-in initialism or abbreviation above-mentioned or that the elsewhere is used mean: AMD: senile macular degeneration; BFGF: basic fibroblast growth factor; BN:Brown-Norway; BRCEC: bovine retina endothelial cell; BSA: bovine serum albumin(BSA); CAM: CAM; Compound 4 (CLT-003): CLT-003; Compound 1:(2, the 6-diisopropylbenzyl)-5-hydroxyl-1H-iso-indoles-1, the 3-diketone; Compound 2:(2, the 6-diisopropylbenzyl)-1H-iso-indoles-1, the 3-diketone; DME: diabetic macular edema; DR: BDR; DMSO: dimethyl sulfoxide (DMSO); EPO: hematopoietin; ERG: electroretinogram; HIF-1: HIF-1; HUVEC: Human umbilical vein endothelial cells; IGF-1: IGF; NV: new vessels forms; OIR: oxygen inductivity PVR; PEG: polyethylene glycol; PET: polyethylene terephthalate; ROP: retinopathy of prematurity; RPE: retinal pigment epithelium; SD:sprague-Dawley; STZ: Streptozotocin; VEGF: VEGF; VEGFR: vascular endothelial growth factor receptor.
Term " Angiogenesis " refers to new vessels grow into tissue or organ.
Term " retinal vessel seepage " refers to that the retinal vessel permeability raises.
" therapeutically effective amount " is defined as in this article: use the therapeutic dose that can produce ideal effect in reference, this dosage meets rational income/risk ratio, and is applicable to any therapeutic treatment. Effective dose may change with the impact of factors, such as seriousness of size, disease or the state of an illness of subject disease or the state of an illness, the patient's that receives treatment special physique and the bodily form thereof etc. For the effective dose of used specific compound, those skilled in the art can rule of thumb need not too much test and just can determine.
Employed term " treatment " word comprises the development that suppresses or stop disease, imbalance or morbid state in the art, eliminates or alleviates it. The treatment of disease or morbid state comprises a kind of symptom of improving at least specified disease or morbid state, even its potential pathologic, physiologic there is no change, for example: treat patient's pain by giving analgestic, even this analgestic is not treated the cause of disease that causes pain.
BDR (DR) is a kind of common microvascular complication of diabetes, and is still severe visual disappearance and blind principal element. Retinal vessel seepage due to the destruction of retina-alveolar-capillary barrier is the early stage and common pathological change of DR. It is generally acknowledged, before the increase of the early stage retinal vessel seepage of DR betides clinical PVR. The retinal vessel seepage often causes diabetic macular edema, and the latter is the first cause of vision loss in diabetic patients.
Diabetic macular edema is gathered to feature with the extracellular fluid in retina Henle ' s layer and the inner nuclear layer, all can take place in any stage of DR development. To the treatment of DR, comprise laser photocoagulation and vitreous excision at present, can stop eyesight to be lost, but most of DR patient's eyesight is not had improvement. Owing to lack early stage and suitable treatment, DR remains visual loss and blind main cause in the industrialized country. Therefore, the new treatment that needs research and development DR/ diabetic macular edema is arranged.
Micromolecular compound 4 (CLT-003) has shown that the treatment diabetic macular edema is effective. Research data confirms that compound 4 has anti-angiogenic generation and anti-inflammatory activity. At Human RPE Cells in Vitro and Oxygen-induced retinopathy (OIR) rat retina, compound 4 can weaken the activation of hypoxia-inducible factor-1 alpha (HIF-1 α), makes VEGF (VEGF) and ICAIU (ICAM-1) down-regulated expression. Compound 4 also significantly reduces the retinal vessel seepage that OIR and STZ induce diabetes rat. In addition, intravitreal injection compound 4 PLGAs (PLGA) nano particle, it continued for six weeks at least to the long-term effect that reduces STZ-diabetic retinal tissue in rat vascular leakage. These find prompting, and this monomer molecule has multi-efficiency, have new drug, particularly diabetic macular edema that great potential becomes the treatment macular edema.
These compounds of recently finding have one or more asymmetric c atoms, and they exist with the form of optical voidness enantiomer, the non-enantiomorph of optical voidness, mixture of enantiomers, non-enantiomer mixture or racemic stereoisomer mixture. These newfound compounds comprise all these isomers and composition thereof.
These newfound compounds all are the compounds relevant with phenylphthalimide analog, have the activity of anti-angiogenic generation and anti-angiogenic seepage. More particularly, the compound of these discoveries is included into phenylphthalimide analog series, and piperidines wherein-2,6-diketo are namely replaced by 2,6-, two different phenylpropyl. As follows:
Figure BSA00000208007300061
In addition, disclosed noval chemical compound has following basic structure:
Figure BSA00000208007300071
R wherein 1Be selected from hydroxyl, hydrogen and amino.
More than, herein or the used chemical compound in elsewhere, be hydroxyl as the R1 in the chemical compound 1, the R1 in the chemical compound 2 is a hydrogen, the R1 in the chemical compound 4 is amino.All cpds is as follows:
Figure BSA00000208007300072
In addition, the noval chemical compound with angiogenesis inhibitor and anti-angiogenic seepage has following basic structure:
Figure BSA00000208007300073
R wherein 1Can be selected from hydroxyl, hydrogen and amino.
As the medicine of effective treatment diabetic macular edema, this chemical compound has following structure:
Figure BSA00000208007300074
In addition, the invention discloses a kind of medicine for preparation treatment aberrant angiogenesis.Say that more particularly instantiation directly is included into the medicine of formation of treatment new vessels and/or vascular leakage.This chemical compound has following basic structure:
Figure BSA00000208007300081
R wherein 1Be selected from hydroxyl, hydrogen and amino.
Though this treatment not exclusively comprises, but especially at retinal vessel abnormity disease, as diabetic renal papillary necrosis, diabetic macular edema, senile degeneration of macula, sickle cell retinopathy, the retinal vein occlusion, retinopathy of prematurity and other forms of by retinal neovascularization forms or the retinal vessel seepage causes retinopathy and disease.In one embodiment, its treatment comprises inhibition VEGF and HIF-1 α, and the latter is the main transcription factor that raises diabetic retina VEGF.In addition, Phthalide acid imide analog also can be used as sodium channel inhibitor, calcium channel blocker, contraceptive, anti-inflammatory agent and anticarcinogen.
On the other hand, Phthalide acid imide analog can synthesize by the method that replaces the glutaramide ring with an aromatic series base.One specific embodiments is: by using reactant 4-nitrophthalic anhydride and 2, the 6-isopropyl aniline produces (2, the 6-diisopropylbenzyl)-5-nitro-1-hydrogen-iso-indoles-1, the 3-diketone, and the latter reprocesses synthetic compound 4.According to this specific embodiments, 4-nitrophthalic anhydride and 2,6-isopropyl aniline and acetic acid reflux 3 hours to produce (2, the 6-diisopropylbenzyl)-5-nitro-1-hydrogen-iso-indoles-1, the 3-diketone, the latter further with hydrogen, palladium carbon and alcohol reflux 2 hours, form chemical compound 4.
Structure-activity relationship studies show that, replaces glutaramide ring in the Thalidomide with an aromatic series base and can generate and have active analog.Especially use 2, the 6-cumenyl replaces the glutaramide ring, can produce angiogenesis inhibitor and the stronger analog of anti-angiogenic seepage activity--- chemical compound 1,2 and 4.
With the external drug screening of endothelial cell proliferation experiment carrying out, prove that this three kinds of chemical compounds--- chemical compound 1,2 and 4 has very strong antiproliferative activity, because they optionally suppress HUVEC and BRCEC growth, its IC50＜3.3 μ M, this is in fact than Thalidomide, existing Phthalide acid imide analog Actimid (CC-4047) and Revimid (CC-5053) (IC 50＞100 μ M) all low.In addition, Phthalide acid imide analog does not suppress the growth of non-endotheliocyte, pericyte (IC50＞32 μ M) for example, and this illustrates that they have the specificity of cell type rather than non-specific cytotoxicity to the inhibition of endothelial cell growth.Wherein a kind of Phthalide acid imide analog-chemical compound 4, to the propagation (IC50≤2.08 μ M) of HUVEC and BRCEC, to the migration (IC50 of＜1 μ M) of HUVEC, the tubulose of HUVEC is formed, and, very strong effect is arranged all to the vascularization (ED50=6.5 μ g/ embryo) in the CAM test.The blood vessel formation against function of chemical compound 4 also is confirmed in the OIR model, and the latter is a generally accepted model of retinal neovascularization formation and proliferative diabetic retinopathy.
Thalidomide and novel Phthalide acid imide analog compare in OIR and STZ-diabetes rat to the influence of retinal vessel seepage and NV.Because the STZ-diabetes rat occurs comprising that the background diabetic retinopathy of vascular leakage becomes, this rat has become an experimental diabetes disease model of widely using.Unusual retinal vessel seepage also appears in the OIR model.Experimental result shows that in two kinds of animal models, new Phthalide acid imide analog obviously is better than Thalidomide to the effect of retinal vessel seepage.
Compare with solvent in the OIR rat and compare, chemical compound 4 and the single agent administration of Thalidomide with 1.0 μ g/ eyes can make the retinal vessel seepage reduce by 40% and 18% respectively.Induce in the diabetes rat to compare with solvent at STZ-to compare, chemical compound 4, Thalidomide, chemical compound 1 are with single agent administration of 1.0 μ g/ eyes, and they make the retinal vessel seepage reduce by 100%, 77% and 61% respectively.After the shot 24,48 hours, chemical compound 4 can be blocked the retinal vessel seepage of diabetes-induced fully.The effect that chemical compound 4 reduces the retinal vessel seepage is dose dependent.The result shows, compares with Thalidomide, and chemical compound 4 shows all in the OIR model but also in the STZ-diabetes model that not only it has stronger effect to the reduction of retinal vessel seepage.
The regulating action of 4 pairs of OIR rat retinas of chemical compound vegf expression is also studied, and the result shows, the expression of chemical compound 4 downward modulation VEGF, and the target spot that chemical compound 4 is described is the VEGF signal path.
Experiment shows that chemical compound 4 can suppress the propagation of HUVEC and BRCEC, but does not suppress non-endotheliocyte, illustrates that its influence is an endothelial cell specific.Select for use chemical compound 4 to assess the potential eye of rat toxicity.ERG and histopathological examination confirm that all chemical compound 4 does not cause the obvious change of rat ocular tissue form and function after a high dose injection.Results suggest, chemical compound 4 there is no overt toxicity under generation angiogenesis inhibitor and the required effective dose of anti-angiogenic seepage effect.
These test demonstration, and the chemical compound 4 of low dosage can suppress NV and anti-angiogenic seepage.Angiogenesis inhibitor and anti-angiogenic seepage Action Specification that chemical compound 4 is stronger, the chemical compound 4 of low dosage is enough to suppress NV and anti-angiogenic seepage, so unlikely cause side effect.
The albuminuria of diabetic nephropathy is the another kind of type of vascular leakage., chemical compound 4 leaks out to outside the blood vessel the albuminuretic medicine of therefore also available its preparation treatment in view of reducing macromole.Vascular leakage is a step important in the neoplasm metastasis process.Blocking-up tumor vessel seepage also is expected to obtain curative effect in the treatment solid tumor.
According to example, chemical compound 4 can reduce the activation of HIF-1 α, and the expression of downward modulation VEGF and ICAM-1.In addition, intravitreal injection and oral administration of compound 4 all can lower the retinal vessel seepage, and prompting 4 pairs of treatments of chemical compound diabetic macular edema has treatment potential.At last, give the chemical compound 4 lasting diabetic retinal tissue in rat vascular leakages that reduce of nano-particle mediation.
Medicine or nutriment composition
In addition, medicine or nutritional labeling comprise disclosed chemical compound, and additional activating agent, carrier, solvent, excipient or adjuvant etc., but those skilled in the art is by reading just these compositions of identification of the existing information that discloses.
Medicine or nutritional labeling preferably comprise a kind of pharmaceutically useful carrier at least.In these ingredients, the one or more chemical compounds that this time disclose are formed " reactive compound ", also are referred to as " activating agent ".Above-mentioned " pharmaceutically useful carrier " comprise solvent, disperse medium, encrusting substance, antibacterium and antifungal, etc. blend absorption delay agent or the like, meet the requirement of medicine management.The additional activity chemical compound also can be included in the ingredient.The ingredient preparation is suitable with predetermined route of administration.Route of administration comprises: injecting drug use absorbs (surface), per mucous membrane and rectally as intravenous injection, intradermal injection, subcutaneous injection, oral (as sucking), percutaneous.The solution or the suspension of injection, Intradermal or subcutaneous administration comprise following ingredients: sterile diluent such as water for injection, saline, fixed oil, Polyethylene Glycol, glycerol, propyleneglycoles or other synthetic solvents; Antibacterial such as benzyl ethanol, methyl metagin; Antioxidant such as ascorbic acid (vitamin C), sodium bisulfite; Chelating agen such as ethylenediaminetetraacetic acid; Buffer such as acetate, citrate or phosphate buffer also have reinforcing agent such as sodium chloride, dextran.The drug administration approach also can comprise through eye: in the vitreous body, under the conjunctiva, behind the anterior chamber, episclera, eyeball, the cone down or subretinal injection, or through surperficial eye drip.PH value usable acid or alkali adjustment, for example hydrochloric acid or sodium hydroxide.Ejection preparation can be encapsulated in ampoule, disposable pipe or the multiple dose vials made by glass or plastics in.
Experimental subject used herein refers to the mankind and non-human primate (as gorilla, Rhesus Macacus, stump-tailed macaque, marmoset), domestic animal (as sheep, cattle, horse, donkey, pig), the animal of accompanying and attending to (as Canis familiaris L., cat), laboratory test catches animal (as fox, deer) with animal (as mice, rabbit, rat, Cavia porcellus, hamster), field and any other can have benefited from the organism of existing disclosure medicine.No matter, all it can be regarded as patient, individuality, animal, host or receptor to liking human still non-human organism body.
The ingredient that is suitable for injecting comprises aseptic aqueous solution or dispersant, and for prepare the sterilized powder that aseptic parenteral solution or suspension are used temporarily.Intravenous suitable carrier comprises normal saline, sterilized water, polyoxyethylene castor oil (BASF, Pa Xipani, New Jersey) or phosphate buffer (PBS).In a word, ingredient must asepticly be easy to use syringe pump with good fluidity.When producing and store, should stablize, should prevent contamination by micro such as antibacterial and fungus during preservation.Carrier can be solvent or dispersant, comprises water, ethanol, polyol (as glycerol, propyleneglycoles, liquid polyetheylene glycol and analog) and suitable mixture thereof.For keeping suitable flowability, can use the bag quilt as lecithin, in dispersant, keep required granular size and use surfactant.The pollution of prophylaxis of microbial can be passed through various antibiotic and antifungal agent, as metagin, methaform, phenol, ascorbic acid (vitamin C), thimerosal and analog.In many cases, preferably comprise isotonic agent in the ingredient, for example sugared, polyhydric alcohol such as mannitol, sorbitol, or sodium chloride.The absorption that prolongs injectable drug can reach by add the medicine that postpones to absorb in composition, for example aluminum monostearate and gelatin.
Aseptic injectable solution can by in suitable solvent with the reactive compound of required dosage with single or make up above-mentioned raw materials and mix aseptic on demand then filtration.Generally speaking, dispersant is by reactive compound is mixed into sterile carrier, and the latter includes basic disperse medium and other required above-mentioned raw materials.For the standby sterilized powder of aseptic injectable solution, preparation method has vacuum drying and lyophilization, so just the powder of output activated feedstock and other desirable feedstock from previous aseptic filtered soln.
Oral medicine generally comprises inert diluent or edible carrier.For oral administration, reactive compound can with mixed with excipients, with tablet, the form of buccal tablet or capsule (as gelatine capsule) is taken.The also available liquid carrier of oral medicine prepares, as collutory.Compatible bridging agent of medicine or adjuvant can be used as composition in the medicine.Tablet, pill, capsule, buccal tablet and analog can comprise any following raw material or similar compound: junctional complex such as microcrystalline Cellulose, tragacanth or gelatin, excipient such as starch or lactose, disintegrating agent such as alginic acid, Sodium Hydroxymethyl Stalcs or corn starch, lubricant such as magnesium stearate or silica gel, fluidizer such as dioxide/silica gel, sweeting agent such as sucrose or glucide, perhaps flavoring agent such as Herba Menthae, another name for Salicylate or the agent of orange flavor.
For inhalation, chemical compound with by pressure vessel, include the allotter of suitable propeller such as carbon dioxide or the form administration of the aerosol in the aerosol apparatus.
Also per mucous membrane or percutaneous but whole body is administered systemically.For per mucous membrane or percutaneous dosing, use the penetrating agent that is suitable for the barrier infiltration in the preparation.This penetrating agent is very universal in the art, and for example, transmucosal drug delivery comprises detergent, bile salts, brown mould acid derivative.Transmucosal drug delivery can be with nasal spray or suppository.For percutaneous dosing, reactive compound is made ointment, ointment, gel or Emulsion, and this is well known in the art.Chemical compound also can be made into suppository (as with conventional suppository bases such as cupu oil and other glyceride) or with the form per rectum administration of retention enema.
Except that other administering modes, chemical compound also can be through eye drip or intraocular injection administration.For putting drops in one's eyes, the composition of new drug comprises one or more excipient that is applicable to eye or nose surface.Excipient is commonly used in the ingredient of eye table, as solution or spray, includes but not limited to: tonicity agent, preservative agent, chelating agen, buffer agent, surfactant and antioxidant.The suitable degree adjustment agent of opening comprises: mannitol, sodium chloride, glycerol, sorbitol and analog.Suitable antiseptic comprises: metagin, benzalkonium chloride, benzene plating bromo-amine, polyquaternary ammonium salt-1 and analog.Suitable chelating agen comprises: sodium ethylene diamine tetracetate, and analog.Suitable reducing has: phosphate, borate, citrate, acetate and analog.Suitable surfactant comprises: ion-type and nonionic surfactant, nonionic better is as polysorbate, poly-castor oil derivative, oxidation octyl phenol yuban (Tai Luoshabai).Suitable antioxidant comprises: sulphite, Ascorbate, BHA and BHT.Can comprise a kind of other activating agent in the prescription of new drug.Except additional preservatives (as polyquaternary ammonium salt-1), except that polyvinylpyrrolidone or polystyrolsulfon acid, should not comprise other any polymer in the new drug.
When containing polyvinylpyrrolidone in the new drug, polyvinylpyrrolidone can reduce the content of peroxide.The polyvinylpyrrolidone of new lot is better than old batch.In addition, specifically, when containing in the composition greater than 0.5% polyvinylpyrrolidone, polyvinylpyrrolidone with should heat (as being heated to more than the room temperature) earlier before olopatadine mixes, reducing the peroxide in the polyvinylpyrrolidone raw material, and peroxide dropped to minimum to the influence of the chemical stability of olopatadine.Long-time heating polyvinylpyrrolidone aqueous solution can significantly reduce the content of peroxide, but can cause polyvinylpyrrolidonesolution solution variable color (yellow or pale brown color).For reducing peroxide and don't making the polyvinylpyrrolidone variable color, the pH value of polyvinylpyrrolidonesolution solution should transfer to pH11～13 before heating.If the pH value of polyvinylpyrrolidonesolution solution raises, the heating of short time can make peroxide level significantly reduce.
A kind of mode of appropriate heating polyvinylpyrrolidonesolution solution is as follows: at first, polyvinylpyrrolidone is dissolved in the solution that is made into 4-6% in the pure water, again pH value is transferred to pH11～13 (effectively the pH value scope is 11-11.5), at 60-121 ℃ of scope internal heating, 65-80 ℃ more satisfactory, preferably 70-75 ℃.30-120 minute (preferably 30 minutes) of continuous high temperature heating.Be cooled to room temperature then, the target P H-number according to olopatadine adds HC1 pH value is transferred to 3.5～8.
For the composition of putting drops in one's eyes, the tonicity contributor that preferably comprises a kind of sufficient dosage is to produce the osmotic pressure (being roughly 150-450m0sm, preferably 250-350m0sm) of ophthalmology's approval.The pH value of new medicament for the eyes should be 4～8, and 6.5～7.5 is relatively good, and preferably 6.8～7.2.
Newly put drops in one's eyes and preferably be contained in the opaque plastics container.The reasonable container of splendid attire medicament for the eyes is the low density polyethylene (LDPE) bottle, and the latter uses ethylene oxide but not the gamma ray sterilization.
For the ocular injection medication, medicine can administration under conjunctiva according to the needs of treatment.A reasonable method that is administered to eye under the conjunctiva is for adopting the injectable formulation that contains ingredient disclosed herein.Another preferably method be the implantation slow release agent.
Through the ingredient of subconjunctival injection, to decide as the case may be, its medicament for the eyes depot formulation comprises a kind of activating agent.The medicament for the eyes depot formulation comprises important purification activating agent microgranule such as Phthalide acid imide analog, and the chemical compound 4 here just so.This particulate constituent can be embedded in the acceptable polymer of good biocompatibility or the lipid and become capsule preparations.Depot formulation can discharge medicine by the speed of appropriateness in a longer time.If polymer or lipid capsule are arranged, after having discharged all activating agents, can fully degrade and discharge from the injection site.Depot formulation can be liquid state, comprises pharmaceutically useful polymer, dissolving or dispersive activating agent.After the injection, polymer can become drug depot through gelation or precipitation form in the injection site.
The solid matter that ophthalmic is implanted also can be designed to be wrapped in the form of polymer, and biodegradable or non-biodegradation.The biological degradation polyalcohol that contains the preparation ophthalmic implantation of new drug has: unconfined aliphatic polyester is as poly-Acetic acid, hydroxy-, bimol. cyclic ester, polylactide, poly-penta-own lactide, poly butyric ester and poly-hydroxypentanoic acid, polyamino acid, polyorthoesters, polyanhydrides, fatty Merlon, polyethers lactone.Illustrating the non-biodegradation polymer that is fit to use is silica gel.
According to carrying out requirement, it avoids the preparing carriers that body is eliminated fast to reactive compound with protection, and for example controlled release agent has the property of implantation and microcapsule medicine-feeding technology.Available biodegradable, nonbiodegradable polymer is as ethylene acetic acid, polyanhydrides, polyhydroxy acid, collagen protein, polyorthoesters and polylactic acid.The method for preparing these controlled release agent is easy the personnel in this area.These raw materials also can obtain by commercial, as having bought from Alza company and Nova pharmaceuticals.Lipid suspension (comprising and the lipid that contains the antigenic monoclonal antibody of cell-specific at infection cell) also can be used as pharmaceutically useful carrier.These all can be prepared by the technician of well known this method, as it is described to be numbered 4,522,811 United States Patent (USP), and the latter also includes reference in.
Make things convenient for administration and dosage homogeneous for reaching, preferably with dosage unitization oral and the intestinal external administration.The dosage unitization of indication is meant the unit of individual variation herein, is applicable to the single dose of being controlled object; Each unit comprises the pre-metering of good reactive compound, with the ideal therapeutic effect of the common generation of required pharmaceutical carrier.
The toxicity of these chemical compounds and therapeutic effect depend on the standard drug Cheng Cheng of cell culture or laboratory animal, as decision LD50 (median lethal dose(LD 50)) and ED50 (median effective dose).The ratio of the dosage of toxicity and therapeutic effect is a therapeutic index, and available LD50/ED50 represents.The compound exhibits medicine of high therapeutic index is effective.The chemical compound that shows toxic and side effects also can use, but notes drug-supplying system of design, and the latter can bring to desired area with chemical compound, makes it to Normocellular potential damage minimum, thereby reduces side effect.
The data that obtain from cell culture test and zoopery can be used for formulating the dosage range that uses this chemical compound for human body.The dosage of chemical compound is preferably within the circulation composition scope, and the latter comprises and follows few poison or nontoxic ED50.Dosage can change in this scope, depends on used dosage form and route of administration.For disclosed any chemical compound, can estimate the treatment effective dose with cell culture experiments earlier.The dosage that reaches the blood circulation concentration range can be formulated as cell culture, formulates by animal model, and the latter comprises IC 50(half suppression ratio).These information can be used to determine more accurately the effective dose used in human body.Blood drug level usable highly effective liquid chromatography for measuring.
Effective therapeutic dose of Ding Yi reactive compound (as effective dosage) can change in the scope of 0.001 to 100g/kg body weight herein, or technicians need not to test other scopes that can realize.Technicians generally admit, and many factors can influence the patient and effectively be treated required drug dose and time, include but not limited to: the seriousness of the disease or the state of an illness, previous treatment, general health or patient age and other disease that taken a disease.
[specific embodiment]
By the following examples and chart, can obtain a more complete understanding to newfound medicine.Its purpose of used embodiment and chart only is in order to say something better, rather than has a mind to be used to limit protection scope of the present invention.When environment change, the variation of profile or equal substitute can be expected.Although used technical term herein, these terms only lay down a definition and unrestricted usefulness.Modification and the change that above discloses information do not broken away from aim of the present invention and related scope, so these restriction meetings are emphasized in separate statement.
Embodiment 1: material and method
Cell culture: unless other explanations are arranged, all cells culture fluid and auxiliary substance are all available from Cellgro company.Human umbilical vein endothelial cells (HUVEC) is obtained from American type culture collection (ATCC), cultivates in EBM-MV2 (Clonetics company) culture fluid.Bovine retina endotheliocyte (BREC) and pericyte with the described method of forefathers separate (Wong, et al.Investig.Opthalmol.Vis.Sci.1987,28:1767-1775).(meat field, national native country) obtains 12 buphthalmos from local slaughterhouse, takes out retina, washes in DMEM 4 times.Retina was by homogenate and at 400xg centrifugal 10 minutes then.The tissue particles of collecting precipitation also is suspended in the separating medium (DMEM adds 100IU/ml penicillin, 100 μ g/ml streptomycins and 250ng/ml amphotericin).Nylon wire (Locker Wire Weavers company limited) with 85 μ m is collected blood capillary, transfer to then in the petri diss that contains the 10ml mixed enzyme, in 37 ℃, hatched 20 minutes, and contained 600 μ g/ml DNase I (Sigma), 165 μ g/ml collagenases (Sigma) and 700 μ g/ml pronase es (EMD) in the mixed enzyme.The nylon wire of blood vessel fragment reuse 53 μ m is collected (Locker Wire Weavers company limited), cleans centrifugal 5 minutes of 400xg in separating medium.For the screening and culturing of pericyte, the blood vessel fragment is suspended in 10ml pericyte grown cultures liquid, and is transferred to 75-cm 2(BDBiosciences) cultivates in the plastics tissue culture flasks.For the screening and culturing of BRCECs, the blood vessel fragment is suspended in 10mlBRCEC grown cultures liquid, transfer to 75-cm then 2Use middle cultivation of plastics tissue culture flasks (BD Biosciences) of glue primordial covering in advance.BRCEC grown cultures liquid adds 10% human serum, 1% glutamate, Glu, 1mg/ml insulin, 550 μ g/ml transfer liquid, 670ng/ml selenium, 100 IU/ml penicillins, 100 μ g/ml streptomycins, 250ng/ml amphotericin, 90 μ g/ml heparin (Sigma) and 15 μ g/ml endothelial cell growth auxiliary substances (Upstate) by DMEM and forms.Cell is cultivated in 37 ℃, 5%CO2, changes culture fluid in per 3 days.Use 0.25% trypsin digestion and cell, to carry out passage at 1: 3.The purity of BRCECs and pericyte uses Dil-Ac-LDL (Biomedical Technologies Inc) and ldl receptor in BRCECs surface combination, the immune labeled detection of smooth muscle antibody (Sigma) respectively.Second filial generation BRCECs and pericyte are stored in the liquid nitrogen container standby.
MTT experiment: with every hole 5x10 4Individual cell with cell inoculation in 24 orifice plates (Nalge Nunc) or in advance with 24 orifice plates of gelatin bag quilt, every group three hole, every hole grown cultures liquid is 400 μ l.After 24 hours, grown cultures liquid is by containing 1%FBS, have or replacing with the Thalidomide of concentration or the culture fluid of Phthalide acid imide analog invariably.Handle after 48 to 72 hours, MTT is added, its final concentration is a 0.5mg/ milliliter culture fluid, hatches 4 hours in 37 ℃, 5%CO2.Adding waits the lysis buffer of capacity then, and according to the operation sequence (Roche Molecular Biochemicals) that the manufacturer recommends, cell is overnight incubation in 37 ℃, 5%CO2.Under wavelength 570nm, measure the absorption value of first distension product (Formazen product), with 750nm as deduction with reference to wavelength.
The endothelial cell migration experiment: BD Matrigel is used in the test of fluorescence staining endothelial cell migration TMAnd BDFalcon TMHTS FluoroBlok TM(BD Biosciences) 24-porous insertion system (Fig. 2 A).This insertion system blocks 3 μ mPET films by fluorescence and forms, and the latter can block the light of 490-700nm wavelength and propagate, and is sealed in porous and inserts in the plate.This makes directly to detect through Matrigel by exometer moves to the cell fluorescence signal that inserts the plate bottom.In this experimental system, under the chemical compound 1,4 and Thalidomide effect of VEGF (4ng/ml) and variable concentrations (0.01-100 μ M), the HUVECs Thalidomide migrates to the bottom, does not contain VEGF in the matched group.The cell migration time is 22 ± 1 hours.Applied Biosystems is used with calcein AM (4 μ g/ml) labeled cell in the migration back
Figure BSA00000208007300161
4000 plate enumerators detect by BD Matrigel down in 485nm excitation wavelength and 530nm wavelength of transmitted light TMThe cell fluorescence of substrate migration.
Chicken allantocherion (CAM) method: the leghorn egg of fertilization was hatched 10 days under 37.5 ℃ of moist environments.With extremely saturated in people VEGF-165 and basic fibroblast growth factor (bFGF) (each 200ng) the adding microbiological test ware, broken duck eye places the former on the CAM on top layer, egg top.Treat that VEGF/bFGF added anti-angiogenic compounds after reaching capacity 8 hours in the same microbiological test ware, idiosome continued to hatch 40 hours again.Take out CAM, use 4% paraformaldehyde PBS liquid-solid fixed immediately, place the Petri culture dish, carry out digital photographing with 7.5 times with Nikon anatomic microscope and Scion photographic system.Then the grid of 1x1-cm is placed on the CAM digital photograph, the blood vessel meansigma methods in the counting 5-7 lattice is as the vascularity amount.
Oxygen inductivity retinopathy (OIR) brings out: bringing out people such as Smith of OIR describes that (Smith, et al.Invest.Ophthalmol.Vis.Sci.1994 have done on basis 35:101-111) partly to improve.In brief, newborn BN rat (Charles River laboratory) is exposed in hyperoxia (75%) environment 5 days (P7-12) in birth back 7 days (P7), returns then in normal oxygen (room air) environment, thereby induces retinopathy.
Streptozotocin (STZ) brings out diabetes: behind the overnight fasting, BN rat (8 week age) give the freshly prepared streptozotocin of once abdominal cavity injection (STZ) (Sigma, 50mg/ this, be dissolved in the 10mM citrate buffer, pH 4.5).Control rats is accepted a citrate buffer injection.STZ injection is measurement of glucose levels after 24 hours, and backward 1 week 1 time, blood sugar level is higher than 350mg/dl and is considered as diabetes rat.The rat that continues 2 all hyperglycemia is used for experiment.
The intravitreal injection chemical compound: Thalidomide and Phthalide acid imide analog compounds 1,2,4 are diluted in BN rat blood serum, filtration sterilization.The BN rat blood serum of Thalidomide, chemical compound 1,2 or the chemical compound 4 left eye intravitreal injection equivalent of OIR and STZ-diabetes rat right eye intravitreal injection 0.5-2.0 μ g/ eye (5 μ l/ eyes, 0.1-0.4mg/ml BN rat blood serum).
High molecular retina fluorescein angiographic: in the retinal vessel radiography, use dextran high molecular fluorescein, as described in the Smith etc. (Smith, et al.Invest.Ophthalmol.Vis.Sci.1994,35:101-111).Be summarized as follows: add acepromazine (5mg/kg body weight) with Animal Anesthesia with ketamine (100mg/kg body weight), be dissolved in the high molecular fluorescein of the 50mg/ml among the PBS then through the left ventricle perfusion.Eyeball is located, enucleated to the labelling eyes, decided 3-24 hour with 4% paraformaldehyde PBS is liquid-solid.Do several otch, then retina is lain in the gelatin bag by on the slide.Under fluorescence microscope, check vascular system.Full retinal area and avascular area territory all use the computerized image analytical system to detect, and calculate every cell mean.
The measurement of vascular permeability: vascular permeability enters amphiblestroid Fluorescein isothiocyanate-albumin or azovan blue (Evans indigo plant) dyestuff-albuminate quantitatively by measuring from blood vessel, this method is at the described (Xu of forefathers, et al.Invest.Ophthalmol.Vis.Sci.2001 has done on basis 42:789-794) partly to improve.Be summarized as follows: Fluorescein isothiocyanate-albumin is injected through femoral vein, circulated 2 hours.Pour into through the left ventricle row then.Careful take out retina and with its homogenate.Measure Fluorescein isothiocyanate-albumin concentration with exometer, do standardization with the total protein concentration in each retina and serum Fluorescein isothiocyanate-albumin concentration.
Azovan blue dyestuff (Sigma company) is dissolved in 0.9% saline (30mg/ml), and sonic oscillation 5 minutes uses the filter (Millipore) of 0.45-μ m to filter then.Anesthetized rat injects azovan blue (30mg/kg) with glass capillary through femoral vein under microscopic examination, inject time is more than 10 seconds.The non-covalent albumin that is incorporated in the blood flow of azovan blue.Azovan blue injects the back rat and obviously becomes blue immediately, and the confirmation dyestuff absorbs and disperses.Rat is placed on warm pad circulated fully to guarantee dyestuff last 2 hour.Open the thoracic cavity then, pour into 1% paraformaldehyde citrate buffer to rat through left ventricle, the latter earlier in 37 ℃ preheating in case vasoconstriction.Under the physical stress of 120mmHg, continue perfusion 10 minutes, to remove the dyestuff in the blood vessel.Enucleate eyeball after the perfusion immediately, the careful retina that takes out under operating microscope.Each sample in 150 μ l Methanamides, 70 ℃ placed 18 hours down, thereby extract the blue dyestuff of Evans.Extract is 70, and 000rpm (rotor type: TLA 100.3), 4 ℃ be centrifugal (Beckman) 20 minutes down.Get the supernatant of 100 μ l and measure absorption value with spectrophotometer DU800 (Beckman) under the 620nm wavelength, the densitometer of azovan blue obtains by the total protein concentration standardization in the standard curve of formulation azovan blue in Methanamide and each sample at last in the extract.The result represents with mg azovan blue/mg total protein concentration.
Immune labeled: the cell of In vitro culture liquid-solid fixed 10 minutes immediately at 4% paraformaldehyde PBS, give a baby a bath on the third day after its birth time with PBS, each 5 minutes, then with 0.5%BSA sealing 20 minutes.One anti-hatching 1 hour, an anti-back of hatching is washed cell 3 times with PBS, resists painted 1 hour with two then.By cell is hatched with Fluorescein isothiocyanate or the red conjugation two of Texas anti-(Jackson Immunoresearch), immune labeled signal is shown.Wash coverslip with PBS, 0.2 μ g/ml DAPI dyeing and mounting.Zeiss 510 confocal laser scanning microscopies with Zeiss fluorescence microscope or band argon-krypton laser are collected fluoroscopic image.
Western blotting: extract albumen with lysate.Equal protein matter from different samples is separated by SDS-PAGE, the western blot analysis VEGF antibody.The immunoblotting signal is by SuperSignalWest Pico chemical luminous substrate (Pierce) colour developing.
Electroretinogram (ERG) record: universe ERG Espion E2 ERG instrument (Diagnosys LLC) record, as (Rohrer as described in the forefathers, Journal of Neuroscience, 1999,19:8919-8913), two kinds of methods are arranged: (A) under dark adaptation and light adaptation, with the crescendo light of 10ms flash of light and (B) under light adaptation, use the 2Hz passage of scintillation light.The BN rat accept respectively intravitreal injection chemical compound 4 (2.0 μ g/ eyes, 5 μ l/ eyes, 0.4mg/ml BN rat blood serum) or equivalent BN rat blood serum.To voltage measurement a wave-amplitude, the trough on top is measured the b wave-amplitude to injection back different time from a ripple to positivity b crest from the baseline to the original minus.By before measuring the flicker amplitude to the trough at scintillation response peak.Data are represented with mean ± standard deviation, check the difference that compares between medication eye and contrast eye with pairing t-.
Retinal tissue is learned and is checked: be 4 potential toxicity of test compounds, 8 interior injection this chemical compound (2.0 μ g/ eyes, 5 μ l/ eyes, 4mg/ml BN rat blood serum) of BN rat difference vitreous body in age in week or equivalent BN rat blood serums.The injection back is put to death in different time and is drawn materials.Get eyeball and place 4% formalin solution fixing, paraffin embedding, section, section comprises that whole retina, its thickness are 6 μ m.Section is checked after HE dyeing.
Design, synthesize and test and detected a series of new Phthalide acid imide analog.Discovery and experimental result are as follows: Figure 16 shows chemical compound 4 route of synthesis.1H-NMR(250MHz，CDCl3)δ1.08(12H，d，J＝6.80Hz)，2.50(2H，hept，J＝6.80Hz)，6.75(1H，dd，J＝1.98Hz，8.25Hz)，6.87(1H，d，J＝1.98Hz)，7.16(2H，d，J＝7.85Hz)，7.31(1H，t，J＝7.85Hz)，7.46(1H，d，J＝8.25Hz).mp?252-253oC(lit.253-254oC)。Figure 17 shows Thalidomide, actimid, revimid, chemical compound 1,2 and 4 different chemical structures.
Embodiment 2: chemical compound 4 is than Thalidomide and two other analog thereof inhibition of endothelial cell proliferation more effectively.
Former generation endotheliocyte (HUVEC and BRCEC) was with variable concentrations compound treatment 3 days.With mtt assay detection by quantitative cell survival rate, calculate the IC of each chemical compound 50(mean ± standard deviation, n=3 see Fig. 1).IC 50Value is represented independently experimental result three times. Chemical compound 1,2,4 inhibition of endothelial cell proliferation are concentration dependent, IC among the HUVEC 50Being respectively 3.3,3.0,2.0 μ M, then is respectively 1.94,3.56,1.83 μ M among the BRCEC.The Thalidomide inhibitory action than three kinds of noval chemical compounds a little less than, its IC in HUVEC and BRCEC 50Be respectively＞100 μ M and＞32 μ M (see figure 1)s.Existing Phthalide acid imide analog, Actimid (CC-4047) and Revimid (CC-5013), in HUVEC experiment, suppress effect a little less than, its IC 50＞100 μ M.Under the equal conditions, these noval chemical compounds fail obviously to suppress pericyte's growth, show that they have specificity to suppressing endothelial cell growth.Above result shows that chemical compound 4 is compared with Thalidomide with its other two kinds of analog has stronger anti-angiogenic rebirth effect.
Fig. 1 is the relatively effect of Thalidomide and new Phthalide acid imide analog on cell proliferation of form.
Embodiment 3: it is strong than Thalidomide that chemical compound 4 suppresses the effect of HUVEC migration
Chemical compound 1,4 and Thalidomide are assessed by cell in vitro migration (invasion and attack) method the effect of endothelial cell migration.The advantage of this method is to improve and is used for efficient screening.This method is the endothelial cell migration experiment based on fluorescence.Use BD Matrigel TMWith BD Falcon TMFluoroBlok TM(MA) 24 porocytes are cultivated the plate system (Fig. 2 A) that inserts for BDBiosciences, Bedford.The 3 μ m that this insertion system is propagated with the light that can stop wavelength 490-700nm intercept the sealing porous insertion plate of ultraviolet polyester film (Fig. 2 A).Like this, utilize exometer bottom read mode just can directly measure to move to and insert the fluorescence signal that the cell bottom the plate sends from matrigel.In this experimental system, human endothelial cell can be attacked and by quantitative with exometer behind the fluorescent dye calcein AM labelling.Shown in Fig. 2 B, chemical compound 1 and 4 suppresses endothelial cell migration, is concentration dependent, corresponding IC 50Be respectively 1 μ M and＜1 μ M.And the IC of Thalidomide 50μ M then＞100.
Fig. 2 has compared chemical compound 1,4 and the Thalidomide inhibitory action to endotheliocyte (HUVEC) migration.Fig. 2 A is the rough schematic of endotheliocyte invasion and attack detection system.Fig. 2 B, cell seeding density is 5 * 10 4/ insert plate, in the porous plate that contains 0.1%BSA EBM-2 liquid, cultivate.Sustainable 6 hours of this combine detection.Recently represent comparative result with matched group (not containing the inhibitor group) with the percentage that suppresses migration.The average result of data represented three experiments is tested triplicate at every turn.Bar diagram is represented mean ± standard deviation.
Embodiment 4: chemical compound 4 can suppress the endotheliocyte tubulose and form
To place 37 ℃, 5%CO with the octal plate of slide 2In, with the matrigel bag by 30 minutes.HUVEC is with 3 * 10 4The density of individual cells/well plantation and at 37 ℃, 5%CO 2Under hatched 16 hours, contain placebo (0.5%DMSO) in the EGM-II culture fluid, 5 μ M chemical compound 4 or Thalidomides.Hatch the back and clean culture plate with PBS, 100% methanol is fixed 10 seconds, DiffQuick II liquid dyeing 2 minutes.Use 2.5 times of object lens digital photographings, analyze tubulose and form, as shown in Figure 3, the representative qualitative picture that tubulose forms experiment shows that chemical compound 4 has the effect that tubulose forms that suppresses.And Thalidomide unrestraint effect.
Fig. 3 shows the influence that 4 pairs of tubuloses of chemical compound form.With solvent, Thalidomide and chemical compound 4 are handled endotheliocyte and are collected presentation graphics after 16 hours.The result shows that chemical compound 4 effectively suppresses tubulose and forms.
Embodiment 5: allantocherion (CAM) Faxian shows chemical compound 4 and Thalidomide, and chemical compound 1 and 2 compares vascularization stronger inhibitory action
The CAM experiment is usually used in the research of angiogenesis inhibitor in the body.The leghorn egg of being fertilized was hatched 10 days under 37.5 ℃ of moist environments.People VEGF-165 and bFGF (each 200ng) are added on the microbiological test ware to saturated, are making a call to an aperture above the egg and this ware is placed on the CAM.VEGF/bFGF after saturated 8 hours, adds anti-angiogenic compounds at the microbiological test ware, the embryo is hatched 40 hours then.After 48 hours, take out CAM, use 4% paraformaldehyde PBS liquid-solid fixed immediately, place petri diss, use 7.5 times of Nikon anatomic microscopes and Scion image system to collect digital image.1 * 1cm grid is put into digital CAM image, with blood vessel meansigma methods in the 5-7 lattice as the counting of vascularity.Fig. 4 demonstration adds 48 hours CAM of chemical compound 4 processing through VEGF-165/bFGF with through VEGF/bFGF respectively.VEGF/bFGF induces the vascularization of CAM bulk.Chemical compound 4 can suppress the inductive vascularization of VEGF/bFGF, its ED when concentration reaches 5 μ g/ embryos 50Be 6.5 μ g/ embryos, and the ED of Thalidomide 50μ g/ embryo then＞100, prompting chemical compound 4 in the CAM experiment can suppress vascularization (Figure 14).
Fig. 4 shows 4 pairs of angiopoietic inhibitory action of chemical compound in the CAM experiment.The left figure of Fig. 4 shows the CAM that 200ngVEGF-165/bFGF handled 48 hours, and on behalf of 200ngVEGF-165/bFGF, right figure add the CAM that 5 μ g/ embryonization compounds 4 are handled.
Figure 14 compares chemical compound 4 in the CAM experiment for form, and Thalidomide and SU5416 are to the effect of angiogenesis, and dosage is represented with " μ g/ embryo ", ED 50Expression can make angiogenesis be reduced to half that simple VEGF/bFGF handles (blood vessel quantity is equivalent to the matched group that not dosing is handled).The ED of Thalidomide 50＞100 μ g/ embryos, the ED of chemical compound 4 and SU5416 50Be respectively 6.5 and 7.8 μ g/ embryos.Data (mean ± standard deviation) are represented 8-16 sample of 2-3 independent experiment.
Embodiment 6: Phthalide acid imide analog suppresses the generation of the HIF-1 α of hypoxia inducible in the PC-3 prostate gland cancer cell
With PC-3 prostate gland cancer cell detection compound 1,2 and 2ME2 (positive control) to anoxia the inhibitory action of inductive HIF-1 alpha expression.Cell is handled with 10 μ M inhibitor (containing 0.1%DMSO) and is spent the night, with DMSO in contrast.Expression (Fig. 5 A) with western blotting analysis PC-3 prostate gland cancer cell HIF-1 α after anoxia and compound treatment.Chemical compound 1 and 2 all significantly suppress anoxia the expression of inductive HIF-1 α, can reach 79-90% (Fig. 5 B).
Fig. 5 shows the influence of Phthalide acid imide analog to the HIF-1 alpha expression.Expression (Fig. 5 A) with western blotting analysis PC-3 prostate gland cancer cell HIF-1 α after anoxia and compound treatment.Quantitative analysis shows that chemical compound 1 and 2 all can suppress anoxia inductive HIF-1 alpha expression (Fig. 5 B).
Embodiment 7: the expression of chemical compound 4 downward modulation OIR rat retina VEGF
VEGF plays a crucial role in diabetic macular edema.The transcriptional activation of the VEGF that HIF-1 α scalable causes because of anoxia.In experiment in vitro, the Phthalide acid imide analog of being tested can significantly suppress the HIF-1 alpha expression of hypoxia inducible, points out these chemical compounds to reduce the retinal vessel seepage by acting on the VEGF signal path.For verifying this hypothesis, detect the expression of VEGF in the OIR rat behind injection chemical compound 4.In lysate, hatch with Ultrasonic Pulverization after, extract the OIR rat retina albumen that normal rat, contrast and chemical compound 4 are handled.Each sample contains equal protein matter and separates with SDS-PAGE, carries out western blot analysis with VEGF antibody.The immunoblotting signal is by SuperSignal West Pico chemical luminous substrate (Pierce) colour developing.The result shows that the OIR rat retina vegf expression of handling through chemical compound 4 reduces (Fig. 6).
Fig. 6 shows chemical compound 4 downward modulation OIR rat retina vegf expressions.The OIR rat retina VEGF level that normal rat, contrast and chemical compound 4 are handled detects (Fig. 6 A) with Western blotting.Fig. 6 B shows the quantitative analysis of vegf expression.Labelling among the figure " normally " is represented the normal BN rat, and " contrast " represents left eye intravitreal injection 5 μ l BN rat blood serums, and " chemical compound 4 " represents right eye intravitreal injection 5 μ l chemical compounds 4 (0.8mM is diluted in the BN rat blood serum).
Retinal vessel is leaked with stronger inhibitory action in 4 pairs of OIR rats of 8: intravitreal injection chemical compounds of embodiment vitreous body
For inducing OIR, the BN rat of will be born back 7 days (P7) is exposed in the oxygen environment 5 days (P7-P12) and is transformed under the normal oxygen condition again and raises.Normal control is raised in room air by rat.Be born back 14 days, intravitreal injection of OIRBN rats underwent, every of right eye are injected 5 μ l (concentration is 0.8mM, is diluted in the BN rat blood serum) Thalidomide respectively, chemical compound 1, chemical compound 2 or chemical compound 4, the BN rat blood serum of left eye injection equivalent.The detection of retinal vessel seepage uses the albumin of marked by fluorescein isothiocyanate as tracer.Be born the back the 16th day (P16) normal non-OIR rat (n=6) contrast as baseline values.The 16th day (P16) in birth back, and relatively to the contrast eye of side injection diluent, Thalidomide handle the eyes retina vascular leakage be reduced to 82% (paired t-test, P＜0.05, n=6).Chemical compound 4 handle the eyes retina vascular leakages be reduced to 61% (paired t-test, P＜0.05, n=6).And under same concentrations, chemical compound 1 and 2 fails obviously to reduce retinal vessel seepage (Fig. 7 A and 7B).The inhibitory action that fluorescein angiographic demonstration chemical compound 4 forms retinal neovascularization under used dosage is weak (Fig. 9), and it is stronger that the effect of 4 pairs of minimizings of prompting chemical compound retinal vessel seepage compares to the effect that the inhibition retinal neovascularization is formed.
For whether the influence of determining 4 pairs of retinal vessel seepages of chemical compound is dose dependent, the OIR rat of P14 is accepted the chemical compound 4 of a shot 0.5,0.75,1.0 μ g/ eyes respectively, and (5 μ l, concentration is respectively 0.10,0.15,0.20mg/ml).Chemical compound 4 and Thalidomide are that 0.75 and 1.0 μ g/ at the moment can significantly reduce vascular leakage (P＜0.05 at dosage, n=6), dosage is that 0.5 μ g/ does not at the moment then have obviously effect (Fig. 7 C and 7D), points out both that the effect of OIR rat aorta seepage is dose dependent.
Fig. 7 comparative compound 1,2,4 (chemical compound 4) and Thalidomide are to the influence of OIR rat retina vascular leakage.Among Fig. 7 A, inject 5 μ l/ eyes (concentration is 0.8mM, is diluted in the BN rat blood serum) Thalidomide in the P14 OIR rat right eye vitreous body respectively, chemical compound 1,2 or 4, left eye injection equivalent BN rat blood serum.Vascular leakage is measured in P16 with the albumin seepage method of marked by fluorescein isothiocyanate, and the retina protein content is with the fd/pr of unit (mean ± standard deviation, n=6) expression.The difference that compares each experimental eye and contralateral control eye with the t check.The normal non-OIR rat retina vascular leakage of P16 is as the baseline values of P16.Among Fig. 7 B, compound injection eye medium vessels leakage values is represented with the percentage ratio of itself and offside diluent contrast eye mean vascular leakage values.The contrast eyes retina mean vascular seepage that diluent is handled counts 100%.Thalidomide and chemical compound 4 reduce retinal vessel seepage 18% and 40% (n=6) respectively.Among Fig. 7 C and the 7D, the dosage of intravitreal injection chemical compound 4 of the OIR rat of P14 acceptance or Thalidomide as shown in the figure.Vascular leakage is measured in P16, and the retina protein content is with the fd/pr of unit (mean ± standard deviation, n=6) expression.Difference with paired t-test comparative experiments group and matched group.Among the figure " normally " represent the vascular leakage level of P16 normal rat.
Embodiment 9: 4 pairs of STZ-diabetic retinal tissue in rat of chemical compound vascular leakage has stronger inhibitory action
Grow up and to inject STZ (50mg/kg, intravenous injection) after one night of BN rat fasting and induce diabetes.Injected back second day and weekly later on monitoring blood sugar level.The rat that blood sugar level is higher than 350mg/dl is diabetes rat and is used for experiment.Onset diabetes is after 2 weeks, and STZ-diabetes rat right eye is the interior injection Thalidomide of vitreous body respectively, chemical compound 1,2 and 4 (5 μ l, concentration is 0.8mM, is diluted in the BN rat blood serum).Injected back 48 hours, and detected the retinal vessel seepage with azovan blue albumen seepage method.The result shows that with injection BN rat blood serum branch hole is compared, Thalidomide, chemical compound 1,2 and 4 injection eyes retina vascular leakages obviously reduce (P＜0.01, n=6) (Fig. 8 A).Thalidomide reduces by 77% with the normal blood vessels seepage, and chemical compound 1 is 61%, and chemical compound 4 then reaches 100% to normal level (baseline values) (Fig. 8 B), shows that chemical compound 4 can block the retinal vessel seepage fully.Be the time effect behind definite chemical compound 4 intravitreal injections, the ORI rat of P14 after administration every injection of 24h and 48h right eye 5 μ l chemical compounds (concentration is 0.8mM, be diluted in the BN rat blood serum), the detection of retinal vessel seepage shows, compare with the contralateral control eye, compound injection eye medium vessels seepage is blocked fully.
For determining chemical compound 4 and Thalidomide amount-result relation to the vascular leakage effect, the STZ diabetes rat is accepted intravitreal injection chemical compound 4 and Thalidomide, and dosage is respectively every 0.5,0.75 and 1.0 μ g (5 μ l/ eyes, concentration be 0.10,0.15 and 0.20mg/ml).Compare with matched group, handle after 2 days, the chemical compound 4 of all dosage all significantly reduces retinal vessel seepage (P＜0.05, n=6) (Fig. 8 C).And Thalidomide is that 0.75 and 1.0 μ g/ at the moment just have inhibitory action at dosage only, and 0.5 μ g/ does not at the moment then have (P＜0.05, n=6) (Fig. 8 D).This observed result shows that chemical compound 4 compares to the effect that Thalidomide and other chemical compound all have stronger reduction retinal vessel seepage not only at the OIR model but also in the experimental diabetes disease model.
Fig. 8 has compared the influence of 1,2,4 pairs of STZ diabetic retinal tissue in rat vascular leakages of chemical compound.Among Fig. 8 A, STZ induces diabetes to give injection 5 μ l (concentration is 0.8mM, is diluted in the BN rat blood serum) Thalidomide in the every vitreum of diabetes rat right eye after two weeks, chemical compound 1,2 or 4, and the BN rat blood serum of left eye injection equivalent is in contrast.Detect the retinal vessel seepage with azovan blue albumen seepage method.Handle after 2 days, with azovan blue concentration in retina albumen total concentration and the blood come standardization (mean ± standard deviation, n=6).The difference that compares each experimental eye and contralateral control eye with the t check.Non-diabetic rat aorta seepage is as the baseline values of vascular leakage." normally " represents the vascular leakage level of normal rat.Among Fig. 8 B, the vascular leakage value of compound injection eye is represented with the percentage ratio that it accounts for the contrast eye vascular leakage value of injection BN rat blood serum.Thalidomide, chemical compound 1 and 4 make the retinal vessel seepage reduce by 77%, 61% and 100% respectively.Among Fig. 8 C and the 8D, onset diabetes is after 2 weeks, and the dosage of STZ diabetes rat intravitreal injection chemical compound as shown in the figure.Detect vascular leakage after handling 48h, with the milligram numerical table of azovan blue in every milligram of retina albumen show (mean ± standard deviation, n=6).The difference that compares each experimental eye and contralateral control eye with the t check." normally " represents the vascular leakage level of normal rat.
Embodiment 10: 4 pairs of OIR rat retinas of chemical compound new vessels is formed with inhibitory action.
Newborn BN rat is exposed in 75% hyperoxia in P7～P12, then raises 4 days so that retinal neovascularization is partly grown in room air.When P16 OIR rat retina new vessels partly forms, a shot 1.0 μ g Thalidomides in the every vitreum of right eye, chemical compound 1,2 or 4 (every 5 μ l, 0.2mg/ml, be diluted in the BN rat blood serum), left eye injection diluent (5 μ l BN rat blood serum) is in contrast.Form with retina shop sheet fluorescein angiographic assessment retinal neovascularization at P20.Compare (Fig. 9 A) with fluorescence microscope retinal blood guard system and with the contralateral control eye.Retina sections observation new vessels situation (Fig. 9 B).The result shows that chemical compound 4 parts suppress OIR rat retina new vessels and form, and chemical compound 1,2 and Thalidomide all do not have obvious inhibitory action.
Fig. 9 shows the retinal vessel radiography behind interior shot Thalidomide of OIR rat vitreous body and the chemical compound.Among Fig. 9 A, OIR rat right eye is intravitreal injection 5 each chemical compound of μ l (concentration is 0.8mM, is diluted in the BN rat blood serum) respectively, left eye injection equivalent BN rat blood serum.Row fluorescein angiographic during P20.Figure medium vessels radiography is represented 3 rats of every group.The rat freshman vascularization that it should be noted that injection chemical compound 4 is few than contrast, and Thalidomide and chemical compound 1,2 fail to reduce new vessels formation under used dosage.Cut sections for microscopic examination show among Fig. 9 B, and the rat of injection chemical compound 4 compares to the rat of injection solvent control treatment, and new vessels forms and reduces before its retina.
Embodiment 11: the species variation of inducing diabetes rat model retinal vessel seepage at OIR and STZ.
The model of having set up retinal vessel seepage longer duration is for the long-term effect that detects new drug.The time-histories of retinal vessel seepage also defines in SD and BN rat OIR and STZ diabetes model.The newborn rat of P7-P12 is exposed to hyperoxia (75%O 2) to induce OIR.The BN rat that grows up is accepted the STZ injection to induce diabetes.Detect the retinal vessel seepage with azovan blue-albumin method.In the OIR-BN rat, vascular leakage reaches the peak from the P12 increase during P16, and this moment, the seepage level was 8.7 times (P=7.5E-06) of normal rat of the same age.During the P18-P22, vascular leakage slowly reduces, and reaches normal level (Figure 10) behind the P30.In the OIR-SD rat, vascular leakage increase slower (P14).Peak value is than BN rat low (2.2 times), and seepage has been reduced to normal level (Figure 10) during P18.Retina VEGF level is different relevant between above observed result and two kinds of kinds.In the STZ-BN rat, behind the STZ injection 24h (1.4 times of high seepages appear; P=0.0292), when 2 weeks, reach (1.8 times of plateaus; P=0.074).High seepage continued for 16 weeks at least behind diabetes-induced.In the STZ-SD rat, injection STZ after 3 days vascular leakage begin to increase (1.3 times; P=0.0271), 1 when week (1.5 times of peakings; P=0.004), 2 weeks the time were reduced to control level (Figure 11).These results show, in OIR and STZ diabetes model, the BN rat is stronger and the persistent period is more of a specified duration than its vascular leakage of SD rat.Therefore, all rat models are all selected the BN rat for use in this project research.These results show that also as described in this project second phase research, the OIR model is suitable for studying the shortterm effect of 4 pairs of retinal vessel seepages of chemical compound, and the STZ diabetes model then is suitable for the research to its long-term effect.
Figure 10 shows the kind difference of rat aorta seepage in the OIR model.BN and SD rat carry out hyperoxia handles and detects its retinal vessel seepage, does standardization with total protein concentration, and (mean ± standard deviation n=4) is represented with the percentage ratio of its shared normal control seepage level of the same age.Be significantly higher than control level person with * number mark.
Figure 11 shows the species variation of STZ diabetes rat model medium vessels seepage.Induce diabetes with BN and SD rat, detect the retinal vessel seepage in described different time points.The total protein concentration standardization of seepage level is with micrograms (mean ± standard deviation, n=4) expression of azovan blue in every milligram of albumen.Be significantly higher than the horizontal person of normal control of the same age with * number mark.
Embodiment 12: BN rat retina VEGF level is than SD rat height during hypoxia response.
For determining whether its more serious retinal neovascularization of OIR BN rat is relevant with retina VEGF high expressed, with rat VEGF enzyme-linked immunosorbent assay kit (R﹠amp; D systems, Inc) detection by quantitative VEGF level, and with the standardization of retina total protein concentration.The result shows that normal BN is similar with the basic VEGF level of SD rat.OIR-SD rat retina VEGF level and normal control SD rat there was no significant difference (Figure 12).And OIR-BN rat retina VEGF level is about 10 times of (P＜0.001, n=4) (Figure 12) of normal control BN rat and OIR SD rat.
Figure 12 shows the VEGF level of OIR BN and SD rat.Retina VEGF level detects with enzyme-linked immunosorbent assay, through the retina protein standardization, with pg/mg represent (mean ± standard deviation, n=4).Be significantly higher than the horizontal person of normal control of the same age with * mark (P＜0.001).
Embodiment 13: BN rat retina VEGF level is than SD rat height in the inductive diabetes model of STZ.
Studies show that the inductive BN diabetes rat of STZ under the similar hyperglycemia state of an illness and the course of disease, its retinal vessel seepage is more serious than the inductive SD diabetes rat of STZ.Be higher than the SD rat for determining whether in the BN rat that retina VEGF level raises due to the diabetes, different time point behind onset diabetes, BN and SD diabetic retinal tissue in rat VEGF level be with the capable semi-quantitative analysis of western blotting, and with non-diabetic rat of the same age separately relatively.The result shows, BN and SD rat retina VEGF foundation level similar (Figure 11).Yet along with STZ induces the generation of diabetes, to 16 weeks, BN diabetic retinal tissue in rat VEGF level was higher than SD diabetes rat (Figure 13) the 3rd day of the course of disease.
These observations show that BN OIR or STZ-diabetic retinal tissue in rat are applicable to the body inner model as research chemical compound 4 mechanisms of action, as study its influence to the VEGF overexpression.
Figure 13 shows BN and SD STZ-diabetic retinal tissue in rat VEGF level.STZ injection 3 days and 1,2,4,8 with 16 weeks after separate BN and SD diabetic retinal tissue in rat.The soluble protein of equivalent VEGF specific antibody labelling.Antibody on the eluting film, the anti-beta-actin antibody labeling of reuse is to carry out standardization to the VEGF level.The retina that these results collect from different time points.
Embodiment 14: chemical compound 4 does not cause any significantly big rathole toxicity
Be 4 potential toxicity of detection compound, 8 age in week normal rat intravitreal injection high doses of compounds 4, every 2 μ g (5 μ l/ eyes, concentration is 0.4mg/ml, is diluted in the BN rat blood serum) or equivalent BN rat blood serum are as negative control.Before the injection and after 1,2,3,4 weeks of injection, with ERG record assessment visual function.Compare with BN rat blood serum injection eye, ERG detects the obvious change (Figure 15 and Figure 18 A-C) that demonstration chemical compound 4 injection eyes are not seen a ripple, b wave-amplitude.
4 weeks after the administration are with the genotoxic potential of histopathologic examination's observation chemical compound 4.The section of retina cross section, HE dyeing, optical microscope is checked down.Compare with offside BN rat blood serum injection eyes retina, under 2 μ g/ eye dosage, the retina (5 μ l/ eyes, concentration is 0.4mg/ml) of chemical compound 4 injection eyes is not seen tangible morphological change or immunoreation (Figure 18 D).
Figure 15 is the influence of 4 pairs of big rathole a ripples of form comparative compound, b ripple.
Figure 18 shows chemical compound 4 processing back rat retina function and morphological analysis.Inject chemical compound 4 (concentration is 0.4mg/ml, is diluted in the BN rat blood serum for 2 μ g/ eyes, 5 μ l/ eyes) or equivalent BN rat blood serum (n=6) 8 ages in week in the BN rat vitreous body respectively.Before the injection and inject 1,2,3,4 week back row ERG and check.Data show compares with injection diluent rat in contrast, and rat ERGa ripple, the b ripple of injection chemical compound 4 do not have remarkable change (Figure 18 A-18C).4 week of injection back execution rat is got the eyeball tissue section, HE dyeing, and microscopically is observed.Pathological examination shows that rat chemical compound 4 handles eye and the contrast eyes retina does not relatively have obvious morphological change (Figure 18 D).
Embodiment 15: chemical compound 4 weakens HIF-1 α and activates.
According to example, 4 pairs of activated influences of HIF-1 α of the experimental data graphic compound of Figure 19.Hatched human retina pigment epithelium cell (ARPE-19) 16 hours with 400 μ M cobaltous chlorides having or do not have under the condition of 0.5,1 or 2 μ M chemical compounds 4.Use the nuclear translocation of immune labeled detection HIF-1 α.HIF-1 α is dyed green, and nuclear is redyed (blueness) by DAPI.Scale is 10 μ M.
Embodiment 16: chemical compound 4 reduces the expression of retinal pigment epithelium VEGF and ICAM-1
According to example, the influence that VEGF and solubility ICAM-1 express in 4 pairs of ARPE-19 cells of experimental data graphic compound of Figure 20.ARPE-19 is respectively through cobaltous chloride, and cobaltous chloride adds chemical compound 4, and TNF-α, TNF-α add chemical compound 4 to be handled.Handle and collect acellular conditioned medium after 18 hours, detect the level of VEGF and solubility ICAM-1 with enzyme-linked immunosorbent assay.Figure 20 A shows the influence to the VEGF level; Figure 20 B shows the influence to solubility ICAM-1 level.*p＞0.05，**p＜0.05，***p＜0.01，****p＜0.001，n＝3。
Embodiment 17: the expression of chemical compound 4 downward modulation OIR rat retina VEGF and ICAM-1.
According to example, the influence that the experimental data graphic compound 4 pairs of OIR rat retinas VEGF of Figure 21 and ICAM-1 express.For inducing OIR, the BN rat is exposed to (75%O in the hyperoxia in birth back 7 days (P7) 2) 5 days (P7-P12), return then under the normal oxygen and raise.OIR rat intravitreal injection chemical compound 4 and its diluent during P14.Inject after 2 days, detect normal rat, diluent and the OIR rat retina VEGF of chemical compound 4 processing and the level of ICAM-1 with enzyme-linked immunosorbent assay.Figure 21 A shows the influence of 4 pairs of vegf expressions of chemical compound; Figure 21 B shows the influence that 4 couples of ICAM-1 of chemical compound express.*P＜0.01，n＝5.
18: intravitreal injection chemical compounds 4 of embodiment can suppress OIR and STZ-diabetic retinal tissue in rat vascular leakage
According to example, the 4 couples of OIR of experimental data graphic compound of Figure 22 and the influence of STZ-diabetic retinal tissue in rat vascular leakage.Among Figure 22 A, the OIR rat is right eye intravitreal injection chemical compound 4 when P14, and the left eye injection solvent compares.Detect vascular leakage with Fluorescein isothiocyanate-albumin seepage method during P16.Chemical compound 4 suppresses the retinal vessel seepage when every injection 0.75 and 1 μ g.Among Figure 22 B, lumbar injection STZ (50mg/kg) induces the BN rat diabetes.After 2 weeks took place in diabetes, diabetes rat right eye intravitreal injection chemical compound 4, omcilon, the left eye injection solvent is in contrast.In injection 2 weeks of back, detect the retinal vessel seepage with azovan blue seepage standard measure.All are all made the retinal vessel seepage obviously reduce by the chemical compound 4 of amount of reagent ((0.5,0.75, and 1 μ g)).Particularly, 1 μ g chemical compound 4 can make the retinal vessel seepage return to the foundation level of normal rat.Under Isodose, omcilon is invalid.*p＜0.05，n＝5。
Embodiment 19: oral administration of compound 4 suppresses STZ-diabetic retinal tissue in rat vascular leakage.
According to example, the influence of 4 pairs of STZ-diabetic retinal tissue in rat of the experimental data of Figure 23 diagram oral administration of compound vascular leakage.STZ induces diabetes after 1 week, and diabetes rat was irritated food chemical compound 4 (200mg/kg/ days once a day) 7 days; The matched group diabetes rat gives the solvent (Semen Maydis oil) of equivalent.Detect the retinal vessel seepage with azovan blue seepage method.Chemical compound 4 processed group and matched group are compared.*p＜0.05，n＝10。
Embodiment 20: the preparation of chemical compound 4 nano-particle
According to example, the electron-microscope scanning image of Figure 24 graphic compound 4PLGA nano-particle.Chemical compound 4PLGA nano-particle is by the preparation of water-in-oil emulsion evaporation.Viscosity at dichloromethane/20% dimethyl formamide that the PLGA (containing 50,65,75,85% lactic acid) of 0.15～1.2dL/g is dissolved in 80%, is made 7% solution.Concentration with 0～22mg/mL adds chemical compound 4 respectively.Then with oil: water is that 1: 2 ratio joins PLGA chemical compound 4 solution in 1～5% the acid polyethylene solution.With mixed solution in ice bath with ultrasonic processor (model: VCX-750,1/2 inch probe) supersound process 1 minute, amplitude is 30%.Vigorous stirring solution spends the night, and makes the oil evaporation.Then that nano-particle is centrifugal twice under 15000g, each centrifugal back is with the water cleaning of 18 megaohms.At last nano-particle is used supersound process 1 minute, it is suspended in 18 megaohm water again.Nano-particle is transferred in the 1.7ml cryovial, freezing spending the night under-80 ℃, lyophilizing is 48 hours then.It is slick and sly spherical that the granule that forms is.The dynamic light scattering analytic process shows that the average hydrodynamic diameter of prepared nano-particle is 239.1 nanometers, and polydispersity index is 0.097 (Figure 25).Chemical compound 4 drug loading are between 10～36%w/w in the nano-particle.
Embodiment 21: chemical compound 4 nano-particle induce the diabetic retinal tissue in rat vascular leakage that long-acting is arranged to STZ.
According to example, Figure 26 graphic compound 4 nano-particle are induced the influence of diabetic retinal tissue in rat vascular leakage to STZ.Among Figure 26 A, STZ induces rat diabetes to take place after 2 weeks, diabetes rat right eye intravitreal injection chemical compound 4 nano-particle (drug loading 13%w/w; 15,30, the 60g/ eye, be dissolved in PBS to 3,6,12mg/ml, the 5ul/ eye is equivalent to 2g, 4g, 8g/ eye chemical compound 4 respectively), the PBS of left eye injection equivalent.The retinal vessel seepage is detected with azovan blue seepage method in injection 2 week backs, the result with azovan blue concentration in retina total protein concentration and the blood carry out standardization (mean ± standard deviation, n=5), with the vascular leakage of non-diabetic rat as baseline values.Give 2,4 at every, during 8g chemical compound 4, the effect that chemical compound 4 nano-particle suppress the retinal vessel seepage is dose dependent.Figure 26 B shows the retinal vessel seepage situation of STZ diabetes rat intravitreal injection chemical compound 4 nano-particle (being equivalent to for 84,2～4 weeks of μ g chemical compound, 64 4,6 weeks of μ g chemical compound).Respectively at 2,4 and 6 week of injection back azovan blue seepage method mensuration retinal vessel seepage.Each experimental group all is equipped with the corresponding solvent matched group.Chemical compound 4 nano-particle are leaked with long lasting inhibitory action to retinal vessel, and sustainable 6 weeks (p＜0.05, n=5).
Embodiment 22: the inhibitory action that 4 couples of BRCEC of chemical compound 4 nano-particle and the chemical compound that is not wrapped grow
Be the inhibitory action of research chemical compound 4 nano-particle, get 24 orifice plates, BRCEC is inoculated in the hole every group three hole in testing preceding 24 hours coated with gelatin bag quilt to endothelial cell growth.Handle cell with contrast nano-particle that does not contain medicine or chemical compound 4 nano-particle (13% drug loading) respectively, concentration is respectively 5,10,20,40 and 80 μ g/ml; And handle cells with the chemical compound 4 of parcel not, concentration is respectively 2,4,8,16 and 32 μ M.In corresponding concentration group, chemical compound 4 nano-particle that the processing of every hole is used and the chemical compound 4 that does not wrap up contain the chemical compound 4 of equivalent.After the drug treating 4 hours, change not contain the fresh medium of nano-particle or chemical compound 4.Use mtt assay quantitative counting viable count after 1,3,5,7,9 days respectively in drug treating.The result shows, (is equivalent to 16 and 32 μ M chemical compounds 4 respectively) under the concentration of 40 and 80 μ g/ml, and chemical compound 4 nano-particle are handled and shown after 1 day that on cell proliferation has significant inhibitory effect, and its inhibitory action continues 9 days.Under same concentrations, the contrast nano-particle does not have any inhibitory action (data are unlisted).In concentration is 8,16 and during 32uM, and the chemical compound 4 of parcel does not handle after 1 day that cell growth has significant inhibitory effect (n=3, p＜0.05) yet, recovers grow (Figure 27 A) after 3 days but cell is grown in drug treating.Above result shows that chemical compound 4 nano-particle have significantly and the inhibitory action that continues the growth of BRCEC.
The chemical compound 4 treatments B RCEC that use contrast nano-particle, chemical compound 4 nano-particle (13%w/w drug loading) respectively and do not wrap up 4 hours, in corresponding concentration group, every hole drug treating compound used therefor 4 nano-particle are identical with the amount of the chemical compound 4 contained chemical compounds 4 that do not wrap up.Renew bright broth out chemical compound afterwards.With mtt assay at time point quantitative assay viable count shown in Figure 27 A.Shown in Figure 27 B, be that the cell growth obviously was suppressed (n=3, p＜0.5) after the chemical compound that do not wrap up of 8,16 and 32 μ M 4 was handled 1 day in concentration, but this inhibitory action disappeared after 3 days.And chemical compound 4 nano-particle (40 and 80 μ g/ml are equivalent to 16 and 32 μ M chemical compounds 4 respectively) have all shown significantly and the cell growth inhibition (n=3, p＜0.5) that continues at all time points of being analyzed.
Embodiment 23: chemical compound 4 synthetic overviews
Shown in scheme 1, with 2, coupling reaction takes place to commercially available 4-nitrophthalic anhydride (5) in 6-diisopropyl aniline (6) in acetic acid, generates (2, the 6-diisopropylbenzyl)-5-nitro-1-hydrogen-iso-indoles-1,3-diketone (7), and output capacity is 91%.The nitro of intermediate product (7) is reduced by transfer hydrogenation, and generation purity is 99.2% API (chemical compound 4), and output capacity is 91%.
Figure BSA00000208007300311
Scheme 1
Synthetic (2, the 6-diisopropylbenzyl)-5-nitro-1-hydrogen-iso-indoles-1, the coupling reaction of 3-diketone is optimization, the output capacity of solid product reaches 91%, purity greater than 99% (high performance liquid chromatography, a/a%).And, even enlarge the synthetic consistent result that also can obtain in proportion.Set up a kind of recrystallization method in order to this intermediate product of purification.Proved already that when containing the 3-nitrophthalic anhydride in the 4-nitrophthalic anhydride raw material, this purification process can successfully be removed this impurity.Based on this, and the unnecessary 4-of purification before use nitrophthalic anhydride.
Can successful synthetic compound 4 by catalytic hydrogenation and transfer hydrogenation.Catalytic hydrogenation is carried out under 45～50psi pressure, and needs the long response time (24 hours); This method needs Special Equipment.Confirmed the catalyst (heating) of adding 10% in ethanol (the proof-spirit degree is 200), transfer hydrogenation can be finished at 3 hours.Enlarging this reaction in proportion, can to obtain purity be 99.2% product, and output capacity is 91%.Preliminary experiment shows, prolongs drying time (89 hours) down at high temperature (70 ℃) and can not cause product to decompose, and can successfully remove residual solvent.
To chemical compound 4 recrystallization, can successfully obtain purity and be 100% chemical compound with ethanol/n-heptane (1: 2), the response rate is 75%.
Example 24: by chemical compound 5 and chemical compound 6 synthetic compounds 7
Scheme 2
According to example, shown in scheme 2, with 2, ((2.0g is in the mixture of acetic acid 10.4mmol) (15mL) 1.2eq) to add the 4-nitrophthalic anhydride for 2.34mL, 12.4mmol for the 6-diisopropyl aniline.With reactant mixture reflux (formation solution), after 12 hours, the temperature of reaction system is slowly reduced to room temperature.Concentrate and mix extremely only surplus residue of slurry.(2 * 50mL) to remove acetic acid to add methanol.With residue with the ethyl acetate/petroleum ether crystalline mixture (1: 2,60mL), filter the solid content that obtains, (3～4mL) wash, and obtain white solid (2.43g, output capacity 66%) after air-dry with petroleum ether.The proton magnetic resonance (PMR) frequency spectrum has confirmed its structure.168～169 ℃ of fusing points, (actual value: 161～162 ℃).
According to example, shown in scheme 2, with 2, ((2.0g is in the mixture of acetic acid 10.4mmol) (15mL) 1.2eq) to add the 4-nitrophthalic anhydride for 2.34mL, 12.4mmol for the 6-diisopropyl aniline.With reactant mixture reflux (formation solution), after 12 hours, the temperature of reaction system is dropped to room temperature.By the isolated by filtration solid, and use 3mLH 2The O flushing.(1: 2,60mL) recrystallization obtained 2.23g white powder (output capacity 61%) with ethyl acetate/heptane with solid.Filtrate is concentrated only surplus residue, and (2 * 25mL) to remove acetic acid to add methanol.When adding methanol for the second time, the solid by filtration that occurs is separated, (1: 2,3mL) recrystallization obtained the 164mg white solid with ethyl acetate/heptane.
According to example, shown in scheme 2, with 2, ((2.0g, (the proof-spirit degree is 200 to ethanol 10.4mmol) to the 6-diisopropyl aniline, in mixture 40mL) 1.2eq) to add the 4-nitrophthalic anhydride for 2.34mL, 12.4mmol.With reactant mixture reflux (formation solution), after 12 hours, the temperature of reaction system is slowly reduced to room temperature.The thin plate chromatography shows to react only finishes 50%.With solution reflux again, continue not find significant change after 3 hours.The reaction solution temperature is reduced to room temperature.Be salmon pink solid (0.93g, output capacity 25%) after the intermediate product crystallization.
According to example, shown in scheme 2, with 2, ((8.0g is in acetate mixture 41.43mmol) for technical grade, purity 90% 1.2eq) to add the 4-nitrophthalic anhydride for 9.4mL, 49.72mmol for the 6-diisopropyl aniline.Reaction system is heated to backflow, forms solution, after 12 hours, is cooled to room temperature.Reactant mixture is concentrated into half of about original volume.Also use the washed with methanol of 8mL by solid collected by filtration.The pale solid that obtains is air-dry, obtain 13.2g (output capacity 90%, purity 99.7%, high performance liquid chromatography a/a%).To reaction intermediate sampling (3.0g) and with its with ethyl acetate/heptane (1: 2,63mL) recrystallization.Collect white solid by filtering, obtain the intermediate product (response rate 72%, purity 99.9%, high performance liquid chromatography a/a%) of 2.17g purification.
According to example, shown in scheme 2, with 2, ((15.0g is in acetate mixture 77.7mmo) (113mL) 1.2eq) to add the 4-nitrophthalic anhydride for 16.5g, 93.2mmol for the 6-diisopropyl aniline.Reaction system reflux (formation solution) slowly cooled to room temperature after 12 hours.Reactant mixture is concentrated approximately to original volume half.Also use the washed with methanol of 15mL by solid collected by filtration.The pale solid that obtains in vacuum environment, is dried to constant weight under 35 ℃, obtain product 24.7g (output capacity 90%, purity 99.7%, high performance liquid chromatography, a/a%).Proton nuclear magnetic resonance spectroscopy has confirmed its structure.
Embodiment 25: by the catalytic hydrogenation of chemical compound 7 synthetic compounds 4
Scheme 3
According to example, shown in scheme 3, (2, the 6-diisopropylbenzyl)-and 5-nitro-1-hydrogen-iso-indoles-1,3-diketone (chemical compound 7,1.0g, 2.84mmol) and the ethyl acetate (20mL) of 10% palladium-carbon catalyst (10mg, 1% (w/w)) under 8～9psi pressure, carry out hydrogenation.With thin layer chromatography monitoring experiment process.Add catalyst (40mg, 5% total amount w/w) in the time of 2 hours.Hydrogenation continues to spend the night.Thin layer chromatography (heptane: ethyl acetate is 60: 40 for silica gel, λ=254nm) shows that two speckles mix, and two speckles keep than identical, but the Ultraluminescence difference.With kieselguhr plate filter reaction mixture, (the proof-spirit degree is 200, and 2mL) the flushing filter plate is concentrated into filter liquor only surplus residue then with ethanol.With the residue crystallization, obtain 275mg yellow solid (output capacity 30%) with ethanol (7mL).Proton NMR spectrometry and high performance liquid chromatography all are confirmed that it is the mixture of two kinds of chemical compounds.
According to example, shown in scheme 3, the chemical compound 7 (0.5 gram) and the ethyl acetate mixture (10mL) of 10% palladium-carbon catalyst (25mg, 5% (w/w)) carry out hydrogenation under 8～9psi pressure.With kieselguhr plate filter reaction mixture,, then filter liquor is concentrated into only surplus residue in the time of 2 hours with ethyl acetate (3mL) flushing filter plate.The proton magnetic resonance (PMR) chromatography shows that residue is the mixture (ratio is about 1: 1) of two kinds of chemical compounds.Further this residue of hydrogenation (pressure 45psi) in the ethyl acetate (20mL) of fresh catalyst (100mg).Separating residual thing after hydrogenation is spent the night, proton magnetic resonance (PMR) chromatography show that second crest significantly weakens (supporting product to transform).
According to example, shown in scheme 3, the ethyl acetate mixture (20mL) of chemical compound 7 (1.0 gram) and 10% palladium-carbon catalyst (100mg, 10% (w/w)) carries out hydrogenation to 23 hour under 45psi pressure.With kieselguhr plate filter reaction mixture, with ethyl acetate (6mL) flushing filter plate.Filter liquor is divided into two equal portions, and first part is concentrated into residue, second part handle with 12 equivalent concentration HCl after with solution concentration to residue.Two parts of solids of contrast under the proton magnetic resonance (PMR) chromatograph, verified, both are respectively purpose product A PI and hydrochlorate.
Embodiment 25: by chemical compound 7 synthetic compound 4--transfer hydrogenations.
According to example, shown in scheme 3, at chemical compound 7 (1.0g, 2.84mmol) and the ethanol of 10% palladium carbon catalyst (0.5g, 5% (w/w)) (the proof-spirit degree is 200,16mL) adds triethylamine (1.7mL in the mixture, 4.40eq) and formic acid (0.55g, 4.22eq).With the mixed liquor reflux, with thin plate chromatography monitoring reaction course.With kieselguhr plate filter reaction mixture, wash filter plate 2 times in the time of 2 hours with ethanol (1.5ml).Stirred the mixture under the room temperature 2 hours, and, obtained 0.49 gram yellow fluorescence solid (output capacity 53%, purity 97.3%, high performance liquid chromatography a/a%) by the isolated by filtration solid.The proton magnetic resonance (PMR) chromatography has confirmed its structure.Repeat to filter, obtain second batch of product (0.11g, purity and first are together)
According to example, shown in scheme 3, at chemical compound 7 (0.5g, 1.42mmol) and the ethanol of 10% palladium carbon catalyst (0.05g, 10% (w/w)) (the proof-spirit degree is 200,8mL) adds triethylamine (0.9mL in the mixture, 4.40eq) and formic acid (0.2g, 4.22eq).(psychrolusia) at room temperature carried out in reaction, with the thin layer chromatography monitoring reaction.In the time of 40 minutes, reactant mixture is filtered by the kieselguhr plate, and with ethanol (3 * lmL) flushing filter plates.Add cosolvent and cool off filtrate, but do not obtain crystallization.Therefore filtrate is concentrated into only surplus residue, with the latter from ethanol/heptane (1: 2,6mL) in crystallization.Filter and extract solid, obtain 0.26 gram yellow solid (output capacity 56%, purity 99.2%, high performance liquid chromatography a/a%)).
According to example, shown in scheme 3, at chemical compound 7 (1.0g, 2.84mmol) and the ethanol of 10% palladium carbon catalyst (0.5g, 5% (w/w)) (the proof-spirit degree is 200,16mL) adds triethylamine (1.8mL in the mixture, 4.40eq) and formic acid (0.5g, 4.22eq).With the mixed liquor reflux, with the high-efficient liquid phase chromatogram technique monitoring course of reaction.With kieselguhr plate filter reaction mixture, (the proof-spirit degree is 200,3 * 1ml) flushing filter plates with ethanol in the time of 3 hours.Stir the mixture under the room temperature and spend the night.0 ℃ was stirred 1 hour down with the mixed liquor cooling in second day.Filter and obtain solid, under vacuum, 35 ℃ of conditions, be dried, obtain 488mg yellow solid (output capacity 53%, purity 99.6%) and filter acquisition second batch of solid (138mg, purity 99.4%, high performance liquid chromatography a/a%) once more.
According to example, shown in scheme 3, at chemical compound 7 (1.0g, 2.84mmol) and the ethanol of 10% palladium carbon catalyst (0.5g, 5% (w/w)) (the proof-spirit degree is 200,16mL) adds triethylamine (1.8mL in the mixture, 4.40eq) and formic acid (0.5g, 4.22eq).At room temperature stirred reaction mixture reacts with the high performance liquid chromatography monitoring.With kieselguhr plate filter reaction mixture, (the proof-spirit degree is 200,6 * 2ml) flushing filter plates (annotating: observe the crystallization under catalyst action of some products, so append cleaning again) with ethanol in the time of 3 hours.Stir the mixture under the room temperature and spend the night.The mixed liquor cooling was stirred 1 hour in 0 ℃ in second day.Filter and obtain solid, under vacuum, 35 ℃ of conditions, be dried, obtain 327mg yellow solid (output capacity 36%, purity 99.7%) and filter acquisition second batch of solid (154mg, purity 99.6%, high performance liquid chromatography a/a%) once more.
According to example, shown in scheme 3, at chemical compound 7 (0.5g, 1.42mmol) and the ethanol of 10% palladium carbon catalyst (0.05g, 10% (w/w)) (the proof-spirit degree is 200,8mL) adds triethylamine (0.9mL in the mixture, 4.40eq) and formic acid (0.2g, 4.22eq).Stirred reaction mixture at room temperature is with the high-efficient liquid phase chromatogram technique monitoring course of reaction.2.5 hour the time with kieselguhr plate filter reaction mixture, with ethanol (standard wine degree 1 * 3mL) flushing filter plate.With filtrate simmer down to grease, with its recrystallization, obtain 247mg product (output capacity 54%, purity 98.8%, high performance liquid chromatography a/a%)) with ethanol.
According to example, shown in scheme 3, chemical compound 7 (5.0g, 14.19mmol) and the ethanol of 10% palladium carbon catalyst (0.25g, 5% (w/w)) (the proof-spirit degree is 200,80mL) add in the mixture triethylamine (6.32mL, 4.40eq).Keep temperature to be lower than 30 ℃, (2.76g, 4.22eq), whole process is no less than 15 minutes dropwise to add formic acid.The heating mixed liquor is to refluxing, with high-efficient liquid phase chromatogram technique monitoring.Added catalyst (0.25 gram, two parts) on the 1st.When high performance liquid chromatography confirmed that reaction is finished, with kieselguhr plate filter reaction mixture, (the proof-spirit degree was 200,2 * 12.5mL) with ethanol.In this solution, add water 80mL.Continue at room temperature to stir, continue 2 hours, stirred 1 hour down at 0 ℃ then.The isolated by filtration solid is also air-dry, obtains the crocus solid, is rough end-product (4.1 grams, output capacity 89%, purity 99.7% (a/a%)).The proton magnetic resonance (PMR) chromatograph has confirmed its structure.
According to example, (1.6: 1,36mL) recrystallization obtained dark yellow solid (1.3 grams, the response rate 65%, purity 100% (a/a%)) to rough end-product with ethanol/water.
According to example, (1: 2,66mL) recrystallization obtained light yellow solid (1.5 grams, the response rate 75%, purity 100% (a/a%)) to rough end-product with ethanol/n-heptane.
Embodiment 26: extensive synthetic compound 4
According to example, 5L three neck round-bottomed flasks in nitrogen down configuration finish, add the 4-nitrophthalic anhydride (295.0g, 1.53mol), acetic acid (2.21L, 7.5mL/g) and 2,6-diisopropyl aniline (326.2g, 1.84mol, 1.2eq).The mixed liquor reflux continues 12 hours, slowly cools to ambient temperature overnight then.Reactant mixture is concentrated into about original volume half (way of distillation is removed about 1.33kg).Cooling mixed liquid is to room temperature, and the isolated by filtration solid is with methanol (3 * 100mL) flushings.Air-dry solid 1～1.5 hour further is dried to constant weight then under vacuum, 40 ℃ of conditions.Product (2, the 6-diisopropylbenzyl)-5-nitro-1-hydrogen-iso-indoles-1,3-diketone are pale solid (488.0g, output capacity 91%, purity 99.3%, high performance liquid chromatography a/a%)).The proton magnetic resonance (PMR) chromatography and 13C nuclear magnetic resonance, NMR chromatography has all confirmed its structure.Fusing point: 167～169 ℃; Elementary analysis: theoretical value: C (68.17, H (5.72), N (7.95); Measured value: C (68.08), H (5.77), N (7.86).
12L three neck round-bottomed flasks are equipped with down in nitrogen and finish, and (the proof-spirit degree is 200,1L) mixed liquor to add the ethanol of 10% palladium carbon catalyst (24.5g, 10% (w/w)).In mixed liquor, successively add (2, the 6-diisopropylbenzyl)-5-nitro-1-hydrogen-iso-indoles-1, the 3-diketone (245.0g, 0.70mol), ethanol (the proof-spirit degree is 200,292L, 3.92L altogether, 16mL/g) and triethylamine (311.7g, 3.08mol, 4.40eq).(4.22eq), the time is no less than 25 minutes for 135.8g, 2.95mol slowly to add formic acid.The mixed liquor reflux is with the high-efficient liquid phase chromatogram technique monitoring course of reaction.Through 3.5 hours, to remove catalyst, (the proof-spirit degree was 200,4 * 175mL) flushing filter plates with ethanol with kieselguhr plate filtering mixt.Remove all microgranules in the filtrate with Whatman filter paper, (the proof-spirit degree is 200,2 * 150mL) as lotion to use ethanol.Add deionized water 3.92L in solution, the time is no less than 45 minutes.The mixed liquor of gained at room temperature stirs and spends the night.Cooling mixed liquid to 0～10 on the secondth ℃ kept 2 hours.The isolated by filtration solid, (the proof-spirit degree is 200,2 * 200mL) flushings with (0～10 ℃) ethanol of pre-cooling.At 40 ℃ of following drying solids of vacuum to constant weight.The proton magnetic resonance (PMR) chromatograph shows the excess ethanol existence in the process, so carried out a small-scale test to weigh further exsiccant effect under high temperature (70 ℃).According to its result (end-product is not degraded), under vacuum, 70 ℃ of conditions, solid further is dried to high performance liquid chromatography and shows that ethanol eliminates, obtained (2 of yellow solid form, the 6-diisopropylbenzyl)-5-amino-1-hydrogen-iso-indoles-1,3-diketone (206.0g, output capacity 91%, purity 99.2%, high performance liquid chromatography a/a%), i.e. chemical compound 4.Proton magnetic resonance (PMR) chromatograph and 13C nuclear magnetic resonance, NMR chromatograph have confirmed its structure.Fusing point: 259～261 ℃; Elementary analysis: theoretical value: C (74.51), H (6.88), N (8.69); Measured value: C (74.70), H (6.86), N (8.59).
At this, the description of method and reagent all is considered to the most practical embodiment and carries out according to existing, but it should be understood that information announcing is not limited in these disclosed embodiment.Be intended to be encompassed in the spirit of described claim and various isomers and the similar approach within the scope herein.The scope of described claim should be given the most generalized explaination to contain all this type of isomer and similar structures.

Claims (10)

1. following materialization compound 4, the application of CLT-003 in preparation treatment retinal edema medicine has the following chemical structure:
Figure FSA00000208007200011
2. application as claimed in claim 1 is characterized in that described material is wrapped in the polylactic-co-glycolic acid copolymer nano granule.
3. application as claimed in claim 1, said retinal edema, this retinal edema is by further qualitative for being macular edema.
4. application as claimed in claim 3, said retinal edema, this macular edema is by further qualitative for being diabetic macular edema.
5. the production method of a pharmaceutical composition, comprise at least: this ingredient comprises a kind of chemical compound that contains following structure at least, and its method is for to be wrapped in chemical compound in the polylactic-co-glycolic acid copolymer nano granule; Administration makes this chemical compound be prolonged the therapeutical effect of diabetes maculopathy behind the nano-particle parcel
Figure FSA00000208007200012
6. the synthetic method of the chemical compound of a kind as following chemical constitution, at least comprise by backflow coupling 4-nitrophthalic anhydride and 2 in acetic acid, 6-two different amfetamine, generate (2, the 6-diisopropylbenzyl)-and 5-nitro-1-hydrogen-iso-indoles-1, the 3-diketone, and make (2 by catalytic hydrogenation or transfer hydrogenation, the 6-diisopropylbenzyl)-and 5-nitro-1-hydrogen-iso-indoles-1, the hydrogenation of 3-diketone.
Figure FSA00000208007200021
7. as the synthetic method in the claim 6, it is characterized in that described catalytic hydrogenation, this catalytic hydrogenation is included in the existence of palladium carbon compound at least down with (2, the 6-diisopropylbenzyl)-5-nitro-1-hydrogen-iso-indoles-1, and the 3-diketone reacts in ethyl acetate.
8. as the synthetic method in the claim 7, it is characterized in that described catalytic hydrogenation reaction, this catalytic hydrogenation reaction carries out being higher than under the pressure of 5psi.
9. as the synthetic method in the claim 6, it is characterized in that described transfer hydrogenation, this hydrogenation comprises at least to be made (2, the 6-diisopropylbenzyl)-5-nitro-1-hydrogen-iso-indoles-1, the 3-diketone reacts under the condition that triethylamine and formic acid exist at the palladium carbon compound.
10. as the synthetic method in the claim 9, it is characterized in that described formic acid, this formic acid is dropwise to add, and reaction temperature should remain on and is not higher than 30 ℃.
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