CN106977467A

CN106977467A - A kind of compound and preparation method thereof and purposes

Info

Publication number: CN106977467A
Application number: CN201710172284.6A
Authority: CN
Inventors: 李卫民; 冯毅凡; 朱贺年; 玉荣
Original assignee: Beijing Yishengtang Medicine Science And Technology Research Co Ltd
Current assignee: Beijing Yishengtang Medicine Science And Technology Research Co Ltd
Priority date: 2017-03-22
Filing date: 2017-03-22
Publication date: 2017-07-25

Abstract

The present invention discloses a kind of compound, and the compound is prepared from by ring-closure reaction under certain conditions by melbine with magnolol.The compound of the present invention, it is inventor under the guidance of tcm clinical medication experience and on the basis of organic chemistry, purposefully, selectively magnolol and melbine are combined together by specific synthetic method, gained compound has the effect of Synergy and attenuation, has more preferable effect in terms of anti-inflammatory, hypoglycemic, antitumor, antibacterial, resisting cardiovascular disease.Meanwhile, the invention also discloses the preparation method of the compound and the compound for preparing the purposes in treatment tumour, hyperglycaemia, high fat of blood, the medicine of cardiovascular and cerebrovascular disease.

Description

A kind of compound and preparation method thereof and purposes

Technical field

The present invention relates to a kind of compound and preparation method thereof, especially with anti-inflammatory, antitumor, hypoglycemic, antibacterial Compound Deng effect and preparation method thereof.

Background technology

Now, with the development of modern pharmacology, molecular biology scheduling theory and related science technology, natural drug is opened Hair approach and means are also being constantly brought forth new ideas.At present, the approach of developing new drug substantially has three, i.e. Folk medicine approach, experience medicine way Footpath and molecular pathways.Wherein with the development of molecular biology, science of heredity and the information processing technology etc., molecular pathways gradually into For the main path of developing new drug.But based on the current national conditions of China, and due to the limitation of technical equipment, current Folk medicine way Footpath, experience medicine approach are the main paths of China's developing new drug, and both approach are based on medicinal plant.

The medicine grown up using medicinal plant as main body, in history to the preventing and curing diseases of the mankind, rehabilitation and life Educate procreation and serve huge effect.Natural drug available sources are a lot of, mostly from plant；It is estimated that the whole world there are about 40 ~50 ten thousand kinds of plants, but it is only few partly carried out chemical composition and its active testing research, China be also plant resources most One of abundant country, therefore fully excavate and develop new natural drug using the compound of nature structure diversity, promote Enter China's medical sci-tech to advance at utmost speed, open up new market, promote the development of China's natural drug, meet medication economics to globalize Challenge has great importance.Medicinal plant is the important sources of developing new drug, at present, in the new drug of whole world approval, close to half Number is natural drug, and medicine comes from natural products.Now, also past 60% population also fully relies on autonomic drug to prevent in the world Control disease.

But with the development of disease, the problems such as viral resistance, body drug resistance, simple certain medicinal plant of dependence, Or ingredient is precursor the new drug researched and developed in medicinal plant, far from people are met to medicine the need for.For certain Specific disease, people are often that, by two or more, or even more medicines are taken simultaneously can just play certain Therapeutic action.

Cortex Magnoliae Officinalis is the branch skin root skin that the sharp leaf bark of official magnolia Magnolia bilola of Magnoliacea plant is dried, and main active is the bark of official magnolia Phenol and honokiol, have pharmacological action in the many-side such as Inhibit proliferaton, antianxiety, antiallergy, anti-inflammatory, antitumor.

Melbine is the classical antidiabetic drug of Western medicine, and Recent study finds that melbine is except with good hypoglycemic Outside effect, also there is a good curative effect in anti-inflammatory, antitumor, regulation blood fat, antibiosis, but melbine half-life period in vivo Short, metabolism is rapid, and lactic acidosis easily occurs for potential renal insufficiency patient, based on factors above, will the two molecule parent nucleus splicing Together, the bioavilability of melbine can be increased.The risk of nephrotic's lactic acidosis is reduced, the two is used The method of chemical synthesis, it is autotelic to be stitched together, see that can its some pharmacological action strengthen, if 1+1 >=2 can be reached, increase Imitate the effect of attenuation, the two anti-inflammatory, pharmacological action etc. of hypoglycemic drop ester can be protruded, these be all it is unknown unpredictable, It is also that, based on this, present inventor is obtained by a large amount of exploratory developments in the prior art without any disclosure or report The following present invention.

The content of the invention

It is an object of the invention to provide a kind of new compound based on above-mentioned technical background, the ratio of the compound is thick The small toxicity of plain phenol, and with the effect such as more preferable anti-inflammatory, antitumor anticancer, hypoglycemic, antibacterial.Meanwhile, the present invention is also The Preparation method and use of the compound is provided.

To achieve the above object, the technical scheme taken of the present invention is：A kind of new compound, the structure of the compound Formula is：

The present invention compound by magnolol and melbine as obtained by specifically reacting and synthesize, compound of the invention The toxicity of magnolol can be significantly reduced, and 1+1 >=2 can be reached, plays a part of Synergy and attenuation, in anti-inflammatory, antitumor There is more preferable effect in terms of anticancer, hypoglycemic and antibacterial.

In addition, the present invention also provides a kind of preparation method of compound as described above, the preparation method includes following step Suddenly：

(1) compound of formula (1) is subjected to bromo-reaction with bromo agent under base catalyst A effects in solvent orange 2 A, obtained The compound of formula (2)；

(2) compound for the formula (2) for obtaining step (1) is under base catalyst B effects, and the compound with formula (3) is molten Ring-closure reaction occurs in agent B, that is, obtains formula of the present invention (4) compound；

The preparation method of compound of the present invention, by specific mode by the parent nucleus cyclization of magnolol and melbine Together, but this cyclization is not blindness, but under the guidance of tcm clinical medication experience and organic chemistry On the basis of, purposefully, selectively the two is combined, it is found that the two can play the effect of Synergy and attenuation after combining, And the method for the present invention is simple to operate, the compound of the present invention can be rapidly and efficiently obtained.

As the preferred embodiment of the preparation method of compound of the present invention, in the step (1), bromo agent is bromine In methyl acetate, bromoacetate, bromoacetic acid butyl ester, methyl chloroacetate, ethyl chloroacetate, butyl chloroacetate, iodoacetic acid methyl esters At least one；Base catalyst A is at least one of sodium carbonate, potassium carbonate；Solvent orange 2 A is acetonitrile, DMF, methanol, acetone, second At least one of alcohol, dimethyl sulfoxide (DMSO), tetrahydrofuran, dichloromethane, dimethyl sulfoxide (DMSO), dioxane.

As the more preferably embodiment of the preparation method of compound of the present invention, the bromo agent in the step (1) For methyl bromoacetate.When the bromo agent selects methyl bromoacetate, two carbon in the methyl bromoacetate molecule both can be with Overcome intermolecular resistance, it is ensured that the generation of nucleophilic substitution, the stability of synthesized compound maintained again, it is to avoid its It is unstable and decompose in vivo.

As the more preferably embodiment of the preparation method of compound of the present invention, the solvent orange 2 A in the step (1) is Methanol.When the solvent orange 2 A in the step (1) selects methanol, the reactant solubility that methanol is reacted the step is preferable, and viscosity Small, low boiling point, polarity is moderate, on the premise of it both ensure that product yield, is easy to post processing, recycling design again.

As the preferred embodiment of the preparation method of compound of the present invention, the base catalyst B in the step (2) For at least one of sodium methoxide, caustic alcohol, metallic sodium；Solvent B is acetonitrile, DMF, ethyl acetate, methanol, ethanol, acetone, pyrrole At least one of pyridine, quinoline, dimethyl sulfoxide (DMSO), tetrahydrofuran, dichloromethane, dimethyl sulfoxide (DMSO), dioxane.

As the more preferably embodiment of the preparation method of compound of the present invention, the base catalysis in the step (2) Agent B is sodium methoxide, and present inventor had found by numerous studies, when the base catalyst B selects sodium methoxide, product Yield apparently higher than other base catalysts.

As the more preferably embodiment of the preparation method of compound of the present invention, the solvent B in the step (2) is Methanol.When the solvent B in the step (2) selects methanol, the reactant solubility that methanol is reacted the step is preferable, and viscosity Small, low boiling point, polarity is moderate, on the premise of it both ensure that product yield, is easy to post processing, recycling design again.

As the preferred embodiment of the preparation method of compound of the present invention, in the step (1), bromo agent and formula (1) mol ratio of compound is 2:1~5:1；The mol ratio of base catalyst A and formula (1) compound is 2:1~6:1；Solvent orange 2 A with The mol ratio of formula (1) compound is 5:1~20:1.

As the preferred embodiment of the preparation method of compound of the present invention, in the step (2), formula (3) chemical combination The mol ratio of thing and formula (2) compound is 0.5:1~5:1；The mol ratio of base catalyst B and formula (2) compound is 0.04:1~ 5:1；The mol ratio of solvent B and formula (2) compound is 5:1~20:1.

As the preferred embodiment of the preparation method of compound of the present invention, bromo-reaction temperature in the step (1) Spend for 30~70 DEG C, the reaction time is 3~12 hours；The temperature of ring-closure reaction is 40~70 DEG C in the step (2), during reaction Between be 1~15 hour.

As the more preferably embodiment of the preparation method of compound of the present invention, bromo-reaction in the step (1) Temperature is 30~70 DEG C, and the reaction time is 8~10 hours；The temperature of ring-closure reaction is 60~70 DEG C in the step (2), reaction Time is 0-15 hours.Present inventor by research find, when the bromo-reaction temperature in the step (1) be 65 DEG C, Reaction time be the ring-closure reaction in 12h, step (2) temperature be 65 DEG C, the reaction time be 12 hours, both can guarantee that yield, There is preferable economy again.

As the preferred embodiment of the preparation method of compound of the present invention, the step (1) is：By formula (1) Compound is added in clean three-necked flask, adds solvent orange 2 A, is added base catalyst A, bromo agent is slowly added to, when the temperature to 67 DEG C when, back flow reaction, HPLC monitoring reactions, raw material fundamental reaction is completely or product stops reaction when not being further added by, while hot suction filtration, Rotation solvent evaporated produces the compound of formula (2).

As the preferred embodiment of the preparation method of compound of the present invention, the step (2) is：By compound (3) add in clean three-necked flask, add solvent B and base catalyst B, stir 30 minutes, then add what step (1) was obtained The compound of formula (2), reacts 0~12 hour, reaction is cooled down after terminating, suction filtration, washing, so in the case where temperature is 60-70 DEG C After dry, with methanol eddy one time, suction filtration, precipitation methylene chloride reflux one time, suction filtration, dichloro filtrate is spin-dried for, filter cake first Alcohol formic acid is dissolved, and places refrigerator overnight, suction filtration, and filtrate decompression is distilled to without methanol, adds suction filtration, neutrality is washed to distillation, Dry, obtain white precipitate, as compound (4), purity 95%.

Finally, present invention also offers compound described above for preparing treatment tumour, cancer, hyperglycaemia, high blood Purposes in fat, the medicine of cardiovascular and cerebrovascular disease, compound of the present invention can be used for prepare treatment tumour, cancer, hyperglycaemia, The medicine of high fat of blood, cardiovascular and cerebrovascular disease.

The compounds of this invention be inventor under the guidance of tcm clinical medication experience and organic chemistry, it is purposeful, Selectively magnolol and melbine are combined together by specific synthetic method, gained compound has Synergy and attenuation Effect, in terms of anti-inflammatory, antitumor, anticancer, hypoglycemic, antibacterial, resisting cardiovascular disease have more preferable effect.This Invent the preparation method of the compound, it is simple to operate, can be rapidly and efficiently obtain the present invention compound.It is of the present invention Purposes of the compound in the medicine for preparing treatment tumour, cancer, hyperglycaemia, high fat of blood, cardiovascular and cerebrovascular disease etc., for system The medicine of standby treatment knurl, cancer, hyperglycaemia, high fat of blood, cardiovascular and cerebrovascular disease etc. provides new compound and selected.

Brief description of the drawings

Fig. 1 is the chemical structural formula of formula of the present invention (4) compound；

Fig. 2 is the high resolution mass spectrum figure of formula of the present invention (4) compound；

Fig. 3 is the infrared spectrogram of formula of the present invention (4) compound；

Fig. 4 is the ultraviolet spectrogram of formula of the present invention (4) compound；

Fig. 5 is the proton nmr spectra carbon spectrogram of formula of the present invention (4) compound；

Nuclear magnetic resonance H-H Correlated Spectroscopy, C-H correlation spectrograms of the Fig. 6 for formula of the present invention (4) compound；

Embodiment

To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair The present invention is described further.

The structural formula difference of the compound of formula used in following examples (1)~(4) is as follows：

Embodiment 1

A kind of embodiment of the compounds of this invention, the chemical structural formula of compound described in the present embodiment as shown in Figure 1, this Compound described in embodiment is prepared from using following preparation method：

(1) compound of formula (1) is added in the three-necked flask of dried and clean, adds sodium carbonate, add methanol, the carbon The mol ratio of sour sodium and formula (1) compound is 2:1, the mol ratio of the methanol and formula (1) compound is 5:1, it is slowly added to bromine The mol ratio of methyl acetate, the methyl bromoacetate and formula (1) compound is 2:1, temperature rises to 65 DEG C or so, back flow reaction 12 Hour, HPLC monitoring reactions stop reaction, while hot suction filtration, filtrate decompression when raw material fundamental reaction is completely or product is not further added by Distillation is complete, obtains the compound of formula (2)；

(2) formula (3) compound is added in clean three-necked flask, formula (3) compound is rubbed with formula (2) compound You are than being 0.5:1, add methanol and sodium methoxide-methanol solution, the mol ratio of the methanol added and formula (2) compound is 20: 1, the sodium methoxide and the mol ratio of formula (2) compound added is 0.04:1, stir 30 minutes, be slowly added to step (1) and obtain Formula (2) compound, temperature be 65 DEG C at react 16 hours, reaction terminate after cooling, suction filtration, drying, be further purified, The compound of formula (4) is obtained, the compounds of this invention is produced.

Embodiment 2

(1) compound of formula (1) is added in the three-necked flask of dried and clean, adds methanol and sodium carbonate, heating is described The mol ratio of sodium carbonate and formula (1) compound is 2:1, the mol ratio of the methanol and formula (1) compound is 20:1, it is slowly added to The mol ratio of methyl chloroacetate, the methyl chloroacetate and formula (1) compound is 3:1, temperature rises to 65 DEG C or so, back flow reaction 12 hours, HPLC monitoring reactions stopped reaction, while hot suction filtration, filtrate subtracts when raw material fundamental reaction is completely or product is not further added by Pressure distillation drying, produces the compound of formula (2)；In this step, the yield of gained formula (2) compound is 80%；

(2) formula (3) compound is added in clean three-necked flask, formula (3) compound is rubbed with formula (2) compound You are than being 1:1；The mol ratio of addition methanol and caustic alcohol-ethanol solution, the ethanol added and formula (2) compound is 20:1, The mol ratio of the caustic alcohol added and formula (2) compound is 0.04:1, stir 30 minutes, be slowly added to what step (1) was obtained The compound of formula (2), reacts 12 hours in the case where temperature is 65 DEG C, and cooling, suction filtration, drying after reaction terminates are further purified, obtained To the compound of formula (4), the compounds of this invention is produced.

Embodiment 3

(1) compound of formula (1) is added in the three-necked flask of dried and clean, adds methanol and sodium carbonate, heating is described The mol ratio of sodium carbonate and formula (1) compound is 2.5:1, the mol ratio of the methanol and formula (1) compound is 20:1, it is slow to add Enter methyl chloroacetate, the mol ratio of the methyl chloroacetate and formula (1) compound is 5:1, temperature rises to 65 DEG C or so, and backflow is anti- Answer 12 hours, HPLC monitoring reactions stop reaction, while hot suction filtration, filtrate when raw material fundamental reaction is completely or product is not further added by Vacuum distillation drying, produces the compound of formula (2)；In this step, the yield of gained formula (2) compound is 80%；

(2) metallic sodium is wrapped with tinfoil, pricked with pin in hole, input methanol solution, metallic sodium all dissolvings are treated, by tinfoil Take out, solution for standby, formula (3) compound is added in clean three-necked flask, formula (3) compound and formula (2) compound Mol ratio be 2:1, with the reacted methanol solution of metallic sodium, the methanol solution added and the mol ratio of formula (2) compound For 5:1, the metallic sodium and the mol ratio of formula (2) compound added is 1:1, stir 30 minutes, be slowly added to step (1) and obtain Formula (2) compound, temperature be 65 DEG C at react 16 hours, reaction terminate after cooling, suction filtration, drying, be further purified, The compound of formula (4) is obtained, the compounds of this invention is produced.

Embodiment 4

(1) will formula (1) compound add dried and clean three-necked flask in, add ethanol, heating, the sodium carbonate with The mol ratio of formula (1) compound is 3:1, the mol ratio of the ethanol and formula (1) compound is 10:1, it is slowly added to bromoacetic acid second The mol ratio of ester, the bromoacetate and formula (1) compound is 2:1, temperature rises to 65 DEG C or so, back flow reaction 8 hours, HPLC monitoring reactions, stop reaction, filtrate decompression distillation drying when raw material fundamental reaction is completely or product is not further added by；Produce formula (2) compound；In this step, the yield of gained formula (2) compound is 80%；

(2) formula (3) compound is added in clean three-necked flask, formula (3) compound is rubbed with formula (2) compound You are than being 5:1, add methanol/ethanol mixed solution and sodium methoxide-methanol solution, the methanol/ethanol mixed solution and formula added (2) mol ratio of compound is 10:1, the sodium methoxide and the mol ratio of formula (2) compound added is 0.05:1, stir 30 points Clock, is slowly added to the compound for the formula (2) that step (1) is obtained, and is reacted 16 hours in the case where temperature is 65 DEG C, reaction terminates rear cold But, suction filtration, drying, are further purified, and obtain the compound of formula (4), produce the compounds of this invention.

Embodiment 5

(1) compound of formula (1) is added in the three-necked flask of dried and clean, adds tetrahydrofuran, heating, the tetrahydrochysene The mol ratio of furans and formula (1) compound is 5:1, it is slowly added to bromoacetate, the bromoacetate and formula (1) compound Mol ratio be 5:1, temperature rises to 70 DEG C or so, back flow reaction 6 hours, HPLC monitoring reactions, raw material fundamental reaction completely or Stop reaction when product is not further added by, filtrate decompression distillation drying produces the compound of formula (2)；In this step, gained formula (2) The yield of compound is 80%.

(2) formula (3) compound is added in clean three-necked flask, adds tetrahydrofuran and sodium methoxide-methanol solution, institute The tetrahydrofuran of addition is 10 with the mol ratio of formula (2) compound:1, the sodium methoxide and the mol ratio of formula (2) compound added For 5:1, stir 30 minutes, be slowly added to the compound for the formula (2) that step (1) is obtained, reacted 16 hours in the case where temperature is 65 DEG C, Cooling, suction filtration, drying after reaction terminates, are further purified, obtain the compound of formula (4), produce the compounds of this invention.

Embodiment 6

(1) compound of formula (1) is directly bought from market, its purity is 90%；

(2) compound of formula (1) is added in the three-necked flask of dried and clean, adds the mixture of acetonitrile and methanol, institute In the mixture for stating acetonitrile and methanol, the volume ratio of the acetonitrile and methanol is 1:4, heating, the mixing of the acetonitrile and methanol The mol ratio of thing and formula (1) compound is 15:1, it is slowly added to bromoacetate, the bromoacetate and formula (1) compound Mol ratio be 2:1, the back flow reaction 12 hours in the case where temperature is 67 DEG C, HPLC monitoring reactions, raw material fundamental reaction is complete or produces Stop reaction when thing is not further added by, while hot suction filtration, filtrate is spin-dried for producing the compound of formula (2)；In this step, gained formula (2) is changed The yield of compound is 70%, is further purified；

(3) formula (3) compound is added in clean three-necked flask, formula (3) compound is rubbed with formula (2) compound You are than being 0.5:1, methanol/ethanol mixed volume compares 1:1, the mixture of the methanol and ethanol and the mol ratio of formula (2) compound For 5:1, sodium methoxide is added, the mol ratio of the sodium methoxide added and formula (2) compound is 5:1, stir 30 minutes, be slowly added to The compound for the formula (2) that step (1) is obtained, reacts 15 hours, cooling, suction filtration, drying after reaction terminates in the case where temperature is 65 DEG C, It is further purified, obtains the compound of formula (4), produce the compounds of this invention.

The compound collection of illustrative plates of the present invention of embodiment 7

It can be seen that by accompanying drawing 2~6,

Formula (4) compound of the present invention, m.p.795.4 DEG C, ESI-MS m/z:476.2322[M+1]⁺；

Formula (4) compound of the present invention,

IR(KBr)ν_max:3225.36,3076.87,2929.34,1680.66,1630.52,1571.7,1402.96, 1275.68,1216.86,1133.94,1056.8,991.23,912.165,808.028(cm^-1)；

Formula (4) compound of the present invention,

¹HNMR(500MHz,DMSO),δ(ppm)7.11(s,1H,C₉-H),7.06(s,1H,C_5’-H),7.05(s,1H,C₆- H),7.02(s,1H,C_8’-H),6.9(d,1H,C₅-H),6.84(s,1H,C_9’-H),6.8(s,2H,NH₂),5.9(s,2H,C₂、 C_2’-H),5.02(s,4H,C₃,C_3’-H),4.7(s,2H,CH₂-O),4.56(s,2H,CH₂-O),3.3(d,4H,C₂、C_2’-H), 3.0(s,6H,(CH₃)₂-N)；

¹³CNMR(500MHz,DMSO),δ(ppm):172.89 (C=O), 170.57 (C=N), 166.55 (N=C-N), 165.07 (N=C-N), 154.49 (C_7’),153.49(C₈),138.0(C₂),137.97(C_2’),131.63(C₄),131.53 (C_4’),131.38(C₆),131.23(C_8’),128.06(C_5’),127.99(C₉),127.47(C_6’),127.14(C₇),115.46 (C₃),115.45(C_3’),112.85(C₅),112.56(C_9’),70.45(O-CH₂-),65.64(O-CH₂),38.69(C₁、_1’), 35.66(CH₃-N),35.47(CH₃-N)。

The cell toxicological test of the compounds of this invention of embodiment 8

First, experiment material, reagent and instrument

Cell：RAW264.7 macrophages (derive from Sheng Ke Yuan Huangze ripples seminar of Guangdong pharmaceutical university).

Experiment reagent：Hyclone (FBS, Gibco), high glucose medium (DMEM, Gibco), 0.25% trypsase (Gibco), penicillin streptomycin is dual anti-(Gibco), phosphate buffer pH=7.4 (PBS, Gibco), and tetrazolium bromide (MTT, together Cloud bio tech ltd), LPS (Escherichia055:B55, Sigma), DMSO (Sigma), brufen (Sigma), LPS (Sigma), distilled water (Watson), Berberine hydrochloride (Hangzhou Great Forest Biomedical Ltd.), Metformin hydrochloride (Guangzhou E-BANG Pharmaceutical Technology Co., Ltd), magnolol (Guangzhou He Cheng enterprises), BM471, HM475, HM568 (Part II experiment system ), 75% ethanol (laboratory autogamy), 95% ethanol (Tianjin Zhi Yuan chemical reagent Co., Ltd), sulfanilamide (SN) (Aladdin) is dense Phosphoric acid, hydrochloride naphthodiamide (Aladdin), cyclooxygenase-2 kit (Bioengineering Research Institute is built up in Nanjing), prostaglandin E2 (Bioengineering Research Institute is built up in Nanjing).

Key instrument：HHS types electric-heated thermostatic water bath (Shanghai Boxun Industrial Co., Ltd.), HF90CO2 incubators (Shanghai Li Shen scientific instrument Co., Ltd), SW-CJ-1FD super-clean benches (Su Jing is safe and sound), KDC-220HR high speed freezing centrifuges (University of Science and Technology Creation Stock Co., Ltd), TDL80-2B low speed centrifuges (Anting Scientific Instrument Factory, Shanghai), microscope, SpectraMax Plus384 ELIASAs (Molecular Devices), VS60-4 microwell plates constant temperature oscillator (irrigates the limited public affairs of glad instrument in Wuxi Department), BP210D assay balances (Sartorius), LDZX-50KBS vertical pressure steam sterilizers (pacify medicine equipment in Shanghai Shen Factory).

2nd, the preparation of solution

The preparation of cell culture medium：DMEM：FBS：Dual anti-=98:10:1.

The preparation of cells frozen storing liquid：FBS：DMSO=9:1.

The compound method of MTT solution:MTT 500mg are weighed, are dissolved in 100ml PBS, the MTT concentration of configuration is 5mg/ Ml, with 0.22 μm of membrane filtration to remove the bacterium in solution, puts -20 DEG C and is kept in dark place.

The preparation of cell complete culture solution：High glucose medium (DMEM)：Hyclone (FBS)：Dual anti-=45：5：0.5.

The preparation of cells frozen storing liquid：FBS：DMSO=9:1.

The preparation of MTT solution：Matching while using, precision weighs MTT powder in right amount, adds PBS, shaking is completely dissolved it, The solution that concentration is 5mg/ml is made, crosses 0.22um miillpore filters, is distributed into some tubules, 4 DEG C save backup, and entirely prepare Journey notes lucifuge.

LPS preparation：Precision pipettes 1mlPBS and added in the LPS Brown Glass Brown glass bottles and jars onlys equipped with 1mg, and ultrasonic dissolution is mixed, complete Total transfer adds 39mlPBS, shakes up, be distributed into a series of tubules, -20 DEG C save backup into 50ml sterile centrifugation tubes, institute It is 25ug/ml to obtain LPS concentration.

The preparation of Metformin hydrochloride solution：Precision weighs Metformin hydrochloride in right amount, adds blank cultures DMEM, matches somebody with somebody Be set to 3200umol/L pastille culture medium mother liquor, be diluted to 5 with blank cultures successively, 10,25,50,100,200,400, 800th, 1600umol/L pastille culture medium.

The preparation of bark of official magnolia phenol solution：Precision weighs magnolol in right amount, adds a small amount of DMSO dissolvings, adds blank culture Base DMEM, is configured to 3200umol/L pastille culture medium mother liquor, be diluted to 5 with blank cultures successively, 10,25,50,100, 200th, 400,800,1600umol/L pastille culture medium (DMSO total amounts are no more than the 0.1% of pharmaceutical culture medium cumulative volume).

The preparation of brufen solution：Precision weighs brufen in right amount, adds a small amount of DMSO dissolvings, adds blank culture Base DMEM, is configured to 1mg/ml brufen mother liquor, and 100ug/ml pastille culture medium is diluted to blank cultures, and (DMSO is total Amount no more than pharmaceutical culture medium cumulative volume 0.1%).

The preparation of magnolol, Metformin hydrochloride mixed solution (H+M, H+2M) solution：Precision weighs magnolol and hydrochloric acid Appropriate melbine, respectively in molar ratio 1:1、1:2 mixing, add a small amount of DMSO dissolvings, add blank cultures DMEM, 3200umol/L magnolol melbine mixed culture medium mother liquor is configured to, H+M, H+2M mother liquor is respectively labeled as, uses successively Blank cultures are diluted to 5,10,25,50,100,200,400,800,1600umol/L magnolol melbine mixed culture Base (DMSO total amounts are no more than the 0.1% of pharmaceutical culture medium cumulative volume).

3rd, experimental method

Cell recovery：15ml sterile centrifugation tubes, add 9ml complete culture solutions, stand-by；Freeze thin is taken out from liquid nitrogen container Born of the same parents' strain, is immediately placed in 37 DEG C of thermostat water baths, and constantly shaking cryopreservation tube completes operation to complete thawing in 1-2min； 75% alcohol wipe sterilize outer tube, be transferred quickly to super-clean bench, by cell suspension be transferred to containing 9ml complete culture solutions from In heart pipe, 1000r/min centrifugation 5min, abandoning supernatant adds appropriate complete medium, is inoculated in 6cm culture dishes, is placed in 37 DEG C, cultivate in the incubator of 5%CO2 and saturated humidity, change culture medium within every two days.

Passage：Culture dish is observed, cell length is treated to the 70%-80% of full ware, discards old culture medium, PBS 3 Secondary, addition 1ml0.25% pancreatin digestion 1min are abandoned in suction, and suction abandons pancreatin, is rapidly added the complete medium prepared, blows and beats repeatedly Ware bottom, makes attached cell blow off completely, and piping and druming is uniform, with 1:3 ratio is passed on.

Cell cryopreservation：Culture dish is observed, cell length is treated to the 80%-90% of full ware, discards old culture medium, PBS 3 Secondary, addition 1ml0.25% pancreatin digestion 1min are abandoned in suction, and suction abandons pancreatin, is rapidly added the complete medium prepared, blows and beats repeatedly Ware bottom, makes attached cell blow off completely, and piping and druming is uniform, will blow and beat uniform cell suspension and is transferred in sterile centrifugation tube, 1000r/min, centrifuges 5min, and abandoning supernatant adds the appropriate frozen stock solution prepared, and piping and druming is uniform, is transferred to cryopreservation tube, often Individual cryopreservation tube liquid feeding 1ml, cryopreservation tube is sealed with sealed membrane, is carried out mark, is put into program temperature reduction box, and program temperature reduction box is put In -86 DEG C of refrigerator overnights, the cell frozen is taken out, is placed in liquid nitrogen container and preserves for a long time.

Cell counter is counted：Tally and cover glass are taken out, 75% alcohol disinfecting is used, after being completely dried, by lid glass Piece is covered on tally, cover glass is placed in counting lattice center；Cell suspension piping and druming is uniform, takes cell suspension 20ul to use Complete culture solution is diluted to 1ml, and piping and druming is uniform, takes cell suspension with range 10ul pipettor, is added in the side of cover glass, Ensure to overflow in cover glass or glass guide channel, in 20 × thing Microscopic observation, adjust focal length, calculate thin in the block plaid of corner Born of the same parents' number, line ball then according to it is upper it is several under do not count, the right not several principles of left number calculate total numbers, and packed cell is according to individual cells meter Number.Cell density Computer Corp. is as follows：(cell number of cell suspension)/ml=(four block plaid total number/4) × 50 × 10⁴。

Mtt assay determines cytoactive：Entitled 3- (4,5- dimethylthiazoles-the 2) -2,5- diphenyltetrazolium bromide bromines of MTT chemistry Salt, is a kind of dyestuff of yellow color, can be reacted with the succinate dehydrogenase in living cells mitochondria, generate the royal purple of water-insoluble Color formazan is crystallized, and is dissolved in DMSO, its absorbance (OD values) is determined at 490nm wavelength, within the specific limits, OD values are got over Greatly, show that cytoactive is stronger, drug toxicity is smaller.

The stable cell cultivated is taken, digestion is counted, and adjusts cell density 10⁴Individual/ml, is inoculated in 96 orifice plates, per hole 100ul, cultivates 24h, and abandoning supernatant, PBS three times adds culture medium (5mg/ml MTT mothers of the 100ul containing MTT per hole Liquid 20ul+80ul blank cultures), 37 DEG C of culture 4h.Careful inhale abandons supernatant (avoiding being drawn onto crystal), is added per hole 150ulDMSO, shakes mixing, and 490nm determines absorbance.

Toxicity research of each medicine to normal RAW264.7 cells：Take the logarithm phase cell, digestion is counted, adjust cell density 10⁵Individual/ml, is inoculated in 96 orifice plates, per hole 200ul, in 37 DEG C, 5%CO₂The 24h of stable culture in incubator, careful inhale is abandoned Clearly, inhaled after packet administration, administration 48h and abandon supernatant, mtt assay determines cytoactive, calculates survival rate.

4th, experimental result

Each medicine is as shown in the table to RAW264.7 cytotoxicity experiment results：

From above-mentioned experimental result, the toxicity reduction of compound 4 is obvious, the toxicity particularly to magnolol in bulk drug Reducing effect is very notable.Itself toxicity of melbine material medicine is relatively low, but the toxicity of noval chemical compound is compared with melbine phase Than also having obvious advantage in higher concentrations, under 40 and 80 μM of concentration, the toxicity of compound 4 is significantly lower than melbine.

The antibacterial effect experiment of the compounds of this invention of embodiment 9

(1) experiment material, laboratory apparatus, reagent

1st, experiment material

Bacterial strain：Resistance candida albicanses 103,100,953 and J28 is clinically separated to be given by Changhai hospital Culture Collection Send, all experiments are drawn plate in husky fort glucose agar medium (SDA) with bacterial strain and activated, and after 30 DEG C are cultivated 2 weeks, choose respectively Take monoclonal draw again plate activation, take second of gained monoclonal to put SDA inclined-planes, cultivate in aforementioned manners after 4 DEG C preserve with It is standby.

Nutrient solution：The preparation of the liquid mediums of RPMI 1640：RPMI 1640 (Gibco BRL) 10g, NaHCO₃2.0g, Morpholine propane sulfonic acid (MOPS) (Sigma) 34.5g (0.165M), plus tri-distilled water 900ml dissolvings, 1N NaOH adjust pH to 7.0 (25 DEG C), tri-distilled water is settled to 000ml, and 0.22 μm of filtering with microporous membrane is degerming, and packing is saved backup after 4 DEG C.

Husky fort agar glucose solid medium (SDA)：Peptone 10g, glucose 40g, agar 18g, plus tri-distilled water 900ml dissolves, and adjusts PH to 7.0, is settled to 1000ml with tri-distilled water, autoclaving (121 DEG C, 15min) is standby after 4 DEG C of preservations With.

YEPD nutrient solutions：Yeast extract 10g, peptone 20g, glucose 20g, plus tri-distilled water 900ml dissolve, and tri-distilled water is fixed Hold to 1000ml, autoclaving (121 DEG C, 15min) is saved backup after 4 DEG C.

2nd, reagent

Fluconazole (Fluconazole, FCZ) parenteral solution is provided by Dalian pharmaceutcal corporation, Ltd of Pfizer, hydrochloric acid magnolol (Berberine Chloride, BBR) is provided by Shanghai Changhai Hospital, dimethyl sulfoxide (DMSO) (Dimethyl Sulphoxide, DMSO) Solution on Chemical Reagents in Shanghai company of Chinese Medicine group produces.

3rd, laboratory apparatus

Multiskan MK3 type enzyme marks detectors (Finland's Labsystems products), water isolation type electro-heating standing-temperature cultivator (on Sea leap medical apparatus and instruments factory), MJX types intelligence mold incubator (Ningbo south of the River instrument plant) THZ-082A Desk type constant-temperatureoscillator oscillators (Shanghai leap medical apparatus and instruments factory)；SW-CT-IF types superpurgative working table (SuZhou Antai Air Tech Co., Ltd.)；It is inverted aobvious Micro mirror (amersham Pharmacia products)；Micro sample adding appliance (Finland's Finnpette products)；96 porocyte culture plates are (red Wheat Nunclon Products)

(2) experimental method

1st, the preparation of fungi suspension

It is a small amount of from the various candida albicans of picking on the SDA culture mediums of 4 DEG C of preservations with inoculation circle before experiment, it is seeded to 1ml YEPD nutrient solutions, in 30 DEG C, 200rpm shaken cultivations, activation 16h makes fungi be in later stage exponential phase of growth.Take the bacterium solution extremely In 1ml YEPD nutrient solutions, counted again after activation 16h with blood cell counting plate, with RPMI 1640 culture mediums in aforementioned manners Bacterial concentration is adjusted to 1 × 10³-5×10³CFU/ml。

2nd, the preparation of drug sensitive reaction plate

Sterile 96 orifice plate is taken, adds the μ l of 1640 fluid nutrient mediums of RPMI 100 to make blank control in every No. 1 hole of row；3-12 holes Respectively plus Fresh the μ l of bacterium solution 100；No. 2 holes add the μ l of bacterium solution 160 and the μ l of test-compound solution 40 respectively；No. 12 holes not pastille Thing, only adds the μ l of bacterium solution 100 to make Growth positive control.2-11 holes carry out doubling dilution, distinguish the final drug concentration in each hole For 64,32,16,8,4,2,1,0.5,0.25 and 0.125 μ g/ml, DMSO contents are below 1% in each hole.Susceptibility is prepared every time A Quality Control bacterium drug sensitive plate is prepared while plate, each drug sensitive plate is in 30 DEG C of insulating box cultures.

3rd, the judgement of minimum inhibitory concentration (MIC)

In 30 DEG C of insulating boxs, each hole OD values are surveyed in 620nm with enzyme micro-plate reader after candida albicans culture 24h.Positive control The OD values control in hole is 0.2 or so, and with positive control boring ratio, the medicine declined with OD values in more than 80% least concentration hole is dense Spend for MIC₈₀(drug concentration when fungi growth 80% is suppressed).As the MIC of medicine₈₀When value exceedes measure concentration range, press Following methods are counted：MIC₈₀When value is higher than 64 μ g/ml of maximum concentration, be calculated as ">64μg/ml”；MIC₈₀It is worth for least concentration Or when below least concentration, do not make difference, it is calculated as "≤0.125 μ g/ml ".The equal operation repetitive of above-mentioned experiment 2 to 3 times, when MIC₈₀Value can accurately repeat or only a poor concentration when just received, and MIC is used as using higher concentration₈₀Value；Work as MIC₈₀Value difference When more than two concentration, then need to test again, untill meeting the requirements.

	Melbine	Magnolol	Compound 4	Penicillin
					MIC₈₀(μg/ml)	10.6±0.61	6.28±0.83	2.82±0.32	1.16±0.61

From MIC₈₀Value is as can be seen that the antibacterial effect of compound 4 is better than monomer, but too late positive control penicillin.

The antitumor anticancer effect test of the compounds of this invention of embodiment 10

It is thin to liver cancer by inquiring into the compounds of this invention by taking hepatoma cell strain HepG 2 and malignant myeloid cell lines K562 as an example In the inhibitory action of born of the same parents strain HepG 2 and malignant myeloid cell lines K562 propagation, to study the antitumor anticancer work of the compound pair With.

This experiment is using Sulforhodamine B (SRB) dyeing, trypan blue row's dye method, comet electrophoresis technology and AO/EB Double dye methods detection the compounds of this invention to the toxicity of hepatoma H22 cells and malignant myeloid cell lines K562, suppress cancer cell and increase The effect of growing, the damage to K562 cell DNAs and the apoptotic effect for inducing K562 cells.

(1) material, instrument and reagent

1st, material and reagent

(DMSO prepares stoste to the compounds of this invention, dilutes before use, it is ensured that DMSO concentration at least dilutes in reaction system For 1 × 10^-2), human hepatoma cell strain He pG2 and human leukemia cell line K562 are quoted from Lanzhou University's Life Science College, training Support base RPM I1640 (U.S. Gibcobr l), yellow acyl rhodamine B (Sulforhodam ineB, S R B, U.S. Sigma), platform Expect blue (Sigma companies), calf serum (Hangzhou Chinese holly), other chemical reagent are that analysis is pure.

2nd, instrument

U.S. Precision Scientific CO₂Incubator, superclean bench, inverted microscope, light microscope, ELIASA (BIO-RADModel 550 and BIO-RAD Model 680), fluorescence microscope, Horizontal electrophoresis tank, high-speed low temperature from Scheming, Costar Tissue Culture Plates.

(2) experimental method

Cell culture：HepG 2 and K562 cells are inoculated in containing in 10% calf serum RPM I1640 culture mediums respectively, are put 37 DEG C, 5%CO₂Cultivated in incubator, growth period cell of taking the logarithm is used to test.

1st, toxic action of the srb assay detection the compounds of this invention to HepG2 cells

Principle：SulforhodamineB (SRB) is a kind of protein combination dye, pink, water-soluble.SRB can be with Combined with the basic amino acid in large biological molecule, it is in good linear relationship in 511nm OD readings and cell number.Therefore can Quantifying as cell number.

Method：The HepG2 cells of exponential phase are with 10 × 10⁴ml^-1Concentration be inoculated in 96 well culture plates, cultivate After 24h, the μ l of the compounds of this invention fresh medium 100 of different diluted concentrations are separately added into respective aperture, continue to cultivate 24, After 48h, nutrient solution is discarded, the 10% μ l of trichloroacetic acid (TCA) 100 are added, stood after 5min, by 4 DEG C of fixed 1h of orifice plate dislocation, Fixer is outwelled, is washed with deionized water 5 times, dries.100 μ l0.4% SRB dyeing 10min are added per hole afterwards, with 1% Acetic acid is washed 5 times, is dried naturally.Then 150 μ l 10mmolL are added per hole^-1Non-buffered Tris alkali lye (unbuffered Tris-base solution, pH10.5) SRB combined with protein is dissolved, it is fully dissolved with oscillator concussion 5min Afterwards, the OD values per hole are determined at 515nm with ELIASA.Seek its cell proliferation rate (%)=(T-T₀)/(C-T₀) × 100, wherein C represents the cell OD values of control group；T represents the cell OD values of dosing group；T₀Represent cell OD when control flat board determines dosing Value.The cell being inoculated with flat board is fixed with TCA at once before dosing.If the final OD values of dosing group are more than T₀；Illustrate thin Born of the same parents still grow after dosing.If the final OD values of dosing group are less than T₀, illustrate that cell is killed after dosing.Utilize recurrence side Journey obtains drug concentration (the 50%inhibition concentration, IC of 50% increment inhibiting rate₅₀).From experimental result See, the final cell number of dosing group is less than T₀, then illustrate that medicine cell growth has cell after inhibitory action, dosing to be killed Extremely.With the IC after the compounds of this invention group and control group processing HepG 2 cell 24h₅₀Carry out each compound of comparison exemplified by value thin to this The toxic action of born of the same parents, IC₅₀Value is as shown in the table：

	Melbine	Magnolol	Compound 4	Cis-platinum
					IC₅₀(μmol/L)	20.54±0.62	40.53±1.85	15.35±1.42	9.54±0.62

Experimental result shows the IC of the compounds of this invention 4₅₀Value is less than monomeric compound before synthesis, i.e., HepG2 cells are pressed down Making strengthens, but still is weaker than positive control drug.

2nd, toxic action of trypan blue row's dye method detection the compounds of this invention to K562 cells

Principle：Living cells has the ability for repelling trypan blue, and due to the destruction of film completeness after cell death, cell is quilt Coloring.Cell suspension containing trypan blue is instilled in white blood cell count(WBC) plate cell, observation under an optical microscope counts living thin Born of the same parents' (cell for not being dyed to blueness).

Method：Concentration is 10 × 10⁴·ml^-1The K562 cell suspensions in exponential phase, with 0.5ml holes^-1Connect Plant in 24 well culture plates.It is placed in 37 DEG C, %CO₂Dosing after preculture 24h in saturated humidity incubator.Continue to cultivate 24,48h Afterwards, take 0.4% trypan blue solution 0.15ml to add in 0.6ml cell suspensions, fully mix, a small amount of mixing is drawn after dyeing 5min Number living cells under liquid, injection blood cell counting plate cell, light microscope, is repeated 3 times, takes its average.Seek its cell proliferation Rate (%)=(T-T₀)/(C-T₀) × 100, wherein C represent the cell number of control group；T represents the cell number of dosing group；T₀Represent Compare cell number when flat board determines dosing.If the final cell number of dosing group is more than T₀, illustrate cell after dosing still Growth.If the final cell number of dosing group is less than T₀, illustrate that cell is killed after dosing.IC is obtained using regression equation₅₀.From Experimental result sees that the final cell number of dosing group is less than T₀, then illustrate that medicine cell growth has inhibitory action, it is thin after dosing Born of the same parents are killed.IC₅₀Value is as shown in the table：

	Cis-platinum	Melbine	Magnolol	Compound 4
					IC₅₀(μmol/L)	20.54±5.63	60.18±7.91	53.25±5.44	32.47±4.86

The blood sugar reducing function effect test of the compounds of this invention of embodiment 11

Using acarbose as positive control drug, external enzyme level experiment is first passed through, the compounds of this invention is determined to glucose The inhibitory action of glycosides enzyme, calculates its IC₅₀Value, if the value is smaller, it is contemplated that carrying out hypoglycemic experiment in animal body.(body herein Outer enzyme level testing program is similar to above-mentioned anti-inflammatory scheme, does not elaborate herein), internal hypoglycemic test method, with high blood Sugared rat is study subject, to investigate the internal blood sugar reducing function of the compounds of this invention.

Hyperglycemic rat is modeled and packet：Cleaning grade SD rats (200 scholar 209) 70 are chosen, male and female half and half, balance is raised 3d, whole blood blood glucose, no pathoglycemia situation are detected before modeling with blood glucose meter.Rat is divided into control group 10, modeling group at random 60, sub-cage rearing gives basal feed and sterile tap water during experiment.Water is can't help in 12h fasting before modeling, in tail vein Inject alloxan (6omg.kg^-1Body weight), control group injects the physiological saline with dosage.5 days rear side blood glucose of Continuous Observation, choosing Fasting blood-glucose is selected in 13~20mmol.L^-1Between test use.

Selection models successfully diabetes rat 40, is randomly divided into 4 groups, every group 10, set up respectively hyperglycemia model group, Magnolol group, the compounds of this invention group and positive control (melbine) group, are remembered with 0.5% picric acid solution mark one, pressed respectively Group sub-cage rearing, male and female are separated.

Administration：2% Tween-80 solution prepares administrable magnolol, the compounds of this invention and melbine solution, and concentration is equal For 10mg.ml^-1.Administration group rat presses 100mg.kg^-1Body weight dose gavage correspondence decoction 10mL.kg^-1, body weight, control group and mould The Tween-80 solution 10mL.kg of type group rat oral gavage 2%^-1Body weight, daily gastric infusion 1 time, continuous gavage 15 days.Zoopery Carried out in standart animal test room, 23 ± 2 DEG C of room temperature, humidity 50~60%, room lighting and dark alternately 12h/d are changed daily Water 1 time, changes bedding and padding for two days 1 time, and each group freely absorbs drinking-water.

Experimental rat blood sugar detection and function of blood sugar reduction evaluation：On the day of each administration group gastric infusion and the 5th after administration, 10th, 15 days tail veins take blood on an empty stomach, and each group rat fasting blood-glucose value is determined with blood glucose meter, compare each group rat fasting blood-glucose value and Blood glucose decline percentage, blood glucose decline percentage=(blood glucose value after blood glucose value-experiment before experiment)/preceding blood glucose value of experiment × 100%.

Statistical procedures：Statistical procedures are carried out to data using SPSS13.0 statistical softwares, all continuous datas are with " Number+standard deviation (x ± s) " represents that standard deviation is calculated by " n-1 " method, the comparison of multigroup sample average, using single factor test Variance analysis, P<0.05 judges that difference has statistical significance, P<0.01 judges that difference has significant statistical significance, as a result It is as shown in the table：

By calculating analysis, the compounds of this invention is compared with magnolol and melbine, P<0.05, it is statistically significant, It is variant, illustrate that the hypoglycemic effect of the compounds of this invention is better than alone magnolol and melbine.

The effect for reducing fat effect test of the compounds of this invention of embodiment 12

The fat Golden Hamster induced using high lipid food is given after the compounds of this invention as study subject, detects body weight, liver Weight, the index such as fat body ratio and blood fat by observing its fat-reducing effect, and then inquires into its effect for reducing fat.

(1) material

Instrument：Supercentrifuge, normal rat experiment cage, electronic scale；

Medicine and reagent：The compounds of this invention, Li Sita difficult to understand, triglyceride reagent box, HDL-C kits, LDL-C reagents Box；

Animal：Male golden yellow gopher 60.

(2) experimental method

Animal model and packet：60 male golden yellow gophers are given basal feed and adapted to after feeding 3 days, randomly select 10 As Normal group, remaining its free choice feeding high lipid food (composition that allows：10% lard, 10% yolk powder, 1% cholesterol, 79% basal feed).Feed four weeks after, extracting vein blood after water 12h, eye socket is can't help in empty stomach fasting, determine its body weight, TC, TG, LDL-C and HDL-C.The successful Golden Hamster of modeling is randomly divided into 5 groups (n=10)：Model group, orlistat group, the present invention The basic, normal, high dosage group of compound, pharmaceutical intervention 4 weeks, Normal group and the isometric physiological saline of the daily gavage of model group, it is difficult to understand Li Sita group daily administration 42mg/kg, the basic, normal, high dosage of the compounds of this invention respectively by daily 23.35,46.70, 70.05mg/kg is administered, and Golden Hamster is freely ingested, drunk water；The illumination rhythm and pace of moving things 12L, 12D (7:00-19:00), room temperature (24 ± 2) DEG C, humidity：(55 ± 10) %.

(3) index determining

1st, body weight, body length and surplus appetite are determined：During testing, each group animal take single cage raise and freely ingest, Drinking-water.A body weight (being weighed before gastric infusion) and surplus appetite are determined, and records result within every 3 days.Surveyed before putting to death under narcosis Fixed its body length (length of nose to anus), waistline, by formula [Lee ' s=(body weight)^-(1/3)x 10³/ body length (cm)] calculate Lee, s index.

2nd, serum TC, TG, LDL-C, HDL-C are determined：Take before blood, water 12h is can't help in empty stomach fasting, with capillary glass tube in eye Vena orbitalis posterior is taken after blood, fasting 2h, 5000r/min centrifugation 10min, takes supernatant, and -20 DEG C of preservations are surveyed using biochemical reagents box Determine TC, TG, LDL-C, HDL-C in serum.

3rd, liver weight, testis and perirenal fat are determined：Take all fat around liver, perinephric fat and testis and claim Weight, calculates the ratio (fat body ratio) of fat/body weight.

(4) data processing

Statistical analysis is carried out to the data obtained with SPSS17.0 softwares, data is calculated and is represented with " mean+standard deviation ", entered Compare between row one-way analysis of variance, group and examined using t, P<0.05 is that difference is statistically significant, P<0.01 is that difference has aobvious Write statistical significance.With the compounds of this invention group after medication four weeks and positive control make hyperlipemia group mouse TC, TG, LDL-C, The effect for reducing fat of the medicine is tentatively probed into exemplified by the percentage that HDL-C declines.

The compounds of this invention (P compared with magnolol and melbine<0.05), have a significant difference, and four with it is positive Comparison medicine Austria Li Sita compares (P<0.01), there is the effect for reducing fat of significant significant difference, i.e. the compounds of this invention better than single Magnolol or melbine, but not as good as positive control drug orlistat.

The compounds of this invention of embodiment 13 is tested the action effect of cardiovascular and cerebrovascular disease

By inquiring into the compounds of this invention to the effect of APOE (-/-) mouse early atherosclerosis (mainly by seeing Examine the influence to the mouse right common carotid artery adventitia removal, vascular collagen and elastic plate), to study the compounds of this invention in the heart Effect in terms of cranial vascular disease.

(1) materials and methods

1st, instrument and equipment：The rotary automatic dehydrators of TISSUE-TEKII:Someiyoshine company, TISSUE-TEK, TECS tissue embedding machines:Someiyoshine company, freezing microtome:German Leica is public, ultra low temperature freezer:SANYO GS company, it is general Logical light microscope:Japanese Nikon companies, polarization microscope:Japanese Olympus companies, fluorescence microscope:Japanese Nikon Company, ImagePro-Plus6.0 image analysis softwares:MacroMedia companies of the U.S., data acquisition and analysis system sPSS17.0:PowerLab companies of the U.S., 7600 automatic analyzers:HIT.

2nd, medicine and reagent

The compounds of this invention (self-control), Atorvastatin：Hangzhou Mo Shadong pharmaceutical Co. Ltds, normal saline solution： Beijing Co., Ltd of Double-Crane Pharmaceutical Co., Ltd, amobarbital sodium：Chemical Reagent Co., Ltd., Sinopharm Group, Hematoxylin-eosin biology dye Toner：Beijing Chemical Plant, the goat anti-rabbit igg of rhodamine mark：Beijing biotech company of Zhong Shan Golden Bridge, the anti-quencher of fluorescence： Beijing biotech company of Zhong Shan Golden Bridge.

Phosphate buffer (phosphate-buffered saline, PBS, 0.01M, pH7.4)：Weigh 8gNaCl, 0.2gKCl、1.44gNa₂HPO₄And 0.24gKH₂PO₄, it is dissolved in 800ml distilled water, adjusts solution ph to 7.4 with HCI, most Afterwards plus distilled water is dissolved to IL.

4% paraformaldehyde liquid：409 paraformaldehydes are weighed, are placed in flask, 800ml 0.l mol/L phosphoric acid buffers are added Liquid, is heated to 60 DEG C, magnetic agitation is completely dissolved powder, finally supplies 0.lmol/L PB to 1000ml, fully mixes.

Picrosirius staining liquid：0.1g sirius reds are dissolved in 100ml picric acid saturated solutions.

Victoria blue dyeing liquor：Victoria blue 2g, dextrin 0.50g, resorcinol 4g, distilled water 200mL, will be above-mentioned 5min is boiled in mixing, then 30% ferric chloride solution boiled is added in upper liquid, continues to boil 2min, is now stirred continuously and is in After colloidal, the cold filtration that reduces internal heat, the residue on paper is placed in 60 DEG C of insulating boxs and dried, dry residue is finally dissolved in 500ml 75% alcohol in, add concentrated hydrochloric acid 5ml and phenol 5g, put room temperature standby.

(2) experimental animal and packet

6 week old male APoE (-/-) mouse 20 by genetic background of C57BL/6, is purchased from Department Of Medicine, Peking University.Raise Support in the animal housing of Sino-Japanese Friendship Hospital for meeting secondary animal feeding standard.Indoor purifying air stream is moved Gas, keeps 22 DEG C~25 DEG C of room temperature, humidity 50% or so, 12 hours light and shade cycles (7am-7Pm).Mouse feeder is in standard mouse cage In, mouse cage, drinking bottle, bedding and padding are through autoclave sterilization.Mouse free water, adaptability feed 6 days after, raise with containing lard 21%, The high lipid food of cholesterol 1.25% and common mixed feed meal 77.75% (60 bore sterilizing treatment with irradiation).Experimental animal operates Process is strictly carried out in accordance with the code of Beijing Union Medical College and Ethics Committee of China-Japan Friendship Hospital, and the processing to animal meets Animal protection ethics.Mouse is randomly divided into 4 groups, the large, medium and small dosage group of blank control group, magnolol, every group each 5, Influence of the various dose group to ApoE (-/-) mouse early atherosclerosis after observation administration.

Medication：According to《Pharmacological experimental methodology》(Xu Shuyun is edited, the third edition in 2002, People's Health Publisher) In people and mouse dosage reduction formula, Experimental agents dosage is converted by body weight.According to being grown up, daily medication is faced The common dose that bed is recommended, is converted to mouse consumption, Atorvastatin group is given by adult and the body weight conversion factor 9.01 of mouse Atorvastatin 3mg/kg/d is given, blank the compounds of this invention group gives physiological saline 0.2ml/kg/d, the compounds of this invention group The compounds of this invention heavy dose group 240mg/kg/d, middle dose group 160mg/kg/d, small dose group 80mg/kg/d are given with gavage Mode be administered, continue 4 weeks.

1st, influence of the compounds of this invention to Ap0E (-/-) mouse early atherosclerosis right common carotid artery adventitia removal

Materials and section：

Materials：After experiment mice fasting 12h, with 1% amobarbital sodium intraperitoneal injection of anesthesia mouse, lain on the back, head Portion and four limbs are fixed on operating table, and longitudinal incision abdomen chest and skin of neck, are successively separated after alcohol disinfecting, cut off tabula, cruelly Reveal heart, inferior caval vein is taken after blood, is irrigated with 0.9% physiological saline by left ventricle, after blood is rinsed well, exposure Right common carotid artery sheath, right common carotid artery of dissociating, is about 1cm, is rinsed with ice physiological saline, after filter paper is blotted, is placed on one On the small circular hardboard for a diameter of 3cm for scribbling OCT frozen section embedding mediums, and OC fourths are coated on whole sample, be placed on Existing filling for Liquid nitrogen precooler about 1min is frozen into solid-state in the small beaker of isopentane, after be placed in -80 DEG C of refrigerators and save backup.

Section：Continuously crosscutting with 6 μm of thickness since right common carotid artery near-end lower section, every 50 μm are left and taken 1 section, will It is placed on slide, and every mouse takes 8 sections.

Pathological examination and analysis：Frozen section row Hematoxylin-eosin (HE) is dyed, around high power Microscopic observation blood vessel The Infiltrating of inflammatory cell.Range of observation is the interstitial tissue of mouse right common carotid artery and surrounding, by dyed tangent plane, The length of vessel that appears is different, it was observed that area it is also different, sample inflammatory cell distribution density degree has very big difference in addition Not, it is unified standard, we choose each intensive region of observed vasculitis cell distribution, and every section is in LEICA It is random on Image analysis system to calculate 8 400 times of object lens visual field positive cells sums as the statistical number of the sample Value.

Frozen section HE staining procedures：1. frozen section, recovers 20min at room temperature；2. cut into slices into HarriS haematoxylins 15min；3. flowing water rinses 5min；4. 0.5% hydrochloride alcohol breaks up 30sec；5. flowing water rinses 5min；6. eosin stains 10min； 7. flowing water rinses 5min；8. gradient alcohol dehydration；9. dimethylbenzene transparent 15min x2 times, middle victory mounting.

Statistical procedures：Using SPSSl7.0 softwares, dosage information is represented using mean ± standard deviation (x scholar S), multigroup ratio Relatively using one-way analysis of variance (one-way ANOVA), compare two-by-two between group and use LSD, P<0.05 is that difference has statistics Meaning.During with heavy dose group, the compounds of this invention group and positive control drug are to mouse right common carotid artery adventitia removal number of cells Influence, to compare influence of the medicine to Ap0E (-/-) mouse early atherosclerosis right common carotid artery adventitia removal, enter And inquire into its effect in terms of cardiovascular and cerebrovascular disease.

Group	Atorvastatin	Magnolol	Melbine	Compound 4
					Inflammatory cell number (individual)	5±2	25±3	29±3	18±2

As a result show：The compounds of this invention (P compared with magnolol and melbine<0.05), there is significant difference, show The compounds of this invention is better than magnolol or melbine to the inhibitory action of mouse right common carotid artery adventitia removal.

2nd, influence of the compounds of this invention to Ap0E (-/-) mouse early atherosclerosis vascular collagen and elastic plate

Experimental animal：Packet is fed, materials are always moved with cutting into slices with to Ap0E (-/-) right neck of mouse early atherosclerosis The experiment of arteries and veins adventitia removal is identical.

Pathological examination and analysis：The ruthless scarlet resisdye Victoria blue dyeing of frozen section day, frozen section sirius red Dyeing is chosen section and dyed with 0.1% picric acid sirius red dye liquor, to assess I types and 111 Collagen Type VIs.Every mouse leaves and takes 8 Section is opened, the collagen content average values of this 8 sections represent the right common carotid artery collagen content value of every mouse.

Picrosirius staining step：(1) 20min is recovered under frozen section room temperature；(2) dimethylbenzene and pure wine are moved into Essence (1:1) 5min or so in mixed liquor；(3) 100%, 95%, 85%, 70% alcohol is entered, at different levels is 5min；(4) distillation washing 3 It is secondary；(5) 60min is contaminated with picric acid sirius red dye liquor；(6) absolute alcohol directly breaks up and dehydration；(7) dimethylbenzene permeabilization, in Property gummy mounting.

Frozen section Victoria blue is dyed：Choose section to dye with Victoria blue dye liquor, to assess elastic fibers point Level and Internal-media thickness, every mouse leave and take 8 sections, and the elastic fibers of this 8 sections is classified and its media thickness average value Represent the classification of right common carotid artery elastic fibers and the value of Internal-media thickness of every mouse.

Victoria blue staining procedure：(1) 20min is recovered under frozen section room temperature；(2) cut into slices and wash in 75% alcohol 1min；(3) 30min or so in Victoria blue dyeing liquor；(4) directly with 95% alcohol color separation several seconds；(5) distilled water is used Wash three times；(6) dimethylbenzene is transparent, neutral gum sealing.

Statistical procedures：Using SPSSl7.0 softwares, dosage information is represented using mean ± standard deviation (x ± S), multigroup ratio Relatively using one-way analysis of variance (one-way ANOVA), compare two-by-two between group and use LSD, P<0.05 is that difference has statistics Meaning.During with heavy dose group, the compounds of this invention group and positive controls are to the type of mouse I, III Collagen Type VI content (%) and elastic force The influence of the sick rate (%) of I grade of fiber investigates the medicine to Ap0E (-/-) mouse early atherosclerosis vascular collagen and bullet The influence of power plate.

As a result show：With I type, III Collagen Type VI content (%) and the sick rate (%) of I grade of elastic fibers for control parameters, the present invention Compound is compared (P with melbine and magnolol<0.05), there is significant difference, and three is compared (P with positive controls< 0.01), there is a significant significant difference, and then show the compounds of this invention to Ap0E (-/-) mouse early atherosclerosis The influence of vascular collagen and elastic plate is better than alone magnolol or melbine, but not as good as positive control drug.

Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should Understand, technical scheme can be modified or equivalent substitution, without departing from the essence of technical solution of the present invention And scope.

Claims

1. a kind of compound, it is characterised in that the structural formula of the compound is：

2. a kind of preparation method of compound as claimed in claim 1, it is characterised in that comprise the following steps：

(1) compound of formula (1) is subjected to bromo-reaction with bromo agent under base catalyst A effects in solvent orange 2 A, obtains formula (2) compound；

(2) compound for the formula (2) for obtaining step (1) is under base catalyst B effects, and the compound with formula (3) is in solvent B Generation ring-closure reaction, that is, obtain formula of the present invention (4) compound；

3. the preparation method of compound as claimed in claim 2, it is characterised in that in the step (1), bromo agent is bromoacetic acid In methyl esters, bromoacetate, bromoacetic acid butyl ester, methyl chloroacetate, ethyl chloroacetate, butyl chloroacetate, iodoacetic acid methyl esters extremely Few one kind；Base catalyst A is at least one of sodium carbonate, potassium carbonate；Solvent orange 2 A is acetonitrile, DMF, methanol, acetone, ethanol, two At least one of methyl sulfoxide, tetrahydrofuran, dichloromethane, dimethyl sulfoxide (DMSO), dioxane.

4. the preparation method of compound as described in Claims 2 or 3, it is characterised in that the base catalyst B in the step (2) For at least one of sodium methoxide, caustic alcohol, metallic sodium；Solvent B is acetonitrile, DMF, ethyl acetate, methanol, ethanol, acetone, pyrrole At least one of pyridine, quinoline, dimethyl sulfoxide (DMSO), tetrahydrofuran, dichloromethane, dimethyl sulfoxide (DMSO), dioxane.

5. the preparation method of compound as claimed in claim 2, it is characterised in that in the step (1), bromo agent and formula (1) The mol ratio of compound is 2:1~5:1；The mol ratio of base catalyst A and formula (1) compound is 2:1~6:1；Solvent orange 2 A and formula (1) mol ratio of compound is 5:1~20:1.

6. the preparation method of compound as described in claim 2 or 5, it is characterised in that in the step (2), formula (3) compound Mol ratio with formula (2) compound is 0.5:1~5:1；The mol ratio of base catalyst B and formula (2) compound is 0.04:1~5: 1；The mol ratio of solvent B and formula (2) compound is 5:1~20:1.

7. the preparation method of compound as claimed in claim 2, it is characterised in that bromo-reaction temperature is in the step (1) 30~70 DEG C, the reaction time is 3~12 hours；The temperature of ring-closure reaction is 40~70 DEG C in the step (2), and the reaction time is 1~15 hour.

8. a kind of compound as claimed in claim 1 is for preparing treatment tumour, cancer, hyperglycaemia, high fat of blood, cardiovascular and cerebrovascular Purposes in the medicine of disease.