CN106924246A - Isoflavonoid prevents or treats the purposes of nerve degenerative diseases - Google Patents

Abstract

The invention discloses isoflavone compounds J37941 under safe dose can dose dependent suppression cell acetylcholine esterase active, protect nerve cell, mitigate nerve cell oxidativestress damage；Regulation neuroinflamation；Have to second, the activity of BuChE and acted on compared with high inhibition；Results of animal proves that J37941 toxicity is relatively low, can pass through blood-brain barrier, and the mouse that can obviously improve caused by hyoscine is dull-witted, can strengthen learning and memory function.A large amount of preclinical study results show that J37941 is expected to turn into the medicine for preventing and/or treating learning memory disorder and Alzheimer disease.

Description

Isoflavonoid prevents or treats the purposes of nerve degenerative diseases

Technical field

The present invention relates to dual choline esterase inhibition, neurocyte protection effect, antiinflammatory action compound, And application of the compound in treatment learning memory disorder and senile dementia.Category pharmaceutical technology field.

Technical background

Alzheimer disease (Alzheimer ' s disease, AD) is that one kind is with behavior, cognitive and memory dysfunction The central nervous system degenerative disease of Clinical symptoms, its characteristic pathological change is turned to：Neuron loss, neurofibril are twined (neurofibrillary tangles, NFTs), senile plaque expelling (senile plaques, SPs).The 65-74 Sui AD morbidity of crowd Rate is about 3%, the 75-84 Sui incidence of disease of crowd can rise to 19%, and more than the 84 years old AD incidence of disease of crowd may be up to 47%. AD is the fourth-largest killer after elderly population relaying cardiovascular disease, tumour, cerebrovascular disease, serious harm elderly population health. As population in the world aging trend is on the rise, if without effective intervening measure, the year two thousand fifty, the AD patient in the whole world will be up to 1 Hundred million, medical system is had an immense impact on.Therefore, research and development associated treatment medicine is significant.

The pathogenesis of AD is complicated, and many factors such as h and E play a role in its progression.According to AD diseases Various pathological biochemistries in change process change, and generate various AD pathology hypothesis, and such as cholinergic hypothesis, A beta hypothesises, poisoning by aluminum is false Say, cholesterol transformant doctrine, calcium channel are damaged hypothesis, inflammatory reaction hypothesis etc..Pathological characters according to senile dementia and Pathogenesis, includes for the therapy approach that old dementia patients are taken：(1) medicine of choline systemic-function is improved.(2) correct Calcium homeostasis is lacked of proper care and oxidation resistant medicine.(3) neurotrophic factor.(4) medicine that interference A β are formed and deposited.(5) anti-nerve Apoptosis agent.

Cholinergic system plays a significant role during learning and memory.Research since the seventies shows：AD patient's brain Inside there is many Cholinergic declines, so as to propose the cholinergic hypothesis (cholinergic of the AD causes of disease hypothesis)：Central cholinergic system dysfunction causes the cognitive decline of AD patient.According to cholinergic hypothesis, strengthen courage The medicine of alkali energy systemic-function, can be used for the treatment of AD.By anticholinesterase (cholinesterase Inhibitor, ChEI) acetyl choline content is improved, strengthen cholinergic nerve meta function, it is the Main Means for treating AD at present.

Clinical conventional ChEI is generally selective acetylcholinesterase (acetylcholinesterase, AChE) suppression at present Preparation, can improve the cognitive function of patient, improving the quality of life of patient and ward.But certain adverse reaction is there is also, Treatment to severe AD is undesirable.Butyrylcholine esterase (butyrylcholinesterase, BuChE) is equal in maincenter and periphery It is distributed, because the physiological function of BuChE is indefinite always, the affinity to acetylcholine (acetylcholine, ACh) is low In AChE.Think the target spot do not treated as AD for a long time.But research in recent years finds BuChE not only alternative AChE hydrolysis ACh, also there is effect in AD processes, and BuChE can be considered a novel targets of AD treatments.Proper proportion ground simultaneously suppress AChE and BuChE is more preferable AD therapeutic schemes.

The content of the invention

The technical problem to be solved in the present invention is to provide a kind of compound as shown in structure formula (I) and its can pharmaceutically connect Salt, its pharmaceutical composition the applying in prevention and/or treatment nerve degenerative diseases medicine is prepared received, are preparing second, fourth Application in acetylcholinesterase double inhibition drugs with function, answering in preparing protection nerve cell, improving oxidative stress medicine With the application in regulation neuroinflamation medicine is prepared is preparing the application in improving learning and memory function medicine.

To solve technical problem of the invention, technical solution of the present invention is as follows：

A kind of compound and its pharmaceutically acceptable salt as shown in structure formula (I) is preparing prevention and/or treatment god Through the application in DD medicine, prepared by the application in second, butyrylcholine esterase double inhibition drugs with function is prepared Protection nerve cell, the application improved in oxidative stress medicine, the application in regulation neuroinflamation medicine is prepared change in preparation Application in kind learning and memory function medicine,

Wherein, HX is selected from organic acid or inorganic acid, and organic acid is selected from maleic acid, caffeic acid, forulic acid, gallic acid, first Sulfonic acid, inorganic acid is selected from hydrochloric acid, sulfuric acid；Wherein, described nerve degenerative diseases are characterized with learning memory disorder；Its In, described nerve degenerative diseases include senile dementia, Parkinson's, Huntington's disease.

A kind of pharmaceutical composition prepare prevention and/or treatment nerve degenerative diseases medicine in application, prepare second, Application in butyrylcholine esterase double inhibition drugs with function, in preparing protection nerve cell, improving oxidative stress medicine Using the application in regulation neuroinflamation medicine is prepared is preparing the application in improving learning and memory function medicine, its feature It is that described pharmaceutical composition includes the compound and its pharmaceutically acceptable salt and pharmacy as shown in structure formula (I) Upper acceptable carrier or excipient,

Wherein, HX is selected from organic acid or inorganic acid, and organic acid is selected from maleic acid, caffeic acid, forulic acid, gallic acid, first Sulfonic acid, inorganic acid is selected from hydrochloric acid, sulfuric acid；

Wherein, described nerve degenerative diseases are characterized with learning memory disorder；

Wherein, described nerve degenerative diseases include senile dementia, Parkinson's, Huntington's disease.

The preparation of the compound (i.e. J37941) shown in above-mentioned formula (I)：

Synthetic route：

a)BrCH₂CH₂Br, K₂CO₃, Acetone；b)K₂CO₃, DMF, N (CH₂CH₂)；

Prepare：

In 50mL single port bottles, addition 3- (4- methoxyphenyls) -7- hydroxyl -4H- chromogen -4- ketone (268.3mg, 1.0mmol), finely ground Anhydrous potassium carbonate powder (414.6mg, 3.0mmol), 15mL acetone, adds 1,2- Bromofumes (866.0 μ L, 10.0mmol), heating reflux reaction overnight, reactant mixture is concentrated under reduced pressure, and residue adds water, stirring, is taken out Filter, filter cake is washed with water, is dried, and obtains white solid 354mg, yield：94.4%.¹H NMR(400MHz,CDCl₃)δ:7.88(s, 1H), 7.46 (d, J=8.8Hz, 2H), 6.98 (d, J=8.8Hz, 2H), 6.40 (dd, J=2.0,15.2Hz, 2H), 4.36 (t, J=6.0Hz, 2H), 3.85 (s, 3H), 3.66 (t, J=6.0Hz, 2H)；¹³C NMR(100MHz,CDCl₃)δ:181.0, 164.0,163.1,160.0,158.0,152.9,130.2,123.9,123.0,114.3,106.8,98.7,93.2,68.3, 55.5,28.4.ESI-MS(m/z):391.38[M+H]⁺

In a 100mL single port bottle, 502.0mg (1.34mmol) 7- (2- bromine oxethyls) -3- (4- methoxybenzenes are added Base) -7- hydroxyl -4H- chromogen -4- ketone, 30mL DMF dissolvings are added, add 926.0mg K₂CO₃Powder and 0.142g (2.0mmol) pyrrolidines, in 100 DEG C of stirring reaction 3h, TLC detections under heating, reaction is finished, and reaction solution is poured into 200mL water In, white solid is separated out, suction filtration, filter cake 100mL water washings obtain white solid 365.0mg, TLC and are shown as single spot, receive Rate 82%.mp：143℃-145℃；ESI-MS:(m/z):367.18(M+1).¹H-NMR(DMSO-d₆):8.40 (s, 1H), 8.02 (d, 1H), 7.52 (d, 2H), 7.15 (s, 1H), 7.08 (d, 1H), 6.96 (d, 1H), 4.22 (t, 2H), 3.77 (s, 3H), 2.80 (t, 2H), 2.49 (m, 4H), 1.67 (m, 4H).Accompanying drawing：36-1 and 36-2.

Advantageous Effects：

There is present invention display J37941 good second, butyrylcholine esterase double inhibition to act on, and nerve cell oxidation should Swash injury protection effect, neuroinflamation adjustment effect can be effectively improved learning and memory function in whole animal level.

Suddenly poison result shows Mouse oral, the oral LD of compound J37941₅₀=550mg/kg, toxicity is far below positive drug Toxicity (the LD of Exelon₅₀=21.75mg/kg).Therefore, the small toxicity of compound J37941, safe.

Brief description of the drawings

Fig. 1, J37941 suppress the amount effect curve of AChE, BuChE

The influence of Fig. 2, J37941 to SY5Y cell viabilities

The influence of Fig. 3, J37941 to SY5Y cells AChE activity

The influence of Fig. 4, J37941 to APPsw-SY5Y cell viabilities

The influence of Fig. 5, J37941 to APPsw-SY5Y cells AChE activity

Fig. 6, J37941 are to Cu²⁺The influence of the APPsw-SY5Y cellular damage model cell vigor of induction

Fig. 7, J37941 are to Cu²⁺The influence of ROS levels in the APPsw-SY5Y cellular damage model cells of induction

The influence that Fig. 8, J37941 secrete to activated microglia IL-1 β

The influence that Fig. 9, J37941 secrete to activated microglia IL-6

Influence (n=13~16) (the #vs control, p of Figure 10, J37941 in Jumping test to learning and memory< 0.5；*vs model,p<0.5；**vs model,p<0.01)

Influence (n=13~16) (the ##vs control, p of Figure 11, J37941 in step-through test to learning and memory< 0.01；*vs model,p<0.5；***vs model,p<0.001)

The influence (n=13~16) of Figure 12, J37941 in water maze laboratory to learning and memory

The 1HNMR collection of illustrative plates (300MHz) of Figure 13, J37941, i.e. WS0902036

The ESI mass spectrums of Figure 14, J37941, i.e. WS0902036

Specific embodiment

Inhibitory action research of the level to ACHE, BuCHE activity in vitro of embodiment 1, J37941

J37941 is its hydrochloride.

Method：AChE inhibitor screening models and BuChE inhibitor screening models are set up using DTNB methods, J37941 is existed Inhibitory activity during 0.08-50 μm of ol/L to AChE, BuChE is evaluated, and calculates IC₅₀.In triplicate, average and SD are calculated Value.

As a result：J37941 has inhibitory action to AChE, BuChE, to the IC of AChE₅₀In 1.24 μm of ol/L, to BuChE's Inhibitory activity is slightly above donepezil, is shown in Table 1.

Table 1, J37941 suppresses the IC of ACHE and BuCHE activity₅₀

Note：The Chinese entitled tetra isopropyl pyrophosphoric acid imines of iso-OMPA, is the instrument medicine of BuChE, herein as sun Property comparison medicine.

Inhibitory action research of the embodiment 2, J37941 to nerve cell AChE activity

(1) to the inhibitory action research of SY5Y nerve cells AChE activity

Method 1：SY5Y cells treat that cell is in logarithmic growth with the DMEM/F12 medium cultures containing 10% hyclone During the phase, by 5*10⁴The density in individual/ml, 100ul/ hole is inoculated in 96 well culture plates.Changed after 24h and trained with serum-free DMEM/F12 Base culture is supported, is changed after 12h with the serum-free DMEM/F12 culture mediums of various concentrations J37941 (0.3-10 μm of ol/L), donepezil And Exelon (final concentration of 10 μm of ol/L) processes identical, MTS methods determine cell viability, DTNB methods detection cell conditioned medium after 24h Middle AChE activity.

As a result：Compared with normal group, J37941 does not make significant difference in the range of administration concentration to cell viability, and positive right (p ＜ 0.05) is significantly reduced according to donepezil administration group cell viability, 2 are shown in Table.J37941 can dose dependent suppression SY5Y The AChE activity of cell, is shown in Table 3.

Influence of the table 2, J37941 to SY5Y cell viabilities

Influence of the table 3, J37941 to SY5Y cells AChE activity

^#Compare P with normal group<0.05,^##Compare P with normal group<0.01

(2) to the inhibitory action research of wild type APPsw transgenosis SY5Y (APPsw-SY5Y) nerve cells AChE activity

Method 2：Wild type APPsw transgenosis SY5Y (APPsw-SY5Y) cells are with containing 10% hyclone, 400ug/ml The DMEM/F12 medium cultures of G418, when cell is in exponential phase, by 5*10⁴The density in individual/ml, 100ul/ hole connects Plant in 96 well culture plates.Changed after 24h with serum-free DMEM/F12 medium cultures, changed after 12h with various concentrations J37941 The serum-free DMEM/F12 culture mediums of (0.3-10 μm of ol/L), donepezil and Exelon (final concentration of 10 μm of ol/L) process phase Together, MTS methods determine cell viability, AChE activity in DTNB methods detection cell conditioned medium after 24h.

As a result：Compared with normal group, J37941 is in the range of administration concentration to wild type APPsw transgenosis SY5Y (APPsw-SY5Y) vigor of cell does not make significant difference, can dose dependent suppression APP-sw SY5Y cells AChE activity. It is shown in Table 4, table 5.

Influence of the table 4, J37941 to APP-sw SY5Y cell viabilities

Influence of the table 5, J37941 to APP-sw SY5Y cells AChE activity

^#Compare P with normal group<0.05,^##Compare P with normal group<0.01

(3) Effect studies of the J37941 on AD cell models

Method：APPsw-SY5Y cells are trained with the DMEM/F12 culture mediums containing 10% hyclone, 400ug/ml G418 Support, when cell is in exponential phase, by 5*10⁴The density in individual/ml, 100ul/ hole is inoculated in 96 well culture plates.After 24h Change and continue to cultivate 12h with serum-free DMEM/F12 culture mediums, add J37941 and 10 μm of ol/L of final concentration of 10,3 μm of ol/L Donepezil, Exelon preincubate 2h after, add the Cu of final concentration of 200 μm of ol/L²⁺Treatment cell, respectively with MTS after 24h Method determines cell viability, DCFH₂- DA fluorescence probe methods determine intracellular ROS contents.

As a result：

1) compared with normal group, Cu²⁺After treatment 24h, model group cell survival rate is significantly reduced (p ＜ 0.01).With model Group is compared, and each dosage group cell survival rates of J37941 are improved, and the cell survival rate of 10 μm of ol/L J37941 administration groups is higher than Positive control donepezil, Exelon group.It is shown in Table 6.

Table 6, J37941 is to Cu²⁺The influence of the APPsw-SY5Y cellular damage model cell vigor of induction

^##Compare P with normal group<0.01

2) compared with normal group, Cu²⁺After treatment 24h, the intracellular ROS fluorescence intensities of model group are significantly raised (p ＜ 0.01). Compared with model group, each dosage group cell ROS fluorescence intensities of J37941 significantly reduce (10 μm of ol/L group p ＜, 0.01,3 μm of ol/L Group p ＜ 0.05), illustrate that J37941 can reduce intracellular reactive oxygen level.It is shown in Table 7.

Table 7, J37941 is to Cu²⁺The influence of the APPsw-SY5Y cellular damage intracellular reactive oxygen contents of induction

^##Compare P with normal group<0.01,^*Compare P with model group<0.05,^**Compare P with model group<0.01

(4) Effect studies of the J37941 to neuroinflamation

Method：The Exelon of final concentration of 10,2 μm of J37941 of ol/L, final concentration of 10 μm of ol/L, nicotine and rat are big After cortex microglia is incubated 2h jointly, in addition to blank control group, model group and administration group add final concentration of 1 μ g/ Ml LPS, continue to collect cell conditioned medium after being incubated 24h, and Elisa methods determine IL-1 β, IL-6 contents in cell supernatant.

As a result：Compared with normal group, after LPS stimulates 24h, IL-1 β, IL-6 contents significantly rise in model group cell supernatant (p ＜ 0.05) high.J37941 can reduce the content of IL-1 β, IL-6 in cell conditioned medium, when dosage is 10 μm of ol/L, on cell IL-1 β, IL-6 contents are significantly reduced (p ＜ 0.01, p ＜ 0.05) compared with model group in clear.It is shown in Table 8.

The influence that table 8, J37941 is secreted to activated microglia born of the same parents IL-1 β, IL-6

^##Compare P with normal group<0.01,^*Compare P with model group<0.05,^**Compare P with model group<0.01

Research of the embodiment 3, J37941 on AD animal models

Method：Mouse study, dysmnesia model are built with hyoscine, is the positive with Exelon (rivastigmine) Control drug, the effect of observation compound J37941.Learning and memory of little mouse function with diving tower, keep away dark, water maze behavioral indexes Evaluate, compare the difference between Normal group, model group and medicine group.

The dosage of compound pharmacodynamic evaluation is tried according to the conventional amount used computational methods combination acute toxicity of medicine Test result determination.Selected during compound J37941 pharmacodynamic evaluations 10mg/kg dosage (high dose), 3mg/kg dosage (in Dosage) and 1mg/kg dosage (low dose), positive drug Exelon group is 3mg/kg.

ICR mouse are used in experiment, for dementia animal model pharmacodynamic evaluation, mouse are randomly divided into 6 groups, normal group, mould Type group, Exelon group (3mg/kg), compound J37941 high doses group, middle dose group and small dose group, every group of mouse 13-15 Only.

Influences of the compound J37941 to hyoscine model mice Jumping test：Hyoscine model group mouse is trained After 24h, the time (incubation period) on diving tower is very short, and incubation period compares with blank group, significant difference (P<0.05).Compound J37941 is high, it is low dose of can be obviously prolonged incubation period, compare with model group, significant difference (P<0.05).The results are shown in Table 9.

Influence (mean ± SEM) of the table 9, J37941 in Jumping test to learning and memory

^#Compare P with normal group<0.05,^*Compare P with model group<0.05,^**Compare P with model group<0.01

Influences of the compound J37941 to hyoscine model mice step-through test：After hyoscine model group training 24h, Time (incubation period) into darkroom is very long, and incubation period compares with blank group, significant difference (P<0.05).Compound J37941 is high Dosage can extend incubation period.

Influence (mean ± SEM) of the table 10, J37941 in step-through test to learning and memory

^##Compare P with normal group<0.01,^*Compare P with model group<0.05,^**Compare P with model group<0.01

Compound J37941 influences on hyoscine model mice water maze laboratory：After mouse injection hyoscine, model Group compares with blank group, and the time that mouse finds platform substantially increases (P<0.05) it is, in compound J37941, low dose of The learning memory disorder that confrontation hyoscine causes, compares, significant difference (P with model group<0.05).

Influence (mean ± SEM) of the table 11, J37941 in water maze laboratory to learning and memory

^##Compare P with normal group<0.01,^*Compare P with model group<0.05,^**Compare P with model group<0.01

Claims (12)

1. a kind of compound and its pharmaceutically acceptable salt as shown in structure formula (I) is preparing prevention and/or treatment nerve Application in DD medicine,

Wherein, HX be selected from organic acid or inorganic acid, organic acid be selected from maleic acid, caffeic acid, forulic acid, gallic acid, methanesulfonic acid, Inorganic acid is selected from hydrochloric acid, sulfuric acid.

2. application of a kind of pharmaceutical composition in prevention and/or treatment nerve degenerative diseases medicine is prepared, it is characterised in that Described pharmaceutical composition is including the compound and its pharmaceutically acceptable salt as shown in structure formula (I) and can pharmaceutically connect The carrier or excipient received,

Wherein, HX be selected from organic acid or inorganic acid, organic acid be selected from maleic acid, caffeic acid, forulic acid, gallic acid, methanesulfonic acid, Inorganic acid is selected from hydrochloric acid, sulfuric acid.

3. according to the application of claim any one of 1-2, it is characterised in that described nerve degenerative diseases are with learning and memory What obstacle was characterized.

4. according to the application of claim any one of 1-2, it is characterised in that described nerve degenerative diseases include senile dementia Disease, Parkinson's, Huntington's disease.

5. a kind of compound and its pharmaceutically acceptable salt as shown in structure formula (I) is preparing acetylcholinesterase, butyryl Application in cholinesterase double inhibition medicine,

Wherein, HX be selected from organic acid or inorganic acid, organic acid be selected from maleic acid, caffeic acid, forulic acid, gallic acid, methanesulfonic acid, Inorganic acid is selected from hydrochloric acid, sulfuric acid.

6. application of a kind of pharmaceutical composition in acetylcholinesterase, butyrylcholine esterase double inhibition medicine is prepared, it is special Levy and be, described pharmaceutical composition includes the compound and its pharmaceutically acceptable salt and medicine as shown in structure formula (I) Acceptable carrier or excipient on,

Wherein, HX be selected from organic acid or inorganic acid, organic acid be selected from maleic acid, caffeic acid, forulic acid, gallic acid, methanesulfonic acid, Inorganic acid is selected from hydrochloric acid, sulfuric acid.

7. a kind of compound and its pharmaceutically acceptable salt as shown in structure formula (I) is preparing protection nerve cell, is improving Application in oxidative stress medicine,

Wherein, HX be selected from organic acid or inorganic acid, organic acid be selected from maleic acid, caffeic acid, forulic acid, gallic acid, methanesulfonic acid, Inorganic acid is selected from hydrochloric acid, sulfuric acid.

8. a kind of application of pharmaceutical composition in preparing protection nerve cell, improving oxidative stress medicine, it is characterised in that institute The pharmaceutical composition stated includes compound as shown in structure formula (I) and its pharmaceutically acceptable salt and pharmaceutically acceptable Carrier or excipient,

Wherein, HX be selected from organic acid or inorganic acid, organic acid be selected from maleic acid, caffeic acid, forulic acid, gallic acid, methanesulfonic acid, Inorganic acid is selected from hydrochloric acid, sulfuric acid.

9. a kind of compound and its pharmaceutically acceptable salt as shown in structure formula (I) is in regulation neuroinflamation medicine is prepared Application,

Wherein, HX be selected from organic acid or inorganic acid, organic acid be selected from maleic acid, caffeic acid, forulic acid, gallic acid, methanesulfonic acid, Inorganic acid is selected from hydrochloric acid, sulfuric acid.

10. a kind of application of pharmaceutical composition in regulation neuroinflamation medicine is prepared, it is characterised in that described drug regimen Thing includes the compound and its pharmaceutically acceptable salt and pharmaceutically acceptable carrier or figuration as shown in structure formula (I) Agent,

Wherein, HX be selected from organic acid or inorganic acid, organic acid be selected from maleic acid, caffeic acid, forulic acid, gallic acid, methanesulfonic acid, Inorganic acid is selected from hydrochloric acid, sulfuric acid.

A kind of 11. compounds and its pharmaceutically acceptable salt as shown in structure formula (I) are preparing improvement learning and memory function Application in medicine,

Wherein, HX be selected from organic acid or inorganic acid, organic acid be selected from maleic acid, caffeic acid, forulic acid, gallic acid, methanesulfonic acid, Inorganic acid is selected from hydrochloric acid, sulfuric acid.

A kind of 12. pharmaceutical compositions are preparing the application in improving learning and memory function medicine, it is characterised in that described medicine Composition include compound as shown in structure formula (I) and its pharmaceutically acceptable salt and pharmaceutically acceptable carrier or Excipient,

Wherein, HX be selected from organic acid or inorganic acid, organic acid be selected from maleic acid, caffeic acid, forulic acid, gallic acid, methanesulfonic acid, Inorganic acid is selected from hydrochloric acid, sulfuric acid.