Novel fatty acid amide hydrolase (FAAH) inhibitor and application thereof in disease treatment

Abstract

The invention discloses a novel potent inhibitor pyridone compound (formula I) of fatty acid amide hydrolase (FAAH) and a treatment effect of the novel potent inhibitor pyridone compound in related diseases, and belongs to the technical field of biological medicines. The biological activity of fatty acid amide hydrolase (FAAH) can be inhibited, the physiological concentration of endogenous arachidonic acid ethanolamine is increased, and the effects of regulating immunity, resisting inflammation and the like are achieved; taking inflammatory bowel disease as an example, the inhibitor can significantly improve various pathological and physiological indexes of an animal level in an inflammatory bowel disease (IBD) model and slow down the progress of the disease course, and is effectively used for treating various types of inflammatory bowel diseases (IBD) including ulcerative colitis (UC) and Crohn disease (CD).

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to pyridone compounds as represented by formula (I), and isotope-labeled substances, optical isomers, and pharmaceutically acceptable salts thereof, pharmaceutical compositions containing the same, and their use as fatty acid amide hydrolase (FAAH) inhibitors in the preparation of drugs for treating or preventing inflammatory bowel disease and neurodegenerative diseases, including peripheral inflammatory diseases, nervous system diseases, and immune-related diseases.

[0002]

[0003] The chemical structure of compound I. Background Art

[0004] FAAH, also known as fatty acid amide hydrolase, is widely expressed in various organs, with the liver, intestines, and brain being the most abundant. Anandamide (an endogenous cannabinoid-like substance) is a chemical substance similar to marijuana secreted endogenously by the human body. It can participate in regulating peripheral inflammation and the occurrence and development of neurodegenerative diseases such as depression, Parkinson's disease, and Alzheimer's disease by stimulating endogenous cannabinoid receptors (Cannabinoid Receptor 1 / 2, CB1 / 2) and temporal receptor potential vanilloid receptor (TRPV1), and has strong biological activity. However, FAAH can rapidly hydrolyze and inactivate anandamide, weakening its biological regulatory effects. It is then metabolized to produce the inflammatory mediator arachidonic acid, which promotes the progression of inflammation in the peripheral and central nervous systems. The mechanism of action of FAAH is shown in Formula II:

[0005]

[0006] Formula II FAAH catalyzes the metabolic inactivation mechanism of anandamide.

[0007] Therefore, inhibiting the biological activity of FAAH is of great significance for the development and progression of inflammation in the peripheral and central nervous systems. Developing potent inhibitors of FAAH will provide drug candidates for the treatment and prevention of various inflammatory diseases.

[0008] Through preliminary screening, the present invention has identified a potent FAAH inhibitor, a pyridone compound (Compound I) with the general structure shown in Formula (I). It has a high affinity for the FAAH protein, with a Kd value of 700 nanomolar. It can inhibit the metabolic process of FAAH hydrolysis of endocannabinoids, increasing endocannabinoid levels and reducing arachidonic acid levels. This, in turn, improves the body's immune status and inflammation levels. In animal models, using DSS-induced ulcerative colitis as an example, Compound I significantly inhibited the expression of inflammatory factors such as IL-1β, IL-6, and TNF-α in DSS-induced ulcerative colitis mice. It also acts on the NLRP3-Caspase1-IL-1β pathway to alleviate colonic tissue damage during IBD and accelerate the repair of colonic epithelial cells in mice with IBD, demonstrating excellent biological activity. Compound I also demonstrated a promising therapeutic effect in a TNBS-induced rat model of Crohn's disease. Therefore, the discovery of a novel FAAH inhibitor, compound I, provides a novel drug candidate for the treatment of multiple types of inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn's disease (CD). Summary of the Invention

[0009] The purpose of the present invention is to provide a fatty acid amide hydrolase (FAAH) inhibitor such as a pyridone compound represented by formula (I) and to verify the application of FAAH inhibition in related diseases.

[0010]

[0011] in,

[0012] R1 is selected from one of H, C1-C3 alkyl, C1-C3 perfluoroalkyl, C3-C5 cycloalkyl, 5-membered or 6-membered heteroaryl, CN, OH, COOH, or halogen;

[0013] R2 is selected from one of H, C1-C3 alkyl, C1-C3 perfluoroalkyl, C3-C5 cycloalkyl, 5-membered or 6-membered heteroaryl, all of which may be optionally substituted with methyl or hydroxyl;

[0014] R3 is selected from H, C1-C3 alkyl, C1-C3 perfluoroalkyl, C3-C5 cycloalkyl, 5-membered or 6-membered heteroaryl, or benzyl, all of which may be optionally substituted with methyl or halogen;

[0015] R4 is selected from H, C1-C5 alkyl, C1-C3 perfluoroalkyl, 5-membered or 6-membered aryl, 5-membered or 6-membered heteroaryl, benzyl, 2-hydroxyethyl or acetyl, tert-butyl, 2-pyridyl, 1-imidazolyl, 1-methyl-2-imidazolyl, or 2-methyl-1-imidazolyl, all of which may be optionally substituted with methyl or hydroxyl.

[0016] Results are selected from:

[0017] 2-[(1-{1-[(4-fluorophenyl)methyl]-1H-benzo[d]imidazol-2-yl}piperidin-4-yl)(methyl)amino]pyrimidin-4(3H)-one;

[0018] 2-[(1-{1-[(2-fluorophenyl)methyl]-1H-benzo[d]imidazol-2-yl}piperidin-4-yl)(methyl)amino]pyrimidin-4(3H)-one;

[0019] 2-[(1-{1-[(4-chlorophenyl)methyl]-1H-benzo[d]imidazol-2-yl}piperidin-4-yl)(methyl)amino]pyrimidin-4(3H)-one;

[0020] 2-[(1-{1-[(2-chlorophenyl)methyl]-1H-benzo[d]imidazol-2-yl}piperidin-4-yl)(methyl)amino]pyrimidin-4(3H)-one;

[0021] 2-[(Methyl)(1-{1-[(4-methylphenyl)methyl]-1H-benzo[d]imidazol-2-yl}piperidin-4-yl)amino]pyrimidin-4(3H)-one;

[0022] 2-[(Methyl)(1-{1-[(2-methylphenyl)methyl]-1H-benzo[d]imidazol-2-yl}piperidin-4-yl)amino]pyrimidin-4(3H)-one;

[0023] 2-[(1-{1-[(phenyl)methyl]-1H-benzo[d]imidazol-2-yl}piperidin-4-yl)(methyl)amino]pyrimidin-4(3H)-one.

[0024] The pyridone compound (I) is 2-[(1-{1-[(4-fluorophenyl)methyl]-1H-benzo[d]imidazol-2-yl}piperidin-4-yl)(methyl)amino]pyrimidin-4(3H)-one (Compound II).

[0025] The present invention provides a FAAH small molecule inhibitor, the chemical structure of which is shown in formula (I), the IC50 of which is 418 nM for inhibiting FAAH from hydrolyzing anandamide, and the affinity with the FAAH protein is 700 nM.

[0026] The present invention also provides a pharmaceutical composition for treating or preventing diseases associated with FAAH, which contains a pyridone compound (I) or its isotope-labeled substance, optical isomer, pharmaceutically acceptable salt, and a pharmaceutically acceptable diluent or carrier.

[0027] The present invention also provides a method for preparing a pharmaceutical composition for treating or preventing diseases associated with FAAH, which comprises preparing a pyridone compound (I) or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable diluent or carrier.

[0028] The present invention also provides a method for treating or preventing diseases associated with FAAH, which comprises treating with an effective amount of a pyridone compound (I) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing any of them.

[0029] Furthermore, the above diseases can be prevented and treated by inhibiting FAAH.

[0030] Furthermore, the above diseases are inflammatory bowel disease, neurodegenerative diseases, peripheral inflammatory diseases, nervous system inflammatory diseases and immune diseases.

[0031] Furthermore, the above disease is DSS / TNBS-induced inflammatory bowel disease (IBD).

[0032] Furthermore, the above-mentioned diseases are Crohn's disease, ulcerative colitis, Parkinson's disease, Alzheimer's disease, neurodegenerative diseases, morphine withdrawal, acute and chronic liver damage, acute and chronic kidney damage, non-alcoholic steatohepatitis (NASH), psoriasis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, acute lung injury, diabetes, colorectal cancer or pancreatic cancer, etc.

[0033] The present invention discloses the following technical effects:

[0034] During early drug screening research, the inventors discovered a potent FAAH enzyme inhibitor, a pyridone compound (I). They hypothesized that the inhibitor may inhibit FAAH by increasing endogenous anandamide levels in the body, thereby exerting anti-inflammatory, immune, and mood-regulating effects. The present invention demonstrates that the pyridone compound (I) alone can inhibit FAAH and exert anti-inflammatory and immune-regulating effects in animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 .Inhibitory activity of compound II against FAAH

[0036] Figure 2 .Inhibition type of compound II on FAAH

[0037] Figure 3 Compound II alleviated the apparent factors such as colon tissue length, DAI score, and body weight changes in DSS-induced IBD mice

[0038] Figure 4Colonoscopy observation of compound II on the effects of DSS-induced IBD mice

[0039] Figure 5 .Study on the pathological changes of colon tissue sections of DSS-induced IBD mice by compound II

[0040] Figure 6 Determination of endogenous cannabinoid content in the intestine of mice after intervention with compound II

[0041] Figure 7 Compound II regulates inflammation-related mRNA in colon tissue of DSS-induced IBD mice DETAILED DESCRIPTION

[0042] Example 1 Inhibitory effect of compound II on FAAH and inhibition mode of compound II

[0043] The present invention comprehensively evaluates the inhibitory effect and inhibition mode of compound II on FAAH. The incubation system is 200 μL, which contains 100 mM potassium phosphate buffer (KH2PO4 / K2HPO4, pH = 7.4), fatty acid amide hydrolase (FAAH), 5 μM deuterated endocannabinoids, and compound II at 0, 10, 20, 50, 100, 200, 500, 1000, 2000, and 5000 nmol. The volume of organic solvent in the reaction system is ≤1%. After pre-incubation for 3 minutes at 37°C, the deuterated endocannabinoids are added to initiate the reaction. After incubation for 30 minutes, 100 μL of glacial acetonitrile is added to terminate the reaction. The reaction is centrifuged at 4°C, 20,000 g for 20 minutes, and the supernatant is analyzed by LC-MS. All incubation reactions are repeated three times. Incubation samples without FAAH and Compound II were used as blank controls to ensure that the metabolites were deuterated endocannabinoids and FAAH-dependent. Figure 1 As shown: IC50 of compound II is 418 micromolar. It is a kinetic combination diagram of the inhibition of FAAH by compound II, as shown Figure 2 As shown: According to the Michaelis theory combined with LB plot analysis, the focus of the double reciprocal method of the LB plot falls on the second quadrant. Based on the above inhibition kinetic characteristics, it is determined that the metabolic inhibition of compound II on FAAH is a mixed inhibition.

[0044] Example 2 Treatment of disease progression of DSS-induced IBD mice by compound II

[0045] After 6-8 week old mice were acclimated to the environment in the cages of the IVC independent circulating air system for one week, they were fully adapted to the new environment and then grouped according to their weight into compound II treatment group, modeling group and blank control group, with 6 mice in each group. The compound II treatment group was given 5 mg / kg compound II by gavage for three days of pre-protection, and then they and the modeling group were given 3% DSS solution to drink freely to induce inflammatory bowel disease. The treatment group was given 5 mg / kg by gavage every day, and the modeling group was given an equal dose of solvent by gavage. The drinking of DSS was stopped on the 7th day, and boiled water was changed to free drinking while the drug was given. The mice were killed on the 9th day for sampling, and the weight changes and DAI scores of the mice were recorded during the process. The colon length was measured, and the results are as follows Figure 3 As shown, the Compound II treatment group was able to improve the rate of weight loss and the DAI score index, and slow down the amount of colon shortening, indicating that Compound II can effectively alleviate the progression of IBD.

[0046] Example 3 Evaluation of Compound II on Tissue Levels in Models

[0047] The extent of inflammation was determined by colon endoscopy. After the inflammation site was fixed, the sections were embedded in paraffin and stained for pathology (H&E staining). Figure 4-5 As shown: Histopathological analysis showed that compound II treatment can improve the damage to the colon epithelium during DSS modeling and protect the integrity of the intestine; this can well demonstrate the therapeutic effect of compound II on inflammatory bowel disease.

[0048] Example 4 Determination of AEA after the action of compound II

[0049] 50 mg of mouse intestinal tissue was added with 300 μl of triple-distilled water and steel balls, and the tissue was homogenized for 10 minutes in a tissue homogenizer. After that, 300 μl of acetonitrile was added for extraction three times. The extracts were combined, centrifuged and concentrated to dryness. The precipitate was re-dissolved with 200 μl of methanol-water (methanol:water=7:3), centrifuged at 20,000 g for 20 minutes, and the supernatant was used for LC-MS analysis to measure the AEA content in the tissue. Figure 6 As shown, the content of endogenous cannabinoids in the intestinal tissue of the drug-treated group increased, further proving that compound II can inhibit FAAH to increase the level of AEA in the body and thus reduce the occurrence and development of inflammation.

[0050] Example 5: Regulatory effect of Compound II on inflammatory-related factors in mouse colon tissue after treatment

[0051] Inflammation is mainly caused by the increased expression of inflammatory-related proteins in the body. Inhibiting the abnormal overexpression of inflammatory factors is one of the important ways to alleviate inflammation. In order to verify the regulatory effect of compound II on inflammatory factors, we used qPCR technology to measure the mRNA of inflammatory-related factors in the colon of IBD mice. The results are as follows: Figure 7As shown, compound II can significantly inhibit the mRNA expression levels of inflammatory factors such as IL-1β, IL-6, and TNF-α, thereby alleviating inflammatory infiltration and tissue damage in inflammatory bowel disease; this also provides a factual basis for its treatment of inflammatory bowel disease.

Claims

1. A pyridone compound represented by formula (I), or its isotope-labeled substance, optical isomer, pharmaceutically acceptable salt, or a pharmaceutical composition containing any one of them, as a novel potent inhibitor of fatty acid amide hydrolase (FAAH). in, R1 is selected from one of H, C1-C3 alkyl, C1-C3 perfluoroalkyl, C3-C5 cycloalkyl, 5-membered or 6-membered heteroaryl, CN, OH, COOH, or halogen; R2 is selected from one of H, C1-C3 alkyl, C1-C3 perfluoroalkyl, C3-C5 cycloalkyl, 5-membered or 6-membered heteroaryl, all of which may be optionally substituted with methyl or hydroxyl; R3 is selected from H, C1-C3 alkyl, C1-C3 perfluoroalkyl, C3-C5 cycloalkyl, 5-membered or 6-membered heteroaryl, or benzyl, all of which may be optionally substituted with methyl or halogen; R4 is selected from H, C1-C5 alkyl, C1-C3 perfluoroalkyl, 5-membered or 6-membered aryl, 5-membered or 6-membered heteroaryl, benzyl, 2-hydroxyethyl or acetyl, tert-butyl, 2-pyridyl, 1-imidazolyl, 1-methyl-2-imidazolyl, or 2-methyl-1-imidazolyl, all of which may be optionally substituted with methyl or hydroxyl.

2. The method according to claim 1, wherein The pyridone compound is selected from the group consisting of: 2-[(1-{1-[(4-fluorophenyl)methyl]-1H-benzo[d]imidazol-2-yl}piperidin-4-yl)(methyl)amino]pyrimidin-4(3H)-one; 2-[(1-{1-[(2-fluorophenyl)methyl]-1H-benzo[d]imidazol-2-yl}piperidin-4-yl)(methyl)amino]pyrimidin-4(3H)-one; 2-[(1-{1-[(4-chlorophenyl)methyl]-1H-benzo[d]imidazol-2-yl}piperidin-4-yl)(methyl)amino]pyrimidin-4(3H)-one; 2-[(1-{1-[(2-chlorophenyl)methyl]-1H-benzo[d]imidazol-2-yl}piperidin-4-yl)(methyl)amino]pyrimidin-4(3H)-one; 2-[(methyl)(1-{1-[(4-methylphenyl)methyl]-1H-benzo[d]imidazol-2-yl}piperidin-4-yl)amino]pyrimidin-4(3H)-one; 2-[(Methyl)(1-{1-[(2-methylphenyl)methyl]-1H-benzo[d]imidazol-2-yl}piperidin-4-yl)amino]pyrimidin-4(3H)-one; 2-[(1-{1-[(phenyl)methyl]-1H-benzo[d]imidazol-2-yl}piperidin-4-yl)(methyl)amino]pyrimidin-4(3H)-one.

3. The use according to claim 1 or 2, characterized in that The inhibitory activity IC of the pyridone compounds on FAAH 50 ≤500nM, and the affinity Kd value for FAAH protein is not less than 700nM.

4. A pharmaceutical composition for treating or preventing a disease associated with FAAH, comprising a pyridone compound according to any one of claims 1 or 2 or an isotope-labeled substance, optical isomer, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent or carrier.

5. A method for preparing a pharmaceutical composition for treating or preventing a disease associated with FAAH, which comprises formulating the pyridone compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 or 2 with a pharmaceutically acceptable diluent or carrier.

6. A method for treating or preventing a disease associated with FAAH, which comprises treating with an effective amount of a pyridone compound according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing any one of them.

7. The composition or method according to any one of claims 4 to 6, characterized in that The disease is prevented or treated by inhibiting FAAH.

8. The composition or method of claim 7, wherein The disease is selected from the group consisting of inflammatory bowel disease, neurodegenerative disease, peripheral inflammatory disease, nervous system inflammatory disease and immune disease.

9. The composition or method of claim 8, wherein The disease is DSS / TNBS-induced inflammatory bowel disease (IBD).

10. The composition or method of claim 9, wherein The diseases include Crohn's disease, ulcerative colitis, Parkinson's disease, Alzheimer's disease, neurodegenerative diseases, morphine withdrawal, acute and chronic liver damage, acute and chronic kidney damage, non-alcoholic steatohepatitis (NASH), psoriasis, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, acute lung injury, diabetes, colorectal cancer or pancreatic cancer, etc.

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