CN102977054A - Application of selective alpha2A receptor stimulant in treating alzheimer disease - Google Patents

Abstract

The invention discloses application of a selective alpha2A receptor stimulant in treating the alzheimer disease. The invention relates to a compound which has beta1-epinephrine acceptor stimulating activity and is represented by a formula (I) or medically accepted salt. The activity of stimulating a beta1-epinephrine receptor is extremely high; EC50 is in the nM level; the EC50 of the compound with the highest activity is 24.34; and the compound can be used for a nerve protection agent and is used for treating the alzheimer disease.

Description

One class selectivity α 2AThe treatment alzheimer's disease purposes of receptor stimulant

Technical field

The present invention relates to a class and have α _2AThe compound of-2-adrenoreceptor agonist activity, this compounds is as α _2A-adrenoceptor receptor stimulant has the activity of good treatment alzheimer's disease.

Background technology

Alzheimer's disease (Alzheimer ' s disease, AD) also claim senile dementia or degenerative brain disorder, is that a common class of senium is chronic, the degeneration of carrying out property neurocyte.Along with the aging of population, AD has become and is only second to cardiovascular diseases, tumour and apoplexy and occupies the 4th the cause of the death, and there are about 2,430 ten thousand people of AD patient in the whole world at present, expects the year two thousand forty whole world and will have AD patient 81,000,000.About the research of the cause of disease, prevention and the treatment target spot of AD becomes one of urgent hot subject in the current world.Pathological characters is mainly amyloid-beta (β-amyloid in patient's AD the brain, A β) neurofibrillary tangles and the neuron loss due to the senile plaque of formation of deposits (Senile Plaque, SP), microtubule-associated protein (Tau albumen) Hyperphosphorylationof.Many investigators inquire into the cause of disease, pathomechanism and the treatment means of AD from aspects such as each different angle such as amyloid beta deposition, inflammatory mediator, vascular factor, cholesterol metabolics in recent years.But up to the present, the AD cause of disease and pathomechanism are not yet fully clear, and be still few to the definite effectively medicine of AD, also do not have the medicine of etiological treatment radical cure AD.

α _2A-adrenoceptor (α _2A-adrenoceptor, α _2A-AR) belong to a kind of hypotype in 7 transmembrane receptor superfamily members of G albumen coupling, extensively be distributed in the histoorgans such as thrombocyte, brain, spinal cord, adipocyte, kidney, spleen, its biological effect is mainly protein mediated by Gi/Go.α _2AAfter activating ,-AR can suppress adenylate cyclase (AC) activity by the adjusting of Gi/Go albumen, reduce the level of cyclic monophosphate (cAMP) in the cell, the protein phosphorylation of arrestin kinases A (PKA) and regulation and control thereof is playing key effect aspect the neurotransmitter release of regulation and control sympathetic nerve and central nervous system norepinephrine neuron.

Along with α _2AThe a large amount of discoveries of-AR in pallium, increasing research steering α _2AThe effect of-AR aspect central nervous system.Had in recent years on a small quantity about α _2A-AR agonist is in treatment schizophrenia, calmness, analgesia, anticonvulsion, anxiety, improvement memory, and there is the report of certain effect the drug habit aspect.In addition, report α is also arranged _2A-AR agonist can be by suppressing the cAMP of prefrontal cortex, hyperpolarization cyclic nucleotide signal pathway and strengthen the working memory that fails in Parkinson's illness.Recent years abroad has several pieces of reports of only a few, thinks α _2A-AR agonist may increase the growth of cortical neuron, further AD be produced therapeutic action by reducing the phosphorylation of microtubule-associated protein 2 (MAP-2) on Serine and threonine residues.

Neuroprotective, neurotrophic agents, nerve regeneration agent are the novel medicines that is used for the treatment of AD of a class; clinical application both at home and abroad shows, brings into use memantine, neurotrophic factor in acute phase or the chronic phase of AD pathology; the neuroprotectives such as mouse nerve growth factor have good therapeutic action.The market share of neuroprotective also increases year by year.At present, the injection nerve growth factor of China's development has been accelerated clinical drug and commercial process behind the kind biological product New Drug Certificate that acquisition SFDA issues.

α _2A-AR agonist stimulates cortical neuron this effect of growing to have great importance for the exploitation neuroprotective, for prevention and the treatment of AD provides new approach.

Summary of the invention

The object of the present invention is to provide a kind of compound for the treatment of alzheimer's disease, this compound is α _2A-AR agonist has prevention or therapeutic action to alzheimer's disease.Compound of the present invention comprises compound or its pharmacy acceptable salt that contains the chemical formula (I) for the treatment of significant quantity.The present invention adopts High Throughput Screening Assay, has set up α _2A-AR agonist high flux screening model also is applied to the compound Large-scale Screening, has searched out the α with chemical formula (I) by screening a collection of compound from the purchase of CHEMDIV company and carrying out functional checking _2A-AR agonist.

Technical scheme of the present invention is: stable transfection α in Chinese hamster ovary cancer cells (CHO) _2AAcceptor is set up α _2A-AR agonist high flux screening model carries out primary dcreening operation, multiple sieve, and the structure activity relationship analysis obtains a class and has α _2AThe drug candidate of receptor agonist activity.Concrete steps are as follows:

Step 1: set up and cultivate stable transfection α _2AThe Chinese hamster ovary celI strain of acceptor.

Step 2: the mensuration of typical curve and optimum cell number are determined.

Step 3: positive drug checking.

Step 4: adopt stable cell strain and the optimal detection condition of groping that compound is carried out α _2AThe high flux screening of receptor stimulant obtains the compound of obvious dose-effect relationship.

Description of drawings

Fig. 1: typical curve and optimum cell number curve (600cell/ μ l)

Fig. 2: positive drug guanfacine (Guanfacine) amount effect curve

Embodiment

Below in conjunction with description of drawings the specific embodiment of the present invention

1. set up and cultivate stable transfection α _2AThe Chinese hamster ovary celI strain of-AR.

The box switching technology (Recombinase-Mediated Cassette Exchange, RMCE) of using recombinase-mediated makes up the g protein coupled receptor cell strain in batches, and makes its stable transfection α _2A-AR cultivates CHO-α _2ACell makes it reach the stable growth state.

2. the mensuration of typical curve and optimum cell number are determined.

For the exactness of confirmatory experiment system and can filter out reliable α _2A-AR agonist needs the bioassay standard curve and determines the optimum cell number when setting up model.Treat CHO-α _2AGrowth of Cells to 80% the time with the PBS peptic cell of 0.5mM EDTA, centrifugal removal supernatant liquid, at last with cell with stimulating damping fluid resuspended for definite optimum cell number.Use the LANCE of PerkinElmer company ^TMCAMP 384Kit test kit, preparation standard cAMP gradient dilution liquid and adenylate cyclase activating agent (forskolin) gradient dilution liquid.The standard curve determination method: with the standardized solution 4 μ mol/L of cAMP ( The cAMP detection kit carries), be the working concentration of 4 times of final concentrations with stimulating the damping fluid stepwise dilution, each 5 μ l of cAMP and antibody-solutions add hatched in 384 orifice plates 45 minutes jointly, added 10 μ l stop buffers, hatched after one hour and detected; Optimum cell is counted detection method: be the working concentration of 4 times of final concentrations with the forskolin mother liquor of 50mM with stimulating the damping fluid stepwise dilution, forskolin adds with each 5 μ l of the antibody-solutions that contains cell hatched in 384 orifice plates 45 minutes jointly, add 10 μ l stop buffers, hatch after one hour and detect at EnVision microwell plate plate reading.According to forskolin amount effect curve window (property is made an uproar than S/B) and the curve cell concn close with the cAMP slope of standard curve as the optimum cell number.Typical curve and optimum cell number determine to see Fig. 1.

3. select α _2A-AR selective agonist Guanfacine verifies and calculates according to amount effect curve its EC to the high flux screening model of setting up ₅₀

The positive drug measuring method: with the Guanfacine mother liquor of 100mM, be the working concentration of 4 times of final concentrations with stimulating the damping fluid stepwise dilution, configuration can cause that optimum cell counts Ratio EC ₉₀The forskolin diluent of effect.Positive drug Guanfacine2.5 μ l adds with the antibody-solutions 5 μ l that contain the optimum cell number hatched in 384 orifice plates 15 minutes jointly, again forskolin diluent 2.5 μ l are added and hatched in 384 orifice plates 30 minutes, add 10 μ l stop buffers and hatch again after one hour with the detection of EnVision microwell plate plate reading.The positive drug amount effect curve is seen Fig. 2.

4. under above-mentioned optimal detection condition, carry out α _2A-AR agonist primary dcreening operation and multiple sieve.

Primary dcreening operation is with to sieve again process identical, primary dcreening operation only selects a concentration that 80,000 compounds are carried out preliminary screening, select again 8 concentration of primary dcreening operation compounds effective gradient dilution to carry out multiple sieve, process is as follows: with Janus full-automatic application of sample workstation diluted compounds and draw 5 μ l and transfer in 384 orifice plates, the antibody-solutions 5 μ l that will contain the optimum cell number with the Multidrop automatic sample adding instrument again add in 384 orifice plates, after both hatch 45 minutes jointly, the Multidrop automatic sample adding instrument adds 10 μ l stop buffers in 384 orifice plates, again hatched one hour, and detected with EnVision microwell plate plate reading.Multiple sieve obtains a series of α of having _2AThe compound of-AR agonism.

Sieve again experimental result

The compound that screening obtains can be summarized as according to the structure of parent nucleus a class, structural formula and EC ₅₀As follows:

The multiple sieve of table 1 obtain to α _2A-AR has a compounds structure of agonism

The multiple sieve of table 2 obtain to α _2A-AR has the compd E C of agonism ₅₀

Claims (3)

1. following formula: compound or its acceptable salt pharmaceutically:

Wherein:

R ₁Be hydrogen atom;

R ₂Be hydrogen atom, C ₁-C ₆The alkyl of straight or branched, replacement or unsubstituted phenyl ,-R ⁴CONR ⁵R ⁶,

-SR ⁵

R ₃Be hydrogen atom, C ₁-C ₆The alkyl of straight or branched, replacement or the benzyl for replacing;

R ⁴Be C ₁-C ₆The alkyl of straight or branched,

R ⁵, R ⁶Be hydrogen, C independently of one another ₁-C ₆The alkyl of straight or branched, replacement or unsubstituted benzyl, replacement or unsubstituted phenyl, the phenyl of wherein said replacement comprise 1～2 substituting group; Described substituting group is halogen, methyl, methoxyl group.

The compound of the claimed formula (I) of claim 1 or its pharmaceutically acceptable salt for the preparation of having as α _2AThe purposes of the pharmaceutical preparation of the pharmacological action of receptor stimulant.

3. according to the purposes of claim 2, as α _2AReceptor stimulant is used for the treatment of alzheimer's disease.

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