A fatty acid amide hydrolase (FAAH) inhibitor and its use in the treatment of disease

Abstract

The application discloses a novel and powerful inhibitor of fatty acid amide hydrolase (FAAH), pyridinone compound (formula I) and therapeutic effect thereof in related diseases, and belongs to the technical field of biological medicine. The inhibitor can inhibit the biological activity of fatty acid amide hydrolase (FAAH), increase the physiological concentration of endogenous arachidonic acid ethanolamine, and play the roles of regulating immunity and resisting inflammation; for example, in inflammatory bowel disease, the inhibitor can significantly improve various pathological and physiological indexes of animals in an inflammatory bowel disease (IBD) model, slow down the progress of the disease, and be effectively used for treating multiple types of inflammatory bowel disease (IBD) including ulcerative colitis (UC) and Crohn's disease (CD).

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to pyridone compounds as shown in formula (I) and their isotopic labels, optical isomers, pharmaceutically acceptable salts, pharmaceutical compositions comprising them, and their use as fatty acid amide hydrolase (FAAH) inhibitors in the preparation of medicaments for the treatment or prevention of inflammatory bowel disease and neurodegenerative diseases, including peripheral inflammatory diseases, nervous system diseases and immune-related diseases.

[0002]

[0003] Figure I shows the chemical structural formula of compound I. Background Technology

[0004] FAAH, also known as fatty acid amide hydrolase, is widely expressed in various organs, especially abundant in the liver, intestines, and brain. Arachidonic acid ethanolamine (an endocannabinoid-like substance) is a cannabis-like chemical substance secreted endogenously in the human body. It can participate in the regulation of peripheral inflammation and the occurrence and development of neurodegenerative diseases such as depression, Parkinson's disease, and Alzheimer's disease by stimulating endocannabinoid receptors (CB1 / 2) and cischronosensitive potential vanillic acid receptor (TRPV1), exhibiting strong biological activity. However, FAAH can rapidly hydrolyze and inactivate arachidonic acid ethanolamine, weakening its biological regulatory effects, and subsequently metabolizing it to produce the inflammatory mediator arachidonic acid, promoting the progression of peripheral and central nervous system inflammation. The mechanism of action of FAAH is as follows: Figure I As shown in Figure I:

[0005]

[0006] Figure II. Mechanism of FAAH-catalyzed metabolic inactivation of arachidonic acid ethanolamine.

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

[0008] This invention patent obtained a potent FAAH inhibitor through preliminary screening. The general structure of the pyridone compound (Compound I), as shown in formula (I), exhibits a high affinity for FAAH protein, with a Kd value of 700 nanomolar. It can inhibit the metabolic process of FAAH hydrolyzing endocannabinoids, increasing the content of endocannabinoids and decreasing the level of arachidonic acid. This, in turn, improves the body's immune status and inflammation level. In animal models, taking DSS-induced ulcerative colitis as an example, Compound I significantly inhibited the expression levels of inflammatory factors such as IL-1β, IL-6, and TNF-α in DSS-induced mice, acting 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 good biological activity. Simultaneously, Compound I also showed good therapeutic effects on a TNBS-induced rat Crohn's disease model. Therefore, the discovery of novel FAAH inhibitor compound I provides a novel drug candidate for the treatment of multiple types of inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD). Summary of the Invention

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

[0010]

[0011] in,

[0012] R1 is selected from 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 H, C1-C3 alkyl, C1-C3 perfluoroalkyl, C3-C5 cycloalkyl, 5-membered or 6-membered heteroaryl, 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, which may optionally be substituted with methyl or halogen;

[0015] R4 is selected from H, C1-C5 alkyl, C1-C3 perfluoroalkyl, 5- or 6-membered aryl, 5- 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 optionally be 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] This invention provides a small molecule inhibitor of FAAH, the chemical structure of which is shown in formula (I). Its IC50 for inhibiting the hydrolysis of arachidonic acid ethanolamine by FAAH is 418 nM, and its affinity for FAAH protein is 700 nM.

[0026] The present invention also provides a pharmaceutical composition for treating or preventing diseases associated with FAAH, comprising a pyridone compound (I) or an isotope thereof, an optical isomer, a 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 related to FAAH, comprising formulating 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, including treatment with an effective amount of a pyridone compound (I) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing any one thereof.

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

[0030] Furthermore, the aforementioned diseases include inflammatory bowel disease, neurodegenerative diseases, peripheral inflammatory diseases, systemic inflammatory diseases, and immune diseases.

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

[0032] Furthermore, the aforementioned diseases include Crohn's disease, ulcerative colitis, Parkinson's disease, Alzheimer's disease, neurodegenerative diseases, morphine withdrawal, acute and chronic liver injury, acute and chronic kidney injury, non-alcoholic lipohepatitis (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 preliminary drug screening studies, the inventors of this invention discovered pyridone compounds (I), potent enzyme inhibitors of FAAH, and hypothesized that they might exert anti-inflammatory, immunomodulatory, mood-regulating, and emotional effects by inhibiting FAAH and increasing the level of endogenous arachidonic acid ethanolamine in the body. This invention demonstrates that pyridone compounds (I) alone can inhibit FAAH and exert anti-inflammatory and immunomodulatory effects in animals. Attached Figure Description

[0035] Figure 1 Compound II's inhibitory activity against FAAH

[0036] Figure 2 The type of inhibition of FAAH by compound II

[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 4Observation of the effects of compound II on DSS-induced IBD in mice via colonoscopy

[0039] Figure 5 Pathological study of colon tissue sections from DSS-induced IBD mice using compound II

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

[0041] Figure 7 Compound II regulates inflammation-related mRNAs in the colonic tissue of DSS-induced IBD mice. Detailed Implementation

[0042] Example 1: Inhibitory effect of compound II on FAAH and the inhibition mechanism of compound II

[0043] This invention comprehensively evaluates the inhibitory effect and mechanism of compound II on FAAH. The incubation system was 200 μL, containing 100 mM potassium phosphate buffer (KH₂PO₄ / K₂HPO₄, pH 7.4), fatty acid amide hydrolase (FAAH), 5 μM of deuterated endocannabinoid, and nanomolar amounts of compound II at concentrations of 0, 10, 20, 50, 100, 200, 500, 1000, 2000, and 5000 nmol. The volume of organic solvent in the reaction system was ≤1%. The reaction was initiated by adding deuterated endocannabinoid after a 3-minute pre-incubation at 37°C. The reaction was terminated by adding 100 μL of ice-cold acetonitrile after 30 minutes of incubation. The mixture was centrifuged at 20000g for 20 minutes at 4°C, and the supernatant was analyzed by LC-MS. All incubation reactions were repeated three times. A blank control was used, containing no FAAH or compound II, to ensure that the metabolites were deuterated endocannabinoids and FAAH-dependent. Figure 1 As shown: The IC50 of compound II is 418 μmol. The kinetic combination diagram of compound II's inhibition of FAAH is shown below. Figure 2 As shown: Based on the Michaelis-Menten theory and LB plot analysis, the focus of the LB plot using the double reciprocal method falls in the second quadrant. Considering the above characteristics of inhibition kinetics, it is determined that the metabolic inhibition of FAAH by compound II is a mixed type of inhibition.

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

[0045] Six- to eight-week-old mice were acclimatized to their new environment for one week in cages with an independent circulating air system in an IVC (Independent Ventilation Control Center). Once fully adapted, the mice were divided into three groups based on body weight: a compound II treatment group, a model group, and a blank control group, with six mice in each group. The compound II treatment group received 5 mg / kg of compound II via gavage for three days as a pre-protection measure. Then, both the treatment and model groups were given free access to 3% DSS solution for inflammatory bowel disease induction. The treatment group received 5 mg / kg of compound II via gavage daily, while the model group received the same dose of the solvent via gavage. On day 7, DSS access was discontinued, and the mice were given free access to cooled boiled water while receiving the compound II treatment. Mice were sacrificed on day 9 for tissue analysis. Changes in mouse body weight and DAI scores were recorded throughout the process. Colon length was measured, and the results are as follows: Figure 3 As shown, the compound II treatment group improved the rate of weight loss and the DAI score index, and slowed the amount of colonic shortening. This indicates that compound II can effectively alleviate the progression of IBD.

[0046] Example 3: Evaluation of Compound II at the Tissue Level in the Model

[0047] The degree of inflammation was determined by colonoscopy. After the inflamed area was fixed, it was embedded in paraffin and sectioned for pathological staining (H&E staining). The results are as follows: Figure 4-5 As shown: Histopathological analysis revealed that compound II treatment could improve the damage to the colonic epithelium during DSS modeling and protect the integrity of the intestine; this provides strong evidence for the therapeutic effect of compound II on inflammatory bowel disease.

[0048] Example 4: Determination of AEA after treatment with compound II

[0049] 50 mg of mouse intestinal tissue was added to 300 μL of triple-distilled water and a steel ball. The tissue was homogenized at low temperature for 10 minutes using a tissue homogenizer. Then, 300 μL of acetonitrile was added for extraction three times. The extracts were combined, centrifuged, concentrated, and dried. The precipitate was reconstituted with 200 μL of methanol-water mixture (methanol:water = 7:3), centrifuged at 20000g for 20 minutes, and the supernatant was analyzed by LC-MS to measure the AEA content in the tissue. Figure 6 As shown, the content of endogenous cannabinoids in the intestinal tissue of the treatment group increased, further demonstrating that compound II can inhibit FAAH from increasing the level of AEA in the body and thus reduce the occurrence and development of inflammation.

[0050] Example 5: Regulation of inflammation-related factors in mouse colon tissue after treatment with compound II

[0051] Inflammation is mainly caused by the increased expression of inflammation-related proteins in the body, and inhibiting the abnormal overexpression of inflammatory factors is one of the important ways to alleviate inflammation. To verify the regulatory effect of compound II on inflammatory factors, we used qPCR to measure the mRNA of inflammation-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. The use of a fatty acid amide hydrolase (FAAH) inhibitor in the preparation of a medicament for the prevention and / or treatment of inflammatory bowel disease (IBD), characterized in that, The FAAH inhibitor is 2-[(1-{1-[(4-fluorophenyl)methyl]-1H-benzis[d]imidazol-2-yl}piperidin-4-yl)(methyl)amino]pyrimidin-4(3H)-one, or a pharmaceutically acceptable salt thereof.

2. The use according to claim 1, characterized in that, The inhibitory activity of the pyridone compounds against FAAH is IC50. 50 ≤500nM, affinity for FAAH protein K d The value is not less than 700 nM.

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