CN111840285A

CN111840285A - GPR17 and/or CSFR1 receptor inhibitors

Info

Publication number: CN111840285A
Application number: CN202010524062.8A
Authority: CN
Inventors: 杨义力
Original assignee: Suzhou Ankangmeng Medical Technology Co ltd
Current assignee: Suzhou Ankangmeng Medical Technology Co ltd
Priority date: 2020-06-10
Filing date: 2020-06-10
Publication date: 2020-10-30

Abstract

The present invention relates to a GPR17 and/or CSFR1 receptor inhibitor. The invention discloses application of at least one of compounds shown as a formula (I) and a formula (II) in preparation of GPR17 and/or CSFR1 receptor inhibitors. The compounds shown in the formula (I) and the formula (II) can act on CSFR1 to inhibit the activation of microglia, act on GPR17 to promote myelin protection, and can be used for preparing medicines for preventing and/or treating central nervous system injury and neurodegenerative diseases with reduced PV neuron number or abnormal function.

Description

GPR17 and/or CSFR1 receptor inhibitors

Technical Field

The present invention relates to compounds acting on GPR17 and/or CSFR1, in particular to a GPR17 and/or CSFR1 receptor inhibitor.

Background

Colony stimulating factor receptor 1(CSFR1) is an important growth factor receptor located on the surface of bone marrow progenitor cells, macrophages and other monocytes that regulates the growth and differentiation of monocytes by binding to CSFR 1. It has been demonstrated that various inhibitors of CSF1R can be used alone or in combination with other drugs in clinical trials of tumors associated with monocyte proliferation, with good progress. It has been reported that PLX3397, an inhibitor of CSFR1, inhibits the activation of microglia in the cortex resulting from hearing loss and the corresponding tinnitus symptoms. Although a great deal of research has been carried out to date to confirm that central system injury, degeneration and various diseases are related to the abnormal activation of microglia, or long-term continuous injury, lesion and afferent loss can cause the activation of microglia, the drugs for treating diseases related to degeneration caused by the excessive activation of cerebral cortex microglia by using the microglia inhibitor have not yet entered the clinic.

The G protein-coupled receptor 17(GPR17) structure is intermediate between the cysteinyl leukotriene receptor group and the purine P2Y subfamily receptor with 28% to 48% amino acid homology. GRP17 is a G protein-coupled receptor that functions primarily through a G protein linked to a Gialpha subunit, while also being linked to a Gqalpha subunit. Based on the above functions, GPR17 was found to be activated by cysteinyl leukotrienes (LTC4 and LTD4) and purines (i.e., uridine diphosphate), while it also mediates dimerization of certain related cysteinyl leukotrienes or purine receptors in cellular responses, and this dimerization was reported to vary with the differential capacity of these ligands to activate GRP17 expression in different cells. In addition, GPR17 can also be activated by emergency signaling and atherosclerosis-promoting sterols as well as synthetic compounds of widely different structures. These are associated with activation of its ligand and interaction with other G protein-coupled receptors. At present, Montelukast which is a relevant signal pathway inhibitor acts on cysteinyl leukotriene receptor 1 and is applied to the long-term and preventive treatment of allergic and non-allergic diseases caused by clinical LTC4 and LTD 4; whereas Cangrelor inhibits the P2Y purine allergic receptor and is approved as an antiplatelet agent in the united states.

GPR17 was first cloned from oligodendrocyte precursor cells of central nervous system tissues. High expression in demyelinated injured central nervous system tissues, GPR17 was induced to be expressed in injured neuronal cells, infiltrated microglia and macrophages, and activated oligodendrocyte precursor cells within 48 hours of injury. Studies on neurological disorders have found that GPR17 is highly expressed in mature oligodendrocyte precursors but not in mature oligodendrocytes, and therefore GPR17 must be downregulated to promote differentiation of precursor cells into oligodendrocytes with the ability to promote myelination and formation. Simultaneous studies have also found a significant increase in GPR17 expression in central nervous system tissues of animal models of ischemia, experimental encephalitis (EAE) and focal demyelination in central tissues of patients suffering from brain injury due to ischemia, trauma and multiple sclerosis. The use of the GPR17 inhibitor, Montelukast, reduced the inflammatory response in a rat model of age-related cognitive impairment, promoted hippocampal nerve regeneration, and improved learning and memory. GPR17 has also been found to be a sensor of central nervous system injury and to be involved in injury repair by clearing and/or promoting remyelination of damaged neurons from various insults, including aging. GPR17 has been proposed as an important candidate drug target for treating human multiple sclerosis and brain trauma, but related drugs have not yet entered clinical application.

Clinical brain tissue diseases are mostly related to cerebral tissue ischemia, hypoxia, trauma, infection, abnormal protein deposition, abnormal neuronal excitability and afferent loss. Recent studies have found that loss of neurotransmission during the acute phase of injury or over a long period of time leads to activation of microglia. Subsequently, microglia differentiate into M1 and M2 type cells, which in turn exert their inflammatory response regulation as well as neuronal trophic and repair functions. In acute reaction period of severe environmental change and injury, the microglia can be differentiated into M1 type cells with inflammation regulation function, and the effects of removing pathogenic microorganisms, foreign matters and abnormal deposited proteins, blocking peripheral tissue infection and the like are achieved. Activation of microglia further triggers activation and response of astrocytes and other inflammation-related cells within central nervous tissue. On the other hand, excessive clearance of microglia can affect the immune balance of the central nervous system, and then 'glial cell inflammatory factor storm' is triggered, so that the function of the microglia is compensated by excessive increase of astrocytes. Abnormal activation of these central nervous system immune cells can lead to severe impairment of the protective function of the myelination of oligodendrocytes, leading to a variety of neuronal dysfunctions, injuries, and death-related degenerative changes. Although the literature reports that myelin protection of neuronal cells is enhanced after the removal of most microglia using CSF1R inhibitors, the mechanism is not yet elucidated and there is a trigger for "glial inflammatory factor storm". However, the residue of abnormally activated microglia may trigger a chronic inflammatory response over a long period of time, further affecting the myelin protection function of neuronal cells and oligodendrocytes. Therefore, the safety of the inhibitor in clinical application needs to be further studied.

In recent years, it has been found that hearing loss is an independent risk factor for the induction of senile dementia, and may be the earliest manifestation of senile dementia, and cognitive ability may be "premature" for 4 years every 10dB of hearing loss, which accelerates the appearance of senile dementia. As people age, their hearing begins to progressively deteriorate, which many find itself as a natural aging process. However, in the elderly, after hearing loss, the signals transmitted into the brain decrease, and over time, degenerative changes occur in the corresponding auditory central parts of the brain. Due to the abstraction and complexity of auditory information, the auditory center is degraded in function, which results in the degradation of the analysis and processing functions of the whole brain. Studies have found that people with mild hearing loss are twice as likely to develop dementia later than normal hearing, 3 times more likely to develop dementia at a later date, and 5 times more likely to develop dementia for people with severe hearing loss. Hearing loss is probably the earliest manifestation of dementia, and the correct intervention rate of China for hearing loss of the elderly is not enough 1/5. Elderly people have reduced hearing, mainly due to degenerative changes in the auditory nervous system, which disrupt signals and communications between neural cells in the brain, causing elderly people to often have to spend more effort listening and often get worse results. Another problem of hearing loss, which is manifested in the difficulty of the average elderly understanding the speech of others in a noisy environment, may arise from two degradations, one being substantial hearing loss and the other being loss of brain's ability to handle the problem. The latter occurrence means that even if the hearing aid is worn (to solve the hearing loss problem), the problem of the auditory system obtaining useful information from the external environment cannot be solved. These findings reveal the important role of neurodegenerative changes in the elderly brain on the hearing impact, so understanding the degenerative mechanisms of the brain at the molecular level will help to find ways and effective strategies that can target the prevention or treatment of hearing loss.

The study found that with age, the number of Parvalbumin (PV) neurons, an important inhibitory neuron, decreased. The reduction in the number or function of the neurons is associated with various neurodegenerative diseases, such as alzheimer disease, but the intrinsic mechanism thereof remains to be elucidated. There is currently no clinically effective agent that effectively inhibits PV neuron damage or reduction in number. Studies have demonstrated that the axon diameter of PV neuronal cells is fine compared to that of normal pyramidal cells, whereas oligodendrocytes have poor recognition and protection functions for the finer axons. Once damaged, subsequent repair may not be accomplished, resulting in dysfunction or death of neuronal cells. Therefore, protection of oligodendrocytes is of paramount importance for the maintenance of PV neuron function.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a GPR17 and/or CSFR1 receptor inhibitor, and the compounds shown in the formula (I) and the formula (II) can act on CSFR1 to inhibit the activation of microglia, act on GPR17 to promote myelin protection, and can be used for preparing a medicine for preventing and/or treating neurodegenerative diseases with central nervous system injury and reduced PV neuron number or abnormal function.

The invention aims to disclose the application of at least one of the compounds shown in the formulas (I) and (II) in preparing GPR17 and/or CSFR1 receptor inhibitors:

wherein A is₁Selected from NH, S or O;

A₂selected from C, N, S or O;

R₁、R₂and R₃Each independently selected from hydrogen, halogen, cyano, azido, nitro or C1-C6 alkoxycarbonylamino;

wherein A is selected from hydrogen, halogen, cyano, azido, nitro or amido bond;

R₅and R₆Each independently selected from hydrogen, halogen, cyano, azido, nitro or pentolyl.

In the formula (I) of the invention, the structural general formula of the C1-C6 alkoxycarbonylamino is R-O-CO-NH-, wherein R represents C1-C6 alkyl, preferably C3-C6 cycloalkyl.

More preferably, the compound of formula (I) comprises the following structural formula:

preferably, the compound of formula (II) comprises the following structural formula:

preferably, at least one of the compounds of formula (I) and formula (II) is used in the preparation of GPR17 and CSFR1 receptor inhibitors.

Further, GPR17 and/or CSFR1 receptor inhibitors are useful for the preparation of a medicament for the treatment and/or prevention of neurodegenerative diseases.

Further, neurodegenerative diseases are caused by inhibitory neuronal damage, and include multiple sclerosis, Alzheimer's disease, dementia, auditory hypersensitivity, migraine, tinnitus secondary to deafness due to noise, neuralgia due to nerve afferent loss, and the like.

Further, a GPR17 receptor inhibitor is useful for the preparation of a medicament for the treatment and/or prevention of a GPR17 mediated dysmyelination related disease.

Further, diseases associated with GPR 17-mediated dysmyelination include multiple sclerosis, alzheimer's disease, dementia, hyperacusis, migraine, secondary tinnitus induced by noise-induced deafness, and the like.

Further, the CSFR1 receptor inhibitor is used for preparing a medicine for treating and/or preventing diseases caused by abnormal activation of microglia.

Furthermore, diseases caused by abnormal activation of microglia include multiple sclerosis, hyperacusis, migraine, secondary tinnitus caused by deafness due to noise, and neuralgia caused by nerve afferent loss.

By the scheme, the invention at least has the following advantages:

the invention discloses compounds acting on GPR17 and CSFR1, which have the functions of promoting myelination and removing microglia, can protect the functions of damaged neuron cells in time, and reduce the influence of the damage on the myelination protection of the neuron, and are particularly important for diseases which are possibly existed simultaneously in inflammation and myelination reduction.

The compound of the invention is used for preparing the medicine for treating clinically relevant diseases caused by abnormal activation of microglia and reduction of myelination mediated by GRP 17; for the preparation of a medicament for the prevention and/or treatment of myelination disorders and syndromes associated with brain tissue damage or neurological and psychiatric disorders, a therapeutically effective amount of a compound of the present invention as defined above may be administered to a patient in need thereof.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.

Drawings

FIG. 1 shows fluorescence measurements of myelin sheaths of neurons in auditory cortex of different experimental groups of mice in example 3.

Detailed Description

The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

EXAMPLE 1 assay of the inhibitory Activity of GPR17 and CSFR1

To test the inhibitory activity of both classes of compounds of the invention on GPR17 and CSFR1, inhibitory activity test experiments were performed as follows. The compounds used therein include Compound I, Compound II, Montelukast (from TCI) and PLX3397 (from Sigma), and other reagents used are commercially available. Montelukast and PLX3397 served as positive controls for GPR17 and CSFR1 receptor inhibitors, respectively. The structural formulas of the compound I and the compound II are as follows in sequence:

the cells used in the experiments included HEK293 cells stably expressing GPR17/Gal6 and CSFR1/Gal6, respectively.

Compound I, Compound II, Montelukast and PLX3397 were dissolved in DMSO to give 4 test drug solutions, which were stored at 4 ℃. HEK293 cells stably expressing GPR17/Gal6 or CSFR1/Gal6 were plated in 96-well plates and cultured overnight. The next day, the culture medium was discarded, 40. mu.L/well of newly prepared chromogenic dye (Fluo-4/AM) was added, and 5% CO was added₂And cultured at 37 ℃ for 40 min. And (3) diluting and uniformly mixing the 4 kinds of medicine solutions to be detected by using a calcium buffer solution to obtain the calcium buffer solution dissolved with the medicine to be detected. The dye in the culture well was aspirated up and discarded, the culture well was washed with newly prepared calcium buffer, 50. mu.L of calcium buffer containing the drug to be tested dissolved therein was added to the culture well, and the mixture was put into a fluorescence detector, and 25. mu.L of calcium buffer containing the known agonist dissolved therein was added at 15 seconds from the time of putting into the fluorescence detector (GPR17 agonist MDL 29951, Santa Cruz Bio Inc.; recombinant human M-CSF protein, 216-MC-025, R)&Company D), and the fluorescence intensity was measured at a wavelength of 525 nm.

The test principle of the above experimental steps is as follows: by establishing a cell line with a target receptor, the activation of the target receptor can cause the activation of Gal6 protein, and further activate phospholipase C (PLC) to generate IP3 and DAG, and the IP3 can be combined with the IP3 receptor on the endoplasmic reticulum and mitochondria, thereby causing the release of calcium ions in cells. Therefore, measurement of the change in intracellular calcium ion concentration is useful as a method for detecting the activation state of a target receptor. Fluo-4/AM is a calcium fluorescent probe indicator, is used for measuring calcium ions, is used as a nonpolar fat-soluble compound, and is dissociated by AM groups under the action of cell lipolytic enzyme after entering cells to release Fluo-4. Because Fluo-4 is a polar molecule that does not readily pass through the lipid bilayer cell membrane, it allows Fluo-4 to remain within the cell, and the level of activation of the Gal6 protein can then be reflected by measuring the intensity of the fluorescence that is excited. If the compound to be screened is capable of activating the target receptor, the calcium ion response may be increased, whereas the calcium ion response may be decreased.

The results of the above experimental procedures are shown in table 1, from which it can be seen that the two positive controls exert their normal inhibitory effects on the activity of GPR17 and CSFR1, respectively, whereas the two compounds of the present invention show stronger binding and inhibitory activities of GPR17 and CSFR1 at low concentrations.

TABLE 1 GPR17 and CSFR1 inhibitory Activity cell assay

Example 2 GPR17 and CSFR1 inhibition of mouse microglial activation assay

To test the function of the two classes of compounds of the present invention in inhibiting microglial activation in vivo, an experiment for inhibiting mouse microglial activation was performed as follows.

A C57BL/6J mouse is taken as an experimental object to construct a mouse hearing impairment model. Specifically, mice were exposed to high frequency noise for 2 hours. The control group and the hearing impaired group were set as controls at the same time. The control group was normal mice. After the mouse hearing impairment model was constructed, 4 compounds used in example 1 were intraperitoneally injected into compound I group, compound II group, Montelukast group, and PLX3397 group, respectively, and the morphological changes of mouse cortical microglia were detected by injecting an equal amount of PBS solution into the hearing impairment group. Frozen sections were prepared from mouse brain tissue and immunofluorescent staining was performed using IBA1 antibody and corresponding secondary antibodies. Morphological observation and statistical analysis were performed after mounting.

The test principle of the above experimental steps is as follows: the hearing impairment causes the activation of microglia in the auditory cortex of the mouse, and the activation of the microglia in the auditory cortex can be reduced after the treatment by using the compound with the function of inhibiting the activation of the microglia.

Table 2 shows the effect of different compounds on the microglia morphology, and in table 2, the meaning of the microglia morphology change is the percentage of the microglia cell body area in the whole area of the microglia, and a larger value of the microglia morphology change indicates that the cell is more likely to be in an activated state. The results confirmed that the auditory cortex microglia of the hearing-impaired mice were activated, consistent with literature reports. In the experiment, the positive control medicament plays a role in inhibiting the activation of microglia, and the two compounds in the invention also have a strong effect of inhibiting the activation of auditory cortex microglia.

TABLE 2 results of the Effect of different compounds on microglial morphology

Example 3 GPR17 and CSFR1 inhibit cortical demyelination in mice

To test the in vivo inhibition of cortical demyelination in mice by the two classes of compounds of the present invention, the following experiments were performed.

A mouse hearing impairment model was constructed as described in example 2, using C57BL/6J mice as the subjects. The control group and the hearing impaired group were set as controls at the same time. The control group was normal mice. After the mouse hearing impairment model was constructed, 4 compounds used in example 1 were intraperitoneally injected into compound I group, compound II group, Montelukast group, and PLX3397 group, respectively, and the change in fluorescence staining intensity of cortical myelin sheath of mouse was detected by injecting an equal amount of PBS solution into hearing impairment group. Frozen sections were prepared from mouse brain tissue and immunofluorescent staining was performed using MBP antibody and corresponding secondary antibody. Morphological observation and statistical analysis were performed after mounting.

The test principle of the above experimental steps is as follows: the hearing impairment results in a reduction in the protection of the myelin sheaths of the auditory cortical neurons in the mouse, and the loss of the myelin sheaths of the auditory cortical neurons can be reduced when treated with a compound having the function of inhibiting the activation of microglia.

Table 3 shows the effect of different compounds on myelin staining fluorescence intensity (MBP), and FIGS. 1a-d show the fluorescence measurements of myelin sheath of auditory cortical neurons of mice in the control group, hearing impaired group, compound I group, and compound II group, respectively. The result shows that the myelin sheath of auditory cortical neuron cells of the hearing-impaired mouse is obviously lost. In the experiment, the positive control medicament plays a role in inhibiting the loss of the myelin sheath of the neuron, and the two compounds in the invention also show a strong effect of inhibiting the reduction of the myelin sheath of the auditory cortex neuron.

TABLE 3 Effect of different Compounds on myelin staining fluorescence intensity (MBP)

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Claims

1. Use of at least one of the compounds of formula (I) and formula (II) in the preparation of a GPR17 and/or CSFR1 receptor inhibitor:

wherein A is₁Selected from NH, S or O;

A₂selected from C, N, S or O;

2. The use of claim 1, wherein the compound of formula (I) comprises the following structural formula:

3. the use of claim 1, wherein the compound of formula (II) comprises the following structural formula:

4. use according to any one of claims 1-3, characterized in that: at least one of the compounds of formula (I) and formula (II) is useful in the preparation of GPR17 and CSFR1 receptor inhibitors.

5. Use according to claim 4, characterized in that: the GPR17 and/or CSFR1 receptor inhibitor is used for preparing a medicament for treating and/or preventing neurodegenerative diseases.

6. Use according to claim 5, characterized in that: the neurodegenerative disease is caused by inhibitory neuron damage, and the neurodegenerative disease comprises one or more of multiple sclerosis, Alzheimer disease, dementia, secondary tinnitus caused by noise-induced deafness, auditory hypersensitivity, migraine and neuralgia caused by nerve afferent deletion.

7. Use according to claim 4, characterized in that: the GPR17 receptor inhibitor is used for preparing a medicament for treating and/or preventing GPR17 mediated dysmyelination related diseases.

8. Use according to claim 7, characterized in that: the GPR17 mediated dysmyelination related diseases comprise one or more of multiple sclerosis, Alzheimer disease, dementia, auditory hypersensitivity, migraine and secondary tinnitus caused by noise-induced deafness.

9. Use according to claim 4, characterized in that: the CSFR1 receptor inhibitor is used for preparing medicines for treating and/or preventing diseases caused by abnormal activation of microglia.

10. Use according to claim 9, characterized in that: the diseases caused by the abnormal activation of the microglia comprise one or more of multiple sclerosis, auditory hypersensitivity, migraine, secondary tinnitus caused by deafness caused by noise and neuralgia caused by nerve afferent deficiency.