CN105622488B - A kind of Methoxycinnamate class compound of 4 cyclammonium alkoxy 3, preparation method and its usage - Google Patents

A kind of Methoxycinnamate class compound of 4 cyclammonium alkoxy 3, preparation method and its usage Download PDF

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CN105622488B
CN105622488B CN201610200276.3A CN201610200276A CN105622488B CN 105622488 B CN105622488 B CN 105622488B CN 201610200276 A CN201610200276 A CN 201610200276A CN 105622488 B CN105622488 B CN 105622488B
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acid
alkoxy
cyclammonium
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compound
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CN105622488A (en
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桑志培
柳文敏
于林涛
马勤阁
陈长中
潘万里
李涛
高利敏
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Nanyang Normal University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D211/20—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by singly bound oxygen or sulphur atoms
    • C07D211/22—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by singly bound oxygen or sulphur atoms by oxygen atoms

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Abstract

The present invention relates to a kind of Methoxycinnamate class compound of 4 cyclammonium alkoxy 3, preparation method and its usage, its preparation method, it is characterised in that：Comprise the following steps：The first step, using forulic acid as initiation material, under the conditions of the first solvent and acylating agent, obtain asafoetide acid chloride compound；Second step, asafoetide acid chloride compound react under the second solvent and the first alkalescence condition with alcohol or phenol, obtain corresponding Glehnilae；3rd step, Glehnilae, with 1 substitution 4 chlorine Alkylpiperidines reaction, generate the Methoxycinnamate class compound of 4 cyclammonium alkoxy 3 under the 3rd solvent and the second alkalescence condition.The compound of the present invention has good result, the including but not limited to nerve degenerative diseases such as vascular dementia, Alzheimer's, parkinsonism, Huntingdon disease, HIV related dementia disorders, multiple sclerosis, progressive lateral sclerosis of spinal cord, neuropathic pain, glaucoma for treating and/or preventing nervus retrogression relevant disease.

Description

A kind of 4- cyclammonium alkoxy -3- Methoxycinnamate classes compound, preparation method and Its purposes
Technical field
The present invention relates to one kind treatment and/or prevention nervus retrogression relevant disease medicine, and in particular to a kind of 4- cyclammonium Alkoxy -3- Methoxycinnamate classes compound, preparation method and its usage.
Background technology
Vascular dementia (Vascular Dementia, VD) is by various types of cranial vascular diseases (including ischemic brain Angiosis, hemorrhagic cerebrovaseular disease, acute and chronic Hypoxic cranial vascular disease etc.) caused by intelligence and cognition dysfunction Clinical syndrome, its main clinical manifestation includes：Cognitive ability, the decline of memory and social-life ability and emotion, The change of personality, it is a kind of chronic progressive disease.In the Asian countries such as China, Japan, vascular dementia is senile dementia First reason；As world population is to the continuous propulsion of aging, cerebrovascular disease is increasing, and Onset of Vascular Dementia rate has The trend being gradually increasing, has a strong impact on work and the quality of life of the elderly, and to society and family bring heavy economy and Mental burden.Therefore, VD turns into current gerontology and an important study hotspot in psychologic medicine field.Vascular is crazy about Stay because pathogenesis is complicated, there is no the medicine that disease can be blocked to develop, clinical treatment is circulated with improving brain blood at present It is metabolized, strengthens based on brain nutrition with brain.
In recent years, research shows both at home and abroad, and cholinergic is also often accompanied by while VD patient shows cerebral damage The exception of system.VD patient's hippocampus ChAT positive neurons and fibre density reduce, under the ChAT activity of intracerebral different parts Drop, the ACh concentration in VD Cerebrospinal Fluid in Patients be significantly lower than normal level, and the degree that reduces of its concentration with it is dull-witted serious Degree is proportionate；And cerebral ischemia can cause intracerebral acetylcholine esterase active to rise；Simultaneously it has also been found that acetylcholinesterase Inhibitor is such as：HuperzineA and Revastigmine can protect neure damage caused by ischemic, and brain can be promoted to lack Neurotrosis and the recovery of brain function after blood.This shows that acetylcholinesteraseinhibitors inhibitors can also be used for the treatment of vascular dementia.
Alzheimer's disease (Alzheimer ' s disease, AD) is the incidence of disease and fatal rate highest disease in the elderly One of disease.Alzheimer's disease international association (Alzheimer ' s disease International, ADI) issue《2015 Global Alzheimer's disease report》Point out, the whole world has had more than 46,000,000 people and suffers from dementia within 2015, it was predicted that to 2050 Year, the whole world will have 1.315 hundred million populations to be perplexed by dementia, wherein the incidence of disease of Chinese Dementia patients has reached 6.61%. With the extension of existent age per capita, this disease has developed into the main burden of society and medical health system, and for society, suffer from Person and family members bring heavy spirit and economic pressures.Thus, it is significant to research and develop new senile dementia medicine. From the point of view of the market demand, Alzheimer's disease international association prediction, to the global marketing of the year two thousand fifty curing senile dementia medicine Volume will be up to 600,000,000,000 dollars；In China, with the rapid rising of the senile dementia incidence of disease, the market of this kind of medicine is also quick swollen It is swollen.
AD is a kind of chronic, characterized by progressive memory and Cognitive function damage multi-pathogenesis, too many levels participates in Complicated nerve degenerative diseases, its key pathological feature are that beta amyloid peptide (β-amyloid peptide, A β) largely deposits The neurofibrillary tangles that senile plaque expelling (Senile plaque, SP), the Protein tau Hyperphosphorylationof of formation are formed (Neurofibrillary tangle, NFT), and degeneration of apoptosis and nerve synapse with neuron etc..In recent years, it is many Researcher is directed to disclosing AD pathogenesis from molecule and cellular level, it is proposed that a variety of hypothesis, such as：Cholinergic neuron Damage, the imbalance of the deposition of amyloid, Protein tau Hyperphosphorylationof, inflammation, free-radical oxidation, metal ion etc., therefore, For these pathogenesis come the novel therapeutic approach and means that develop, it will be hopeful to alleviate and improve AD patient the state of an illness.Mesh Preceding clinically effective treatment AD medicine mainly has two classes：(1) cognitive function is caused to lose based on neurotransmitter acetylcholine deficiency The cholinergic hypothesis of tune, patient's intracerebral levels of acetylcholine is improved using acetylcholinesteraseinhibitors inhibitors, such as：Tacrine、 Donepezil、Ravastigmine、Galantamine；(2) subtracted using N-methyl-D-aspartate (NMDA) acceptor inhibitor Few damage of the glutamate to nerve cell, such as：Memantine Hydrochloride.But there is action target spot in these medicines Single, the problems such as toxic side effect is more, not good enough to the long-term efficacy of AD patient.
Therefore, research and development with novel chemical structure, new mechanism of action, multiaction target spot, less toxic side effect it is anti- Neurodegenerative disease therapeutic agent not only conforms with the active demand of social senilization's process, and with before good market Scape.
The content of the invention
The invention provides a kind of 4- cyclammonium alkoxy -3- Methoxycinnamate classes compound, preparation method and its use On the way, develop it is a kind of treat and/or prevention nervus retrogression relevant disease medicine, including but not limited to vascular dementia, Ah Er Cihai Mo's diseases, parkinsonism, Huntingdon disease, HIV related dementia disorders, multiple sclerosis, progressive lateral spinal sclerosis The nerve degenerative diseases such as disease, neuropathic pain, glaucoma.
In order to solve the above technical problems, the technical solution adopted by the present invention is：
A kind of 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds, the chemical structure of general formula such as (I) of the compound It is shown：
In formula：M represents 1-12；
R1Represent C1~C12Alkyl, benzyl, substituted benzyl；
X represents C1~C12The benzene substituted on alkyl, benzyl or substituted benzyl, phenyl ring by the 1-4 groups being selected from the group Base：F、Cl、Br、I、C1-4Alkyl, C1-4Alkoxy, trifluoromethyl, trifluoromethoxy, nitro, cyano group, substituent can be in phenyl ring Any possible position.
Further, a preferred embodiment of the present invention is：R1Represent benzyl, 2- methoxy-benzyls, 3- methoxy-benzyls, 4- Methoxy-benzyl, 2- trifluoro-methoxybenzyls, 3- trifluoro-methoxybenzyls, 2- methyl-benzyls, 4- methyl-benzyls, 2- trifluoromethyls Benzyl, 3- trifluoromethyl benzyls, methyl or ethyl.
Present invention provides a kind of the pharmaceutically acceptable of 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds Salt, described pharmaceutically acceptable salt is 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds and hydrochloric acid, hydrogen bromine Acid, nitric acid, sulfuric acid, phosphoric acid, C1-6Aliphatic carboxylic acid, oxalic acid, benzoic acid, salicylic acid, maleic acid, fumaric acid, butanedioic acid, tartaric acid, Citric acid, C1-6The salt that the reaction of alkyl sulfonic acid, camphorsulfonic acid, benzene sulfonic acid or p-methyl benzenesulfonic acid is generated.
A kind of preparation method of 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds of the present invention, including it is as follows Step：
The first step, using forulic acid as initiation material, under the conditions of the first solvent and acylating agent, obtain forulic acid acyl chlorides chemical combination Thing；
Second step, asafoetide acid chloride compound react under the second solvent and the first alkalescence condition with alcohol or phenol, obtain phase The Glehnilae answered；
3rd step, Glehnilae under the 3rd solvent and the second alkalescence condition with 1- substitution -4- chlorine alkyl piperidines Pyridine is reacted, and generates 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds, and its reaction equation is as follows：
Further, a preferred embodiment of the present invention is：In the described first step：First solvent is：Tetrahydrofuran, N, N- Dimethylformamide, dimethyl sulfoxide (DMSO), C3-8Aliphatic ketone, benzene, toluene, acetonitrile or C1-8Alkane, preferred solvent be dichloromethane or Toluene；
Acylating agent is：Thionyl chloride or oxalyl chloride, preferably acylating agent are thionyl chloride；
Forulic acid：The molar feed ratio of acylating agent is 1.0：1.0~10.0, preferably molar feed ratio is 1.0：1.0~ 5.0；
Reaction temperature is 25~150 DEG C, and preferable reaction temperature is 25~100 DEG C；
Reaction time is 1~20 hour, and preferred reaction time is 1~10 hour.
Further, a preferred embodiment of the present invention is：In described second step：Alkali used in first alkalescence condition is：Alkali Metal or alkaline earth metal carbonate, alkali metal or alkali metal bicarbonates, C1-8The alkali metal salt of alcohol, triethylamine, tri-n-butylamine, One or more than one kinds of trioctylamine, pyridine, N-methylmorpholine, N- methyl piperidines, triethylene diamine and TBAH Mixture, preferably alkali be saleratus, sodium acid carbonate and triethylamine one or more than one kinds of mixture；
Second solvent is ether, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide (DMSO), dichloromethane, chloroform, C3-8 Aliphatic ketone, benzene, toluene, acetonitrile and C5-8The one or more kinds of mixture of alkane, preferably reaction dissolvent are dichloromethane, first Mixture one or more than one kinds of benzene, acetonitrile and chloroform；Asafoetide acid chloride compound：Alcohol or phenol：The molar feed ratio of alkali For 1.0:1.0~10.0:1.0~10.0, preferably molar feed ratio is 1.0:1.0~5.0:1.0~5.0；
Reaction temperature is 25~150 DEG C, and preferable reaction temperature is 25~100 DEG C；
Reaction time is 1~72 hour, and preferred reaction time is 1~24 hour.
Further, a preferred embodiment of the present invention is：In the 3rd described step, alkali used in the second alkalescence condition is：Alkali Earth metal hydroxide, alkali carbonate, alkaline earth metal carbonate, alkali metal hydrogencarbonate, alkali metal bicarbonates, C1-8The alkali metal salt of alcohol, triethylamine, tri-n-butylamine, trioctylamine, pyridine, N-methylmorpholine, N- methyl piperidines, triethylene diamine and Mixture one or more than one kinds of TBAH, preferably alkali are：Potassium carbonate, sodium carbonate, triethylamine and pyridine one Kind or more than one mixture；
3rd solvent is ether, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide (DMSO), dichloromethane, chloroform, C3-8 Aliphatic ketone, benzene, toluene, acetonitrile and C5-8One or more than one kinds of mixture in alkane, preferred solvent be toluene, acetone or Acetonitrile；
Glehnilae：1- substitution -4- chlorine Alkylpiperidines：The molar feed ratio of alkali is 1.0:1.0~10.0: 1.0~10.0, preferably molar feed ratio is 1.0:1.0~5.0:1.0~5.0；
Reaction temperature is 25~150 DEG C, and preferable reaction temperature is 25~100 DEG C；
Reaction time is 1~72 hour, and preferred reaction time is 1~24 hour.
A kind of pharmaceutically acceptable salt of 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds of the present invention Preparation method, comprise the following steps：
4- cyclammonium alkoxy -3- Methoxycinnamate class compounds and acetone are stirring evenly and then adding into acid, temperature rising reflux Stirring reaction 15-30 minutes, reaction are cooled to room temperature after terminating, and remove solvent, residue acetone recrystallization, filtering analysis under reduced pressure The solid gone out, produce the pharmaceutically acceptable salt of 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds.
The pharmaceutically acceptable salt of the 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds of the present invention is used to make Purposes in the standby medicine treated and/or prevent nervus retrogression relevant disease.
Further, a preferred embodiment of the present invention is：Described 4- cyclammonium alkoxy -3- Methoxycinnamate classes The pharmaceutically acceptable salt of compound accounts for gross weight than 10%~99.5% in medicine.
Invention also discloses the 4- cyclammonium alkoxy -3- methoxycinnamates that a kind of pharmaceutical composition includes therapeutically effective amount The pharmaceutically acceptable salt of acid esters compound, the pharmaceutical composition can be further containing one or more pharmaceutically acceptable Carrier or excipient." therapeutically effective amount " refers to cause the researcher or targeted tissue of doctor, system or animal The amount of the medicine or medicament of biology or medicine reaction；" composition " refer to by by more than one materials or component mix and Into product；" pharmaceutically acceptable carrier " refers to pharmaceutically acceptable material, composition or carrier, such as：Liquid Or solid-filling agent, diluent, excipient, solvent or packing material, they carry or transported certain chemical substance.Institute of the present invention Its preferable ratio of the pharmaceutical composition of offer is 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds or its pharmacy Upper acceptable salt accounts for gross weight than 10%~99.5% as active component, and remainder is to account for gross weight than less than 90%.
Beneficial effects of the present invention：
The compound of the present invention is respectively provided with the effect of significantly inhibiting to acetylcholinesterase, and its IC50 is 0.01 μM~5 μM, right The inhibitory activity of acetylcholinesterase is much higher than the inhibitory activity to butyrylcholine esterase, illustrates chemical combination disclosed in this invention Thing has certain selective inhibitory to acetylcholinesterase.
The compound of the present invention has significant protective effect to the PC12 cellular damages of hydrogen peroxide-induced, and 10- 5Antioxidation activity under mol/L concentration is better than forulic acid.
The compound of the present invention is to A β1-42The aggregation of auto-induction is respectively provided with the effect of significantly inhibiting, its IC50For 0.1 μM~ 20μM。
Compound disclosed in this invention causes mouse to obtain memory disorders to hyoscine has the improvement of dose dependent Effect, significant difference (p is relatively respectively provided with model group<0.01).
Compound disclosed in this invention is respectively provided with the effect of being obviously improved to ethanol induced mice memory representational role obstacle, There is significant difference (p compared with model group<0.01).
Embodiment
Below in conjunction with the specific embodiment of the invention, technical scheme is clearly and completely described, shown So, described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.Based on the reality in the present invention Example is applied, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made, is all belonged to In the scope of protection of the invention.
Embodiment 1-15
A kind of preparation method of 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds, comprises the following steps：
The first step, forulic acid, the first solvent, catalytic amount DMF and acylating agent, the addition of catalyst are added in reaction bulb For the 0.05% of forulic acid weight, temperature rising reflux stirring reaction, remove solvent under reduced pressure, obtain asafoetide acid chloride compound；
Second step, above-mentioned asafoetide acid chloride compound is all dissolved in the second solvent, added used in the first alkalescence condition Alkali and ethanol, stirring reaction, reaction process are tracked with TLC；After reaction terminates, remove solvent under reduced pressure, second is added in residue Solvent, washed successively with saturated aqueous sodium carbonate and saturated sodium-chloride water solution, organic layer mistake after anhydrous sodium sulfate drying Filter, removes solvent under reduced pressure, residue purifies (eluent through column chromatography：Chloroform), obtain corresponding Glehnilae；
3rd step, Glehnilae is dissolved in the 3rd solvent, adds alkali and 1- used in the second alkalescence condition (2 '-methoxyl group) benzyl -4- chloroethene phenylpiperidines, temperature rising reflux stirring reaction, reaction process are tracked with TLC, after reaction terminates, are taken advantage of Heat filtering, a small amount of acetone wash filter cake, and solvent is evaporated off in filtrate decompression, and residue purifies (eluent through column chromatography：Dichloromethane： Methanol=30：1v/v), corresponding 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds are obtained, its chemical constitution passes through1H-NMR,13C-NMR and ESI-MS confirmations.
Embodiment 1-14 concrete technology condition is shown in Table 1-4.
Embodiment 15
Substantially the same manner as Example 7, the difference of the first step is：Catalyst used is DMF, pyridine and tri-chlorination The mixture of aluminium, DMF additions are the 0.03% of forulic acid weight, pyridine addition is forulic acid weight 0.01%, trichlorine Change 0.005% that aluminium addition is forulic acid weight.The difference of other steps is shown in Table 1-4.
The first step process conditions of table 1 and result
Process conditions have serious influence to the yield of product as shown in Table 1, select suitable process conditions to product Yield has the important meaning.The first solvent uses dichloromethane as shown in Table 1：The volume ratio 2 of toluene:1, acylating agent uses two Chlorine sulfoxide, molar feed ratio 1:3, reaction temperature is 70 DEG C, under conditions of the reaction time is 6h, yield 86.8%, and yield It is very high.
From embodiment 15, under the conditions of same process, the selection of catalyst has material impact to yield, using urging Agent be DMF, pyridine and alchlor mixture, DMF additions are the 0.03% of forulic acid weight, pyridine addition be Ah The 0.01% of Wei's acid weight, under conditions of alchlor addition is the 0.005% of forulic acid weight, yield is up to 89.6%.
The second step process conditions of table 2 and result
Process conditions have serious influence to the yield of product as shown in Table 2, select suitable process conditions to product Yield has important influence.The second solvent uses dichloromethane as shown in Table 2：Toluene：Chloroform=2:1：2, the first alkaline bar Alkali used in part is saleratus：Pyridine is 2:1, molar feed ratio 1:3:2, reaction temperature is 70 DEG C, and the reaction time is 6h's Under the conditions of, yield 87.8%, yield highest, and change any of the above conditions and yield can all be had an immense impact on.
The three step process condition of table 3 and result
Process conditions have serious influence to the yield of product as shown in Table 3, select suitable process conditions to product Yield has important influence.The 3rd solvent uses dichloromethane as shown in Table 3：Toluene：Chloroform=2:1：2, the second alkaline bar Alkali used in part is potassium carbonate：Pyridine is 2:3, molar feed ratio 1:3:2, reaction temperature is 70 DEG C, and the reaction time is 6h bar Under part, yield 95.3%, yield highest, and change any of the above conditions and yield can all be had an immense impact on.
Embodiment 16
Concrete technology condition is same as Example 7, and for difference again with investigating different substituents, specific substituent is shown in Table 5, Gained 4- amine alkoxy -3- Methoxycinnamate class compounds, its chemical constitution pass through1H-NMR,13C-NMR and ESI-MS are true Card.
The different substituents of table 5 are tested
Embodiment 17
A kind of preparation side of the pharmaceutically acceptable salt of 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds Method, comprise the following steps：
Take the 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds in table 5 respectively and acetone stir after plus Enter acid, temperature rising reflux stirring reaction 15-30 minutes, reaction is cooled to room temperature after terminating, removes solvent, residue acetone under reduced pressure Recrystallization, filters the solid of precipitation, produces the pharmaceutically acceptable of 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds Salt, its chemical constitution warp1HNR and ESI-MS confirmations.
Embodiment 18
Bioactivity screening experiment is carried out using the product prepared by embodiment 7
(1) acetylcholinesterase and butyrylcholine esterase inhibitory activity
1.0mmol/L acetylthiocholine iodides are sequentially added into 96 orifice plates or thio BuCh (is purchased from Sigma Company) 30 μ L, pH7.4 the μ L of PBS 40, the μ L of testing compound solution 20 (DMSO contents be less than 1%) and 10 μ L acetyl Cholinesterase (rat brain cortex 5% is homogenized supernatant, and pH 7.4 phosphate buffer makees homogenate medium), after finishing mixing, 37 DEG C be incubated 15min, into each hole add mass fraction be 0.2% thio-bis- (2- nitros) benzoic acid of 5,5'- bis- (DTNB, purchase From Sigma companies) solution 30 μ L colour developing, with ELIASA measure 405nm at each hole optical density (OD values), with being not added with testing sample Blank well compare, calculate compound to the inhibiting rate of enzyme [enzyme inhibition rate=(1- sample sets OD values/blank group OD values) × 100%]；Select five to six concentration of compound, determine its enzyme inhibition rate, and with the negative logarithm of the compound molar concentration with The inhibiting rate linear regression of enzyme, molar concentration when trying to achieve 50% inhibiting rate are the IC of the compound50.Measurement result shows, Compound disclosed in the embodiment of the present invention is respectively provided with the effect of significantly inhibiting to acetylcholinesterase, its IC50For 0.01 μM~5 μM, and positive control medicine --- ICs of the Rivastigmine to acetylcholine ester enzyme level50For 6.3 μM；Measurement result also table Bright, the compound disclosed in this project embodiment is much higher than to butyrylcholine esterase to the inhibitory activity of acetylcholinesterase Inhibitory activity, illustrate that compound disclosed in this invention has certain selective inhibitory to acetylcholinesterase.
(2) compound is to H2O2The protective effect screening of the PC12 cellular damages of induction
DMEM nutrient solution of the PC12 cells containing 10% calf serum, with 1 × 105Individual/mL density is inoculated in the culture of 96 holes On plate, inoculation volume is 100mL/ holes, is subsequently placed into containing 5%CO237 DEG C of constant incubators in cultivate.After culture 24 hours, Add the compound (final concentration of 10 of respective concentration in administration group-5Mol/L, 10-6Mol/L) 10mL/ holes, preincubate 2 hours are (right Add 10 μ L/ hole PBS respectively with damage group according to group, its volume is kept equal).After PC12 cell incubations 2 hours, administration group with 100 μ Μ H are separately added into damage group2O2The μ L/ holes of agent 10 (control group adds 10 μ L/ hole PBS) are damaged, after 30 minutes, by each group The RPMI1640 nutrient solutions that nutrient solution changes no calf serum into continue to be put into constant incubator and cultivated 24 hours, cultivate liquid Product thinks 100 μ L/ holes.After continuing culture 24 hours, each group adds 5mg/mL, the μ L/ holes of MTT 100, carries out living cells dyeing.Treat The μ L/ holes of 100%DMSO terminate liquids 100 are added after 3 hours, in each group, fully dissolving mixes.Determined under 490nm wavelength each The OD values of group, test result is repeated 3 times, and with Duncan ' s test method statistics, each group numerical value is expressed as mean ± S.E.M., Using control group as 100%, administration group and damage class value are represented with the percentage of control group.Measurement result shows that the present invention is implemented Compound disclosed in example has significant protective effect to the PC12 cellular damages of hydrogen peroxide-induced, and 10-5Mol/L is dense Antioxidation activity under degree is better than forulic acid.
(3) compound suppresses A beta peptide aggregation determinations of activity
Take 20 μ L A β1-42The A β of the μ of solution+20 L testing compound solution, 20 μ L1-42The μ L PBSs of solution+20 The μ L PBSs (containing 25%DMSO) of (containing 2%DMSO), 20 μ L PBSs (containing 2%DMSO)+20 are in the orifice plate of black 96 In, compound and A β1-42Ultimate density be 25 μM.37 DEG C of incubation 24h, then add 160 μ L and contain 5 μM of thioflavine Ts 50mM glycine-NaOH buffer (pH=8.5), Varioskan Flash Multimode are used immediately after shaking 5s Reader (Thermo Scientific) multi-function microplate readers determine fluorescence under 446nm excitation wavelengths and 490nm launch wavelengths Value；Aβ1-42The fluorescent value of+testing compound is recorded as IFi, A β1-42The fluorescent value of+PBS is recorded as IFc, comprise only The fluorescent value of PBS is recorded as IF0, A β are suppressed by compound1-42The inhibiting rate calculation formula of self assemble is：100- (IFi-IF0)/(IFc-IF0)*100.Each compound two multiple holes of each concentration mensuration.Measurement result shows that the present invention is implemented Compound disclosed in example is to A β1-42The aggregation of auto-induction is respectively provided with the effect of significantly inhibiting, its IC50For 0.1 μM~20 μM, Inhibiting rate 65-90%, and positive control medicine --- curcumin and forulic acid are under 25 μM of concentration to A β1-42Auto-induction is assembled Inhibiting rate be respectively 56.2% and 28.3%.
(4) blood-brain barrier passes through merit rating (PAMPA-BBB)
Pig brain phosphatide (PBL, purchased from Avanti Polar Lipids, Inc.) is dissolved in (20mg/ in dodecane (Sigma) ML), 4 μ L are taken to be added dropwise on the lipophilicity filter membrane of receptor hole with Biomimetic membrane.350 μ L PBS/EtOH are added in receptor hole (70:30) buffer solution, added in donor hole 200 μ L samples liquid (compound, which is dissolved in DMSO, obtains 5mg/mL storing solutions, then With 50 times of PBS/EtOH (70:30) it is 100mg/mL that buffer solution, which is diluted to ultimate density,.96 hole filter plates are placed in PVDF receiver boards (Millipore) on, immobilized artificial membrane is just touched donor liquid, be thusly-formed the confession that sandwich structure-bottom is determinand Body fluid, centre are artificial phospholipid's films, and drug molecule to be measured spreads from donor hole, through immobilized artificial membrane, enter upper strata receptor hole In.25 DEG C of static 18h, after incubation, donor plate is gently removed, draws respectively by body fluid and donor liquid, uses Varioskan Flash Multimode Reader ELIASAs determine concentration, each hole of sample four, and independent test three times, tests 10 wavelength OD values under (250-450nm), effective transmissivity (P is drawn according to formulae).PAMPA effective permeabilities Pe(cm·s-1) calculate It is as follows：
Pe=-VdVa/[(Vd+Va)At]ln(1-drugacceptor/drugequilibrium)
Wherein, VdIt is the volume in donor hole, VaIt is the volume of receptor hole, A is the area of artificial phospholipid's film, when t represents infiltration Between, drug acceptor are the absorbances of donor boreliquid, and drug equilibrium are Theoretical Equilibrium absorbances, as a result with Mean value ± standard error (Stand error, S.E.) represents.Test result shows, presently disclosed under the experiment condition Compound be respectively provided with good blood-brain barrier and pass through ability, and be better than forulic acid.
(5) influence of the compound to hyoscine induced mice memory acquisition disturbance
SPF level ICR male mices, 25-30g, are randomly divided into：Normal group, model group, by reagent high and low dose group (5.0, 2.5mg/kg), every group of 10 animals.Disposable gavage gives test medicine, and blank group and model group give solvent 0.5%CMC- Na, administered volume are 0.1ml/10g；45min after medicine, normal group mouse peritoneal injection physiological saline, remaining each group animal are equal Hyoscine (5mg/kg) is injected, administered volume is 0.1ml/10g；After modeling 30min, mouse is put into non-electro photoluminescence Y fans Palace carries out Behavior test.Mouse is put in an arm end during test, allows it freely to walk 8min in labyrinth, records its entrance The number and alternate frequency of each arm, alternately rate is calculated according to below equation：Alternately rate %=[alternate frequency/(always enter indegree- 2)] × 100, as a result represented with mean ± standard deviation, group difference uses one-way analysis of variance.Measurement result shows, at this Under experiment condition, compound disclosed in this invention causes mouse to obtain memory disorders to hyoscine, and there is dose dependent to change Kind effect, significant difference (p is relatively respectively provided with model group<0.01).
Behavioral experiment finishes takes brain by mouse broken end immediately, with precooling normal saline flushing, is rapidly separated out on ice chest Cerebral hippocampal tissue, hippocampal tissue weight is weighed, add 9 times of 4 DEG C of physiological saline that 10% homogenate, 3500r.min is made-1, 4 DEG C of centrifugations 15min, -20 DEG C of storage supernatants are to be measured, and total protein concentration is determined by Coomassie brilliant blue.Exist according to method as defined in kit AChE contents are determined under 412nm wavelength, AChE vigor is expressed as U/mg.The ACh that ChAT vigor is catalyzed by ChAT is synthesized React to determine.Operating method is determined under 412nm wavelength also according to the explanation of kit, ChAT vigor with U/mg come Represent.Measurement result shows, under the experiment condition, compound disclosed in this invention can strengthen acetylcholine transferase (ChAT) vigor, significant difference (p is relatively respectively provided with blank group<0.01)
(6) influence of the compound to ethanol induced mice reproducibility dysmnesia
SPF level ICR male mices, 25-30g, are randomly divided into：Normal group, model group, by reagent high and low dose group (5.0, 2.5mg/kg), Rivastigmine group (3mg/kg), every group of 10 animals.Daily gavage is given to test medicine, blank group and model group Give solvent 0.5%CMC-Na, administered volume is 0.1ml/10g, successive administration 32 days；During administration 1~24 day, daily medicine 30min afterwards, model group and each administration group gavage 0.1ml/10g ethanol (15%w/v), successive administration 24 days, remove ethanol entrance Ethanol cleans the phase, and medicine continues to give；Carry out animal Jumping test within 31,32 days in administration, 45min is trained or surveyed after medicine Examination experiment, allows mouse to be placed in diving tower instrument during training, puts down gently in being powered on platform, when animal is under platform up-regulation with biped simultaneously Copper grid are contacted to get an electric shock, are considered as wrong reaction, the normal avoiding reaction after mouse is shocked by electricity is to escape onto platform, is recorded small Mouse escapes the incubation period to platform, and records electric shock number in 5min, in this, as school grade.Surveyed after 24 hours Examination, record mouse jump off the time (incubation period) shocked by electricity and its number (errors number) to be shocked by electricity in 5min for the first time, with This is as memory representational role evaluation index.Test result represents that group difference uses single factor test variance with mean ± standard deviation Analysis.Test result shows that compound disclosed in this invention is remembered to ethanol induced mice under the experiment condition reproduces work( Energy obstacle is respectively provided with the effect of being obviously improved, and has significant difference (p compared with model group<0.01).
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention God any modification, equivalent substitution and improvements made etc., should be included in the scope of the protection with principle.

Claims (7)

  1. A kind of 1. 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds, it is characterised in that：The chemical constitution of the compound Formula is such as shown in (I)：
    In formula：M represents 1-12；
    R1Represent C1~C12Alkyl, benzyl, substituted benzyl；
    X represents C1~C12The phenyl substituted on alkyl, benzyl or substituted benzyl, phenyl ring by the 1-4 groups being selected from the group：F、 Cl、Br、I、C1-4Alkyl, C1-4Alkoxy, trifluoromethyl, trifluoromethoxy, nitro, cyano group, substituent can be in any of phenyl ring Possible position.
  2. 2. a kind of 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds according to claim 1, its feature exist In：Described R1Represent benzyl, 2- methoxy-benzyls, 3- methoxy-benzyls, 4- methoxy-benzyls, 2- trifluoro-methoxybenzyls, 3- trifluoro-methoxybenzyls, 2- methyl-benzyls, 4- methyl-benzyls, 2- trifluoromethyl benzyls, 3- trifluoromethyl benzyls, methyl or second Base.
  3. 3. a kind of 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds as claimed in claim 1 can pharmaceutically connect The salt received, it is characterised in that：Described pharmaceutically acceptable salt is 4- cyclammonium alkoxy -3- Methoxycinnamate class chemical combination Thing and hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, C1-6Aliphatic carboxylic acid, oxalic acid, benzoic acid, salicylic acid, maleic acid, fumaric acid, Butanedioic acid, tartaric acid, citric acid, C1-6The salt that the reaction of alkyl sulfonic acid, camphorsulfonic acid, benzene sulfonic acid or p-methyl benzenesulfonic acid is generated.
  4. 4. a kind of preparation method of 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds as claimed in claim 1, its It is characterised by：Comprise the following steps：
    The first step, using forulic acid as initiation material, under the conditions of the first solvent and acylating agent, obtain asafoetide acid chloride compound；
    Second step, asafoetide acid chloride compound react under the second solvent and the first alkalescence condition with alcohol or phenol, obtain corresponding Glehnilae；
    3rd step, Glehnilae are anti-with 1- substitution -4- chlorine Alkylpiperidines under the 3rd solvent and the second alkalescence condition Should, generate 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds；
    In the described first step：First solvent is：Tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide (DMSO), C3-8Aliphatic ketone, Benzene, toluene, acetonitrile or C1-8Alkane；
    Acylating agent is：Thionyl chloride or oxalyl chloride；
    Forulic acid：The molar feed ratio of acylating agent is 1.0：1.0~10.0；
    Reaction temperature is 25~150 DEG C；
    Reaction time is 1~20 hour；
    In described second step：Alkali used in first alkalescence condition is：Alkali metal or alkaline earth metal carbonate, alkali metal or alkaline earth gold Belong to bicarbonate, C1-8The alkali metal salt of alcohol, triethylamine, tri-n-butylamine, trioctylamine, pyridine, N-methylmorpholine, N- methyl piperidines, three Mixture one or more than one kinds of ethylene diamine and TBAH；
    Second solvent is ether, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide (DMSO), dichloromethane, chloroform, C3-8Fat Ketone, benzene, toluene, acetonitrile and C5-8The one or more kinds of mixture of alkane；Asafoetide acid chloride compound：Alcohol or phenol：Alkali rubs Your rate of charge is 1.0:1.0~10.0:1.0~10.0；
    Reaction temperature is 25~150 DEG C；
    Reaction time is 1~72 hour；
    In the 3rd described step, alkali used in the second alkalescence condition is：Alkaline earth metal hydroxide, alkali carbonate, alkaline earth gold Belong to carbonate, alkali metal hydrogencarbonate, alkali metal bicarbonates, C1-8It is the alkali metal salt of alcohol, triethylamine, tri-n-butylamine, three pungent It is mixed one or more than one kinds of amine, pyridine, N-methylmorpholine, N- methyl piperidines, triethylene diamine and TBAH Compound；
    3rd solvent is ether, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide (DMSO), dichloromethane, chloroform, C3-8Fat Ketone, benzene, toluene, acetonitrile and C5-8One or more than one kinds of mixture in alkane；
    Glehnilae：1- substitution -4- chlorine Alkylpiperidines：The molar feed ratio of alkali is 1.0:1.0~10.0:1.0~ 10.0；
    Reaction temperature is 25~150 DEG C；
    Reaction time is 1~72 hour.
  5. 5. a kind of 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds as claimed in claim 3 can pharmaceutically connect The preparation method for the salt received, it is characterised in that comprise the following steps：
    4- cyclammonium alkoxy -3- Methoxycinnamate class compounds and acetone are stirring evenly and then adding into acid, temperature rising reflux stirring React 15-30 minutes, reaction is cooled to room temperature after terminating, and removes solvent under reduced pressure, residue acetone recrystallization, filters precipitation Solid, produce the pharmaceutically acceptable salt of 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds.
  6. 6. 4- cyclammonium alkoxy -3- Methoxycinnamate class compounds prepared by claim 5 is pharmaceutically acceptable Salt is used to prepare the purposes in the medicine for the treatment of and/or prevention nervus retrogression relevant disease.
  7. 7. purposes according to claim 6, it is characterised in that：Described 4- cyclammonium alkoxy -3- Methoxycinnamates The pharmaceutically acceptable salt of class compound accounts for gross weight than 10%~99.5% in medicine.
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