Use of a compound in the modulation of tgr5 receptor activity and in the treatment of neuroprotection or depression

Abstract

The application of a compound in regulating TGR5 receptor activity and in treating neuroprotection or depression, wherein the compound L2301 is screened from a small molecule library, and the compound can specifically activate the TGR5 receptor. Pharmacodynamic experiments show that the L2301 can penetrate the blood-brain barrier, has a protective effect on PC12 cells induced by glutamate, and implies that the compound has a function of protecting nerve cells. In the open field experiment and the elevated plus maze experiment, the L2301 can obviously improve the behavior of the depressive mice, and exhibits an excellent anti-depression effect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a compound that modulates TGR5 receptor activity and its application in neuroprotection or the treatment of depression. Background Technology

[0002] Mental illness is a disease characterized by disordered nervous activity, causing abnormal changes in a patient's psychology, emotions, cognition, and behavior. Depression is one of the most common mental illnesses, clinically manifested as prolonged low mood and lack of pleasure / interest.

[0003] The causes of depression are complex, and its pathogenesis and prevention have always been a hot topic in this field of research. Classical treatment for depression involves a combination of psychotherapy and medication, with selective serotonin reuptake inhibitors (SSRIs) being the most prominent, including fluoxetine, paroxetine, and citalopram. However, the clinical antidepressant effects of these drugs often appear several weeks or even months after starting medication; moreover, they only alleviate symptoms in 70%-80% of patients with depression. Long-term use of antidepressants, due to side effects and reduced efficacy, often fails to effectively control clinical symptoms, prompting researchers to explore new therapeutic targets beyond serotonin.

[0004] The TGR5 receptor, a bile acid membrane receptor, was first discovered by Maruyama and Kawamata in 2002. Composed of 330 amino acids and containing seven transmembrane domains, it belongs to the GPCR family. TGR5 is mainly distributed in the gallbladder, intestine, adipose tissue, and muscle tissue. TGR5 plays an important regulatory role in various physiological processes, such as energy metabolism, immune inflammatory responses, hepatobiliary function, and gastrointestinal function, and has potential therapeutic value in metabolic diseases such as diabetes and obesity.

[0005] Recent studies have shown that TGR5 is highly expressed in the brain tissue of humans and rodents, and is expressed in neurons, astrocytes, and microglia. Numerous studies have confirmed that TGR5 in the hypothalamus can mediate central anorexia, protecting the body from obesity. TGR5 in the hippocampus activates CA3 pyramidal neuronal activity, producing an antidepressant effect. Regulating TGR5 can also inhibit lipopolysaccharide-induced cognitive impairment, playing a neuroprotective role. Therefore, TGR5 also holds promise as an effective target for treating depression.

[0006] Currently, reported TGR5 receptor agonists can be classified into three types based on their structure: steroids, triterpenoids, and synthetic small molecules. However, most of these existing compounds have difficulty crossing the blood-brain barrier, thus limiting their effectiveness in the prevention and treatment of central nervous system diseases.

[0007] Therefore, developing novel TGR5 agonists for the prevention or treatment of central nervous system disorders such as neuroprotection, anxiety, and depression is of great research value. We used a computer-aided drug design supercomputing platform for virtual screening and verified through in vitro activity experiments that compound L2301 possesses strong TGR5 agonist activity. In vivo experiments showed that L2301 can cross the blood-brain barrier, improve glutamate-induced damage in PC12 cells, and exert a neuroprotective effect. Simultaneously, animal experiments showed that L2301 can significantly improve the behavior of CUMS-induced depressed mice. This molecule is the first to be discovered to possess TGR5 agonist activity, has a novel structure, and exhibits significant antidepressant activity, making it of great research value. Summary of the Invention

[0008] To overcome the shortcomings of the prior art, the present invention aims to provide a compound for regulating TGR5 receptor activity and its application in neuroprotection or the treatment of depression. This compound can cross the blood-brain barrier and protect against glutamate-induced damage to PC12 cells, suggesting that it has a neuroprotective function. In open field and elevated cruciate maze tests, this compound significantly improved the behavioral performance of depressed mice, demonstrating excellent antidepressant effects.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A compound for regulating TGR5 receptor activity has the following structural formula (Ⅰ): (I).

[0010] Furthermore, the compound shown in formula (I) is used to prepare drugs that regulate the activity of the TGR5 receptor.

[0011] Furthermore, the compound shown in formula (I) is used to prepare agonists that regulate the activity of the TGR5 receptor.

[0012] Furthermore, the compound shown in formula (I) is used in the preparation of drugs for the prevention or treatment of mental illnesses characterized by reduced expression or activity of the TGR5 receptor.

[0013] Furthermore, drugs prepared from compounds shown in formula (I) are used to treat mental illnesses, including neuronal damage, anxiety, depression, and other neurological disorders caused by inflammation, oxidative stress, and neuroexcitability damage.

[0014] Furthermore, the drug prepared from the compound shown in formula (I) has neuroprotective and repairing effects on glutamate-induced PC12 cell damage.

[0015] Furthermore, the compound of formula (I) or a pharmaceutically acceptable salt thereof may be used in the preparation of drugs for treating neurodegenerative diseases or for antiaging.

[0016] Furthermore, when a compound of formula (I) or a pharmaceutically acceptable salt thereof is used in the preparation of a medicament for the treatment and / or prevention of neurological diseases, it may also include a pharmaceutical excipient or a prodrug or solvate of the compound.

[0017] Furthermore, the antidepressant preparations made from the compounds shown in formula (I) are in the form of solution, colloidal solution, emulsion / suspension, oral instantaneous film, oral liquid, capsule, injection or transdermal absorption formulation.

[0018] The beneficial effects of this invention are: This invention provides compounds of formula (I) that can modulate TGR5 receptor activity. The effect of these compounds on the regulation of TGR5 receptor activity is disclosed for the first time.

[0019] This invention provides the use of the compound of formula (I) for the prevention or treatment of pathophysiological diseases characterized by reduced expression or activity of the TGR5 receptor. This is the first public disclosure of this use of the compound.

[0020] This invention provides the use of the compound shown in formula (I) in the preparation of medicaments for the treatment and / or prevention of neuroprotective / anxiety / depression and other neurological disorders. This is the first disclosure of the use of this compound in this respect, broadening the range of options for mental illnesses.

[0021] This invention provides a compound as shown in formula (I), abbreviated as L2301, which can significantly activate TGR5, cross the blood-brain barrier, and exhibit significant neuroprotective and repair functions. Simultaneously, animal experimental results show that L2301 demonstrates a good effect in improving depressive behavior. This compound has lower toxicity than the existing TGR5 agonist INT-777, and its antidepressant effect is comparable to fluoxetine. Therefore, L2301 is a novel TGR5 agonist with the potential to be developed into a novel antidepressant drug. Attached Figure Description

[0022] Figure 1 The diagram shows the docking results of compound L2301 with the TGR5 receptor, where A. is a three-dimensional diagram of the docking between compound L2301 and TGR5 protein; and B. is a two-dimensional diagram of the docking between compound L2301 and TGR5 protein.

[0023] Figure 2 The image shows the ADMET prediction results for compound L2301.

[0024] Figure 3The graph shows the results of the blood-brain barrier permeability detection of compound L2301. A and B are the HPLC chromatograms (A) and LC-MS chromatograms (B) of compound L2301 standard; C is the HPLC chromatogram of brain homogenate from mice after injection of L2301; and D is the HPLC chromatogram of brain homogenate from the control mouse group.

[0025] Figure 4 Compound L2301 dose-dependently activates TGR5, whose EC5 50 =12.5 μM, including: A. Functional assay of stable transgenic strains activated by the TGR5-specific agonist INT-777; B. Determination of the half-maximal effective concentration (HMC) of TGR5 activated by compound L2301.

[0026] Figure 5 To investigate the neuroprotective effect of compound L2301 on glutamate-damaged PC12 cells, the study included: A. Verification of the toxicity of compound L2301 on PC12 cells; B. The damaging effects of different concentrations of glutamate on PC12 cells; C. The protective effect of compound L2301 against glutamate (200 μM)-induced damage to PC12 cells. *P < 0.05 vs Control; # P < 0.05, ## P < 0.01 vs Glu.

[0027] Figure 6 The effect of compound L2301 on the morphological changes of PC12 damaged by glutamate.

[0028] Figure 7 In the open field experiment, compound L2301 improved CUMS-induced depressive behavior in mice, with the following results: A. There was no significant difference in the total distance traveled by mice in each group during the open field experiment; B. L2301 significantly increased the time spent in the central region by CUMS mice; C. L2301 significantly increased the distance traveled in the central region by CUMS mice. *P < 0.05 vs Control; # P < 0.05, ## P < 0.01 vs CUMS.

[0029] Figure 8 In the elevated cross maze test, compound L2301 improved CUMS-induced depressive behavior in mice. Specifically, A. L2301 significantly increased the proportion of mice remaining in the open arms during the CUMS test; B. L2301 significantly increased the proportion of mice entering the open arms during the CUMS test. *P < 0.05 vs Control; # P < 0.05 vs CUMS. Detailed Implementation

[0030] The present invention will be further described below with reference to the embodiments, but the present invention is not limited to the following embodiments.

[0031] Further explanation of the use of compound (Ⅰ) in regulating TGR5 receptor activity, preventing or treating pathophysiological diseases characterized by reduced TGR5 expression or activity, and in the preparation of drugs for the treatment and / or prevention of mental illness / neuroprotection / anxiety / depression.

[0032] Example 1. Dissolution and use of the compound; Compound (Ⅰ), abbreviated as L2301, was purchased from Shanghai Taoshu Biotechnology Co., Ltd. (Shanghai, China), catalog number: V023-3768, with a purity > 98%. The compound powder was dissolved in DMSO to prepare a 50 mM solution, which was then aliquoted and stored at -80°C for later use. Before use, it was diluted with physiological saline to the working concentration.

[0033] Example 2. Docking of compound L2301 with the TGR5 receptor; Experimental Methods: Using Discovery Studio 2021 software, the TGR5 protein crystal structure (PDB ID: 7CFN) downloaded from the PDB database was imported, and the structure of L2301 was plotted in Discovery Studio. First, the energy of the small molecule was minimized using the "PrepareLigands" module, resulting in 10 conformations. Then, the protein was dehydrated and hydrogenated using "Prepare Protein". Referring to existing literature, the active pocket of the receptor in the protein was defined, and the CDOCKER module in "Receptor-Ligand Interactions" was used to dock the TGR5 protein and compound L2301. After the program ran, the results were output, and the docking results were analyzed according to the docking mode and interaction mode.

[0034] Experimental Results: Docking results showed that the docking energy (CDOCKERENERGY) between compound L2301 and the receptor protein TGR5 was -6.0 kcal / mol, indicating high binding activity between the compound and the receptor. Compound L2301 formed hydrogen bonds with the amino acid residue Tyr 240 of the TGR5 protein, formed Pi-Pi stacking with Phe96, and formed Pi-Alkyl interactions with Leu71, Phe 161, Leu 166, Val170, Leu244, and Leu266, increasing the hydrophobic interaction between L2301 and the TGR5 receptor. A three-dimensional diagram of the interaction between compound L2301 and TGR5 is shown below. Figure 1A, a two-dimensional planar diagram of the ligand-protein interaction generated with the TGR5 receptor is shown below. Figure 1 B.

[0035] Example 3. Drug-likeness evaluation of the compound; Experimental Methods: Using the ADMET module in Discovery Studio software, the structure of compound L2301 was input. In the "Small Molecules" module, "Calculate Molecular Properties" was selected, and ADMET descriptors was clicked to set the parameters. After clicking run, the absorption, distribution, and toxicity of the compound were predicted, and data on the water solubility, passive intestinal absorption, blood-brain barrier penetration, and hepatotoxicity of compound L2301 at 25℃ were obtained.

[0036] Experimental Results: The water solubility value (logSW) of compound L2301 was -5.26, indicating that compound L2301 has good water solubility and is easily absorbed by the intestine. The blood-brain barrier permeability of compound L2301 was 0.586, demonstrating good blood-brain barrier penetration. The hepatotoxicity assay value of compound L2301 was -6.40, indicating that the compound has no hepatotoxicity. ADMET prediction results are shown below. Figure 2 .

[0037] Example 4. Detection of blood-brain barrier permeability of compound L2301; Experimental methods: Mice were administered 2 mg / kg of compound L2301 via the tail vein, while control mice were injected with the same volume of physiological saline via the tail vein. After 30 min, the mice were perfused with PBS, and subsequently, the brains were separated, weighed, and ground. Brain tissue samples were centrifuged and analyzed by LC-MS.

[0038] Experimental results: The peak elution time of the standard sample of compound L2301 was 1.91 min. After in vivo transport in mice, the peak elution time of compound L2301 was 1.98 min, comparable to that of the standard. The control group showed no peak at approximately 1.90 min, indicating that compound L2301 can cross the blood-brain barrier. (See attached figures). Figure 3 .

[0039] Example 5. Activation effect of compound L2301 on TGR5 receptor; Construction of stable TGR5 expression cell lines: HEK293 cells were cultured in DMEM containing 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in an incubator with 5% CO2. Subsequently, HEK293 cells were seeded into 60 mm culture dishes. The next day, when the cell density was approximately 70% and the cells were in good condition, liposome transfection was performed using Lipofectamine 2000 with 2 µg of hTGR5 plasmid (pRK-HA-human-TGR5) according to the manufacturer's instructions. After 48 hours, the transfected cell lines were sequentially screened using fresh medium containing 2 μg / mL puromycin and a flow cytometry sorter to obtain monoclonal cell lines.

[0040] Functional assay of TGR5 stably expressing cell lines: The TGR5-specific agonist INT-777 was prepared to a final concentration of 10... -10 M, 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, 10 -5 M, 10 -4 M. The cAMP Gs dynamic kit was prepared according to the manufacturer's protocol (PerkinElmer (Cisbio). TGR5 stably expressing cells were resuspended in 1×stimulation buffer + 500 μM 3-isobutyl-1-methylxanthine (IBMX). Approximately 8000 cells per well were added to 384-well plates at 5 μL / well, and INT-777 at different concentrations was added at 5 μL / well, with three replicates. The cells were stimulated at 37°C for 40 min. cAMP-d2 and cAMP-Eu were diluted with lysis buffer and added sequentially at 5 μL / well to the cells, and the reaction was carried out at room temperature for 1 h. Time-resolved fluorescence resonance energy transfer (HTRF) was detected using a multi-well microplate reader with excitation wavelength of 320 nm and emission wavelengths of 620 nm and 665 nm. The cAMP concentration was compared by calculating the 620 / 665 nm fluorescence ratio.

[0041] Experimental results: The TGR5-specific agonist INT-777 produces cAMP in a dose-dependent manner, with a calculated EC50 of 0.82 μM. (See attached table). Figure 4 Value A is close to the reported value in the literature, indicating that the TGR5 stably expressing cell line was successfully constructed.

[0042] The agonistic effect of L2301 on TGR5: The stock solution of compound L2301 was diluted 2-fold with physiological saline, and its activation effect on the TGR5 receptor was determined according to the manufacturer's protocol of the cAMP Gs dynamic kit (PerkinElmer (Cisbio)). The activation of TGR5 by compound L2301 showed a good concentration-dependent relationship. The calculated half-maximal effective concentration (IC50) of compound L2301 was 12.6 μM. (See attached table). Figure 4 B.

[0043] Example 6. Protective effect of compound L2301 against glutamate-induced PC12 damage; PC12 cell culture: Rat adrenal chromaffin cells PC12 were cultured in DMEM containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin in a constant temperature incubator containing 5% CO2 at 37°C.

[0044] Toxicity assay of the compound: PC12 cells in the logarithmic growth phase were evenly seeded in 96-well plates at a density of 5000 cells per well, with 100 μL of cell culture medium added to each well. After cell attachment, 100 μL of serum-free DMEM diluted with different concentration gradients of compound L2301 or blank culture medium was added to each well. Eight gradients and five replicates were set up in the experiment. The drug was prepared using a 2-fold dilution method, and a cell-free blank control group was also included. After drug addition, the cells were incubated in a cell culture incubator for 24 hours. Then, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for another 30-40 minutes. The absorbance at 450 nm was measured using a microplate reader, and the OD value was recorded. Cell viability % = (average OD of compound / average OD of blank solvent) × 100%.

[0045] To investigate the protective effect of L2301 against glutamate-induced PC12 cell damage: PC12 cells in the logarithmic growth phase were collected, digested, dispersed, and counted. Cells were seeded at a density of 5000 cells / well in 96-well plates (100 μL per well). After culturing at 37℃ and 5% CO2 for 24 h, the old medium was discarded, and glutamate (final concentration 200 μM) prepared in serum-free DMEM medium and different concentrations of L2301 were added for 24 h. 10 μL of CCK-8 was added to each well, and the cells were incubated together for 30 min. After shaking, the OD value was measured at 450 nm using a microplate reader. Cell viability % = (average OD compound / average OD blank solvent) × 100%.

[0046] Experimental Results: Compound L2301 only damaged PC12 cells at higher concentrations (50 μM and 100 μM), with inhibition rates of 24.7 ± 0.1% and 36.3 ± 0.6%, respectively. The inhibition rate of L2301 on PC12 cells was significantly lower than that of the positive control drug INT-777. Furthermore, L2301 showed a protective rate of 27.4 ± 0.3% against glutamate-damaged PC12 cells, which was superior to that of the positive control drug INT-777, indicating that the drug has fewer toxic side effects and possesses neuroprotective effects. (See attached figures). Figure 5 The morphological diagram of the protective effect of compound L2301 against glutamate-induced damage in PC12 cells is shown below. Figure 6 .

[0047] Example 7: Open field test and high-cost cross maze test to evaluate the antidepressant effect of compound L2301; Experimental animals: 48 male C57BL / 6 (8 weeks old) mice were purchased from the Experimental Animal Center of Air Force Medical University, weighing approximately 20-25g.

[0048] Experimental Methods: After one week of acclimatization, mice were randomly divided into 6 groups of 8 mice each. A mouse model of depression was established using chronic unpredictable mild stress (CUMS). Seven stimuli were administered: 5 min of swimming in 4°C ice water, 24 h of moist bedding, 24 h of fasting and water restriction, 5 min of tail clamping, 4 h of restraint in 50 mL centrifuge tubes (with several 2 mm diameter holes for ventilation), 12 h of nighttime flash lighting, and 12 h of nighttime noise playback. Mice were given one stimulus per day, with no consecutive administration of the same stimulus. The control group received no intervention. After 4 weeks, the model mice were divided into a model group, low, medium, and high L2301 groups, and a fluoxetine positive control group. Mice in the low, medium, and high L2301 groups and the fluoxetine group were administered the corresponding drugs by gavage according to their body weight. The control group and the model group were administered physiological saline by gavage once daily for two weeks. Behavioral tests were then performed.

[0049] Open field detection: Using the Tracking Master V3.0 data acquisition and analysis system from Shanghai Vanbi Intelligent Technology, mice in each group were placed in an open field (40×40×40 cm), and their spontaneous activities, dwell time in the central and peripheral areas, and movement distance were recorded and analyzed within 15 minutes.

[0050] Elevated cross maze test: Mice were placed facing open arms at the central intersection of an elevated cross maze. The Tracking Master V3.0 data acquisition and analysis system was used to record the mice's free movement trajectory over 5 minutes. The number of times the mice entered open arms and closed arms, and the dwell time in open and closed arms were analyzed. The percentage of time spent in open arms and the percentage of times the mice entered were calculated. Percentage of time spent in open arms (%) = (Open arm dwell time / (Open arm dwell time + Closed arm dwell time)) × 100%; Percentage of times the mice entered open arms (%) = Number of times the mice entered open arms / (Number of times the mice entered open arms + Number of times the mice entered closed arms) × 100%.

[0051] Experimental Results: Open field experiments showed no difference in the total movement distance among the six groups of mice, indicating that the spontaneous movement ability of the mice in each group was not impaired. However, compared with the normal group, the number of times the CUMS model mice entered the central region and the time spent in the central region were both reduced. Medium and high doses of compound L2301 significantly increased the number of times CUMS mice entered the central region; the high-dose group, like fluoxetine, increased the time spent in the central region in depressed mice, and the effect of the high-dose group was similar to that of fluoxetine. High-valence maze experiments showed that the proportion of time spent in the open arms and the proportion of times the CUMS mice entered the open arms were significantly lower than those in the normal group. Compared with the CUMS group, the high-dose group of L2301 significantly increased the proportion of time spent in the open arms and the number of times the CUMS mice entered the open arms, and the improvement effect of the high-dose group in the high-valence maze was comparable to that of fluoxetine.

Claims

1. The use of a compound of formula (I) in the preparation of a neuroprotective drug for treating depression or glutamate-induced neuronal damage, the compound having the following structural formula (I): （Ⅰ）； The depression mentioned is chronic, unpredictable, mild stress-induced depression; the glutamate-induced neuronal damage mentioned is glutamate-induced PC12 cell damage.

2. An application according to claim 1, characterized in that, The compound shown in formula (I) exerts its therapeutic effect on depression and its neuroprotective effect against glutamate-induced neuronal damage by activating the TGR5 receptor.

3. An application according to claim 1, characterized in that, The compound shown in formula (Ⅰ) has neuroprotective and cell repair activities against glutamate-induced PC12 cell damage.

4. An application according to claim 1, characterized in that, The drug is formulated as a solution, colloidal solution, emulsion, or suspension.

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