CN104945281B - Flavone acetate derivatives, and pharmaceutical composition, preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of medical chemical industry, and relates to flavone acetate derivatives, and a pharmaceutical composition, preparation method and application thereof, particularly compounds disclosed as Formula I or Formula II. The compounds have strong actions on inducing tumor cells to generate TNF-alpha and breaking vessels in the chick chorioallantoic membrane, are an effective tumor vessel inhibitor or breaker, and have the prospects in preparing antineoplastic drugs.

Description

Flavone acetic acid analog derivative, its pharmaceutical composition, Preparation Method And The Use

Technical field

The invention belongs to field of medicine and chemical technology, is related to a kind of flavone acetic acid analog derivative, its pharmaceutical composition, its preparation side Method and purposes.

Background technology

Tumor needs to provide oxygen, nutriment for it and remove metabolite in time by functional blood vessel network.Except Part blood vessel can be obtained by integrating with host blood vessel, tumor must also build the blood of oneself by forming new vesselses net Guard system could constantly advolution.If providing oxygen and nutriment without vascular system, the growth of solid tumor will not surpass Cross 1mm³.In view of its important function in tumor development, tumor vessel has become an important target of antineoplaston Point.

Tumor vessel disrupting agent can be quick and selectively destroy tumor vascular medicine.It utilizes tumor vessel The difference existed with Normal tissue vascular optionally destroys tumor vessel, or by being capable of the tumor vascular part of specific bond Toxin, blood coagulation derivant, apoptosis induction molecule etc. are transported to into tumor vessel, so as to quickly and selectively damaging or blocking The tumor vessel for completing has been built.For treatment blood vessel has formed tumor, tumor vessel disrupting agent has significant curative effect.Small molecule Tumor vessel disrupting agent can be quick, widely destroys the tumor vessel for having been formed, and becomes the focus of research.At present by The tumor vessel disrupting agent generally studied includes Colchicine, combretastatin A-4 P, flavone acetic acid class etc., has been applied to non-little The solid tumors such as cell lung cancer, breast carcinoma treatment (Disrupting Tumour Blood Vessels, Nature Reviews, June 2005 Volume 5：423-435).

Flavonoid tumor vessel disrupting agent is one kind of small molecule tumor vessel disrupting agent, generally believes that it is to pass through at present Directly destroy tumor vascular endothelial cell and promote tumor tissue cell release tumor necrosis factor α (TNF-α) etc. to play indirectly Destroy tumor vascular effect.Flavone acetic acid (FAA) is the flavonoid tumor vessel disrupting agent being found earliest, and it is by induction Tumor cell produces TNF-α, significantly raises the concentration of the TNF-α in tumor microenvironment, makes the vascular endothelial cell of tumor by local Generation apoptosis plays a role, and also can avoid the general toxic reaction of TNF-α.But FAA is only in mouse tumor model Effectively, do not have antitumaous effect in clinical trial.

DMXAA (DMXAA) is the FAA derivants with greater activity, and activity is better than FAA (research finds that FAA is only effective in animal level), studies have shown that it only has the reversible toxicity of slight dosage.Additionally, in III DMXAA fails the time-to-live for significantly extending patient in clinical trial phase.

Still needing at present will develop new antitumoral compounds.

The content of the invention

The present inventor has obtained a kind of new flavone acetic acid analog derivative through in-depth study and performing creative labour. Surprisingly, it was found that such compound is effective tumor vessel disrupting agent, produce with stronger inducing tumor cell Raw TNF-α effect, and chick chorioallantoic membrane can be used for preparing antitumor drug when the destruction of medium vessels.By This provides following inventions：

One aspect of the present invention is related to the compound shown in Formulas I, or its officinal salt or ester,

Wherein：

R₁Selected from H, C₁-C₆Alkyl；

When the aceticoceptor in female ring is located at R₁During position, R₁Do not exist；

R₂Selected from the phenyl that phenyl, one or more (such as 2,3,4 or 5) hydroxyls replace, and selected from following base Group：

Specifically, for R₂Be except phenyl ring it Outer above-mentioned other substituent groups, R₂It is the unsubstituted any site on the phenyl ring in R2 with the link position of female ring；

R₃Selected from H ,-OH.

Another aspect of the present invention is related to the compound shown in Formula II, or its officinal salt or ester,

Wherein：

R₁Selected from H, C₁-C₆Alkyl；

When the aceticoceptor in female ring is located at R₁During position, R₁Do not exist；

R₂Selected from the phenyl that phenyl, one or more (such as 2,3,4 or 5) hydroxyls replace, and selected from following base Group：

Specifically, for R₂It is except phenyl ring Outside above-mentioned other substituent groups, R₂It is the unsubstituted any site on the phenyl ring in R2 with the link position of female ring；

R₃Selected from H ,-OH；

Represent singly-bound or double bond.

Formulas I or Formula II compound according to any one of the present invention, or its officinal salt, wherein,

R₁Independently selected from H, C₁-C₃Alkyl；Specifically, the C₁-C₃Alkyl is methyl, ethyl, propyl group or isopropyl；It is excellent Selection of land, R₁It independently is H or methyl.

Formulas I or Formula II compound according to any one of the present invention, or its officinal salt, wherein,

R₂Independently selected from Where the dotted line signifies that R₂With the link position of female ring.

Formulas I or Formula II compound according to any one of the present invention, or its officinal salt, wherein it is preferred to, the formula I or Formula II compound do not include following 4 compounds：

And

Formulas I or Formula II compound according to any one of the present invention, wherein, the ester is the aceticoceptor shape in female ring Into ester；Specifically, it is the aceticoceptor and C₁- C₆The ester that saturated monohydroxy alcohol (alkanol) is formed；Specifically, the C₁- C₆ Saturated monohydroxy alcohol is methanol, ethanol, propanol, isopropanol, n-butyl alcohol, amylalcohol or hexanol.

The present inventors have additionally discovered that, the ester of the present invention is also effective tumor vessel disrupting agent, with stronger induced tumor Cell produces TNF-α effect, and chick chorioallantoic membrane can be used for preparing antineoplastic agent when the destruction of medium vessels Thing.

Described Formulas I or Formula II compound, or its officinal salt or ester, wherein, the Formulas I or Formula II compound are selected from down The table 1 in face：

Table 1：The part of compounds of the present invention

Another aspect of the invention is related to the preparation method of the compound of formula I any one of the present invention, including following Step：

(1) preparation of A fragments：

(2) preparation of B2, B3, B4, B5, B6 fragment：

(3) with ethanol as solvent, in the presence of 60% potassium hydroxide aqueous solution, by A fragments respectively with B₁Fragment isB₂Fragment, B₃Fragment, B₄Fragment, B₅Fragment or B₆Fragment reaction obtains compound of formula I；

Wherein, dotted portion represents R₁。

Preparation method according to any one of the present invention, wherein, the step (1) includes：

A) withObtain with paraformaldehyde and concentrated hydrochloric acid reaction for raw material

B) in organic solvent (such as toluene), in the presence of reagent (such as tetrabutyl ammonium bromide), make With Cyanogran. reaction, obtainFurther hydrolysis, obtains A fragments(for example)。

Preparation method according to any one of the present invention, wherein, the step (2) includes：

c)B₁FragmentWith B₂FragmentCan directly buy；

d)B₂Fragment is reacted with bromo iso-amylene under the conditions of solution of potassium carbonate, B when obtaining₃Fragment

E) B is made₃Fragment is reacted with methoxy chlorine, obtains B₄Fragment

F) by B₄Fragment respectively with p-methyl benzenesulfonic acid, chloro- 5, the 6- dicyanos-benzoquinones of 2,3- bis-, obtain B₅FragmentB₆Fragment

Detailed teachings of the invention and existing synthesis chemical knowledge, those skilled in the art can easily close Into the compound of formula I of the present invention.

Another aspect of the invention is related to the preparation method of the Formula II compound any one of the present invention, and it includes this The method of the preparation compound of formula I any one of invention, and also including：

(4) Formula II compound is obtained by compound of formula I；Specifically comprise the steps：

By compound of formula I Jing DMSO/I₂Reaction under/microwave condition obtains flavone compound (such as reference implementation example 17)；The reaction under TFA/ microwave conditions obtains flavone alkyl compound (such as reference implementation example 35)；In hydrogen peroxide/NaOH bars Reaction under part obtains Formula II compound (such as reference implementation example 53).

In one embodiment, the present invention relates to prepare the preparation method of the compound shown in formula I, Formula II.Specifically Ground is said, the invention provides the method for preparing the compound shown in formula I, Formula II, comprises the following steps：

1) synthesis of A fragments

Such as reaction scheme, with o-hydroxyacetophenone initiation material, it is obtained into 2- hydroxyls with paraformaldehyde and concentrated hydrochloric acid reaction Base -3- chloromethyls -1-Phenylethanone. and 2- hydroxyls -5- chloromethyls -1-Phenylethanone., by 2- hydroxyls -3- chloromethyls -1-Phenylethanone. toluene is dissolved in In, 2- hydroxyl -3- cyano-acetophenones are obtained with Cyanogran. reaction in the presence of tetrabutyl ammonium bromide, by itself and strong sulfuric acid response Obtain A₁Fragment 2- (3- acetyl group -2- hydroxyphenyl) acetic acid, same method obtains A₂Fragment 2- (3- acetyl group -4- hydroxyphenyl) second Acid.

With 2- hydroxy-5-methyls base -1-Phenylethanone. as raw material, operational approach ibid, obtains A₃Fragment 2- (3- acetyl group -2- hydroxyls Base -5- tolyls) acetic acid.

2) synthesis of B fragments

Such as reaction scheme, B₂Fragment is reacted with bromo iso-amylene in wet chemical, obtains B₃Fragment；It is with acetone Solvent, B₃Fragment is reacted with methoxy chlorine, obtains B₄Fragment；With toluene as solvent, B₄Fragment respectively with 2,3- bis- chloro- 5, 6- dicyanos-benzoquinone, p-methyl benzenesulfonic acid reaction, respectively obtain B₅Fragment, B₆Fragment.

C) with ethanol as solvent, in the presence of 60% potassium hydroxide aqueous solution, A₁Fragment, A₂Fragment, A₃Fragment respectively with B₁Fragment, B₂Fragment, B₃Fragment, B₄Fragment, B₅Fragment, B₆Fragment reaction obtains compound of formula I, and compound of formula I is by corresponding Reaction obtains Formula II compound.

Embodiment is also shown with regard to preparing the more detailed data of formula I, II compound.

Another aspect of the invention is related to a kind of pharmaceutical composition, and it includes the Formulas I or formula any one of the present invention II compound or pharmaceutically acceptable salt thereofs, and optional pharmaceutically acceptable adjuvant；Alternatively, it also includes lipopolysaccharide.

Term " compositionss " means to include the product of each specified composition comprising specified amount, and directly or indirectly from specifying Any product that the combination of each specified composition of amount is produced.

The compounds of this invention that generally pharmaceutical composition of the present invention contains 0.1-90 weight % and/or its pharmaceutically can connect The salt received.Pharmaceutical composition can be prepared according to methods known in the art.When for this purpose, if it is desired, can be by the present invention Compound and/or its pharmaceutically acceptable salt are combined with one or more solid or liquid pharmaceutical excipients and/or adjuvant, system Into can be used as the appropriate administration form of people or dosage form.

The compound or the pharmaceutical composition containing it of the present invention can be administered in a unit, and route of administration can be intestinal Road or non-bowel, such as oral, muscle, subcutaneous, nasal cavity, oral mucosa, skin, peritoneum or rectum.Form of administration such as tablet, Capsule, drop pill, aerosol, pill, powder, solution, suspensoid, Emulsion, granule, liposome, transdermal agent, buccal tablet, bolt Agent, lyophilized injectable powder etc..Can be ordinary preparation, slow releasing preparation, controlled release preparation and various particulate delivery systems.In order to by unit Form of administration makes tablet, can widely use various carriers well known in the art.It is with regard to the example of carrier, for example diluent With absorbent, such as starch, dextrin, calcium sulfate, Lactose, Mannitol, sucrose, Sodium Chloride, glucose, carbamide, Calcium Carbonate, white pottery Soil, Microcrystalline Cellulose, aluminium silicate etc.；Wetting agent and binding agent, such as water, glycerol, Polyethylene Glycol, ethanol, propanol, starch slurry, paste Essence, syrup, Mel, glucose solution, mucialga of arabic gummy, gelatine size, sodium carboxymethyl cellulose, lac, methylcellulose, phosphoric acid Potassium, polyvinylpyrrolidone etc.；Disintegrating agent, for example, be dried starch, alginate, agar powder, laminaran, sodium bicarbonate and Chinese holly Rafter acid, Calcium Carbonate, Polyethylene oxide, sorbitan fatty acid ester, dodecyl sodium sulfate, methylcellulose, ethyl cellulose etc.； Disintegration inhibitor, such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil and fat etc.；Absorption enhancer, such as quaternary ammonium salt, 12 Alkyl sodium sulfate etc.；Lubricant, such as Pulvis Talci, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, poly- second Glycol etc..Tablet can also further be made coated tablet, such as sugar coated tablet, thin membrane coated tablet, ECT, or it is double-deck Piece and multilayer tablet.In order to administration unit is made into pill, various carriers well known in the art can be widely used.With regard to carrier Example is, such as diluent and absorbent, such as glucose, Lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidine Ketone, Gelucire, Kaolin, Pulvis Talci etc.；Binding agent such as arabic gum, Tragacanth, gelatin, ethanol, Mel, liquid sugar, rice paste Or batter etc.；Disintegrating agent, such as agar powder, it is dried starch, alginate, dodecyl sodium sulfate, methylcellulose, ethyl cellulose Element etc..In order to administration unit is made into suppository, various carriers well known in the art can be widely used.With regard to the example of carrier It is, for example Polyethylene Glycol, lecithin, cocoa butter, higher alcohol, the ester of higher alcohol, gelatin, semi-synthetic glyceride etc..In order to give Capsule is made by prescription unit, and effective ingredient compound or pharmaceutically acceptable salt thereof is mixed with above-mentioned various carriers, and will thus obtain Mixture be placed in hard obviously capsule or soft capsule.Also effective ingredient compound or pharmaceutically acceptable salt thereof can be made microcapsule Agent, is suspended in aqueous medium and forms suspensoid, also can be fitted in hard capsule or make injection application.In order to by administration unit Make injection preparation, such as solution, Emulsion, lyophilized injectable powder and suspensoid, it is possible to use all dilutions commonly used in the art Agent, for example, water, ethanol, Polyethylene Glycol, 1,3-PD, the isooctadecanol of ethoxylation, polyoxygenated isooctadecanol, polyoxy Ethylene Span etc..In addition, in order to prepare isotonic injection, appropriate chlorination can be added in injection preparation Sodium, glucose or glycerol, further, it is also possible to add conventional cosolvent, buffer agent, pH adjusting agent etc..

Additionally, if desired, can also add in pharmaceutical preparation coloring agent, preservative, spice, correctivess, sweeting agent or Other materials.

The present invention compound, or the dosage of its officinal salt depend on many factors, for example to be prevented or be controlled The property for the treatment of disease and the sex of the order of severity, patient or animal, age, body weight and individual reaction, particular compound used, Route of administration and administration number of times etc..Above-mentioned dosage with ingle dose form or can be divided into several, such as two, three or four dosage shapes Formula is administered.

The actual dose level of each active component in pharmaceutical composition of the present invention can be changed, so as to the reactive compound of gained Amount effectively can obtain required therapeutic response for concrete patient, compositionss and administering mode.Dosage level must be according to materialization The patient's condition and medical history of the activity of compound, route of administration, the order of severity for treating the patient's condition and patient to be treated is selecting. But, the way of this area is, the dosage of compound from the beginning of less than the level for obtaining required therapeutic effect and requiring, gradually Increasing dosage, until obtaining required effect.

Another aspect of the invention be related to any one of Formulas I or Formula II compound or pharmaceutically acceptable salt thereof or this Purposes of the bright pharmaceutical composition in the medicine described in following any one is prepared：

1) medicine for for example raising TNF ＆ alpha levels is adjusted；

2) inducing tumor cell produce tumor necrosis factor α medicine '

3) destroy tumor vessel or suppress the medicine of Tumor angiogenesis；

4) medicine of tumor cell or vascular endothelial cell is suppressed；And

5) purposes in the medicine for the treatment of and/or prevention and/or adjuvant therapy of tumors；

Specifically, the tumor cell is colon cancer cell (such as DLD-1 human colon cancer cells), the macrophages of RAW 264.7 Cell, breast cancer cell (such as MDA-MB-231 breast cancer cells) or cervical cancer cell (such as Hela cervical cancer cells).

Specifically, the tumor is pulmonary carcinoma, breast carcinoma, cervical cancer or colon cancer.

Another aspect of the invention is related to a kind of method selected from described in following any one, including using effective dose this The step of pharmaceutical composition of Formulas I or Formula II compound or pharmaceutically acceptable salt thereof or the present invention any one of bright：

1) method for for example raising TNF ＆ alpha levels is adjusted in vivo or in vitro；

2) method that in vivo or in vitro inducing tumor cell produces tumor necrosis factor α；And

3) method for suppressing tumor cell for example to suppress tumor cell proliferation and/or migration in vivo or in vitro.

In one embodiment of the invention, above-mentioned method in vitro is non-treatment purpose.

Another aspect of the invention be related to a kind for the treatment of and/or prevent and/or adjuvant therapy of tumors method, including using The pharmaceutical composition of Formulas I or Formula II compound or pharmaceutically acceptable salt thereof or the present invention any one of the present invention of effective dose The step of；Specifically, the tumor is pulmonary carcinoma, breast carcinoma, cervical cancer, colon cancer.

Term " effective dose " is referred to can be realized treating, prevent, mitigate and/or alleviating disease of the present invention in experimenter Or the dosage of disease.

Term " experimenter " can refer to patient or other receive the present composition to treat, prevent, mitigate and/or delay Solve the animal of disease of the present invention or disease, particularly mammal, such as people, Canis familiaris L., monkey, cattle, horse etc..

Term " disease and/or disease " refers to a kind of condition of the experimenter, the condition and institute of the present invention State disease and/or disease is relevant.

It is to be understood that total consumption per day of the compounds of this invention or pharmaceutical composition must be by attending physician in reliable medical science Make decision in determination range.For any specific patient, specific treatment effective dose level must be according to many factors Fixed, the factor includes treated obstacle and the order of severity of the obstacle；The activity of the particular compound for being adopted；Adopted Concrete composition；Age of patient, body weight, general health, sex and diet；The administration of the particular compound for being adopted Time, route of administration and excretion rate；The treatment persistent period；It is applied in combination with the particular compound for being adopted or medicine used at the same time Thing；And similar factor known to medical field.For example, the way of this area is that the dosage of compound is from less than needed for obtaining Therapeutic effect and desired level starts, gradually increasing dosage, until obtaining required effect.It is, in general, that the change of the present invention Compound can be between 0.001-1000mg/kg body weight/days, such as between 0.01- for the dosage that mammal is particularly people 100mg/kg body weight/days, such as between 0.01-10mg/kg body weight/days.

Compound of the invention can effectively prevent and/or treat various diseases of the present invention or disease.

Another aspect of the invention is related to selected from following midbody compound：

And

Another aspect of the invention be related to heretofore described arbitrary midbody compound prepare antitumor drug or Person is preparing Formulas I or the purposes in Formula II compound described in any one of the present invention.

The beneficial effect of the invention

The compound of the present invention only has the effect that stronger inducing tumor cell produces TNF-α, and Embryo Gallus domesticus fine hair is urinated Cyst membrane, when the destruction of medium vessels, is effective tumor vessel inhibitor or disrupting agent, with for preparing antineoplastic agent The prospect of thing.Additionally, the compound of the present invention is independent of LPS approach also there is TNF-α to promote secretory action.

Description of the drawings

Fig. 1：Control (control), DMXAA, the tumor body picture of the compound of embodiment 2.Top rule can read tumor body Size.

Fig. 2：Tumor volume changes.*P<0.05.n=3-5 Mus/group.mean±SEM.

Fig. 3：(Hoshest 33342 is dyeed) is acted on to angiolysis.Fig. 3 A, control (control)；Fig. 3 B, DMXAA； Fig. 3 C, the compound of embodiment 2.

Specific embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is unreceipted concrete in embodiment Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, are Can pass through city available from conventional products.

The fusing point of compound is determined by RY-1 melting point apparatus, the non-calibration of thermometer.Mass spectrum is by Micromass ZabSpec high scores Resolution mass spectrograph (resolution 1000) is determined.¹H-NMR is determined by JNM-ECA-400 SUPERCONDUCTING NMRs instrument, operating frequency¹H-NMR 400MHz。

In the context of the present invention, using following abbreviation：

MOM:- methoxy

DMSO：Dimethyl sulfoxide

DDQ:The chloro- 5,6- dicyanos-benzoquinones of 2,3- bis-

TsOH:P-methyl benzenesulfonic acid

Intermediate is prepared below by preparation example 1-7.

Preparation example 1：The preparation of 2- hydroxyls -3- chloromethyls -1-Phenylethanone.

Reaction process：

Step 1：The preparation of o-hydroxyacetophenone derivant

Under mechanical agitation, o-hydroxyacetophenone 178g (1.30mol), paraformaldehyde 39.5g (1.30mol) and concentrated hydrochloric acid 800mL reacts 7 hours at 50-60 DEG C.Organic faciess, water phase are separated, water phase is extracted with toluene 200mL, dissolved with toluene 600mL Reaction system layer oily matter, merges organic faciess, and organic faciess use successively saturation NaHCO₃Aqueous solution 300mL × 2 time, saturated common salt Water 300mL is washed, anhydrous sodium sulfate drying.Filter, Rotary Evaporators are steamed after solvent, and to residual oil thing decompression steaming is carried out Evaporate, obtaining main distillate fraction has 211.4g, be the mixing of 2- hydroxyls -3- chloromethyls -1-Phenylethanone. and 2- hydroxyls -5- chloromethyls -1-Phenylethanone. Thing.Ratio according to 1g～5mL is added thereto to normal hexane, heating for dissolving.It is 2- hydroxyl -5- chloromethanes that crystal is separated out after cooling Base -1-Phenylethanone. 155.1g；Filtrate cold preservation (4 DEG C) after filtration has grease to separate out, and pours out supernatant, concentrates and placed after about half A large amount of crystal can be separated out, this crystal is 2- hydroxyls -3- chloromethyls -1-Phenylethanone. 38.3g.Total recovery 80.8%.

Preparation example 2：The preparation of 2- hydroxyls -5- cyano methyls -1-Phenylethanone.

Reaction process：

2- hydroxyls -5- chloromethyls -1-Phenylethanone. 37g (0.2mol) is dissolved in the middle of 500mL toluene (heating dissolution), adds four Butylammonium bromide 3.2g (20mmol), is stirred at room temperature down, and thereto Deca Cyanogran. 12.5g (0.255mol) is dissolved in 80mL water Central solution.Drip off within 20 minutes, 70 DEG C are reacted 1 hour.

Cooling, organic faciess are dried with 500mL × 2 water washing, organic faciess, are filtered, and are concentrated under reduced pressure to give a large amount of solids.Use stone Oily ether/ethyl acetate (5:1) recrystallization, obtains sterling 31.6g, yield 89.2%.

The preparation of 2- hydroxyls -3- cyano methyls -1-Phenylethanone. is with reference to aforesaid operations.

Preparation example 3：The preparation of 2- (3- acetyl group -4- hydroxyphenyl) acetic acid

Reaction process：

2- hydroxyls -5- cyano-acetophenone 8.7g (34.5mmol), adds 33% sulphuric acid 75mL (1:2 volume ratios dilute To), back flow reaction 1.5 hours.Cooling, adds cold water 100mL, places, and separates out a large amount of yellow solids.Washing is filtered, crude product is used Hot water recrystallization, heat filters off the removal of impurity, and filtrate separates out a large amount of faint yellow solids, is filtrated to get pure products 8.3g.Yield 87.7%.

The preparation of 2- (3- acetyl group -2- hydroxyphenyl) acetic acid is with reference to aforesaid operations.

Preparation example 4：The preparation of 4- hydroxyl -3- (3- methylene -2- alkene) benzaldehyde

Reaction process：

Potassium carbonate 140g (1mol) is dissolved in 200g water, after heat release terminates, adds para hydroxybenzene aldehyde 61.0g (0.5mol), stir to being completely dissolved.Again Deca bromo iso-amylene 60.0mL (0.51mol), drips off in 5 minutes.It is anti-under room temperature Should, TLC is detected 12 hours.

Reactant liquor is mutually discarded with the extraction of ether 250mL × 2, water；Ether layer merge, with 5% aqueous sodium carbonate 200mL × 3 washings, water is mutually discarded；Washed with 0.5N sodium hydrate aqueous solutions 200mL × 3 again, organic faciess are discarded；Water is acidified with concentrated hydrochloric acid Afterwards, with the extraction of ether 200mL × 3, it is dried, chromatographs post separation, petrol ether/ethyl acetate (5:1) 14.1g colorless oils, are obtained Thing, places slow solidification.Yield 14.8%.

Preparation example 5：The preparation of 4- (methoxymethoxy) -3- (3- methylene -2- alkene) benzaldehyde

Reaction process：

4- hydroxyl -3- (3- methylene -2- alkene) benzaldehyde 1.9g (10mmol), is dissolved in the middle of 50mL acetone, adds carbon Sour potassium 2.0g, ice bath stirring is lower to add methoxy chlorine 2.0mL (20mmol), drips off within 10 minutes, ice bath is then removed, normal The lower reaction of temperature 8 hours.Decompression is spin-dried for solvent, adds ether 30mL, respectively with water 20mL, 0.5N sodium hydroxide solution 20mL, full With saline solution 20mL washings, it is dried, filters, chromatographs post separation, petrol ether/ethyl acetate (8:1), colorless oil 2.1g is obtained, Placement gradually solidifies, yield 83.1%.

Preparation example 6：The preparation of 2,2- dimethyl -2H- .alpha.-5:6-benzopyran -6- formaldehyde

Reaction process：

4- (methoxymethoxy) -3- (3- methylene -2- alkene) benzaldehyde 1.90g (10mmol) is dissolved in 30mL toluene and works as In, DDQ (2,3- bis- chloro- 5,6- dicyanos-benzoquinone) 2.70g (12mmol) is added, it is heated to reflux 5 hours.Mistake after cooling Filter, filter cake is washed with toluene 10mL, and filtrate is washed successively with 15mL water, unsaturated carbonate potassium solution, saturated nacl aqueous solution.It is organic Mutually it is dried, filters, chromatographs post separation, petrol ether/ethyl acetate (10:1) product 0.80g, yield 44.4%, are obtained.

Preparation example 7：The preparation of 2,2- diformazan .alpha.-5:6-benzopyran -6- formaldehyde

Reaction process：

4- (methoxymethoxy) -3- (3- methylene -2- alkene) benzaldehyde 1.90g (10mmol) is dissolved in toluene 30mL and works as In, p-methyl benzenesulfonic acid 0.20g is added, flow back 3 hours.Cooled and filtered, filter cake is washed with toluene 10mL, and filtrate uses successively 15mL Water, unsaturated carbonate potassium solution, saturated nacl aqueous solution washing.Organic faciess are dried, and filter, and chromatograph post separation, petroleum ether/acetic acid second Ester (10:1) product 1.58g, yield 83.2%, are obtained.

Embodiment 2：2- (2- hydroxyl -3- (3- (4- hydroxy phenyls) acryloyl group) phenyl) acetic acid

By 2- (3- acetyl group -2- hydroxyphenyl) acetic acid 1.00g (5.18mmol) and para hydroxybenzene aldehyde 0.63g (5.18mmol) Mixing, adds ethanol 6mL dissolvings, adds 60% potassium hydroxide aqueous solution 8mL, room temperature reaction 4 hours.Diluted with frozen water 50mL, Extracted with ether 20mL, ether is mutually discarded.Under ice bath stirring, water hydrochloric acid is acidified to pH ≈ 2, there is a large amount of bright yellow solid analysis Go out, filtration drying, obtain 0.89g products, yield 92.2%.

¹H-NMR(DMSO-d⁶,400MHz)：3.586 (2H, s ,-CH ₂- COOH), 6.843-6.981 (3H, m, Ar-H× 3), 7.498-7.517 (1H, dd, Ar-H), 7.806-7.893 (4H, m, Ar-H× 4), 8.253-8.273 (1H, m, Ar-H), 10.232 (1H, s ,-OH), 12.292 (1H, s ,-OH), 13.492 (1H, s ,-COOH)。ESI-MS[M+1]:299.1。

Embodiment 3：2- (4- hydroxyl -3- (3- (4- (hydroxy phenyl) acryloyl group) phenyl) acetic acid

The present embodiment compound is prepared with reference to the synthetic method of embodiment 2, except for the difference that using 2- (3- acetyl group -4- hydroxyls Phenyl) acetic acid 1.00g (5.18mmol) replaces corresponding initiation material, obtains product 1.45g, yield 94.1%.

¹H-NMR(DMSO-d⁶,400MHz)：3.603 (2H, s ,-CH ₂- COOH), 6.778-7.018 (3H, m, Ar-H× 3), 7.326-7.465 (2H, m, Ar-H× 2), 7.658-7.823 (3H, m, Ar-H× 3), 8.143-8.148 (1H, d, J= 2Hz, Ar-H), 10.231 (1H, s ,-OH), 12.367 (1H, s ,-OH), 12.729 (1H, s ,-COOH)。ESI-MS[M+1]: 298.0。

Embodiment 4：2- (2- hydroxyl -3- (3- (4- hydroxy phenyls) acryloyl group) -5- aminomethyl phenyls) acetic acid

The present embodiment compound is prepared with reference to the synthetic method of embodiment 2, except for the difference that 2- (3- acetyl group -2- hydroxyl -5- Tolyl) acetic acid 1.00g (4.83mmol) replaces corresponding initiation material, obtains product 1.43g, yield 88.5%.

¹H-NMR(DMSO-d⁶,400MHz)：2.325 (3H, s ,-CH ₃), 3.552 (2H, s ,-CH ₂- COOH), 6.855- 6.877 (2H, d, J=8.8Hz, Ar-H× 2), 7.336-7.340 (1H, d, J=1.6Hz, Ar-H), 7.818-7.934 (4H, M, Ar-H× 4), 8.093-8.096 (1H, d, J=1.2Hz, Ar-H), 10.235 (1H, s ,-OH), 12.280 (1H, s ,-OH), 13.310 (1H, s ,-COOH)。ESI-MS[M+1]:313.0。

Embodiment 9：2- (3- (3- (2,2- dimethyl -6- bases) acryloyl group) -2- hydroxy phenyls) acetic acid

The present embodiment compound is prepared with reference to the synthetic method of embodiment 2, except for the difference that using 2- (3- acetyl group -4- hydroxyls Phenyl) acetic acid 1.65g (8.55mmol) and 4- (methoxymethoxy) -3- (3- methylene -2- alkene) benzaldehyde 2.00g (8.55mmol) replace corresponding initiation material, obtain product 1.30g, yield 37.14%.

¹H-NMR(DMSO-d⁶,400MHz)：1.688-1.730 (6H, d, J=16.8Hz ,-CH ₃× 2), 3.323 (1H, S ,-CH ₂One of), 3.391 (3H, s ,-CH ₃), 3.618 (2H, s ,-CH ₂- COOH), 5.276-5.317 (3H, m ,-OCH ₂；- CH), 6.943-6.964 (1H, d, J=8.4Hz, Ar-H), 7.111-7.133 (1H, d, J=8.8Hz, Ar-H), 7.444- 7.471 (1H, dd, Ar-H), 7.713-7.775 (2H, m, Ar-H× 2), 7.814-7.893 (2H, m, Ar-H× 2), 8.110- 8.115 (1H, d, J=2Hz, Ar-H), 12.591 (2H, s ,-OH；-COOH)。ESI-MS[M+1]:411.5。

Embodiment 10：2- (2- (2- hydroxyl -3- (3- (4- (methoxymethoxy) -3- (3- methyl but-2-ene bases) phenyl) Acryloyl group) -5- aminomethyl phenyls) acetic acid

The present embodiment compound is prepared with reference to the synthetic method of embodiment 2, except for the difference that using 2- (3- acetyl group -2- hydroxyls Base -5- tolyls) acetic acid 1.77g (8.55mmol) and 4- (methoxymethoxy) -3- (3- methylene -2- alkene) benzaldehyde 2.00g (8.55mmol) replaces corresponding initiation material, obtains product 1.71g, yield 47.2%.

¹H-NMR(DMSO-d⁶,400MHz)：1.695-1.741 (6H, d, J=18.4 ,-CH ₃× 2), 2.341 (3H, s ,- CH ₃), 3.334-3.415 (5H, m ,-CH ₂-CH；-OCH₃), 3.562 (2H, s ,-CH ₂- COOH), 5.287-5.326 (3H, m ,- OCH ₂；-CH₂-CH), 7.116-7.139 (1H, d, J=9.2Hz, Ar-H), 7.357-7.360 (1H, d, J=1.2Hz, Ar-H), 7.776-7.859 (3H, m, Ar-H× 3), 7.943-7.982 (1H, d, J=15.6Hz, Ar-H), 8.089 (1H, s, Ar-H), 12.296 (1H, s ,-OH), 13.245 (1H, s ,-COOH)。ESI-MS[M+1]:425.2。

The synthesis of embodiment 1,5,6,7,8,11,12,13,14,15,16,68 is with reference to embodiment 2, such as table 2 below.

Table 2：With reference to embodiment it is 2-in-1 into part of compounds

Embodiment 17：2- (2- (4- hydroxy phenyls) -4- oxo -4H- .alpha.-5:6-benzopyran -8- bases) acetic acid

By 2- (2- hydroxyl -3- (3- (4- hydroxy phenyls) acryloyl group) phenyl) acetic acid 89.4mg (0.3mmol), it is dissolved in In the middle of 2mL DMSO, 3mg I are added₂, microwave reaction 10 minutes, 150 DEG C of design temperature.Extracted with ethyl acetate 15mL and water 15mL Take, organic faciess are dried, filter, concentration chromatographs post separation, methylene chloride/methanol (20:1) yellow solid 67mg, yield, are obtained 54.4%.

¹H-NMR(DMSO-d⁶,400MHz)：3.987 (2H, s ,-CH ₂- COOH), 6.859-6.943 (3H, m, Ar-H× 3), 7.400-7.438 (1H, t, J=7.6Hz, Ar-H), 7.714-7.732 (1H, dd, Ar-H), 7.920-7.961 (3H, m, Ar-H× 3), 10.284 (1H, s ,-OH), 12.544 (1H, s ,-COOH).ESI-MS[M+1]:297.1。

Embodiment 18：2- (3- hydroxyl -2- (4- hydroxy phenyls) -4- oxo -4H- .alpha.-5:6-benzopyran -8- bases) acetic acid

The present embodiment compound is prepared with reference to the synthetic method of embodiment 17, except for the difference that using 2- (4- hydroxyl -3- (3- (4- hydroxy phenyls) acryloyl group) phenyl) acetic acid 0.3g (1.01mmol)

For initiation material, product 0.16mg, yield 60.4% are obtained.

¹H-NMR(DMSO-d⁶,400MHz)：3.757 (2H, s ,-CH ₂- COOH), 6.862-6.955 (3H, m, Ar-H× 3), 7.699-7.701 (2H, d, J=0.8Hz, Ar-H× 2), 7.922-7.975 (3H, m, Ar-H× 3), 10.318 (1H, s ,- OH), 12.444 (1H, s ,-COOH).ESI-MS[M+1]:297.1。

Embodiment 20：2- (2- (4- hydroxyl -3- (3- methyl but-2-ene bases) phenyl) -4- oxo -4H- .alpha.-5:6-benzopyran -8- Base) acetic acid

The present embodiment compound is prepared with reference to the synthetic method of embodiment 17, except for the difference that using 2- (2- hydroxyl -3- (3- (4- hydroxyl -3- (3- methyl but-2-ene bases) phenyl) acryloyl group) phenyl) acetic acid 0.25g (0.68mmol) be initiation material, Obtain product 135mg, yield 54.4%.

¹H-NMR(DMSO-d⁶,400MHz)：1.719 (6H, s ,-CH ₃× 2), 3.981 (2H, s ,-CH ₂- COOH), 5.340-5.378 (1H, t, J=7.6Hz ,-CH₂-CH), 6.851-6.951 (2H, m, Ar-H× 2), 7.402-7.439 (1H, T, J=7.2Hz, Ar-H), 7.711-7.804 (3H, m, Ar-H× 3), 7.933-7.956 (1H, dd, Ar-H), 10.266 (1H, S ,-OH), 12.585 (1H, s ,-COOH).ESI-MS[M+1]:365.2。

Embodiment 22：2- (2- (4- hydroxyl -3- (3- methyl but-2-ene bases) phenyl) -6- methyl -4- oxo -4H- benzos Pyrans -8- bases) acetic acid

The present embodiment compound is prepared with reference to the synthetic method of embodiment 17, except for the difference that using 2- (2- hydroxyl -3- (3- (4- hydroxyl -3- (3- methyl but-2-ene bases) phenyl) acryloyl group) -5- aminomethyl phenyls) acetic acid 0.26g (0.68mmol) is Beginning raw material, obtains product 134mg, yield 51.8%.

¹H-NMR(DMSO-d⁶,400MHz)：1.715 (6H, S ,-CH ₃× 2), 2.410 (3H, S, Ar-CH ₃), 3.335 (2H, S ,-CH ₂- CH), 3.929 (2H, S ,-CH ₂- COOH), 5.350-5.356 (1H, t, J=1.2Hz ,-CH₂-CH), 6.808- 6.943 (2H, m, Ar-H× 2), 7.538-7.543 (1H, d, J=2Hz, Ar-H), 7.737-7.779 (3H, m, Ar-H×3), 10.245 (1H, s ,-OH), 12.580 (1H, s ,-COOH).ESI-MS[M+1]:379.1。

Embodiment 26：2- (2', 2'- dimethyl -4- oxo -2'H, 4H-2,6'- .alpha.-5:6-benzopyran -8- bases) acetic acid

The present embodiment compound is prepared with reference to the synthetic method of embodiment 17, except for the difference that using 2- (3- (3- (2,2- bis- Methyl -2H- .alpha.-5:6-benzopyran -6- bases) -2- acryloyl groups) -2- hydroxy benzeness) and acetic acid 110mg (0.3mmol) be initiation material, obtain Product 54.4mg, yield 49.8%.

¹H-NMR(DMSO-d⁶,400MHz)：1.318-2.222 (6H, m ,-CH ₃× 2), 2.852-2.918 (1H, m ,-CH ₂ One of), 3.056-3.091 (1H, m ,-CH ₂One of), 3.230-3.319 (3H, m ,-CH；-CH ₂), 4.003 (2H, s ,-CH ₂- COOH), 6.891-6.927 (2H, d, J=5.2Hz, Ar-H× 2), 7.431-7.450 (1H, t, J=7.6Hz, Ar-H), 7.892-7.961 (4H, m, Ar-H× 4), 12.646 (1H, s ,-COOH).ESI-MS[M+1]:365.1。

The synthesis of embodiment 19,21,23,24,25,27,28,29,30,31 is with reference to embodiment 17, such as Table 3 below.

Table 3：With reference to the part of compounds of the synthesis of embodiment 17

Embodiment 35：2- (2- (4- hydroxy phenyls) -4- oxo benzodihydropyran -8- bases) acetic acid

By 2- (2- hydroxyl -3- (3-4- hydroxy phenyls) acryloyl group) phenyl) acetic acid 0.25g (0.84mmol), it is dissolved in 2mL tri- In the middle of Fluoroethanoic acid, microwave reaction 10 minutes, 80 DEG C of design temperature.After with the dilution of ethyl acetate 20mL, washed with water 15mL × 2, had Machine is mutually dried, filtering and concentrating, chromatographs post separation, methylene chloride/methanol (35:1) yellow solid 0.11g, yield 44%, are obtained.

¹H-NMR(DMSO-d⁶,400MHz)：2.796-2.846 (1H, dd ,-CH ₂One of), 3.120-3.195 (1H, m ,-CH ₂It One), 3.580 (1H, s ,-CH ₂- COOH), 5.482-5.521 (1H, s ,-CH), 6.772-6.801 (2H, m, Ar-H× 2), 7.016- 7.054 (1H, t, J=7.6Hz, Ar-H), 7.305-7.327 (2H, d, J=8.6Hz, Ar-H), 7.506-7.529 (1H, dd, Ar-H), 7.690-7.715 (1H, dd, Ar-H), 9.579 (1H, s ,-OH), 12.335 (1H, s ,-COOH)ESI-MS[M+1]:299.1。

Embodiment 38：2- (2- (4- hydroxyl -3- (3- methyl but-2-ene bases) phenyl) -4- oxo benzodihydropyran -8- Base) acetic acid

The present embodiment compound is prepared with reference to the synthetic method of embodiment 35, except for the difference that using 2- (2- hydroxyl -3- (3- (4- hydroxyl -3- (3- methyl but-2-ene bases) phenyl) acryloyl group) phenyl) acetic acid 120mg (0.33mmol) be initiation material, Obtain product 83mg, yield 69.2%.

¹H-NMR(DMSO-d⁶,400MHz)：1.284 (6H, s ,-CH ₃× 2), 1.758-1.791 (2H, t, J=6.8Hz ,- CH ₂- CH), 2.811-2.853 (2H, dd ,-CH ₂), 3.118-3.193 (1H, m ,-CH ₂- CH), 3.586 (2H, s ,-CH ₂- COOH), 5.475-5.507 (1H, dd ,-OH), 6.722-6.743 (1H, d, J=8.4Hz, Ar-H), 7.019-7.057 (1H, M, Ar-H), 7.189-7.232 (2H, m, Ar-H× 2), 7.509-7.527 (1H, dd, Ar-H), 7.694-7.713 (1H, dd, Ar-H), 12.276 (1H, s ,-COOH).ESI-MS[M+1]:367.1。

Embodiment 39：2- (2- (4- hydroxyl -3- (3- methyl but-2-ene bases) phenyl) -4- oxo benzodihydropyran -6- Base) acetic acid

The present embodiment compound is prepared with reference to the synthetic method of embodiment 35, except for the difference that using 2- (4- hydroxyl -3- (3- (4- hydroxyl -3- (3- methyl but-2-ene bases) phenyl) acryloyl group) phenyl) acetic acid 120mg (0.33mmol) be initiation material, Obtain product 81mg, yield 67.8%.

¹H-NMR(DMSO-d⁶,400MHz)：1.283 (6H, s ,-CH ₃× 2), 1.756-1.988 (2H, t, J=6.8Hz ,- CH ₂- CH), 2.739-2.755 (3H, m ,-CH ₂,-CH), 3.227-3.316 (1H, m ,-CH₂-CH), 3.592 (2H, s ,-CH ₂- COOH), 5.484-5.516 (1H, dd ,-OH), 6.727-6.748 (1H, d, J=8.4Hz, Ar-H), 7.001-7.023 (1H, D, J=8.8Hz, Ar-H), 7.215-7.264 (2H, m, Ar-H× 2), 7.447-7.474 (1H, dd, Ar-H), 7.669- 7.674 (1H, d, J=2Hz, Ar-H).ESI-MS[M+1]:367.2。

Embodiment 40：2- (2- (4- hydroxyl -3- (3- methyl but-2-ene bases) phenyl -6- methyl -4- oxo benzo dihydro pyrroles Mutter -8- bases) acetic acid

The present embodiment compound is prepared with reference to the synthetic method of embodiment 35, except for the difference that using 2- (2- hydroxyl -3- (3- (4- hydroxyl -3- (3- methyl but-2-ene bases) phenyl) acryloyl group) -5- aminomethyl phenyls) acetic acid 125mg (0.33mmol) is Beginning raw material, obtains product 84.2mg, yield 67.2%.

¹H-NMR(DMSO-d⁶,400MHz)：1.173 (6H, s ,-CH ₃× 2), 1.753-1.786 (2H, t, J=6.8Hz ,- CH ₂- CH), 2.273 (3H, S, Ar-CH ₃), 2.743-2.820 (3H, m ,-CH,-CH ₂), 3.081-3.156 (1H, m ,-CH₂- CH), 5.419-5.452 (1H, dd, Ar-OH), 6.715-6.736 (1H, d, J=8.4Hz, Ar-H), 7.176-7.222 (2H, M, Ar-H× 2), 7.341-7.346 (1H, d, J=2Hz, Ar-H), 7.497-7.500 (1H, d, J=1.2Hz, Ar-H), 12.311 (1H, s ,-COOH).ESI-MS[M+1]:381.2。

The synthesis of the synthesis reference embodiment 35 of embodiment 36,37,44,45,46,47,48,49, such as table 4 below.

Table 4：With reference to the part of compounds of the synthesis of embodiment 35：

Embodiment 41：2- (2- (4- (methoxymethoxy) -3- (3- methyl but-2-ene bases) phenyl -4- oxos benzos two Hydrogen pyrans -8- bases) acetic acid

2- (2- hydroxyl -3- (3- (4- (methoxymethoxy) -3- (3- methyl but-2-ene bases) phenyl) acryloyl group) benzene Base) acetic acid 123mg (0.3mmol), it is dissolved in the middle of 6mL ethanol, sodium acetate 180mg is added, instilling several dripping makes sodium acetate complete Dissolving.Back flow reaction 2 days.The dilution of ethyl acetate 20mL is added, is washed with water 15mL × 2, organic faciess are dried, filtering and concentrating, layer Analysis post separation, methylene chloride/methanol (35:1) yellow solid 33mg, yield 26.8%, are obtained.

¹H-NMR(DMSO-d⁶,400MHz)：1.684-1.690 (6H, d, J=2.4Hz ,-CH ₃× 2), 2.886-2.893 (1H, dd ,-CH ₂One of), 3.105-3.137 (1H, m ,-CH ₂One of), 3.283-3.383 (7H, m ,-CH ₂× 3 ,-CH), 3.599 (2H, s ,-CH ₂- COOH), 5.244-5.272 (3H, t, J=6.4Hz ,-OCH ₃), 5.537-5.569 (1H, dd ,-CH₂- CH), 7.044-7.069 (2H, t, J=7.6Hz, Ar-H× 2), 7.276-7.296 (2H, t, J=8.0Hz, Ar-H×2), 7.518-7.537 (1H, d, J=7.6Hz, Ar-H), 7.692-7.711 (1H, d, J=7.6Hz, Ar-H), 12.329 (1H, s ,- COOH).ESI-MS[M+1]:411.1。

Embodiment 42：2- (2- (4- (methoxymethoxy) -3- (3- methyl but-2-ene bases) phenyl -4- oxos benzos two Hydrogen pyrans -6- bases) acetic acid

The present embodiment compound is prepared with reference to the synthetic method of embodiment 41, except for the difference that using 2- (4- hydroxyl -3- (3- (4- (methoxymethoxy) -3- (3- methyl but-2-ene bases) phenyl) acryloyl group) phenyl) acetic acid 123mg (0.3mmol) is Initiation material, obtains product 31mg, yield 25.2%.

¹H-NMR(DMSO-d⁶,400MHz)：1.683 (6H, s ,-CH ₃× 2), 2.746-2.795 (1H, dd ,-CH₂-CH), 3.286-3.380 (6H, m ,-CH ₂× 3), 3.591 (2H, S ,-CH ₂- COOH), 5.237-5.256 (3H, S ,-CH ₃), 5.542- 5.567 (1H, dd ,-CH₂-CH), 7.008-7.029 (2H, m, Ar-H× 2), 7.305-7.318 (2H, m, Ar-H× 2), 7.451-7.478 (1H, dd, Ar-H), 7.664-7.669 (1H, d, J=2Hz, Ar-H), 12.291 (1H, s ,-COOH).ESI- MS[M+1]:411.1。

Synthesis of the synthesis of embodiment 43 with reference to embodiment 41.

Embodiment 53：2- (3- hydroxyl -2- (4- hydroxy phenyls) -4- oxo -4H- .alpha.-5:6-benzopyran -8- bases) acetic acid

2- (2- hydroxyl -3- (3- (4- hydroxy phenyls) acryloyl group) phenyl) acetic acid 0.25g (0.84mmol), is dissolved in 6mL In the middle of methanol.It is stirred at room temperature down, adds 16% sodium hydrate aqueous solution (about 5N) 1.2mL, system color is red by xanthochromia.Again plus Enter 30% hydrogen peroxide 0.15mL, system color is gradually taken off, react 1 hour, strong absorption is shown at ultraviolet 365nM.Use frozen water 20mL dilutes, and is then acidified to pH 2, has a large amount of bright yellow solids to separate out, and filters, and obtains product 0.18g, yield 69.2%.

¹H-NMR(DMSO-d⁶,400MHz)：3.989 (2H, s ,-CH₂- COOH), 6.924-6.947 (2H, m, Ar-H× 2), 7.387-7.426 (1H, t, Ar-H), 7.696-7.717 (1H, dd, Ar-H), 8.012-8.094 (3H, m, Ar-H×3), 9.399 (1H, s ,-OH), 10.126 (1H, s ,-OH), 12.632 (1H, s ,-COOH)。ESI-MS[M+1]:313.1。

Embodiment 54：2- (3- hydroxyl -2- (4- hydroxy phenyls) -4- oxo -4H- .alpha.-5:6-benzopyran -6- bases) acetic acid

The present embodiment compound is prepared with reference to the synthetic method of embodiment 53, except for the difference that using 2- (4- hydroxyl -3- (3- (4- hydroxy phenyls) acryloyl group) phenyl) acetic acid 200mg (0.67mmol) be initiation material, obtain product 0.16g, yield 76.2%.

¹H-NMR(DMSO-d⁶,400MHz)：3.771 (2H, s ,-CH ₂- COOH), 6.936-6.958 (2H, d, J=2Hz, Ar-H× 2), 7.671-7.681 (2H, t, J=2Hz, Ar-H× 2), 8.089-8.120 (3H, d, J=8.4Hz, Ar-H× 3), 9.337 (1H, s, Ar-OH), 10.084 (1H, s ,-OH), 12.433 (1H, s ,-COOH)。ESI-MS[M+1]:313.0。

Embodiment 59：2- (3- hydroxyl -2- (4- (methoxymethoxy) -3- (3- methyl but-2-ene bases) phenyl -4- oxygen Generation -4H- benzodihydropyran -8- bases) acetic acid

The present embodiment compound is prepared with reference to the synthetic method of embodiment 53, except for the difference that using 2- (2- hydroxyl -3- (3- (4- (methoxymethoxy) -3- (3- methyl but-2-ene bases) phenyl) acryloyl group) phenyl) acetic acid 200mg (0.5mmol) is Initiation material, obtains product 133.7mg, yield 64.7%.

¹H-NMR(DMSO-d⁶,400MHz)：1.717-1.738 (6H, d, J=8.4Hz ,-CH ₃× 2), 3.350-3.414 (5H, m ,-CH ₂-CH；-OCH ₃), 3.990 (2H, s ,-CH ₂- COOH), 5.324-5.341 (3H, m ,-OCH ₂；-CH₂-CH), 7.193-7.215 (1H, d, J=8.8Hz, Ar-H), 7.397-7.435 (1H, t, J=7.6Hz, Ar-H), 7.708-7.730 (1H, dd, Ar-H), 8.019-8.061 (3H, m, Ar-H× 3), 9.491 (1H, s ,-OH), 12.617 (1H, s ,-COOH)。 ESI-MS[M+1]:425.1。

Embodiment 60：2- (3- hydroxyl -2- (4- (methoxymethoxy) -3- (3- methyl but-2-ene bases) phenyl) -4- oxygen Generation -4H- .alpha.-5:6-benzopyran -6- bases) acetic acid

The present embodiment compound is prepared with reference to the synthetic method of embodiment 53, except for the difference that using 2- (4- hydroxyl -3- (3- (4- (methoxymethoxy) -3- (3- methyl but-2-ene bases) phenyl) acryloyl group) phenyl) acetic acid 200mg (0.5mmol) is Initiation material, obtains product 132.9mg, yield 64.3%.

¹H-NMR(DMSO-d⁶,400MHz)：1.707-1.733 (6H, d, J=10.4Hz ,-CH ₃× 2), 3.314-3.410 (5H, m ,-CH ₂× 2 ,-CH), 3.773 (2H, s ,-CH ₂- COOH), 5.275-5.326 (3H, s ,-OCH ₃), 7.198-7.221 (1H, d, Ar-H), 7.686 (2H, s, Ar-H× 2), 7.991-8.043 (3H, m, Ar-H× 3), 9.404 (1H, s ,-OH), 12.366 (1H, s ,-COOH).ESI-MS[M+1]:425.1。

The synthesis reference embodiment 53 of embodiment 50,51,52,55,56,57,58,61,62,63,64,65,66,67, such as Table 5 below.

Table 5：With reference to the part of compounds of the synthesis of embodiment 53

Pharmacology test and activity data

Such as following test example 1-3.

Test example 1：TNF-α concentration in the AlphaLISA methods detection cells and supernatants of RAW 264.7

Experimental technique is as follows：Take the logarithm the cells of RAW 264.7 of trophophase, with the RPMI- containing 10% hyclone 1640 culture medium are configured to cell suspension, are inoculated with 96 well culture plates, and (2500 RAW264.7 cells are contained) per the μ l of hole 100, 37 DEG C are placed in, 5%CO₂In incubator, after adding target compound effect certain hour, 96 orifice plates are centrifuged into 2000r, 10min, it Afterwards 150 μ l supernatants are taken per hole, it is frozen standby in -20 DEG C per a 50 μ l in being dispensed into three EP.Subsequently according to AlphaLISA TNF-α test kit illustrates, prepares calibration curve solution, and takes 5 μ l supernatants and be inoculated in White OptiPlate-384 orifice plates, Then per hole add AlphaLISA Anti-Analyte Acceptor beads (the μ g/ml of final concentration 10) and μ l (the Acdeptor beads of Biotinylated Antibody Anti-Analyte (final concentration 1nM) mixed liquor 20： Biotinylated Antibody Anti-Analyte：1X buffer=1:2:198), add after 23 DEG C of incubation 60min μ l (the Donor beads of Donor beads (final concentration of 40 μ g/ml) 25：1X buffer=1:61.5), 23 DEG C of lucifuge incubations Detected using EnVision after 30min.

Experimental result such as table 6.

Test example 2：MTS methods determine the cytotoxicity of compound

Experimental technique is as follows：Take the logarithm the cells of mouse monokaryon macrophage strain RAW 264.7 of trophophase, with containing 10% The RPMI-1640 culture medium of hyclone is configured to cell suspension, is inoculated in 96 well culture plates, and (2500 are contained per the μ l of hole 100 Tumor cell), 37 DEG C are placed in, 5%CO₂In incubator.Add and act on after target compound after certain hour, add MTS molten per hole The μ l of liquid 10,37 DEG C are incubated 4 hours.Its light absorption value is determined at 490nm wavelength with enzyme-linked immunosorbent assay instrument.

Cell survival rate (%)=(administration group OD- blank group OD)/matched group OD × 100%

Experimental result such as table 6.

Table 6：Part of compounds activity experiment result

"-" is represented and detected.

Target compound concentration be 30 μ g/ml, LPS concentration be 2.5ng/ml, with DMSO or DMSO with LPS to compare；“a” Represent that target compound independent role promotes the secretion rate of the cell TNF secretion-α of RAW 264.7；" b " is represented and LPS (lipopolysaccharide Target compound shares the secretion rate for promoting the cell TNF secretion-α of RAW 264.7 with LPS when 2.5ng/ml) sharing；" c " is represented Growth inhibition effect of the target compound independent role to the cells of RAW 264.7；" d " represents that target compound shares right with LPS The growth inhibition effect of the cells of RAW 264..

As a result show, Formulas I, Formula II compound have anti-tumor activity, therefore Formulas I, Formula II compound can be used as antitumor Medicine is used in antineoplastic research, with the potentiality for being developed into antitumor drug.

Additionally, general rush TNF-α secretion is all that, by LPS approach, experimental result shows, the compound of the present invention is disobeyed Bad LPS approach can also produce TNF-α and promote secretory action.

Test example 3：Chick chorioallantoic membrane model investigates the impact of compound on vascular

Experimental technique is as follows：Aseptically, 10 μ l respective concentration solution title compounds are spread evenly across On Whatman filter paper, negative control is coated with the 0.3%DMSO solution of same volume, will fertilization hatching egg be placed in 37 DEG C, it is 60% relative Hatched in the constant-temperature incubation case of humidity, after 6 days, the CAM films of Dan Pei lower floors are leaked cruelly.Load sample filter paper is placed in into CAM and ovum Then blood vessel is sealed compared with multiple location with sterile transparent glue at yellow cyst membrane, continues to cultivate to 8 days, and removing is covered in saturating on Embryo Gallus domesticus Gelatin cloth.Medicine-feeding part is shot, picture, statistics administration group and matched group vessel area is processed, two groups of blood vessel faces are calculated Product is accounted for the percentage ratio of the CAM gross areas and (Embryo Gallus domesticus is urinated using Adobe Photoshop CS5, the softwares of Image-Pro Plus 6.0 Cyst membrane blood vessel carries out visualization and follow-up area quantitative, n=2).

Angiolysis rate (Distrupting Ratio, %)=[1- (experimental group vessel areas/solvent control group blood vessel face Product)] × 100%.

Experimental result such as table 7.

Table 7：Impact of the target compound to chick chorioallantoic membrane blood vessel

The concentration of target compound is 10 μ g/ Embryo Gallus domesticus, with DMSO as control.

Experimental result shows that the compound of the present invention can promote tumor cell secretion TNF-α, while can also significantly subtract Few chick chorioallantoic membrane number of blood vessel.Therefore, compound of the invention can be used in preparing antitumor drug.

Test example 4：To the tumor vascular destruction of pulmonary carcinoma tumor-bearing mice

Experimental procedure：

(1) foundation of the subcutaneous lotus knurl models of mice LLC

By the C of the indoor Lewis lung cancer cell lotus knurl of experiment₅₇The de- neck of BL/6 mices is put to death, and soaking disinfection 1 in 75% ethanol～ 2min.Separate its subcutaneous tumor mass in aseptic operating platform afterwards, normal saline flushing 2 times is placed in advance through high temperature sterilize 150～200 mesh stainless steel sifts are online, and shears are fully ground after shredding.The underlying glass for filling appropriate physiological saline solution of screen cloth Culture dish collects the cell after grinding, and all Cell saps collected are incorporated in centrifuge tube, 1500rpm centrifugation 5min. Supernatant is abandoned, with appropriate physiological saline solution is resuspended cell suspension is made, fully piping and druming is uniform, is transferred to 25cm²In culture bottle, envelope Membrana oralis sealing is stand-by.

With 75% cotton ball soaked in alcohol cleaning disinfection C₅₇BL/6 right side of mice armpit skins, 1ml syringes draw the cell for preparing Suspension injection mice oxter is subcutaneous, every 0.2ml (every Mus all note fully mixing when Cell sap is drawn), stops 2～3s of pin Syringe needle is overturn afterwards pulls out pin.After inoculation is finished, the body weight change and tumor bulk-growth situation of each group mice is recorded every other day.By formula V=D (major diameter) × d²(minor axis)/2 calculate tumor volume.If animal occurs death, its time-to-live and optionally anatomic observation are recorded Its in-vivo tumour infects situation.

(2) administration and detection

When Tumor diameter length is to about 10mm, remove not into tumor animal.Residue is all to be fitted into tumor Mus by tumorous size Work as adjustment, make every group of sample try one's best mean allocation.The corresponding lumbar injection DMXAA (20mg/kg) of reagent group, the compound of embodiment 2 (20mg/kg), the 1%DMSO of matched group lumbar injection respective volume.All mices tail vein injection fluorescence dye after administration 3h Material Hoechst 33342 (800 μ g/mouse, 0.1ml).The de- neck of mice is put to death after injection 1min, is peeled off tumor body and is taken pictures, claims Weight, calculates tumor volume, and is wrapped with masking foil rapidly, frozen that frozen section is carried out in liquid nitrogen, in inverted fluorescence microscope Lower observation.

Experimental result：As shown in accompanying drawing 1,2,3 (3A, 3B, 3C).

As a result show, the compound of embodiment 2 can destroy tumor vessel, reduce tumor blood perfusion.

Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.Root According to disclosed all teachings, various modifications and replacement can be carried out to those details, these change in the guarantor of the present invention Within the scope of shield.The four corner of the present invention is given by claims and its any equivalent.

Claims (12)

1. the preparation method of compound of formula I, comprises the steps：

(1) preparation of A fragments：

(2)B₂、B₃、B₄、B₅、B₆The preparation of fragment：

(3) with ethanol as solvent, in the presence of 60% potassium hydroxide aqueous solution, by A fragments respectively with B₁Fragment is B₂Fragment, B₃Fragment, B₄Fragment, B₅Fragment or B₆Fragment reaction obtains compound of formula I；

Wherein, dotted portion represents R₁；

The compound of formula I is as follows：

Wherein：

R₁Selected from H, C₁-C₆Alkyl；

When the aceticoceptor in female ring is located at R₁During position, R₁Do not exist；

R₂Selected from phenyl, the phenyl replaced by one or more hydroxyls, and selected from following group：

2. method according to claim 1, wherein, R₂Selected from the phenyl replaced by 2,3,4 or 5 hydroxyls.

3. method according to claim 1, wherein, R₂When being other substituent groups in addition to phenyl ring, R₂With the connection of female ring Position is R₂In phenyl ring on unsubstituted any site.

4. method according to claim 1, it meets any one or multinomial in following (1)-(3) item,

(1)R₁Independently selected from H, C₁-C₃Alkyl；

(2)R₂Independently selected from Where the dotted line signifies that R₂With the link position of female ring；

(3) compound of formula I does not include following compound：

5. method according to claim 4, wherein, in (1st) item, the C₁-C₃Alkyl is methyl, ethyl, propyl group or different Propyl group.

6. method according to claim 4, wherein, in (1st) item, R₁It independently is H or methyl.

7. method according to claim 1, wherein, the compound of formula I is selected from：

8. method according to any one of claim 1 to 7, wherein, the step (1) includes：

A) withObtain with paraformaldehyde and concentrated hydrochloric acid reaction for raw material

B) in organic solvent, in the presence of reagent, makeWith Cyanogran. reaction, obtain Further hydrolysis, obtains A fragments

9. method according to claim 8, wherein, b) described in organic solvent be toluene.

10. method according to claim 8, wherein, b) described in reagent be tetrabutyl ammonium bromide.

11. methods according to claim 8, wherein, b) described in A fragmentsFor

12. methods according to any one of claim 1 to 7, wherein, the step (2) includes：

c)B₁FragmentWith B₂FragmentCan directly buy；

d)B₂Fragment is reacted with bromo iso-amylene under the conditions of solution of potassium carbonate, B when obtaining₃Fragment

E) B is made₃Fragment is reacted with methoxy chlorine, obtains B₄Fragment

F) by B₄Fragment respectively with p-methyl benzenesulfonic acid, chloro- 5, the 6- dicyanos-benzoquinones of 2,3- bis-, obtain B₅FragmentB₆Fragment