CN102977111B

CN102977111B - Beta1-epinephrine receptor analgesia with effect of improving Alzheimer disease

Info

Publication number: CN102977111B
Application number: CN201210543597.5A
Authority: CN
Inventors: 张陆勇; 何玲; 严明; 苏冉
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2012-12-12
Filing date: 2012-12-12
Publication date: 2015-04-15
Anticipated expiration: 2032-12-12
Also published as: CN102977111A

Abstract

The invention relates to a compound with beta1-epinephrine receptor analgesia activity in the chemical formula (I) and pharmaceutically acceptable salts thereof. The compound has strong activity of in-vitro agonizing beta1-epinephrine receptor, and the EC50 is in an nM level, wherein the EC50 of the compound with the strongest activity is 24.340.94nM, so that the compound can be used as a neuroprotective agent for treating Alzheimer disease.

Description

One class has the β1-adrenergicreceptor agonist improving alzheimer disease effect

Technical field

The present invention relates to the compound that a class has β1-adrenergicreceptor agonist activity, this compounds has the activity of well treatment alzheimer's disease as β 1 adrenoceptor agonists.

Background technology

Alzheimer's disease (Alzheimer ' s disease, AD), also known as senile dementia or degenerative brain disorder, is that a senium common class is chronic, the degeneration of Progressive symmetric erythrokeratodermia neurocyte.Along with the aging of population, AD has become and has been only second to cardiovascular diseases, tumour and apoplexy and the cause of the death occupying the 4th.About the cause of disease of AD and pathogeny are still not clear so far, there is no specific treatment medicine at present.The pathological hallmark of AD is amyloid-beta (β-amyloid, A β) form senile plaque and NFT-tau phosphorylation at born of the same parents' external sediment, simultaneously, neuroinflamation has become the 3rd the key pathological mark causing AD, in AD process, Neuroinflammation participates in the early stage pathogenesis of AD.Neuroinflamation process in brain mediated primarily of microglia, microglia after activation can discharge a large amount of precursor inflammatory factors, causes adjacent cells inflammation and death in brain, the deposition of aggravation amyloid-beta, thus initiation neuron dysfunction, cause neurodegenerative generation.

β1-adrenergicreceptor (β-adrenoceptor, β 1-AR) is the member of classical g protein coupled receptor family, and its biological effect mediates primarily of the Gs-cAMP-PKA signal path of classics.After β 1-AR activates by Gs albumen regulate can activated adenyl cyclase (AC) active, increase the level of cAMP in cell, the protein phosphorylation of activated protein kinase A (PKA) and regulation and control thereof, plays key effect in the neurotransmitter regulator regulating and controlling sympathetic nerve and central nervous system norepinephrine neuron.β 1-AR is relevant with the outstanding plasticity-(synaptic plasticity) of central nervous system (CNS), the activation of β 1-AR can cause long term potentiation (the long-term potentiation of hippocampus and amygdala, LTP), the memory organization of animal is strengthened.Foreign study shows, endogenous norepinephrine (NA) can make the levels such as proinflammatory inflammation factor IL-1 β, IL-6 and tumor necrosis factor TNF-alpha decline, also can the excitatory toxicity that causes of antagonist ectoglia cell L-glutamic acid, produce neuroprotective; Meanwhile, β 1-AR agonist also can tyrosine phosphatase (STEP) translation skill of increase cortico-striatal enrichment of dose-dependently, and this enzyme can strengthen cognition, improve learning and memory.These have studied in prompting brains the metabolism of β 1-AR and adjustment may be fallen ill with AD, prevention and therapy has certain relation, but current this type of medicine that there is no clinically occurs.

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 β1-adrenergicreceptor agonist, has prevention or therapeutic action to alzheimer's disease.Compound of the present invention comprises compound or its pharmacy acceptable salt of the chemical formula (I) containing treatment significant quantity.The present invention adopts High Throughput Screening Assay, establish β1-adrenergicreceptor agonist high flux screening model and be applied to compound Large-scale Screening, the β1-adrenergicreceptor agonist with chemical formula (I) has been searched out, simultaneously to its AD pharmacodynamics of being correlated with and Mechanism Study by screening a collection of compound from the purchase of CHEMDIV company and carrying out functional authorization.

Technical scheme of the present invention is: stable transfection β 1 adrenoceptor in Chinese hamster ovary cancer cells (CHO), set up β 1 adrenoceptor agonists high flux screening model, carry out primary dcreening operation, multiple sieve, Structure-activity analysis, obtains the drug candidate that a class has anti-alzheimer disease.Concrete steps are as follows:

Step one: the Chinese hamster ovary celI strain of setting up and cultivating stable transfection β 1 adrenoceptor.

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

Step 3: positive drug is verified.

Step 4: adopt stable cell strain compound to be carried out to the high flux screening of β 1 adrenoceptor agonists with the optimal detection condition groped, obtain the compound of pronounced amount effect relationship.

Accompanying drawing illustrates:

Fig. 1: typical curve and optimum cell number curve

Fig. 2: positive drug xamoterol (Xamoterol) amount effect relation curve

Fig. 3: the EC of compound 1-6 ₅₀

Fig. 4: the EC of compound 7-12 ₅₀

Embodiment

Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described

1. set up and cultivate CHO-β 1 cell strain of stable transfection β 1 adrenoceptor.

Box switching technology (the Recombinase-Mediated Cassette Exchange of application recombinase-mediated, RMCE) batch builds g protein coupled receptor cell strain, and make its stable transfection β 1 adrenoceptor, cultivate CHO-β 1 cell and make it reach stable growth state.

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

For confirmatory experiment system exactness and reliable β 1 adrenoceptor agonists can be filtered out, need bioassay standard curve when Modling model and determine optimum cell number.Until CHO-β 1 Growth of Cells to 80% time with the PBS peptic cell of 0.5mM EDTA, centrifugal segregation supernatant liquid, finally stimulates damping fluid resuspended for determining optimum cell number in cell.Use the LANCE of PerkinElmer company ^tMcAMP 384Kit test kit, preparation standard cyclic monophosphate (cAMP) gradient dilution liquid and adenylate cyclase activating agent (forskolin) gradient dilution liquid.Standard curve determination method: by standardized solution 50 μm of ol/L of cAMP ( cAMP detection kit carries), be the working concentration of final concentration 4 times with stimulation damping fluid stepwise dilution, cAMP and each 5 μ l of antibody-solutions adds in 384 orifice plates hatches 45 minutes jointly, adds 10 μ l stop buffers, hatches after one hour and detects; Optimum cell number detection method: the working concentration by the forskolin mother liquor of 50mM stimulation damping fluid stepwise dilution being final concentration 4 times, forskolin adds in 384 orifice plates with each 5 μ l of antibody-solutions containing cell hatches 45 minutes jointly, add 10 μ l stop buffers, hatch after one hour and detect on EnVision microwell plate plate reading.According to forskolin amount effect curve window (to-noise ratio S/B) and the curve cell concn close with cAMP slope of standard curve as optimum cell number.Typical curve and optimum cell number are determined to see Fig. 1.

3. select the positive drug Xamoterol of β 1 adrenoceptor agonists to verify set up β 1 suprarenin screening active ingredients model and calculate its EC according to amount effect curve ₅₀.

Positive drug measuring method: by the Xamoterol mother liquor of 100mM, the working concentration that stepwise dilution is final concentration 2 times is cushioned with stimulating, positive drug Xamoterol 5 μ l adds in 384 orifice plates with the antibody-solutions 5 μ l containing optimum cell number hatches 45 minutes jointly, add 10 μ l stop buffers to hatch with the detection of EnVision microwell plate plate reading after one hour, positive drug amount effect curve is shown in Fig. 2 again.

4. carry out under above-mentioned optimal detection condition β 1 adrenoceptor agonists primary dcreening operation and multiple sieve.

Primary dcreening operation is identical with multiple sieved journey, primary dcreening operation only selects a concentration to carry out preliminary screening to 80,000 compounds, primary dcreening operation compounds effective gradient dilution 8 concentration are selected to carry out multiple sieve again, process is as follows: draw 5 μ l transfer in 384 orifice plates by Janus full-automatic application of sample workstation diluted compounds, with Multidrop automatic sample adding instrument, the antibody-solutions 5 μ l containing optimum cell number is added in 384 orifice plates again, after both hatch 45 minutes jointly, 10 μ l stop buffers add in 384 orifice plates by Multidrop automatic sample adding instrument, again hatch one hour, detect with EnVision microwell plate plate reading.Multiple sieve obtains a series of compound with D1 receptor agonism.Processing data, computerized compound EC ₅₀, the EC of each compound ₅₀see Fig. 3, Fig. 4.

Sieve experimental result again

Screen the compound obtained and can be summarized as a class according to the structure of parent nucleus, structural formula and EC ₅₀as follows:

2-phenyl-5H-chromeno[2，3-d]pyrimidine-4-thiol

Table 1 sieves compounds structure β1receptor being had to agonism obtained again

Table 2 sieves compounds EC β1receptor being had to agonism obtained again ₅₀

Claims

1. formula (I) or its pharmaceutically acceptable salt for the preparation of the purposes of pharmaceutical preparation with the pharmacological action of β 1 adrenoceptor agonists:

Its Chinese style (I) compound is following particular compound:

Compound 1, R1 is H, R2, and to be H, R3 be r4 is H;

Compound 2, R1 is-CH ₃, to be H, R3 be R2 r4 is H;

Compound 3, R1 is-CH ₃, to be H, R3 be R2 r4 is F;

Compound 4, R1 is H, R2, and to be H, R3 be r4 is Cl;

Compound 5, R1 is H, R2 is-CH ₃, R3 is r4 is-CH ₃;

Compound 6, R1 is-CH ₃, to be H, R3 be R2 r4 is-CH ₃;

Compound 7, R1 is H, R2 is-CH ₃, R3 is r4 is

Compound 8, R1 is-CH ₃, to be H, R3 be R2 r4 is

Compound 9, R1 is H, R2 is-CH ₃, R3 is r4 is H;

Compound 10, R1 is H, R2 is-CH ₃, R3 is r4 is H;

Compound 11, R1 is H, R2, and to be H, R3 be r4 is H;

Compound 12, R1 is H, R2, and to be H, R3 be r4 is H.

2. pharmaceutical applications according to claim 1, the pharmaceutical preparation with the pharmacological action of β 1 adrenoceptor agonists is used for the treatment of alzheimer disease.