CN1245162C - Synergistic precursor medicine compound for treating Alzheimer disease - Google Patents

Abstract

The present invention relates to a synergistic precursor medicine compound or compound salt for treating Alzheimer's disease, a preparation method thereof, a medicine composition containing the compound or the compound salt, and an application of the compound or the compound salt in the preparation of medicines for treating Alzheimer's disease. More specifically, the compound is formed by coupling nicotinic acid and metrifonate through ester linkage, namely (1-dimethyl phosphoryl-2, 2, 2-trichloro)-ethyl-1-alcohol nicotinic acid ester. The compound or the compound salt of the present invention has favorable effect on treating Alzheimer's disease.

Description

The collaborative prodrug compound of treatment Alzheimer's disease

Invention field

The present invention relates to collaborative prodrug compound of Alzheimer's disease and preparation method thereof, also relate to the pharmaceutical composition and the application of described chemical compound in the medicine of preparation treatment Alzheimer's disease that contain described chemical compound.Specifically, described chemical compound is formed through the ester bond coupling by nicotinic acid and metrifonate, i.e. (1-solutions of dimethyl phosphoryl base-2,2,2-trichlorine)-ethyl-1-alcohol nicotinate (NMF).

Background of invention

Alzheimer's disease (Alzheimer ' s disease, be called for short AD) and be the main type of senile dementia, be a kind of central nervous system degenerative disease based on cognitive disorder of carrying out property and memory infringement, be attended by personality change and behavior disorder.Among the one's mid-60s old people sickness rate be more than 10%, 85 years old among the old people sickness rate be 50%.The present old people more than 60 years old of China has reached 1.3 hundred million, and AD patient is about 1,300 ten thousand, and quantity constantly rises with aging trend, and can be for classification, the negligible amounts of the medicine of clinical use, and curative effect is also not satisfactory.Along with progressively carrying out of family planning policy, a pair of middle aged Mr. and Mrs family will have four old men to look after and to support; And AD patient more needs others nurse and treatment than other Senile disease, and this huge pressure to family and society is conspicuous.

Because the cause of disease of Alzheimer's disease (AD) and pathomechanism still imperfectly understands and quite complicated, thereby the angle that can set about studying and therapeutic scheme are more, and wherein Metrifonate (metrifonate) is the more promising AChE inhibitor of a script by the exploitation of Bayer company.This medicine has the history of three more than ten years as schistosomicide and ancylostome medicine, side effect is slight, better tolerance, be listed in the essential drugs (essential drug) of WHO, it increases new indication as old medicine directly under the jurisdiction of a municipal government in the application of Alzheimer's disease curative always.This medicine has originally been finished the III clinical trial phase, show that its cognitive function and behavior disorder to the patient all has improvement, and submitted application for quotation to U.S. FDA and the EMEAD of European Union, but 20 many cases generation muscle weaknesses are arranged owing in follow-up III clinical trial phase in 1998, find to take among the 3000 routine patients of this product, though Bayer company has found reason and solution subsequently, the U.S. does not ratify yet.

Studies show that nicotinic acid deficiency is one of clinical common factor of alzheimer disease.Nicotinic acid is vitamin the most stable in the vitamin B complex, be converted into nicotiamide in vivo rapidly, become nadide and coenzyme II ingredient then, participate in the formation of the synthetic and energy-rich phosphate bond of zymolysis, lipid metabolism, acetone acid metabolism, the pentose of glucose in the body etc., has physiologically active widely, as effects such as blood vessel dilating, blood lipid regulation.Prolonged application nicotinic acid can reduce the incidence rate or the mortality rate of coronary heart disease, myocardial infarction and other cardiovascular and cerebrovascular diseases, but because the needed therapeutic dose of nicotinic acid big (3-6g/ days), and easily produce flush, pruritus, bring out Peptic Ulcers, increase the weight of side effect such as diabetes and gout, limited its clinical practice.

In order to strengthen the activity of medicine; reduce toxic and side effects; on the basis of adopting prodrug design principle and structure principle of hybridization; we have synthesized (1-solutions of dimethyl phosphoryl base-2; 2; the 2-trichlorine)-ethyl-1-alcohol nicotinate (NMF); it has comprised the whole chemical constitutions of two parent drug metrifonates and nicotinic acid; can be two independently parent drugs by enzyme hydrolysis in vivo; collaborative performance pharmacodynamics effect; thereby become a kind of collaborative prodrug (Mutual Prodrug), bring into play dual, collaborative pharmacological action and reach long-acting, slow release and reduce toxic purpose.

Summary of the invention

For this reason, an object of the present invention is to provide medical compounds or its salt of the treatment Alzheimer's disease shown in a kind of following formula I:

The molecular formula of The compounds of this invention: C ₁₀H ₁₀Cl ₃NO ₅P=362.5

The physicochemical property of The compounds of this invention: MP88.4-88.8 ℃; Water insoluble, be soluble in chloroform, ether, ethanol, acetone, ethyl acetate, diluted acid.

It is evident that, have the chirality carbon atom in the The compounds of this invention, therefore, have optical isomer.Raceme and optical isomer all are included among the scope of the present invention.Chemical compound of the present invention can be unformed powder or crystal form.

Salt of the present invention is the inorganic acid salt or the acylate of formula I chemical compound, for example includes but not limited to hydrochlorate, nitrate, sulfate, hydrobromate, phosphate, formates, acetate, benzoate, malate, maleate, citrate etc.Be preferably hydrochlorate, sulfate or citrate.

Another object of the present invention provides the synthetic method of described compound or its salt.Synthetic method of the present invention is diversified.In general, the method for carboxyl and hydroxy esterification all can be can be used for the synthetic of The compounds of this invention in this area.Specifically, the present invention is undertaken by the carboxyl in the nicotinic acid and the hydroxyl in the metrifonate are carried out esterification.Esterification process includes but not limited to the method or the mixed anhydride method of chloride method, active ester.Synthetic schemes exemplifies as follows:

Synthetic schemes 1: chloride method

Synthetic schemes 2,3: the method for active ester

Synthetic schemes 4: mixed anhydride method

The salt of The compounds of this invention is normally obtained by basic nitrogen in the The compounds of this invention and acid generation salt-forming reaction.Described acid can be to have tart any material usually.For example include but not limited to mineral acid example hydrochloric acid, nitric acid, sulphuric acid, hydrobromic acid, phosphoric acid etc., organic acid such as formic acid, acetic acid, benzoic acid, malic acid, maleic acid, citric acid etc.

Compound or its salt of the present invention can also be formed targeted drug by the chemical bond coupling with dihydropyridine or other drug molecule or zymolyte etc., as the brain targeted drug.

A further object of the present invention is a kind of pharmaceutical composition for the treatment of Alzheimer's disease, wherein contains the formula I compound or its salt of the present invention and the pharmaceutically suitable carrier for the treatment of effective dose.Described pharmaceutically suitable carrier be to The compounds of this invention or its salt do not have a negative impact at the operable any excipients of drug world, diluent or additive.The concrete dosage form that can prepare according to desired route of administration is selected the carrier that uses.Compound or its salt of the present invention can pass through oral or parenteral administration.Parenteral administration can comprise injection, suction, transdermal or rectally.The dosage form of described pharmaceutical composition includes but not limited to tablet, capsule, suspending agent, solution, liposome, microsphere or suppository etc.What described carrier can exemplify is adhesive, disintegrating agent, lubricant, isotonic agent, surfactant, antiseptic, sweeting agent, flavouring agent, coloring agent etc., for example starch, sucrose, sodium chloride, glucose, cyclodextrin, magnesium stearate, phospholipid, cholesterol, corn wet goods.The compounds of this invention or the content of its salt in compositions can change within a large range, as long as can reach the purpose of treatment and be convenient to the preparation of pharmaceutical preparation and patient's use.For example, calculate, contain The compounds of this invention or its salt of 0.1-99.9% by the weight of compositions.Preferred 1-99%.In addition, compositions of the present invention can also contain other can with other active component of The compounds of this invention or its salt administering drug combinations, for example help the active component such as the AChE inhibitor of Alzheimer's disease treatment.Special needs to be pointed out is that the physical combination product of nicotinic acid and metrifonate realizes that purpose of the present invention also is fully passable, also should be included within the scope of the present invention.The selection of the proportioning between two components also is conspicuous for a person skilled in the art, for example, calculates by weight, and nicotinic acid and metrifonate proportioning are 1: 20-20: 1.Described physical combination product can be formulated in combination together pharmaceutical preparation or respectively the preparation but medicine box packaging together.

Pharmaceutical composition of the present invention can prepare by conventional method, for example each component of compositions is mixed, and perhaps mixes in certain sequence according to this area conventional method to prepare.As when preparing tablet, active component and adhesive mixing granulation can be added disintegrating agent and lubricant and compacting in flakes.This is known to those skilled in the art.

Another object of the present invention provides the application of physical combination product in the medicine of preparation treatment Alzheimer's disease of formula I compound or its salt or nicotinic acid and metrifonate.As being confirmed in this description, compound or its salt of the present invention has the effect of good curing Alzheimer's disease.Route of administration is diversified, as oral, injection, suction etc.Dosage can change within a large range, and this depends on approach, patient's body situation, age, sex of administration etc.In general, the dosage of The compounds of this invention or its salt is 0.1-100mg/ days, kg body weight, preferred 1-50mg/ days, kg body weight, more preferably 10-30mg/ days, kg body weight.

Cell in vitro is learned experiment confirm NMF can significantly promote the former fetus nourishing Corium Mus layer neuronal activity of being commissioned to train, and increases neurocyte survival quantity, and nerve injury due to the alginic acid of antagonism excitatory amino acid people from sea.Animal test results shows in the body, and NMF can significantly improve the cognitive competence of the large and small Mus of memory cognitive disorder due to the scopolamine, and drug effect is better than metrifonate; NMF can obviously improve some aging index of mouse aging due to the D-galactose, makes it to return near normal level or no longer develops to old and feeble direction.In sum, NMF can be applicable to treat Alzheimer's disease, and drug effect is better than metrifonate, and toxic and side effects is little.

Specific embodiments

Write a Chinese character in simplified form note:

NMF:(1-solutions of dimethyl phosphoryl base-2,2, the 2-trichlorine)-ethyl-1-alcohol nicotinate

DCC: dicyclohexylcarbodiimide

DMF:N, N '-dimethyl formamide

TEA: triethylamine

CDI:1,1 '-carbonyl dimidazoles

The DMAP:4-dimethylamino naphthyridine

AD: Alzheimer's disease

Embodiment 1

Chloride method preparation (1-solutions of dimethyl phosphoryl base-2,2,2-trichlorine)-ethyl-1-alcohol nicotinate (NMF)

Under dry anhydrous condition, install drying device and gas absorbing device, the 8mL thionyl chloride is added in the reaction bulb, be cooled to about 0 ℃ with ice-water bath, stir, to wherein adding 0.8g (6.5mmol) nicotinic acid, and splash into 5-7 and drip dry DMF, oil bath slowly is warming up to about 77 ℃ and refluxes, and removes the excess chlorination sulfoxide behind the 3h under reduced pressure, gets nicotinoyl chlorine hydrochlorate (light yellow solid).Cryosel is bathed and add an amount of anhydrous methylene chloride down in exsiccant reaction bulb, strong agitation, successively drip the 20mL dichloromethane solution of anhydrous TEA of 10mL and 1.7g (6.6mmol) metrifonate, reaction 3h, filter, filtrate decompression concentrates the back and adds an amount of ethyl acetate, water, 0.1N HCl, saturated NaHCO successively ₃, saturated common salt washing 3 times; anhydrous magnesium sulfate drying; concentrating under reduced pressure; rapid column chromatography (silica gel H; cleaning mixture: petroleum ether-acetone), get (1-solutions of dimethyl phosphoryl base-2,2; the 2-trichlorine)-and ethyl-1-alcohol nicotinate 1.2g (light yellow solid), petroleum ether-acetone mixed solvent recrystallization gets colourless bulk crystals.Productive rate: 51.0%.

Embodiment 2

DCC/DMAP method (method of active ester) preparation NMF

2.46g (0.02mol) be dissolved among the exsiccant 120mL DMF under nicotinic acid and 5.15g (0.02mol) the metrifonate room temperature, add 0.30g (0.002mol) DMAP, stir.In 0-5 ℃ of 50mL DMF solution that drips 6.19g (0.03mol) DCC, stirred overnight at room temperature is filtered, and concentrating under reduced pressure adds an amount of ethyl acetate, filters, successively water, 0.1N HCl, saturated NaHCO ₃, saturated common salt washing 3 times; anhydrous magnesium sulfate drying; concentrating under reduced pressure; rapid column chromatography (silica gel H; cleaning mixture: petroleum ether-acetone), get (1-solutions of dimethyl phosphoryl base-2,2; the 2-trichlorine)-and ethyl-1-alcohol nicotinate 4.70g (light yellow solid), petroleum ether-acetone mixed solvent recrystallization gets colourless bulk crystals.Productive rate: 65.0%.

Embodiment 3

CDI/DMAP method (method of active ester) preparation NMF

0.62g (5mmol) nicotinic acid is dissolved in the exsiccant 25mL DMF solution, adds 0.81g (0.002mol) CDI and 0.08g (0.5mmol) DMAP fast, in 0-5 ℃ of stirring reaction 1h.Drip the 25mL DMF solution of 1.29g (5mmol) metrifonate, stirred overnight at room temperature, concentrating under reduced pressure adds an amount of ethyl acetate, filters, successively water, 0.1N HCl, saturated NaHCO ₃, saturated common salt washing 3 times; anhydrous magnesium sulfate drying; concentrating under reduced pressure; rapid column chromatography (silica gel H; cleaning mixture: petroleum ether-acetone), get (1-solutions of dimethyl phosphoryl base-2,2; the 2-trichlorine)-and ethyl-1-alcohol nicotinate 1.26g (light yellow solid), petroleum ether-acetone mixed solvent recrystallization gets colourless bulk crystals.Productive rate: 70.0%.

Embodiment 4

Mixed anhydride method prepares NMF

Cryosel is bathed down 1.0g (8.12mmol) nicotinic acid, an amount of dichloromethane and TEA is added reaction bulb successively, slowly drips 1.1mL (8.59mmol) anhydrous benzene sulfonic acid chloride, keeps 0 ℃ of stirring reaction 1h.Slowly drip the mixed solution that 2.50g (9.71mmol) metrifonate is dissolved in an amount of dichloromethane and TEA, stirring at room reaction 3h, concentrating under reduced pressure adds an amount of ethyl acetate, filters, successively water, 0.1N HCl, saturated NaHCO ₃, saturated common salt washing 3 times; anhydrous magnesium sulfate drying; concentrating under reduced pressure; rapid column chromatography (silica gel H; cleaning mixture: petroleum ether-acetone), get (1-solutions of dimethyl phosphoryl base-2,2; the 2-trichlorine) ethyl-1-alcohol nicotinate 2.2g (light yellow solid), petroleum ether-acetone mixed solvent recrystallization gets colourless bulk crystals.Productive rate: 74.4%.IR (KBr, ν): 3081,1745,1267,1049; ¹HNMR (CDCl ₃, δ): 9.31 (s, 1H, PyC ₂-H), 8.84 (d, 1H, PyC ₆-H), 8.38 (d, 1H, PyC ₄-H), 7.46 (t, 1H, PyC ₅-H), 6.12 (d, 1H, CH), 3.85 (q, 6H, OCH ₃); EI-MS (m/z): 362 (M ⁺). purity: 99.0% (HPLC mensuration).

Embodiment 5

NMF is to the influence of In vitro culture normal tire Corium Mus layer neuronal activity

Get conceived 15 days normal kunming mice, the dislocation of craning one is put to death, and with iodine tincture and 75% alcohol disinfecting, dissects in the aseptic condition lower leaf, takes out fetal mice, places the dissociation solution D of ice bath ₁-SGH (D ₁Be the PUCK liquid of no calcium ion, SGH is sucrose-glucose solution and HEPES buffer).Anatomical isolation goes out tire Mus cerebral cortex under the stereomicroscope, rejects blood vessel and tunicle, is cut into 1mm roughly ³Fragment, with 80mg/L Dnasel effect 10min, agglomerate is organized in the soft piping and druming of thin bore dropper of scraping with flame then, and it is fully disperseed, and leaves standstill the back and draws upper strata cell suspension.Centrifugal back is with containing the blood serum medium (DF that contains 10% hyclone ₁₂) cell density is transferred to is 1.0 * 10 ⁶Individual/mL, be inoculated in and wrap in advance by in 96 orifice plates of PLL, every hole 100 μ L, in 37 ℃, 5% CO ₂Cultivate in the incubator.Postvaccinal the 3rd day of neurocyte, every hole adds the DF of the NMF of 50 μ L variable concentrations ₁₂(final concentration is followed successively by 10 μ mol L to liquid ^-1, 1 μ mol L ^-1, 0.1 μ mol L ^-1, 10nmol L ^-1, 1nmol L ^-1, 0.1nmol L ^-1), matched group adds isopyknic DF ₁₂Continue to cultivate 3 days, every hole adds MTT (Sigma) 15 μ L (1.5g L ^-1), hatch 4h for 37 ℃, mirror is observed purple acicular crystal down, careful supernatant discarded, every hole adds DMSO 100 μ L, places 10min under the room temperature, after treating that purple crystal all dissolves under the mirror, measure the MTT absorbance value with Dynatech MR400EL ISA readout instrument, detect wavelength 570nm, reference wavelength 630nm.Contrast with parent drug nicotinic acid, metrifonate respectively with method NMF.The result all checks through SPSS 10.0 software One-wayANOVA (one factor analysis of variance).Compare with matched group, the NMF of each dosage group all can significantly promote normal tire Corium Mus layer neuronal activity, wherein 1 μ mol L ^-1, 0.1 μ mol L ^-1, 10nmol L ^-1NMF promote the active rate of increase respectively up to 34.7%, 37.4%, 36.7% (seeing Table 1).On the contrary, metrifonate suppresses normal mouse cortical neuron activity (seeing Table 2), and nicotinic acid does not then have influence (seeing Table 3) to normal tire Corium Mus layer neuronal activity.Above results suggest NMF can significantly promote it normally is the cortical neuron activity.

Table 1 NMF is to the influence of normal tire Corium Mus layer neuronal activity (n=10, x ± s)

Group	Absorbance (OD)	Active increment rate (%)
			10μmol·L -1	0.176±0.006 ***	19.7
1μmol·L -1	0.198±0.017 ***	34.7
			0.1μmol·L -1	0.202±0.014 ***	37.4
10nmol·L -1	0.201±0.014 ***	36.7
			1nmol·L -1	0.167±0.013 ***	13.6
0.1nmol·L -1	0.159±0.009 *	8.2
			10pmol·L -1	0.165±0.007 **	12.2
Contrast (DF 12)	0.147±0.008	0

^*P＜0.05vs contrast, ^*P＜0.01vs contrast, ^{* *}P＜0.001vs contrast

Table 2 metrifonate is to the influence of normal tire Corium Mus layer neuronal activity (n=6, x ± s)

Group	Absorbance (OD)
		10μmol·L -1	0.083±0.005 ***
1μmol·L -1	0.087±0.006 ***
		0.1μmol·L -1	0.089±0.010 ***
10nmol·L -1	0.101±0.006

Contrast (DF 12)

0.109±0.017

^{* *}P＜0.001vs contrast

Table 3 nicotinic acid is to the influence of normal tire Corium Mus layer neuronal activity (n=10, x ± s)

Group	Absorbance (OD)
		10μmol·L -1	0.119±0.025
1μmol·L -1	0.115±0.017
		0.1μmol·L -1	0.092±0.025 *
10nmol·L -1	0.106±0.025
		Contrast (DF 12)	0.112±0.013

^*P＜0.05vs contrast

Embodiment 6

NMF is to the influence of In vitro culture normal tire Corium Mus layer neuron survival rate

Normal tire Corium Mus layer neuron cultural method contains the blood serum medium (DF that contains 10% hyclone with embodiment 5 ₁₂) cell density is transferred to is 4.0 * 10 ⁴/ cm ², be inoculated in and wrap in advance by in 24 plates of PLL, every hole 0.5mL, in 37 ℃, 5% CO ₂Cultivate in the incubator.Postvaccinal the 2nd day of neurocyte, every hole adds the DF of cytosine arabinoside ₁₂(final concentration is 10 μ mol L to liquid ^-1) effect 24h, to suppress non-neuronal hyper-proliferative.The 3rd day every hole adds the DF of the NMF of 50 μ L variable concentrations ₁₂(final concentration is followed successively by 0.1 μ mol L to liquid ^-1, 10nmol L ^-1, 1nmol L ^-1), matched group adds isopyknic DF ₁₂Change liquid every day 1 time, drug effect concentration is the same.Be cultured to the 7th day, the culture fluid in each hole of sucking-off adds platform and expects orchid, carries out the survived neuronal counting under the phase contrast microscope, and 5 visuals field of meter, every hole amount to 6 holes.The result all checks through SPSS 10.0 software One-way ANOVA (one factor analysis of variance).NMF can significantly promote normal tire Corium Mus layer neuronal survival, compares 10nmol L with matched group ^-1, 1nmol L ^-1NMF the survival rate of cortical neuron is improved 39.3%, 73.5% (seeing Table 4) respectively.Above results suggest NMF can show and promote normal tire Corium Mus layer neuronal survival, obviously reduces cell mortality.

Table 4 NMF is to the influence of normal tire Corium Mus layer neuron survival rate (n=6, x ± s)

Group	Survival rate (%)	Survival increment rate (%)
			0.1μmol·L -1	25.56±5.48	0
10nmol·L -1	34.27±4.53 ***	39.3
			1nmol·L -1	42.69±2.48 ***	73.5
Contrast (DF 12)	24.61±3.86	0

^{* *}P＜0.001vs contrast

Embodiment 7

NMF is to the protective effect of neurovirulent cortical neuron due to extra large people's alginic acid

Normal tire Corium Mus layer neuron cultural method and NMF adding method thereof are with embodiment 6, and cortical neuron is cultured to the 7th day, and the culture fluid in each hole of sucking-off is used D ₁-SGH liquid is washed 2 times, adds the D of extra large people's alginic acid ₁(final concentration is 50 μ mol L to-SGH liquid ¹).Use the D of no extra large people's alginic acid behind the processing 30min ₁-SGH liquid is washed 3 times, reuse DF ₁₂Culture fluid is washed 1 time, adds DF in every hole ₁₂Culture fluid 0.5mL.The normal control group except that not adding extra large people's alginic acid, the complete same experimental group of treatment conditions.Through 18-24h, add platform and expect blue dyeing, carry out the survived neuronal counting under the phase contrast microscope, 5 visuals field of meter, every hole amount to 6 holes.The result all checks through SPSS 10.0 software One-way ANOVA (one factor analysis of variance).Phase contrast microscope is observed down, compares with the normal control group, and the cell diopter of extra large people's alginic acid group descends, granule increases, and occurs the buoyant dead cell that is refractivity successively, its projection of webbed neurocyte that the interweaves shape that becomes to break gradually, mortality rate also obviously raises, and shows 50 μ mol L ¹People from sea alginic acid has stronger neurotoxicity to the neuron of cultivating.NMF can significantly resist the nerve injury due to extra large people's alginic acid, compares 0.1 μ mol L with extra large people's alginic acid group ¹, 10nmol L ¹, 1nmol L ¹NMF to the damage cortical neuron protective rate be respectively 77.30%, 80.10%, 84.15% (seeing Table 5).

Table 5 NMF is to the influence of normal tire Corium Mus layer neuron survival rate (n=6, x ± s)

Group	Survival rate (%)	Protective rate (%)
			NMF(0.1μmol·L -1)	51.56±1.91 ***	77.30
NMF(10nmol·L -1)	52.37±1.51 ***	80.10
			NMF(1nmol·L -1)	53.55±0.88 ***	84.15
People from sea alginic acid (50 μ mol L -1)	29.08±0.34 ###	0
			Normal control group (DF 12)	40.00±1.35

^{* *}P＜0.001vs people from sea alginic acid; ###P＜0.001vs normal control group.

Embodiment 8

NMF is to the improvement effect of scopolamine induced mice learning memory disorder

Kunming mouse male and female half and half, 20min before training every day removes normal group intraperitoneal injection of saline and model group lumbar injection 3mg kg ^-1Beyond the scopolamine hydrobromide, all the other respectively organize mouse peritoneal injection 3mg kg ^-1In the time of scopolamine hydrobromide, NMF (2.8mg kg is injected in the abdominal cavity respectively ^-1, 14.1mgkg ^-1, 70.3mg kg ^-1) and metrifonate 50mg kg ^-1Every mice uses MORRIS water maze autographic apparatus (being provided by Beijing Jie Riou Science and Technology Ltd.) to position sail training twice every day, continuous 6 days, carries out space exploration and tests in the 7th day.Broken end was got brain in the 8th day, and the acetylcholine enzymatic activity is measured according to the description of acetylcholine enzyme reagent kit in the back of weighing, and protein quantification carries out with the Coomassie brilliant blue colorimetry.The result all carries out the multifactor analysis of variance of repeated measure through the Repeated measure of SPSS10.0 software General linear model, and wherein mice training in 6 days grand mean achievement is checked with One-way ANOVA (one factor analysis of variance).

As shown in table 6, compare 3mg kg with normal group ^-1The scopolamine model group has the preclinical trend of the mice orientation navigation of prolongation, and the swimming track illustrates that based on random coil shape blindly scopolamine causes mice space learning dysmnesia.Compare 70.3mg kg with model group ^-1NMF can significantly shorten the average latency of learning memory disorder mice due to the scopolamine, and seeks tactful purposiveness and obviously strengthen, and trend formula and orthoscopic ratio increase; Etc. the metrifonate of molar concentration DeGrain then, illustrate that NMF can significantly improve scopolamine induced mice orientation navigation obstacle, and usefulness is higher than metrifonate.

Table 6 NMF is to the preclinical influence of scopolamine induced mice orientation navigation (n=10, x ± s)

Group	First day (s)	Second day (s)	The 3rd day (s)	The 4th day (s)	The 5th day (s)	The 6th day (s)	Six days grand mean achievements (s)
								Normal group	60±0	45.6±12.4	50.8±14.2	42.9±18.9	41.0±19.6	32.5±19.3	45.4±7.0
Model group	60±0	55.7±7.5	57.8±7.1	42.8±13.8	50.2±13.5	36.6±20.2	50.5±5.3
								NMF 2.8 mg·kg -1	60±0	57.0±6.9	50.3±9.6	43.5±20.8	45.4±11.6	28.3±14.1	47.4±5.4
NMF 14.1 mg·kg -1	60±0	51.3±12.2	46.8±11.4 *	37.3±15.9	32.3±14.1 *	39.3±19.2	44.5±7.0
								NMF 70.3 mg·kg -1	60±0	43.8±19.6	43.8±12.7 *	41.5±19.2	37.0±17.0	22.1±9.9	41.3±9.6 **
Metrifonate 50mgkg -1	60±0	53.4±51.1	47.9±14.9	43.9±16.7	35.8±16.2 *	38.3±20.1	46.5±8.2

^*P＜0.05vs model group, ^*P＜0.001vs model group

As shown in table 7, compare 3mg kg with normal group ^-1The memory frequency of scopolamine model group has downward trend, illustrates that scopolamine causes mice space exploration ability obstacle.Compare 70.3mg kg with model group ^-1NMF can significantly improve the memory frequency of dysmnesia mice due to the scopolamine, and etc. the metrifonate of molar concentration DeGrain then, in addition, NMF does not have obvious influence to the mouse memory scoring average.Above results suggest explanation NMF can significantly improve scopolamine induced mice location and explore obstacle, and usefulness is higher than metrifonate.

Table 7 NMF is to the influence of scopolamine induced mice space exploration memory frequency and memory scoring average (n=10, x ± s)

Group	Average memory frequency (%)	Average memory scoring average (%)
			Normal group	19.1±5.6	11.8±4.5
Model group	16.0±6.0	9.9±3.9
			NMF 2.8mg·kg -1	15.9±5.0	8.1±3.9
NMF 14.1mg·kg -1	15.5±5.6	8.7±3.1
			NMF 70.3mg·kg -1	22.2±4.3 *	8.4±3.4
Metrifonate 50mgkg -1	16.3±7.0	10.0±4.7

^*P＜0.05vs model group

As shown in table 8, compare 3mg kg with normal group ^-1The scopolamine model group can significantly cause cerebral atrophy.Compare 70.3mg kg with model group ^-1The NMF group can significantly increase the brain coefficient; Etc. the metrifonate of molar concentration DeGrain then, illustrate that NMF can significantly resist scopolamine induced mice cerebral atrophy, and usefulness is higher than metrifonate.Compare 3mg kg with normal group ^-1The scopolamine model group can significantly increase the activity of acetylcholinesterase, causes dysmnesia.Compare 70.3mg kg with model group ^-1NMF group can acetylcholine esterase inhibition activity, etc. the degree of the metrifonate acetylcholine esterase inhibition of molar concentration is big, is useful to improving memory

Table 8 NMF is to the influence of scopolamine induced mice brain coefficient (n=10, x ± s)

Group	Brain coefficient (g/g)	Acetylcholinesterase (μ molmg -1)
			Normal group	0.0165±0.0010 *	2.52±1.13 ***
Model group	0.0151±0.0013	5.49±2.80
			NMF 2.8mg·kg -1	0.0156±0.0017	5.00±2.29
NMF 14.1mg·kg -1	0.0163±0.0017	2.22±0.79 ***
			NMF 70.3mg·kg -1	0.0170±0.0023 *	2.11±1.17 ***
Metrifonate 50mgkg -1	0.0156±0.0014	3.14±1.00 **

^*P＜0.05vs model group

Embodiment 9

NMF (lumbar injection) is to the improvement effect of SD learning and memory in rats obstacle due to the scopolamine

SD rat male and female half and half, experimental technique and dosage, method are with embodiment 8.By shown in the table 9, compare 3mg kg with normal group ^-1Scopolamine model group significant prolongation rat orientation navigation incubation period, and the swimming track illustrates that based on marginal mode at random blindly scopolamine has damaged rat space learning memory ability.Compare 2.8mg kg with model group ^-1NMF can significantly shorten the average latency of learning memory disorder rat due to the scopolamine, and seeks tactful purposiveness and obviously strengthen, and trend formula and orthoscopic ratio increase; Etc. the metrifonate of molar concentration DeGrain then, illustrate that NMF can significantly improve rat orientation navigation obstacle due to the scopolamine, and usefulness is much higher than metrifonate.

Table 9 NMF is to the preclinical influence of rat orientation navigation due to the scopolamine (n=10, x ± s)

Group	First day (s)	Second day (s)	The 3rd day (s)	The 4th day (s)	The 5th day (s)	Five days grand mean achievements (s)
							Normal group	79.5±30.9	48.0±35.6	31.6±21.1	29.7±17.4 **	20.4±13.1 ***	41.8±12.2 ***
Model group	95.8±41.3	79.9±38.2	61.3±49.0	70.7±44.7	70.8±38.4	75.7±31.7
							NMF 2.8 mg·kg -1	87.5±35.6	69.3±45.8	42.9±40.2	31.8±16.1 **	29.3±22.3 **	52.1±214 *
NMF 14.1 mg·kg -1	102.8±19.4	57.1±49.4 *	52.2±44.4	48.7±34.5	39.9±22.8 *	60.2±24.5
							NMF 70.3 mg·kg -1	114.1±12.9	89.2±40.2 *	57.3±25.6	29.4±21.4 **	52.8±41.3	68.5±19.8
Metrifonate 50mgkg -1	120.0±0	112.8±22.9	87.0±35.7	61.7±44.7	65.7±41.1	89.4±20.6

^*P＜0.05vs model group, ^{* *}P＜0.001vs model group

As shown in table 10, compare 3mg kg with normal group ^-1The memory branch rate of scopolamine model group has downward trend, illustrates that scopolamine has formed damage to mice space exploration ability.Compare 14.1mg kg with model group ^-1NMF can significantly improve the memory scoring average of rat, illustrate that the U.S. song of cigarette can significantly improve due to the scopolamine rat location and explore obstacle, and usefulness is much higher than metrifonate.

Table 10 NMF is to the influence of rat space exploration memory scoring average due to the scopolamine (n=10, x ± s)

Group	Average memory scoring average (%)
		Normal group	16.8±8.5 ***
Model group	4.1±4.3
		NMF 2.8mg·kg -1	5.0±4.3
NMF 14.1mg·kg -1	11.2±3.8 *
		NMF 70.3mg·kg -1	8.4±4.5
Metrifonate 50mgkg -1	7.7±6.1

^*P＜0.01vs model group, ^{* *}P＜0.001vs model group

Embodiment 10

NMF (filling stomach) is to the improvement effect of SD female rats learning memory disorder due to the scopolamine

Experimental technique and dosage are with embodiment 8.Medication is for irritating stomach.By shown in the table 11, with model group relatively, 14.1,70.3mg kg ^-1NMF all can significantly shorten the average latency of learning memory disorder SD female rats due to the scopolamine, seeks tactful purposiveness and obviously strengthens, and trend formula and orthoscopic ratio increase; Etc. the metrifonate of molar concentration DeGrain then, illustrate that NMF can significantly improve SD male and female rat orientation navigation obstacle due to the scopolamine, and usefulness is much higher than metrifonate.

Table 11 NMF is to the preclinical influence of SD female rats orientation navigation due to the scopolamine (n=10, x ± s)

Group	First day (s)	Second day (s)	The 3rd day (s)	The 4th day (s)	The 5th day (s)	The 6th day (s)	Six days grand mean achievements (s)
								Normal group	76.2±20.4	45.8±19.6 *	39.6±24.0 *	25.3±13.5 *	19.7±9.1 ***	18.0±11.4	45.4±7.0
Model group	120.0±0.0	87.9±32.2	84.4±36.8	85.2±36.5	103±31.7	80.0±38.4	50.5±5.3
								NMF 2.8 mg·kg -1	105.4±29.4	90.3±32.9	65.7±33.8	67.3±38.1	65.8±36.8 *	49.2±30.1	47.4±5.4
NMF 14.1 mg·kg -1	105.9±22.7	85.2±413	72.3±27.1	58.9±43.5 *	51.6±37.3 ***	45.1±39.1 *	44.5±7.0 **
								NMF 70.3 mg·kg -1	109.7±18.2	61.4±29.4	78.3±44.5	54.6±46.4 *	42.7±35.3 *	59.3±40.4	41.3±9.6 *
Metrifonate 50mgkg -1	104.3±34.2	87.3±45.7	114.4±12.0	77.4±44.4	49.0±48.1 **	50.2±46.5	46.5±8.2

^*P＜0.05vs model group, ^*P＜0.01vs model group, ^{* *}P＜0.001vs model group

As shown in table 12, compare with model group, NMF has the trend that improves SD female rats memory scoring average, but does not have statistical significance, and usefulness is higher than metrifonate.

Table 12 NMF is to the influence of SD female rats space exploration memory scoring average due to the scopolamine (n=8, x ± s)

Group	Average memory scoring average (%)
		Normal group	13.5±3.8 *
Model group	8.0±5.4
		NMF 2.8mg·kg -1	8.4±5.0

NMF 14.1mg·kg -1	8.7±3.6
		NMF 70.3mg·kg -1	8.9±4.5
Metrifonate 50mgkg -1	7.5±6.1

^*P＜0.01vs model group

Embodiment 11

NMF (filling stomach) is to the improvement effect of SD male rat learning memory disorder due to the scopolamine

Remove model group and administration group lumbar injection 7mg kg ^-1Beyond the scopolamine hydrobromide, all the other experimental techniques and dosage are with embodiment 8.Medication is for irritating stomach.By shown in the table 13, compare 70.3mg kg with model group ^-1NMF can significantly shorten the average latency of learning memory disorder SD male rat due to the scopolamine, seeks tactful purposiveness and obviously strengthens, and trend formula and orthoscopic ratio increase; Etc. the metrifonate of molar concentration DeGrain then, illustrate that NMF can significantly improve SD male rat orientation navigation obstacle due to the scopolamine, and usefulness is much higher than metrifonate.

Table 13 NMF is to the preclinical influence of SD male rat orientation navigation due to the scopolamine (n=10, x ± s)

Group	First day (s)	Second day (s)	The 3rd day (s)	The 4th day (s)	The 5th day (s)	The 6th day (s)	Six days grand mean achievements (s)
								Normal group	86.8±25.0 ***	44.7±32.2 ***	28.7±17.9 ***	35.0±26.6 ***	21.5±11.4 ***	17.0±11.6 ***	38.9±9.5 ***
Model group	115.2±15.3	101.2±22.1	78.4±47.6	84.9±39.7	74.1±38.7	82.2±44.4	89.3±29.5
								NMF 2.8 mg·kg -1	120±0	111.0±20.0	98.6±36.9	86.9±27.0	74.6±36.1	54.0±32.6	90.8±15.0
NMF 14.1 mg·kg -1	120±0	88.1±44.6	103.1±20.6	90.5±37.7	90.8±40.5	88.3±40.8	96.8±22.3
								NMF 70.3 mg·kg -1	97.1±19.0 **	93.8±36.8	67.7±40.6	55.0±36.1	46.2±37.1	22.1±9.9	66.8±22.0 *
Metrifonate 50mgkg -1	120±0	103.8±26.3	102.2±23.8	102.5±34.9	84.8±43.4 *	38.3±20.1	99.7±15.0

^*P＜0.05vs model group, ^{* *}P＜0.001vs model group

As shown in table 14, compare 70.3mgkg with model group ^-1NMF has the trend that improves SD male rat memory scoring average, but not statistically significant, usefulness is higher than metrifonate.

Table 14 NMF is to the influence of SD male rat space exploration memory scoring average due to the scopolamine (n=8, x ± s)

Group	Average memory scoring average (%)
		Normal group	35.1±12.2 *
Model group	18.7±19.6
		NMF 2.8mg·kg -1	16.1±10.6
NMF 14.1mg·kg -1	10.0±13.0
		NMF 70.3mg·kg -1	20.0±17.0
Metrifonate 50mgkg -1	10.4±11.6

^*P＜0.01vs model group

Embodiment 12

NMF is to the influence of subacute mouse aging aging index due to the D-galactose

Kunming mouse, male and female half and half are irritated stomach NMF (2.8mg kg every day respectively ^-1, 14.1mg kg ^-1, 70.3mg kg ^-1), SHUXUENING 100mg kg ^-1, each Mus lumbar injection every day D-galactose 80mg kg of while ^-1After the successive administration modeling 42 days, detect relevant aging index respectively.The result adopts the Repeated measure of SPSS 10.0 software General linear model to carry out the multifactor analysis of variance and One-way ANOVA (one factor analysis of variance) statistical disposition of repeated measure.

As shown in Table 15, NMF has that mouse aging reaches the preclinical trend of bank (table 15) due to the remarkable shortening D-galactose, and is similar to SHUXUENING.

Table 15 NMF is to the preclinical influence of subacute mouse aging orientation navigation due to the D-galactose (n=10, x ± s)

Group	First day (s)	Second day (s)	The 3rd day (s)	The 4th day (s)	The 5th day (s)	Five days grand mean achievements (s)
							Normal group	60.0±0	42.7±14.6	36.9±21.8	37.3±16.7	32.6±10.8	41.9±12.8
The D-galactose	60±0	52.8±15.6	48.6±10.7	473±18.0	41.6±17.9	50.1±12.4
							NMF 2.8mg·kg -1	60±0	50.6±10.5	47.6±15.0	47.3±14.1	38.3±21.0	48.8±12.1

NMF 14.1mg·kg -1	60±0	46.9±18.5	45.5±14.7	42.8±19.5	36.2±11.6	46.3±12.9
							NMF 70.3mg·kg -1	60±0	46.8±18.5	43.8±12.7	31.9±15.2	31.9±15.4	42.9±12.4
SHUXUENING 100mgkg -1	60±0	46.4±14.9	423±183	42.8±19.5	35.0±16.5	45.3±13.8

Shown in table 16, compare with normal physiological saline group, the body weight of model group mouse aging is obviously lighter, and hair is sparse, the color and luster dimness.Show that the D-galactose suppresses the body weight gain of aging model mice.NMF promotes the body weight gain of mouse aging, and hair color gloss is shinny, and wherein administration effect in 28 days is the most obvious.

Table 16 NMF is to the influence of subacute mouse aging body weight due to the D-galactose (n=10, x ± s)

Group	The 7th day (s)	The 14th day (s)	The 21st day (s)	The 28th day (s)	The 35th day (s)	The 42nd day (s)
							Normal group	31.0±5.7	32.0±4.3	34.8±3.3	35.6±4.4	37.1±4.8	37.5±3.6
Model group	26.5±4.7#	25.1±5.0##	30.6±3.2#	29.5±3.2##	32.1±4.6#	33.0±4.9#
							NMF 2.8mg·kg -1	26.9±3.9	29.4±3.8 *	31.1±2.6	33.2±3.3 *	35.4±3.3	34.9±4.0
NMF 14.1mg·kg -1	31.3±3.7 *	33.6±4.5 **	34.6±4.7 *	36.5±5.5 **	37.9±5.5 *	36.9±5.4
							NMF 70.3mg·kg -1	29.2±4.9	30.2±3.9 *	32.9±4.9	34.7±6.2 *	35.7±5.6	34.8±5.1
SHUXUENING 100mgkg -1	30.6±3.1 *	32.9±3.3	35.5±3.7 **	36.3±3.9 ***	38.3±5.1 *	37.8±4.7 *

##P＜0.01, #P＜0.05vs normal group

^{* *}P＜0.001, ^*P＜0.01, ^*P＜0.05vs model group

Shown in table 17, compare with normal physiological saline group, the thymus coefficient and the spleen coefficient of model group mouse aging all have obvious minimizing, show that atrophy appears in the immune internal organs of D-galactose aging model mice.NMF can significantly improve the thymic mouse and the spleen mice of mouse aging, shows good immunological enhancement.NMF does not have obvious influence to brain, liver, kidney mice.

Table 17 NMF is to the influence of subacute mice organ coefficient due to the D-galactose (n=10, x ± s)

Group	Brain (mgg -1)	Liver (mgg -1)	Kidney (mgg -1)	Thymus (mgg -1)	Spleen (mgg -1)
						Normal group	11.8±2.3	53.4±14.2	12.8±3.0	2.8±0.9##	4.0±1.4#
Model group	12.6±1.9	50.9±7.5	12.1±1.8	1.4±1.2	3.5±0.5
						NMF 2.8mg·kg -1	11.9±1.2	43.6±3.4	12.0±2.2	2.2±2.4	3.5±2.2
NMF 14.1mg·kg -1	12.0±2.0	46.8±3.0	12.7±3.0	24±0.4 *	4.5±1.0 **
						NMF 70.3mg·kg -1	12.2±1.8	48.2±2.5	13.8±1.8	2.4±0.7 *	4.1±0.5 *
SHUXUENING 100mgkg -1	11.3±2.0	53.6±3.4	13.9±2.8	2.8±0.8 **	4.3±0.8 *

##P＜0.01, #P＜0.05vs normal group

^{* *}P＜0.001, ^*P＜0.01, ^*P＜0.05vs model group

Shown in table 18, compare with normal physiological saline group, the content of the MDA of model mouse aging, MAO activity and lipofuscin level obviously raise, and the content of SOD level, liver GSH obviously reduces.NMF can significantly reduce serum malonaldehyde (MDA) content, cerebral tissue MAO activity and lipofuscin level, and improves serum superoxide dismutases (SOD) level, and the GSH level is had raising trend, shows good antioxidation.

Table 18 NMF is to the influence of subacute mouse aging biochemical indicator due to the D-galactose (n=10, x ± s)

Group	SOD (NU·mL -1)	MDA (nmol·mL -1)	GSH (mg·mgprot -1)	MAO (U·h -1·mgprot -1)	Lipofuscin (μ gg -1)
						Normal group	305.0±102.0#	28.3±19.1#	49.2±2.6#	6.1±3.9#	0.5±0.4#
Model group	169.4±102.3	47.2±21.8	37.7±4.5	10.3±4.3	2.4±1.8
						NMF 2.8mg·kg -1	218.3±55.7	37.2±11.3	38.4±3.2	8.3±5.8	2.0±2.0
NMF 14.1mg·kg -1	311.8±110.0 *	24.7±11.4 **	38.3±4.1	9.57±5.4	0.5±0.7 *
						NMF 70.3mg·kg -1	290.3±128.6	16.4±5.26 ***	40.3±17.1	5.4±3.3 *	0.3±0.3 *
SHUXUENING 100mgkg -1	335.2±137.5 *	25.9±19.4 *	38.0±7.2	1.8±0.7 *	0.4±0.4 *

#P＜0.05vs normal group, ^*P＜0.05vs model group

Mice long term injections D-galactose, it is disorderly to cause that general metabolism mixes, produce each organ dysfunction decline, show as hypomnesis, each immune organ is degenerated, because free radical is piled up, damaged membrane, MDA, MAO and lipofuscin level increase, and SOD, GSH level reduce, these variations are similar to aged mouse, and our experiment has also confirmed the above-mentioned pathological change of subacute aging model mice due to the D-galactose.Experimental result shows, NMF can significantly improve some aging index of mouse aging due to the D-galactose, make it to return near normal level or no longer old and feeble direction and develop, prompting NMF is in that to improve the body substance metabolism disorderly and delay aspect tool such as body aging and have certain effect.

Embodiment 13

Tablet (ten thousand amounts)

Component	Weight
		NMF	100g
Starch	700g
		Starch slurry (10%)	10g
Magnesium stearate	8g
		Total amount	818g

Preparation method: NMF is pulverized and crosses 100 mesh sieves, after weighing, starch mix homogeneously with crossing 100 mesh sieves adds 10% starch slurry and makes soft material, granulates with 16 order nylon mesh, wet grain is in 60 ℃ of dryings, dried granule and magnesium stearate mix homogeneously are again through 16 mesh sieve granulate, behind assay, the calculating slice is heavy, selects 6mm scrobicula or flat punch die tabletting promptly.

Embodiment 14

Capsule (ten thousand amounts)

Component	Weight
		NMF	100g
Starch	700g
		Starch slurry (10%)	10g
Total amount	810g

Preparation method: NMF is pulverized and crosses 100 mesh sieves, after weighing, starch mix homogeneously with crossing 100 mesh sieves adds 10% starch slurry and makes soft material, granulates with 16 order nylon mesh, wet grain is in 60 ℃ of dryings, dried granule is sub-packed in hard capsule case No. 3 again through 16 mesh sieve granulate, covers joint, flatten, polishing gets final product.

Embodiment 15

Injection

Component	Amount
		The hydrochlorate of NMF	0.5g
Sodium chloride	8.5g
		Water for injection	Add to 1000mL

Preparation method: take by weighing hydrochlorate, the sodium chloride of NMF respectively, be dissolved in an amount of water for injection, add adding to the full amount of water for injection, stir evenly, filter, the filtrate fill in ampoule, sealing by fusing, in 100 ℃ of flowing steam sterilization 30min promptly.

Claims (12)

1, the compound or its salt shown in the formula I:

2, the compound or its salt of claim 1 is its raceme.

3, the compound or its salt of claim 1 is its optical isomer.

4, claim 1,2 or 3 compound or its salt are unformed powder.

5, claim 1,2 or 3 compound or its salt are crystal forms.

6, claim 1,2 or 3 compound or its salt, described salt is the inorganic acid salt or the acylate of formula I chemical compound.

7, the compound or its salt of claim 6, described salt are hydrochlorate, nitrate, sulfate, hydrobromate, phosphate, formates, acetate, benzoate, malate, maleate or the citrates of formula I chemical compound.

8, the compound or its salt of claim 7, described salt are hydrochlorate, sulfate or the citrates of formula I chemical compound.

9, the synthetic method of the compound or its salt of claim 1, this method comprise carries out esterification with carboxyl in the nicotinic acid and the hydroxyl in the metrifonate, when needing and the pharmaceutically acceptable acid salify.

10, the method for claim 9, described esterification are to be undertaken by the method for chloride method, active ester or mixed anhydride method.

11, the application of the arbitrary described compound or its salt of claim 1-6 in the medicine of preparation treatment Alzheimer's disease.

12, the application of claim 11, described medicine are oral, injections, suck medicine.