CN102731522B - Paeoniflorin compound with inhibitory activity against abnormal expression of cyclooxygenase-2, its preparation method and application - Google Patents

Abstract

The invention belongs to the field of pharmaceutical chemistry and specifically relates to a paeoniflorin compound with an inhibitory activity against abnormal expression of cyclooxygenase-2, a synthetic method thereof and an application in preparation of nonsteroidal anti-inflammatory drugs and drugs for treating and preventing cerebral apoplexy and/or Parkinson's disease. The paeoniflorin compound has a structure as shown in the following general formula I.

Description

There is cyclooxygenase-2 unconventionality expression and suppress an active peoniflorin compounds, Its Preparation Method And Use

Technical field

The invention belongs to pharmaceutical chemistry field, more specifically, relate to a kind of COX-2 of having unconventionality expression suppress active peoniflorin compounds, its synthetic method and prepare nonsteroidal anti-inflammatory drug and be used for the treatment of with the medicine of preventing brain stroke and/or parkinsonism in purposes.

Background technology

Cyclooxygenase (cyclooxygenase, COX) be a kind of bifunctional enzyme, there is cyclooxygenase and catalase activity, it is the rate-limiting enzyme that catalytic film phosphatide arachidonic acid (arachidonic acid, AA) is converted into prostaglandins (prostanoid).Cyclooxygenase participates in comprising the multiple physiological and pathological approach such as inflammation, pain, vasculogenesis and blood pressure regulation, is important metabolic enzyme in body.

There is two kinds of isozyme: COX-1 and COX-2 in COX.COX-1 almost in a organized way in intrinsic expression, mediate the generation of low-level physiological prostaglandin(PG), Main Function is protection and regulates intestines and stomach and hematoblastic normal physiological function; COX-2 is a kind of cyclooxygenase of induction type, after being subject to stimulating such as somatomedin, tumour stimulating factor, hormone, bacteriotoxin and cytokine etc., can, by abduction delivering rapidly, mainly participate in the generation of pathologic prostaglandin(PG).

Many evidences show that the prostaglandin(PG) in COX-2 source plays an important role in inflammatory reaction, and inflammatory reaction has participated in the generation of multiple pathologic process, and therefore COX-2 may be served as the potential treatment target spot of many inflammation related diseases.

COX-2 has a large amount of abduction deliverings in inflammation model and rheumatic arthritis patient's synovial bursa tissue, can synthesize some pathologic prostaglandin(PG)s as PGE by metabolism arachidonic acid ₂(prostaglandin E ₂), be attended by the generation of active oxygen radical (reactive oxygen species, ROS) simultaneously, cause amplification and the enhancing of inflammatory reaction, cause pain, fever, the symptom such as red and swollen.At the inflammatory model of a series of rats, as arthritis model, carrageenin cause in capsule model and sufficient sole of the foot edema model, the enzymic activity that selectivity suppresses COX-2 all has obvious antiphlogistic effects, shows that induction type cyclooxygenase COX-2 really plays the effect of outbalance in inflammatory reaction.

The pathological effect of COX-2 in various central nervous system diseases also has report widely.First, the abduction delivering of COX-2 in brain is relevant with acute neurotoxicity.In several hours of epileptic seizures or post-stroke, the abduction delivering of COX-2 and PGE in cerebral tissue ₂generation all significantly increase (being mainly derived from microglia), PGE ₂can discharge a large amount of L-glutamic acid by induction astroglia cell, activate postsynaptic neuron calcium channel open, thereby cause that neurone intracellular calcium overload causes damage and apoptosis.Separately there are some researches show, in neurone, cross the excititoxic effect of expressing COX-2 and also can strengthen glutamate induction.In addition, the chronic inflammatory diseases activity in COX-2 and cerebral tissue is closely related, and this chronic inflammatory reaction meeting increases the weight of to worsen the neuronal damage of nerve degenerative diseases.Epidemiological investigation is found non-selective or optionally nonsteroidal anti-inflammatory agent (the nonsteroidal anti-inflammatory drugs of COX-2 of long-term taking; NSAIDs) crowd suffers from A Ercaimo disease (Alzheimer ' s disease; AD) and parkinsonism (Parkinson ' s disease; PD) risk obviously reduces, and shows that anti-inflammatory drug may provide provide protection to a certain degree in the evolution of nerve degenerative diseases.The mechanism of action of COX-2 in nerve degenerative diseases is not also perfectly clear at present, except the Prostaglandin PGE of the proinflammatory disease of catalysis ₂outside generation, PGD ₂and PGJ ₂also may cause the generation of some cell responses relevant with nerve retrograde affection, wherein PGJ ₂except having neurotoxicity, or the inductor of oxidative stress (oxidative stress), it can reduce neurone glutathion inside (glutathione, GSH) content, reduce the vigor of Selenoperoxidase, thereby the resistance of oxidation of cell is declined.COX-2 also can produce ROS in the generative process of catalysis prostaglandin(PG), further strengthens response to oxidative stress, and this is also considered to cause an important factor of brain injury and anoxic; ROS also can cause the oxidation of Dopamine HCL, and this process has close relationship with the generation development of parkinsonism.

Non-steroidal anti-inflammatory drugs (Non-Steroidal Anti-Inflammatory Drugs, NSAIDs) refers to that a large class has the non-steroid medicine of anti-inflammatory, pain relieving and refrigeration function.The acetylsalicylic acid (Aspirin) of German Bayer AG in 1899 exploitation is first NSAID product, and at the main medicine that becomes anti-inflammatory treatment half a century thereafter.1971, Vane and colleague thereof found that by research NSAIDs is the activity by suppressing COX, play a role thereby blocking-up arachidonic acid is converted into prostaglandin(PG).At present, according to the not homospecificity to the effect of two kinds of COX isozymes, mainly NSAIDs is divided into four large classes: (1) COX-1 selective depressant (Asprin); (2) non-selective COX inhibitor (indomethacin); (3) COX-2 relative selectivity inhibitor (meloxicam); (4) specific C OX-2 selective depressant (celecoxib).Wherein COX-1 selective depressant and non-selective inhibitor are owing to having suppressed the activity of COX-1 of intrinsic expression; reduce physiological, to there is the prostaglandin(PG) of intestines and stomach provide protection generation; therefore life-time service can cause more serious intestines and stomach damage, and the NSAIDs of this quasi-tradition also can cause the side effect such as dysfunction and bleeding tendency of liver, kidney simultaneously.NSAIDs of new generation taking COX-2 selective depressant as representative, by the COX-2 of induction type being carried out to special inhibition and don't affecting the activity of COX-1, therefore can bring into play significant anti-inflammatory action, can reduce again the toxic side effect that traditional NSAIDs brings.Found first since 1991 COX-2 to 1998 first selective depressant celecoxibs at the year end (Celecoxib) as medicine by U.S. FDA approval listing, being enough to soon of the research and development of the anti-inflammatory drug taking COX-2 as target and promotion rate illustrates that the discovery of COX isozyme is a landmark breakthrough in the development history of nonsteroidal anti-inflammatory agent.At present, COX-2 selective N SAIDs is mainly used in the treatment of the diseases such as rheumatoid arthritis, osteoarthritis and acute pain.

But in September, 2004, Merck drugmaker announces to recall voluntarily in the whole world COX-2 selective N SAID rofecoxib (Rofecoxib) of the said firm's research and development, reason is the side effect that can cause cardiovascular aspect due to this medicine of long-term taking.In October, 2002, one section of research report in the famous medical journal " lancet " (The Lancet) of Britain is pointed out, the adenomatous polyp of the rofecoxib of 3 years prevention by a definite date (Adenomatous Polyp Prevention on Vioxx, APPROVe) data of follow-up investigation shows, taking medicine every day, to exceed ratio that the crowd of 25 milligrams suffers from severe coronary artery disease be 1.7 times of the crowd of not taking medicine.So far, the safety problem of COX-2 selectivity anti-inflammatory drug starts to cause people's attention.Think that at present COX-2 selective depressant causes that the mechanism of cardiovascular side effects is mainly because the Shearing stress energy rapid induction vascular endothelial cell of physiological range is expressed COX-2 and maintains a lower level, the prostacyclin PGI that these a small amount of endogenous CO X-2 catalysis of expressing produce ₂there is vasodilation and antiplatelet aggregative activity, thus the thromboxane TXA that in balance thrombocyte, COX-1 catalysis produces ₂effect, the use of COX-2 selective depressant causes this physiological equilibrium to be broken, the effect of thromboxane is exaggerated, cause elevation of the blood pressure, atherogenesis accelerate and atherosclerotic plaque rupture process in the enhancing of thrombosis reaction, therefore strengthened the risk of cardiovascular injury.

Rofecoxib event prompt we: be necessary different cyclooxygenases role and mechanism thereof under different physiology or pathological conditions to do further research, to promote the exploitation of the medicine that has more selectivity and greater security.The mechanism of action of COX-2 selective N SAIDs is mainly the activity by suppressing COX-2 enzyme, thus inflammation-inhibiting medium PGE ₂generation, reduce inflammation and react and alleviate various symptoms.Express if can suppress the COX-2 of proinflammatory disease material induction, can reduce equally PGE ₂generation, reach the similar antiphlogistic effects with COX-2 selective N SAIDs.

The applicant discloses peoniflorin and has had the effect of the nerve degenerative diseases such as treatment and preventing brain stroke and parkinsonism in previous granted patent (number of patent application 200410025717.8), and little by further studies have shown that its cardiovascular side effects, there is clinical value.But, we are carrying out finding in drug development research to peoniflorin, and this compound does not show activity in testing in vitro, only have in vivo in animal model, could show active, and peoniflorin administration could be effectively in advance, these characteristic limitations the one-tenth property of medicine of peoniflorin.And the inventor studies peoniflorin active mechanism on this basis, find that it is active relevant with the unconventionality expression that suppresses COX-2, instead of the activity of inhibition COX-2, and utilize COX-2 external model to carry out screening active ingredients to peoniflorin structure of modification product, find the peoniflorin compounds of a class novelty, it is active that wherein Compound D G has good COX-2 unconventionality expression inhibition, and confirm that by experimentation on animals this compound also has good restraining effect to the unconventionality expression of COX-2 in vivo, and experimentation on animals also proves that it can realize by the mode of pre-administration and rear administration effect for the treatment of and cerebral apoplexy and sequela and parkinsonism.Therefore, DG is not only suitable for being developed to and is used for the treatment of with the medicinal application of preventing brain stroke and sequela and parkinsonism in clinical, and can the treatment for various inflammation for the preparation of NSAID (non-steroidal anti-inflammatory drug).

Summary of the invention

Technical problem

The object of this invention is to provide a class have COX-2 unconventionality expression suppress active peoniflorin compounds, its synthetic method and prepare nonsteroidal anti-inflammatory drug and be used for the treatment of with preventing brain stroke and/or parkinsonism medicine in purposes.

Technical scheme

To achieve these goals, one aspect of the present invention provides a kind of COX-2 of having unconventionality expression to suppress the active peoniflorin compounds for nervous system disorders effects such as various inflammation treatments and treatment and preventing brain stroke and Parkinson's disease, and it has the structure shown in following general formula I:

Wherein, R ₁for example, for hydrogen, benzoyl, naphthoyl, pyridine formyl radical, benzenesulfonyl, phenylacetyl, C3-C8 cycloalkyl formyl radical (hexanaphthene formyl radical), diamantane formyl radical, C1-C6 alkyl acyl or furancarbonyl;

R ₂for C1-C6 alkyl acyl (for example, ethanoyl, positive propionyl, isopropyl acyl group, positive butyryl radicals, isobutyryl, tertiary butyryl radicals), C1-C6 alkoxy C 1-C6 alkyl (for example methoxymethyl), C1-C6 alkyl sulphonyl (for example, methylsulfonyl), benzenesulfonyl, C3-C8 cycloalkyl formyl radical (for example hexanaphthene formyl radical, cyclopropane formyl radical), benzoyl, C1-C6 alkyl (for example, methyl) or hydrogen;

R ₃for example, for example, for example, for hydrogen, C1-C6 alkyl acyl (, ethanoyl), C1-C6 alkyl (, methyl) or C1-C6 alkyl sulphonyl (, methylsulfonyl);

1～3 group that described phenyl is not necessarily selected from amino, nitro, halogen or C1-C6 alkoxyl group replaces.

The preferred structure of compound of the present invention is as follows:

Another aspect of the present invention also provides the preparation method of described peoniflorin compounds.

Can adopt synthetic the application's of method of various routines in chemical field compound, the inventor is doing after research widely, preferred following synthetic method.

The synthetic method of the compound of formula I is as follows:

Step a: first by starting raw material peoniflorin 1, TERT-BUTYL DIMETHYL CHLORO SILANE TBSCl, imidazoles and solvent synthetic 6 '-O-tertiary butyl dimethylsilyl peoniflorin 2 at a certain temperature;

Wherein, described solvent is one or more in acetonitrile, toluene, benzene, ethanol, DMF; The consumption of solvent is 0.5～10 times of peoniflorin 1 weight; Temperature of reaction is 0～40 DEG C; Reaction times is 0.5～3 hour;

Wherein, preferred reaction conditions is as follows: solvent is DMF, and the consumption of solvent is 5～7 times of peoniflorin 1 weight; Temperature of reaction is 25 DEG C; Reaction times is 1 hour;

Step b: compound 2 is removed benzoyl synthetic compound III with alkali in solvent;

Wherein, taking the mixed solvent of methyl alcohol and water as reaction solvent, mixed volume ratio is 100:1～50:50, and the consumption of reaction solvent is 1～20 times of compound compound 2 weight; Alkali is selected from sodium hydroxide, calcium hydroxide and lithium hydroxide, and the consumption of alkali is 1/400～1/100 of compound 2 weight; Temperature of reaction is 0～100 DEG C; Reaction times is 0.5～10 hour;

Step c: compound III and TrocCl be synthetic compound IV in solvent and under certain temperature, and described TrocCl is 2,2,2-, tri-chloroethyl carbonyl chlorine;

Wherein, solvent is pyridine, and solvent load is 1-10 times of compound III weight; The consumption of TrocCl is 1%～10% of compound III weight; Temperature of reaction is 0～100 DEG C; Reaction times is 0.5～10 hour;

Steps d: the synthetic compound V under certain temperature and solvent action by compounds Ⅳ and acylating agent;

Wherein, described acylating agent is C1-C6 alkyl acyl chloride, SULPHURYL CHLORIDE, benzene sulfonyl chloride, C1-C6 alkoxy C 1-C6 alkyl alcohol, C3-C8 cycloalkyl formyl chloride, Benzoyl chloride or the C1-C6 alkyl alcohol of C1-C6; Described solvent is one or more in pyridine, toluene, benzene, DMF; The consumption of solvent is 0.5～10 times of compounds Ⅳ weight; The consumption of acylating agent is 2～10 times of mole number of compounds Ⅳ; Temperature of reaction is 0～100 DEG C; Reaction times is 0.5～10 hour;

Wherein, preferred reaction conditions is as follows: solvent is anhydrous pyridine, and the consumption of solvent is 5～6 times of compounds Ⅳ weight; Acylating agent is formyl chloride, Acetyl Chloride 98Min., propionyl chloride, benzene sulfonyl chloride, cyclohexyl acyl chlorides or Benzoyl chloride, and its consumption is 8 times of compounds Ⅳ mole number; Temperature of reaction is 40～80 DEG C; Reaction times is 5～8 hours;

Step e: compound V and excess acid are removed tertiary butyl dimethoxy silylation and obtained compound VI in solvent and under certain temperature;

Wherein, solvent is acetonitrile, and acid is selected from hydrofluoric acid or hydrochloric acid; The consumption of acid is excessive; Temperature of reaction is 0～100 DEG C; Reaction times is 0.5～10 hour;

Wherein, preferred reaction conditions is as follows: the ratio of hydrofluoric acid-acetonitrile be (1:20 (volume ratio), its consumption is 10～15 times of compound 5 weight; Temperature of reaction is 0～20 DEG C; Reaction times is 0.5～2 hour;

Step f: compound VI and triphenylphosphine, imidazoles, iodinating agent be synthetic compound VII in solvent;

Wherein, described solvent is one or more in pyridine, toluene, benzene, DMF; The consumption of solvent is 10～60 times of compound VI weight; Iodinating agent is selected from pure iodine, hydroiodic acid HI and pentaiodo phosphorus, and the consumption of iodinating agent is 1～4 times of compound VI weight; Temperature of reaction is 0～100 DEG C; Reaction times is 0.5～10 hour;

Wherein, preferred reaction conditions is as follows: solvent is benzene, and the consumption of solvent is 30～40 times of compound VI weight; Iodinating agent is pure iodine, and its consumption is 2 times of compound VI mole number; Temperature of reaction is 60～80 DEG C; Reaction times is 1～3 hour;

Step g: compound VIII and metallic zinc are removed glucosyl group synthetic compound IX in solvent;

Wherein, taking ethanol and water mixed solvent as reaction solvent, mixed volume ratio is 100:1～50:50, and the consumption of reaction solvent is 1～20 times of compound VIII weight; Temperature of reaction is 0～100 DEG C; Reaction times is 0.5～10 hour;

Wherein, preferred reaction conditions is as follows: the volume ratio of the mixing of ethanol and water mixed solvent is 95:5, and the consumption of solvent is 10～15 times of compound VIII weight; Temperature of reaction is 70～90 DEG C; Reaction times is 1～3 hour;

Step h: compound IX is reacted under certain temperature and obtained compound X in solvent with acylating agent;

Wherein, solvent is the mixed solvent of methylene dichloride and triethylamine, its blending ratio is 100:1～100:10, described acylating agent is Benzoyl chloride, naphthoyl chloride, pyridine formyl chloride, benzene sulfonyl chloride, phenyllacetyl chloride, C3-C8 cycloalkyl formyl chloride, diamantane formyl chloride, C1-C6 alkyl acyl chloride or furoyl chloride, and described acylating agent consumption is 1～3 times of triethylamine volume; Temperature of reaction is 0～100 DEG C; Reaction times is 0.5～10 hour;

Step I: compound X generates with excessive acylation reaction the compound that formula I represents in solvent;

Wherein, described acylating agent is C1-C6 alkyl acyl chloride, C1-C6 alkyl alcohol or C1-C6 alkyl sulfonyl chloride, and reaction solvent is pyridine, and consumption is 1/20～1/10 of compound X weight, and temperature of reaction is 0～100 DEG C; Reaction times is 0.5～10 hour.

Another aspect of the present invention provides the purposes of described peoniflorin compounds in the medicine of preparing cox 2 inhibitor, further for preparing nonsteroidal anti-inflammatory drug and for the preparation of the purposes in the medicine for the treatment of and preventing brain stroke and sequela and parkinsonism.

Cyclooxygenase COX is the rate-limiting enzyme that catalytic film phosphatide arachidonic acid (AA) is converted into prostaglandins, and it is mainly metabolized to PGH2 (PGH by cyclooxygenase and catalase activity by AA ₂); Dissimilar cell has the synthetic path in different downstreams, can be further by PGH ₂change into final meta-bolites, as endotheliocyte generates prostacyclin (PGI ₂), thrombocytopoiesis thromboxane (TXA ₂), scavenger cell generate PGE ₂deng, thereby exercise different physiology or pathologic function.Induction type cyclooxygenase COX-2 is as a kind of immediate early gene (IEG) albumen, can be by the abduction delivering rapidly such as many somatomedins, proinflammatory disease material, tumour stimulating factor, hormone, bacteriotoxin and cytokine, there is a large amount of research evidences to show, in the pathologic process of numerous disease, be all attended by the overexpression of COX-2, as inflammation, nerve degenerative diseases, cancer etc.; And the abduction delivering of COX-2 equally also can cause the generation of disease or the deterioration of the course of disease, as amplified inflammatory reaction by positive feedback effect, induce acute nerve excitability toxicity, increasing the weight of neuronic damage in nerve retrograde affection process by the proinflammatory or the generation active oxygen radical (ROS) that postpone, this shows that the unconventionality expression of COX-2 and the generation of these diseases development are high-positive correlation relation.Therefore the abnormal induction that, suppresses COX-2 is expressed an alternative thinking that becomes this class disease for the treatment of.

In cerebral apoplexy process, generation pathology inflammation is followed the rising of COX-2, and peoniflorin reduces cerebral apoplexy process midbrain injury by the inflammation that suppresses pathology.Accordingly, the inventor suppresses COX-2 pathologic to the various peoniflorin compounds of structure of modification and crosses the evaluation of expression activity Pharmacodynamics in vitro.We find that the activity of DG is best, concentration range dose-effect relationship at 1～50 μ M is obvious, and pre-treatment, co-processing and aftertreatment can play suppress COX-2 cross expressional function, illustrate that the therapeutic time window of DG is larger, this has improved the possibility as Treatment of Cerebral Stroke medicine.In onset concentration range DG to the enzymic activity of COX-1 and COX-2 all without obvious restraining effect, this has got rid of the COX-1 of DG normal tissue existence and the impact of COX-2 function, got rid of gi tract and cardiovascular systems aspect are had side effects may, in this point, be better than current non-steroidal anti-inflammatory drugs (NSAIDs).And the inventor also confirms by experimentation on animals, DG has good anti-inflammatory activity and treatment cerebral apoplexy activity.Therefore,, from DG pharmacodynamic evaluation, DG is reacted and is had good clinical value to treating cerebral apoplexy and parkinsonism aspect and various inflammation treatments aspect by inflammation-inhibiting.

Brief description of the drawings

Fig. 1 shows the THP-1 cell expressing COX-2 albumen of LPS (lipopolysaccharides) induction differentiation;

Fig. 2 shows that DG suppresses the expression of the COX-2 albumen of LPS induction in differentiation THP-1 cell, does not affect the intrinsic expression of COX-1 albumen;

Fig. 3 shows that DG suppresses the time-effect relationship of COX-2 abduction delivering;

Fig. 4 shows the toxic action of DG to THP-1 cell;

Fig. 5 shows the impact that DG generates for the PGE2 of LPS induction in differentiation THP-1 cell;

Fig. 6 shows the impact of DG on COX-1 (A) and COX-2 (B) enzymic activity;

Fig. 7 shows the impact of the COX-2 genetic transcription of DG on LPS induction;

Fig. 8 shows the effect of DG in the cerebral ischemia of anti-intraluminal middle cerebral artery occlusion in rats embolism initiation;

Fig. 9 a shows that DG can obviously dwindle the area of the cerebral infarction of intraluminal middle cerebral artery occlusion in rats embolism initiation, difference significance compared with physiological saline group;

Fig. 9 b is the scatter diagram that shows each treated animal brain infarction area.

Embodiment

Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail, but the present invention is not limited to these drawings and Examples.

Specific embodiment

1. instrument and equipment

The multi-functional microplate reader of NOVOstar (BMG LABTECH, Germany);

Electrophoresis apparatus and half-dried electric transfer groove (Bio-Rad Laboratories, Hercules, CA);

PCR instrument (MJ Research, South San Francisco, CA);

Gel imaging instrument (Fu scientific & technical corporation);

Olympus IX51 research grade inverted fluorescence microscope (Olympus, Japan);

Infrared spectra: Perkin-Elmer 577 type infrared spectrophotometers;

Mass spectrum: Finnigan MAT-95, Finnigan LCQ-DECA type mass spectrograph (low resolution ESI).

Nuclear magnetic resonance spectrum: Varian Mecury Plus-300 type nuclear magnetic resonance analyser, Bruker AM-300 type nuclear magnetic resonance analyser, δ (ppm), taking TMS as interior mark;

LC-MS:Agilent 1100 liquid phases are coupled bruker esquire mass spectrograph;

Analysis mode HPLC instrument: Waters 2690 Separate Model, Waters PDA 996 detectors are coupled Alltch ELSD 2000 detectors, Millennium 2000 operating systems, XTerra ^tMrP18column (4.6 × 250mm, 5 μ m, Waters), flow velocity 1.0ml/min, CH ₃cN (Merck, Germany), H ₂o (Robust Purewater, Robust).

2. reagent and material:

Column chromatography silica gel: 200～300 order silica gel and silica gel H are Haiyang Chemical Plant, Qingdao and produce;

TLC prefabricated thin laminate: HSGF254 is that chemical plant, Yantai produces;

Dextrane gel Sephadex LH-20:Pharmacia BiotechAB, Uppsala, Sweden;

Color developing detection method: 10% sulfuric acid 4-hydroxyl-3-methoxylbenxaldehyde solution, iodine, UV254 and UV365 ultraviolet lamp;

In experiment, peoniflorin used is that Yeyang seminar of Shanghai Pharmaceutical Inst., Chinese Academy of Sciences separates and obtains from the radix paeoniae rubrathe, and through UV, IR, NMR and MS are accredited as peoniflorin;

RPMI 1640, penicillin, Streptomycin sulphate and foetal calf serum (fetal bovine serum, FBS) are purchased from Gibco-BRL (Grand Island, NY); Sodium.alpha.-ketopropionate is purchased from Sigma (St.Louis, MO);

COX-1, COX-2, β-actin, I-κ B α, NF-κ B p65 antibody and horseradish peroxidase (horseradish peroxidase, HRP) mark two is anti-purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); ERK, JNK, p38MAPK, IKK antibody and corresponding phosphorylation antibody are purchased from Cell Signaling Technology (Beverly, MA); Alexa Fluor 488 goat anti-mouse iggs are purchased from Molecular Probes (Eugene, OR);

Bio-Rad Protein Assay Kit is purchased from Bio-Rad Laboratories (Hercules, CA); LightShift Chemiluminescent EMSA Kit is purchased from Pierce (Rockford, IL);

PGE2ELISA Kit is purchased from R & D Systems (Minneapolis, MN);

Buddhist ripple ester (Phorbol-12-myristate-13-acetate, PMA), LPS (from Escherichia coli serotype 0111:B4), NS-398, arachidonic acid (AA) and carrageenin (carrageenan) are purchased from Sigma (St.Louis, MO); Proteinase inhibitor leupeptin (leupeptin), Trypsin inhibitor,Trasylol (aprotinin), pepstatin (pepstatin) and phenylmethylsulfonyl fluoride (phenylmethylsulphonylfluoride, PMSF) purchased from Calbiochem (San Diego, CA); Immobilon-P transfer transfer film (PVDF) is purchased from Millipore Corporation (Bedford, MA); Enhanced chemiluminescence reagent (Enhanced chemiluminescence reagents, ECL) is purchased from Pierce (Rockford, IL); Medical X-ray film is purchased from Eastman Kodak Company (Rochester, NY); TRIzol reagent is purchased from Invitrogen (Carlsbad, CA); M-MLV ThermoScript II is purchased from Promega (Madison, WI); Agarose is purchased from BioWest (France); DakoCytomation fluorescence is installed substratum (fluorescent mounting medium) purchased from DakoCytomation (Carpinteria, CA).

Laboratory animal is male mice in kunming, body weight 20 ± 2g, purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center; SD rat, body weight 250～300g, purchased from Shanghai western pul Bi Kai laboratory animal company limited, animal is placed in (21 ± 1 DEG C of temperature in Shanghai Pharmaceutical Inst., Chinese Academy of Sciences's Animal House; Humidity 60%), nature circulation at daytime/night, with food and the water raising of standard.In this article, unless stated otherwise, the ratio that relates to solvent is volume ratio.

Embodiment 1 Compound D G synthetic method

Embodiment 1.1

Peoniflorin (1) (4.8g, 10mmol) is dissolved in DMF (30mL), adds TBSCl (1.5g, 10mmol) and imidazoles (1.4g, 20mmol), stirring at room temperature 1 hour, and TLC monitors reaction.Question response is complete, adds water, ethyl acetate extraction, and the solution washing of the saturated NaCl aqueous solution for ester layer, anhydrous magnesium sulfate drying, filters, concentrated.Purification by silica gel column chromatography for resistates (methylene chloride/methanol=15/1 (V/V)), obtains the compound 2 of 5.5g colorless oil.Productive rate: 93%.

Compound 2:ESI-MS:[M+Na]+: 617. ¹h-NMR (CDCl ₃, 300Hz, δ ppm): 8.18-7.83 (m, 2H), 7.54 (m, 1H), 7.48-7.31 (m, 2H), 5.51 (s, 1H), 4.82-4.35 (m, 3H), 3.94 – 3.16 (m, 5H), 2.59 (m, 1H), 2.37 (m, 1H), 2.24 – 1.74 (m, 3H), 1.34 (s, 3H), 0.87 (9H, s), 0.04 (3H, s), 0.03 (3H, s).

Embodiment 1.2

By compound 2 (6.0g, about 10mmol) be dissolved in anhydrous pyridine (30mL), add excessive acetic anhydride (approximately 8 times of molar equivalents), be heated to 60 DEG C with oil bath, stirring reaction 8 hours, TLC confirms that reaction finishes, to add in reaction solution appropriate dilute hydrochloric acid with in and pyridine, add ethyl acetate extraction, the solution washing of the saturated NaCl aqueous solution for organic layer, anhydrous magnesium sulfate drying, filters, concentrated.Purification by silica gel column chromatography for resistates (chloroform/sherwood oil=2/1 (V/V)) obtains the compound 3 of 4.6g oily.Productive rate is about 70%.

Compound 3:ESIMS m/z 785 ([M+Na] ⁺). ¹h-NMR (CDCl ₃, 300Hz, δ ppm): 8.02 (2H, m), 7.59 (1H, m), 7.47 (2H, m), 5.53 (1H, s), 5.14 (1H, d, J=9.6Hz), 5.02 (1H, d, J=9.5Hz), 4.96 (1H, d, J=7.6Hz), 4.80 (1H, d, J=7.9Hz), 4.61 (1H, d, J=12.1Hz), 4.50 (1H, d, J=12.0Hz), 3.70 (1H, m), 3.60 (1H, m), 3.42 (1H, m), 2.82 (1H, dd, J=6.8, 1.5Hz), 2.52 (1H, d, J=12.4Hz), 2.47 (1H, dd, J=11.2, 7.0Hz), 2.17 (1H, d, J=11.1Hz), 2.10 (3H, s), 2.06 (3H, s), 2.05 (3H, s), 1.98 (3H, s), 1.97 (1H, m), 1.40 (3H, s), 0.89 (9H, s), 0.04 (3H, s), 0.03 (3H, s).

Embodiment 1.3

Compound 3 (700mg, 0.91mmol) is dissolved in to 5%HF/CH ₃in CN (10mL), ice bath stirs 1 hour, and TLC confirms that reaction finishes, and adds saturated NaHCO ₃be neutralized to neutrality, add a small amount of water (10ml) and pump acetonitrile with Rotary Evaporators.With appropriate ethyl acetate extraction, the solution washing of the saturated NaCl aqueous solution for ester layer, anhydrous magnesium sulfate drying, filters, concentrated.Purification by silica gel column chromatography for resistates (chloroform/methanol=100/1 (V/V)), obtains white powder solid 380mg, is compound 4.Productive rate: 65%.

Compound 4: molecular formula: C ₃₁h ₃₆o ₁₅, M _w: 648, white amorphous powder.HRESIMSm/z671.1959 ([M+Na] ⁺, calculate molecular weight 671.1952). ¹h-NMR (CDCl ₃, 300Hz, δ ppm): 8.02 (2H, m), 7.61 (1H, m), 7.47 (2H, m), 5.53 (1H, s), 5.14 (1H, d, J=9.6Hz), 5.02 (1H, d, J=9.5Hz), 4.96 (1H, d, J=7.6Hz), 4.80 (1H, d, J=7.9Hz), 4.61 (1H, d, J=12.1Hz), 4.50 (1H, d, J=12.0Hz), 3.70 (1H, m), 3.60 (1H, m), 3.42 (1H, m), 2.82 (1H, dd, J=6.8, 1.5Hz), 2.52 (1H, d, J=12.4Hz), 2.47 (1H, dd, J=11.2, 7.0Hz), 2.17 (1H, d, J=11.1Hz), 2.10 (3H, s), 2.06 (3H, s), 2.05 (3H, s), 1.98 (3H, s), 1.97 (1H, m), 1.40 (3H, s).

Embodiment 1.4

Compound 4 (400mg, 0.65mmol) is dissolved in benzene (15ml), adds wherein triphenylphosphine (510mg, 1.95mmol), imidazoles (132mg, 1.95mmol), iodine grain (330mg, 1.3mmol), reflux 2 hours, adds water and ethyl acetate extraction, the aqueous solution (the same) washing of the saturated NaCl aqueous solution for organic layer, anhydrous magnesium sulfate drying, filters, concentrated.Gained solid is removed triphenylphosphine with ethyl acetate-sherwood oil mixed solution recrystallization repeatedly.All the other are purification by silica gel column chromatography (pure chloroform) for resistates, continues to utilize sephadex lh-20 (chloroform/methanol=1/1 (V/V)) to obtain 340mg amorphous powder compound 5.Productive rate: 70%.

Compound 5:ESIMS m/z:383 ([M+Na] ⁺), ¹h NMR (300MHz, CDCl ₃) δ 8.03 (m, 2H), 7.61 (m, 1H), 7.48 (m, 2H), 5.52 (s, 1H), 5.11 (t, J=9.3Hz, 1H), 5.01 (dd, J=9.5, 7.8Hz, 1H), 4.86 (t, J=9.4Hz, 1H), 4.79 (d, J=7.8Hz, 1H), 4.58 (d, J=12.0Hz, 1H), 4.47 (d, J=12.0Hz, 1H), 3.48 (m, 1H), 3.25 (dd, J=11.0, 2.3Hz, 1H), 3.07 (dd, J=10.9, 9.2Hz, 1H), 2.86 (d, J=6.2Hz, 1H), 2.58 (d, J=12.4Hz, 1H), 2.52-2.40 (m, 2H), 2.10 (s, 3H), 2.05 (s, 6H), 1.98 (m, 1H), 1.98 (s, 3H), 1.45 (s, 3H).

Embodiment 1.5

Compound 5 (750mg) is dissolved in ethanol/water mixed solvent (95/5,10ml), adds excessive Zn powder, reflux 1 hour.Reaction solution suction filtration obtains filtrate, and after filtrate is spin-dried for, by ethyl acetate and water dispenser for enriched material, organic layer is used the aqueous solution (the same) washing of the saturated NaCl aqueous solution again, and anhydrous magnesium sulfate drying, filters, concentrated.Purification by silica gel column chromatography for resistates (chloroform/methanol=50/1), continues to utilize sephadex lh-20 (chloroform/methanol=1/1 (V/V)) to separate, and obtains colorless oil product D G (6) 210mg, productive rate: 59%.

DG: molecular formula: C ₁₉h ₂₀o ₇, M _w: 360.Colorless oil.HRESIMS m/z 383.1105 ([M+Na] ⁺calculate molecular weight 383.1107); ESIMS m/z:383 ([M+Na] ⁺), 743.1 ([2M+Na] ⁺). ¹h-NMR (CD ₃oD, 300Hz, δ ppm): 8.03 (2H, m), 7.61 (1H, m), 7.47 (2H, m), 5.53 (1H, s), 4.69 (1H, d, J=12.0Hz), 4.61 (1H, d, J=12.0Hz), 2.74 (1H, d, J=6.0Hz), 2.49 (1H, d, J=12.4Hz), 2.13 (2H, m), 2.04 (3H, s), 1.92 (3H, dd, J=12.5,1.8Hz), 1.30 (3H, s). ¹³c NMR (100MHz, CDCl ₃): δ 170.4 (s), 168.4 (s), 134.8 (d), 131.5 (s), 131.1 (d, 2C), 130.1 (d, 2C), 110.3 (s), 103.4 (d), 87.3 (s), 84.3 (s), 70.8 (s), 62.0 (t), 44.9 (q), 42.8 (t), 28.0 (t), 21.8 (d), 18.6 (q).

Embodiment 2 compound 1-5～1-35's is synthetic

Experiment 2.1

Compound 5 (710mg, 1mmol) is dissolved in methylene dichloride (2mL) and is placed in round-bottomed bottle, adds the appropriate (600mg of silver suboxide, about 3mmol), then add methyl iodide (2mL), condensing reflux, stirring reaction 10 hours, TLC monitoring reaction finishes.By reacting liquid filtering, remove silver suboxide and other precipitations, filtrate is concentrated.Enriched material is dissolved in 10ml ethanol/water (95:5), adds excessive Zn powder, reflux 1 hour.Reaction solution suction filtration obtains filtrate, and filtrate is spin-dried for to rear ethyl acetate and the water dispenser used, and organic layer is again with saturated NaCl solution washing, and anhydrous magnesium sulfate drying, filters, concentrated.Resistates is with purification by silica gel column chromatography (chloroform/methanol=50/1), continue to utilize sephadex lh-20 (sephadex LH-20) (chloroform/methanol=1/1 (V/V)) to separate, obtain colorless oil product 1-5160mg, productive rate: 52%.

1-5:MF:C ₁₈h ₂₀o ₆, MW:332. colorless oil .HRESIMS m/z 355.1172 ([M+Na] ⁺calculate molecular weight 355.1158); ¹h NMR (300MHz, CDCl ₃) δ 8.04 (m, 2H), 7.57 (m, 1H), 7.45 (m, 2H), 5.52 (s, 1H), 4.70 (1H, d, J=12.0Hz), 4.61 (1H, d, J=12.0Hz), 3.36 (s, 3H), 2.55 (d, J=4.9Hz, 1H), 2.23 (dd, J=10.5,6.5Hz, 1H), 2.15 (d, J=12.6Hz, 1H), 1.88 (m, 2H), 1.42 (s, 3H).

Experiment 2.2

1-5 (33mg, 0.1mmol) is dissolved in to methanol aqueous solution (4:1,5mL), and ice bath is cooled to 0 DEG C.Add LiOH (3mg), stir 30 minutes, reaction process remains 0 DEG C.TLC detects and confirms that reaction finishes, and adds appropriate dilute hydrochloric acid that reaction system is neutralized to neutrality, adds a small amount of water (10ml) and pumps methyl alcohol with Rotary Evaporators.With appropriate ethyl acetate extraction, ester layer is with saturated NaCl solution washing, and anhydrous magnesium sulfate drying, filters, concentrated.Resistates, with purification by silica gel column chromatography (chloroform/methanol=50/1 (V/V)), obtains 14mg colorless oil 1-6.Productive rate: 62%.

MDGB (1-6): MF:C ₁₁h ₁₆o ₅, MW:228, colorless oil.HRESIMS m/z 251.0909 ([M+Na] ⁺calculate molecular weight: 251.0895); ¹h NMR (300MHz, CD ₃oD) δ 5.34 (s, 1H), 3.88 (d, J=12.1Hz, 1H), 3.83 (d, J=12.1Hz, 1H), 3.37 (s, 3H), 2.62 (dd, J=6.4,1.7Hz, 1H), 2.00 (d, J=10.4Hz, 1H), 1.97-1.80 (m, 2H), 1.73 (d, J=10.4Hz, 1H), 1.24 (s, 3H).

Experiment 2.3

Get compound 2 (600mg, 1mmol) and be dissolved in methanol aqueous solution (4:1,10mL), ice bath is cooled to 0 DEG C.Add LiOH (3mg), stir 30 minutes, reaction process remains 0 DEG C.TLC detects and confirms that reaction finishes, and adds appropriate dilute hydrochloric acid that reaction system is neutralized to neutrality, adds a small amount of water (10ml) and pumps methyl alcohol with Rotary Evaporators.With appropriate ethyl acetate extraction, ester layer is with saturated NaCl solution washing, and anhydrous magnesium sulfate drying, filters, concentrated.Resistates, with purification by silica gel column chromatography (chloroform/methanol=5/1 (V/V)), obtains 290mg colorless oil 1-35a.Productive rate: 62%.

1-35a：ESIMS?m/z:490([M+Na] ⁺); ¹H?NMR(300MHz,CD ₃OD)δ5.26(s,1H),4.54(d,J=7.7Hz,1H),3.93(m,3H),3.72(m,1H),3.40–3.15(m,3H),2.38(m,2H),2.07(m,1H),1.80(m,2H),1.32(s,3H),0.91(9H,s),0.04(3H,s),0.03(3H,s)。

Experiment 2.4

Get 1-35a (5.0g, 10mmol) and be dissolved in two mouthfuls of flasks that fill 30mL pyridine, argon shield, and flask is cooling with cryosel bath, the TrocCl of 350mg was slowly splashed within 4 hours in reaction soln, reaction solution continues stirring reaction 4 hours.Till all disappearing by TLC detecting reactant.Add appropriate dilute hydrochloric acid for and pyridine, add ethyl acetate extraction, organic layer is with saturated NaCl solution washing, anhydrous magnesium sulfate drying, filters, concentrated.Resistates silica gel simple purification, (this reaction is difficult to avoid not occurring excessive Trocization for substrate 1-35a to isolate the product of polarity maximum, be difficult to too ensure all to occur the Trocization of 8 hydroxyls, but the product of polarity maximum name a person for a particular job contain a large amount of 1-35b).This product is dissolved in pyridine, adds 6mL diacetyl oxide, be heated to 60 DEG C, stirring reaction 4 hours.Add appropriate dilute hydrochloric acid for and pyridine, add ethyl acetate extraction, organic layer is with saturated NaCl solution washing, anhydrous magnesium sulfate drying, filters, the concentrated 1-35c crude product that to obtain.This crude product is dissolved in to 5%HF/CH ₃cN (30mL), ice bath stirs 1 hour, and TLC confirms that reaction finishes, and adds saturated NaHCO ₃be neutralized to neutrality, add a small amount of water (20ml) and pump acetonitrile with Rotary Evaporators.With appropriate ethyl acetate extraction, ester layer is with saturated NaCl solution washing, and anhydrous magnesium sulfate drying, filters, concentrated.Resistates, with purification by silica gel column chromatography (chloroform/methanol=100/1 (V/V)), obtains 3.0g colorless oil 1-35d, and overall yield is about 40%.

1-35d：ESIMS?m/z:741,743([M+Na] ⁺) ¹H?NMR(300MHz,CDCl ₃)δ6.20(s,1H),5.31(m,1H),5.04(m,1H),4.81(m,3H),4.47(d,J=12.5Hz,1H),4.25(d,J=12.1Hz,2H),3.63(m,3H),2.87(d,J=7.4Hz,1H),2.70(m,3H),2.09(m,1H,ovrelapped),2.08(s,3H),2.05(s,3H),1.99(s,3H),1.96(s,3H),1.44(s,4H)。

Experiment 2.5

Compound 1-35d (1.4g, 1.9mmol) is dissolved in benzene (10ml), adds triphenyl phosphorus (1.5g, about 3e.q.), imidazoles (0.4g, about 3e.q.), iodine grain (0.9g, about 2e.q.), stirs fully.Reflux 1 hour, adds water and ethyl acetate extraction, and organic layer is concentrated.In resistates, contain 1-35e.Resistates is dissolved in 30ml ethanol/water (95:5), adds excessive Zn powder, reflux 1 hour.Reaction solution suction filtration obtains filtrate, and after filtrate is spin-dried for, by ethyl acetate and water dispenser for enriched material, organic layer is again with saturated NaCl solution washing, and anhydrous magnesium sulfate drying, filters, concentrated.Resistates is with purification by silica gel column chromatography (chloroform/methanol=50/1), continue to utilize sephadex lh-20 (sephadex LH-20) (chloroform/methanol=1/1 (V/V)) to separate, obtain 4mg colorless oil 1-35 (synthetic R ₁the important intermediate of series derivates).Two step overall yields: 6.1%.

1-35:MF:C ₁₂h ₁₆o ₆, MW:256. colorless oil .HRESIMS m/z 279.0826 ([M+Na] ⁺calculate molecular weight 279.0845); ESIMS m/z:279.1 ([M+Na] ⁺), 535.0 ([2M+Na] ⁺). ¹h NMR (300MHz, DMCO) δ 5.36 (s, 1H), 3.99 (d, J=11.9Hz, 1H), 3.90 (d, J=11.9Hz, 1H), 2.55 (dd, J=8.2,4.7Hz, 1H), 2.45 (d, J=12.1Hz, 1H), 2.04 (m, 2H), 2.01 (s, 3H), 1.82 (d, J=12.0Hz, 1H), 1.25 (s, 3H). ¹³c NMR (100MHz, CDCl ₃): δ 168.4 (s), 109.2 (s), 102.3 (d), 85.7 (s), 83.2 (s), 71.3 (s), 58.5 (t), 43.5 (q), 41.9 (t), 27.4 (t), 21.0 (d), 17.9 (q).

Experiment 2.6

Get 1-35 (20mg, 0.08mmol) and add in the two-mouth bottle that fills 2mL methylene dichloride, add triethylamine (30 μ L), under argon shield, reaction system is placed in to cryosel and bathes cooling.Use microsyringe in 5 minutes, slowly benzene sulfonyl chloride (20 μ L) to be dropped in reaction solution again.Stirring reaction 2 hours.Reaction solution is distributed between methylene dichloride and water, and organic layer is with saturated NaCl solution washing, and anhydrous magnesium sulfate drying, filters, concentrated.Purification by silica gel column chromatography for resistates (chloroform/methanol=50/1), continuing preparative TLC purifying (petrol ether/ethyl acetate=1/1 (V/V)) separates, obtain 10mg colorless oil product 1-18 (R1BS), productive rate is about 35%.

1-18:HRESIMS m/z 419.0792 ([M+Na] ⁺calculate molecular weight 419.0777); ¹h NMR (300MHz, CDCl ₃) δ 7.93 (m, 2H), 7.70 (m, 1H), 7.59 (m, 2H), 5.31 (s, 1H), 4.40 (m, 2H), 2.61 (d, J=8.5Hz, 1H), 2.43 (m, 2H), 2.13 (m, 2H), 2.08 (s, 3H), 1.96 (d, J=12.4Hz, 1H), 1.31 (s, 3H).

Experiment 2.7

Get 1-35 (10mg, 0.04mmol) add in 2mL methylene dichloride, add DMAP (3mg, catalytic amount) and anisic acid (7mg, about 0.05mmol), under room temperature, stir about 10 minutes, adds DCC (12.4mg, about 0.06mmol), continue to stir 2 hours.TLC monitoring reaction finishes.First, by the sedimentation and filtration generating in reaction, after filtrate is concentrated, utilize purification by silica gel column chromatography (chloroform/methanol=50/1), obtain 10mg colorless oil product 1-7, productive rate 66%.

1-7:MF:C ₂₀h ₂₂o ₈; MW:390; HRESIMS m/z 413.1230 ([M+Na] ⁺calculate molecular weight 413.1212); ¹h NMR (300MHz, CDCl ₃) δ 7.96 (J=10.9Hz, 2H), 6.91 (d, J=10.9Hz, 2H), 5.54 (s, 1H), 4.72 (d, J=12.2Hz, 1H), 4.65 (d, J=12.2Hz, 1H), 3.86 (s, 3H, OMe), 3.09 (s, 1H, OH), 2.72 (d, J=6.0Hz, 1H), 2.51 (d, J=12.3Hz, 1H), 2.14 (m, 2H), 2.09 (s, 3H), 2.00 (dd, J=12.3,1.7Hz, 1H), 1.34 (s, 3H).

Experiment 2.8---experiment 2.17

Experimental implementation is basic identical with experiment 2.7, and difference is to change the raw material anisic acid of reaction into other carboxylic acids (RCOOH).From testing 2.8 carboxylic acids that use respectively to experiment 2.17 in above-mentioned sign, synthesize thus 1-8 to 1-17, reaction yield is at 60%-70%.1-14 and 1-15 are white powder solid, and other product is colorless oil.

1-8:MF:C ₂₀h ₂₂o ₇; MW:374; HRESIMS m/z 397.1262 ([M+Na]+calculating molecular weight 397.1263); 1H NMR (300MHz, CDCl3) δ 7.40-7.19 (m, 5H), 5.41 (s, 1H), 4.47 (s, 2H), 3.67 (s, 2H), 2.55 (d, J=6.4Hz, 1H), 2.45 (d, J=12.3Hz, 1H), 2.13 (bs, 1H), 2.08 (s, 3H), 2.00-1.88 (m, 2H), 1.28 (s, 3H).

1-9:MF:C ₁₉h ₂₁nO ₇; MW:375; HRESIMS m/z 398.1234 ([M+Na]+calculating molecular weight 398.1216); 1H NMR (300MHz, CDCl3) δ 7.82 (d, J=8.6Hz, 2H), 6.62 (d, J=8.6Hz, 2H), 5.52 (s, 1H), 4.70 (d, J=11.7Hz, 1H), 4.62 (d, J=12.1Hz, 1H), 2.71 (d, J=6.3Hz, 1H), 2.50 (d, J=12.3Hz, 1H), 2.18-1.96 (m, 3H), 2.09 (s, 3H), 1.38 (s, 3H).

1-10:MF:C ₁₉h ₂₆o ₇; MW:366; HRESIMS m/z 389.1590 ([M+Na]+calculating molecular weight 389.1576); 1H NMR (300MHz, CDCl3) δ 5.42 (s, 1H), 4.46 (s, 2H), 2.61 (d, J=6.0Hz, 1H), 2.48 (d, J=12.2Hz, 1H), 2.34 (ddd, J=11.2,7.6,3.5Hz, 1H), (2.08 s, 3H), 2.18 – 1.97 (m, 3H), 1.95 – 1.05 (m, 10H), 1.34 (s, 3H).

1-11:MF:C ₁₉h ₁₉nO ₉; MW:405; HRESIMS m/z 428.0960 ([M+Na]+calculating molecular weight 428.0958); 1H NMR (300MHz, CD3OD) δ 8.32 (m, 2H), 8.24 (m, 2H), 5.55 (s, 1H), 4.74 (d, J=12.1Hz, 1H), 4.68 (d, J=12.1Hz, 1H), 2.78 (d, J=5.8Hz, 1H), 2.49 (d, J=12.3Hz, 1H), 2.12 (m, 2H), 2.03 (s, 3H), 1.92 (m, 1H), 1.30 (s, 3H).

1-12:MF:C ₂₃h ₃₀o ₇; Mw:418; HRESIMS m/z 441.1895 ([M+Na]+calculating molecular weight 441.1889); 1H NMR (300MHz, CDCl3) δ 5.41 (s, 1H), 4.49 (d, J=12.1Hz, 1H), 4.47 (d, J=12.1Hz, 1H), 2.63 (d, J=6.1Hz, 1H), 2.49 (d, J=12.3Hz, 1H), 2.13 (m, 1H), 2.09 (s, 3H), 2.01 (m, 5H), 1.89 (m), 1.77 – 1.64 (m), 1.34 (s, 3H).

1-13:MF:C ₁₉h ₁₉o ₇f; MW:378; HRESIMS m/z 401.1026 ([M+Na]+calculating molecular weight 401.1013); 1H NMR (300MHz, CDCl3) δ 8.04 (m, 2H, coupled with F), 7.12 (m, 2H, coupled with F), 5.55 (s, 1H), 4.72 (d, J=12.2Hz, 1H), 4.68 (d, J=12.2Hz, 1H), 2.73 (d, J=6.0Hz, 1H), 2.51 (d, J=12.2Hz, 1H), 2.14 (m, 2H), 2.09 (s, 3H), 2.00 (dd, J=12.2,1.8Hz, 1H), 1.36 (s, 3H).

1-14:MF:C ₁₄h ₁₈o ₇; MW:298; HRESIMS m/z 321.0938 ([M+Na]+calculating molecular weight 321.0950); 1H NMR (300MHz, CDCl3) δ 5.45 (s, 1H), 4.44 (s, 2H), 2.63 (d, J=4.6Hz, 1H), 2.49 (d, J=12.2Hz, 1H), 2.15 (d, J=10.9Hz, 1H), 2.11 (s, 3H), 2.09 (s, 3H), 2.09 (m, 1H), 1.98 (dd, J=12.2,1.6Hz, 1H), 1.35 (s, 3H).

1-15:MF:C ₂₃h ₂₂o ₇; MW:410; HRESIMS m/z 433.1271 ([M+Na]+calculating molecular weight 433.1263); 1H NMR (300MHz, CDCl3) δ 8.59 (s, 1H), 8.02 (dd, J=8.6,1.7Hz, 1H), 7.96 (d, J=7.9Hz, 1H), 7.87 (d, J=8.6Hz, 2H), 7.58 (m, 2H), 5.62 (s, 1H), 4.82 (d, J=12.2Hz, 1H), 4.75 (d, J=12.2Hz, 1H), 2.78 (m, 1H), 2.53 (d, J=12.3Hz, 1H), 2.18 (m, 2H), 2.10 (s, 3H), 2.02 (dd, J=12.2,1.7Hz, 1H), 1.38 (s, 3H).

1-16:MF:C ₁₈h ₁₉nO ₇; MW:361; HRESIMS m/z 384.1067 ([M+Na]+calculating molecular weight 384.1059); 1H NMR (300MHz, CDCl3) δ 8.68 (d, J=4.8Hz, 1H), 8.16 (d, J=7.8Hz, 1H), 7.89 (t, J=7.2Hz, 1H), 7.53 (dd, J=7.6,4.8Hz, 1H), 5.55 (s, 1H), 4.80 (d, J=12.0Hz, 1H), 4.75 (d, J=11.9Hz, 1H), 2.66 (m, 1H), 2.52 (d, J=12.2Hz, 1H), 2.17 (d, J=5.2Hz, 2H), 2.09 (s, 3H), 2.01 (d, J=12.6Hz, 1H), 1.40 (s, 3H).

1-17:MF:C ₁₇h ₁₈o ₈; MW:350; HRESIMS m/z 373.0895 ([M+Na]+calculating molecular weight 373.0899); 1H NMR (300MHz, CDCl3) δ 7.60 (d, J=0.8Hz, 1H), 7.23 (d, J=3.5Hz, 1H), 6.52 (m, 1H), 5.52 (s, 1H), 4.73 (d, J=12.3Hz, 1H), 4.67 (d, J=12.2Hz, 1H), 2.71 (d, J=5.2Hz, 1H), 2.51 (d, J=12.4Hz, 1H), 2.16 (m, 2H), 2.09 (s, 3H), 2.00 (d, J=12.4Hz, 1H), 1.35 (s, 3H).

Experiment 2.18

Compound 5 (71mg; 0.1mmol) be dissolved in (5mL) in triethylamine; add methoxychlor methyl ether (MOMCl) appropriate (about 2e.q.) with microsyringe, nitrogen protection stirring reaction 3 hours under room temperature, TLC monitoring reaction finishes.By ethyl acetate and water dispenser for reaction solution, organic layer is again with saturated NaCl solution washing, concentrated.Enriched material is dissolved in 10ml ethanol/water (95:5), adds excessive Zn powder, reflux 1 hour.Reaction solution suction filtration obtains filtrate, and filtrate is spin-dried for to rear ethyl acetate and the water dispenser used, and organic layer is again with saturated NaCl solution washing, and anhydrous magnesium sulfate drying, filters, concentrated.Resistates, with purification by silica gel column chromatography (chloroform/methanol=50/1), continues to utilize sephadex lh-20 (chloroform/methanol=1/1 (V/V)) to separate, and obtains 12mg colorless oil product 1-20, productive rate: 32%.

1-20:MF:C ₁₉h ₂₂o ₇; MW:362; HRESIMS m/z 385.1258 ([M+Na]+calculating molecular weight 385.1263); 1H NMR (300MHz, CDCl3) δ 8.01 (dd, J=8.4,1.3Hz, 2H), 7.57 (ddd, J=8.7,2.6,1.3Hz, 1H), 7.42 (m, 2H), 5.41 (s, 1H), 4.87 (d, J=6.6Hz, 1H), 4.69 (d, J=12.2Hz, 1H), 4.60 (d, J=12.2Hz, 1H), 4.53 (d, J=6.6Hz, 1H), 3.27 (s, 3H), 2.92 (d, J=7.0Hz, 1H), 2.67 (m, 2H), 2.44 (dd, J=11.0,7.2Hz, 1H), 2.00 (d, J=11.0Hz, 1H), 1.39 (s, 3H).

Experiment 2.19

Compound 5 (71mg, 0.1mmol) is dissolved in (5mL) in methylene dichloride, adds triethylamine (30 μ L; about 3e.q.), then add propionyl chloride (20 μ L, about 2e.q.); under room temperature nitrogen protection, stir 30 minutes, TLC monitoring reaction finishes.By ethyl acetate and water dispenser for reaction solution, organic layer is again with saturated NaCl solution washing, concentrated.Enriched material is dissolved in 10ml ethanol/water (95:5), adds excessive Zn powder, reflux 1 hour.Reaction solution suction filtration obtains filtrate, and filtrate is spin-dried for to rear ethyl acetate and the water dispenser used, and organic layer is again with saturated NaCl solution washing, and anhydrous magnesium sulfate drying, filters, concentrated.Resistates, with purification by silica gel column chromatography (chloroform/methanol=50/1), continues to utilize sephadex lh-20 (chloroform/methanol=1/1 (V/V)) to separate, and obtains 20mg colorless oil product 1-19, productive rate 54%.

1-19:MF:C ₂₀h ₂₂o ₇; MW:374; HRESIMS m/z 397.1261 ([M+Na]+calculating molecular weight 397.1263); 1H NMR (300MHz, CDCl3) δ 8.00 (m, 2H), 7.57 (m, 1H), 7.42 (dd, J=10.7,4.8Hz, 2H), 5.55 (s, 1H), 4.73 (d, J=12.2Hz, 1H), 4.67 (d, J=12.2Hz, 1H), 2.71 (d, J=6.2Hz, 1H), 2.51 (dd, J=12.2,2.8Hz, 1H), 2.37 (q, J=7.5Hz, 2H), 2.14 (m, 2H), 1.98 (m, 1H), 1.34 (s, 3H), 1.11 (t, J=7.5Hz, 3H).

Experiment 2.20～2.27

Experimental implementation is basic identical with experiment 2.19, and difference is to change propionyl chloride wherein into other acyl chlorides (or SULPHURYL CHLORIDE).Indicate in the drawings from testing 2.20 acyl chlorides or the SULPHURYL CHLORIDE (RCl) that use respectively to experiment 2.27, synthesized thus 1-21 to 1-28, productive rate is at 50%-70%.Product is colorless oil.

1-21:MF:C ₂₁h ₂₄o ₇; MW:388; HRESIMS m/z 411.1432 ([M+Na]+calculating molecular weight 411.1420); 1H NMR (300MHz, CDCl3) δ 8.02 (m, 2H), 7.59 (m, 1H), 7.45 (m, 2H), 5.56 (s, 1H), 4.75 (d, J=12.2Hz, 1H), 4.70 (d, J=12.2Hz, 1H), 2.69 (d, J=6.2Hz, 1H), 2.60 (m, 1H), 2.55 (d, J=11.0Hz, 1H), 2.21 (d, J=10.9Hz, 1H), 2.13 (dd, J=11.2,6.7Hz, 1H), 1.97 (dd, J=12.2,1.5Hz, 1H), 1.37 (s, 3H), 1.18 (d, J=2.6Hz, 3H), 1.16 (d, J=2.7Hz, 3H).

1-22:MF:C ₁₈h ₂₀o ₈s; MW:396; HRESIMS m/z 419.0786 ([M+Na]+calculating molecular weight 419.0777); 1H NMR (300MHz, CDCl3) δ 8.02 (m, 2H), 7.60 (m, 1H), 7.46 (m, 2H), 5.59 (s, 1H), 4.72 (s, 2H), 3.19 (s, 3H), 3.13 (m, 1H), 2.36 (m, 2H), 2.08 (dd, J=11.1,6.4Hz, 1H), 1.95 (d, J=11.1Hz, 1H), 1.39 (s, 3H). this compound room temperature is exposed in air unstable, easily decomposes, and needs refrigeration.

1-23:MF:C ₂₂h ₂₆o ₇; MW:402; HRESIMS m/z 425.1581 ([M+Na]+calculating molecular weight 425.1576); 1H NMR (300MHz, CDCl3) δ 8.02 (m, 1H), 7.59 (m, 1H), 7.45 (m, 1H), 5.55 (s, 1H), 4.75 (d, J=12.3Hz, 1H), 4.70 (d, J=12.3Hz, 1H), 2.67 (d, J=6.2Hz, 1H), 2.52 (d, J=12.2Hz, 1H), 2.21 (d, J=10.9Hz, 1H), 2.13 (dd, J=11.0,6.3Hz, 1H), 1.94 (dd, J=12.3,1.6Hz, 1H), 1.38 (d, J=6.3Hz, 3H), 1.19 (s, 9H).

1-24:MF:C ₂₃h ₂₂o ₈s; MW:458; HRESIMS m/z 481.0925 ([M+Na]+calculating molecular weight 481.0933); 1H NMR (300MHz, CDCl3) δ 8.04-7.97 (m, 2H), 7.97-7.88 (m, 2H), 7.67-7.37 (m, 6H), 5.49 (s, 1H), 4.68 (s, 2H), 3.09 (d, J=6.2Hz, 1H), 2.41 (d, J=12.5Hz, 1H), 2.35 (m 1H), 2.08 (dd, J=8.4,4.2Hz, 1H), 1.97 (d, J=11.1Hz, 1H), 1.37 (s, 3H).This compound room temperature is exposed in air unstable, easily decomposes, and needs refrigeration.

1-25:MF:C ₂₁h ₂₄o ₇, MW:388, HRESIMS m/z 411.1432 ([M+Na]+calculating molecular weight 411.1420), 1H NMR (300MHz, CDCl3) δ 8.02 (m, 2H), 7.59 (m, 1H), 7.46 (t, J=7.6Hz, 1H), 5.56 (s, 1H), 4.75 (d, J=12.2Hz, 1H), 4.70 (d, J=12.2Hz, 1H), 2.77 (s, 1H, OH), 2.72 (d, J=6.1Hz, 1H), 2.53 (d, J=12.2Hz, 1H), 2.33 (t, J=7.3Hz, 2H), 2.21 (d, J=10.8Hz, 1H) .2.19 (m, 1H), 1.99 (dd, J=12.2, 1.7Hz, 1H), 1.64 (m, 2H), 1.37 (s, 2H), 0.95 (t, J=7.4Hz, 3H).

1-26:MF:C ₂₁h ₂₂o ₇; MW:386; HRESIMS m/z 409.1270 ([M+Na]+calculating molecular weight 409.1263); 1H NMR (300MHz, CDCl3) δ 8.02 (m, 2H), 7.60 (t, J=7.4Hz, 1H), 7.46 (t, J=7.6Hz, 2H), 5.56 (s, 1H), 4.75 (d, J=12.3Hz, 1H), 4.69 (d, J=12.2Hz, 1H), 2.73 (d, J=7.2Hz, 1H), 2.52 (d, J=12.3Hz, 1H), 2.13 (m, 2H), 2.01 (d, J=12.3Hz, 1H), 1.65 (m, 1H), 1.37 (s, 3H), 1.00 (m, 2H), 0.96 (s, 2H).

1-27:MF:C ₂₄h ₂₈o ₇; MW:428; HRESIMS m/z 451.1717 ([M+Na]+calculating molecular weight 451.1733); 1H NMR (300MHz, CDCl3) δ 8.03 (d, J=7.6Hz, 2H), 7.59 (t, J=7.4Hz, 1H), 7.46 (t, J=7.6Hz, 2H), 5.55 (s, 1H), 4.75 (d, J=12.3Hz, 1H), 4.70 (d, J=12.2Hz, 1H), 2.68 (d, J=6.0Hz, 1H), 2.52 (d, J=12.2Hz, 1H), 2.34 (m, 1H), 2.21 (d, J=11.0Hz, 1H), 2.12 (dd, J=10.8,6.3Hz, 1H), 1.96 (d, J=12.3Hz, 1H), 1.90 – 1.15 (m, 10H), 1.36 (s, 2H).

1-28:MF:C ₂₄h ₂₂o ₇; MW:422; HRESIMS m/z 445.1272 ([M+Na]+calculating molecular weight 445.1263); 1H NMR (300MHz, CDCl3) δ 8.03 (m, 4H), 7.59 (t, J=6.9Hz, 2H), 7.45 (t, J=7.7Hz, 4H), 5.63 (s, 1H), 4.79 (d, J=12.2Hz, 1H), 4.74 (d, J=12.2Hz, 1H), 2.87 (d, J=6.3Hz, 1H), 2.69 (d, J=12.3Hz, 1H), 2.34 (d, J=10.8Hz, 1H), 2.21 (dd, J=11.0,6.5Hz, 1H), 2.11 (d, J=12.2Hz, 1H), 1.41 (s, 3H).

Experiment 2.28

Get DG (36mg, about 0.1mmol) be dissolved in anhydrous pyridine (3mL), add excessive acetic anhydride (100 μ L), reaction system is heated to 60 DEG C with oil bath, stirring reaction 6 hours, TLC confirms that reaction finishes, in reaction solution, add appropriate dilute hydrochloric acid for and pyridine, add ethyl acetate extraction, organic layer is with saturated NaCl solution washing, anhydrous magnesium sulfate drying, filters, concentrated.Resistates obtains 36mg oily product 1-29 with purification by silica gel column chromatography (chloroform/sherwood oil=2/1 (V/V)).Productive rate is about 90%.

1-29:MF:C ₂₁h ₂₂o ₈; MW:402; HRESIMS m/z 425.1227 ([M+Na]+calculating molecular weight 425.1212); 1H NMR (300MHz, CDCl3) δ 8.03 (m, 2H), 7.59 (t, J=7.4Hz, 1H), 7.46 (t, J=7.5Hz, 2H), 5.61 (s, 1H), 4.74 (d, J=12.2Hz, 1H), 4.64 (d, J=12.1Hz, 1H), 2.88 (d, J=6.7Hz, 1H), 2.72 (d, J=11.8Hz, 1H), 2.66 (d, J=12.3Hz, 1H), 2.43 (dd, J=11.8,6.6Hz, 1H), 2.10 (s, 3H), 2.04 (m, 1H), 2.03 (s, 3H), 1.33 (s, 3H).

Experiment 2.29

Get DG (36mg, about 0.1mmol) and be dissolved in methylene dichloride (1mL) and be placed in round-bottomed bottle, add the appropriate (60mg of silver suboxide, about 0.3mmol), then add methyl iodide (1mL), condensing reflux, stirring reaction 8 hours, TLC monitoring reaction finishes.By reacting liquid filtering, remove silver suboxide and other precipitations, filtrate is concentrated.Resistates obtains 30mg white powder product 1-30 with purification by silica gel column chromatography (chloroform/sherwood oil=2/1 (V/V)).Productive rate is 81%.

1-30:MF:C ₂₀h ₂₂o ₇; MW:374; HRESIMS m/z 397.1267 ([M+Na]+calculating molecular weight 397.1263); 1H NMR (300MHz, CDCl3) δ 8.04 (m, 2H), 7.57 (m, 1H), 7.45 (t, J=7.6Hz, 2H), 5.59 (s, 1H), 4.70 (d, J=12.2Hz, 1H), 4.62 (d, J=12.2Hz, 1H), 3.36 (s, 3H), 2.75 (dd, J=6.6,1.7Hz, 1H), 2.53 (d, J=12.3Hz, 1H), 2.34 (dd, J=10.8,6.7Hz, 1H), 2.15 (d, J=10.7Hz, 1H), 2.09 (s, 3H), 1.97 (dd, J=12.2,1.9Hz, 1H), 1.41 (s, 3H).

Experiment 2.30

Get DG (36mg, about 0.1mmol) and be dissolved in methylene dichloride (1mL), add triethylamine (30 μ L, about 3e.q.), then add methane sulfonyl chloride (25 μ L, about 2e.q.), stirring reaction 1 hour, TLC monitoring reaction finishes.Reaction solution is distributed in water and methylene dichloride, and organic layer is with saturated NaCl solution washing, and anhydrous magnesium sulfate drying, filters, concentrated.Resistates obtains 34mg oily product 1-31 with purification by silica gel column chromatography (chloroform), and productive rate is 81%.

1-31:MF:C ₂₀h ₂₂o ₉s; MW:438; HRESIMS m/z 461.0889 ([M+Na]+calculating molecular weight 461.0882); 1H NMR (300MHz, CDCl3) δ 8.02 (dd, J=5.3,3.2Hz, 2H), 7.58 (m, 1H), 7.44 (m, 2H), 5.60 (s, 1H), 4.77 (d, J=12.2Hz, 1H), 4.70 (d, J=12.2Hz, 1H), 3.02 (s, 3H), 3.01 (m, 1H), 2.92 (dd, J=7.0,1.8Hz, 1H), 2.61 (t, J=11.5Hz, 2H), 2.07 (s, 3H), 2.04 (m, 1H), 1.50 (s, 3H).

Experiment 2.31

Compound 5 (710mg; 1mmol) be dissolved in methylene dichloride (5mL) and be placed in two-mouth bottle; under argon shield, first add 2; 6-lutidine (2,6-lutidine) (approximately 200 μ L), stirring at room temperature 10 minutes; add again TIPSOTf (450mg; 1.5mmol), stirring at room temperature 1 hour, TLC monitors reaction.Question response is complete, removes argon shield, adds water, dichloromethane extraction, and organic layer washs with saturated NaCl, and anhydrous magnesium sulfate drying filters, concentrated.Resistates, with silica gel column chromatography simple purification (methylene dichloride/sherwood oil=1/1 (V/V)), obtains compound 5a crude product.Again 5a crude product is dissolved in 10ml ethanol/water (95:5), adds excessive Zn powder, reflux 1 hour.Reaction solution suction filtration obtains filtrate, and filtrate is spin-dried for to rear ethyl acetate and the water dispenser used, and organic layer is again with saturated NaCl washing, and anhydrous magnesium sulfate drying, filters, concentrated.Resistates is with purification by silica gel column chromatography (chloroform/methanol=50/1), continue to utilize sephadex lh-20 (sephadex LH-20) (chloroform/methanol=1/1 (V/V)) to separate, obtain colorless oil product 5b 260mg, productive rate: 55%.

5b：ESIMS?m/z?497([M+Na]+).1H-NMR(CDCl3,300Hz,δppm):8.01(d,J=7.0,5.6Hz,2H),7.57(t,J=7.4Hz,1H),7.42(m,2H),5.47(s,1H),4.72(d,J=12.1Hz,1H),4.62(d,J=12.1Hz,1H),2.53(d,J=6.3Hz,1H),2.24-1.78(m,4H),1.33(s,3H),1.03(m,18H)。

Experiment 2.32

Get compound 5b (94mg, about 0.2mmol) be dissolved in anhydrous pyridine (3mL), add excessive acetic anhydride (100 μ L), reaction system is heated to 60 DEG C with oil bath, stirring reaction 6 hours, TLC confirm reaction finish, in reaction solution, add appropriate dilute hydrochloric acid for and pyridine, add ethyl acetate extraction, organic layer is concentrated.Enriched material is dissolved in 5%HF/CH ₃cN (10mL), ice bath stirs 1 hour, and TLC confirms that reaction finishes, and adds saturated NaHCO ₃be neutralized to neutrality, add a small amount of water (10ml) and pump acetonitrile with Rotary Evaporators.With appropriate ethyl acetate extraction, ester layer is with saturated NaCl solution washing, and anhydrous magnesium sulfate drying, filters, concentrated.Resistates, with purification by silica gel column chromatography (chloroform/methanol=100/1 (V/V)), obtains 40mg colorless oil 1-32.Productive rate is about 55%.

1-32:MF:C ₁₉h ₂₀o ₇; MW:360; HRESIMS m/z 383.1116 ([M+Na]+calculating molecular weight 383.1107); 1H NMR (300MHz, CDCl3) δ 8.02 (m, 2H), 7.58 (m, 1H), 7.45 (m, 2H), 5.53 (s, 1H), 4.72 (d, J=12.1Hz, 1H), 4.62 (d, J=12.1Hz, 1H), 2.65 (d, J=6.3Hz, 1H), 2.43 (d, J=11.5Hz, 1H), 2.32 (m, 2H), 2.00 (s, 3H), 1.96 (m, 1H), 1.32 (s, 3H).

Experiment 3.33

Get compound 5b (94mg, about 0.2mmol) be dissolved in methylene dichloride (2mL) and be placed in round-bottomed bottle, add the appropriate (100mg of silver suboxide, about 0.5mmol), add again methyl iodide (2mL), condensing reflux, stirring reaction 8 hours, TLC monitoring reaction finishes.By reacting liquid filtering, remove silver suboxide and other precipitations, filtrate is concentrated.Enriched material is dissolved in 5%HF/CH3CN (10mL), and ice bath stirs 1 hour, and TLC confirms that reaction finishes, and adds saturated NaHCO3 to be neutralized to neutrality, adds a small amount of water (10ml) and pumps acetonitrile with Rotary Evaporators.With appropriate ethyl acetate extraction, ester layer is with saturated NaCl solution washing, and anhydrous magnesium sulfate drying, filters, concentrated.Resistates, with purification by silica gel column chromatography (chloroform/methanol=50/1 (V/V)), obtains 33mg colorless oil 1-33.Productive rate is about 49%.

1-33:MF:C ₁₈h ₂₀o ₆; MW:332; HRESIMS m/z 355.1165 ([M+Na]+calculating molecular weight 355.1158); 1H NMR (300MHz, CDCl3) δ 8.04 (d, J=7.7Hz, 2H), 7.57 (m, 1H), 7.44 (m, 2H), 5.52 (s, 1H), 4.68 (d, J=7.0Hz, 1H), 4.61 (d, J=7.0Hz, 1H), 3.34 (s, 3H), 2.55 (d, J=6.5Hz, 1H), 2.21 (m, 1H), 2.17 (s, 1H), 1.90 (dd, J=14.4,1.8Hz, 1H), 1.87 (d, J=12.0Hz, 1H), 1.42 (s, 3H).

Experiment 2.34

Get compound 5b (94mg, about 0.2mmol) and be dissolved in methylene dichloride (1mL), add triethylamine (60 μ L), add again methane sulfonyl chloride (50 μ L, about 2e.q.), stirring reaction 1 hour, TLC monitoring reaction finishes.Reaction solution is distributed in water and methylene dichloride, and organic layer is concentrated.Enriched material is dissolved in 5%HF/CH3CN (10mL), and ice bath stirs 1 hour, and TLC confirms that reaction finishes, and adds saturated NaHCO3 to be neutralized to neutrality, adds a small amount of water (10ml) and pumps acetonitrile with Rotary Evaporators.With appropriate ethyl acetate extraction, ester layer is with saturated NaCl solution washing, and anhydrous magnesium sulfate drying, filters, concentrated.Resistates, with purification by silica gel column chromatography (chloroform/methanol=50/1 (V/V)), obtains 30mg colorless oil 1-34, and productive rate is about 38%.

1-34:MF:C ₁₈h ₂₀o ₈s; MW:396; HRESIMS m/z 419.0793 ([M+Na]+calculating molecular weight 419.0777); 1H NMR (300MHz, CDCl3) δ 8.03 (m, 2H), 7.58 (m, 1H), 7.44 (m, 2H), 5.52 (s, 1H), 4.76 (d, J=12.2Hz, 1H), 4.68 (d, J=12.2Hz, 1H), 3.02 (s, 3H), 2.91 (dd, J=11.4,6.9Hz, 1H), 2.72 (dd, J=6.9,1.8Hz, 1H), 2.28 (dd, J=12.0,9.0Hz, 2H), 1.99 (dd, J=12.7,1.9Hz, 1H), 1.48 (s, 3H).

Embodiment 3 compound activity tests

Test 3.1 peoniflorin modification active testings:

3.1.1LPS the foundation of (lipopolysaccharides) induction THP-1 cell expressing COX-2 albumen model

(contain 2g/L NaHCO at RPMI RPMI-1640 ₃, 2.5g/L glucose, 0.11g/L Sodium.alpha.-ketopropionate, 0.0625g/L penicillin, 0.1g/L Streptomycin sulphate and 10%FBS) in cultivator monocyte THP-1 (American Type Culture Collection, Manassas, VA).Incubator is set to 37 DEG C, 5%CO ₂, 95% air and saturated humidity.THP-1 cell is with 1 × 10 ⁶the density of/mL is inoculated in 12 orifice plates (every hole 1mL system), breaking up 48 hours containing in the training liquid of 100nM PMA, then uses instead without the training liquid of PMA and continues to cultivate 24 hours, preparing experiment.Stimulate the THP-1 cell of differentiation with 1 μ g/mL LPS, extract cell protein at different time points and carry out Diagnosis of Sghistosomiasis notation (Western Blot) detection.Result demonstration, LPS processing just can detect in cell for 1 hour a small amount of COX-2 protein expression, significantly increases and until still lasting rising (referring to Fig. 1) in 16 hours at 4 hours expression amounts.The inventor considers many-sided factor, as experimental period, COX-2 abduction delivering amount and medicine inhibition etc., and finally selected 4 hours treatment times as the protein induced expression model of COX-2.

3.1.2 the screening of peoniflorin structure of modification compound

The aforementioned synthetic each compound (final concentration is 10 μ M) obtaining is processed after the THP-1 cell 2h of differentiation, add again 1 μ g/mL LPS irritation cell 4h, extract cell protein and carry out Diagnosis of Sghistosomiasis notation and detect to screen COX-2 abduction delivering is had to inhibiting compound.Meanwhile, whether we have cytotoxicity with the each candidate compound of MTT colorimetric determination, to get rid of compound, the restraining effect of the protein induced expression of COX-2 are caused owing to having reduced cytoactive and protein synthesis of C 3 A.Experimental result is shown in table 1, the COX-2 of LPS induction is expressed to inhibited compound and have 13 (significant difference of having compared with LPS treatment group, P<0.05); Wherein there are 7 compounds to find that there is the cytotoxic effect (significant difference of having compared with DMSO solvent control group in various degree in MTT detects, P<0.05), therefore our finishing screen is selected Compound D G and is carried out further research work.

Table 1. peoniflorin structure of modification Compound C OX-2 abduction delivering suppresses screening active ingredients result

COX-2 protein expression detects with immunoblotting.LPS induction group is control group, and other is respectively organized comparative data with it and represents n=3 with mean value ± S.D ^*p<0.05; For there being significant difference, ^*p<0.01 is for there being significant differences.It is blank that tetrazolium bromide colorimetry for cell viability (MTT) method detects DMSO treatment group.Data represent that with mean value ± S.D n=3#P<0.05 is for having significant difference ##P<0.01 for there being significant differences, compared with DMSO group.

Make exemplary biological activity test with Compound D G below, further to study the biological activity of peoniflorin compounds.

Embodiment 3.2DG inside and outside suppresses COX-2 unconventionality expression activity research

Embodiment 3.2.1DG suppresses the quantitative dose-effect relationship of COX-2 abduction delivering

The DG (1,5 and 10 μ M) of different concns was hatched THP-1 cell through PMA differentiation after 2 hours, added the LPS irritation cell 4 hours of 1 μ g/mL, extracted cell protein and carried out the detection of Diagnosis of Sghistosomiasis notation.Experimental result shows, DG can suppress the expression (referring to Fig. 2) of the COX-2 albumen of LPS induction dose-dependently, wherein the DG of 1 μ M is not clearly (inhibiting rate is 25.5%) to the restraining effect of COX-2 abduction delivering, but there is significant difference (P<0.05 compared with LPS treatment group, compared with LPS treatment group), the DG of 5 μ M, 10 μ M can significantly reduce the abduction delivering of COX-2 albumen, and (inhibiting rate is respectively 80.6% and 85.3%, P<0.01, compared with LPS treatment group).Meanwhile, DG does not affect (referring to Fig. 2) to the intrinsic expression of COX-1 albumen.

Embodiment 3.2.2DG suppresses the time-effect relationship research of COX-2 abduction delivering

Add again the LPS irritation cell 4 hours of 1 μ g/mL as pretreated group through the THP-1 cell different time (1～24 hour) of PMA differentiation with the DG preincubate of 10 μ M; DG and LPS add in training liquid incubated cell 4 hours as co-processing group simultaneously; After LPS irritation cell different time, (1～3 hour) adds DG to hatch as aftertreatment group, examines or check with this time-effect relationship that DG suppresses COX-2 abduction delivering.Experimental result demonstration, the pretreated time of DG, the longer restraining effect to COX-2 abduction delivering was stronger, and the COX-2 that pre-treatment can suppress LPS induction for 8 hours above substantially completely crosses expression, and COX-2 expressing quantity approaches background level; Co-processing and aftertreatment group restraining effect relatively a little less than, LPS stimulates after 3 hours and gives the abduction delivering (P>0.05, compared with LPS treatment group) (referring to Fig. 3) that DG can not suppress COX-2 albumen again.

The toxic action research of embodiment 3.2.3.DG to THP-1 cell

Methyl-sulphoxide for DG (DMSO) is mixed with the storage liquid of 10mM, after packing, at-20 DEG C, saves backup.THP-1 cell is with 2 × 10 ⁵the density in/hole is inoculated in 24 orifice plates, and after PMA differentiation, (final concentration of compound is 10 μ M in the training liquid that contains each compound, to hatch 24 hours; Solvent control group is 1 ‰ DMSO).The mensuration of cell viability adopts tetramethyl-azo azoles salt (MTT) colorimetry: add MTT (final concentration is 0.5mg/mL), 37 DEG C of incubators continue cultivation and suck supernatant after 4 hours, every hole adds 0.5mL DMSO, after abundant dissolving to be crystallized, every hole is got 100 μ L solution and is added 96 orifice plates, and NOVOstar microplate reader 595nm measures light absorption value.Cytoactive is carried out data statistics with compound treatment group cell viability with respect to the per-cent of DMSO solvent control group cell viability.Every kind for the treatment of group is done 3 multiple holes, 3 repeated experiments.

The inventor is hatched through the THP-1 cell of PMA differentiation 72 hours with the DG that concentration is respectively 1 μ M, 5 μ M, 10 μ M and 50 μ M, then detects by MTT colorimetry.Found that, DG incubated cell in the concentration range of 1～10 μ M does not have toxic action for 72 hours substantially, high dosage DG (50 μ M) long time treatment can produce cytotoxicity, after 72 hours, cell survival rate is about 48.6% (P<0.01, compared with DMSO blank group) (referring to Fig. 4).Therefore, DG belongs to safe dose scope at 10 μ M with planted agent, and can get rid of its restraining effect to the protein induced expression of COX-2 is to decline because toxic action causes cell viability, thereby reduces the synthetic possibility of COX-2 albumen.

The mechanisms of anti-inflammatory of embodiment 4: Compound D G

Embodiment 4.1.DG reduces the generation of (prostaglandin E2) PGE2 of LPS induction

The THP-1 cell breaking up with 1 μ g/mL LPS thorn regular menstruation during early pregnancy PMA 16 hours, collects training liquid supernatant, measures the concentration of training PGE2 in liquid with ELISA Kit (R & D).The PGE2 of training liquid sample and mouse monoclonal antibody and HRP mark incubated at room 2 hours in the enzyme plate that has been coated with goat anti-mouse antibody.Wash after plate 4 times, add reaction substrate solution, room temperature lucifuge is hatched 30 minutes.Add stop buffer termination reaction, 450nm measures light absorption value, calculates the content of PGE2 in each sample according to typical curve.Every kind for the treatment of group is done 3 multiple holes, 3 repeated experiments.

Hatch through the THP-1 cell of PMA differentiation 2 hours with the DG that concentration is respectively 1 μ M, 5 μ M and 10 μ M, then add the LPS irritation cell 16 hours of 100ng/mL, collect training liquid supernatant and detect the amount of the PGE2 of emiocytosis in training liquid.Experimental result is consistent with the inventor's expection, and DG can reduce the generation of the PGE2 of LPS induction dose-dependently, and wherein the PGE2 growing amount of 10 μ M DG treatment group is 0.34 ± 0.10ng/10 ⁶cell count, with LPS treatment group (0.84 ± 0.08ng/10 ⁶cell count) to compare, minimizing amplitude can reach 60% (P<0.01, compared with LPS treatment group) (referring to Fig. 5).

Embodiment 4.2.DG is to COX enzymic activity unrestraint effect

COX-1 enzyme assay: THP-1 cell is with 1 × 10 ⁶the density in/hole is inoculated in 12 orifice plates, after PMA differentiation, at 37 DEG C, hatch 30 minutes (blank group and arachidonic acid model group add corresponding dilution DMSO) containing in the HHBS of medicine (Hank ' s Hepes balanced salt solution) damping fluid, adding final concentration is to react 15 minutes at 37 DEG C of the arachidonic acids (blank group adds corresponding dilution dehydrated alcohol) of 10 μ M.Draw supernatant damping fluid, centrifugal 5 minutes of 2500rpm room temperature, the concentration of collecting supernatant ELISA Kit (R & D) mensuration PGE2.

Result shows, COX-1 selective depressant Asprin and COX-2 selective depressant NS-398 (Sigma company of the U.S.) thus can effectively suppress respectively the generation of the enzymic activity blocking-up PGE2 of COX-1 and COX-2, and DG does not have obvious restraining effect (referring to Fig. 6) to the enzymic activity of COX-1 and COX-2 in 1～50 μ M concentration range.

Embodiment 4.3.DG suppresses the COX-2 genetic transcription of LPS induction

By TRIzol reagent extracted total RNA.Each sample is got 3 μ gRNA M-MLV reversed transcriptive enzymes and is carried out reverse transcription and obtain cDNA, then carries out pcr amplification.The special primer of people COX-2 gene and GAPDH gene order synthesizes (sequence is in table 2) by Invitrogen.PCR reaction conditions is: denaturation 5 minutes at 94 DEG C, (sex change 30 seconds at 94 DEG C, anneals 30 seconds at 62 DEG C, 72 DEG C downward-extension 45 seconds) × 30 circulations (COX-2) or 24 circulations (GAPDH), 72 DEG C of downward-extensions 5 minutes, preserve at 4 DEG C.PCR product detects through 1% agarose gel electrophoresis, and gel imaging instrument is taken pictures, quantitative with Image-Pro Plus 5.1 software analysis.

Table 2. people COX-2 gene and GAPDH gene order

Experimental result shows, 1 μ g/mL LPS stimulates the THP-1 cell of differentiation after 4 hours, can cause COX-2 genetic transcription significantly to increase, DG pre-treatment can suppress the transcriptional level of COX-2mRNA dose-dependently, wherein the inhibiting rate of 10 μ M DG can reach 62.6% (P<0.01, compared with LPS treatment group) (referring to Fig. 7).This shows the restraining effect of DG to LPS induction COX-2 protein expression, is that the transcriptional level by reducing COX-2 gene is realized at least partly.

Embodiment 5.DG causes the research of the anti-inflammatory action in mouse toes swelling inflammatory model at carrageenin

DG is mixed with the concentrated solution of 400mg/mL with DMSO, after mixing, be diluted to respectively 1mg/mL, 2mg/mL and 4mg/mL with physiological saline with isopyknic tween 20 (Tween-20).Indometacin Enteric-coated Tablets (25mg/ sheet) is mixed with the concentrated solution of 50mg/mL with DMSO, with normal saline dilution to 0.5mg/mL.

Animal is divided into model control group, indomethacin (5mg/kg) positive drug control group, DG 10mg/kg group, DG 20mg/kg group and DG 40mg/kg at random and organizes totally 5 groups, every group of 10 animals, each number of animals is also weighed.Each administration group abdominal injection relative medicine 0.2mL (model control group injection is containing the physiological saline of 1%DMSO), caused inflammation in the right back toes subcutaneous injection of mouse carrageenin (0.2%, w/v) 20 μ l after 30 minutes.After 4 hours, put to death animal, cut left and right metapedes from ankle, weigh, carry out statistical study using left and right sufficient weight difference as foot swelling deciding degree index.

Experimental result demonstration, the DG of 10～40mg/kg can alleviate the mouse toes swelling that carrageenin causes dose-dependently, and wherein the DG of 40mg/kg is compared with model control group, and it can reach 33.6% to the inhibiting rate of swelling degree; Positive antiphlogiston indomethacin (5mg/kg) can reach 39.6% (table 3) to the inhibiting rate of toes swelling.This result proves, consistent with external experimental result, DG has stronger anti-inflammatory activity in vivo equally.

Table 3.DG causes the anti-inflammatory action in mouse toes swelling inflammatory model at carrageenin

Data represent with mean value ± S.D. (n=10; ^*p<0.05 and ^*p<0.01, compared with physiological saline control group).

The treating cerebral ischemia evaluation experimental animal of embodiment 6.SM6-DG in intraluminal middle cerebral artery occlusion in rats cerebral infarction models

Male SD rat, body weight 220～240g.Purchased from Shanghai western pul Bi Kai laboratory animal company limited.The qualified numbering of project: SIMM-AM-FLY-2010-05.

Medicine preparation

DG mother liquor is achromaticity and clarification clear solution, content of dispersion 20mg/ml.Before use mother liquor is mixed by 1:9 with physiological saline, vortex 1～2min immediately, makes the uniform solution containing DG 2mg/ml after mixing, and can use by injection for intravenous.At room temperature (more than 25 DEG C) are stable.Blank preparation does not contain the solvent control of DG.Actual administration concentration 8mg/kg, about 1ml.

Experiment reagent material

MCAO fastens line (diameter 0.26mm, round end diameter 0.34 ± 0.02mm) purchased from Shadong Biological Technology Co., Ltd., Beijing;

TTC (Wo Kai) is purchased from traditional Chinese medicines group, and 2%TTC, prepares with PBS before use.

Experimental technique

Animal is divided at random 4 groups by 1, physiological saline control group, blank preparation control group, DG 8mg/kg aftertreatment, DG 8mg/kg pre-treatment, pseudo-operation group.(n≥4)

2 modelings: animal abdominal injection Chloral Hydrate (350mg/kg) anesthesia, dorsal position is fixed.Neck sterilization, makes center otch to the left.Blunt separation musculi colli, exposes the total vein of left side neck, wears two pull wires.Separate outside arteria carotis communis, neck and internal carotid artery, ligation external carotid artery and arteria carotis communis near-end, wear a pull wire simultaneously and be placed between the two, then closes internal carotid artery with bulldog clamp folder, between arteria carotis communis near-end and ligature, cut an osculum, insert bolt line ligation from otch to far-end and fix.Decontrol bulldog clamp, will fasten line and only slowly push 2cm in neck, tighten ligature.Not plug line is organized in pseudo-operation

3 administrations: the total venal distal ligation of neck, insert transfusion needle, tighten proximal part.Give physiological saline, DG and blank preparation with the speed vein of 4ml/h, administration finishes to extract syringe needle, ligation near-end.The first modeling of rear administration group, then intravenously administrable; Pre-administration group, first intravenously administrable, then modeling.

4 fill with again: after modeling, 1h extracts and fastens the about 0.5cm of line.

5 Behavioral assessments: adopt 5 grades of scoring methods, 0 point of normal asymptomatic person meter; Catch rat tail, held unsettled observation, offside forelimb can not fully stretch and be designated as 1 point, shows to have slight focal neural disappearance; Animal tendency offside, revolver are 2 points, show the focal nerve injury of moderate; It is 3 points that animal is swung to offside, indicates serious focal nerve injury; Can not spontaneously walk, and be 4 points with the disturbance of consciousness in various degree.

The functional enzyme groupization dyeing of 6 cerebral ischemias: 72h after filling with again, by rat sacrificed by decapitation, get immediately brain, after-20 DEG C of quick-frozen 30min, take out, brain is done continuous coronal section (the thick 2mm of sheet).Brain sheet is placed in 37 DEG C of 2%TTC (pH7.2) and hatches 15min dyeing, sucks staining fluid, and brain sheet is fixed with 4% paraformaldehyde solution.Digital camera is to the brain sheet (see figure 8) of taking pictures.Measure the infarct size of brain and the area of full brain sheet, obtain infarcted region and account for the per-cent of offside brain area, the software of measurement is Image-Pro Plus5.1.

Experimental result

1 survival rate comparison

Table 4

2TTC coloration result

Referring to Fig. 9, in Fig. 9 a, statistics show, DG 8mg/kg aftertreatment group reduces approximately 20% compared with physiological saline group cerebral ischemia area, and exist significant difference ( ^*, p<0.05, t inspection); Fig. 9 b is the scatter diagram of each treated animal brain infarction area.

In sum, cyclooxygenase COX is the rate-limiting enzyme that catalytic film phosphatide arachidonic acid (AA) is converted into prostaglandins, and it is mainly metabolized to PGH2 (PGH by cyclooxygenase and catalase activity by AA ₂); Dissimilar cell has the synthetic path in different downstreams, can be further by PGH ₂change into final meta-bolites, as endotheliocyte generates prostacyclin (PGI ₂), thrombocytopoiesis thromboxane (TXA ₂), scavenger cell generate PGE ₂deng, thereby exercise different physiology or pathologic function.Induction type cyclooxygenase COX-2 is as a kind of immediate early gene (IEG) albumen, can be by the abduction delivering rapidly such as many somatomedins, proinflammatory disease material, tumour stimulating factor, hormone, bacteriotoxin and cytokine, there is a large amount of research evidences to show, in the pathologic process of numerous disease, be all attended by the overexpression of COX-2, as inflammation, nerve degenerative diseases, cancer etc.; And the abduction delivering of COX-2 equally also can cause the generation of disease or the deterioration of the course of disease, as amplified inflammatory reaction by positive feedback effect, induce acute nerve excitability toxicity, increasing the weight of neuronic damage in nerve retrograde affection process by the proinflammatory or the generation active oxygen radical (ROS) that postpone, this shows that the unconventionality expression of COX-2 and the generation of these diseases development are high-positive correlation relation.Therefore the abnormal induction that, suppresses COX-2 is expressed an alternative thinking that becomes this class disease for the treatment of.

In cerebral apoplexy process, generation pathology inflammation is followed the rising of COX-2, and peoniflorin reduces cerebral apoplexy process midbrain injury by the inflammation that suppresses pathology.Accordingly, the inventor suppresses COX-2 pathologic to peoniflorin compounds DG and crosses expression activity and carried out Pharmacodynamics in vitro evaluation.Find, DG is obvious in the concentration range dose-effect relationship of 1～50 μ M, and pre-treatment, co-processing and aftertreatment can play suppress COX-2 cross expressional function, illustrate that the therapeutic time window of DG is larger, this has improved the possibility as Treatment of Cerebral Stroke medicine.In onset concentration range DG to the enzymic activity of COX-1 and COX-2 all without obvious restraining effect, this has got rid of the COX-1 of DG normal tissue existence and the impact of COX-2 function, got rid of gi tract and cardiovascular systems aspect are had side effects may, in this point, be better than current non-steroidal anti-inflammatory drugs (NSAIDs).And the inventor also confirms by experimentation on animals, this anti-inflammatory activity is really effective.Therefore,, from DG pharmacodynamic evaluation, DG is reacted treatment cerebral apoplexy and parkinsonism aspect is had to good clinical value by inflammation-inhibiting.

Claims (7)

1. a peoniflorin compounds, it has the structure shown in formula I:

Wherein,

Work as R ₁during for hydrogen, benzoyl, naphthoyl, pyridine formyl radical, benzenesulfonyl, phenylacetyl, C3-C8 cycloalkyl formyl radical, diamantane formyl radical, C1-C6 alkyl acyl or furancarbonyl;

R ₂for C1-C6 alkyl acyl, C1-C6 alkoxy C 1-C6 alkyl, C1-C6 alkyl sulphonyl, benzenesulfonyl, C3-C8 cycloalkyl formyl radical or benzoyl;

R ₃for hydrogen, C1-C6 alkyl acyl, C1-C6 alkyl or C1-C6 alkyl sulphonyl;

Or

Work as R ₁during for hydrogen, benzoyl, naphthoyl, pyridine formyl radical, benzenesulfonyl, phenylacetyl, C3-C8 cycloalkyl formyl radical, diamantane formyl radical, C1-C6 alkyl acyl or furancarbonyl;

R ₂for C1-C6 alkyl or hydrogen;

R ₃for C1-C6 alkyl acyl, C1-C6 alkyl or C1-C6 alkyl sulphonyl;

1～3 group that described phenyl is not necessarily selected from amino, nitro, halogen or C1-C6 alkoxyl group replaces.

2. peoniflorin compounds according to claim 1, wherein, in formula I,

Work as R ₁during for hydrogen, benzoyl, naphthoyl, pyridine formyl radical, benzenesulfonyl, phenylacetyl, hexanaphthene formyl radical, diamantane formyl radical, ethanoyl or furancarbonyl;

R ₂for ethanoyl, positive propionyl, isopropyl acyl group, methoxymethyl, isobutyryl, methylsulfonyl, tertiary butyryl radicals, benzenesulfonyl, positive butyryl radicals, hexanaphthene formyl radical, cyclopropane formyl radical or benzoyl;

R ₃for hydrogen, ethanoyl, methyl or methylsulfonyl;

Or

Work as R ₁during for hydrogen, benzoyl, naphthoyl, pyridine formyl radical, benzenesulfonyl, phenylacetyl, hexanaphthene formyl radical, diamantane formyl radical, ethanoyl or furancarbonyl;

R ₂for methyl or hydrogen;

R ₃for ethanoyl, methyl or methylsulfonyl;

1～3 group that described phenyl is not necessarily selected from amino, nitro, halogen or C1-C6 alkoxyl group replaces.

3. peoniflorin compounds according to claim 2, wherein, the compound of formula I is the compound with following structure:

4. a preparation method for peoniflorin compounds, it comprises the steps:

Step a: first by starting raw material peoniflorin 1, TERT-BUTYL DIMETHYL CHLORO SILANE TBSCl, imidazoles and solvent synthetic 6 '-O-tertiary butyl dimethylsilyl peoniflorin 2 at a certain temperature;

Wherein, described solvent is one or more in acetonitrile, toluene, benzene, ethanol, DMF; The consumption of solvent is 0.5～10 times of peoniflorin 1 weight; Temperature of reaction is 0～40 DEG C; Reaction times is 0.5～3 hour;

Step b: compound 2 is removed benzoyl synthetic compound III with alkali in solvent;

Wherein, taking the mixed solvent of methyl alcohol and water as reaction solvent, mixed volume ratio is 100:1～50:50, and the consumption of reaction solvent is 1～20 times of compound 2 weight; Alkali is selected from sodium hydroxide, calcium hydroxide and lithium hydroxide, and the consumption of alkali is 1/400～1/100 of compound 2 weight; Temperature of reaction is 0～100 DEG C; Reaction times is 0.5～10 hour;

Step c: compound III and TrocCl neutralize synthetic compound IV at a certain temperature at solvent, described TrocCl is 2,2,2-, tri-chloroethyl carbonyl chlorine;

Wherein, solvent is pyridine, and solvent load is 1-10 times of compound III weight; The consumption of TrocCl is 1%～10% of compound III weight; Temperature of reaction is 0～100 DEG C; Reaction times is 0.5～10 hour;

Steps d: the synthetic compound V under certain temperature and solvent action by compounds Ⅳ and acylating agent;

Wherein, described acylating agent is C1-C6 alkyl acyl chloride, SULPHURYL CHLORIDE, benzene sulfonyl chloride, C1-C6 alkoxy C 1-C6 alkyl alcohol, C3-C8 cycloalkyl formyl chloride, Benzoyl chloride or the C1-C6 alkyl alcohol of C1-C6; Described solvent is one or more in pyridine, toluene, benzene or DMF; The consumption of solvent is 0.5～10 times of compounds Ⅳ weight; The consumption of acylating agent is 2～10 times of mole number of compounds Ⅳ; Temperature of reaction is 0～100 DEG C; Reaction times is 0.5～10 hour;

Step e: compound V and excess acid are removed tertiary butyl dimethoxy silylation and obtained compound VI in solvent and under certain temperature;

Wherein, solvent is acetonitrile, and acid is selected from hydrofluoric acid or hydrochloric acid; The consumption of acid is excessive; Temperature of reaction is 0～100 DEG C; Reaction times is 0.5～10 hour;

Step f: compound VI and triphenylphosphine, imidazoles, iodinating agent be synthetic compound VII in solvent;

Wherein, described solvent is one or more in pyridine, toluene, benzene or DMF; The consumption of solvent is 10～60 times of compound VI weight; Iodinating agent is selected from pure iodine, hydroiodic acid HI and pentaiodo phosphorus, and the consumption of iodinating agent is 1～4 times of compound VI weight; Temperature of reaction is 0～100 DEG C; Reaction times is 0.5～10 hour;

Step g: compound VIII and metallic zinc are removed glucosyl group synthetic compound IX in solvent;

Wherein, taking ethanol and water mixed solvent as reaction solvent, mixed volume ratio is 100:1～50:50, and the consumption of reaction solvent is 1～20 times of compound VIII weight; Temperature of reaction is 0～100 DEG C; Reaction times is 0.5～10 hour;

Step h: compound IX is reacted under certain temperature and obtained compound X in solvent with acylating agent;

Wherein, solvent is the mixed solvent of methylene dichloride and triethylamine, its blending ratio is 100:1～100:10, described acylating agent is Benzoyl chloride, naphthoyl chloride, pyridine formyl chloride, benzene sulfonyl chloride, phenyllacetyl chloride, C3-C8 cycloalkyl formyl chloride, diamantane formyl chloride, C1-C6 alkyl acyl chloride or furoyl chloride, and described acylating agent consumption is 1～3 times of triethylamine volume; Temperature of reaction is 0～100 DEG C; Reaction times is 0.5～10 hour;

Step I: compound X generates with excessive acylation reaction the compound that formula I represents in solvent;

Wherein, described acylating agent is C1-C6 alkyl acyl chloride, C1-C6 alkyl alcohol or C1-C6 alkyl sulfonyl chloride, and reaction solvent is pyridine, and consumption is 1/20～1/10 of compound X weight, and temperature of reaction is 0～100 DEG C;

Reaction times is 0.5～10 hour,

Wherein,

R ₁for hydrogen, benzoyl, naphthoyl, pyridine formyl radical, benzenesulfonyl, phenylacetyl, C3-C8 cycloalkyl formyl radical, diamantane formyl radical, C1-C6 alkyl acyl or furancarbonyl;

R ₂for C1-C6 alkyl acyl, C1-C6 alkoxy C 1-C6 alkyl, C1-C6 alkyl sulphonyl, benzenesulfonyl, C3-C8 cycloalkyl formyl radical, benzoyl, C1-C6 alkyl or hydrogen;

R ₃for hydrogen, C1-C6 alkyl acyl, C1-C6 alkyl or C1-C6 alkyl sulphonyl;

1～3 group that described phenyl is not necessarily selected from amino, nitro, halogen or C1-C6 alkoxyl group replaces.

5. preparation method according to claim 4, wherein,

The reaction conditions of step a is: solvent is DMF, and the consumption of solvent is 5～7 times of peoniflorin 1 weight; Temperature of reaction is 25 DEG C; Reaction times is 1 hour;

The reaction conditions of steps d is: solvent is anhydrous pyridine, and the consumption of solvent is 5～6 times of compounds Ⅳ weight; Described acylating agent is formyl chloride, Acetyl Chloride 98Min., propionyl chloride, benzene sulfonyl chloride, cyclohexyl acyl chlorides or Benzoyl chloride, and its consumption is 8 times of compounds Ⅳ mole number; Temperature of reaction is 40～80 DEG C; Reaction times is 5～8 hours;

The reaction conditions of step e is: the volume ratio of hydrofluoric acid-acetonitrile is 1:20, and its consumption is 10～15 times of compound V weight; Temperature of reaction is 0～20 DEG C; Reaction times is 0.5～2 hour;

The reaction conditions of step f is: solvent is benzene, and the consumption of solvent is 30～40 times of compound VI weight; Iodinating agent is pure iodine, and its consumption is 2 times of compound VI mole number; Temperature of reaction is 60～80 DEG C; Reaction times is 1～3 hour;

The reaction conditions of step g is: the volume ratio of the mixing of ethanol and water mixed solvent is 95:5, and the consumption of solvent is 10～15 times of compound VIII weight; Temperature of reaction is 70～90 DEG C; Reaction times is 1～3 hour.

6. the purposes of the compound shown in general formula I in the medicine of preparing cox 2 inhibitor;

R ₁for hydrogen, benzoyl, naphthoyl, pyridine formyl radical, benzenesulfonyl, phenylacetyl, C3-C8 cycloalkyl formyl radical, diamantane formyl radical, C1-C6 alkyl acyl or furancarbonyl;

R ₂for C1-C6 alkyl acyl, C1-C6 alkoxy C 1-C6 alkyl, C1-C6 alkyl sulphonyl, benzenesulfonyl, C3-C8 cycloalkyl formyl radical, benzoyl, C1-C6 alkyl or hydrogen;

R ₃for hydrogen, C1-C6 alkyl acyl, C1-C6 alkyl or C1-C6 alkyl sulphonyl;

1～3 group that described phenyl is not necessarily selected from amino, nitro, halogen or C1-C6 alkoxyl group replaces.

7. the compound shown in general formula I is preparing nonsteroidal anti-inflammatory drug and for the preparation of the purposes in the medicine for the treatment of and preventing brain stroke and/or parkinsonism;

R ₁for hydrogen, benzoyl, naphthoyl, pyridine formyl radical, benzenesulfonyl, phenylacetyl, C3-C8 cycloalkyl formyl radical, diamantane formyl radical, C1-C6 alkyl acyl or furancarbonyl;

R ₂for C1-C6 alkyl acyl, C1-C6 alkoxy C 1-C6 alkyl, C1-C6 alkyl sulphonyl, benzenesulfonyl, C3-C8 cycloalkyl formyl radical, benzoyl, C1-C6 alkyl or hydrogen;

R ₃for hydrogen, C1-C6 alkyl acyl, C1-C6 alkyl or C1-C6 alkyl sulphonyl;

1～3 group that described phenyl is not necessarily selected from amino, nitro, halogen or C1-C6 alkoxyl group replaces.