CN1160331C

CN1160331C - Process for preparing photoactivated (-) yellow skin amide

Info

Publication number: CN1160331C
Application number: CNB001246305A
Authority: CN
Inventors: 量黄; 黄量; 张均田; 吴克美; 孙万儒
Original assignee: Institute of Materia Medica of CAMS and PUMC
Current assignee: GUANGZHOU NUOHAO PHARMACEUTICAL CO Ltd
Priority date: 2000-09-28
Filing date: 2000-09-28
Publication date: 2004-08-04
Anticipated expiration: 2020-09-28
Also published as: CN1345721A

Abstract

一种制备光活(-)黄皮酰胺或其衍生物的方法，以氯乙酰氯与手性醇R*OH形成手性醇的氯乙酸酯；再与苯甲醛反应得光活的芳基缩甘油酸手性醇酯；经酯交换得相应的苯基缩水甘油酸甲酯；与相应的胺缩合和氧化得(+)-(2S，3R)-N-甲基-N-苯甲酰甲基-3-苯基缩水甘油酰胺；在碱性条件下环合，得光活(-)黄皮酰胺酮，再经硼氢化钠还原可得光活(-)黄皮酰胺或其衍生物。及其在制备促智，抗衰老作用药物的应用。A method for preparing photoactive (-) xanthamide or derivatives thereof, using chloroacetyl chloride and chiral alcohol R*OH to form a chiral alcohol chloroacetate; then reacting with benzaldehyde to obtain a photoactive aryl group Glycidic acid chiral alcohol ester; transesterification to the corresponding phenyl glycidic acid methyl ester; condensation with the corresponding amine and oxidation to (+)-(2S,3R)-N-methyl-N-benzoyl Methyl-3-phenylglycidyl amide; cyclization under alkaline conditions to obtain photoactive (-) ketamide ketone, and then reduce it with sodium borohydride to obtain photoactive (-) ketamide or its derivatives . And its application in the preparation of nootropic and anti-aging drugs.

Description

Light (-)-Clausenamide preparation method alive

Technical field

The present invention relates to the potentiality pharmaceutical use that a kind of light Clausenamide preparation method alive and light Clausenamide alive are used for the treatment of senile dementia.

Background technology

The mean lifetime of China's population at present surpasses 70 years old, and the average life span during than liberation has increased by one times.External scientific research prediction: during by 2025, the ratio of children below 15 years old will account for 18.6% of total population, and the elderly of 65 years old and over-65s will be above virgin number, reach 18.8%, this numeral shows, surplus 20 year, per 5 philtrums just have 1 the elderly.Degenerative brain disorder (Alzheimer ' s Disease) mostly occurs year surplus 50.The multiple embolism dementia or the senile dementia that cause because of cerebrovascular disease mostly occurred after 60 years old.As seen, because the aging of population, the sickness rate of degenerative brain disorder and senile dementia also will increase.The elderly and distinctive neurodegenerative disorders thereof---various dementias will experience two kinds of death, at first are spiritual death, after be sensual death, suffer untold misery, bring heavy burden more for society and family.Aging population is considered to be only second to war, pestilence, famine, energy resource shortage and influence social development and stabile unfavorable factor.

Control medicament categories old and feeble and the treatment senile dementia is various, as cerebral vasodilator, help to provide energy and improve intelligence by improving cerebral blood flow (CBF), but real valuable cerebral vasodilator must have high selectivity, do not influence the brain metabolism, do not have " stealing blood " phenomenon, platelet aggregation-against and anti thrombotic action are arranged.Though the calcium antagonist nimodipine meets above-mentioned some condition, it only acts on the L-passage in the voltage-dependent ca channel, and N type and T type calcium channel are not had influence.Strengthen in the medicine of choline system function, the Ach precursor only has faint therapeutic action, though Ach receptor stimulant and cholinesterase inhibitor have certain effect, acts on ofer short durationly, and toxic side effect is bigger.Multiple neuropeptide and nerve growth factor once were considered to the hope of treatment dementia, but clinical effectiveness is not good, may be mainly be difficult to enter in the brain and play a role by hemato encephalic barrier owing to this class material, (piracetam is domestic to be produced the 2-Pyrrolidone ethanamide, the trade(brand)name piracetam) after the appearance, belong to the not novel nootropics (nootropil of a class of arguement in early days in the document, this speech be from Greece speech noo (brain) and tropein (to) derivation), reported both at home and abroad in recent years, this medicine slightly or does not still have final conclusion to all types of dysmnesia and senile dementia effect, a major cause is that this medicine is a water-soluble cpds, low by the hemato encephalic barrier rate, be difficult for focusing on target spot and play a role.

The applicant isolates the compound of the gamma-lactam skeleton that contains the piracetam medicine for the first time from rutaceae Calusena lansium [Clausena Lansium (lour) Skells], be referred to as Clausenamide (Clausenamide).Clausenamide is the gamma-lactam with four chiral centres, and natural product are raceme, and the synthetic method of racemization Clausenamide was once applied for European patent, and its application number is EP0414020.Chinese patent application 86107090,90107145.5 and 90107144.7.

In the prior art, the preparation route and the method for racemization (±)-Clausenamide are as follows:

But this method can not be directly used in and prepare optically active Clausenamide or derivatives thereof that has of the present invention.

Summary of the invention

In order to overcome the deficiencies in the prior art part, the object of the present invention is to provide a kind of optical activity Clausenamide or derivatives thereof;

The another object of the present invention purpose is to provide a kind of preparation method of optical activity Clausenamide;

Another object of the present invention is to provide a kind of this optically active compound (-)-Clausenamide in the short intelligence of preparation, the application of anti-aging effects medicine.

Now to the preparation method of (-), (+)-Clausenamide, short intelligence is after anti-aging effects and possible mechanism of action thereof are reported in.

Compound of the present invention has the structure shown in the following general formula (I), and its absolute configuration is that (5R 6S), has left-handed photosensitiveness, wherein R for 3S, 4R ₁And R ₂Can be H, halogen, C _1-6Alkyl,

Alkoxyl group, inferior methylenedioxy group, nitro; Also can be polysubstituted; R ₁And R ₂Can be identical, also can be different.R ₁, R ₂During=H, be (-)-Clausenamide, in addition, the present invention also comprises the light of the generation in preparation process intermediate alive.

Herein ^*Be expressed as photoactive substance.

The invention still further relates to a kind of preparation and have the method for the photoactive compound shown in general formula I, it is a kind of asymmetric from the beginning (de novo) synthesis method, and this this synthesis method adopts intramolecularly to induce asymmetric D arzen ' s reaction, with light R alive ^*Be part.It may further comprise the steps:

(1) with chloroacetyl chloride and chiral alcohol R ^*OH forms the chloracetate (1) of chiral alcohol ^*

Described chiral alcohol comprises (+)-menthol, (+)-8-phenyl menthol or (+)-8-betanaphthyl menthol.

The chloracetate of the chiral alcohol that (2) obtains and phenyl aldehyde or R ₁The phenyl aldehyde reaction that replaces obtains aryl that corresponding light the lives R-Glyceric acid chirality alcohol ester (2a) that contracts ^*, (2b) ^*

(3) through transesterify get (+)-(2S, 3R)-3-phenyl glycidyl acid methyl esters (2) ^*,

(4) (+) that obtains-(2S, 3R)-3-phenyl glycidyl acid methyl esters (2) ^*With corresponding amine condensation get (+)-(2S, 3R)-N-methyl-N-(beta-hydroxy-styroyl)-3-phenyl glycidyl acid amides (3) ^*, again through oxidation get (+)-(2S, 3R)-N-methyl-N-phenacyl-3-phenyl glycidyl acid amides (4) ^*

(5) described (+)-(2S, 3R)-N-methyl-N-phenacyl-3-phenyl glycidyl acid amides (4) ^*Cyclization gets light (-)-Clausenamide ketone (5) alive under alkaline condition ^*Described alkali comprises lithium hydroxide, sodium hydroxide, potassium hydroxide, diisopropylamine lithium, butyllithium, tetraalkyl oxyammonia and trialkylamine.

The solvent that uses during cyclization is low alkyl alcohol or their mixtures such as water, methyl alcohol, ethanol, as first alcohol and water etc.

(6) light Clausenamide ketone (5) alive ^*Can get light Clausenamide or derivatives thereof alive through sodium borohydride reduction.

Should be noted that in above-mentioned method described chiral alcohol comprises (+)-menthol, (+)-8-phenyl menthol or (+)-8-betanaphthyl menthol.The used alkali of cyclization step comprises lithium hydroxide, sodium hydroxide, potassium hydroxide, diisopropylamine lithium, butyllithium, tetraalkyl oxyammonia and trialkylamine.The solvent that uses during cyclization is low alkyl alcohol or their mixtures such as water, methyl alcohol, ethanol.

Light of the present invention Clausenamide or derivatives thereof alive can also make through another step.In other words, light of the present invention Clausenamide and derivative thereof alive can obtain by the intermediate Split Method.

1. chemical method for splitting

In the method for the invention, can utilize the C of racemization Clausenamide ketone compound (5) ₃The hydroxyl of position becomes ester with the resolving agent chiral acid, then through recrystallization or a pair of epimer of chromatographic separation gained.

This resolving agent chiral acid comprises: L-fluoroacetic acid in the Meng, camphorsulfonic acid, (-)-S-phthalyl L-Ala or N-tolysulfonyl proline(Pro), preferably (-)-S-phthalyl L-Ala.

This method may further comprise the steps: the C that utilizes intermediate racemization Clausenamide ketone compound (5) ₃The hydroxyl of position becomes ester with chiral acid, gets a pair of epimer through recrystallization or chromatographic separation then.

Specifically, be with (-)-S-phthalyl L-Ala, at SOCl ₂Existence under; in toluene with racemization Clausenamide reactive ketone; obtain (-)-(3S; 4R; 5R)-5-benzoyl-1-methyl-4-phenyl-3-[(S)-α-(phthalimide-based) propionyloxy] pyrrolidin-2-one and (+)-(3S; 4R, 5R)-5-benzoyl-1-methyl-4-phenyl-3-[(S)-α-(phthalimide-based) propionyloxy] pyrrolidin-2-one.Get the epimer of optically-active for (-) through recrystallization, the latter is at NaBH ₄With hydrolysis under the existence of NaOH, through concentrating under reduced pressure, recrystallization obtains (-) of the present invention-Clausenamide.

In addition, racemization Clausenamide ketone is dropped into the CH of oxygen Acetyl Chloride 98Min. in (-) Meng ₂Cl ₂In the solution, room temperature reaction, reactant be through recrystallization, (-)-(3S, 4R, 5R)-5-benzoyl-3-oxygen acetoxy in the Meng-1-methyl-4 Phenylpyrrolidines-2-ketone.The latter is with the tosic acid hydrolysis, light (-)-Clausenamide ketone of living, then, according to above-mentioned steps (b) through NaBH ₄Reduction makes (-)-Clausenamide.

2 enzymatic resolution methods: can utilize the differential responses speed of enzyme to enantiomer, the method that the 3-phenyl glycidyl of racemization acid methyl esters can be split through enzyme make (+)-(2S, 3R)-3-phenyl glycidyl acid methyl esters (2) ^*, wherein,

Employing has the fungi that is selected from that produces the lytic enzyme ability, bacterium, yeast and actinomycetic microorganism are in substratum, and pH is 5-8, temperature is 10-50 ℃, be preferably 20～40 ℃ of following aerobic fermentations, the mycelia that is produced carries out the stereo selective hydrolysis reaction to compound racemization-3-phenyl glycidyl acid methyl esters, and the pH value of its hydrolysis reaction is between 5～10, temperature is 10-50 ℃, and separating and extracting obtains the photolytic activity Clausenamide.

Use in the present invention's enzymatic resolution method, the microorganism with the ability that produces lytic enzyme can be a fungi, bacterium, yeast, and actinomycetes.These bacteriums comprise as achromobacter (Achromobacter.sp), Alcaligenes (Alcaligenes.sp), Arthrobacter (Archrobacter, sp), genus bacillus (Bacillus sp), tyrothricin (Breubacillus, sp), rod bacillus (Corynebacterium, sp), Erwinia (Erwinia sp), pseudomonas (Pseudomonas sp) belongs to, zymic candiyeast (Candida sp) and complete red (Pichiasp), rhodotorula (Rhadatorula sp), Chinese sweet-smelling grass yeast (Hansenula sp) belongs to, the aspergillus in the fungi (Aspergillus), Mucor (Mucor sp), aspergillus oryzae (Aspergillus, sp), mould (Penicillium), promise Ka Shi (Nocardia sp) in head mold (Rhizopus sp) and the actinomycetes, streptomycete bacterial strains such as (Streptomycessp) all has this enzymatic productivity.These bacterial classifications are easy to obtain from the market.

In substratum, also can add tensio-active agent, to increase the solubleness of substrate in buffered soln, tensio-active agent comprises polyoxyethylene glycol, tween, polyvinyl alcohol, cetyl trimethylammonium bromide, and surfactant concentrations is controlled at 0.05～5%, is preferably 0.5～2%.

In above-mentioned stereo selective hydrolysis reaction, the concentration of substrate can be controlled between 0.05～10%, is preferably 0.5～5%, and pH is 5～11, is preferably 6～9.

Described stereo selective hydrolysis reaction can be carried out in the two-phase in damping fluid and organic solvent, described organic solvent comprises: benzene,toluene,xylene, ether, ethyl acetate, isopropyl ether, tetracol phenixin, chloroform, preferably toluene or dimethylbenzene or isopropyl ether.

Described damping fluid comprises phosphate buffered saline buffer, borate buffer solution, carbonate buffer solution, citrate buffer solution, O-phthalic acid buffer, tartrate damping fluid, diethyl barbituric acid sodium damping fluid, 2,4,6-trimethylpyridine-hydrochloride buffer, 2-amino-2-methylol-(1,3)-and propylene glycol-hydrochloride buffer, citric acid-Sodium phosphate dibasic damping fluid and N-ethylmorpholine-hydrochloride buffer.

In other words, cultivate mentioned microorganism, can produce the said hydrolyzed enzyme, the cell that fermentation produces all can be used as the enzyme source.Use the carbon source that comprises well known to those skilled in the art, the substratum of nitrogenous source and suitable inductor and inorganic salt commonly used.Fermentation preferably under 20～40 ℃, is carried out under the situation of aerobic at heating or normal temperature, and the pH value is between 5～8.

Stereoselective hydrolysis reaction can carry out in appropriate solvent.Described organic solvent comprises: benzene,toluene,xylene, ether, ethyl acetate, isopropyl ether, tetracol phenixin, chloroform, preferably toluene or dimethylbenzene or isopropyl ether.

The concentration of substrate can be controlled between 0.05～10%, preferably between 0.5～5%.Reaction can be carried out in room temperature or under the situation of heating, and preferably between 10～50 ℃, the pH value of reaction solution also has very big influence to hydrolysis reaction to Heating temperature, so will regulate the pH value in the reaction process and be controlled between 5～10, preferably between 6～9.

The enzymically hydrolyse reaction of racemize beta-phenyl glycidic acid methyl esters can be carried out in buffered soln, also can be at buffered soln with as toluene, benzene, ether, ethyl acetate, isopropyl ether, dimethylbenzene, tetracol phenixin, carry out in the two-phase organic solvents such as chloroform, preferred organic is a toluene, dimethylbenzene, isopropyl ether.

Optically active beta-the separation and purification of phenyl glycidyl acid methyl esters from reaction system can be undertaken by ordinary method well known to those skilled in the art.

Owing to have phenylacetic aldehyde to generate in the reaction process, so product must wash with the aqueous solution of saturated sodium bisulfite.

If reaction is water and organic phase two alternate carrying out, then organic phase to be separated, water layer merges organic layer with organic solvent extraction, and with saturated aqueous solution of sodium bisulfite washing, drying concentrates.If reaction is to carry out at aqueous phase, then with organic solvent extraction, and with saturated aqueous solution of sodium bisulfite washing, drying concentrates.

Therefore, can obtain (+)-beta-phenyl glycidic acid methyl esters (2) according to above-mentioned enzymatic resolution method ^*, can be used as the key intermediate of synthetic (-)-Clausenamide.

Is 5～10mgkg through (-)-Clausenamide that the inventive method obtains to the required oral dosage of memory improvement effect of normal mice or dysmnesia mouse ^-1, and (+)-Clausenamide is invalid.In the prior art, once to the facilitation of (±), (+), (-)-Clausenamide study, memory and strengthen the long time-histories of cynapse strengthen (LTP) effect and with relatively the studying in great detail of piracetam (Piracetam).

The oral effective dose of piracetam is 500mgkg ^-1(Dall R.Demographic andepidemiological trends today.In:Wolff HP et al eds.Drug research and drugdevelopment in 21 ' st century.Berlin:Spring-verlag.1998:25-26.), the intensity that former (-)-Clausenamide improves memory effect is 50～100 times of piracetam, (-)-Clausenamide also can strengthen the amplitude that the LTP that high-frequency electrical stimulation causes was transmitted and strengthened on cynapse basis, and (+)-Clausenamide and piracetam do not have influence to LTP.

The compounds of this invention can be with oral methods or the medication of parenteral road.Oral medication can be tablet, capsule, Drug coating, and non-have injection and suppository etc. through the intestines drug formulation.These preparations are according to the known method preparation of those skilled in the art.For making tablet, capsule, the used auxiliary material of Drug coating is the auxiliary agent of conventional usefulness, starch for example, gelatin, gum arabic, silica, polyoxyethylene glycol, the used solvent of liquid dosage form for example has water, ethanol, propylene glycol, plant oil such as Semen Maydis oil, peanut oil, olive wet goods.Containing in the preparation of The compounds of this invention also can have other auxiliary agents, tensio-active agent for example, lubricant, disintegrating agent, sanitas, correctives, pigment etc.

At tablet, capsule, Drug coating, the dosage that contains general formula of the present invention (I) compound in injection or the suppository is to calculate with the compound amount that exists in the unit dosage form.

On above-mentioned working foundation, the inventor has obtained new progress, substantially illustrated the mechanism of action of (-)-Clausenamide facilitation learning and memory and LTP, observe (-)-(β-A) causes amnemonic improvement effect and anti-nerve cell apoptosis effect to Clausenamide to β-amyloid.

Description of drawings:

Fig. 1 is the chemical synthesis route figure of the inventive method, wherein,

(+) 2 expressions: (+)-(2S, 3R)-3-phenyl glycidyl acid methyl esters;

(+) 3 expressions: (+)-(2S, 3R)-N-methyl-N-(beta-hydroxy-styroyl)-3-phenyl glycidyl acid amides;

(+) 4 expressions: (+)-(2S, 3R)-N-methyl-N-phenacyl-3-phenyl glycidyl acid amides;

(-) 5 expressions: (-)-Clausenamide ketone; (-)-(3S, 4R, 5R)-3-hydroxyl-5-phenacyl-1-methyl-4-Phenylpyrrolidine-2-ketone; (-) I represents: (-)-Clausenamide; (-)-(3S, 4R, 5R, 6S)-3-hydroxyl-5-(a-hydroxybenzyl)-1-methyl-4-Phenylpyrrolidine-2-ketone.

Fig. 2 is the dynamic change figure of four kinds of typical search strategies frequency of occurrences in the training of Morris water maze.Wherein 1,2,3,4, on behalf of control group, model group, model, 5,6 and 7 add (-)-Clausenamide 8mg/kg treatment group, model (-)-Clausenamide 40mg/kg treatment group, model (+)-Clausenamide 8mg/kg treatment group, model (+)-Clausenamide 40mg/kg treatment group, model respectively adds piracetam 400mg/kg treatment group.

Fig. 3 is the differentiation Butut that rat dentate gyrus tongue shape nerve fiber germinates, wherein

The A contrast, B (-)-Clausenamide 8mgkg ^-1, C (-)-Clausenamide 40mgkg ^-1, D (+)-Clausenamide 8mgkg ^-1, E (+)-Clausenamide 40mgkg ^-1Compare with control group, ^*P＜0.05 reaches ^*P＜0.01.

The differentiation Butut that Fig. 4 germinates for rat hippocampus CA3 district tongue shape nerve fiber, wherein,

The A contrast, B (-)-Clausenamide 8mgkg ^-1, C (-)-Clausenamide 40mgkg ^-1, D (+)-Clausenamide 8mgkg ^-1, E (+)-Clausenamide 40mgkg ^-1Compare with control group, ^*P＜0.05 ^*P＜0.01.

The distribution plan of the relative quantity that Fig. 5 expresses in hippocampal pyramidal cell and tegumental cell for bdnf protein, wherein, A contrast, B (-)-Clausenamide 8mgkg ^-1, C (-)-Clausenamide 40mgkg ^-1, D (+)-Clausenamide 8mgkg ^-1, E (+)-Clausenamide 40mgkg ^-1

Among the figure ^*And ^*Expression is compared with control group, and P＜0.05 reaches ^*P＜0.01.

The following word " Meng " that uses of this paper should be " Meng " with grass-character-head.

Describe the present invention in detail below with reference to embodiment, but infinite purpose.

Embodiment

Embodiment 1 (+)-2S, 3R-3-phenyl glycidyl acid (+)-8-8-naphthalene replaces alcohol ester in the Meng (2a) ^* :

NaH (80%) 430mg is added among the 20ml exsiccant THF N ₂The protection lower magnetic force stirs, and slowly drips 1g phenyl aldehyde and 2.3g (+)-8-betanaphthyl alcohol in Meng chloracetate (1a) ^*10ml THF solution, finish, stirring at room 40 hours (contains the 1ml glacial acetic acid) in the frozen water with reaction solution impouring 30ml, with ether (40ml * 3) extraction, use saturated NaHSO successively ₃The aqueous solution, saturated NaHCO ₃The aqueous solution, saturated NaCl solution washing adds anhydrous Na ₂SO ₄Drying is filtered, be evaporated to dried, 2.9g oily matter., get the 2.16g white solid through silica gel column chromatography.

Yield 79.0%, mp:38～40 ℃, [α] ¹⁵ _D=+35.8 ° of (C1.31 CHCl ₃).NMR 500MHz，δ，ppm：7.03-6.4(12H，m)，4.96(1H，td，J＝2.2Hz，10.7Hz)，3.96(1H，d，J＝1.27Hz)，2.64(1H，d，J＝1.2Hz)，1.48(3H，s)，0.98(3H，d，J＝6.48Hz)，0.9-2.2(9H，m)

Embodiment 2. (+)-2S, 3R-3-phenyl glycidyl acid (+) alcohol ester in the Meng (2b) ^*

With stirring evenly among the anhydrous THF of NaH (80%) 3.27g adding 100ml, drip 11.6g phenyl aldehyde and 11.85g Mono Chloro Acetic Acid (+) alcohol ester in the Meng (1b) ^*The 100ml anhydrous THF solution, finish, stirring at room 10 hours, suction filtration, filtrate decompression is concentrated into dried, adds an amount of ether dissolution, uses saturated NaHSO successively ₃The aqueous solution, saturated NaHCO ₃The aqueous solution, the saturated NaCl aqueous solution is washed till neutrality, adds anhydrous Na ₂SO ₄Drying is filtered, and filtrate decompression is concentrated into dried, the freezing curing of gained oily matter, and recrystallization three times gets white crystals 7.62g.Yield 35%, mp:62～63 ℃, [α] ¹⁵ _D=+167 ° of (C1.0 CHCl ₃).

Embodiment 3 (+)-2S, 3R-3-phenyl glycidyl acid methyl esters (2) ^*

With 4.28g compound (2a ^*Or 2b ^*) being dissolved in 80ml methyl alcohol, magnetic agitation adds the 200mg sodium methylate, reacts after 6 hours, drip the 0.2ml glacial acetic acid, concentrating under reduced pressure is done, and adds the 100ml ether and stirs filtration in 30 minutes, is evaporated to dried, get 4.3g oily matter,, get 1.33g oily compound (2) through silica gel column chromatography ^*Yield: 75%, [α] ¹⁵ _D=+170.9 ° of (C1.33 CHCl ₃).

Embodiment 4 (+)-2S, 3R-N-methyl-N-(beta-hydroxyphenyl ethyl)-3-phenyl glycidyl acid amides (3) ^*

With 1.78g compound (2) ^*, 1.51g N-methyl-beta-hydroxyphenyl ethamine is dissolved in respectively in the 5ml methyl alcohol, mixes after being cooled to-20 ℃, adds the 100mg sodium methylate, places 2 days in the refrigerator of dissolving back; Drip 0.2m1 2N hydrochloric acid, concentrating under reduced pressure is done, white solid, filter 2.3g compound (3) ^*Yield: 77.3%, mp:93～94 ℃, [α] ¹⁵ _D=+70.2 ° of (C0.684 CH ₃OH).

Embodiment 5 (+)-2S, 3R-N-methyl-N-α-phenacyl-3-phenyl glycidyl acid amides (4) ^*

With 2.97g compound (3) ^*, the anhydrous CuSO of 2.2g ₄With 6.32g KMnO ₄Join 100mlCH successively ₂Cl ₂CH is filtered, used to middle the stirring 3 hours ₂Cl ₂Wash, concentrating under reduced pressure does, 2.9g oily matter, recrystallization, 2.3g compound (4) ^*Yield: 78.0%, mp:86～87 ℃, [α] ¹⁵ _D=+140.2 ° of (C0.5CH ₃OH).

Synthetic (5) of embodiment 6 (-)-Clausenamide ketone ^*

With 2.95g compound (4) ^*Powder joins 6.6mg LiOHH ₂In the 30ml aqueous solution of O, magnetic agitation is 8 hours in 30～35 ℃ of water-baths, cooling, filtration, drying, and recrystallization gets 1.5g compound (5) ^*Yield: 50.8%, mp:196～198 ℃, [α] ¹⁵ _D=-338 ° of (C0.6 CH ₃OH).

Synthetic (-) I of embodiment 7 (-)-Clausenamide

With 2.95g levorotatory compound (5) ^*Join in the 200ml methyl alcohol, the magnetic agitation dissolving adds 1.52gNaBH ₄, reacted 3 hours; Drip 1ml 2N hydrochloric acid, concentrating under reduced pressure is done, and uses CH ₂Cl ₂Washing adds anhydrous Na ₂SO ₄Drying is filtered, and concentrating under reduced pressure is done, and recrystallization gets 2.4g compound (I) ^*Yield: 80.8%, mp:160～161 ℃, [α] ¹⁵ _D=-144 ° of (C0.2 CH ₃OH).

Embodiment 8. is through fractionation preparation (-)-Clausenamide (-) I of racemization Clausenamide ketone (±) 5

3g (-) alcohol in Meng fluoroacetic acid is dissolved in 10g SOCl ₂In, reflux 5 hours, decompression steams SOCl ₂, add 5ml toluene, pressure reducing and steaming toluene again, resistates is the heavy 3.5g of brown oil, and it is dissolved in 50mlCH ₂Cl ₂, dropping into 2g racemization Clausenamide ketone (±) 5 again, the ice bath cooling adds the 1ml anhydrous pyridine, stirring at room 4 hours.TLC shows 2 points, and reactant is used 2N hydrochloric acid successively, saturated NaHCO ₃The aqueous solution, the saturated NaCl aqueous solution is washed anhydrous Na ₂SO ₄Drying gets brown solid 5.4g, through leafing and recrystallization, gets two kinds of white crystals (+7) ^*(7) ^*

(+7) ^*: 0.86g, yield 24.8% (theoretical yield 50%), mp:152～153 ℃, [α] ²⁰ _D=+156 ° of (C1.1 CHCl ₃);

(7) ^*: 1.06g, yield 30.9% (theoretical yield 50%), mp:177～178 ℃, [α] ¹⁸ _D=-249 ° of (C0.21 CHCl ₃).

Refluxed 2 hours in 30ml methyl alcohol as catalyzer with tosic acid respectively, boil off methyl alcohol, organic layer is used dilute hydrochloric acid successively, and diluted alkaline is washed to neutrality, by (+7) ^*Get (+)-Clausenamide ketone (+5) ^*, yield 82%, mp:193～194 ℃, [α] ¹⁸ _D=+339 ° of (C0.21 CH ₃OH).By (7) ^*Get (-)-Clausenamide ketone (5) ^*, yield 88%, mp:193～194 ℃, [α] ¹⁸ _D=-340 ° of (C0.22 CH ₃OH).

With (-)-Clausenamide ketone (5) ^*0.59g, according to the identical method of embodiment 7, with 0.11g NaBH ₄Reduction, recrystallization gets 0.5g (-)-Clausenamide (I) ^*, yield 83%, mp:161～162 ℃, [α] ¹⁸ _D=-145 ° of (C0.24 CH ₃OH).

Concrete preparation flow is as follows:

Embodiment 9. is through Split Method preparation (-)-Clausenamide of raceme Clausenamide ketone (5)

With 3.56g (-)-S-phthalyl L-Ala, 5g SOCl ₂Join in the 50ml toluene reflux 5 hours, pressure reducing and steaming SOCl ₂, add 20ml toluene again, be evaporated to driedly, add 100ml exsiccant CH ₂Cl ₂, with being cooled in the ice bath below 0 ℃, adding 4g racemization Clausenamide ketone (5), magnetic agitation drips the 1.6g pyridine, finishes stirring at room 10 hours, adds 30ml water, divides and gets organic layer, uses 30mlCH ₂Cl ₂Extraction merges organic phase, uses 30ml 2N hydrochloric acid successively, saturated NaHCO ₃The aqueous solution, the saturated NaCl aqueous solution is washed, and adds anhydrous Na ₂SO ₄Drying is filtered, and concentrating under reduced pressure is done, and recrystallization gets (6) ^*: prismatic crystal 1.42g, yield: 21.0% (theoretical amount 50%), mp:189～190 ℃, [α] ¹⁵ _D=-241 ° of (C0.5 CHCl ₃); (+6) ^*: mp:153～154 ℃, [α] ¹⁵ _D=+128.5 ° of (C2.1 CHCl ₃).

With 4.96g prismatic crystal (6) ^*Join in the 200ml methyl alcohol, the magnetic agitation dissolving adds 1.52g NaBH ₄, room temperature reaction 5 hours adds 1ml 2N HCl, concentrating under reduced pressure is done, recrystallization, the 2.38g of (-)-Clausenamide+(I), yield 81%, mp:161～163 ℃, [α] ²⁰ _D=-144.4 ° of (C0.46 CH ₃OH).

Its concrete preparation flow is as follows:

Embodiment 10. fungal enzyme method for splitting preparation (+)-2S, 3R-3-phenyl glycidyl acid methyl esters (2) ^*

To contain 3% sucrose, 1% peptone, 0.5% potassium primary phosphate, 0.5% polyoxyethylene glycol, 1% sweet oil 30ml substratum places the flask of 250ml, and sterilization is 30 minutes under 120 ℃ and a normal atmosphere.After the cooling, (under 30 ℃, rotating speed 220rpm cultivated 40 hours, with the thalline centrifugation for Aspergillus, sp) bacterium to insert the aspergillus oryzae of a transfering loop in this substratum.And thalline is added in the phosphate buffer soln that the pH value is 7 the 10ml beta-phenyl glycidic acid methyl esters that contains 150mg 30 ℃ of reactions 72 hours down.Reaction finishes, twice of the ethyl acetate extraction of adding 20ml.Merge organic layer, and with the solution washing of saturated sodium bisulfite, saturated sodium hydrogen carbonate solution washing, anhydrous MgSO ₄Dry.Concentrate 66mg oily matter.This oily matter is through chirality gas chromatographic analysis (G-TA type chiral column, column length are 10 meters, and nitrogen is carrier gas, and flow velocity is 2.5ml/min, and furnace temperature is 105 ℃), and its ee% value is 98%, [α] _D ²⁰=+180 ° of (c=1, CHCl ₃).

Embodiment 11. actinomycetes method for splitting preparation (+)-2S, 3R-3-phenyl glycidyl acid methyl esters (2) ^*

To contain 1% sucrose, 2% peptone, 0.3% potassium primary phosphate, 1% polyoxyethylene glycol, 2% sweet oil, the substratum of 30ml places the flask of 250ml, and sterilization is 30 minutes under 120 ℃ and a normal atmosphere.After the cooling, insert promise Ka Shi (Nocardia sp) bacterium of a transfering loop in this substratum, 30 ℃, rotating speed 250rpm cultivated 36 hours, with the thalline centrifugation.And thalline is added in the phosphate buffer soln that the pH value is 7.5 the 10ml racemization beta-phenyl glycidic acid methyl esters (2) that contains 200mg 30 ℃ of reactions 72 hours down.Reaction finishes, twice of the ethyl acetate extraction of adding 20ml.Merge organic layer, and with the solution washing of saturated sodium bisulfite, saturated sodium hydrogen carbonate solution washing, anhydrous MgSO ₄Dry.Concentrate 80mg oily matter.This oily matter is through chirality gas chromatographic analysis (G-TA type chiral column, column length are 10 meters, and nitrogen is carrier gas, and flow velocity is 2.5ml/min, and furnace temperature is 105 ℃), and its ee% value is 98%, [α] _D ²⁰=+180 ° of (C=1 CHCl ₃).

Embodiment 12. yeast enzymatic resolution methods preparation (+)-2S, 3R-3-phenyl glycidyl acid methyl esters (2) ^*

To contain 4% sucrose, 2% peptone, 1% potassium primary phosphate, 2% polyoxyethylene glycol, 1% sweet oil, the substratum of 30ml places the flask of 250ml, and sterilization is 30 minutes under 120 ℃ and a normal atmosphere.After the cooling, insert candiyeast (Candida sp) bacterium of a transfering loop in this substratum, 30 ℃ of following rotating speed 200rpm cultivated 40 hours, with the thalline centrifugation.And thalline is added in the phosphate buffer soln that the pH value is 8 the 30ml racemization beta-phenyl glycidic acid methyl esters (2) that contains 300mg 30 ℃ of reactions 72 hours.Reaction finishes, twice of the ethyl acetate extraction of adding 30ml.Merge organic layer, and with the solution washing of saturated sodium bisulfite, saturated sodium hydrogen carbonate solution washing, anhydrous MgSO ₄Dry.Concentrate 120mg oily matter.This oily matter is through chirality gas chromatographic analysis (G-TA type chiral column, column length are 10 meters, and nitrogen is carrier gas, and flow velocity is 2.5ml/min, and furnace temperature is 105 ℃), and its de% value is 98%, [α] _D ²⁰=+180 ° of (c=1, CHCl ₃).

Embodiment 13 bacterial enzyme method for splitting preparations (+)-2S, the 3R-3-phenyl methyl glycerate (2) that contracts ^*

To contain 2.5% Trisodium Citrate, 1% soy peptone, 0.5% potassium primary phosphate, 2.5% polyoxyethylene glycol, the substratum of 1% sweet oil 30ml places the flask of 250ml, and sterilization is 30 minutes under 120 ℃ and a normal atmosphere.After the cooling, insert the achromobacter (Achromobacter.sp) of a transfering loop in this substratum, 40 ℃ of following rotating speed 200rpm cultivated 40 hours, with the thalline centrifugation.And thalline is added in the phosphate buffer soln that the pH value is 7 the 10ml racemization beta-phenyl glycidic acid methyl esters (2) that contains 150mg 30 ℃ of reactions 72 hours.Reaction finishes, twice of the ethyl acetate extraction of adding 20ml.Merge organic layer, and with the solution washing of saturated sodium bisulfite, saturated sodium hydrogen carbonate solution washing, anhydrous MgSO ₄Dry.Concentrate 66mg oily matter.This oily matter is through chirality gas chromatographic analysis (G-TA type chiral column, column length are 10 meters, and nitrogen is carrier gas, and flow velocity is 2.5ml/min, and furnace temperature is 105 ℃), and its de% value is 99%, [α] _D ²⁰=+181 ° of (C=1 CHCl ₃).

Embodiment 14 bacterial enzyme method for splitting preparation (+)-2S, 3R-3-phenyl glycidyl acid methyl esters (2) ^*

To contain 2% lactic acid, 1.5% sulfuric acid amine, 0.5% potassium primary phosphate, 0.5% polyoxyethylene glycol, the substratum of 0.1% sweet oil 30ml places the flask of 250ml, and sterilization is 30 minutes under 120 ℃ and a normal atmosphere.After the cooling, insert the genus bacillus (Bucillus.sp) of a transfering loop in this substratum, 50 ℃ of following rotating speed 220rpm cultivated 40 hours, with the thalline centrifugation.And thalline is added in the phosphate buffer soln that the pH value is 10 the 10ml racemization beta-phenyl glycidic acid methyl esters (2) that contains 150mg 50 ℃ of reactions 72 hours.Reaction finishes, twice of the ethyl acetate extraction of adding 20ml.Merge organic layer, and with the solution washing of saturated sodium bisulfite, saturated sodium hydrogen carbonate solution washing, anhydrous MgSO ₄Dry.Concentrate 66mg oily matter.This oily matter is through chirality gas chromatographic analysis (G-TA type chiral column, column length are 10 meters, and nitrogen is carrier gas, and flow velocity is 2.5ml/min, and furnace temperature is 105 ℃), and its de% value is 98%, [α] _D ²⁰=+181 ° of (C=1 CHCl ₃).

Embodiment 15 X-luminescent crystal diffraction approaches are measured the absolute configuration of (-)-Clausenamide

In order to determine the absolute configuration of (-)-Clausenamide, adopt the monocrystalline of the compound (7) among the embodiment 9:

Because the absolute configuration of (-)-lid fluoroacetic acid is known, the monocrystalline x-ray diffraction measurement result according to compound (7) shows compound (5) ^*The following structural formula of absolute configuration shown in.

With (-) 7 compound hydrolysis, get (-)-Clausenamide (-) I through sodium borohydride reduction, according to being (+), be configured as 3R by the optically-active of Sharpless method of reducing gained Clausenamide ^*, 4S ^*, 5S ^*, 6R ^*(+) I, and its absolute value is identical with (-) I of gained, and opposite in sign, fusing point are also consistent, thus C ₆The configuration that 6S should be arranged, i.e. (-)-Clausenamide (+) I should have 3S, 4R, 5R, the configuration of 6S.

The short intelligence activity that draws (-)-Clausenamide through experiment is 5 times of (+)-Clausenamide at least, and (+)-Clausenamide does not almost have biological activity with regard to effect of the present invention; (-)-Clausenamide not only on study of behaviour, also proves the effect of its facilitation learning and memory and enhancing neural plasticity aspect electric physiology; (-)-Clausenamide not only has very strong nootropic effect, and has the effect of potential control senile dementia, and it shows anti-nerve cell apoptosis and inhibition Tau albumen peroxophosphoric acidization; From biochemical, aspects such as molecular biology and morphological structure are illustrated beyond the nootropic effect mechanism of (-)-Clausenamide except, prove aspect morphological structure that particularly it can stimulate the germination of hippocampus tongue shape nerve ending; Research through to (-) (+)-Clausenamide proves that its meta-bolites also has biological activity.

Experimental example 1 (-)-Clausenamide strengthens the mechanism of rat hippocampus synaptic transmission in dentate gyrus

Adopt anesthetized rat, stimulating electrode is buried in entorhinal area anterior perforated substance path, recording electrode buries in hippocampal dentate (DG) granular cell layer, with population spike (PS) as the excitatoty index of DG granulosa cell group, LTP is induced by the high-frequency electrical stimulation of 10 string 200Hz, every train is made up of the square wave stimulation of 5 wide 0.2ms of ripple, (-), the solubility promoter DMSO of (+)-Clausenamide and respective concentration gives by the basket pipe of implanting tricorn, the PS of record 30min represent the basic cynapse transmission level before the administration, in 5 minutes relative medicine or the solubility promoter of 5 μ l is slowly injected tricorn then.

1. studies have shown that, induce a kind of nmda receptor and the dependent LTP of VDCC with the 200Hz high frequency stimulation at anesthetized rat DG position.It is that VDCC is dependent and irrelevant with nmda receptor that (-)-Clausenamide strengthens anesthetized rat hippocampus DG cynapse transmittance process, high frequency stimulation inductive LTP and (-)-Clausenamide enhanced cynapse transduction activity are all with dependent phosphoprotein phosphatase Calcineurin of hippocampus and the active raising of Calpain, (see table 1 and table 2) prompting participates in LTP and (-)-Clausenamide protein kinase of being not only to cynapse transduction activity enhancement, and phosphoprotein phosphatase arranged, illustrating that protein phosphorylation and dephosphorylation process all strengthen to some extent, may be to be coordinated mutually at last and the result that complements one another determines the height of cynapse level by these two processes.

The Calcineurin activity of table 1. rat layer and hippocampal tissue

Calcineurin activity (umol Pi/hr/ μ g albumen)

Group

The cortex hippocampus

Contrast 8.3+0.4 7.8 ± 0.6

AP5 7.9±0.7 8.1±0.7

Nimodipine 8.4 ± 0.5 7.7 ± 0.8

High-frequency electrical stimulation (HFS) 9.2 ± 1.1 8.5 ± 0.7

APS+HFS 8.8±0.7 9.6±0.5 ^*

Nimodipine+HFS 7.7 ± 1.4 9.8 ± 0.4 ^*

APS+ nimodipine+HFS 8.0 ± 0.9 7.9 ± 0.7

(-)-Clausenamide 11.2 ± 0.9 ^*10.4 ± 0.5 ^*

(-)-Clausenamide+APS 11.6 ± 1.4 ^*10.1 ± 0.8 ^*

(-)-Clausenamide+nimodipine 9.1 ± 1.2 8.3 ± 0.6

^*Compare P＜0.05 with control group

The Calpain activity of table 2. rat layer and hippocampal tissue

Calpain (Δ A595nm/hr/mg albumen)

Group

The cortex hippocampus

Contrast 1.5 ± 0.2 7.8 ± 0.6

AP5 1.6±0.4 1.6±0.6

Nimodipine 1.7 ± 0.3 1.8 ± 0.5

HFS 1.8±0.7 4.3±0.7 ^**

APS+HFS 1.7±0.4 2.9±0.8 ^*#

Nimodipine+HFS 1.8 ± 0.5 2.6 ± 0.7 ^*#

APS+ nimodipine+HFS 1.7 ± 0.4 1.8 ± 0.6

(-)-Clausenamide 2.6 ± 0.5 ^*2.7 ± 0.8 ^*

APS+ (-)-Clausenamide 2.7 ± 0.6 ^*2.6 ± 0.7 ^*

Nimodipine+(-)-Clausenamide 1.6 ± 0.6 1.9 ± 0.6

^*And ^*Expression is compared with control group, and P＜0.05 and P＜0.01# compare P＜0.05 with the HFS group.

2. studies have shown that there are a small amount of moss fibers end phenomenon of sprouting slightly in normal adult rat hippocampus CA3 district and DG.(-)-Clausenamide 8mgkg ^-1And 40mgkg ^-1Sprouting of the moss fibers tip of these two hippocampuss of promotion, dose-dependently ground, and same dosage (+)-Clausenamide does not significantly influence it, and the notable difference of prompting (-), (+)-this effect of Clausenamide may be Different Effects is transmitted in its long term administration to the hippocampus cynapse a morphological base (the results are shown in Fig. 3, Fig. 4)

(3.BDNF Brain Derived Neurotrophic Factor) expressed in normal rat cortical neuron and hippocampus granulosa cell and pyramidal cell in large quantities, and (-)-Clausenamide (8,40mgkg ^-1) dose-dependently increases the expression of BDNF in these two zones, (+)-Clausenamide (40mgkg ^-1) it also there is slight increase effect, prompting (-) (+)-Clausenamide long term administration may be relevant with its difference between the effects to the bdnf protein expression to the Different Effects of hippocampus cynapse transduction activity.The results are shown in Figure 5:

4. glutamate receptor causes effect among the LTP at (-)-Clausenamide.Excitatory neurotransmitter L-glutamic acid content in central nervous system is abundant, it discharged from the presynaptic, activated multiple glutamate receptor of postsynaptic, occupied critical role in LTP, G albumen coupling acceptor mGluR does not directly regulate electrical signal, but activates multiple second messenger's cascade reaction.We have observed mGluR antagonist (±) MCPG (-)-Clausenamide have been brought out influence at body hippocampal dentate LTP, the result shows, (±) MCPG (10 μ mol, icv) suppress HFS and (-)-Clausenamide inductive LTP, 15min intracerebroventricular injection (±)-MCPG can reverse the LTP that has set up after giving HFS and (-)-Clausenamide, the mGluR acceptor of prompting MCPG sensitivity is that (-)-Clausenamide is induced and to keep LTP necessary, and (-)-Clausenamide causes that by the mGluR acceptor cynapse propagation function strengthens.

Experimental example 2 (-) and (+)-Clausenamide causes amnemonic improvement effect to β-A

Senile plaque is degenerative brain disorder patient's a main europathology feature.Its nucleus is a kind of polypeptide of being made up of 40-42 amino acid, thisly is called as amyloid-beta (it has neurotoxic effect to the polypeptide of β-A) by a large amount of studies confirm that.A segment β-A (25-35) who is made up of 11 its acid of ammonia (25-35) has neurotoxic effect similar to β-A and self-cohesion ability in β-A structure, the same with β-A, the neurotoxicity of condensed state β-A (25-35) is strong than its solubilised state, utilizes and has successfully caused the dysmnesia model of animal in the Morris water maze to rat intracerebroventricular injection condensed state β-A (25-35).

Intracerebral ventricle injection 15nmol β-A (25-35) back began to adopt the space learning memory capability of Morris water maze training test rat on the 15th day, trained each 3min every day 4 times.It is latent period that the result finds the required time of platform with animal, represents towards wrong angle (animal body major axis direction and place of entry are to the angle of line between platform) and the animal searching strategy that platform adopted.

Beginning in the 2nd day of training, the latent period of β-A (25-35) model group, obviously comparison was shone group leader (F=3.04, P＜0.05) to the 5th day, difference the most remarkable (F=13.7, P＜0.01).Since the 3rd day, (-)-Clausenamide 8mgkg ^-1Group and 40mgkg ^-1Obviously shorten (F=2.68, P＜0.05 and F=2.73, P＜0.05) than β-A (25-35) model group the latent period of group.(+)-Clausenamide 8 and 40mgkg ^-1Group and piracetam 400mgkg ^-1Group trains latent period and β-A (25-35) model group of each day not to have significant difference.The mistake of each treated animal reduces gradually towards the increase of angle along with frequency of training, since the mistake of the 3rd day model group towards angle obviously greater than control group, (-)-Clausenamide 8mgkg ^-1And 40mgkg ^-1Group is significantly less than model group.(+)-Clausenamide 8mgkg with dosage ^-1And 400mgkg ^-1There was no significant difference between piracetam group and model group.(seeing Table 3)

Table 3. β-AP (25-35) is to reaching the effect of (-), (+)-Clausenamide towards the influence of wrong angle in the training of Morris water maze.Represent with MEAN ± S.D. towards wrong angle.

The training fate

Group

1 2 3 4 5

A 93.6±34.6 76.8±29.8 47.3±15.1 29.6±10.7 15.4±20.4

B 106.3±29.8 98.7±30.4 73.5±20.3 ^* 60.6±23.8 ^* 46.7±19.4 ^*

C 89.7±29.4 72.3±19.8 46.5±21.2# 30.4±14.8# 17.6±9.5#

D 101.5±24.3 83.5±21.8 51.2±18.7# 28.4±13.2# 20.7±8.7#

E 99.4±31.7 93.5±29.6 82.1±26.7 70.2±21.5 59.2±18.3

F 112.4±32.8 102.5±28.7 94.2±22.5 83.1±26.5 65.4±20.8

G 92.5±28.9 87.3-24.2 74.6±23.7 62.5±19.6 44.5±14.3

^*P＜0.05vs control group: #P＜0.05vs model group.A: control group B: model group C: model (-)

-Clausenamide 8mg/kg treatment group D: model (-)-Clausenamide 40mg/kg treatment group E: model (+)

-Clausenamide 8mg/kg treatment group F: model (+)-Clausenamide 40mg/kg treatment group G: model piracetam 400mg/kg treatment group.

The mode of four kinds of typical search platforms of rat in this experiment: random mode, marginal mode, trend formula and orthoscopic have all been observed.Each treated animal is all mainly sought platform with marginal mode and random mode strategy when the training beginning, and along with the increase of frequency of training, the occurrence rate of these two kinds of search strategies descends gradually.Since the 3rd day, it is fast than other group that the speed and the degree of this decline are organized in normal control group and the treatment of (-)-Clausenamide.Simultaneously, the appearance of trend formula and orthoscopic increases gradually.It the results are shown in Figure of description 2.

Experimental example 3 (-)-Clausenamide is to the apoptotic restraining effect of PC12 of Bax c DNA high expression level

1, builds up the PC12 clone of Bax α stably express first

At first removed Bax α cDNA3 ' end and added Poly a-signal sequence and other sequence of part with restriction enzyme, made up intermediate carrier Pbluescript SK-Bax α (PA) and Bax α cDNA has been cut out from middle carrier with restriction endonuclease then, being connected with carrier for expression of eukaryon PcDNA3 with single direction obtains PcDNA3Bax α recombinant plasmid.The Bax α cDNA of sequence reorganization contains-and 5 ' the untranslated end of 50bp (5 '-UTR), 3 ' the untranslated end (3 '-UTR) of the open reading frame of 576bp (ORF) and 137bp.Change pcDNA3-Bax α over to the PC12 cell with lipofectamine liposome transfection method, select the resistant cell clone with G-418.Western blotting and immunohistochemical method all show the high-caliber expression of Bax α albumen in the PC-12 clone, so the PC12 cytokine of Bax α stably express has successfully been set up in this research first.

2, the anti-apoptotic effect of the foundation of Bax α high expression level PC12 clone apoptosis model and (one) Clausenamide

Original position end mark (TUNEL) and flow cytometer are observed and are shown that 6-hydroxydopamine (6-OHDA) 100 μ mol/L can induce the apoptosis damage of Bax α high expression level PC-12 clone, with change the unloaded cell ratio of apoptosis obviously rise (49.96%vs32.9%) of comparing, illustrate that Bax α plays an important role in apoptotic process, give (one) Clausenamide 10 ^-7～10 ^-5Can make the apoptosis ratio drop to 36 respectively behind the mol/L from 49.96%.23%, 28.1% and 9.5%.

3, (-)-Clausenamide anti-apoptotic Its Mechanisms

(1) Bax α high expression level PC12 cell is after 6-OHDA induces 24 hours, and the level of Bcl-2 anti-apoptotic gene has to a certain degree and descends in the cell, gives (one) Clausenamide 10 ^-7With 10 ^-6Behind/the mol/L, the proteic level of Bcl-2 is obviously raise.

(2) Bax α high expression level PC12 cell is after the 6-OHDA effect 24 hours, the content of GSH obviously reduces (9.84 ± 1.20 vs, 14.98 ± 1.18nmol/mg pro) in the plastosome, the content of MDA obviously raise (2.46 ± 0.19 vs, 2.01 ± 0.12nmol/mg pro) give (one) Clausenamide 10 ^-710 ^-610 ^-5Can make significantly the raise content of (12.29 ± 0.99,15.10 ± 0.95,17.78 ± 1.04nmol/mg pro) MDA of the content of GSH in the plastosome obviously descend (1.76 ± 0.17,1.54 ± 0.13,1.33 ± 0.11nmol/mgpro) behind the mol/L

(3) (one) Clausenamide is to mitochondrial membrane potential and plastosome electron transport chain prozyme I, the active influence of IV.

Bax α high expression level PC12 cell is compared with the unloaded cell of commentaries on classics and is had higher mitochondrial membrane potential (95.08 VS 83.03).After 6-OHDA induces 12 hours, the mitochondrial membrane potential of Bax α high expression level PC12 cell obviously reduced (95.08 vs 24.6) and gives (one) Clausenamide 10 ^-7With 10 ^-6Can make mitochondrial membrane potential obviously raise (30.7 ± 3.3,56.0 ± 5.0) behind the mol/L.In addition, Bax α high expression level PC12 cell after 6-OHDA induces 12 hours, active significantly reduce (1.44 ± 0.16 vs, 1.68 ± 0.14 μ mol/mg pro/min) of plastosome electron transport chain prozyme I, the activity of prozyme IV also significantly reduces (0.09 ± 0.019vs, 0.15 ± 0.022 μ mol/mg pro/min).Give (one)

Clausenamide

10 ^-7, 10 ^-6, 10 ^-5Behind the mol/L, can make active raise (1.57 ± 0.12,1.98 ± 0.05,2.16 ± 0.2 μ mol/mg pro/min) of plastosome electron transport chain prozyme I.And make active raise (0.12 ± 0.015,0.15 ± 0.02,0.18 ± 0.01 μ mol/mg pro/min) of prozyme IV.

(4) (one) Clausenamide is to the influence of in-vitro separation mitochondrial cytochrome C release

Cell is after the stimulation that is subjected to apoptotic signal, cytochrome C enters into and activates dead program in the cytoplasm, this research at first obtains Bax α albumen with the separation of solid metallic ion method of purification, after it is joined isolating plastosome, can make mitochondrial cytochrome C content reduce (74.43 ± 5.69 μ g/mg pro.vs, 50.2 ± 30.65 μ g/mg pro), (one)

Clausenamide

10 ^-7, 10 ^-6, 10 ^-5Mol/L can make cytochrome C discharge and be reduced to 58.73 ± 3.77,61.7 ± 5.3 and 67.95 ± 7.6 μ g/mg pro respectively.

Experimental example 4 (±), (-), (+)-Clausenamide improve the comparison of memory effect

The present invention adopts disposable avoidance conditioning diving tower method and darkness avoidance test to observe (±), (-), (+)-Clausenamide to improving the test of comparing property of memory impairments with the water maze method of repeatedly study.

Oral pharmaceutical once before diving tower method and darkness avoidance test lay in experiment.The water maze method was tested 5 days altogether, trained preceding intraperitoneal injection of drugs every day once, and continuous 5 days, the result represented with latent period, errors number.

Table 4 causes the effect of memory acquisition disturbance because of the injection Daturamine to diving tower method and darkness avoidance test for piracetam and (-)-Clausenamide.

Table 4

Medicine	Dosage mg/kg	Errors number	Errors number	Latent period (X ± g)	Latent period (X ± g)
Medicine	Dosage mg/kg	Errors number	Errors number	Latent period (X ± g)	Latent period (X ± g)			Darkness avoidance test	The diving tower method	Darkness avoidance test	The diving tower method
0.9％Nacl		12.7±4.6	0.9±1.0	24.4±59	184±105			Darkness avoidance test	The diving tower method	Darkness avoidance test	The diving tower method
0.9％Nacl		12.7±4.6	0.9±1.0	24.4±59	184±105	Piracetam	500	4.1±3.2 ^***	0.7±0.5	107±110 ^**	180±110
(-)-Clausenamide	5	5.7±4.7 ^***	0.5±0.5	47.9±90 ^**	186±129	Piracetam	500	4.1±3.2 ^***	0.7±0.5	107±110 ^**	180±110
(-)-Clausenamide	5	5.7±4.7 ^***	0.5±0.5	47.9±90 ^**	186±129		10	6.5±5.0 ^***	0.2±0.4 ^**	59.4±91 ^**	296±97 ^**
	50	7.1±3.6 ^***	0.2±0.4 ^**	17.1±12.4	265±91 ^**		10	6.5±5.0 ^***	0.2±0.4 ^**	59.4±91 ^**	296±97 ^**

N=10x ± SD ^*＜0.05 ^{* *}P＜0.01 VS 0.9%Nacl (Daturamine)

Table 5 is that piracetam and (-), (±)-Clausenamide are to the learning and memory latent period of water maze and the influence of memory process.

Table 5

Drug dose mg/kg second day the 3rd day the 4th day

Reach the latent period (second) that reaches platform latent period (second) of platform

0.9％Nacl 32.4±29.2 25.8±18.4 26.1±38.4

Piracetam 500 32.2 ± 22.6 18.6 ± 9.9 ^*16.7 ± 16.3 ^*

(-)-Clausenamide 10 28.7 ± 35.2 17.4 ± 10.9 ^*12.1 ± 6.9 ^*

(±)-Clausenamide 10 35.8 ± 35.2 23.9 ± 22.6 17.1 ± 14.3

(±)-Clausenamide 100 36.6 ± 37.0 25.7 ± 13.8 14.6 ± 10.4 ^*

N＝10，x±SD ^**P＜0.05 ^***P＜0.01 VS 0.9％NaCl

Table 6 is that piracetam, (-), (±)-Clausenamide are to causing the effect of memory acquisition disturbance because of injection Daturamine (10mg/kg) in the water maze laboratory.

Table 6

Medicine N dosage (mg/kg) errors number

Daturamine 10 15.9 ± 11.3

Piracetam 9 500 23.5 ± 25.0

(-)-Clausenamide 10 10 9.4 ± 5.9 ^*

(±)-Clausenamide 8 10 16.6 ± 23.9

(±)-Clausenamide 8 100 9.11 ± 3.8 ^*

^*P＜0.05 VS Daturamine group

To be (-), (+)-Clausenamide cause the effect of memory acquisition disturbance to keeping away Daturamine in the dark experiment with table 7

Table 7

Drug dose (mg/kg) latent period (second) errors number

0.9％Nacl 152.1±64.2 2.0±3.8

Daturamine 10 35.8 ± 76.4 ^*14.1 ± 8.9 ^*

(+)-Clausenamide 10 138.2 ± 86.7 ^*6.7 ± 11.0 ^*

(-)-Clausenamide 50 140.6 ± 96.4 ^*5.4 ± 4.1 ^*

(±)-Clausenamide 10 43.0 ± 65.8 11.3 ± 21.5

(±)-Clausenamide 50 79.9 ± 63.6 14.4 ± 12.3

^*P＜0.05 ^*P＜0.01 VS Daturamine group ^*P＜0.01 VS 0.9%Nacl group

(-), (+)-Clausenamide was irritated in training and is fed administration in preceding 30 minutes.The Daturamine group is in preceding 10 minutes intraperitoneal injections of training.

Claims

1. method for preparing compound shown in general formula (I),

May further comprise the steps:

(1) forms the chloracetate of chiral alcohol with chloroacetyl chloride and chirality alcohol roh;

The reaction of the chloracetate of the chiral alcohol that (2) obtains and phenyl aldehyde obtains aryl that corresponding light the lives R-Glyceric acid chirality alcohol ester that contracts;

(3) through transesterify get (+)-(2S, 3R)-3-phenyl glycidyl acid methyl esters;

(4) (+) that obtains-(2S, 3R)-3-phenyl glycidyl acid methyl esters gets (+)-(2S with corresponding amine condensation, 3R)-N-methyl-N-(beta-hydroxy-styroyl)-3-phenyl glycidyl acid amides, (+) that obtains-(2S, 3R)-N-methyl-N-(beta-hydroxy-styroyl)-3-phenyl glycidyl acid amides through oxidation get (+)-(2S, 3R)-N-methyl-N-phenacyl-3-phenyl glycidyl acid amides;

(5) described (+)-(2S, 3R)-N-methyl-N-phenacyl-3-phenyl glycidyl acid amides cyclization under alkaline condition gets light (-)-Clausenamide ketone of living;

(6) light Clausenamide ketone alive can get the compound shown in the general formula (I) through sodium borohydride reduction,

It is characterized in that the chirality alcohol roh described in the step (1) is selected from (+)-menthol, (+)-8-phenyl menthol, (+)-8-betanaphthyl menthol.

2. the method for claim 1 is characterized in that, the used alkali of cyclization described in the step (5) is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, diisopropylamine lithium, butyllithium, tetraalkyl oxyammonia and trialkylamine.

3. the method for claim 1 is characterized in that, the used solvent of cyclization described in the step (5) is selected from water, methyl alcohol, ethanol or their mixture.

4. method for preparing compound shown in general formula (I),

May further comprise the steps:

(1) chloracetate and phenyl aldehyde reaction obtains racemization 3-phenyl glycidyl acid methyl esters;

(2) hydrolysis of racemization 3-phenyl glycidyl acid methyl esters get (+)-(2S, 3R)-3-phenyl glycidyl acid methyl esters;

(3) (+) that obtains-(2S, 3R)-3-phenyl glycidyl acid methyl esters and corresponding amine condensation get (+)-(2S, 3R)-N-methyl-N-(beta-hydroxy-styroyl)-3-phenyl glycidyl acid amides,

(4) (+) that obtains-(2S, 3R)-N-methyl-N-(beta-hydroxy-styroyl)-3-phenyl glycidyl acid amides through oxidation get (+)-(2S, 3R)-N-methyl-N-phenacyl-3-phenyl glycidyl acid amides;

It is characterized in that, the hydrolysis of the racemization 3-phenyl glycidyl acid methyl esters described in the step (2) gets (+)-(2S, 3R)-3-phenyl glycidyl acid methyl esters is the hydrolysis reaction that adopts lytic enzyme, wherein, in substratum, adopt have produce the lytic enzyme ability be selected from fungi, bacterium, yeast and actinomycetic microorganism in substratum, temperature is 10～50 ℃, aerobic fermentation carries out the stereo selective hydrolysis reaction to compound racemization-3-phenyl glycidyl acid methyl esters under between the pH value 5～8, the pH value of its hydrolysis reaction is between 5～10, and temperature is 10～50 ℃.

5. method as claimed in claim 4 is characterized in that, the used alkali of cyclization described in the step (5) is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, diisopropylamine lithium, butyllithium, tetraalkyl oxyammonia and trialkylamine.

6. method as claimed in claim 4 is characterized in that, the used solvent of cyclization described in the step (5) is water, methyl alcohol, ethanol or their mixture.

7. method as claimed in claim 4 is characterized in that, described fungi be selected from aspergillus (Aspergillus), Mucor (Mucor sp), aspergillus oryzae (Aspergillus, sp), mould (Penicillium) and head mold (Rhizopus sp).

8. method as claimed in claim 4, it is characterized in that, described bacterium is selected from achromobacter (Achromobacter.sp) genus, Alcaligenes (Alcaligenes.sp) genus, Arthrobacter (Archrobacter.sp) genus, genus bacillus (Bacillus sp) genus, tyrothricin (Breubacillus, sp) genus, excellent bacillus (Corynebacterium, sp) genus, Erwinia (Erwinia sp) genus and pseudomonas (Pseudomonas sp) genus.

9. method as claimed in claim 4 is characterized in that, described yeast is selected from candiyeast (Candida sp) and belongs to, finishes red (Pichia sp) genus, rhodotorula (Rhadatorulas sp) genus and Chinese sweet-smelling grass yeast (Hansenula sp) genus.

10. method as claimed in claim 4 is characterized in that, described actinomycetes are selected from promise Ka Shi (Nocardia sp) and the streptomycete (Streptomyces sp) in the actinomycetes.

11. method as claimed in claim 4, it is characterized in that described lytic enzyme be selected from aspergillus oryzae (Aspergillus, sp), Nocardia bacteria (Nocardia sp), candidiasis (Candida sp), achromobacter (Achromobacter.sp) or genus bacillus (Bacillussp).

12. method according to claim 11 is characterized in that, described temperature is 20～40 ℃.

13. method as claimed in claim 11 is characterized in that, described culture condition is to add tensio-active agent in substratum.

14. method as claimed in claim 13 is characterized in that, described tensio-active agent is selected from polyoxyethylene glycol, tween, polyvinyl alcohol and cetyl trimethylammonium bromide.

15. method as claimed in claim 14 is characterized in that, described surfactant concentrations is 0.05～5%.

16. method as claimed in claim 15 is characterized in that, described surfactant concentrations is 0.5～2%.

17. method as claimed in claim 4, its feature can be carried out in the two-phase in organic solvent and damping fluid at described hydrolysis reaction.

18. method as claimed in claim 17, its feature is selected from benzene,toluene,xylene, ether, ethyl acetate, isopropyl ether, tetracol phenixin and chloroform at described organic solvent.

19. method as claimed in claim 18, its feature is selected from toluene, dimethylbenzene and isopropyl ether at described organic solvent.

20. method as claimed in claim 17, its feature is selected from phosphate buffered saline buffer, borate buffer solution, carbonate buffer solution, citrate buffer solution, O-phthalic acid buffer, tartrate damping fluid, diethyl barbituric acid sodium damping fluid, 2 at described damping fluid, 4,6-trimethylpyridine-hydrochloride buffer, 2-amino-2-methylol-(1,3) propylene glycol-hydrochloride buffer, citric acid-Sodium phosphate dibasic damping fluid and N-ethylmorpholine-hydrochloride buffer.

21. method according to claim 4 is characterized in that, the 3-phenyl glycidyl acid methyl esters concentration of described racemization is 0.05～10%.

22. method according to claim 21 is characterized in that, the 3-phenyl glycidyl acid methyl esters concentration of described racemization is to be 0.5～5%.

23. the application of compound in the preparation medicine for senile dementia shown in general formula (I).