CN103739642A - Carbamate derivates of scutellarin and seutellarein, and application thereof - Google Patents

Abstract

The invention discloses flavonoid compounds which can treat neurodegenerative disease and have the structures represented by the following structure general formula I, II and III, and preparation methods and an application thereof. Physicochemical properties and in-vitro metabolism experiments prove that the compounds have better physicochemical and metabolic properties compared with scutellarin and seutellarein, and can be applied in preparation of drugs for treating the neurodegenerative disease.

Description

Scutellarin and aglycon carbamate derivatives and application thereof

Technical field

The present invention relates to pharmaceutical field, especially a kind of scutellarin and aglycon carbamate derivatives and application thereof.

Background technology

Nerve degenerative diseases (Neurodegenerative disease) is the morbid state that brain and cord cell neurone are lost, As time goes on this disease worsens, finally cause disordered brain function (Appel S.H.et al.Adv Neurol.1996,69,153-159) nerve degenerative diseases mainly comprise Alzheimer (Alzheimer ' s Disease, AD), Parkinson's disease (Parkinson ' s disease, PD) and amyotrophic lateral sclerosis (Amyotrophic Lateral Sclerosis, ALS) etc.Their common feature is irreversible damage, the death of specific region neuronal function, so far without the Perfected process for the treatment of this type of disease.Along with China enters aging society, nerve degenerative diseases sickness rate sharply rises, and becomes lethal one of the main reasons.Therefore, the treatment of such disease is one of important medical problem urgently to be resolved hurrily.

Oxidative stress is due to the redox equilibrium imbalance in body, has produced too much reactive oxygen species (Reactive oxygen species, ROS) and makes body face potential damage.Research report shows, mitochondrial defects and oxidative stress have participated in pathogenic process (the Gackowski D.et al.JNeurol Sci.2008 of nerve degenerative diseases, 266 (1-2): 57-62), AD, PD and ALS show cell injury (Barnham K.J.et al.Nat Rev Drug Discov.2004,3 (3): 205-214) that the impaired and excessive free radicals of electron transport chain causes.Cause the reason of above pathology to be superoxide-dismutase (the Superoxide dismutase of patient's frontal cortex and hippocampus under nerve retrograde affection pathological conditions, SOD), Selenoperoxidase (Glutathione peroxide, GSH-Px) and glutathione reductase (Glutathione reductase, GSH-Rd) reducing all appears in level, cause causing in cell overbalance between oxidizing substance and antioxidant system, thereby cause excessive active oxygen (ROS) in brain cell to pile up, produce lipid peroxidation, the latter can change the mobility and the ability of seeing through of neuron membrane, thereby enzyme or function of receptors that infringement cell function or cytolemma connect.Research discovery simultaneously, because brain cell Mitochondrial DNA (mtDNA) lacks histone protection, the oxidative damage therefore ROS being caused is extremely sensitive, and this has accelerated the process of nerve degenerative diseases ^[11].Therefore, suppress the generation of oxyradical, promote it to remove, and suppress the neuronal damage that it causes, significant to treatment nerve degenerative diseases.

Herba Erigerontis is the herb of composite family bitter fleabane platymiscium Erigeron breviscapus (Vant.) Hand.-Mazz. (Erigerm breviscapus), is mainly distributed in the ground such as Yunnan, Guizhou of Southwestern China portion, has the effects such as promoting blood circulation and removing blood stasis, clearing and activating the channels and collaterals.Scutellarin in Herba Erigerontis (scutellarin) is topmost flavonoid activeconstituents.Pharmacodynamics test shows that scutellarin can be by significantly reducing the content of cerebral tissue Peroxidation Product mda (MDA), rising superoxide-dismutase (SOD) and Selenoperoxidase (GSH-Px) level, suppress the generation of active oxygen (ROS), and can significantly improve the neuronic form of hippocampus, alleviate the peroxide injury of cerebral tissue ^[12-15].At present, the various preparations of scutellarin are widely used in treatment cardiovascular and cerebrovascular diseases clinically, have very high pharmaceutical use and economic worth.But physico-chemical property and absorption no better than one ought to due to scutellarin, cause it can not effectively see through hemato encephalic barrier (blood-brain barrier, BBB), realize medicine effectively enrichment in cerebral tissue, thereby a little less than making it to cerebrovascular disease therapy effect, do not make its advantage be fully utilized and bring into play.

Clinical pharmacodynamics and pharmacokinetic are found, Scutellarein (seutellarein) is absorption and the drug effect form in scutellarin body, comparing its oral relative bioavailability with scutellarin is 301.8%, Scutellarein has and the similar drug effect of scutellarin, and drug effect is significantly better than second element.Although aglycon has compared with the better drug effect of scutellarin and oral absorption character, its deficiency remains absolute bioavailability lower (7.0%), a little less than the penetrating ability of hemato encephalic barrier, brain targeting selective action a little less than.

The structural modification type of carrying out for the deficiency of scutellarin at present has: on scutellarin glycosyl, introduce macromolecular carrier, or be ester derivative by carboxyl derivation in glycosyl structure, or on the hydroxyl of Scutellarein B ring 4 '-position, introduce polyoxyethylene glycol ester group, N, N-two replacement aminomethyl phenyl manthanoate and A ring 8-position are introduced aminomethyl and are formed Scutellarein 8-aminomethyl derivative.Although above-mentioned transformation has obtained certain improvement in pharmacodynamics and pharmacokinetics, above structure of modification is Shortcomings all, does not therefore all enter clinical study.

Research shows: carboxylamine ester structure is because the both sides at carbonyl exist respectively N, O atom, so such Compound Phase has higher chemistry and zymetology stability for carboxylic acid ester compound, but more easily degraded again for acid amides.Conventionally carbamate derivates keeps stable in intestinal absorption process, and its process that is converted into former medicine is under Procaine esterase mediation, to carry out in cell, therefore can improve drug absorption and disease therapeuticing effect.This layout strategy is by successfully for Irinotecan ; Capecitabine

with Bambuterol design.Carbamate strategy also has application on flavonoid glycoside compound structural modification: on Quercetin, introduce glycine (PJ.Mulholland et al.J.Ann.Oncol.2001,12,245) water-soluble increase after, transformation period is 2-3 times of Quercetin, and after quiet note, AUC does not produce toxicity under 10 times of Quercetins and 400mg dosage.Other Quercetin carbamate is water-soluble, stable all increase, the penetrating rate of mdck cell improve 2-5 doubly (Kim et al.Bioorg.Med.Chem.2009,17,1164-1171).In Tricin, introduce after carbamate stability higher (t1/22-3h) in cell pyrolysis liquid, Tricin (T-Ala-Glu) can be absorbed by the oligopeptides transporter active transport of Epithelium of intestinal villus cell, cell permeability improves 2.6-6.3 doubly, and oral maximum plasma concentration is 45 times (Ninomiya M.et al.J.Med.Chem.54 (2011) 1529-1536) of former medicine.

Summary of the invention

The object of the invention is: a kind of scutellarin and aglycon carbamate derivatives and application thereof are provided, this compound oral administration biaavailability is high, and there is the effect selectivity of anti-nerve degenerative diseases, solve the oral administration biaavailability existing in scutellarin clinical application lower, the problem that drug effect is undesirable.

The present invention is achieved in that scutellarin and aglycon carbamate derivatives thereof, and its structural formula is:

Wherein, R is that carbonatoms is 1_4 alkyl, the aryl that carbonatoms is 7 or carboxylic alkyl.

The application in the medicine of preparation treatment nerve degenerative diseases of scutellarin and aglycon carbamate derivatives thereof.

The preparation method of scutellarin and aglycon carbamate derivatives thereof, comprises the following steps:

1) scutellarin-6 " synthesizing of methyl esters _ 4 '-carbamate derivates;

By the amino acid isocyanic ester of scutellarin methyl esters and tertiary butyl protection in non-protonic solvent, under nitrogen protection, under evaluation condition, react and obtain scutellarin-6 take triethylamine " carbamate derivates of methyl esters 4 '-tertiary butyl protection; these gains remove the tertiary butyl through trifluoroacetic acid below at 0 ℃, obtain target compound;

2) preparation of Scutellarein 4 '-amino amino manthanoate

The amino acid salts hydrochlorate that the tertiary butyl is protected and two _ 4 nitrophenyl carbonate are in non-protonic solvent, reaction produces after active ester intermediate, add 6,7-ketal protection Scutellarein intermediate, continue reaction and obtain 6, the amino amino manthanoate of Scutellarein _ 4 of 7-ketal protection '-tertiary butyl protection, these gains remove the tertiary butyl through trifluoroacetic acid below at 0 ℃, obtain target compound;

3) preparation of Scutellarein 7-methyl ether 4 '-amino amino manthanoate

1. Scutellarein 7-methyl ether is synthetic

The Scutellarein that is 1: 3.7 by mol ratio and anhydrous sodium acetate are under excessive diacetyl oxide existence condition, and at 135-140 ℃, heating reflux reaction prepares the Scutellarein of full acidylate; The Scutellarein of the full acidylate that is 1: 16 by mol ratio and Anhydrous potassium carbonate, under methyl iodide existence condition, reflux in anhydrous propanone, the Scutellarein 7-methyl ether of the full acidylate of reaction preparation, and temperature of reaction is 55-60 ℃; Gains 0 ℃ of low temperature in the saturated methanol solution of ammonia removes below ethanoyl reaction and prepares Scutellarein 7-methyl ether, and the mol ratio of ammonia and methyl alcohol is 1: 1.38;

2. Scutellarein 7-methyl ether 4 '-amino amino manthanoate is synthetic

By the amino acid isocyanic ester of Scutellarein 7-methyl ether and tertiary butyl protection in non-protonic solvent, the mol ratio of Scutellarein 7-methyl ether and amino acid isocyanic ester is 1: 1.5, under nitrogen protection, under catalyzer condition, react the carbamate derivates that 10h obtains the protection of the Scutellarein 7-methyl ether 4 '-tertiary butyl take triethylamine, the trifluoroacetic acid that is 10% through mass concentration by these gains removes the tertiary butyl below at 0 ℃, obtains target compound;

In step 1) in the molar ratio of amino acid isocyanic ester of scutellarin methyl esters and tertiary butyl protection be 1.0～3.5: 1, temperature of reaction is 30-80 ℃, the reaction times is 4～48.

The molar ratio 1.0～2.0: 1 of the amino acid isocyanic ester of scutellarin methyl esters and tertiary butyl protection, temperature of reaction is 45-65 ℃, the reaction times is 5～15.

In step 2) in the amino acid salts hydrochlorate of tertiary butyl protection and the molar ratio of two _ 4 nitrophenyl carbonate be 0.5～2.5: 1, at 10～40 ℃, react 5～36 hours.

In step 3) in the 1. molar ratio 1: 8～12 of Scutellarein and diacetyl oxide; The mol ratio of Scutellarein and anhydrous sodium acetate is 1: 3～4, and in 10～30 minutes reaction times, reaction need be carried out under anhydrous condition; The molar ratio 1: 15～20 of full acidylate aglycon and methyl iodide, reflux time 10～15 hours; While sloughing acyl group, in full acidylate aglycon 7-methyl ether and the saturated methyl alcohol of ammonia, NH3 molar ratio is 1: 1.2～1.5,5.5～6 hours reaction times.

In step 2) in the amino acid salts hydrochlorate of tertiary butyl protection and the molar ratio of two _ 4 nitrophenyl carbonate be 0.8～1.5: 1, at 15～25 ℃, react 6～24 hours.

Described non-protonic solvent is anhydrous tetrahydro furan.

Representative compound of the present invention can be following compound:

1) 4,5,6-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene-7-glucuronide _ 4-oxygen carbonylamino acetic acid

2) 4,5,6-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene-7-glucuronide _ 4-oxygen carbonylamino 2-methyl acetic acid

3) 4,5,6-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene-7-glucuronide _ 4-oxygen carbonylamino 2-sec.-propyl acetic acid

4) 4,5,6-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene-7-glucuronide _ 4-oxygen carbonylamino 2-benzylacetic acid

5) 4,5,6-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene-7-glucuronide _ 4-oxygen carbonylamino 2-carboxymethyl acetic acid

6) 4,5,6-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene-7-glucuronide _ 4-oxygen carbonylamino 2-propyloic acetic acid

7) 4,5,6-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene-7-glucuronide _ 4-oxygen carbonylamino 2-(2-methyl-propyl) acetic acid

8) 4,5,6-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene-7-glucuronide _ 4-oxygen carbonylamino 2-(3-methyl-propyl) acetic acid

9) 5,6,7-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene 4-oxygen carbonylamino-2-carboxymethyl acetic acid

10) 5,6,7-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene 4-oxygen carbonylamino 2-sec.-propyl acetic acid

11) 5,6-dihydroxyl-7-methoxyl group _ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene 4-oxygen carbonylamino-2-methyl acetic acid

12) 5,6-dihydroxyl-7-methoxyl group _ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene 4-oxygen carbonylamino-2-carboxymethyl acetic acid

13) 5,6-dihydroxyl-7-methoxyl group _ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene 4-oxygen carbonylamino-2-propyloic acetic acid

The structural formula of the compounds of this invention (1) is as follows, and the structural formula of its representation compound is referring to table 1:

The 1-8 structural formula of table 1 compound of the present invention (1)

The structural formula of the compounds of this invention (2) is as follows, the structural formula of its representation compound referring to

Table 2:

The structural formula of of the present inventionization of table 2 platform thing (2) 9-10

The structural formula of the compounds of this invention (3) is as follows, and the structural formula of its representation compound is referring to table 2:

Table 3 representation compound 11-13 of the present invention structural formula

The method of preparing above-claimed cpd comprises the following steps:

(1) scutellarin-6 " preparation of methyl esters

Under the condition of-10 ℃, in 50mL anhydrous methanol, drip 10mL sulfur oxychloride, insulation reaction 10min, makes methanolic hydrochloric acid solution; At 0 ℃, methanolic hydrochloric acid solution is slowly added drop-wise in the scutellarin (0.010mol) of 60mL dry DMF dissolving, stirs 15min, remove ice bath, answer for 40 ℃, while putting plate without raw material, stopped reaction; Use saturated NaHCO ₃adjust pH=7, suction filtration, washing twice, reduced vacuum is dried to obtain yellow solid (3.60g, 75.6%);

(2) preparation of the amino acid isocyanic ester of tertiary butyl protection

The amino acid salts hydrochlorate of 2.5mmol tertiary butyl protection will be added in the dry three-necked bottle of 250ml, add the dry Cl2CH2 of 50mL and 0.8mL pyridine, the triphosgene solution that adds 500mg to dissolve with 3mLCH2Cl2 react 15min at-10 ℃ after, 0 ℃ of following reaction 2 hours under nitrogen protection, with the 0.1mol/L that adds trash ice, (object that adds trash ice is to keep low temperature, prevent compound decomposition, M represents mol/L) hydrochloric acid soln wash fast twice, with adding the saturated NaCl solution of trash ice, wash fast one time, to organic layer, add desiccant dryness, filter, filtrate decompression evaporate to dryness, obtain yellow oil,

(3) scutellarin-6 " synthesizing of methyl esters _ 4 '-carbamate derivates

In 100mL dry reaction bottle, add 1.37mmol second element methyl esters, 15mL dry DMF makes it entirely molten, after the amino acid isocyanic ester (2.05mmol) of tertiary butyl protection being dissolved with the anhydrous THF of 10mL, add reaction system, add triethylamine (0.205mmol, 28.7 μ L), the lower 50 ℃ of reactions of nitrogen protection 10 hours, remove solvent under reduced pressure, silica gel column chromatography separating purification, obtains scutellarin-6 " carbamate derivates of methyl esters 4 '-tertiary butyl protection; With 0 ℃ of following reaction of trifluoroacetic acid 5～6 hours, slough the tertiary butyl, obtain target compound;

(4) preparation of Scutellarein

The 5g scutellarin work in-process of take are raw material, back hydrolysis 20h in the sulphuric acid soln of 500mL (5g/500mL) 8%, suction filtration after naturally cooling, filter cake acetone extraction, merge acetone layer, steaming desolventizes, and obtains Scutellarein crude product, gains are used solvent recrystallization again, obtain purity higher than 90% Scutellarein;

(5) Scutellarein ketal protection intermediate preparation;

Scutellarein and catalytic amount 4-dimethylamino pyridine (DMAP) are dissolved in to appropriate N; in dinethylformamide (DME); add and the equimolar dichloro diphenyl methane of aglycon; system is warming up to 160-180 ℃; reaction 2h, after naturally cooling steams solvent resistates silica gel column chromatography separating purification; obtain 6,7-ketal protection Scutellarein intermediate:

The amino acid salts hydrochlorate 0.15mmol of tertiary butyl protection is joined to 50mL is dry to be revolved in bottle, with the anhydrous THF of 10mL, dissolve, add two _ 4 nitrophenyl carbonate (NPC) 0.15mmol and N, N-diisopropylethylamine (DIPEA) 0.30mmol, after stirring at room reaction 12h, add Scutellarein ketal protection intermediate, stirring at room reaction 12h; Reaction solution concentrating under reduced pressure, resistates silica gel column chromatography separating purification, obtain the amino amino manthanoate of Scutellarein _ 4 '-tertiary butyl protection of ketal protection, take CH2Cl2 as solvent, 0 ℃ of following reaction of trifluoroacetic acid slough simultaneously hexichol ketal protected with tertiary butyl protecting group obtain ultimate aim compound;

(6) Scutellarein 7-methyl ether is synthetic

By Scutellarein 1.56mmol and anhydrous sodium acetate 5.78mmol, at excessive diacetyl oxide (20mL) heating reflux reaction 10min, stopped reaction; after cooling, add excessive water; filter, filter cake silica gel column chromatography separating purification, obtains the Scutellarein of full acidylate.The Scutellarein 0.91mmol of full acidylate, Anhydrous potassium carbonate 8.15mmol, methyl iodide 0.9mL, anhydrous propanone 20mL, back flow reaction 20h, filters, and filtrate decompression evaporate to dryness silica gel column chromatography separating purification obtains the Scutellarein 7-methyl ether of full acidylate.According to the method for bibliographical information (Mi Kyoung Kim et al.J.Med.Chem.2010; 53; 8597-8607) the Scutellarein 7-methyl ether of full acidylate 0 ℃ of reaction 6h in the saturated methanol solution of ammonia; stopped reaction evaporate to dryness; silica gel column chromatography separating purification, obtains Scutellarein 7-methyl ether.

(10) Scutellarein 7-methyl ether 4 '-amino amino manthanoate is synthetic

In 100mL dry reaction bottle, add Scutellarein 7-methyl ether, the anhydrous THF of 15mL makes it entirely molten, after the amino acid isocyanic ester of tertiary butyl protection being dissolved with the anhydrous THF of 10mL, add reaction system, add triethylamine, under nitrogen protection, at 50 ℃, react 10 hours, remove solvent under reduced pressure, silica gel column chromatography separating purification, obtains the carbamate derivates that Scutellarein 7-methyl ether 4 '-tertiary butyl is protected; With reacting 17h under TFA/CH2Cl2 low temperature, slough the tertiary butyl, obtain target compound:

The preparation method of target compound 1-8 is as shown in flow process (I) particularly,

Wherein, a: pyridine (Pyr) ,-10～0 ℃; B: 50 ℃ of triethylamines (TEA); C: trifluoracetic acid (TFA), 0 ℃.

The embodiment of flow process (I) is described in detail as follows:

3) by scutellarin-6 of above-mentioned gained, " carbamate of methyl esters _ 4 '-tertiary butyl protection is dissolved in trifluoracetic acid (TFA), and 0 ℃ is reacted 5～6 hours, obtains target compound 1-8;

The preparation method of target compound 9-10 is as shown in flow process (II) particularly,

Wherein, a: two _ 4 nitrophenyl carbonate (NPC), DIPEA (DIPEA), room temperature; B:N, N-diisopropylethylamine (DIPEA), room temperature; C: trifluoracetic acid (TFA), 0 ℃.

The embodiment of flow process (II) is described in detail as follows:

1) take the amino acid salts hydrochlorate of tertiary butyl protection is reaction raw materials; according to literature method (James S.Nowick et al.J.Org.Chem.1992; 57; 7364-7366); take pyridine as catalyzer; after-10 ℃ of reaction 15min, add triphosgene, under nitrogen protection, low-temp reaction is 2 hours, obtains the amino acid isocyanic ester of tertiary butyl protection.

2) the amino acid isocyanic ester of tertiary butyl protection and second element methyl esters are reaction raw materials; according to literature method (Wuet al.Letters in Organic Chemistry; 2005; 2; 535-538); the triethylamine (TEA) of take is catalyzer, and 50 ℃ are reacted 10 hours, obtain scutellarin-6 " the carbamate derivates that methyl esters _ 4 '-tertiary butyl is protected.

The preparation method of target compound 11-13 is as shown in flow process (III) particularly,

The embodiment of flow process (III) is described in detail as follows:

1) take Scutellarein as raw material, anhydrous sodium acetate catalysis, back flow reaction 10min in excessive diacetyl oxide, obtains obtaining the Scutellarein of full acidylate;

2) Scutellarein of full acidylate under the catalysis of Anhydrous potassium carbonate with methyl iodide back flow reaction 20h, obtain the Scutellarein 7-methyl ether of full acidylate;

3) the Scutellarein 7-methyl ether of full acidylate 0 ℃ of reaction 6h in the saturated methanol solution of ammonia, obtains Scutellarein 7-methyl ether;

4) to take triethylamine (TEA) be catalyzer for the amino acid isocyanic ester of tertiary butyl protection and second element methyl esters and Scutellarein 7-methyl ether, and 50 ℃ of reactions 10 hours, obtain the carbamate derivates that Scutellarein 7-methyl ether _ 4 '-tertiary butyl is protected;

5) carbamate of Scutellarein 7-methyl ether _ 4 '-tertiary butyl protection is with CH ₂cl ₂for solvent, 0-25 ℃ of reaction of trifluoroacetic acid (TFA) 17 hours, obtains target compound 11-13.

Owing to having adopted technique scheme, compared with prior art, the present invention utilizes 4 in Scutellarein prodrug structure '-carbamate structure fragment to improve its oral administration biaavailability, the effect selectivity of anti-nerve degenerative diseases, adopt carbamate prodrugs layout strategy lower as solving the oral administration biaavailability existing in scutellarin clinical application, undesirable these two the not enough breaches of drug effect, physico-chemical property and the In vitro metabolism of compound of the present invention experimental results show that, this compounds has compared with scutellarin and the better physics and chemistry of aglycon and metabolisming property, can be used for the application in preparation treatment nerve degenerative diseases medicine.Material source of the present invention is extensive, is easy to obtain, with low cost, and result of use is good.

Accompanying drawing explanation

Fig. 1 is scutellarin methyl esters carbamate derivatives degradation curve (37 ± 1 ℃) in PBS (pH7.4and pH2.0);

In Fig. 1,4a_4e represents respectively amino acid moiety counter structure 4a:Ala in scutellarin 6 " methyl esters 4 "-carbamate derivatives; 4b:Val; 4c:Phe; 4d:Glu; 4e:Asp;

Fig. 2 is scutellarin methyl esters carbamate derivatives degradation curve (37 ± 1 ℃) in blood plasma;

In Fig. 2,4a_4e represents respectively amino acid moiety counter structure 4a:Ala in scutellarin 6 " methyl esters 4 "-carbamate derivatives; 4b:Val; 4c:Phe; 4d:Glu; 4e:Asp.

embodiment

One, target compound is synthetic

Embodiments of the invention 1:4, the preparation of 5,6-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene-7-glucuronide _ 4-oxygen carbonylamino acetic acid:

In 100ml dry reaction bottle, add second element methyl esters (770mg, 1.6mmol), 15ml dry DMF makes it entirely molten, after the glycine isocyanic ester (381mg2.43mmol) of tertiary butyl protection being dissolved with the anhydrous THF of 10ml, add reaction system, add triethylamine (34.02 μ L, 0.243mmol), the lower 50 ℃ of reactions of nitrogen protection 10 hours, remove solvent under reduced pressure, silica gel column chromatography separating purification, eluent is trichloromethane: methyl alcohol=15: 1 (volume ratio), off-white color scutellarin-6 " the glycine carbamate solid 374.6mg of methyl esters 4 '-tertiary butyl protection, productive rate 37%,

¹H-NMR(400MHz，DMSO-d ₆)：δ13.05(brs，1H)，10.46(brs，1H)，8.18(t，1H，J=5.6Hz)，7.97(d，2H，J=8.8Hz)，7.12(s，1H)，6.95(d，2H，J=8.8Hz)，6.92(s，1H)，5.53(d，1H，J=5.6Hz)，5.34(brs，2H)，5.20(brs，1H)，4.21(d，1H，J=9.6Hz)，3.73(d，2H，J=6.0Hz)，3.65(s，1H)，1.42(s，9H)。

Upper step is obtained to compound (158mg, 0.24mmol) with 3mL trifluoroacetic acid, dissolve and add in reaction flask, ice bath reaction 5.5 hours, TLC monitoring is without raw material, and stopped reaction, removes trifluoroacetic acid under reduced pressure, by chloroform-sherwood oil system, wash, centrifugal, obtain oyster pressed powder 112.8mg, productive rate 81.46%.

¹H?NMR(400MHz，DMSO-d ₆)：δ13.06(brs，1H)，10.46(brs，1H)，8.16(t，1H，J=6Hz)，7.99(d，2H，J=9.2Hz)，7.12(s，1H)，6.97(d，2H，J=8.8Hz)，6.91(s，1H)，5.37(d，2H，J=7.6Hz)，4.23(d，1H，J=9.6Hz)，378(d，2H，J=6.4Hz)，3.67(s，3Hz).ESI-MS(m／z)[M+H] ⁺：578.2，[M-H] ^-：576.2.

Embodiments of the invention 2:4, the preparation of 5,6-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene-7-glucuronide _ 4-oxygen carbonylamino 2-methyl acetic acid

In 100mL dry reaction bottle, add second element methyl esters (650mg, 1.37mmol), 15mL dry DMF makes it entirely molten, after the L-Ala isocyanic ester (351mg2.05mmol) of tertiary butyl protection being dissolved with the anhydrous THF of 10mL, add reaction system, add triethylamine (28.7 μ L, 0.205mmol), the lower 50 ℃ of reactions of nitrogen protection 10 hours, remove solvent under reduced pressure, silica gel column chromatography separating purification, eluent is trichloromethane: methyl alcohol=15: 1 (V/V), off-white color scutellarin-6 " the phenylalanine ammonia carbamate solid 457.8mg of methyl esters 4 '-tertiary butyl protection, productive rate 51.6%.

¹H-NMR(400MHz，DMSO-d ₆)：δ13.05(brs，1H)，10.46(brs，1H)，8.25(d，1H，J=7.2Hz)，7.97(d，2H，J=8.8Hz)，7.12(s，1H)，6.95(d，2H，J=8.8Hz)，6.90(s，1H)，5.54(d，1H，J=4.8Hz)，5.36(brs，2H)，508(brs，1H)，4.21(d，1H，J=9.2Hz)，3.99(m，1H)，1.40(s，9H)，1.31(d，3H，J=7.2Hz)。

Upper step is obtained to compound (158mg0.24mmol) dissolves and adds in reaction flask with 3mL trifluoroacetic acid, ice bath reaction 5.5 hours, TLC monitoring is without raw material, stopped reaction, remove trifluoroacetic acid under reduced pressure, with the washing of chloroform-sherwood oil system, centrifugal, obtain oyster pressed powder 124.04mg, productive rate 87.45%.

¹H?NMR(400MHz，DMSO-d ₆)：δ13.06(brs，1H)，10.46(brs，1H)，8.23(d，1H，J=7.6Hz)，7.99(d，2H，J=8.8Hz)，7.13(s，1H)，6.97(d，2H，J=9.2Hz)，6.92(s，1H)，5.37(d，1H，J=7.2Hz)，4.23(d，1H，J=9.6Hz)，3.67(s，3H)，3.53-3.34(m，3H)，1.36(d，3H，J=7.6Hz).ESI-MS(m／z)[M+H] ⁺：592.3，[M-H] ^-：590.3.HRMS?calcd.for?C ₂₆H ₂₆NO ₁₅[M+H] ⁺592.1302，found592.1290。

Embodiments of the invention 3:4, the preparation of 5,6-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene-7-glucuronide _ 4-oxygen carbonylamino 2-sec.-propyl acetic acid:

In 100mL dry reaction bottle, add second element methyl esters (680.68mg1.34mmol), 15mL dry DMF makes it entirely molten, the figured silk fabrics amino acid isocyanic ester (428mg tertiary butyl being protected with the anhydrous THF of 10mL, 2.15mmol) after dissolving, add reaction system, add triethylamine (28.0 μ L, 0.200mmol), the lower 50 ℃ of reactions of nitrogen protection 10 hours, remove solvent under reduced pressure, silica gel column chromatography separating purification, eluent is trichloromethane: methyl alcohol=15: 1 (volume ratio), off-white color scutellarin-6 " the α-amino-isovaleric acid carbamate solid 398.1mg of methyl esters 4 '-tertiary butyl protection, productive rate 44%.

¹H?NMR(400MHz，DMSO-d ₆)：δ13.07(brs，1H)，10.48(brs，1H)，8.14(d，1H，J=8.4Hz)，7.99(d，2H，J=8.8Hz)，7.13(s，1H)，6.97(d，2H，J=8.8Hz)，6.92(s，1H)，5，53(brs，1H)，5.38(brs，2H)，512(brs，1H)，4.21(d，1H，J=9.2Hz)，3.87-3.83(m，1H)，3.67(s，3H)，2.12-2.07(m，1H)，1.44(s，9H)，0.96(d，6H，J=9.2Hz)。

Upper step is obtained to compound (81.52mg, 0.12mmol) with 3mL trifluoroacetic acid, dissolve and add in reaction flask, ice bath reaction 5.5 hours, TLC monitoring is without raw material, and stopped reaction, removes trifluoroacetic acid under reduced pressure, by chloroform-sherwood oil system, wash, centrifugal, obtain yellowish pink pressed powder 62.7mg, productive rate 84.4%.

¹H?NMR(400MHz，DMSO-d ₆)：δ13.06(brs，1H)，10.46(brs，1H)，8.05(d，1H，J=8.0Hz)，7.95(d，2H，J=8.8Hz)，7.18(s，1H)，6.97(d，2H，J=8.8Hz)，6.91(s，1H)，5.29(d，1H，J=7.6Hz)，4.36(d，1H，J=9.2Hz)，3.93-3.90(m，1H)，3.67(s，3H)，3.37-3.29(m，3H)，2.15-2.10(m，1H)，0.97-0.95(m，6H).ESI-MS(m／z)[M+H] ⁺：620.3，[M-H] ^-：618.4.HRMS?calcd.for?C ₂₈H ₃₀NO ₁₅[M+H] ⁺620.1615，found620.1623.

Embodiments of the invention 4:4, the preparation of 5,6-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene-7-glucuronide-4-oxygen carbonylamino 2-benzylacetic acid:

In 100mL dry reaction bottle, add second element methyl esters (685mg, 1.44mmol), 15mL dry DMF makes it entirely molten, phenylalanine isocyanic ester (the 534mg tertiary butyl being protected with the anhydrous THF of 10mL, 2.16mmol) after dissolving, add reaction system, add triethylamine (30.167 μ L, 0.215mmol), lower 50 ℃ of nitrogen protection is answered 10 hours, remove solvent under reduced pressure, silica gel column chromatography separating purification, eluent is trichloromethane: methyl alcohol=20:1 (V/V), class oyster scutellarin-6 " the phenylalanine carbamate solid 502.2mg of methyl esters 4 '-butyl protection, productive rate 48.2%.

¹H?NMR(400MHz，DMSO-d ₆)：δ13.03(brs，1H)，10.46(brs，1H)，8.33(d，1H，J=6.8Hz)，7.97(d，2H，J=8.4Hz)，7.31-7.24(m，5H)，7.12(s，1H)，6.95(d，2H，J=8Hz)，6.90(s，1H)，5.54(brs，1H)，5.40(d，1H，J=13.2Hz)，5.08(brs，1H)，4.21(d，1H，J=9.2Hz)，4.14(d，1H，J=7.2Hz)，3.64(s，3H)，3.0(d，2H)，1.32(s，9H)。

Upper step is obtained to compound (159mg, 0.22mmol) with 3mL trifluoroacetic acid, dissolve and add in reaction flask, ice bath reaction 5.5 hours, TLC monitoring is without raw material, and stopped reaction, removes trifluoroacetic acid under reduced pressure, by chloroform-sherwood oil system, wash, centrifugal, obtain greyish-green pressed powder 125.9mg, productive rate 85.8%.

¹H?NMR(400MHz，DMSO-d ₆)：δ13.01(brs，1H)，10.46(brs，1H)，8.24(d，1H，J=8.4Hz)，7.98(d，2H，J=8.8Hz)，7.32-7.24(m，5H)，7.12(s，1H)，6.97(d，2H，J=8.8Hz)，6.90(s，1H)，5.37(d，1H，J=7.2Hz)，4.23(d，1H，J=9.2Hz)，3.67(s，3H)，3.36-3.26(m，3H)，3.17-3.10(m，1H)，2.96-2.91(m，1H)；ESI-MS(m／z)[M+H] ⁺：668.3，[M-H] ^-：666.5.HRMS?calcd.for?C ₃₂H ₃₀NO ₁₅?[M+H]+668.1615，tound668.1596。

Embodiments of the invention 5:4, the preparation of 5,6-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene-7-glucuronide _ 4-oxygen carbonylamino 2-carboxymethyl acetic acid:

In 100mL dry reaction bottle, add second element methyl esters (517mg, 1.08mmol), 15mL dry DMF makes it entirely molten, aspartic acid isocyanic ester (the 441mg tertiary butyl being protected with the anhydrous THF of 10mL, 1.62mmol) after dissolving, add reaction system, add triethylamine (22.62 μ L, 0.16mmol), lower 50 ℃ of nitrogen protection is answered 10 hours, remove solvent under reduced pressure, silica gel column chromatography separating purification, eluent is trichloromethane: methyl alcohol=20: 1 (volume ratio), white scutellarin-6 " the aspartic acid carbamate solid 331.58mg of methyl esters 4 '-butyl protection, productive rate 41.06%.

¹H?NMR(400MHz，DMSO-d ₆)：δ13.06(brs，1H)，10.45(brs，1H)，8.29(d，1H，J=8.4Hz)，7.98(d，2H，J=8.8Hz)，7.13(s，1H)，6.96(d，2H，J=8.8Hz)，6.91(s，1H)，5.53(d，1H，J=5.2Hz)，5.39-5.35(m，2H)，5.09(brs，1H)，4.29_4.27(m，1H)，4.21(d，1H，J=9.2Hz)，3.64(s，3H)，2.72-2.70(m，1H)，2.60-2.58(m，1H)，1.40(s，18H)。

Upper step is obtained to compound (94mg, 0.126mmol) with 3mL trifluoroacetic acid, dissolve and add in reaction flask, ice bath reaction 5.5 hours, TLC monitoring is without raw material, and stopped reaction, removes trifluoroacetic acid under reduced pressure, by chloroform-sherwood oil system, wash, centrifugal, obtain pale solid powder 57.8mg, productive rate 72.2%.

¹H?NMR(400MHz，DMSO-d ₆)：δ13.05(brs，1H)，10.45(brs，1H)，8.30(d，1H，J=8.4Hz)，7.97(d，2H，J=8.8Hz)，7.10(s，1H)，6.96(d，2H，J=8.8Hz)，6.90(s，1H)，5.35(d，1H，J=7.2Hz)，4.36(m，1H)，4.22(d，1H，J=9.6Hz)，3.67(s，3H)，3.37-3.26(m，3H)，2.79-2.74(m，1H)，2.66-2.62(m，1H)；ESI-MS(m／z)[M+H] ⁺：636.3，[M-H] ^-：634.3.HRMS?calcd.for?C ₂₇H ₂₆NO ₁₇[M+H]+636.1201，found636.1181.HRMS?calcd.for?C ₂₇H ₂₆NO ₁₇[M+H]+636.1201，found636.1181。

Embodiments of the invention 6:4, the preparation of 5,6-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene-7-glucuronide _ 4-oxygen carbonylamino 2-propyloic acetic acid:

In 100mL dry reaction bottle, add second element methyl esters (405mg, 0.85mmol), 15mL dry DMF makes it entirely molten, L-glutamic acid isocyanic ester (the 367mg tertiary butyl being protected with the anhydrous THF of 10mL, 1.28mmol) after dissolving, add reaction system, add triethylamine (17.8 μ L, 0.127mmol), lower 50 ℃ of nitrogen protection is answered 10 hours, remove solvent under reduced pressure, silica gel column chromatography separating purification, eluent is trichloromethane: methyl alcohol=20:1 (V/V), obtain slightly yellow scutellarin-6 " the aspartic acid carbamate light yellow solid 347.7mg that methyl esters 4 '-butyl is protected of off-white color, productive rate 53.7%.

¹H?NMR(400MHz，DMSO-d ₆)：δ13.07(brs，1H)，10.46(brs，1H)，8.24(d，1H，J=6.0Hz)，7.97(d，2H，J=9.2Hz)，7.12(s，1H)，6.96(d，2H，J=72Hz)，6.93(s，1H)，5.38-5.35(m，2H)，5.08(brs，1H)4.21(d，1H，J=9.6Hz)，4.00-3.95(m，1H)，3.66(s，3H)，2.40-2.31(m，2H)，?1.98-21.93(m，1H)，1.82-1.79(m，1H)，1.41(s，9H)，1.40(s，9H)。

Upper step is obtained to compound (150mg, 0.197mmol) with 3mL trifluoroacetic acid, dissolve and add in reaction flask, ice bath reaction 5.5 hours, TLC monitoring is without raw material, and stopped reaction, removes trifluoroacetic acid under reduced pressure, by chloroform-sherwood oil system, wash, centrifugal, obtain white-yellowish solid powder 101.2mg, productive rate 79.2%.

¹H?NMR(400MHz，DMSO-d ₆)：δ13.06(brs，1H)，10.46(brs，1H)，8.22(d，1H，J=8.0Hz)，7.99(d，2H，J=8.4Hz)，7.13(s，1H)，6.97(d，2H，J=8.8Hz)，6.90(s，1H)，5.53(d，1H，J=5.6Hz)，5.37-5.36(m，2H)，4.23(d，1H，J=9.6Hz)，4.08_4.02(m，1H)，3.67(s，3H)，2.42-2.36(m，2H)，2.08-2.02(m，1H)，1.89-1.82(m，1H)；ESI-MS(m／z)[M+H] ⁺：650.3，[M-H] ^-：648.3.HRMScalcd.for?C ₂₈H ₂₈NO ₁₇[M+H]+650.1357，found650.1352。

Embodiments of the invention 7:4, the preparation of 5,6-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene-7-glucuronide _ 4-oxygen carbonylamino 2-(2-methyl-propyl) acetic acid:

" the leucine isocyanic ester of methyl esters and tertiary butyl protection is as reaction raw materials is according to embodiment 1 method preparation to take scutellarin-6.Obtain oyster solid target product, productive rate 49.8%.

¹H?NMR(400MHz，DMSO-d ₆)：δ13.05(brs，1H)，10.43(brs，1H)，8.18(d，1H，J=8.4Hz)，7.97(d，2H，J=8.8Hz)，7.12(s，1H)，6.96(d，2H，J=8.8Hz)，6.88(s，1H)，5.52(d，1H，J=4.8Hz)，5.37-5.35(m，2H)，5.06(d，1H，J=4.4Hz)，4.20(d，1H，J=9.2Hz)，3.97-3.92(m，1H)，3.65(s，3H)1.77-1.70(m，1H)，1.62-1.56(m，1H)，1.52-1.45(m，1H)，1.41(s，9H)，0.93(d，3H，J=6.8Hz)，0.91(d，3H，J=6.4Hz).

The compound that more than step obtains is that reaction raw materials is according to being similar to embodiment 1 method preparation.Obtain sallow green solid target product, productive rate 87.8%.

¹H?NMR(400MHz，DMSO-d ₆)：δ13.05(brs，1H)，10.46(brs，1H)，8.16(d，1H，J=8Hz)，7.99(d，2H，J=9.2Hz)，7.13(s，1H)，6.97(d，2H，J=8.8Hz)，6.92(s，1H)，6.92(brs，1H)，5.38(d，2H，J=7.2Hz)，5.36((brs，1H)，4.22(d，1H，J=9.2Hz)，4.06(m，1H)，1.79-1.59(m，1H)，1.57-1.51(m，1H)，0.95(d，3H，J=6.8Hz)，0.91(d，3H，J=6.4Hz).ESI-MS(m／z)[M+H] ⁺：634.3，[M-H] ^-：632.3.

Embodiments of the invention 8:4, the preparation of 5,6-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene-7-glucuronide _ 4-oxygen carbonylamino 2-(3-methyl-propyl) acetic acid:

" the Isoleucine isocyanic ester of methyl esters and tertiary butyl protection, as reaction raw materials is according to embodiment 1 method preparation, obtains oyster solid target product, productive rate 40.8% to take scutellarin-6.

¹H?NMR(400MHz，DMSO-d ₆)：δ13.06(brs，1H)，10.44(brs，1H)，8.12(d，1H，J=8.4Hz)，8.02(d，2H，J=8.4Hz)，7.16(s，1H)，7.01(d，2H，J=8.4Hz)，6.93(s，1H)，5.52(d，1H，J=?5.6Hz)，5.36-5.35(m，2H)，5.11(d，1H，J=4.4Hz)，4.20(d，1H，J=9.6Hz)，3.89-3.85(m，1H)，3.65(s，3H)，1.81-1.58(m，1H)，1.52-1.46(m，2H)，142(s，9H)，0.91(d，3H，J=6.8Hz)，0.89(d，3H，J=7.6Hz)。

By scutellarin-6 that obtain before, " the Isoleucine carbamate solid of methyl esters 4 '-tertiary butyl protection is that reaction raw materials is according to being similar to embodiment 1 method preparation.Obtain yellow-green colour solid target product, productive rate 88.4%.

¹H?NMR(400MHz，DMSO-d ₆)：δ13.06(brs，1H)，10.44(brs，1H)，8.10(d，1H，J=8.4Hz)，7.99(d，2H，J=8.8Hz)，7.13(s，1H)，6.97(d，2H，J=8.8Hz)，6.92(s，1H)，5.38(d，1H，J=7.2Hz)，4.22(d，1H，J=9.2Hz)，3.97(m，1H)，3.42-3.27(m，3H)，1.85(m，1H)，1.51-1.44(m，1H)，1.31-1.23(m，1H)，0.94(d，3H，J=6.8Hz)，0.88(d，3H，J=7.6Hz).ESI-MS(m／z)[M+H] ⁺：634.3，[M-H] ^-：632.3。

Embodiments of the invention 9:5, the preparation of 6,7-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene 4-oxygen carbonylamino-2-carboxymethyl acetic acid:

L-Ala hydrochloride (27.25mg with tertiary butyl protection; 0.15mmol) He two _ 4 nitrophenyl carbonate (NPC) are (45.6mg0.15mmol) reaction raw materials; N; N-diisopropylethylamine (38.7mg; 0.3mmol) for catalyzer room temperature stirring reaction, after 12 hours, add 6; 7-ketal protection Scutellarein intermediate (66mg, 0.15mmol), stirring at room reaction 12 hours.Remove solvent under reduced pressure, silica gel column chromatography separating purification, eluent is trichloromethane, obtains the phenylalanine ammonia carbamate yellow oil 49.65mg of Scutellarein _ 4 '-tertiary butyl protection of 6,7-ketal protection, productive rate is 53.2%.

¹H?NMR(400MHz，DMSO-d ₆)：δ12.76(s，1H)，7.86(d，2H，J=8.4Hz)，7.63-7.60(m，4H)，7.41-7.38(m，6H)，6.98(d，2H，J=8.8Hz)，6.64(s，1H)，6.62(s，1H)，5.74(d，1H，J=7.6Hz)，4.321(m，1H)，1.46(d，3H，J=0.8Hz)。

Under condition of ice bath, the compound (20mg, 0.032mmol) that upper step is obtained is dissolved in the CH that 3ml is dry ₂cl ₂in, add 0 ℃～25 ℃ reactions of 1.5mL trifluoroacetic acid (TFA) 17 hours, remove solvent under reduced pressure, obtain Scutellarein 4 '-phenylalanine ammonia carbamate glassy yellow pressed powder 10.6mg, productive rate 82.5%.

¹H-NMR(400MHz，DMSO-d ₆)：δ12.65(brs，1H)，8.30(d，1H，J=3.6Hz)，8.09(d，2H，J=8.8Hz)，7.30(d，2H，J=8.8Hz)，6.91(s，1H)，6.60(s，1H)，4，08(m，1H)，1.34(d，3H，J=7.6Hz).ESI-MS(m／z)[M+H] ⁺：402.2。

Embodiments of the invention 10:5, the preparation of 6,7-trihydroxy-_ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene 4-oxygen carbonylamino 2-sec.-propyl acetic acid:

α-amino-isovaleric acid hydrochloride (31.455mg with tertiary butyl protection; 0.15mmol) He two _ 4 nitrophenyl carbonate (NPC) (45.6mg; 0.15mmol) be reaction raw materials; N; N-diisopropylethylamine (38.7mg, 0.3mmol) adds 6 for catalyzer room temperature stirring reaction after 12 hours, 7-ketal protection Scutellarein intermediate (66mg; 0.15mmol), stirring at room reaction is 12 hours.Remove solvent under reduced pressure, silica gel column chromatography separating purification, eluent is trichloromethane, obtains the α-amino-isovaleric acid carbamate yellow oil 46.7mg of Scutellarein _ 4 '-tertiary butyl protection of 6,7-ketal protection, productive rate is 47.8%.

¹H-NMR(400MHz，DMSO-d ₆)：δ12.76(s，1H)，7.93(d，2H，J=8.8Hz)，7.63-7.59(m，4H)，7.43-7.37(m，6H)，7.33(d，2H，J=8.4Hz)，6.65(s，1H)，6.62(s，1H)，5.66(d，1H，J=9.2Hz)，4.27_4.23(m，1H)，2.26-2.22(m，1H)，1.50(s，9H)，1.04-1.02(m，3H)，0.96-0.93(m，3H)。

Under condition of ice bath, the compound (20mg, 0.03mmol) that upper step is obtained is dissolved in the CH that 3mL is dry ₂cl ₂in, add 0 ℃～25 ℃ reactions of 1.5mL trifluoroacetic acid (TFA) 17 hours, remove solvent under reduced pressure, obtain Scutellarein 4 '-phenylalanine ammonia carbamate glassy yellow pressed powder 10.4mg, productive rate 80.4%.

¹H-NMR(400MHz，DMSO-d ₆)δ：12.76(brs，1H)，8.08(d，1H，J=8.8Hz)，7.90(d，2H，J=8.4Hz)，6.91(d，2H，J=8.8Hz)，6.72(s，1H)，6.56(s，1H)，3.93-3.89(m，1H)，2.15-2.02(m，1H)，0.95-0.93(m，3H)，0.87-0.84(m，3H).ESI-MS(m／z)[M+H] ⁺：431.3。

Embodiments of the invention 11:5, the preparation of 6-dihydroxyl-7-methoxyl group _ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene 4-oxygen carbonylamino-2-methyl acetic acid:

By Scutellarein 7-methyl ether (200mg; 0.67mmol) being placed in the anhydrous THF of 15mL makes it entirely molten; by the L-Ala isocyanic ester (171mg of tertiary butyl protection; after 1mmol) being dissolved in the anhydrous THF of 10mL, add reaction system; after add triethylamine (10 μ L; 0.07mmol); the lower 50 ℃ of reactions of nitrogen protection 10 hours; remove solvent under reduced pressure; silica gel column chromatography separating purification; with CH2Cl2:CH3OH=50:1 (V/V), be eluent, obtain the L-Ala manthanoate yellow oil 143.9mg of Scutellarein 7-methyl ether 4 '-tertiary butyl protection, productive rate 45.6%.

¹H-NMR(400MHz，DMSO-d ₆)：δ10.27(brs，1H)，8.06(d，1H，J=7.2Hz)，7.90(d，2H，J=8.8Hz)，6.91(d，2H，J=8.8Hz)，6.62(s，1H)，6.57(s，1H)，3.97-3.94(m，1H)，3.81(s，3H)，1.42(s，9H)，1.31(d，3H，J=7.6Hz)。

Under condition of ice bath, upper step gained compound (11mg, 0.023mmol) is dissolved in to the CH that 3mL is dry ₂cl ₂in, add 0.36mL trifluoroacetic acid (TFA) 0 ℃～25 ℃ reactions 17 hours, remove solvent under reduced pressure, obtain Scutellarein 7-methyl ether 4 '-phenylalanine ammonia carbamate glassy yellow pressed powder 8.1mg, productive rate 84.8%;

¹H-NMR(400MHz，DMSO-d ₆)：δ10.28(brs，1H)，8.02(d，1H，J=7.6Hz)，7.91(d，2H，J=8.8Hz)，6.93(d，2H，J=8.8Hz)，6.63(s，1H)，6.58(s，1H)，4.06_4.02(m，1H)，3.82(s，3H)，1.35(d，3H，J＝7.6Hz).ESI-MS(m／z)[M+H] ⁺：414.0；[M-H] ^-：412.2。

Embodiments of the invention 12:5, the preparation of 6-dihydroxyl-7-methoxyl group _ 4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene 4-oxygen carbonylamino-2-carboxymethyl acetic acid:

Scutellarein 7-methyl ether (206mg; 0.69mmol); the anhydrous THF of 15mL makes it entirely molten; after the aspartic acid isocyanic ester (281mg, 1.035mmol) of tertiary butyl protection being dissolved with the anhydrous THF of 10mL, add reaction system, add triethylamine (14 μ L; 0.1mmol) the lower 50 ℃ of reactions of nitrogen protection are 10 hours; remove solvent under reduced pressure, silica gel column chromatography separating purification, uses CH ₂cl ₂: CH ₃oH=100: 1 (V/V) is eluent, obtains the aspartic acid manthanoate yellow oil 201mg that Scutellarein 7-methyl ether 4 '-tertiary butyl is protected, productive rate 51%.

¹H-NMR(400MHz，DMSO-d ₆)：δ12.42(brs，1H)，11.28(brs，1H)，7.67(d，1H，J=8.8Hz)，6.92(d，2H，J=8.8Hz)，6.67(s，1H)，6.46(s，1H)，4.71_4.67(m，1H)，3.99(s，3H)，2.97-2，91(m，1H)，2.85-2.79(m，1H)，1.49(s，9H)，1.48(s，9H)。

Under condition of ice bath, upper step gained compound (14mg, 0.025mmol) is dissolved in to the CH that 3mL is dry ₂cl ₂in, add 0 ℃～25 ℃ reactions of 0.36mL trifluoroacetic acid (TFA) 17 hours, remove solvent under reduced pressure, by ethyl acetate, wash away impurity, obtain Scutellarein 7-methyl ether 4 '-aspartic acid carbamate ivory buff pressed powder 8mg, productive rate 70%.

¹H-NMR(400MHz，DMSO-d ₆)：δ10.29(brs，1H)，8.41(d，1H，J=7.2Hz)，7.91(d，1H，J=8.8Hz)，7.45(s，1H)，6.92(d，2H，J=8.4Hz)，6.61(d，1H，J=12.8Hz)，4.43_4.36(m，1H)，3.97(s，3H)，2.86-2.75(m，1H)，2.71-2.66(m，1H).ESI-MS(m／z)[M-H] ^-：459.2。

Embodiments of the invention 13:5, the preparation of 6,7-trihydroxy--4-oxygen-2-(4-hydroxy phenyl) _ 4H-1-chromene _ 4-L-glutamate:

Scutellarein 7-methyl ether (230mg; 0.77mmol); the anhydrous THF of 15mL makes it entirely molten; after the L-glutamic acid isocyanic ester (329mg, 1.16mmol) of tertiary butyl protection being dissolved with the anhydrous THF of 10mL, add reaction system, add triethylamine (16 μ L; 0.1mmol) the lower 50 ℃ of reactions of nitrogen protection are 10 hours; remove solvent under reduced pressure, silica gel column chromatography separating purification, uses CH ₂cl ₂: CH ₃oH=100:1 (V/V) is eluent, obtains the L-glutamic acid manthanoate yellow oil 196.8mg of Scutellarein 7-methyl ether 4 '-tertiary butyl protection, productive rate 43.7%.

¹H-NMR(400MHz，DMSO-d ₆)δ：12.37(brs，1H)，11.34(brs，1H)，7.65(d，1H，J=3.6Hz)，7.64(d，2H，J=8.8Hz)，6.90(d，2H，J=8.8Hz)，6.63(s，1H)，6.46(s，1H)，4.44_4.38(m，1H)，3.95(s，3H)，2.46-2.35(m，1H)，2.19(s，1H)，2.01-1.96(m，1H)，1.50(s，9H)，1.45(s，9H)。

Under condition of ice bath, upper step is obtained to compound (14mg, 0.024mmol) and be dissolved in the CH that 3ml is dry ₂cl ₂in, add 0 ℃～25 ℃ reactions of 0.36ml trifluoroacetic acid (TFA) 17 hours, remove solvent under reduced pressure, by ethyl acetate, wash away impurity, obtain the Scutellarein 7-methyl ether 4 '-shallow white solid powder of L-glutamic acid carbamate 9mg, productive rate 79%.

¹H-NMR(400MHz，DMSO-d ₆)δ：12.37(brs，1H)，11.34(brs，1H)，8.37(d，1H，J=3.6Hz)，7.91(d，2H，J=8.4Hz)，7.45(s，1H)，6.92(d，2H，J=8.4Hz)，6.63(s，1H)，4.18(m，1H)，3.98(s，3H)，2.43-2.41(m，2H)，2.07-2.02(m，1H)，1.89(m，1H).ESI-MS(m／z)[M-H] ^-：475.2。

Two, target compound physico-chemical property research

1, experiment material and instrument

Test compounds is provided by Guiyang Medical College pharmaceutical college; Formic acid, acetonitrile (chromatographically pure) are purchased from Tianjin Kermel Chemical Reagent Co., Ltd.; The triple quadrupole rods tandem mass spectrometry instrument of superelevation liquid phase (UPLC-MS) ACQUITY TQD (Low-resolution mass spectrometer, Waters).

2, test method

(1) measuring method of different pH and plasma stability

Take 1mg sample, add 20 μ L DMSO to make it entirely molten, add 2mL methyl alcohol, 2mL water, be settled to 5mL, get 200 μ L and add respectively different pH, in cell pyrolysis liquid and blood plasma 2mL, 37 degree are hatched, by different time points sampling, every sub-sampling 100 μ L, add 1% formic acid 50 μ L-acetonitrile 400 μ L centrifugal, get supernatant sample introduction, sampling volume is 1 μ L.Test-results is in Table 4.

(2) measuring method of solubleness

Mark bent preparation

Precision samples 1mg in 5mL volumetric flask, and methanol constant volume is ultrasonic, make it entirely molten, get 20 μ L in 1ml volumetric flask, be settled to scale, obtain the solution that concentration is 4 μ g/mL, being made into successively concentration is the solution of 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL, 0.25 μ g/mL.Sampling volume is 1 μ L, records corresponding peak area, take concentration as X-coordinate, and peak area is ordinate zou, tries to achieve typical curve equation.

The mensuration of solubleness

Get 2_4mg target compound in 5mL centrifuge tube, add 50 μ L distilled water, ultrasonic one-tenth turbid solution, with 0.45 μ m filtering with microporous membrane, filtrate is diluted corresponding multiple, sample introduction, and sampling volume is 1 μ L, record peak area, bring corresponding typical curve equation into, try to achieve solubleness.Test-results is in Table 4.

UPLC-MS testing conditions

LC analysis condition

Waters Van Guard BEH G18 guard column (2.1mm * 5mm, 1.7 μ m); Mobile phase composition: the acetonitrile of A phase-0.1% formic acid, the water of B phase-0.1% formic acid; Gradient elution: 0～2min10%A, 2～3min90%A, 3min10%A; Flow velocity: 0.35mL/min; Column temperature: 45 ℃; Sampling volume: 1 μ L.

MS analysis condition

Waters ACQUITY TQD, Z-Spray, ESI ion source, is connected with Waters ACQUITY UPLC system.Collision gas: Ar; Deionization and dry gas: N ₂; Ion source temperature: 120 ℃; Desolvation temperature: 350 ℃; Taper hole gas velocity: 50L/h; Desolvation gas velocity 650L/h; Collision gas velocity: 0.16mL/min; Capillary voltage: 3kv; Many reactive ions monitoring pattern (MRM).

3, test-results

Scutellarin, Scutellarein and scutellarin 6 " the typical curve equation of methyl esters 4 '-carbamate derivatives: (1/X weight) Y-peak area X-compound concentration

Scutellarin Y=896.737X-138.155 r ²=0.9973

Scutellarein Y=1038.92X_41.184 r ²=0.9997

Scutellarin Ala manthanoate (1) Y=2856.56X-106.142 r ²=0.9987

Scutellarin Val manthanoate (2) Y=5007.99X-98.11732 r ²=0.9999

Scutellarin Phe manthanoate (3) Y=1882.25X-147.532 r ²=0.9922

Scutellarin Asp manthanoate (4) Y=1356.54X-63.8712 r ²=0.9982

Scutellarin Glu manthanoate (5) Y=2179.22X-74.0496 r ²=0.9979

The physico-chemical property of table 4 scutellarin methyl esters carbamate derivates

4, result and discussion

Experimental result explanation, synthesized second element methyl esters amino formate compounds of the present invention is stable under acidic conditions, unstable under alkaline condition.Although short at blood plasma and pH=7 transformation period, " methyl esters key ruptures very soon, and 4 '-amido linkage slowly ruptures, and discharges former medicine can to find out its hydrolysis law, 6 from UPLC-MS/MS figure.

Remove second element methyl esters phenylalanine manthanoate solubleness lower, other 4 compounds are all good than scutellarin solubleness, second element methyl esters L-Ala manthanoate solubleness has improved 24 times compared with scutellarin, and second element methyl esters aspartic acid manthanoate solubleness has improved 28 times compared with scutellarin.

Three, target compound PC12 cellular oxidation injury protection activity research

1, experiment material and instrument

PC12 cell strain is provided by Inst. of Medicinal Biological Technology, Chinese Academy of Medical Sciences; Test compounds is provided by Guiyang Medical College pharmaceutical college; Vitamin-E is purchased from Sigma company; DMEM high glucose medium is purchased from Gibco company; Foetal calf serum is purchased from BIOCHROM AG; MTT is purchased from Sigma company; Microplate reader is Bole 680Model.

2, test method

(1) cell toxicity test

Adopt tetrazole reduction method, the restraining effect of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide, MTT) detection of drugs to PC12 cell growth itself.By PC12 cell dissociation, be individual cells suspension, adjusting cell density is 3～4 * 10 ⁴individual/ml, is inoculated in 96 orifice plates (100 μ l/ hole), 37 ℃, 5%CO ₂incubator overnight incubation.After cell attachment, suck supernatant, add the nutrient solution containing concentration gradient dilution medicine (6.25 μ M, 12.5 μ M, 25 μ M, 50 μ M), put in incubator and continue to cultivate.24h after dosing, sucks supernatant, adds the serum free medium containing MTT (0.25mg/ml), cultivates 4 hours, sucks nutrient solution, adds 100 μ l DMSO vibrations to make Formazan dissolving crystallized, 490nm measure each hole light absorption value to calculate cell survival rate.Test-results is in Table-1.

(2) the PC12 cytoprotection test to Hydroperoxide injury

Choose logarithmic phase PC12 cell, with every hole 100 μ l, 3～4 * 10 ⁴individual/ml kind is planted in 96 well culture plates, and culture plate is put to 37 ℃, 5%CO ₂in incubator, hatch 24h.Experiment is divided into Normal group, positive controls, dosing group.Filter after substratum, get cell plate, carefully suck nutrient solution, change liquid dosing.The every hole of Normal group adds the DMEM nutrient solution of 100 μ l; The every hole of model group adds contains 800 μ MH ₂o ₂nutrient solution 100 μ l, the every hole of positive controls adds contains 800 μ M H ₂o ₂and the nutrient solution 100 μ l of 10 μ M vitamin-Es; The every hole of dosing group adds contains 800 μ MH ₂o ₂and the nutrient solution 100 μ l of 2.5,5,10 μ M test-compounds, after effect PC12 cell 6h, survey its survival rate.Suck supernatant, add the serum free medium containing MTT (0.25mg/ml), cultivate 4 hours, suck nutrient solution, add 100 μ l DMSO vibrations to make Formazan dissolving crystallized, 490nm measure each hole light absorption value to calculate cell survival rate test-results in Table-2.

3, test-results

(1) compound is to PC12 cell toxicity test result

Experimental result explanation, scutellarin and series compound thereof are with 6.25-50 μ M effect PC12 cell 24h, less to its growth effect.Wherein SC and scutellarin-6 " methyl esters 4 '-aspartic acid carbamate, scutellarin-6 " methyl esters 4 '-phenylalanine ammonia carbamate, scutellarin-6 " methyl esters 4 '-L-glutamic acid carbamate scutellarin-6 " methyl esters 4 '-leucine aminopeptidase carbamate cell growth do not make significant difference.(table-1)

(2) provide protection of compound to the PC12 cell of Hydroperoxide injury

By MTT, investigate cell survival rate and evaluate scutellarin and the provide protection of series compound to the PC12 cell of Hydroperoxide injury thereof.Experimental result explanation, the scutellarin of 1-25 μ M and series compound and H ₂o ₂jointly with effect PC12 cell 6h, the oxidative damage that test-compound all can protect superoxide to produce PC12 cell in the mode of acellular poison.Wherein " methyl esters 4 '-aspartic acid carbamate effect is better than positive drug vitamin-E, scutellarin-6, and " methyl esters 4 '-leucine aminopeptidase carbamate acts on and is better than positive drug vitamin-E under high density in scutellarin-6.(table-2)

Tab.2The?liability?ofthe?SC’s?compounds?on?PC12cells?treated?with?the?defined?concentrations?for6h.(n=6).

Tab.1The?anti-proliferation?effects?ofthe?SC’s?compounds?on?PC12cells?treated?with?the?defined?concentrations?for24h?by?MTT?assay.Each?value?indicates?the?mean±S.E.of?three?samples(n=6)，

Each?value?indicates?the?mean±S.E.of?two?samples

Embodiments of the invention 14: the medicine of preparation treatment nerve degenerative diseases, get the compound that 5g embodiment 1 prepares, by its medical starch 75g, Microcrystalline Cellulose 20g, medical starch is first dry, cross 120 mesh sieves, then mix with Microcrystalline Cellulose, cross twice 120 mesh sieves, insert in hard capsule, make 1000 capsules of the present invention.Every hard capsule is containing main ingredient composition 0.5mg.

Embodiments of the invention 15: the medicine of preparation treatment nerve degenerative diseases, get the compound that 5g embodiment 10 prepares, by itself and hypromellose 6g, carboxymethylstach sodium 10g, Microcrystalline Cellulose 8g, lactose 115g, starch 50g, Magnesium Stearate 1g; Main ingredient and auxiliary material are fully mixed in rear input homogenizer, and it is appropriate that spraying adds water, whole grain, and moisture controlled is 3～4%, and then compressing tablet, makes 1000, film coating.

Embodiments of the invention 16: the medicine of preparation treatment nerve degenerative diseases, get the compound that 5g embodiment 13 prepares, add 400ml Macrogol 200 to dissolve, add again appropriate distilled water to dilute, and then add appropriate sucrose and adjust volume to 1000ml, stir evenly, filter, every of filling one-tenth 10ml or 20ml, sterilising packaging.

Unless dated especially, term used herein has to give a definition:

" alkyl " represents saturated or undersaturated, substituted or non-substituted straight chain, branched alkane hydrocarbon chain, concrete enumerates as methyl, ethyl, propyl group, sec.-propyl, butyl, isobutyl-, sec-butyl, the tertiary butyl, amyl group, isopentyl, neo-pentyl, tert-pentyl, 1-methyl butyl, 2-methyl butyl, 2-methyl-propyl, hexyl, isohexyl, 1-methyl amyl, 2-methyl amyl, 3-methyl amyl, 2-methyl butyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl etc.In these groups, the alkyl that the carbonatomss such as methyl, ethyl, propyl group, sec.-propyl, butyl of take are 1_4, as good, be take methyl, sec.-propyl, 2-methyl-propyl, 3-methyl-propyl, carboxymethyl and propyloic as best.

Claims (9)

1. scutellarin and an aglycon carbamate derivatives thereof, is characterized in that: its structural formula is:

Wherein, R is that carbonatoms is 1-4 alkyl, the aryl that carbonatoms is 7 or carboxylic alkyl.

2. a scutellarin as claimed in claim 1 and aglycon carbamate derivatives thereof the application in the medicine of preparation treatment nerve degenerative diseases.

3. a preparation method for scutellarin as claimed in claim 1 and aglycon carbamate derivatives thereof, is characterized in that: comprise the following steps:

1) scutellarin-6 " synthesizing of methyl esters-4 '-carbamate derivates;

By the amino acid isocyanic ester of scutellarin methyl esters and tertiary butyl protection in non-protonic solvent, under nitrogen protection, under evaluation condition, react and obtain scutellarin-6 take triethylamine " carbamate derivates of methyl esters 4 '-tertiary butyl protection; these gains remove the tertiary butyl through trifluoroacetic acid below at 0 ℃, obtain target compound;

2) preparation of Scutellarein 4 '-amino amino manthanoate

The amino acid salts hydrochlorate that the tertiary butyl is protected and two-4 nitrophenyl carbonate are in non-protonic solvent, reaction produces after active ester intermediate, add 6,7-ketal protection Scutellarein intermediate, continue reaction and obtain 6, the amino amino manthanoate of Scutellarein-4 of 7-ketal protection '-tertiary butyl protection, these gains remove the tertiary butyl through trifluoroacetic acid below at 0 ℃, obtain target compound;

3) preparation of Scutellarein 7-methyl ether 4 '-amino amino manthanoate

1. Scutellarein 7-methyl ether is synthetic

The Scutellarein that is 1:3.7 by mol ratio and anhydrous sodium acetate are under excessive diacetyl oxide existence condition, and at 135-140 ℃, heating reflux reaction prepares the Scutellarein of full acidylate; The Scutellarein of the full acidylate that is 1:16 by mol ratio and Anhydrous potassium carbonate, under methyl iodide existence condition, reflux in anhydrous propanone, the Scutellarein 7-methyl ether of the full acidylate of reaction preparation, and temperature of reaction is 55-60 ℃; Gains 0 ℃ of low temperature in the saturated methanol solution of ammonia removes below ethanoyl reaction and prepares Scutellarein 7-methyl ether, and the mol ratio of ammonia and methyl alcohol is 1:1.38;

2. Scutellarein 7-methyl ether 4 '-amino amino manthanoate is synthetic

By the amino acid isocyanic ester of Scutellarein 7-methyl ether and tertiary butyl protection in non-protonic solvent, the mol ratio of Scutellarein 7-methyl ether and amino acid isocyanic ester is 1:1.5, under nitrogen protection, under catalyzer condition, react the carbamate derivates that 10h obtains the protection of the Scutellarein 7-methyl ether 4 '-tertiary butyl take triethylamine, the trifluoroacetic acid that is 10% through mass concentration by these gains removes the tertiary butyl below at 0 ℃, obtains target compound;

。

4. the preparation method of scutellarin as claimed in claim 3 and aglycon carbamate derivatives thereof; it is characterized in that: in step 1), the molar ratio of the amino acid isocyanic ester of scutellarin methyl esters and tertiary butyl protection is 1.0 ~ 3.5:1; temperature of reaction is 30-80 ℃, and the reaction times is 4 ~ 48.

5. as claim: the preparation method of the scutellarin as described in 4 and aglycon carbamate derivatives thereof; it is characterized in that: the molar ratio 1.0 ~ 2.0:1 of the amino acid isocyanic ester of scutellarin methyl esters and tertiary butyl protection; temperature of reaction is 45-65 ℃, and the reaction times is 5 ~ 15.

6. the preparation method of scutellarin as claimed in claim 3 and aglycon carbamate derivatives thereof; it is characterized in that: in step 2) in the amino acid salts hydrochlorate of tertiary butyl protection and the molar ratio of two-4 nitrophenyl carbonate be 0.5 ~ 2.5:1, at 10 ~ 40 ℃, react 5 ~ 36 hours.

7. the preparation method of scutellarin as claimed in claim 3 and aglycon carbamate derivatives thereof, is characterized in that: 1. molar ratio 1:8 ~ 12 of Scutellarein and diacetyl oxide in step 3); The mol ratio of Scutellarein and anhydrous sodium acetate is 1:3 ~ 4, and in 10 ~ 30 minutes reaction times, reaction need be carried out under anhydrous condition; Molar ratio 1:15 ~ 20 of full acidylate aglycon and methyl iodide, reflux time 10 ~ 15 hours; NH in full acidylate aglycon 7-methyl ether and the saturated methyl alcohol of ammonia while sloughing acyl group ₃molar ratio is 1:1.2 ~ 1.5,5.5 ~ 6 hours reaction times.

8. the preparation method of scutellarin as claimed in claim 6 and aglycon carbamate derivatives thereof; it is characterized in that: in step 2) in the amino acid salts hydrochlorate of tertiary butyl protection and the molar ratio of two-4 nitrophenyl carbonate be 0.8 ~ 1.5:1, at 15 ~ 25 ℃, react 6 ~ 24 hours.

9. the preparation method of scutellarin as claimed in claim 3 and aglycon carbamate derivatives thereof, is characterized in that: described non-protonic solvent is anhydrous tetrahydro furan.

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