CN102977054A - Use of a class of selective α2A receptor agonists for treating Alzheimer's 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

Use of a class of selective α2A receptor agonists for treating Alzheimer's disease

technical field

The present invention relates to a class of compounds with α _2A -adrenoceptor agonist activity. As α _2A -adrenergic receptor agonists, the compound has excellent activity in treating Alzheimer's disease.

Background technique

Alzheimer's disease (Alzheimer's disease, AD), also known as senile dementia or Alzheimer's disease, is a common type of chronic and progressive neurodegeneration in old age. With the aging of the population, AD has become the fourth cause of death after cardiovascular disease, tumor and stroke. Currently, there are about 24.3 million AD patients in the world, and it is estimated that by 2040, there will be a total of 81 million AD patients worldwide. Research on the etiology, prevention and therapeutic targets of AD has become one of the urgent international hot topics. The pathological features in the brain of AD patients are mainly senile plaques (Senile Plaque, SP) formed by the deposition of β-amyloid (A β) and neurofibrillary tangles caused by hyperphosphorylation of microtubule-associated protein (Tau protein). Nodules and neuronal loss. In recent years, many researchers have explored the etiology, pathological mechanism and treatment of AD from various aspects such as amyloid deposition, inflammatory mediators, vascular factors, and cholesterol metabolism. But so far, the etiology and pathological mechanism of AD have not been fully understood, and there are still few effective drugs for AD, and there is no drug for the root cause of AD.

α _2A -adrenoceptor (α _2A -adrenoceptor, α _2A -AR) belongs to a subtype of G protein-coupled seven transmembrane receptor superfamily members, widely distributed in platelets, brain, spinal cord, fat The biological effects of cells, kidney, spleen and other tissues and organs are mainly mediated by Gi/Go protein. α _2A -AR can inhibit the activity of adenylyl cyclase (AC) through the regulation of Gi/Go protein after activation, reduce the level of intracellular cyclic adenosine monophosphate (cAMP), and inhibit protein kinase A (PKA) and its regulated proteins Phosphorylation, plays a key role in regulating neurotransmitter release from sympathetic and central nervous system norepinephrine neurons.

With the discovery of a large number of α _2A -AR in the cerebral cortex, more and more studies have turned to the role of α _2A -AR in the central nervous system. In recent years, there are a few reports about α _2A -AR agonists being effective in the treatment of schizophrenia, sedation, analgesia, anticonvulsant, anxiolytic, memory improvement, and drug addiction. In addition, it has also been reported that α _2A -AR agonists can enhance the declining working memory in Parkinson's disease by inhibiting cAMP in the prefrontal cortex and hyperpolarizing the cyclic nucleotide signaling pathway. In recent years, there have been very few foreign reports that α _2A -AR agonists may increase the growth of cortical neurons by reducing the phosphorylation of microtubule-associated protein 2 (MAP-2) on serine and threonine residues, further Have a therapeutic effect on AD.

Neuroprotective agents, neurotrophic agents, and nerve regeneration agents are a new class of drugs for the treatment of AD. Clinical applications at home and abroad have shown that memantine, neurotrophic factors, and rat nerve growth agents are used in the acute or chronic phase of AD lesions. Factors and other neuroprotective agents have good therapeutic effects. The market share of neuroprotective agents is also increasing year by year. At present, the nerve growth factor for injection developed in my country has accelerated the process of clinical and commercialization of the drug after obtaining the new drug certificate of a class of biological products issued by SFDA.

The role of α _2A -AR agonists in stimulating the growth of cortical neurons is of great significance for the development of neuroprotective agents, and provides a new way for the prevention and treatment of AD.

Contents of the invention

The object of the present invention is to provide a compound for treating Alzheimer's disease, which is an α _2A -AR agonist, and has preventive or therapeutic effects on Alzheimer's disease. The compounds of the present invention include a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. The present invention uses high-throughput screening technology to establish a high-throughput screening model for α _2A -AR agonists and apply it to large-scale screening of compounds. By screening a batch of compounds purchased from CHEMDIV and performing functional verification, a compound with the chemical formula ( I) α _2A -AR agonists.

The technical scheme of the present invention is: stably transfect _α2A receptor in Chinese hamster ovarian cancer cells (CHO), establish a high-throughput screening model of _α2A -AR agonist, conduct primary screening, secondary screening, structure-activity relationship analysis, A class of candidate drugs with _α2A receptor agonistic activity is obtained. Specific steps are as follows:

Step 1: establishing and culturing a CHO cell line stably transfected with _α2A receptor.

Step 2: Determination of standard curve and determination of optimal cell number.

Step 3: Positive drug verification.

Step 4: Perform high-throughput screening of _α2A receptor agonists on compounds using stable cell lines and explored optimal detection conditions to obtain compounds with obvious dose-effect relationship.

Description of drawings

Figure 1: Standard curve and optimal cell number curve (600cell/μl)

Figure 2: The positive drug Guanfacine (Guanfacine) dose-effect curve

Detailed ways

The specific embodiment of the present invention is described below in conjunction with accompanying drawing

1. Establish and culture a CHO cell line stably transfected with α _2A -AR.

Apply recombinase-mediated cassette exchange technology (Recombinase-Mediated Cassette Exchange, RMCE) to construct G protein-coupled receptor cell lines in batches, and make them stably transfect α _2A -AR, and cultivate CHO-α _2A cells to achieve stable growth state.

2. Determination of standard curve and determination of optimal cell number.

In order to verify the correctness of the experimental system and to screen out reliable α _2A -AR agonists, it is necessary to determine the standard curve and determine the optimal cell number when establishing the model. When the CHO- _α2A cells grew to 80%, the cells were digested with 0.5mM EDTA in PBS, the supernatant liquid was removed by centrifugation, and finally the cells were resuspended with stimulation buffer to determine the optimal cell number. The LANCE ^TM cAMP 384Kit kit from PerkinElmer was used to prepare standard cAMP gradient dilutions and adenylate cyclase activator (forskolin) gradient dilutions. Standard curve determination method: the standard solution of cAMP 4μmol/L ( The cAMP detection kit comes with), dilute step by step with stimulation buffer to the working concentration of 4 times the final concentration, add 5 μl each of cAMP and antibody solution to a 384-well plate and incubate for 45 minutes, add 10 μl stop solution, incubate for one hour and then detect ; Optimum cell number detection method: Dilute the 50mM forskolin mother solution with stimulating buffer step by step to a working concentration of 4 times the final concentration, add 5 μl each of forskolin and cell-containing antibody solution to a 384-well plate and incubate for 45 minutes, add 10 μl Stop solution, incubated for one hour and assayed on the EnVision Microplate Reader. According to the window of forskolin dose-effect curve (sex-to-noise ratio S/B) and the cell concentration whose slope is similar to that of the cAMP standard curve, the optimum cell number was used. See Figure 1 for the standard curve and optimal cell number determination.

3. Select α _2A -AR selective agonist Guanfacine to verify the established high-throughput screening model and calculate its EC ₅₀ according to the dose-effect curve.

Positive drug determination method: Dilute the 100mM Guanfacine stock solution with stimulation buffer step by step to the working concentration of 4 times the final concentration, and prepare the forskolin dilution solution that can cause the optimal cell number Ratio EC ₉₀ effect. Add 2.5 μl of the positive drug Guanfacine and 5 μl of the antibody solution containing the optimal cell number to a 384-well plate and incubate for 15 minutes, then add 2.5 μl of forskolin dilution solution to the 384-well plate and incubate for 30 minutes, add 10 μl of stop solution and incubate for another hour Detection was performed with an EnVision microplate reader. The positive drug dose-effect curve is shown in Figure 2.

4. Carry out primary screening and secondary screening of α _2A -AR agonists under the above-mentioned optimal detection conditions.

The primary screening process is the same as the re-screening process. Only one concentration is selected for preliminary screening of 80,000 compounds in the primary screening, and then the compound that is effective in the primary screening is gradually diluted to 8 concentrations for re-screening. The process is as follows: dilute with Janus automatic sampling workstation Pipette 5 μl of the compound and transfer it to a 384-well plate, then add 5 μl of the antibody solution containing the optimal number of cells into the 384-well plate with the Multidrop autosampler, and after the two are incubated together for 45 minutes, the Multidrop autosampler will stop with 10 μl The solution was added to a 384-well plate, incubated for another hour, and detected with an EnVision microplate reader. A series of compounds with α _2A -AR agonism were obtained through rescreening.

Re-screening test results

The screened compounds can be summarized into one category according to the structure of the parent nucleus, and the structural formula and EC ₅₀ are as follows:

Table 1 The structure of a class of compounds with agonistic effect on α _2A -AR obtained by rescreening

Table 2 EC ₅₀ of compounds with agonistic effect on α _2A -AR obtained by rescreening

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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