Background technique
Neurodegenerative disease refers to the disease general name generated by chronic progressive central nervous tissue retrograde degeneration,Including Alzheimer's disease (Alzheimer ' s disease, AD), Parkinson's disease (Parkinson ' s disease,PD), Huntington's disease (Huntington disease, HD), amyotrophic lateral sclerosis (Amyotrophic lateralSclerosis, ALS) and multiple sclerosis (Multiple sclerosis, MS) etc., pathogenesis and oxidative stress,It neuroinflamation and damages accordingly closely related.Oxidative stress is by active oxygen (Reactive oxygen species, ROS)Free radical mediated, including superoxide anion, hydrogen peroxide and hydroxyl radical free radical etc..Under normal physiological conditions, ROS generates waterIt is flat to be in dynamic balance state with antioxidant ability of organism, when the generation of ROS is more than that cellular anti-oxidant capacity can then aoxidizeStress (Oxidative stress), and brain is particularly sensitive to oxidative stress, to induce a variety of the nervous system diseases.Separately haveThe study found that vascular dementia, HIV associated dementia, neuropathic pain, glaucoma, cerebral arterial thrombosis, hemorrhagic apoplexyAnd neurotrosis caused by brain trauma etc. is also related to the oxidative stress of body and neuroinflamation.
Vascular dementia (Vascular Dementia, VD) is by various types of cranial vascular diseases (including ischemicCerebrovascular disease, hemorrhagic cerebrovaseular disease, acute and chronic Hypoxic cranial vascular disease etc.) caused by intelligence and cognitive function barrierThe clinical syndrome hindered.Vascular dementia there is no the drug that can block disease development due to pathogenesis complexity at present, clinicalTreatment is reinforced based on brain nutrition with improving brain blood circulation and brain metabolism.Recent studies indicate that being showed in VD patientAlso the exception of cholinergic system is often accompanied by while cerebral damage.VD patient's hippocampus ChAT positive neuron and fiberDensity lowers, and the ChAT activity decline of intracerebral different parts, the acetylcholine concentration in VD Cerebrospinal Fluid in Patients is significantly lower than justOrdinary water is flat, and the degree that its concentration reduces is positively correlated with dull-witted severity；And cerebral ischemia can lead to intracerebral acetylCholinesterase activity rises；Simultaneously it has also been found that some acetylcholinesterase inhibitors can protect the damage of neuron caused by ischemicWound, and the recovery of neurotrosis and brain function after cerebral ischemia can be promoted.
Alzheimer's disease (senile dementia) is a kind of maincenter based on progressive cognitive disorder and memory damageNervous system degenerative disease, disease incidence become the frequently-occurring disease for being only second to cardiovascular disease and cancer in trend is risen year by yearDisease, having gone up in developed countries such as America and Europes is the 4th of the cause of death.With the quickening of population in the world aging process, hairSick rate is in obvious the ascendant trend, " A Er announced according to Alzheimer's Disease International in December, 2013The global implication of Ci Haimo disease: 2013-2050 " report in point out, AD will become the coming few decades whole world face maximum healthChallenge, to the year two thousand thirty, patient numbers will rise to 76,000,000 by 44,000,000 in 2013, and to the year two thousand fifty, this numerical value is up toSurprising 1.35 hundred million.Since AD clinical manifestation is memory capability, capacity of orientation, thinking and judgement decline and daily lifeViability reduces, or even abnormal Behavioral and psychological symptom occur etc., keep patient care difficulty larger, is brought to society and family heavyBurden.The drug that approved is used to treat light/moderate AD at present has acetylcholinesterase (AChE) inhibitor, and is used for severeAD treatmentNMethyl-DAspartic acid (NMDA) receptor antagonist.But clinical use shows that these drugs can suffer from by improvingLevels of acetylcholine or the exitotoxicity of excitatory amino acid is inhibited to alleviate AD symptom in person's body, but not can effectively prevent orReverse the course of disease, but also can cause hallucinations, misunderstanding, dizziness, headache, nausea, hepatotoxicity, loss of appetite and stool frequencyThe serious toxic side effect such as numerous, thus long-term efficacy is not satisfactory.Therefore, clinically there is an urgent need to research and develop have both AD symptom improve andThe new A D therapeutic agent that the course of disease changes.
AD belongs to disease caused by many factors, and pathogenesis is complicated, and pathogenesis does not illustrate also completely so far.But it studiesShow patient's intracerebral levels of acetylcholine decline,βThe excessive of amyloid protein generates and the blood platelet in deposition, the cerebrovascularAggregation, metal ion metabolic disorder, Ca2+Dysequilibrium,tauNeurofibrillary tangles caused by protein hyperphosphorylation, glutamic acidReceptor active is excessively high, oxidative stress generates a large amount of active oxygens (ROS) and many factors such as free radical and Neuroinflammation existIt plays an important role in the pathogenic process of AD.For above-mentioned pathogenic factors, researcher is set using traditional " one target of a medicine " drugStratagem omit, it was found that largely to a certain target spot have high activity and highly selective drug, such as: anticholinesterase andNFirstBase-DAspartate receptor agonist etc..But that there are action target spots is single for these drugs, clinical use toxic side effect is more, rightNot the problems such as long-term efficacy of AD patient is not good enough.
In recent years, with constantly illustrating to AD pathogenesis, it is found that the occurrence and development of AD have multimachine system, multifactorIt is the characteristics of effect, again interrelated between different mechanisms to influence each other, constitute complicated network during AD occurrence and developmentRegulator control system.Obviously, it is current inevitable choice that research and development can act on the therapeutic agent of multiple links in AD pathologic process simultaneously.Based on the above results, researcher propose " multiple target point targeted drug " (Multitarget-directed Ligands,MTDLs) strategy researches and develops anti-neurodegenerative disease drug.So-called " multiple target point drug " refers to single chemical entities while acting onMultiple target spots in disease network can produce synergistic effect to the effect of each target spot, so that gross effect is greater than each single-action and the sum of answers,Such compound is also referred to as " Multifunctional " or " Multipotential " drug.Multiple target point drug is combined with multiple medicineUsing and the main distinction of compound medicine be: dosage can be reduced, therapeutic effect is improved, avoid the phase interaction between drugWith and thus bring toxic side effect, uniform pharmacokinetic properties, be easy to use etc..Therefore, research and development have novelChemical structure, novel mechanism of action, and with multiple target effect, less toxic side effect anti-neurodegenerative disease therapeutic agent notOnly meet the urgent need of social senilization's process, and there are good market prospects.A large amount of clinical researches are it has proven convenient that AChEAD patient symptom, short curative effect affirmative can be effectively relieved in inhibitor；Therefore, it is usually needed in the anti-AD drug of design multiple target pointRetain the AChE inhibitory activity (inhibiting the enzyme most important to AD patient symptom is improved) of compound, and increases by one on this basisIt is a or it is multiple have the function of pharmacology synergistic effect other targets or, to reach multiple target point AD therapeutic effect.Obviously, design is concurrentNow there is acetylcholine esterase inhibition simultaneously, inhibitβThe excessive generation of amyloid protein and deposition, anti-oxidation stress and anti-The multiple target point AD therapeutic agent of monoamine oxidase-B is still research direction important at present.
Summary of the invention
Present invention aims at open a kind of double Mannich alkaloid compounds of novel 3- methoxyl group -4-HC(I) and its pharmaceutically acceptable salt.
Another object of the present invention is to disclose the double Mannich alkaloid compounds (I) of such 3- methoxyl group -4-HCAnd its pharmaceutically acceptable salt preparation method.
Include the double Mannich bases chemical combination of such 3- methoxyl group -4-HC another object of the present invention is to openThe pharmaceutical composition of object (I) and its pharmaceutically acceptable salt.
Still a further object of the present invention is to disclose the double Mannich alkaloid compounds (I) of such 3- methoxyl group -4-HCAnd its pharmaceutically acceptable salt has multiple target effect, can be used for preparing treatment and/or prevention nervous system related disorder medicinePurposes in object, including but not limited to vascular dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, HIV phaseClose dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain, glaucoma, cerebral arterial thrombosis, hemorrhagicThe diseases such as neurotrosis caused by cerebral apoplexy and brain trauma.
The chemical structure of the double Mannich alkaloid compounds (I) of 3- methoxyl group -4-HC provided by the present invention is logicalFormula are as follows:
In formula: R1、R2、R3And R4Each independently represent C1~C12Alkyl, benzyl, substituted benzyl, NR1R2Or NR3R4Also illustrate that fourHydrogen pyrrole radicals, morpholinyl, piperidyl, 4- by C1~C12Piperidyl replaced alkyl, the 4- piperidyls replaced by benzyl,The thio piperidyl of 4-, piperazinyl, 4- by C1~C12Piperazinyl replaced alkyl, 4- replaced benzyl or substituted benzylPiperazinyl, any possible position of OH in molecule on B ring in corresponding phenyl ring；" substituted benzyl " refers on phenyl ring by 1-Benzyl replaced 4 groups selected from the group below: F, Cl, Br, I, C1-4Alkyl, C1-4Alkoxy, trifluoromethyl, trifluoro methoxyBase, dimethylamino, these substituent groups any possible position on the phenyl ring of benzyl.
The double Mannich alkaloid compounds (I) of 3- methoxyl group -4-HC proposed by the invention can be by with lower sectionMethod is prepared:
In formula: R1~R4And the definition of hydroxy position and the double Mannich bases chemical combination of 3- methoxyl group -4-HC on B ringThe general formula of the chemical structure of object (I) is identical.
With corresponding 3- methoxyl group -4- hydroxyl -5- amine methyl acetophenone class compound (1) and hydroxy benzaldehyde Mannich baseClass compound (2) is starting material, and the direct polycondensation under solvent and alkaline condition obtains corresponding 3- methoxyl group -4- hydroxyl Cha ErThe double Mannich alkaloid compounds (I) of ketone.Wherein, alkali used is reacted are as follows: alkali metal hydroxide, alkaline earth metal hydroxide, alkaliMetal carbonate, alkaline earth metal carbonate, alkali metal hydrogencarbonate, alkali metal bicarbonates, C1-8The alkali metal salt of alcohol hasMachine tertiary amines or quaternary ammonium bases (such as: triethylamine, tri-n-butylamine, trioctylamine, pyridine,NMethyl morpholine,NMethyl piperidine, triethyleneDiamines, tetrabutylammonium hydroxide), preferred alkali are as follows: potassium hydroxide, sodium hydroxide, potassium carbonate, triethylamine or pyridine；Used in reactionSolvent are as follows: C1-8Fatty alcohol, ether, tetrahydrofuran, 2- methyltetrahydrofuran,N,NDimethylformamide, dimethyl sulfoxide, twoChloromethanes, chloroform, Isosorbide-5-Nitrae-dioxane, benzene, toluene or acetonitrile, preferred solvent are as follows: methanol, ethyl alcohol, isopropanol,N,NDimethylFormamide, acetonitrile, tetrahydrofuran, methylene chloride or toluene；3- methoxyl group -4- hydroxyl -5- amine methyl acetophenone class compound(1): hydroxy benzaldehyde Mannich alkaloid compound (2): the molar feed ratio of alkali is 1.0:1.0 ~ 3.0:1.0 ~ 20.0, is preferably rubbedYour feed ratio is 1.0:1.0 ~ 2.0:1.0 ~ 10.0；Reaction temperature is 0 ~ 150 DEG C, and preferable reaction temperature is room temperature ~ 120 DEG C；InsteadIt is 1 ~ 120 hour between seasonable, preferred reaction time is 2 ~ 72 hours.
Starting material of the invention --- 3- methoxyl group -4- hydroxyl -5- amine methyl acetophenone class compound (1) and hydroxy benzenesFormaldehyde Mannich alkaloid compound (2) can be made with the common technology in this field, it may be assumed that with corresponding 3- methoxyl group -4- hydroxy benzenesEthyl ketone or hydroxy benzaldehyde compound are substrate, under acid catalysis with formaldehyde (or paraformaldehyde), corresponding secondary amine class chemical combinationObject is prepared through conventional Mannich reaction.
Contain in double Mannich alkaloid compound (I) molecules of 3- methoxyl group -4-HC of gained according to the method described aboveThere is amino, which can be made it by pharmaceutically conventional salifying method with any suitable acid and pharmaceutically may be used in alkalinityThe salt of receiving, the acid are as follows: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, amidosulfonic acid, C1-6Aliphatic carboxylic acid (such as: formic acid,Acetic acid, propionic acid etc.), trifluoroacetic acid, stearic acid, flutter acid, oxalic acid, benzoic acid, phenylacetic acid, salicylic acid, maleic acid, fumaric acid, amberAmber acid, tartaric acid, citric acid, malic acid, lactic acid, hydroxymaleic acid, pyruvic acid, glutamic acid, ascorbic acid, lipoic acid, C1-6AlkaneBase sulfonic acid (such as: methane sulfonic acid, ethylsulfonic acid), camphorsulfonic acid, naphthalene sulfonic acids, two sulphur of benzene sulfonic acid, p-methyl benzenesulfonic acid or 1,4- fourthAcid.
Pharmaceutical composition disclosed in this invention includes one or more 3- methoxyl group -4- hydroxyl Cha Er of therapeutically effective amountThe double Mannich alkaloid compounds (I) of ketone or its pharmaceutically acceptable salt, the pharmaceutical composition can be further containing a kind of or moreKind pharmaceutically acceptable carrier or excipient." therapeutically effective amount ", which refers to, causes researcher or the targeted group of doctorIt knits, the amount of the drug of the biology of system or animal or medicine reaction or medicament；" composition " refer to by by more than oneProduct made of substance or component mix；" pharmaceutically acceptable carrier " refers to pharmaceutically acceptable substance, combinationObject or carrier, such as: liquid or solid filler, diluent, excipient, solvent or packing substance, they carry or transport certainChemical substance.Its ideal ratio of pharmaceutical composition provided by the present invention is the double Mannich of 3- methoxyl group -4-HCAlkaloid compound (I) or its pharmaceutically acceptable salt account for total weight than 5%~99.5% as active constituent, and rest part is to account forTotal weight is than 95% or less.
The double Mannich alkaloid compound (I) of 3- methoxyl group -4-HC disclosed in this invention and its pharmaceutically may be usedThe salt of receiving has carried out following bioactivity screening.
(1) the double Mannich alkaloid compounds (I) of 3- methoxyl group -4-HC are to acetylcholinesterase and BuChThe inhibitory activity of esterase
1.0 mmol/L acetylthiocholine iodides are sequentially added into 96 orifice plates or iodine bisulfide (is purchased from for BuChSigma company) 1%) and 10 30 μ L, 40 μ L of PBS buffer solution of pH7.4,20 μ L(DMSO content of testing compound solution is less thanμ L acetylcholinesterase (rat brain cortex 5% is homogenized supernatant, and the phosphate buffer of pH7.4 makees homogenate medium) or BuChEsterase (25% supernatant of rat blood serum, pH7.4 phosphate buffer make homogenate medium) solution, after finishing mixing, 37 DEG C of incubations0.2% 5,5 '-two thio-bis- (2- nitrobenzoic acid) (DTNB is purchased from Sigma company) solution are added into each hole by 15min30 μ L colour developing, with the optical density (OD value) in each hole at microplate reader measurement 405nm, compared with the blank well that sample to be tested is not added, meterCompound is calculated to the inhibiting rate (enzyme inhibition rate (%)=(1- sample sets OD value/blank group OD value) × 100%) of enzyme；Select compoundFive to six concentration, measure its enzyme inhibition rate, and linearly return with the inhibiting rate of the negative logarithm of the compound molar concentration and enzymeReturn, molar concentration when acquiring 50% inhibiting rate is the IC of the compound50.Measurement result shows that institute is public in the embodiment of the present inventionThe double Mannich alkaloid compounds (I) of the 3- methoxyl group -4-HC opened, which all have acetylcholinesterase, significantly inhibits workWith IC50For 1.2 nM ~ 12.0 μM；And the double Mannich alkaloid compounds (I) of such 3- methoxyl group -4-HC are rightThe inhibitory activity of acetylcholinesterase is significantly higher than the inhibitory activity (selectivity is greater than 10 times or more) to butyrylcholine esterase, saysBright compound disclosed in this invention has selective inhibitory to acetylcholinesterase.Measurement result is also shown that 3- methoxyParent nucleus --- dyhydroxyl chalcone compound (that is: the CH of the double Mannich alkaloid compounds (I) of base -4-HC2NR1R2And CH2NR3R4Indicate that H, the OH of B ring are located at compound represented when any possible position of corresponding phenyl ring simultaneously) and hydroxylThe IC that benzaldehyde Mannich alkaloid compound (2) inhibits acetylcholinesterase50It is all larger than 100 μM.In further research,It was also found that in the double Mannich alkaloid compounds (I) of such 3- methoxyl group -4-HC, if by CH2NR1R2OrCH2NR3R4One of both is replaced with H, then respective compound significantly reduces the inhibitory activity of acetylcholinesterase and (at least dropsLow 3 times or more).
(2) antioxidant activity (side ORAC-FL of the double Mannich alkaloid compounds (I) of 3- methoxyl group -4-HCMethod)
Reference literature (Qiang, X.M.et al.Eur. J Med. Chem.Side 2014,76,314-331) reportedMethod is measured, it may be assumed that 6- hydroxyl -2,5,7,8- tetramethyl primary colours alkane -2- carboxylic acids (Trolox) be made into the PBS buffer solution of pH7.4The solution of 10-80 μm of ol/L, fluorescein (fluorescein) are made into the solution of 250 nmol/L with the PBS buffer solution of pH7.4,2,2 '-azo diisobutyl amidine dihydrochlorides (AAPH) use the preceding solution that 40 mmol/L are made into the PBS buffer solution of pH7.4.The compound solution and luciferin solution of 50-10 μm of ol/L are added into 96 orifice plates, mixes, 37 °C of incubation 15min, is addedAAPH solution makes every 200 μ L of hole total volume, mixes, is immediately placed on Varioskan Flash Multimode ReaderIn (Thermo Scientific) instrument, 90 min of METHOD FOR CONTINUOUS DETERMINATION under 485 nm excitation wavelengths and 535 nm launch wavelengths.It calculatesArea AUC under fluorescence decay curve out, wherein with 1-8 μm of ol/L'sTroloxAs standard, sample to be tested is not added as blank,The antioxidant activity results expression of compound isTroloxEquivalent, its calculation formula is [(AUC Sample-AUCblank)/(AUCTrolox-AUC blank)] ×[(concentration ofTrolox/concentration ofSample)], each compound measures 3 multiple holes every time, and every group of experiment is independent in triplicate.Measurement result shows present invention realityThe antioxidant activity for applying the double Mannich alkaloid compounds (I) of 3- methoxyl group -4-HC disclosed in example isTrolox1.6~4.0 times, illustrate such compound have strong anti-oxidative activity.
(3) the double Mannich alkaloid compounds (I) of 3- methoxyl group -4-HC are to Ab1-42The inhibition of self assemble is livingProperty
Reference literature (Qiang, X.M.et al.Eur. J Med. Chem.Side 2014,76,314-331) reportedMethod is measured, it may be assumed that pretreated Aβ1-42It is made into stock solution with DMSO, is diluted to using preceding with the PBS buffer solution of pH7.450μM；Untested compound is made into 2.5 mM stock solutions with DMSO, is diluted to respective concentration with the PBS buffer solution of pH7.4 using preceding,Take the A of 20 μ Lβ1-42The A of the testing compound solution of+20 μ L of solution, 20 μ Lβ1-42The PBS buffer solution of+20 μ L of solution (contains 2%DMSO) in 96 orifice plates, for 24 hours, the glycine-NaOH that the 50mM that 160 μ L contain 5 μM of thioflavine Ts is then added is slow for 37 °C of incubationsFliud flushing (pH=8.5) is measured under 446 nm excitation wavelengths and 490 nm launch wavelengths with multi-function microplate reader immediately after shaking 5sFluorescent value；Aβ1-42The fluorescent value of+untested compound is denoted as IFi, Aβ1-42The fluorescent value of+PBS buffer solution is denoted as IFc, contain onlyThe fluorescent value of PBS buffer solution is denoted as IF0, compound inhibition Aβ1-42The inhibiting rate of self assemble are as follows: 100- (IFi-IF0)/(IFc-IF0)*100；Five to six concentration for selecting compound, measure its inhibiting rate, each each concentration repetition measurement of compound three times, withCurcumin is positive control.Measurement result shows that the methoxyl group of the 3- disclosed in the embodiment of the present invention -4-HC is double gracefulBuddhist nun wishes alkaloid compound (I) to Aβ1-42Self assemble all has remarkable inhibiting activity, to A under 25.0 μM of concentrationβ1-42ItselfThe inhibiting rate of aggregation is all larger than 50.0%；And inhibiting rate of the curcumin under same concentrations is 41.3%, it is clinically widely usedAnti- AD drug: donepezil, Rivastigmine, memantine hydrochloride, compound (I) parent nucleus --- dyhydroxyl chalcone chemical combinationObject (that is: CH2NR1R2And CH2NR3R4Indicate that H, the OH of B ring are located at change represented when any possible position of corresponding phenyl ring simultaneouslyClose object) and hydroxy benzaldehyde Mannich alkaloid compound (2) under 25.0 μM of concentration to Aβ1-42The inhibiting rate of self assembleRespectively less than 15%.
(4) the double Mannich alkaloid compounds (I) of 3- methoxyl group -4-HC are living to the inhibition of monoamine oxidase A and BProperty
Recombined human MAO-A is made into 12.5 μ g/mL sample liquids with 7.4 kaliumphosphate buffer of pH of 100 mM, MAO-B is made into75 μ g/mL sample liquids.20 μ L of testing compound solution, 80 μ L of monoamine oxidase are added into 96 orifice plate of black, mixes, 37 °C is incubated for 15 min in the place of being protected from light, and 200 μM of Amplex Red reagents is added, 2U/mL horseradish peroxidase, 2 mM are to hydroxylPhenyl ethylamine (inhibiting MAO-A) or 2 mM benzene methanamines (inhibiting MAO-B) initiation reaction, 37 °C of 20 min of incubation, in multifunctional enzyme markOn instrument, to fix 545 nm of excitation wavelength, survey 590 nm and locate fluorescent emission intensity, with kaliumphosphate buffer instead of MAO-A orMAO-B is blank；The inhibiting rate calculation formula of compound inhibition monoamine oxidase are as follows: 100- (IFi)/(IFc) * 100, in formula,IFiAnd IFcRespectively there is the difference of the fluorescence intensity and blank fluorescence intensity under inhibitor and no inhibitor.Each compound is every3 multiple holes of secondary measurement, every group of experiment are independent in triplicate.Five to six concentration for selecting compound, measure its enzyme inhibition rate, andWith the inhibiting rate linear regression of the negative logarithm of the compound molar concentration and enzyme, molar concentration when acquiring 50% inhibiting rate isThe IC of the compound50.Measurement result shows the double Mannies of the methoxyl group of the 3- disclosed in the embodiment of the present invention -4-HCUncommon alkaloid compound (I) all has the effect of significantly inhibiting, IC to MAO-B50It is 0.05 μM ~ 15.0 μM；And MAO-A is inhibitedIC5030.0 μM are above, illustrates that compound disclosed in this invention has selective inhibitory to MAO-B.Test is also sent outIt is existing, in the double Mannich alkaloid compounds (I) of such chalcone, if by CH2NR1R2Or CH2NR3R4One of both is replaced with H, thenRespective compound significantly reduces (at least reduction 2 times or more) to the inhibitory activity of MAO-B；In addition, hydroxy benzaldehyde MannichThe IC that alkaloid compound (2) inhibits MAO-B50It is above 80.0 μM.
(5) the double Mannich alkaloid compounds (I) of 3- methoxyl group -4-HC obtain the memory of hyoscine induced miceObtain the influence of obstacle (by taking embodiment compound 2-3 as an example)
SPF grades of ICR male mices, 25-30g are randomly divided into: normal group, model group, positive controls, the high low dosage of test drugGroup (15.0mg/kg, 2.5mg/kg), every group of 10 animals.Test medicine is given in disposable stomach-filling, and blank group and model group are givenSolvent 0.5%CMC-Na, administered volume are 0.1ml/10g；45 min after medicine, it is normal to organize mouse peritoneal injecting normal saline,Remaining groups of animals injects hyoscine (5mg/kg), and administered volume is 0.1ml/10g；After 30 min of modeling, mouse is putEnter the labyrinth non-electro photoluminescence Y and carries out Behavior test.Mouse is put in an arm end when test, it is allowed to travel freely 8 in labyrinthMin records its number and alternate frequency for entering each arm, according to following formula calculating alternating rate: alternating rate %=[alternate frequency/(always entering number -2)] × 100, it is as a result indicated with mean ± standard deviation, group difference uses one-way analysis of variance.Measurement knotFruit shows that the double Mannich alkaloid compounds (I) of the 3- tested under the experiment condition methoxyl group -4-HC (are implementedExample compound 2-3) cause the acquired memory disorders of mouse that there is dose-dependent improvement result hyoscine, with model group ratioMore have statistical difference (p < 0.01), and activity be significantly higher than under same molar ratio clinical application object Rivastigmine (p <0.01).
Method is led in the preparation of the double Mannich alkaloid compounds (I) of 1 3- methoxyl group of embodiment -4-HC
The corresponding 3- methoxyl group -4- hydroxyl -5- amine methyl acetophenone class compound (1) of 2.0 mmol, 2.5 are added in reaction flaskThe corresponding hydroxy benzaldehyde Mannich alkaloid compound (2) of mmol and 30 ml ethyl alcohol are added dropwise to 30% KOH water after mixing evenly15.0 mmol of solution is stirred at room temperature reaction 2.0~48.0 hours (reaction process is tracked with TLC)；After reaction, it is cooled toRoom temperature adjusts reaction solution pH to highly acid with 10% aqueous hydrochloric acid solution, then with saturated sodium bicarbonate aqueous solution adjust reaction solution pH toAlkalescent removes ethyl alcohol under reduced pressure, and 80 mL deionized waters are added in residual solution, is extracted in three times with 240 mL methylene chloride, organicIt is laminated and after washed with saturated sodium-chloride water solution, filtered after being dried over anhydrous sodium sulfate, evaporating solvent under reduced pressure, residue is through columnChromatographic purifying (eluent: methylene chloride: acetone=6:1 v/v) obtains the double Mannich of corresponding 3- methoxyl group -4-HCAlkaloid compound (I), yield 30.0%-62.2%, chemical structure pass through1H-NMR、13C-NMR and ESI-MS confirmation；Gained meshThe purity for marking object is all larger than 96.5% through HPLC measurement.The object structure being prepared using above-mentioned logical method is as follows:
；
Part of compounds1H-NMR data are as follows:
1H NMR (CDCl3): 7.69 (d,J = 15.6 Hz, 1H), 7.51 (s, 1H), 7.49 (d,J =15.6 Hz, 1H), 7.38 (s, 1H), 7.11 (s, 1H), 7.01-6.97 (m, 2H), 3.94 (s, 3H),3.79 (s, 2H), 3.74 (s, 2H), 2.68-2.59 (m, 8H), 1.14-1.08 (m, 12H)；
1H NMR (CDCl3): 7.72 (d,J = 15.6 Hz, 1H), 7.54 (s, 1H), 7.51 (d,J =15.6 Hz, 1H), 7.40 (s, 1H), 7.15 (s, 1H), 7.04-6.99 (m, 2H), 3.96 (s, 3H),3.79 (s, 2H), 3.71 (s, 2H), 2.64 (brs, 8H), 1.69-1.66 (m, 8H), 1.51 (brs,4H)。