A kind of apiolin-O- alkyl amine compound, preparation method and applicationTechnical field
The invention belongs to field of pharmaceutical chemistry technology, and in particular to a kind of apiolin-O- alkyl amine compound, preparation sideMethod and application.
Background technique
Alzheimer's disease (Alzheimer ' s disease, AD, senile dementia) is that a kind of recognized with progressive hindersHinder with memory damage based on central nervous system degenerative disease, with rapidly aging, the elderly population of population in the worldHealth problem have become current social significant problem in the urgent need to address.Alzheimer's disease (Alzheimer ' sDisease, AD) it is one of disease incidence and the highest disease of lethality in the elderly.Alzheimer's disease international association" 2015 global Alzheimer's diseases report " of (Alzheimer ' s disease International, ADI) publication point out,The whole world has had more than 46,000,000 people and suffers from dementia within 2015, it was predicted that the year two thousand fifty, the whole world will have 1.315 hundred million populations byDull-witted puzzlement, wherein the disease incidence of Chinese Dementia patients has reached 6.61%.With the extension of existent age per capita, this diseaseHave developed into the main burden of society and medical health system, and for society, patient and family members bring heavy spirit andEconomic pressures.The drug that approved is used to treat light/moderate AD at present has acetylcholinesterase (AChE) inhibitor, and is used forN-methyl-D-aspartate (NMDA) receptor antagonist of severe AD treatment, but clinical use shows that these drugs can be by mentioningHigh patient's body levels of acetylcholine inhibits the exitotoxicity of excitatory amino acid to alleviate AD symptom, but cannot effectively hinderOnly or the course of disease is reversed, but also can caused hallucinations, misunderstanding, dizziness, headache, nausea, hepatotoxicity, loss of appetite and bigJust the serious toxic side effect such as frequently, thus long-term efficacy is not satisfactory.Therefore, clinically there is an urgent need to research and develop with novel effectThe AD therapeutic agent of mechanism.
AD belongs to disease caused by many factors, and pathogenesis is complicated, does not illustrate its pathogenesis completely also so far, but studyShow the decline of patient's intracerebral levels of acetylcholine, excessive generation and the deposition, the excessive phosphoric acid of tau- albumen of beta-amyloid proteinNeurofibrillary tangles caused by changing, oxidative stress generate a large amount of active oxygen (ROS) and free radical and neural confirmatory reaction etc.Many factors are played an important role in the pathogenic process of AD.For above-mentioned pathogenic factors, researcher is using a traditional " medicine oneTarget " drug design strategies, it was found that largely there is high activity and highly selective drug to a certain target spot.Such as: cholinesterase suppressionPreparation and N-methyl-D-aspartate receptor antagonist etc., but that there are action target spots is single for these drugs.Clinical use poison is secondary to be madeWith the problems such as more, not good enough to the long-term efficacy of AD patient.
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.Based on the above results, researcher proposes " multiple target point targeted drug " (Multitarget-directedLigands, MTDLs) strategy researches and develops anti-neurodegenerative disease drug.So-called " multiple target point targeted drug " refers to single chemistryEntity acts on multiple target spots in disease network simultaneously, can produce synergistic effect to the effect of each target spot, is greater than gross effectEach single-action the sum of is answered, and such medicine is also referred to as " Multifunctional " or " Multipotential " drug.Multiple target point drug withMultiple medicine use in conjunction and the main distinction of compound medicine are: can reduce dosage, improve therapeutic effect, avoid between drugInteraction and thus bring side effect, uniform pharmacokinetic properties, be easy to use etc..Therefore, research and development have newType chemical structure, novel mechanism of action, and the anti-Alzheimer disease drug with multiple target effect, less toxic side effect not only accords withThe urgent need of social senilization's process is closed, and there are good market prospects.Up to the present, multiple target point inhibitor attractsThe concerns of more researchers.Although the advantage of multiple target point is that clearly, how multiple target spot functions are at same pointIt is combined in son, and how to select most suitable therapy target is still a key point.
Summary of the invention
To overcome drawbacks described above, the first object of the present invention is to provide a kind of apiolin-O- alkyl amine compound.
The second object of the present invention is to provide a kind of preparation method of apiolin-O- alkyl amine compound.
The third object of the present invention, which also resides in, to be provided a kind of apiolin-O- alkyl amine compound and moves back in preparation treatment nerveApplication in row disease medicament.
The fourth object of the present invention, which also resides in, provides a kind of pharmaceutical composition for treating neurodegenerative disease, including celeryThe pharmaceutically acceptable salt of element-O- alkyl amine compound or itself and acid synthesis.
A kind of apiolin-O- alkyl amine compound, general formula of the chemical structure are as follows:
Wherein: n is 2~12；
R1Indicate H or C1~C12Alkyl；R2Indicate C1~C12Alkyl, benzyl, substituted benzyl, 1,2,3,4- tetrahydro acridine-Chloro- -9 base of 1,2,3,4- tetrahydro acridine of the chloro- 1,2,3,4- tetrahydro acridine -9- base of 9- base, 6-, 8- or 6,8- bis- chloro- 1,2,3,4- tetrahydro acridine -9- base；
Or the NR1R2Indicate N- demethylation-galanthamine base, nafoxidine base, morpholinyl, piperidyl, 4- quiltsC1~C12Piperidyl replaced alkyl, the 4- piperidyls replaced benzyl or substituted benzyl, piperazinyl, 4- by C1~C12Piperazinyl replaced alkyl, the 4- piperazinyls replaced benzyl or substituted benzyl；1,2,3,4- tetrahydro isoquinolyl,Any position is by C1~C121,2,3,4- tetrahydro isoquinolyl or any position replaced alkyl are by benzyl or substituted benzylReplaced 1,2,3,4- tetrahydroisoquinoline；
Or O (the CH2)nNR1R2It indicatesM indicates 0-10, R3Indicate H, C1~C12Alkyl, benzyl or substituted benzyl.
Preferably, the substituted benzyl is F, Cl, Br, I, C1-4Alkyl, C1-4Alkoxy, trifluoromethyl, trifluoro methoxyAny one in base, dimethylamino, nitro and cyano, two kinds, three kinds or four kinds of groups are in the nuclear substituted benzyl of benzene.
A kind of preparation method of apiolin-O- alkyl amine compound, comprising the following steps:
(1) with apiolin and two bromoalkane Br (CH2)nBr is starting material, anti-under the first solvent and the first alkaline conditionIt answers, obtains 2,7- dibromo alkoxy apiolin；
(2) 2,7- dibromo alkoxy apiolin under the second solvent and the second alkaline condition from different secondary amine NR1R2Reaction isObtain target compound apiolin-O- alkyl amine compound.
It chemically reacts general formula are as follows:
Wherein: n, R1、R2Definition it is identical as the general formula of the chemical structure of apiolin-O- alkyl amine compound (I).
Preferably, first alkaline condition or the second alkaline condition are alkaline earth metal hydroxide, alkali gold with alkaliBelong to carbonate, alkaline earth metal carbonate, alkali metal hydrogencarbonate, alkali metal bicarbonates, C1-8It is the alkali metal salt of alcohol, organicIt is one or more of in tertiary amines and quaternary amine bases.
Preferably, first solvent or the second solvent are ether, tetrahydrofuran, n,N-Dimethylformamide, twoMethyl sulfoxide, methylene chloride, chloroform, C3-8Aliphatic ketone, benzene, toluene, acetonitrile and C5-8One or more of alkane.
Preferably, apiolin described in step (1): two bromoalkane Br (CH2)nBr: the molar ratio feed ratio of alkali is 1:2~20:2~20；Reaction temperature is 25~150 DEG C, and the reaction time is 10~48h.
Preferably, 2,7- dibromo alkoxy apiolin described in step (2): secondary amine NR1R2: the molar feed ratio of alkali is1:1~10:1~10；Reaction temperature is 25~150 DEG C, and the reaction time is 10~48h.
A kind of application of apiolin-O- alkyl amine compound in preparation treatment neurodegenerative disease drug.
Preferably, the neurodegenerative disease be Alzheimer's disease, vascular dementia, parkinsonism, Huntingdon disease,HIV related dementia disorders, multiple sclerosis, progressive lateral sclerosis of spinal cord, neuropathic pain or glaucoma neurologicalProperty disease.
A kind of pharmaceutical composition for treating neurodegenerative disease, including apiolin-O- alkyl amine compound or itsWith the pharmaceutically acceptable salt of acid synthesis.
Pharmaceutical composition disclosed in this invention includes one or more apiolin-O- alkyl amines of therapeutically effective amountObject (I) or its pharmaceutically acceptable salt are closed, which can be further containing one or more pharmaceutically acceptableCarrier or excipient." therapeutically effective amount ", which refers to, causes researcher or targeted tissue, system or the life of animal of doctorObject or the drug or dosage of medicine reaction；" composition " refers to the production by mixing more than one substances or componentProduct；" pharmaceutically acceptable carrier " refers to pharmaceutically acceptable substance, composition or carrier, such as: liquid or solidFiller, diluent, excipient, solvent or packing substance etc.；Its ideal ratio of pharmaceutical composition provided by the present invention isApiolin-O- alkyl amine compound (I) or its pharmaceutically acceptable salt as active constituent account for total weight ratio be 2%~99.0%.
Preferably, the acid is hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, C1-6Aliphatic carboxylic acid, oxalic acid, benzoic acid,Salicylic acid, maleic acid, fumaric acid, succinic acid, tartaric acid, citric acid, C1-6Alkyl sulfonic acid, camphorsulfonic acid, benzene sulfonic acid or to firstBenzene sulfonic acid.
Positive beneficial effect of the invention:
1. apiolin-O- alkyl amine compound of the present invention introduces amine alkoxy fragments on apiolin skeleton, designAnd it finds while having stronger anti-acetylcholinesterase and butyrylcholine esterase, anti-oxidation stress, complexing of metal ion, inhibit A βIt is excessive generate the multiple target point AD therapeutic agent balanced with deposition and activity, can be used for preparing treatment and/or prevent alzheimer 'Silent disease, vascular dementia, parkinsonism, Huntingdon disease, HIV related dementia disorders, multiple sclerosis, progressive lateral spinal sclerosisThe neurodegenerative diseases such as disease, neuropathic pain or glaucoma.
2. apiolin-O- alkyl amine compound of the present invention is to acetylcholinesterase and butyrylcholine esterase IC50Respectively0.002-10.8 μM, 0.003-4.3 μM, inhibitory activity is significant；Apiolin-O- alkyl amine compound of the present invention is dense at 25.0 μMTo A β under degree1-42The inhibiting rate IC of self assemble50Much smaller than curcumin, 70% or more inhibiting rate, there is remarkable inhibiting activity；This hairThe antioxidant activity of bright disclosed apiolin-O- alkyl amine compound is 1.2-1.8 times of Trolox, illustrates chemical combination of the present inventionObject has strong anti-oxidative activity；Apiolin-O- alkyl amine compound of the present invention is 10-5Mol/L~10-6It is right under mol/L concentrationThe PC12 cellular damage of hydrogen peroxide-induced has significant protective effect；Apiolin-O- alkyl amine compound of the present invention is equalShow have strong complexing to metal ion；The compounds of this invention apiolin-O- alkyl amine compound is in 25.0 μM of concentrationUnder to Cu2+The A β of induction1-4262.8% or more the inhibiting rate of self assemble, is significantly higher than curcumin, the compounds of this invention pairCu2+The A β of induction1-42The inhibitory activity of self assemble is high.
3. the present invention can significantly improve AlCl3The motor function of zebra fish AD is induced, it is equal compared with model control groupWith statistical difference；The compounds of this invention apiolin-O- alkyl amine compound is to hyoscine induced mice memory disordersIt is significantly improved effect.
Detailed description of the invention
Fig. 1 is apiolin-O- alkyl amine compound of the present invention to H2O2The protective effect of the PC12 cellular damage of induction is examinedSurvey result figure；
Fig. 2 is that apiolin-O- alkyl amine compound of the present invention and complexing of metal ion act on testing result figure；
Fig. 3 is apiolin-O- alkyl amine compound of the present invention to AlCl3Induce zebra fish Alzheimer's disease modelPreventive and therapeutic effect testing result figure；
Fig. 4 is shadow of the apiolin-O- alkyl amine compound of the present invention to hyoscine induced mice memory acquisition disturbanceRing testing result figure.
Specific embodiment
Below with reference to some specific embodiments, the present invention is further described.
The general formula of the chemical structure of the apiolin-O- alkyl amine compound of 1-128 of the embodiment of the present invention is as follows:
Wherein, n, R1、R2It is shown in Table 1.
Apiolin-O- alkyl amine the compound of 1 1-128 of the embodiment of the present invention of table
The compounds of this invention structural analysis data
I-44:Light yellow oil, 83.7%yield, 99.5%HPLC purity.1H NMR(400MHz,CDCl3) δ 12.80 (s, 1H, OH), 7.79 (d, J=8.4Hz, 2H, 2 × Ar-H), 7.35 (d, J=7.2Hz, 4H, 4 × Ar-), H 7.30 (t, J=7.6Hz, 4H, 4 × Ar-H), 7.23 (t, J=7.6Hz, 2H, 2 × Ar-H), 6.94 (d, J=8.4Hz,2H,2×Ar-H),6.54(s,1H,Ar-H),6.42(s,1H,Ar-H),6.30(s,1H,Ar-H),3.99-3.95(m,4H,2×OCH2),3.61(s,4H,2×phCH2),2.59-2.50(m,8H,4×NCH2),1.84-1.80(m,4H,2×CH2),1.70-1.64(m,4H,2×CH2), 1.08 (t, J=7.2Hz, 6H, 2 × CH3).13C NMR(100MHz,CDCl3)δ182.4,164.9,164.0,162.1,157.7,139.3,129.0(5C),128.2(5C),128.0(2C),127.0(2C),123.3,115.0(2C),105.4,98.5,93.0(2C),68.4,68.0,58.0(2C),52.5,52.4,47.3(2C),26.9,26.7,23.4,23.3,11.6(2C).HR-ESI-MS:Calcd.for C41H48N2O5[M+H]+:649.3597,found:649.3643。
I-49:Light yellow oil, 65.2%yield, 98.1%HPLC purity.1H NMR(400MHz,CDCl3) δ 12.80 (s, 1H, OH), 7.80 (d, J=8.8Hz, 2H, 2 × Ar-H), 7.31-7.24 (m, 10H, 10 × Ar-H),6.97 (d, J=8.8Hz, 2H, 2 × Ar-H), 6.52 (s, 1H, Ar-H), 6.45 (d, J=2.0Hz, 1H, Ar-H), 6.33 (d, J=2.4Hz, 1H, Ar-H), 4.06-4.01 (m, 4H, 2 × OCH2),3.51(s,4H,2×phCH2),2.53-3.40(m,20H,10×NCH2),1.86-1.80(m,4H,2×CH2),1.73-1.64(m,4H,2×CH2).HR-ESI-MS:Calcd.forC45H54N4O5[M+H]+:731.4128,found:731.4143。
I-53:Light yellow oil, 71.8%yield, 98.2%HPLC purity.1H NMR(400MHz,CDCl3) δ 12.79 (s, 1H, OH), 7.80 (d, J=8.4Hz, 2H, 2 × Ar-H), 7.29-7.25 (m, 4H, 4 × Ar-H),7.20-7.12 (m, 6H, 6 × Ar-H), 6.97 (d, J=8.4Hz, 2H, 2 × Ar-H), 6.55 (s, 1H, Ar-H), 6.45 (s,1H,Ar-H),6.32(s,1H,Ar-H),4.06-4.02(m,4H,2×OCH2), 2.98 (d, J=10.8Hz, 4H, 2 ×phCH2), 2.54 (d, J=6.8Hz, 4H, 2 × NCH2), 2.43 (t, J=7.2Hz, 4H, 2 × NCH2), 1.95 (t, J=11.2Hz,4H,2×NCH2),1.84-1.80(m,4H,2×CH2),1.72-1.64(m,8H,4×CH2),1.57-1.53(m,2H,2×CH),1.42-1.31(m,4H,2×CH2).HR-ESI-MS:Calcd.for C47H56N2O5[M+H]+:729.4223,found:729.4267。
I-54:Light yellow oil, 78.2%yield, 98.0%HPLC purity.1H NMR(400MHz,CDCl3) δ 12.86 (s, 1H, OH), 7.79 (d, J=8.4Hz, 2H, 2 × Ar-H), 7.17-7.10 (m, 8H, 8 × Ar-H),6.96 (d, J=8.0Hz, 2H, 2 × Ar-H), 6.56 (s, 1H, Ar-H), 6.44 (s, 1H, Ar-H), 6.32 (s, 1H, Ar-H),4.08-4.02(m,4H,2×OCH2),3.81(s,2H,phCH2),3.77(s,2H,phCH2),2.97-2.95(m,4H,2×phCH2),2.93-2.86(m,4H,2×NCH2),2.72-2.67(m,4H,2×NCH2),1.87-1.83(m,8H,4×CH2).HR-ESI-MS:Calcd.for C41H44N2O5[M+H]+:645.3284,found:645.3306。
The preparation method of apiolin-O- alkyl amine compound described in embodiment 1, comprising the following steps:
(1) apiolin of 5.0mmol, the corresponding two bromoalkanes Br (CH of 15mmol are added in reaction flask2)nBr、10mmolPotassium carbonate and 50mL acetonitrile, after mixing evenly, 65 DEG C are stirred to react for 24 hours；After reaction, evaporated under reduced pressure solvent, in residual solution100mL deionized water is added, is extracted in three times with 300mL methylene chloride, organic layer is washed after merging with saturated sodium-chloride water solutionIt washs, evaporated under reduced pressure solvent, residue obtains accordingly 2,7- through column chromatographic purifying (eluent: petroleum ether: acetone=100:1v/v)Dibromo alkoxy apiolin；
(2) 2,7- dibromo alkoxy apiolin, the corresponding secondary amine NR of 4.0mmol of 3.0mmol are added in reaction flask1R2、4.5mmol potassium carbonate and 30mL acetonitrile, after mixing evenly, 65 DEG C are stirred to react for 24 hours；After reaction, evaporated under reduced pressure solvent, it is residual100mL deionized water is added in extraction raffinate, is extracted in three times with 300mL methylene chloride, organic layer uses saturated sodium chloride water after mergingSolution washing, evaporated under reduced pressure solvent, residue obtain accordingly through column chromatographic purifying (eluent: petroleum ether: acetone=50:1v/v)Apiolin-O- alkyl amine compound (I), chemical structure passes through1H NMR、13C NMR and ESI-MS confirmation.
The preparation method of apiolin-O- alkyl amine compound described in embodiment 2-128 is substantially the same manner as Example 1,The same thing is not repeated, and difference is shown in Table 2 and 3.
One of apiolin-O- alkyl amine compounds process for production thereof parameter of 2 1-10 of the embodiment of the present invention of table
The two of the apiolin-O- alkyl amine compounds process for production thereof parameter of 3 1-10 of the embodiment of the present invention of table
Biological activity test
1. the acetylcholinesterase and butyrylcholine esterase of apiolin-O- alkyl amine compound (I) of the present invention inhibit to liveProperty
1.0mmol/L acetylthiocholine iodide is sequentially added into 96 orifice plates or thio BuCh (is purchased from SigmaCompany) 30 μ L, pH=8.0 40 μ L of PBS buffer solution, 20 μ L of testing compound solution (DMSO content is less than 1%) and 10 μ L electricity(0.045U is purchased from for eel acetylcholinesterase (EeAChE) or butyrylcholine esterase (equineserum BuChE, eqBuChE)Sigma company), after adding mixing, 37 DEG C of incubation 15min, into each hole be added mass fraction be 0.2% 5,5'- bis- it is thio-Bis- 30 μ L colour developings of (2- nitro) benzoic acid (DTNB is purchased from Sigma company) solution, with the light in each hole at microplate reader measurement 405nmDensity (OD) value calculates compound to the inhibiting rate [enzyme inhibition rate=(1- sample of enzyme compared with the blank well that sample to be tested is not addedProduct group OD value/blank group OD value) × 100%]；Five to six concentration for selecting compound, measure its enzyme inhibition rate, and with the changeThe inhibiting rate linear regression of the negative logarithm and enzyme of object molar concentration is closed, molar concentration when acquiring 50% inhibiting rate is the chemical combinationThe IC of object50, with Rivastigmine (Rivastigmine) and donepezil (Donepezil) for positive control, testing result is shown in Table4。
Cholinesterase inhibition, antioxidant activity, the auto-induction A β of 4 the compounds of this invention of table1-42Aggregation inhibits to liveProperty and Cu2+The A β of induction1-42Self assemble inhibitory activity
Measurement result shows the parent nucleus of the compounds of this invention --- apiolin is to acetylcholinesterase and butyrylcholine esteraseThe IC of inhibition50500 μM are all larger than, apiolin-O- alkyl amine compound (I) of the present invention is to acetylcholinesterase and BuChEsterase IC50Respectively 0.002-10.8 μM, 0.003-4.3 μM, inhibitory activity is significant, with positive control Rivastigmine/Donepezil testing result is quite or more preferably.
2. apiolin-O- alkyl amine compound (I) of the present invention is to A β1-42The inhibitory activity of self assemble
The method that reference literature (Sang, Z.P.et al.Eur.J.Med.Chem.2015,94,348-366) is reported intoRow measurement, it may be assumed that pretreated A β1-42It is made into stock solution with DMSO, is diluted to 50 μM with the PBS buffer solution of pH7.4 using preceding；Untested compound is made into 2.5mM stock solution with DMSO, is diluted to respective concentration with the PBS buffer solution of pH7.4 using preceding, takes 20 μ LA β1-42The testing compound solution of+20 μ L of solution, 20 μ L A β1-42+ 20 μ L PBS buffer solution of solution (containing 2%DMSO), 20 μ LPBS buffer solution (containing 2%DMSO)+20 μ L PBS buffer solution (containing 25%DMSO) are in 96 orifice plate of black, compound and A β1-42'sUltimate density is 25 μM.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 DEG C of incubationsFliud flushing (pH=8.5) is existed with VarioskanFlash Multimode Reader multi-function microplate reader immediately after shaking 5sFluorescent value is measured under 446nm excitation wavelength and 490nm launch wavelength；Aβ1-42The fluorescent value of+untested compound is recorded as IFi, Aβ1-42The fluorescent value of+PBS buffer solution is recorded as IFc, the fluorescent value for containing only PBS buffer solution is recorded as IF0, A is inhibited by compoundβ1-42The inhibiting rate calculation formula of self assemble are as follows: 100- (IFi-IF0)/(IFc-IF0)×100；Each each concentration of compoundMeasure two multiple holes；Using curcumin as positive control, testing result is shown in Table 4.
Measurement result shows curcumin to auto-induction A β1-42The IC of the inhibition of aggregation50For 26.8 μM of (inhibiting rates43.1%), now widely used anti-AD medicine donepezil is under 25.0 μM of concentration to A β1-42The inhibiting rate of self assemble is less than20.0%, apiolin-O- alkyl amine compound of the present invention is under 25.0 μM of concentration to A β1-42The inhibiting rate IC of self assemble50Much smaller than curcumin, 70% or more inhibiting rate, there is remarkable inhibiting activity.
3. the Antioxidative Activity Determination (ORAC-FL method) of apiolin-O- alkyl amine compound (I) of the present invention
Reagent and instrument: 6- hydroxyl -2,5, (trolox changes 7,8- tetramethyl primary colours alkane -2- carboxylic acids purchased from the uncommon love (Shanghai) of ladderAt industrial development Co., Ltd) solution of 10-80 μm of ol/L is made into the PBS buffer solution (pH7.4) of 75mM；Fluorescein(fluorescein, purchased from uncommon love (Shanghai) the chemical conversion industry Development Co., Ltd of ladder) is matched with the PBS buffer solution (pH7.4) of 75mMAt the solution of 250nmol/L；2,2 '-azo diisobutyl amidine dihydrochlorides (AAPH, it is limited purchased from splendid remote chemistry scientific and technological (Shanghai)Company) using the preceding solution that 40mmol/L is made into the PBS buffer solution (pH7.4) of 75mM；Microplate reader is VarioskanFlash Multimode Reader(Thermo Scientific)。
Measurement experiment method: 20 μ L of compound solution and the fluorescein that 50 or 10 μm of ol/L are added into 96 orifice plate of black are molten120 μ L of liquid is mixed, 37 DEG C of incubation 15min, and 60 μ L of AAPH solution is added, makes every 200 μ L of hole total volume, is mixed, is immediately placed onIn Varioskan Flash Multimode Reader instrument, under 485nm excitation wavelength and 535nm launch wavelength per minuteFirst order fluorescence value is measured, METHOD FOR CONTINUOUS DETERMINATION 90min calculates area AUC under fluorescence decay curve by instrument automatically.Wherein with 1-The trolox of 8 μm of ol/L is as standard, sample to be tested is not added as blank.The antioxidant activity results expression of compound isThe equivalent of trolox, calculation formula are as follows: [(AUC Sample-AUC blank)/(AUC Trolox-AUC blank)] ×[(concentration of Trolox/concentration of sample)].Each compound measures 3 again every timeHole, testing result are shown in Table 4.
Measurement result shows that the antioxidant activity of apiolin-O- alkyl amine compound disclosed by the invention is Trolox1.2-1.8 times, the compounds of this invention have strong anti-oxidative activity.
4. apiolin-O- alkyl amine compound (I) of the present invention is to H2O2The protective effect of the PC12 cellular damage of induction is sievedChoosing
DMEM culture solution of the PC12 cell containing 10% calf serum, with 1 × 105A/mL density is inoculated in the culture of 96 holesOn plate, inoculation volume is the hole 100mL/, is subsequently placed into containing 5%CO237 DEG C of constant incubators in culture.After culture 24 hours,Add the compound (final concentration of 10 of respective concentration in administration group-5Mol/L, 10-6Mol/L) the hole 10mL/, preincubate 2 hours (rightAdd 10 μ L/ hole PBS respectively with damage group according to group, its volume is made to keep equal).After PC12 cell incubation 2 hours, administration group with100 μ Μ H are separately added into damage group2O2It damages 10 hole μ L/ of agent (control group adds 10 μ L/ hole PBS), after 30 minutes, by each groupThe RPMI1640 culture solution that culture solution changes no calf serum into, which continues to be put into constant incubator, to be cultivated 24 hours, and liquid is cultivatedProduct thinks 100 holes μ L/.After continuing culture 24 hours, 5mg/mL is added in each group, and the hole MTT100 μ L/ carries out living cells dyeing.To 3After hour, 100 hole μ L/ of 100%DMSO terminate liquid is added in each group, sufficiently dissolution mixes.Each group is measured under the wavelength of 490nmOD value, test result is repeated 3 times, and with Duncan ' s test method statistic, each group numerical value is expressed as mean ± S.E.M., withControl group is 100%, and administration group and damage class value with the percentage of control group indicate that testing result is shown in Fig. 1.
Measurement result shows apiolin-O- alkyl amine compound of the present invention 10-5Mol/L~10-6Under mol/L concentrationThere is significant protective effect to the PC12 cellular damage of hydrogen peroxide-induced.
5. the measurement of apiolin-O- alkyl amine compound (I) of the present invention and complexing of metal ion effect
CuCl is dissolved with methanol2.2H2O、ZnCl2、FeSO4.7H2O、AlCl3And untested compound, it is made into 75 μm of ol/L's100 μ L testing compound solutions and 100 μ l metal ion solutions are added into 96 orifice plates for solution, mix, are stored at room temperature 30 pointsClock records the ultraviolet absorption curve within the scope of 200-600nm on multi-function microplate reader, and with 100 μ L testing compound solutionsIt is control, the Red Shift Phenomena of observation metal ion and the maximum absorption band of untested compound mixed liquor with 100 μ L methyl alcohol mixed liquorsAnd the intensity of maximum absorption band, testing result are shown in Fig. 2.
Measurement result shows that apiolin-O- alkyl amine compound of the present invention shows have Qiang Luohe to metal ionEffect.
6. apiolin-O- alkyl amine compound (I) of the present invention is to Cu2+The A β of induction1-42The inhibitory activity of self assemble
At room temperature by A β1-42Trifluoroacetate (1mg) is dissolved in 1ml hexafluoroisopropanol (HFIP), ultrasonic 5minAfterwards, for 24 hours, reduced pressure at room temperature removes solvent, is dissolved again with DMSO, is made into 200 μM of A β for incubation at room temperature1-42Stock solution, in -80 DEG CStorage, is diluted to 50 μM with the PBS buffer solution (pH=7.4) of 50mM using preceding；Untested compound is dissolved with DMSO is made into 2.5mMStock solution is diluted to 50 μM with the PBS buffer solution (pH=7.4) of 50mM using preceding.Take 20 μ L PBS buffer solution (containing 2%DMSO)The A β of+20 μ LPBS buffers (containing 25%DMSO) (blank group), 20 μ L1-42+ 20 μ L PBS buffer solution of solution (contains 2%DMSO)The A β of (control group), 20 μ L1-42Solution (test group), in 96 orifice plate of black.37 DEG C are incubated for for 24 hours, and 20 μ are added into test groupThe testing compound solution of L, compound and A β1-42Ultimate density be 25 μM.37 DEG C are incubated for for 24 hours again, and 160 μ L are then addedThe glycine-NaOH buffer (pH=8.5) of 50mM containing 5 μM of thioflavine Ts uses Varioskan after shaking 5s immediatelyFlash Multimode Reader multi-function microplate reader measures fluorescent value under 446nm excitation wavelength and 490nm launch wavelength；Aβ1-42The fluorescent value of+untested compound is recorded as IFi, A β1-42The fluorescent value of+PBS buffer solution is recorded as IFc, it is slow to contain only PBSThe fluorescent value of fliud flushing is recorded as IF0, A β is inhibited by compound1-42The inhibiting rate calculation formula of self assemble are as follows: 100- (IFi-IF0)/(IFc-IF0) * 100, each each two multiple holes of concentration mensuration of compound, using curcumin as positive control, testing resultIt is shown in Table 4.
Measurement result shows that curcumin inhibiting rate is 53.8%, the compounds of this invention apiolin-O- alkyl amine compoundTo Cu under 25.0 μM of concentration2+The A β of induction1-4262.8% or more the inhibiting rate of self assemble, is significantly higher than curcumin, this hairBright compound is to Cu2+The A β of induction1-42The inhibitory activity of self assemble is high.
7. apiolin-O- alkyl amine compound (I) prepared by the present invention is to AlCl3Induce zebra fish Alzheimer's diseaseThe preventive and therapeutic effect of model (by taking compound I-44 as an example).
3dpf wild type AB system zebra fish is randomly selected in six orifice plates, with alchlor (AlCl3) induce zebra fish AhWurz sea is write from memory disease model (hereinafter referred to as AD zebra fish), water-soluble respectively to give 0.09 μ g/mL of I-44,0.26 μ g/mL and 0.78 μG/mL concentration, 8 μM of positive control drug donepezil, while Normal group (untreated) and model control group (AlCl are set3Group), every 30 tail zebra fish of experimental concentration group.After administration 3 days, each experimental group zebra fish is observed and recorded respectively with behavioural analysis instrument and is existedThe move distance of 3 light and shade periods (that is: dark 10min, illumination 10min replace 3 periods), analyzes zebra fish in 60minThe move distance of 60min, it is for statistical analysis with model control group with move distance, with statistical significance evaluation compound to spotThe preventive and therapeutic effect of horse fish alzheimer's disease model, testing result are shown in Fig. 3.
Test result shows that compared with Normal group (7303mm), the move distance of model control group is aobvious for 5203mmWriting reduces, and shows model foundation success；Compared with model control group, positive controls donepezil can then be such that move distance increasesTo 6358mm (p < 0.01), illustrate that the response function to AD zebra fish has improvement result, the high, medium and low dosage group of drug I-44Move distance significantly increase (p < 0.001) compared with model control group, illustrate that compound I-44 can significantly improve AlCl3InductionThe motor function of zebra fish AD, all has statistical difference compared with model control group.
8. apiolin-O- alkyl amine compound (I) prepared by the present invention hinders hyoscine induced mice Memory acquisitionThe influence (diving tower passive avoidance test) (by taking compound I-44 as an example) hindered.
SPF grades of ICR male mices, 25-30g are randomly divided into: normal group, model group, test drug high (50mg/kg), in(20mg/kg), low dose group (5.0mg/kg), every group of 10 animals.Test medicine, blank group and model are given in disposable stomach-fillingGroup gives solvent 0.5%CMC-Na, and administered volume is 0.1ml/10g；45min after medicine, normal mouse peritoneal of organizing inject physiologySalt water, remaining groups of animals inject hyoscine (3mg/kg), and administered volume is 0.1mL/10g；20min is carried out after modelingStep dow n test training, animal is put into reaction chamber and adapts to 3min, passes to 36V alternating current immediately after.Training 5min, and record everyThus secondary mouse is used as school grade by the number (errors number) to shock by electricity.It is tested afterwards for 24 hours, every mouse assay5min records the errors number jumped off in the incubation period and 5min of platform by the number of animals and first time to shock by electricity.As a result intoRow statistical analysis.All data use mean value ± standard error (Stand error, S.E.) to indicate.It is soft using SPSS11.5Part analysis.The neat selection one-way analysis of variance of variance (One-way ANOVA).Measurement data compares using single factor test variance pointThe comparison of analysis, each group mean is examined using t, and testing result is shown in Fig. 4.
Measurement result shows that compared with the control group the incubation period of model group diving tower passive avoidance test test is obviously shortened(p < 0.01) shows model foundation success；Compared with model group, the prolongation of latency (p < 0.05) of drug height, middle dose group, andDrug low dose group and donepezil group incubation period significantly shorten (p < 0.01), and the incubation period of drug low dose group (5mg/kg) is261.8s, the compounds of this invention apiolin-O- alkyl amine compound have significantly hyoscine induced mice memory disordersImprovement result.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, this field is commonOther modifications or equivalent replacement that technical staff makes technical solution of the present invention, without departing from technical solution of the present inventionSpirit and scope, be intended to be within the scope of the claims of the invention.